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| InsectaCentral ID | Study Name | Study Type | Study Description | Publication repository | Repository (e.g. PubMed) ID | Author Last Name | Author First name | Target Name | Target sequence | Target Taxonomic Order | Target Taxonomic family | Target Taxonomic genus | Target Taxonomic species | Target Taxonomy NCBI ID | Target alignment region | Target Repository Name | Target Repository Accession | Construct Name | ConsTruCTSequenCe | Construct type | Construct in-vitro method | Construct purification method | Construct annealing method | Construct protocol | Construct Repository Name | Construct Repository ID | Animal Tax Order | Animal Tax family | Animal Tax genus | Animal Tax species | Animal Tax NCBI id | Animal Stock Center | Animal Dev stage | Colony Infection | Indiv Infection | Colony Origin | Delivery Construct Concentration (µg/µl) | Delivery method | Delivery buffer | Delivery adjuvant name | Delivery adjuvant amnt (µl/mg insect) | Delivery Control method | Delivery replicates | Delivery organ | Control method detail | Assay dev stage | Assay detection method | Assay tissue | Assay processing detection | Assay detection time (hours) | Assay average silencing | Assay accurate silencing | |
| An internal ID which is publicly accessible at http://insectacentral.org/genes4all/experiment/view/ + ID | A unique name provided by the submitter | The type of study. Currently only RNAi | A description of the study, as provided by the submitter. In the case of published work, this is the abstract. | If published, this is the NCBI PubMed ID | Last name of communicating author | First name of communicating author | Unless explicitly set by the submitter, this is derived from the study name | The sequence of the target gene | The phylogenetic order of the species used to acquire the target gene | The phylogenetic family of the species used to acquire the target gene | The phylogenetic genus of the species used to acquire the target gene | The species epithet of the species used to acquire the target gene | The NCBI taxonomy identifier of the species used to acquire the target gene | Which part of the target gene aligns to the RNA construct | Publicly accessible location to acquire the target gene information | The accession number of the relevant repository | A unique name for the RNA construct; unless explicitly set by the submitter it is derived from the Study Name | RNAConsTruCTsequenCeinDNAalphabeT;CaseswiThouTasequenCeviolaTeTheMIARErequiremenTs | What type of RNA construct | If the construct was constructed in-vitro with a kit | How was the construct purified | How was the construct allowed to anneal to the target gene message | The detailed protocol for generating the RNA construct | If the construct is available on a public repository | The identifier in the relevant repository | The phylogenetic order of the experimental animals | The phylogenetic family of the experimental animals | The genus name of the experimental animals | The species name of the experimental animals | The NCBI taxonomy ID of the experimental animals | If the animals were derived from a stock center | Developmental stage of the experimental animals | If the colony, from which the experimental animals are derived, was infected and the infection identified. Unknown relates to cases where infection existed but the nature was unknown | Whether the actual experimental individual was infected | The origin of the experimental: directly collected from the field or an established laboratory colony | The concentration of the construct in the buffer | Method of delivery | Buffer used in delivery | If an additional adjuvant was used to facilitate delivery | Amount/concentration of adjuvant used | If and how a control was implemented | how many replicates, if any, were attempted with the same conditions | Experimental animals target organ. | Detailed control protocol | The developmental stage at which the detection assay was performed | What type of method was used to detect transcriptional differences | Which tissue was used in the assay | Was the processing of the construct checked. If it was, was it detected? | After how many hours past construct delivery was the assay performed? | A subjective measure of silencing as provided by the author when exact details are no longer available | If available, an accurate measure of transcription reduction using the assay method | ||
| 3 | Silencing of Hemolin in Hyalophora cecropia development | RNAi | There is increasing evidence of an intimate connection between participants in the innate immune system and in development. Molecules involved in the determination of dorso-ventral polarity in Drosophila have related counterparts in the signalling pathways for immune gene activation in both insects and mammals. Hemolin from the Giant silkmoth, Hyalophora cecropia, identified as a bacteria-inducible molecule and a member of the immunoglobulin superfamily, is present as protein and transcripts in oocytes and embryos. We used RNA interference (RNAi) to investigate H. cecropia gene function in vivo and demonstrated that Hemolin is crucial for the normal development of embryos. When RNAi-females were mated, no larvae emerged from their eggs and when dissected, the eggs revealed malformed embryos. Western blot analysis confirmed the lack of Hemolin gene products. We conclude that Hemolin is necessary for development, since the silencing of Hemolin gene expression leads to embryonic lethality. | pubmed | 12000646 | Faye | Ingrid | Silencing of Hemolin in Hyalophora cecropia development target | CTGGAGTATAAGGCACGGGTACACAATGGCGTTCAAGAGTATAGCAGTTTTAAGCGCCTGCATAATTGTGGGTTCAGCGCTTCCCGTGGATAAATACCCGGTGCTGAAAGACCAGCCGGCCGAGGTTCTCTTCAGGGAGAATAACCCAACGGTCCTCGAATGTATCATCGAAGGGAACGATCAGGGAGTCAAGTACTCCTGGAAAAAGGATGGGAAATCCTACAACTGGCAGGAGCATAACGCCGCTCTTCGCAAGGATGAAGGATCTTTGGTATTCCTGAGACCGCAAGCCTCAGACGAGGGTCACTACCAGTGCTTCGCTGAAACCCCAGCCGGTGTTGCCAGCTCGAGGGTGATCAGCTTCAGGAAGACTTACCTAATCGCGTCGCCAGCAAAGACACACGAGAAAACGCCAATCGAAGGCAGGCCTTTCCAATTGGACTGCGTCCTCCCTAACGCTTACCCTAAACCCTTGATTACTTGGAAGAAACGTTTGTCCGGAGCCGATCCTAACGCTGACGTGACTGACTTTGATCGCCGCAATCACAGCTGGACTGACGGAAACCTCTACTTTACAATCGTCACTAAAGAGGACGTCAGTGACATTTATAAATACGTATGCACCGCCAAGAACGCAGCGGTTGACGAAGAGGTAGTTTTGGTGGAGTATGAAATCAAGGGAGTGACAAAAGACAACTCTGGGTACAAGGGTGAGCCGGTCCCTCAATACGTAAGCAAGGATATGATGGCTAAAGCTGGTGACGTTACCATGATATACTGTATGTATGGAAGCAATCCTATGGGTTATCCTAACTACTTCAAGAATGGTAAGGACGTGAATGGAAACCCTGAAGACCGTATCACCCGCCACAATAGAACCTCAGGCAAACGTCTCCTCTTCAAGACAACACTGCCAGAAGACGAGGGCGTATACACTTGTGAAGTCGACAATGGAGTCGGCAAACCCCAGAAACACAGTTTGAAATTGACTGTAGTCAGTGCACCGAAGTACGAACAGAAACCGGAAAAGGTGATCGTCGTCAAACAAGGACAGGATGTCACGATCCCTTGCAAGGTGACCGGTCTGCCAGCGCCCAACGTCGTCTGGAGCCATAACGCGAAGCCTCTAAGCGGTGGTAGAGCTACGGTCACCGACAGTGGTCTGGTCATCAAAGGCGTAAAAAATGGTGACAAGGGATACTACGGCTGCAGGGCTACTAACGAGCATGGAGATAAATACTTCGAGACCCTTGTACAAGTTAACTAAACAGGTTAACTTGAATGTGGACTGTCAAAATACGTAAAAAAGACATAAATGACAGTTGTGCGTCAACATCAAACTATAAGAACTTTTTAGGTTATATTATAATCGAAATAAGTAATAAAATAAAATTATTATAAAATAT | Lepidoptera | Saturniidae | Hyalophora | cecropia | 7123 | cds | GenBank | M63398.1 | Silencing of Hemolin in Hyalophora cecropia development RNAi construct | ATGGCGTTCAAGAGTATAGCAGTTTTAAGCGCCTGCATAATTGTGGGTTCAGCGCTTCCCGTGGATAAATACCCGGTGCTGAAAGACCAGCCGGCCGAGGTTCTCTTCAGGGAGAATAACCCAACGGTCCTCGAATGTATCATCGAAGGGAACGATCAGGGAGTCAAGTACTCCTGGAAAAAGGATGGGAAATCCTACAACTGGCAGGAGCATAACGCCGCTCTTCGCAAGGATGAAGGATCTTTGGTATTCCTGAGACCGCAAGCCTCAGACGAGGGTCACTACCAGTGCTTCGCTGAAACCCCAGCCGGTGTTGCCAGCTCGAGGGTGATCAGCTTCAGGAAGACTTACCTAATCGCGTCGCCAGCAAAGACACACGAGAAAACGCCAATCGAAGGCAGGCCTTTCCAATTGGACTGCGTCCTCCCTAACGCTTACCCTAAACCCTTGATTACTTGGAAGAAACGTTTGTCCGGAGCCGATCCTAACGCTGACGTGACTGACTTTGATCGCCGCAATCACAGCTGGACTGACGGAAACCTCTACTTTACAATCGTCACTAAAGAGGACGTCAGTGACATTTATAAATACGTATGCACCGCCAAGAACGCAGCGGTTGACGAAGAGGTAGTTTTGGTGGAGTATGAAATCAAGGGAGTGACAAAAGACAACTCTGGGTACAAGGGTGAGCCGGTCCCTCAATACGTAAGCAAGGATATGATGGCTAAAGCTGGTGACGTTACCATGATATACTGTATGTATGGAAGCAATCCTATGGGTTATCCTAACTACTTCAAGAATGGTAAGGACGTGAATGGAAACCCTGAAGACCGTATCACCCGCCACAATAGAACCTCAGGCAAACGTCTCCTCTTCAAGACAACACTGCCAGAAGACGAGGGCGTATACACTTGTGAAGTCGACAATGGAGTCGGCAAACCCCAGAAACACAGTTTGAAATTGACTGTAGTCAGTGCACCGAAGTACGAACAGAAACCGGAAAAGGTGATCGTCGTCAAACAAGGACAGGATGTCACGATCCCTTGCAAGGTGACCGGTCTGCCAGCGCCCAACGTCGTCTGGAGCCATAACGCGAAGCCTCTAAGCGGTGGTAGAGCTACGGTCACCGACAGTGGTCTGGTCATCAAAGGCGTAAAAAATGGTGACAAGGGATACTACGGCTGCAGGGCTACTAACGAGCATGGAGATAAATACTTCGAGACCCTTGTACAAGTTAACTAA | dsRNA | phenol_chloroform | subsequent to transcription | The template for synthesis of full length (1.4 kb) sense and antisense Hemolin RNA was amplified from the HcP4 pBIIKS+ plasmid (Bettencourt et al., 1999) by PCR, using T7 antisense and T3 sense sequencing primers. The PCR product was treated with Proteinase K and SDS, phenol/chloroform extracted and ethanol precipitated. The antisense strand Hemolin RNA was synthesized using the T7 MEGAscript kit (Ambion), according to manufacturerâs instructions. The sense strand was synthesized using the same conditions, but adding T3 instead of T7 polymerase. The DNA templates were removed with RNase free DNaseI (1 U/Îĵl) and the reactions stopped by addition of EDTA pH 8 (20 ÎĵM). After precipitation, the RNA was dissolved in DEPC treated water and the annealing of the complementary strands was performed in 750 mM NaCl, 75 mM sodium citrate, by heating to 65â70 °C for 15 min and cooling overnight at room temperature. | InsectaCentral | Silencing of Hemolin in Hyalophora cecropia development RNAi construct | Lepidoptera | Saturniidae | Hyalophora | cecropia | 7123 | local resource | pupa | field_collected | 0.01 | injection | Lepidopteran Ringer | 0.033 | non_specific_dsRNA | pupa | W.blot phenotype | whole organism | not checked | 720 | high | |||||||||
| 4 | silencing of APN1 in Spodoptera exigua | RNAi | InsectaCentral | Unpublished | Herrero | S | silencing of APN1 in Spodoptera exigua target | GAAATTATACAGCTTTGTTATTCGTTTTCAAACCAGCGTCAAAATGGCGAATCGCTGGTTTAGCCTCATCTTAGGGGTCATTGTACTCCAGTCTGTACTGGCGTTTGGCCCAATCGATGTAACGGACGCCGAATGGATTGAATACATGAATCTGATGAGCAACTCTAATTATCGGTTAGGAACAGAAACTGAACCAATTAATTACAAAGTAAAACTAACACCGAATTTAGTTAGTTTTACGTTTGAAGGTGAGGTTACAATACAGGTTAGAGTTACCAGTCAGAATCCAGTCAACGAAATCATACTTCACTGCAATAGTTTGACGATCAGTTCCGTGTTAGTTACACTGCCATCCCAAACGCAAAATCTCGCTACGGGTAATACTTTTCAATGCGAAGATGGTACTGACTTTTTAAGGATTCCAACTTTAATTCAACTAACTTCTAACATTGAGTACGTCGTCGTCACAATGTCGTTTACTGGGGTTCTGACAAATACCATGAGAGGTTTCTACAGAAGTTGGTACTATGACAGCACAATGCAAAAGAGATGGATGGCAACGACACAGTTCCAGCCTGGCCATGCTCGTCAAGCATTCCCTTGCTACGATGAACCTCGTTTCAAGGCCACGTTCGATATAACACTGGTTAGAGACAACAACGCCCAATCCAAGCCTTCGATATCAAACATGCCCATAAAAGATACAGATAGTACTGTTACTGGGAAAATAGCGGAAACCTTCTACACTACTCCTAAAATGTCCACCTACTTGCTGGCTTTCATAGTATCTGACTACATACCAGTCGCAGTTGGCACGACCCCTCAACGACCATTCACTATCTATGCTAGAGATAACATTAAAGATACTGGCAAATATTCTTTGGAAGTTGGAGAAAAACTTCTGAATTTGATGGAAGAATATACTGCTTTCCCTTATTATGGAATGGGTGATCATATGGAAATGAAACAAGCTGCTATCCCAGACTTTAGCGCTGGTGCTATGGAAAACTGGGGCCTGTTAACCTACAGGGAAGCTCTCATTTTATACGATCCTCAAAACACCAACAACTTTTACAAACAACGTATAGCTAACATTATTTCTCATGAAATTGCACACATGTGGTTCGGTAACCTGGTCACGTGCGCCTGGTGGGACACTCTTTGGCTAAACGAAGGTTTTGCTAGATTCTACCAGTACTACTTGACAGACAAGGCTGAACCAGAAATGGGATTCCCCACACGTTTCATAGTCGAGCAGTTGCAAGTTTCCTTGCTATCCGACTCCTTCGCATCTGCCCACCCCCTCACCAACCCTGACGTGTCAGACAAGGATTCAGTACGAGCACACTTTTCTACCATCACTTATGCTAAAGGTGCCTCTATACTCAGAATGACACAGCACCTCCTTGGTGAAGAAACTTACCAGAAGGGTCTTCAGGCCTACCTTAAGGACCGAAAATATAATACTGCTGAACCAGAAGACTTATTTAGAAACTTGGACGCTAATGCAGGCAATTCCCTGGCTAGTTACGAGGGTATGACTATCAGTCGCTACTTCAAATCATGGTCAGAAAAAGCAGGACATCCTCTGTTGACAGTTAACATTGACCACGCTAGCGGACGAATGACTGTAGGACAACATCAATTCGATATCAACAATGGCGTGTCTTCGAATAATGGTTTATGGGACATTCCTCTAACATGGACTAGGGCTGGAGCTCCCGATTTTAACAACCTCAAGCCTTCTGAATTCCTTAGTGGCCCATTAAAGATCATCGACCGTGGAAGCGTTGGACGAGAATGGGTCATTTTCAACAAACAGCAATCTGGTTTCTACAGAGTAAACTACGACCAGGCTACTTGGACACTTATTACTCAAGCTTTAAGAAGTAACAACAGAGCAGTCATACACGAATACAACCGCGCTCAGATTGTCGATGATGTGTTCGTGCTGGCCAGATCCAACATCATGTCTTATATGAGAGCGTTGAATATTCTTTCCTTCCTCGAATTTGAAGACCAGTACGCTCCTTGGATTGCTGCTATTACTGGATACAACTTTGCGCTTCGAAGATTGGCTCACGACAATATCGCTCTTCAAAGTCTGAAGGACATAATCTTTGCTTCGAGCACGGCGGTCGTCCAGCGTCTCGGCTTCATCGAAGGTACCAATGGTAACTTCATGGATGACTTACTCCGTATGCATGTGATGACCTTCCTTTGCAACGCTGGACATGAACAATGTTCCAACGTAGCCACACAGCGCTTCCAGGCTTGGAGGCAGAATGGTGATCGCATTCCACCAAACATGCGCCCTTGGGTGTACTGCGGTGGTCTTCGTAATGGAAATGAAGCGGACTTTGACTTTTTCTGGCGACGTTACTTGGACGAAAACTTATCTAACGAGAAAGTCGTGATGCTTGGTGCAGCAGGTTGTACAGGAAATACAGTTGCCTTGCACAAATTCCTGGACGTAATCGTATCCACACCTTCTATCAACGAAGACGAAGACATCAGACCTCAAGACTACAGTGCTGCTATAAGCTCTGCTGTCACTAGCAACGAAGCCAATACAATGAAAGTGATCGAATGGCTTATGAATCACCCTCAACATCTTGACACAGCAAACGGCATCAGTCTTTTGTCGACTGCTACAAGCCGATTGTTGACCCAGAGTGATATTCTAAGGGTCGAAACCTGGTTGAATACTGCTACCCAACTCAAAGCGGAGGCCATTCAAGCGGCTAGAGCCGGTATTGCTACATCAACGGCAAACATTCAATGGTATCAAAGAAGGCAACATGAGTTCAAAGCTTACTTCGACACAGGATACTTTGAAGAAGGTTTTGAAGTTCCTCCTTCCTCCAGTACTACAACTAAAGCTACCCCTTCCACACCTGAAGATACCCCTTCCACACCTGAAGATACCCCTTCCACACCTGAAGATACCCCTTCCACACCTGGTGATACCACATCAGACGCTACTATTACTGACGAACCAGGACCGAGCTCTACTGAACCAACCACGACCGCGGAGCCCGGCTCTGCGAACATTGCTTCTCTTAGCTTCTTCACTTTGATAATCACAGTTATCATCAACATGGTTTAATATAAAAGCGTTCTGTTGAAGACTATTAATAAAGTAATCATCCAAATAAGTGAAAACACCCTTCCCTAAGTTTGTTTCTCAGTAATATTCAGTGGTTGTTTTTAAATAAAGACGAATTTGTTTGTTACTTTTTAAATGTAGCATGCATAATATTTTTTATTAATAATATAAATAAATATATTTATTAACGTCATAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Spodoptera | exigua | 7107 | cds | InsectaCentral | silencing of APN1 in Spodoptera exigua target | silencing of APN1 in Spodoptera exigua RNAi construct | ATGGCGAATCGCTGGTTTAGCCTCATCTTAGGGGTCATTGTACTCCAGTCTGTACTGGCGTTTGGCCCAATCGATGTAACGGACGCCGAATGGATTGAATACATGAATCTGATGAGCAACTCTAATTATCGGTTAGGAACAGAAACTGAACCAATTAATTACAAAGTAAAACTAACACCGAATTTAGTTAGTTTTACGTTTGAAGGTGAGGTTACAATACAGGTTAGAGTTACCAGTCAGAATCCAGTCAACGAAATCATACTTCACTGCAATAGTTTGACGATCAGTTCCGTGTTAGTTACACTGCCATCCCAAACGCAAAATCTCGCTACGGGTAATACTTTTCAATGCGAAGATGGTACTGACTTTTTAAGGATTCCAACTTTAATTCAACTAACTTCTAACATTGAGTACGTCGTCGTCACAATGTCGTTTACTGGGGTTCTGACAAATACCATGAGAGGTTTCTACAGAAGTTGGTACTATGACAGCACAATGCAAAAGAGATGGATGGCAACGACACAGTTCCAGCCTGGCCATGCTCGTCAAGCATTCCCTTGCTACGATGAACCTCGTTTCAAGGCCACGTTCGATATAACACTGGTTAGAGACAACAACGCCCAATCCAAGCCTTCGATATCAAACATGCCCATAAAAGATACAGATAGTACTGTTACTGGGAAAATAGCGGAAACCTTCTACACTACTCCTAAAATGTCCACCTACTTGCTGGCTTTCATAGTATCTGACTACATACCAGTCGCAGTTGGCACGACCCCTCAACGACCATTCACTATCTATGCTAGAGATAACATTAAAGATACTGGCAAATATTCTTTGGAAGTTGGAGAAAAACTTCTGAATTTGATGGAAGAATATACTGCTTTCCCTTATTATG | dsRNA | MEGAscript TM RNAi kit | column-based | subsequent to transcription | The fragment was cloned in the pGemT-easy vector that was later use for PCR amplification using M13F and M13R primers. The PCR product was purified using QIAquick PCR Purification Kit (Qiagen). RNA synthesis, annealing and purification steps were performed following the MEGAscript TM RNAi Kit instructions. | InsectaCentral | silencing of APN1 in Spodoptera exigua RNAi construct | Lepidoptera | Noctuidae | Spodoptera | exigua | 7107 | local resource | larva | lab_colony | 5 | injection | 10%Phenol red in water | 1 | buffer | 10 | fat-body | larva | N.blot | midgut | not checked | 48 | none | |||||||
| 5 | Silencing of Hemolin in Hyalophora cecropia immunity | RNAi | Melanization is regulated by the prophenoloxidase cascade and functions as a response to intruding microorganisms in invertebrates. When injecting dsRNA of the lepidopteran immune protein hemolin in pupae of Hyalophora cecropia (Lepidoptera: Saturniidae), we observed a significant reduction in phenoloxidase activity after 24 h, but not after 72 h. The link between hemolin and the prophenoloxidase system suggests that hemolin is a pattern recognition protein important for the triggering of the prophenoloxidase cascade in the defence against bacterial infections. | pubmed | 17129606 | Faye | Ingrid | Silencing of Hemolin in Hyalophora cecropia immunity target | CTGGAGTATAAGGCACGGGTACACAATGGCGTTCAAGAGTATAGCAGTTTTAAGCGCCTGCATAATTGTGGGTTCAGCGCTTCCCGTGGATAAATACCCGGTGCTGAAAGACCAGCCGGCCGAGGTTCTCTTCAGGGAGAATAACCCAACGGTCCTCGAATGTATCATCGAAGGGAACGATCAGGGAGTCAAGTACTCCTGGAAAAAGGATGGGAAATCCTACAACTGGCAGGAGCATAACGCCGCTCTTCGCAAGGATGAAGGATCTTTGGTATTCCTGAGACCGCAAGCCTCAGACGAGGGTCACTACCAGTGCTTCGCTGAAACCCCAGCCGGTGTTGCCAGCTCGAGGGTGATCAGCTTCAGGAAGACTTACCTAATCGCGTCGCCAGCAAAGACACACGAGAAAACGCCAATCGAAGGCAGGCCTTTCCAATTGGACTGCGTCCTCCCTAACGCTTACCCTAAACCCTTGATTACTTGGAAGAAACGTTTGTCCGGAGCCGATCCTAACGCTGACGTGACTGACTTTGATCGCCGCAATCACAGCTGGACTGACGGAAACCTCTACTTTACAATCGTCACTAAAGAGGACGTCAGTGACATTTATAAATACGTATGCACCGCCAAGAACGCAGCGGTTGACGAAGAGGTAGTTTTGGTGGAGTATGAAATCAAGGGAGTGACAAAAGACAACTCTGGGTACAAGGGTGAGCCGGTCCCTCAATACGTAAGCAAGGATATGATGGCTAAAGCTGGTGACGTTACCATGATATACTGTATGTATGGAAGCAATCCTATGGGTTATCCTAACTACTTCAAGAATGGTAAGGACGTGAATGGAAACCCTGAAGACCGTATCACCCGCCACAATAGAACCTCAGGCAAACGTCTCCTCTTCAAGACAACACTGCCAGAAGACGAGGGCGTATACACTTGTGAAGTCGACAATGGAGTCGGCAAACCCCAGAAACACAGTTTGAAATTGACTGTAGTCAGTGCACCGAAGTACGAACAGAAACCGGAAAAGGTGATCGTCGTCAAACAAGGACAGGATGTCACGATCCCTTGCAAGGTGACCGGTCTGCCAGCGCCCAACGTCGTCTGGAGCCATAACGCGAAGCCTCTAAGCGGTGGTAGAGCTACGGTCACCGACAGTGGTCTGGTCATCAAAGGCGTAAAAAATGGTGACAAGGGATACTACGGCTGCAGGGCTACTAACGAGCATGGAGATAAATACTTCGAGACCCTTGTACAAGTTAACTAAACAGGTTAACTTGAATGTGGACTGTCAAAATACGTAAAAAAGACATAAATGACAGTTGTGCGTCAACATCAAACTATAAGAACTTTTTAGGTTATATTATAATCGAAATAAGTAATAAAATAAAATTATTATAAAATAT | Lepidoptera | Saturniidae | Hyalophora | cecropia | 7123 | cds | GenBank | M63398.1 | Silencing of Hemolin in Hyalophora cecropia immunity RNAi construct | ATGGCGTTCAAGAGTATAGCAGTTTTAAGCGCCTGCATAATTGTGGGTTCAGCGCTTCCCGTGGATAAATACCCGGTGCTGAAAGACCAGCCGGCCGAGGTTCTCTTCAGGGAGAATAACCCAACGGTCCTCGAATGTATCATCGAAGGGAACGATCAGGGAGTCAAGTACTCCTGGAAAAAGGATGGGAAATCCTACAACTGGCAGGAGCATAACGCCGCTCTTCGCAAGGATGAAGGATCTTTGGTATTCCTGAGACCGCAAGCCTCAGACGAGGGTCACTACCAGTGCTTCGCTGAAACCCCAGCCGGTGTTGCCAGCTCGAGGGTGATCAGCTTCAGGAAGACTTACCTAATCGCGTCGCCAGCAAAGACACACGAGAAAACGCCAATCGAAGGCAGGCCTTTCCAATTGGACTGCGTCCTCCCTAACGCTTACCCTAAACCCTTGATTACTTGGAAGAAACGTTTGTCCGGAGCCGATCCTAACGCTGACGTGACTGACTTTGATCGCCGCAATCACAGCTGGACTGACGGAAACCTCTACTTTACAATCGTCACTAAAGAGGACGTCAGTGACATTTATAAATACGTATGCACCGCCAAGAACGCAGCGGTTGACGAAGAGGTAGTTTTGGTGGAGTATGAAATCAAGGGAGTGACAAAAGACAACTCTGGGTACAAGGGTGAGCCGGTCCCTCAATACGTAAGCAAGGATATGATGGCTAAAGCTGGTGACGTTACCATGATATACTGTATGTATGGAAGCAATCCTATGGGTTATCCTAACTACTTCAAGAATGGTAAGGACGTGAATGGAAACCCTGAAGACCGTATCACCCGCCACAATAGAACCTCAGGCAAACGTCTCCTCTTCAAGACAACACTGCCAGAAGACGAGGGCGTATACACTTGTGAAGTCGACAATGGAGTCGGCAAACCCCAGAAACACAGTTTGAAATTGACTGTAGTCAGTGCACCGAAGTACGAACAGAAACCGGAAAAGGTGATCGTCGTCAAACAAGGACAGGATGTCACGATCCCTTGCAAGGTGACCGGTCTGCCAGCGCCCAACGTCGTCTGGAGCCATAACGCGAAGCCTCTAAGCGGTGGTAGAGCTACGGTCACCGACAGTGGTCTGGTCATCAAAGGCGTAAAAAATGGTGACAAGGGATACTACGGCTGCAGGGCTACTAACGAGCATGGAGATAAATACTTCGAGACCCTTGTACAAGTTAACTAA | dsRNA | T7 MEGAscript kit Ambion | phenol_chloroform | subsequent to transcription | The template for synthesis of full length (1.4 kb) sense and antisense Hemolin RNA was amplified from the HcP4 pBIIKS+ plasmid (Bettencourt et al., 1999) by PCR, using T7 antisense and T3 sense sequencing primers. The PCR product was treated with Proteinase K and SDS, phenol/chloroform extracted and ethanol precipitated. The antisense strand Hemolin RNA was synthesized using the T7 MEGAscript kit (Ambion), according to manufacturerâs instructions. The sense strand was synthesized using the same conditions, but adding T3 instead of T7 polymerase. The DNA templates were removed with RNase free DNaseI (1 U/Îĵl) and the reactions stopped by addition of EDTA pH 8 (20 ÎĵM). After precipitation, the RNA was dissolved in DEPC treated water and the annealing of the complementary strands was performed in 750 mM NaCl, 75 mM sodium citrate, by heating to 65â70 °C for 15 min and cooling overnight at room temperature. | InsectaCentral | Silencing of Hemolin in Hyalophora cecropia immunity RNAi construct | Lepidoptera | Saturniidae | Hyalophora | cecropia | 7123 | local resource | pupa | bacteria | field_collected | 0.01 | injection | PBS | 0.033 | non_specific_dsRNA | 12 | fatbody, hemolymph | Control pupae were injected with 100 microliter dsGFP in LR (10 ng/ml). | pupa | enzymatactivity | hemolymph | not checked | 24 | high | ||||
| 7 | Manduca sexta hemolymph proteins | RNAi | pubmed | 17868866 | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha3 target | ATAAGTAAAGTTACTGGCCGATGGAACGGTCATATGTCAATATAAACATTAAAATCGTGTACAAAAAACTGATTATAAGTTAAAATTAATTATTGTGAATAGTGATAAATTATTGTTTCTTAAAATGGTTTAAAATAGATTATATCTAAAGCTGACAATTACGGAGTGTTTATCACAAGCCTGGGATGATATTTGAACTGCAAAATGGCATGTGTAAAACTATTTCATTGATTTGTGTTTTCAATAAGACTTAATTTTGATATGTGAAAAGTGATAACATAAGTTAAAACAAGACCTCTTTTTGAAGTCGGTTAAAAAGGACAAGATGTACAAGTTATTTGCGGCTATAGTGATTTTGATGTGCGTGGAAGTGCGCACGAGTGGTATATTTCACGAAAATACCAAAATAACGTTTCATCCAGATGTAGACTCTGGATTTTTCGGATACTCCGTGTTTGTATATGAATATGGGATTATGGTGAGTGCTCCAAAAGCTAGGAGCAAGGTCAAGCCTACCGTGTTCTCTGGATTGGTCTACAACTGCTCTATCAACATCGTTGATGACGTCAGTGACGTCATGTGCTACCCCATGGATTCTGATTCAGTTTCAGACCGTTCCAGAAGTTACTACAGATATAAGTTCTTGATTGATTTCTTTAGAGATGACATGTGGTATGGAGCTTCTATGGGCGCTTTGTCCAACAATAAAATTTTGATCTGCGCACCAAGATATGCAGAACTTTTCAAAGATAACCATATTCTGCCTTACGGCGCTTGTTACATGCACAAAAAGGGACGGGAGATGCCTGTCTACCCTTTAGCGGATAAAGGTCATCTGGCTTATATGATCGATGGAAAGAGGAAGGAATACGGTGACTACGGGACGCATTTGAACTTCTACGCTAATGGACAAGTGGGGATGTCCATGAAAGTTACAGAAAGTAACACGGTTATAATCGGCGCCCCTGGACTTTTGCACTGGACTGGTGGCATAGTAAACTACAAATTCAACCCTGCAGATGATAGTATATACTTCGCCAAACAAACTACAACCAATCCGTACTTCACCAAGGAACTGGGGCCAGATCATTATTTCGGATATAGCGTAGAGTCTGGTATTTTCGAAAAATTCGGTCAGGTGCTATACGTCGGCGGAGCACCTCGGTCTAATGGAACCGGACAAGTGTTAATATTCGAACCAGCAGTAAAAGAAAACGCTCCTCTGAAAATCCAAGGTGTTCTCAAAGGACCTCAGCTGGGTGCCTATTTCGGGGCAAGTTTATGTTGCACGGACATTGATGGTGATGGAAGATCAGACCTTTTAGTCGGGGCTCCCAACTATGTGCAGAATGATGGAGGATTGCCTTATGATCAAGGAGCCGTGTTTGTTTATATGGCTTCTGATTTATCTGTAAAGGAAAAAGTGAAGGGTCCCAACTTTATATTAGAGCCTGTTGGTTACGTAATCGGTTCGGGACAAAGTGGCGCGCGATTTGGAACAGCTATTGCTGGTCTGGGAGATATAGACGGGGATGGATATAAAGATATTGCCATCGGCGCCCCGTGGGAAGACGATGGAGTAGGAGCAGTATACATTTACAGAGGAACTACTAGAGGGCTCAAGAGTCAATACGTCCAGAGAATAGCTGGAGATCACGCACAAGGCTTCGGCTGGTCCATAGCTAAAGGATTTGATGTGGACCATAATAATTGCAGTGATTTAGTAGTAGGAGCTTTTAAAAGCAACACAGTGAGTCTATATCGTTGCGTGCCGACAATACAAGTGCATGCTTCTATAAAAGTGCCAGACGCTATGAACCTGCCACAGAATTCGACATCATTCACAGCCATGTTCTGCTTGACTGTGCCAGCGAAGCATATGTGGTCTCATGTTAAACTTGATCTAAAAGCTCGTATAGTAATAGACCCAGAGCAGAATAGAGCGAGGGTTTCCGGAGACTCGGAGTACAACATCACCGTCAAGCCGGGGAACGATATCTGTGGAGAGCAAACCGTTGAAGTTACTCCGACCGCAGATTTGTCAAGGCCAATATCAATCAAGTTTGAACTGGAGCCGTTGGAGCTACTTCAAGATAATTCATCAACGTTCCTTAACGAAGCAGCGAGATTGTCAGAGGACTCGGAGCTGCAGTCATCATTCCTCATCCAACTGGTTAGGGACTGTGGAGACGATCTCATTTGCACACCATGGCTGGTCATGACTTGGGATGCTTTGGTCAACCCATACATCCCAGGGTCGGAAAAGCCTTTAGGAGGAAGACTGACTGTTCTGAACAAAGAGGAACCTGCCTACGGAGCCAAGGTCTACATAACCCTCCCAGCAACACCAATAAGAGTCCCAAGCGAATGTTCTTTGAAGGAATTAAACATGACTTGCAATATACCAGCTCCTTTAGAGCGAGGTGATGAAATAACTTGGGATATAGAACTGGAGTACACCGCACGAATGCCCTATGAGGATAATTTGAGGCTCGTGGCTGTACTAGAGGATTCTTTGTACAGTCGAAATATCACGGATGAACCTGTTAAAGAGCTAGTGATTGATATTGTCCCTCAAGCTAACTTTACTGTGAGTGGCAAACCACTTCCAAACGGAACAATAACTGTGACAAGACCAATGTTATATGAAACTGAAAATATAACCTTTTCCCATCATTACGAGACTATCAATTACGGACCGTCGGATTGGTACAGATTGGAAGGAGACATTTTTATACCAGAACATATAAATTTATCTAGTCCTATCAAAGGATGTTCTTGGAATTGGAGCTCTGAGTGCGGCTGGTCTATTCCCGCCAAAGTTTCTGTTCCAATAGTACTGGCACTGCGTTTTGATCTTGCCCAATATGTTTCAGGAGACATGAAGGAAAACACGACATTTAACGTAACTACAAAATTAAGTTTTTGGATAAAGGACCAATTCTACGCATCGGAAGTAACAGCTACTCTGATACTAGAACCAGGTCCACCAACTCTCTGGTATCTTATAGCGGCATTGATAGTTGGTCTACTGCTGCTTGCGCTCATTATTTTAATTTTATATAAGCTCGGATTCTTCTCACGAACACAACGTGATAAACTAAAGGAACTTCAAGAACAGACAGCTCAACAAGACCAAGCTGAGACGAGCTGCAGTTCCTCAGAACTTCTAGACATTAATGACTCCACAAGGGAATTATTAATAGAGGACTCAGAATGATGATGTAGCGGCTAAAACGCGTTTCGTTTCGTTTTTGCGTTTTTGATGTGAAGGAATCGAGAAAACGTTTTTTGCTGCAAATGTTTAGCATACTGTTAAGTTTCCCAACGTTGTATCTCAAAAAACAGAATTATTAGTTCATTAATGAAGGCATGATTGATGTATAGTCCGAAACAATATTTTTTAATCTTCTAGTCATTTTTTACCCAATGCAATATGTAAATAGAAGTTTATTTGGGTTTAAAACATTGGTCTAAAAATTTCGTATTTGCTAGAGCCGTTTGGAAAACAAATTCACGAACAATATAAATAAAACGGTTCAAACCCATACAAGTTTAAACAACACACGTGAATTTGCTATTGAACGTGTGAAATAACATTTTAATGAGATAATAAAAATAGTTTTATTAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | DQ531846 | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha3 RNAi construct | ACCTGCCACAGAATTCGACATCATTCACAGCCATGTTCTGCTTGACTGTGCCAGCGAAGCATATGTGGTCTCATGTTAAACTTGATCTAAAAGCTCGTATAGTAATAGACCCAGAGCAGAATAGAGCGAGGGTTTCCGGAGACTCGGAGTACAACATCACCGTCAAGCCGGGGAACGATATCTGTGGAGAGCAAACCGTTGAAGTTACTCCGACCGCAGATTTGTCAAGGCCAATATCAATCAAGTTTGAACTGGAGCCGTTGGAGCTACTTCAAGATAATTCATCAACGTTCCTTAACGAAGCAGCGAGATTGTCAGAGGACTCGGAGCTGCAGTCATCATTCCTCATCCAACTGGTTAGGGACTGTGGAGACGATCTCATTTGCACACCATGGCTGGTCATGACTTGGGATGCTTTGGTCAACCCATACATCCCAGGGTCG | dsRNA | MEGAsript RNAi Kit (Ambion) | column-based | simultaneously with transcription | (1) Amplify gene by PCR with forward primer (TAATACGACTCACTATAGGGAGACCTGCCACAGAATTCGACATC) and reverse primer (TAATACGACTCACTATAGGGAGACGACCCTGGGATGTATGGGT). (2) PCR product was recovered and used as template for preparation of dsRNA using MEGAsript RNAi Kit (Ambion). | InsectaCentral | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha3 RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | ddH2O | 0.05 | non_specific_dsRNA | 3 | abdominal hemocoels | Green fluorescent protein (GFP) dsRNA was purchased from Dharmacon and used as a control. | larva | qPCR phenotype | hemocytes | not checked | 120 | low | 50 | |||||
| 9 | Ubx silencing in Bicyclus anynana | RNAi | We attempted to inject dsRNA against a xxxbp fragment of Ubx cloned from the butterfly Bicyclus anynana. The dsRNA was produced in vitro using xxx. The RNA was injected at a concentration of xxx into the hemocoel of the larvae, at xxx position. xx% of the injected larvae survived to adulthood. There was no phenotype observed in the adult butterfly. | InsectaCentral | Unpublished | Popadic | Aleksandar | Ubx silencing in Bicyclus anynana target | AATCACACATTTTATCCCTGGATGGCCATTGCAGGAGCGAACGGCCTGAGAAGACGAGGGAGACAAACCTATACAAGATATCAAACGCTAGAGTTAGAGAAGGAATTCCACACGAATCACTACCTCACGCGGAGGAGACGTATAGAAATGGCGCACGCGCTCTGTCTCACGGAAAGACAAATCAAAATATGGTTCCAAAACCGCCGT | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | cds | InsectaCentral | Ubx silencing in Bicyclus anynana target | Ubx silencing in Bicyclus anynana RNAi construct | AATCACACATTTTATCCCTGGATGGCCATTGCAGGAGCGAACGGCCTGAGAAGACGAGGGAGACAAACCTATACAAGATATCAAACGCTAGAGTTAGAGAAGGAATTCCACACGAATCACTACCTCACGCGGAGGAGACGTATAGAAATGGCGCACGCGCTCTGTCTCACGGAAAGACAAATCAAAATATGGTTCCAAAACCGCCGT | dsRNA | Ambion MEGAscript T3/T7 High yield transcription kit | isopropanol precipitation | subsequent to transcription | Subcloning: A 207 bp fragment of Bicyclus Ubx containing the homeo-domain was generated using degenerate primers targeted to the highly conserved amino acid motifs FYPWM (5â AYC ACA CRT TYT AYC CCT GGA 3â) and WFQNRR (5â GCT CTA GAC GIC GRT TTT GRA ACC A 3â). The partial cDNA fragment was subsequently ligated into a TOPO TA sequencing vector (Invitrogen, Cat. # 45-0071). This vector contains NOT-I and PME-I restriction sites to linearize the plasmid and also contains T3 and T7 priming sites to generate ssRNA molecules. Generation of ssRNA: 10ug of Bicyclus Ubx plasmid was linearized with either NOT-I or PME-I and then subsequently cleaned using a standard phenol-chloroform reaction. Following digestion, the following reaction was performed to generate Bicyclus Ubx ssRNA: 1.0ug NOT-I or PME-I linearized DNA 2.0uL ATP solution 2.0uL CTP solution 2.0uL GTP solution 2.0uL UTP solution 2.0uL 10X reaction Buffer 2.0uL T3 or T7 enzyme to 20uL Nuclease free water *NOTE: T3 enzyme is used with NOT-I digested DNA and T7 enzyme is used with PME-I digested DNA Mix reagents well and incubate at 37°C for 16 hours Recovery of RNA: 1. Add 115uL of nuclease free water and 15uL Ammonium Acetate Stop solution (supplied with kit) 2. Extract with an equal volume of phenol-chloroform and then with an equal volume of chloroform. Recover acqueous phase and transfer into a new tube. 3. Precipitate the RNA by adding 1 volume of isopropanol and mix well. 4. Chill the mixture for at least 20 minutes at -20°C. Centrifuge at 4°C for 15 minutes at maximum speed to pellet the RNA. Carefully remove the supernatant and resuspend the RNA in 60uL of nuclease-free water. Annealing of ssRNA molecules: Mix equal molar amounts of ssRNA transcripts and run in a PCR-type rxn that initiates at 85°C, and then slowly cools as follows: 55°C for 20 minutes; 40°C for 10 minutes; 30°C for 5 minutes. | InsectaCentral | Ubx silencing in Bicyclus anynana RNAi construct | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | local resource | larva | lab_colony | 3.4 | injection | Nuclease free water | Inert dye | 1.5 | buffer | 168 | Injected in the dorsal A1 region and aimed anteriorly to target the T3 and T2 segments | Third larval staged Bicyclus anynana were anesthetized on a CO2 pad and injected with a solution containing nuclease free water and an inert injection dye. The injection was targeted to the dorsal A1 region of the larvae with the needle aimed anteriorly so the injected solution would accumulate in the T3/T2 region of the larvae. An identical injection procedure was performed for our dsUbx injections. | adult | phenotype | adult wing pattern | not checked | 240 | none | ||||
| 13 | Spodoptera frugiperda RNAi | RNAi | Contributing authors to the study: Magnus Lundmark, Daniela Nowara, Frank hauser, Cornelis Grimmelikhuijzen, Merete Albrechtsen. This study entails an initial round of dsRNA injections with five different gene targets chosen to induce mortality phenotypes. Two of these targets that appeared to give a faint deleterious effect (inhibitor of apoptosis protein, IAP, and diuretic hormone receptor, DHR) were chosen for a second series of injections. In the second injection series, larger numbers of larvae were injected and qPCR used to examine possible depletion of transcript in the IAP target. The five different S. frugiperda gene targets where: ⢠Inhibitor of apoptosis protein. AF186378 ⢠Diuretic hormone receptor. FJ374694 ⢠Predicted mRNA for vacuolar ATPase subunit A. Spodobase contig Sf2M05515-5-1. ⢠Predicted mRNA for vacuolar ATPase subunit D. Spodobase contig Sf2M04683-5-1 ⢠Predicted mRNA for ribosomal protein S9. Spodobase contig Sf2L01134-5-1 In none of the targets above a significant mortality phenotype was established. Neither was a depletion of transcript detected in the IAP target using q-pcr. | InsectaCentral | Unpublished | Lundmark | Magnus | Spodoptera frugiperda RNAi target | AGTTTTAGTCCGAACGCCGACGAGTGACGCATGTTACGAGCCTACTGTACTGACTCGACTCGAACCGCGATCGATCGTGGACCGCTGTAAACGTCACTTCGTTTCGTTCGTTAGTCGCGAGTTTCGCACTCATGTTGGAGAGTTGTGTTGTTTGTTTATCAGTCCGTTAACGTTTGACCAGTGAGAGTGAGAACAGTTTTTAAAACCTAGTCATAAACAATCAATTTGATGTGGTCGTGTTCCTTACCTTGTTGGAATACAAAAAAATCTGGATTACAAATGGATATTACCAAAGTGGCATCCAATGGCTCCTCCTCAACATTAACGCTATTCAAGAGCGGATCGCTTGAGGCTAAAATTCGACCTCTCGCGCCACTAATGCTGCCGACGCCAAGTTACGACTCCAACGCCGGCTCTCCATCTTTGTCTCCATCCACGCCTTGCTCTTCATCTTCTTTCTCCATTGATAAAACCGACAACCACGACACCTTCGGCTTCAGTGCGGACACAGTTGATATGAGAAAAGAGGATGAACGTATGAAAACATTTGAAAAATGGCCCGTAAGTTTTCTATCCGGAGAGCAACTTGCTCGAAATGGATTTTACTACCTCGGCCGTAGAGATGAAGCCCGTTGCGCTTTCTGTAAAGTGGAGATTATGAGGTGGGTGGAGGGCGATGACCCTGCGAAGGACCATCAGCGTTGGGCGCCACAGTGCCCATTTGTGCGCAAATTGAACGGTACTGCAGCAGCAGACACGGGTAGTTCGGGCCAGGACGAGTGTGGTGCCCGCGCCGCTCCCTCCGGTACCTCTCCGCCGCGTATGGCCGGTCCCGTGCACCCACGATATGCATCTGAAGCCGCACGACTACGCAGTTTTAAAGACTGGCCACGATGCATGCGACAAAAACCTGAAGAACTCGCCGAGGCTGGCTTTTTTTACACTGGTCAGGGAGACAAAACCAAGTGTTTTTATTGCGATGGTGGATTAAAAGATTGGGAGAACCATGACGTACCCTGGGAACAACACGCAAGGTGGTTTGACCGTTGCGCCTACGTGCAATTGGTGAAGGGTCGAGAATACGTTCAAAAGGTGATTTCTGAAGCTTGTGAGGTATCCGCGTCAGAAGCGGAACGTGATGTAGCACCCGCACGGACTGCCGAGCCAAGCCCGCCAGCAGAGGCGCCAGAAAACTCAGTCGATGACTCAAAGTTGTGTAAAATCTGTTATGCTGAAGAGCGTAACGTGTGCTTCGTGCCGTGCGGCCACGTGGTGGCTTGCGCCAAGTGCGCGCTGGCGGCCGACAAGTGCCCCATGTGCCGCAGGACGTTTCAAAATGCAGTGCGGTTATATTTCTCGTGAGAAGAGCCACCAATTTGCAGGCTCAATTCTAGTCTTAAGGGACGACCGGACAGAGCTGTGTGCTGAAACCATGAGCTCGTATGTCACGTTTCCACAGGCGGAGAAACCTATTAAAACTTATTGTTTGATGGTTTCGATAGGAACTCGCTCGCTAGTGTAGCAAAAGACGAAATTGAAAGGTTCCTCAGGAGTAAGACAGATATACTTAGTTATAGTGAATTATAAAATACATAGCATACATTAAGATTATATATTGATGACGCTTATTACTTTAATTCACGTTCCAATTTGACGTGTTATTACAATTATTTATTTTACACAGATGTAACGAAAACTTTGTGTGAGATGTAACATTTACACCGAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | GenBank | AF186378 | Spodoptera frugiperda RNAi RNAi construct | TCCAACGCCGGCTCTCCATCTTTGTCACCATCCACGCCTTGTTCTTCATCTTCTTTCTCCATTGATAAAACCGACAACCACGACACCTTCGGCTTCAGTGCGGACACAGTTGATATGAGAAAAGAGGATGAACGTATGAAAACATTTGAAAAATGGCCCGTAAGTTTTCTATCCGGAGAGCAACTTGCTCGAAATGGATTTTACTACCTCGGCCGTGGAGATGAAGTCCGTTGCGCTTTCTGTAAAGTGGAGATTATGAGGTGGGTGGAGGGCGATGACCCTGCGAAGGACCATCAGCGTTGGGCGCCACAGTGCCCATTTGTGCGCAAATTGAACGGTACTGCAGCAGCAGACACGGGTAGTTCGGGCCAGGACGAGTGTGGTGCCCGCGCCGCTCCCTCCGGTACCTCTCCGCCGCGTATGGCCGGTCCCGTGCACCCACGATATGCATCTGAAGCCGCACGACTACGCAGTTTTAAAGACTGGCCACGATGCATGCGACAAAAACCTGAAGAACTCGCCGAGGCTGGCTTTTTTTACACTGGTCAGGGAGACAAAACCAAGTGTTTTTATTGCGATGGTGGATTAAAAGATTGGGAGAACCATGACGTACCCTGGGAACAACACGCAAGGTGGTTTGACCGTTGCGCCTACGTGCAATTGGTGAAGGGTCGAGAATACGTTCAAAAGGTGATTTCTGAAGCTTGTGAGGTATCCGCGTCAGAAGCGGAACGTGATGTAGCACCCGCACGGACTG | dsRNA | salt precipitation | subsequent to transcription | PCR products were generated with the use of T7-adapter primers and used as templates for the in vitro transcription reaktions. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to kit protocol. The mixture was incubated at 37 degree Celsius over night. After LiCl precipitation (as per kit protocol) the dsRNA was eluted in pure water and the heated to 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. dsRNA concentration was quantified using ND-1000 Spectrophotometer (nanodrop). Annealing was examined by gel electrophoresis of the product. | InsectaCentral | Spodoptera frugiperda RNAi RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | local resource | larva | lab_colony | 1.4 | injection | H2O | none | 0.015 | buffer non_specific_dsRNA | 32 | Directly into haemoceul through the base of first abdominal pseudoleg | In total 3µl fluid was injected into each larvae in a single injection (4µg total amount dsRNA of IAP or TC-LAC2 control per animal). In the first round of injections (all 5 gene targets) and equal amount of sterile water with dye was injected as control. In the second injection series (IAP and DHR targets) non specific dsRNA (T castaneum laccase-2) was injected as control. No injection related mortality was detected. | larva | qPCR | whole organism | not checked | 144 | none | |||||
| 14 | silencing of APN1 in Spodoptera exigua-eggs injected | RNAi | InsectaCentral | Unpublished | Herrero | S | silencing of APN1 in Spodoptera exigua target | GAAATTATACAGCTTTGTTATTCGTTTTCAAACCAGCGTCAAAATGGCGAATCGCTGGTTTAGCCTCATCTTAGGGGTCATTGTACTCCAGTCTGTACTGGCGTTTGGCCCAATCGATGTAACGGACGCCGAATGGATTGAATACATGAATCTGATGAGCAACTCTAATTATCGGTTAGGAACAGAAACTGAACCAATTAATTACAAAGTAAAACTAACACCGAATTTAGTTAGTTTTACGTTTGAAGGTGAGGTTACAATACAGGTTAGAGTTACCAGTCAGAATCCAGTCAACGAAATCATACTTCACTGCAATAGTTTGACGATCAGTTCCGTGTTAGTTACACTGCCATCCCAAACGCAAAATCTCGCTACGGGTAATACTTTTCAATGCGAAGATGGTACTGACTTTTTAAGGATTCCAACTTTAATTCAACTAACTTCTAACATTGAGTACGTCGTCGTCACAATGTCGTTTACTGGGGTTCTGACAAATACCATGAGAGGTTTCTACAGAAGTTGGTACTATGACAGCACAATGCAAAAGAGATGGATGGCAACGACACAGTTCCAGCCTGGCCATGCTCGTCAAGCATTCCCTTGCTACGATGAACCTCGTTTCAAGGCCACGTTCGATATAACACTGGTTAGAGACAACAACGCCCAATCCAAGCCTTCGATATCAAACATGCCCATAAAAGATACAGATAGTACTGTTACTGGGAAAATAGCGGAAACCTTCTACACTACTCCTAAAATGTCCACCTACTTGCTGGCTTTCATAGTATCTGACTACATACCAGTCGCAGTTGGCACGACCCCTCAACGACCATTCACTATCTATGCTAGAGATAACATTAAAGATACTGGCAAATATTCTTTGGAAGTTGGAGAAAAACTTCTGAATTTGATGGAAGAATATACTGCTTTCCCTTATTATGGAATGGGTGATCATATGGAAATGAAACAAGCTGCTATCCCAGACTTTAGCGCTGGTGCTATGGAAAACTGGGGCCTGTTAACCTACAGGGAAGCTCTCATTTTATACGATCCTCAAAACACCAACAACTTTTACAAACAACGTATAGCTAACATTATTTCTCATGAAATTGCACACATGTGGTTCGGTAACCTGGTCACGTGCGCCTGGTGGGACACTCTTTGGCTAAACGAAGGTTTTGCTAGATTCTACCAGTACTACTTGACAGACAAGGCTGAACCAGAAATGGGATTCCCCACACGTTTCATAGTCGAGCAGTTGCAAGTTTCCTTGCTATCCGACTCCTTCGCATCTGCCCACCCCCTCACCAACCCTGACGTGTCAGACAAGGATTCAGTACGAGCACACTTTTCTACCATCACTTATGCTAAAGGTGCCTCTATACTCAGAATGACACAGCACCTCCTTGGTGAAGAAACTTACCAGAAGGGTCTTCAGGCCTACCTTAAGGACCGAAAATATAATACTGCTGAACCAGAAGACTTATTTAGAAACTTGGACGCTAATGCAGGCAATTCCCTGGCTAGTTACGAGGGTATGACTATCAGTCGCTACTTCAAATCATGGTCAGAAAAAGCAGGACATCCTCTGTTGACAGTTAACATTGACCACGCTAGCGGACGAATGACTGTAGGACAACATCAATTCGATATCAACAATGGCGTGTCTTCGAATAATGGTTTATGGGACATTCCTCTAACATGGACTAGGGCTGGAGCTCCCGATTTTAACAACCTCAAGCCTTCTGAATTCCTTAGTGGCCCATTAAAGATCATCGACCGTGGAAGCGTTGGACGAGAATGGGTCATTTTCAACAAACAGCAATCTGGTTTCTACAGAGTAAACTACGACCAGGCTACTTGGACACTTATTACTCAAGCTTTAAGAAGTAACAACAGAGCAGTCATACACGAATACAACCGCGCTCAGATTGTCGATGATGTGTTCGTGCTGGCCAGATCCAACATCATGTCTTATATGAGAGCGTTGAATATTCTTTCCTTCCTCGAATTTGAAGACCAGTACGCTCCTTGGATTGCTGCTATTACTGGATACAACTTTGCGCTTCGAAGATTGGCTCACGACAATATCGCTCTTCAAAGTCTGAAGGACATAATCTTTGCTTCGAGCACGGCGGTCGTCCAGCGTCTCGGCTTCATCGAAGGTACCAATGGTAACTTCATGGATGACTTACTCCGTATGCATGTGATGACCTTCCTTTGCAACGCTGGACATGAACAATGTTCCAACGTAGCCACACAGCGCTTCCAGGCTTGGAGGCAGAATGGTGATCGCATTCCACCAAACATGCGCCCTTGGGTGTACTGCGGTGGTCTTCGTAATGGAAATGAAGCGGACTTTGACTTTTTCTGGCGACGTTACTTGGACGAAAACTTATCTAACGAGAAAGTCGTGATGCTTGGTGCAGCAGGTTGTACAGGAAATACAGTTGCCTTGCACAAATTCCTGGACGTAATCGTATCCACACCTTCTATCAACGAAGACGAAGACATCAGACCTCAAGACTACAGTGCTGCTATAAGCTCTGCTGTCACTAGCAACGAAGCCAATACAATGAAAGTGATCGAATGGCTTATGAATCACCCTCAACATCTTGACACAGCAAACGGCATCAGTCTTTTGTCGACTGCTACAAGCCGATTGTTGACCCAGAGTGATATTCTAAGGGTCGAAACCTGGTTGAATACTGCTACCCAACTCAAAGCGGAGGCCATTCAAGCGGCTAGAGCCGGTATTGCTACATCAACGGCAAACATTCAATGGTATCAAAGAAGGCAACATGAGTTCAAAGCTTACTTCGACACAGGATACTTTGAAGAAGGTTTTGAAGTTCCTCCTTCCTCCAGTACTACAACTAAAGCTACCCCTTCCACACCTGAAGATACCCCTTCCACACCTGAAGATACCCCTTCCACACCTGAAGATACCCCTTCCACACCTGGTGATACCACATCAGACGCTACTATTACTGACGAACCAGGACCGAGCTCTACTGAACCAACCACGACCGCGGAGCCCGGCTCTGCGAACATTGCTTCTCTTAGCTTCTTCACTTTGATAATCACAGTTATCATCAACATGGTTTAATATAAAAGCGTTCTGTTGAAGACTATTAATAAAGTAATCATCCAAATAAGTGAAAACACCCTTCCCTAAGTTTGTTTCTCAGTAATATTCAGTGGTTGTTTTTAAATAAAGACGAATTTGTTTGTTACTTTTTAAATGTAGCATGCATAATATTTTTTATTAATAATATAAATAAATATATTTATTAACGTCATAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Spodoptera | exigua | 7107 | cds | InsectaCentral | silencing of APN1 in Spodoptera exigua target | silencing of APN1 in Spodoptera exigua RNAi construct | ATGGCGAATCGCTGGTTTAGCCTCATCTTAGGGGTCATTGTACTCCAGTCTGTACTGGCGTTTGGCCCAATCGATGTAACGGACGCCGAATGGATTGAATACATGAATCTGATGAGCAACTCTAATTATCGGTTAGGAACAGAAACTGAACCAATTAATTACAAAGTAAAACTAACACCGAATTTAGTTAGTTTTACGTTTGAAGGTGAGGTTACAATACAGGTTAGAGTTACCAGTCAGAATCCAGTCAACGAAATCATACTTCACTGCAATAGTTTGACGATCAGTTCCGTGTTAGTTACACTGCCATCCCAAACGCAAAATCTCGCTACGGGTAATACTTTTCAATGCGAAGATGGTACTGACTTTTTAAGGATTCCAACTTTAATTCAACTAACTTCTAACATTGAGTACGTCGTCGTCACAATGTCGTTTACTGGGGTTCTGACAAATACCATGAGAGGTTTCTACAGAAGTTGGTACTATGACAGCACAATGCAAAAGAGATGGATGGCAACGACACAGTTCCAGCCTGGCCATGCTCGTCAAGCATTCCCTTGCTACGATGAACCTCGTTTCAAGGCCACGTTCGATATAACACTGGTTAGAGACAACAACGCCCAATCCAAGCCTTCGATATCAAACATGCCCATAAAAGATACAGATAGTACTGTTACTGGGAAAATAGCGGAAACCTTCTACACTACTCCTAAAATGTCCACCTACTTGCTGGCTTTCATAGTATCTGACTACATACCAGTCGCAGTTGGCACGACCCCTCAACGACCATTCACTATCTATGCTAGAGATAACATTAAAGATACTGGCAAATATTCTTTGGAAGTTGGAGAAAAACTTCTGAATTTGATGGAAGAATATACTGCTTTCCCTTATTATG | dsRNA | MEGAscript TM RNAi kit | column-based | subsequent to transcription | The fragment was cloned in the pGemT-easy vector that was later use for PCR amplification using M13F and M13R primers. The PCR product was purified using QIAquick PCR Purification Kit (Qiagen). RNA synthesis, annealing and purification steps were performed following the MEGAscript TM RNAi Kit instructions. | InsectaCentral | silencing of APN1 in Spodoptera exigua RNAi construct | Lepidoptera | Noctuidae | Spodoptera | exigua | 7107 | local resource | embryo | lab_colony | 5 | injection | 10% Phenolred in water | 1 | buffer | 20 | early embryo | larva | N.blot | midgut | not checked | 48 | none | |||||||
| 18 | period gene silencing | RNAi | InsectaCentral | Unpublished | Bebas | Piotr | period gene silencing target | ACGTGTTTCAATGTCCTGAGCCGGAAAAATCAATGAAAGTTCCCGAAGAAGAAAAAAACAAGGCACAAATGCTGAGAGAGACCATTATTCGAACAATGAATGAGGCTCTGACTAAACCCGCGGAAATAGCGAAGCAACAGATGAGCAAGCGTTGTCAAGATCTTGCTTCGTTCATGGAGAGCTTAATGGAGGAAGTGCCCAAGACTGATGAGGAATTGCGCTTAGAGATTCACGATCCTGATCACAGCTATTATGAAAGGGACTCAGTGATGCTTGGTGGGATCTCCCCCCACCACGACTAGGTCATAGCTGTTTCCTGATCTC | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | cds | NCBI | AF315349.1 | period gene silencing RNAi construct | ACAAGGCACAAATGCTGAGAGAGACCATTATTCGAACAATGAATGAGGCTCTGACTAAACCCGCGGAAATAGCGAAGCAACAGATGAGCAAGCGTTGTCAAGATCTTGCTTCGTTCATGGAGAGCTTAATGGAGGAAGTGCCCAAGACTGATGAGGAATTGCGCTTAGAGATTCACGATCCTGATCACAGCTATTATGAAAGGGACTCAGTGATGCTTGGTGGGATCTCCCCCCACCACGACTAGGTCATAGCTGTTT | dsRNA | New England Biolabs | phenol_chloroform | subsequent to transcription | Total RNA from Testis-Upper Vas Deferens complexes of 2-day-old S. littoralis males was extracted with TRIzol Reagent (Invitrogen) and treated with RQ1 DNase I (Promega, Madison, WI). First-strand synthesis from total RNA was performed with M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, MA) and random hexamers (Promega). The S. littoralis period sense and antisense RNA probes were designed based on the published sequence (GenBank accession number AF315349). A 258-nt fragment of this sequence was amplified by PCR, using Takara Ex Taq DNA polymerase (Takara Bio, Shiga, Japan) and the following primers: 5-ACAAGGCACAAATGCTGAGAGAG (sense), 5â²-AAACAGCTATGACCTAGTCGTGG (antisense). To generate sense and antisense probes, SP6 and T7 RNA polymerase promoter sequences were added to the sense and antisense primers, respectively. The PCR product was than reamplified using this new pair of primers, gel purified, and transcribed in vitro using appropriate RNA polymerases (New England Biolabs). Products were treated with RQ1 DNase I and extracted with phenol/ chloroform. Sense and antisense RNA strands were mixed in equal amounts, heated at 95°C for 1 min, and slowly cooled at room temperature for 18 h. The efficiency of in vitro transcription was verified on agarose gels. | InsectaCentral | period gene silencing RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | adult | lab_colony | 2 | injection | PBS | 2 | buffer non_specific_dsRNA | 108 | testes | Based on the same strategy (as described for period gene dsRNA synthesis), control nonspecific dsRNA was synthesized using the gene sequence for ribulose-1,5-bisphosphate carboxylase small subunit (SSU) from Nicotiana tabacum. A fragment of SUU gene was amplified using the following primers: 5â²-TCCATTGCCAGC AACGGCGGAAG (sense), 5â²-TCCAAGCAAGGAACCCATCC (antisense) based on the sequence from GenBank (accession number M32419). As the additional control we used insects injected with PBS. | adult | qPCR N.blot | undetected | 8 | high | 95 | ||||||
| 20 | Silencing of Hemolin in Antheraea pernyi | RNAi | Hemolin is one of the haemolymph proteins most strongly induced upon bacterial infection in Lepidoptera. When we applied RNA interference (RNAi) to suppress Hemolin expression in the Chinese oak silk moth Antheraea pernyi , we discovered that Hemolin is induced by double-stranded RNA (dsRNA) per se. As dsRNA is recognized as a virus pattern molecule, we then investigated the effect of a baculovirus ( Ap NPV) infection. We found that Hemolin is induced and expressed with similar kinetics as upon dsRNA injection. Notably, no Attacin gene expression or antibacterial activity was recorded. When baculovirus and high amounts of dsRNA were coinjected, the viral symptoms appeared earlier with Hemolin dsRNA than with GFP dsRNA. This indicates that silencing of hemolin affected the progress of the viral infection. | pubmed | 15271212 | Faye | Ingrid | Silencing of Hemolin in Antheraea pernyi target | ATGGCGTCCAAAAGTATTGCTGTGTTGAGTGCGTGCATAATATTAGGTGCAGCACTTCCTGTGGATAAGCAGCCGGTGATGAAAGAACAGCCCTCTGAGGTCCTCTACAAAGAGGGCAAACCAGCAATCATCGAATGCTTTACAGAGGGAAAAGAGGAGGGCAACAAATACTTCTGGCAAAAGGATGGGAAATCTTTCGATTTGCAACAAAATCACGCCACCATGCGCAAAGACGAAGGATCCCTTGTCTTCCTGAACCCCCAAGCGTCAGACGAAGGCCAATACCAGTGCTTCGTTGAAACCCCGGCCAGCATAGCTAGCTCAAGGGTGATCAGCTTCAGAAAGACTTACCTCATCGCCGCCCCGGTCAAATCCCATGAGAAGACTCCCGTGGAAGGGAAACCATTCCAATTAGACTGCGTTGCCCCCGACGCGTACCCGAAACCGGAGATCTATTGGAAGAAACGCTTGTCCGGCGCTGATCCCAATGCTGACTCGACGAGCTTCAACCGTCGCATCACCGCCGGGTCTGACGGAAACCTCTACTTCGAGACnGTGACCAAGGATGACGTGAGCGACATTAACATATACGTCTGCGTGGCTAAAAACGCTGCCGTCAATGAGGAAGTGCCATTAGTTGAGTACGTCATCAAGGGGGTGACCAAAGATACCTCCGGCTACAATGGCGAGCTGGTACCTCAATACCTGAGCAAGGATATGATGGCGAAGGCTGGGGATACTACCATGATTTACTGCATGTACGGTGGCGACCCACACGCCTACCCTAAATACTCCAAGGACGGAAAGCGAGTCGGTGAGAAATCCGGCGATCGCGTCACAGCGCACAACAGAACCTCCGGCAAGCGCCTCCTCATCCAGGACACAAACGAAGGAGACGCCGGCAAATACACTTGCGAAGTCGACAATGGAAAGGGCGCGGCACAGACGCACTCTATGACATTGACTGTAGTCAGTGCACCGAAATACGAAGTGAAACCTGAAAAGGTAGTAATCGTCAAGACCGGACAAGACGTGACGATCCCTTGCAAGGTGACCGGCAAGCCGGAGCCCAAGGTCATCTGGACTCATAACGCCAAGCCCATCAGCGGAGACAGGTTCGAGGTCAGCGAAAACGGTCTAGTCATCAAGGGTGTGCAAAAGAGCGACAAGGGTTACTACGGCTGCAGAGCCATCAACGAATACGGGGACGAATACGTCGAGTCCTTGGTCCAAGTGAACTAA | Lepidoptera | Saturniidae | Antheraea | pernyi | 7119 | cds | InsectaCentral | Silencing of Hemolin in Antheraea pernyi target | Silencing of Hemolin in Antheraea pernyi RNAi construct | CGGTGATGAAAGAACAGCCCTCTGAGGTCCTCTACAAAGAGGGCAAACCAGCAATCATCGAATGCTTTACAGAGGGAAAAGAGGAGGGCAACAAATACTTCTGGCAAAAGGATGGGAAATCTTTCGATTTGCAACAAAATCACGCCACCATGCGCAAAGACGAAGGATCCCTTGTCTTCCTGAACCCCCAAGCGTCAGACGAAGGCCAATACCAGTGCTTCGTTGAAACCCCGGCCAGCATAGCTAGCTCAAGGGTGATCAGCTTCAGAAAGACTTACCTCATCGCCGCCCCGGTCAAATCCCATGAGAAGACTCCCGTGGAAGGGAAACCATTCCAATTAGACTGCGTTGCCCCCGACGCGTACCCGAAACCGGAGATCTATTGGAAGAAACGCTTGTCCGGCGCTGATCCCAATGCTGACTCGACGAGCTTCAACCGTCGCATCACCGCCGGGTCTGACGGAAACCTCTACTTCGAGACnGTGACCAAGGATGACGTGAGCGACATTAACATATACGTCTGCGTGGCTAAAAACGCTGCCGTCAATGAGGAAGTGCCATTAGTTGAGTACGTCATCAAGGGGGTGACCAAAGATACCTCCGGCTACAATGGCGAGCTGGTACCTCAATACCTGAGCAAGGATATGATGGCGAAGGCTGGGGATACTACCATGATTTACTGCATGTACGGTGGCGACCCACACGCCTACCCTAAATACTCCAAGGACGGAAAGCGAGTCGGTGAGAAATCCGGCGATCGCGTCACAGCGCACAACAGAACCTCCGGCAAGCGCCTCCTCATCCAGGACACAAACGAAGGAGACGCCGGCAAATACACTTGCGAAGTCGACAATGGAAAGGGCGCGGCACAGACGCACTCTATGACATTGACTGTAGTCAGTGCACCGAAATACGAAGTGAAACCTGAAAAGGTAGTAATCGTCAAGACCGGACAAGACGTGACGATCCCTTGCAAGGTGACCGGCAAGCCGGAGCCCAAGGTCATCTGGACTCATAACGCCAAGCCCATCAGCGGAGACAGGTTCGAGGTCAGCGAAAACGGTCTAGTCATCAAGGGTGTGCAAAAGAGCGACAAGGGTTACTACGGCTGCAGAGCCATCAACGAATACGGGGACGAATACGTCGAGTCCTTGGTCCAAGTGAAC | dsRNA | MegaScript Ambion | ethanol precipitation | subsequent to transcription | The open reading frame of Hemolin from A. pernyi (104â1270 nt, GENBANK accession no. AY529704) was amplified by PCR. Each forward and reverse primer contained a T7 RNA polymerase binding site (TTA ATA CGA CTC ACT ATA GGG AGA) at the 5â² end. The PCR product was purified using QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). The sense and antisense Hemolin RNA were made with MEGAScript Kit (Ambion, Austin, Texas) according to the manufacturerâs instructions. Both sense and antisense RNA were mixed, ethanol-precipitated and then re-suspended in DEPC treated water. The RNA was annealed with annealing buffer by incubation at 65 °C for 30 min in a water bath, followed by slow cooling to room temperature overnight. The dsRNA was analyzed by 1% agarose gel electrophoresis to ensure that the majority of the dsRNA existed as a single band of approximately 1100 bp. The dsRNA was stored at â20 °C until use. | GenBank | AY529704 | Lepidoptera | Saturniidae | Antheraea | pernyi | 7119 | local resource | pupa | virus | field_collected | 0.2 | injection | PBS | 0.01 | buffer non_specific_dsRNA | 3 | fatbody | The pupae were injected with 50 Îĵl PBS containing 10 Îĵg GFP dsRNA. Also, 50 Îĵl of PBS without dsRNA was used as a control. | pupa | phenotype | whole organism looking through pupal window | not checked | 240 | high | ||||
| 25 | Silencing of phenoloxidase in Spodoptera frugiperda | RNAi | This protocol has also been used for silencing prophenoloxidase activating enzyme (PPAE). In both cases, there was not any difference in larval survival after challenge with E. coli when larvae were treated with either dsRNA or PBS, meaning that, under our experimental conditions, injection of dsRNA directed to either phenoloxidase or PPAE did not induce a higher susceptibility of S. frugiperda to a non-pathogenic bacteria such as E. coli. | InsectaCentral | Unpublished | Duvic | Bernard | Silencing of phenoloxidase in Spodoptera frugiperda target | GACAGTCGAGCCGTGGATACTCACGGCTTGAAACCAAAATAAAATAGTTTTTATTTAATTAATTTTATTTAGTTCTTTTTTGTTTTTTTTTTTGGTAAAAATCTTGTGTATGCAAGATGTCGGACGCAAAGAAGAACCTGCTGTTATTCTTCGACCGGCCCTCGGAGCCGTGCTTCATGCAGAAGGGGGAAGAGAAGGCTGTCTTCGAGATCCCTGAACACTACTACCCGGAGAAGTACAAGGCACTAACGAGCACCATCGCCAACCGTTTCGGAGATGACGCCGGCCGCTCCATCCCCGTGCGTAACATCGCGCTCCCCAACCTGGCGCAGCCCATGGAGCTGCCCTATAATGACCAGTTCTCTCTCTTCGTGCCCAAACACAGGAGACTGGCGGGCAAGCTCATCGACATCTTCATGGGCATGCGTGACCTGGAGGACCTGCAGTCTGTGTGCTCGTACTGCCAGCTGCGCATCAACCCGTACATGTTCAACTACTGCCTCTCCGTCGCCATACTGCACAGGCCCGACACCAAAGGACTGAACATCCCGACGTTCGCGGAGACCTTCCCCGACAAGTTCATGGACCCCAAGGTGTTCCGCAAGGCCAGGGAAGTCAGCAACGTCGTCACCTCCGGAGTCAGGATGCCAGTAACAATCCCGGTGAACTACACGGCGAACGACTCGGAGCCTGAACAGCGCGTGGCGTACTTCCGCGAGGACATCGGCATCAACCTGCACCACTGGCACTGGCACCTGGTGTACCCGTTCGACTCCGCCGACCGGTCCATCGTCAACAAGGACAGGCGGGGAGAGCTCTTCTATTATATGCATCAGCAGATCATTGCTAGGTACAACATGGAGCGCATGTGCAACGGCCTGTCCCGCGTGGCGCGCTACCAGAACTTCCGCGACCCCATCGAGGAGGGATACTTCCCCAAGCTCGACTCGCAGGTCGCCAGCAGAGCCTGGCCGCCGAGGTTTGCGGGCACCACCATCCGCGACCTGGACCGTCCGGTGGACCAGATCCGCGCCGACGTGTCCCAGCTGGAGACCTGGAGGGACCGCTTCGTACAGGCTGTGGAGACTCTCTCCGTCACTCTGCCCAACGGTCGGCAGATGCCTCTGGACGAAGAGCGCGGCATCGACATCCTGGGCAACATGATGGAGTCGTCCATCATCAGCCCCAACCGAGGGTACTACGGGGACCTCCACAACATGGGACACGTCTTCATCTCCTACTCACACGACCCTGACCATCGCCATCTGGAACAATTCGGCGTGATGGGAGACTCGGCGACGGCGATGCGCGACCCCGTGTTCTACCGCTGGCACTCGTACATCGACGACCTGTTCCAGCTCTACAAGGTCAAGCTCACCCCCTACGGGGACGACAAGTTGGACTTCCCCGGCGTGCGCGTGTCGAGCGTGTCGCTGGAGGGCGCGGCGGGACGGAACACGCTGGGCACGTTCTGGGAGCTCAGCACCGTGGACCTCGGCCGCGGACTCGACTTCACGCCGCGCGGCTCTGTGCTCGCCCGCTTCACGCATCTGCAGCATCAGGACTTCAACTACGTTATCGAGGTGAACAACACGTCGGGCCAGTCAGTGATGGGCACGGTCCGCATCTTCATGGCGCCGGTGCAGGACGAGCGCGGCGCCCCGCTGTCCTTCGACGACCAGCGCCGTTCCATGATCGAACTCGACAAGTTCACCGCTGGGTTACGTCCGGGCAACAACACGATCCGCCACCGCAGCGTGGACTCGTCCGTCACCATTCCCTACGAGAGGACCTTCCGCGACCAGTCTGCGCGTCCAGGCGACCCTGGCTCGGAGGAGGCGGCAGAGTTCGACTTCTGCGGCTGTGGATGGCCACACCACATGCTGATCGCCAAGGGCAACCAGCAAGGGTACCCCGTCGTACTCTTCGCCATGGTCTCCAACTGGGCTGAGGACAGGGTGGAACAAGACCTGGTAGGCTCCTGCAACGACGCGGCCTCGTACTGCGGTATCCGGGACCGCAAGTACCCGGACCGCCGCGCCATGGGCTTCCCCTTCGACCGGCCGTCCCCCGCCCAGAGCCTGACCGACTTCCTGCGCCCCAACATGGCCATGCAGAACTGTTCCATCCGGTTCACAGACACCACCATCCCGAGGCAGCAAAGACGGTAGGTTGTGGGAGTATCAGGAGAAGGAGTAAGGGGTGAAAGGGAAACGTTGATAGGAGATCATTATTCCTTTATAGAAAGTTTAGATTGTGAATGTATTGTAAACATCTTGGACAGTTGTTTTCCCCCTTCTTCTATATTTATTTATTTATATCCAGTAACAACACAACACATTTAGGTACACCTTTCCTACACAGTGTAGGTAAGGCGGTAGGTAGGCATCTAACTAAAAGGTTCTAGATTAGCAACATAGATGGCGCTATTATACAATACCGAATTAATAACTTATTTTAGAAAATGAGATATAAAACCAGGCTTTTCTTACTAATATTGATATTTCAATACATTAGATAAGTCTAGATATTTATTAGTCAGTTATTGTACTAGTGGTGCCATCTATTAGTAGTTAACTGAACTGTTGGGTGTGTAGGTCTGCCTCGGTTTCGTCCGATTCGTCCGATTCGTCCGATTCGTCCATATCGTCGGCCTCGTCTGGTCAAGATGGCCACCACAAGCCGTGTGGCTGTCCAGACGAAACTATCACTCCTGCTGAAATAGCAGCTATCATTAAGAAGCCATATTTTGGTGACAAGTGCAAACGTACTAGACATTACGATTATATTTTCACACCTAATCCTATTGATAAAAAAATGGATACTTCCAAAAAAGCTTAAAGAATATCCTTCATGATATCCTTCTAAAAAACAAAATTCATTCGTGATAACATTCACATTGAGGCAGATTTACAAATCCAAACAATATTTCTCACTCTTACCTAATTGAACCATATTATTCATTAATCGTAATATTGTTTCATTTGGTATTTAGCACTTTCTTTTTATATAAATTTAAAACATCTTTTCACCGATTTTCTGTGTAACAAAGAATATTTAAACATCTTTAGTTGGCAGTAGCGACCTTCCTTCTGCCATATATTCGAGACTGTTTGTTGCTAACACAGGCAAATTTTAAGATAATATATATCCTCTTGTATATTATTTAATACAGCATTATAATAATATTGTATATGTATTCATTCAACTGTCTTATTTATATTGTTTGGCTGTTACTGGTACGACCTGGCATTGACAAAGGACTCCGATTGTAGCTGTAGGTAATTATCTAAGAGTAGTTGTAAGTAAACGGGTCTGTAAATATACTAAGTAGTTGTTAGTTCTATTGTACATATTGTGTAAATACTTGAATTCTTGAGACTGAATAAATTAGCCTTTTACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | Silencing of phenoloxidase in Spodoptera frugiperda target | Silencing of phenoloxidase in Spodoptera frugiperda RNAi construct | TTCTATTATATGCATCAGCAGATCATTGCTAGGTACAACATGGAGCGCATGTGCAACGGCCTGTCCCGCGTGGCGCGCTACCAGAACTTCCGCGACCCCATCGAGGAGGGATACTTCCCCAAGCTCGACTCGCAGGTCGCCAGCAGAGCCTGGCCGCCGAGGTTTGCGGGCACCACCATCCGCGACCTGGACCGTCCGGTGGACCAGATCCGCGCCGACGTGTCCCAGCTGGAGACCTGGAGGGACCGCTTCGTACAGGCTGTGGAGACTCTCTCCGTCACTCTGCCCAACGGTCGGCAGATGCCTCTGGACGAAGAGCGCGGCATCGACATCCTGGGCAACATGATGGAGTCGTCCATCATCAGCCCCAACCGAGGGTACTACGGGGACCTCCACAACATGGGACACGTCTTCATCTCCTACTCACACGACCCTGACCATCGCCATCTGGAACAATTCGGCGTGATGGGAGACTCGGCGACGGCGATGCGCGACCCCGTGTTCTACCGCTGGCACTCGTACATCGACGACCTGTTCCAGCTCTACAAGGTCAAGCTCACCCCCTACGGGGACGACAAGTTGGACTTCCCCGGCGTGCGCGTGTCGAGCGTGTCGCTGGAGGGCGCGGCGGGACGGAACACGCTGGGCACGTTCTGG | dsRNA | phenol_chloroform | simultaneously with transcription | cDNA encoding phenoloxidase 1 was cloned into pTriplex vector. Then the sequence used to generate dsRNA was PCR-amplified using primers containing Sac I restriction site in order to clone this fragment into L4440 vector. E. coli HT115 cells were electroporated with our construct and the dsRNA was produced after a 2h-induction with 0.4 mM IPTG. | InsectaCentral | Silencing of phenoloxidase in Spodoptera frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | Spodoptera | local resource | larva | lab_colony | 0.005 | injection | PBS | 45 | buffer | 5 | Hemocoel | PBS was injected instead of dsRNA to S. frugiperda larvae. On the other hand, when checking for a potentially induced phenotype, 20 µl of PBS per larvae was injected in place of E. coli. | larva | W.blot phenotype | Plasma, hemocytes | not checked | 24 | low | |||||||
| 33 | Mamestra midgut RNAi Hegedus-Erlandson-Toprak | RNAi | Midgut genes encoding several peritrophic matrix proteins were successfully knocked-down in a midgut culture system | InsectaCentral | Unpublished | Hegedus | Dwayne | Mamestra midgut RNAi Hegedus-Erlandson-Toprak target | ACTCTCTGGTAGATCTAGCAAAATGAAGTTTCTTGGTTTACTAGCGCTACTGCTGGTTGCTTCGGCAGCCATTGCCGACGATTCTTCTTCAAAAGAGGAAGCGGGCCTGAAGGAAGCGGAAGAATGCACTCCTGAAACTGTGTGTGAACTGCCTAATTGCAGATGTTCGAGTACTAACATCCCTGGAGGATTGCAGCCTAAAGATACACCACAGTTTGTCACAGTCACCTTCGACGACGGCATCAACGTCAACAATATCCTCACATACCGTAATACTCTTTACAACCGTCGCAACTCCAATGGCTGCCCTGCCGGAGCCACTTTCTACGTCAGCCACGAGTACACAAACTATGTCATCGTCAATGAATTGTACAACCAAGGCTTTGAAATCGCTCTACACTCCATCAGTCACCAGACCCCACAGACTTACTGGTTTGAAGCTACCAAAGATGATATGAAGAGGGAATTTGGAGACCAGAAGATTCAAATAGCTCACTTTGCAAACATTCCTTATGAATCCATCAAGGGTCTCCGCATTCCCTTCCTCCAAATGACGGGTAATGCCAGCTTTGAAATCATGAAAGAATATGGCTTGGAGTACGACTGCACCTGGCCCACTACATCCCACACAAACCCTGGACTATGGCCTTACACCCTGGACTACGCTTCCACCCAGGACTGCATTGTTCCTCCTTGCCCGACTGCGTCCTTCCCTGGAACATGGGTTAAACCTCTGGTAACCTGGTCTGATCTTCAGGGAGTTGCTTGCTCGTTTGTGGATGCATGTTTCTTCATCCCCGACCGCGCCGACGAAGATGCATGGTACAAGTTCATCCTCACCAACTTCGAGAGGCACTACTTGGGTAACCGCGCTCCCTTCGGCTTCTTCGTCCACGAGGCTTTCTTGTCAGCTTTCCCTGCCGTTCGTGGTGCCTTCGTCCGTTTCTTGGATTTGATCAACAACCTTCCTGATACGTTCATGGTGAACTCCCACGAAGTTATTGACTGGGTCAAGAACCCCGTTCCAATTGATAAGTACAAGGCCCAAGGCTGCCGTAGATTCAACCCCAGAGCCTGCGTTGCTAGAAGCTGCGGTCCTCTTAACTCTGGCCACAATGGAATGGACTACTGGATGCAAATCTGTAACGTTTGCCCCCGTGTCTACCCATGGCTCGGCAACCCTCTTGGACAATAAATCTAAGACCGAATAAGTTTAATAATAAATAAAGATTTCGATCTAAATAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Mamestra | configurata | 174822 | cds | NCBI | EU660852 | Mamestra midgut RNAi Hegedus-Erlandson-Toprak RNAi construct | GGACTATGGCCTTACACCCTGGACTACGCTTCCACCCAGGACTGCATTGTTCCTCCTTGCCCGACTGCGTCCTTCCCTGGAACATGGGTTAAACCTCTGGTAACCTGGTCTGATCTTCAGGGAGTTGCTTGCTCGTTTGTGGATGCATGTTTCTTCATCCCCGACCGCGCCGACGAAGATGCATGGTACAAGTTCATCCTCACCAACTTCGAGAGGCACTACTTGGGTAACCGCGCTCCCTTCGGCTTCTTCGTCCACGAGGCTTTCTTGTCAGCTTTCCCTGCCGTTCGTGGTGCCTTCGTCCGTTTCTTGGATTTGATCAACAACCTTCCTGATACGTTCATGGTGAACTCCCACGAAGTTATTGACTGGGTCAAGAACCCCGTTCCAATTGATAAGTACAAGGCCCAAGGCTGCCGTAGATTCAACCCCAGAGCCTGCGTTGCTAGAAGCTGCGGTCCTCTTAACTCTGGCCACAATGGAATGGACTACTGGATGCA | dsRNA | ethanol precipitation | subsequent to transcription | Two clones coding opposite strands of target gene was obtained from pGEM-T easy cloning and linearized for use as a template for being converted into RNA using Promega T7 RiboMAX⢠Express RNAi System. The RNA strands were annealed to form a duplex RNA. Twenty microgram of dsRNA was applied into 2 ml of Grace`s media containing three midguts obtained from 3rd instar Mamestra configurata larvae. At 24 h intervals, three midguts were collected to extract the total RNA. Expression of the target genes was examined by RT-PCR. In parallel, total RNA from control midgut tissues(no dsRNA)was also extracted for examination of the target gene expression. Tubulin was used a house keeping gene in all conditions. | NCBI | EU660852 | Lepidoptera | Noctuidae | Mamestra | configurata | 174822 | local resource | larva | lab_colony | 0.01 | injection | Grace`s | 250 | buffer | 1 | Midgut primary tissue culture | Same as above | larva | qPCR | Midgut primary cell culture | not checked | 24 | high | 95 | |||||
| 34 | Manduca sexta hemolymph proteins Kanost lab-HP6 | RNAi | InsectaCentral | Unpublished | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-HP6 target | TTTGTGTCTCTTGTGTATTTTATATTTGAAAAGAAAATACTTTAAGATCATTAGGTTTCCTGATATACAAATACATGGATCTTCTAACTTTAGAAATGATGTCACTATATTTTAACCTTTTATGAACTAAAATGCATAAATAACTGCACTGCGCTCGAAAGTTCACAAATAATACCGAACCGGTTTAGACATCTTATAAATTTTTGTTTAGAGTAACAGGAATTTTGTAGAAAACTTATTTTTTTATTCAAGATGTGGTTAATGGTGAATAATATAATACTGTGTTTATTAATAACTAATTCCATCATCGCCGAAAACGTTGGTGATGAATGTACTCCAAGCTCAAGTACTGGTGATGGTACATGTACATTGGTTTCTGATTGTCCAGCAGCAATAAGGGCGATCAAAAATAAAAGGTTTCATGAGTTCCAAAGATGTGGATTCGATGGTTTTCAAGAAATTGTATGCTGTCCTCTTACAACAGACAAATTCGGTGCAACCGAGACTTCACCAAAAGTTGCACAAAGGATTACTGACAGAGAATGCAAAACGATCCTCGCCGGCACGATACCGCCGCTAGACTTACACATACTCGGCGGTGAAGAGGCATCGCTTGGGGAATTTCCACATATGGTGGCGCTTGGCTTCGATAATGGTGGCGGTGAATACAGATTTGACTGCGGCGGCTCCTTGATATCTAACTACTATGTGTTGACCGCGGCGCACTGCATCGATACGGCTGACAGGGAACCGCCATCTGTAGTGCGAGCGGGAGTCGTTAATATTGGGGGTCCTGCGTGGGACGACGAGACAGACTATCGTGTCGCCGAAACTATATTACACCCGAACTACACTCGTCGAGAGAAATACCATGACGTGGCGTTGTTGAGGTTAGACAGACCTGTCCAGTTTTCAAGCACTCTAAATGCTGTGTGCCTCTTCAGTTCAAACGAAAACCCGACATCTAAACTAACAATAACCGGATGGGGAAGAACAAGCAACACTCGAGACATCAAGAGTAGTAAGTTGCTGAAAGCAGATGTGGTTGTAGTTCCAAGTGACAAATGCGGTGAATCATATACCAATTGGCGGAAATTACCGCACGGAATCTCACAGGAGATGATGTGCGCGGGAGATCCGAAAGGCGTGCGTGACACGTGTCAGGGCGACTCTGGCGGGCCTCTTCAGCTAATGGAGAAGGACGGTTTGTATCGGCTGGTGGGCGTGACGTCATTCGGGCGCGGCTGCGGTTCGTATGTACCAGGAGTGTACACGCGTGTCAGCAATTATCTAGGCTGGATAGAAAGTATTGTTTGGCCTAATTAATGTAATTGTGATTTCAGTAGAGAATTATGGTACAAAATACTTTACGCGTGCACTTTATAATATACAGGTGAATTTTTCAAGTGGGTGTGGAGGGACCATTCCTACAGGGAATGGTGAAAAACGGTTAAAAGAGTCTTAAGACTGATTTAAAGAAAAAAAAATATCATTATGTATATGAAAAATTTCTTGTCGCAGCCAGTATTCAATCCCGGCAACTTGGATAATATTAATGATTATTGCTTTTTCATATTCACCATTTTGCATTTGGCCATCCACACTTTCTTGACAGAATCACCATATTTACGTCATTCTTCTTCTCAGTCGTACACTCCTGGCGGAGTGGTCGTGGTTATGGATTGTCTGAAAAATTGATTGCGACTTTTGCGAGTCTCCTCCATCTCTCTCTCTTCATGGCACTGCGTGTGCATTCAGTAAGGGAACGAACACCTTGCAAACGATTTAATAACGTCTGTCCATTTGGTGGGTATTCGGCCGCGAGGAAGTTTACCTTTACTTACGTCATATGTATTTTTAATACAACAATTGACCTTACATTACCAACAGATCCTACTTCAATCTATTCTATCAACCAGATTGTCATATACTTAATAGATTTTCTTTTAAATTATATGTTACAAGTCTAGAAATTGACAGCAATGACCTATTACACAAATGATTAACTTAACAGTAGGTCAAAAATAAAAAACATTATTCTAAGAAATGTGCCTCGTGTTGTGTCACTATGTGTTTATACTTATTGACCATAATATGTACCTATTGTAAGTAGTGTAATTAAATTTATAACACTAAAAATGTATATTAAACAAGGTAATGTGTCTTGCCTACTATTTTGTCTGATCTCTTGTTTCTTTATAATTAATTAACTACTATGATTACTTTCGAATAATTTTATTACTGTTTATTCATAAATATGTGAATGTTTAGTTTACACTCTTGTAAAGAATGAATCCATAATAATCAGCATTTTTTAAATTCTTACCTAAGAGTAACCTATTTTCAACAATTGACTACCACTTTTTATTGCCAAGAATTTAAAAGCAAAAGAAACATAAATAGATAAATACTCACTTGTTTAGGTACTGACTATTAAGTAATATTTACTGTATGCTGAAATGTGTCAAACTAATATTGTACTTTTTTAAAAAAATAAAATATCCTTTTTCTAAAGCATTTAGCTATTTGCAGATATTATTTGAGACTGACAAAAGAAACTAGAACGAATCACCTACTACAAGACATGTAGAGAAATAATGTAAATAGTTTTAGTTGTATTCAGTTCGATTGCATTTAATGTAATAGCAATTACCGTAGTGTTCGCGACTGACAACACTTCCAAAAAGGTGGTCCATTTGTCGATGGTCACTGATCCTTTGGCTGATTGATGCATAAACACTTGCAACTTTATCGCATTCACGATTGTTTCAAAGACGATTCGTGTATTTGGATTTCGAAACCGAAATGCAGCTGTTTTCGAAATGCATGCAATTTCACTGTTGCCAGATGGTGAGGTTACTCCACAAATTTAACGCCATGGGGAAATTTGGTGTTTGGTGGTATTTTTAAAGCGAACAAATTTACTCATTTGTCAGCAAATATAATCCGTTTTCTGAGACCTCTTAACTGAATTTTGGGAGATTTCGTTGAAGTCATCTGGCAACCATGTCCGATTTCATTCCATTTTATGCCAATTAGATTAATCTGGAAGGCTGGTTAATCCGCTCCAATAGTGTCCACCAGAGAGCATATCTTCCTTATGATATTTAACTTTTATTTTATATGAAATTTATTATTAATTGAATTAATTGTAGATAAGGGTAAGAGAAGGCATGGTGACACAAGTGGTTTTTCACTTAACAACTAGAATATATTGTATAAATCATTTTTTTTTTAAATTGCGTTCAAATAAGGTTACTTTTCTTTGACTGTGCCAAGTTTTACACTGTTTGCCTTCTTACCCTGTCTAGGCATTTTTAGGGCTAGTCATGAAGTAACAATGCTCTCTATAATTTTGTAATTTGTTCAATAAAATGATTGTACCAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | AY672782 | Manduca sexta hemolymph proteins Kanost lab-HP6 RNAi construct | ATGTGGTTAATGGTGAATAATATAATACTGTGTTTATTAATAACTAATTCCATCATCGCCGAAAACGTTGGTGATGAATGTACTCCAAGCTCAAGTACTGGTGATGGTACATGTACATTGGTTTCTGATTGTCCAGCAGCAATAAGGGCGATCAAAAATAAAAGGTTTCATGAGTTCCAAAGATGTGGATTCGATGGTTTTCAAGAAATTGTATGCTGTCCTCTTACAACAGACAAATTCGGTGCAACCGAGACTTCACCAAAAGTTGCACAAAGGATTACTGACAGAGAATGCAAAACGATCCTCGCCGGCACGATACCGCCGCTAGACTTACACATACTCGGCGGTGAAGAGGCATCGCTTGGGGAATTTCCACATATGGTGGCGCTTGGCTTCGATAATGGTGGCGGTGAATACAGATTTGACTGCGGCGGCTCCTTGATATCTAACTACTATGTGTTGACCGCGGCGCACTGCATCGATACGGCTGACAGGGAACCGCCATCTGTAGTGCGAGCGGGAGTCGTTAATATTGGGGGTCCTGCGTGGGACGACGAGACAGACTATCGTGTCGCCGAAACTATATTACACCCGAACTACACTCGTCGAGAGAAATACCATGACGTGGCGTTGTTGAGGTTAGACAGACCTGTCCAGTTTTCAAGCACTCTAAATGCTGTGTGCCTCTTCAGTTCAAACGAAAACCCGACATCTAAACTAACAATAACCGGATGGGGAAGAACAAGCAACACTCGAGACATCAAGAGTAGTAAGTTGCTGAAAGCAGATGTGGTTGTAGTTCCAAGTGACAAATGCGGTGAATCATATACCAATTGGCGGAAATTACCGCACGGAATCTCACAGGAGATGATGTGCGCGGGAGATCCGAAAGGCGTGCGTGACACGTGTCAGGGCGACTCTGGCGGGCCTCTTCAGCTAATGGAGAAGGACGGTTTGTATCGGCTGGTGGGCGTGACGTCATTCGGGCGCGGCTGCGGTTCGTATGTACCAGGAGTGTACACGCGTGTCAGCAATTATCTAGGCTGGATAGAAAGTATTGTTTGGCCTAATTAA | dsRNA | MEGAscript RNAi Kit (Ambion) | column-based | subsequent to transcription | (1) Amplify gene by PCR with forward primer (ATGTGGTTAATGGTGA) and reverse primer (ACAAACCGATTAATT) (2) PCR product was recovered and ligated to pCR4-TOPO TA sequencing vector, and transformed to TOP 10 competent cells (3) Identify positive clone and amply with T3 (ATTAACCCTCACTAAAGGGA) and T7 (TAATACGACTCACTATAGGG) primers. (4) PCR product was recovered and used as template to synthesize sense and anti-sense ssRNAs using T7 and T3 in vitro translation kit (Ambion). (5) ssRNAs were annealed at room temperature overnight to get dsRNA. | NCBI | AY672782 | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | unknown | unknown | lab_colony | 0.003 | injection | ddH2O | 0.03 | buffer | 3 | hemolymph | larva | qPCR | not checked | 18 | none | ||||||
| 35 | Manduca sexta hemolymph proteins Kanost lab-IML2 | RNAi | InsectaCentral | Unpublished | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-IML2 target | GGGAGCGGGGGGCTGCAACGAACGACTCTTGCGAGTCGTGTGATAGATTGAATTATGTACAAATCATTTATTTTTATTTGCGTTTATTTCACTTCATCGATTGTTTCGACCAATCATGTGAATTTTCGATGCGACTACAAATACTTAGATGTAATAGACGGATGGATGAAACTGCATGAGATTCCTGCGAACTGGCATGAGGCAAGGTTGAGATGCCATCTTGAGGGAGCCGTTTTAGCTTCTCCTTTGAATTCGAATTTGAAATTCGCTATGGCATCAATGATGATCCTTAAAACTCCCAAACAAAGCGTTTTTACAGGAATCCATGCGACCTTCTCAAGAGGAGACTTTTTCTCTGTTGAAGGAATACCTTTAAAAAAGATACCTCACAAATGGGCTCCATCAGAGCCAGGCAACTGGAACGACCAAGAAAATTGCCTGACCATGCATTTCGACGGAAATCTAGCCGCAAAATCATGCTCTGCTACATTCAACTATATATGTTACAAGAAACGTATCCCTGACATGGTGGTCACTGAATGCGGAACCGTAGATTCCAAATACGTCCACTATGACCGTACAAACAGCTGCTACAAATTCCATGGCGTACCTCGGACCTGGTCGAGAGCATACATGACCTGTGCTTGCCGAAGGTGGATACTTGACTATCATTACAGTGAGAAGGAAGCAGGAATCATCAGAGAAATTTTCGCACAACACCTTCCAGCGTCTATGGTCGGTAATTTTTGGAAAGATATGGCTTTTGTGGGATTCCACGATTGGGGAGAACATGGAACCTGGTTAACAGTTCAAGGCCAAACACTGGAAGAAGCAGGTTACGCCAAATTTGCTCCAGGTGAGCCAAACAATGCAACTACAGGAGAATACTGCGGCGGGGTTTATCGTACTGGGTTGTTGGATGATATCTGGTGTGAAAATGTGTACGCGTTTATTTGCGAAAAGGACCCAAACAGTCTTCTCTGTGATCCTACAAGTGATAGTTTCGATGATATTATTGACATAAGAAATGTAAACTAGGATTGTTATAACAAAAAAAATACATGTTTATTGTATTATTTTGGTATAATGATGATCTATGTAAATGATCTCTGTATGTTTTATTTCACATTAACTATGTAAAATGTTTTATTAGTGTATAGACAAACAATTATTATAATTAATCATTGTACTACTGTTTATAAAATATGAAATAAAATTATTAGTCCTCAATTTAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | AF242202 | Manduca sexta hemolymph proteins Kanost lab-IML2 RNAi construct | GACTCTTGCGAGTCGTGTGATAGATTGAATTATGTACAAATCATTTATTTTTATTTGCGTTTATTTCACTTCATCGATTGTTTCGACCAATCATGTGAATTTTCGATGCGACTACAAATACTTAGATGTAATAGACGGATGGATGAAACTGCATGAGATTCCTGCGAACTGGCATGAGGCAAGGTTGAGATGCCATCTTGAGGGAGCCGTTTTAGCTTCTCCTTTGAATTCGAATTTGAAATTCGCTATGGCATCAATGATGATCCTTAAAACTCCCAAACAAAGCGTTTTTACAGGAATCCATGCGACCTTCTCAAGAGGAGACTTTTTCTCTGTTGAAGGAATACCTTTAAAAAAGATACCTCACAAATGGGCTCCATCAGAGCCAGGCAACTGGAACGACCAAGAAAATTGCCTGACCATGCATTTCGACGGAAATCTAGCCGCAAAATCATGCTCTGCTACATTCAACTATATATGTTACAAGAAACGTATCCCTGACATGGTGGTCACTGAATGCGGAACCGTAGATTCCAAATACGTCCACTATGACCGTACAAACAGCTGCTACAAATTCCATGGCGTACCTCGGACCTGGTCGAGAGCATACATGACCTGTGCTTGCCGAAGGTGGATACTTGACTATCATTACAGTGAGAAGGAAGCAGGAATCATCAGAGAAATTTTCGCACAACACCTTCCAGCGTCTATGGTCGGTAATTTTTGGAAAGATATGGCTTTTGTGGGATTCCACGATTGGGGAGAACATGGAACCTGGTTAACAGTTCAAGGCCAAACACTGGAAGAAGCAGGTTACGCCAAATTTGCTCCAGGTGAGCCAAACAATGCAACTACAGGAGAATACTGCGGCGGGGTTTATCGTACTGGGTTGTTGGATGATATCTGGTGTGAAAATGTGTACGCGTTTATTTGCGAAAAGGACCCAAACAGTC | dsRNA | MEGAsript RNAi Kit (Ambion) | column-based | subsequent to transcription | (1) Amplify gene by PCR with forward primer (GACTCTTGCGAGTCGTGTGA) and reverse primer (GACTGTTTGGGTCCTTTTCG) (2) PCR product was recovered and ligated to pCR4-TOPO TA sequencing vector, and transformed to TOP 10 competent cells (3) Identify positive clone and amply with T3 (ATTAACCCTCACTAAAGGGA) and T7 (TAATACGACTCACTATAGGG) primers. (4) PCR product was recovered and used as template to synthesize sense and anti-sense ssRNAs using T7 and T3 in vitro translation kit (Ambion). (5) ssRNAs were annealed at room temperature overnight to get dsRNA. | NCBI | AF242202 | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | unknown | unknown | lab_colony | injection | ddH2O | 0.03 | buffer | 2 | hemolymph | larva | qPCR W.blot | hemolymph, fat-body | not checked | 18 | none | ||||||
| 36 | Manduca sexta hemolymph proteins Kanost lab-PPO2 | RNAi | InsectaCentral | Unpublished | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-PPO2 target | GGAACTGTGAAGTGGGTATACTAAAAAGTTTTTTTTTTATTTATTTAAAAATACGTTTATCAGACACCATGGCTGATATTTTTGATAGTTTCGAACTCCTCTACGACCGTCCAGGAGAACCTATGATTAACACCAAAGGGGAAGACAAAGTTCTATTCGAACTCACTGAACAATTTCTGACCCCGGAATACGCCAACAATGGTCTGGAGTTGAATAACCGCTTCGGTGATGAGGAGGAGGTGTCTCGTAAAATAATATTGAAGAATCTTGACAAGATTCCAGAGTTTCCTAAAGCTAAACAACTCCCAAACGATGCCGATTTCTCCCTTTTCCTGCCCAGCCATCAAGAAATGGCTAATGAAGTCATTGATGTCCTAATGAGTGTAACTGAGAACCAACTACAAGAACTCCTGTCTACTTGTGTGTATGCCCGAATCAATCTCAACCCGCAGCTGTTCAACTACTGCTACACTGTTGCCATTATGCACAGACGTGACACGGGTAAGGTTCGTGTACAGAACTATGCAGAAATTTTCCCTGCAAAGTTTTTGGACTCTCAAGTATTCACCCAGGCCCGTGAAGCTGCAGCAGTCATCCCGAAAACTATTCCTCGAACTCCTATCATCATTCCACGAGACTACACTGCTACCGACTTGGAAGAAGAGCATCGCCTGGCGTACTGGCGTGAAGACCTCGGTATCAATCTTCACCATTGGCATTGGCATCTCGTTTACCCGTTCTCTGCTAGCGACGAGAAAATTGTAGCCAAAGACCGTCGCGGGGAGCTGTTCTTCTACATGCACCAGCAAATCATTGCCAGATACAACTGCGAGCGTTTGTGCAATTCTCTGAAGAGGGTAAAGAAATTCAGCGACTGGCGCGAGCCTATCCCGGAGGCATATTACCCTAAACTAGACAGCTTGACTTCAGCCCGCGGCTGGCCGCCACGCCAAGCTGGTATGCGTTGGCAAGACCTGAAGAGACCTGTGGACGGTCTAAACGTTACAATTGATGACATGGAACGTTACAGGAGGAACATTGAAGAGGCTATTGCTACTGGTAACGTTATATTGCCAGACAAATCTACCAAAAAGTTGGATATCGATATGCTCGGCAACATGATGGAAGCAAGCGTCCTGTCACCCAACCGCGATTTGTATGGCTCCATCCACAACAACATGCACAGCTTCAGCGCGTACATGCACGACCCCGAACATCGATACCTCGAATCATTCGGTGTGATCGCTGATGAAGCGACGACGATGCGCGATCCGTTCTTCTACCGCGTGCATGCGTGGGTCGATGACATCTTCCAGAGTTTCAAAGAGGCCCCTCATAACGTGCGCCCATACAGTCGCTCTCAGCTCGAGAACCCTGGCGTTCAAGTGACATCCGTCGCGGTGGAGTCTGCTGGCGGCCAACAGAACGTGCTGAACACTTTCTGGATGCAGAGCGATGTGAACCTCTCTAAAGGTTTAGATTTCTCGGACCGCGGGCCGGTGTACGCGCGCTTCACTCATCTCAACCACAGACCTTTCCGTTACGTTATAAAGGCGAACAACACTGCGTCCGCTCGCCGCACGACCGTGCGCATCTTCATAGCCCCGAAGACAGACGAGCGCAACCTGCCGTGGGCTCTGTCCGACCAACGCAAGATGTTCATTGAGATGGATAGATTCGTAGTACCCTTGAGCGCTGGCGAAAACACAATTACTCGTCAGTCCACAGAATCGTCACTGACAATTCCGTTCGAACAGACGTTCCGCGACCTTTCCATCCAGGGAAGTGACCCTAGGCGTTCGGAACTCGCCGCATTTAATTACTGCGGGTGTGGCTGGCCGCAGCACATGCTCGTGCCTAAGGGAACGGTCGGCGGTGTTGCCTATCAACTCTTTGTAATGCTTTCCAACTACGAACTGGATAAGATTGAGCAACCAGACGGCAGGGAACTGAGTTGCGTCGAAGCTTCCATGTTCTGTGGCTTGAAGGATAAGAAGTACCCAGACGCACGTCCGATGGGCTATCCGTTCGACCGGCCATCCAACTCTGCTACCAACATCGAAGACTTCAGCGCGATGTCCAACATGGGACTGCAAGACATAGTCATCAAACTATCCGATGTAACCGAACCAAACCCTAGAAACCCACCCGCTTAAACTACCAAGCTTGCTAAATCATTGTGAATTTACTATCACAAAGGATTTTTGTTTATAATGATACTTTATGATACTTAAGTATTACATTTATAATTGAATTTTAAGCTTGAATTATCCTCATAGTGTTTTTCTTTTAATTTTACTTTTCTTTAAAAATAAAAAATGAAAGGCTGATAGTGTTTATCTTGTAAATTCTTAAGTGTAAATATGTTATATATGTTTGTAGAGTTATGTAACTATTATTGACCCATTTTATAAGGAATAAGACAATAAATAAAACAATGCATTTAT | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | L42556 | Manduca sexta hemolymph proteins Kanost lab-PPO2 RNAi construct | AAACAACTCCCAAACGATGCCGATTTCTCCCTTTTCCTGCCCAGCCATCAAGAAATGGCTAATGAAGTCATTGATGTCCTAATGAGTGTAACTGAGAACCAACTACAAGAACTCCTGTCTACTTGTGTGTATGCCCGAATCAATCTCAACCCGCAGCTGTTCAACTACTGCTACACTGTTGCCATTATGCACAGACGTGACACGGGTAAGGTTCGTGTACAGAACTATGCAGAAATTTTCCCTGCAAAGTTTTTGGACTCTCAAGTATTCACCCAGGCCCGTGAAGCTGCAGCAGTCATCCCGAAAACTATTCCTCGAACTCCTATCATCATTCCACGAGACTACACTGCTACCGACTTGGAAGAAGAGCATCGCCTGGCGTACTGGCGTGAAGACCTCGGTATCAATCTTCACCATTGGCATTGGCATCTCGTTTACCCGTTCTCTGCTAGCGACGAGAAAATTGTAGCCAAAGACCGTCGCGGGGAGCTGTTCTTCTACATGCACCAGCAAATCATTGCCAGATACAACTGCGAGCGTTTGTGCAATTCTCTGAAGAGGGTAAAGAAATTCAGCGACTGGCGCGAGCCTATCCCGGAGGCATATTACCCTAAACTAGACAGCTTGACTTCAGCCCGCGGCTGGCCGCCACGCCAAGCTGGTATGCGTTGGCAAGACCTGAAGAGACCTGTGGACGGTCTAAACGTTACAATTGATGACATGGAACGTTACAGGAGGAACATTGAAGAGGCTATTGCTACTGGTAACGTTATATTGCCAGACAAATCTACCAAAAAGTTGGATATCGATATGCTCGGCAACATGATGGAAGCAAGCGTCCTGTCACCCAACCGCGATTTGTATGGCTCCATCCACAACAACATGCACA | dsRNA | MEGAsript RNAi Kit (Ambion) | column-based | simultaneously with transcription | (1) Amplify gene by PCR with forward primer (TAATACGACTCACTATAGGAAACAACTCCCAAACGATGC) and reverse primer (TAATACGACTCACTATAGGTGTGCATGTTGTTGTGGATG) (2) PCR product was recovered and used as template for amplification with T7 primer (TAATACGACTCACTATAGG) (3) PCR product was recovered and used as template to synthesize dsRNAs using MEGAsript RNAi Kit (Ambion). | NCBI | L42556 | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | unknown | unknown | lab_colony | 0.0024000000000000002 | injection | ddH2O | 0.06 | buffer | 2 | hemolymph | larva | qPCR W.blot | hemolymph, fat-body | not checked | 20 | none | |||||
| 37 | Silencing of Cecropin D in Spodoptera frugiperda | RNAi | Silencing of one PGRP as well as Immulectin I were performed in parallel to that of Cec D. In all cases, there was a significant decrease in respective mRNA transcript but no associated phenotype such as a higher susceptibility to an infection with E. coli when analysing the insect survival. | InsectaCentral | Unpublished | Duvic | Bernard | Silencing of Cecropin D in Spodoptera frugiperda target | GGTACTTGTTCATACATCAGTTTCATTTTGAGCTTTCTCCAAGTAACAAGGTGCACGGTAGAGGTAAAGCCAAGTTGCAAAAAAAGCAAAAATGAATTCCAAAATCATAATTTTCCTGTGCATCTGCTTCCTAGCTGTGTCTACAGTATCAGCGTGGGACCTCTTTAAAGAAATTGAAGGAGTTGGCCAAAGGGTCCGTGATGCTGTCATCAGTGCAGGACCTGCAGTAGACGTACTAACTAAGGCTAAAAAGCTGGCTGATGGATCCAGCGAAGAAGACTAGAAGACCATCAGGTCAAACTACCCTGATGGGAGTATTGTCAAAGAAAATTATACCAAATGTTTATTTGTATACGATACAGTTTTGTAAATACACATCTCATGTTGGTAATCAACTGCATTTAATCCTTTATTGTATTTTTTTAGTATTTTTTTTTATGTGAATCATTTCATTAAACGTATGTATTAAGCTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | Silencing of Cecropin D in Spodoptera frugiperda target | Silencing of Cecropin D in Spodoptera frugiperda RNAi construct | AACAAGGTGCACGGTAGAGGTAAAGCCAAGTTGCAAAAAAAGCAAAAATGAATTCCAAAATCATAATTTTCCTGTGCATCTGCTTCCTAGCTGTGTCTACAGTATCAGCGTGGGACCTCTTTAAAGAAATTGAAGGAGTTGGCCAAAGGGTCCGTGATGCTGTCATCAGTGCAGGACCTGCAGTAGACGTACTAACTAAGGCTAAAAAGCTGGCTGATGGATCCAGCGAAGAAGACTAGAAGACCATCAGGTCAAACTACCCTGATGGGAGTATTGTCAAAGAAAATTATACCAAATGTTTATTTGTATACGATACAGTTTTGTAAATACACATCTCATGTTGGTAATCAACTGCAT | dsRNA | MEGAscript RNAi Kit - Ambion | column-based | simultaneously with transcription | According to the manufacturer's instructions. cDNA encoding Cec D was cloned into pTriplex vector. Then the sequence used to generate dsRNA was PCR-amplified using primers including a T7 promoter. | InsectaCentral | Silencing of Cecropin D in Spodoptera frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | Spodoptera | local resource | larva | lab_colony | 0.1 | injection | PBS | 42.5 | buffer | Haemocoel | PBS was injected instead of dsRNA to S. frugiperda larvae. On the other hand, when checking for a potentially induced phenotype, 20 µl of PBS per larvae was injected in place of E. coli. | larva | phenotype N.blot | Whole larvae, fat-body and hemocytes | not checked | 24 | high | 90 | ||||||
| 38 | Spofr neuropeptide | RNAi | allatoregulating neuropeptides of Spodoptera frugiperda expression was downregulated by RNAi injecting or feeding dsRNA to the penultimate larval stage or injecting into adults shortly after emergence. RNAi affected the expression of these genes, mortality, development, oviposition, juvenile hormone and ecdysteroid titer(Meyering-Vos et al.,(2006) Functional analysis of the allatostatin-A type gene in the cricket Gryllus bimaculatus and the armyworm Spodoptera frugiperda, Insect Biochem. Mol. Biol. 36, 492-504; Griebler et al., (2008) J. Insect Physiol. 54, 997-1007.) | pubmed | 16731345 | Meyering-Vos | Martina | Spofr neuropeptide target | CTAATACGACTCACTATAGGGCAAGCAGTGGTAAACGCAGAGTACGCGGGGACAGCTGTTAGCTGGCGGGCTTCAAGCACGCCGCATTAACATCGCGTGTGCCAAACCTTACGTGACTACGAACACATAAGAATGCTGTACCCATCAATTCCGGTTTGCTTCCTCGTGATTGGAGTAGCACTCTGCGCTCCAGAGAGGATGCAGAACGAACCAGACCCTCACGACACTCCGGTGCATGAGGGCACTGAGCCACACAGTGACCACATTGCCCCTCTTGAGAAGAGATCCCCTCACTACGACTTTGGGTTGGGCAAGAGGGCTTACAGCTACGTGTCAGAATATAAACGACTACCTGTCTACAACTTTGGACTGGGCAAGAGATCCAGGCCCTACTCCTTTGGCCTGGGCAAACGTTCAGTTGACGAGGACCAGTCCAGCGAGAGCCAGCCTCTGACCAGCGACCTGGACCAAGCTGCCTTAGCTGAATTCTTCGATCAGTATGATGATGCCGGTTACGAGAAGCGCGCTCGACCTTACAGCTTTGGCCTCGGCAAACGCTTCGCTGACGACGAAACTTCCGAAGAAAAGCGGGCAAGGGCATACGACTTTGGACTGGGCAAGCGGCTACCGATGTACAACTTTGGTTTGGGCAAGCGAGCGAGGAGCTCAACTTTGGCTTGGGCAAGCGATTGAGCAGCAAATTCAACTTTGGTTTAGGCAAAAGGGAGAGGGACATGCACGGTTTCAGTTTCGGCCTGGGCAAAAGGGTCCATAAGTTTACGGCCGAAATATGGACTTCTGTTGAGGTCTTAAATAAAATTCTATAATCGTCCTAATTGAATTTAATATGAATAAAGAATAACTTACTTAACAATGTTAATGTCCATGGGCGGCGCTGATCGCTTACCATCAGGTGACTCGTTTGCTCGTTTGCCTCCTATTCCAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | Spofr neuropeptide target | Spofr neuropeptide RNAi construct | CCTCACTACGACTTTGGGTTGGGCAAGAGGGCTTACAGCTACGTGTCAGAATATAAACGACTACCTGTCTACAACTTTGGACTGGGCAAGAGATCCAGGCCCTACTCCTTTGGCCTGGGCAAACGTTCAGTTGACGAGGACCAGTCCAGCGAGAGCCAGCCTCTGACCAGCGACCTGGACCAAGCTGCCTTAGCTGAATTCTTCGATCAGTATGATGATGCCGGTTACGAGAAGCGCGCTCGACCTTACAGCTTTGGCCTCGGCAAACGCTTCGCTGACGACGAAACTTCCGAAGAAAAGCGGGCAAGGGCATACGACTTTGGACTGGGCAAGCGGCTACCGATGTACAACTTTGGTTTGGGCAAGCGAGCGAGGAGCTCAACTTTGGCTTGGGCAAGCGATTGAGCAGCAAATTCAACTTTGGTTTAGGCAAAAGGGAGAGGGACATGCACGGTTTCAGTTTCGGCCT | dsRNA | T7- High Yield Transcript Megascript Kit / Ambion | salt precipitation | subsequent to transcription | setup of reaction according to kit, precipitation by LiCl, denaturation at 95 °C for 5 min, annealing at room temperature | InsectaCentral | Spofr neuropeptide RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | local resource | larva | lab_colony | 0.4 | injection | noctuide ringer | 4 | buffer non_specific_dsRNA | 3 | either noctuide ringer was injected, additionally a non specific control was set for sulfakinin dsRnA derived from cDnA of Gryllus bimaculatus | larva | qPCR RT-PCR (semi-quantitative) | brain | not checked | 96 | high | 74 | |||||
| 39 | RNAi analysis of Ultrabithorax in Bombyx mori 1 | RNAi | pubmed | 19908062 | Niimi | Teruyuki | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCATACTTTCTACCCTTGGATGGCCATAGCAGGAGCGAACGGCCTCAGGAGACGAGGAAGACAAACCTACACTAGATATCAAACGCTAGAATTAGAGAAAGAGTTCCACACGAACCACTACCTTACGCGAAGGAGACGCATAGAGATGGCGCACGCGTTGTGCCTCACGGAGAGGCAAATCAAAATATGGTTCCAGAACCGAAGGATGAAGTTAAAGAAAGAGATCCAGGCTATAAAGGAGTTGAACGAGCAGGAGAAACAGGCGCAGGCGCAGAAGGCGGCAGCGGCTGCTGCGGCGGCCGCGGCTGCTGCCCAGGGACACCCCGAACATTAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | RNAi analysis of Ultrabithorax in Bombyx mori 1 RNAi construct | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCATACTTTCTACCCTTGGATGGCCATAGCAGGAGCGAACGGCCTCAGGAGACGAGGAAGACAAACCTACACTAGATATCAAACGCTAGAATTAGAGAAAGAGTTCCACACGAACCACTACCTTACGCGAAGGAGACGCATAGAGATGGCGCACGCGTTGTGCCTCACGGAGAGGCAAATCAAAATATGGTTCCAGAACCGAAGGATGAAGTTAAAGAAAGAGATCCAGGCTATAAAGGAGTTGAACGAGCAGGAGAAACAGGCGCAGGCGCAGAAGGCGGCAGCGGCTGCTGCGGCGGCCGCGGCTGCTGCCCAGGGACACCCCGAACATTAA | dsRNA | MEGAscript T7 kit (Ambion) | salt precipitation | subsequent to transcription | Preparation of dsRNA A 765 bp fragment (the ORF of Bm-Ubx cDNA), obtained using Bm-Ubx-5 (5'-ATGAACTCTTACTTCGAGCA-3') and Bm-Ubx-8 (5'-TTAATGTTCGGGGTGTCCCT-3') as primers (See Dev. Genes Evol., 219, 437-444 (2009), Fig. S1; Electronic Supplementary Material) were subcloned into the EcoR V site of the pBluescript KSïĞ vector. For the production of a template for in vitro transcription, PCR was performed using plasmids containing Bm-Ubx-ORF (the 765 bp PCR product), two universal primers (T7-KS, 5'-TAATACGACTCACTATAGGGAGACCACTCGAGGTCGACGGTATC-3'; T7-SK, 5'-TAATACGACTCACTATAGGGAGACCACCGCTCTAGAACTAGTGGATC-3') containing the T7 polymerase promoter sequence at their 5' ends, and AmpliTaq Gold (Perkin Elmer). As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). Sense and antisense transcripts were simultaneously synthesized using 1 Îĵg PCR product and a MEGAscript T7 kit (Ambion) according to the manufacturerâs instructions. After DNase I treatment, RNA precipitated with LiCl was dissolved in RNase free water. The RNA solution was heated at 65°C for 30 min and cooled slowly to room temperature for annealing of the dsRNA. The concentrations of dsRNA for Bm-Ubx-ORF and DsRed were 1.2 Îĵg/µl. The quality of the dsRNA was examined by agarose gel electrophoresis and small aliquots of the dsRNA were stored at â80°C until use. Injection of dsRNA into B. mori embryos (N4 strain) Injections were performed under a dissection microscope (Stemi 2000, Carl Zeiss). B. mori embryos (N4) were collected within 5 h of oviposition to perform dsRNA injection during the syncytial blastoderm stage. The dsRNA was injected ventrally into an egg using a micromanipulator (Narishige) and FemtoJet (Eppendorf). The injection into an embryo was performed with a special glass needle (uMPm-02, Natsume Optical Corporation). | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 1 RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | embryo | lab_colony | 1.2 | injection | RNase free water | None | 0.035 | non_specific_dsRNA | 227 | B. mori embryos (N4 strain) were collected within 5 h of oviposition to perform dsRNA injection during the syncytial blastoderm | As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). | embryo | phenotype | whole pharate first instar larvae | not checked | 336 | high | 16.7 | ||||
| 40 | RNAi analysis of Ultrabithorax in Bombyx mori 2 | RNAi | pubmed | 19908062 | Niimi | Teruyuki | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCATACTTTCTACCCTTGGATGGCCATAGCAGGAGCGAACGGCCTCAGGAGACGAGGAAGACAAACCTACACTAGATATCAAACGCTAGAATTAGAGAAAGAGTTCCACACGAACCACTACCTTACGCGAAGGAGACGCATAGAGATGGCGCACGCGTTGTGCCTCACGGAGAGGCAAATCAAAATATGGTTCCAGAACCGAAGGATGAAGTTAAAGAAAGAGATCCAGGCTATAAAGGAGTTGAACGAGCAGGAGAAACAGGCGCAGGCGCAGAAGGCGGCAGCGGCTGCTGCGGCGGCCGCGGCTGCTGCCCAGGGACACCCCGAACATTAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | RNAi analysis of Ultrabithorax in Bombyx mori 2 RNAi construct | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCAT | dsRNA | MEGAscript T7 kit (Ambion) | salt precipitation | subsequent to transcription | Preparation of dsRNA A 435 bp fragment (encoding the N-terminus sequences to the conserved YPWM motif) was obtained using Bm-Ubx-5 and Bm-Ubx-6 (5'-ATGATTAGTAGGCTGTTGGT-3') as primers (See Dev. Genes Evol., 219, 437-444 (2009), Fig. S1; Electronic Supplementary Material) were subcloned into the EcoR V site of the pBluescript KSïĞ vector. For the production of a template for in vitro transcription, PCR was performed using plasmids containing Bm-Ubx-HDless (the 445 bp PCR product, which does not contain homeobox; Fig. S1; Electronic Supplementary Material), two universal primers (T7-KS, 5'-TAATACGACTCACTATAGGGAGACCACTCGAGGTCGACGGTATC-3'; T7-SK, 5'-TAATACGACTCACTATAGGGAGACCACCGCTCTAGAACTAGTGGATC-3') containing the T7 polymerase promoter sequence at their 5' ends, and AmpliTaq Gold (Perkin Elmer). As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). Sense and antisense transcripts were simultaneously synthesized using 1 Îĵg PCR product and a MEGAscript T7 kit (Ambion) according to the manufacturerâs instructions. After DNase I treatment, RNA precipitated with LiCl was dissolved in RNase free water. The RNA solution was heated at 65°C for 30 min and cooled slowly to room temperature for annealing of the dsRNA. The concentrations of dsRNA for Bm-Ubx-HDless and DsRed were 1.2 Îĵg/µl. The quality of the dsRNA was examined by agarose gel electrophoresis and small aliquots of the dsRNA were stored at â80°C until use. Injection of dsRNA into B. mori embryos (N4 strain) Injections were performed under a dissection microscope (Stemi 2000, Carl Zeiss). B. mori embryos (N4 strain) were collected within 5 h of oviposition to perform dsRNA injection during the syncytial blastoderm stage. The dsRNA was injected ventrally into an egg using a micromanipulator (Narishige) and FemtoJet (Eppendorf). The injection into an embryo was performed with a special glass needle (uMPm-02, Natsume Optical Corporation). | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 2 RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | embryo | lab_colony | 1.2 | injection | RNase free water | None | 0.035 | non_specific_dsRNA | 225 | B. mori embryos (N4) were collected within 5 h of oviposition to perform dsRNA injection during the syncytial blastoderm stage. | As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). | embryo | phenotype | whole pharate first instar larvae | not checked | 336 | high | 34.3 | ||||
| 41 | RNAi analysis of Ultrabithorax in Bombyx mori 3 | RNAi | InsectaCentral | Unpublished | Niimi | Teruyuki | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCATACTTTCTACCCTTGGATGGCCATAGCAGGAGCGAACGGCCTCAGGAGACGAGGAAGACAAACCTACACTAGATATCAAACGCTAGAATTAGAGAAAGAGTTCCACACGAACCACTACCTTACGCGAAGGAGACGCATAGAGATGGCGCACGCGTTGTGCCTCACGGAGAGGCAAATCAAAATATGGTTCCAGAACCGAAGGATGAAGTTAAAGAAAGAGATCCAGGCTATAAAGGAGTTGAACGAGCAGGAGAAACAGGCGCAGGCGCAGAAGGCGGCAGCGGCTGCTGCGGCGGCCGCGGCTGCTGCCCAGGGACACCCCGAACATTAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | RNAi analysis of Ultrabithorax in Bombyx mori 2 RNAi construct | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCAT | dsRNA | MEGAscript T7 kit (Ambion) | salt precipitation | subsequent to transcription | Preparation of dsRNA A 435 bp fragment (encoding the N-terminus sequences to the conserved YPWM motif) was obtained using Bm-Ubx-5 and Bm-Ubx-6 (5'-ATGATTAGTAGGCTGTTGGT-3') as primers (See Dev. Genes Evol., 219, 437-444 (2009), Fig. S1; Electronic Supplementary Material) were subcloned into the EcoR V site of the pBluescript KSïĞ vector. For the production of a template for in vitro transcription, PCR was performed using plasmids containing Bm-Ubx-HDless (the 445 bp PCR product, which does not contain homeobox; Fig. S1; Electronic Supplementary Material), two universal primers (T7-KS, 5'-TAATACGACTCACTATAGGGAGACCACTCGAGGTCGACGGTATC-3'; T7-SK, 5'-TAATACGACTCACTATAGGGAGACCACCGCTCTAGAACTAGTGGATC-3') containing the T7 polymerase promoter sequence at their 5' ends, and AmpliTaq Gold (Perkin Elmer). As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). Sense and antisense transcripts were simultaneously synthesized using 1 Îĵg PCR product and a MEGAscript T7 kit (Ambion) according to the manufacturerâs instructions. After DNase I treatment, RNA precipitated with LiCl was dissolved in RNase free water. The RNA solution was heated at 65°C for 30 min and cooled slowly to room temperature for annealing of the dsRNA. The concentrations of dsRNA for Bm-Ubx-HDless and DsRed were 1.2 Îĵg/µl. The quality of the dsRNA was examined by agarose gel electrophoresis and small aliquots of the dsRNA were stored at â80°C until use. Injection of dsRNA into B. mori embryos (N4 strain) Injections were performed under a dissection microscope (Stemi 2000, Carl Zeiss). B. mori embryos (N4 strain) were collected within 5 h of oviposition to perform dsRNA injection during the syncytial blastoderm stage. The dsRNA was injected ventrally into an egg using a micromanipulator (Narishige) and FemtoJet (Eppendorf). The injection into an embryo was performed with a special glass needle (uMPm-02, Natsume Optical Corporation). | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 2 RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 0.124 | injection | RNase free water | in vivo jetPEI (Polyplus transfection) | 0.114 | non_specific_dsRNA | 2 | 4.96 Îĵg of dsRNA in 40 Îĵl solution was injected laterally into A2 segment of each 0-day-old female pupa (N4 strain) (approximate | As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). | larva | phenotype | first instar larvae | not checked | 480 | none | |||||
| 42 | RNAi analysis of Ultrabithorax in Bombyx mori 4 | RNAi | InsectaCentral | Unpublished | Niimi | Teruyuki | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCATACTTTCTACCCTTGGATGGCCATAGCAGGAGCGAACGGCCTCAGGAGACGAGGAAGACAAACCTACACTAGATATCAAACGCTAGAATTAGAGAAAGAGTTCCACACGAACCACTACCTTACGCGAAGGAGACGCATAGAGATGGCGCACGCGTTGTGCCTCACGGAGAGGCAAATCAAAATATGGTTCCAGAACCGAAGGATGAAGTTAAAGAAAGAGATCCAGGCTATAAAGGAGTTGAACGAGCAGGAGAAACAGGCGCAGGCGCAGAAGGCGGCAGCGGCTGCTGCGGCGGCCGCGGCTGCTGCCCAGGGACACCCCGAACATTAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | RNAi analysis of Ultrabithorax in Bombyx mori 2 RNAi construct | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCAT | dsRNA | MEGAscript T7 kit (Ambion) | salt precipitation | subsequent to transcription | Preparation of dsRNA A 435 bp fragment (encoding the N-terminus sequences to the conserved YPWM motif) was obtained using Bm-Ubx-5 and Bm-Ubx-6 (5'-ATGATTAGTAGGCTGTTGGT-3') as primers (See Dev. Genes Evol., 219, 437-444 (2009), Fig. S1; Electronic Supplementary Material) were subcloned into the EcoR V site of the pBluescript KSïĞ vector. For the production of a template for in vitro transcription, PCR was performed using plasmids containing Bm-Ubx-HDless (the 445 bp PCR product, which does not contain homeobox; Fig. S1; Electronic Supplementary Material), two universal primers (T7-KS, 5'-TAATACGACTCACTATAGGGAGACCACTCGAGGTCGACGGTATC-3'; T7-SK, 5'-TAATACGACTCACTATAGGGAGACCACCGCTCTAGAACTAGTGGATC-3') containing the T7 polymerase promoter sequence at their 5' ends, and AmpliTaq Gold (Perkin Elmer). As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). Sense and antisense transcripts were simultaneously synthesized using 1 Îĵg PCR product and a MEGAscript T7 kit (Ambion) according to the manufacturerâs instructions. After DNase I treatment, RNA precipitated with LiCl was dissolved in RNase free water. The RNA solution was heated at 65°C for 30 min and cooled slowly to room temperature for annealing of the dsRNA. The concentrations of dsRNA for Bm-Ubx-HDless and DsRed were 1.2 Îĵg/µl. The quality of the dsRNA was examined by agarose gel electrophoresis and small aliquots of the dsRNA were stored at â80°C until use. Injection of dsRNA into B. mori embryos (N4 strain) Injections were performed under a dissection microscope (Stemi 2000, Carl Zeiss). B. mori embryos (N4 strain) were collected within 5 h of oviposition to perform dsRNA injection during the syncytial blastoderm stage. The dsRNA was injected ventrally into an egg using a micromanipulator (Narishige) and FemtoJet (Eppendorf). The injection into an embryo was performed with a special glass needle (uMPm-02, Natsume Optical Corporation). | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 2 RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 0.124 | injection | RNase free water | in vivo jetPEI (Polyplus transfection) | 0.023 | non_specific_dsRNA | 2 | 0.99 Îĵg of dsRNA in 8 Îĵl solution was injected laterally into A2 segment of each 0-day-old female pupa (N4 strain) (approximatel | As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). | larva | phenotype | first instar larvae | not checked | 480 | none | |||||
| 43 | RNAi analysis of Ultrabithorax in Bombyx mori 5 | RNAi | InsectaCentral | Unpublished | Niimi | Teruyuki | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCATACTTTCTACCCTTGGATGGCCATAGCAGGAGCGAACGGCCTCAGGAGACGAGGAAGACAAACCTACACTAGATATCAAACGCTAGAATTAGAGAAAGAGTTCCACACGAACCACTACCTTACGCGAAGGAGACGCATAGAGATGGCGCACGCGTTGTGCCTCACGGAGAGGCAAATCAAAATATGGTTCCAGAACCGAAGGATGAAGTTAAAGAAAGAGATCCAGGCTATAAAGGAGTTGAACGAGCAGGAGAAACAGGCGCAGGCGCAGAAGGCGGCAGCGGCTGCTGCGGCGGCCGCGGCTGCTGCCCAGGGACACCCCGAACATTAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | RNAi analysis of Ultrabithorax in Bombyx mori 1 RNAi construct | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCATACTTTCTACCCTTGGATGGCCATAGCAGGAGCGAACGGCCTCAGGAGACGAGGAAGACAAACCTACACTAGATATCAAACGCTAGAATTAGAGAAAGAGTTCCACACGAACCACTACCTTACGCGAAGGAGACGCATAGAGATGGCGCACGCGTTGTGCCTCACGGAGAGGCAAATCAAAATATGGTTCCAGAACCGAAGGATGAAGTTAAAGAAAGAGATCCAGGCTATAAAGGAGTTGAACGAGCAGGAGAAACAGGCGCAGGCGCAGAAGGCGGCAGCGGCTGCTGCGGCGGCCGCGGCTGCTGCCCAGGGACACCCCGAACATTAA | dsRNA | MEGAscript T7 kit (Ambion) | salt precipitation | subsequent to transcription | Preparation of dsRNA A 765 bp fragment (the ORF of Bm-Ubx cDNA), obtained using Bm-Ubx-5 (5'-ATGAACTCTTACTTCGAGCA-3') and Bm-Ubx-8 (5'-TTAATGTTCGGGGTGTCCCT-3') as primers (See Dev. Genes Evol., 219, 437-444 (2009), Fig. S1; Electronic Supplementary Material) were subcloned into the EcoR V site of the pBluescript KSïĞ vector. For the production of a template for in vitro transcription, PCR was performed using plasmids containing Bm-Ubx-ORF (the 765 bp PCR product), two universal primers (T7-KS, 5'-TAATACGACTCACTATAGGGAGACCACTCGAGGTCGACGGTATC-3'; T7-SK, 5'-TAATACGACTCACTATAGGGAGACCACCGCTCTAGAACTAGTGGATC-3') containing the T7 polymerase promoter sequence at their 5' ends, and AmpliTaq Gold (Perkin Elmer). As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). Sense and antisense transcripts were simultaneously synthesized using 1 Îĵg PCR product and a MEGAscript T7 kit (Ambion) according to the manufacturerâs instructions. After DNase I treatment, RNA precipitated with LiCl was dissolved in RNase free water. The RNA solution was heated at 65°C for 30 min and cooled slowly to room temperature for annealing of the dsRNA. The concentrations of dsRNA for Bm-Ubx-ORF and DsRed were 1.2 Îĵg/µl. The quality of the dsRNA was examined by agarose gel electrophoresis and small aliquots of the dsRNA were stored at â80°C until use. Injection of dsRNA into B. mori embryos (N4 strain) Injections were performed under a dissection microscope (Stemi 2000, Carl Zeiss). B. mori embryos (N4) were collected within 5 h of oviposition to perform dsRNA injection during the syncytial blastoderm stage. The dsRNA was injected ventrally into an egg using a micromanipulator (Narishige) and FemtoJet (Eppendorf). The injection into an embryo was performed with a special glass needle (uMPm-02, Natsume Optical Corporation). | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 1 RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 0.033 | injection | RNase free water | DOTAP liposomal transfection reagent (Roche) | 0.14300000000000002 | non_specific_dsRNA | 3 | 1.67 Îĵg of dsRNA in 50 Îĵl solution was injected laterally into A2 segment of each 0-day-old female pupa (N4 strain) (approximate | As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). | larva | phenotype | first instar larvae | not checked | 480 | none | |||||
| 44 | RNAi analysis of Ultrabithorax in Bombyx mori 6 | RNAi | InsectaCentral | Unpublished | Niimi | Teruyuki | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCATACTTTCTACCCTTGGATGGCCATAGCAGGAGCGAACGGCCTCAGGAGACGAGGAAGACAAACCTACACTAGATATCAAACGCTAGAATTAGAGAAAGAGTTCCACACGAACCACTACCTTACGCGAAGGAGACGCATAGAGATGGCGCACGCGTTGTGCCTCACGGAGAGGCAAATCAAAATATGGTTCCAGAACCGAAGGATGAAGTTAAAGAAAGAGATCCAGGCTATAAAGGAGTTGAACGAGCAGGAGAAACAGGCGCAGGCGCAGAAGGCGGCAGCGGCTGCTGCGGCGGCCGCGGCTGCTGCCCAGGGACACCCCGAACATTAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 1 target | RNAi analysis of Ultrabithorax in Bombyx mori 1 RNAi construct | ATGAACTCTTACTTCGAGCAGGGTGGTTTTTACGGGGCCCATGGAGTGCACCAGGGCGGCGGTGGTGGAGACCAGTACCGCGGCTTCCCTCTGGGCCTCACGTATGCACAGCCACACGCTTTGCACCAGCCTCGTCCTCAGGATTCACCGTACGACGCGTCTGTCGCGGCGGCCTGTAAGCTCTATGCTGGAGAGCAGCAATATCCTAAGGCAGATTGTTCAAAGCCAGGCGGTGAGCAGCAGAATGGCTATGGTGGGAAAGAAGCCTGGGGCTCAGGTCTGGGAGCACTAGTGAGGCCGGCAGCATGCACTCCTGAAGCTCGATACAGCGAGTCGTCAAGTCCTGGTAGAGCACTTCCGTGGGGCAACCAGTGTGCACTTCCGGGATCAGCAGCATCAGCCGCGCAGCCAGTGCACCAACAGCCTACTAATCATACTTTCTACCCTTGGATGGCCATAGCAGGAGCGAACGGCCTCAGGAGACGAGGAAGACAAACCTACACTAGATATCAAACGCTAGAATTAGAGAAAGAGTTCCACACGAACCACTACCTTACGCGAAGGAGACGCATAGAGATGGCGCACGCGTTGTGCCTCACGGAGAGGCAAATCAAAATATGGTTCCAGAACCGAAGGATGAAGTTAAAGAAAGAGATCCAGGCTATAAAGGAGTTGAACGAGCAGGAGAAACAGGCGCAGGCGCAGAAGGCGGCAGCGGCTGCTGCGGCGGCCGCGGCTGCTGCCCAGGGACACCCCGAACATTAA | dsRNA | MEGAscript T7 kit (Ambion) | salt precipitation | subsequent to transcription | Preparation of dsRNA A 765 bp fragment (the ORF of Bm-Ubx cDNA), obtained using Bm-Ubx-5 (5'-ATGAACTCTTACTTCGAGCA-3') and Bm-Ubx-8 (5'-TTAATGTTCGGGGTGTCCCT-3') as primers (See Dev. Genes Evol., 219, 437-444 (2009), Fig. S1; Electronic Supplementary Material) were subcloned into the EcoR V site of the pBluescript KSïĞ vector. For the production of a template for in vitro transcription, PCR was performed using plasmids containing Bm-Ubx-ORF (the 765 bp PCR product), two universal primers (T7-KS, 5'-TAATACGACTCACTATAGGGAGACCACTCGAGGTCGACGGTATC-3'; T7-SK, 5'-TAATACGACTCACTATAGGGAGACCACCGCTCTAGAACTAGTGGATC-3') containing the T7 polymerase promoter sequence at their 5' ends, and AmpliTaq Gold (Perkin Elmer). As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). Sense and antisense transcripts were simultaneously synthesized using 1 Îĵg PCR product and a MEGAscript T7 kit (Ambion) according to the manufacturerâs instructions. After DNase I treatment, RNA precipitated with LiCl was dissolved in RNase free water. The RNA solution was heated at 65°C for 30 min and cooled slowly to room temperature for annealing of the dsRNA. The concentrations of dsRNA for Bm-Ubx-ORF and DsRed were 1.2 Îĵg/µl. The quality of the dsRNA was examined by agarose gel electrophoresis and small aliquots of the dsRNA were stored at â80°C until use. Injection of dsRNA into B. mori embryos (N4 strain) Injections were performed under a dissection microscope (Stemi 2000, Carl Zeiss). B. mori embryos (N4) were collected within 5 h of oviposition to perform dsRNA injection during the syncytial blastoderm stage. The dsRNA was injected ventrally into an egg using a micromanipulator (Narishige) and FemtoJet (Eppendorf). The injection into an embryo was performed with a special glass needle (uMPm-02, Natsume Optical Corporation). | InsectaCentral | RNAi analysis of Ultrabithorax in Bombyx mori 1 RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 0.243 | injection | RNase free water | None | 0.057 | non_specific_dsRNA | 2 | 4.85 Îĵg of dsRNA in 20 Îĵl solution was injected laterally into A2 segment of each 0-day-old female pupa (N4 strain) (approximate | As a negative control, DsRed ORF (678 bp) was amplified using two primers with a T7 promoter sequence at the 5' end (T7-DsRed5, 5'-TAATACGACTCACTATAGGGAGACCACATGGTGCGCTCCTCCAAG-3'; T7-DsRed3, 5'-TAATACGACTCACTATAGGGAGACCACCTACAGGAACAGGTGGTG-3'). | larva | phenotype | first instar larvae | not checked | 480 | none | |||||
| 46 | Bicyclus anynana Dll dsRNA injections | RNAi | In an attempt to knock down expression of the Distal-less (Dll) gene in Bicyclus anynana, 0.3-2.8 micrograms of a 375 bp double stranded RNA (dsRNA) corresponding to the B. anynana Dll gene was synthesised using the Megascript RNAi kit (AMBION) and injected into larvae and pupae. Animals were allowed to eclose and inspected for phenotypic modifications of a) the adult eyespot on the wing, and b) offspring embryos of mated females. No injection resulted in phenotypic modification of the adults or their offspring. | InsectaCentral | Unpublished | Oostra | Vicencio | Bicyclus anynana Dll dsRNA injections target | CGCGAGTTGGTTGTGTCGGGTTACCTCGGAGGGGTAACGCTATTAAAATATCTACAAACATTACCTTTAATTGGACAGAAATTTGTGCTTGTGTGTGTGTATCTCGTTTTTTTTTTAGTATCCAACTGTCAGTGACGATCTGCCGTTTTTTCGAAAGCTGATGACTGTTATCATTCCTGTTTAGTGTGTGGTGATAGTCAGTGATTTCTGCGATTCGAAAATGACCGCCGATTAGAATTGCGTACGCCGATTTCGTTCGGACCGATGGTGATGGTGATTAAAAATGACATCAGTGAGGTGATAGCCGAATCGCGGGCAAATCGCGCAAAGAGCACAGACGCCACGTGCTCTATAATTGCGAAATTGTTTATGGATCGTTAATTAATCTATAATTATATTTCTGTCCCGCTCCCGCTAATTGCCGTCAATCATATTTTGATTTCGCATTACCAAGTGGCGTTTTTAATTCGGATTTTCTTTGCTCTACATATTTTTTTAAAACTCTAAACTACTTTTAACTCCATACAAAGCAAAAAATCGGTTATAAATAATTAGAAACACAATAAGCACCATAAAGAAATTATAAAAATCCAAAATATTATCTGTGAATAGACAAGTGGTGAATTTTTTAAATTTGAAACAATATCTAGTGATTTTTTTTTAAATTGTGATAGTGATGCTGTGTTTCCACGTTTGAGGCCAAACAAGACCGCGCCACAAGCGGCCGGGTAGCGTTCGGAGCAGTCCAGAAATCCCTCAAAATTACACGAATTCAGTCCCCAAACAGCAAACCCACCACGCTGAGTTTCTCAGATCCCTTTGGGCCTCCTCAGTCCACGGACGGGGGGGGCCCGTCGACCCCGCAGCCCGCCATGACCACCCAGGAGCTGGACCACCAACACCACCACCTGGGAGGTTCCCAAACCCCCCACGATATATCCAACTCCACGAATTCCACCCCCACCAACGTTTCCTCGAAATCCGCCTTCATAGAGTTACAGCAACACGGTTACGGGCCTTTCAAAGGTGGTTACCAACATCCCCACCATTTCGGCAGCCCGGGGGGTCAGCAGAACCCCCACGAGGCGTCGGGGTTCCCGAGCCCTAGGTCCTTAGGTTATCCCTTTCCTCCTATGCACCAAAATACGTACGGATATCACATAGGCTCCTACGCTCCACAATGCGCAAGTCCGCCCAAAGATGAAAAATGTGGTCTATCCGACGACCCTGGGCTGCGGGTGAATGGCAAGGGCAAGAAGATGCGCAAGCCGCGCACCATCTACTCCAGCTTGCAGCTGCAGCAGCTCAACAGGCGGTTCCAGAGGACACAGTATCTAGCACTGCCGGAGCGAGCTGAGCTGGCTGCTAGCTTGGGCCTGACGCAGACGCAGGTGAAGATCTGGTTCCAGAACAGACGCAGTAAATACAAAAAGATGATGAAGGCCGCGCAAGTAGGCGCCGTACCGCCCGGCCTCGGCCTACCGCCGGGCAGTCCACCCAACAACAACCAACTTTTACATGGTGGCGGCGGCAGTTCCAGCGGTTCCCAACATTCGCCGTCGGGCTACGCGGGCGGGCCGGCGCAAGCCCACTCGCCCACGCCCAGCTCCACGCCCGTGTCCGAGCTGTCCCCGGGCCTGTCGCCCACGGCCACGCCGTGGGATGTTAAGCAGCCCCCTCAGCCCTCATGGGACGTGAAGGTTGGATATCCCACGGCCGGTCGGTCCCCTGATGGCACCTCCTGTGATGTGAAGCCTCCGCACCAGCAGTCGTGGGATCCAAGGGTCGGATACGGCGCGCCCCCCGGCCCTTGGGACATGAAGGGCGCGCACGCTCACGCTCTACACCACCAGGGGGCGCCGCAGCCGCACCCCCCCTACGTGCCGCAGTACTCCTGGTACCAGGCTGATGCCAACCCTGGCCTCTTGACGGTCTGGCCGGCCGTGTAACGACCATCGGAACAATCAAAATGACACACACGCTAAAAATATAATAATTTGCAAGTATAATTCACTTAAAACTTATAAAACACACAAAATGGCGTACCTAATTCCTAAACGAAAGGACGCCACAACCAAGATTATTGTTAAGAATGTTATTTAATAGTTATAAAAAATAATTGTACATTTTTTGTATTAACAATAAACTATAAATAAATAGATGTATAATGTTTTTAATGTAATTGTAAATTTTCATGAGGATAAGTATTAAAAGAAATATACAATTCAAAAGTTCCAGTGCCCATGTTGGTCAGACTTATTAAAATTATATTTTTTTTGTCACAAAATTGTATAATTTTTTGGTGTCAAAGTTGTGTATGCCATTACACCCAGTTTAGAAATAGATAAATCAAAAATGCGTATGCAATTTTGACTGATGTATATAACAATATTACATGTGAAAGGTGGTTGTATCCTATTTTATTTTGTATTTTTAAATTCCCGTTATTGTAAATAGGTTAAAAATTAATGAAATACTATGCTGTAAATTTCTGTATTTGTGAATAACTGTTTAATTGTGCAATAAATTCATGTAATTTTAATGAACCCTTCTTCATAGGTGAAAATTCTTCATGATATATATACTTATATTATATATTTGTGAATTTAACCAGAATTAATGTAACGTACTGCAAGTAACGTGGCCACGGTATAAAACGTGAGTTTTYTAAACCGCAGTAAAATTTTAAATATTAACAAAACCCCTGGAGTATTACGATTATTTCTAAGGCTATTTCTATTTCTTATTTTTATGTTTGTCATTTTTATAGCCGTCAATAAAATCATATGCGATTATTATTACAGCATTAAAAATAATCGTCAACCGATACTTTTTTTTCAAAGTAACTTTGACAAAAAAAAACGTTTTTTTCTAGCAGCAATTGTGTTCAACCCCAATACCACATAATATTTAAATATAAAATAAGAATACAAAACCTTTAATGTTGTAGGTACTAGAAAGTATTAAACATTTTTTTTTTCAAAATTACTTCGAAAAGCAAAGTTGGTAAAATTTAATTGTAATCTTTTTTTATTTTGCCCATTTAACTAACATTAATTTAATTGTGTTCTGTATTAATAATAACCTCACCTGAAATGAAGTTATGGTGCTGATTTATATAACAATTTAACAAAGTTGTGTTTTAATAATAAAAACACAATTTTTGTGTGTTATTATTTAACACATGAAATAAACCAGTGATTTTACGAGTTCTTTGGAATTAATGATGGCTGACAGTATTCTATCATTTCTTTATGAAATTATATTTTTTTTAAACTAAGCTTTAAAACATGATTGTGTTTATTTATTTAAATTTAATCAATAAATCAAACGGGAACACAAACCAGTCTGTTGTGATACATAATTTTAAGATTATTTTTTGTAGACAAAATATAATTTGTTTTTTTCTTTGGTTAACTATTAACAAAATGTGTATTTATTACATAATGTTTGTGCAAAAATTGTGTTCTGAATGCAAAATCTATATTAAGCTATTTTTATTTCTAATGCAAACTGAATTTATATTTTGTAAATACTGTAGTTTATTTTGAGATAACACATCACATAATATAAAAATATAATCATTAAAATAAAGTTTTTTTAATACAAAATATTTGTGGAAAAATTACAATATACTCGTATTATTAAATTTAAAATTCTAAAGGAAAATTAATGATTTTTTTTGTCTATTTTATTGAATATTTAGTAACGCATAATATAATTGTTATGTGTACAACCAAATAGCAAAGTCAGTATAAAATTTCCAGAATAAAGTAATTATTTGAAACATTTATCATTTTTGGTTGTTTTCTAATAATTTGAAAATGTAGTGTGTAAATCTAGGAAAATCTTGATGCATAAATTGAAAATCATTTTCAGATTATTATTAAAATATTCTTATAATTATGTTGTACTCGAGATAACTAAGGTGKTATTTTAGTAAATATAAAGGTGKTTTATTGCTTTTAAATTGTYTCCCAATAGACTMCAGTATATAAAAAAAAGGTGTTACGGGGTGTTTTTTTGTAAATATGTTTTGTAATATATAGAGAGTATAAAATATACATATAAATATAGKTTAGATAGTATAATTACTTTATAACTACMATGTATTATAAGGAAAATGGRAATGAGAAAAAAGTGAATTAATTTCTTTGTCCAATAAAAATTG | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | cds | GenBank | AF404825.1 | Bicyclus anynana Dll dsRNA injections RNAi construct | ACGAATTCAGTCCCCAAACAGCAAACCCACCACGCTGAGTTTCTCAGATCCCTTTGGGCCTCCTCAGTCCACGGACGGGGGGGGCCCGTCGACCCCGCAGCCCGCCATGACCACCCAGGAGCTGGACCACCAACACCACCACCTGGGAGGTTCCCAAACCCCCCACGATATATCCAACTCCACGAATTCCACCCCCACCAACGTTTCCTCGAAATCCGCCTTCATAGAGTTACAGCAACACGGTTACGGGCCTTTCAAAGGTGGTTACCAACATCCCCACCATTTCGGCAGCCCGGGGGGTCAGCAGAACCCCCACGAGGCGTCGGGGTTCCCGAGCCCTAGGTCCTTAGGTTATCCCTTTCCTCCTATGCACCAA | dsRNA | AMBION Megascript RNAi kit | column-based | subsequent to transcription | The 375 bp template for the synthesis of dsRNA corresponding to the B. anynana Dll gene (Genbank accession # AF404825.1) was prepared by amplifying cDNA that was previously synthesised from total RNA of embryo and wing tissue of different stages. In 100 Îĵl PCR reactions, 10 Îĵl template was incubated with 2 U Taq DNA polymerase in its recommended buffer (QIAGEN), 10 ÎĵM Dll-specific primers containing T7 promotor sequence and 2.5 mM of each dNTP. After exploring different PCR cycling conditions to maximise DNA yield, the reaction was set up according to the following optimised conditions: 94 oC for 3 minutes, 5 x (94 oC for 30 seconds, 56 oC for 30 sec., 72 oC for 45 sec.) and 28 x (94 oC for 30 seconds, 62 oC for 30 sec., 72 oC for 45 sec.) followed by 72 oC for 10 minutes. This yielded a specific product of 375 bp, the expected size, as verified by checking on 1.1% agarose gel. After cleanup over a filter cartridge (PROMEGA) concentration was quantified by measuring absorbance at 260nm with a spectrophotometer. Reactions yielded 3.4 - 3.9 Îĵg PCR product, which was diluted to the desired concentration for dsRNA synthesis. Using the Megascript RNAi kit (AMBION), 1 Îĵg PCR product was used as template for in vitro transcription of dsRNA in a single reaction by incubating it with 2 Îĵl T7 RNA polymerase in its recommended buffer and 8 Îĵl dNTPs for 16 h at 37 oC, according to manufactorerâs instructions. Subsequently, the dsRNA was checked on 1.1% agarose gel to verify size and quality, quantified by measuring absorbance at 260nm with a spectrophotometer and stored at -20oC. Afterwards, dsRNA was thawed, incubated for 5 min at 75 oC and (on bench) slowly cooled to room temperature, allowing dsRNA to anneal properly. Subsequently, dsRNA was digested for 1 h at 37 oC with DNase I and RNase according to manufacturerâs instructions to degrade contaminating DNA and single strand RNA. Finally, dsRNA was purified using the provided filter cartridge, eluted in 100 Îĵl PBS (pH 7), checked on 1.1% agarose gel to verify size and quality, quantified by measuring absorbance at 260nm with a spectrophotometer and stored at -20oC. | InsectaCentral | Bicyclus anynana Dll dsRNA injections RNAi construct | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | evolutionary biology lab, institute of biology leiden, leiden university, the netherlands | pupa | microsporidia | lab_colony | 0.7 | injection | PBS | 0.02 | buffer | 19 | Pupae were injected between the 3rd and 4th abdominal segment in the dorsal abdomen. | In parallel to the 19 injected pupae, 41 other pupae were injected with the same volume of PBS without dsRNA. They were treated in the same way, and their phenotypes were also assayed in the same way. | adult | phenotype | not checked | 160 | none | |||||
| 47 | Mamestra IIM1 gene midgut RNAi Hegedus-Erlandson-Toprak | RNAi | McIIM1 gene encoding a PM-associated mucin was successfully knocked down in a midgut primary cell culture system. | InsectaCentral | Unpublished | Hegedus | Dwayne | Mamestra IIM1 gene midgut RNAi Hegedus-Erlandson-Toprak target | CATAACCAAAGTGGTATTTGTGACTGTGTGTGCAAAATACGAACATCCAAAATAAAGGAATGATAAAAACCCTTCTACTCGTGACGGCCTTAGCCCTCGTGCAGGCTCGTCCCAACGAAGATGCAGACTTGACCAACGGCAGGTTATATGAAGTCCATGATGACTGTCCTCCTGCTGAAGTTCACTTCTTGCTGCCCCACGAATACGACTGCACCAAGTTCTACTACTGTGAATATGGTCTGAAGTATATCGAACCCAGAAACTGTGCTTCTGGTACTGAGTTCAACGCTGAGATCCAGGTTTGTGTTCATCCAAGCTCTTCAGGATGCTCTTTACCCGGATTCTCCACATTAGCACCAGGTGAAACTGCTGCACCCACCGCAGCTCCTACAGCCGCACCCACTGCAGCTCCTACAGCCGCTCCCACTGCAGCCCCTACCGCTGCACCCACTGCGGCCCCTACCGCTGCACCCACTGCGGCCCCTACCGCTGCACCCACTGCAGCCCCTACTGCTGCACCCACTGCAGCCCCTACCGCAGCTCCTACTGCAGCCCCAACCGCTGCACCCACAGCAGCTCCTACTGCAGCTCCAACGGCTGCTCCCACCGCAGCTCCTACTGCAGCCCCACCAGTAACTTCAGACCCTGACGATTGCGAGACCCTAGATAATGGTTGCCCTGTCGACTTCACCATCCACAAGCTTATTCCTCATGAGGAGTACTGCCACCTGTTCTACTACTGTGACAAAGGCGAGCTGCTCCTGAGGTCCTGCCCTCAGCCTCTTTACTTCGACCCTGCTACAGAGGTCTGCGTCTGGTCTTGGGAAACAGACTGTGTGAACGATGGCCCCTACACATACCCAACTACCGTTGCTCCCGAGATTGGAACCACTTCAGCTCCTGGAGACAATGACATTGGTGATGTCTTGGACAACGGCTGTCCCGTCGACTTCAGCATCATTCACCACCTCCCCCACGAAGAATGTGAGAAGTACTATCAGTGTGACGCTGGTAAGAAGATCGAGAGGAATTGTGCTCCTGGAACCGTTTTCAACTTCGCAGCACAGGCCTGTGACTGGCCCTTCAATGTGCCACACTGTGCTGGAAGCGCCGGCGCTACCGCAGCTCCTACTACCGAAGCAGACAGCGAAGAAATACCTCTCCCCAATGACCCTGACAGTTGGGAGTCTCTCCCCAACGGTTGCCCAGTCGACTCCAGCATCAGCCACCTGGTGCCTCATGAATCTGACTGCGACAAGTACTACGTTTGCGACAATGGAAGATTAGTCCAGCTCGGATGTCCTGCTGGAACTCACTTCAGCCCCTCCCAACAGTTTTGCACATGGCCTCACGAGGCTGGCTGTGAACACTGGACTGGTGGCGGTTGCACCACCCCAGGCAATGGTGGTGGCAGCTGTGGAGGATCAACAGCTGCACCCGTCGACCCGACAACCCCTGTTGCCGTCGTCACGAGCACTTCAGCCCCAATTTCAGACCCATCCACTTCAGCACCCAACGAACCATCGACACCTGTTGCCGTCGTCACGACAACCACTTCAGCCCCCATTTCAGTCCCATCAACTTCAGCACCCAACGACCCAACGACACCTGTTGTAAATAGCAGCGAGGAAATCCCACTCCCAAACGACCCAGAAGACCTTCTTCCCAACGGCTGCCCAGCTGACTTCGAGGTCGACCTCCTCTTGCCTCATGAGACTGACTGCGACAAGTTTTACTACTGCGTTCATGGAGAGATAGTTGAGTTCCCCTGTGCTCCTGGAACCCATTTCAGCCCAGCTCTGCAGGCATGCACATGGCCCCAAGAGGCTGGCTGTGAGCACTGGTCCGAACCATCAACAGTTGCACCCGAAATAACCGTCACTGCAGTTACAAGCACACTATCCGTTGCTCCTGACACCACAGCCGCTGTTCCAAACACACCCACCGTGGCTCCCGAAACCACCACAGCATCTGTTACAAACGCACCCACCGTGGCTCCCGAAACCACCACAGCAGCTGTCACAAACGCACCAACTGTGGCTCCCGAAACCACCACAGCAGTTGTCACAAACGCACCAACTGTGGCTCCCGAAACCACCACAGCAGTTGTCACAAACGCACCAACTGTTGCTCCCGTTCCAGACCCCACCACTGTCGGAACAACCGCAAACCCTGCTTGCCCTGAATGCCTTCCCGGCCCAGTAAACCCAGCTGACAAATGCAAGGAAGAATGCAACGTTGCTCCATGGGCCCACGCAGAGTGCGACAAATACTACACTTGTGTCGGAGACGAGTTCAGAGTAAACGCCTGTGCTGAAGGTCTTCACTTCAACCCCAGCACACTCACTTGTGACTTCATCTGCAATGCTGGTTGCGTCAGGAACATCCCTCAGATCACTAGACATGTCGAGGGTATGTTGATGTTCATACCTCACGACTTCAACAACAGGGATGTCATTGACCTGATTGAACACGAACTGAATGCAGAACTTTAAATCAAATTGAAANGACAAAGCAAAGTTTTCGGTGAATAATAATAATGTTGTTATTTAAATTATTTTATTATTGTTAAATAAATTAACTATAAATATTTATATGTCTGTTTTGAAATTGTATTCACGAGATTGATTACTATTTTTTAAAGTTTAAGAAAAATAATTTGTTTTAATACTGTTTTTTCTATCTTTCGGCAATATAGACTTGACTTCTTATTCATATATTTTTTTTGTTTATTTTAAAATACCTCTCTTCTTTATTTGCATGTCAATGCGACACGAGGAGAATTAAATTAAATAAAAATAGTTTTAAATTAAATTAATAGGAAAAAACACGTGTCTTTAAAAAAAGAAACTGAAAAAAAGGTTAACTCATATCGATTGTTGAACCTGTTAGCGTTATTATAAAATGAGATTAAGAGTTTTAAATAAACTTGTCTTTATTTTGACTACGATAAAAAAAAA | Lepidoptera | Noctuidae | Mamestra | configurata | 174822 | cds | NCBI | AY057052 | Mamestra IIM1 gene midgut RNAi Hegedus-Erlandson-Toprak RNAi construct | CCTGTCGACTTCACCATCCACAAGCTTGTTCCTCATGAGGAGTACTGCCACCTGTTCTACTACTGTGATAAAGGCGAGCTGGTGCTGAGTTCCTGCCCAGAGCCTCTTTACTTCGACCCTAAGGCACAGGTCTGCGTCTGGTCTTGGGCAACTGATTGTGTAAACAATGGACCCTACACATACCCAACAACCGCTGCTCCTGAGGTTGAAAACAGCACAGCTCCTGGAACCATTGACATCGGAGAAGTTTTGGACAACGGCTGTCCCTCTGACATCCACATTCATCACCATCTGCCTCACGAGGAATGTGAAAAGTTTTATCAGTGTAACTTCGGACAGAAGGTCGAACGGGATTGTGCTCCCGGAACCGTTTTCCACTTTGAAATCCAGGTCTGTGACTGGCCTCGTAACGTGCCACGTTGTGCTGGAAGTGCTGGGGCTACAGCCAGACCCCAAACCACCCCTGAAGCCAGCAGCGAAGAAATACCAACCTCAAATGACCCAGTAGAATGGGAGTCTCTCCCCAACGGCTGCCCAGTTGACTCCAGCATC | dsRNA | Promega | ethanol precipitation | subsequent to transcription | Two clones coding opposite strands of target gene was obtained from pGEM-T easy cloning and linearized for use as a template for being converted into RNA using Promega T7 RiboMAX⢠Express RNAi System. The RNA strands were annealed to form a duplex RNA. Twenty microgram of dsRNA was applied into 2 ml of Grace`s media containing three midguts obtained from 3rd instar Mamestra configurata larvae. At 24 h intervals, three midguts were collected to extract the total RNA. Expression of the target genes was examined by RT-PCR. In parallel, total RNA from control midgut tissues(no dsRNA)was also extracted for examination of the target gene expression. Tubulin was used a house keeping gene in all conditions. | NCBI | AY057052 | Lepidoptera | Noctuidae | Mamestra | configurata | 174822 | local resource | larva | lab_colony | 0.01 | injection | dd water | 250 | buffer | 1 | larva | qPCR | midgut | not checked | 72 | low | 95 | ||||||
| 48 | Mamestra IIM4 gene midgut RNAi Hegedus-Erlandson-Toprak | RNAi | McIIM4 gene encoding a peritrophic matrix mucin was succesfully knocked down in a primary midgut cell culture system of M. configurata by 72 h p.i. | InsectaCentral | Unpublished | Hegedus | Dwayne | Mamestra IIM4 gene midgut RNAi Hegedus-Erlandson-Toprak target | AATAGCTAAAGTAGTCTAGTGACTCGTGTGTGCAAAATATAGGACATCTAGTTAAGGAATAATGTACAAAACTCTATTCTTCCTGACGGCCTTAGCCATCGTGCAGGCTCGTCTCAACGAAGACGCAGTCGTGACCAATGTCAGGTTAAATAAAGTCCCCGTTCAAATTGCTAACGCTGACGATGACTGCGAGACTTTAGACAACGGGTGTCCTGTCGACTTCACCATCCACAAGCTTGTTCCTCATGAGGAGTACTGCCACCTGTTCTACTACTGTGATAAAGGCGAGCTGGTGCTGAGTTCCTGCCCAGAGCCTCTTTACTTCGACCCTAAGGCACAGGTCTGCGTCTGGTCTTGGGCAACTGATTGTGTAAACAATGGACCCTACACATACCCAACAACCGCTGCTCCTGAGGTTGAAAACAGCACAGCTCCTGGAACCATTGACATCGGAGAAGTTTTGGACAACGGCTGTCCCTCTGACATCCACATTCATCACCATCTGCCTCACGAGGAATGTGAAAAGTTTTATCAGTGTAACTTCGGACAGAAGGTCGAACGGGATTGTGCTCCCGGAACCGTTTTCCACTTTGAAATCCAGGTCTGTGACTGGCCTCGTAACGTGCCACGTTGTGCTGGAAGTGCTGGGGCTACAGCCAGACCCCAAACCACCCCTGAAGCCAGCAGCGAAGAAATACCAACCTCAAATGACCCAGTAGAATGGGAGTCTCTCCCCAACGGCTGCCCAGTTGACTCCAGCATCCACCACCTGCTGCCTCACGAATCTGTCTGCGACAAGTACTATGCATGCGATAACGGAAGATTAGTTGAAATCGGATGTGCTTCTGGAACTCATTTCAGCCCTGCTCAGCAGGTCTGCACATGGCCTCACGAGGCTGGCTGTGAACACTGGACTGGTGGTGGCGGATGCTCCACCCCAGGAGACAATGGTGGTAGCTGTGGAGGATCAACAGCTGTACCCATCAGCCCAACGACCCCTGTGTCCAACATCTCAACCACCCCTGTGCCAGACAACATAACAACAACTACAACTAAAACCCCTGAGACAACAACCCCTGTTTCCGACAACTCAACAACCCCTGTTTATGAGATCACAACAACCCCTGTTTCCGATAACTCAACAACCCCTGTATCCGACAACTCAACAACCCCTGTTTACGAGATTACAACAACCCCTGTTTCCGATAACTCAACAACCCCTGTATCCGACAACTCAACAACCCCTGTTTACGAGATCACAACAACCCCTGTTTCCGATAACTCAACAACCCCTGTTTCCGACAACTCAACAACCCCTGTTTACGAGATCACAACAACCCCTGTTTCCGATAACTCAACAACCCCTGTTTCCGACAACTCAACAACCCCTGTTTACGAGATCACAACAACCCCTGTTTCCGATAACTCAACAACCCCTGTTTCCGACAACTCAACAACCCCTGTTTACGAGATCACAACAACCCCTGTTTCCGACATCGAAACAACAACTACAACTAAAACCCCTGAGGTATCCACAACAACAACATCAGCACCTGACTGCGATACTGAGGGTACAGTAACTGAAGACCCAATTACCGAAGACTCAACTCCGGAATCTAACAGTAGCGAAGAGGTACCTGCCCCAGCTGACCCCGAGTTACCCGGTACCACCACAAACTCGCCACCTGCACCTAGCTGCCCTGAATGCCCAACCGTCCCGCTTACCCCAGCAGAGAAATGCAAACAAGGTTGCAACGTTGCCCCATGGGCCCATGCAGAGTGCGACAAATACTACTCTTGCATCGGAAACGAGTTTCGTCTTAACATCTGCTCTGAGGGACTTCATTTCAATCCCAGTACGCTTACTTGTGACTTCATTTGCAATGCTGGTTGCGATAGGAATATCCCACAGGTCACCAGACATGAAGATGGTATGTTAATTTTCGTACCACACAGTTTCAATAACAAAGCTAATATGCTTGAGTTGATAGAACATGAACTGAATGAAGAATTCTAGACCAAAACGAAATTTTTTAGCAACCGAAAATGTTCTGTGTAAAATTATGATTGTTAAATTATTTGATTTCGAAATTGTATCGAAAATTGGTGCATTTAAACGTCCTGGATAAATAATTTTCTTTGAATATTGTATAGCATTGTCTTTCACTATTTATAGATTTGCCCTTTATTTATTTAATTTTAAATGTTATTTTTAAATGGTTCATAAATACCTCTCTTTATTATTGGCATGCCAATGAGACAAGAGGCGAATTAAACTGATTAAAAATAGTTTAAAATTTTTTATTCAAATAAAATAAGTTTAAAAATTTTAACTCATAATTTTAAATAACTAGTTACCGTTAAACCTGTAGAGATATGTAGAAAATTTTGTAGCATAATAGTAAGTGTTTTAAATTATTTTATTTTAAATTTCCAATGGTGCAAATAAGAAAACCTTATATGGAGCCATAAGAGGAGCCTCAGGAGTATATTTGAGCCCTTTGGGGGTGAAACATTTGGTTCGTGGGGTTCGAGTGCTCATCGAGTTTTTCATGACATCATTAAATGGTTAGTGGACTTTTCGCGAGGCCAGAGGGCTGGCCTTTTCAGTCAAAAAATCAGCCCTTCAACGTGGGAATGCTGCTAGTATTTTTGGGCAAATTTTGCAATGACAGCGATGTGGACAATTTTATTGATTCCCAATGAATTAAAGAATAAAGAAGCCAGGGATAGCCCCTTTTTTACCCTCGTTTGTACATAATTTGTTATTATTATCATTTTTGTATTTTAAGTTATTTATTATTTCTTTGATACTGATAATAAATATAAACATGAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Mamestra | configurata | 174822 | cds | NCBI | FJ670569 | Mamestra IIM4 gene midgut RNAi Hegedus-Erlandson-Toprak RNAi construct | CCTGTCGACTTCACCATCCACAAGCTTGTTCCTCATGAGGAGTACTGCCACCTGTTCTACTACTGTGATAAAGGCGAGCTGGTGCTGAGTTCCTGCCCAGAGCCTCTTTACTTCGACCCTAAGGCACAGGTCTGCGTCTGGTCTTGGGCAACTGATTGTGTAAACAATGGACCCTACACATACCCAACAACCGCTGCTCCTGAGGTTGAAAACAGCACAGCTCCTGGAACCATTGACATCGGAGAAGTTTTGGACAACGGCTGTCCCTCTGACATCCACATTCATCACCATCTGCCTCACGAGGAATGTGAAAAGTTTTATCAGTGTAACTTCGGACAGAAGGTCGAACGGGATTGTGCTCCCGGAACCGTTTTCCACTTTGAAATCCAGGTCTGTGACTGGCCTCGTAACGTGCCACGTTGTGCTGGAAGTGCTGGGGCTACAGCCAGACCCCAAACCACCCCTGAAGCCAGCAGCGAAGAAATACCAACCTCAAATGACCCAGTAGAATGGGAGTCTCTCCCCAACGGCTGCCCAGTTGACTCCAGCATC | dsRNA | ethanol precipitation | subsequent to transcription | Two clones coding opposite strands of target gene was obtained from pGEM-T easy cloning and linearized for use as a template for being converted into RNA using Promega T7 RiboMAX⢠Express RNAi System. The RNA strands were annealed to form a duplex RNA. Twenty microgram of dsRNA was applied into 2 ml of Grace`s media containing three midguts obtained from 3rd instar Mamestra configurata larvae. At 24 h intervals, three midguts were collected to extract the total RNA. Expression of the target genes was examined by RT-PCR. In parallel, total RNA from control midgut tissues(no dsRNA)was also extracted for examination of the target gene expression. Tubulin was used a house keeping gene in all conditions. | NCBI | FJ670569 | Lepidoptera | Noctuidae | Mamestra | configurata | 174822 | local resource | larva | lab_colony | 0.01 | injection | ddwater | 250 | buffer | midgut | larva | qPCR | midgut | not checked | 72 | low | 95 | |||||||
| 49 | Mamestra PM1 gene midgut RNAi Hegedus-Erlandson-Toprak | RNAi | Mamestra PM1 gene encoding a peritrophin matrix protein was successfully knocked-down in a primary midgut cell culture system by 72 h p.i. | InsectaCentral | Unpublished | Hegedus | Dwayne | Mamestra PM1 gene midgut RNAi Hegedus-Erlandson-Toprak target | AGTTCTAGTGCAACAATCACGAAAAATACAGTCTCATAGGATCTATTAAATGAAAGACGCCGTAATATTACTGCTATGTGCAGTTGCACTAGCACAAGGGCTCAACCAAAGCCCAGATCATCGCCGCCCATGCAATTGTGACCCGTCTGAAGCGCAGCAGATCTGCCAGGCGAACTACGATAACGATGATGTATTAATAGCACACGAAAACTGCGACCAGTTCTACAAATGTGCAAATGGAAAACCAGTAGCGTACTTTTGTCCAAATAACCTTCGGTATGACCCATTCTCTGAAACATGCGAGTGGCCTGACTCCGTTGACTGCGGCAATAGGCCTATTTCAGATGGCCCTGACAAGGGAGAGGATAACGACAGCGACGATGTCAGCGACGTCGACAATGACTGGACTTGTAATTGCAACCCTGGTGAAGCGCCATCCATCTGTGCAGCTGAAGGCTCCAACGGAATCCTTGTAGCACATCAAAACTGCAATCAATTCTACAAGTGCGCCGAAGGTAGACCAGTGACGTTCGATTGTTCACCGACTCTTTTATACAATCCCTACAAAGAAGAGTGCGATTGGGCGCACAATGTTGAATGTGGTGATAGAGTTATTCCCGACCTCAAAGAAGATGATAGCAGTGATGATGACAACAACAGCACAGAAAACGATGGTACTTGCAATTGCAATCCAGAAGAAGCTCCAGCCATCTGTGCAGCTCCTGGTTCGGAAAGTCAATTAATTGCACATGAGAACTGCAATAAATACTACATTTGCAACCATGGCCTACCAGTTGCTGTTTCATGCGTCGGAGATTTACTATTCAATCCCTACACTAGGGAATGCGACTGGCCAAGAAATGTTGACTGCGGAGACAGGCTTGTTCCTGAAACTGAATGTACAGGTTGTAACGACAATGCCAGCAACGACGCTAGCTGTGATGGTGATGATGATGACAGCGTTCCAACCCCAGGTGTTACCAACCCAGCTGTGACCAACCCAGATCCTGAATCCTCTGACAGTGGCAGCTCTGAAATTCGCCCACCAGGTGATGATGTTGTGGTACCCCCCCGACCACCCGGTACATGCAATTGCAATCCAGGGGAGGCACCTTCCATCTGCGCATCTGAAGATTCTGATGGCGTCCTAGTCGCACATGAAAATTGTAACCAATTCTACAAATGTGACCACGGAAAACCTGTCGTACTCTCTTGCTACGGTGACTTACTGTACAATCCCTACACTGAACAATGCGACTGGCCGGAGAATGTGGACTGTGGAGACAGAGTGATCCCAGACCCAGACGACAGCGTTATCACCCCCGGTGTGACCAACCCAGGTGTGACTAACCCAGGTGTAACCAACCCAGGTGTGACAAACCCAGCCGATACCACCCCAGGCAATAACTGCGATCCTAGCGAAGCTCCTGCTATCTGCGCAGCAGATGATTCCGAAGGAGTTTTGGTAGCACACGAGAATTGTAACCAGTTCTACATGTGCTCAGGAGGTAAACCAGTAGCATTGAAATGCCCACCAAATCTTCTATTCAACCCTGCGAAAGACAAATGTGACTGGCCGGAGAATGTAGACTGCGGAGACAGAGTCGTCCCAGATCCTGAATCCTCTGACAGTGGCAGCTCTGAAATTCGTCCACCAGGTGATGATGTCGTGGCACCCACCCGACCACCCGGTACGTGCAATTGCAATCCAGGGGAGGCACCTTCCATCTGCGCGGCTGAAGATTCTGATGGCGTCCTAGTCGCTCATGAAAATTGTAACCAATTCTACAAGTGTGACCACGGAAAACCTGTCGTTCTCTCTTGTTACGGTGGCTTACTGTACAATCCCTACACTGAACAATGCGACTGGCCGGAAAATGTTGACTGTGGAGACAGAGTGATCCCAGACCCAGACGACAGCGTTATCACCCCCGGTGTAACCAACCCAGGTATGACTAACCCAGGTGTGACCAACCCAGGTGTAACCAACCCAGCCGATACTACCCCAGGCAATAACTGCGATCCTAGCGAAGCTCCTGCTATATGCGCAGCAGATGATTCTGAAGGAGTTTTGGTAGCTCACGAGAATTGTAACCAGTTCTACATGTGCTCAGGAGGTAAACCAGTAGCATTGAAATGCCCACCAAATCTTCTATTCAACCCTGCGAAAGACCAATGTGACTGGCCGGAGAATGTAGACTGCGGAGACAGAGTCATCCCAGATCCTGAATCCTCTGACAGTGGCAGCTCTGAAATTCGCCCACCAGGTGATGATGTTGTGGTACCCCCCCGACCACCCGGTACGTGCAATTGCAATCCAGGGGAGGCACCTTCCATCTGCGCATCTGGAGATTCGGATGGCGTCCTAGTCGCACATGAAAATTGTAACCAATTCTACAAATGTGACCACGGAAAACCTGTCGTACTCTCTTGCTACGGTGACTTACTGTACAATCCCTACACTGAACAATGCGACTGGCCGGAGAATGTGGACTGTGGAGACAGAGTGATCCCAGACCCAGACGACAGCGTTATCACCCCCGGTGTGACCAACCCAGGTGTGACTAACCCAGGTGTAACCAACCCAGGTGTGACAAACCCAGCCGATACCACCCCAGGCAATAACTGCGATCCTAGCGAAGCTCCTGCTATCTGCGCAGCAGATGATTCCGAAGGAGTTTTGGTAGCACACGAGAATTGTAACCAGTTCTACATGTGCTCAGGAGGTAAACCAGTAGCATTGAAATGCCCACCAAATCTTCTATTCAACCCTGCGAAAGACCAATGTGACTGGCCGGAGAATGTAGACTGCGGAGACAGAGTCATCCCAGATCCTGAATCCTCTGACAGTGGCAGCTCTGAAATTCGCCCACCAGGTGATGATGTTGTGGTACCCCCCCGACCACCCGGTACGTGCAATTGCAATCCAGGGGAGGCACCTTCCATCTGCGCATCTGAAGATTCGGATGGCGTCCTAGTCGCTCATGAAAATTGTAACCAATTCTACAAGTGTGACCACGGAAAACCTGTCGTACTCTCTTGCTACGGTGACTTACTTTACAATCCTTACACTGAACAATGCGACTGGCCGGAGAATGTGGACTGTGGAGACAGAGTGATCCCAGACCCAGACGACAGCGTTATCACCCCCGGTGTGACCAACCCAGGTGTGACTAACCCAGGTGTAACCAACCCAGGTGTGACAAACCCAGCCGATACCACCCCAGGCAATAACTGCGATCCTAGCGAAGCTCCTGCTATATGCGCAGCAGATGATTCCGAAGGAGTTTTGGTAGCTCACGAGAATTGTAACCAGTTCTACATGTGCTCAGGAGGTAAACCAGTAGCATTGAAATGCCCACCAAATCTTCTATTCAACCCTGCGAAAGACCAATGTGACTGGCCGGAGAATGTAGACTGCGGAGACAGAGTCATCCCAGATCCTGAATCCTCTGACAGTGGCAGCTCTGAAATTCGCCCACCAGGTGATGATGTTGTGGTACCCCCCCGACCACCCGGTACGTGCAATTGCAATCCAGGGGAGGCACCTTCCATCTGCGCATCTGGAGATTCGGATGGCGTCCTAGTCGCTCATGAAAATTGTAACCAATTCTACAAGTGTGACCACGGAAAACCTGTCGTACTCTCTTGCTACGGTGACTTACTTTACAATCCTTACACTGAACAATGCGACTGGCCGGAGAATGTGGACTGTGGAGACAGAGTGATCCCAGACCCAGACGACAGCGTTATCACCCCCGGTGTGACCAACCCAGGTGTGACTAACCCAGGTGTAACCAACCCAGGTGTGACAAACCCAGCCGATACCACCCCAGGCAATAACTGCGATCCTAGCGAGGCCCCAGCCATCTGCGCAGCAGATGATTCCGAAGGAGTTTTGGTAGCTCACGAGAATTGTAACCAGTTCTACATGTGCTCAGGAAGTAAACCAGTAGCATTGAAATGCCCACCAAATCTTCTATTCAACCCTGCGAAAGACCAATGTGACTGGCCGGAGAATGTAGACTGCGGAGACAGAGTCATCCCAGATCCTGAATCCTCTGACAGTGGTAGCTCTGAAATTCGCCCACCAGGTGATGATGTCGTGGCACCCACCCGACCACCCGGTACGTGCAATTGCAATCCAGGGGAGGCACCTTCCATCTGCGCGGCTGAAGATTCTGATGGCGTCCTAGTCGCTCATGAAAATTGTAACCAATTCTACAAGTGTGACCACGGAAAACCTGTCGTTCTCTCTTGTTACGGTGGCTTACTGTACAATCCCTACACTGAACAATGCGACTGGCCGGAGAATGTGGACTGTGGAGACAGAGTGATCCCAGACCCAGACGACAGCGTTATCACCCCTGGTGTGACCAACCCAGGTGTGACTAACCCAGGTGTGACCAACCCAGGTGTAACCAACCCAGCCGATACTACCCCAGGCAATAACTGCGATCCTAGCGAAGCTCCTGCTATCTGCGCAGCTGATGATTCCGAAGGAGTTTTGGTAGCACACGAGAATTGTAATCAGTTTTACAAGTGTTCTGGGGGTAAACCAGTAGCATTAACATGTCCACCAAATCTTCTGTTCAACCCTAATAAGGACCAATGTGATTGGCCGGAGAATGTAGACTGCGGAGACAGAGTCATCCCAAATCCTGAATCCTCTGACAGTGGCAGCTCTGAAATTCGCCCACCGGGTGATGATGTCCCCCCGCAACCACCCGTTGTAGACAGTAACGAAGATTGTTCTGGAATCAGTGACGAGAACGGCAGTCCATGCAACTGTGACCCTGATCAAGCTCCTTCTATTTGCGCTGTAGATAACTCTGAAGGTGTTTTGATAGCTCACGAAAACTGTAACCAGTTCTACCAATGCGTCAACGGTAGGCCAATTCCATTGAAATGCCCGGTAAATACATTATATAATCCTGTTTCACAAGTGTGTGACTGGGCGTTCAATGTTGAATGTGGCGATAGAATAATTCCGGACCCTGAAGAAAATGTAAGCGAGAGTAATGAAGACGACAGCAAGGAAGAAGAACCAATCGTCGGGCCGTGTAACTGTAATCCTGAAGAAGCTCCCGCAATTTGTGCAGTCGATGGATCTAGTGGCGTACAAATTGCTCATGAAAATTGTAACCAATTCTACATTTGCGACCATGGTAGACCTGTGGCTTTTACTTGCAATGGATTCCTATTGTACAACCCCTACACTGAAAGGTGTGACTGGCCAGAACATGTTCAATGTGGTGACAGAGTGATACCAGAACCTGGAAACGAGAGTGATGAAAACGATAGCAATGAAGACAACATCAGCAACCCTAATGACGACCCCAGTCAAGCTCCCACGATCTGTGCTGGCAATGGTTCTGAAGGAGTATTAGTAGCCCATGAAAACTGTGACCAATATTACATTTGCTCTGGAGGCGTGCCTGTCTCGCGACCATGTAACGATGGGCTTTTATACAACCCCTATAATCAACGTTGTGACTGGCCCAGTAATGTTGTTTGTGGTGACAGAATTGTCCCTGATGATTGTGCATGCAACCCCAGAAATGCGCCCGCTTTGTGTGCTAAACCAGGATCTCAAGGAAAATTGGTCGCCCATGAGAATTGTAACCAGTTCTACATATGCTCCAACTCAGTTCCCGTGTCGCAAACCTGTCCCGCCAGCTTGGTGTATAACCCAGACAGGGAGTTCTGCGACTGGCCGCAAAATGTTAACTGTGAAAACAGATTACTATCATATGCCTCTCTCAACAAACATTGGCAGTCACGTCAAACTCTCAGAAACTAAATTATGTATGTATTAAGTTTATACTAAAACTGTGTTATTAATCTTATACAATTATGCGATATCAGTATTTATAGTCTTTAGACTAGATATTTAAAATTTAACAGTATTGTTTTCTTGCCTTGTCTTAATTTCGGATTCGGATCCAAAATATATAATTATGAAAAATAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Mamestra | configurata | 174822 | cds | NCBI | AY277403 | Mamestra PM1 gene midgut RNAi Hegedus-Erlandson-Toprak RNAi construct | GTGCGCCGAAGGTAGACCAGTGACGTTCGATTGTTCACCGACTCTTTTATACAATCCCTACAAAGAAGAGTGCGATTGGGCGCACAATGTTGAATGTGGTGATAGAGTTATTCCCGACCTCAAAGAAGATGATAGCAGTGATGATGACAACAACAGCACAGAAAACGATGGTACTTGCAATTGCAATCCAGAAGAAGCTCCAGCCATCTGTGCAGCTCCTGGTTCGGAAAGTCAATTAATTGCACATGAGAACTGCAATAAATACTACATTTGCAACCATGGCCTACCAGTTGCTGTTTCATGCGTCGGAGATTTACTATTCAATCCCTACACTAGGGAATGCGACTGGCCAAGAAATGTTGACTGCGGAGACAGGCTTGTTCCTGAAACTGAATGTACAGGTTGTAACGACAATGCCAGCAACGACGCTAGCTGTGATGGTGATGATGATGACAGCGTTCCAACCCCAGGTGTTACCAACCCAGCTGTGACCAAC | dsRNA | Promega | ethanol precipitation | subsequent to transcription | Two clones coding opposite strands of target gene was obtained from pGEM-T easy cloning and linearized for use as a template for being converted into RNA using Promega T7 RiboMAX⢠Express RNAi System. The RNA strands were annealed to form a duplex RNA. Twenty microgram of dsRNA was applied into 2 ml of Grace`s media containing three midguts obtained from 3rd instar Mamestra configurata larvae. At 24 h intervals, three midguts were collected to extract the total RNA. Expression of the target genes was examined by RT-PCR. In parallel, total RNA from control midgut tissues(no dsRNA)was also extracted for examination of the target gene expression. Tubulin was used a house keeping gene in all conditions. | NCBI | AY277403 | Lepidoptera | Noctuidae | Mamestra | configurata | 174822 | local resource | larva | lab_colony | 0.01 | injection | ddwater | 250 | buffer | 1 | midgut | larva | qPCR | midgut | not checked | 72 | low | 95 | |||||
| 50 | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha1 | RNAi | pubmed | 17553801,17868866 | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha1 target | GAAACAGTCGTAGTGTTGGCTTCGTTGAAATAACAACCATGATGTTACTATGGCTATTGCTGATGGCGCAACTGGCTTGGCCGACATCAGCACTGTACCACGAAAAATCTTTGAAAGTATTTCAACCAGGAGATCTCAAGTCACCACTGAGAAAAGAGGCCAACTTTGGATTTAGCATCGCTTATCAATCTGCTACTCAAAGTTTGATCATAAGCGCTCCAACAGCGGATTACATCGGCAGAATATATTCTTGCGACATTGAACTGAAAAGAAATTGTACTGTCGTGCCGTATGACATCAAGAGAGACGACCCTCTGTATAACCATGATTACTGGCTGGGTGCGACAGTGAAGGCGGGCCCTGACTATTTTGTCACGTGTGCTCCGCGTTACGTCGAAGTGGGTAGTTCGTACAACCTACCCGGTGAATTCAGCCATTGTTATAGATCATCCAAGCACTCCAAACTACTCCCTATTAGTCAAATAAGTAGCCAACAGCGAAGAGTTAACACAAAAAAATTCGAGGATGAAATGGATTCATTTGGATGGAGTATGGATTTGAAGGACAGGAAAATTCTATTTGGAGGCCCTGCTATGTTCTCAGGACGTGCAATGGCATCTGCCGATTCAGCGATAACATCAATAAGTGGACATTCGAAAGGTGTAGACGTCAAATACAATTTAGGTTACAGTGTCGCAATAGGCGCTTTCTTTTCTGACAAAATCGTTTATGCTGTTGGATCGCCTTTTGGACAAAACGGCCGCGGAAAGGTGGTTTTCTTCAATAAGGATTGGAAAGTAATGTCGTCTATAAGTCATAAAAATATTGGCACGATGTTTGGCGCGGTGCTGGCGACTGCCCATATATTCAGTGATTCACTGACTGACTTGCTCGTGGGAGCGCCGACCGAGGCTGCAAATCACGACTACGACACCGGAGCTGTTTACTATTACAAAACTGGGAGTCGACGTAGTGTACAAATGAGCATTGAACATACAAAAATTATTAGTGGGTACCAACCGGGATCGCTCTTCGGCAGCGCAATCATAAGTGTCGGCGACTTGAATGGCGACGGAAAAGATGAAATAGCGATTGCTGCGCCGTACGAGGATGAGGGCAAAGGCGCTGTTTACCTCTACTGCGGGTCCTCATTCTTGGATGCATCATCGAATGTGTTGCTTCAACGACTCCAGCCAGAAGGCCTCCACACCTTCGGCTTGAGCCTGTCCTCTCCCAGCGACTACGATGAAAATGGATGCAATGAGCTGGCGATTGGTGCCCCACAAGACAATGCAGTGGTGTTGTACAAGTGCTTGGCGTCTGTTGTGCTGACAGTCTTCGCCGTATTTCCAAACCTACGGGACCGCAGAAATGTAAAAAGTCCGTATCCTGATAGAAATGAAACTTATTTCGTTTTCGATTCGTGTTTTGACGTCAAATACCCGAAGAAACTAAAAACTGTTATAGCGAAAATTGAAGTAACCGTCCAAATTAAACACCCGGACGCAACCTTAGCGATGCCTCACAAAGACGGAAAGTACAATGTCACACTAGATATAAAGAAAACACGGTATTGTGAAAAAATTGGAGTAAGCACGCCTGAGCACGGCAACTACAACATAAAAATATTTTACAATATACAAGCTCGTTTAGTAAACTCTCCGATCAACGAGACGGATTTCGACGGCAAACGCGTCATCCTGAGCGGGCGTAGTGAGTTATCAATATCGGACAATGTATGGGCCGCTGAGTGTAAGGTCCAGAATGCTTGCAAAGCAGATTTGAGCCTGAAGATAGCGACCTCGCCGCGGGGATCATACATGATCGGGTCTATGGAGCCTATAAAAGTGAATATGATATTAGAAAACAAAGGTGAAATAGCTTACGACGCCTGCGTGGAACTGCAGCTAGAGGGAGCGGCAATGAAGAAAACACCAACTACCTCCTGCACTCATCACCCACTTACTAACACGTTGAAATGTGCACCCAGCTATGCACTGAAGACCGATAAATCCTGGGAAACTACAGACATAATACTAAAAACAGAAACCTTGACAAACAAAAATAAGCAAATTAATTTAACGTCACGTTTATACGAGCATTGTACTGATCCTGCAACATTTAAAGAACAAAACTATACTATTACTGTACTAGCGAATTCTGAAGGGATTACTGTAAATGGAAAAACAAACATCGGTGACGTAGTGAGCATGACGACATTAGAAGTAACTGATTCAGGAAAACAGTTTCAACATAATTACATGATACAAAACGACGGGATCACATGGGAGGATGTGACTTGTGAAATCATATTGCCCAAGTTGCCCTACGTCAACTACCCACAAAAAGCTGTTACGGTTTTTATTCAGCAAAATAGTTTCGAGTGTAATATGACTGACGATTTGAGCCCTGACTACATAACTGCAAATTGTAAGCTGAGTAGCATCAAACAGAACATAATTACTTTCATCATTGTCTTAATACAAGTGCCGCCAACAACGCTCGATGACATTCTCTATAAGCAAAATGTGACCATAAAATCAACATTGAAGCTTCACTTTGACGATGGCGACAAAACATACAGTGTGGCGACCGAAGTGATGCTGAGAGACACAGGCATACCCTGGTGGATAATATTATTGGCGGCGCTAATTGGCCTCTTGATACTAATCATCCTTATACTGATTCTTCGCCAGTATGGTTTCCTTAAAAGGAAAGAAAGGAAAAAGCTACAAGAGTTAAGGAAGAGTGTACGGAGACAAACGGTTAGACGCTCCATGATGCAGCAGCAGGTAGAAGATCGTCAAAGATTAACCGATGTACCGATAGAAGCGACTGAAACTGACGTACCGTGCGGCATAGAACCAGACCATAGAGTAGAAGTAAATATTAAATGATTTTACCGCATTAGCAACGTGCCATTCACTACTTATTTACCACTAGAGTTGGCGTTTGGTGTTACATATTATAAGTCCATTAATATTTCCATATATATTATAAGTCCATAAATTAAACTACATTTGCTGACTTTGTGCCAGAAATCATCCATTAATTATAACTCATATTTTTACCAAAAATCCTGGTTTTAGTTTACTGACTTTTTTTATAATTTTGTAGTTTAATGTAAGTATGAGTGTATTTCTGACTGAAAGTATCATGTTGCCGCTGTGACGAATTCTCTTAAGAGTCATAAGTAGTGGCATTAGGGCATGTAAAATTACTTTTTATTTATATTTATTTTCTTCGAAACTTTGAATTTTTTTATTTTAAATCTACGCTGAAGTACCTTATTGTAAAGTGTTATTTCCAAATAGGTAAATATGTAGTTTTATTTAGTAACGCAATATAAAAATGACCCTGTATTTTTATCTTGCATCATGATTAATAGTGCATTGTAATTTATGTATTATTCATTAATTAAGGGCTGATTTGTCAATCGTCAGACATCTATTTAACCTAACTGAATAATAAAAGTGTGCCAATTGATAACTTAAAATAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | DQ531844 | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha1 RNAi construct | AGCGCAATCATAAGTGTCGGCGACTTGAATGGCGACGGAAAAGATGAAATAGCGATTGCTGCGCCGTACGAGGATGAGGGCAAAGGCGCTGTTTACCTCTACTGCGGGTCCTCATTCTTGGATGCATCATCGAATGTGTTGCTTCAACGACTCCAGCCAGAAGGCCTCCACACCTTCGGCTTGAGCCTGTCCTCTCCCAGCGACTACGATGAAAATGGATGCAATGAGCTGGCGATTGGTGCCCCACAAGACAATGCAGTGGTGTTGTACAAGTGCTTGGCGTCTGTTGTGCTGACAGTCTTCGCCGTATTTCCAAACCTACGGGACCGCAGAAATGTAAAAAGTCCGTATCCTGATAGAAATGAAACTTATTTCGTTTTCGATTCGTGTTTTGACGTCAAATACCCGAAGAAACTAAAAACTGTTATAGCGAAAATTGAAGTAACCGTCCAAATTAAACACCCGGACG | dsRNA | MEGAsript RNAi Kit (Ambion) | column-based | simultaneously with transcription | (1) Amplify gene by PCR with forward primer (TAATACGACTCACTATAGGGAGAGCGCAATCATAAGTGTCGGC) and reverse primer (TAATACGACTCACTATAGGGAGACGTCCGGGTGTTTAATTTGG). (2) PCR product was recovered and used as template for preparation of dsRNA using MEGAsript RNAi Kit (Ambion). | InsectaCentral | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha1 RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | ddH2O | 0.05 | non_specific_dsRNA | 3 | abdominal hemocoels | Green fluorescent protein (GFP) dsRNA was purchased from Dharmacon and used as a control. | larva | qPCR phenotype | hemocytes | not checked | 120 | low | 50 | |||||
| 51 | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha3 | RNAi | pubmed | 17868866 | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha3 target | ATAAGTAAAGTTACTGGCCGATGGAACGGTCATATGTCAATATAAACATTAAAATCGTGTACAAAAAACTGATTATAAGTTAAAATTAATTATTGTGAATAGTGATAAATTATTGTTTCTTAAAATGGTTTAAAATAGATTATATCTAAAGCTGACAATTACGGAGTGTTTATCACAAGCCTGGGATGATATTTGAACTGCAAAATGGCATGTGTAAAACTATTTCATTGATTTGTGTTTTCAATAAGACTTAATTTTGATATGTGAAAAGTGATAACATAAGTTAAAACAAGACCTCTTTTTGAAGTCGGTTAAAAAGGACAAGATGTACAAGTTATTTGCGGCTATAGTGATTTTGATGTGCGTGGAAGTGCGCACGAGTGGTATATTTCACGAAAATACCAAAATAACGTTTCATCCAGATGTAGACTCTGGATTTTTCGGATACTCCGTGTTTGTATATGAATATGGGATTATGGTGAGTGCTCCAAAAGCTAGGAGCAAGGTCAAGCCTACCGTGTTCTCTGGATTGGTCTACAACTGCTCTATCAACATCGTTGATGACGTCAGTGACGTCATGTGCTACCCCATGGATTCTGATTCAGTTTCAGACCGTTCCAGAAGTTACTACAGATATAAGTTCTTGATTGATTTCTTTAGAGATGACATGTGGTATGGAGCTTCTATGGGCGCTTTGTCCAACAATAAAATTTTGATCTGCGCACCAAGATATGCAGAACTTTTCAAAGATAACCATATTCTGCCTTACGGCGCTTGTTACATGCACAAAAAGGGACGGGAGATGCCTGTCTACCCTTTAGCGGATAAAGGTCATCTGGCTTATATGATCGATGGAAAGAGGAAGGAATACGGTGACTACGGGACGCATTTGAACTTCTACGCTAATGGACAAGTGGGGATGTCCATGAAAGTTACAGAAAGTAACACGGTTATAATCGGCGCCCCTGGACTTTTGCACTGGACTGGTGGCATAGTAAACTACAAATTCAACCCTGCAGATGATAGTATATACTTCGCCAAACAAACTACAACCAATCCGTACTTCACCAAGGAACTGGGGCCAGATCATTATTTCGGATATAGCGTAGAGTCTGGTATTTTCGAAAAATTCGGTCAGGTGCTATACGTCGGCGGAGCACCTCGGTCTAATGGAACCGGACAAGTGTTAATATTCGAACCAGCAGTAAAAGAAAACGCTCCTCTGAAAATCCAAGGTGTTCTCAAAGGACCTCAGCTGGGTGCCTATTTCGGGGCAAGTTTATGTTGCACGGACATTGATGGTGATGGAAGATCAGACCTTTTAGTCGGGGCTCCCAACTATGTGCAGAATGATGGAGGATTGCCTTATGATCAAGGAGCCGTGTTTGTTTATATGGCTTCTGATTTATCTGTAAAGGAAAAAGTGAAGGGTCCCAACTTTATATTAGAGCCTGTTGGTTACGTAATCGGTTCGGGACAAAGTGGCGCGCGATTTGGAACAGCTATTGCTGGTCTGGGAGATATAGACGGGGATGGATATAAAGATATTGCCATCGGCGCCCCGTGGGAAGACGATGGAGTAGGAGCAGTATACATTTACAGAGGAACTACTAGAGGGCTCAAGAGTCAATACGTCCAGAGAATAGCTGGAGATCACGCACAAGGCTTCGGCTGGTCCATAGCTAAAGGATTTGATGTGGACCATAATAATTGCAGTGATTTAGTAGTAGGAGCTTTTAAAAGCAACACAGTGAGTCTATATCGTTGCGTGCCGACAATACAAGTGCATGCTTCTATAAAAGTGCCAGACGCTATGAACCTGCCACAGAATTCGACATCATTCACAGCCATGTTCTGCTTGACTGTGCCAGCGAAGCATATGTGGTCTCATGTTAAACTTGATCTAAAAGCTCGTATAGTAATAGACCCAGAGCAGAATAGAGCGAGGGTTTCCGGAGACTCGGAGTACAACATCACCGTCAAGCCGGGGAACGATATCTGTGGAGAGCAAACCGTTGAAGTTACTCCGACCGCAGATTTGTCAAGGCCAATATCAATCAAGTTTGAACTGGAGCCGTTGGAGCTACTTCAAGATAATTCATCAACGTTCCTTAACGAAGCAGCGAGATTGTCAGAGGACTCGGAGCTGCAGTCATCATTCCTCATCCAACTGGTTAGGGACTGTGGAGACGATCTCATTTGCACACCATGGCTGGTCATGACTTGGGATGCTTTGGTCAACCCATACATCCCAGGGTCGGAAAAGCCTTTAGGAGGAAGACTGACTGTTCTGAACAAAGAGGAACCTGCCTACGGAGCCAAGGTCTACATAACCCTCCCAGCAACACCAATAAGAGTCCCAAGCGAATGTTCTTTGAAGGAATTAAACATGACTTGCAATATACCAGCTCCTTTAGAGCGAGGTGATGAAATAACTTGGGATATAGAACTGGAGTACACCGCACGAATGCCCTATGAGGATAATTTGAGGCTCGTGGCTGTACTAGAGGATTCTTTGTACAGTCGAAATATCACGGATGAACCTGTTAAAGAGCTAGTGATTGATATTGTCCCTCAAGCTAACTTTACTGTGAGTGGCAAACCACTTCCAAACGGAACAATAACTGTGACAAGACCAATGTTATATGAAACTGAAAATATAACCTTTTCCCATCATTACGAGACTATCAATTACGGACCGTCGGATTGGTACAGATTGGAAGGAGACATTTTTATACCAGAACATATAAATTTATCTAGTCCTATCAAAGGATGTTCTTGGAATTGGAGCTCTGAGTGCGGCTGGTCTATTCCCGCCAAAGTTTCTGTTCCAATAGTACTGGCACTGCGTTTTGATCTTGCCCAATATGTTTCAGGAGACATGAAGGAAAACACGACATTTAACGTAACTACAAAATTAAGTTTTTGGATAAAGGACCAATTCTACGCATCGGAAGTAACAGCTACTCTGATACTAGAACCAGGTCCACCAACTCTCTGGTATCTTATAGCGGCATTGATAGTTGGTCTACTGCTGCTTGCGCTCATTATTTTAATTTTATATAAGCTCGGATTCTTCTCACGAACACAACGTGATAAACTAAAGGAACTTCAAGAACAGACAGCTCAACAAGACCAAGCTGAGACGAGCTGCAGTTCCTCAGAACTTCTAGACATTAATGACTCCACAAGGGAATTATTAATAGAGGACTCAGAATGATGATGTAGCGGCTAAAACGCGTTTCGTTTCGTTTTTGCGTTTTTGATGTGAAGGAATCGAGAAAACGTTTTTTGCTGCAAATGTTTAGCATACTGTTAAGTTTCCCAACGTTGTATCTCAAAAAACAGAATTATTAGTTCATTAATGAAGGCATGATTGATGTATAGTCCGAAACAATATTTTTTAATCTTCTAGTCATTTTTTACCCAATGCAATATGTAAATAGAAGTTTATTTGGGTTTAAAACATTGGTCTAAAAATTTCGTATTTGCTAGAGCCGTTTGGAAAACAAATTCACGAACAATATAAATAAAACGGTTCAAACCCATACAAGTTTAAACAACACACGTGAATTTGCTATTGAACGTGTGAAATAACATTTTAATGAGATAATAAAAATAGTTTTATTAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | DQ531846 | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha3 RNAi construct | ACCTGCCACAGAATTCGACATCATTCACAGCCATGTTCTGCTTGACTGTGCCAGCGAAGCATATGTGGTCTCATGTTAAACTTGATCTAAAAGCTCGTATAGTAATAGACCCAGAGCAGAATAGAGCGAGGGTTTCCGGAGACTCGGAGTACAACATCACCGTCAAGCCGGGGAACGATATCTGTGGAGAGCAAACCGTTGAAGTTACTCCGACCGCAGATTTGTCAAGGCCAATATCAATCAAGTTTGAACTGGAGCCGTTGGAGCTACTTCAAGATAATTCATCAACGTTCCTTAACGAAGCAGCGAGATTGTCAGAGGACTCGGAGCTGCAGTCATCATTCCTCATCCAACTGGTTAGGGACTGTGGAGACGATCTCATTTGCACACCATGGCTGGTCATGACTTGGGATGCTTTGGTCAACCCATACATCCCAGGGTCG | dsRNA | MEGAsript RNAi Kit (Ambion) | column-based | simultaneously with transcription | (1) Amplify gene by PCR with forward primer (TAATACGACTCACTATAGGGAGACCTGCCACAGAATTCGACATC) and reverse primer (TAATACGACTCACTATAGGGAGACGACCCTGGGATGTATGGGT). (2) PCR product was recovered and used as template for preparation of dsRNA using MEGAsript RNAi Kit (Ambion). | InsectaCentral | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha3 RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | ddH2O | 0.05 | non_specific_dsRNA | 3 | abdominal hemocoels | Green fluorescent protein (GFP) dsRNA was purchased from Dharmacon and used as a control. | larva | qPCR phenotype | hemocytes | not checked | 120 | low | 50 | |||||
| 53 | Manduca sexta hemolymph proteins Kanost lab-Integrin beta1 | RNAi | pubmed | 17553801,15804572 | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-Integrin beta1 target | ATTTATTTGAATACAACGATGTGGAATATATATTCTATAGTGTTTGTGTCGCTATGTTTAAAATTAATCAATTGTCAATCTGTATGTAATCACTTAGGGACATGCGGGGAGTGTATAGGTTTCAGCAGCGGAACGGAGCGATGCATTTGGTGTCAACAGGAGACGCTGGATAATTATACAAGTAGGTGCCAACCCGAAAGCTATTTAAAAAAAGAAGGATGGTGCGACTCGAAATTCATTGAAAATCCTAAAAAAGTTCAATTAATTGAAGTGGACAAAGATTTTGGTTCGACTATGGATGATTTAAAAATTCAGTTTAAACCTCAAGTGATGAGAATGAAAGCGCGTCCCGGTACTAAATTATATTTCAACATGTCGTATAAGCCGGCCGAGCACTTTCCTTTAGATGTTTATTATCTCATGGACACCTCTTATACAATGACAATGCATAGAGAGGCGCTCATTAGTCAAGCAAACCAAATATATAAAGAACTTACAAGTTTAACTAATAATGTTCAACTTGGCGTTGGCAGTTTTGTAGAAAAACCAGGCTACCCGTACTTCGACAAAAACAAACAAGAAAGCGTCGCATTTATAAATGTATTGCCGTTAACAAAAAATATTAAACAGTTTACACAAAGTGTTCAAAATATGTCTTTTGGTTCGAATTATGACGATATGGAAGCTGGGCTGGATGCCCTAATGCAAGTCATGACTTGTGAGAAAGAAATAGGGTGGAGGCCTGGCTCAAGGCGTATAATTGTTCTGTGCACCGATTCCCCATATCACAGCGCTGGTGACGGCAAAATGATAGGCATTATCAAACCCAACGACATGTTATGCCACTTAAAGGAACAAAAATATGAAGCAGAAATGGCCCAAGATTATCCATCTGTGAGTAAAATAAATAAAGTAGCAAAGCAAGGAAAATTCGGTATCATATTCGCTGCTTTGGCTGAGGTCCGTGATGTTTATACATTGTTAGCGGAACAAATAGTCGGAGCTGAGTACGCCGAACTGAAGAAACAGAAGTCAAATATTGTAGAGATCATTATAAAAGCGTACCAACGCAGCGTTCGAAGTATCAAATTGGATTACGACATCCCCTCCTTCGTTAGACTGAAACTTAATCAAAGTTGTGACGGGACACCAATTAATTGTGCCAGCACCTATGAAAATCCAGTGGTTACAATTCCGGCTATTCTAGAGGTTAAAGAATGTCCTAAAGAAAATAAAACACATGAGCTTGTTATTAACCCTGTGTCTTTAAATGACAAATTAATAATTAAATTGGAAGTCATCTGTAAATGTGAATGTGAAGTCAAAAGTGATATAAGTTCAAGATGTAATAATGCAGGATATATACAGTGTGGTATCTGCAAGTGTCTCGATTCAAGTTATGGCGACGAATGTCAGTGCAGCGTTACATCTTCGGGGGTGGCTAATAAGGAGAAAGATGACGCCAAATGCCGTAAGGATCTAAATGACATAGTACTGTGTAGTGGGAAAGGCGTATGTATGTGCGGTAAATGTACCTGTAACCCTGATCGTTCAGGAAAATATTGCGAATTTGACGATAAGGCATGCGATAATCTTTGCTCAAACCATGGGATTTGTACCTTAGGCTCATGCCAGTGCGATAGCGGTTGGTCAGGAAATGATTGCGGTTGTCCAACTAGTAACACAGACTGCTACGCTCAATACTCTGAGGAGGTTTGTTCTGGTAATGGTGAATGTGTATGCGGAAAATGCCAATGTGCGAAGGTTAAAGGAAAAAACGAAACGTACACAGGAGTATTTTGTGACACATGCAATGACTGCCAATCAAAATATTGTAAAGCCCTCGAACCCAATGTAGAATGTAACTACAAACAAGGTCTAGAAGCTTGTGATAAGATTTACAACAACACAGAAAACAATGTTGTTATAAAAATGGTCAACAAAACAGAAATTAATTCGCCTAAATGGAGTGGCGCTACTTGGTGCAAAAAAGTAATAGAGGACGGCAGTTTTATAATATTCAGATATTATCATAACGCAACGACACACGGGTTACATATAATCATTCAAACGGAACCAGAGGCACCCCCAAGAGGAAATAAGTGGATTGCCCTCATCAGTTGCATAGTGGCTGTAGTACTCATTGGCTTGTTGACGTTGATTGCGTGGAAGATCCTCGTGGACTTGCACGATAAAAGAGAGTATGCCAAGTTTGAAGAAGAATCACGTTCTCGAGGATTTGATGTGTCGTTAAATCCCTTATATCAAGAGCCGGAGATCAACTTCTCCAATCCCGTATACAATGCAAATGCTTCACACTAGTATTTATATAACAAAATCCGAGGAAGAGAGGCGACGTGTTTCCGTGATCATAAGGAAAATACAATAAATAATTATTTGTGTTAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | AY630342 | Manduca sexta hemolymph proteins Kanost lab-Integrin beta1 RNAi construct | GTTTAAACCTCAAGTGATGAGAATGAAAGCGCGTCCCGGTACTAAATTATATTTCAACATGTCGTATAAGCCGGCCGAGCACTTTCCTTTAGATGTTTATTATCTCATGGACACCTCTTATACAATGACAATGCATAGAGAGGCGCTCATTAGTCAAGCAAACCAAATATATAAAGAACTTACAAGTTTAACTAATAATGTTCAACTTGGCGTTGGCAGTTTTGTAGAAAAACCAGGCTACCCGTACTTCGACAAAAACAAACAAGAAAGCGTCGCATTTATAAATGTATTGCCGTTAACAAAAAATATTAAACAGTTTACACAAAGTGTTCAAAATATGTCTTTTGGTTCGAATTATGACGATATGGAAGCTGGGCTGGATGCCCTAATGCAAGTCATGACTTGTGAGAAAGAAATAGGGTGGAGGCCT | siRNA | MEGAsript RNAi Kit (Ambion) | column-based | simultaneously with transcription | (1) Amplify gene by PCR with forward primer (TAATACGACTCACTATAGGGAGACAGTTTAAACCTCAAGTGATGAG) and reverse primer (TAATACGACTCACTATAGGGAGAGGCCTCCACCCTATTTCTTTC). (2) PCR product was isolated by agarose electrophoresis and purified using the QIAquick gel extraction kit (Qiagen). (3) This DNA was used as template for preparation of dsRNA using MEGAsript RNAi Kit (Ambion). (4) The dsRNA was digested with RNase III (Ambion) to prepare short dsRNA fragments (small interfering RNA, siRNA). | InsectaCentral | Manduca sexta hemolymph proteins Kanost lab-Integrin beta1 RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | ddH2O | 0.04 | non_specific_dsRNA | 4 | abdominal hemocoels | Green fluorescent protein (GFP) siRNA was purchased from Pharmacon and used as a control. | larva | qPCR phenotype | not checked | 120 | high | 70 | ||||||
| 54 | Manduca sexta hemolymph proteins Kanost lab-Neuroglian | RNAi | pubmed | 17448535 | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-Neuroglian target | GTTGAACGAACGAACGACAGCGAATCTCAGAGGCCCCGCCTATAAAATATCCGTATTTAAGAATTATAGATCGTTTTCGCGATGTTATTATAATAAAACACATACCTAGATACACGAGTTGTACACGGCACCAGTTGTGCCACCATGGGTACGACGTTTCTGTGTTTTATCGCGCTAGCGGCGCAGGCGGCGGCGTTACTGACGTCACCACCGAAGATGGTGAAGCAGCCGACGCAAGAGGAGATCCTGTTCCAAGTGGCGCAGGCCGGCGAGGTGGACAAGCCGTTCATCATAGAGTGCAGAGGCGAGGCTGAGGGAGAGCCGGGACCTAAATACCGATGGATCAAAAACGGCAAACCGTTCGAGTACACGAGCTATGACAACAGAATCCTGCAGCAGTCCGGGCGAGGTACCCTCGTGGTCAGCAAGCCCAGAGACGAGGATCTCGGACAGTACCAGTGCTTCGCCTACAACGAGTGGGGTACGGCCGCCTCCAACTCCGTGTTCGTGAGGAGGGCTGAACTCAACTCCTTCAAAGAGACCGATGGCCAACAAGTCGTGAAGGCTGAGGAGGGAAAACCATTCAAACTCACCTGCGAACCTCCTGATGGCCACCCGAAGCCCAAAGTGTACTGGATGTTGCAAGGAGACCAGGGTCAATTAAAGACCATCAACAACTCCCGCATGACATTGGACCCCGAAGGAAACTTGTGGTTCTCGAACGTTACTCGCAACGATGCCAGTGTTGACTTCGCATACATTTGTACGGCAAACTCGATCTTCAGAAATGAATACAAGTTCGGCAACAAGATCTATTTGGATGTAACCCAGACTGGTATCTCGCCTACCCTGAACAGGCATGCGCCGGAGCGCCAGTACACCACCACGAAAATAGAGAAAGCTCTCAAAGGGAAGAAGGTCGAACTGTACTGCATTTATGGTGGAACGCCGCTGCCTCAAATCGTGTGGAAGAAGGACGGGCACAACATCATACCCTCAGCGAGTATCACGCAGGACAACTACGGCAAGACGCTCGTCATCAAGTACCCGAACTACGAAGACAGCGGCACGTACACCTGCGAGGTCAGCAACGGCGTCGGTACCGCGCTGTCCTACTCCATCCAGCTTAATATTGAAGCGGCACCATTCTTCACGGTGGAACCGGACGTCCAGAACCTGGCTGAGGGCGAGACCGCGGTGATCCGGTGCGAGGCCGGCGGCACCCCGGTCCCCAAGATCACGTGGATCCACAACGGCAAACCCATCGAGCAAGCGGAACCGAACCCTCGCCGGCAGGTCACCGCGAACTCCATCGTCATCACTGACCTGGTGAAGAAGGACACCGGCAACTACGGTTGTAACGCGACAAGCTCCATCGGCTACGTGTACAAGGATGTCTATATCAACGTTCAATCGATCCCGCCTGAAATCAAGGAGGGTCCCGAGAACCTGACCAAGGTGGACGGTTCCGAGGCTGTGCTCAAGTGCAGAGTGTTCGGAGCGCCGAAGCCAATCGTCAAGTGGATGAGGGATGACGTCGACATCACCGGGGGGAAATACAACATCACGTCGGAGGGTGACCTGGTCATCCGCGACGTGTCGTTCACAGACGTCGGGACCTACCAGTGTTACGCGAAGAACAAGTTCGGAGAGAAATCGGCGTTCGGATCTCTCGCCGTCAAGAAGCGCACCGTGATTACCGACAAGCCGGAAGACTACGAAGTGGCCGCGGGTTCGTCCGCCACATTCCGATGCAATGCCAACGCGGACGACTCGCTGAAGCTGACGATAGTGTGGCTGAACGATGGGCAGCTGATCGACTTCGAAAACCAGCCGCGCTTCCGCATGACCAACGACTACTCGCTGCTGATATCGGACACCACCGAGCTTGACTCCGGACAGTATACTTGTATCGCCAAGACCGCGATCGATGAGGCGCGTGCGCAGGCCACTCTTACTGTGCAAGACAAGCCGAACCCGCCGGCGCTGGACGGCGTGGAGTGCGGCGCGGCGACGGCGACGCTGCGCTGGCGCTCCATGGGCGACAACCGCGCGCCCGTCGTGCGCTACCAGATACACTACAACACCAGCTTCACGCCCGACTCCTGGGCCGCCGCCGCCGACCACGTGCCCGCCATCGACACCTCCTGGACCGTGCAGCTCAGCCCCTGGGCCAACTACACCTTCCGAGTGATCGCAGTGAACAAAATCGGTCCTTCGAGCCCGTCGTCGCACTCGGACGTGTGCACCACGCAGCCCGACGTGCCTTACAAGAACCCCGACAACGTCAAGGGCGAAGGCTCCGACCCCACCAACATGGTCATCTCTTGGTCGAAAATGCCACAAATCGAACACAACGGTCCCGGGTTCTACTACCTGGTGTCGTGGCGGCGCAACATCACCGGCGACGAGTGGAACAAGGAGCAGGTCCGCGACTGGCAGCAGACGGAGTATATAGTCACCAACACGCCCACCTTCCAGCCGTACAAAATCAAGGTTACTGCCGTGAACTTCAAAGGCACGTCAAACGTGACGCCTGTAGAGGTGATCGGCTGGTCGGGCGAGGACACGCCCCTGCAAGCTCCCGCCAACTTCACACTGGTGCAGGTCACCACCGGCACCACTGCGCTGCTCAGCTGGAATGCAGTTGCTCCGGAATCCGTCCGCGGACACTTCAAAGGATACAAGATACAGACCTGGACTGATGGTGAAGAGGACCGGCTCAAGGAAATCCTCGTTAAGGCAGATTCCACGTCCGCACTGGTGACTAAATTCAAGCCGTTCAAGAAGAACAACGCCCGCATCCTGGTCTACAATGGACGCTTCAACGGCCCTCCCAGCGATATCCTCAGCTTTGAAACGCCTGAAGGCAAGCCTGGCACTGTCCGCACCTTCGGAGTCTATCCCATTGGATCATCCGCCATGTTGCTGAAGTGGGAGAAACCTGTGGACGAGAATGGAGTGCTGACTGGGTACAAAATATACTACCAGAAGGTTACTGGAACATCCTTGGGACCTCTTCAGGAACGTAAGAAGGAAATCGACCCTAAATTCGACCGTGCTAAGCTTGCTGGATTGGAACCCAATACCAAGTACAGGATCATCATCAGAGCGAAGACCAAGGCCGGAGAAGGTGACGAGTACTACGTGGAACAGACGACGAAGTCCGCTGTTACGGCTAAGCCAGAGATACCACTGTTCGAGACCAGAACCTTATCGGCTAAAGAGGGAACTGCTCATATTCTGGTTAGATGGATTCCTTCGTTGGACGGCCACGCTGGCTCGCATTTCGTGGCATGGTACAAGCTGAAAGGCATGCCGGACTGGCTGAAGACTAACGACATCACGGACGACGACTACGTCATCCTGACGGGGCTGGAGCCGGGCCAGGTGTACGAGGTGAAGGTCACCGCTCACGACGGCGAGTACTTCAGCACCAGTGAGATCAAAGACGTCGACACTACTATTGACGGTCCTATAGTGAAGCCGGACGAGAAGATGGCCGCCGCCGGGTGGTTCATCGGCGTGATGCTGGCGCTGGCGTTCCTGCTGCTGGTCCTGGTGCTGGTGTGCGTGGTGCGGCGCAACCGCGGCGGCAAGTACGACGTGCACGACCGCGAGCTCGCGCACGGCCGCCGCGACTACGCCGAGGGCGGCTTCCACGAGTACACGCACCCCCTGGACAACAAGTCGCGGCACTCGATGAGCAGCGGAACTAAGCCCGGCCCGGAGAGCGACACCGACTCCATGGCCGAGTACGGCGAGGGCGAGACTGGTCGGTTCACGGAGGACGGTTCGTTCATCGGGCAGTACGTGCCGGGCGCGCGCGTGCTGCCGCCGGCGCCGGGCGCGCGCGCGCCGCTGTCGCCGCCCTCGCCGCCCGCGCCCGCGCCCGCGCCCGCCGCGCCCGCCGCGCCGCACCCGCCCACATACGTCTAAGCGCACCGTCTACTGACTAATGTGGCTAGAATACATCGCGTAAGCTCTTTTAATGTCTCCGTACATGAATAAATGCAATTCTGTAGGTAATCAACTATACATTATGTATTGATTT | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | U50719 | Manduca sexta hemolymph proteins Kanost lab-Neuroglian RNAi construct | ACGATAGTGTGGCTGAACGATGGGCAGCTGATCGACTTCGAAAACCAGCCGCGCTTCCGCATGACCAACGACTACTCGCTGCTGATATCGGACACCACCGAGCTTGACTCCGGACAGTATACTTGTATCGCCAAGACCGCGATCGATGAGGCGCGTGCGCAGGCCACTCTTACTGTGCAAGACAAGCCGAACCCGCCGGCGCTGGACGGCGTGGAGTGCGGCGCGGCGACGGCGACGCTGCGCTGGCGCTCCATGGGCGACAACCGCGCGCCCGTCGTGCGCTACCAGATACACTACAACACCAGCTTCACGCCCGACTCCTGGGCCGCCGCCGCCGACCACGTGCCCGCCATCGACACCTCCTGGACCGTGCAGCTCAGCCCCTGGGCCAACTACACCTTCCGAGTGATCGCAGTGAACAAAATCGGTCCTTCGAGCCCGTCGTCGCACTCGGACGTGTGCACCACGCAGCCCGACGTGCCTTACAAGAACCCCGACA | dsRNA | MEGAsript RNAi Kit (Ambion) | column-based | simultaneously with transcription | (1) Amplify gene by PCR with forward primer (TAATACGACTCACTATAGGGAGAGACGATAGTGTGGCTGAACG) and reverse primer (TAATACGACTCACTATAGGGAGATGTCGGGGTTCTTGTAAGGC). (2) PCR product was isolated by agarose electrophoresis and purified using the QIAquick gel extraction kit (Qiagen). (3) This DNA was used as template for preparation of dsRNA using MEGAsript RNAi Kit (Ambion). | InsectaCentral | Manduca sexta hemolymph proteins Kanost lab-Neuroglian RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | ddH2O | 0.04 | non_specific_dsRNA | 5 | abdominal hemocoels | Green fluorescent protein (GFP) siRNA was purchased from Dharmacon and used as a control. | larva | qPCR phenotype | hemocytes | not checked | 96 | low | 50 | |||||
| 55 | Papilio xuthus oviposition stimulant receptor silencing | RNAi | We make a success of gene silencing in Swallowtail butterfly, Papilio xuthus, by dsRNA injection to pupae. | InsectaCentral | Unpublished | Ozaki | Katsuhisa | Papilio xuthus oviposition stimulant receptor silencing target | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | cds | InsectaCentral | Papilio xuthus oviposition stimulant receptor silencing target | Papilio xuthus oviposition stimulant receptor silencing RNAi construct | dsRNA | MEGAscript T7 kit / Ambion | ethanol precipitation | simultaneously with transcription | About 400bp of target region of the gene was amplified by T7 promoter adding specific primers. dsRNA were synthesized by MEGAscript T7 kit following manufacture's protocol. | InsectaCentral | Papilio xuthus oviposition stimulant receptor silencing RNAi construct | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | local resource | pupa | lab_colony | injection | 1 | buffer | 1 | thorax of pupae | Same amounts of ultra pure water is injected. | adult | qPCR | fore-leg tarsi | not checked | 120 | high | 95 | ||||||||
| 56 | helicoverpa feeding | RNAi | RNAi experiments to knockdown H. armigera genes via feeding or embryo microinjection delivery of dsRNA. | InsectaCentral | Unpublished | Collinge | Derek | Ecdysone Receptor dsRNA feeding target | GCAGCACAGATGGCGAGGCGAGGCGGCAGAAGAAAGGCCCAGCGCCGAGGCAGCAAGAAGAGCTATGTCTTGTCTGCGGTGACAGAGCCTCCGGATATCACTACAACGCGCTCACATGTGAAGGGTGTAAAGGTTTCTTCAGGCGGAGTGTAACCAAAAATGCAGTGTACATATGCAAATTCGGCCATGCTTGCGAAATGGATATGTATATGCGGAGAAAATGTCAGGAGTGTCGGTTGAAGAAATGTCTTGCGGTAGGCATGAGGCCCGAGTGCGTGGTGCCGGAGAACCAGTGTGCAATGAAACGGAAAGAGAAAAAGGCGCAGAGGGAAAAAGACAAATTGCCCGTCAGTACGACGACAGTAGACGATCACATGCCTCCCATCATGCAATGTGATCCTCCGCCCCCAGAGGCCGCTAGAATTCTGGAATGTTTGCAGCACGAGGTGGTGCCGCGGTTCCTCAATGAGAAGCTCATGGAACAGAACAGATTGAAG | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | InsectaCentral | Ecdysone Receptor dsRNA feeding target | Ecdysone Receptor dsRNA feeding RNAi construct | GCAGCACAGATGGCGAGGCGAGGCGGCAGAAGAAAGGCCCAGCGCCGAGGCAGCAAGAAGAGCTATGTCTTGTCTGCGGTGACAGAGCCTCCGGATATCACTACAACGCGCTCACATGTGAAGGGTGTAAAGGTTTCTTCAGGCGGAGTGTAACCAAAAATGCAGTGTACATATGCAAATTCGGCCATGCTTGCGAAATGGATATGTATATGCGGAGAAAATGTCAGGAGTGTCGGTTGAAGAAATGTCTTGCGGTAGGCATGAGGCCCGAGTGCGTGGTGCCGGAGAACCAGTGTGCAATGAAACGGAAAGAGAAAAAGGCGCAGAGGGAAAAAGACAAATTGCCCGTCAGTACGACGACAGTAGACGATCACATGCCTCCCATCATGCAATGTGATCCTCCGCCCCCAGAGGCCGCTAGAATTCTGGAATGTTTGCAGCACGAGGTGGTGCCGCGGTTCCTCAATGAGAAGCTCATGGAACAGAACAGATTGAAG | dsRNA | Ambion silencer siRNA cocktail kit | ethanol precipitation | simultaneously with transcription | Primers were designed to amplify a ~500bp region of each of the candidate genes for silencing. Gene fragments were PCR amplified and cloned into pGEM-T-Easy (Promega) following the manufacturerâs instructions. Once ligated into pGEM-T-Easy, the vector was transformed by electroporation into DH10β cells and plated on LB ampicillin selective plates. PCR colony screening confirmed the correct size inserts in the multiple cloning site of pGEM-T-Easy (Promega). Cells were selectively liquid cultured and plasmids isolated using a commercial mini-prep kit (Invitrogen). pGEM-T-Easy containing gene fragments and the pL4440 vector plasmid DNA were digested separately with PstI and SacII restriction enzymes, resolved on a 1% agarose gel after which appropriate restriction fragments were excised and gel purified with an Eppendorf gel purification kit. A ligation reaction was performed between each of the gene fragments and the digested pL4440 vector. Recombinant plasmids were colony screened and isolated as described above. Inserts were sequenced to confirm the correct fragment had been inserted. An Ambion silencer siRNA cocktail kit was used for the production of dsRNA and siRNA. Using the two inverted T7 promoters flanking the multiple cloning site in the pL4440 vector, T7 RNA polymerase was used to produce single stranded sense and antisense RNAs that were subsequently hybridised to produce dsRNA. RNaseIII digested siRNA (RsiRNA) were produced through RNaseIII digestions of the dsRNA following the Ambion kit instructions. DICER-digested siRNAs (DsiRNAs) were generated by incubating the dsRNA with Turbo-DICER (Genlantis) following the manufacturerâs instructions. | InsectaCentral | Ecdysone Receptor dsRNA feeding RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | 0.30000000000000004 | feeding | 0.1 | buffer non_specific_dsRNA | 21 | Four negative controls were also set up where neonates were fed a mixture of either dsRNA for non-endogenous EGFP, milliQ water, nuclease free water (Ambion) or elution solution (Ambion siRNA cocktail kit) mixed with green food dye. | larva | qPCR phenotype | whole organism | not checked | 48 | none | |||||||
| 57 | Sl-Halloween genes (shadow) | RNAi | InsectaCentral | Unpublished | Iga | Masatoshi | Sl-Halloween genes (shadow) target | ATGATTTGTAACAGGAATAAAGCTTTATTTAAACTCATAAAATTTAATAAGAATTTATCATACGATGCGTCTCCATCACCCAAGTCGATTGAATTTATGCCTCGTCCAAAATCTCTGCCCATTGTAGGAACTAAATTGGATTTCCTAGCAGCAGGAGGTGGCTCTAAATTGCACGAATATGTCGACTTTCGTCATAAACAATTAGGTCCCATATTTTGTGATAAATTAGGAGGGAGAATAGATTTAGTATTTGTTAGTGATCCCGCACTAATAAAAACACTATTTCTAAATCTTGAAGGAAAATATCCCATACATATATTACCAGAACCATGGGAATTGTACGAAAAGCTTTATGGGGCCAAAAGAGGCTTATTTTTCATGAATGGAGAAGAATGGTTAGAAAATCGAAGAGTTGTTAACAAACATTTATTAAAAGAAAATTCAGAAAAATTATTCGATAATCCAGTTACAAATACTATCAACCAATTGGTACAGAAATGGATTATTGAAGCTAAAAAGGGCTTTTATGTGCCAAACTTAGAGACCGAATTCTATCGACTGTCTATTGATGTGATAATATCAGTAATGCTAGGAAGTTCTATCTTCCATAAGTTTAGCGTACACAGTGATGCTTTATTGACTGCATTTGCAGAAGAAGTCAAGAAAATTTTTCAGACCACAACGAAATTGTATGGGTGGCCTGTTAACATGTGTCAGAAGATGAATTTAAAAGTGTGGAGAGATTTCAAAAAATCTGTTGATATATCACTATTCCTAGCTAACAAAATTGTCGAGGAGATGATAAATAATAAACAACCTAAAGACGGACTGATTAATCTTCTTATTGAAGAAAATCTTAAACCAGAAATCATTAAAAGGATTATCGTAGATTTTGTCATCGCTGCTGGTGATACGACATCTTATACTACAATATGGACATTGTATCTATTGTCCAAGAACAAAGATGTGAGACAAGAACTATTTAAAAGAAATTCTATAGCAAACTATGCGATAAAGGAGTCTATGAGATTATATCCTGTTGCTCCGTTTTTGACCAGGATTCTGCCCAAAGAATGCATCTTCGGACCTTACAAACTTAAAGGAGGCACTCCAATAATAGTTTCCATTTACACATCTGGAAGAGACGAGCAATACTTTAGTAGAGCTACAGAATACCTGCCTTACCGCTGGGACCGCATGGATATAAGAAGGAATGATATTGTAAACCATGTGTCTTCTGCATCATTGCCATTTGCTCTTGGGGCCCGGTCATGTATAGGCAAAAAGCTAGCAATGCTACAAATGAAAGAACTTATAACTCAGATGGTACAAAACTTTGAATTTGAATGTATAAACAAAGATGAAGTTACTTCAAAAACATCTCAGGTACTTGTGCCCAGCCAAAAA | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | cds | InsectaCentral | Sl-Halloween genes (shadow) target | Sl-Halloween genes (shadow) RNAi construct | AATGGTTAGAAAATCGAAGAGTTGTTAACAAACATTTATTAAAAGAAAATTCAGAAAAATTATTCGATAATCCAGTTACAAATACTATCAACCAATTGGTACAGAAATGGATTATTGAAGCTAAAAAGGGCTTTTATGTGCCAAACTTAGAGACCGAATTCTATCGACTGTCTATTGATGTGATAATATCAGTAATGCTAGGAAGTTCTATCTTCCATAAGTTTAGCGTACACAGTGATGCTTTATTGACTGCATTTGCAGAAGAAGTCAAGAAAATTTTTCAGACCACAACGAAATTGTATGGGTGGCCTGTTAACATGTGTCAGAAGATGAATTTAAAAGTGTGGAGAGATTTCAAAAAATCTGTTGATATATCACTATTCCTAGCTAACAAAATTGTCGAGGAGATGATAAATAATAAACAA | dsRNA | MEGAscript RNAi kit /Ambion | column-based | simultaneously with transcription | Followed the kit protocol. | InsectaCentral | Sl-Halloween genes (shadow) RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | larva | lab_colony | 0.5 | injection | TE | 0.015 | non_specific_dsRNA | GFP dsRNA was synthesized as same as target gene and used as a control. | larva | RT-PCR (semi-quantitative) phenotype | Prothoracic glands, Testis | not checked | 72 | none | ||||||||
| 58 | Sl-Halloween genes (shadow)2 | RNAi | InsectaCentral | Unpublished | Iga | Masatoshi | Sl-Halloween genes (shadow)2 target | ATGATTTGTAACAGGAATAAAGCTTTATTTAAACTCATAAAATTTAATAAGAATTTATCATACGATGCGTCTCCATCACCCAAGTCGATTGAATTTATGCCTCGTCCAAAATCTCTGCCCATTGTAGGAACTAAATTGGATTTCCTAGCAGCAGGAGGTGGCTCTAAATTGCACGAATATGTCGACTTTCGTCATAAACAATTAGGTCCCATATTTTGTGATAAATTAGGAGGGAGAATAGATTTAGTATTTGTTAGTGATCCCGCACTAATAAAAACACTATTTCTAAATCTTGAAGGAAAATATCCCATACATATATTACCAGAACCATGGGAATTGTACGAAAAGCTTTATGGGGCCAAAAGAGGCTTATTTTTCATGAATGGAGAAGAATGGTTAGAAAATCGAAGAGTTGTTAACAAACATTTATTAAAAGAAAATTCAGAAAAATTATTCGATAATCCAGTTACAAATACTATCAACCAATTGGTACAGAAATGGATTATTGAAGCTAAAAAGGGCTTTTATGTGCCAAACTTAGAGACCGAATTCTATCGACTGTCTATTGATGTGATAATATCAGTAATGCTAGGAAGTTCTATCTTCCATAAGTTTAGCGTACACAGTGATGCTTTATTGACTGCATTTGCAGAAGAAGTCAAGAAAATTTTTCAGACCACAACGAAATTGTATGGGTGGCCTGTTAACATGTGTCAGAAGATGAATTTAAAAGTGTGGAGAGATTTCAAAAAATCTGTTGATATATCACTATTCCTAGCTAACAAAATTGTCGAGGAGATGATAAATAATAAACAACCTAAAGACGGACTGATTAATCTTCTTATTGAAGAAAATCTTAAACCAGAAATCATTAAAAGGATTATCGTAGATTTTGTCATCGCTGCTGGTGATACGACATCTTATACTACAATATGGACATTGTATCTATTGTCCAAGAACAAAGATGTGAGACAAGAACTATTTAAAAGAAATTCTATAGCAAACTATGCGATAAAGGAGTCTATGAGATTATATCCTGTTGCTCCGTTTTTGACCAGGATTCTGCCCAAAGAATGCATCTTCGGACCTTACAAACTTAAAGGAGGCACTCCAATAATAGTTTCCATTTACACATCTGGAAGAGACGAGCAATACTTTAGTAGAGCTACAGAATACCTGCCTTACCGCTGGGACCGCATGGATATAAGAAGGAATGATATTGTAAACCATGTGTCTTCTGCATCATTGCCATTTGCTCTTGGGGCCCGGTCATGTATAGGCAAAAAGCTAGCAATGCTACAAATGAAAGAACTTATAACTCAGATGGTACAAAACTTTGAATTTGAATGTATAAACAAAGATGAAGTTACTTCAAAAACATCTCAGGTACTTGTGCCCAGCCAAAAA | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | cds | InsectaCentral | Sl-Halloween genes (shadow)2 target | Sl-Halloween genes (shadow) RNAi construct | AATGGTTAGAAAATCGAAGAGTTGTTAACAAACATTTATTAAAAGAAAATTCAGAAAAATTATTCGATAATCCAGTTACAAATACTATCAACCAATTGGTACAGAAATGGATTATTGAAGCTAAAAAGGGCTTTTATGTGCCAAACTTAGAGACCGAATTCTATCGACTGTCTATTGATGTGATAATATCAGTAATGCTAGGAAGTTCTATCTTCCATAAGTTTAGCGTACACAGTGATGCTTTATTGACTGCATTTGCAGAAGAAGTCAAGAAAATTTTTCAGACCACAACGAAATTGTATGGGTGGCCTGTTAACATGTGTCAGAAGATGAATTTAAAAGTGTGGAGAGATTTCAAAAAATCTGTTGATATATCACTATTCCTAGCTAACAAAATTGTCGAGGAGATGATAAATAATAAACAA | dsRNA | MEGAscript RNAi kit /Ambion | column-based | simultaneously with transcription | Followed the kit protocol. | InsectaCentral | Sl-Halloween genes (shadow) RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | larva | lab_colony | 0.5 | injection | TE | 0.015 | non_specific_dsRNA | GFP dsRNA was synthesized as same as target gene and used as a control. | pupa | phenotype | not checked | 120 | none | |||||||||
| 59 | Sl-Halloween genes (shadow)3 | RNAi | InsectaCentral | Unpublished | Iga | Masatoshi | Sl-Halloween genes (shadow) target | ATGATTTGTAACAGGAATAAAGCTTTATTTAAACTCATAAAATTTAATAAGAATTTATCATACGATGCGTCTCCATCACCCAAGTCGATTGAATTTATGCCTCGTCCAAAATCTCTGCCCATTGTAGGAACTAAATTGGATTTCCTAGCAGCAGGAGGTGGCTCTAAATTGCACGAATATGTCGACTTTCGTCATAAACAATTAGGTCCCATATTTTGTGATAAATTAGGAGGGAGAATAGATTTAGTATTTGTTAGTGATCCCGCACTAATAAAAACACTATTTCTAAATCTTGAAGGAAAATATCCCATACATATATTACCAGAACCATGGGAATTGTACGAAAAGCTTTATGGGGCCAAAAGAGGCTTATTTTTCATGAATGGAGAAGAATGGTTAGAAAATCGAAGAGTTGTTAACAAACATTTATTAAAAGAAAATTCAGAAAAATTATTCGATAATCCAGTTACAAATACTATCAACCAATTGGTACAGAAATGGATTATTGAAGCTAAAAAGGGCTTTTATGTGCCAAACTTAGAGACCGAATTCTATCGACTGTCTATTGATGTGATAATATCAGTAATGCTAGGAAGTTCTATCTTCCATAAGTTTAGCGTACACAGTGATGCTTTATTGACTGCATTTGCAGAAGAAGTCAAGAAAATTTTTCAGACCACAACGAAATTGTATGGGTGGCCTGTTAACATGTGTCAGAAGATGAATTTAAAAGTGTGGAGAGATTTCAAAAAATCTGTTGATATATCACTATTCCTAGCTAACAAAATTGTCGAGGAGATGATAAATAATAAACAACCTAAAGACGGACTGATTAATCTTCTTATTGAAGAAAATCTTAAACCAGAAATCATTAAAAGGATTATCGTAGATTTTGTCATCGCTGCTGGTGATACGACATCTTATACTACAATATGGACATTGTATCTATTGTCCAAGAACAAAGATGTGAGACAAGAACTATTTAAAAGAAATTCTATAGCAAACTATGCGATAAAGGAGTCTATGAGATTATATCCTGTTGCTCCGTTTTTGACCAGGATTCTGCCCAAAGAATGCATCTTCGGACCTTACAAACTTAAAGGAGGCACTCCAATAATAGTTTCCATTTACACATCTGGAAGAGACGAGCAATACTTTAGTAGAGCTACAGAATACCTGCCTTACCGCTGGGACCGCATGGATATAAGAAGGAATGATATTGTAAACCATGTGTCTTCTGCATCATTGCCATTTGCTCTTGGGGCCCGGTCATGTATAGGCAAAAAGCTAGCAATGCTACAAATGAAAGAACTTATAACTCAGATGGTACAAAACTTTGAATTTGAATGTATAAACAAAGATGAAGTTACTTCAAAAACATCTCAGGTACTTGTGCCCAGCCAAAAA | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | cds | InsectaCentral | Sl-Halloween genes (shadow) target | Sl-Halloween genes (shadow) RNAi construct | AATGGTTAGAAAATCGAAGAGTTGTTAACAAACATTTATTAAAAGAAAATTCAGAAAAATTATTCGATAATCCAGTTACAAATACTATCAACCAATTGGTACAGAAATGGATTATTGAAGCTAAAAAGGGCTTTTATGTGCCAAACTTAGAGACCGAATTCTATCGACTGTCTATTGATGTGATAATATCAGTAATGCTAGGAAGTTCTATCTTCCATAAGTTTAGCGTACACAGTGATGCTTTATTGACTGCATTTGCAGAAGAAGTCAAGAAAATTTTTCAGACCACAACGAAATTGTATGGGTGGCCTGTTAACATGTGTCAGAAGATGAATTTAAAAGTGTGGAGAGATTTCAAAAAATCTGTTGATATATCACTATTCCTAGCTAACAAAATTGTCGAGGAGATGATAAATAATAAACAA | dsRNA | MEGAscript RNAi kit /Ambion | column-based | simultaneously with transcription | Followed the kit protocol. | InsectaCentral | Sl-Halloween genes (shadow) RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | larva | lab_colony | 0.67 | injection | TE | 0.045 | non_specific_dsRNA | GFP dsRNA was synthesized as same as target gene and used as a control. | pupa | phenotype | not checked | 120 | none | |||||||||
| 60 | Sl-Halloween genes (shadow)4 | RNAi | InsectaCentral | Unpublished | Iga | Masatoshi | Sl-Halloween genes (shadow) target | ATGATTTGTAACAGGAATAAAGCTTTATTTAAACTCATAAAATTTAATAAGAATTTATCATACGATGCGTCTCCATCACCCAAGTCGATTGAATTTATGCCTCGTCCAAAATCTCTGCCCATTGTAGGAACTAAATTGGATTTCCTAGCAGCAGGAGGTGGCTCTAAATTGCACGAATATGTCGACTTTCGTCATAAACAATTAGGTCCCATATTTTGTGATAAATTAGGAGGGAGAATAGATTTAGTATTTGTTAGTGATCCCGCACTAATAAAAACACTATTTCTAAATCTTGAAGGAAAATATCCCATACATATATTACCAGAACCATGGGAATTGTACGAAAAGCTTTATGGGGCCAAAAGAGGCTTATTTTTCATGAATGGAGAAGAATGGTTAGAAAATCGAAGAGTTGTTAACAAACATTTATTAAAAGAAAATTCAGAAAAATTATTCGATAATCCAGTTACAAATACTATCAACCAATTGGTACAGAAATGGATTATTGAAGCTAAAAAGGGCTTTTATGTGCCAAACTTAGAGACCGAATTCTATCGACTGTCTATTGATGTGATAATATCAGTAATGCTAGGAAGTTCTATCTTCCATAAGTTTAGCGTACACAGTGATGCTTTATTGACTGCATTTGCAGAAGAAGTCAAGAAAATTTTTCAGACCACAACGAAATTGTATGGGTGGCCTGTTAACATGTGTCAGAAGATGAATTTAAAAGTGTGGAGAGATTTCAAAAAATCTGTTGATATATCACTATTCCTAGCTAACAAAATTGTCGAGGAGATGATAAATAATAAACAACCTAAAGACGGACTGATTAATCTTCTTATTGAAGAAAATCTTAAACCAGAAATCATTAAAAGGATTATCGTAGATTTTGTCATCGCTGCTGGTGATACGACATCTTATACTACAATATGGACATTGTATCTATTGTCCAAGAACAAAGATGTGAGACAAGAACTATTTAAAAGAAATTCTATAGCAAACTATGCGATAAAGGAGTCTATGAGATTATATCCTGTTGCTCCGTTTTTGACCAGGATTCTGCCCAAAGAATGCATCTTCGGACCTTACAAACTTAAAGGAGGCACTCCAATAATAGTTTCCATTTACACATCTGGAAGAGACGAGCAATACTTTAGTAGAGCTACAGAATACCTGCCTTACCGCTGGGACCGCATGGATATAAGAAGGAATGATATTGTAAACCATGTGTCTTCTGCATCATTGCCATTTGCTCTTGGGGCCCGGTCATGTATAGGCAAAAAGCTAGCAATGCTACAAATGAAAGAACTTATAACTCAGATGGTACAAAACTTTGAATTTGAATGTATAAACAAAGATGAAGTTACTTCAAAAACATCTCAGGTACTTGTGCCCAGCCAAAAA | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | cds | InsectaCentral | Sl-Halloween genes (shadow) target | Sl-Halloween genes (shadow)4 RNAi construct | AATGGTTAGAAAATCGAAGAGTTGTTAACAAACATTTATTAAAAGAAAATTCAGAAAAATTATTCGATAATCCAGTTACAAATACTATCAACCAATTGGTACAGAAATGGATTATTG | dsRNA | MEGAscript RNAi kit /Ambion | column-based | subsequent to transcription | Followed the kit protocol. | InsectaCentral | Sl-Halloween genes (shadow)4 RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | larva | lab_colony | 0.25 | injection | TE | 0.03 | non_specific_dsRNA | GFP dsRNA was synthesized as same as target gene and used as a control. | pupa | phenotype | not checked | 120 | none | |||||||||
| 61 | Sl-Halloween genes (disembodied) | RNAi | InsectaCentral | Unpublished | Iga | Masatoshi | Sl-Halloween genes (disembodied) target | ATGTACAAGTCATCTAAAATATTTTCCAATCGTTTGAAATATGTGAATTTAGTGTCGAACGTAGCGAAGTATGGGAGTGGCAAGACACAGAACTGTAATGTAAGGAGTTTTGAAGAAATCCCAGGACCTAAAAGTTATCCCATAATAGGGACGCTTCATCAATATGCACCATTTATTGGTGACTACGATGTTGAAACTCTAGACAAAAATGCTTGGTTGAATTATCGCCGCTATGGCAGCCTTGTTCGTGAAACACCAGGCGTCAACGTCCTGCATGTTTATGATCCAGAAGATATAGAAACAGTATTCCGTCAAGATCATCGGTTCCCAGCAAGGAGAAGCCATATTGCGATGTACCACTATCGTATGAACAAACCTGATGTGTATAAAACTGGTGGATTACTGAGCACAAACGGTGAAGAATGGTGGAGACTAAGGAGTACATTTCAAAAGAATTTCACCAGCCCGAAAAGTGCTAAAAGTCACATAGAAAGTACTGAGATTGTGATAAGAGAATTTATTAACTGGATAAAAGAAAGAAATGTAACGCACAATGAAGATATTTTGCCGTATTTGAATAGATTGAATTTGGAAGTTATTGGTGTTGTAGCATTCAATGAGCGTTTTAGAAGCTTTTCTCCGGAAGAACAAGATCCGACGTCCAGAAGTAGCAAAACAATTGATGCGGCATTTGGTTCCAATTGTGGAATTATGAAACTCGATAAAGGATTTATGTGGAAGATCTTCCAAACTCCTGTTTATAAGAGACTTGCTGATTCACAAACGTATTTAGAAAAGGTATCTACAGATATCTTATACAATCGAATTCATTATTTTGAACAACCTGAAGATGGCGATATTTCTTTGTTGGGATCTTTTCTAAAGCAGCCCAATGTGGACTTAAAGGACGTCATTGGAGTGATGGTTGATATTCTTATGGCTGCTATAGACACGACGGCATATTCAACCAGCTTTGCATTATATCATATTGGAAGAAATCCCGAAGTTCAGCAGAAAATGTTCGAAGAAATCTCCACTCTTCTTCCCACAGATGATGCCAAAATAACTCCTGATATTTTATCAAAAGCAACTTACGTACGAGCTTGTCTCAAAGAAAGTTTGCGACTTAACCCTGTGTCGGTTGGCATTGGTCGTTTAACTCAAAAAGATTTCGTATTAAGGGGTTATCTAATTCCAGAAGGGACAGTCATTGTCACTCAAAATTTTGTGGCTTCTCGAATGCCGCAGTATGTAAAGGATCCATTAAAATTCAAACCTGAACGGTGGATAAGAGACTCGGAGAGTTATGAAAACATTCATCCATTCCTGAGTTTGCCATTTGGTTTTGGACCACGGTCTTGTATAGCAAGGAGATTAGCAGAACAAAATATATGTATTTTCTTAATGCGGTTAATTCGTGATTTTAACGTAACATGGATGGGTGAAGATCTTGGTATTAAAACCTTACTTATAAATAAACCAGACAAACCCGTCTCTTTGTCATTTACTTCAAGAATCGTG | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | cds | InsectaCentral | Sl-Halloween genes (disembodied) target | Sl-Halloween genes (disembodied) RNAi construct | TTATTGGTGTTGTAGCATTCAATGAGCGTTTTAGAAGCTTTTCTCCGGAAGAACAAGATCCGACGTCCAGAAGTAGCAAAACAATTGATGCGGCATTTGGTTCCAATTGTGGAATTATGAAACTCGATAAAGGATTTATGTGGAAGATCTTCCAAACTCCTGTTTATAAGAGACTTGCTGATTCACAAACGTATTTAGAAAAGGTATCTACAGATATCTTATACAATCGAATTCATTATTTTGAACAACCTGAAGATGGCGATATTTCTTTGTTGGGATCTTTTCTAAAGCAGCCCAATGT | dsRNA | MEGAscript RNAi kit /Ambion | column-based | simultaneously with transcription | Followed the kit protocol. | InsectaCentral | Sl-Halloween genes (disembodied) RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | larva | lab_colony | 0.6000000000000001 | injection | TE | 0.028 | buffer | pupa | phenotype | not checked | 120 | none | ||||||||||
| 67 | HSG Epiphyas postvittana APN3 RNAi | RNAi | Epiphyas postvittana were fed with naked dsRNA and the effects on the insect and on the protein levels corresponding to the target gene were examined. | InsectaCentral | Unpublished | Gatehouse | Heather Sian | HSG Epiphyas postvittana APN3 RNAi target | GCTTTCCCTTGCTACGATGAGCCTAGCTTCAAAGCTACTTTCGACATCACCATCCGCCGCCCCGCTGCTTACCGCAGTTGGTCTTGTACTCGCATTGCCAGCACTGCGCCTAGCACTACCCCTCCCAACTATGAAGACGACATCTACCACAGGACGCCTATAATGTCAACGTACCTCTTGGCTCTGATTGTTGCTGAGTACGATAGCTTAACGGTCAACAACGCGCAAGGCCAGTTAATCTACGAAGTCATTGCTCGACCTAACGCTATTAGCACAGGCCAGGGACAGTATGCCTTAGACGTGGGTCAGGATCTTCTCGCTGAAATGAACGACCACACTAATTACAACTTCTACACCATGAACCCGAATCTGAAGATGACTCAAGCGTCGATTCCCGACTTCTCTGCCGGTGCTATGGAAAACTGGGGACT | Lepidoptera | Tortricidae | Epiphyas | postvittana | 65032 | cds | GenBank | AF276241 | HSG Epiphyas postvittana APN3 RNAi RNAi construct | TAATACGACTCACTATACGAGCTCTCCCATATGGTCGACGGTATCGATAAGCTTGATTGCTTTCCCTTGCTACGATGAGCCTAGCTTCAAAGCTACTTTCGACATCACCATCCGCCGCCCCGCTGCTTACCGCAGTTGGTCTTGTACTCGCATTGCCAGCACTGCGCCTAGCACTACCCCTCCCAACTATGAAGACGACATCTACCACAGGACGCCTATAATGTCAACGTACCTCTTGGCTCTGATTGTTGCTGAGTACGATAGCTTAACGGTCAACAACGCGCAAGGCCAGTTAATCTACGAAGTCATTGCTCGACCTAACGCTATTAGCACAGGCCAGGGACAGTATGCCTTAGACGTGGGTCAGGATCTTCTCGCTGAAATGAACGACCACACTAATTACAACTTCTACACCATGAACCCGAATCTGAAGATGACTCAAGCGTCGATTCCCGACTTCTCTGCCGGTGCTATGGAAAACTGGGGACTAATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCACTAGTGATATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTA | dsRNA | phenol_chloroform | simultaneously with transcription | DsRNA was produced as detailed in Gatehouse et al. (2004, Amylase activity in honey bee hypopharyngeal glands reduced by RNA interference. Journal of Apicultural Research 43(1):9-13). Briefly, genes were cloned into a modified pGEM vector between two opposing T7 promotors and this âdouble-T7â plasmid transformed into pLysS BL21 cells, which produce T7 polymerase when induced with IPTG. This means that any dsRNA produced will include vector fragments between the T7 promotor and the gene fragment, and also vector fragments beyond the gene and possibly beyond the second T7 promotor. The E. postvittana Aminopeptidase-3 (APN3) fragment clone was obtained from Richard Newcomb (Plant and Food Research, Auckland, New Zealand) in pBluescript SK-, T-tailed into the Eco RV site. The fragment was excised with Not I and Sal I and ligated into the Not I to Sal I region of the double T7 plasmid. Total nucleic acid was purified from induced cells by a no RNaseA variation of a standard miniprep. Thus dsRNA preparations also contained E. coli RNA, mRNA and plasmid DNA. Control RNA preparations were produced by the same method from uninduced cells and thus contained E. coli RNA, mRNA and plasmid DNA, but no dsRNA. Concentration of dsRNA was estimated as approx 1011 molecules of target dsRNA in 1 ul of 10mg/ml RNA preparations (Gatehouse et al., 2004). | InsectaCentral | HSG Epiphyas postvittana APN3 RNAi RNAi construct | Lepidoptera | Tortricidae | Epiphyas | postvittana | 65032 | local resource | larva | lab_colony | 0.066 | feeding | 10% sucrose solution | 0.25 | buffer non_specific_dsRNA | 3 | 3rd instar E. postvittana larvae were held without food for 24 hours before individual larvae were fed a 0.25 uL droplet of one of three treatments: 10% sucrose solution (control 1), 10mg/ml non-induced sample in 10% sucrose (control 2), and 10mg/ml induced sample in 10% sucrose (target dsRNA). The sucrose solution was diethyl procarbonate (DEPC) treated before use. Induced and uninduced RNA was prepared as above with the final resuspension in a 10% DEPC-treated sucrose solution, the induced preparation estimated at 10^11 molecules dsRNA per uL, ie. 2.5 x 10^10 molecules dsRNA per larva (approximately 0.066ug/ul), and the uninduced preparation at a corresponding concentration. The larvae were placed, one at a time, in a small Petri dish which facilitated beading when the virus droplet came into contact with the surface. The 0.25uL droplet was partially ejected from the tip of pipette until it beaded, and was then offered to the larva. As the larva started to consume the droplet, it was fully ejected from the pipette tip onto the surface of the Petri dish. Once the droplet had been fully consumed (generally within 5 seconds), the larva was placed into an AA cup containing approximately 0.5mL of leafroller assay diet until dissection. Care was taken not to cause the larvae to regurgitate its stomach contents. Any larvae that did not consume the whole droplet were discarded. 30 larvae per treatment were tested and at least two biological replicates used. | larva | enzymatactivity phenotype | midgut tissue assayed for changes in APN activity at 24, 48 and 168 hours post-delivery, whole organism for phenotypic changes | not checked | 48 | none | |||||||
| 68 | Ecdysone Receptor dsRNA feeding | RNAi | dsRNA Caterpillar Feeding | InsectaCentral | Unpublished | Collinge | Derek Phillip | Ecdysone Receptor dsRNA feeding target | GCAGCACAGATGGCGAGGCGAGGCGGCAGAAGAAAGGCCCAGCGCCGAGGCAGCAAGAAGAGCTATGTCTTGTCTGCGGTGACAGAGCCTCCGGATATCACTACAACGCGCTCACATGTGAAGGGTGTAAAGGTTTCTTCAGGCGGAGTGTAACCAAAAATGCAGTGTACATATGCAAATTCGGCCATGCTTGCGAAATGGATATGTATATGCGGAGAAAATGTCAGGAGTGTCGGTTGAAGAAATGTCTTGCGGTAGGCATGAGGCCCGAGTGCGTGGTGCCGGAGAACCAGTGTGCAATGAAACGGAAAGAGAAAAAGGCGCAGAGGGAAAAAGACAAATTGCCCGTCAGTACGACGACAGTAGACGATCACATGCCTCCCATCATGCAATGTGATCCTCCGCCCCCAGAGGCCGCTAGAATTCTGGAATGTTTGCAGCACGAGGTGGTGCCGCGGTTCCTCAATGAGAAGCTCATGGAACAGAACAGATTGAAG | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | InsectaCentral | Ecdysone Receptor dsRNA feeding target | Ecdysone Receptor dsRNA feeding RNAi construct | GCAGCACAGATGGCGAGGCGAGGCGGCAGAAGAAAGGCCCAGCGCCGAGGCAGCAAGAAGAGCTATGTCTTGTCTGCGGTGACAGAGCCTCCGGATATCACTACAACGCGCTCACATGTGAAGGGTGTAAAGGTTTCTTCAGGCGGAGTGTAACCAAAAATGCAGTGTACATATGCAAATTCGGCCATGCTTGCGAAATGGATATGTATATGCGGAGAAAATGTCAGGAGTGTCGGTTGAAGAAATGTCTTGCGGTAGGCATGAGGCCCGAGTGCGTGGTGCCGGAGAACCAGTGTGCAATGAAACGGAAAGAGAAAAAGGCGCAGAGGGAAAAAGACAAATTGCCCGTCAGTACGACGACAGTAGACGATCACATGCCTCCCATCATGCAATGTGATCCTCCGCCCCCAGAGGCCGCTAGAATTCTGGAATGTTTGCAGCACGAGGTGGTGCCGCGGTTCCTCAATGAGAAGCTCATGGAACAGAACAGATTGAAG | dsRNA | Ambion silencer siRNA cocktail kit | ethanol precipitation | simultaneously with transcription | Primers were designed to amplify a ~500bp region of each of the candidate genes for silencing. Gene fragments were PCR amplified and cloned into pGEM-T-Easy (Promega) following the manufacturerâs instructions. Once ligated into pGEM-T-Easy, the vector was transformed by electroporation into DH10β cells and plated on LB ampicillin selective plates. PCR colony screening confirmed the correct size inserts in the multiple cloning site of pGEM-T-Easy (Promega). Cells were selectively liquid cultured and plasmids isolated using a commercial mini-prep kit (Invitrogen). pGEM-T-Easy containing gene fragments and the pL4440 vector plasmid DNA were digested separately with PstI and SacII restriction enzymes, resolved on a 1% agarose gel after which appropriate restriction fragments were excised and gel purified with an Eppendorf gel purification kit. A ligation reaction was performed between each of the gene fragments and the digested pL4440 vector. Recombinant plasmids were colony screened and isolated as described above. Inserts were sequenced to confirm the correct fragment had been inserted. An Ambion silencer siRNA cocktail kit was used for the production of dsRNA and siRNA. Using the two inverted T7 promoters flanking the multiple cloning site in the pL4440 vector, T7 RNA polymerase was used to produce single stranded sense and antisense RNAs that were subsequently hybridised to produce dsRNA. RNaseIII digested siRNA (RsiRNA) were produced through RNaseIII digestions of the dsRNA following the Ambion kit instructions. DICER-digested siRNAs (DsiRNAs) were generated by incubating the dsRNA with Turbo-DICER (Genlantis) following the manufacturerâs instructions. | InsectaCentral | Ecdysone Receptor dsRNA feeding RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | 0.30000000000000004 | feeding | 0.1 | buffer non_specific_dsRNA | 21 | Four negative controls were also set up where neonates were fed a mixture of either dsRNA for non-endogenous EGFP, milliQ water, nuclease free water (Ambion) or elution solution (Ambion siRNA cocktail kit) mixed with green food dye. | larva | qPCR phenotype | whole organism | not checked | 48 | none | |||||||
| 69 | Juvenile Hormone Esterase Receptor dsRNA feeding | RNAi | InsectaCentral | Unpublished | Collinge | Derek Phillip | Juvenile Hormone Esterase Receptor dsRNA feeding target | CGCGGCAAGAACATCCTCTCTTAGTATGATTACCAGCGCAGAATGCGAGACATCCCGCAATCGACTACCCAACTTTGGTTTCGTCAATAAGATAAAAGACAATCCTGCAATCATAATACCGCCCAGAGTTTTATTAATGACACCACCACAACTAGTGATAGATTTGAAAGAGTCTATTGAGAGAAGGTACTACAACGATTCAATAAGTATCGATAACTTTGTGAAATCATGTTCGGACGGCTTCTATGAGTACCCTGCATTGAAACTGGCGCAAAAACGTGCTGAGACTGGTGGAGCTCCACTGTACTTGTACCGGTTCGGATACGAGGGTCAGAGCAGCATCATCAAGGAGGTAATGGAGCTGGACTACGATGGCGCTGGCCACATTGAAGACCTAACCTATGTATTTAGGGCGAACTC | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | InsectaCentral | Juvenile Hormone Esterase Receptor dsRNA feeding target | Juvenile Hormone Esterase Receptor dsRNA feeding RNAi construct | CGCGGCAAGAACATCCTCTCTTAGTATGATTACCAGCGCAGAATGCGAGACATCCCGCAATCGACTACCCAACTTTGGTTTCGTCAATAAGATAAAAGACAATCCTGCAATCATAATACCGCCCAGAGTTTTATTAATGACACCACCACAACTAGTGATAGATTTGAAAGAGTCTATTGAGAGAAGGTACTACAACGATTCAATAAGTATCGATAACTTTGTGAAATCATGTTCGGACGGCTTCTATGAGTACCCTGCATTGAAACTGGCGCAAAAACGTGCTGAGACTGGTGGAGCTCCACTGTACTTGTACCGGTTCGGATACGAGGGTCAGAGCAGCATCATCAAGGAGGTAATGGAGCTGGACTACGATGGCGCTGGCCACATTGAAGACCTAACCTATGTATTTAGGGCGAACTC | dsRNA | Ambion silencer siRNA cocktail kit | ethanol precipitation | simultaneously with transcription | Primers were designed to amplify a ~500bp region of each of the candidate genes for silencing. Gene fragments were PCR amplified and cloned into pGEM-T-Easy (Promega) following the manufacturerâs instructions. Once ligated into pGEM-T-Easy, the vector was transformed by electroporation into DH10β cells and plated on LB ampicillin selective plates. PCR colony screening confirmed the correct size inserts in the multiple cloning site of pGEM-T-Easy (Promega). Cells were selectively liquid cultured and plasmids isolated using a commercial mini-prep kit (Invitrogen). pGEM-T-Easy containing gene fragments and the pL4440 vector plasmid DNA were digested separately with PstI and SacII restriction enzymes, resolved on a 1% agarose gel after which appropriate restriction fragments were excised and gel purified with an Eppendorf gel purification kit. A ligation reaction was performed between each of the gene fragments and the digested pL4440 vector. Recombinant plasmids were colony screened and isolated as described above. Inserts were sequenced to confirm the correct fragment had been inserted. An Ambion silencer siRNA cocktail kit was used for the production of dsRNA and siRNA. Using the two inverted T7 promoters flanking the multiple cloning site in the pL4440 vector, T7 RNA polymerase was used to produce single stranded sense and antisense RNAs that were subsequently hybridised to produce dsRNA. | InsectaCentral | Juvenile Hormone Esterase Receptor dsRNA feeding RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | 0.30000000000000004 | feeding | 0.1 | buffer non_specific_dsRNA | 21 | Four negative controls were also set up where neonates were fed a mixture of either dsRNA for non-endogenous EGFP, milliQ water, nuclease free water (Ambion) or elution solution (Ambion siRNA cocktail kit) mixed with green food dye. | larva | qPCR phenotype | whole organism | not checked | 48 | none | ||||||||
| 70 | Ultraspiracle dsRNA feeding | RNAi | InsectaCentral | Unpublished | Collinge | Derek Phillip | Ultraspiracle dsRNA feeding target | GCTACTGCTAATGCTGTAATTGATCAAATTATCGTTTTATTACGAGTGTGTTTCATAACATCTTCAAAGACATTTGTGTACAGCGAAGGGGTGTATTGTTTCATCTTCATATAGGTATATTTAGTTCAACGACCTTGTTCCTGACAGGTTCATCGTTAGCCACTGAACTATTCTATGAAAGTGGTATATCGTAGACGATAGATAAATTAAATCCAATGATGGAGCCCTCGAGAGATTCAGGGCTAAACTTGGAGGGAGGTTTTATGTCGCCGATGTCACCACCGGAGATGAAGCCAGACACGGCGATGCTAGACGGCCTGCGAGACGACTCCACCCCACCCCCAGCTTTCAAGAACTACCCCCCGAACCATCCCCTAAGTGGTTCTAAGCACCTCTGTTCTATATGTGGAGACAGAGCGTCGGGAAAACATTATGGAGTATACAGTTGTGAAGGTTGCAAAGGTTTTTTCAAACGGACGGTAAGA | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | InsectaCentral | Ultraspiracle dsRNA feeding target | Ultraspiracle dsRNA feeding RNAi construct | GCTACTGCTAATGCTGTAATTGATCAAATTATCGTTTTATTACGAGTGTGTTTCATAACATCTTCAAAGACATTTGTGTACAGCGAAGGGGTGTATTGTTTCATCTTCATATAGGTATATTTAGTTCAACGACCTTGTTCCTGACAGGTTCATCGTTAGCCACTGAACTATTCTATGAAAGTGGTATATCGTAGACGATAGATAAATTAAATCCAATGATGGAGCCCTCGAGAGATTCAGGGCTAAACTTGGAGGGAGGTTTTATGTCGCCGATGTCACCACCGGAGATGAAGCCAGACACGGCGATGCTAGACGGCCTGCGAGACGACTCCACCCCACCCCCAGCTTTCAAGAACTACCCCCCGAACCATCCCCTAAGTGGTTCTAAGCACCTCTGTTCTATATGTGGAGACAGAGCGTCGGGAAAACATTATGGAGTATACAGTTGTGAAGGTTGCAAAGGTTTTTTCAAACGGACGGTAAGA | dsRNA | Ambion silencer siRNA cocktail kit | ethanol precipitation | simultaneously with transcription | Primers were designed to amplify a ~500bp region of each of the candidate genes for silencing. Gene fragments were PCR amplified and cloned into pGEM-T-Easy (Promega) following the manufacturerâs instructions. Once ligated into pGEM-T-Easy, the vector was transformed by electroporation into DH10β cells and plated on LB ampicillin selective plates. PCR colony screening confirmed the correct size inserts in the multiple cloning site of pGEM-T-Easy (Promega). Cells were selectively liquid cultured and plasmids isolated using a commercial mini-prep kit (Invitrogen). pGEM-T-Easy containing gene fragments and the pL4440 vector plasmid DNA were digested separately with PstI and SacII restriction enzymes, resolved on a 1% agarose gel after which appropriate restriction fragments were excised and gel purified with an Eppendorf gel purification kit. A ligation reaction was performed between each of the gene fragments and the digested pL4440 vector. Recombinant plasmids were colony screened and isolated as described above. Inserts were sequenced to confirm the correct fragment had been inserted. An Ambion silencer siRNA cocktail kit was used for the production of dsRNA and siRNA. Using the two inverted T7 promoters flanking the multiple cloning site in the pL4440 vector, T7 RNA polymerase was used to produce single stranded sense and antisense RNAs that were subsequently hybridised to produce dsRNA. | InsectaCentral | Ultraspiracle dsRNA feeding RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | 0.30000000000000004 | feeding | 0.1 | buffer non_specific_dsRNA | 21 | Four negative controls were also set up where neonates were fed a mixture of either dsRNA for non-endogenous EGFP, milliQ water, nuclease free water (Ambion) or elution solution (Ambion siRNA cocktail kit) mixed with green food dye. | larva | qPCR phenotype | whole organism | not checked | 48 | none | ||||||||
| 71 | Chitin Synthase dsRNA feeding | RNAi | InsectaCentral | Unpublished | Collinge | Derek Phillip | Chitin Synthase dsRNA feeding target | TATTACAACGCGGCTACCGAGTCGAGTACTCGGCCGCCTCCGATGCGTACACGCACTGTCCGGAACAATTCGACGAGTTCTTCAACCAGCGACGACGATGGGTACCCTCTACTATGGCCAACATATTTGATCTGCTGGCAGATGCTAAAAGGACTATCTCTATTAATGATAATATTTCCACGCTTTATATAATGTATCAGTCCATGTTAATGTTCGGCACAATCCTGGGCCCCGGGACCATATTCCTGATGATGGTGGGAGCGATGAACGCCATCACTCAGATGAGCATGTCCAACGCGCTGATACTCAACTTGGTGCCCATTCTCATATTCATTGTAGTCTGTATGACTTGTAAGTCTGAAACGCAGCTATTCCTGGCGAGCTTGATAACCTGCG | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | InsectaCentral | Chitin Synthase dsRNA feeding target | Chitin Synthase dsRNA feeding RNAi construct | TATTACAACGCGGCTACCGAGTCGAGTACTCGGCCGCCTCCGATGCGTACACGCACTGTCCGGAACAATTCGACGAGTTCTTCAACCAGCGACGACGATGGGTACCCTCTACTATGGCCAACATATTTGATCTGCTGGCAGATGCTAAAAGGACTATCTCTATTAATGATAATATTTCCACGCTTTATATAATGTATCAGTCCATGTTAATGTTCGGCACAATCCTGGGCCCCGGGACCATATTCCTGATGATGGTGGGAGCGATGAACGCCATCACTCAGATGAGCATGTCCAACGCGCTGATACTCAACTTGGTGCCCATTCTCATATTCATTGTAGTCTGTATGACTTGTAAGTCTGAAACGCAGCTATTCCTGGCGAGCTTGATAACCTGCG | dsRNA | Ambion silencer siRNA cocktail kit | ethanol precipitation | simultaneously with transcription | Primers were designed to amplify a ~500bp region of each of the candidate genes for silencing. Gene fragments were PCR amplified and cloned into pGEM-T-Easy (Promega) following the manufacturerâs instructions. Once ligated into pGEM-T-Easy, the vector was transformed by electroporation into DH10β cells and plated on LB ampicillin selective plates. PCR colony screening confirmed the correct size inserts in the multiple cloning site of pGEM-T-Easy (Promega). Cells were selectively liquid cultured and plasmids isolated using a commercial mini-prep kit (Invitrogen). pGEM-T-Easy containing gene fragments and the pL4440 vector plasmid DNA were digested separately with PstI and SacII restriction enzymes, resolved on a 1% agarose gel after which appropriate restriction fragments were excised and gel purified with an Eppendorf gel purification kit. A ligation reaction was performed between each of the gene fragments and the digested pL4440 vector. Recombinant plasmids were colony screened and isolated as described above. Inserts were sequenced to confirm the correct fragment had been inserted. An Ambion silencer siRNA cocktail kit was used for the production of dsRNA and siRNA. Using the two inverted T7 promoters flanking the multiple cloning site in the pL4440 vector, T7 RNA polymerase was used to produce single stranded sense and antisense RNAs that were subsequently hybridised to produce dsRNA. | InsectaCentral | Chitin Synthase dsRNA feeding RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | 0.30000000000000004 | feeding | 0.1 | buffer non_specific_dsRNA | 21 | Four negative controls were also set up where neonates were fed a mixture of either dsRNA for non-endogenous EGFP, milliQ water, nuclease free water (Ambion) or elution solution (Ambion siRNA cocktail kit) mixed with green food dye. | larva | qPCR phenotype | whole organism | not checked | 48 | none | ||||||||
| 72 | Chitin Synthase 1156R dsRNA feeding | RNAi | InsectaCentral | Unpublished | Collinge | Derek Phillip | Chitin Synthase 1156R dsRNA feeding target | GAACTTGGGAGCAGCGTGTGGACGTATCCATCCTGTGGGCTCTGGCTTCATGGCTTGGTATCAAATGTTCGAGTACGCTATTGGTCATTGGCTGCAAAAGGCGACGGAACACATGATCGGCTGCGTACTCTGTAGTCCTGGATGCTTCTCTCTGTTCAGAGGAAAGGGCTTTGATGGACGACAACGTCATGAGAAATACACACTGACTTCTCACGAAGCCCGCCATTACGTACAATACGATCAAGGTGAGGACCGTTGGCTCTGCACATTACTATTACAACGCGGCTACCGAGTCGAGTACTCGGCCGCCTCCGATGCGTACACGCACTGTCCGGAACAATTCGACGAGTTCTTCAACCAGCGACGACGATGGGTACCCTCTACTATGGCCAACATATTTGATCTGCTGGCAGATGCTAAAAGGACTATCTCTATTAATGATAATATTTCCACGCTTTATATAATGTATCAGTCC | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | InsectaCentral | Chitin Synthase 1156R dsRNA feeding target | Chitin Synthase 1156R dsRNA feeding RNAi construct | GAACTTGGGAGCAGCGTGTGGACGTATCCATCCTGTGGGCTCTGGCTTCATGGCTTGGTATCAAATGTTCGAGTACGCTATTGGTCATTGGCTGCAAAAGGCGACGGAACACATGATCGGCTGCGTACTCTGTAGTCCTGGATGCTTCTCTCTGTTCAGAGGAAAGGGCTTTGATGGACGACAACGTCATGAGAAATACACACTGACTTCTCACGAAGCCCGCCATTACGTACAATACGATCAAGGTGAGGACCGTTGGCTCTGCACATTACTATTACAACGCGGCTACCGAGTCGAGTACTCGGCCGCCTCCGATGCGTACACGCACTGTCCGGAACAATTCGACGAGTTCTTCAACCAGCGACGACGATGGGTACCCTCTACTATGGCCAACATATTTGATCTGCTGGCAGATGCTAAAAGGACTATCTCTATTAATGATAATATTTCCACGCTTTATATAATGTATCAGTCC | dsRNA | Ambion silencer siRNA cocktail kit | ethanol precipitation | simultaneously with transcription | Primers were designed to amplify a ~500bp region of each of the candidate genes for silencing. Gene fragments were PCR amplified and cloned into pGEM-T-Easy (Promega) following the manufacturerâs instructions. Once ligated into pGEM-T-Easy, the vector was transformed by electroporation into DH10β cells and plated on LB ampicillin selective plates. PCR colony screening confirmed the correct size inserts in the multiple cloning site of pGEM-T-Easy (Promega). Cells were selectively liquid cultured and plasmids isolated using a commercial mini-prep kit (Invitrogen). pGEM-T-Easy containing gene fragments and the pL4440 vector plasmid DNA were digested separately with PstI and SacII restriction enzymes, resolved on a 1% agarose gel after which appropriate restriction fragments were excised and gel purified with an Eppendorf gel purification kit. A ligation reaction was performed between each of the gene fragments and the digested pL4440 vector. Recombinant plasmids were colony screened and isolated as described above. Inserts were sequenced to confirm the correct fragment had been inserted. An Ambion silencer siRNA cocktail kit was used for the production of dsRNA and siRNA. Using the two inverted T7 promoters flanking the multiple cloning site in the pL4440 vector, T7 RNA polymerase was used to produce single stranded sense and antisense RNAs that were subsequently hybridised to produce dsRNA. | InsectaCentral | Chitin Synthase 1156R dsRNA feeding RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | 0.30000000000000004 | feeding | 0.1 | buffer non_specific_dsRNA | 21 | Four negative controls were also set up where neonates were fed a mixture of either dsRNA for non-endogenous EGFP, milliQ water, nuclease free water (Ambion) or elution solution (Ambion siRNA cocktail kit) mixed with green food dye. | larva | qPCR phenotype | whole organism | not checked | 48 | none | ||||||||
| 73 | Chitin Synthase 1156R3 dsRNA feeding | RNAi | InsectaCentral | Unpublished | Collinge | Derek Phillip | Chitin Synthase 1156R3 dsRNA feeding target | TATTACAACGCGGCTACCGAGTCGAGTACTCGGCCGCCTCCGATGCGTACACGCACTGTCCGGAACAATTCGACGAGTTCTTCAACCAGCGACGACGATGGGTACCCTCTACTATGGCCAACATATTTGATCTGCTGGCAGATGCTAAAAGGACTATCTCTATTAATGATAATATTTCCACGCTTTATATAATGTATCAGTCCATGTTAATGTTCGGCACAATCCTGGGCCCCGGGACCATATTCCTGATGATGGTGGGAGCGATGAACGCCATCACTCAGATGAGCATGTCCAACGCGCTGATACTCAACTTGGTGCCCATTCTCATATTCATTGTAGTCTGTATGACTTGTAAGTCTGAAACGCAGCTATTCCTGGCGAGCTTGATAACCTGCGCATACGCAATGGTAATGATGTTAGTCATAGTTGGGATAGTTCTTCAAATCGTAGAAGACGGATGGCTGGCCCCGTCCAGTTTGTTCACGGCCGTCATATTCGGGACTTTCTTCGTGACGGCGGCACTTCATCCTCAGGAGATCATATGTTTGCTGTACCTAACTGTGTACTATGTGACCATTCCGAGTATGTACATGTTGCTCATTATATACTCGCTATGCAATCTCAACAACGTGTCATGGGGTACTAGGGAGGTGGTGCAGAAGAAAACGGCTAAGGAAATGGAACAAGAACGCAAAGAAGCAGAGGAAGCTAAGAAGAAGATGGACGAGAAGAGCATACAGAAGTGGTTCGGCAAGAGTGATGAGAC | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | InsectaCentral | Chitin Synthase 1156R3 dsRNA feeding target | Chitin Synthase 1156R3 dsRNA feeding RNAi construct | TATTACAACGCGGCTACCGAGTCGAGTACTCGGCCGCCTCCGATGCGTACACGCACTGTCCGGAACAATTCGACGAGTTCTTCAACCAGCGACGACGATGGGTACCCTCTACTATGGCCAACATATTTGATCTGCTGGCAGATGCTAAAAGGACTATCTCTATTAATGATAATATTTCCACGCTTTATATAATGTATCAGTCCATGTTAATGTTCGGCACAATCCTGGGCCCCGGGACCATATTCCTGATGATGGTGGGAGCGATGAACGCCATCACTCAGATGAGCATGTCCAACGCGCTGATACTCAACTTGGTGCCCATTCTCATATTCATTGTAGTCTGTATGACTTGTAAGTCTGAAACGCAGCTATTCCTGGCGAGCTTGATAACCTGCGCATACGCAATGGTAATGATGTTAGTCATAGTTGGGATAGTTCTTCAAATCGTAGAAGACGGATGGCTGGCCCCGTCCAGTTTGTTCACGGCCGTCATATTCGGGACTTTCTTCGTGACGGCGGCACTTCATCCTCAGGAGATCATATGTTTGCTGTACCTAACTGTGTACTATGTGACCATTCCGAGTATGTACATGTTGCTCATTATATACTCGCTATGCAATCTCAACAACGTGTCATGGGGTACTAGGGAGGTGGTGCAGAAGAAAACGGCTAAGGAAATGGAACAAGAACGCAAAGAAGCAGAGGAAGCTAAGAAGAAGATGGACGAGAAGAGCATACAGAAGTGGTTCGGCAAGAGTGATGAGAC | dsRNA | Ambion silencer siRNA cocktail kit | ethanol precipitation | simultaneously with transcription | Primers were designed to amplify a ~500bp region of each of the candidate genes for silencing. Gene fragments were PCR amplified and cloned into pGEM-T-Easy (Promega) following the manufacturerâs instructions. Once ligated into pGEM-T-Easy, the vector was transformed by electroporation into DH10β cells and plated on LB ampicillin selective plates. PCR colony screening confirmed the correct size inserts in the multiple cloning site of pGEM-T-Easy (Promega). Cells were selectively liquid cultured and plasmids isolated using a commercial mini-prep kit (Invitrogen). pGEM-T-Easy containing gene fragments and the pL4440 vector plasmid DNA were digested separately with PstI and SacII restriction enzymes, resolved on a 1% agarose gel after which appropriate restriction fragments were excised and gel purified with an Eppendorf gel purification kit. A ligation reaction was performed between each of the gene fragments and the digested pL4440 vector. Recombinant plasmids were colony screened and isolated as described above. Inserts were sequenced to confirm the correct fragment had been inserted. An Ambion silencer siRNA cocktail kit was used for the production of dsRNA and siRNA. Using the two inverted T7 promoters flanking the multiple cloning site in the pL4440 vector, T7 RNA polymerase was used to produce single stranded sense and antisense RNAs that were subsequently hybridised to produce dsRNA. | InsectaCentral | Chitin Synthase 1156R3 dsRNA feeding RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | 0.30000000000000004 | feeding | 0.1 | buffer non_specific_dsRNA | 21 | Four negative controls were also set up where neonates were fed a mixture of either dsRNA for non-endogenous EGFP, milliQ water, nuclease free water (Ambion) or elution solution (Ambion siRNA cocktail kit) mixed with green food dye. | larva | qPCR phenotype | whole organism | not checked | 48 | none | ||||||||
| 75 | RP S4 silencing in S frugiperda | RNAi | Ribosomal protein S4 was chosen for 3 dsRNA feeding experiments in Spodoptera frugiperda. The animals were kept separately in a 5cm x 3.5cm x 3.5cm compartment. The dsRNA was provided on a small piece of maize leave. The first experiment was started with L4. They were fed twice with 3µg of dried dsRNA, respectively. The time period between the two feedings was 2 days. The second experiment was started with L1. They were fed twice with 3µg of dried dsRNA, respectively. The time period between the two feedings was 7 days. The third experiment was started with L3. They were fed eight times with 3µg of dried dsRNA, respectively. The time period between the two feedings was 24 hours. None of the animals showed the expected lethal phenotype or any other obvious phenotype. Author contributing to these experiments are Daniela Nowara, Magnus Lundmark, Frank Hauser, Merete Albrechtsen, Cornelis J. Grimmelikhuijzen. | InsectaCentral | Unpublished | Nowara | Daniela | RP S4 silencing in S frugiperda target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | RP S4 silencing in S frugiperda target | RP S4 silencing in S frugiperda RNAi construct | CAAGGTGGACGGTAAAGTGAGGACAGACCCCACGTACCCTGCTGGCTTCATGGATGTTGTTTCTATTGAGAAGACAAATGAATTGTTCCGCCTCATCTATGATGTAAAAGGTCGTTTTACAGTACACCGTATCACGCCTGAAGAAGCTAAGTACAAGCTCTGCAAGGTGAAGCGTGTGTCCACTGGCCCTAAGAATGTGCCATTCCTGGTGACACATGACGGACGTACCATCCGCTACCCCGACCCACTCATTAAAGTCAATGACTCTGTGCAACTTGACATTGCCACCTCCAAGATCATGGACTTCATCAAGTTTGAGTCAGGCAATCTGTGCATGATCACTGGCGGTAGGAACTTGGGAAGAGTGGGCACCATTGTGTCCCGTGAGAGACATCCCGGATCATTTGACATTGTACATATTAAGGATTCCACAGGACACACTTTTGCCACCAGATTAAACAACGTCTTCATCATTGGCAAGGGCACGAAGGCTTACATTTCTCTCCCGCGCGGCAAGGGAGTGCGTCTCACCATCGCAGAGGAGCGAGACAAGCG | dsRNA | Ambion Megascript T7 Kit | other | subsequent to transcription | A PCR product which was generated using T7-adapter primer was used as template for the in vitro transcription. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to the kits protocol. The mixture was incubated at 37 degree Celsius over night. Accordingly, the whole reaction was heated at 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. The dsRNA was used for the feeding experiments without any further purification. | InsectaCentral | RP S4 silencing in S frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | bayer | larva | lab_colony | feeding | megascript kit buffer | maize leaf | non_specific_dsRNA | 3 | larva | phenotype | whole organism | not checked | none | ||||||||||
| 76 | CHS B silencing in S frugiperda | RNAi | Chitin synthase B was chosen for the dsRNA feeding experiment in Spodoptera frugiperda. The animals were kept separately in a 5cm x 3.5cm x 3.5cm compartment. The dsRNA was provided on a small piece of maize leave. The experiment was started with L4. They were fed twice with 3µg of dried dsRNA, respectively. The time period between the two feedings was 2 days. 15 animals were fed. None of the animals showed the expected phenotype or any other obvious phenotype. Author contributing to these experiments are Daniela Nowara, Magnus Lundmark, Frank Hauser, Merete Albrechtsen, Cornelis J. Grimmelikhuijzen. | InsectaCentral | Unpublished | Nowara | Daniela | CHS B silencing in S frugiperda target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | CHS B silencing in S frugiperda target | CHS B silencing in S frugiperda RNAi construct | GCGGGCGCATCCATCCTGTGGGCTCAGGCTTCATGGCATGGTATCAAATGTTCGAGTACGCTATTGGTCATTGGCTGCAAAAGGCGACTGAACACATGATTGGCTGTGTACTCTGTAGCCCTGGATGCTTCTCCCTCTTCAGAGGAAAGGCTTTGATGGACGACAACGTTATGAAGAAATATACCTTAACTTCCCACGAGGCACGACACTATGTGCAATACGATCAAGGCGAGGACCGTTGGTGCACGCTACTGCTGCAGCGCGGGTACCGCGTGGAGTACAGCGCGGTGTCGGACGCGTACACGCACTGCCCCGAGCACTTCGACGAGTTCTTCAACCAGCGCCGCCGCTGGGTGCCCTCCACGCTCGCCAACATCTTCGACCTGCTCGGCAGCGCCAAGCTCACCGTCAAGTCCAACGACAACATCTCCACCCTCTATATAGTCTATCAGTTCATGTTGATAGTGGGTACGGTGTTGGGTCCCGGCACGATCTTCCTGATGATGGGGGGAGCCATGAACGCCATCATTCAGATCAGCAACGCGTACGCGATGATGTTGAACCTCGTACCACTCGTCATCTTCCTTATAGTCTGTATGACTTGTCAGTCAAAGACGCAGCTCTTCCTCGCTAACCTCATAACATGCGCATACGCAATGGTGATGATGATCGTGATAGTGGGGATAGTTCTGCAGATAGTGGAGGATGGATGGCTGGCTCCGTCCAGTATGTTCACAGCTTTAATATTCGGTACATTCTTCGTCACCGCGGCACTACA | dsRNA | Ambion Megascript T7 Kit | other | subsequent to transcription | A PCR product which was generated using T7-adapter primer was used as template for the in vitro transcription. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to the kits protocol. The mixture was incubated at 37 degree Celsius over night. Accordingly, the whole reaction was heated at 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. The dsRNA was used for the feeding experiments without any further purification. | InsectaCentral | CHS B silencing in S frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | bayer | larva | lab_colony | feeding | megascript buffer diluted with water | maize leaf | non_specific_dsRNA | larva | phenotype | whole organism | not checked | none | |||||||||||
| 77 | RP S9 silencing in S frugiperda | RNAi | Ribosomal protein S9 was chosen for the dsRNA feeding experiment in Spodoptera frugiperda. The animals were kept separately in a 5cm x 3.5cm x 3.5cm compartment. The dsRNA was provided on a small piece of maize leave. The experiment was started with L1. They were fed twice with 3µg of dried dsRNA, respectively. The time period between the two feedings was 7 days. 19 animals were fed. None of the animals showed the expected lethal phenotype or any other obvious phenotype. dsRNA against GFP and water were used as constrols. Author contributing to these experiments are Daniela Nowara, Magnus Lundmark, Frank Hauser, Merete Albrechtsen, Cornelis J. Grimmelikhuijzen. | InsectaCentral | Unpublished | Nowara | Daniela | RP S9 silencing in S frugiperda target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | RP S9 silencing in S frugiperda target | RP S9 silencing in S frugiperda RNAi construct | GTCCAGGCGAACAATGAATGAAGGGATGTTCACCACTTGTTTGCGTACACGGATGTGCCTCTGTCTGATGAGGATACGTGCGTGATGGATGGACTTGGCGAGACCAGCCTTGAACACCTGGGTCTGCAGACGGCGCTCCAAGAAGTCCTCGATCTTCAGACCGAGCACGTAATCGAGCTTCATCTGTTTCTCATCCAACACGCCGATCCTGACAAGACGACGCAAAAGCGCGTTACCTTCGAACAACCTCTTGGGGTCCTTCTCTTCGAGGGTGAGCAGCTCACGGGCAGCCTTACGGATGCGGGCAAGCGTGTACTTGACGCGCCATACCTCACGCTTGTTGCGCAGACCATACTCTCCGATGATCTTCAACTCCTGGTCGAGACGCGCCTTCTCGAAGGGACGACGAGGCGTCACGTAGGTTTTGGAGAATACCGACGGTACCCAAGGGCGAATTCTGCAGATATCCATCACACT | dsRNA | Ambion Megascript T7 Kit | other | subsequent to transcription | A PCR product which was generated using T7-adapter primer was used as template for the in vitro transcription. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to the kits protocol. The mixture was incubated at 37 degree Celsius over night. Accordingly, the whole reaction was heated at 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. The dsRNA was used for the feeding experiments without any further purification. | InsectaCentral | RP S9 silencing in S frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | bayer | larva | lab_colony | feeding | megascript buffer | maize leaf | non_specific_dsRNA | larva | phenotype | whole organism | not checked | none | |||||||||||
| 78 | ATPase A silencing in S frugiperda | RNAi | Vacuolar ATPase subunit A was chosen for the dsRNA feeding experiment in Spodoptera frugiperda. The animals were kept separately in a 5cm x 3.5cm x 3.5cm compartment. The dsRNA was provided on a small piece of maize leave. The experiment was started with L1. They were fed twice with 3µg of dried dsRNA, respectively. The time period between the two feedings was 7 days. 15 animals were fed. None of the animals showed the expected lethal phenotype or any other obvious phenotype. dsRNA against GFP and water were used as constrols. Author contributing to these experiments are Daniela Nowara, Magnus Lundmark, Frank Hauser, Merete Albrechtsen, Cornelis J. Grimmelikhuijzen. | InsectaCentral | Unpublished | Nowara | Daniela | ATPase A silencing in S frugiperda target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | ATPase A silencing in S frugiperda target | ATPase A silencing in S frugiperda RNAi construct | CATGGTGTACTTCTCCTTCTCGCCGTCGAACTCCGTCTCCAACACTACGTCAGTGACTTTGTAGTTGCCGGAGGGCGCGACGTAGGTGACGGTACCCTTGGCCTTGGGTGGGATCAACATCTTATGCTTAACCAATGTGCTCTCGTGTACGATACCGTACAAGTCTCCTCCGGTGATGTGGGAGCCGACCTTAACATTCAAGGGGTTGAATTCCCAGGTGACATCACGTCCAAGGCAGGGTACGTTGACACCCTTGGGGATGTAGATGGACTGTGTGAGCTCGTTGATGTCCTTCAGTGGCCGCTGGATACCGTCAAAGATGGAGCCGAGGATACCAGGACCCAGCTCTACGGACAGGGGCTTGCCGGTACGCAGCACGGGGTCACCTACAGTTACACCTGATGTTTCTTCGTATACCTGGATGGTGGCCATGTCACCCTCAAGACGGATGATCTCTCCGACCAGCTCGTGGTAGCCGACACGCACCAACTCGTACATAGCCGATCCGGACATTTTCTCCGCCGTTACGACGGGTCCGGATACGGCGAAGACGAATCCGAACTTTTCCTCCCTGTCCTCATCA | dsRNA | Ambion Megascript T7 Kit | other | subsequent to transcription | A PCR product which was generated using T7-adapter primer was used as template for the in vitro transcription. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to the kits protocol. The mixture was incubated at 37 degree Celsius over night. Accordingly, the whole reaction was heated at 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. The dsRNA was used for the feeding experiments without any further purification. | InsectaCentral | ATPase A silencing in S frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | bayer | larva | lab_colony | feeding | Megascript buffer | maize leaf | non_specific_dsRNA | larva | phenotype | whole organism | not checked | none | |||||||||||
| 79 | ATPase D silencing in S frugiperda | RNAi | 40-kDa V-ATPase subunit D was chosen for the dsRNA feeding experiment in Spodoptera frugiperda. The animals were kept separately in a 5cm x 3.5cm x 3.5cm compartment. The dsRNA was provided on a small piece of maize leave. The experiment was started with L1. They were fed twice with 3µg of dried dsRNA, respectively. The time period between the two feedings was 7 days. 5 animals were fed. None of the animals showed the expected lethal phenotype or any other obvious phenotype. dsRNA against GFP and water were used as constrols. Author contributing to these experiments are Daniela Nowara, Magnus Lundmark, Frank Hauser, Merete Albrechtsen, Cornelis J. Grimmelikhuijzen. | InsectaCentral | Unpublished | Nowara | Daniela | ATPase D silencing in S frugiperda target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | ATPase D silencing in S frugiperda target | ATPase D silencing in S frugiperda RNAi construct | CTGTGCCGTGGTTTCAAGTGTGGAATCCTGAAGCAGGCTGACTACCTTAACTTGGTGCAATGCGAGACCCTGGAAGATCTAAAGCTTCATCTGCAGGGCACCGACTACGGTACGTTCCTTGCTAATGAGCCCAGCCCACTTTCCGTCTCTACAATTGACGATAAGCTTCGCGAGAAGCTCGTAATTGAATTCCAGCATCTAAGGAATCATTCAGTGGAGCCTCTCTCTACCTTCTTGGACTTCATTACTTACAGTTACATGATTGACAACATCATCTTGTTGATCACTGGTACTCTACACCAGAGGCCCATTTCTGAGCTCATTCCCAAATGCCACCCCTTGGGCTCCTTCGAGCAAATGGAAGCCATCCATGTTGCTGCAACACCTGCTGAACTGTACAATGCAGTCCTTGTCGACACCCCATTGGCCCCCTTCTTTGTGGACTGCATCAGTGAGCAAGACTTGGATGAGATGAATATTGAGATCATTCGTAACACTCTGTACAAGGCTTACTTGGAAGCCTTCTATGATTTCTGCAAGCAGATTGGTGAAACTACTGCTGATGTTATGTGTGAAATACTGGCCTTTGAAGCTGATCGCCGTGCAATCATCATTACTATCAACTCTTTCGGGACAGAATTGTCCAAGGATGACCGTGCTAAGCTGTACCCACGCTGTGGCAAACTGAACCCTGATGGATTGGCTGCTCTTGCCAGAGCTGACGATTACGAGCAGGTTAAGGCTGTTGCTGAGTATTATGCTGAGTACCAAGCATTGTTTGAGGGCGCTGGCACTAAT | dsRNA | Ambion Megascript T7 Kit | other | subsequent to transcription | A PCR product which was generated using T7-adapter primer was used as template for the in vitro transcription. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to the kits protocol. The mixture was incubated at 37 degree Celsius over night. Accordingly, the whole reaction was heated at 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. The dsRNA was used for the feeding experiments without any further purification. | InsectaCentral | ATPase D silencing in S frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | bayer | larva | lab_colony | feeding | megascript kit buffer | maize leaf | non_specific_dsRNA | larva | phenotype | whole organism | not checked | none | |||||||||||
| 80 | ATP synthase B silencing in S frugiperda | RNAi | H+transporting ATP synthase beta subunit was chosen for 3 dsRNA feeding experiments in Spodoptera frugiperda. The animals were kept separately in a 5cm x 3.5cm x 3.5cm compartment. The dsRNA was provided on a small piece of maize leave. The first experiment was started with L4. They were fed twice with 3µg of dried dsRNA, respectively. The time period between the two feedings was 2 days. 17 animals were fed. The second experiment was started with L1. They were fed twice with 3µg of dried dsRNA, respectively. The time period between the two feedings was 7 days. 20 animals were fed. The third experiment was started with L2. They were fed six times with 3µg of dried dsRNA, respectively. The time period between the two feedings was 2 days. 16 animals were fed. A pool of these animals was analyzed by qPCR. None of the animals showed the expected lethal phenotype or any other obvious phenotype. Author contributing to these experiments are Daniela Nowara, Magnus Lundmark, Frank Hauser, Merete Albrechtsen, Cornelis J. Grimmelikhuijzen. | InsectaCentral | Unpublished | Nowara | Daniela | ATP synthase B silencing in S frugiperda target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | ATP synthase B silencing in S frugiperda target | ATP synthase B silencing in S frugiperda RNAi construct | TGGCCACAAGGACGGTAGTAAATAATAGTTCCGACAAGGCACCCCTAGTTGCAGCGCCCGTGGTGAACAAACGTGATTATGCGGCCAAAGCTGCAGCAGGCAAGGGACAAGGTAAGGTGGTAGCTGTTATTGGTGCCGTCGTGGACGTACAGTTCGAAGACAACCTACCGCCCATTCTAAATGCCCTCGAAGTACAAAACCGTCAACCCCGGCTTGTGCTGGAGGTGGCGCAGCACTTGGGAGAGAACACCGTCCGTACCATCGCTATGGACGGTACCGAGGGTCTGGTCCGTGGCCAGCCCGTCCTCGACTGCGGCTCGCCCATTCGTATCCCCGTGGGCGCTGAGACCCTCGGCCGCATCATCAACGTAATCGGAGAGCCCATCGACGAGCGTGGCCCCATCCCCACTGACAAGACTGCCGCTATCCACGCTGAGGCGCCCGAGTTCGTGGACATGTCCGTGCAGCAGGAGATTCTGGTCACTGGCATCAAGGTCGTCGACCTGCTCGCCCCCTACGCCAAGAGAGGTAAGATCGGTCTGTTCGGAGGTGCCGGAGTCGGTAAGACTGTACTTATCATGGAGCTGATCAACAACGTCGCCAAGGC | dsRNA | Ambion Megascript T7 Kit | other | subsequent to transcription | A PCR product which was generated using T7-adapter primer was used as template for the in vitro transcription. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to the kits protocol. The mixture was incubated at 37 degree Celsius over night. Accordingly, the whole reaction was heated at 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. The dsRNA was used for the feeding experiments without any further purification. | InsectaCentral | ATP synthase B silencing in S frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | bayer | larva | lab_colony | feeding | megascript kit buffer | maize leaf | non_specific_dsRNA | 3 | larva | qPCR phenotype | whole organism | not checked | none | ||||||||||
| 81 | APN1 dsRNA injection in O nubilalis | RNAi | InsectaCentral | Unpublished | Crava | Cristina M | APN1 dsRNA injection in O nubilalis target | GAAGATAATGGCACGCTCCTGGGTACTTTTGCTGGCGGGGGTTGCCCTCCTCCAGGGCGTATGCACCTTCAATCCGTTACCGCTTCTAGAGGAAGAAGAGGCATGGGAAAAATTCAGCAGGGAACTGAACGATGCCTCTTTCCGACTGCCGACGACGACGATGCCGATGCACTATGAACTGTCCCTGACACCGTACTTTGAAGACGAAGAAAGACCATTCACTTTCGATGGAACAGTCGCGATCTACACGAGCGCTACGGAAGAGAATGTCTCTGAAATTGTGATACACTGCAATGATCTCACTATTCACAGTCTGACTGTAGAGCACACAGACGCTGAGGGTGTGGTTCAGCAAATAGCTGCTCCTCAGACCTACGAGTGTGAAGCTCCTCAAAGCTTCTTGAGAATTGCGACCATAGAGCCATTGCAAGTTGGACAAGAGTACATCATCAGATCGAGCTTCACTGGCAATCTTCAATCCAATATGAATGGGTTCTACAGGAGTTGGTACAGAGACAGCACCACCACAAGATGGATGGCCACCACTCAGTTCCAACCTGGGTACGCGCGGCAAGCTTTCCCCTGCTATGACGAGCCCGGCTTCAAGGCCACATTCGACATAACCATGAACAGGGAGCCAGACTTCAGTCCCACCATCTCTAACATGCCTATAAAAACTACTGAAAACACAACTGATGGCCGTATATCCGAAACCTTCTACACGACTCCTATCACCTCCACATACCTGCTTGCCTTCATCGTCTCCCATTACGATAAGGTGGAAACTAACAACGATGAAGATAGACCTTTTGACATCTATGCCCGTGACAATGCCAATGGAACCGGTGCATGGTCCTTAGAAATAGGAATGAAGCTTCTAGAAGCCATGGAGAATTACACTGATTACCCTTACTACACGATGGCAGAAAAAATTAACATGAAACAAGCTGCTATTCCTGATTTCAATGCGGGTGCTATGGAAAACTGGGGTCTTTTGACTTACAGAGAAGCCTTAATCCTATACGACCCACTGAACTCCAACCACTTTTACAAGCAGCGTGAGGCCAATATCGTGTCCCACGAGATTGCCCACATGTGGTTTGGGAACCTCGTCACCTGCGCGTGGTGGGGGAACCTGTGGCTCAACGAAGGCTTCGCCCGTTTCTATCAATACTTCTTGACCGGTTCTGTTGCACCAGAGCTGGGATATGAGAGGAGATTCATGGTGGAGCAGTACATATCTGCCTTGTCAGTGGATTCAGTTGACTCTGCTCATGCGCTTACCAACCCAGATGTTTACAACCCGACAACTGTTTGGAACCATTTCTCTACCATCACCTACGCAAGAGGAGCGTGCATCTTGAGAATGACCCAGTACCTTCTTGGTCAAGAGACTTACGTTAAGGGACTTCGCAGCTATCTGAAGGAAAGGGCATTCGACGTAGCCGAGCCTCACCACCTCTTCAACGCTCTTGACGCTGCAGCCAGAGAAGACGGTGCTTTAGCCGCTTATGGTGGCATCACCATCGACCGCTACTTCAGGACTTGGTCTGAGAAGGCTGGACATCCAATGCTTTCTGTTACTATTGATCAACGCACTGGGACTATGCAAGTAACCCAGGCTCGTTGGGAAAGGACTACTGGTTCATCTGCCTTCCCTGGTATTTGGGATATTCCGATTACTTGGACCAGAGAGGGCGCTGCCGATTTTGATGATCTCAAACCGTCGCAGTTCTTGACAGCCCAAAGCACAGTAATTGAAAGAGGCACAGAAGGTCTCGAATGGGTGATTTTCAACAAACAAGCCTCTGGTTTCTACCGAGTGAACTACGATGACACCAACTGGGCTCTAATTGTGCAAGCTTTAAGGAACAATCCCAATGTAATACACGAATTCAACCGTGTTCAGATTATCGACGATCTATTCAACATGGGTAGAGCCGGAGTCAGGCCTTACCAGCAAGTGCTCAACCTTCTGTCGTTCCTCCAATTCGATGACGAGTACGGACCCTGGATCGCCGCTATCGATGGTTTCAATTTCGTGCTGAGAAGGCTTGCGCATGATGCACCTAACTTGCAGAAGCTGAGGGATATTATTAACAAAGAACTGAGTGTCGCTGTGACTGGTCGACTGGGCTACGTCGAGGTGGAGAATGAAACCTTCATGGATGGAATCTTGAGGATGTATCTCATGAACTTCCTTTGCGACAATGGACACGAGGAATGCATTGCAACTGGCATAGAGAAATTCGCAGAATGGAAGGCGGGAGGATTCATTCCAGCAAACATGCGACCATGGGTGTACTGCACTGGACTCCGTTTCGGTGACGCAGAAGATTTCGAATTCTTCTGGAACCGCTATCTGCAGGAAGACTTAGCCGGCGAACAAATTGTCATGCTTCAAAACGCTGGTTGCACCAGGGACGAGGCCAGCCTACGGAAATTCCTGGATGCAATCGTATCTCAACAGAACATTGTAAGAGACCAAGATTTCACGACCGCACTTAACTCCGCTGTATCCAAAAACGAATACAACACGATGAGAGCTTTCGCATGGCTCAAGGATAATGTGAACCAGACTACAGAAGCTTTAGGCGGCATTGCTACTCCATTGAGCTACATTGTTTCACGGCTGTTGAATGAACAGGACATGGCAGAGGTTCAAACTTGGCTCGACGAGAACCAAGACTCGATTGGAACAGCGTACAATACCGGTGTTAACGGGATCGCCTCATCCAGAAACAATCTGGCGTGGTCGGCCAGACGCATGCCTGAAGTCTACGAGTATTTCGACAACGGAGTATATGTGGAATACGAAATCGAAGACCCCACACAAGAAGAGGTGACCACAGAAGAATCTGTAACGGTCACTCCCCCAGAACTTGATATTGAAGTAACAACTGAAGTTGATGATGAATCAGATTCAGCTAACATCGCAGCGTTGAGCATTTTCACTCTTATTATAAGGGTCTCTATTAATCTAATTAGTTAAAGCAAAGTTAAATAATTTTTGGATATTGAAATCAAGTACAATAAAATGAAATGATTTTAAGAAAACAAATACAGGGACATTGTTGATATACTTAAACCGTTGATTGTTATTTATAATATTTTGTAATAAATGTGCCCCAGAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Crambidae | Ostrinia | nubilalis | 29057 | cds | GenBank | EU878375 | APN1 dsRNA injection in O nubilalis RNAi construct | TCAGCCTTCCCTGGTATTTGGGATATTCCGATTACTTGGACCAGAGAGGGCGCTGCCGATTTTGATGATCTCAAACCGTCGCAGTTCTTGACAGCCCAAAGCACAGTCATTGAAAGAGGCACAGAAGGTCTCGAATGGGTGATTTTCAACAAGCAAGCCTCTGGTTTCTACCGAGTGAACTACGATGACACCAACTGGGCTCTAATTGTGCAAGCTTTAAGGAACAATCCCAATGTAATACACGAATTCAACCGTGTTCAGATTATCGACGATCTATTCAACATGGGTAGAGCCGGAGTCAGGCCTTACCAGCAAGTGTTCAACCTTCTGTCGTTCCTGCAATTCGATGACGAGTACGGGCCCTGGATCGCCGCTATCGATGGTTTCAATTTCGTGCTGAGAAGGCTTGCGCATGATGAAGCTAACTTGCAGAAGCTGAGGGATA | dsRNA | Megascript T7 Kit, Ambion | column-based | subsequent to transcription | Manufacturer's protocol | InsectaCentral | APN1 dsRNA injection in O nubilalis RNAi construct | Lepidoptera | Crambidae | Ostrinia | nubilalis | 29057 | local resource | larva | unknown | unknown | lab_colony | 0.5 | injection | Water | 0.18 | non_specific_dsRNA | 21 | Non specific dsRNA was injected at the same amount and concentration of specific dsRNA. ds unspecific RNA was prepared at the same time and with the same kit used for specific dsRNA (Ambio Megascript T7). Specific and unspecific dsRNA injections were performed at the same time and with larvae from the same box. | larva | qPCR | Midgut | not checked | 96 | none | |||||
| 82 | HSG Epiphyas postvittana Actin RNAi | RNAi | Feeding 3rd instar Epiphyas postvittana larvae with actin dsRNA to study RNAi effects. | InsectaCentral | Unpublished | Gatehouse | Heather Sian | HSG Epiphyas postvittana Actin RNAi target | CAGAGCAAGAGAGGTATCCTCACTCTCAAATACCCCATCGAGCACGGCATCATCACCAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTACAACGAGCTGCGCGTGGCTCCCGAGGAGCATCCCATTCTCCTGACGGAGGCGCCGCTGAACCCCAAGGCCAATCGCGAGAAGATGACTCAGATCATGTTCGAGACATTCAACTGCCCCGCGATGTATGTCGCCATCCAGGCCGTGCTCTCCCTGTACGCCTCCGGTCGTACCACCGGCATCGTGCTGGACTCCGGCGATGGTGTCTCCCACACTGTCCCCATCTACGAAGGTTTCGCGCTACCCCACGCTATCCTGCGTCTGGACTTGGCCGGTCGCGACTTGACCGACTACCTCATGAAGATCCTGACCGAGCGAGGTTACTCCTTCACCACCACCGCTGAGCGGGAAATCGTCCGTGATATCAAGGAGAAGTTGTGCTACGTGGCGCTGGACTTCGAGCAGGAGATGCAGACGGCGGCCGCCTCCACCTCCCTGGAGAAGTCGTACGAGCTTCCTGACGGGCAGGTCATCACCATCGGCAACGAGAGGTTCCGTTGCCCCGAAGCCCTATTCCAGCCCTCTTTCGTGGGTATGGAATCCTGTGGCGTCCATGAGACCGTGTACAACTCCATCATGAAGTGCGACGTCGACATCCGCAAGGACCTGTACGCCAACACCGTACTGTCCGGTGGCACCACCATGTACCCCGGTATCGCCGACAGGACGCAGAAGGAGATCACCGCCCTGGCTCCCTCCACCATCAAGATCAAGATCATCGCTCCCCCGGAGAGGAAATACTCCGTATGGATCGGTGGA | Lepidoptera | Tortricidae | Epiphyas | postvittana | 65032 | cds | GenBank | DQ211891 | HSG Epiphyas postvittana Actin RNAi RNAi construct | TAATACGACTCACTATAGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATTCAGAGCAAGAGAGGTATCCTCACTCTCAAATACCCCATCGAGCACGGCATCATCACCAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTACAACGAGCTGCGCGTGGCTCCCGAGGAGCATCCCATTCTCCTGACGGAGGCGCCGCTGAACCCCAAGGCCAATCGCGAGAAGATGACTCAGATCATGTTCGAGACATTCAACTGCCCCGCGATGTATGTCGCCATCCAGGCCGTGCTCTCCCTGTACGCCTCCGGTCGTACCACCGGCATCGTGCTGGACTCCGGCGATGGTGTCTCCCACACTGTCCCCATCTACGAAGGTTTCGCGCTACCCCACGCTATCCTGCGTCTGGACTTGGCCGGTCGCGACTTGACCGACTACCTCATGAAGATCCTGACCGAGCGAGGTTACTCCTTCACCACCACCGCTGAGCGGGAAATCGTCCGTGATATCAAGGAGAAGTTGTGCTACGTGGCGCTGGACTTCGAGCAGGAGATGCAGACGGCGGCCGCCTCCACCTCCCTGGAGAAGTCGTACGAGCTTCCTGACGGGCAGGTCATCACCATCGGCAACGAGAGGTTCCGTTGCCCCGAAGCCCTATTCCAGCCCTCTTTCGTGGGTATGGAATCCTGTGGCGTCCATGAGACCGTGTACAACTCCATCATGAAGTGCGACGTCGACCATATGGGAGAGCTCGTATAGTGAGTCGTATTA | dsRNA | phenol_chloroform | simultaneously with transcription | DsRNA was produced as detailed in Gatehouse et al. (2004, Amylase activity in honey bee hypopharyngeal glands reduced by RNA interference. Journal of Apicultural Research 43(1):9-13). Briefly, genes were cloned into a modified pGEM vector between two opposing T7 promotors and this âdouble-T7â plasmid transformed into pLysS BL21 cells, which produce T7 polymerase when induced with IPTG. This means that any dsRNA produced will include vector fragments between the T7 promotor and the gene fragment, and also vector fragments beyond the gene and possibly beyond the second T7 promotor. The E. postvittana actin fragment clone was obtained from Vernon Ward (University of Otago, New Zealand) in pGEM-T Easy. The fragment was excised with Nco I and Pst I and ligated into the Nco I to Pst I region of the double T7 plasmid. Total nucleic acid was purified from induced cells by a no RNaseA variation of a standard miniprep. Thus dsRNA preparations also contained E. coli RNA, mRNA and plasmid DNA. Control RNA preparations were produced by the same method from uninduced cells and thus contained E. coli RNA, mRNA and plasmid DNA, but no dsRNA. Concentration of dsRNA was estimated as approx 10^11 molecules of target dsRNA in 1 ul of 10mg/ml RNA preparations (Gatehouse et al., 2004). | InsectaCentral | HSG Epiphyas postvittana Actin RNAi RNAi construct | Lepidoptera | Tortricidae | Epiphyas | postvittana | 65032 | local resource | larva | lab_colony | 0.08600000000000001 | feeding | 10% sucrose solution | 0.25 | buffer non_specific_dsRNA | 2 | 3rd instar E. postvittana larvae were held without food for 24 hours before individual larvae were fed a 0.25 uL droplet of one of three treatments: 10% sucrose solution (control 1), 10mg/ml non-induced sample in 10% sucrose (control 2), and 10mg/ml induced sample in 10% sucrose (target dsRNA). The sucrose solution was diethyl procarbonate (DEPC) treated before use. Induced and uninduced RNA was prepared as above with the final resuspension in a 10% DEPC-treated sucrose solution, the induced preparation estimated at 10^11 molecules dsRNA per uL, ie. 2.5 x 10^10 molecules dsRNA per larva (approximately 0.086ug/ul), and the uninduced preparation at a corresponding concentration. The larvae were placed, one at a time, in a small Petri dish which facilitated beading when the virus droplet came into contact with the surface. The 0.25uL droplet was partially ejected from the tip of pipette until it beaded, and was then offered to the larva. As the larva started to consume the droplet, it was fully ejected from the pipette tip onto the surface of the Petri dish. Once the droplet had been fully consumed (generally within 5 seconds), the larva was placed into an AA cup containing approximately 0.5mL of leafroller assay diet until dissection. Care was taken not to cause the larvae to regurgitate its stomach contents. Any larvae that did not consume the whole droplet were discarded. 30 larvae per treatment were tested and at least two biological replicates used. | larva | W.blot phenotype | midgut tissue assayed for changes in actin levels at 24, 48 and 168 hours post-delivery, whole organism for phenotypic changes | not checked | 48 | none | |||||||
| 83 | Papilio xuthus ebony RNAi | RNAi | Collaborator: Haruhiko Fujiwara | InsectaCentral | Unpublished | Futahashi | Ryo | Papilio xuthus ebony RNAi target | CTTTGCCTTTGTGCATACTGCATCGGCTCAAGAGAACGCGCGGGTTTTTTTTTCTGTGACAGTGATAGCTAAATATTGTGCCAGTGACATTTTTTTTAAAAGTGAATTATTTTAAACTGCACAATGGGATCACTGCCGCGCGTTTCTGTGGTCACCGGGCAAAGAGTGTCAGCACCGGCTGGACCTCTGACCAATATGATGGACAATCTAACAGCTAGAGACGCCACTGCTCTCATTTATAAAGATGAGACAAGTTGCGTTCATGTGAGCTACGCGGAACTGCAGGCCCGGACTAAyGCyCTGGGCCGGGCCATCGCCGCCCATGCGCkCCCTGTCGGACCTAACAGGGATGGTGATTTCGTTATAGCCGTATGCATGGAGCCCACGCATAATACAATAATGACTCTGTTAGCGACATGGAAGGCGGGTGCAGCGTATGTTCCGATGGACCCGAGCTTCCCGCAGGGCGGGATCTCACACATACTAAAGGATGCGGAGCCATCCCTTGTAATATACGATAACACAGCAAATCCCTCTATGTTTTCCGGGAGTGGAATACCGGCGGTATCATTTGACGAACTATCTTTAGAAGCCAGTGGCTTACCGGGAGATAAGCCGAGCGATTCAGAAATGCTATTACAGACGACTGCCGAGAGTATTGCTATAGTTCTTTATACTTCTGGAAGCACGGGGATACCGAAAGGTGTGCGTCTGCCATATTCTGCTATCTGCAACCGTCTTTGGTGGCAATTCCGTACCTTCCCGTACACCGAACACGAAAGGTTCTGCGTTTGGAAGACCGCTTTAACTTTCGTAGACTCTGTATGCGAGATCTGGGGACCTTTGCTGCACGGCAGGACCCTAATTATCCTATCTAGAGAGACGACCCGAGATCCTCAGAAATTGGTTCGGGTCCTAGCGGAAAATGAGGTCCATCGGTTGGTATTGGTCCCGACGCTACTTCGGTCTATTCTAATGTACCTCTCTCTAAACCGATCCGAAAGACCACTACAATCCCTTAAACTATGGGTTTGTTCTGGTGAAACTCTCAGCAAGGAACTTGCGGGACAATTCTTyCAGTACTTCGGTGATAATGAAGGCTACAAATTGGCTAACTTCTACGGAAGTACAGAAGTCATGGGAGATGTAACATTTTATGTTCTGGAACGTTCTCAGCAGTTGGACATACATCCTACGATACCGATAGGCAAGCCAGTCGACAACTGCGCCATCTATCTACTAGACGAAGAGATGAACCCTGCTCGyGAAGCAGAGCCTGGCGAAGTkTGGGTTGCGGGCAGTAATCTAGCTGCAGGATACGTCGGCGGACAAGCGCCTGAGAAGTTTTGCTACAATCCTCACGCCTCGCATCCAGATTTTGGCAAGCTATACCGCACTGGAGACTTCGGTGTACTGCACAAAGGAATGATCCTGTACGCGGGTAGAACTGACTCACAAGTCAAAATCCGAGGTCACAGAGTAGATCTATTGGAAGTGGAAACCGCGGTGTCAGCCGTGGAAGGAATCGAGAAAGCGGTGGTCCTCTGCTATGGACTGGAGCGTGGCAACCCTGAAGTGCTCGCCTTTGTGACCATATCACCTGACGCGAGGCTCTCAGCACACCATATTGAGGCAGCCCTGAAGAACTCTCTCACACATTATATGCTACCACAGGTGATTGTGATAGAGGCTGTTCCATTGTTGGTGAACGGCAAAGTAGACAGACAGGCTCTACTCAAGATGTACGAGAACACTAACAACAATGATGACTCAGAGATATCTTTCGACATTGATTACACTGGAGTGGAAGCAGAGAAGTTAGAAGCAGCTAAGGTCCTCTTCGAGACGGTAGGAGAGGTCCTCGGGAGGGCAGCAAGAGGGACACTCTCGGTTAGAGCGGGATTTTACGAGCTGGGAGGGAATTCCCTTAATTCTATATACACCATCACTAGACTGAGAGACAGAGGATATTAyATTGATATTAGTGAGTTTCTGGGAGCTGCCAACCTGGGCGATGTrTTrGCCCACATGAGCTCCGGTCCAGACCACGAGACCGGCGGCTACGACCACAAGTTTACAGCAGTGGCCATGAATGAAGATCATGAGCAGCAAGTCATCGACATGATCGTATCATCGTTCTACGAGAAGGCGGAGTTGGAACAGTTTTTGAAACATGAAATCGAAACrGAGGATTAyGCGCACTGCATCmGAGCTTGyTGGGCCGCGCTGTTGCAGGCTAGGCTCAGCGTTGTGCTAGAAGATCATTCTGGAACACCTGTAGCGGTTGCAyTGAATTTTGACGCACGGGACGAGCCTGAGATTGAATTAAAyGGCGGTCTTGCCAAAATCATGACATTCCTTGAATTCGTTGAGAGCTCAGTTAGAGATACCATGCTGCCTGAAGGAAAGGGAACGATTCTGCACTCATTCATGATGGCAACAGCATCCTCATTGAGCCCACAAGACAACGTGGCGGCAATACGTGCCCTGGAACATGCCACTATGAGGATTGCACGCGATAGACGATTCCATGGAGTGTTCACTACAAACACAAGCCCGTTGACTCAGCAACTGGGTACTGACGTGCTAGGCTTCCAAACTTTACTGGATTACCAAATAAACGAATACATCGACCCTAACGGAGACAGAATTTTCGGAAAAGCGCCTGATGACATGAGAGCTATCGTCTGCTGGAAACCTATTGAATAGTTTTGCTATTTAAAAAATATAGGTATCTTTACTCTATGGTATTAGTAATGTTAAGGCATAGACTAATGCACTTCCTAATAGAATAAAAAGACATCCAAAGTAATAGCAATGTTATGAGTTGTGTGAATTCGTTTTAAGTTTTTTTTTTTAACTCGACATCAATTTAAACCTCTATGGCCAGTCGACGATAAGCAAGAGTTATATAAAAAAATGTTTTGTTTTTTAGTTTGCACTGCCTAAATATAGGATATAATTTTATCTGCATTAAATATAGCAACATTTTGTAATATGTCTATTATTGTGAAAAAAATACAATAAAATTTATGACGCAAAAAATCGGAAAGATTGGTCTTAAAGTATCCAGGACATTCCAAGTAGTCTCGTTAGGCTTTATTGGCTAGCACTAAACAATTATTATTAATTAGATTAAATATAATAGTGTGATATATAGATTTAGGTTACTTAGTATTTATATTACATAATTAAAGTACAAAATTGTAATGTGTATGATTATAATTGTTAGTAAACCACAGTTATAACATATTTTAAGTTAACATTGTAATATTGTTCATAATAATTTCATAAGTTCGTAAATTACAAAAATCTTTTATGGCAACATTGCAAGTTTGTGGAGAAAGCTATAACAATTATGATTAGTTAAAACATTCCCAGTTTTGTAAACAGTGATAAATATTATATTATCGTAGTATAGTGTTAATTTTGACAGTCATCGGATTTCTAAATAAGATCTCCACTTCAAGAATATTGTGATAAATATTATTTTTGTTTAATTTTAATTGTTACGAATTATTCTATAATTATTATTTCAAAATAAAACTGTAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | cds | GenBank | AB195255 | Papilio xuthus ebony RNAi RNAi construct | TTCTGGAAGCACGGGGATACCGAAAGGTGTGCGTCTGCCATATTCTGCTATCTGCAACCGTCTTTGGTGGCAATTCCGTACCTTCCCGTACACCGAACACGAAAGGTTCTGCGTTTGGAAGACCGCTTTAACTTTCGTAGACTCTGTATGCGAGATCTGGGGACCTTTGCTGCACGGCAGGACCCTAATTATCCTATCTAGAGAGACGACCCGAGATCCTCAGAAATTGGTTCGGGTCCTAGCGGAAAATGAGGTCCATCGGTTGGTATTGGTCCCGACGCTACTTCGGTCTATTCTAATGTACCTCTCTCTAAACCGATCCGAAAGACCACTACAATCCCTTAAACTATGGGTTTGTTCTGGTGA | dsRNA | column-based | simultaneously with transcription | Target region was amplified using gene specific primers by PCR, and PCR product was subcloned into pGEM-T-easy Vector. For the production of template for in vitro transcription, insert of the plasmid was amplified by PCR using vector primers containing the T7 polymerase promoter sequence at their 5â ends. PCR product was purified using QIAquick PCR purification columns. Sense and antisense transcripts were simultaneously synthesized using MEGAscript T7 Kit (Ambion) according to the manufacturer's instructions. | InsectaCentral | Papilio xuthus ebony RNAi RNAi construct | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | local resource | larva | field_collected | injection | non_specific_dsRNA | 20 | epidermis | larva | phenotype | epidermis | not checked | 12 | low | 30 | |||||||||
| 85 | Papilio xuthus TH RNAi | RNAi | collaborator: Haruhiko Fujiwara | InsectaCentral | Unpublished | Fujiwara | Haruhiko | Papilio xuthus TH RNAi target | CTGATTGCAATCCAAAGTTTAGTTAGCTACTGTAATACGGCAGCTCGAGTCGTAGACAGACAGGCAACAGCCAGTGGTCGAGACAAGAAAGCTCTCGCAGACATACGCTAGCTAAACGCGCAGTGAACTCACGTTCAGGCCCTACCGTTACCAGTGGAGTGTCCTTAGAAGAAATGGCCGTAGCAGCTGCCCAGAAGAACCGCGAAATGTTCGCCATCAAGAAATCCTACAGCATTGAGAACGGCTACCCATCCCGACGCCGTTCCCTCGTGGACGACGCTCGCTTCGAAACGCTGGTTGTCAAACAGACCAAACATAGTGTGCTCGAAGAGGCTCGTGCTCGTGCCAACGACTCTGGCTTGGACTCGGAATTCATCCAAGACGCCACGCACATTGGCAACGATGACAACACCCCGACCGTCGAAGATGGCACTCAGCAAGACGAAACCAAGAACGGTCACCTTGCAGATGCAGATATCGGTGACGATGCTGCCAACGCAGATGAAGACTATACTCTAACTGAAGAGGAAGTTATTCTGCAAAATGCGGCAAGCGAAAGCCCCGAAGCGGAGCAAGCTATCCAACAAGCCGCGTTGCTGCTAAGAATGCGTGACGGCATGGGCACCCTCGCCCGCATTCTGAAGACCATCGACAACTACAAGGGATGCGTCGAACACCTCGAGACCCGCCCCTCGCAAGCCCCCGGCGTACAGTTCGATGCTCTCGTCAAAGTGAGCATGTCTCGCATCAACTTGCTCCAGCTCATCCGATCTCTTCGACAGTCAACATCGTTCGCGGGCGTCGATCTGATGTCCGACAACAACATCTCCTCAAAGACCCCATGGTTCCCTCGCCACGCTTCCGACCTAGACAACTGCAACCACCTCATGACCAAATATGAACCGGAACTCGACATGAACCATCCCGGTTTCGCGGACAAGGACTACAGGGAGCGCAGAAAGCAGATCGCCGAAATCGCGTTCGCGTACAAATACGGAGACCCGATCCCGTCCATCACCTACACCGAGTCCGAGAATGCTACCTGGCAGCGAGTGTTCAACACTGTGCTGGACTTGATGCCCAAGCACGCCTGCCGTGAGTACAAGGCTGCGTTCGGCAAGCTGCAAGCAGCAGACATCTTCGTGCCTCACCGCATTCCTCAACTGGAGGATGTAAGCAACTTCCTGAGGAAGCACACCGGTTTCACGCTGCGCCCTGCAGCTGGGCTTCTCACsGCCCGCGATTTCCTCGCTTCCCTCGCCTTCCGTGTCTTCCAGTCCACACAGTACGTGCGTCACGCCAACTCGCCCTTCCACACACCCGAACCTGATTGCATCCACGAATTACTTGGTCACATCCCACTTCTGGCAGACCCAAGCTTCGCTCAATTCTCTCAAGAAATTGGTCTTGCATCTCTTGGCGCCTCCGATTCAGAAATCGAGAAGCTCTCAACGGTGTACTGGTTCACCGTTGAATTCGGCTTGTGCAAGGAGAATCAGCAACTGAAGGCTTACGGTGCCGCTCTCCTATCCTCGATTGGAGAACTGCTGCACGCCCTCAGTGACAAACCTGAACTTAGGGCCTTCGAGCCAGCTTCCACCTCTGTACAGCCTTACCAAGATCAAGAATATCAGCCCATCTACTACGTAGCTGAGAGCTTCGAAGATGCCAAAGAAAAATTCAGACGCTGGGTGTCAACAATGTCACGACCTTTCGAGGTGCGCTTCAACCCGCACACCGAACGCGTCGAGGTTTTGGACTCCGTTGACAAACTGGAGACCCTCATCTGGCAGCTGAACACGGAGATGCTGCACCTTACCAATGCCGTCAAGAAGCTGAAGGGCCTGCACTTTGAATAAAGCACCTACACACTAACACAAATCAATAGTACAGTGCAAGTGGTGACGCCACAATGTATAGCTAGTGATATTGTAGTTAAGACTGCGGAATGCGTGGAGCGTGAATGTCGAGAGAGCGAGTGTCGAACCATCGTATGGTACTTCATCGAATCCTTTTGTACCTTATTATTTATTATGCCGAGCTCATTGCCCGGGTCTCTGAAGGCGGGTGCCACCTGATTGCCCATTTAAGGGTGTACACCTGAACTTCACGAGTCCTTAATAGTATTAGTATTAAATGATAATTTAATAAGAAACACACATAATTAATAAAAAACGGAGCACCAAAAAGTAAAAAAAAAATGAAACAGAAAAATATATGTGTTTAAGTGGAAAAAAAAAAAAAAAAA | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | cds | GenBank | AB178006 | Papilio xuthus TH RNAi RNAi construct | TACTGGTTCACCGTTGAATTCGGCTTGTGCAAGGAGAATCAGCAACTGAAGGCTTACGGTGCCGCTCTCCTATCCTCGATTGGAGAACTGCTGCACGCCCTCAGTGACAAACCTGAACTTAGGGCCTTCGAGCCAGCTTCCACCTCTGTACAGCCTTACCAAGATCAAGAATATCAGCCCATCTACTACGTAGCTGAGAGCTTCGAAGATGCCAAAGAAAAATTCAGACGCTGGGTGTCAACAATGTCACGACCTTTCGAGGTGCGCTTCAACCCGCACACCGAACGCGTCGAGGTTTTGGACTCCGTTGACAAACT | dsRNA | column-based | simultaneously with transcription | Target region was amplified using gene specific primers by PCR, and PCR product was subcloned into pGEM-T-easy Vector. For the production of template for in vitro transcription, insert of the plasmid was amplified by PCR using vector primers containing the T7 polymerase promoter sequence at their 5â ends. PCR product was purified using QIAquick PCR purification columns. Sense and antisense transcripts were simultaneously synthesized using MEGAscript T7 Kit (Ambion) according to the manufacturer's instructions. | InsectaCentral | Papilio xuthus TH RNAi RNAi construct | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | local resource | larva | field_collected | 10 | injection | RNase free water | 100 | non_specific_dsRNA | 20 | epidermis | larva | phenotype | epidermis | not checked | 12 | none | |||||||
| 86 | Papilio xuthus DDC RNAi | RNAi | collaborator: Haruhiko Fujiwara | InsectaCentral | Unpublished | Fujiwara | Haruhiko | Papilio xuthus DDC RNAi target | GGCGTCCGTTGACAACGTGATTTTACGCTCTGCAAAATTTTTACTTAAACACCTTTTAAATATCAATAAAAGAAATATTGATTGCATATTTAACGAGTGTTGAATTAAAATCGTTTTGTGAAAGTACGAAwrATACAATCAGAAACAATGGAAGCGGGAGATTTCAAGGATTTCGCTAAAGCGATGACTGACTACATCGCTGAATATTTGGAGAATATAAGAGACAGGCCCGTCGTTCCTTTAGTGAAGCCAGGCTACTTGCGGCCCCTGGTGCCGGAGCAGGCGCCGGATAAGGCAGAGCCCTGGACCGCGGTGATGGCGGACATAGAGCGCGTGGTGATGTCCGGAGTGACGCACTGGCACTCGCCGCGCTTCCACGCCTACTTCCCAACCGCCAACTCTTATCCTGCCATCGTCGCCGACATGTTGAGCGGTGCGATCGCCTGCATTGGATTTACATGGATCGCTAGTCCCGCGTGCACTGAGCTGGAGGTCGTGATGTTGGATTGGCTGGGCCAGATGCTCGGGCTTCCGGAGTGCTTCCTCGCGCGGTCGGGAGGCGAGGCAGGCGGCGTCATCCAGGGCACGGCCAGCGAGGCCACCCTGGTCGCACTGCTCGGTGCCAAGTCCCGCACCATGCAGCGAGTCAAGGAACAACATCCCGAATGGACCGACACCGAAATCCTCTCAAAACTTGTAGGCTACTGTAACAAACAAGCGCATTCATCGGTTGAACGAGCCGGTCTTTTGGGGGGCGTGAAGATGCGTTCCTTGAAGCCTGACGGCAAGCACCGTCTGCGGGGCGACATCTTGCAGGAGGCTATCGATGAAGATATTAAGAAAGGCCTTATACCATTCTACGTTGTCGCCACACTGGGCACTACATCTTCGTGTACGTTTGATGCCCTGGATGAAATAGGCGAAGTGTGCGCGGCCCGCGACGTTTGGCTyCACGTGGACGCGGCCTACGCAGGATCAGCATTCATATGCCCCGAGTACCGCTACCTAATGAAGGGGGTGGAAAAAGCCGATTCGTTTAATTTCAATCCTCACAAATGGATGCTGGTTAACTTCGACTGCTCCGCTATGTGGCTCAAACAGCCGCGCTGGATTGTAGACGCGTTCAATGTCGACCCGTTGTACTTGAAACACGACCAACAAGGGTCCGCGCCCGACTACCGCCACTGGCAGATCCCGTTGGGACGTCGGTTCAGAGCGTTGAAGTTGTGGTTCGTCTTGAGATTGTACGGCGTTGAAAATCTACAGAAACATATCAGGAAGCACATCGCTTTGGCTCACCTCTTTGAAAAGTTGTGCACGTGTGACGAAAGATTTGAAATAGTGGAGGAAGTGACGATGGGTCTCGTTTGCTTTAGACTGAAGGGAAGCAACGATATCAACGAGGAGCTGCTCCGGCGTATCAACGGCCGCGGAAAGATTCATCTCGTACCTTCAAAAATTGACGACGTTTATTTCCTGAGATTAGCTATTTGCTCGCGTTTCTCGGAAGAGAGTGATATTCATATATCGTGGGAGGAAATCAAGAACTCTGCCGACGAAGTACTTGCCCAAAAGTAAAGTATTTTATTTATAAGTCAATTAATGAACCGATAAAATGATATTTATGATAAACTCTTACAATCTATAGTATATCTATGTAATCTAATAATCTTAAATTATTGTTAACACGTAGTAGATGTCTATATACACCTTAATTGATAACAAAACAAAAGAAATAGAAAACTGACATAAAAAATACGCAGTATTAAAAAAAAAGTAAATGTAACTATGTATGTGTATCTAATTCGTAGAAATGTTACAATATACAGTAGTGTTAGCTTTTGTTCATTTTCATTAATATAAATATTAATGTGGCCAAAGATAAAAGTGCTCACATTACTTGTTGCTTTGATATGCCCCATTATTTTCCACTTTAACTTTGTCTCTTCGAATAATTTTACCCATATTATTTTTTTGCTAGGATTAGTTGCTATTATTAAGGTATTATGTGATTATTTATTGAAAAAGTTAAATGTTAATAAACAGGGATTTAGAAATAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | cds | GenBank | AB178007 | Papilio xuthus DDC RNAi RNAi construct | TGCCAAGTCCCGCACCATGCAGCGAGTCAAGGAACAACATCCCGAATGGACCGACACCGAAATCCTCTCAAAACTTGTAGGCTACTGTAACAAACAAGCGCATTCATCGGTTGAACGAGCCGGTCTTTTGGGGGGCGTGAAGATGCGTTCCTTGAAGCCTGACGGCAAGCACCGTCTGCGGGGCGACATCTTGCAGGAGGCTATCGATGAAGATATTAAGAAAGGCCTTATACCATTCTACGTTGTCGCCACACTGGGCACTACATCTTCGTGTACGTTTGATGCCCTGGATGAAATAGGCGAAGTGTGCGCGGCCCGCGACGTTTGGCTyCACGTGGACGCGGCCTACGCAGGATCAGCATTC | dsRNA | column-based | simultaneously with transcription | Target region was amplified using gene specific primers by PCR, and PCR product was subcloned into pGEM-T-easy Vector. For the production of template for in vitro transcription, insert of the plasmid was amplified by PCR using vector primers containing the T7 polymerase promoter sequence at their 5â ends. PCR product was purified using QIAquick PCR purification columns. Sense and antisense transcripts were simultaneously synthesized using MEGAscript T7 Kit (Ambion) according to the manufacturer's instructions. | InsectaCentral | Papilio xuthus DDC RNAi RNAi construct | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | local resource | larva | field_collected | 10 | injection | RNase free water | 100 | non_specific_dsRNA | 20 | epidermis | larva | phenotype | epidermis | not checked | 12 | none | |||||||
| 87 | Papilio xuthus bilin binding protein RNAi | RNAi | collaborator: Haruhiko Fujiwara | InsectaCentral | Unpublished | Futahashi | Ryo | Papilio xuthus bilin binding protein RNAi target | TAAAGTGAGGCCGTTAGCGCTCCGwAACAAGCCGAATTTATTTAAAAAAAAAGTAGCATGTTTCGCTTCGTAACTATCGCAGTGTTGTTCGCTGCGGCGACTTCTGAAGTCATATTTGAGGGGCCATGTCCyGATATCAAGACGGTTGACAATTTTGAATTTGAAGCTTACGGAGGAACATGGTACGAAATGGCAAAGTACCCTAACGCAGGGGAAGAAAACACAAAGGGCAAATGyACAATCGCTGAATACACAGTAAACGGTGACAAGGGCAAGGTTAAGAACTCGCACGTCATTGATGCGGTCCGACACTACATCTCyGGTGATCTGACACTCGTCGCTCCTGGCAAGATTATGCTTACTTACACATTTGGAGGTCAATCCAAAAAyTCTTACCTGAACATCTTGGATACGGACTACAAGAGCTACTCTATCGGATACAGCTGCAAATACTTCAAGGATGGTAACAAACATCAAGTCTTCGCATGGATCAAGTCAAGGAGCAAGAAACTTGATTGCGAAGCCAAATACAAGATCGACAACTTTTTAAGGACCTCCAAAGTATTGGATTCyGCCAAATTCGTTGTwAACGAACACACTGACGCCGCTTGTTCsGCACCCACCACCAAAACCATCACTGAGTTCCTTAAGTGAATAGACATATAGTTAAGTAGATAAAATAGGTACATATTGTCAATAAAAAACTTTAAAATGCAATAAATTCAATTGTTCCATTAACAGTAAATTACAGATAAAAATAATATAACACAAAAAAAAAAAAAAA | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | cds | GenBank | AB264632 | Papilio xuthus bilin binding protein RNAi RNAi construct | ACCCTAACGCAGGGGAAGAAAACACAAAGGGCAAATGyACAATCGCTGAATACACAGTAAACGGTGACAAGGGCAAGGTTAAGAACTCGCACGTCATTGATGCGGTCCGACACTACATCTCyGGTGATCTGACACTCGTCGCTCCTGGCAAGATTATGCTTACTTACACATTTGGAGGTCAATCCAAAAAyTCTTACCTGAACATCTTGGATACGGACTACAAGAGCTACTCTATCGGATACAGCTGCAAATACTTCAAGGATGGTAACAAACATCAAGTCTTCGCATGGATCAAGTCAAGGAGCAAGAAACTTGATTGCGAAGCCAAATACAAGATCGACAACTTTTTAAGGACCTCCAAAGTATTGGATTCyGCCAAATTCGTTGTwAACGAACACACTGACGCCGCTTGTTCsGCACCCACCACCAAAACCATCACTGAGTTCCTTAAGTGA | dsRNA | column-based | simultaneously with transcription | Target region was amplified using gene specific primers by PCR, and PCR product was subcloned into pGEM-T-easy Vector. For the production of template for in vitro transcription, insert of the plasmid was amplified by PCR using vector primers containing the T7 polymerase promoter sequence at their 5â ends. PCR product was purified using QIAquick PCR purification columns. Sense and antisense transcripts were simultaneously synthesized using MEGAscript T7 Kit (Ambion) according to the manufacturer's instructions. | InsectaCentral | Papilio xuthus bilin binding protein RNAi RNAi construct | Lepidoptera | Papilionidae | Papilio | xuthus | 66420 | local resource | larva | field_collected | 10 | injection | RNase free water | 100 | non_specific_dsRNA | 20 | epidermis | larva | phenotype | epidermis | not checked | 48 | none | |||||||
| 88 | Bombyx mori ebony RNAi | RNAi | InsectaCentral | Unpublished | Futahashi | Ryo | Bombyx mori ebony RNAi target | ATTCATACCTGCACTATTGCACAATTCAGTCGCGCGCGTGTTAACTGAAGGGACGTTTTGTGAGTGTGTTCTGTGTTTACGGTGCATTTGCGTTGTGCAATTTAAAAAAAAAAAACAAAAAACAAAAACTAAGTACGATGGGCTCCTTGCCTCGCGTCGCTGTGGTGACTGGACCCAGAGCGGCCGTCCCGACTGCCCCCCTCACTCACCACCTCGGCCCTTTAGCCAAGAGCGACAGGACAGCTCTTATTTACAAAGACGAGATCTCCAGCGTTCAGGTCAGTCATGCAGAGCTTGCAGCGAGGACTAATGTCCTGGCGAGAGCTATCAGCGAGAGGGCCAGGACTGTGGGACCGAACAGAGATGGTGACTACGTAATTGCTGTTTGTATGCAGCCCACACACAATACGATAATGGCGTTGTTAGCGACGTGGAAGGCAGGAGCAGCGTACGTACCAATGGAGCCGAGCTTCCCTCAGGGCAGGATCACTCACATACTGAAGGACGCCGAACCTTCCCTTGTCATTTATGATGATAGCGCGGACCCCTCAATGTTCAAGGAAAGCGGAGTACCGTTCGCGTCATTCGAAGAGTTAGCGCAGGAAGCTACCAGCCACTCAGCCGACGAACCAATGGACGCGGAGACACTGGCGCCACTTACAAGTGACAGCATCGCTATCGTACTCTACACTTCTGGAAGCACCGGCATTCCGAAAGGTGTGCGTTTGTCGTACTCGGCAATCTGCAATCGTCTCTGGTGGCAGTTCCGCACGTTCCCCTACTCCGACTCCGAAGAGATCTGCGTTTGGAAGACCGCGCTCACCTTCGTGGATTCCGTCTGCGAGATATGGGGACCGCTGCTGCACGGGAGAGCCTTGCTAATACTGTCGAGGGAGACCACCAGGGATCCTCAGAAACTTGTCAACGTCCTCGCTGATAATCAGATTGAAAGACTCGTGTTGGTTCCAACTCTGCTACGTTCCATCCTGATGTACCTGGCGTTGGGCCCCGCCTCACGACCGCTGCGGCGTCTCAAGCTGTGGGTCTGCTCCGGGGAAACCCTCAGCAAGGAGCTGGCTTCGGAGTTCTTTAAGTACTTCGGCAATGAGGACGGATACAAGCTCGCCAACTTCTACGGAAGCACTGAAGTCATGGGCGACGTGACGTATTACGTCTTGGAAGGCAGCGACCAATTGGATTTGTACCCGACTATTCCTATTGGGAGGCCCTTGGACAATTGTGCGATATACCTGTTGGATGAGGAGTTGAGCCCGACCCGTGATTCCGAGCCGGGCGAGGTTTGGGTCGCGGGAGCTAATCTAGCCGCTGGATACGTTGGAGGCGGCGGCGCGAACCGTTTCTGTGATAACCCTCACGCCACTCAACCTGAATTCCAACGTTTATACCGCACAGGAGATTTCGGGACTCTTGTTAAGGGCGCGGTGCTGTACGCCGGTCGCACGGACGCCCAGGTCAAGATTCGAGGACACCGCGTGGATCTGCTGGAAGTGGAAAGAGCGCTGGCCCAAGTCCCAGGAGTTGACAAGTGCGTGGTGCTATGCTACGGTTTGGACAGAGGCAACCCTGAGATCCTCGGCTTCGTCACAACCAAGCCGGGCGCCCGGACCAGCGCGCAGCACATCGAGGGCAAGCTCAGAAGCGCCCTGACCCACTACATGATGCCACAGGTAATCGAGGTTGAAAGCATTCCTCTGCTGGTAAACGGCAAAGTGGACCGGCAGGGTCTCCTTAAGATGTACGAGAACACGAACAACAATGACGACGCCGAGGTAGCCGTCGACATTGACTGCAGTGGCGCGGGGCTCGCGGACCTGGAGGCAGCCCAGGCCCTCTTGGAGACGGTGGGCGCCGTGCTCGGCAGGGCCGCCAGAGGAACGCTGTCTCTGAGGTCCGGCTTCTATGAGCTGGGAGGGAACTCTCTGAACTCCATCTACACTATCACCAAGTTGAGGGACAGAGGTTACTATGTGGAAATCAGCGACTTCCTGGGAGCTTCCACTCTGGGCGAGGTTCTGTCGAAGATGTCGACGGATCCTAATGGCGGCGCCGACCCGAAGGAGGCCACGTTCACAGCCGTGCCCATGAGGGATGAGCACAAGAAGGAAGTTATTGACATGGTGGAGTCCTCGTTCTACGAGAAAGCCGAGCTGGAGCAGTTCTTGAAACACGAGATCGAGACCAGGGACTACGCCGACTGCATCGACGCCTGCTGGCCGGCGCTGCTGCAGGCCGGACTGAGTGTCGTCCTCAATGACGTGAACAGCGAACCAGTAGCAGTGGCTTTGAACTTCGACGTGAGAGACGAACCTGAGATCGAGTTGTCTGGAGGACTCGCCAAGATCATGGGATTTTTGGAGTACGTGGAGGGTTCCGTGAGGGACACCATGCTGCCCCATGGGAAAGGTGCTGTGCTGCATTCGTTCATGATGGCGACGAGTGAGCGTCTGAGCGCACGAGACAATGTGTCCGCCATCCGCGCTCTGGAGCTAGCCACAGACCGAGTAGCGAGACAGAGACGCTTCAGAGGCGTGTTCACGACCAACACGAGTCCGCTTACGCAGGACAATGACCACAACATACGCAGCTACAGATTTTGCAGCAAGATGAATTTATACAGCGGGTGTCTTACAAAAGAAAAAGCTCGTTTGGCTCACAATAAGCCCTACCACCACCGCTAAAGAACTTGAGGCTTATGAATCGGACGGGAAAGGTGGGGCAGAACTGGCTTAGACAGTTGGGGACGAAATAGAAGCGATATTACCCGATTGTCGGCGATTGCTATTCTCTCAACTAGATATAACTATGAAATGCTCTAAGTATGCTAGTATTGTGCATAGAAACTCATGTGTTAGGATAGGCCATTTTGTTAATTAGGTATATCTTTGTTAATATATAGTGTACAACAACAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AB439000 | Bombyx mori ebony RNAi RNAi construct | ATCGCTATCGTACTCTACACTTCTGGAAGCACCGGCATTCCGAAAGGTGTGCGTTTGTCGTACTCGGCAATCTGCAATCGTCTCTGGTGGCAGTTCCGCACGTTCCCCTACTCCGACTCCGAAGAGATCTGCGTTTGGAAGACCGCGCTCACCTTCGTGGATTCCGTCTGCGAGATATGGGGACCGCTGCTGCACGGGAGAGCCTTGCTAATACTGTCGAGGGAGACCACCAGGGATCCTCAGAAACTTGTCAACATTGAAAGACTCGTGTTGGTTCCAACTCTGCTACGTTCCATCCTGATGTACCTGGCGTTGGGCCCCGCCTCACGACCGCTGCGGCGTCTCAAGCTGTGGGTCTGCTCCGGGGAAACCCTCAGCAAGGAGCTGGCTTCGGAGTTCTTTAAGTACTTCGGCAATGAGGACGGATACAAGCTCGCCAACTTCTACGGAAGCACTGAAGTCATGGGCGACGTGACGTATTACGTCTTGGAAGGCAGCGACCAATTGGATTTGTACCCGACTATTCCTATTGGGAGGCCCTTGGACAATTGTGCGATATACCTGTTGGATGAGGAGTTGAGCCCGACCCGTGATTCCGAGCCGGGCGAGGTTTGGGTCGCGGGAGCTAATCTAGCCGCTGGATACGT | dsRNA | column-based | simultaneously with transcription | Target region was amplified using gene specific primers by PCR, and PCR product was subcloned into pGEM-T-easy Vector. For the production of template for in vitro transcription, insert of the plasmid was amplified by PCR using vector primers containing the T7 polymerase promoter sequence at their 5â ends. PCR product was purified using QIAquick PCR purification columns. Sense and antisense transcripts were simultaneously synthesized using MEGAscript T7 Kit (Ambion) according to the manufacturer's instructions. | InsectaCentral | Bombyx mori ebony RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | 10 | injection | RNase free water | 100 | non_specific_dsRNA | 20 | epidermis | larva | phenotype | epidermis | not checked | 72 | none | ||||||||
| 89 | Bombyx mori yellow RNAi | RNAi | InsectaCentral | Unpublished | Futahashi | Ryo | Bombyx mori yellow RNAi target | AGTCGAACACAAGCTGCCGCGAACATAGGCTAGTTTTGAGTAAATAAAATGGCAGCGAAGTTTTTAGTTTTCTGTGGGCTAGTCTCACTAGCATCAGCAACGATAAAGCTCCAAGAAATATTCTCGTGGAACGTGGTCGACTGGAACTACCCGGACCAGTTCTCGAAGCAGCAGGCTCTCAGGACTGGTGCTCTGATACCAGAGAACGCACTGCCCGTTGGTATCGAAAGGTGGAGGAACAAATTGTTCGTCAGCGTTCCTAGGTGGCGCTCAGGTATTCCAGCTACTTTGAATTACATTCCACTGGACGCTCCATATGAACCATCCCCGAAATTAACGCCCTACCCGAGCTTCGAAGGCAACGAATTAGGCAATTGTCAAACAGGACTGACTACTGTATACAGGGTCAAAGCAGACCAGTGTGATCGTCTTTGGGTATTAGACGTTGGCACTTATGGATACGACAACGTTACAAATGTGTGCCCGTACACGCTCAATGTATTTGACTTGAACACTGATCAAATCATCCGTAAATACGTGTTACGACCAGAAGACATTGTCTCTACGACTTTCATTGCCAACATCGCTCTCGATATCGGTACCAGCTGTGAGGACACCTTTGCCTACTTCTCCGATGAACTGGGCTATGGTCTTATCGCTTACTCGTGGGAACAGAACAAGTCATGGAGATTCAGCCACAGCTATTTCATGCCCGACCCTCTCGTCGGTGATTTCAATATCGCTGGCTTGAATTTCCAGTGGGGAGCTGAAGGTATCTTCGGCATCACGGCGTCTCCTATCGGTGCCGATGGTTACAGAACTCTGTACTTCTCACCTCTGAGCAGTCACACAGAATTTTCCGTGTCGACCCGTATCTTGAGAGACGAAACTAAAGTGAAAGGTTCCTACAAAGACTTCTCAGTTGTCGGAGTACGTGGTCCTGACGGTCATACGACAGCCAAAGTAATGGACGACTCCGGTGTCCAGCTTTTCAACTTGATCGACCAAAACGCTGTCGGCTGTTGGAGATCGTCCCTGCCCTACAAACCTCAAAATATCGGAATTGCCGATAAAGACGACGTTGGCCTTGTTTTCCCCGTAGACGTCAAAATTGATGATGAGAAAAATGTATGGGTTATGTCAGACCGTATGGCTGTATTCCTTGAAGCCGAATTGGACTATAGTGATATTAACTTCAGGGTTTATACTGCACCTTTAGATACCCTAATTCAAGGAACCGTTTGTGAAGCGCCATTGCCAGTGCTTCAGGCACAGCAAATACTTAAGCCACTCCAACCGATCAAGGCTCTTTCTCGAGCGAAACTCTACAATCATGGCCTGGGAGTAACAGCAACTCCTTTCTCTGAAGCCGTTGCGGTATACACACCAGGCTCAACGAAATCTGGACTGACAGGTTCTTATAGACAACCGATTATAAAGAACACTGCTCCTAAAGTTCAGAACTACATTTACATTCCGAAACAAACTTCATCCGCTTATACATCTGGTCAATTTACCAGGCCGAAAAGTACCAACAAGAATCCGTGGTGGAATAAAGCTACCAACTACAATTACGAAGTTTTTGAACCTTGAAAAACATTGTTTGATTCAAATTCTCAATTCCTAGATTAAAAACTGTAAAATTATAGGACATTATGTATTAAATGTGAGGCTTTCTATTGCAATAAATAAATATAGAGCGTTTAAGCAGAGACAATTTCGTAAAAGTGTCGTAAACTCTTTATAATAAACTTTCAGAAACCATTTACAAACAAAACTTCTAGTACACTATAAAATTTATCAATGAATGAATCCATTTGGTGATAAAAAAATTGTGATGGTTGTAATATTATTGTTGTTTAGTGTTATTATGTATCTTTTATTACTTATTTAATTTTTTTTATAAATTTGTAATAAGTACTAGGTCGGTACAAGATTGTTGATTTTGTAATAAAAGTTGTTATCTATAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AB438999 | Bombyx mori yellow RNAi RNAi construct | AACGCACTGCCCGTTGGTATCGAAAGGTGGAGGAACAAATTGTTCGTCAGCGTTCCTAGGTGGCGCTCAGGTATyCCAGCTACTTTGAAyTACATTCCACTGGACGCTCCATATGAACCATCCCCGAAATTAACGCCCTACCCGAGCTTCGAAGGCAACGAATTAGGCAATTGTCAAACAGGACTGACTACTGTATACAGGGTCAAAGC | dsRNA | column-based | simultaneously with transcription | Target region was amplified using gene specific primers by PCR, and PCR product was subcloned into pGEM-T-easy Vector. For the production of template for in vitro transcription, insert of the plasmid was amplified by PCR using vector primers containing the T7 polymerase promoter sequence at their 5â ends. PCR product was purified using QIAquick PCR purification columns. Sense and antisense transcripts were simultaneously synthesized using MEGAscript T7 Kit (Ambion) according to the manufacturer's instructions. | InsectaCentral | Bombyx mori yellow RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | 10 | injection | RNase free water | 100 | non_specific_dsRNA | 20 | epidermis | larva | phenotype | epidermis | not checked | 72 | none | ||||||||
| 91 | ATPase H silencing in S frugiperda | RNAi | Vacuolar ATPase subunit H was chosen for the dsRNA feeding experiment in Spodoptera frugiperda. The animals were kept separately in a 5cm x 3.5cm x 3.5cm compartment. The dsRNA was provided on a small piece of maize leave. The experiment was started with L3. They were fed eight times with 3µg of dried dsRNA, respectively. The time period between two feedings was 24 hours. 17 animals were fed. None of the animals showed the expected lethal phenotype or any other obvious phenotype. Author contributing to these experiments are Daniela Nowara, Magnus Lundmark, Frank Hauser, Merete Albrechtsen, Cornelis J. Grimmelikhuijzen. | InsectaCentral | Unpublished | Nowara | Daniela | ATPase H silencing in S frugiperda target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | ATPase H silencing in S frugiperda target | ATPase H silencing in S frugiperda RNAi construct | CCGCCCAACTGCTCGATAATGTGTTTGCCTCGTGGATAGTGGCGGACGTATTCACCGATGTCGTAGCAGGCGACGGCGAGTACGACGGGGTCGTTACTCCTCTCCAGCAGGTGCACCAAGGTACGGAGCAACTCCTGGCCGCGCTCGTTCAGACGAGCAGCGTTCTCGCGCCAGAACTTAGCTGATTTGTGGACGGGTGACCATTCCAGACGGCCGCTCTTGACTTCAGTAGCGTACTGGTCGAATGAGCTGAGGTCTTGTACTGAAGTCTGCAGGCGTTCGTTCAGGAAGTCCACATCGTTCATGATGTCTTCATCGTCAGAGCGCTTCTGCTGCAGGATCATCAATTGCTTCAGGACCTTGCACTGTACCATGGCGATGCAGTGCTCCTTGGCAACCTGATGGTCTTCAGGTTTTTCGATCAGGTTCCTGAACACAGCGAGCACGATGCGCGTGACCTTCTCCTTGACGGAGTCGCTGAGGATGTCGGCGAGGATGGGGATGGCATTGAACTTGTTCATCTTCTCGGCCAACAGGGGGTTGAATGTCAGCACCCACAAGCAGAACACAAGCTGGTATTGCACCTGGAAGTTGACTCTGGAAGCCAGAATAGACAGCAAGGTGGAGATGCCGTCAACAGACAGGAAAGCAAAGCGGTACTCGTCGACGCGCAGCATCATTTGCAGGCAGCGGGC | dsRNA | Ambion Megascript T7 Kit | other | subsequent to transcription | A PCR product which was generated using T7-adapter primer was used as template for the in vitro transcription. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to the kits protocol. The mixture was incubated at 37 degree Celsius over night. Accordingly, the whole reaction was heated at 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. The dsRNA was used for the feeding experiments without any further purification. | InsectaCentral | ATPase H silencing in S frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | bayer | larva | lab_colony | feeding | megascript buffer | maize leaf | non_specific_dsRNA | larva | qPCR phenotype | whole organism | not checked | none | |||||||||||
| 92 | G protein silencing | RNAi | Heterotrimeric G protein alpha subunit type s was chosen for 3 dsRNA feeding experiments in Spodoptera frugiperda. The animals were kept separately in a 5cm x 3.5cm x 3.5cm compartment. The dsRNA was provided on a small piece of maize leave. The first experiment was started with L1. They were fed twice with 3µg of dried dsRNA, respectively. The time period between the two feedings was 7 days. 19 animals were fed. The second experiment was started with L2. They were fed six times with 3µg of dried dsRNA, respectively. The time period between the two feedings was 2 days. 12 animals were fed. A pool of these animals was analyzed by qPCR. None of the animals showed the expected lethal phenotype or any other obvious phenotype. Author contributing to these experiments are Daniela Nowara, Magnus Lundmark, Frank Hauser, Merete Albrechtsen, Cornelis J. Grimmelikhuijzen. | InsectaCentral | Unpublished | Nowara | Daniela | G protein silencing target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | G protein silencing target | G protein silencing RNAi construct | TCGCGCAGCACCATGTTGTAGGAGGAGCAGGCCGTCACGAAGATGATGGCCGTCACGTCGTTGAAGCACTGGATCCACTTGCGACGCTCGTCGCGTTGCCCGCCCACGTCGAACATGTGGAAATTGACTTTATCGACGACGAACTGCGTCTCGAAGATGCCGGATGTGAGCACTCGGCAGCGGAGGATGTCCTGCTCCGTCGGAGTGTAGTTCGGCTGCTTTATTATATGCACTTGATCCAGGAAATACTTGGCGCAGTCGATGAGCTGGTACTCGTTGCTGCGCTCGTAGGTGCGCTGCACGCCGGCGTCCTGCCACAGCGCCTCCGTGTGCTCGTAGAACTCCATCGGGTAGTCGAAGTCCGGCTGCGACGCTACGTCCTGTATATAGTCGACGCGCGCCTTATTCTCGGGTTTTTCAAGCGGTATAGGGGGCGTGAGGGTGCTCATCGCGCCCGTGATTGTAAGTATAGCATCTCTAATATTCTTCTTGATGTCCTCGATCTTCTCCCGGCGCTCCTTGTC | dsRNA | Ambion Megascript T7 Kit | other | subsequent to transcription | A PCR product which was generated using T7-adapter primer was used as template for the in vitro transcription. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to the kits protocol. The mixture was incubated at 37 degree Celsius over night. Accordingly, the whole reaction was heated at 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. The dsRNA was used for the feeding experiments without any further purification. | InsectaCentral | G protein silencing RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | bayer | larva | lab_colony | feeding | megascript buffer | maize leaf | non_specific_dsRNA | 2 | larva | qPCR phenotype | whole organism | not checked | none | ||||||||||
| 93 | IAP silencing in S frugiperda | RNAi | Inhibitor of apoptosis protein (IAP) was chosen for the dsRNA feeding experiments in Spodoptera frugiperda. The animals were kept separately in a 5cm x 3.5cm x 3.5cm compartment. The dsRNA was provided on a small piece of maize leave. The experiment was started with L2. They were fed six times with 3µg of dried dsRNA, respectively. The time period between the two feedings was 2 days. 12 animals were fed. A pool of these animals was analyzed by qPCR. None of the animals showed the expected lethal phenotype or any other obvious phenotype. Author contributing to these experiments are Daniela Nowara, Magnus Lundmark, Frank Hauser, Merete Albrechtsen, Cornelis J. Grimmelikhuijzen. | InsectaCentral | Unpublished | Nowara | Daniela | IAP silencing in S frugiperda target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | IAP silencing in S frugiperda target | IAP silencing in S frugiperda RNAi construct | TCCAACGCCGGCTCTCCATCTTTGTCACCATCCACGCCTTGTTCTTCATCTTCTTTCTCCATTGATAAAACCGACAACCACGACACCTTCGGCTTCAGTGCGGACACAGTTGATATGAGAAAAGAGGATGAACGTATGAAAACATTTGAAAAATGGCCCGTAAGTTTTCTATCCGGAGAGCAACTTGCTCGAAATGGATTTTACTACCTCGGCCGTGGAGATGAAGTCCGTTGCGCTTTCTGTAAAGTGGAGATTATGAGGTGGGTGGAGGGCGATGACCCTGCGAAGGACCATCAGCGTTGGGCGCCACAGTGCCCATTTGTGCGCAAATTGAACGGTACTGCAGCAGCAGACACGGGTAGTTCGGGCCAGGACGAGTGTGGTGCCCGCGCCGCTCCCTCCGGTACCTCTCCGCCGCGTATGGCCGGTCCCGTGCACCCACGATATGCATCTGAAGCCGCACGACTACGCAGTTTTAAAGACTGGCCACGATGCATGCGACAAAAACCTGAAGAACTCGCCGAGGCTGGCTTTTTTTACACTGGTCAGGGAGACAAAACCAAGTGTTTTTATTGCGATGGTGGATTAAAAGATTGGGAGAACCATGACGTACCCTGGGAACAACACGCAAGGTGGTTTGACCGTTGCGCCTACGTGCAATTGGTGAAGGGTCGAGAATACGTTCAAAAGGTGATTTCTGAAGCTTGTGAGGTATCCGCGTCAGAAGCGGAACGTGATGTAGCACCCGCACGGACTG | dsRNA | Ambion Megascript T7 Kit | other | subsequent to transcription | A PCR product which was generated using T7-adapter primer was used as template for the in vitro transcription. About 100ng PCR product were mixed with NTPs, buffer and enzyme according to the kits protocol. The mixture was incubated at 37 degree Celsius over night. Accordingly, the whole reaction was heated at 70 degree Celsius for 10 minutes and then slowly cooled to room temperature. The dsRNA was used for the feeding experiments without any further purification. | InsectaCentral | IAP silencing in S frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | bayer | larva | lab_colony | feeding | megascript buffer | maize leaf | non_specific_dsRNA | larva | qPCR phenotype | whole organism | not checked | none | |||||||||||
| 96 | RNAi Manduca sexta ATPase E | RNAi | pubmed | 19815067 | Whyard | Steven | RNAi Manduca sexta ATPase E target | GGCTCTTTATCGGTTGTTTTTCGTGTTAGTGGTGCTAAATAATAACCTACAGCCATGGCGCTCAGCGATGCCGATGTTCAAAAGCAGATCAAGCATATGATGGCCTTCATCGAACAAGAGGCCAATGAAAAGGCCGAAGAAATCGATGCTAAGGCGGAGGAGGAGTTCAACATCGAAAAGGGCCGTCTGGTTCAGCAGCAACGCCTCAAGATCATGGAATACTATGAAAAGAAAGAAAAACAAGTAGAGCTCCAGAAAAAGATTCAATCATCGAACATGCTGAACCAGGCTCGTCTGAAGGTACTAAAAGTGCGTGAGGACCACGTACGTAACGTACTGGACGAGGCCCGCAAGCGCCTGGCTGAGGTACCCAAGGACATCAAGCTGTACTCAGATCTCCTGGTCACTCTCATTGTGCAGGCTCTCTTCCAGCTTGTGGAGCCCACAGTGACCCTCCGCGTGCGACAAGCCGACAAAGCTCTGGTTGAATCTCTGCTTGGCAGAGCCCAACAGGATTACAAGGCCAAGATCAAGAAGGATGTCGTGCTCAAGATCGACAATGAGAACTTCCTTCCCCCTGACACCTGTGGAGGAATTGAGCTGATTGCTGCCAAGGGACGCATCAAGATCAGCAACACGCTGGAGTCGCGCCTGGAGCTGATCGCGCAGCAGCTGCTGCCGGAGATCCGTAACGCGCTGTTCGGACGCAACCCTAACCGCAAATTCACCGACTAAGCCCCAACGGTGACCCACATTCACTCGTTATACTAACAACATATTTAAATTTCATAATATGTAGAAGTAAAATAATATTATAAGTGTTTTCTTTTGCCTAATTAATTATATCGTATAATGCGTACAATTATATCTGTATTAGTATGTATTGATGAATGGTCCCGTTAAAGACACTCGTACAAATGCGATCATGTGGATTTCTGATTCTCGGCATGTCATTGTTCACTCGACTGTACATTAGTTTCGCCAATTAACCAACATGCAAATGTTAAATGTTTTATTATAACAATTCATTCGTATAGCATCATGTCTATGTATTTAGGGAACGTAGTTTATAGGATTTGAGAACAGTTAAGACGTTACCACATTTAGGCGAAATAATATATGTCGCTAAGTATTTCATAGAAATTGAGGCACCTATCTGATACAGTATTATAGCACAATGACAGTTTGAGCAGTAGATGGTTAACGTATCTAAAACTATTGCGTTTCAAATCTTCCATATTTAATGTTACTGAATGTTTTGAATATTTATCGTGTTTACCTGTTTATTGGCTGATGTAGGAGGCATTGAGGTCTGTAGGTTTGAGACTTGATGGCAGCATCCCATGCTGTTTCTACTTAAATCGAAGCAAAGATTTGGCTGGTGGCAGTGTGGTGTCAAGTGCCAACTCTACAGCTGCATAACATTCCTACCTCGTGTTCCAACTGGCAATATGATATTTTGGCTTGCTTAGCAGATGCAAAGAGCACCTTAGCTCTTATAAAATAAGATACAAGTGTAATTTCCGTTATGGCAATGTTAAAAATTCTTATGTAAATATATAGATGTGTTGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | X67131 | RNAi Manduca sexta ATPase E RNAi construct | AAGGCCAAGATCAAGAAGGATGTCGTGCTCAAGATCGACAATGAGAACTTCCTTCCCCCTGACACCTGTGGAGGAATTGAGCTGATTGCTGCCAAGGGACGCATCAAGATCAGCAACACGCTGGAGTCGCGCCTGGAGCTGATCGCGCAGCAGCTGCTGCCGGAGATCCGTAACGCGCTGTTCGG | dsRNA | T7 RiboMAX Express; Promega | column-based | simultaneously with transcription | Total RNA was isolated from first or second instar insects using a Qiagen RNA extraction kit. Superscript II reverse transcriptase (Invitrogen) was used to make first strand cDNA using random primers. The cDNA was used as template to PCR-amplify 185 bp portions of the coding sequence of the E-subunit of the vATPase gene. The PCR product was gel-purified with a QIAquick PCR Purification kit (Qiagen), ligated into the pGEM T-Easy vector (Promega), and the identity of the DNA fragment were confirmed by DNA sequencing. The vATPase gene fragment was subsequently excised from the pGEM T-Easy plasmid using EcoRI and ligated into the EcoRI restriction site of the dsRNA transcription vector pL4440 (kindly provided by Andrew Fire), which contains two convergent T7 promoters that flank the plasmidâs multiple cloning site. To obtain sufficient DNA template for in vitro transcription, the vATPase gene fragment was PCR-amplified along with the flanking convergent T7 promoter sequences from the dsRNA expression plasmid, using primers pL4440T7-F: 5âCCACCTGGCTTATCGAAA3â and pL4440T7-R: 5âTAAAACGACGGCCAGTGA3â. Double-stranded RNA was prepared using a T7 RiboMAX Express Large Scale RNA production System (Promega) according to the manufacturerâs specifications. DsRNA concentrations were determined using standard spectophotometric techniques. | InsectaCentral | RNAi Manduca sexta ATPase E RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.5 | feeding | water | 25 | non_specific_dsRNA | 3 | β-glucuronidase (gus) dsRNA was fed to the larvae as a negative control. The gus gene was amplified by PCR from the pBacPAK8-GUS plasmid (Clontech) and a 0.4 kb PCR product was cloned into the dsRNA transcription plasmid pL4440, and dsRNA was synthesized as described above. | larva | qPCR phenotype | gut | not checked | 48 | low | 34 | ||||||
| 97 | Bicyclus anynana white RNAi | RNAi | InsectaCentral | Unpublished | Saenko | Suzanne | Bicyclus anynana white RNAi target | CTTTyTGTTACGAAAGAGCCTATGCTAATTAAAGTCCGGTTTTTGCAAACTATTATGGTATCTATTCTTATCGGGGTGATTTACTTTGGACAACATTTGGATCAAGACGGCGTGATGAACATAAAyGGnGCGAThTTCATGTTCCTTACCAATATGACGTTCCAGAACATCTTCGCAGTTATCAACGTATTCTGTTCAGAGCTGCCAATATTCATTCGCGAGCACCATTCAGGGATGTACCGAGCTGACGTGTATTTCCTCTCGAAGACGCTCGCCGAGGCGCCGGTGTTCGCGACTATCCCTCTGGTCTTCACCACCATCGCCTACTACATGATAGGCCTCAACCCCGCCCCAGAGAGATTCTTCATCGCTTCGGGCTTGGCTGCTCTTATTACCAACGTTGCTACTTCGTTTGGTTACCTGATATCTTGCGCGAGCAGCAGCGTGAGCATGGCCGCGTCAGTGGGGCCGCCCATCATCATCCCGTTCATGCTGTTTGGAGGATTCTTCCTTAACTCAGGTTCCGTGCCGCCATATCTGGGCTGGATATCGTACCTGTCGTGGTTCCGGTACGGCAACGAGGCGCTGCTCATCAACCAGTGGTCCGGCGTGGAGAGCATCGCCTGCACCAGGGAGAACTTCACCTGTCCCGCTAGCGGGGAGGTGGTCTTGACTACGCTTAGCTTTGCTGAGAAGGACTTCACGATGGACGTTGTGAACATGGTCCTGCTCTTCATCGGGTTCCGTCTGCTCGCCTACTTCGCCCTGCTGTGGAGGACGCGACGCGGAAAGTAGACCTAAGCACAAACATACCTCTTACTGCCAACGAGT | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | cds | InsectaCentral | Bicyclus anynana white RNAi target | Bicyclus anynana white RNAi RNAi construct | AGTCCGGTTTTTGCAAACTATTATGGTATCTATTCTTATCGGGGTGATTTACTTTGGACAACATTTGGATCAAGACGGCGTGATGAACATAAACGGAGCAATATTCATGTTCCTTACCAATATGACGTTCCAGAACATATTCGCAGTTATCAACGTATTCTGTTCAGAGCTGCCAATATTCATTCGCGAGCACCATTCAGGGATGTACCGAGCTGACGTGTATTTCCTCTCGAAGACGCTCGCCGAGGCGCCGGTGTTCGCGACTATCCCTCTGGTCTTCACCACCATCGCCTACTACATGATAGGCCTCAACCCCGCCCCAGAGAGATTCTTCATCGCTTCGGGCTTGGCTGCTCTTATTACCAACGTTGCTACTTCGTTTGGTTACCTGATATCTTGCGCAAGCAGCAGCGTGAGCATGGCCGCGTCAGTGGGACCGCCCATCATCATCCCGTTCATGCTGTTTGGAGGATTCTTCCTTAACTCAGGCTCCGTGCCGCCATACCTGGGCTGGATATCGTACCTGTCGTGGTTCCGGTACGGCAACGAGGCGCTGCTCATCAACCAGTGGTCCGGCGTGGAGAGCATCGCCTGCACCAGGGAGAACTTCACCTGTCCCGCTAGCGGGGAGGTGGTCTTGACTACGCTTAGCTTTGCTGAGGACTTCACGATGGACGTTGTGAACTGGTCCTGCTC | dsRNA | MEGAscript RNAi Kit (Ambion) | column-based | subsequent to transcription | For the production of a template for in vitro transcription, PCR with vector-specific M13F and M13R primers was performed on a plasmid containing white insert and the promotor sequences for the T7 and SP6 polymerases. After cleaning with Wizard SV Gel and PCR Clean-Up System (Promega, Cat. # A9282), 1 µg of the PCR product was used as a template for transcription reactions with T7 and SP6 enzymes. Sense and antisense transcripts were synthesized using the MEGAscript RNAi Kit (Ambion, Cat. # AM1626), according to the manufacturerâs instructions. After purification in sterile 1xPBS, the quality of dsRNA was verified on an agarose gel, and its concentration measured with NanoDrop spectrophotometer (Thermo Scientific). | InsectaCentral | Bicyclus anynana white RNAi RNAi construct | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | local resource | pupa | microsporidia | microsporidia | lab_colony | 0.12 | injection | 1x PBS | 0.13 | buffer | 8 | pupal wing | same volumes of 1xPBS were injected in pupae in a similar manner(N = 7) | adult | phenotype | not checked | 120 | none | |||||
| 98 | Bicyclus anynana Antennapedia RNAi | RNAi | InsectaCentral | Unpublished | Saenko | Suzanne | Bicyclus anynana RNAi target | GTGCCGGGCAGTCCGCCCCTGGAGCAAGCTCAGCAGATGCCGCACCACATGCACCCGCAGCAGCACATGCAGCACGGCATGCCACCGCACCAGCAGCACGTCATGTACCCGGTGGACGACATGCAGCACCAGACGCAGATGCCGCCCATGCACCAGCAGTCCATGCACCCGCAGCAGGCGCCGCCTCAACAACCCCCGCCGAATACGAACGCGTCGCTCCCCAGTCCACTGTACCCTTGGATACGAAGTCAATTTGAACGAAAGCGGGGACGGCAAACGTACACCCGGTACCAGACCCTCGAGTTGGAGAAGGAGTTCCACTTCAACCGATACCTGACGCGGAGAAGACGGATCGAGATCGCGCACGCCCTCTGTCTCACCGAGCGCCAAATCAAGATCTGGTTCCAGAACCGGCGCATGAAGTGGAAAAAGGAGAACAAGACCAAGGGC | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | cds | InsectaCentral | Bicyclus anynana RNAi target | Bicyclus anynana RNAi RNAi construct | GTGCCGGGCAGTCCGCCCCTGGAGCAAGCTCAGCAGATGCCGCACCACATGCACCCGCAGCAGCACATGCAGCACGGCATGCCACCGCACCAGCAGCACGTCATGTACCCGGTGGACGACATGCAGCACCAGACGCAGATGCCGCCCATGCACCAGCAGTCCATGCACCCGCAGCAGGCGCCGCCTCAACAACCCCCGCCGAATACGAACGCGTCGCTCCCCAGTCCACTGTACCCTTGGATACGAAGTCAATTTGAACGAAAGCGGGGACGGCAAACGTACACCCGGTACCAGACCCTCGAGTTGGAGAAGGAGTTCCACTTCAACCGATACCTGACGCGGAGAAGACGGATCGAGATCGCGCACGCCCTCTGTCTCACCGAGCGCCAAATCAAGATCTGGTTCCAGAACCGGCGCATGAAGTGGAAAAAGGAGAACAAGACCAAGGGC | dsRNA | MEGAscript RNAi Kit, Ambion | column-based | subsequent to transcription | For the production of a template for in vitro transcription, PCR with vector-specific M13F and M13R primers was performed on a plasmid containing Antp insert and the promotor sequences for the T7 and SP6 polymerases. After cleaning with Wizard SV Gel and PCR Clean-Up System (Promega, Cat. # A9282), 1 µg of the PCR product was used as a template for transcription reactions with T7 and SP6 enzymes. Sense and antisense transcripts were synthesized using the MEGAscript RNAi Kit (Ambion, Cat. # AM1626), according to the manufacturerâs instructions. After purification in sterile 0.5xPBS, the quality of dsRNA was verified on an agarose gel, and its concentration measured with NanoDrop spectrophotometer (Thermo Scientific). | InsectaCentral | Bicyclus anynana RNAi RNAi construct | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | local resource | larva | microsporidia | microsporidia | lab_colony | 366 | injection | 0.5xPBS | 0.0143 | buffer | 23 | haemolymph | adult | phenotype | not checked | none | |||||||
| 99 | Bicyclus anynana ddc RNAi | RNAi | InsectaCentral | Unpublished | Saenko | Suzanne | Bicyclus anynana ddc RNAi target | AGGGCAGTGAGCGCGAGTCAGTGGTCAGGGTTAGTTATTTACTGCGAAAGCCCGGTTGGCTCATACATAATTCAGTGTCGGTTTAATAAGTTAAGTGTTCCTTTCGATCGAAAAAAGTATTAGTTGACGCAATGGAAGCTAAGGAGTTCAAGGAATTCTCGTCCGCGATGACTAACTATATCGCCGAATATTTAGAGAACATACGCGACAGACAAGTGGTGCCATCAGTGAAGCCTGGCTATCTGCGTCCACTGGTGCCGGAGCAGGCGCCTGAGAAGCCCGAACCCTGGACGGCTGTCATGGATGACATCGAGCGCGTGATCATGTCCGGGGTCACACACTGGCACTCCCCGCGTTTCCACGCTTACTTCCCTACCGCTAACTCGTATCCATCCATCGTAGCTGATATGCTTAGCGGAGCTATTGCGTGCATCGGATTTACTTGGATTGCTAGTCCCGCTTGCACTGAACTGGAAGTAGTGATGTTGGATTGGCTGGGTCAGATGATTGGACTCCCCGAAGAGTTCCTAGCGCGCTCTGGCGGCGAAGCAGGCGGCGTGATCCAAGGCACGGCCAGTGAAGCTACTCTGGTCGCGCTTCTTGGAGCGAAGTCCCGTACCATGCATAGATTGAAGGAGCAACACCCTGAATGGACAGAAGTTGAAATTCTTTCCAAACTTGTGGGATATTGTAACAAACAAGCCCATTCGTCTGTAGAGCGCGCTGGTCTTCTGGGTGGAGTGAAACTTCGCAGTTTAGCGCCTGACAATAAGCGACGTCTACGTGGGGACACCCTAAAGGAGGCCATAGAAGAGGATACTCGTAATGGACTTATACCTTTTTATGTCGTGGCGACACTAGGAACGACTTCTTCTTGCGCTTTCGACGCATTAGATGAGATCGGTGATGTTTGCAAAGAAAAAGACATTTGGTTGCATGTTGATGCTGCTTACGCTGGTTCAGCCTTCATTTGCCCGGAATACCGTTATCTGATGAAGGGAGTGGAGAAGGCGGATTCCTTCAATTTCAACCCACACAAATGGATGCTGGTCAATTTTGACTGTTCCGCTATGTGGCTCCAG | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | cds | InsectaCentral | Bicyclus anynana ddc RNAi target | Bicyclus anynana ddc RNAi RNAi construct | GACAAGTGGTGCCATCAGTGAAGCCTGGCTACCTGCGTCCACTGGTGCCGGAGCAGGCGCCTGAGAAGCCCGAACCCTGGACGGCTGTCATGGATGACATCGAGCGCGTGATCATGTCCGGGGTCACACACTGGCACTCCCCGCGCTTCCACGCTTACTTCCCTACCGCTAACTCGTACCCATCCATCGTAGCTGATATGCTTAGCGGAGCTATTGCGTGCATCGGATTTACTTGGATTGCTAGTCCCGCTTGCACTGAACTGGAAGTAGTGATGTTGGATTGGCTGGGTCAGATGATTGGACTCCCCGAAGAGTTCCTAGCGCGATCTGGCGGCGAAGCAGGCGGCGTGATCCAAGGCACGGCCAGTGAAGCTACTCTGGTCGCGCTTCTTGGAGCGAAGTCCCGTACCATGCATAGATTGAAGGAGCAACACCCTGAATGGACAGAAGTTGAAATTCTTTCCAAACTTGTGGGATATTGTAACAAGCAAGCCCATTCGTCTGTAGAGCGCGCTGGTCTTCTGGGTGGAGTGAAACTTCGCAGTTTAGCGCCCGACAATAAGCGACGTCTACGTGGGGACACCCTAAAGGAGGCTATAGAAGAGGATACTCGTAATGGACTTATACCTTTTTATGTCGTGGCGACACTAGGAACGACTTCTTCTTGCGCTTTCGACGCATTAGATGAGATCGGTGATGTTTGCAAAGAAAAAGATATTTGGTTGCATGTTGATGCTGCTTACGCTGGTGCAGCCTTC | dsRNA | MEGAscript RNAi Kit (Ambion) | column-based | subsequent to transcription | For the production of a template for in vitro transcription, PCR with vector-specific M13F and M13R primers was performed on a plasmid containing ddc insert and the promotor sequences for the T7 and SP6 polymerases. After cleaning with Wizard SV Gel and PCR Clean-Up System (Promega, Cat. # A9282), 1 µg of the PCR product was used as a template for transcription reactions with T7 and SP6 enzymes. Sense and antisense transcripts were synthesized using the MEGAscript RNAi Kit (Ambion, Cat. # AM1626), according to the manufacturerâs instructions. After purification in sterile 0.5xPBS, the quality of dsRNA was verified on an agarose gel, and its concentration measured with NanoDrop spectrophotometer (Thermo Scientific). | InsectaCentral | Bicyclus anynana ddc RNAi RNAi construct | Lepidoptera | Nymphalidae | Bicyclus | anynana | 110368 | local resource | pupa | microsporidia | microsporidia | lab_colony | injection | 0.5x PBS | 0.04 | buffer | 12 | pupal wing | adult | phenotype | not checked | none | ||||||||
| 101 | Spofr allatostatin C | RNAi | pubmed | 18541256 | Meyering-Vos | Martina | Spofr allatostatin C target | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | InsectaCentral | Spofr allatostatin C target | Spofr allatostatin C RNAi construct | GCTGCGCCAATGGAGGCGGACGATGAGACGGCTGAGAACACCCTCGTGGCGCATCCCGATGGTGACATGGAGCTCTCAGGCCCCTGGGATGCTATCAACACTGCCGCTCTACGCAAACTGCTGCTGCAACTTGATGCAGAGGACAGGATGGGCGGGGTGACCCGCTCGTGGCCCCAAGCTGAGCCCCGCGGTTGGGGTCTGCGGGCGTTGGACAGCCGTCTGGCGCGGCAGTGGAGGGCAGACAAGCGGCAGGTGCGATTCCGCCAGTGCTACTTCAACCCAATTTCCTGCTTCCGCAAGTGA | dsRNA | T7-Megascript Kit / Ambion | salt precipitation | subsequent to transcription | according to instructions of the kit, licl precipitation, denature at 95 °C 5 min, anneal at room temperature | InsectaCentral | Spofr allatostatin C RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | local resource | adult | lab_colony | 0.6000000000000001 | injection feeding | noctuide ringer | 5 | buffer | 3 | noctuide ringer was injected into or fed to larvae as control in the same volume as dsRNA | larva | qPCR | brain, gut | not checked | 48 | high | 80 | |||||||
| 102 | Spofr Allatotropin2 | RNAi | pubmed | 18541256 | Meyering-Vos | Martina | Spofr Allatotropin2 target | CAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCGAATTCACTAGTGATTCTAATACGACTCACTATAGGGCAAGCAGTGGTAAAACGCAGAGTACGCGGGAACCCAATTTCCTGCTTCCGCAAGAAGAAGCTGAAGGATTAAATTATTCTATTTCTTTCAATTGCATAGCATTACCAATTTACC | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | NCBI | AJ566903 | Spofr Allatotropin2 RNAi construct | CAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCGAATTCACTAGTGATTCTAATACGACTCACTATAGGGCAAGCAGTGGTAAAACGCAGAGTACGCGGGAACCCAATTTCCTGCTTCCGCAAGAAGAAGCTGAAGGATTAAATTATTCTATTTCTTTCAATTGCATAGCATTACCAATTTACC | dsRNA | T7-Megascript kit /Ambion | salt precipitation | simultaneously with transcription | according to the instructions of the manufacturer, precipitation by liCl, denature of dsRNA by 5 min 95°C, anneal at room temperature | InsectaCentral | Spofr Allatotropin2 RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | local resource | adult | lab_colony | 0.5 | injection | noctuide ringer | 2 | buffer | 20 | noctuide ringer was injected into animals in same volume as dsRNA | adult | qPCR | brain | not checked | 96 | high | 88 | ||||||
| 104 | Sl-ILP1 | RNAi | InsectaCentral | Unpublished | Iga | Masatoshi | Sl-ILP1 target | ACGCGGGGACATTCAAGCTTCAGCGTTTGAAACAGTCACATCTACCAGTTCTTCAACCCCCTCATCATCATGAAGTTCTACATCGTCTTTGCCTTGATCCTGGCCTGTGCTGCATGCTACGCTTCTCGGGAAGGAACTGATTTCTACTGCGGACGGAACCTGGCTATCACTATAGCAAAATTATGCTATAATTCTGAGATCGCTAAGCGTGATGCGGGCTGGTGGTTGCCTCCCCAAGGCATCAGGGCTCTGAACGGCGTTCGAGGCAAACGAGGGCCTGTTGACGAGTGCTGCGATAAACCCTGCTCCCTTAATGAACTCCTGTCTTACTGCTAAACTTGGTCCGATGTTGCTGTTAAATGTTGATTGTAATAAATGAGATTTATCTCTTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | 3utr 5utr cds | InsectaCentral | Sl-ILP1 target | Sl-ILP1 RNAi construct | GACATTCAAGCTTCAGCGTTTGAAACAGTCACATCTACCAGTTCTTCAACCCCCTCATCATCATGAAGTTCTACATCGTCTTTGCCTTGATCCTGGCCTGTGCTGCATGCTACGCTTCTCGGGAAGGAACTGATTTCTACTGCGGACGGAACCTGGCTATCACTATAGCAAAATTATGCTATAATTCTGAGATCGCTAAGCGTGATGCGGGCTGGTGGTTGCCTCCCCAAGGCATCAGGGCTCTGAACGGCGTTCGAGGCAAACGAGGGCCTGTTGACGAGTGCTGCGATAAACCCTGCTCCCTTAATGAACTCCTGTCTTACTGCTAAACTTGGTCCGATGTTGCTGTTAAATG | dsRNA | MEGAscript RNAi kit /Ambion | column-based | simultaneously with transcription | Followed the kit protocol. | InsectaCentral | Sl-ILP1 RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | larva | lab_colony | 1 | injection | TE | 0.015 | buffer | larva | RT-PCR (semi-quantitative) phenotype | Brain | not checked | 48 | none | |||||||||
| 105 | Sl-ILP2 | RNAi | InsectaCentral | Unpublished | Iga | Masatoshi | Sl-ILP2 target | ACGCGGGGATACATCTATAAACTTTCCCTAAAATCACACCGCAGCTGAAACGTAAAACATGAAGCTTATCCTGGTATCCTGTCTTCTGACCCTGGCGTATGCTGCAGCAAACCAGACCCCCGTGATCCTGTGTGGACGGCAGCTGGCTAACGCTAGAGTATTGCTGTGTTATGGGAAAGAATATGTCAATAAGAGGACAAGGATCAGCCCAGTTTCAGACTTTATGCCTGGCGAAAAAATGAATGAAATCGATTGGCCGTGGAGCGGGCGTCGAGGATCCTTAAGTGCCAACTGGTCACGATACAAGAGAGACGGACTGGTCGACGAGTGCTGCCTCAAGCCCTGCACTACCGACGTCATCCTTAACTACTGCTAAACGAAAATCAACCTTAAACCAAAGAAAATCAGAATGAAGACAAAAATTTTCTAACGCAAGAAAGTTGTCGGAAAACTTGCGGCAACCCTGAGAAGACTTAAGCAAGATGGCGTCATTTCATTCTGAACGCTAATGTACGTATATTTATTTAGATGTAAGCTAGTTACGTAACTAGTCCACATAAATAATTGTATATTTTATACTATAAACCTAATAAACGACCGAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | 3utr 5utr cds | InsectaCentral | Sl-ILP2 target | Sl-ILP2 RNAi construct | TCCTGGTATCCTGTCTTCTGACCCTGGCGTATGCTGCAGCAAACCAGACCCCCGTGATCCTGTGTGGACGGCAGCTGGCTAACGCTAGAGTATTGCTGTGTTATGGGAAAGAATATGTCAATAAGAGGACAAGGATCAGCCCAGTTTCAGACTTTATGCCTGGCGAAAAAATGAATGAAATCGATTGGCCGTGGAGCGGGCGTCGAGGATCCTTAAGTGCCAACTGGTCACGATACAAGAGAGACGGACTGGTCGACGAGTGCTGCCTCAAGCCCTGCACTACCGACGTCATCCTTAACTACTGCTAAACGAAAATCAACCTTAAACCAAAGAAAATCAGAATGAAGACAAAAATTTTCTAACGCAAGAAAGTTGTCGGAAA | dsRNA | MEGAscript RNAi kit /Ambion | column-based | simultaneously with transcription | Followed the kit protocol. | InsectaCentral | Sl-ILP2 RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | larva | lab_colony | 1 | injection | TE | 0.015 | buffer | larva | RT-PCR (semi-quantitative) phenotype | Brain | not checked | 48 | none | |||||||||
| 106 | Sl-ILP2-2 | RNAi | InsectaCentral | Unpublished | Iga | Masatoshi | Sl-ILP2 target | ACGCGGGGATACATCTATAAACTTTCCCTAAAATCACACCGCAGCTGAAACGTAAAACATGAAGCTTATCCTGGTATCCTGTCTTCTGACCCTGGCGTATGCTGCAGCAAACCAGACCCCCGTGATCCTGTGTGGACGGCAGCTGGCTAACGCTAGAGTATTGCTGTGTTATGGGAAAGAATATGTCAATAAGAGGACAAGGATCAGCCCAGTTTCAGACTTTATGCCTGGCGAAAAAATGAATGAAATCGATTGGCCGTGGAGCGGGCGTCGAGGATCCTTAAGTGCCAACTGGTCACGATACAAGAGAGACGGACTGGTCGACGAGTGCTGCCTCAAGCCCTGCACTACCGACGTCATCCTTAACTACTGCTAAACGAAAATCAACCTTAAACCAAAGAAAATCAGAATGAAGACAAAAATTTTCTAACGCAAGAAAGTTGTCGGAAAACTTGCGGCAACCCTGAGAAGACTTAAGCAAGATGGCGTCATTTCATTCTGAACGCTAATGTACGTATATTTATTTAGATGTAAGCTAGTTACGTAACTAGTCCACATAAATAATTGTATATTTTATACTATAAACCTAATAAACGACCGAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | 3utr 5utr cds | InsectaCentral | Sl-ILP2 target | Sl-ILP2 RNAi construct | TCCTGGTATCCTGTCTTCTGACCCTGGCGTATGCTGCAGCAAACCAGACCCCCGTGATCCTGTGTGGACGGCAGCTGGCTAACGCTAGAGTATTGCTGTGTTATGGGAAAGAATATGTCAATAAGAGGACAAGGATCAGCCCAGTTTCAGACTTTATGCCTGGCGAAAAAATGAATGAAATCGATTGGCCGTGGAGCGGGCGTCGAGGATCCTTAAGTGCCAACTGGTCACGATACAAGAGAGACGGACTGGTCGACGAGTGCTGCCTCAAGCCCTGCACTACCGACGTCATCCTTAACTACTGCTAAACGAAAATCAACCTTAAACCAAAGAAAATCAGAATGAAGACAAAAATTTTCTAACGCAAGAAAGTTGTCGGAAA | dsRNA | MEGAscript RNAi kit /Ambion | column-based | simultaneously with transcription | Followed the kit protocol. | InsectaCentral | Sl-ILP2 RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | larva | lab_colony | 2 | injection | TE | 0.056 | buffer | larva | RT-PCR (semi-quantitative) phenotype | Brain | not checked | 48 | none | |||||||||
| 108 | Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a non-midgut gene | RNAi | Data entered by Jennie. Background: RNA interference (RNAi) induced through double stranded RNA (dsRNA) has been used widely to study gene function in insects. Recently, it has been reported that gene knockdown in several insects can be induced successfully through feeding with dsRNA. However, it is still unknown whether phenotypic silencing of genes not expressed in the midgut occurs after ingestion of insect dsRNA. Principal Findings: Using chitin synthase gene A (SeCHSA) as the target gene, which is expressed in the cuticle and tracheae of the lepidopteran pest Spodoptera exigua, we showed that the growth and development of S. exigua larvae fed Escherichia coli expressing dsRNA of SeCHSA was disturbed, resulting in lethality. In the 4th and 5th larval instars, prepupae, and pupae, the mean survival rates of insects fed the dsRNA-containing diet were 88.64%, 74.24%, 68.43% and 62.63% respectively. The survival rates in the 5th instar larvae, prepupae and pupae stages were significantly lower than those of all controls, and significant lethality differences were also found between dsSeCHSA treatment and dsControl or ddH2O control in the 4th instar larvae. The effects of ingesting bacterially expressed dsRNA on transcription of the target gene, tissue structure, and survival rates of insects were dose-dependent. Conclusions: Our results suggest that SeCHSA dsRNA may be useful as a means of insect pest control. | pubmed | 19593438 | Tian | Honggang | Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a non-midgut gene target | GAGTGAGCGCGCGGCCTACCGACCGAACGCACGCGCAAACCATGCGCAGCGACGCTGTCCCGCGAGTGCACTCCTCGTTACATTGTGAAGTGATATACAGTATAAACACGGATTACAACAACGCTTATCTCCACAACATACTGAACTAATATTAATTAAAAATGGCGACGTCAGGAGGGAAACGGCGGGAAGAGGGCAGCGATAACTCCGATGATGAGCTTACACCGCTCGCTAACGATATTTATGGCGGAAGCCAAAGGACAGTACAAGAAACGAAAGGATGGGACGTGTTCCGGGAGTTTCCACCGAAGCAGGACAGTGGGTCTATGGAGACTCAGAAATGTTTGGAGTTCACAGTGCGGTTGCTGAAGGTGACGGCATATCTAGTCGTCTTCATTGCGGTCCTCGGGTCCGGAGTCGTAGCAAAGGGCAGCACGCTCTTTATGACATCACAGCTAAAAAAGGACAGGCGGATTGCGTATTGTAATAGGAATTTAGGTAGGGATAAACAATTTATAGTAAGTCTTCCAGACGAGGAACGAGTGGCTTGGATGTGGGCTATTTTGGCTGCGTTCGCCATACCAGAAATAGGAACACTCATTAGATCAGTGAGGATATGCTTTTTCAAAACTTCTAGACGACCGACTAGCACACAATTCGCTGTGATTTTCATAGCGGAGACGTTGCATACGATAGGAATGGGTCTCCTATTCTTTTTGATCCTACCAGAACTGGACGTGGTCAAAGGAGCAATGATTACGAACTGCCTTTGCATAATTCCTGCCGTTTTGGGCTTGCTCTCACGCAACTCCAGAGATTCGAAACGGTTCATAAAAGTGATTGTGGACATGGCGGCAATAGTTGCGCAAGTCACTGGATTCATTGTATGGCCACTATTAGAGAATAAACCTGTACTATGGTTGATACCAGTCGCATCACTATGCATATCCCTTGGATGGTGGGAAAACTATGTCACACGTCAGAGTCCGATAGGTATAATCAAAAGCCTTGGCAGATTAAAAGATGAATTAACCTTCACTCGCTACTACACGTACCGTTTTATATCTGTCTGGAAAATCTTGGTCTTCCTCATGTGCATTCTCTTCAGCATTTGGCTCGAAGGTGACGAGCCTGCCATGTTTTTCCAACTGTTCAATGCCGGTTTTGGACCACACAGTATCGTTGTCGAAGAGGTACAAATTCAATTAGGCGGGACCGTCATTCCTGATTTAGCTAATGTTACTTTAACCGGAGACTCAGTTGAGGTCGCAGCTGCTTACAAATCCGCATTCTACGTGATGCTTATCCAAATATTTGCAGCGTATATCTGCTACATATTTGGAAAGTTCGCTTGTAAGATCCTCATCCAAGGCTTCAGTTACGCGTTCCCCATCAATCTCGTCATTCCATTGGTGGTCAACTTGTTGATTGCCGCGTGTGGTATCAGAAATGGTGACAATTGCTATTTCCATGGGACAGTTCCCGATTATCTTTACTTCGAGAGTCCACCAGTGTTTACGCTAAGCGATTTCATATCTCGTCAAATGGCATGGATATGGCTACTATGGCTATTGTCGCAAACATGGATCACCATACACATCTGGACACCAAAAGCTGAACGTTTGGCCTCTACGGAGAAGTTATTCGTGATGCCAATGTACAACGGTTTACTTATTGATCAGAGTATGGCCTTAAACAGAAAGAGGAATGATCAAAGAGATGTTAAGACTGAGGACCTCGCAGAAATAGAAAAAGAAAAAGGCGACGAATACTATGAAACTATATCAGTTCACACGGATAACACTGGGTCTTCTCCAAAAGCTATTAAGTCATCAGATCAGATCACCAGGATATATGCATGCGCTACTATGTGGCACGAAACTAAAGACGAGATGATGGAGTTCTTGAAGTCCATTCTTCGGTTAGACGAGGATCAGTGCGCTCGGCGTGTAGCTCAAAAGTATTTACGAGTCGTTGACCCTGATTACTATGAATTCGAAACACATATCTTCTTAGACGACGCCTTCGAAATATCAGATCACAGTGACGATGATTCTCAGGTGAATCGATTCGTAAAACTGCTTGTTGACACTATCGATGAAGCGGCTTCCGAGGTACATCAGACGAACATTCGTATTCGACCACCTAAGAAGTATCCCGCGCCTTACGGAGGACGATTGACGTGGGTACTGCCAGGAAAGACGAAGATGATTTGTCACTTGAAGGATAAGGCAAAGATTCGTCACAGGAAACGTTGGTCTCAGGTGATGTACATGTACTACCTACTCGGTCACCGACTAATGGAGCTGCCAATATCTGTGGATCGTAAAGAAGTTATGGCTGAGAACACCTATCTGCTGACCCTGGACGGAGACATCGATTTCCAACCTCATGCTGTACGTTTGCTTATCGATTTGATGAAGAAGAACAAGAATCTGGGAGCTGCTTGCGGTCGTATTCATCCCGTAGGCTCTGGCCCTATGGTGTGGTATCAAATGTTCGAGTATGCCATTGGTCATTGGCTGCAAAAGGCAACTGAACACATGATTGGCTGTGTACTGTGTAGCCCTGGCTGCTTCTCCCTCTTCAGAGGAAAGGCTTTGATGGACGACAACGTAATGAAGAAGTATACATTGAGGTCTGATGAAGCTCGGCATTACGTACAGTACGATCAAGGGGAAGATCGATGGTTATGTACGCTGTTACTTCAACGAGGTTACCGTGTAGAGTACTCAGCTGCCTCCGACGCCTACACTCACTGTCCCGAAGGTTTTAACGAGTTCTACAACCAACGTCGTCGTTGGGTGCCTTCCACCATCGCCAACATTATGGACTTGCTTGCCGATTGCAAACACACCATCAAGATTAACGATAACATCTCCAGTCCTTATATCGCATACCAGATGATGTTGATGGGTGGTACAATCTTGGGTCCCGGAACTATATTCCTTATGTTGGTGGGTGCCTTCGTGGCCGCTTTCCGTATTGACAACTGGACTTCTTTCGAATATAACTTGTATCCCATTTTGATCTTCATGTTTGTATGTTTTACGATGAAATCCGAAATTCAATTGCTCGTGGCTCAGATATTATCGACGGCATACGCCATGATTATGATGGCTTTTATAGTCGGTACCGCGCTTCAGTTAGGCGAGGACGGTATCGGATCTCCTTCGGCTATATTTTTGATATCACTTTCGAGTTCGTTCTTCATAGCCGCTTGCTTGCATCCGCAGGAGTTCTGGTGTATTGTACCGGGAATTATTTATCTTTTATCTATACCCTCTATGTACTTGCTTTTGATTTTATATTCGATTATAAATCTTAACGTAGTATCTTGGGGTACTCGAGAAGTAGCTGTTAAGAAGACGAAGAAGGAAATCGAAGCAGAAAAGAAAGAAGCAGAATTAGCAAAAAAATCGGCAAAACAGAAGTCTTTGTTAGGTTTCCTTCAAGGAGTAAACAGCAATGAAGAAGAAGGATCTATAGAATTCTCGTTCGCCGGTCTATTCAAGTGTCTGTTATGCACGCATCCAAAAGGAAACGAAGAGAAAGTGCAACTCTTGCATATTGCATCTACTCTAGAGAAGTTGGAAAAGAAATTAGAAACTGTTGAGAGGGCTGTTGATCCTCACGGCATTAGCAGAGGACGTAAACTGTCGGTTGGACCAAGAGGTAGCACCACTGGAGATCATGGTTTGGACGCTCTAGCTGAAGGACCAGAAGAGGATAACGACTCAGATTCTGAAACTGACACACTTTCTACTGTGCCAAGAGAAAAGAGAGATGATCTCATAAACCCATACTGGATTGAGGATCCTGAGTTGAAAAAGGGTGAAGTAGACTTTTTGAGTCCCGCCGAATTATCTTTCTGGAAAGATCTCATTGACAAATATTTATACCCTATTGATGCTAACAAGGAGGAGCAGGCCCGTATATCCAAGGATCTGAAAGAATTGAGAGACTCGTCTGTTTTTTCTTTCTTTATGGTCAATGCTCTCTTTGTATTGATTGTATTCTTGCTACAACTGAACAAGGACAACCTTCACATTAAGTGGCCCTTCGGAGTTAAAACTAACATAACATACGATGAGGTTACTCAAGAGGTGTTAATATCAAAGGAATATCTGCAACTGGAGCCTATTGGTTTAGTGTTTGTATTCTTCTTCGCCTTGATCCTGGTGATACAGTTCTCCGCCATGTTGTTCCATAGATTTGGAACCCTTTCACACATATTAGCGTCGACAGAACTGAACTGGTTCTGTTCTAAGAAGTCCGACGACTTGTCTCAAGACGCACTATTAGATAAGAATGCAATAGCAATAGTAAAAGACCTGCAGAAATTGAATGGTCTGGACGACGATTATGACAATGACTCGGGCTCGGGTCCTCATAACGTCGGCAGAAGAAAGACTATTCACAATTTGGAGAAGGCGAGACAGAAGAAGAGGAATATAGGCACACTGGATGTGGCCTTCAAGAAGAGATTCTTCAATATGAACGCCAACGATGGACCAGGTACACCAGTTCTAAATCGTAAGATGACGTTGAGAAGGGAAACGCTAAAGGCTTTAGAGACGAGAAGGAATTCAGTGATGGCGGAAAGAAGAAAATCCCAGATGCAAACACTTGGTGCCAATAATGAATATGGAGTGACAGGAATGCTCAATAATAACTTAGGTGTCGGGCCGCGGCACAGGACATCTAATGCCAACATATCAGTGAAAGATGTTTTCGGCGAGCCAAACGGAGGTCAAGTCAACCGAGGCTACGAAACCACCATAGGCGACGAAGATGACACAAACTCAATGAGATTACAACCTAGACAAAACCAAGTTTCCTTCCAGGGTAGATTCTAAAAGAGTTGCCAAACTAAGTGCCTAGGTTAAACAAATGTAGATACTAGAAATATAGACTGTAAAATTTTTTACAATGAAGAATCTCAGCAAATGTTTGCGGGAATCATGTATTGCTTGTGATATATACTTTTTTTTAGGTCGTGACAGCAATGTGCTGTCATTAAATACCAACATCGGACAATTTACAGCTTTATTGTAAGATAAAACATACGAATTTACACTGCCACTTAACTTTACATTATTGATCTTTTTATACTTATTTAAAATAATATCTTTTGTATTAAAAGATTTTTGTTTTAGTTTTTATGAAAGTAGACATCGAATTAGTGTTGTATGTTTTTAATGGGG | Lepidoptera | Noctuidae | Spodoptera | exigua | 7107 | cds | NCBI | DQ062153 | Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a non-midgut gene RNAi construct | TTGTATTCTTCTTCGCCTTGATCCTGGTGATACAGTTCTCCGCCATGTTGTTCCATAGATTTGGAACCCTTTCACACATATTAGCGTCGACAGAACTGAACTGGTTCTGTTCTAAGAAGTCCGACGACTTGTCTCAAGACGCACTATTAGATAAGAATGCAATAGCAATAGTAAAAGACCTGCAGAAATTGAATGGTCTGGACGACGATTATGACAATGACTCGGGCTCGGGTCCTCATAACGTCGGCAGAAGAAAGACTATTCACAATTTGGAGAAGGCGAGACAGAAGAAGAGGAATATAGGCACACTGGATGTGGCCTTCAAGAAGAGATTCTTCAATATGAACGCCAACGATGGACCAGGTACACCAGTTCTAAATCGTAAGATGACGTTGAGAAGGGAAACGCTAAAGGCTTTAGAGACGAGAAGGAATTCAGTGATGGCGGAAAGAAGAAAATCCCAGATGCAAACACTTGGTGCCAATAATGAATATGGAGTGACAGGAATGCTCAATAATAACTTAGGTGTCGGGCCGCGGCACAGGACATCTAATGCCAACATATCAGTGAAAGATGTTTTCGGCGAGCCAAACGGAGGTCAAGTCAACCGAGGCTACGAAACCA | dsRNA | phenol_chloroform | simultaneously with transcription | In order to construct a plasmid that expresses dsRNA corresponding to S. exigua chitin synthase A (SeCHSA, GenBank accession no. DQ062153), a 635 bp fragment (dsSeCHSA) was amplified by RT-PCR using total RNA as a template. The forward primer was SeA3F (59-TCG CCATGG TTG TAT TCT TCT TCG CCT TG-39), which spans nucleotides 4156â4175, and the reverse primer was SeA3R (59-GAC AAGCTT GTC GCC TAT GGT GGT TTC GT-39), which spans nucleotides 4790â4771. The underlined portions of sequence are NcoI and HindIII restriction sites, respectively. Amplification reactions comprised 30 cycles of 94uC for 40 s, 58uC for 40 s, and 72uC for 40 s, with a final extension step of 72uC for 8 min. PCR products were confirmed by separation on 1% agarose gels and visualized by ethidium bromide staining. The PCR product was then cloned into the plasmid L4440 [4] between the NcoI and HindIII sites. The L4440 plasmid, which was provided by Andrew Fire (Stanford University, CA, USA) and obtained from Addgene Inc. (Cambridge, MA, USA), has two T7 promoters in inverted orientation flanking the multiple cloning site [13]. The resulting recombinant vector L4440-SeA3 was introduced into competent HT115(DE3) cells [4,9] lacking RNase III. The HT115(DE3) bacterium has the following genotype: F-, mcrA, mcrB, IN (rrnDrrnE) 1, rnc14::Tn10 (DE3 lysogen: lavUV5 promoter -T7 polymerase); the RNase III gene is disrupted by a Tn10 transposon carrying a tetracycline-resistance marker [9]. This bacterium can be induced to express T7 polymerase in the presence of isopropyl b-D-thiogalactoside (IPTG). The HT115(DE3) was provided by Lisa Timmons (University of Kansas, KS, USA) & Andrew Fire and obtained from CGC (Caenorhabditis Genetics Center, Minneapolis, MN, USA). To produce dsRNA, single colonies of HT115(DE3) bacteria containing dsSeCHSA or cloned L4440 were grown for 14 h with shaking in LB with 100 mg/ml ampicillin plus 12.5 mg/ml tetracycline at 37uC. The culture was diluted 100-fold in 100 ml of 26YT medium and allowed to grow to OD595 = 0.4. Synthesis of T7 polymerase was induced by addition of IPTG to 0.4 mM and the bacteria were incubated with shaking for an additional 4 h at 37uC. The expressed dsRNA was extracted as described previously [4,26]. The length of the dsRNA was confirmed by electrophoresis on 1% agarose gel. To prepare bacterial cells that express dsRNA, the procedures described above were used. Bacterial cells were collected from 100 ml IPTG-induced culture by centrifugation at 10,000 g for 2 min, resuspended in 0.4 ml (2506), 2 ml (506) or 10 ml (106) sterile water, and then used for S. exigua feeding bioassays. | InsectaCentral | Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a non-midgut gene RNAi construct | Lepidoptera | Noctuidae | Spodoptera | exigua | 7107 | local resource | larva | lab_colony | feeding | sterile water | non_specific_dsRNA | 12 | An unrelated gene of the D. melanogaster, white gene (DmWhite) (GenBank accession no. X51749), was selected for use as a control dsRNA. | larva | RT-PCR (semi-quantitative) phenotype | whole organism, cuticle, trachea | not checked | high | ||||||||||
| 109 | A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head | RNAi | The suboesophageal body of insects was identified over a century ago in the silkworm embryo, but its biological function is still unknown. We discovered that this tissue is differentiated in the earliest embryonic stages of the cabbage armyworm and secretes the insect cytokine, growth-blocking peptide (GBP), transiently from 24 to 60 h after oviposition when gastrulation is in progress. Over-expression of GBP, achieved by microinjection of the GBP gene driven by a cytomegalovirus (CMV) constitutive promoter, resulted in complex deformities of the procephalon (embryonic head). Severe abnormal phenotypes of the head structure were produced by silencing the GBP expression in the embryo by treating with GBP double-stranded RNA: the procephalon-containing optic lobes diminished and completely separated into bilateral halves. This indicates that GBP secreted from the suboesophageal body plays an essential role in the formation of the procephalic domain during early embryogenesis. The cytokine-induced fusion of bilateral procephalic lobes is thought to be evolutionarily conserved at least in insects, because of the widespread occurrence of the suboesophageal body in insect embryos. | pubmed | 15652706 | Tsuzuki | Seiji | A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head target | GAATTCTGTAAGTTGTAATCTCATCAGCATCTCAGGTTTCAGAGCAACTTGCTGCCGAAATACAATTAGGCGCATCACAGTTACGCTGCTTTAAGTTTCTAAATCCCAAACAGAGTTCCAGCTACTTTAGGACACAACAATTTTTTTTCAAAGCGAAAGTAAAATAAAAACGATGTTGTAACCGTCGCAACAAAGTCATACAATCAATATTGCTGTGAGAAATAAAACGATTTTAAGAAATGACATCATTTCTTATTACATACATTCCTTTCACATCATAATTTAATTTTACTCAATATGATTCTTGTCCTAAAAACAATACAGCAAAATGTTGACCGCTGCTACGCAACGTATCGTTATTACAACCCATACCAAGAGATAGCCACATTCCTTTTATTCAATCCGGCCAGCCGCAATACGACGTATTTTACAAATTGACCGCGACATCTATAAGTAGATACCTAACTCATCTTCTTATCTATAACATGTCACAAATTCATTTTGCAACATTTGACCTCATTATCGGAAATAACACAAAAATAAATTAAGATTCAAAATAGTTATTGCTTTCACGATTGTAAAGTTCTTGATACTACGTTACAGATCAATACCAGTAGTGGCTTGCATTTGCCCCTGACGATTAATTTATTCCACTCATAACATACTGTAGGCATGCATTTTTGTTAGTCAATTGACAATAGACGCAATGATAGAAGTCAATTGGCGTAATGGGATCAAATGGTCCATATAAATGTGAATATCTTGCAGCGCGAACTGTATTGATTAAAAACTCCCTCTTCATATTCTTCCTCTGAAACTAGATCCAGACAGACGTTATGAAAATACTGTGATGGATAAATTAGTTTTTGTTGTTGTGTTTTTATACTGCTTCGCATTTTCAAGCCAAGGATTAGTTTTGCATAAAGCGGTCGAGGATCAAACAAACTTTTACGAAGTCGATACAATAAAAGAGTCAGACGAACGTCCTATCATAGATTACCTGAAGGATAGTGAAGACAAATCTGAGCTCGCGTACATTATACTTGTGCCAAAAAACAGTCAAGAAAATTACAACAAATACATCATAATTAAAGCAAAACCAAAATTACTAGACCTCGAAGCGTATTCTCTAGTTCATCGTGTTGATAAATCTAAATTAATGGTTATGTGGCTCAATGTGTACTGCCAAATGCATCCGGACTGTGACAAGAACAAGTTAAATGAAGATCTGATGAAGGTACGCCCTTATTTAGAGTTTTAAAGCACAGTGAGAANAGAAGTACTTAANAACGATAACGATGGAACAACGGACGATTCTGACGGGGAAAATTAAATTGTAAATACGCAAACAGTCTAGTGGCACATAATCCACGTTGAAGTAATGTAAGTCTTGAACAAAGTGTGTGGACGAAAAGTAAATGAGAAGTCCTATCATTTTAATGAGTGTAAGTAGGTGACCCAGGCGTTCGAGACTAGGATTGACCCACATTCTGATGAATTGAAGATTTGAACAAGATCAGTTCATAGTTGTTGTTGCTAGATCGTGCGTGTTACTTCGTATTTTTATTTTGACTCTAAAATATAACCAGTGTTGTCTTAAGTGAGTTAATTTACATGTGATTCTCATAAGTGATTTTGTGTGTTTAACTTTAAAGATAAACACCTTAGAGTGGCGTGTTGCTACATTCTTGGATATTATTGATTTTATAATTAGCCAATGTGTTCAGAAATATGTAAAGTAAAATAAACAAATAGTTCAATAGAGATAAATTAACATCATGTTGGCGAATGTTATCGTATTTGTGTAGCGGCCTGGTAATTATTGGCTGATAAATGTACAGCGGGCAGCGATATGTTTTAGTATTGTAAAATGATTCTGTGTCGACACGTTTTATTATATTTAGTTTATTAATCAGCTTCCTAGTTTGTGCTGCCAGCACTTCGTTCACTGGACAGTTCATATTAGATATATTCGGGAACCCAATTAAAGACCTCAAGAATACATTTGTNAAACCAGAGAAAAAGGGAGGTTCCAAAACAAGTAGTGTACGAGAAGACTTATCTGCTGATGGAGCTAATCCTAAACCAAAAATGATAAACGTGCCGCGTAATATTGAGAGGTGTGGCGCAGGCCTAGCCATGGACGTGAACGGAGTGTGCAGACAACCATGGTGAAACGCCGCACTGACTATGTATACGTATAGTTGCGACGTAGTATGCTCGATGCACTACAATAAAATGGACGCGAAATGTGTGTTGTTTCTAATAGTGATGGTTCANAGCATGTGTTGTCTGCCTGAGCCAAGAGTTGTTTTTAATTTTCACGACAAACTTCGGATGGCGACTACGACTCCGGTCGAGGAATTGACGCAACATAGTCNAATAATACGAGTACCAGAACTGGAATGTCCCTTGGGGCAGAGAAGGGACATTCTANGAAACTGCAGGCAGAGACTTTAAAGGTACTGTTTTTTTATTATTTGGCGACATTATCCTCATAGGTAGTTACTAAAATCTAGGTAATTTAGTGAATAACTAAATTACCTAGATTTTAGTAACTGGTGTTAGTTTATCAATTGTATATAAAATAAACCGATTTAGTCATAACGAGTAGCGTGACCTGTGACATTTATTATCCATACCCATTTATAATTATGTTGCATGAATACAAAAATAATAAACGATATCTATCTTAGTGTGAAGATATTTTGAAATTATAGTACACTGAATTGTAAAAAAAGAACATATATTTTTGTGTGTTCACGAGTAGATACTTTTTTGAGAATAGATTATATTTACTTGAGACCTATGCACTTAAAAACACCTACATTACTCTAACTAAAACTGTTTTATTTCAGATGGTGCTTTTGATATATTACGTTGAAGACTTGGTGAAAAATAATTTGCCGACAGGAAAATATCTAGTATTTAAAGAGGATAATTGAATGTACTCGTATTAACTCTTAAGTATTTAATCAGTTCTCGGAATATGTTTGTAAGATAAATTTTAAAAGGTCAATTGAGTTATCTTATAAAGCGAAGTCATTCTCGATACTTCAGTCAAGAGAAAATTATCTGAACAGACGTGACACGAACATTTTAATTTCACAAAATGAAATTAACTATTACCATTTTATTTTGTGTTATTTTAACTCTACAATATAACGGTGCTGATGCGCAATTCAAGGATTTATTCGGGAAGATCCATGATTCAGTACATGGGACAGCAGATAAGGTGAAACAGGATTTAAATACCATATTCAATCCAAATGCGAAGAAGCAACAGGAGAACAAAGATGAGTCCTCGAATATTCACTTTGTTGAAGATGAGTCGGAAGAAGACTTTGCCGCTGCTGCAGCTGCTAGGAAACCAGTTGAAGTAACTCCTGCACCAGAGGCCAGTGAGACAACAATAGAACCGGTGACCAGTAGCACTCCACTTGCATCTTCGGAGAATGCGACCACACTTGCACCCTCCACGACCACTGTTAAAGGCCGCGAGAACTTTGCGGGCGGATGTCTCACCGGCTTCATGCGCACTCCTGACGGAAGATGCAAACCCACCTTTTAGCAATTAACTACTAAATAGCTAAGACATGCCAAGGTAACGCAAACTTTTACCGTAATGTTAGTCTGTTACGTCTGCATCTTATTTGTTTGTGTGTAACAATGTAAGTAGTTATTGTTATTTTACGACTTTTAACTGTATACATGTTTAAATATCCATTGTAGAGTATATTTGTTGATTTCTTTGTTGTAATTAAGGTGTTATACGTAAAATAAAATACTTAGACAGTTCTTAATAGATTTGTTAATGAAAGTTTTACATAAATAGTGAAATTATTGAAATGGAATTAAAGTTTTTGAATAATTCAAAAGTTTAAAACCTTTATTTAGCTTCCTGTCTCAACCTTACGGAAAGTAGANCACTGTATATTATCCAGTTCTTCTAAGTCTAAATATGATTTTAAGTGCTATTGGAAATTATTACTTAAAGGGGATTTTATATTTATCATGACTCTTTGTGTATTTTCGATTGCCCAGATAAGCAATTGTTTATACTTAATAGATAAGGCACGCCCTCAGTTGAAAATCCACTTTAATGAAGTTATATTATTCCTTGAAATGCTAACAGTCAATAAAAATAAGTTGTCAATTCACAAAAATAATTTAGACAAGAATTTCAGTTAATTTCAGTCTTCAAATTAAGTAAGGTTTTAAAAACAACCTAAAGTCTAACAACGACTGTGACTAATTGGATAATCATTTTATTTGACCAGTCTAAATTAAATATAGTATTCTTAATTAATGAATAAACATGTTAGACATTAAAAATTCATAGTTTAAAGTAATATTCTTATTTCCTAACTTANGGCCCAGTAATGGGTACTCTTTCACTGGGGTGTTTTTTTTACTTTTGTCACTAAATTTGAATTAAAATTACTCTTCCGGAGTGACAGCTACTGCCTGAAATATTGGATAAGCTGGGGCCCTTGTTGGTGGTTGCTTACTTGTATTGTCATTTACTTTATCTCCGAAATATTCATGTAGAACAGCTACCAGTTGTTGCTTGTTTTTGTCATTTTCTTCTTTATTCTCTTCTTTATTACCTTCGGGAACCGCATTGTTGTTTGGTCTATCATAATTGTTTCCAGCGTCATTGCCTACATTGTCTTCGTAGTTAGGTTGTCCGTTTTGTGTTGGCCCGTTGTTGTTTGGTGGTCCTCTATTGCCGGGAGGTCCTCTGTTGGCTGGTGGACCTCTGTTGTCTGGTGGGCCTCTGTTGTCCGGTGGACCTCTGTTGTCTGGTGGCCCTCTGTTAGCTGGTGGTCCTCTGTTTGCAGGCGGTCCTCTATTAGGATTACCATTACCTTTATTTCGTCCCCAACCTTGGGGCGACCGTTTGTCTATAAATAATAAAACACATGATTTAATTTTAGGATATTATTTAATTAAGCATCAATTTTTATATAGATTTATAAAAACTGATGCTTTTTTAAAGGCTGGCTTAGCTCATACATGCACGAAATTTTATGATGATAGGTTCCTATAGGAAAAAATATTGTTCATACAAGTAACCGTTTTTGCATTTGTGTAAAAAGTCTAATAGTCTATGTCTGTTCTAACTTATAACACGAACTACTATAGCTGTGTCATGTTTTTTTTTGTATCTCTAAGTATATGCTTAATACAAATAATAACATATTATATTAATCTTTAGACCTTTAACGTATCTTTGTATTATTCTAAGTTTAATAACCTTGTGTACAACAGGTACCTACGTTTAAAGACATTGGTAAACATTTAACTAATAAATAACACATAAAAAAAATCAAAACTTTATTAAAAAAATATATATAAAACAGAGTTATAATTAATAAGCTTCTTTTCTTTCGTAGTTTTGTTATTGCATAGGTTTTGCAAACATAGGTCACAAGCTTTAATTTAATACTGACAAAATGTAGCTGTATATTTAAAATCAATCCTCCGGCGTAACAGGAGCAGCTTGAAATATAAACGGAGACAGTGGTCGAGCCGTTGTTACAGTTACATCCCTTTTAATGGCTTCAGCTGTCTTCTGTTCATCATCATTAAATCTAACGTCTAGCTGGTACTGATCATTATCATCATCAGCTTCAGTTGTCGAANATGTTTGAGTAACATCAATCACTTTACCCGAATGATCTTCATTCTGCGAGTTTTTTTTGTTTTCATTGTCTTGGTCGTAATATTGTTCTCGCTGAAGTGGTGGTCTTCGATCATTANGCGGTCGTCGATCGTATGGAGGTCTATTATCAAAACGATCTGGTGTTCTATCTCGGGGTGGAGGTCGCCNGTCATCANAACGGTGTGGGGGTGGGGGTGGTCTATTATCATAACGGTCNGGGGGTCCGCCGTGATGTGGAGGTGGTCNACGATCATATCGATCTGGTGGTCCGTCTCGGGGAGGTGGTGGTCCACGATCANACCGATCAGGTTGTCCGTCTCGGGGTGGTGGCCCACCANGGNGCCGAGGCGGCCCACCATAATCCAGGGGTGGTCCCCGATGGTGCCGTGGTGGTTCGTCATATCGTGGTGGTCGAC | Lepidoptera | Noctuidae | Mamestra | brassicae | 55057 | cds | pubmed | AB126696 | A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head RNAi construct | GTGCTGATGCGCAATTCAAGGATTTATTCGGGAAGATCCATGATTCAGTACATGGGACAGCAGATAAGGTGAAACAGGATTTAAATACCATATTCAATCCAAATGCGAAGAAGCAACAGGAGAACAAAGATGAGTCCTCGAATATTCACTTTGTTGAAGATGAGTCGGAAGAAGACTTTGCCGCTGCTGCAGCTGCTAGGAAACCAGTTGAAGTAACTCCTGCACCAGAGGCCAGTGAGACAACAATAGAACCGGTGACCAGTAGCACTCCACTTGCATCTTCGGAGAATGCGACCACACTTGCACCCTCCACGACCACTGTTAAAGGCCGCGAGAACTTTGCGGGCGGATGTCTCACCGGCTTCATGCGCACTCCTGACGGAAGATGCAAACCCACCT | dsRNA | MEGAscript RNAi kit (Ambion) | other | simultaneously with transcription | For RNAi (Fire et al., 1998), the 0.4 kb-DNA fragment containing an ORF of GBP gene was amplified by PCR with a set of primers, which tagged T7 RNA polymerase-binding site (50-taatacgactcactatagggagacggtgctgatgcgcaattca-30/50- taatacgactcactatagggagaggtgggtttgcaccttccg-30; 1.3kb cDNA position 558â578/940â959). The PCR product purified using Gene pure (Nippongene) was used as a template for in vitro synthesis using a Megascript RNAi kit (Ambion). | InsectaCentral | A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head RNAi construct | Lepidoptera | Noctuidae | Mamestra | brassicae | 55057 | local resource | embryo | lab_colony | 0.5 | injection | water | non_specific_dsRNA | Mock-treated embryos in RNAi were injected with dsRNA of GFP | embryo | RT-PCR (semi-quantitative) | whole organism | not checked | 100 | high | ||||||||
| 111 | Immune upregulation of novel antibacterial proteins from silkmoths | RNAi | Entered by Jennie Study on immune proteins in domesticated and wild silkmoths Bombyx mori and Antheraea mylitta, respectively, led to identification of a new class of antimicrobial proteins. We designated them as lysozyme-like proteins (LLPs) owing to their partial similarity with lysozymes. However, lack of characteristic catalytic amino acid residues essential for muramidase activity in LLPs puts them functionally apart from classical lysozymes. Two LLPs, one from B. mori (BLLP1) and the other from A. mylitta (ALLP1) expressed in a recombinant system, exhibited a broad-spectrum antibacterial action. Further investigation of the antibacterial mechanism revealed that BLLP1 is bacteriostatic rather than bactericidal against Escherichia coli and Micrococcus luteus. Substantial increase in hemolymph bacterial load was observed in B. mori upon RNA interference mediated in vivo knockdown of BLLP1. We demonstrate that the antibacterial mechanism of this protein depends on peptidoglycan binding unlike peptidoglycan hydrolysis or membrane permeabilization as observed with lysozymes and most other antimicrobial peptides. To our knowledge, this is the first report on functional analysis of novel, noncatalytic lysozyme-like family of antibacterial proteins that are quite apart functionally from classical lysozymes. The present analysis holds promise for functional annotation of similar proteins from other organisms. | pubmed | 17550822 | Ghande | Archana S | Immune upregulation of novel antibacterial proteins from silkmoths target | TTCGAGGATCCGGGTACCATGGGAAAGATACGATCGAAATGCAGAAGTTAATTATTTTCGCTTTGGTTGTCCTCTGCGTTGGTTCTGAAGCCAAAACGTTCACGAGATGCGGTCTCGTGCATGAGCTGAGGAAGCATGGCTTCGAAGAAAATCTCATGAGGAACTGGGTATGTCTGGTCGAGCACGAGAGCAGCCGTGACACGTCCAAGACGAACACGAACCGTAACGGCTCGAAGGACTACGGATTGTTCCAGATCAACGACCGGTACTGGTGCAGCAAAGGCGCCAGTCCGGGCAAAGACTGCAACGTTAAGTGCTCCGACCTCCTGACTGACGACATTACTAAGGCAGCGAAATGCGCTAAGAAAATTTACAAACGTCACCGCTTCGATGCCTGGTACGGTTGGAAGAACCACTGCCAGGGCTCTCTGCCTGATATTAGCAGCTGCTAAATTCCACTGTGAGCTTCAGCTAGACTTTCGATTCGATCTCCTCGAAACAGAACTGTGTGTTTAGTGCGAGTTTTTTAACGTTCTCGATAGCGTAAAAGTGTAATGAAATTTGTATGTAGTTGGAACAGCGCCCCTAGCGGCAAACGTAAGCAAACATTCCAACTCCATATAAATTTAAGTTAACTTTTACGTTATCGAGAATGTTAAAAAACTCGCACTAAGCACGCAGATTGCGTATTCAAATCTGGCGCGCAATCGCTTCGACAAAAATTCGGGGATATCATTCAATTCATTTACTCGTAACATTTTAGACATTTAAACAAACACTTCTCACAATGTTTTTTTTTGAAGCTACCTCGATCGATTTAATTGGAAAACATTGATCTATTCGAACTGATTGTCATAAAGCTGTTTTTTTTTTCCTTTTTAATGCTGTAAGAAGATACCTTCAAAATAAAGGATGATATTTCCTAAAGATAATTCAAGTCAAAATAGATTTTGATCGTAATGAGTTTCTTCACATAAACACTACCGTGCCAGATTTAATTCGTCTTCATGAAAATACACGGCGTTTCTCGATTTTCTAGCTGATACTGACGCTAATGTGAACATTATTAATTGAAGTAGCTTAAAATCATTTTAGTGTGTGATTGTTGAGTTAATTTGTTTTCTGACGATCCTTTTCGTCAATTGGAAAATTGTGTCGCTAAAATAAGCCAGAACTTTTAATTGAATTGAAGCCGCGAACGAATTTATTAATGCCCGTCTGCTAATTAAATTGTTATTTGGTAAAATGTGATAATTAGGTTGATTTTAATTTTTTGTTATTTCATTTGGTACCC | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | L37416.1 | Immune upregulation of novel antibacterial proteins from silkmoths RNAi construct | dsRNA | MEGAscript T7&SP6 kit (Ambion) | phenol_chloroform | subsequent to transcription | BLLP1 cDNA (357 bp) was cloned into pCRII-TOPO vector (Invitrogen) and amplified with M13 forward and reverse primers. This template with flanking T7 and SP6 promoters was utilized for in vitro transcription reaction and sense and antisense RNA strands were generated with the T7 and SP6 Megascript kits (Ambion). The DNA template was removed from the transcripts by DNAse treatment and the RNA products were subsequently purified by Trizol extraction (Invitrogen) followed by isopropanol precipitation. The complementary single stranded RNAs were dissolved in DEPC treated water, combined in equimolar amounts in 1X insect buffer saline (IBS, composition: NaClâ160mM, KClâ10mM, CaCl2â4mM) and annealed by heating to 95 1C and slow cooling overnight at room temperature. Similarly, dsRNA specific to green fluorescent protein (GFP) was synthesized as a non-specific control. The dsRNA formation was confirmed by agarose gel electrophoresis and the concentration was determined spectrophotometrically. To determine the in vivo role of BLLP1 in immunity, we knocked down the BLLP1 expression by RNAi and then assessed the effects on immune function by an experiment mentioned below. | InsectaCentral | Immune upregulation of novel antibacterial proteins from silkmoths RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | injection | Insect buffered saline | non_specific_dsRNA | 5 | 1 microgram/larvae | dsGFP | larva | RT-PCR (semi-quantitative) | Fat body | not checked | 24 | low | ||||||||
| 112 | RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding | RNAi | Entered by Jennie RNA interference (RNAi) or gene silencing is typically induced in insects by the injection of double-stranded RNAs (dsRNAs), short interfering RNAs, or through the use of hairpin constructs in transgenic insects. Here we demonstrate in the horticultural pest, Epiphyas postvittana (Lepidoptera: Tortricidae), that RNAi can be triggered by oral delivery of dsRNA to larvae. Transcript levels of a larval gut carboxylesterase gene (EposCXE1) were reduced to less than half that of controls within 2 days of being fed EposCXE1 dsRNA. Transcript levels of the pheromone binding protein gene (EposPBP1) were reduced in adult antennae by feeding larvae EposPBP1 dsRNA. Knockdown of Epos-PBP1 transcripts was observed for the first 2 days after adult eclosion but recovered to wild-type levels at 4 days posteclosion. The potential mechanisms involved in the initiation, movement and amplification of the silencing signal are discussed. | pubmed | 16756557 | Turner | C T | RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding target | CTACCCTCCAATGATTGAAACGGAACTGTTGCTTAATTCAAGTGGCCTGCCATTGTACCATTATTTCCTCACATATGATGGATGGAGGAACATCGGCAAATTTACCTCTGGCTCCATCTTTAGAAACTCGCCTGGGATGTCGCATGCAGATGACCTGTTTTACTTGTTTTACCAATCTTTTATTCCTGCGTGGTTTGAGATGGAAATGATCAATAAAATGACAACCTTGTGGACTAATTTTGCAAAGCATGGTGACCCAACCCCAGAAATCTCGGAGCTGCTGCCGCGACGCTGGCTTTCCTCCAGCAAGGAGGCGCCGCAGGCTTTAATAATAGATAAGGACTTATCTACTATCCCACTATGGCATAGGGAATCATTGAAATATTGGAAAGACTTATACTCAAAATACAGAAAGAAATAAATGAACGCAAAACCTATAGAGGCGAGTGCTGTGGAAGATTTACCAGAGACCTAATGTACAAGCTTGACTTGGAACGTCACTGGCATAAAATCACTGACAAGCAATTTCTGAGTTAATGTCTATTGTTGTTTGGTCATGCTAAAATAAATATGTTGTAATTAAAAAAAAAAAA | Lepidoptera | Tortricidae | Epiphyas | postvittana | 65032 | cds | GenBank | DQ209211 | RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding RNAi construct | dsRNA | Ambion kit | phenol_chloroform | subsequent to transcription | To synthesize dsRNA, 1 Îĵg of PCR product was used as a transcription template with 7.5 mM each of ATP, CTP, UTP, GTP, 20 units of T7 polymerase and supplied reaction buffer containing 40 mM TrisâHCl (pH 7.9), 6 mM MgCl2, 2 mM spermidine, 10 mM DTT (Ambion, Austin, TX, USA). Reactions were incubated for 2 h at 37 °C and then stopped by heating to 65 °C for 5 min. Transcription reactions were analysed by agarose gel electrophoresis and compared with reactions containing no T7 polymerase as a negative control. DNase I (Invitrogen) was used to degrade the remaining transcription template according to the manufacturerâs instructions, and dsRNA was purified by phenol/chloroform extraction and ethanol precipitation (Sambrook & Russell, 2001). dsRNA was then resuspended in RNase-free water and quantified spectrophotometrically. Standard precautions were used for manipulating RNA (Sambrook & Russell, 2001). | InsectaCentral | RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding RNAi construct | Lepidoptera | Tortricidae | Epiphyas | postvittana | 65032 | local resource | larva | lab_colony | 4 | feeding | RNase free water | buffer | (0.25 microlitres/insect) | Control insects were fed 0.25 Îĵl droplets of 10% w/v sucrose solution containing no dsRNA. | larva | qPCR | Gut | not checked | 168 | low | 35 | |||||||
| 113 | RNA interference in the light brown apple moth, PBP | RNAi | Entered by Jennie RNA interference (RNAi) or gene silencing is typically induced in insects by the injection of double-stranded RNAs (dsRNAs), short interfering RNAs, or through the use of hairpin constructs in transgenic insects. Here we demonstrate in the horticultural pest, Epiphyas postvittana (Lepidoptera: Tortricidae), that RNAi can be triggered by oral delivery of dsRNA to larvae. Transcript levels of a larval gut carboxylesterase gene (EposCXE1) were reduced to less than half that of controls within 2 days of being fed EposCXE1 dsRNA. Transcript levels of the pheromone binding protein gene (EposPBP1) were reduced in adult antennae by feeding larvae EposPBP1 dsRNA. Knockdown of Epos-PBP1 transcripts was observed for the first 2 days after adult eclosion but recovered to wild-type levels at 4 days posteclosion. The potential mechanisms involved in the initiation, movement and amplification of the silencing signal are discussed. | pubmed | 16756557 | Turner | C T | RNA interference in the light brown apple moth, PBP target | ATGATGAACCATAAGGAATTAGTTTTGTTCGCGGTAGTATGCCTGAGTTTGTACCAGGCAGTAGAACCGTCGCAAGATGTAGTTAAAGACATGTCTTTGAACTTTCGTAAGGGACTAGACGCATGCAAAAAAGAGGTGAGCTTTATAATACGGTAAACTTCTTAGTGCCATGCTAATGCCTTATAGTTAGTTGACACCGCTCTGTCACCCCTATTGCTAATGACTTAACATTCAAGATGGCAACTGGGACTGACAAGGGTATGATCTTAGTAATGTAGTTGGTTCATACACACTTTTTTTGTTCGCGTGTCTTCTGTGGACTTTGCAAAAGTTAAGAGCATTTCTACATAAGATATTTTATTTATGTCTCCATCCACATGTAGGAGATCTTGCCATATTTTAGCCCATTTAATACTTTTAAGTCTTAGGTTTGATGCCTCTAAGTCGTTCATTTTGAGGCCACAGGAGAGACAACTTTTGTTTCGATTTGTGTGTATGACCCAGTTCTATAAAGTTTACGTTTATTATCTGCCGAAATTTTCGCCAGTAAATCACGAATACGGTAACAATACAAACCATATTGCATTTTGAAAAACTATCTTAGTCTTAACCCAGTATTCTTTAAGACAGCTTTCAGTGTCACGCTGGTCGTCGGCGAAGAGAGATCAGCGCACTGCTGTTGAACCTTAAAACGCTTCTAGGATATAATACATATTGGCCTGTCAGCACCAATGGTCAGCGTGCAATCAGCGTGACATTAGAAGCAGTGGGCTTATTGTCTAACGCAATTGGTTTCGGTATGACATTCGCCGTTTTTTTTGGCAAATGCTAAAAGATCATGCGAGAAACAGATGGAAGGGTGACAAATGAAGCCAACTGCTGTAGACAATGTGGGCTCTCTTCCTCTGGGAGCGCGCAACTCAGCGTAGCCAACCGCACACTACGTGCCAAGGCTGCGCCCAGTAGCCGCGCGATCAGAGCCTTGGTATGCAGGTGGCTACCGCCGAGTCGCGCGCTCCCAGAGGAACGACTACGAACTAGCCCGAAACTAAGGCTAAACTCGACTTATATACGTTTTAACACATTATGTCTCAGTCTCACGGTAGTTTCATGATAAAAATGTGGCCTCTGTTTAACAAATGCAAAACCATTCCCAGCTGAACCTCCCAGACACGATCAATTCAGACTTCAACCGCTTCTGGAACGACGACCACGTAGTGACCAACAGAGATACGGGATGCGCCATCATGTGCTTGTCCAGCAAACTGGAGCTAGTGTCGGACACCGGCCTGCATCACGGGAACACGTTAGAATACGCCAAACAGCACGGAGCTGGTGAGGACACGGCTCTTTCTTCATCATTATTTTTAAGAGTTCCACTCTTGTCGGTGGAGTTTCTTCCAATCATCTCCTAAGCGCTAACTCCTTGCCCTCCTGCCTGATACGATACGACACCGGCTTTTTCCCTTATCTGGTCCATTGTAGCTACGTCTAGGGCGACTTCCTCTATAGAAGCTGCAAAAAAATTAGGTCTCCCTAAGTGGGTTTCCCTAGCCTAGAGAATTTTGGTATCTTTACCAAAAATATGCAACGTTGAAAGACTTGGAAGGAAGCAACGAGTTGGGCCTCTATTGGACACCGGTTCCAGCAATTCCAAAGAGACTCGAGGCACATTTGAATCGTGGTACTCTTAGTGGGGTGTGAAATACAGATGTGGCCTGCGCCTCACTGCGATATTGCTGGGAACTTTCCCCTGTTTTTGTTATGCTAAAGCTTAGCTAAAGCAAGCAAGGCAAAGCTTATACTTATTGATACGTATTACTCCGGCTGGATACCTTTATCTATACCTATAAAACAGTAACGTGACTGACTGACAGACAATGCACAGGCTAAACCACCGGAGCTAGAGACTTCAAATTTGGCAGAAATGTAGCTTGAGTATCCTAGAGGTGCACTAAGAAGGGATTTCCTAAAATTCCTATGGGATAGGGAGATAGCCAGTTTTTTATTCTTTCACGGGAGCGAAGCTAGTTAGAAATAATTCAGCTTATTAACTATTACCTTATTCCAGACGACACTGTGGCCCAGCAAATAGTAGATCTCCTCCACTCCTGTGCGCAGGCGGTACCAGACCTCGAGGACCCTTGCTTGAAAGTGCTGGAATGGGCCAAATGTTTCAAGGCCGAGATCCACAAGCTCAACTGGGCACCGTCCGCGGAAGTTATGGCTGCAGAAATGCTGGCTGAAGTATAA | Lepidoptera | Tortricidae | Epiphyas | postvittana | 65032 | cds | NCBI | AF416587 | RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding RNAi construct | dsRNA | Ambion kit | phenol_chloroform | subsequent to transcription | To synthesize dsRNA, 1 Îĵg of PCR product was used as a transcription template with 7.5 mM each of ATP, CTP, UTP, GTP, 20 units of T7 polymerase and supplied reaction buffer containing 40 mM TrisâHCl (pH 7.9), 6 mM MgCl2, 2 mM spermidine, 10 mM DTT (Ambion, Austin, TX, USA). Reactions were incubated for 2 h at 37 °C and then stopped by heating to 65 °C for 5 min. Transcription reactions were analysed by agarose gel electrophoresis and compared with reactions containing no T7 polymerase as a negative control. DNase I (Invitrogen) was used to degrade the remaining transcription template according to the manufacturerâs instructions, and dsRNA was purified by phenol/chloroform extraction and ethanol precipitation (Sambrook & Russell, 2001). dsRNA was then resuspended in RNase-free water and quantified spectrophotometrically. Standard precautions were used for manipulating RNA (Sambrook & Russell, 2001). | InsectaCentral | RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding RNAi construct | Lepidoptera | Tortricidae | Epiphyas | postvittana | 65032 | local resource | larva | lab_colony | 4 | feeding | RNase free water | buffer | (0.25 microlitres/insect) | Control insects were fed 0.25 Îĵl droplets of 10% w/v sucrose solution containing no dsRNA. | larva | qPCR | adult antennae | not checked | 48 | low | 30 | |||||||
| 114 | Bombyx mori abdominal B (abd-B) | RNAi | Pterygotes lack abdominal appendages except for pleuropods and prolegs. The larvae of some holometabolous insects develop prolegs, which are used for locomotion. We analyzed the role of the homeotic genes abd-A and Abd-B in lepidopteran proleg development using mutant analysis and embryonic RNAi in the silkworm Bombyx mori. The EMu mutant developed extra prolegs in its posterior abdomen and showed the misexpression of both genes, suggesting their involvement in proleg formation. The depletion of Abd-B by embryonic RNAi caused the development of extra prolegs on all segments posterior to A6, indicating the suppressive function of Abd-B. The abd-A RNAi animals failed to develop prolegs. These results indicate that abd-A and Abd-B are involved in proleg development in B. mori. | pubmed | 19389347 | Tomita | Shuichiro | Bombyx mori abdominal B (abd-B) target | ATAAACGATCCGCGGCCATAGTAGAGGTGCACGCGCGAGAATCGATCCGAATCGCCGGCGATAAACGCATCTCCCGCGAGGCGATCGTCGCGCGCCGCGTCGCCGCCGAGCGATGAGTTCCAAGTTCATCATCGATAGCATGCTCCCAAAATACCACCAGCAATTCCACCACCAGAATTTATTTGCCGGTGCCGGAGCGTCGCCTATAGAGGCGTCGCTGTCGTCTTCGCTGTCATCGTCGCTGTCTACCTCGCTGTCTAGTTCGCTGTCTGGCGGACTGGGTGCGGCAGCGCTGGGAGCAGGCTCACCGGGTGCCGGGAGCCCGCAGAGGTCTTCATCTTCATCGTCGGCGTCGCCGGGGGCGCCGGCGAGAATGTATCCGTACGTGTCGCATCACCAGCAGTTCGGGGGCTCAGTGCCGTTCTCGGCGGGCGGGGGACTGTCAGCAGCAGATGACAAGAGTTGTCGATACCCGACCGCCGTGGGTCCTGATCCGATGGTGAATTACGCTCTGGGTCAGCACAATGGAGGCGCAGCTGTGTCTGCTGCGTCGGCGAGCATGGCGGCAGCGGCTCAGTTTTACCACCAGGCAGCAGCTTCAGCGGCCTCCGCAGCTAACGCAGCTACCGCGGACGCCATGGGGGTGGCCTGCGCTCAGCCCTCAGCACAGACGCTCCCTGAAATCCCCCGATACCCGTGGATGTCCATCACTGATTTTCCATTTCCAGATTGGATGAACCCCTTCGACCGCGTGGTCTGCGGTGAGTTCAATGGTCCAAACGGATGTCCGCGAAGGCGAGGACGACAAACTTACACTAGGTTCCAGACTCTAGAACTAGAAAAGGAATTCCACTTCAATCACTATCTGACGCGCCGCCGCAGGATAGAAATAGCACACGCGCTATGCCTCACCGAGAGACAGATCAAAATATGGTTTCAAAATCGACGCATGAAACTAAAGAAAGAACTACGAGCCGTGAAAGAGATAAACGAACAGGCTCGCCGCGAAAGAGAGGAACAGGACAGAATGAAGCAGCAGCAACAAGAGAAGCAGGCCAAGCTGGAAGGCCAGCATCACGGACACCACGTCACACATCATCACGACCCGATGAAGATGCCTATCGATAAGGGCTCCAATGACCTACTCAAAGTGAACAAGGTCCCCACGTAAGGTGTCCCCGACACCGAATAGAAAATCTTGACTGATTCTGTATCATATAGTGTGAAATGTATGGAAGTGTTGCGTCCAAAGGGACCCGGCGGTGTTAGTCGATTCGTATCCACTAGTTTTATATTTTGCGGCTGATTCGGAAGTGTCGTCTTGTTGTGAAGGTGTGTCGTGACTATTGTTTTGTAATGTTCCTTTAGTGCAGAGGACTCGCCTCGATGTCGGTCGCTGGGTGGACAGGCTGGTGATGTGCTTTAAGCATTTATTTTGTTGCCATAACCACACCTTGTATTGCAATAATATTTTACTAAAATTTACAAAAGCGTTAGAATCGCTCTATGATTTACTTAGGAAGCCCGAGTGAACTTCGGTGTTGTTAGTTAAGATTTAAAATCTGTGAATCTCATAGTATTTATTGCTACCACTTACTCTAATCCATTTAGTTATTTTTTTATTAATAGAAATTATATTATGTACATTTTGATGTATATTTTATTATTTGTCCGCTGTTAGCGGGCTGGTTATGTTTTGGTCGAATTTTTTTATGATTGTAAAATTTTGTTGTATTTTATCAGTTGATTTCAAAAGAGTTACTTTTATTTGACCAGATTGTGATTCATTTTCTGATAGTTAAGTCCGTGAATTGTTATATTATTATCATTTGATTCTTTAATTTTTATAGTATAAATATTATCAGAATAAATACCTGCATAGTTATTACTATTAGGAGTTAAATTTAAAAACAAAATACGTATGGAAGTACAATCTCTTTAGGTCTTCGTCGAGTGAAATATTTCTTTTTAAAATTTCCGTCTTCAGGAACGATAGGTATTTAAAAATTAGATCGTATTTTTTGATGATTCTAGTGTAAAATGTCTCGTTCGTAAATAAAACTGGTCGATCAAAATGAACAAAAAGAGATCGTTATTTCGATCGTCATAACGACGTTGTCCAAGTTCTTTGAAGTCATCCGATGTTAAAAAAATATATACGTAACAATGATACATATAATAAATTACAATAATGTGACGTTAATATAAATTTTACACGTATAATTAAAAGTTGAGTAGTTTAAGTGTATTATATAAGCATATAGGTTCTACTTTGTATGAAGTATTGCTAAGTCTAGGTACCTAAAACGTAACTAACT | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | AB461860 | Bombyx mori abdominal B (abd-B) RNAi construct | dsRNA | CUGA T7 in vitro transcription kit (Nippon Gene) & MEGAscript SP6 kit (Ambion) | other | subsequent to transcription | dsRNAs for abd-A and Abd-B were synthesized using a CUGA T7 In Vitro Transcription Kit (Nippon Gene) and a MEGAscript SP6 Kit (Ambion; Supplementary Figs.1A, B). The dsRNAswere diluted to 1 Îĵg/ Îĵl for injection. pnd homozygous (non-diapausing) eggswere sorted on slides 5â8 h after oviposition and punctured on the dorsal side with a sharp-pointed tungsten needle (Narishige). A glass needle filled with the RNA solution was then pushed through the hole, and the RNA was injected using a YOU-1manipulator and IM-30 injector (Narishige). The injected eggswere sterilized with formalin vapor and then incubated at 25 °C until dissection. abd-A and Abd-B dsRNA-injected individuals were assessed to confirm the knockdown of the corresponding genes by in situ hybridization and immunostaining (data not shown). | InsectaCentral | Bombyx mori abdominal B (abd-B) RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | embryo | lab_colony | 1 | injection | non_specific_dsRNA | ? (unknown volume delivered) | Buffer and egfp dsRNA injections were used as a negative controls | embryo | phenotype | proleg (NB detection by in situ hybridisation and immunostaining, silencing results not shown) | not checked | low | ||||||||||
| 115 | Bombyx mori abdominal A (abd-A) | RNAi | Entered by Jennie Pterygotes lack abdominal appendages except for pleuropods and prolegs. The larvae of some holometabolous insects develop prolegs, which are used for locomotion. We analyzed the role of the homeotic genes abd-A and Abd-B in lepidopteran proleg development using mutant analysis and embryonic RNAi in the silkworm Bombyx mori. The EMu mutant developed extra prolegs in its posterior abdomen and showed the misexpression of both genes, suggesting their involvement in proleg formation. The depletion of Abd-B by embryonic RNAi caused the development of extra prolegs on all segments posterior to A6, indicating the suppressive function of Abd-B. The abd-A RNAi animals failed to develop prolegs. These results indicate that abd-A and Abd-B are involved in proleg development in B. mori. | pubmed | 19389347 | Tomita | Shuichiro | Bombyx mori abdominal A (abd-A) target | ATGATGAACGGGGTCGGCGGCGCGCTCTACGAGGAGGCGCACGCGTCGCCGCCCGGTGGCTCACCCCCAGCAGCTCCTGCGGCCGCACCACCTTCGGCCTCCAGCGCCTCGCCCGCTTCAGTCGGCTCCAACCCACCACAACCGCCGTTGCACATTCCGGCTAAGCGCTACGAGCCCGAGCCCGGCGTCATCCGGCATGCTCAGCAGCCCTGGGGCTATCCTCCAGATGCTCCCGGCTCATTCGAACATCAGTACCCCAGCGCCCCTACATATTATAATTTACCTGTCGAAAGAGAGCGCAAGTCGACTCTCCCCTTTTGGCCAACCAGCAGTGGTGAATACAAACCATACGCTGATGGAGGCTGTCATCAGGGTTTCTCGCAGCCCTGCTGGAACTATCCGTATGGCACACCTAGAGGCGATCAACCTCTGCCCTACGTCAGCGGAGAAGAACGACGAGCTGCAGTCTCCGAGGCGTCCGGGTTCTCTCATGATGCATATGGTCTACGAAATTACGCACCAGAGTCAGTTTCTGGCGCACCGTATCCGCCACCAGGTTCTCTACCCGGTTCTCTGTCAATGTCGGTCGGAGTCGGGGTGGGGTGTGGATCCTCGAACCCTCTGGATTGGACTGGTCAAGTGACGGTGCGCAAAAAACGCAAACCATACTCGAAATTTCAAACTCTCGAACTGGAAAAGGAATTCCTTTTCAACGCGTATGTCTCCAAACAAAAGAGATGGGAGCTCGCGAGAAATCTGAATCTAACCGAGAGACAAGTGAAGATATGGTTCCAGAATAGACGAATGAAAAATAAGAAGAACTCACAGCGACAGGCGGCGCAAGCTGCCCAGAACAACAACAACAATTCAAATGCGAACAACCACAACCACCACGCGGGCCACCACCACGCACCGCACCACGTGGCGCTGCACCATCCACCGCCTGCGAAACATCATCAGTGACCCGCGCCCTTCTCTGGGACTTACCCCCTTCATCACGGTATAGCCAGCTAAATTGTATGATTCATGAAGGAAGTACCTATCGTAGGGCCTAAATGTGGAATGTGGTCGTACTTTAAGCGAGATTATAATCACGTCCAATGTTTGTTACCTGATAGACTAGAATTGACCGGAGGAACGTTGTATGCGCTATATCGTTTGTATCGCAACGTGTTGGACGCTAAGTCAGTGCCGCGTGCGCCGCAGGTCCCAGAGAAACTCGTACTAAGATTATTGTGTTGTCGATTTGTGTATAGTATTAATTACGAAATTATGTTCTGTAATTGTATCGATTAAGTACGTTAGTAGATAACGATTCCTGATGTGAGTTATGTCTAGTAATAGCGCTGATTATTGTATTTGAAAGGTTATTTAAGTACGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | AB461858 | Bombyx mori abdominal B (abd-B) RNAi construct | dsRNA | CUGA T7 in vitro transcription kit (Nippon Gene) & MEGAscript SP6 kit (Ambion) | other | subsequent to transcription | dsRNAs for abd-A and Abd-B were synthesized using a CUGA T7 In Vitro Transcription Kit (Nippon Gene) and a MEGAscript SP6 Kit (Ambion; Supplementary Figs.1A, B). The dsRNAswere diluted to 1 Îĵg/ Îĵl for injection. pnd homozygous (non-diapausing) eggswere sorted on slides 5â8 h after oviposition and punctured on the dorsal side with a sharp-pointed tungsten needle (Narishige). A glass needle filled with the RNA solution was then pushed through the hole, and the RNA was injected using a YOU-1manipulator and IM-30 injector (Narishige). The injected eggswere sterilized with formalin vapor and then incubated at 25 °C until dissection. abd-A and Abd-B dsRNA-injected individuals were assessed to confirm the knockdown of the corresponding genes by in situ hybridization and immunostaining (data not shown). | InsectaCentral | Bombyx mori abdominal B (abd-B) RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | embryo | lab_colony | 1 | injection | non_specific_dsRNA | ? (unknown volume delivered) | Buffer and egfp dsRNA injections were used as a negative controls | embryo | phenotype | proleg (NB detection by in situ hybridisation and immunostaining, silencing results not shown) | not checked | low | ||||||||||
| 117 | Identification and function of Abdominal-A in the silkworm, Bombyx mori | RNAi | Entered by Jennie Abdominal-A (adb-A) is a key gene in the development of insects. To understand its function in the silkworm, we cloned 1193 bp of the abd-A gene of Bombyx mori (Bmabd-A), including the complete coding sequence and part of the 3â² untranslated region sequence. Bmabd-A has at least three mRNA splice variants with coding sequences of lengths 1032, 1044 and 1059 bp, encoding 343, 347 and 352 amino acids, respectively. Each splice variant of Bmabd-A has three exons and differs only in second exon size. Bmabd-A was expressed at low levels in unfertilized eggs, but increased gradually in fertilized eggs after laying 22 h. Bmabd-A expression decreased in ant silkworms (newly hatched silkworms). After RNA interference for Bmabd-A , the embryos had two mutant phenotypes, either completely or partially absent abdominal feet from the third to sixth abdominal segments, suggesting that Bmabd-A is responsible for normal development of the third to sixth abdominal segments during embryonic development. | pubmed | 19320756 | Pan | M H | Identification and function of Abdominal-A in the silkworm, Bombyx mori target | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | Identification and function of Abdominal-A in the silkworm, Bombyx mori target | Identification and function of Abdominal-A in the silkworm, Bombyx mori RNAi construct | dsRNA | T7/SP6 RiboMAX Express; Promega | other | subsequent to transcription | dsRNA was synthesized using the RibomaxTM Large Scale RNA Production System-T7 (Promega) and RibomaxTM Large Scale RNA Production System-SP6 (Promega) based on the Bmabd-A gene fragment amplified by primers P1 and P2. The sense and antisense RNAs were mixed, digested and precipitated, resulting in dsRNA. Three different doses of dsRNA were injected into 2â4-h-old embryos by micro-injector (TABLE genes4all_1). After disinfecting the injected silkworm eggs, they were incubated (25 °C, relative humidity: 70â80%). Eggs that failed to hatch were counted and dissected. Embryos that successfully hatched were counted and classified by mutant phenotype. Mutant phenotypes were described and digitally imaged with a stereoscope (OLYMPUS SZX12; Olympus, Tokyo, Japan). | InsectaCentral | Identification and function of Abdominal-A in the silkworm, Bombyx mori RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | embryo | lab_colony | 6 | injection | buffer | (50 ng/egg) | water | embryo | phenotype | whole organism (levels of silencing results not given) | not checked | low | |||||||||||
| 118 | RNA interference cytochrome P450, CYP6BG1, from the diamondback moth | RNAi | Entered by Jennie We have previously reported that a cytochrome P450, CYP6BG1, from Plutella xylostella was found to be overexpressed in 4th instars of a permethrin resistant strain and inducible in the susceptible counterpart. The findings suggested potential involvement of CYP6BG1 in permethrin resistance, hence warranted a functional analysis. To assess the functional link of the gene to permethrin resistance, we adopted RNA interference-mediated gene silencing (RNAi) by dsRNA droplet feeding. Here, real time PCR analyses show that oral delivery of dsRNA can efficiently reduce the expression of CYP6BG1. Knockdown of CYP6BG1 transcript was evident in midgut and larval tissues enclosed in carcass. As a consequence of knockdown, a significant reduction in resistance of larvae fed CYP6BG1 dsRNA was observed after 24 and 48 h of exposure to permethrin. In addition, CYP6BG1 dsRNA feeding to larvae led to reduced total P450 activities of microsomal preparations toward model substrates p-nitroanisole and benzyloxyresorufin. These results indicate that the overexpressed CYP6BG1 participate in enhanced metabolism of permethrin, thereby, resistance. The knockdown of a non-overexpressed P450, CYP6BF1v4, from the same resistant P. xylostella strain did not lead to changes in the level of resistance to permethrin, supporting further the specific involvement of CYP6BG1 in the resistance. | pubmed | 18957322 | Bautista | Anita M | RNA interference cytochrome P450, CYP6BG1, from the diamondback moth target | CCCGTCCCCGTGGTACAAGACCCCGCGCTGCTCAAGCTCATCACCACCAAGGACTTCTACTACTTCAACGGACGCGAGGTCTCCGAGCACACGCACAAGGAAACCTTGACCAGAAACTTGTTCTTTACTTACGGAGACAAATGGAAGGTGCTCAGGCAAAACCTCACGCCACTCTTCTCTTCGGCCAAGATGAAGAACATGTTCCCTTTGATAGAAAAGTGTACACATGACCTCGAAAAGCTGTTGGATGAACAGAGTAAATCGAATAAAAGCGGGGTGGACATCAGGTCGGTGATATCCCGGTTCACCATGGACTGCATCGGCGCCTGCGCGTTCGGGATCAACACCGGAGTCATGCAAAAGGACTCCGAAGTAAACCCTTTCAGACAAATCGGTGATAAGATTTTCGAGGTGTCAATAATAAGAGGCTTTCTAACTGCTTTTAGGTCGATATGGCCGTCCCTCTTTTACTCTTTGAGACTCTCTATGTTTCCGCCACAAATACCTGCGTTCTTCCACTCAGTATTGATGACTGTATTCAAGGAGCGCAGCCTGACGGCATCGTCCCGGCAAGACTTTGTAGACATGATCCTGGCTCTAAAGAAGAGCAACTACATAACTGGCGACAGCGTGAACAACATGAAAGGTGGAAATGACAAACTGCAGATAAAGGTAGACGATGAGTTTCTGGTGGCGCAGTGTGTTCTGTTCTTTGCGGCTGGCTTCGAGACTTCTTCGACCACATCTTCGTTTACCCTGTTTGAGTTGGCGAAGCAGCCCGAGCTGCAGCAGCGGGTCATCGACGAGGTGGACTCCTACCTGTCCACGCGCAGTGAAGTTGGCTACGACTGTACCACTGAGCTTCCATTCCTGGAGCAATGCATAGAGGAGACTCTGCGACTGTACCCGGTTCTACCAGTCGTCACTCGTGAGGTGGTGGAGGACTACAAGCTCCCCAGCGGCCTGACCCTCGAGAAGGGCCTCCGAGTCCACATCCCCATGACGCACCTGCACCGCAACCCCAAGTACTTCCCAGACCCTCCGGAGTACATCCCTGATCGGTTTTCGCCGGAGAATAAGAAGAATATTGTGCCGTTCACGTACTTCCCGTTTGGTGAAGGACCCAGAATATGTATAGGAATGCGGTTCGCCAAGATGCAGATGATGGCGGGTCTGGTGACCCTGTTCAAGAGCTACCGAGTGGAGCTGGCCCCCGACATGCCCACGGAGGTGTCGTACTCCTCCACGGCCATGGTCACCCAGTTCACCACCGGCATCAAGCTGATACTGGTGCCTCGCGTGAAGAATGCTAATTAG | Lepidoptera | Plutellidae | Plutella | xylostella | 51655 | cds | NCBI | AB372008 | RNA interference cytochrome P450, CYP6BG1, from the diamondback moth RNAi construct | dsRNA | MEGAscript RNAi kit (Ambion) | other | subsequent to transcription | A double-stranded RNA (345 bp) corresponding to a portion of the CYP6BG1 was synthesized using a method that eliminates the cloning step (Schepers, 2005). Here, templates for in vitro transcription were produced by adding T7 promoter sequences to each 50 end of cDNA template prepared by SMART-RACE (Clontech) using PCR. Poly A RNA was used as a starting material in SMART-RACE cDNA synthesis. The primers (TABLE genes4all_1) used to produce a cDNA with T7 promoter sequences were designed based on the nucleotide sequences flanking 226â570 positions of CYP6BG1 cDNA fragment (GenBank accession no. AB372008). Because there is a high risk of cross-suppression or co-suppression between closely related P450s, the primers targeted the sequences outside the signature motifs of P450s for more specificity (Supplementary figure 1). Also, the primer sets used did not exhibit homology with P. xylostella and other insect P450 nucleotide sequences by BLASTn search. The PCR products of 345 bp were examined on agarose gel prior to in vitro transcription to verify that the products show a single band and the expected sizes. The two amplification products possessing a single T7 promoter sequence on opposite ends were combined (1 mg: 2 mg), and were used directly for in vitro transcription outlined in MEGAScript RNAi kit (Ambion, Inc.) instruction manual. All dsRNA preparations were quantified spectrophotometrically and stored at 20 C until use. | InsectaCentral | RNA interference cytochrome P450, CYP6BG1, from the diamondback moth RNAi construct | Lepidoptera | Plutellidae | Plutella | xylostella | 51655 | local resource | larva | lab_colony | feeding | elution solution (es, 10 mM TrisâCl, pH 7, 1 mM EDTA, provided in the RNAi kit) | buffer | (250ng/insect, 0.3 microlitre) | elution solution | larva | qPCR | whole organism | not checked | 24 | high | 55 | ||||||||
| 119 | cadherin RNAi in diamondback moth Plutella xylostella | RNAi | Entered by Jennie Two laboratory diamondback moth (DBM) strains were used to study the effects of injecting cadherin gene double-stranded RNA (dsRNA) on the growth and development of Plutella xylostella (L.). Specifically, the susceptible strain named DBM.1Ac-S and the low resistant strain DBM.1Ac-R selected with Cry1Ac toxin were studied. The third larvae of the two strains were injected dsRNA of cadherin gene and their corresponding controls, DBM.1Ac-RH and DBM.1Ac-SH, were both injected diethypyrocarbonate (DEPC)-treated water respectively. The basic biological properties such as death rate, hatching ratio, fecundity, weight of pupa and eclosion rate of the strains mentioned above were likewise studied. Meanwhile, the length and width of the egg and pupa were also measured. The results showed that the cadherin gene dsRNA injection resulted in a significant increase of the death rate and sex ratio. On the other hand, hatching ratio, fecundity, weight of pupa, eclosion rate and adult longevity for male and female of treatments decreased compared to their corresponding controls. As such, there was no significant difference on the length of egg and pupa in between treatments and the corresponding controls. However, their width increased inversely with their corresponding controls. Hence, the results suggest that cadherin gene dsRNA injection retarded the larval growth and development of P. xylostella. Also, these results can help reveal the function of cadherin gene through the RNA interference technique. | wiley interscience digital object identifier (doi) | 10.1111/j.1439-0418.2008.01346. | Yang | Z X | cadherin RNAi in diamondback moth Plutella xylostella target | ATGGGAGCTGATGCCCGAATCTCAGCCATCTATTTACTTTTTATCGCCATCCCGTTAATTCTAGCACAAGATTGCTCATATTTCGTAGCAGTTCCGAGAGAAGACAAACCAGAGCAAGATAACCCAGATTTTTCAGGTACACCCTGGTCGTCGCGGCCGCTGCTGCCGCCTCCAGACCGCGATGACACCTGCATCGGATCCATGTCTCAGACCGGAAACACTGTGATCCACAACATCTACATGGACGAGTCTATTGAGGGAGATGTCATCATTGCAAGGCTCAATTATGAAGATACTGGCACGCCCACCATCGGCACTTTCCTGGGCCAGGGCCCGAGGGACCTGCTGGGGCCGGTGATACGCCGGGTCCCCGAGCTGGACAACGCCTGGCACCTCGTCATTACACAGAAGCAGGACTATGAGACTCCGATAATGCGGAGCTACATCTTCCCGATCTCCGTGAGCGGCGAGGCCACCTCGGCGCGCGTGCAGCTCACCATCGTCAACATCGACGACAACCCCCCCTTCATCCAAGCCTTGGACGCCTGCTTCGTCAACGAGCTGACGGAGGCGCCTCTGCTCACGGACTGCGTGTACGACGTGACTGACGCCGACGGCTACATCAGCACGGGCTTCATGACCTACAGCCTCAGCAGCGACCGCGGCGACGAGCTGCTGTTCGACGTCGACTCGCAAGAGGTTGAGAACGACCAGTACCGCCTCAGGATGACCATCAAGCTGTTGCAGCCGCTGAACTATGAGACCAACATGATACACATCTTCTCTGTTACTGCGTTTGACTCGTTGCCCAACAACCACACGGCGAGCATCTCCGTGCGTGTTCAGAACGTGGAGTCCCGGCCGCCGCGCTGGCAGGAGATCTGGGCCGTGCAGCAGTTCGATGAGAAGACCAACCAGAGCTTCTCCGTCAGGGCCATCGACGGAGACACCGGCCTGGACCGCCCCATCGCTTACAGACTGGAGAAAAACGAGACATACACTTTCTTTGACATCGAAACCATCGGGGGTGGTAAAGATGGAGCCATCTTTACTGTAACAGGCATAGACCGGGATACTCTGCAACAAGAAGTCTTCCAGGTGTCCATCATAGCATACAAGGCTCACAACGAGGCATTTTTTACGGAGACCAATGTGGTAATTATAGTGAACGACGTGAACGACCAGAGGCCTGAGCCGCTGCATAAAGAATACCGAACGGAAATTCGTGAGGAAACGCCACTGACGATATCATTTGATAACGACTTCGGATTTCATGATCAGGATTTGGGTGACAACGCGCGATACGAGGTGAAGCTGAAGAGCGTCTCTCCAGCGGGCGTGGCCGACGCGTTCTACATCGCGCCCGGAGTCGGCTACCAGCGACAGACCTTCACCATGGGCACCATCGTCCACTCGATGCTCGACTACGAGGTGCCCGAGTTCCAGAGCATCATCGTCAATGTGACAGCTACGGACCTAAACGACCCGTCTCTAGTGGGAGAGGCGATGGTGTACATCGATCTGGTGAACTGGAATGACGAGGAGCCGATATTCGAGCTGCCGACGTACCGAGCCGCCTTCAACGAGACCGAAGGCCAAGGATATGTCGTCGCCACCGTGCTCGCCAAAGACAGGGATATTGGAGATGTCGTCAAACACTCGCTGCTGGGCAACGCCGCAAGTTACCTGGCCATCGACGAGCTCACGGGCGAGGTCACCGTCGCCGTCGACAACGCCTTCGACTACCACCGACAGAACGAGCTGGTTGTACAGATTCGTGCTGAAGACACCCTGGGGGAGCCATACCACATCACCACCACCCAGCTGGTCATAGAGCTCTATGACGTCAACAACACACCGCCAACCATTCGCTTACCTAGCGAGAGCCCTCGCGTGGAGGAGAACGTGCCCTCAGGGTTCGAGATCACACGCGGGGTGACGGCCAGCGACCCCGACACCACCGCCGAGCTGCGCTTCGAGATCGACTGGGACGCGTCCAGCGCCACCAAGCAGGGACGCAACGCCGACCGGGCGGAGTTCGTCGAGTGCGTCAAAATCGAAACCATTTACCCAGATGAGGAAAATAAAGGAAACGCTATCGGACTCATAGTGGCCAACGAGATCCGGCCCGGAGTCACCATCGACTTCGAGGAGTTTGAGGTCCTGTACTTGTCCGTCAAGGTTACAGACAAGAACACCGTCATAGGAGATCCTGACGATCAATTGATGTTCACAATCATCATCATCGACATGAACGACAACCCGCCAGTGTGGTCGGCGGGCGCGCTGGACGCCTCGTTCAGGGTGCGAGAGGTCGCCAGCACGGGCGTGGTGATCGGCTCCGTGCTCGCCACTGATATCGACGGACCGCTGTACAACCAGATACGATACTCTATTAGAGAGCGAGAGGACACGCCAGCCGGCCTGGTCAAGATCGACCGTCTGACGGGCCAGATCGATGTGGACGCCGACCAAGCCATCGACGCGGACGAGCCGCCGCGCCCCGCGCTCCACTACACCGTGATCGCCAGCGATGAGTGTGACTACGAGGATAAGGAGGACTGCCCGCCTCATAAACACTACTTTGAGACTGAGGGGAATATAACGATCGCGATCACAGACACGAACAACAAGCCGCCGCAGGTGCTCACCAGCGACTTCACCGTGTACGTGTGGGAGAACGCCACCAACGGCACCGACGTGCTGGCACTAGAGGCCATGGATGCAGACAGAGACGACATATACAGCTTCATCCGCTACCAGATCAACTTCGCGTTCAACAACCGCATCCGCGCGTTCTTCGACGTGGAGCTCGACACGGGGCTCATCTTCGTGCATTTCACCACGGACGAGGTGCTGGATCGCGACGGTGATGAGCCGGAGCACAGGATCTTCTTCAACCTCATCGACAACTTTTATTCTGATGGCGACGGCAACAGGAACCAGGCGGAGCTGGAGGTGCTGGTGGTGCTGCTAGACGTGAACGACAACGCGCCGGAGCTACCACCGCGGGACCAGCTCGCCTGGACCGTCTCCGAGAACCTAGACCAGGGTATCCGTCTGGTCCCGGACATCGAGGCGCCGGACCGCGACGAGCCCAACACCGACAACTCCCGGGTCCAGTACGAGATCCTGGACCTCAGCCTCGTGAACCGCGACCTGGAGCTGCCAGAACTCTTCACCATGGTCAATCTGGACAACAAGACCGGCGAGCTCGAGACCACCATGCCCCTCAAGGGCTTCTGGGGCACCTACGATATACACATACGGGCGTTTGACCTCGGGGTGCCGCAGCAGGCGTCCGAAGAGACGTACGTGCTGACGGTGGCTCCCTACAACTACCACGCGCCGCAGTTCGTGTTCCCCGCGCCCGGCGCCGTGCTGAGACTCGCCAGGGAAACAGCTTTGGCAAACCTGGAACTGGTGGACGGAACTAAACTGAGGAAGGTGGAGGCCACTGACGAGGACGGGCTGGAGGCCGGCGAGGTCACCTACTCCATCGTAGGCAGCGATGAAGCTATGGAATATTTTACTATTATTGCATCTGACGGTACTCTGTTGCTTACTTCGGCAATCCAGGATGAAGTAAAGAGATTTGAGATGACGATCAGGGCCACCGACGGCGGCACGGACCCGGGCCCTATTCATACTGACGTCACGTTCACCCTGCTGTTTGTGCCCACGCGCGCCGACCCTGAGTTCCGCCCCAACCAGGCTGAAGTCTCTTTCTTTGAAAAAGAACAAGGCCTCACGGAGGCTTTCCAGCTGCCACAGGCTGTGGACCTCAAGAATGAAGGATGCACCGCCGACGCCGGCGATTGCTACTCCGTATACTACAGGATTGTTTCGGATATCAGCGAGTCATTCAAGGTGGACGCTGAGAAGAACATTATATCGCTGACGCGTGAGTTGGACCGGGCCGACGGGGTGCGACACATCGTGACTGTGGCCGCCAGCAACCAGCCTGACGCCACGGACAACCCCACCAACGTGCTCACTGTCACCGTGTTTGTGAGGGAAGCTAATCCGCGGCCGATTTTCGAAAATGAAGTGTACACCGCTGGCATCTCCACCATGGACTCTATCAACAGGGAACTATTTACGGTTAAGGCAACCCACACAGAGAACCTATCAATAAAATACACGATAGACCCAAGCTCGATGGTAGCGGACACCTCTCTACAGAGCGTGCAGGGGTCGGCCTTCGAGCTGGACGCTGACTCCGGAGTCCTGACCCTCAAGATCAAGCCGACGGCTTCCATGCGGGGCATGTTCGAGTTCGAGGTCATCGCCACGGATACTGAGCAAGCTACGGACCGTGCGGAGGTGAAGGTGTACATCGTGTCGGACAACAACCGCGTCTCCTTCCTCTTCCAGAACCAACTCACGCAAGTGGAGCAGTACAGGGACTTTATAGCGCAAACCCTGTCGGCGGGTTTCAAGATGGCGTGCAACATCGACGAGGTGATTCCGTCGACGGACGCGGGCGGCTCCCCGGTGGAGGGGGTCACGGAGGTGCGCGCTCACTTCATCAGGGACAACCAGCCGGTCACTGCTGATGTCATTGAGGCACTCCGGAGCGACGTGGAACTGCTGACCAACATCCAGCGCACACTCAACACCAACCTGCTGGTGCTCACTGACTTCGTGACGGACATCAGCCCCTCGGTGACGGCGGACGCGGCGCGCGTCATCATCTACCTGTTGGGCGCGCTGTCAGCCGCGCTCGCGCTCGCGCTCGCTTGTCTCGTGCTGCTGGCTGCTTATTGGTTTAGGACGAGGGCATTGAACCGGCGCCTGGAAGCACTATCCACCACTAAGTTCGGCTCCATGGACTCGGGGCTGAACCGCGTGGGCAACATCAGCGTGCCCGGCACCAACAAGCACGCAGTCGAGGGCTCCAACCCGATCTGGAACGAGCAGATCAGGGCGCCCGACTTCGACGCTATCAGTGAAGATTCGGACAACTCTGACCTCATCGGCATCGAGGATCTGCCTCAGTTCAGATACAACTACTTCCCTCCCTCGCCGGGACAAGCGAGGAATGCAGAACCTAAGAATGAACAAGAGGATGAGCTGCCGAGCCACAGCAACAACTTCAAGTTCAATGCTACACCGTTCAGCCAGAACTTTGGATCACAGAAATTCTAATATTTCTAATATTTATGTTAAATATTTTACAAATTGTATAATAATCTATGAAAAAACATAATAATACTCTAAGTATTGAAACTTATACACATACCTGTCCAAAAAGTATAATAATCATAACTAATAATAAGTAAAGTGCCTTACTTCATAAGTATTAATTATTATGACTATAACCCCATGTAAATAAAATTATTGCAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Plutellidae | Plutella | xylostella | 51655 | cds | NCBI | EF541176 | cadherin RNAi in diamondback moth Plutella xylostella RNAi construct | dsRNA | MEGAscript RNAi kit (Ambion) | other | simultaneously with transcription | The midgut was dissected from the third instars of 30 P. xylostella and homogenized in TRIzoL reagent (Invitrogen, Carlsbad, CA, USA). The extract was treated with DNase (RNase free) (NEB, Beverly, MA, USA) to avoid DNA contamination. The total RNA was subjected to gel electrophoresis and quantified using a spectrophotometer (UV-2550; SHIMADZU, Kyoto, Japan). The first-strand cDNA was synthesized according to the manual of SuperScript (Carlsbad, MA, USA) III First-Strand Synthesis System for RT-PCR Kit (Invitrogen, Carlsbad, CA, USA). The forward primer 5¢-TAATACGACTCACTATAGGGCTTCGAGATCGACTGG- 3¢ and the reverse primer 5¢-TAATACGACTCACTATAGGGGGTTGTAAGTGGTCCG- 3¢ used to amplify cadherin gene were designed based on its complete sequences. GenBank accession numbers EF541176, EF569598 and AY564235 were used. A 446 bp fragment with 97% and 98% similarity to E-cadherin gene of H. armigera was amplified and cloned into the pEasy-T1 vector (TransGen, Beijing, China). The said fragments are those with GenBank numbers AY515302 and AY351904, from 1973 to 2379 bp, respectively in cadherin cDNA complete sequence of EF541176. The PCR product was purified using the PCR purification kit (Bioteke, Beijing, China). After sequencing confirmation, it was used as a template to generate corresponding dsRNA by MEGAscript RNAi Kit (Ambion, Austin, TX, USA). The dsRNA was suspended in 50 ll diethypyrocarbonate (DEPC)-treated water, subjected to 1% gel electrophoresis and quantified using a spectrophotometer (UV-2550; SHIMADZU). Finally, the dsRNA was thawed on ice prior to injection. | InsectaCentral | cadherin RNAi in diamondback moth Plutella xylostella RNAi construct | Lepidoptera | Plutellidae | Plutella | xylostella | 51655 | local resource | larva | lab_colony | 1.36 | injection | DEPC water | buffer | (75 nl injected) | DEPC water | larva | RT-PCR (semi-quantitative) | Midgut (no internal control gene used for RT-PCR) | not checked | 48 | high | ||||||||
| 120 | RNAi B mori bombeil-2 | RNAi | Entered by Jennie The insect brain is the center of developmental control, from which ecdysone governs brain morphogenesis and regulates gene expression cascades associated with molting and metamorphosis. In order to identify the 20-hydroxyecdysone (20E)-inducible genes responsible for molting and metamorphosis, we constructed a 20E-induced subtraction complementary DNA library from the fifth instar larval brain of the silkworm Bombyx mori. We isolated 10 genes, designated as bombeil-1 to bombeil-10, three of which did not show any sequence similarity to previously identified Bombyx genes. Whole-mount in situ hybridization revealed that all of these bombeil messenger RNAs were exclusively located in two pairs of lateral neurosecretory cells in the larval brain, known as prothoracicotropic hormone (PTTH)-producing cells. RNA-interference knockdown targeting bombeil-2 resulted in larvalâpupal molt defects, and adult wing and leg malformations. These results, together with the cell-specific co-localization of bombeil transcripts with PTTH, suggest that bombeil genes play important roles during larvalâpupalâadult development. | pubmed | 18835445 | Hussain | Monwar | RNAi B mori bombeil-2 target | ACATGTGACGCACAAGTTAATTCTCACATACGATTCAAATAGCATACGTTGCTTTGTTTAACTTTAATTATAAGTAGTTATATTCACCGATATAAAAGGGCATAATTAGCGCCGGCATTAATATCTCGGTTTCCTAAGTAGAGGTGATATAGTTAGAGCTTCGGAAGCTTCGAGAATTATTTCTGATGTAAATATATTTTATTGTGAGTTTGCGCGTTTATGCGGTTCGATAAATTTACGTAAGTCTAGGGGGCGTTCGGTCGAGCGGTTCAGTCAATTAATCCGTGCTCTCGCGGCGCGAGACGGACGTCTCTTATCTCAATGCACCACTAAACTTCATAATTTCATTTTAATTGTAGGCGAAAATAATTTATTGTCGATAGTGATCATTTAGTAGTAATAAGTAGGCCAAATCGTTAACAAATTCGGACGAATTACGTATGTGATAGTGAGAATGCTAAAGCTGCACGCGCTTCGGCCGT | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | AB372867 | RNAi B mori bombeil-2 RNAi construct | ACATGTGACGCACAAGTTAATTCTCACATACGATTCAAATAGCATACGTTGCTTTGTTTAACTTTAATTATAAGTAGTTATATTCACCGATATAAAAGGGCATAATTAGCGCCGGCATTAATATCTCGGTTTCCTAAGTAGAGGTGATATAGTTAGAGCTTCGGAAGCTTCGAGAATTATTTCTGATGTAAATATATTTTATTGTGAGTTTGCGCGTTTATGCGGTTCGATAAATTTACGTAAGTCTAGGGGGCGTTCGGTCGAGCGGTTCAGTCAATTAATCCGTGCTCTCGCGGCGCGAGACGGACGTCTCTTATCTCAATGCACCACTAAACTTCATAATTTCATTTTAATTGTAGGCGAAAATAATTTATTGTCGATAGTGATCATTTAGTAGTAATAAGTAGGCCAAATCGTTAACAAATTCGGACGAATTACGTATGTGATAGTGAGAATGCTAAAGCTGCACGCGCTTCGGCCGT | dsRNA | T7/SP6 RNA polymerase (Takara) | phenol_chloroform | subsequent to transcription | Plasmid DNA was purified from a clone containing a 482 base pair (bp) fragment of the bombeil-2 transcript isolated from the subtraction cDNA library. The plasmid was linearized with PstI and NcoI. Sense and antisense RNAs were synthesized in vitro using T7 RNA polymerase (Takara, Otsu, Japan) and SP6 RNA polymerase (Takara), respectively. To generate dsRNA, equal amounts of sense and antisense RNAs were mixed, heated at 95 C for 5 min, and gradually cooled to 25 C. The solution was then treated withRNaseA (Nacalai Tesque, Kyoto, Japan) and DNase RQ1 (Promega) for 45 min at 37 C. The dsRNA was purified with phenol/chloroform followed by ethanol precipitation, and then dissolved in water. A 722-bp fragment of dsEGFP was used as a control. The enhanced green fluorescent protein (EGFP) sequence was derived from the pEGFP-N3 vector (Clontech, Palo Alto, CA), sub-cloned into the pGEM-7Zf() vector (Promega), and the dsRNA was prepared according to the protocol described above. A 5-mg sample of the dsRNA of bombeil-2 (dsbombeil-2) in 10 ml insect Ringerâs solutionwas injected into V2 larvae through the first pro-leg. Ringerâs solution and dsEGFP (5 mg) in Ringerâs solution were injected as controls. After injection, the larvae were maintained under normal rearing conditions until adult eclosion. To examine the effects of bombeil-2 RNAi on brain development during the larvalâpupal transformation, the brains were dissected at stage P2. To evaluate the knockdown effect of RNAi, total RNA was purified from the brains, wing discs (WDs), and leg discs (LDs) of V4 larvae. A 1-mg sample of total RNA was reverse-transcribed, and the resulting cDNA was amplified for 30 or 35 cycles using a thermal cycle as follows: 94 C for 30 s, 60 C for 30 s, and 72 C for 30 s for EcR-A, EcR-B1, and RpL32; 94 C for 30 s, 64 C for 30 s, and 72 C for 30 s for bombeil-2; and 94 C for 30 s, 59 C for 30 s, and 72 C for 45 s for PTTH. The primers for PCR were designed from the nucleotide sequences (TABLE genes4all_1). Therewas no amplification without reverse transcriptase, even after 40 cycles of PCR (data not shown). | InsectaCentral | RNAi B mori bombeil-2 RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | injection | ringers solution | non_specific_dsRNA | 10 microlitre/insect (5 micrograms/insect) | dsGFP | larva | RT-PCR (semi-quantitative) | brain, wing disc and leg discs | not checked | 48 | high | ||||||||
| 121 | RNAi Cry1Ab M sexta | RNAi | Entered by Jennie The evolution of insect resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins that are widely used in sprays and transgenic crops. Resistance to Bt toxins in some insects is linked with mutations that disrupt a toxin-binding cadherin protein. We show that susceptibility to the Bt toxin Cry1Ab was reduced by cadherin gene silencing with RNA interference in Manduca sexta, confirming cadherinâs role in Bt toxicity. Native Cry1A toxins required cadherin to form oligomers, but modified Cry1A toxins lacking one a-helix did not. The modified toxins killed cadherin-silenced M. sexta and Bt-resistant Pectinophora gossypiella that had cadherin deletion mutations. Our findings suggest that cadherin promotes Bt toxicity by facilitating toxin oligomerization and demonstrate that the modified Bt toxins may be useful against pests resistant to standard Bt toxins. | pubmed | 17975031 | Soberon | Mario | RNAi Cry1Ab M sexta target | GACCAATCGGAGTGTGGTGAATTTTTGGAAAATATTTTGTGCGGTTCCTTTAGTTGTGTAATATAGTACTTTAGTTACAAATTTGGAATAATTTGGCAGCAAAACCATCTGCAGCAACAAAATCATCTGCAGCTGCGAAATCATCTGCAGCAGCAAAAGCATCTTCAGGAGCGAGAAAAGCCCCAAATAATGTGAGATGGCAGTTGACGTCCGAATCGCTGCCTTCCTGCTGGTGTTTATAGCGCCTGCAGTTTTAGCTCAAGAGAGATGTGGGTATATGACCGCCATCCCAAGGCTACCACGACCGGATAATTTGCCAGTACTAAATTTTGAAGGCCAGACATGGAGTCAGAGGCCCCTGCTCCCCGCCCCGGAGCGGGATGACCTGTGCATGGACGCCTACCACGTGATAACAGCCAACCTCGGCACGCAGGTCATCTACATGGATGAAGAGATAGAAGACGAAATCACCATCGCCATACTTAATTATAACGGACCATCAACTCCGTTCATTGAACTGCCATTTTTATCCGGTTCGTACAATCTGCTGATGCCGGTCATCAGGAGAGTTGACAACGGGGAGTGGCATCTCATCATCACGCAAAGACAGCATTACGAGTTGCCCGGCATGCAGCAGTACATGTTCAATGTGCGCGTGGACGGCCAGTCGCTGGTGGCAGGCGTGTCTCTCGCTATCGTCAACATAGATGACAACGCGCCCATCATACAAAACTTCGAGCCTTGCCGGGTTCCTGAACTGGGCGAGCCAGGGTTGACAGAATGCACATACCAAGTATCGGACGCGGACGGACGGATCAGCACAGAGTTCATGACGTTCAGGATCGACAGCGTTCGTGGCGACGAGGAGACCTTCTACATCGAACGGACGAATATCCCCAACCAATGGATGTGGCTAAATATGACCATAGGCGTTAATACCTCGCTCAACTTCGTCACCAGTCCGCTGCATATATTCAGCGTGACAGCCCTGGACTCGCTCCCGAACACCCACACGGTGACTATGATGGTGCAAGTGGCGAATGTGAACAGCCGTCCGCCGCGCTGGCTGGAGATCTTCGCTGTCCAACAGTTTGAAGAGAAATCTTACCAAAACTTCACAGTGAGGGCGATCGACGGAGACACTGAGATCAATATGCCTATCAACTACAGGCTGATCACAAATGAGGAAGACACATTCTTCAGCATTGAGGCCCTGCCTGGTGGAAAAAGCGGGGCTGTATTCCTCGTGTCGCCAATTGACCGCGACACACTGCAACGAGAGGTGTTTCCACTTACGATCGTCGCTTACAAATATGATGAGGAGGCCTTCTCCACATCAACAAACGTGGTCATCATTGTGACAGACATCAACGACCAAAGACCTGAACCTATACACAAGGAATATCGACTGGCAATCATGGAGGAGACGCCCCTGACCCTCAACTTCGATAAAGAATTCGGATTTCATGATAAGGATTTAGGTCAAAACGCTCAGTACACGGTGCGTCTAGAGAGCGTGGACCCTCCAGGCGCTGCTGAGGCATTCTACATAGCGCCTGAAGTCGGCTACCAGCGACAGACCTTCATCATGGGCACCCTCAATCACTCCATGCTGGATTACGAAGTGCCAGAGTTTCAGAGTATTACGATTCGGGTGGTAGCGACCGACAACAACGACACGAGGCACGTGGGCGTCGCGTTGGTTCACATTGACCTCATCAATTGGAACGATGAGCAGCCGATCTTCGAACACGCCGTGCAGACCGTCACCTTCGACGAGACTGAAGGCGAGGGGTTCTTCGTCGCCAAGGCGGTTGCACACGACAGAGACATCGGGGATGTCGTCGAGCATACTTTATTGGGTAACGCTGTTAACTTCCTGACCATCGACAAACTCACCGGCGACATCCGCGTCTCAGCTAACGACTCCTTCAACTACCATCGAGAAAGTGAATTATTTGTGCAGGTGCGAGCTACAGACACGCTGGGCGAACCCTTCCACACGGCGACGTCACAGCTGGTCATACGACTAAATGACATCAACAACACGCCACCCACCTTACGGCTGCCTCGAGGCAGTCCCCAAGTGGAGGAGAACGTGCCTGATGGCCACGTCATCACCCAGGAGTTACGCGCCACCGACCCCGACACCACGGCCGATCTGCGCTTCGAGATAAACTGGGACACCTCTTTCGCCACCAAGCAAGGCCGCCAGGCTAACCCCGACGAGTTTAGGAATTGCGTGGAAATCGAGACCATCTTCCCCGAGATTAACAACCGGGGACTGGCTATCGGCCGCGTTGTAGCGCGCGAAATCAGACACAACGTGACCATAGACTACGAGGAGTTTGAGGTCCTCTCCCTCACAGTGAGGGTGCGTGACCTTAACACCGTCTACGGAGACGACTACGACGAATCGATGCTCACAATAACTATAATCGATATGAACGACAACGCGCCGGTGTGGGTGGAGGGGACTCTGGAGCAGAACTTCCGAGTCCGCGAGATGTCGGCGGGCGGGCTCGTGGTGGGCTCCGTGCGCGCGGACGACATCGACGGACCGCTCTACAACCAAGTGCGATACACCATTTTCCCTCGTGAAGACACAGATAAGGACCTGATAATGATCGACTTCCTCACGGGTCAAATTTCCGTGAACACAAGCGGCGCCATCGACGCGGATACTCCTCCACGCTTCCACCTCTACTATACAGTGGTCGCTAGTGACCGATGCTCGACAGAAGATCCTGCAGATTGCCCCCCTGACCCGACTTATTGGGAAACCGAAGGAAATATCACAATCCACATCACCGACACGAACAACAAGGTCCCGCAGGCGGAAACGACTAAGTTCGATACCGTCGTGTATATTTACGAGAACGCAACCCACTTAGACGAGGTGGTCACTCTGATAGCCAGTGATCTTGACAGAGACGAAATATACCACACGGTGAGCTACGTCATCAATTATGCAGTGAACCCTCGACTGATGAACTTCTTCTCCGTGAACCGAGAGACCGGCCTGGTGTACGTGGACTATGAGACCCAGGGTAGTGGCGAGGTGCTGGACCGTGATGGTGATGAACCAACGCACCGTATCTTCTTCAACCTCATCGACAACTTCATGGGGGAAGGAGAAGGTAACAGAAATCAGAACGACACAGAAGTTCTCGTTATCTTGTTGGATGTGAATGACAATGCTCCTGAATTGCCACCGCCGAGCGAACTCTCTTGGACTATATCTGAGAACCTTAAGCAGGGCGTCCGTCTTGAACCACATATCTTCGCCCCGGACCGCGACGAGCCCGACACAGACAACTCCAGGGTCGGCTACGAGATCCTGAACCTCAGCACGGAGCGGGACATCGAAGTGCCGGAGCTGTTTGTGATGATACAGATCGCGAACGTCACGGGAGAGCTGGAGACCGCCATGGACCTCAAGGGATATTGGGGGACGTACGCTATACATATACGGGCATTCGACCACGGCATTCCGCAAATGTCCATGAACGAGACATATGAGCTGATCATCCATCCGTTCAACTACTACGCGCCTGAGTTCGTCTTCCCGACCAACGATGCCGTCATACGACTTGCGAGGGAACGAGCTGTAATCAATGGAGTTCTAGCGACAGTGAACGGAGAGTTCTTGGAGCGGATATCGGCGACTGATCCGGACGGACTCCACGCGGGCGTCGTCACCTTCCAAGTGGTAGGCGATGAGGAATCACAACGGTACTTTCAAGTAGTTAACGATGGCGAGAACCTCGGCTCGTTGAGGTTACTGCAAGCCGTTCCAGAGGAGATCAGGGAGTTCCGGATAACGATTCGCGCTACAGACCAGGGAACGGACCCAGGACCGCTGTCCACGGACATGACGTTCAGAGTTGTTTTTGTGCCCACGCAAGGAGAACCTAGATTCGCGTCCTCAGAACATGCTGTCGCTTTCATAGAAAAGAGTGCCGGCATGGAAGAGTCTCACCAACTTCCTCTAGCACAAGACATCAAGAACCATCTCTGTGAAGACGACTGTCACAGCATTTACTATCGTATTATCGATGGCAACAGCGAAGGTCATTTCGGCCTGGATCCTGTTCGCAACAGGTTGTTCCTGAAGAAAGAGCTGATAAGGGAACAAAGTGCCTCCCACACTCTGCAAGTGGCGGCTAGTAACTCGCCCGATGGTGGCATTCCACTTCCTGCTTCCATCCTTACTGTCACTGTTACCGTGAGGGAGGCAGACCCTCGTCCAGTGTTTGTGAGGGAATTGTACACCGCAGGGATATCCACAGCGGACTCCATCGGCAGAGAGCTGCTCAGATTACATGCGACCCAGTCTGAAGGCTCGGCCATTACTTATGCTATAGACTACGATACAATGGTAGTGGACCCCAGCCTGGAGGCAGTGAGACAGTCGGCTTTCGTACTGAACGCTCAAACCGGAGTGCTGACGCTTAATATCCAGCCCACGGCCACGATGCATGGACTGTTCAAATTCGAAGTCACAGCTACTGACACGGCCGGCGCTCAGGACCGCACCGACGTCACCGTGTACGTGGTATCCTCGCAGAACCGCGTCTACTTCGTGTTCGTCAACACGCTGCAACAGGTCGAAGACAACAGAGACTTTATCGCGGACACCTTCAGCGCTGGGTTCAACATGACCTGCAACATCGACCAAGTGGTGCCCGCTAACGACCCCGTCACCGGCGTGGCGCTGGAGCACAGCACGCAGATGCGCGGCCACTTCATACGGGACAACGTACCCGTACTCGCTGATGAGATAGAACAGATCCGTAGTGACCTAGTCCTCCTGAGCTCGATACAAACAACGCTGGCGGCGCGATCGCTGGTGTTGCAGGACTTGTTGACCAACTCCAGCCCGGACTCGGCGCCTGACTCGAGCCTCACGGTGTACGTGCTGGCCTCACTGTCTGCTGTGCTCGGTTTCATGTGCCTTGTGCTACTGCTTACCTTCATCATCAGGACTAGAGCGCTAAACCGACGGTTGGAAGCCCTGTCGATGACGAAGTACGGCTCACTGGACTCTGGATTGAACCGCGCCGGCATCGCCGCCCCCGGCACCAACAAACACACTGTGGAAGGCTCCAACCCTATCTTCAATGAAGCAATAAAGACGCCAGATTTAGATGCCATTAGCGAGGGTTCCAACGACTCTGATCTGATCGGCATCGAAGATCTTCCGCACTTTGGCAACGTCTTCATGGATCCTGAGGTGAACGAAAAGGCAAATGGTTATCCCGAAGTCGCAAACCACAACAACAACTTCGCTTTCAACCCGACTCCCTTCTCGCCTGAGTTCGTTAACGGACAGTTCAGAAAGATCTAGAAGATAACAACACTAGTTAAGATCATTAATTTTGGAGTTTGGAATTAAGATTTTTGAAAGGATAGTTGTGATAAGCCTGTGATTTTTAAAACTGTAATTGAAAAAAAAAATTGAGACCTCCATTTAAGCTCTTGCTCTCATCTCATCAAATTTTATAAAATGCCATTAGTCATTAAGATACTCGATTTAATTTAAGATTATTTAAGATATTATGTAAAATAAATATATTGTC | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | AF319973 | RNAi Cry1Ab M sexta RNAi construct | GCTGCCTTCCTGCTGGTGTTTATAGCGCCTGCAGTTTTAGCTCAAGAGAGATGTGGGTATATGACCGCCATCCCAAGGCTACCACGACCGGATAATTTGCCAGTACTAAATTTTGAAGGCCAGACATGGAGTCAGAGGCCCCTGCTCCCCGCCCCGGAGCGGGATGACCTGTGCATGGACGCCTACCACGTGATAACAGCCAACCTCGGCACGCAGGTCATCTACATGGATGAAGAGATAGAAGACGAAATCACCATCGCCATACTTAATTATAACGGACCATCAACTCCGTTCATTGAACTGCCATTTTTATCCGGTTCGTACAATCTGCTGATGCCGGTCATCAGGAGAGTTGACAACGGGGAGTGGCATCTCATCATCACGCAAAGACAGCATTACGAGTTGCCCGGCATGCAGCAGTACATGTTCAATGTGCGCGTGGA | dsRNA | T7 RNA polymerase - HiScribeTM transcription system (HiScribeTM, RNAi Transcription kit, Roche, Indianapolis, IN) | other | simultaneously with transcription | RTPCR from cDNA produced from RNA samples obtained from 5th instar larvae with primers Rev: 5â-GCTCTAGAGCTGCCTTCCTGCTGGTGTTTA-3â and For: 5â- GGAATTCCTCCACGCGCACATTGAACAT-3â that amplified a 442 bp fragment (corresponding to a BT-R1 fragment A8-D155) from nt 218 to nt 660 of the BT-R1 sequence (Acc.AF319973). The 442 bp PCR fragment was digested with XbaI and EcoRI and cloned into a previously digested pLITMUS 28i (HiScribeTM, New England Biolabs, Beverly, MA) vector containing two T7 promoters flanking the multi-cloning site. This enabled amplification of the cloned fragment by using a T7 oligonucleotide. The PCR product was purified with QIAquik PCR purification kit protocol (Quiagen Valencia, CA). In vitro transcription of both DNA strands of the insert was performed with T7 RNA polymerase using the HiScribeTM transcription system (HiScribeTM, RNAi Transcription kit, Roche, Indianapolis, IN) as reported by the manufacturers, yielding dsRNA. Injection of larvae was performed with a Brinkmann micromanipulator coupled to a 10 Îĵl VWR Digital Microdispensor (VWR Scientific, San Francisco CA). Twelve hours after hatching, first instar larvae were chilled on ice for 5 min and injected with dsRNA in 100 nl of final volume directly into the hemolymph using 10 Îĵl replacement capillaries for microdispensors (Thoman Scientific,Swedesboro, NJ). The needle was inserted at the dorsal side in the last segment of the abdomen, parallel to the longitudinal axis of the insect, minimizing damage of internal organs. The dsRNA was injected and the needle was left inside the insects for a few seconds before removal (S7). RNAi-treated larvae were injected with 100 ng or 1 Îĵg of cadherin dsRNA in the first and second set of experiments, respectively. Control larvae were injected with water only (100 nl, the same final volume as treated larvae). After injections of cadherin dsRNA or water only, all larvae were held on artificial diet without toxin for 12 hours before bioassays in which they were fed on diet with or without toxin as detailed below. | InsectaCentral | RNAi Cry1Ab M sexta RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | injection | water | buffer | 100 nl (100 ng or 1 Îĵg) | water | larva | W.blot | midgut | not checked | 144 | high | ||||||||
| 122 | Silencing of acetylcholinesterase gene of Helicoverpa armigera by siRNA | RNAi | Entered by Jennie RNA interference is an effective means of regulation of gene expression both in vitro and in vivo. We studied the effect of siRNA on larval development by selective targeting of the acetylcholinesterase (AChE) gene of Helicoverpa armigera. Chemically synthesized siRNA molecules were directly fed to H. armigera larvae along with the artificial diet. The siRNA treatment resulted in specific gene silencing of AChE and consequently brought about mortality, growth inhibition of larvae, reduction in the pupal weight, malformation and drastically reduced fecundity as compared to control larvae. Our studies suggest some novel roles for AChE in growth and development of insect larvae and demonstrate that siRNA can be readily taken up by insect larvae with their diet. | pubmed | 19135057 | Kumar | Maneesh | Silencing of acetylcholinesterase gene of Helicoverpa armigera by siRNA target | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | pubmed | siRNA in conserved region of alignment AY142325, AF369793, AY686704, AY686705 | Silencing of acetylcholinesterase gene of Helicoverpa armigera by siRNA RNAi construct | GCUAUCGUAUUCCAAUAUATTUAUAUUGGAAUACGAUAGCTT | siRNA | other | subsequent to transcription | All the available nucleotide sequences of H. armigera AChE gene (GenBank accession numbers: AY142325, AF369793, AY686704, AY686705) were retrieved from the NCBI GenBank database and a homology search was carried out using Megalign (DNASTAR) software to define the conserved regions. Many siRNAs were designed based upon the conserved region and tested in silico to ascertain the fulfillment of different parameters for maximum silencing as described in the literature. The one satisfying all the criteria and giving maximum score was used in the present study. The 21-nucleotide siRNA was designed based on a conserved cDNA sequence of the AChE gene of H. armigera with the sense strand being 50 GCUAUCGUAUUCCAAUAUATT 30 and the antisense sequence 50 UAUAUUGGAAUACGAUAGCTT 30 and were chemically synthesized (Microsynth Inc., Switzerland). | InsectaCentral | Silencing of acetylcholinesterase gene of Helicoverpa armigera by siRNA RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | feeding | non_specific_dsRNA | siRNA (25, 37.5 and 50 nM) | Unrelated siRNA of ornithine decarboxylase (odc) gene of Chlamydomonas reinhardti origin was used to check the specificity of the RNAi effect (sense strand sequence 50 UACAUUUAACAGCGUAAAAAUGTT 30 and antisense strand 50CAUUUUUACGCUGUUAAAUGUATT 30). NCBI BLAST search was carried out to check the resemblance of both siRNA sequences, if any, with other known genes of Helicoverpa, humans and livestock. | larva | RT-PCR (semi-quantitative) | whole organism | not checked | high | ||||||||||||
| 123 | RNAi ecdysis-triggering hormone B mori | RNAi | Ecdysis-triggering hormone (ETH) is an integration factor in the ecdysis process of most insects, including Bombyx mori (silkworm). To understand the function of the ETH gene in silkworm, we developed an effective approach to knockdown the expression of ETH in vivo based on RNA interference (RNAi) and a binary UAS/GAL4 expression system that has been successfully used in other insect species. Two kinds of transgenic silkworm were established with this method: the effector strain with the ETH RNAi sequence under the control of UAS and the activator strain with the GAL4 coding sequence under the control of Bombyx mori cytoplasmic actin3. By crossing the two strains, double-positive transgenic silkworm was obtained, and their ETH expression was found to be dramatically lower than that of each single positive transgenic parent. Severe ecdysis deficiency proved lethal to the double-positive transgenic silkworm at the stage of pharate second instar larvae, while the single positive transgenic or wild-type silkworm had normal ecdysis. This UAS/GAL4 RNAi approach provides a way to study the function of endogenous silkworm genes at different development stages. | pubmed | 18776991 | Dai | Hongjiu | RNAi ecdysis-triggering hormone B mori target | GTAAACAGTAACTATTTAAGATGACTTCAAAATTGACCATGATGTTGTTCACGTTGAGTCTAATGTTTATCGCCGGGTTAGATGGTTCGTTCATCAAACCTAATAACGTACCGAGGGTCGGCAGAAGCAACGAAGCATTTGACGAGGACGTTATGGGATACGTGATCAAATCAAATAAAAACATTCCGCGTATGGGTAGAAGAAATTACGACTCGGGAAATCATTTCGACATTCCAAAGGTCTACAGTTTACCGTTCGAATTTTATGGAGACAACGAAAAAAGTTTGAATAATGATGATGCCGAGGAATACTATGCAAAAAAAATGGGAAGCATGAAGAAATAAACGCGTCGGATTTCGGATATTTTATTCATTCGTTGAATTAACGAATCCATGATAACTCCTGTTCTAGTCAATCGCTTAAAATAAATTCTTGTATGCATGCAGAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | NM_001172272 | RNAi ecdysis-triggering hormone B mori RNAi construct | ATGACTTCAAAATTGACCATGATGTTGTTCACGTTGAGTCTAATGTTTATCGCCGGGTTAGATGGTTCGTTCATCAAACCTAATAACGTACCGAGGGTCGGCAGAAGCAACGAAGCATTTGACGAGGACGTTATGGGATACGTGATCAAATCAAATAAAAACATTCCGCGTATGGGTAGAAGAAATTACGACTCGGGAAATCATTTCGACATTCCAAAGGTCTACAGTTTACCGTTCGAATTTTATGGAGACAACGAAAAAAGTTTGAATAATGATGATGCCGAGGAATACTATGCAAAAAAAATGGGAAGCATGAAGAAA | hairpinRNA | other | simultaneously with transcription | To generate the effector vector containing cDNA coding for ETH RNAi, complete ETH code sequence was first cloned from silkworm. Total RNA was extracted from the epitracheal gland of the fifth instar larvae with RNeasy mini isolation kit (Qiagen, Shanghai, China) and reversetranscribed by SuperScript II (Takara) with oligo(dT) primer in a reaction volume of 10 Îĵl. As the template for PCR amplification of ETH cDNA, 2 Îĵl of reverse transcription (RT) product was used with 30 cycles of 94 ÂşC for 30 s, 60 ÂşC for 40 s and 72 ÂşC for 30 s. The primer sequences were (up1) 5'-CTGTCGACATGACTTCAAAATTGACAATGATG- 3', (down1) 5'-GTCTGCAGTTTCTTCATGCTTCCCATTTTTTT- 3', (up2) 5'-ACGGGCCCATGACTTCAAAATTGACAATGATG- 3', and (down2) 5'- GTCCGCGGTTTCTTCATGCTTCCCATTTTTTT-3', which contained SalI, PstI, ApaI and SacII sites, respectively (underlined). The two PCR fragments (with primer pair of up1/down1 or up2/down2) were treated with respective restriction enzymes and ligated tail to tail into multiple cloning site of the vector psiRNA to form the vector psiETH. | InsectaCentral | RNAi ecdysis-triggering hormone B mori RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | embryo | lab_colony | injection | buffer | Transgenic RNAi | NB no control method used (transgenic system) | larva | qPCR | whole organism | not checked | high | 90 | ||||||||||
| 124 | Bmrunt RNAi Bombyx mori | RNAi | Entered by Jennie. Pair-rule genes (genes that are expressed only in alternate segments, odd or even) play an important role in translating the broad gradients of upstream genes into dual segment periodicity for body plan patterning in Drosophila. However, homologues of pairrule genes show a remarkable diversity of expression patterns and functions in other insects. We cloned the homologue of runt in the silkworm Bombyx mori, an intermediate germband-type insect. Whole-mount in situ hybridization revealed three stripes arose one by one before gastrulation at the blastoderm stage. Five additional stripes were then generated sequentially as the growth zone elongated. Eight stripes appeared in a pair-rule manner with two-segment periodicity, each of which was confined to the posterior of an odd-numbered parasegment. The weaker segmental secondary stripes emerged de novo in even-numbered parasegments. The Bmrunt transcript vanished before blastokinesis and was then expressed again in the whole embryo. RNA interference for Bmrunt caused severely truncated, almost completely asegmental defects. This cadual-like phenotype suggests that Bmrunt does not function as a pair-rule gene in silkworm segmentation. Bmrunt is required for formation of most body segments and axis elongation in B. mori. | pubmed | 18615617 | Liu | Wenbin | Bmrunt RNAi Bombyx mori target | ATCAGTCGAAACCGAGTTCCCGCGGCACGCGTCCCTTGGCCGCTACTTCGGAGCGCGTCGTGAGTCTTCGGCTTGCGGTCGCGCGACCGCCCGTTTATTTTTCCGAGTCCCCCGCCCGGCGTCTTGCCGTCCGCGCCCAACAAGTGGTGTAGACTGTAGACGGTGACTCGCAGCTCACACGACGTCTCGCGCTCGCACTTTACACAACTTTTCCGAGTGCTCCCCGCGCACGCAACGGTCGCCGCGAGTTCGAACTTCGGTCGCGTGTTTTTAAAACTTTAATCGAAGTGTTCAGACCTCAGTGGCGTGTCAGAGAAGTGTTCGGGGGTAGTTTGAAGTCGATGCACCTCCCGCACGCGAGTCCTGCCGTCAGGATGGCAGACGTTTACGCTCATATCCACGAGTACTACCGGCAGAGTCACGGAGAGTTAGTACAGACTGGTTCTCCGGCTGTCTTGTGCTCTGCGTTACCTGGCCATTGGAGGTCGAATAAATCATTACCGTTGGCTTTTAAAGTAGTGGCACTAGATGACGTTCAAGACGGCACTCTAGTTACGATAAAGGCTGGAAACGACGAGAACGTGATGGCCGAACTCAGGAACTGTACCGCGGTGATGAAGAACCAAGTGGCAAAGTTCAACGACCTGAGGTTTGTCGGTCGGAGCGGACGAGGCAAGTCCTTCACCCTCACCATCACCATCAGCTCATTTCCTTCACAAGTCGCCACCTACACCAAAGCAATCAAAGTAACGGTCGATGGACCAAGGGAGCCGAGAACAAAACAAAATTATGGCTACGGACACCCAGGACCATTCAGTCCATTCCTTCTGAATCCCGGATGGTTAGATGCGGCGTATCTCAACTACGCCTGGGCCGACTACTTCAGGCCCCCTCAAATGCGAGAGCCTTCCACGCTTATCAAAGGTGGAGCCGCGCCGCTTACTACGCCACCTGTGACGATACCTGGTGCTGATTTATTCCCCTTTCCTCCGGCTGTTACGAATTTACCACCTGGTGGCCTAATACCTCCACCCGGTGCATTCTTACCACCGAACGGTCTTCTACCTTTCCCACCGCACCCGGCGGAACTAGCACTAAAAAGTTTACCCCCTGAACTGTCTCTAAAGAGCGGTTTAACTCCTTATGAAGCGTTACGACAATTTCAAAACAATGTTTCTTCAATGGACACGTCTAGTGCACGACTATCACCGACAAGTAGTAGACAGAGCGGTAGTCCGAGAAGTATGGCGAACGCTAGTCCTGATCGATCGAAAACAGATTCGAAATCGGAAGCGAATTCGATACACGACGCAACGATCACTGATGAATCGGACGAAGAACCTATCGAAGTTGTAAAGTCCGCTTTCCATCCAACTAGGCCAGCGAATTTAGAGCTTCAAGAAATGAAAAGGGTTCAGGCAGCGGATTCAACAGTTTCAGATAGGCCACGAACACGCAATGAACTGAAATCAACTTCACAGCGTACTACCAGAGTACTTTCCACCAGTCCAACGTCAACCAAAATCGCTAACGGAACAATACCTAGTCACAAATCTGTGTGGCGGCCATATTGACAGTTTCAGTGTTAGTGCTAAACATAGTATAGTTACCGGCTTCGCAGAGCGGGTACGTTGAAATGTGATACAACCATGAGCTTGTAAATACTTAGTCATAACATAGACATAGCAAATATTTATTGTTAGAATTTAAATGAGAATTTCAGCTGAAATAGATTCGAGTGCTAATAAATGTTTAAATTGTGTAATTCTTGTTAATTTTATTAGTTTTATTGCTACCGTATTCAGGCTTTCATTGTATAACTGTGTATATGTAGTTTTGTATATTGTGTACAGAGAATATTAAATTTGGAATTTAATATCATTATCATTAAAAATGACTGATAAATTTAAAACGTTGTAGTAAAGACATTAAGCTTAATGTAAATAATGATTATTTATGACCAGAATGTACAGTAATTATTATTGAAAATAGTCATCGTAATCATAATGTAAATACGAATTATCGATATAAAAGAATTTATCCGAACAAAAAAAAAAAAAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | EU113258 | Bmrunt RNAi Bombyx mori RNAi construct | TGTGCTCTGCGTTACCTGGCCATTGGAGGTCGAATAAATCATTACCGTTGGCTTTTAAAGTAGTGGCACTAGATGACGTTCAAGACGGCACTCTAGTTACGATAAAGGCTGGAAACGACGAGAACGTGATGGCCGAACTCAGGAACTGTACCGCGGTGATGAAGAACCAAGTGGCAAAGTTCAACGACCTGAGGTTTGTCGGTCGGAGCGGACGAGGCAAGTCCTTCACCCTCACCATCACCATCAGCTCATTTCCTTCACAAGTCGCCACCTACACCAAAGCAATCAAAGTAACGGTCGATGGACCAAGGGAGCCGAGAACAAAACAAAATTATGGCTACGGACACCCAGGACCATTCAGTCCATTCCTTCTGAATCCCGGATGGTTAGATGCGGCGTATCTCAACTACGCCTGGGCCGACTACTTCAGGCCCCCTCAAATGCGAGAGCCTTCCACGCTTATCAAAGGTGGAGCCGCGCCGCTTACTACGCCACCTGTGACGATACCTGGTGCTGATTTATTCCCCTTTCCTCCGGCTGTTACGAATTTACCACCTGGTGGCCTAATACCTCCACCCGGTGCATTCTTACCACCGAACGGTCTTCTACCTTTCCCACCGCACC | dsRNA | MEGAscript RNAi T7 kit (Ambion) | other | simultaneously with transcription | dsRNA was constructed with a MEGAscript RNAi T7 kit (Ambion). The dsDNA template was amplified with primers TAATACGACTCACTATAGGGAGATCTTGTGCTCTGCGTTACCTGG (runi forward) and TAATACGACTCACTATAGGGAGAGGTGCGGTGGGAAAGGTAGAA (runi reverse). This 626-bp fragment including part of the RUNT domain sequence was confirmed by sequencing. | InsectaCentral | Bmrunt RNAi Bombyx mori RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | embryo | lab_colony | injection | non_specific_dsRNA | 10nl (unknown concentration) | dsGFP | embryo | RT-PCR (semi-quantitative) | whole organism | not checked | 96 | high | |||||||||
| 125 | RNAi B mori bursicon | RNAi | Entered by Jennie. We studied the role of the bursicon gene in wing expansion. First, we investigated its expression at different developmental stages in the silkworm, Bombyx mori. Bursicon gene was expressed at low levels in larvae, high levels in pupae, and low levels again in adults. Then, we injected the double-stranded bursicon RNA into B. mori pupae to test RNA interference. The level of bursicon mRNA was reduced significantly in pupae, and a deficit in wing expansion was observed in adults. In addition, the differential display reverse transcription polymerase chain reaction (DD-RT-PCR) was used to reveal differences in the expression of transcripts in response to the inhibition of bursicon. In conclusion, bursicon plays a key role in the stereotyped behavioral program involved in wing expansion. | pubmed | 17270178 | Huang | Jianhua | RNAi B mori bursicon target | ATGTCCGTATTGAATACTTTTTTAGTCATAGTGGCTTTAATCTTATGTTACGTAAATGATTTCCCTGTTACTGGGCATGAAGTTCAACTACCTCCAGGTACAAAATTCTTTTGCCAGGAATGTCAAATGACCGCAGTCATTCATGTTTTAAAACACCGAGGATGTAAACCTAAAGCAATTCCGTCGTTTGCTTGCATTGGAAAATGCACAAGCTACGTTCAGGTATCGGGTAGTAAAATATGGCAAATGGAGCGTACCTGCAACTGCTGTCAAGAATCAGGAGAAAGAGAAGCTACAGTTGTCTTGTTTTGTCCGGATGCTCAAAATGAAGAAAAGCGATTCAGAAAGGTTTCAACGAAAGCTCCGCTTCAATGCATGTGTCGTCCTTGCGGAAGTATAGAAGAGAGTTCCATTATTCCTCAGGAAGTTGCTGGATACTCCGAAGAAGGACCGCTGTATAACCATTTCAGGAAGTCTCTTTAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | NM_001098375.1 | RNAi B mori bursicon RNAi construct | GGGCATGAAGTTCAACTACCTCCAGGTACAAAATTCTTTTGCCAGGAATGTCAAATGACCGCAGTCATTCATGTTTTAAAACACCGAGGATGTAAACCTAAAGCAATTCCGTCGTTTGCTTGCATTGGAAAATGCACAAGCTACGTTCAGGTATCGGGTAGTAAAATATGGCAAATGGAGCGTACCTGCAACTGCTGTCAAGAATCAGGAGAAAGAGAAGCTACAGTTGTCTTGTTTTGTCCGGATGCTCAAAATGAAGAAAAGCGATTCAGAAAGGTTTCAACGAAAGCTCCGCTTCAATGCATGTGTCGTCCTTGCGGAAGTATAGAAGAGAGTTCCATTATTCCTCAGGAAGTTGCTGGATACTCCGAAGAAGGACCGCTG | dsRNA | MEGAscript RNAi kit (Ambion) | other | simultaneously with transcription | To produce dsRNA for RNA interference, a 383-bp PCR fragment of Burs-a was amplified using cDNA described above as template and primers containing the T7 promoter sequence (in italics) 50-TAA TAC GAC TCA CTA TAG GGA GAG GGC ATG AAG TTC AAC TAC CT-30 and 50-TAA TAC GAC TCA CTA TAG GGA GAC AGC GGT CCT TCT TCG G-30. The PCR products were purified using a PCR purification kit and used as template to generate dsRNA using a MEGAscript RNAi kit (Cat. No. 1626; Ambion, Austin, TX, USA) in vitro. Then, the solution was treated with DNase I and RNase for 1 h at 37 C. The purified dsRNA was added to 200 ll 1 · TE buffer and checked on a 1% agarose gel to ensure that the product consisted of a single 383-bp band. | InsectaCentral | RNAi B mori bursicon RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | injection | non_specific_dsRNA | (20 microgram / insect, 2 microlitres vol) | dsRNA desat2 | pupa | qPCR | not checked | 168 | high | 80 | |||||||||
| 126 | RNAi nodular A mylitta | RNAi | Entered by Jennie. Insect immune system comprises of both humoral and cellular defenses. Nodulation is one of the major, yet very poorly understood cellular responses against microbial infections in insects. Through screening for novel immune genes from an Indian saturniid silkmoth Antheraea mylitta, we identified a protein up-regulated in hemolymph within minutes upon bacterial challenge. We have shown here, for first time, the involvement of this novel protein in mediating nodulation response against bacteria and hence designated it as Noduler. Noduler possessed a characteristic reeler domain found in several extracellular matrix vertebrate proteins. Noduler was shown in vitro to bind a wide range of bacteria, yeast, and also insect hemocytes. Furthermore, Noduler specifically bound LPS, lipotechoic acid, and -1, 3 glucan components of microbial cell walls. RNAinterference mediated knock-down of the Noduler resulted in significant reduction in the number of nodules and consequent increase in bacterial load in larval hemolymph. The results suggest that the Noduler is widely conserved and is involved in very early clearance of bacteria by forming nodules of hemocytes and bacterial complexes in insects. The results would promote further studies for understanding of the crucial but hitherto overlooked nodulation mechanism in insects and also provide cues for the study of similar mammalian proteins whose function is not understood. | pubmed | 17982085 | Ghande | Archana S | RNAi nodular A mylitta target | CGGACTTCAGATATCGTGTGGGATAGTTATTAAACAGTATTGCCCAAGAAATATGATGTTCGCGTACATAGTAGCTGTCGTGTCAGCGTTGGCGCTTACCAGTGCTTTCCCTACTGGTGCACCACGTAGCGCTTGCTTCGACATGATACCCGGCCACTTCGCTAACCCAAAATTGGAACCTGCGCCTTACACTATCACCACGCCTATTAGCGCGGTGAAAGGTGGTAATTCCGTTGAAGTTACAATCAGCGGCAAGACACCTGAAGACACCATGCGTGGTATCCTACTCGAGGCTCGACAAGGAGACAACATCGTCGGCACGTGGACCGTACCTCCTGGTGATGACTTCTCACAACCCATGAACTGTGGAGAACCTAATAACGCCGTCACCCACAAACGCCATTCTGAGTCAGCGGACAAGCAGACCGTGTCATACGTTTGGACTGCTCCCAGTGATTTGGAAGGCGACGTCGTATTCATGGTCACCATAGTAAAGGATTACTCCAACTTCTGGGTCAGACAAACCTCGGCACCCGTAAAAATTTTAAGTCACCATTAATCGCGTGAACCTTACATTAAGTATTTGTACTTTGTAGTTTTATAAGAAACAACAAATTTATAAAACCATTGTGTATTTATAAGCAATAAACTATATATTTTTATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACAAAAAAAACAAAAAAAAAA | Lepidoptera | Saturniidae | Antheraea | mylitta | 34739 | cds | NCBI | DQ666501.1 | RNAi nodular A mylitta RNAi construct | ATGATGTTCGCGTACATAGTAGCTGTCGTGTCAGCGTTGGCGCTTACCAGTGCTTTCCCTACTGGTGCACCACGTAGCGCTTGCTTCGACATGATACCCGGCCACTTCGCTAACCCAAAATTGGAACCTGCGCCTTACACTATCACCACGCCTATTAGCGCGGTGAAAGGTGGTAATTCCGTTGAAGTTACAATCAGCGGCAAGACACCTGAAGACACCATGCGTGGTATCCTACTCGAGGCTCGACAAGGAGACAACATCGTCGGCACGTGGACCGTACCTCCTGGTGATGACTTCTCACAACCCATGAACTGTGGAGAACCTAATAACGCCGTCACCCACAAACGCCATTCTGAGTCAGCGGACAAGCAGACCGTGTCATACGTTTGGACTGCTCCCAGTGATTTGGAAGGCGACGTCGTATTCATGGTCACCATAGTAAAGGATTACTCCAACTTCTGGGTCAGACAAACCTCGGCACCCGTAAAAATTTTAAGTCACCATTAA | dsRNA | MEGAscript T7&SP6 kit (Ambion) | phenol_chloroform | subsequent to transcription | The dsRNA specific to Noduler was synthesized corresponding to entire protein coding region (507 bp) using primers: forward primer, 5 -ATGA TGTTCGCGTACATAGTAGCTG-3 ; reverse primer, 5 -TTAATGGTGA CTTAAAATTTTTACGGGTG-3 (Fig. 1A); and cloned into pCRIITOPO vector (Invitrogen Life Technologies) followed by amplification with M13 forward and reverse primers. This template with flanking T7 and SP6 promoters was used for in vitro transcription reaction and sense and antisense RNA strands were generated with the T7 and SP6 Megascript kits as prescribed by the manufacturer (Ambion). The DNA template was removed from the transcripts by DNase treatment and the RNA products were subsequently purified by Trizol extraction (Invitrogen Life Technologies) followed by isopropanol precipitation. The complementary single stranded RNAs were dissolved in DEPC treated water, combined in equimolar amounts in 1 insect buffer saline (IBS; composition: NaCl - 160 mM, KCl - 10 mM, CaCl2 - 4 mM) and annealed by heating to 95°C and slow cooling overnight at room temperature. Similarly, dsRNA specific to full-length green fluorescent protein (GFP) was synthesized as a nonspecific control. The dsRNA formation was confirmed by agarose gel electrophoresis and the concentration was determined spectrophotometrically. | InsectaCentral | RNAi nodular A mylitta RNAi construct | Lepidoptera | Saturniidae | Antheraea | mylitta | 34739 | local resource | larva | lab_colony | injection | non_specific_dsRNA | (100 micrograms/insect) | dsGFP | larva | W.blot | hemolymph | not checked | 24 | high | |||||||||
| 127 | Aminopeptidase-N RNAi in Helicoverpa armigera | RNAi | Entered by Jennie. Aminopeptidase-N (APN) and cadherin proteins located at the midgut epithelium of Helicoverpa armigera have been implicated as receptors for the Cry1A subfamily of insecticidal proteins of Bacillus thuringiensis. Ligand blot analysis with heterologously expressed and purified H. armigera Bt receptor with three closely related Cry1A proteins tentatively identified HaAPN1 as an interacting ligand. However, to date there is no direct evidence of APN being a functional receptor to Cry1Ac in H. armigera. Sf21 insect cells expressing HaAPN1 displayed aberrant cell morphology upon overlaying with Cry1Ac protein. Down-regulating expression of HaAPN1 by RNA interference using double-stranded RNA correlated with a corresponding reduction in the sensitivity of HaAPN1-expressing cells to Cry1Ac protein. This clearly establishes that insect cells expressing the receptor recruit sensitivity to the insecticidal protein Cry1Ac, and their susceptibility is directly dependent on the amount of HaAPN1 protein expressed. Most importantly, silencing of HaAPN1 in H. armigera in vivo byRNAinterference resulted in reduced transcript levels and a corresponding decrease in the susceptibility of larvae to Cry1Ac. BIAcore analysis of HaAPN1/Cry1Ac interaction further established HaAPN1 as a ligand for Cry1Ac. This is the first functional demonstration of insect aminopeptidase-N of H. armigera being a receptor of Cry1Ac protein of B. thuringiensis. | pubmed | 17213205 | Sivakumar | Swaminathan | Aminopeptidase-N RNAi in Helicoverpa armigera target | TTTTTTTTTTGACCAGGGTGGAGTCACAATGGCGAACCGCTGGTACACCCTCCTTTTGGGGGCAGCTCTTCTGCAGAGCGCCCTCTCTTTCGGTCCTATTGAAGTGACAGACGACGAATGGGCTGAATACAGAAACCTGATGCGGGACCCTGCTTACCGCCTGCCCACGACTACGAAGCCTAGCAACTACGCCATCAACCTTACACCATACTTCACTGGCTCCACATTAGCTTTCACCTTTGAGGGTTCAGTAGCCATCACCATTACGGCCACGCAAGCTAATGTCAACGAAATTGTGCTCCATTGCAATGACTTGACCATCGAATCGGTCACGGTGGCTACAGTAGCTAGCCCGAATGTTAATCTTGCGGCAAGCGGACAGACTTTTGTCTGCGACCCTGTCTACAGTTTCCTAAGAATAAGGACCGCAGGAGCATTGGCTGTTGACACGAATTACGTAATCAGGAGTACCTTCAGGGGCAACCTTCAAACGAACATGAGAGGTTTTTACAGGAGTTGGTATTACGACTCTAGTCGTGAAAAGAGATGGATGGCAACGACCCAATTCCAGCCTGGCCACGCTCGCCAAGCCTTCCCCTGCTACGACGAACCAGGATTCAAAGCCACTTTCGATATTACCATCAACAGGGAAGCCGACTTCAGCCCAACCCTTTCCAACATGCCCATTAGAACCACAACTAACCTCGCAACCGGCAGAGTTGCTGAGACGTTCCACACCACTCCCGAAACATCAACGTACTTGATTGCTTTTATAGTATCGCACTATAGTCAAGTAGCTTCAAACAACAACCAGCAGAGGCCTTTTCATATCTATGCTAGAGACAATGTTGGGGTCCATGGGAACTTCGCCTTGGAAATTGGAGTGCCTCTCTTGGAAGTCATGGAGCGCTATACAGAAATACCTTACTATGGCATGGCTCAAAACATGAACATGAAGCAAGCTGCTATCCCTGACTTCTCAGCTGGTGCCATGGAGAACTGGGGACTTTTGACTTACAGGGAGGCTTTGATTCTGTTTGATCCAGTGAATACCAACAACTTCTACAGACAGCGTATCGCCAACATCATTTCTCACGAAATCGCTCACATGTGGTTTGGAAACCTCGTCACATGCGCTTGGTGGGACAACCTTTGGCTGAACGAAGGTTTTGACACGATTCTACCAGTACTACTTGACTGGGTGGTCGCTCCTGAAATGGGCTTCGAAACTCGTTTCATAGTGGAACAGCTGCACGTGTCGATGTTGTCTGACTCCCTTGACTCTGCTCACGCCCTCACCAACCCCAATGTGAACGACCCTACTACTGTCAGCGCACACTTCTCCACCATCACTTATGCCAAAGGCGCCAGCATCATCAGAATGACACAACACTTACTGGGCAACAACACTTTTGTGAAAGGCCTTAGGACTTACTTGAAAGACAATGCCTACGGTGTCGCTGAGCCCCGTCACTTGTTCACTGCCTTAGACGCTGCTGCAACCGCAGACAATCGTCTCGCCAACTATGGTGGTATGACCATCGATCGCTACTTCAGAAGCTGGTCAGAGAAAGCAGGTCATCCATTGTTGACTGTGTCCATTGACCACTCCTCTGGACGTATGACCATTATTCAAACCCGATTTGAGCGCAATAGTGGTGTATCAACAGCGACCGACAGTCTTTGGGACATCCCCATTACTTGGACTAGGGCAGGATCTATTGACTTTGACAACCTGAAACCTACGCAATTCATCAGTGGTGTTTTGACTATCATCGACAGAGGAACCACTGGCAGGGAATGGGTTATTTTCAATAAGCAGCAAACTGGATTCTATAGAGTTAACTACGATCAAATCACTTGGGGTCTCATCACTCAAGCTCTTAGGAGTAACGTGAGGCTATCAATCCATGAATATAACCGTGCTCAGATCGTCGATGACGTTATGTTGTTAGCTCGAGCTGGCATTATGACCTACAGCAGAGCCTTGAACATTCTCTCCTTCCTCAAATTTGAAGATCAATATGCTCCTTGGGGCGCACGTATTACTGGATTCAACTTCGCCCTCCGAAGATTAGCTCATGATGCTACAGCTCTCCAGAAACTGAGGAATGAAATCTTGGATTTGAGCACGGCCATCGTTAATCGTTTGGGCTTCAGCGAGCCAGCTGTTAGCAATTTCATGGACGACCTTCTCCGCATGAACGTCATGACTTTCCTTTGTGACATCGGCCACCAGGGGTGCATCACTGCTGCTAGAACTAGCTTTGCTACCTGGAAGAACGGTGGAGTTGTCCCACCCAACATGCGTCCATGGGTGTATTGCAATGGAGTGCGCTACGGAGATCAATCTGACTTCACTCACTTGTGGACTAGTACAAACAATCTGACGTTGCTAACGATCAAGTTGGTTCATGCTGTTCGGCTTTGTCTTGGTTGCACTCTTAAACCAGGCCAGCTTGAGATATTCCTCAATGACATCGTGTCTGGTGGCATGACTCGGCCTCAAGACCACAGCGCAGCTACCGTAGCGGCTGTCCGCAGTAATGAAGTGAACACTATGAGAGTGTTCACATGGCTGCAGGCTAATGTGCAACAGACTATAAGCACTCTGGGAAGGGTCAGTCCTATTTTGAACGAAATCACAGCGGGCCTATTGAATGAAGCCCAAATCACTGCAGCTCACACTAGTGCCACAAATGGCATCGCCACGTCTAGGTCCAACATTCAGTGGTACACACAGAGGGTTGCGGAATTCAACGTATACTTTGAAACTGGATATGTTGAAGAGAACTTCGCTGATCCTACAACTACCAGTACGACTACGACTACGACAACTACTACGACCACTACAGCAGCTCCTACGACTACGACTACGACAGAGGCGCCTACGACAACAACTACGACCACTACGGAAGCGCCTACGACCACAACTACAGAAGCTACAACAACACCTGTACCTGGCTCAGCAAACATCGCCACTCTTAGCATCGTCACAATGATCGTGACTCTCGTTGTTAATATGGCTTAAATTTTATTTGTATTTTAATATATAAAGAACTGTATTTTAACAATAGTATAACATAATGTAATATATCTTGTAACTATATTTATGACAATAATATTAATATTTTTTTATTAAACTGATATTAAAATATATTGGTTCATTTTTTTCTAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | NCBI | AF521659 | Aminopeptidase-N RNAi in Helicoverpa armigera RNAi construct | GTTAGCTCGAGCTGGCATTATGACCTACAGCAGAGCCTTGAACATTCTCTCCTTCCTCAAATTTGAAGATCAATATGCTCCTTGGGGCGCACGTATTACTGGATTCAACTTCGCCCTCCGAAGATTAGCTCATGATGCTACAGCTCTCCAGAAACTGAGGAATGAAATCTTGGATTTGAGCACGGCCATCGTTAATCGTTTGGGCTTCAGCGAGCCAGCTGTTAGCAATTTCATGGACGACCTTCTCCGCATGAACGTCATGACTTTCCTTTGTGACATCGGCCACCAGGGGTGCATCACTGCTGCTAGAACTAGCTTTGCTACCTGGAAGAACGGTGGAGTTGTCCCACCCAACATGCGTCCATGGGTGTATTGCAATGGAGTGCGCTACGGAGATCAATCTGACTTCACTCACTTGTGGACTAGTACAAACAATCTGACGTTGCTAACGATCAAGTTGGTTCATGCTGTTCGGCTTTGTCTTGGTTGCACTCTTAAACCAGGCCAGCTTGAGATATTCCTCAATGACATCGTGTCTGGTGGCATGACTCGGCCTCAAGACCACA | dsRNA | T7 and SP6 polymerases (MBI Fermentas) | phenol_chloroform | subsequent to transcription | The full-length cDNA of haapn1 (AF521659) from the midgut of H. armigera larvae was already reported by us (16). A 565-bp internal fragment of haapn1 was obtained by PCR using the primers HaAPN1â53F 5 -GTTAGCTCGAGCTGGCATT- 3 and HaAPN1â54R 5 -TGTGGTCTTGAGGCCGAGTCAT- 3 . The truncated fragment of H. armigera haapn1 was subcloned in pGEM-Te and used for the preparation of dsRNA. As a control, the gene for falcipain of P. falciparum cloned in pGEM-T was used as described earlier (18). The pGEM-Te-cloned fragments were amplified by PCR using vector-specific universal and reverse primers (Promega). The PCR product was purified (Qiagen GmbH) and used as DNA template for dsRNA preparation after the in vitro transcription procedure described by us (18). The T7 and SP6 RNA polymerases (MBI Fermentas) were used to generate single strand sense RNA and antisense RNA, respectively, from the DNA. To make dsRNA, equal amounts of sense RNA and antisense RNA were mixed, heated to 65 °C, and annealed by slow cooling over 4 h followed by DNase (Invitrogen) treatment for 15 min at 37 °C. The dsRNA was extracted with phenol-chloroform and precipitated overnight with ice-cold ethanol in the presence of 0.3 M sodium acetate, pH 5.4, at 20 °C. The dsRNA pellet was washed with 75% ethanol and resuspended in DEPC-treated water to a concentration of 1 g/ l dsRNA. | InsectaCentral | Aminopeptidase-N RNAi in Helicoverpa armigera RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | 1 | injection | DEPC water | (6 microlitres) | Unknown control. | larva | qPCR | Midgut | not checked | 48 | high | 45 | |||||||
| 129 | RNAi alpha-integrin M sexta | RNAi | Entered by Jennie. In their encounters with foreign intruders, the cells of the insect innate immune system, like those of the mammalian immune system, exhibit both humoral and cell-mediated responses. Some intruders can be dispatched by the humoral immune system alone, but many must be phagocytosed by individual hemocytes or encapsulated by interacting hemocytes. Surface proteins of hemocytes control the abrupt transition of hemocytes from resting, nonadherent cells to activated, adherent cells during these cell-mediated responses.Twoof these surface proteins, an integrin and a tetraspanin, interact during this adhesive transition. As demonstrated with a hemocyte adhesion assay and a surface plasmon resonance assay, the large extracellular loop of tetraspanin D76 binds to a hemocyte-specific integrin of Manduca sexta. The interaction between the large extracellular loop domain and hemocyte-specific integrin is interrupted not only by a monoclonal antibody (MS13) that binds to a domain of -integrin known to be a ligand-binding site for cell adhesion but also by double-stranded -integrin RNA. Transfected S2 cells expressing tetraspanin mediate adhesion of hemocytes. A monoclonal antibody to tetraspanin D76 perturbs the cell-mediated immune response of encapsulation. These studies involving antibody blocking, RNA interference, and binding assays imply a trans interaction of integrin and tetraspanin on hemocyte surfaces. | pubmed | 17553801 | Zhuang | Shufei | RNAi alpha-integrin M sexta target | GAAACAGTCGTAGTGTTGGCTTCGTTGAAATAACAACCATGATGTTACTATGGCTATTGCTGATGGCGCAACTGGCTTGGCCGACATCAGCACTGTACCACGAAAAATCTTTGAAAGTATTTCAACCAGGAGATCTCAAGTCACCACTGAGAAAAGAGGCCAACTTTGGATTTAGCATCGCTTATCAATCTGCTACTCAAAGTTTGATCATAAGCGCTCCAACAGCGGATTACATCGGCAGAATATATTCTTGCGACATTGAACTGAAAAGAAATTGTACTGTCGTGCCGTATGACATCAAGAGAGACGACCCTCTGTATAACCATGATTACTGGCTGGGTGCGACAGTGAAGGCGGGCCCTGACTATTTTGTCACGTGTGCTCCGCGTTACGTCGAAGTGGGTAGTTCGTACAACCTACCCGGTGAATTCAGCCATTGTTATAGATCATCCAAGCACTCCAAACTACTCCCTATTAGTCAAATAAGTAGCCAACAGCGAAGAGTTAACACAAAAAAATTCGAGGATGAAATGGATTCATTTGGATGGAGTATGGATTTGAAGGACAGGAAAATTCTATTTGGAGGCCCTGCTATGTTCTCAGGACGTGCAATGGCATCTGCCGATTCAGCGATAACATCAATAAGTGGACATTCGAAAGGTGTAGACGTCAAATACAATTTAGGTTACAGTGTCGCAATAGGCGCTTTCTTTTCTGACAAAATCGTTTATGCTGTTGGATCGCCTTTTGGACAAAACGGCCGCGGAAAGGTGGTTTTCTTCAATAAGGATTGGAAAGTAATGTCGTCTATAAGTCATAAAAATATTGGCACGATGTTTGGCGCGGTGCTGGCGACTGCCCATATATTCAGTGATTCACTGACTGACTTGCTCGTGGGAGCGCCGACCGAGGCTGCAAATCACGACTACGACACCGGAGCTGTTTACTATTACAAAACTGGGAGTCGACGTAGTGTACAAATGAGCATTGAACATACAAAAATTATTAGTGGGTACCAACCGGGATCGCTCTTCGGCAGCGCAATCATAAGTGTCGGCGACTTGAATGGCGACGGAAAAGATGAAATAGCGATTGCTGCGCCGTACGAGGATGAGGGCAAAGGCGCTGTTTACCTCTACTGCGGGTCCTCATTCTTGGATGCATCATCGAATGTGTTGCTTCAACGACTCCAGCCAGAAGGCCTCCACACCTTCGGCTTGAGCCTGTCCTCTCCCAGCGACTACGATGAAAATGGATGCAATGAGCTGGCGATTGGTGCCCCACAAGACAATGCAGTGGTGTTGTACAAGTGCTTGGCGTCTGTTGTGCTGACAGTCTTCGCCGTATTTCCAAACCTACGGGACCGCAGAAATGTAAAAAGTCCGTATCCTGATAGAAATGAAACTTATTTCGTTTTCGATTCGTGTTTTGACGTCAAATACCCGAAGAAACTAAAAACTGTTATAGCGAAAATTGAAGTAACCGTCCAAATTAAACACCCGGACGCAACCTTAGCGATGCCTCACAAAGACGGAAAGTACAATGTCACACTAGATATAAAGAAAACACGGTATTGTGAAAAAATTGGAGTAAGCACGCCTGAGCACGGCAACTACAACATAAAAATATTTTACAATATACAAGCTCGTTTAGTAAACTCTCCGATCAACGAGACGGATTTCGACGGCAAACGCGTCATCCTGAGCGGGCGTAGTGAGTTATCAATATCGGACAATGTATGGGCCGCTGAGTGTAAGGTCCAGAATGCTTGCAAAGCAGATTTGAGCCTGAAGATAGCGACCTCGCCGCGGGGATCATACATGATCGGGTCTATGGAGCCTATAAAAGTGAATATGATATTAGAAAACAAAGGTGAAATAGCTTACGACGCCTGCGTGGAACTGCAGCTAGAGGGAGCGGCAATGAAGAAAACACCAACTACCTCCTGCACTCATCACCCACTTACTAACACGTTGAAATGTGCACCCAGCTATGCACTGAAGACCGATAAATCCTGGGAAACTACAGACATAATACTAAAAACAGAAACCTTGACAAACAAAAATAAGCAAATTAATTTAACGTCACGTTTATACGAGCATTGTACTGATCCTGCAACATTTAAAGAACAAAACTATACTATTACTGTACTAGCGAATTCTGAAGGGATTACTGTAAATGGAAAAACAAACATCGGTGACGTAGTGAGCATGACGACATTAGAAGTAACTGATTCAGGAAAACAGTTTCAACATAATTACATGATACAAAACGACGGGATCACATGGGAGGATGTGACTTGTGAAATCATATTGCCCAAGTTGCCCTACGTCAACTACCCACAAAAAGCTGTTACGGTTTTTATTCAGCAAAATAGTTTCGAGTGTAATATGACTGACGATTTGAGCCCTGACTACATAACTGCAAATTGTAAGCTGAGTAGCATCAAACAGAACATAATTACTTTCATCATTGTCTTAATACAAGTGCCGCCAACAACGCTCGATGACATTCTCTATAAGCAAAATGTGACCATAAAATCAACATTGAAGCTTCACTTTGACGATGGCGACAAAACATACAGTGTGGCGACCGAAGTGATGCTGAGAGACACAGGCATACCCTGGTGGATAATATTATTGGCGGCGCTAATTGGCCTCTTGATACTAATCATCCTTATACTGATTCTTCGCCAGTATGGTTTCCTTAAAAGGAAAGAAAGGAAAAAGCTACAAGAGTTAAGGAAGAGTGTACGGAGACAAACGGTTAGACGCTCCATGATGCAGCAGCAGGTAGAAGATCGTCAAAGATTAACCGATGTACCGATAGAAGCGACTGAAACTGACGTACCGTGCGGCATAGAACCAGACCATAGAGTAGAAGTAAATATTAAATGATTTTACCGCATTAGCAACGTGCCATTCACTACTTATTTACCACTAGAGTTGGCGTTTGGTGTTACATATTATAAGTCCATTAATATTTCCATATATATTATAAGTCCATAAATTAAACTACATTTGCTGACTTTGTGCCAGAAATCATCCATTAATTATAACTCATATTTTTACCAAAAATCCTGGTTTTAGTTTACTGACTTTTTTTATAATTTTGTAGTTTAATGTAAGTATGAGTGTATTTCTGACTGAAAGTATCATGTTGCCGCTGTGACGAATTCTCTTAAGAGTCATAAGTAGTGGCATTAGGGCATGTAAAATTACTTTTTATTTATATTTATTTTCTTCGAAACTTTGAATTTTTTTATTTTAAATCTACGCTGAAGTACCTTATTGTAAAGTGTTATTTCCAAATAGGTAAATATGTAGTTTTATTTAGTAACGCAATATAAAAATGACCCTGTATTTTTATCTTGCATCATGATTAATAGTGCATTGTAATTTATGTATTATTCATTAATTAAGGGCTGATTTGTCAATCGTCAGACATCTATTTAACCTAACTGAATAATAAAAGTGTGCCAATTGATAACTTAAAATAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | DQ531844.1 | RNAi alpha-integrin M sexta RNAi construct | GCGCAATCATAAGTGTCGGCGACTTGAATGGCGACGGAAAAGATGAAATAGCGATTGCTGCGCCGTACGAGGATGAGGGCAAAGGCGCTGTTTACCTCTACTGCGGGTCCTCATTCTTGGATGCATCATCGAATGTGTTGCTTCAACGACTCCAGCCAGAAGGCCTCCACACCTTCGGCTTGAGCCTGTCCTCTCCCAGCGACTACGATGAAAATGGATGCAATGAGCTGGCGATTGGTGCCCCACAAGACAATGCAGTGGTGTTGTACAAGTGCTTGGCGTCTGTTGTGCTGACAGTCTTCGCCGTATTTCCAAACCTACGGGACCGCAGAAATGTAAAAAGTCCGTATCCTGATAGAAATGAAACTTATTTCGTTTTCGATTCGTGTTTTGACGTCAAATACCCGAAGAAACTAAAAACTGTTATAGCGAAAATTGAAGTAACCGTCCAAATTAAACACCCGGACG | dsRNA | MEGAscript RNAi kit (Ambion) | other | simultaneously with transcription | Double-stranded RNA (dsRNA) was prepared from a template that consisted of a 468-bp PCR product amplified from the M. sexta integrin- 1 cDNA (nucleotides 1039â1506), using primers that each contained a T7 RNA polymerase promoter sequence at their 5 ends. The sequences for these primers are as follows: forward primer 5 -TAATACGACTCACTATAGGGAGAGCGCAATCATAAGTGTCGGC- 3 ; reverse primer 5 -TAATACGACTCACTATAGGGAGACGTCCGGGTGTTTAATTTGG- 3 . The PCR product was used as template for preparation of dsRNA using MEGAscript RNAi kit (Ambion), following its isolation and extraction after agarose gel electrophoresis. Double-stranded RNA of the M. sexta 1-integrin subunit was prepared as described previously | InsectaCentral | RNAi alpha-integrin M sexta RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | injection | non_specific_dsRNA | (5 micrograms) | dsGFP | larva | qPCR | hemocytes | not checked | high | 40 | |||||||||
| 131 | RNAi beta-integrin M sexta | RNAi | Entered by Jennie. In their encounters with foreign intruders, the cells of the insect innate immune system, like those of the mammalian immune system, exhibit both humoral and cell-mediated responses. Some intruders can be dispatched by the humoral immune system alone, but many must be phagocytosed by individual hemocytes or encapsulated by interacting hemocytes. Surface proteins of hemocytes control the abrupt transition of hemocytes from resting, nonadherent cells to activated, adherent cells during these cell-mediated responses.Twoof these surface proteins, an integrin and a tetraspanin, interact during this adhesive transition. As demonstrated with a hemocyte adhesion assay and a surface plasmon resonance assay, the large extracellular loop of tetraspanin D76 binds to a hemocyte-specific integrin of Manduca sexta. The interaction between the large extracellular loop domain and hemocyte-specific integrin is interrupted not only by a monoclonal antibody (MS13) that binds to a domain of -integrin known to be a ligand-binding site for cell adhesion but also by double-stranded -integrin RNA. Transfected S2 cells expressing tetraspanin mediate adhesion of hemocytes. A monoclonal antibody to tetraspanin D76 perturbs the cell-mediated immune response of encapsulation. These studies involving antibody blocking, RNA interference, and binding assays imply a trans interaction of integrin and tetraspanin on hemocyte surfaces. | pubmed | 17553801 | Zhuang | Shufei | RNAi beta-integrin M sexta target | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | AY630342.1 | RNAi beta-integrin M sexta RNAi construct | GTTTAAGCCGCAAGTGATGAGAATGAAAGCGCGTCCCGGTACTAAATTATATTTCAACATGTCGTATAAGCCGGCCGAGCACTTTCCTTTAGATGTTTATTATCTCATGGACACCTCTTATACAATGACAATGCATAGAGAGGCGCTCATTAGTCAAGCAAACCAAATATATAAAGAACTTACAAGTTTAACTAATAATGTTCAACTTGGCGTTGGCAGTTTTGTAGAAAAACCAGGCTACCCGTACTTCGACAAAAACAAACAAGAAAGCGTCGCATTTATAAATGTATTGCCGTTAACAAAAAATATTAAACAGTTTACACAAAGTGTTCAAAATATGTCTTTTGGTTCGAATTATGACGATATGGAAGCTGGGCTGGATGCCCTAATGCAAGTCATGACTTGTGAGAAAGAAATAGGGTGGAGGCCT | dsRNA | MEGAscript RNAi kit (Ambion) | other | simultaneously with transcription | Double-stranded RNA (dsRNA) was prepared from a template that consisted of a 468-bp PCR product amplified from the M. sexta integrin- 1 cDNA (nucleotides 1039â1506), using primers that each contained a T7 RNA polymerase promoter sequence at their 5 ends. The sequences for these primers are as follows: forward primer 5 -TAATACGACTCACTATAGGGAGAGCGCAATCATAAGTGTCGGC- 3 ; reverse primer 5 -TAATACGACTCACTATAGGGAGACGTCCGGGTGTTTAATTTGG- 3 . The PCR product was used as template for preparation of dsRNA using MEGAscript RNAi kit (Ambion), following its isolation and extraction after agarose gel electrophoresis. Double-stranded RNA of the M. sexta 1-integrin subunit was prepared as described previously | InsectaCentral | RNAi beta-integrin M sexta RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | injection | non_specific_dsRNA | (5 micrograms) | dsGFP | larva | qPCR | hemocytes | not checked | high | 40 | ||||||||||
| 132 | RNAi beta-integrin M sexta 2 | RNAi | Entered by Jennie. Upon encountering an object recognized as foreign, insect hemocytes aggregate in multiple layers on the surfaces of the object in a process known as encapsulation. For encapsulation to occur, hemocytes must switch from their usual nonadherent state to an adherent state, presumably by regulating the activity of adhesion proteins. Although detailed knowledge exists regarding the adhesion receptors for cells of the mammalian immune system, comparable information on adhesion molecules of insect hemocytes and their function in immune responses is extremely limited. We report here the identification of an integrin present exclusively on the surface of hemocytes in the tobacco hornworm, Manduca sexta. Monoclonal antibodies MS13 and MS34, which bind to plasmatocytes and block encapsulation, were used for immunoaffinity chromatography to isolate their corresponding hemocyte antigen, which was revealed to be the same integrin b subunit. A cDNA for this M. sexta integrin b1 was cloned and characterized. Integrin-b1 mRNA was detected by Northern analysis in hemocytes and not in other tissues tested. MS13 and MS34 were demonstrated to bind to a recombinant fragment of integrin b1 consisting of the I-like domain, consistent with their blocking of a ligand-binding site and subsequent disruption of plasmatocyte adhesion. Injection of double stranded integrin-b1 RNA into larvae resulted in decreased integrin b1 expression in plasmatocytes and significantly suppressed encapsulation. These results indicate that activation of ligand-binding by the hemocyte-specific integrin plays a key role in stimulating plasmatocyte adhesion leading to encapsulation. | pubmed | 15804572 | Levin | David M | RNAi beta-integrin M sexta target | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | AY630342.1 | RNAi beta-integrin M sexta 2 RNAi construct | GTTTAAGCCGCAAGTGATGAGAATGAAAGCGCGTCCCGGTACTAAATTATATTTCAACATGTCGTATAAGCCGGCCGAGCACTTTCCTTTAGATGTTTATTATCTCATGGACACCTCTTATACAATGACAATGCATAGAGAGGCGCTCATTAGTCAAGCAAACCAAATATATAAAGAACTTACAAGTTTAACTAATAATGTTCAACTTGGCGTTGGCAGTTTTGTAGAAAAACCAGGCTACCCGTACTTCGACAAAAACAAACAAGAAAGCGTCGCATTTATAAATGTATTGCCGTTAACAAAAAATATTAAACAGTTTACACAAAGTGTTCAAAATATGTCTTTTGGTTCGAATTATGACGATATGGAAGCTGGGCTGGATGCCCTAATGCAAGTCATGACTTGTGAGAAAGAAATAGGGTGGAGGCCT | dsRNA | other | simultaneously with transcription | Double stranded RNA (dsRNA) was prepared from a template that consisted of a 477 bp PCR product amplified from the integrin-b1 cDNA, using primers that contained T7 RNA polymerase promoter sequences at their 50 ends. The following primer sequences from the integrin cDNA were designed with T7 promoters at their 50 ends. Forward primer: 50TAATACGACTCACTATAGGGAGACAGTTTAAACCTCAAGTGATGAG30 (underlined sequence represents residues 315-335 in the 2426 bpin tegrin-b1 cDNA). Reverse primer: 50TAATACGACTCACTATAGGGAGAGGCCTCCA CCCTATTTCTTTC30 (underlined residues represent the reverse complement of residues 723â744 in the integrinb1 cDNA). The 477 bpPC R product was isolated by agarose electrophoresis and purified using the QIAquick gel extraction kit (Qiagen). This DNA was used as template for preparation of dsRNA, using MEGAscript RNAi kit (Ambion), following manufacturerâs instructions for synthesis and purification of dsRNA. | InsectaCentral | RNAi beta-integrin M sexta 2 RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | injection | non_specific_dsRNA | (5 micrograms) | dsGFP | larva | RT-PCR (semi-quantitative) | hemocytes | not checked | 48 | none | |||||||||||
| 133 | RNAi beta-integrin M sexta 2 siRNA | RNAi | Entered by Jennie. Upon encountering an object recognized as foreign, insect hemocytes aggregate in multiple layers on the surfaces of the object in a process known as encapsulation. For encapsulation to occur, hemocytes must switch from their usual nonadherent state to an adherent state, presumably by regulating the activity of adhesion proteins. Although detailed knowledge exists regarding the adhesion receptors for cells of the mammalian immune system, comparable information on adhesion molecules of insect hemocytes and their function in immune responses is extremely limited. We report here the identification of an integrin present exclusively on the surface of hemocytes in the tobacco hornworm, Manduca sexta. Monoclonal antibodies MS13 and MS34, which bind to plasmatocytes and block encapsulation, were used for immunoaffinity chromatography to isolate their corresponding hemocyte antigen, which was revealed to be the same integrin b subunit. A cDNA for this M. sexta integrin b1 was cloned and characterized. Integrin-b1 mRNA was detected by Northern analysis in hemocytes and not in other tissues tested. MS13 and MS34 were demonstrated to bind to a recombinant fragment of integrin b1 consisting of the I-like domain, consistent with their blocking of a ligand-binding site and subsequent disruption of plasmatocyte adhesion. Injection of double stranded integrin-b1 RNA into larvae resulted in decreased integrin b1 expression in plasmatocytes and significantly suppressed encapsulation. These results indicate that activation of ligand-binding by the hemocyte-specific integrin plays a key role in stimulating plasmatocyte adhesion leading to encapsulation. | pubmed | 15804572 | Levin | David M | RNAi beta-integrin M sexta 2 siRNA target | ATTTATTTGAATACAACGATGTGGAATATATATTCTATAGTGTTTGTGTCGCTATGTTTAAAATTAATCAATTGTCAATCTGTATGTAATCACTTAGGGACATGCGGGGAGTGTATAGGTTTCAGCAGCGGAACGGAGCGATGCATTTGGTGTCAACAGGAGACGCTGGATAATTATACAAGTAGGTGCCAACCCGAAAGCTATTTAAAAAAAGAAGGATGGTGCGACTCGAAATTCATTGAAAATCCTAAAAAAGTTCAATTAATTGAAGTGGACAAAGATTTTGGTTCGACTATGGATGATTTAAAAATTCAGTTTAAGCCGCAAGTGATGAGAATGAAAGCGCGTCCCGGTACTAAATTATATTTCAACATGTCGTATAAGCCGGCCGAGCACTTTCCTTTAGATGTTTATTATCTCATGGACACCTCTTATACAATGACAATGCATAGAGAGGCGCTCATTAGTCAAGCAAACCAAATATATAAAGAACTTACAAGTTTAACTAATAATGTTCAACTTGGCGTTGGCAGTTTTGTAGAAAAACCAGGCTACCCGTACTTCGACAAAAACAAACAAGAAAGCGTCGCATTTATAAATGTATTGCCGTTAACAAAAAATATTAAACAGTTTACACAAAGTGTTCAAAATATGTCTTTTGGTTCGAATTATGACGATATGGAAGCTGGGCTGGATGCCCTAATGCAAGTCATGACTTGTGAGAAAGAAATAGGGTGGAGGCCTGGCTCAAGGCGTATAATTGTTCTGTGCACCGATTCCCCATATCACAGCGCTGGTGACGGCAAAATGATAGGCATTATCAAACCCAACGACATGTTATGCCACTTAAAGGAACAAAAATATGAAGCAGAAATGGCCCAAGATTATCCATCTGTGAGTAAAATAAATAAAGTAGCAAAGCAAGGAAAATTCGGTATCATATTCGCTGCTTTGGCTGAGGTCCGTGATGTTTATACATTGTTAGCGGAACAAATAGTCGGAGCTGAGTACGCCGAACTGAAGAAACAGAAGTCAAATATTGTAGAGATCATTATAAAAGCGTACCAACGCAGCGTTCGAAGTATCAAATTGGATTACGACATCCCCTCCTTCGTTAGACTGAAACTTAATCAAAGTTGTGACGGGACACCAATTAATTGTGCCAGCACCTATGAAAATCCAGTGGTTACAATTCCGGCTATTCTAGAGGTTAAAGAATGTCCTAAAGAAAATAAAACACATGAGCTTGTTATTAACCCTGTGTCTTTAAATGACAAATTAATAATTAAATTGGAAGTCATCTGTAAATGTGAATGTGAAGTCAAAAGTGATATAAGTTCAAGATGTAATAATGCAGGATATATACAGTGTGGTATCTGCAAGTGTCTCGATTCAAGTTATGGCGACGAATGTCAGTGCAGCGTTACATCTTCGGGGGTGGCTAATAAGGAGAAAGATGACGCCAAATGCCGTAAGGATCTAAATGACATAGTACTGTGTAGTGGGAAAGGCGTATGTATGTGCGGTAAATGTACCTGTAACCCTGATCGTTCAGGAAAATATTGCGAATTTGACGATAAGGCATGCGATAATCTTTGCTCAAACCATGGGATTTGTACCTTAGGCTCATGCCAGTGCGATAGCGGTTGGTCAGGAAATGATTGCGGTTGTCCAACTAGTAACACAGACTGCTACGCTCAATACTCTGAGGAGGTTTGTTCTGGTAATGGTGAATGTGTATGCGGAAAATGCCAATGTGCGAAGGTTAAAGGAAAAAACGAAACGTACACAGGAGTATTTTGTGACACATGCAATGACTGCCAATCAAAATATTGTAAAGCCCTCGAACCCAATGTAGAATGTAACTACAAACAAGGTCTAGAAGCTTGTGATAAGATTTACAACAACACAGAAAACAATGTTGTTATAAAAATGGTCAACAAAACAGAAATTAATTCGCCTAAATGGAGTGGCGCTACTTGGTGCAAAAAAGTAATAGAGGACGGCAGTTTTATAATATTCAGATATTATCATAACGCAACGACACACGGGTTACATATAATCATTCAAACGGAACCAGAGGCACCCCCAAGAGGAAATAAGTGGATTGCCCTCATCAGTTGCATAGTGGCTGTAGTACTCATTGGCTTGTTGACGTTGATTGCGTGGAAGATCCTCGTGGACTTGCACGATAAAAGAGAGTATGCCAAGTTTGAAGAAGAATCACGTTCTCGAGGATTTGATGTGTCGTTAAATCCCTTATATCAAGAGCCGGAGATCAACTTCTCCAATCCCGTATACAATGCAAATGCTTCACACTAGTATTTATATAACAAAATCCGAGGAAGAGAGGCGACGTGTTTCCGTGATCATAAGGAAAATACAATAAATAATTATTTGTGTTAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | AY630342.1 | RNAi beta-integrin M sexta 2 siRNA RNAi construct | GTTTAAGCCGCAAGTGATGAGAATGAAAGCGCGTCCCGGTACTAAATTATATTTCAACATGTCGTATAAGCCGGCCGAGCACTTTCCTTTAGATGTTTATTATCTCATGGACACCTCTTATACAATGACAATGCATAGAGAGGCGCTCATTAGTCAAGCAAACCAAATATATAAAGAACTTACAAGTTTAACTAATAATGTTCAACTTGGCGTTGGCAGTTTTGTAGAAAAACCAGGCTACCCGTACTTCGACAAAAACAAACAAGAAAGCGTCGCATTTATAAATGTATTGCCGTTAACAAAAAATATTAAACAGTTTACACAAAGTGTTCAAAATATGTCTTTTGGTTCGAATTATGACGATATGGAAGCTGGGCTGGATGCCCTAATGCAAGTCATGACTTGTGAGAAAGAAATAGGGTGGAGGCCT | siRNA | MEGAscript RNAi kit (Ambion) | other | simultaneously with transcription | Double stranded RNA (dsRNA) was prepared from a template that consisted of a 477 bp PCR product amplified from the integrin-b1 cDNA, using primers that contained T7 RNA polymerase promoter sequences at their 50 ends. The following primer sequences from the integrin cDNA were designed with T7 promoters at their 50 ends. Forward primer: 50TAATACGACTCACTATAGGGAGACAGTTTAAACCTCAAGTGATGAG30 (underlined sequence represents residues 315-335 in the 2426 bpin tegrin-b1 cDNA). Reverse primer: 50TAATACGACTCACTATAGGGAGAGGCCTCCA CCCTATTTCTTTC30 (underlined residues represent the reverse complement of residues 723â744 in the integrinb1 cDNA). The 477 bpPC R product was isolated by agarose electrophoresis and purified using the QIAquick gel extraction kit (Qiagen). This DNA was used as template for preparation of dsRNA, using MEGAscript RNAi kit (Ambion), following manufacturerâs instructions for synthesis and purification of dsRNA. The dsRNA was digested with RNase III (Ambion), following manufacturerâs instructions, to prepare short dsRNA fragments (small interfering RNA, siRNA). | InsectaCentral | RNAi beta-integrin M sexta 2 siRNA RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | injection | non_specific_dsRNA | (5 micrograms) | dsGFP | larva | RT-PCR (semi-quantitative) | hemocytes | not checked | low | ||||||||||
| 135 | RNAi moricin Manduca sexta | RNAi | RNAi of moricin (an antimicrobial peptide in hemocytes). | InsectaCentral | Unpublished | Garbutt | Jennie | RNAi moricin Manduca sexta target | AATTGCAAGAGTCGTCTGTAAAATATTGTTAAAATGAAGTTAACAAGTTTATTTATTTTTGTTATTGTCGCGTTGTCGCTATTATTTTCGAGTACCGACGCAGCACCGGGTAAAATTCCCGTGAAGGCTATAAAGCAGGCTGGCAAGGTCATTGGAAAAGGTCTACGAGCAATAAATATTGCTGGCACCACACACGATGTTGTTAGTTTCTTCAGGCCTAAGAAGAAGAAGCACTAGACGTGTAATGTTTAAACGATAACTTTACAAATTGATATTATATAGTAATTACTTAATAGTGTTTTGTAATGGTATTTATGTTATGTACGAAAATTGCAACTAATTTTATGATGTTTTGACTAAGTTATGCCTGCCGTTCTCCCTTCGGAATCTTCTGTGAATTCTCAGTTAAACAATGTAAAATAATTACCTTTTGCATAAAATATTCAACAAGACATGGAATATTATTTTAACGAAACTGCTTTCTTTAACCTTTGTCCTCATTTCAAAATAACTTTTGTATTCACATCAAAGTTAATGGTCCCGAACTTTATACAGTGCATCGTTTATTATTCATCTTTCAATATAGTATGACATATTGTAATGCATAATGATGGATGGTTTGCTAATTATTTGAATTTCGCATTATAGGCTGTGTTAGAATATAATAAAAAAGTAAAAAATATGTGGCGGGTAAAATGATACCTCATATTGTATTATCGTTATTTTAAACTTTAATAATAAAGCATTTGTAATTACGGAATAAAGCATTTTATTATATCTTAAACAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | AY232301.1 | RNAi moricin Manduca sexta RNAi construct | GCGTTGTCGCTATTATTTTCGAGTACCGACGCAGCACCGGGTAAAATTCCCGTGAAGGCTATAAAGCAGGCTGGCAAGGTCATTGGAAAAGGTCTACGAGCAA | dsRNA | T7 RNA polymerase - Ambion | salt precipitation | simultaneously with transcription | Primers designed with T7 5' ends. PCR reactions conducted using cDNA as template. PCR product used as template for transcription reaction (left to proceed overnight). dsRNA treated with RNase and precipitated with LiCl. | InsectaCentral | RNAi moricin Manduca sexta RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 2 | injection | DEPC water | 0.05 | non_specific_dsRNA | 6 | dsRNA for eGFP. | larva | qPCR | hemocytes | not checked | 24 | none | ||||||
| 137 | Gloverin | RNAi | InsectaCentral | Unpublished | ELEFTHERIANOS | IOANNIS | Gloverin target | CTATACGACTCACTATAGGGCAAGCAGTGGTAACTGCGCGTATAGTTTTTCCAGATGACGATCAAGATGTTGCGCCAAGGCGTGAGCAACCTAAAATAACCACAAAGGCACCTACGACAGAGGCACCGACCACGACAACCACTGATGAAGTTATTTTTTATAGCAATTCTTTTCGCTGCCATCGTCGCTTGCGCGTGCGCTCAAGTGTCGATGCCCCCGCAATACGCTCAGATATATCCAGAATATTACAAGTACTCCAAACAAGTCCGCCATCCCAGAGACGTGACCTGGGACAAGCAAGTCGGCAACAATGGGAAGGTCTTCGGAACTCTGGGACAGAATGACCAGGGTCTTTTCGGTAAAGGAGGCTATCAACACCAATTCTTCGATGATCACCGCGGCAAACTGACAGGACAGGGTTACGGGTCCAGGGTCCTCGGACCTTACGGAGACAGCACCAACTTCGGCGGCCGGCTTGACTGGGCCAACAAGAATGCTAACGCTGCTCTTGATGTGACCAAGAGCATTGGCGGTAGGACTGGGCTGACTGCCAGTGGATCAGGCGTGTGGCAACTTGGGAAGAACACGGATTTATCTGCGGGAGGCACTCTGTCTCAGACGCTTGGACATGGGAAGCCTGATGTCGGCTTCCAAGGTCTCTTCCAGCATAGATGGTGATATCTCTTTCTTCCTTTCGGTTATTGTCTTTTTGTAAAATATTTCATGTTTCTTCTTGAATTGAAATAAATGTCGGTATACCCCTCTAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | AM293324 | Gloverin RNAi construct | ATGCTGGAAGAGACCTTGGAAGCCGACATCAGGCTTCCCATGTCCAAGCGTCTGAGACAGAGTGCCTCCCGCAGATAAATCCGTGTTCTTCCCAAGTTGCCACACGCCTGATCCACTGGCAGTCAGCCCAGTCCTACCGCCAATGCTCTTGGTCACATCAAGAGCAGCGTTAGCATTCTTGTTGGCCCAGTCAAGCCGGCCGCCGAAGTTGGTGCTGTCTCCGTAAGGTCCGAGGACCCTGGACCCGTAACCCTGTCCTGTCAGTTTGCCGCGGTGATCATCGAAGAATTGGTGTTGATAGCCTCCTTTACCGAAAAGACCCTGGTCATTCTGTCCCAGAGTTCCGAAGAC | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | Gloverin cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: GLOV_F: 5'-ATGCTGGAAGAGACCTTGGA-3' and GLOV_R: 5'-GTCTTCGGAACTCTGGGACA-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for gloverin. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | Gloverin RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | unknown | unknown | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat-body | not checked | 18 | none | ||||
| 139 | Heliothis virescens pheromone gland | RNAi | 1. Background information In the biosynthesis of sex pheromones, fatty-acyl reductase (FAR) is a key enzyme that is necessary for the production of oxygenated functional groups (Tillman 1999; Jurenka 2004; Moto et al. 2003). This enzyme converts fatty-acyl pheromone precursors to corresponding alcohols. Recently, a substrate specific FAR was identified from pheromone glands of Bombyx mori (Moto et al. 2003), and injection of dsRNA corresponding to the identified FAR resulted in a successful reduction of the precursor of bombykol in Bombyx mori (Ohnishi et al. 2006). In Ostrinia nubilalis the sex pheromone gland specific FAR was found to be the key enzyme that is responsible for the E and the Z ratio variation (Lassance et al. submitted). In Heliothis virescens (Hv) we recently found four FARs, and the one that was specifically expressed in the sex pheromone glands was most similar to the O. nubilalis FAR (Vogel et al. 2010). We conducted RNAi experiments in vitro and in vivo with this FAR to attempt to silence sex pheromone production in the sex pheromone gland of Hv. RNAi tests in vivo RNAi experiments were also conducted in vivo. To determine the effect of RNAi in vivo, the same dsRNA of the sex pheromone specific FAR of experiments in vitro were used. One-day old pupae were injected in the abdominal tip with 5µg dsRNA of FAR, using a 10 ml microsyringe (Hamilton). As a control, pupae were injected with 5 µl buffer (EDTA,Tris). To assess the time point when FAR may be switched off by dsRNA, the pheromone production and the transcript level of FAR was observed at different time points. Pheromone glands were extracted 0 to 6 hours (day 0) and after 24 to 36 hours (day 1) after females emerged from their pupal stage. Extracted sex pheromone glands were used for GC analysis to analyze the pheromone profile or for RNA extraction to determine differential transcription levels of FAR. For GC analysis, emerged female moths were injected with PBAN to stimulate pheromone production (see section below). Phenotype: in vivo The in vivo effect and the functionality of dsRNA on pheromone production was examined by comparing the production the entire 5-component pheromone profile. This total pheromone profile and the total amount of pheromone produced was compared to the pheromone production in in vivo glands without dsRNA. In the in vivo experiments, adult females moths were injected with 7.5 pmol PBAN (synthetic HezPBAN from Heliothis zea, Peninsula Laboratories, San Carlos, CA) 90 minutes before gland extraction, as described in detail in Groot et al. (2005). The sex-pheromone glands were extracted in 50 ul hexane, with pentadecane as internal standard, and analyzed in a HP7890 gas chromatograph as described in Groot et al. (2005; 2009). qRT-PCR experiment in vivo First-strand cDNA was synthesized using VersoTM SYBRR Green 2-Step QRT-PCR Kit Plus ROX Vial (Thermo Scientific, ABgeneR UK) according to the manufacturerâs instructions, starting with 800 ng of total RNA. Quantitative real-time PCR was performed in a total volume of 25 ml using a 96-well micro well plates (ABgeneR , UK). One ml of cDNA (20 ng), 12 ml 2-Step QPCR SYBR green (ABgeneR , UK), 0.5 ml ROX Reference Dye (ABgeneR , UK), 10 pmol of forward and reverse primer and 9.5 ml RNAse free water (Ambion) were added to each microwell. The reaction for comparative quantification was run at 95°C for 15 min and 40 cycles at 95°C for 15 s, 56°C for 30 s and 72°C for 30 s and for SYBR green analysis the reaction was run with an additional meltingcurve. All PCR reactions were performed in duplicates. To evaluate the amplification, two housekeeping genes, Rps 18 and EiFa4, were used as a reference. The reactions were run on Stratagene Mx3000P QPCR System. For the comparative quantification analysis of qRT-PCR, I used the computer program qBASE. Results: Phenotypic assay The total amount of pheromone produced per gland (150 ng) did not differ between the dsRNA injected and control individuals, assayed at 0-6 hrs after emergence. The total amount of phermone per gland increased to 220 ng in individuals 24-36 hrs after emergence, and did not differ between dsRNA injected and control individuals. Results: qRT-PCR The relative amount of FAR mRNA increased 1.6-fold in dsRNA-injected individuals relative to control individuals, assayed at 0-6 hrs after emergence. The relative amount of FAR mRNA returned to 1.2-fold of the control value, and did not differ between dsRNA injected and control individuals. | InsectaCentral | Unpublished | Barthel | Andrea | Heliothis virescens pheromone gland target | ATTGTTCATCGGATCGCCTGGACATCCACTAGTCCTGAAAGACACACAGAGATAATCATAATAATTCATTCCTTAGCCTTTCACAAAAAAAACATCATTTATCAAAGTGAAGGTGGTTTTAAAAATTATCAAAATGGTTGTTTTGACTTCTAAAGAAACGAAACCTTCAGTAGCTGAATTTTATGCGGGAAAATCTGTATTTATTACGGGAGGTACTGGATTCCTTGGAAAGGTATTCATAGAGAAACTTCTGTACAGCTGTCCTGATATCGTCAATATTTACATGCTCATACGAGAGAAGAAGGGGCTATCTGTTAGCGAGAGAATAAAACAGTTTCTTGATGACCCGCTCTTCACAAGACTGAAAGACAAAAGACCAGCTGACTTAGAGAAGATTGTACTTATACCTGGAGATATTACTGCTCCTGACCTGGGTATCACTGCTGCAAATGAAAAAATGCTTATTGAGAAGGTATCGGTGATTATTCACTCGGCTGCTACGGTGAAGTTTAATGAGCCTCTCCCTACGGCTTGGAAGATCAACGTGGAAGGGACCAGGATGATGCTGGCTTTGAGTAGAAGAATGAAGCGGATTGAGGTTTTCATTCACATATCGACAGCATACACGAACACAAACAGGGAAGTGGTTGACGAGATCCTGTATCCAGCCCCAGCTGACATCGACCAAGTTTATCAGTATGTCAAGGAGGGAATTTCTGAGGAAGACACTGAGAAAATACTGAATGGTCGTCCGAATACATACACGTTCACGAAAGCGCTGACTGAGCATTTGGTTGCTGAGAACCAAGCCTACGTACCAACTATTATCGTGAGGCCGTCTGTCGTGGCAGCAATAAAAGATGAGCCGTTAAAAGGTTGGTTAGGCAACTGGTTTGGAGCGACCGGTCTCACAGTGTTCACCGCTAAGGGTCTCAACCGAGTCATCTACGGTCATTCTAATTACATCGTAGACCTGATTCCAGTGGATTATGTGGCTAATCTGGTGATTGCTGCTGGGGCTAAGAGTAACACATCAAGTGAGTTGAAGGTGTATAACTGCTGCAGCAGTTCCTGCAATCCCGTCAAAATTGGCACGCTGATGAGCATGTTTGCTGACGATGCCATCAAACAGAAGTCTTATGCCATGCCGCTACCGGGGTGGTACATATTCACGAAGTACAAGTGGCTGGTGCTACTTCTGACGTTTCTCTTCCAAGTTATACCGGCGTATATCACGGATCTCTCCAGGCACTTGGTTGGGAAGAGTCCACGGTATATAAAACTCCAATCACTGGTGAATCAAACGCGTTCTTCAATCGACTTCTTCACGAATCACTCCTGGGTGATGAAGGCAGACAGAGTGAGAGAGCTGTATGCGTCTCTTTCTCCCGCAGACAAGTACTTGTTCCCCTGCGATCCCGTCAACATTAACTGGACACAATACTTACAAGATTACTGTTGGGGCGTCCGAAATTTTTTGGAGAAAAAAACGTAAAAAATATATAAAATWWATTATAATATTTTTTTGTTTGTTTTTAAGGACTGTATAGTATGGAGGTTGAGCAATGGCTAATTTTTGTAATAAAAGTATTATTTTGATGAAAATATTAATTGTTAAGTAATGATAAGACATATCTAGGTGTTTCCATGTTTTGCCGTAGTTGGGTAGAAATGTTAAATGCGGATAGTAGTAGGTTAATTAGAATGTAGTWATATATAAAAAAATCTTATCACATAATTTATTTATGGATTCCATTAACTTTATTTAATWAATTTTTCSAACAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Heliothis | virescens | 7102 | cds | GenBank | EZ407233 | Heliothis virescens pheromone gland RNAi construct | TTAGGCAACTGGTTTGGAGCGACCGGTCTCACAGTGTTCACCGCTAAGGGTCTCAACCGAGTCATCTACGGTCATTCTAATTACATCGTAGACCTGATTCCAGTGGATTATGTGGCTAATCTGGTGATTGCTGCTGGGGCTAAGAGTAACACATCAAGTGAGTTGAAGGTGTATAACTGCTGCAGCAGTTCCTGCAATCCCGTCAAAATTGGCACGCTGATGAGCATGTTTGCTGACGATGCCATCAAACAGAAGTCTTATGCCATGCCGCTACCGGGGTGGTACATATTCACGAAGTACAAGTGGCTGGTGCTACTTCTGACGTTTCTCTTCCAAGTTATACCGGCGTATATCACGGATCTCTCCAGGCACTTGGTTGGGAAGAGTCCACGGTA | dsRNA | MEGAscriptR RNAi kit (Ambion Inc.) | column-based | simultaneously with transcription | Construction of dsRNA One forward PCR primers and two reverse primers (see in Sequence, point 3) were used to synthesize a region of the fatty acid reductase gene that contained a T7 promoter region (TAA TAC GAC TCA CTA TAG GG) on both the sense and antisense strands, followed by sequences specific for the targeted genes. The PCR reaction was done under the following cycling conditions: 94_C for 2 min followed by 5 cycles of 94_C for 30 s, 65_C for 30 s, 72_C for 1 min, 35 cycles of 94_C for 30 s, 77_C for 30 s, 72_C for 1 min a final extension of 72_C for 3 min. The PCR products were purified with the MiniEluteR PCR Purification Kit (QIAGEN) and were then used as templates to synthesize dsRNA. The dsRNA was produced using the MEGAscriptR RNAi kit (Ambion Inc., Austin, TX) according to the manufactures recommendations to obtain the corresponding long dsRNA without an additional annealing step. The in vitro transcription products were assessed for integrity on a 1% agarose gel, diluted in 6xOrange Loading Dye Solution (Fermentas) and the concentration was calculated by measuring its absorbance at 260nm. | InsectaCentral | Heliothis virescens pheromone gland RNAi construct | Lepidoptera | Noctuidae | Heliothis | virescens | 7102 | local resource | adult | unknown | unknown | lab_colony | 1 | injection | TE | none | 0.018500000000000003 | buffer | 24 | tip of female one-day-old pupal abdomen | As a control, pupae were injected with 5 µl buffer (EDTA,Tris). | adult | qPCR phenotype | female sex pheromone gland | not checked | 240 | none | ||
| 141 | invivo knockdown of select genes in plusiine noctuids-PSP | RNAi | this study was performed to see whether we will be able to knock-down in the expression of an constitutive gene in soybean looper. | InsectaCentral | Unpublished | Strand | Mike | invivo knockdown of select genes in plusiine noctuids-PSP target | ATGAAATTAACAATTAATATTTTATTTTGCTTAATTCTAATTTCTCAATACAACTCCGCAAATGGAAACTTAAGGGATCTATTTAATAACGTACGAGGATCGATAAGTTCATCGGCAAATAAAATAAGGCAAGATGTGAAGACTTTATTTCATCCGAGTGACAAATCTGGGAATAAGGAATCATCGAACATTGTTTTTGTTGAAGATAAAGATGAAGGGGCAGTGGGTCCGGCTCGGGACAACAAACCTGTAGCAGTCACACCTGCACCTGTTGTTAGTACCACTACCCAGGCTAGTGCACCAACCGTGGCGACTAATGGTACTGCAACTGGAGGCAAGGACGACAAAGGCCGAGAGAACTTCAACGGTGGCTGTCTCGCCGGCTACATGCGAACCGCTGATGGAAGATGCAAACCCACCTTTTGATGTTTAACTGTGTTTTTATGTGTTGCAACTGTTTATACCTATTTAGCATTTACTTTTAATTTTTTTTTCTTTATTTAGTGTTCTATATTATGTGTTAATAAGAAGTGTAATCTTTTCAAAATTTATAAAATAAGTTAAATCATAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Chrysodeixis | includens | cds | pubmed | AF062489.1 | invivo knockdown of select genes in plusiine noctuids-PSP RNAi construct | TAATACGACTCACTATAGGGCCGCAAATGGAAACTTAAGGGATCTATTTAATAACGTACGAGGATCGATAAGTTCATCGGCAAATAAAATAAGGCAAGATGTGAAGACTTTATTTCATCCGAGTGACAAATCTGGGAATAAGGAATCATCGAACATTGTTTTTGTTGAAGATAAAGATGAAGGGGCAGTGGGTCCGGCTCGGGACAACAAACCTGTAGCAGTCACACCTGCACCTGTTGTTAGTACCACTACCCAGGCTAGTGCACCAACCGTGGCGACTAATGGTACTGCAACTGGAGGCAAGGACGACAAAGGCCGAGAGAACTTCAACGGTGGCTGTCTCGCCGGCTACCCTATAGTGAGTCGTATTA | dsRNA | phenol_chloroform | subsequent to transcription | The targeted DNA sequence was PCR amplified using a forward and reverse primer with T7 promoter at the end of each primer. Set up a 20 micro liter reaction with 8 micro liter of PCR product and incubate at 37 degree celsius for 6 hours. After 6h,incubate at 75 degree celsius for 5 minutes and leave at room temperature for 2-3 hours for cooling down which facilitates annealing. Add 1 micro liter of Dnase and incubate at 37 degree celsius for 15 minutes. Then a phenol-chloroform extraction was performed. | InsectaCentral | invivo knockdown of select genes in plusiine noctuids-PSP RNAi construct | Lepidoptera | Noctuidae | Chrysodeixis | includens | local resource | larva | lab_colony | 1 | injection | Nuclease free water | 2 | buffer | 6 | Injected into hemocoel through proleg | Control animals were injected with 1xPBS | larva | RT-PCR (semi-quantitative) | fat body,hemocytes | not checked | none | |||||||||
| 142 | invivo knockdown of select genes in plusiine noctuids-Alpha integrin1 | RNAi | InsectaCentral | Unpublished | Strand | Mike | invivo knockdown of select genes in plusiine noctuids-Alpha integrin1 target | ATACTCGGCCGGCGGACGGCAACCGCGCTGGGGGTCTCGACGTCGCACGACGCAACTATCACGAAAGTCGCGATGTGCCAAATAATAACTGGAAAACAGTGAATTGGTGCGATAACACGATTTATTACACGAATATATTACGAACGGTGTACACCATGGCTCGAGCTAGGTGCGCTTGTGTTTTATTTATGTGCATTTTATATAGTGTCACAAGTTTTAATCTGGAACCGAGGATTCCTGTGATAAAGTTCGGAGAGCCAGGCTCTTATTTCGAATTTTCCGTTGCGGAACACCTCACCATCAGCGGAGATGGATCGCAAACTAGTTGGTTATTAGTGGGAGCGCCGTTGGGTCAAAATCTGCAGCCAAACACAACGAAATCTGGAGCGTTATGGAAGTGTGGCGTCTCTCCGTCCACCGGCGACTGCATACAAATAATCACAGATGGGAAAAGGACGAAGTACGGCAAGATAGACACGAATTTGGAGTCAAATAACCTGACTGCTCCGTACTCGGATGAGATTAAGGAGGGTCAATGGCTGGGAGTGTCGGTGAGGAGCCAAGGCCCGGGGAAAAAAGCCGCCGTCTGCGCACACAGATATATTCGCAAACAGGGAGAGCTGCAGTTTGGCCAGGGTCTGTGCTACACTCTAAGCAACGAATTAGAACTATTAGATGTTATGGAGCCGTGCAGAGGGAGATCAGTAGTAAGAGAGCACGAGGAATACGGTTTCTGTCCAGTGGGAACTAGCAGTTCTCTTCTAGATGATGACACGTCGTTGATGGGCAGCCCGGGACCGTATACCTGGAGGGGGACCATCTTCGCCCAGGACACGAGGGACGATGTCCTGGAGAGAGACAACGTGGTGTACATGGCCCCGGTACAAGATGGCGCTAGCCCTGTAGAAAAGTATAGTTATCTAGGTATGTCAGTTACCAGTGGGAACTTCTTCGGCGGTGACAGAGCATCGTACGTGGCCGGAGCGCCTCGGGCGCACGGCACGGGCGAGGTCGTCATCTTCAGCAAGCTGGCTACAGAAGACTGGGGCCGAACCGACCTGAATATACTGAACTTCACGCTTCTACTCAAAGGCGAACAGTTTGGCTCTAACTTCGGATATGGAGTAGCCAGCGCTGATGTTAATGGAGATGGGTTGCCTGACCTATTAGTAGGCGCCCCTTTCTATTTCTCCCGCGACGTGGGTGGCGCTGTTTACTTATACCTAAACGTAAACTCTAGCCTACAGCAACAGTATAATGTCAAGTTAACGGGCAAACAAGAATCACAATTTGGTATCGCTATTGCTAACGCCGGGGACTTGAACAAGGACGGTTGTCAAGACATAGCCATTGGAAGCCCATACGAAGGCAATGGTGTGGTGTACATCTATATGGGAGATAGAGAGAACGGTCTGAATACTAAACCAGATCAGGTCATAACTGCTGAAACGTTGCCGACTGTGATGAAAACCTTTGGTTATGCTTTATCTGGTGGAGTAGATTTAGATGCGAATGGCTACCCTGATTTGTTAGTCGGGGCATACGAAAATAGTAGCGTTGCTTTAATACGAACACGACCAATTATCGACATTAAGACGTCGATTAAACCATCGAACAGTATCATTAACATTGATCCCACGATCCACGGCTGCGAGCGCGACCCTGGCTCCAACTACACCTGCTTCCACTTCGAAGCCTGCTGCATCATCGAGTCTCTAGTCAAAACCTCACAATCGAACATCCATCGACTCAACTACGTCATCGAAGCTGAGACATTCCCCGGTGGAAGGAAATATTCCAGGGTCTTCTTTGACTCCGACAAGAGTAATATTGTCAATAAGACTATTATGCTAGGCAAAGATGTTGAAGACTGCAGAGAGCATATCGTGTATTTGAAAAACAATACTAGAGATATACAAACTCCGCTCAAGTTCCAGTTAACTTACATGCTAGTCCACGCGGAGCCTCGCTACTCGTCATCAGGCCCTCTCCCCGATGTGGACCAGTACCCCGTGCTGAACGCCACCGCCAGCTCCGTGTTCGCCGCCAACTTCCTTAACGACTGCGGTGCTGATGTCGTCTGTATCAGTGATCTAGTGGTCGATCCTAAGCTGTTGCTGTCGTTGACAAAAGACAACCAAACATATTCTCTTACACTGGGACAAGAAGAGGAGATAAAGTTGTCGATAGCAGTAGACAACTACGGCGAGTCGGCGTATGGAGCACAGCTGTTCGTCACCCACCCTCCCAGCTTGCACTACATCGCTGTCAACATTAGCGATAAGCACGTGATCTGTACAAGTTTCAACAAGACGACAGTGACCTGTATGCTGGAGAATCCTTTCAAGAAGCAAGCGGACGGTTCTCCGCCAATCACGATGCGGTTCGATGCGCGGGCGCTGGAGGACAACGACCAGTCTGTCGTGTTCACTGTCTGGGCAAACTCCACGTCTAAGGAACTGCATGCTGGGAAGAGACCCGTGCAGGTCGAAGCGTTGGTTATTAAGAATGCTGAACTTCTTATAAAGGGTGTGGCACGACCCGAGCAAGTGTTCTATGGTGGTGAAATAAAGGGCGAGTCTGCTATGACGTACTTTGATGACATCGGTACAAGAGTGGTGCATACATATCAGGTATTCAACGAAGGTCCATGGCATGTGTCGTCAGTGCAGGTGGTGATACAATGGCCACATCAGTTAGCATCAGACGGGCCGCAGGGCAAATGGCTGCTCTACCCTGAAGATGTTCCTACCGTCGATGGAGAAGCTGACCAAAACGGCGAATGTTTCGCACGAGAGATAAACCCTTTGAACTTAACACCACGGCCTGGCGCCCCGGAGCCTCTCGAGAACTTAGAGATGGATCCTTTCCTAAGCACCGGCAAGTTAAGCGTCAGCTCCAATGAGAAGTCCCACAACGAAACCAGGAGGAAGTTCATGGAGTACTCCAGTTCTAGCTCTTATACTAGCAGTAATAAAGTCCGACGGAAAAGAGATGAGGAAGACGCTGTTAAAGCGGAAGCCTACACTGATAGAAAGCAAGTTATTAATTTGAACTGCCAGCAGCGCACAGCGAAGTGCATCCAGTTCCAGTGTGTAATCTACAAGCTGGGTCGCATGAAGACCAGCACGATCAGTGTGCGCGCGCGCCTGTGGAACGCCACGCTTGTCGAGGACTTTGCCCGCGCCGCGAGCGTCGACATCGCCTCCACCGCCTACATCAGCATCCCCGCCCACTTCAATATACACCAGAATGAACAGTCCGATGATAAAACTACTGTGAACACAGTCGCATATCCAGACTTGAAGATAAGTGAACCGACAGAGGTTCCTCTGTGGGTGATCATCATATCAGTGATAGTGGGGCTCATAGTGCTGGTGCTGCTCATCATTGCGCTGTGGAAGCTCGGCTTCTTCAAGCGCAGCCGGCCGGACCCCACGCTCTCCGGCAACCTGGAGAAAAACAACCACGAGTCCAGCCCTTTCATCGGTAGAGACCGGAATAGTGTTCGATAGCCCTCGGCCCTCTACGTAGTGGACAGAACGTTCCAGTGCGAAAAACCCGCCCAAGTGTAAATAAACTGTTGTAATGTTTACCCCTTTTCATGCGTTACGAACTGAAAAGCCGACGACTATCTGTCGGTCGGAAGCAGTAAAACTTTGTTTTGCATACTTTTGCTGCCAGATAGTCGGCACTGGCTATGGCCCGGTCGCCACCGAAACGCGTGATGGAGTTGTGCACAAGTATTTAGTACGTAATATCTCTGACTGTGATGTCTTCTTAGAGGAAATTTGTGTGTATGTGATGTTCTAAAGTAATGGTTCTATGTTTTGTTGTTGACGCACGGTACGATAGTATATTGTTTTATTGTAATTGCATTTCTTTAGCGCGTAATGGGAACTTGTTTTAGATTGTTTGTTTATATATTCTGATGGATTATGTATTTTTCTGTAAATAATTACTTGCCTTATTATTTATGAATAGGTAGTTTAAATGTCTACTTAATTTGACTAGATTTTTTACTAGTCACAAAATGTAATAATGTTTATGTGTAGGTATGATAGATATATCGTCCTTAACTTAAGTGGAATTTAGTCACAATTGATTTTGGACACTAAAATTACTGAAAATGTATATCGTCCGGTAGGTACTTACACAATGTTTTTGACTTTTTATATGAATAACTTCCCCTTGTACTGACGTCATACTTATGAAATGCAAAATGAAAAGTGTCATGAATTAGATATGTAACTTTAAGTATTGTAAATATGATTGTTTCCATGTCATTAGATTTTATACAAAATTTTATATTTGCAACAAGAGGCATACATTTTACACGTTCACTGCCAAGTGCTGTATCCCTGCAAACACGATCACTTAGTATTTTATGTCATTTTAATAAAATTACGACTACTTAAATAACCCTGGTTGTGATTATTTCTGTTATGAACTACATATGATACCTACTTAATAGTACATTTTAGTTTTGAATAAAATTGTGCCCTTTTT | Lepidoptera | Noctuidae | Chrysodeixis | includens | cds | pubmed | AY237585.1 | invivo knockdown of select genes in plusiine noctuids-Alpha integrin1 RNAi construct | TAATACGACTCACTATAGGGTAGCGCGTAATGGGAACTTGTTTTAGATTGTTTGTTTATATATTCTGATGGATTATGTATTTTTCTGTAAATAATTACTTGCCTTATTATTTATGAATAGGTAGTTTAAATGTCTACTTAATTTGACTAGATTTTTTACTAGTCACAAAATGTAATAATGTTTATGTGTAGGTATGATAGATATATCGTCCTTAACTTAAGTGGAATTTAGTCACAATTGATTTTGGACACTAAAATTACTGAAAATGTATATCGTCCGGTAGGTACTTACACAATGTTTTTGACTTTTTATATGAATAACTTCCCCTTGTACTGACGTCATACTTATGAAATGCAAAATGAAAAGTGTCATGAATTAGATATGTAACTTTAAGTATTGTAAATATGATTGTTTCCATGTCATTAGATTTTATACAAAATTTTATATTTGCAACAAGAGGCATACATTTTACACGTCCCTATAGTGAGTCGTATTA | dsRNA | MEGAscript RNAi kit, Ambion Inc. | phenol_chloroform | subsequent to transcription | The targeted gene sequence was PCR amplified using a forward and reverse primer with T7 promoter at the end of each primer. Set up a 20 micro liter reaction with 8 micro liter of PCR product and incubate at 37 degree celsius for 6 hours. After 6h,incubate at 75 degree celsius for 5 minutes and leave at room temperature for 2-3 hours for cooling down which facilitates annealing. Add 1 micro liter of DNase and incubate at 37 degree celsius for 15 minutes. Then a phenol-chloroform extraction was performed. | InsectaCentral | invivo knockdown of select genes in plusiine noctuids-Alpha integrin1 RNAi construct | Lepidoptera | Noctuidae | Chrysodeixis | includens | local resource | larva | lab_colony | 1 | injection | Nuclease free water | 2 | buffer | 6 | Injected into hemocoel through proleg | Control animals were injected with 1xPBS | larva | RT-PCR (semi-quantitative) | fatbody,hemocytes,gut | not checked | 120 | none | ||||||||
| 143 | invivo knockdown of select genes in plusiine noctuids-Alpha integrin2 | RNAi | InsectaCentral | Unpublished | Strand | Mike | invivo knockdown of select genes in plusiine noctuids-Alpha integrin2 target | TGAGTACTGCGCCGACCGCGGGCCGCGACACATGCGTCTGCCGTGCGCCTCCCGAACACGAACATCGTAAAAAGTTACTCGAACGACATGAGGACGAATTATGTAAATTATAAATAAGTGTGCGAATTAATTTTTATAATTATTTTTCTGTGATCTGTGAATTATTGTGCATCTGACAATGGCGTTAGCGTTGTGTGTATTTTTGTGCGCTTGCGCTAGTGTTTTAGGATTTAATGTGGACATACCCTCAAGGGTGGTTTATAAGGGTAATCCGAATTCTATGTTTGGATTTACGGTCCAGGCGCACGTCGATGGCGATAGGAAAATGATCCTGGTGGGAGCTCCGGAAGACCAGCCGTACTCCGCGGTGGACTTCAACGTGACGAACCCTGGGGCCGTGTACCGCTGCGAGCCAGGTTACAGGAACTACGCCAACGAGGGCAGCGCACGCGCACTCGAGCGGTGCTACCCTATGCAGTTCGACAAACATCCAAGAAACAACGAGGATAGGCAAGGTCGCATCATCGACCAGAAGTCGAGACAATGGTTCGGGGCCACACTCACCAGTACCGGCAGGAATGGCGCTATCATGGCGTGTGCCCCTCGCTACGTGTCCTTCGTGTCAGCCAAGCTGAACCAGCGGGACCCCGTCGGCACCTGCTTCGTGGCCAACTCTCCAGACCTTACCGACGTCAAGGAGTTCTCTCCATGCCGAACTTCGACCCACGGCCAACGCAAGACTGGCATGTGCCAAGCCGGTTTCTCAGCTGCCATATCCAAGGATGGACAGCGACTCTTCATGGGTGCCCCCGGCAGCTTCTTCTGGCAAGGTCAGCTGATGACTCAGTCGATGAGCAGTCGGCCCGCACTGATCGCTACACCGGAGGCTAGTCCTAAGTACGACGACTCCTACATGGGTTATTCGATGACTGTCGGAGATTTCGCTGGACCTGGTATTCAGAGTGTTGCCGTTGGTGTTCCTAGAGGAGCTTCTTTGAAAGGATTGGTCGTTCTTTACACGTGGGAACTCCAAAACATCAAGAACATCAGTGGCTCCCAGATCGGTGCCTACTTCGGTTACAGCCTTGCCTCCGGCGACATTGATGGCGATGGTTTCGATGACGTCATCGTCGGTGCTCCTATGTTCACCCGTGCTAAGACTGATGGCTTTGAACACGGAAGAATCTATGTTATCTACCAGGATAAGGATAGGTCTTTCACAAGGAGCCACGCTAGGACCGGTGAAGTATCTCGAGGCAGATTTGGTTTGGCTGTCACATCTCTAGGCGACATCAACTACGATGGCTTTGGAGACATAGCTGTCGGCGCCCCGTACGGCGGCGAGAATGGCCGAGGTGTGGTGTTCATCTACCACGGCAGTGAGCTCGGCATCCACGAGAAGTACTCTCAGGCCATCACCGCCGAGGAGATCTCTCCCTCTCTTTCCACCTTCGGCTTCTCCCTCTCTGGTGGCGTAGACTTGGATAACAATAACTATACTGACTTGGCTGTTGGTGCTTATAAGTCAGATAGTGTTGTGTTTTTGAAATCCCGTCCCGTAGTGAAAGTATCAGCTGACGTGAAGTTCCTAGGAGACAGTAAGCTGATCTCCCTGACCGACAAGAAGTGTCACCTGGCCAACGGCACGACGGTAGCGTGCGCGCAGCTCATGTTCTGTCTCACCTACGGAGGTGTCAATGTGGACCAGCAGATAAAGTTCGAGGTGGTCCTGGACCTGGACTCGCGACAGACGGCCACGAAGAGGCTGTTCCTGGCCGAGACCCGTGAGACCACGTACAAGACGCAGATGCTGCTGACGCAAGGACAGCAGGAGTGCAAGGATATCACCGTCTACTTAGATGAGGAGATCCGAGACAAGCTAACACCGATAGAAGTGAAGATGTCATACGACCTGGTGAACCAGCCGTCTGGAAACATTGTCCCGCCGGTCCTCGACCAGACCAGGAGCATCGTCCACGCGGACTCCCTCAACATTCAGAAGAATTGCGGACCAGACAATATCTGTGTTCCGGACCTGAGAATGGCTGCCACTACCTCAACCATAAACTACGTGCTTGGCTCCGGTGAAAACGTTAACATTGACGTCAAGGTGGAAAATTCTGGAGAAGACGCCTTCGAAGCTGCTTATTTCCTAGTCATCCCGCCAGGAGTGACGTACGCCAAGATGGAGCGTTTGGACTCACAGGAATCTGAAACACCTATCTACTGCTCCATCACGAATAGGTTCGGTGATGGAAACAGCACGCTGAAATGTGATCTGGGAAATCCTATGGCCAGTGAGCAGAAGGTGAACTTCCGCTTGGTCCTGGAAGTGGAGGCTCGCGTGACGGCGCTGAGCTTCGACATGGAGGCCAACTCCACCAACCCCGAGCAGGGCACGCTCTACGATAACACCTTGCACATGAACATCGGCGTCGTTGTCAGAGCACAACTCTCTATTATCGGTACCTCAGACCCACCCGAGCTGCATTACAACGCGTCGCTCTACGAGATTGAGAACATCAAGGATGACACAAAACTCGGACCCCAACTGATCCACAAATACAACATCAAGAACGAGGGACCTTTCACTGTCGACGAGACTGAAATATACTTCATGTGGCCCTACCAGACGCTCGAAGGTGAAAACCTGATGTACATGCTGGTGCAGCCACAATGGCTCGGTAACGTTAAATGCGACGTGGCGCGACACATCAACCCTGAGAATCTCTTCGTGCAGAACCCCTACGCTTTCCTATTGAGCAAGGAGAAAGAAGCCATGATGAGCAGCGACCTGTACACTGCATCCCAAACATCAGGTGGCAGTGGATATTATGGACAAGTGAACTGGGACTCCAGCAGTGGTCAAAGCTTTTCATCTGGCGGTCAGTTTAGCCAGGGATCTGGTCAGTTTAGTCAAGGCTCGGGTCAGTATGGTACCCATCAAGGATCAGGTGGCTACCAAACTGGAAGCTCCAGCTCCGGCCACTTCAGTGGTAGCTCTTCGAGTGGTCAAGGAGCAGGAGGACAGGTTTATGCCCAAGGATCTACTCACTATGGCGGAAGCCAAGCTGGGCAAACGGGTGGCGCCGGAAACACTTGCAGCTACAACAAAACTTGGTCCAACTCATACGGAGAAGGCCTTTCTGCTGAAGAGCAAGAACAGATCAGGAGAAACCTTGCCGCTGCTGTCGCCCAAGGAGGTTACTCCCACGGTAACCAAGGAGGTGTTCAAGGAACCTACTCCCAAAGCGGTTACGGGCAAGGTCAAGGCCAGATCGTAAAGGCGAAGAACAAGACCATTGTGTATGACTCGAACCACAACATCATATCTGAGACTGAATCCAGTACTGAGTATGGAAGCTTAGGACATGAGGGAGAGCCTGGAAGCTCATTCAATGTATATGCTAACCAAGGCGGTGTCAACCTAGCTTCATCACAAGGCAGCGGCAATTCTTGGCAAAGCCATTCGCAAGGGTCAAACTCTTACCAAGGCTCTGGTGGATACTCCCAAGGATACGGAAACTACAACCAAGGCTCCGGAAGTTATCAGGGTTCAGCCTCTCAAGGCCAAGGATCGAGAGGATTCGCTCAAGGCTCTGGCGGAGGTTTCGTCCATGGCTCATGGGCCACTACTGAAACTGTCAACCCTGATATCTCTAAAGCTGGCTCCTCAACATTCAGAGGATCGTCAAGTGTGACAGTAGTCGGTGACGAAGAAGAAGACAAACTCGAAGGTTTTGGAGCACACGCTGCGTACAACCCTAATAATGAATTTAGATATGGTATTGCCGATGTTACTGGTGCTGGCAGTCGTGGAAGTGGTTCACAAAGTGGAAGCCATCAAGCCAGTGGGGGTGGATACAGCTCTCAAGCCGGCGGTGGCAGCTACCAGGCAGGTGGCGGTAGCTATCAAGCTGGAGGAGGTAGCTACCAAGCTGGTGGAGGTAGTTACCAAGCTGGAGGAGGTAGTTACCAGACCGGCGGCGGAAGCTATCAAACTGGAGGTGGAAGCTACCAATCTGGTGGCTCTGGATACTCCTACTCTTCACAGTCAGGTGGCCATACATCATACAGCACTCACTCAAGCAGCGGTTACGGCACAAGCGATCGTCGAGAGAACACCAGGAGACGAAGACAGGATGAGGTTGATCCTCAACTGAAGAAGATTCTGGAGAAATGCGAGGAGAAATACAAGTGCGAAGTGTTGAGGTGTACGACTGGCCGCATGGTGAAGGGACAGGAAGCTTGGTTCGCGCTCCGGTCCAGGATCAACGCGTCCGTTCTTAACGAGATCTCCAAAGACCGTCCGGTCATCCTATCCACGCTGGCCGCGACCCGCGTATCCCGCCTGCCGATGGTCGGGCGGCCGTCGGACCCCGCGTGGCACACCGCCGAGGCGCAGACCGTGATCACGCCGCAGCTGGAAGCGAGGGATAGTGGCACTATACCGCCGTGGGTGGTCGTGCTGGCCGCTGTGGTGGGAGCGTTACTGCTTCTGCAGCTTACCTTCGCCCTTTACAAGTGCGGTTTCTTCAAGCGCAACCGGCCCTCGGACCACGCTGAGCGTCAACCCCTGAACGGCCGCGACGAGCATCTTTGATATCCGAGACCGGGGCGTAGCACGAGCGACGCGGCCGACACGGGCGGCACGAGCGACGCGGATGCGGCCAACACGTCCGACACGCGCGACAGCTCCTCCACTGACGCCGCCAGTGACGTCACCAGCCGGGACTCTACTAGCTTTGACAGATATTGACCGCACCGCTTCTAGTGACGTCACGCTCGCCAATATGGCGCTATCTAAATATTTGTAGAGTCGCGGTTTATTTGTAGTATTCTAAACTAACACTTTGAGCTTATTTTAATTTAATTTAAAAATAAGCCCAAAATGTACAGTCTGTAAAGTTGTCCGATCATCATTGTAGAATAGAGAAGCTATATTATTCTTGTTGCAATAGTTTTAACCTCATGACATCACGAAATATTTAATCTTTCCATGTCTTTTTCACAAAGAAAATGGGAAGGTCGACTGTTTTAAAAAAATACATAACATCGTAATGTAACTAGTCACGGTAAACAATTAATTTTCGGACGACATATTAG | Lepidoptera | Noctuidae | Chrysodeixis | includens | cds | pubmed | AY237586.1 | invivo knockdown of select genes in plusiine noctuids-Alpha integrin2 RNAi construct | TAATACGACTCACTATAGGGCTGGCCGCATGGTGAAGGGACAGGAAGCTTGGTTCGCGCTCCGGTCCAGGATCAACGCGTCCGTTCTTAACGAGATCTCCAAAGACCGTCCGGTCATCCTATCCACGCTGGCCGCGACCCGCGTATCCCGCCTGCCGATGGTCGGGCGGCCGTCGGACCCCGCGTGGCACACCGCCGAGGCGCAGACCGTGATCACGCCGCAGCTGGAAGCGAGGGATAGTGGCACTATACCGCCGTGGGTGGTCGTGCTGGCCGCTGTGGTGGGAGCGTTACTGCTTCTGCAGCTTACCTTCGCCCTTTACAAGTGCGGTTTCTTCAAGCGCAACCGGCCCTCGGACCACGCTGAGCGTCAACCCCTGAACGGCCGCGACGAGCATCTTTGATATCCGAGACCGGGGCGTAGCACGAGCGACGCGGCCGACACGGGCGGCACGAGCGACGCGGATGCGGCCAACACGTCCGACACGCGCGACAGCTCCTCCACTGACGCCGCCAGTGACGTCACCAGCCGGGACTCTACTAGCCCCTATAGTGAGTCGTATTA | dsRNA | MEGAscript RNAi kit, Ambion Inc. | phenol_chloroform | subsequent to transcription | The target DNA sequence was PCR amplified using a forward and reverse primer with T7 promoter at the end of each primer. Set up a 20 micro liter reaction with 8 micro liter of PCR product and incubate at 37 degree celsius for 6 hours. After 6h,incubate at 75 degree celsius for 5 minutes and leave at room temperature for 2-3 hours for cooling down which facilitates annealing. Add 1 micro liter of DNase and incubate at 37 degree celsius for 15 minutes. Then a phenol-chloroform extraction was performed. | InsectaCentral | invivo knockdown of select genes in plusiine noctuids-Alpha integrin2 RNAi construct | Lepidoptera | Noctuidae | Chrysodeixis | includens | local resource | larva | lab_colony | 1 | injection | Nuclease free water | 2 | buffer | 6 | Injected into hemocoel through proleg | Control animals were injected with 1xPBS. | larva | RT-PCR (semi-quantitative) | fatbody,hemocytes,gut | not checked | 120 | none | ||||||||
| 144 | invivo knockdown of select genes in plusiine noctuids.Betaintegrin1 | RNAi | InsectaCentral | Unpublished | Strand | Mike | invivo knockdown of select genes in plusiine noctuids.Betaintegrin1 target | CGCCCGGGCAGGTCAAAGTACTGTGAACCTTTAAGGTAAACCAGTTTTAGAGTGTTAATTACTTAGTAATTTAATGCAGTTTCCAACNAAAAGACTGATATTTTTATAGTTATGTTATTAAAAGGATAACTGTGATAGTGCTAATCAAGTGGACAATCAGTGTTTTCAAAATTTGATCTGAAAACGAATCTGTAGTTTATATTAAACAAAATGTACATACATCGATCGAGTGTATTACGACTGTGTGTGTGGGTGAGCCTGTTGGCGTTATGTTGGGGCCAGCGGGCGGAGCAGCTCCTCGCACAGAACCCTTGCTCGAGCAAGACGACGTGCAGCGACTGCATCAGGACGGCCAGCTGTGCGTGGTGCTTCGCTTCCGACTTCAATGGCCCGCGCTGCTTCAACCCCGCCATGGAGCGCGGCGGCACGGCCGGCTGCGACGAGGCCTACATCTTTAACCCCGACAACCAGCGATCTGTGGACCCTAGATATAATATGGAGCTAAGTAGAGCCAAATCTCGCATGGGAATGGCCGGTGGTTCATTCGAAGAGTCTATGAGCTCAAAGGGCAGCAGTTTCTCTGGCTCAAGTGCCTCTGGGTCAGCTGCAGCCGCTGCAGGTTCAGGAGAGAGCTTGGTACAAATGAAGCCACAAAGAGTCTCACTTAATCTAAGAATGAACCAAATGCAGAAGCTGACATTTGCTTACTCCCGGGCGCAAGACTATCCCGTCGACTTGTACTATCTCATGGACTTGAGTAGATCTATGAAGAACGACAAGGAGAAGCTCAGTACACTGGGCAGTTTGCTCTCGGATACCATGAAGAACATGACTTCCAATTTCTGGATCGGCTTCGGTTCATTCGTAGACAAACTTGTCATGCCTTATGTATCGACTGTGCCTAAGAATTTGATTTCGCCCTGTGATGGCTGCGCAGCACCTTACGGATACCAAAACCAGATGTCACTCAGCAATGACACCAACTTCTTTGTGAAAGCGGTAGCCAACGCCGACGTGTCTGGTAACTTGGATGCTCCCGAGGGCGGTTTCGACGCCATCATGCAAGCCGTGGTGTGCAAGCAGCAGATAGGTTGGCGCGACCAGGCTAGACGACTGCTCGTATTCTCAACTGACGCAGGATTCCATTATGCTGGCGATGGAAAGCTCGGTGGTATCGTACAACCTAATGACGGGGAATGCCACATGAAGGGCAACACATACACCCACTCGACTCTACAGGACTACCCTAGCATCTCACAGATTAATCACAAGGTGAAGAAACACGCTATCAACGTGATATTCGCGGTGACGGCGGAGCAGATCAGCGTGTACGAACAGCTGAGCAAGCACATCGAGGGCTCCAGTACTGGAGTACTCAGCAACGACTCGGACAACATCGTAGATTTAGTACGGGAACAATACAACAAAATTACGTCAGCTGTGGAAATGAAAGATTCATCGAGTGACGCCGTGCAAATAGTGTATTACTCATCTTGTTTGAGCGGCAAGGAACTCATCCAGACTAACAAGTGCGACGGATTGAAGGTGGGAGATGTTGTGGAGTTCACAGCTGAGATCACCCTGAAGGAATGCCCCAAGGACCGCAGCAAGTGGAGGCAGACCCTCGACATCTCCCCCGTCGGTATCTCCGACAGCCTGGTCGTTGACCTCGAGATGGTGTGCGACTGTCCCTGCGAACAGCCTGGACATCATGCTTACAACGACAGCCCACTAGTATGCAGCGGCGAGGGAGTATCCGCGTGCGGCGTGTGTGTGTGCGCGCCGGGAAGGTTCGGCAAGAGCTGCGAGTGTTCGGCGCACGGCGGCGTGTCGGCCGAGCAGGAGCGCGGCTGTCGCCCCACCAACGCCAGCTCCGGCCCGCTCTGCTCCAACCGCGGCACCTGCATCTGCGGCGTCTGCGAGTGCAACAAGATGGACGACCCGCTCAAGGTGATCTCCGGTCCGTTCTGCGAGTGTGACAACTTCACGTGCGACATGAACAAGGGCCTACTGTGCTCGGGCCCCGACCACGGCGAGTGCGTGTGCGGCAAGTGCAGCTGTACTGCGGACTACACCGGGCCCGCCTGCCAGTGCCTCAAGGATCAGACGCCTTGCCGGTCACCTGAAAACAACGAAATCTGCAGTGGAAACGGACAATGTGTATGTGGACAATGTATGTGTAACTCTGACGATGACCGCCACTATAGTGGCAAATACTGCGAGAAATGCCCTACATGCCCCGGCCGTTGCGGCGAGTTCAAGGACTGTGTCCTGTGCGAGGTACACAAGCGCGGCCCCAAGTACAACGCTGACACCGACACTTGTGGCGACTGTGCCTTGTTCCCTATCGTTGAAGAAGGCAAAATTGAAGCTAACGAATCTCGAAACGAACACCTGTGCAGTTTCTACGACGACGAAGACTGTCTCTACGTGTACGTGTACTCGTACAACGAGAACCAGCAGCTCGTGATCAGGGCGCAGAAGGAACGCGAGTGTCCTAAGAAGGTACCAATCCCGGGAATAGTACTGGGTGTGATAGCAGCCATCGTGCTCGTCGGGCTCGCCCTGCTTATGCTGTGGAAGATGGCGACCACCAGCCATGATCGCAGGGAGTTCGCGCGCTTCGAGAAGGAACGCATGATGGCCAAGTGGGACACGGGCGAGAACCCCATTTACAAACAAGCGACGTCTACCTTCAAAAACCCTACGTATGCTGGAAAATAATGCTATTGTATAAAACAGTCAGAAGTCTATTGTAAGATAGTCTCAAAACAAACGTACAATTATTATGCCGCATTTATATAATGAACAAGTCGACAGTATTTCATGAAATTTTCACGATTTATAGAATTTTTATATAAAAAAGTTTTAGAGCAATAAAAATCGTTTATAGAGCTGCCTTAAAAATGTGCCTTTTCATAGATCTTATGAAATGTTAAGCGTTCAAGTTCATACGCGAAACGCTAATAAAGTATATTGTAGCAATACTC | Lepidoptera | Noctuidae | Chrysodeixis | includens | cds | pubmed | AY237588.1 | invivo knockdown of select genes in plusiine noctuids.Betaintegrin1 RNAi construct | TAATACGACTCACTATAGGGGTACAACGCTGACACCGACACTTGTGGCGACTGTGCCTTGTTCCCTATCGTTGAAGAAGGCAAAATTGAAGCTAACGAATCTCGAAACGAACACCTGTGCAGTTTCTACGACGACGAAGACTGTCTCTACGTGTACGTGTACTCGTACAACGAGAACCAGCAGCTCGTGATCAGGGCGCAGAAGGAACGCGAGTGTCCTAAGAAGGTACCAATCCCGGGAATAGTACTGGGTGTGATAGCAGCCATCGTGCTCGTCGGGCTCGCCCTGCTTATGCTGTGGAAGATGGCGACCACCAGCCATGATCGCAGGGAGTTCGCGCGCTTCGAGAAGGAACGCATGATGGCCAAGTGGGACACGGGCGAGAACCCCATTTACAAACAAGCGACGTCTACCTTCAAAAACCCTACGTATGCTGGACCCTATAGTGAGTCGTATTA | dsRNA | MEGAscript RNAi kit, Ambion Inc. | phenol_chloroform | subsequent to transcription | The target DNA sequence was PCR amplified using a forward and reverse primer with T7 promoter at the end of each primer. Set up a 20 micro liter reaction with 8 micro liter of PCR product and incubate at 37 degree celsius for 6 hours. After 6h,incubate at 75 degree celsius for 5 minutes and leave at room temperature for 2-3 hours for cooling down which facilitates annealing. Add 1 micro liter of Dnase and incubate at 37 degree celsius for 15 minutes. Then a phenol-chloroform extraction was performed. | InsectaCentral | invivo knockdown of select genes in plusiine noctuids.Betaintegrin1 RNAi construct | Lepidoptera | Noctuidae | Chrysodeixis | includens | local resource | larva | lab_colony | 1 | injection | Nuclease free water | 2 | buffer | 6 | Injected into hemocoel through proleg | Control animals were injected with 1xPBS. | larva | RT-PCR (semi-quantitative) | fatbody,hemocytes,gut | not checked | 120 | none | ||||||||
| 146 | Spodoptera litteralis RNAi beta-actin | RNAi | Entered by Jennie. Release of sperm bundles from moth testes is controlled by the local circadian oscillator. The mechanism which restricts migration of sperm bundles to a few hours each day is not understood. We demonstrate that a daily cycle of sperm release is initiated by the migration of folded apyrene sperm bundles through a cellular barrier at the testis base. These bundles have conspicuous concentrations of actin filaments at their proximal end. Inhibition of actin polymerization by cytochalasin at a specific time of day inhibited sperm release from the testis. Likewise, application of double-stranded actin CMLS, Cell. Mol. Life Sci. 60 (2003) 1744â1751 1420-682X/03/081744-8 DOI 10.1007/s00018-003-3139-z Âİ Birkh¤user Verlag, Basel, 2003 CMLS Cellular and Molecular Life Sciences RNA specifically inhibited sperm release. This RNA-mediated interference (RNAi) lowered the pool of actin mRNA in tissues involved in sperm release. The decline in mRNA levels resulted in the selective depletion of Factin from the tip of apyrene sperm bundles, suggesting that this actin may be involved in the initiation of sperm release. Combined results of RNAi experiments at physiological, cellular and molecular levels identified unique cells that are critically involved in the mechanism of sperm release. | pubmed | 14513839 | Gvakharia | B O | Spodoptera litteralis RNAi beta-actin target | AGTCGACAATGGCTCCGGCATGTGCAAGGCCGGTTTCGCCGGCGACGACGCGCCCCGCGCCGTCTTCCCATCCATCGTAGGTCGCCCTCGTCACCAGGGTGTGATGGTTGGTATGGGTCAGAAGGACTCCTACGTAGGCGATGAGGCCCAGAGCAAGAGAGGTATCCTCACCCTGAAGTACCCCATCGAGCACGGTATCATCACCAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTACAACGAGCTGCGCGTCGCCCCTGAGGAACACCCAGTCCTCCTGACTGAGGCTCCCCTCAACCCTAAGGCCAACAGGGAGAAGATGACCCAGATCATGTTTGAGACCTTCAACTCCCCCGCCATGTACGTCGCCATCCAGGCTGTGCTCTCTCTGTACGCCTCTGGTCGTACCACCGGTATCGTCCTGGACTCCGGTGATGGTGTCTCCCACACCGTCCCCATCTACGAAGGTTACGCTCTGCCCCACG | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | cds | NCBI | Z46873 | Spodoptera litteralis RNAi beta-actin RNAi construct | GAGCAAGAGAGGTATCCTCACCCTGAAGTACCCCATCGAGCACGGTATCATCACCAACTGGGACGACATGGAGAAGATCTGGCACCACACCTTCTACAACGAGCTGCGCGTCGCCCCTGAGGAACACCCAGTCCTCCTGACTGAGGCTCCCCTCAACCCTAAGGCCAACAGGGAGAAGATGACCCAGATCATGTTTGAGACCTTCAACTCCCCCGCCATGTACGTCGCCATCCAGGCTGTGCTCTCTCTGTACGCCTCTGGTCGTACCACCGGTATCGTCCTGGACT | dsRNA | T3 and T7 polymerase (unknown kit company) | other | subsequent to transcription | Based on the sequence of the b-actin gene from S. littoralis (AN Z46873), we amplified a fragment of this sequence by PCR using the following oligonucleotide primers 5¢-GAGCAAGAGGTATC (sense primer) and 5¢-AGTCCAGGACGATAC (antisense primer). A T3 promoter sequence was added to the 5¢ end of the sense primer, and a T7 promoter was added to the 5¢ end of the antisense primer. To produce dsRNA corresponding to the b-actin gene, the DNA template described above was transcribed in vitro in two separate reactions with T3 and T7 RNA polymerases. After 1.5 h of transcription, the DNA template was destroyed with DNAse to stop the reaction. The efficiency of in vitro transcription was verified on agarose gels. Sense and antisense RNA strands were mixed in equal amounts, heated at 95°C for 1 min, and slowly cooled at room temperature for 18 h. A small aliquot of annealed RNA was run on an agarose gel to confirm that most RNA has been annealed. Usually almost 100% of RNA used showed a shift in electrophoretic mobility, corresponding to dsRNA. | InsectaCentral | Spodoptera litteralis RNAi beta-actin RNAi construct | Lepidoptera | Noctuidae | Spodoptera | littoralis | 7109 | local resource | unfertilized egg | lab_colony | injection | non_specific_dsRNA | testis-uvd (upper vas deferens) complex | unfertilized egg | qPCR | testis-uvd (upper vas deferens) complex | not checked | 1 | high | 55 | |||||||||
| 147 | silkmoth | RNAi | Insect immune system comprises of both humoral and cellular defenses. Nodulation is one of the major, yet very poorly understood cellular responses against microbial infections in insects. Through screening for novel immune genes from an Indian saturniid silkmoth Antheraea mylitta, we identified a protein up-regulated in hemolymph within minutes upon bacterial challenge. We have shown here, for first time, the involvement of this novel protein in mediating nodulation response against bacteria and hence designated it as Noduler. Noduler possessed a characteristic reeler domain found in several extracellular matrix vertebrate proteins. Noduler was shown in vitro to bind a wide range of bacteria, yeast, and also insect hemocytes. Furthermore, Noduler specifically bound LPS, lipotechoic acid, and beta 1, 3 glucan components of microbial cell walls. RNAinterference mediated knock-down of the Noduler resulted in significant reduction in the number of nodules and consequent increase in bacterial load in larval hemolymph. The results suggest that the Noduler is widely conserved and is involved in very early clearance of bacteria by forming nodules of hemocytes and bacterial complexes in insects. The results would promote further studies for understanding of the crucial but hitherto overlooked nodulation mechanism in insects and also provide cues for the study of similar mammalian proteins whose function is not understood. The Journal of Immunology, 2007, 179: 6943â 6951. | pubmed | 17982085 | Javaregowda | Nagaraju | silkmoth target | CGGACTTCAGATATCGTGTGGGATAGTTATTAAACAGTATTGCCCAAGAAATATGATGTTCGCGTACATAGTAGCTGTCGTGTCAGCGTTGGCGCTTACCAGTGCTTTCCCTACTGGTGCACCACGTAGCGCTTGCTTCGACATGATACCCGGCCACTTCGCTAACCCAAAATTGGAACCTGCGCCTTACACTATCACCACGCCTATTAGCGCGGTGAAAGGTGGTAATTCCGTTGAAGTTACAATCAGCGGCAAGACACCTGAAGACACCATGCGTGGTATCCTACTCGAGGCTCGACAAGGAGACAACATCGTCGGCACGTGGACCGTACCTCCTGGTGATGACTTCTCACAACCCATGAACTGTGGAGAACCTAATAACGCCGTCACCCACAAACGCCATTCTGAGTCAGCGGACAAGCAGACCGTGTCATACGTTTGGACTGCTCCCAGTGATTTGGAAGGCGACGTCGTATTCATGGTCACCATAGTAAAGGATTACTCCAACTTCTGGGTCAGACAAACCTCGGCACCCGTAAAAATTTTAAGTCACCATTAATCGCGTGAACCTTACATTAAGTATTTGTACTTTGTAGTTTTATAAGAAACAACAAATTTATAAAACCATTGTGTATTTATAAGCAATAAACTATATATTTTTATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACAAAAAAAACAAAAAAAAAA | Lepidoptera | Saturniidae | Antheraea | mylitta | 34739 | cds | NCBI | DQ666501.1 | silkmoth RNAi construct | ATGATGTTCGCGTACATAGTAGCTGTCGTGTCAGCGTTGGCGCTTACCAGTGCTTTCCCTACTGGTGCACCACGTAGCGCTTGCTTCGACATGATACCCGGCCACTTCGCTAACCCAAAATTGGAACCTGCGCCTTACACTATCACCACGCCTATTAGCGCGGTGAAAGGTGGTAATTCCGTTGAAGTTACAATCAGCGGCAAGACACCTGAAGACACCATGCGTGGTATCCTACTCGAGGCTCGACAAGGAGACAACATCGTCGGCACGTGGACCGTACCTCCTGGTGATGACTTCTCACAACCCATGAACTGTGGAGAACCTAATAACGCCGTCACCCACAAACGCCATTCTGAGTCAGCGGACAAGCAGACCGTGTCATACGTTTGGACTGCTCCCAGTGATTTGGAAGGCGACGTCGTATTCATGGTCACCATAGTAAAGGATTACTCCAACTTCTGGGTCAGACAAACCTCGGCACCCGTAAAAATTTTAAGTCACCATTAA | dsRNA | Megascript Kit (Ambion) | isopropanol precipitation | subsequent to transcription | The dsRNA corresponding to entire protein coding region (507 bp) using primers: forward primer 5â-ATGATGTTCGCGTACATAGTAGCTG-3; reverse primer 5-TTAATGGTGACTTAAAATTTTTACGGGTG-3 and cloned into pCRIITOPO vector (Invitrogen Life Technologies) followed by amplification with M13 forward and reverse primers. This template with flanking T7 and SP6 promoters was used for in vitro transcription reaction. Sense and antisense RNA strands were generated with the T7 and SP6 Megascript kits as prescribed by the manufacturer (Ambion). The DNA template was removed from the transcripts by DNase treatment and the RNA products were subsequently purified by Trizol extraction (Invitrogen Life Technologies) followed by isopropanol precipitation. The complementary single stranded RNAs were dissolved in DEPC treated water, combined in equimolar amounts in insect buffer saline (IBS: NaCl - 160 mM, KCl - 10 mM, CaCl2 - 4 mM) and annealed by heating to 95°C and slow cooling overnight at room temperature. Similarly, dsRNA specific to full-length green fluorescent protein (GFP) was synthesized as a nonspecific control. The dsRNA formation was confirmed by agarose gel electrophoresis and the concentration was determined spectrophotometrically. | InsectaCentral | silkmoth RNAi construct | Lepidoptera | Saturniidae | Antheraea | mylitta | 34739 | local resource | larva | field_collected | 10 | injection | Insect buffer saline | 10 | non_specific_dsRNA | 8 | hemolymph | For control, dsRNA specific to full-length green fluorescent protein (GFP) was cloned into pCRIITOPO vector (Invitrogen Life Technologies) followed by amplification with M13 forward and reverse primers. This template with flanking T7 and SP6 promoters was used for in vitro transcription reaction. Sense and antisense RNA strands were generated with the T7 and SP6 Megascript kits as prescribed by the manufacturer (Ambion). The DNA template was removed from the transcripts by DNase treatment and the RNA products were subsequently purified by Trizol extraction (Invitrogen Life Technologies) followed by isopropanol precipitation. The complementary single stranded RNAs were dissolved in DEPC treated water, combined in equimolar amounts in insect buffer saline (IBS: NaCl - 160 mM, KCl - 10 mM, CaCl2 - 4 mM) and annealed by heating to 95°C and slow cooling overnight at room temperature. The dsRNA formation was confirmed by agarose gel electrophoresis and the concentration was determined spectrophotometrically. | larva | W.blot phenotype | hemolymph | not checked | 18 | high | |||||
| 148 | TGBomb | RNAi | InsectaCentral | Unpublished | Tomita | Shuichiro | TGBomb target | ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA | Hydroida | Aequoreidae | Aequorea | victoria | 6100 | cds | InsectaCentral | TGBomb target | TGBomb RNAi construct | TACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG | dsRNA | MEGAScript (Ambion) | other | subsequent to transcription | The template DNA was prepared by PCR so that T7 and SP6 promoters are attached for the transcription of anti-sense and sense transcripts, respectively. After transcription, template DNA was digested by DNase which was included in the kit. RNA polymerases were inactivated by adding EDTA then two reactions were mixed. RNAs are annealed by incubating at 80C for 15min then cooling down to the room temperature. dsRNA was purified by standard ethanol precipitation followed by gel filtration with Sephadex G25 spin column. The quality and quantity of dsRNA was checked by gel electrophoresis. | InsectaCentral | TGBomb RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | 1 | injection | Distilled Water | 0.1 | buffer | 10 | larva | W.blot phenotype | whole organism for flurescent, hemocytes for western-blot | not checked | 24 | none | ||||||||
| 149 | RNAi B mori CBP | RNAi | Entered by Jennie. We examined the role of carotenoid-binding protein (CBP) in yellow cocoon pigmentation. First, using yellow or white cocoon races, we investigated the linkage between the yellow pigmentation and CBP expression. CBP was expressed only in the silk gland of the yellow cocoon races, which utilize carotenoids for cocoon pigmentation. Furthermore, CBP expression in the silk glands of day 1â7 fifth instar larvae matched the period of carotenoid uptake into the silk gland. Finally, we gave double-stranded CBP RNA to Bombyx mori (B. mori) larvae to induce RNA interference. The significantly reduced expression of CBP in the silk gland of fifth instar larva was confirmed on day 4 and a decrease in yellow pigmentation was observed in the cocoon. We showed that CBP plays a key role in the yellow cocoon pigmentation caused by carotenoids. | pubmed | 15178318 | Tabunoki | Hirok | RNAi B mori CBP target | GTTGGAAACCGTTCACCGGATGAGGCGGTTGTGTGCGCGTTAAAAAAAATCGATCCCAAAAGCGATGTGTAGCTCCGTGCGTTGTAATAAGTAGTGTAAAAAATATTGCACGGCCTTTTGTATTGCACAAAGACGCGATATTAAAATACGAATTTAGATACAAGGCTAACAACTCTGGTTGGCCTTTGTGAATTTTGTGAATTAATTTGATAATTTCGCGTCGAATAAAGAATAGTTACGTACCCGCAATCAAGCTTATAAAAACACGGTTGCAATATAAAAAAAAAAAAGAAACCCTAAGCTCTTGAAGTGTTATAAAAAAATAAGCAAGCAAAGACAATTAAAATGGCCGACTCTACGTCGAAAAGCGCGCCAAAAATCAACGAGGAAACCGCCGAAAGACAATTAAACGAATTGATAAACGTGGACAGTCACGATGAGTACGAGAATAGGCTGAGCAGGATCAGTAGCGCCCTAGCGAACTGGATGAAATCAGTTTTCAATATGGACACCACGACCAAGGAGGAGTTCGATCCTGTTTGGCTATCGATCGATGAGTACAAGAGTCAGGCTAACGAGAGCATGGCGAACGCTTGGCGCATCATCACGCTGCCGAACTGGACGGTGGAGAAGCGAGGGACCGTCAGGGGAGACGTCGTGGAGTCAAGGAAAGTTGAAGGCTTCGGCAAGGTTTATAGATTTACGGGCGTCGTCAACTGTCCCGCCAGGTTTCTATACGAGGAGTTCAAGAACAACCTGACGAAGTTGCCCGAATGGAATCCGACGATATTGAAATGCGAAATAATTAAGGAAATCGGCGATGGCGTGGACCTATCGTACCAGGTGACGGCTGGCGGGGGACGAGGCATAATAACCCCTCGTGACTTCGTGATCCTGCGCCGGACCGCGCTACTCTCCCGGGAGGGACGCGTCGTCGACGACAATCCCCACGGATACATCAGTAGCGGAGTCAGCGTTCAAGTTCCGGGGTATCCGCCCCTCAAGGAGATGGTTAGGGGACACAATAAAGTCGGTTGCTGGTACCTCCAGCCTCGGACCGTGCAGACTCCTGGAGGAAAAATCGAGGACCAGGCCCTGTTCCAATGGTTGATGTGTTGTGATCTCAAAGGTAAAATACCTCAATTCGTGTTGGACGTCGCTTTCGCCACTGTGATGCTGGACTACATCGTGCACGTCAGGAAGTTCGTGGCCGAAGCGAAGGCCAGAGCCGAAATCTGACCTTGAAACGTCCAAAAGCTGCATCTGTTTACATGCATATTTTGCACTGGTGGTAGGACCTCTTGTGAGTCCGCGCGGGTAGGTACCACCACCCTGCCTATTTCTGCGCCGTGAAGCAGTAATGCGCTTCGGTTCGAAGGGCGGGGCAGCCGTTGTAACTAAACTTGAGACCTTAGAACTTATGTCTCAAGGTGGGTGGCATTTACTTTGTGGATGTCTATGGGCTCAAGTAACCACTTAACACCAGGTGGGCTGTGAGCTCGTCCACCCATTTAAGCTATAAAAAAAAACTACCCTCAAAATTCGACTGACAAGTGAGGGGAAGGTTTGCGGGAGAGTCCCGGTGACTCAGGACACTCTGTCCTCAGGACTGATAGTATTCGTGACCCGTCGTAAGAATACTCATTTGGATCATGATAACAAAAATATCTGATAAATAACTCATGGATACCTTGAAAATCGTTCGCGTGTCTGTATGTCCTCTAATATACCAGAATGGGTAGCATTGATGTATGTCGAGATCGCGGCGTTGATGTTGTGGTCAGGGGACCACTAGGACATCCGGCTACAATTAGTTTGATAAAGTAATGGCATTGAAACTCCCGAAAATCGCGGGCTTACGGAATATCAAACGTTGTAAGTGAACCCGCAATGGAAGACGAAGGTAGCTGATATTTTGTTACATTGCGCGCCCCTATGACGGCTTTACTAAGAACCAATGTTCACCCATCGGCCGGATTAAGTTGATTTAAAGATTTTGTAGCTTTAGATACATCAGCGATCGGGCTTATTTTTAATACGCTTTTATTAGCTTCAGACGTATGTATGTTAGTATGTAACGGAATCTTTGAACATGATTTTGACCCCCTTCAAAACGTCGGATTAACTCGAAATTTGGTATACTTATTAAGGACCGATGACAATTCAATATTAAAATGAAAAATAATTGAAAAAATTGAAATTAAACTAAAAAAAAAAAAAAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | AB062740 | RNAi B mori CBP RNAi construct | ATGGCCGACTCTACGTCGAAAAGCGCGCCAAAAATCAACGAGGAAACCGCCGAAAGACAATTAAACGAATTGATAAACGTGGACAGTCACGATGAGTACGAGAATAGGCTGAGCAGGATCAGTAGCGCCCTAGCGAACTGGATGAAATCAGTTTTCAATATGGACACCACGACCAAGGAGGAGTTCGATCCTGTTTGGCTATCGATCGATGAGTACAAGAGTCAGGCTAACGAGAGCATGGCGAACGCTTGGCGCATCATCACGCTGCCGAACTGGACGGTGGAGAAGCGAGGGACCGTCAGGGGA | dsRNA | MEGAscript T7&T3 kit (Ambion) | phenol_chloroform | subsequent to transcription | To produce the CBP for RNA interference, a 308-bp PCR fragment of CBP was amplified using CBP cDNA1 as template and the primers 50-ATGGCCGACTCTACGTCGAAAAGC G-30 and 50-TCCCCTGACGGTCCCTCGCTTCTCC- 30, and cloned into p123-T vector. To produce aminopeptidase N-1 (APN-1) dsRNA for RNA interference, a 458-bp PCR fragment of APN-1 was amplified using APN-1 cDNA2 as template and the primers 50-TTCTTCTGGAATCGCTACCTTCAGGAAGAT- 30 and 50-TAGCCT GACTCGAAGTAGTTCGTGAATTCT- 30, and cloned into p123-T vector. The cloned fragments were amplified usingM13 forward and reverse primers. The PCR products were purified using a PCR purification kit and used as template to generate sense (sRNA) and antisense (asRNA) RNA using a T7 and T3 MEGA script kit. To generate dsRNA, equal amounts of sRNA and asRNA were mixed, heated at 95 C for 3 min, and gradually cooled to 25 C. Then, the solution was treated with RNase for 1 h at room temperature. The dsRNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in PBS. One microgram of dsRNA was analyzed using 1% agarose gel electrophoresis to ensure that the majority of the dsRNA existed as a single band of approximately 300 or 450 bp, respectively. | InsectaCentral | RNAi B mori CBP RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | injection | non_specific_dsRNA | (30 micrograms/insect) | dsAPN-1 (aminopeptidase1) | larva | W.blot | midgut, silk glands | not checked | 96 | high | 80 | ||||||||
| 150 | silkmoth-1 | RNAi | Insect immune system comprises of both humoral and cellular defenses. Nodulation is one of the major, yet very poorly understood cellular responses against microbial infections in insects. Through screening for novel immune genes from an Indian saturniid silkmoth Antheraea mylitta, we identified a protein up-regulated in hemolymph within minutes upon bacterial challenge. We have shown here, for first time, the involvement of this novel protein in mediating nodulation response against bacteria and hence designated it as Noduler. Noduler possessed a characteristic reeler domain found in several extracellular matrix vertebrate proteins. Noduler was shown in vitro to bind a wide range of bacteria, yeast, and also insect hemocytes. Furthermore, Noduler specifically bound LPS, lipotechoic acid, and beta-1, 3 glucan components of microbial cell walls. RNAinterference mediated knock-down of the Noduler resulted in significant reduction in the number of nodules and consequent increase in bacterial load in larval hemolymph. The results suggest that the Noduler is widely conserved and is involved in very early clearance of bacteria by forming nodules of hemocytes and bacterial complexes in insects. The results would promote further studies for understanding of the crucial but hitherto overlooked nodulation mechanism in insects and also provide cues for the study of similar mammalian proteins whose function is not understood. The Journal of Immunology, 2007, 179: 6943â 6951. | pubmed | 17982085 | Javaregowda | Nagaraju | silkmoth target | CGGACTTCAGATATCGTGTGGGATAGTTATTAAACAGTATTGCCCAAGAAATATGATGTTCGCGTACATAGTAGCTGTCGTGTCAGCGTTGGCGCTTACCAGTGCTTTCCCTACTGGTGCACCACGTAGCGCTTGCTTCGACATGATACCCGGCCACTTCGCTAACCCAAAATTGGAACCTGCGCCTTACACTATCACCACGCCTATTAGCGCGGTGAAAGGTGGTAATTCCGTTGAAGTTACAATCAGCGGCAAGACACCTGAAGACACCATGCGTGGTATCCTACTCGAGGCTCGACAAGGAGACAACATCGTCGGCACGTGGACCGTACCTCCTGGTGATGACTTCTCACAACCCATGAACTGTGGAGAACCTAATAACGCCGTCACCCACAAACGCCATTCTGAGTCAGCGGACAAGCAGACCGTGTCATACGTTTGGACTGCTCCCAGTGATTTGGAAGGCGACGTCGTATTCATGGTCACCATAGTAAAGGATTACTCCAACTTCTGGGTCAGACAAACCTCGGCACCCGTAAAAATTTTAAGTCACCATTAATCGCGTGAACCTTACATTAAGTATTTGTACTTTGTAGTTTTATAAGAAACAACAAATTTATAAAACCATTGTGTATTTATAAGCAATAAACTATATATTTTTATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACAAAAAAAACAAAAAAAAAA | Lepidoptera | Saturniidae | Antheraea | mylitta | 34739 | cds | NCBI | DQ666501.1 | silkmoth RNAi construct | ATGATGTTCGCGTACATAGTAGCTGTCGTGTCAGCGTTGGCGCTTACCAGTGCTTTCCCTACTGGTGCACCACGTAGCGCTTGCTTCGACATGATACCCGGCCACTTCGCTAACCCAAAATTGGAACCTGCGCCTTACACTATCACCACGCCTATTAGCGCGGTGAAAGGTGGTAATTCCGTTGAAGTTACAATCAGCGGCAAGACACCTGAAGACACCATGCGTGGTATCCTACTCGAGGCTCGACAAGGAGACAACATCGTCGGCACGTGGACCGTACCTCCTGGTGATGACTTCTCACAACCCATGAACTGTGGAGAACCTAATAACGCCGTCACCCACAAACGCCATTCTGAGTCAGCGGACAAGCAGACCGTGTCATACGTTTGGACTGCTCCCAGTGATTTGGAAGGCGACGTCGTATTCATGGTCACCATAGTAAAGGATTACTCCAACTTCTGGGTCAGACAAACCTCGGCACCCGTAAAAATTTTAAGTCACCATTAA | dsRNA | Megascript Kit (Ambion) | isopropanol precipitation | subsequent to transcription | The dsRNA corresponding to entire protein coding region (507 bp) using primers: forward primer 5â-ATGATGTTCGCGTACATAGTAGCTG-3; reverse primer 5-TTAATGGTGACTTAAAATTTTTACGGGTG-3 and cloned into pCRIITOPO vector (Invitrogen Life Technologies) followed by amplification with M13 forward and reverse primers. This template with flanking T7 and SP6 promoters was used for in vitro transcription reaction. Sense and antisense RNA strands were generated with the T7 and SP6 Megascript kits as prescribed by the manufacturer (Ambion). The DNA template was removed from the transcripts by DNase treatment and the RNA products were subsequently purified by Trizol extraction (Invitrogen Life Technologies) followed by isopropanol precipitation. The complementary single stranded RNAs were dissolved in DEPC treated water, combined in equimolar amounts in insect buffer saline (IBS: NaCl - 160 mM, KCl - 10 mM, CaCl2 - 4 mM) and annealed by heating to 95°C and slow cooling overnight at room temperature. Similarly, dsRNA specific to full-length green fluorescent protein (GFP) was synthesized as a nonspecific control. The dsRNA formation was confirmed by agarose gel electrophoresis and the concentration was determined spectrophotometrically. | InsectaCentral | silkmoth RNAi construct | Lepidoptera | Saturniidae | Antheraea | mylitta | 34739 | local resource | larva | field_collected | 10 | injection | Insect buffer saline | 10 | non_specific_dsRNA | 8 | hemolymph | For control, dsRNA specific to full-length green fluorescent protein (GFP) was cloned into pCRIITOPO vector (Invitrogen Life Technologies) followed by amplification with M13 forward and reverse primers. This template with flanking T7 and SP6 promoters was used for in vitro transcription reaction. Sense and antisense RNA strands were generated with the T7 and SP6 Megascript kits as prescribed by the manufacturer (Ambion). The DNA template was removed from the transcripts by DNase treatment and the RNA products were subsequently purified by Trizol extraction (Invitrogen Life Technologies) followed by isopropanol precipitation. The complementary single stranded RNAs were dissolved in DEPC treated water, combined in equimolar amounts in insect buffer saline (IBS: NaCl - 160 mM, KCl - 10 mM, CaCl2 - 4 mM) and annealed by heating to 95°C and slow cooling overnight at room temperature. The dsRNA formation was confirmed by agarose gel electrophoresis and the concentration was determined spectrophotometrically. | larva | W.blot phenotype | hemolymph | not checked | 18 | high | |||||
| 151 | RNAi B mori white | RNAi | Entered by Jennie. Injection of double-stranded RNA (dsRNA) corresponding to the silkworm white gene (Bmwh3) into preblastoderm eggs of the wild-type silkworm induced phenotypes similar to those observed with mutants of the white egg 3 locus (10â19.6). The induced phenotypes were characterized by the presence of white eggs and translucent larval skin. Northern analysis showed that the expression of the endogenous Bmwh3 gene in the injected embryos was distinctly depressed. Furthermore, the injection of the GFP dsRNA inhibited the expression of the GFP gene from a plasmid coinjected with the dsRNA but did not depress the expression of the Bmwh3 gene. These findings demonstrate that sequence-specific RNA interference occurred in the silkworm. We conclude from the results that the RNA interference can be applied as a tool for the analysis of the gene function in the lepidopteran insects. | pubmed | 12000640 | Quan | G X | RNAi B mori white target | ACATCGCTCTGTGGCATCGCAGCACTCGCGCGCCGTTGCCTAACCTGCACATTTCCCGCCAAATCAAAATAAAAAAATTGTGACGTAACTAACAATAATCTCGTGCTGTGTGTATGTGTGTGTGAAATCATGAGTTTGATCGTATGATTAATATTGTTATAGTTTAATTTTGTGATCAACATTAAAAATGACTGCCGGAAACGAAGAACAAGAGCCCCTAATATCAACTTCGGTTGATAATCAGCGCGTAACATACAACAACTCCCCACAGGATGGTCAAACGCCAAATGACTCGCCACGATCAAGTGTCGGTGAAGTCACTGTGGCCATACCTCAAAATCGCAACTACGGAGCCATCGGAGGTATTGAAAAAGTTACTTACACGTGGGCCGATGTCAACGCTTTCGCCACAGAGTCCAGATCGAGAGGAAGAAGGTTCTGGAGCTTTTGGAAAAATTCTTCGGATCGCATGTTCCAGCAAAGAAAGCAGTTGCTTAGAAATGTAAACGGAGCGGCCTATCCTGGTGAACTGCTGGCCATCATGGGATCGTCGGGAGCCGGAAAGACCACACTCCTCAATACGCTTACGTTCAGAACGCCCGGTGGTGTTGTCGCCACTGGTACCAGGGCGTTGAATGGGCAACCGGCCACTCCTGACGCTTTGACAGCGCTGTCGGCTTATGTCCAGCAACAGGATCTGTTCATAGGCACTTTGACCGTGCGAGAGCATTTGGTATTTCAGGCGATGGTGAGAATGGATCGCCACATACCATACGCTCAGAGAATGAAGAGAGTTCAAGAAGTTATTCAAGAGTTGGCGCTGTCGAAATGCCAGAACACAGTGATCGGTATACCCGGTCGACTGAAGGGCATATCGGGGGGTGAGATGAAGCGGCTTTCCTTCGCCAGTGAAGTCCTCACGGATCCTCCTCTCATGTTCTGCGACGAACCCACCTCTGGGCTTGATTCATTTATGGCCCAAAACGTTATACAGGTACTGAAAGGCTTGGCACAGAAGGGCAAGACCGTGGTCTGCACAATTCACCAGCCCTCTTCAGAGCTTTACGCTATGTTTGACAAGCTCCTCATAATGGCTGACGGAAGAGTTGCTTTCTTAGGATCATCTGATGAAGCTTTCCAGTTCTTCAAAGAGCTGGGCGCCGCGTGTCCCGCTAACTACAACCCGGCTGACCACTTCATCCAACTCCTCGCCGGAGTACCTGGAAGAGAGGAGGTCACGAGGCACACCATAGATACAGTTTGCACGGCCTTCGCCAAATCCGAGATCGGGTGTAGGATAGCTGCGGAAGCTGAAAATGCGCTCTATAATGAACGTAAGATACAGGCCGGGCTAGCGGACGCGCCGTGGGCCATGTCGTCGACGACGCGAGCCCGCCGCTCCCCGTACAAGGCGTCGTGGTGCACGCAGTTCCGGGCCGTGCTGTGGCGCTCCTGGCTCTCCGTCACCAAGGAGCCGATGCTCATCAAAGTTCGCTTCCTACAGACCATTATGGTGTCGATACTGATAGGCGTCATATACTTCGGCCAGAACTTGGACCAGGACGGGGTGATGAACATCAACGGGGCGATCTTCATGTTCCTAACGAACATGACGTTCCAGAACATATTCGCTGTTATTAACGTATTCTGCTCTGAACTACCAATCTTCATCAGAGAGCACCACTCGGGAATGTATCGAGCCGACGTCTATTTCTTATCGAAGACGTTAGCCGAAGCTCCCGTTTTCGCCACTATACCATTAGTCTTCACAACTATAGCTTATTACATGATCGGATTGAACCCAGATCCGAAGAGGTTCTTCATCGCTTCAGGATTGGCTGCTCTGGTTACGAACGTCGCTACTTCGTTCGGTTATTTAATATCCTGTGCCAGCAGCAGCGTCAGTATGGCAGCCTCCGTTGGTCCTCCAATCATCATCCCCTTTATGCTATTTGGTGGATTCTTCTTGAATTCTGGGTCCGTCCCACCGTACTTAAGTTGGATATCGTATCTATCCTGGTTCCACTACGGCAACGAAGCGCTTCTCATCAACCAGTGGGCCGGCGTGGAGACTATCGCCTGCACAAGGGAGAACTTCACCTGTCCAGCTTCGGGACAGGTCGTACTGGAGACGCTTAGCTTTTCACAGGACGATTTCGCGATGGACGTGGTGAACATGATCTTGTTATTCGTCGGCTTCAGATTTCTGGCGTACCTCGCCCTGCTGTGGAGGACGCGCAGGGCCAAGTGACCCCCCAAACGTCCAGGGCCCCCCAGTCCCCCCGGTCCCCCCAACCCCCCCGTCCCTTCCTAACACCCAACTACGAACAAATCATCAATCGTATTCCGATATCTTTTTACCTGTATTTGTTTTTTGAGAATAAATTTATTGGAAATTTTATTTAGA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | AB445460 | RNAi B mori white RNAi construct | CTACGGAGCCATCGGAGGTATTGAAAAAGTTACTTACACGTGGGCCGATGTCAACGCTTTCGCCACAGAGTCCAGATCGAGAGGAAGAAGGTTCTGGAGCTTTTGGAAAAATTCTTCGGATCGCATGTTCCAGCAAAGAAAGCAGTTGCTTAGAAATGTAAACGGAGCGGCCTATCCTGGTGAACTGCTGGCCATCATGGGATCGTCGGGAGCCGGAAAGACCACACTCCTCAATACGCTTACGTTCAGAACGCCCGGTGGTGTTGTCGCCACTGGTACCAGGGCGTTGAATGGGCAACCGGCCACTCCTGACGCTTTGACAGCGCTGTCGGCTTATGTCCAGCAACAGGATCTGTTCATAGGCACTTTGACCGTGCGAGAGCATTTGGTATTTCAGGCGATGGTGAGAATGGATCGCCACATACCATACGCTCAGAGAATGAAGAGAGTTCAAGAAGTTATTCAAGAGTTGGCGCTGTCGAAATGCCAGAACACAGTGATCGGTATACCCGGTCGACTGAAGGGCATATCGGGGGGTGAGATGAAGCGGCTTTCCTTCGCCAGTGAAGTCCTCACGGATCCTCCTCTCATGTTCTGCGACGAACCCACCTCTGGGCTTGATTCATTTATGGCCCAAAACGTTATACAGGTACTGAAAGGCTTGGCACAGAAGGGCAAGACCGTGGTCTGCACAATTCACCAGCCCTCTTCAGAGCTTTACGCTATGTTTGACAAGCTCCTCATAATGGCTGACGGAAGAGTTGCTTTCTTAGGATCATCTGATGAAGCTTTCCAGTTCTTCAAAGAGCTGGGCGCCGCGTGTCCCGCTAACTACAACCCGGCTGACCACTTCATCCAACTCCTCGCCGGAGTACCTGGAAGAGAGGAGGTCACGAGGCACACCATAGATACAGTTTGCACGGCC | dsRNA | T7 RiboMAX Express; Promega | phenol_chloroform | subsequent to transcription | To produce the dsRNAs for Bmwh3 and GFP, PCR fragments for each were cloned into pGEM-T plasmid (Promega). A 925 bp DNA fragment of the silkworm white gene (Bmwh3, Abraham et al., 2000), was amplified using Bmwh3 cDNA as template with the primers CTACGGAGCCATCGGAGGTATT and GGCCGTGCAAACTGTATCTATG. A 706 bp DNA fragment of the green fluorescent protein (GFP) gene was PCR amplified using the plasmid pPIGA3GFP (Tamura et al., 2000) as the template with the primers TGGTGAGCAAGGGCGAGGAG and TCGTCCATGCCGAGAGTGAT. Plasmids with PCR fragments inserted in both directions for the Bmwh3 and GFP genes were isolated for the synthesis of the dsRNAs. The sense and antisense RNAs were synthesized for each gene target using the RiboMAX Large Scale RNA Production Systems-T7 (Promega). The plasmids were linearized by the digestion with the restriction enzyme PstI and used as the template for the RNA synthesis. Following the RNA synthesis, the reaction mix was extracted with phenol/chloroform, precipitated with ethanol and dissolved in RNase-free water. Equal molar amounts of the sense and antisense RNAs were mixed in the 10 mM Tris-HCl (pH 7. 5) /20 mM NaCl buffer, and heated at 95 °C for 1 min. To anneal the dsRNA, the heated RNA solution was kept at 25 °C for 12â16 h. The annealed dsRNA was treated with RNaseA (1 Îĵg/ml) at 37 °C for 30 min, extracted with phenol/ chloroform, precipitated with ethanol and dissolved in distilled water. The formation of the dsRNA was confirmed by non-denaturing agarose gel electrophoresis. The injection solutions were prepared by dilution of the dsRNAs in distilled water. | InsectaCentral | RNAi B mori white RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | embryo | lab_colony | injection | non_specific_dsRNA | dsGFP | embryo | N.blot | whole organism | not checked | 96 | high | ||||||||||
| 152 | Cathepsin B RNAi Bombyx larvae | RNAi | Entered by Jennie. Background: Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. Results: Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage. Conclusion: Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis. | pubmed | 17062167 | Gui | Zheng | Cathepsin B RNAi Bombyx larvae target | ACTGTTGGCCTCTGGACTTACTGCGTTGTTTTTCGATTGACAAGACGTTACGGTCACAGTGTTCTATTTTAATAAATCAATAAATAACAATAGTCAATTTCTCATAAGCAAGTCGCCTAGTTGCGGCTTCTAAAGACAACAATGGGAAAGATATCTTTATTTTTTTTGGCGCTGATCGCCAGCTCCGTAATGGCACTATATAGGGTGCCATTACATCGTATGAAAACTGCGAGAACCCACTTTCATGAGGTTGGCACTGAACTGGAGCTGTTGAGATTAAAATACGATGTGACTGGCCCTTCACCCGAACCATTGTCAAATTATCTTGATGCTCAGTACTACGGAGTGATCAGTATCGGCACGCCGCCGCAGTCGTTCAAGGTGGTATTCGACACCGGATCCTCCAACCTCTGGGTGCCTTCCAAAAAGTGCCACTACACCAACATCGCTTGTTTGCTGCACAACAAGTACGACAGCCGCAAGTCCAAGTCGTACGTCGCGAACGGCACCCAGTTCGCGATACAGTACGGCTCCGGCAGCCTCTCCGGCTTCCTCTCCACTGATGATGTCACCGTGGGCGGGCTCAAGGTGCGGCGCCAGACGTTCGCCGAGGCCGTGTCGGAGCCCGGGCTCGCTTTCGTGGCCGCCAAGTTCGACGGGATCCTCGGGATGGCCTTCAGCACTATCGCAGTGGACCATGTGACCCCGGTGTTCGACAACATGGTGGCTCAGGGACTCGTGCAGCCCGTCTTCTCGTTCTATCTCAACAGGGACCCCGGGGCGACAACGGGCGGCGAGTTGCTGCTGGGCGGCTCCGACCCCGCCCACTACCGAGGCGACCTCGTACGGGTGCCCCTACTCCGGGACACGTACTGGGAGTTCCACATGGACTCTGTCAATGTCAACGCTAGCCGCTTCTGTGCCCAGGGCTGCTCGGCCATCGCCGACACGGGCACGTCGCTGATCGCCGGGCCCTCCAAGGAGGTGGAAGCCCTCAACGCGGCGGTGGGCGCCACGGCCATCGCCTTCGGGCAGTACGCCGTGGACTGCAGCCTCATACCGCACCTGCCGCGAGTCACCTTTACCATCGCCGGGAACGACTTCACGCTCGAGGGCAACGACTACGTGCTCCGGGTGGCACAAATGGGGCACACGGTGTGCCTGTCCGGCTTCATGGCGCTGGACGTGCCGAAGCCCATGGGCCCGCTGTGGATCCTGGGGGACGTGTTCATCGGCAAGTACTACACGGAGTTCGACGCGGGGAACCCGCCAGCCTCGGGTTCGCCGCCCGCCGTGTGAGCCGGGCGACCCGCTTGCATTAATAAACATTTTTCATTAATTAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | AY297160 | Cathepsin B RNAi Bombyx larvae RNAi construct | CCGCAGTCGTTCAAGGTGGTATTCGACACCGGATCCTCCAACCTCTGGGTGCCTTCCAAAAAGTGCCACTACACCAACATCGCTTGTTTGCTGCACAACAAGTACGACAGCCGCAAGTCCAAGTCGTACGTCGCGAACGGCACCCAGTTCGCGATACAGTACGGCTCCGGCAGCCTCTCCGGCTTCCTCTCCACTGATGATGTCACCGTGGGCGGGCTCAAGGTGCGGCGCCAGACGTTCGCCGAGGCCGTGTCGGAGCCCGGGCTCGCTTTCGTGGCCGCCAAGTTCGACGGGATCCTCGGGATGGCCTTCAGCACTATCGCAGTGGACCATGTGACCCCGGTGTTCGACAACATGGTGGCTCAGGGACTCGTGCAGCCCGTCTTCTCGTTCTATCTCAACAGGGACCCCGGGGCGACAACGGGCGGCGAGTTGCTGCTGGGCGGCTCCGACCCCGCCCACTACCGAGGCGACCTCGTACGGGTGCCCCTACTCCGGGACACGTACTGGGAGTTC | dsRNA | MEGAscript RNAi kit (Ambion) | other | subsequent to transcription | The MEGAscript RNAi kit (Ambion) was used to generate double-stranded RNA (dsRNA) corresponding to nucleotides 226 to 741 of the BmCatD cDNA. T7 promoter sites were added to the PCR primers BmCatD-Fi (5'- GCGTAATACGACTCACTATAGGGAGACCGCAGTCGTTCAAGGTGGTA- 3') and BmCatD-Ri (5'- GCGTAATACGACTCACTATAGGGAGAGAACTCCCAGTACGTGTCCCG- 3'). PCR reactions were conducted to generate complementary templates with a single T7 promoter site. T7 RNA polymerase was used to transcribe single- stranded RNA (ssRNA) from each DNA template over 4 h at 37°C. BmCatD dsRNA was produced by mixing solutions containing equal amounts of complementary ssRNA, incubating at 75°C for 5 min, and allowing the solution to cool down to room temperature. DNA and ssRNA were removed from the solution by digestion with DNase I and RNase at 37°C for 1 h. The dsRNA was purified using purification cartridges provided in the kit and dsRNA was eluted with two successive 50 Îĵl washings of pre-heated (95°C) 10 mM Tris-HCl (pH 7.0) containing 1 mM EDTA. Finally, total dsRNA was quantified from the A260. BmCatD dsRNA (â1 mg ml-1) was injected into larvae or pupae of B. mori (injection volume â50 Îĵl/individual) using a sterile needle. After injection, all individuals were covered with paraffin. | InsectaCentral | Cathepsin B RNAi Bombyx larvae RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | 1 | injection | buffer | (50 microlitres/insect) | untreated insects? | larva | N.blot | fat body | not checked | 24 | low | ||||||||
| 153 | TO RNAi P interpunctella embryo | RNAi | Entered by Jennie. Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi) provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database under NCBI Accession # AY427951. | pubmed | 15861231 | Fabrick | Jeffrey A | TO RNAi P interpunctella embryo target | GGACACTGACATGGACTGAAGGAGTAGAAAGTATCGAGTGACCACGCGCCCGGCAAGTGCGCGCGCGTCCTAATTCGAAATTCAAACTTTATTTATTTTTTAGTGTTTAACTTAGAACTACAAAATGGCCTGTCCTATGAGGTCAATGAACGACTCGTCGCAGGACGGCAGCTGCCTGGGCAACGAAGCCGGCATGCTGTACGGGGAGTACCTGATGCTGGACAAGCTCCTCGCAGCACAGCGGATGCTCAGCGCCGAGTCCTCGCGGCCGGCGCACGACGAGCATCTGTTCATTGTCACCCATCAAGCGTACGAGTTATGGTTCAAGCAGATAATATATGAAGTGGACTCTGTCAGGTCGTTGCTGGACGTTGAAGGTCTGGACGAGAGCCACACCATGGAGATACTGAAGCGGCTAAACAGGATTGTGTTAATTCTTAAGCTTCTTGTGGACCAAGTCATGATCTTAGAGACCATGACCCCTCTGGACTTCATGGACTTCCGGAACTACCTGAGGCCAGCTTCCGGCTTCCAGAGCCTTCAGTTCCGGCTATTGGAAAATAAGTTGGGGCTAAAACAGGCGTTGCGCGTGAAGTACAATCAGAATTACCAGACAGTGTTTGGGGATGACCCTGCAGCTATGGACGCCTTGCACAAATCAGAAGAAGAACCAGCGCTCCTGGCGCTGATCGAGCGGTGGCTAGAACGAACCCCAGGGCTGCAAACGCACGGCTTCAACTTCTGGGGCAAGTTCCAGGCCGCCGTCAACAAAACTATTACAGACGACATTGAGGCCGCTATGACGGAACCCAACGAAACAGTCCGTCGTCACAGGCTGCAGGACGCGGAAAACCGACGCGAGATCTACCGGTCTATCTTCGATCCAGCCGTGCACGATGCCCTCAGATCCAGGGGGGAAAGGAGACTGTCGCACCGCGCGCTGCAAGGCGCCATCATGATCACGATCTACCGCGACGAGCCGCGTTTCTCGCAGCCGCACCAGCTGCTCACGCTGCTCATGGACATCGACAGCCTCATCACCAAGTGGCGCTATAACCACGTGATCATGGTGCAGCGTATGATTGGCTCGCAGCAACTCGGCACGGGCGGCTCCTCAGGGTACCAGTACCTTCGCTCCACTCTCAGTGACCGTTACAAGGTATTCTTGGATCTATTTAACCTCTCAACCTTCCTCCTCCCCCGCTCCCTCATCCCGCCCCTCGACGACGGCATGAAGAAGAGTCTCAACCTCACCTGGGGCGACAACGCCAGGGAGAATGGGAAAGAAACACACGACGGAGAGCTTAATAACTGCATGTTGAAATTGGAGTTGGCTTGATCTGATTATGTCATGTACTGTATATAATACTGTAAGAGAGGTAATCTGGGGGCTACCATGAAAAATAAAACACGTAGTGCGTCATTACGTCTAAAAAAAAAAAAAAAAAAA | Lepidoptera | Pyralidae | Plodia | interpunctella | 58824 | cds | NCBI | AY427951 | TO RNAi P interpunctella embryo RNAi construct | CTGGCGCTGATCGAGCGGTGGCTAGAACGAACCCCAGGGCTGCAAACGCACGGCTTCAACTTCTGGGGCAAGTTCCAGGCCGCCGTCAACAAAACTATTACAGACGACATTGAGGCCGCTATGACGGAACCCAACGAAACAGTCCGTCGTCACAGGCTGCAGGACGCGGAAAACCGACGCGAGATCTACCGGTCTATCTTCGATCCAGCCGTGCACGATGCCCTCAGATCCAGGGGGGAAAGGAGACTGTCGCACCGCGCGCTGCAAGGCGCCATCATGATCACGATCTACCGCGACGAGCCGCGTTTCTCGCAGCCGCACCAGCTGCTCACGCTGCTCATGGACATCGACAGCCTCATCACCAAGTGGCGCTATAACCACGTGATCATGGTGCAGCGTATGATTGGCTCGCAGCAACTCGGCACGGGCGGCTCCTCAGGGTACCAGTACCTTCGCTCCACTCTCAGTGACCGTTACAAGGTATTCTTGGATCTATTTAACCTCTCAACCTTCCTCCTCCCCCGCTCCCTCATCCCGCCCCTCGACGACGGCATGAAGAAGAGTCTCAACCTCACCTGGGGCGACAACGCCAGGGAGAATGGGAAAGAAACACACGACGGAGAGCTTAATAACTGCATGTTGAAATTGGAGTTGGCT | dsRNA | MEGAscript RNAi kit (Ambion) | other | subsequent to transcription | The MEGAscript⢠RNAi kit (Ambion, www.ambion.com) was used to generate double-stranded RNA corresponding to nucleotides 680 to 1,336 of the Plodia tryptophan oxygenase. T7 promoter sites were added to the PCR primers VerF5 (5' CTGGCGCTGATCGAGCGGTGG 3') and VerR6 to generate the primers T7VerF5 (5' TAATACGACTCACTATAGGGACTGGCGCTGATCGAGCGGTGG 3') and T7VerR6 (5' ATTATGCTGAGTGATATCCCTAGCCAACTCCAATTTCAACATGCA 3'). Two separate PCR reactions were conducted to generate complementary templates with a single T7 promoter site (T7VerF5+VerR6 and VerF5+T7VerR6). T7 RNA polymerase was used to transcribe single-stranded RNA from each DNA template over 4 h at 37° C. double-stranded RNA was produced by mixing solutions containing equivalent amounts of complementary ssRNA, incubating at 75° C for 5 minutes, and allowing the solution to cool to room temperature. DNA and single-stranded RNA were removed from the solution by digestion with DNase I and RNase at 37° C for 1 h. The double-stranded RNA was purified using provided purification cartridges as per the manufacturerâs instructions. Double-stranded RNA was eluted with two successive 50 Îĵl washes of 95° C pre-heated 10 mM Tris-HCl pH 7 containing 1 mM EDTA (ethylenediamine tetraacetate). Total double-stranded RNA was quantified from A260. | InsectaCentral | TO RNAi P interpunctella embryo RNAi construct | Lepidoptera | Pyralidae | Plodia | interpunctella | 58824 | local resource | embryo | lab_colony | 1 | injection | 10 mM Tris-HCl pH 7 containing 1 mM EDTA | buffer | (100pl volume delivered) | Control is non-treated insects. | larva | RT-PCR (semi-quantitative) | whole organism | not checked | 96 | low | |||||||
| 158 | Heliothis virescens pheromone gland cultured in vitro | RNAi | 1. Background information In the biosynthesis of sex pheromones, fatty-acyl reductase (FAR) is a key enzyme that is necessary for the production of oxygenated functional groups (Tillman 1999; Jurenka 2004; Moto et al. 2003). This enzyme converts fatty-acyl pheromone precursors to corresponding alcohols. Recently, a substrate specific FAR was identified from pheromone glands of Bombyx mori (Moto et al. 2003), and injection of dsRNA corresponding to the identified FAR resulted in a successful reduction of the precursor of bombykol in Bombyx mori (Ohnishi et al. 2006). In Ostrinia nubilalis the sex pheromone gland specific FAR was found to be the key enzyme that is responsible for the E and the Z ratio variation (Lassance et al. submitted). In Heliothis virescens (Hv) we recently found four FARs, and the one that was specifically expressed in the sex pheromone glands was most similar to the O. nubilalis FAR (Vogel et al. 2010). We conducted RNAi experiments in vitro and in vivo with this FAR to attempt to silence sex pheromone production in the sex pheromone gland of Hv. Phenotype: in vitro The in vitro effect and the functionality of dsRNA on pheromone production was examined by comparing the production of the major sex pheromone component (Z11-16:ALD) and a minor component (16:ALD) to obtain a pheromone profile. This pheromone profile and the total amount of pheromone produced was compared to the pheromone production in in vitro glands without dsRNA. In the in vitro experiments, sex pheromone glands of Hv were extracted after being in the medium and analyzed by GC (gas chromatography). PBAN (synthetic HezPBAN from Heliothis zea, Peninsula Laboratories, San Carlos, CA) was used to stimulate pheromone production in vitro. A concentration of 0.5 pmol was used to induce pheromone production. The PBAN stock solution (200 pmol/ml in 50% methanol and 1N HCl) was diluted with Graceâs insect medium to obtain the optimal solvent of PBAN for in vitro applications. Because studies have shown that PBAN stimulates a maximum of pheromone production after 120 min of incubation, we used this incubation time. Results: Phenotypic assay in vitro The total amount of pheromone produced per gland increased to 18 ng in dsRNA treated glands, relative to 11 ng in control glands, when assayed 3 hrs after incubation with dsRNA. The total amount of pheromone per gland dropped to 2ng by 6 hrs, in both dsRNA-treated and control glands. | InsectaCentral | Unpublished | Barthel | Andrea | Heliothis virescens pheromone gland target | ATTGTTCATCGGATCGCCTGGACATCCACTAGTCCTGAAAGACACACAGAGATAATCATAATAATTCATTCCTTAGCCTTTCACAAAAAAAACATCATTTATCAAAGTGAAGGTGGTTTTAAAAATTATCAAAATGGTTGTTTTGACTTCTAAAGAAACGAAACCTTCAGTAGCTGAATTTTATGCGGGAAAATCTGTATTTATTACGGGAGGTACTGGATTCCTTGGAAAGGTATTCATAGAGAAACTTCTGTACAGCTGTCCTGATATCGTCAATATTTACATGCTCATACGAGAGAAGAAGGGGCTATCTGTTAGCGAGAGAATAAAACAGTTTCTTGATGACCCGCTCTTCACAAGACTGAAAGACAAAAGACCAGCTGACTTAGAGAAGATTGTACTTATACCTGGAGATATTACTGCTCCTGACCTGGGTATCACTGCTGCAAATGAAAAAATGCTTATTGAGAAGGTATCGGTGATTATTCACTCGGCTGCTACGGTGAAGTTTAATGAGCCTCTCCCTACGGCTTGGAAGATCAACGTGGAAGGGACCAGGATGATGCTGGCTTTGAGTAGAAGAATGAAGCGGATTGAGGTTTTCATTCACATATCGACAGCATACACGAACACAAACAGGGAAGTGGTTGACGAGATCCTGTATCCAGCCCCAGCTGACATCGACCAAGTTTATCAGTATGTCAAGGAGGGAATTTCTGAGGAAGACACTGAGAAAATACTGAATGGTCGTCCGAATACATACACGTTCACGAAAGCGCTGACTGAGCATTTGGTTGCTGAGAACCAAGCCTACGTACCAACTATTATCGTGAGGCCGTCTGTCGTGGCAGCAATAAAAGATGAGCCGTTAAAAGGTTGGTTAGGCAACTGGTTTGGAGCGACCGGTCTCACAGTGTTCACCGCTAAGGGTCTCAACCGAGTCATCTACGGTCATTCTAATTACATCGTAGACCTGATTCCAGTGGATTATGTGGCTAATCTGGTGATTGCTGCTGGGGCTAAGAGTAACACATCAAGTGAGTTGAAGGTGTATAACTGCTGCAGCAGTTCCTGCAATCCCGTCAAAATTGGCACGCTGATGAGCATGTTTGCTGACGATGCCATCAAACAGAAGTCTTATGCCATGCCGCTACCGGGGTGGTACATATTCACGAAGTACAAGTGGCTGGTGCTACTTCTGACGTTTCTCTTCCAAGTTATACCGGCGTATATCACGGATCTCTCCAGGCACTTGGTTGGGAAGAGTCCACGGTATATAAAACTCCAATCACTGGTGAATCAAACGCGTTCTTCAATCGACTTCTTCACGAATCACTCCTGGGTGATGAAGGCAGACAGAGTGAGAGAGCTGTATGCGTCTCTTTCTCCCGCAGACAAGTACTTGTTCCCCTGCGATCCCGTCAACATTAACTGGACACAATACTTACAAGATTACTGTTGGGGCGTCCGAAATTTTTTGGAGAAAAAAACGTAAAAAATATATAAAATWWATTATAATATTTTTTTGTTTGTTTTTAAGGACTGTATAGTATGGAGGTTGAGCAATGGCTAATTTTTGTAATAAAAGTATTATTTTGATGAAAATATTAATTGTTAAGTAATGATAAGACATATCTAGGTGTTTCCATGTTTTGCCGTAGTTGGGTAGAAATGTTAAATGCGGATAGTAGTAGGTTAATTAGAATGTAGTWATATATAAAAAAATCTTATCACATAATTTATTTATGGATTCCATTAACTTTATTTAATWAATTTTTCSAACAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Heliothis | virescens | 7102 | cds | GenBank | EZ407233 | Heliothis virescens pheromone gland RNAi construct | TTAGGCAACTGGTTTGGAGCGACCGGTCTCACAGTGTTCACCGCTAAGGGTCTCAACCGAGTCATCTACGGTCATTCTAATTACATCGTAGACCTGATTCCAGTGGATTATGTGGCTAATCTGGTGATTGCTGCTGGGGCTAAGAGTAACACATCAAGTGAGTTGAAGGTGTATAACTGCTGCAGCAGTTCCTGCAATCCCGTCAAAATTGGCACGCTGATGAGCATGTTTGCTGACGATGCCATCAAACAGAAGTCTTATGCCATGCCGCTACCGGGGTGGTACATATTCACGAAGTACAAGTGGCTGGTGCTACTTCTGACGTTTCTCTTCCAAGTTATACCGGCGTATATCACGGATCTCTCCAGGCACTTGGTTGGGAAGAGTCCACGGTA | dsRNA | MEGAscriptR RNAi kit (Ambion Inc.) | column-based | simultaneously with transcription | Construction of dsRNA One forward PCR primers and two reverse primers (see in Sequence, point 3) were used to synthesize a region of the fatty acid reductase gene that contained a T7 promoter region (TAA TAC GAC TCA CTA TAG GG) on both the sense and antisense strands, followed by sequences specific for the targeted genes. The PCR reaction was done under the following cycling conditions: 94_C for 2 min followed by 5 cycles of 94_C for 30 s, 65_C for 30 s, 72_C for 1 min, 35 cycles of 94_C for 30 s, 77_C for 30 s, 72_C for 1 min a final extension of 72_C for 3 min. The PCR products were purified with the MiniEluteR PCR Purification Kit (QIAGEN) and were then used as templates to synthesize dsRNA. The dsRNA was produced using the MEGAscriptR RNAi kit (Ambion Inc., Austin, TX) according to the manufactures recommendations to obtain the corresponding long dsRNA without an additional annealing step. The in vitro transcription products were assessed for integrity on a 1% agarose gel, diluted in 6xOrange Loading Dye Solution (Fermentas) and the concentration was calculated by measuring its absorbance at 260nm. | InsectaCentral | Heliothis virescens pheromone gland RNAi construct | Lepidoptera | Noctuidae | Heliothis | virescens | 7102 | local resource | adult | unknown | unknown | lab_colony | 1 | injection | Grace's Medium | 5 | buffer | 25 | in vitro tissue culture of pheromone gland (1 mg), added to make total volume 50 ul medium | RNAi tests in vitro Sex pheromone glands were excised from 1-7 day-old virgin females and cultured singly in a micro well of a 96-microwell plate (MICROTESTTM Tissue Culture Plate 96 well Flat Bottom with Low Evaporation Lid), each well with 50 µl culture medium. All tissue cultures were kept in an incubator at 27° C. Graceâs insect medium supplemented with 5% antibiotics and JH (50µg/ml) was selected as tissue medium in all assays. Contamination of the tissue culture medium was determined on an agar plate with LB-medium (lysogeny broth medium). The dsRNA of FAR1 (EZ407233) as described above was added directly into the tissue culture medium (1-5 µl /well). To amplify the possible effect of RNAi in vitro, we tested lipofectamine in combination with dsRNA in tissue culture of the pheromone glands. This reagent is used to introduce a transfection of dsRNA into cells of tissue culture. | adult | phenotype | in vitro cultured adult female sex pheromone gland | not checked | 3 | none | |||
| 162 | Cadherin-RNAi | RNAi | A truncation mutation in cadherin causes BT resistance in Heliothis virescens. The orthologous cadherin was cloned into pGEM-T-easy vector (Promega) and double stranded RNA was synthesis for injection into the haemolymph. 1ug of RNA was injected into third instar larvae and the RNAi result was followed by growth rate and real time RT-PCR. Controls were injected with water. Larvae were fed on diet impregnated with sub-lethal concentrations of BT (0.2, 1 and 2 ug/mL of diet) or normal diet, and the increase in weigh was determined 7 days post injection. Midguts were dissected out from larvae post injection (12, 24 and 48 hours) and real time RT-PCR was done to measure the endogenous cadherin mRNA level. Larvae receiving cadherin dsRNA has a higher increase in weigh compared to control larvae on BT diet. However, real time RT-PCR showed that there is an elevated level of cadherin mRNA in larvae that received the dsRNA. This is not due to contamination from the injected RNA as the real time RT-PCR primers were designed upstream of the region used to generate dsRNA. | InsectaCentral | Unpublished | Wee | Choon Wei | Cadherin-RNAi target | ATGGCAGTCGACGTGAGAATATTCACGGCAGCGGTTTTTATACTCGCTGCTCACTTCACTTTCGCACAAGATTGTAGCTACATGGTAGCAATACCCAGACCAGAGCGACCAGATTTTCCAAGTCAAAATTTCGATGGAATACCATGGAGTCAGTATCCCTTGATACCAGTGGAGGGTAGAGAAGACGTGTGTATGAACGAGTTCGAGCCAGGTAACCAAAACCCTGTTACCGTCATCTTCATGGAAGAGGAGATCGAAGGGGATGTGGCCATCGCACGGCTCAACTATCGAGGTACCAATACTCCGATCATTGTATCTCCATTTAGCTTTGGTACTTTTAACATGTTGGGGCCGGTCATACGTAGAATACCTGAGAACGGTGGTGACTGGCATCTCGTCATTACACAGAGACAGGACTACGAGACACCGGGCATGCAGCAGTACATATTCGACGTGAGGGTAGACGATGAGCCGCTAGTGGCCACGGTCATGCTGCTCATCGTCAACATTGATGACAACGATCCTATCATACAGATGTTCGAGCCTTGTGATATTCCTGAACGCGGTGAAACAGGCATTACATCATGCAAGTATACAGTGAGCGATGCTGACGGTGAGATCAGTACTCGCTTCATGAGGTTTGAAATTTCAAGCGATCGAGACGATGACGAATATTTCGAACTCGTTAGAGAAAATATTCAAGGACAGTGGATGTATGTTCATATGAGAGTTCACGTCAAAAAACCTCTTGATTATGAGGAAAACCCGCTGCATTTGTTTAGAGTTACAGCTTATGATTCCCTACCAAACACACATACAGTAACAATGATGGTGCAAGTAGAGAACGTTGAGAACAGACCGCCGCGATGGGTGGAGATATTTGCTGTCCAGCAGTTCGATGAGAAGACGGAGCGGTCCTTCAGGGTTCGAGCCATCGATGGTGATACAGGAATCGATAAACCTATCTTCTATAGGATCGAAACTGAAAAAGGAGAGGAAGACTTGTTCAGCATTCAAACGATAGAAGGTGGTCGAGAAGGTGCTTGGTTTAACGTCGCTCCAATAGACAGGGACACTCTTGAGAAGGAAGTTTTCCACGTGTCCATAATAGCGTACAAATATGGCGATAATGACGTGGAAGGCAGTTCGTCGTTCCAGTCGAAAGCCGATGTGGTCATCATCGTGAACGATGTCAATGATCAGGCTCCGTTGCCTTTCCGGGAAGAGTACTCCATTGAAATTATGGAGGAAACTGCGATGACACTGAACTTAGAAGACTTTGGGTTCCACGATAGAGATCTTGGTCCTCACGCCCAATACACAGTGCACCTAGAAAGCATCCATCCCCCCCGAGCTCACGAGGCGTTCTACATAGCGCCGGAGGTTGGCTACCAGCGCCAGTCCTTCATCATGGGCACGCAGAACCATCACATGCTGGACTTCGAAGTACCAGAGTTCCAGAATATACAGCTAAGGGCCATAGCGATAGACATGGACGATCCCAAATGGGTTGGTATTGCGATAATCAACATTAAACTGATCAACTGGAACGATGAGCTGCCGATGTTCGAGAGTGACGTGCAAACTGTCAGCTTCGACGAGACAGAGGGCGCTGGCTTCTATGTGGCTACTGTTGTGGCGAAGGACCGGGATGTTGGTGATAAAGTCGAACACTCTCTAATGGGTAACGCAGTAAGCTACTTGAGGATCGACAAGGAAACCGGCAAGATCTTCGTCACAGAAAACGAAGCTTTCAACTATCACAGACAGAACGAGCTTTTTGTGCAGATACGAGCTGATGACACGCTGGGCGAGCCATACAACACCAACACCACCCAGTTGGTGATCAAGCTTCGGGACATTAACAACACCCCTCCCACGCTCAGACTGCCTCGCTCCACTCCATCAGTGGAAGAGAACGTACCCGACGGTTTCGTGATCCCCACGCAGCTGAACGCCACGGACCCCGACACTACAGCCGAGCTGCGCTTCGAGATCGACTGGGAGAACTCCTATGCCACCAAGCAGGGACGGAATACTGACTCTAAGGAATATATCGGTTGTATAGAAATCGAGACGATATACCCGAATATAAACCAGCGCGGCAACGCCATCGGCCGCGTGGTGGTGCGAGAGATCCGGGACGGCGTCACCATAGACTATGAGATGTTTGAGGTTCTATACCTCACCGTCATTGTAAGGGATCTCAACACCGTTATTGGGGAAGACCATGATATATCCACATTCACGATCACGATAATAGACATGAACGACAACCCTCCCCTGTGGGTGGAAGGCACCCTGACGCAAGAGTTCCGCGTGAGAGAGGTCGCCGCTTCAGGAGTGGTTATAGAATCCGTACTGGCTACTGATATCGATGGACCGCTGTATAACCAAGTGCGGTATACTATCACTCCCAGACTCGATACTCCAGAAGACCTAGTGGACATAGACTTCAACACGGGTCAGATCTCCGTGAAGTTACACCAGGCCATCGACGCGGACGAGCCGCCGCGACAGAACTTGTATTATACCGTCATAGCTAGTGACAAGTGTGACCTCCTCACTGTTACTGAGTGTCCTCCTGACCCTACTTACTTTGAGACGCCGGGAGAGATCACGATCCACATAACGGACACGAACAACAAGGTGCCTCAAGTGGAAGACGACAAGTTCGAGGCGACGGTGTACATCTACGAGGGCGCGGACGACGGAGAACATGTCGTGCAGATCTACGCCAGCGATCTCGATAGAGATGAAATCTACCACAAAGTGAGCTACCAGATCAACTACGCGATCAACTCTCGTCTCCGTGACTTCTTCGAGATGGACCTAGAGACAGGCCTCGTGTACGTCAACAACACCGCCGGCGAGCTGCTGGACAGGGACGGCGACGAGCCCACACACCGCATCTTCTTCAATGTCATCGATAACTTCTATGGAGAAGGAGACGGTAACCGCAATCAGAACGAGACACAAGTGTTGGTAGTGTTACTGGACATCAACGACAACTATCCGGAACTGCCTGAAACAATCCCATGGGCTATCTCTGAGAGCTTAGAGCAGGGTGAGCGAGTACAGCCAGAAATCTTCGCCCGGGACCGCGACGAACCAGGAACAGACAACTCCCGCGTCGCCTACGCCATCACCGGCCTCGCCAGTACCGACCGGGACATACAAGTGCCTGATCTCTTCAACATGATCACCATAGAGAGGGACGGGGGTATTGATCAAACTGGAATACTTGAGGCAGCTATGGATTTGAGAGGCTATTGGGGCACTTATGAAATTGATATTCAGGCGTACGACCACGGAATACCTCAAAGGATTTCAAATCAGAAGTACCCGCTGGTCATCAGACCTTACAACTTCCACGACCCAGTGTTCGTGTTCCCACAACCTGGATCCACCATCAGACTGGCGAAGGAGCGAGCAGTAGTCAACGGTATACTCGCTACAGTGGACGGAGAATTTCTGGACAGAATCGTTGCTACCGACGAAGATGGTTTAGAAGCAGGACTCGTTACATTCTCTATCGCGGGAGATGATGAAGGTTCTCAGTTCTTCGACGTGTTGAACGACGGAGTGAACTCAGGCTCACTCACCCTCACGCGGCTCTTCCCAGAAGACTTCCGAGAGTTTCAGGTGACGATTCGTGCTACGGACGGTGGAACTGAGCCTGGTCCAAGGAGTACGGACTGCTTGGTGACCGTAGTGTTTGTTCCCACCCAGGGAGAACCCGTGTTCGAGATTAGTACCTACACGGTCGCTTTTGTTGAAAAAGATGAGGGTATGGAGGAGAGGGCGGAACTGCCTCGCGCCTCAGACCCGAGGAACATCATGTGTGAAGATGATTGTCACGACACCTATTACAGCATTGTAGGGGGCAATCCGGGAGAACACTTCAGAGTAGACCCTCGTACCAACGTGCTGACCCTCGTGAGGCCGCTGGACCGCTCCGAACAGGAGACACACACCCTCATCATAGGAGCCAGCGACACTCCCAACCCGGCCGCCGTCCTGCAGGCTTCTACACTCACCGTCACTGTTAACGTTCGAGAAGCGAACCCGCGACCAGTGTTCCAGAGAGCACTCTACACAGCTGGCATCTCTGCTGGCGATTTCATCGAAAGAAATCTGCTGACTGTAGTAGCGACACATTCAGAAGGTCTGCCCATCACTTACACTCTAATACAAGAGTCTATGGAAGCAGACCCCACACTCGAAGCTGTTCAGGAGTCAGCCTTCATCCTCAACCCTGAGACTGGAGTCCTGTCCCTCAACTTCCAGCCAACCGCCGCCATGCACGGCATGTTTGAGTTCGAAGTCGAAGCCACTGATTCAAGGAGAGAAACTGCCCGCACGGAAGTGAAGGTGTATCTGATATCGGACCGCAACCGAGTGTTCTTCACGTTCAATAACCCGCTGCCTGAAGTCACTCCCCAGGAAGATTTCATAGCGGAGACGTTCACGGCATTCTTCGGCATGACGTGCAACATCGACCAGACGTGGTGGGCCAGCGACCCCGTCACCGGCGCCACCAGGGACGACCAGACTGAAGTCAGGGCTCATTTGATCAGGGACGACTTACCCGTGCCTGCTGAGGAGATTGAACAGTTGCGCGGTAACCCAACCCTAGTGAACAGTATCCAACGAGCCCTAGAAGAACAGAACTTACAGCTGGCCGACCTGTTCACCGGCGAGACGCCCATCCTGGGAGGAGATGCTCAGGCGCGAGCTCTGTACGCGCTGGCGGCGGTAGCTGCGGCACTCGCGCTGATCGTCGTTGTGCTGCTTATTGTGTTCTTTGTTAGGACTAGGACACTGAACCGGCGCTTGCAAGCTCTGTCCATGACCAAGTACAGTTCGCAAGACTCGGGGCTGAACCGCGTGGGTCTGGCGGCTCCGGGCACCAACAAGCACGCTGTCGAGGGCTCCAACCCCATCTGGAACGAAACGTTGAAGGCTCCGGACTTTGATGCTCTAAGTGAGCAGTCATACGACTCAGACCTGATCGGCATCGAAGACTTGCCGCAGTTCAGGAACGACTACTTCCCGCCTGAAGAGGGCAGCTCCATGCGAGGAGTCGTCAATGAACATGTGCCTGAATCAATAGCGAACCATAACAACAACTTCGGGTTTAACTCTACTCCCTTCAGCCCAGAGTTCGCGAACACACAGTTCAGAAGATAA | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | InsectaCentral | Cadherin-RNAi target | Cadherin-RNAi RNAi construct | TTCAACATGATCACCATAGAGAGGGACGGGGGTATTGATCAAACTGGAATACTTGAGGCAGCTATGGATTTGAGAGGCTATTGGGGCACTTATGAAATTGATATTCAGGCGTACGACCACGGAATACCTCAAAGGATTTCAAATCAGAAGTACCCGCTGGTCATCAGACCTTACAACTTCCACGACCCAGTGTTCGTGTTCCCACAACCTGGATCCACCATCAGACTGGCGAAGGAGCGAGCAGTAGTCAACGGTATACTCGCTACAGTGGACGGAGAATTTCTGGACAGAATCGTTGCTACCGACGAAGATGGTTTAGAAGCAGGACTCGTTACATTCTCTATCGCGGGAGATGATGAAGGTTCTCAGTTCTTCGACGTGTTGAACGACGGAGTGAACTCAGGCTCACTCACCCTCACGCGGCTCTTCCCAGAAGACTTCCGAGAGTTTCAGGTGACGATTCGTGCTACGGACGGTGGAACTGAGCCTGGTCCAAGGAGTACGGACTGCTTGGTGACCGTAGTGTTTGTTCCCACCCAGGGAGAACCCGTGTTCGAGATTAGTACCTACACGGTCGCTTTTGTTGAAAAAGATGAGGGTATGGAGGAGAGGGCGGAACTGCCTCGCGCCTCAGACCCGAGGAACATCATGTGTGAAGATGATTGTCACGACACCTATTACAGCATTGTAGGGGG | dsRNA | column-based | subsequent to transcription | 695 bps of the cadherin gene (CDS) was cloned into pGEM-T-Easy Vector (Promega). Sense and antisense RNA of the gene were generated using T7 and SP6 DNA dependent RNA polymerases. The in vitro transcribed RNA samples were checked on agarose gel and 260/280nm ratio. RNA samples were purified using Qiagen column and equal amount of RNA were annealed to form dsRNA. Concentration was adjusted to 1ug/uL. | InsectaCentral | Cadherin-RNAi RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | lab_colony | 1 | injection | water | 1 | larva | qPCR phenotype | midgut | not checked | none | |||||||||||
| 166 | membrane-bound trehalase | RNAi | Background: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1) and membrane-bound (Tre-2) trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. Principal Findings: The membrane-bound trehalase of Spodoptera exigua (SeTre-2) was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1) and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi) of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA) and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB) expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. Conclusions: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2 has an important role in CHSB expression and chitin synthesis in the midgut. | pubmed | 20405036 | Zhang | Wenqing | membrane-bound trehalase target | ATGTATAAGAAAATGTGGTGTGTTTTTATTGCGATACTTGGTGTGGCTGCGGGGATGGACAGAAGCCATTTACCGCCAACCTGCTCTAGCAATATCTACTGTCATGGCCCTCTTCTGGACACGGTGCAGATGGCGGGGCTGTACAATGACTCCAAGACCTTCGTTGACATGAAACTCAAGCTCTCCGCTAACATCACCATGGACCATTTCCACGAAATGATGGCCAGGACAGGTTCACATCCGACCAAAGCTGACATCCAGGAGTTCGTCAATCAGAACTTTGACCCCGAAGGATCTGAGTTCGAGGACTGGAGACCTACAGACTGGAAGGATAATCCTGCGTTTCTCCAAAATATCAAGGATCCACTGCTGCACGAATGGGCAGCCGACCTGAACCGCTTGTGGCTGCAGTTGGGCAGAAAGATGAAGCCAGATGTGAAGGAGAACCAGGATCTGTACTCCATAATATACGTGGACAACCCTGTGATTGTGCCTGGTGGTCGTTTCCGTGAATTCTATTACTGGGACTCCTACTGGATAATCAAAGGTTTACTGCTGTCTGAGATGAGGTCCACTGCCAGGGGCATGGTCTCCAACTTCATGGATATCGTGGAAAGGATCGGCTTCATACCTAACGGGGGCAGGATATACTATGCTATGAGATCTCAACCTCCCCTCCTAATTCCGATGGTGAAGCTAATATTGGATGACATGGATGACATCGAGTTCCTTAGACAACACATCCATACTTTGGATAGAGAGTATGATTACTGGATGACGAACCATACTGTTGAAGTCCACCATAACGGCCACAGATACACTCTGGCGAGGTACTTCGATCAATCACAAGGACCCAGACCTGAGAGTTACAAAGAAGACGTTGATGTTGCCAGACATTTCGACACGAATGACAAGAAAGAAGAATTATACGCTGAGTTGAAAGCTGCGGCTGAATCCGGGTGGGACTTCTCATCAAGGTGGTTCATTCTTAATGGCACCAACAAAGGTAACCTAACGAACCTGAAAACGCGGTCTATCATCCCAGTAGACCTGAATGCCATCATGTGTTGGAACGCCCAGCTTCTCCGAGATTTCCATACCAGACTCGGAAATGTCGATAAGGCAGAATATTACAGAAACGTGCACGCCAGATTCATGGATGCTATTGAACAGGTGCTCTGGCACGAAGATGTGGGTGTCTGGCTGGACTACAGCCTGGAGTCTGGCAGGCGTCGGGATTACTTCTATCCATCCAACGTGTCGCCGCTTTGGGCAGTTTGCTACGACCAGGCGAGGAAGGATTACTACGTGAACCGCGTCGTCAATTATTTGGATAAAGTTAAAGTAGACATATTCGACGGAGGCATACCAACGACTTTTGAGCACTCAGGAGAGCAATGGGACTATCCGAATGCCTGGCCACCTCTACAGTACATTGTGGTTATGGGTCTGGCGAACACTGGCCAGCCAGAAGCCGTGCGTCTGGCCAGCGAGATCGCCACTAAATGGGTCAGGTCGAATTTCGAAGTTTGGAAACAGAAGACTGCTATGCTTGAAAAGTACGACGCGACAATTTTCGGTGGTCTCGGCGGTGGTGGCGAGTATGTCGTACAAACTGGCTTCGGATGGACCAATGGCGTTATAATGGCTATGTTAAACCGATGGGGAGATACAATGACTTCAGCGGACGCGTTCGGTACGGGCGCCGCCGCCGACTCCGGGGCCGTGTACGGCGCTCACGTCGGCGCCAGCGGGGTCGCCACGGCCATTCTTGTTGTTCTGGCTTCCTTGGCAGCTGGGACTCTTGGGTTGATGGTGTACAGAAAACGTAAAGATTACATTCGAGTCTCTGGCGGTGAAGATTACAAGCTTCTCTCCCGAAAACCCTACACGGAACTGAGAAGCCTCAACGGGGCATCGAACCCACGTCAACGATGA | Lepidoptera | Noctuidae | Laphygma | exigua | cds | GenBank | membrane-bound trehalase target | membrane-bound trehalase RNAi construct | GCCAGGACAGGTTCACATCCGACCAAAGCTGACATCCAGGAGTTCGTCAATCAGAATTTCGACCCCGAAGGATCTGAGTTCGAGGACTGGAGACCTACAGACTGGAAGGATAATCCTGCGTTTCTCCAAAACATCAAGGATCCACTGCTGCACGAATGGGCAGCCGACCTGAACCGCTTGTGGCTGCAGTTGGGCAGAAAGATGAAGCCAGATGTGAAGGAGAACCAGGATCTGTACTCCATCATATACGTGGACAACCCTGTGATTGTGCCTGGTGGTCGTTTCCGTGAATTCTATTACTGGGACTCCTACTGGATAATCAAAGGTTTACTGCTGTCTGAGATGAGGTCCACTGCCAGGGGCATGGTCTCCAACTTCATGGATATCGTGGAAAGGATCGGCTTCATACCTAACGGGGGCAGGATATACTATGCTATGAGATCTCAACCTCCCCTCCTAATTCCGATGGTGAAGC | dsRNA | T7 RiboMAXTM Express RNAi System Kit (Promega) | ethanol precipitation | subsequent to transcription | according to the manufacturerâs instructions | InsectaCentral | membrane-bound trehalase RNAi construct | Lepidoptera | Noctuidae | Laphygma | exigua | local resource | larva | lab_colony | 1 | injection | DEPC | 0.5 | buffer non_specific_dsRNA | 3 | hemolymph | larva | qPCR RT-PCR (semi-quantitative) W.blot phenotype enzymatic_activity | not checked | high | ||||||||||
| 167 | Helicoverpa armigera acetylcholinesterase | RNAi | RNA interference is an effective means of regulation of gene expression both in vitro and in vivo. We studied the effect of siRNA on larval development by selective targeting of the acetylcholinesterase (AChE) gene of Helicoverpa armigera. Chemically synthesized siRNA molecules were directly fed to H. armigera larvae along with the artificial diet. The siRNA treatment resulted in specific gene silencing of AChE and consequently brought about mortality, growth inhibition of larvae, reduction in the pupal weight, malformation and drastically reduced fecundity as compared to control larvae. Our studies suggest some novel roles for AChE in growth and development of insect larvae and demonstrate that siRNA can be readily taken up by insect larvae with their diet. | InsectaCentral | Unpublished | Rajam | Manchikatla Venkat | Development of insect resistant transgenic crops using RNAi approach target | CATCTATGCAATATAGGGTTGGGGCGTTCGGATTTTTATACTTGAATAAATATTTCTCGCCGGGCAGTGAAGAGGCGCCGGGAAATATGGGCTTATGGGATCAACAACTCGCTATTCGTTGGATTAAAGATAATGCTCGTGCTTTTGGTGGTGACCCAGAATTGATAACTTTGTTTGGAGAGTCGGCAGGCGGGGGAAGCGTGAGTTTGCATATGCTTTCACCAGAGATGAAGGGATTATTCAAAAGAGGCATCTTGCAATCTGGAACGTTAAACGCTCCATGGAGTTGGATGACAGGAGAGAGAGCACAAGACATAGGAAAAGTGTTAGTAGATGACTGTAATTGCAATAGCAGCCTGCTAGCTGCCGATCCGAGCTTAGTGATGGACTGTATGCGCGGAGTTGATGCAAAAACTATATCCGTACAGCAGTGGAATTCCTACACTGGCATATTGGGATTCCCGTCAGCTCCCACGGTTGACGGCGTGTTTTTGCCCAAAGACCCGGACACAATGATGAAAGAAGGCAATTTCCATAATACCGAGGTGCTTCTTGGAAGTAATCAAGACGAAGGAACCTATTTCTTACTATACGATTTCCTTGACTACTTCGAGAAAGACGGCCCCAGCTTCTTGCAACGCGAGAAATTTCTAGAAATTGTCGACACTATTTTCAAGGATTTCTCCAAAATAAAGAGGGAAGCTATCGTATTCCAATATACGGACTGGGAGGAAATTACCGATGGCTATCTGAACCAGAAAATGATA | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | cds | ncbi genbank | gi:51173171 | Helicoverpa armigera acetylcholinesterase RNAi construct | GCTATCGTATTCCAATAUATT | siRNA | Microsynth Inc., Switzerland | other | subsequent to transcription | custom synthesized | InsectaCentral | Helicoverpa armigera acetylcholinesterase RNAi construct | Lepidoptera | Noctuidae | Helicoverpa | armigera | 52318 | local resource | larva | virus | lab_colony | 0.05 | feeding | 0.05 | non_specific_dsRNA | Gut | larva | RT-PCR (semi-quantitative) phenotype enzymatic_activity | Whole organism | not checked | 72 | high | 80 | ||||||
| 168 | BLLP | RNAi | Study on immune proteins in domesticated and wild silkmoths Bombyx mori and Antheraea mylitta, respectively, led to identification of a new class of antimicrobial proteins. We designated them as lysozyme-like proteins (LLPs) owing to their partial similarity with lysozymes. However, lack of characteristic catalytic amino acid residues essential for muramidase activity in LLPs puts them functionally apart from classical lysozymes. Two LLPs, one from B. mori (BLLP1) and the other from A. mylitta (ALLP1) expressed in a recombinant system, exhibited a broad-spectrum antibacterial action. Further investigation of the antibacterial mechanism revealed that BLLP1 is bacteriostatic rather than bactericidal against Escherichia coli and Micrococcus luteus. Substantial increase in hemolymph bacterial load was observed in B. mori upon RNA interference mediated in vivo knockdown of BLLP1. We demonstrate that the antibacterial mechanism of this protein depends on peptidoglycan binding unlike peptidoglycan hydrolysis or membrane permeabilization as observed with lysozymes and most other antimicrobial peptides. To our knowledge, this is the first report on functional analysis of novel, noncatalytic lysozyme-like family of antibacterial proteins that are quite apart functionally from classical lysozymes. The present analysis holds promise for functional annotation of similar proteins from other organisms. | pubmed | 17982085 | Javaregowda | Nagaraju | silkmoth-1 target | ATGGTCCAATCACCACATCTCGGTCTACTGTTTCTGGCCGTGACCCTAGTCCACAGCTCCGAAGGGCACGCGAAGGTCTTCACGAGATGCCAACTATCCAGAGAGCTTTTAAGATACAACTTTCCAAGAGCTTTGATCCCGACTTGGGTATGCCTCATAGAGCATATGATCAGCAGAACAACCGAGAAGATTACGAACCACAACAACTCCTACTCTAGCTACGGATTGTTCCAGATTAACAACAAAGATTGGTGCAAGAAAGGCAGGAAGGGCGGAAATTGCAACATGAAATGTGAAGATCTCCTGAACGAAGACCTGGCCGATGACGTTAGATGCGCAAAACGGGTATACGATAGAATCGGATTCAAGGCCTGGCCGTCTTCTTACTCCTACTGCAAGCAGAAGAACCTACCAGACATCTCCAGATGCTGA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | silkmoth-1 target | silkmoth-1 RNAi construct | ATGGTCCAATCACCACATCTCGGTCTACTGTTTCTGGCCGTGACCCTAGTCCACAGCTCCGAAGGGCACGCGAAGGTCTTCACGAGATGCCAACTATCCAGAGAGCTTTTAAGATACAACTTTCCAAGAGCTTTGATCCCGACTTGGGTATGCCTCATAGAGCATATGATCAGCAGAACAACCGAGAAGATTACGAACCACAACAACTCCTACTCTAGCTACGGATTGTTCCAGATTAACAACAAAGATTGGTGCAAGAAAGGCAGGAAGGGCGGAAATTGCAACATGAAATGTGAAGATCTCCTGAACGAAGACCTGGCCGATGACGTTAGATGCGCAAAACGGGTATACGATAGAATCGGATTCAAGGCCTGGCCGTCTTCTTACTCCTACTGCAAGCAGAAGAACCTACCAGACATCTCCAGATGCTGA | dsRNA | Megascript Kit (Ambion) | isopropanol precipitation | subsequent to transcription | BLLP1 cDNA (357 bp) was cloned into pCRII-TOPO vector (Invitrogen) and amplified with M13 forward and reverse primers. This template with flanking T7 and SP6 promoters was utilized for in vitro transcription reaction and sense and antisense RNA strands were generated with the T7 and SP6 Megascript kits (Ambion). The DNA template was removed from the transcripts by DNAse treatment and the RNA products were subsequently purified by Trizol extraction (Invitrogen) followed by isopropanol precipitation. The complementary single stranded RNAs were dissolved in DEPC treated water, combined in equimolar amounts in insect buffer saline (IBS, composition: NaClâ160mM, KClâ10mM, CaCl2â4mM) and annealed by heating to 95 1C and slow cooling overnight at room temperature. The dsRNA formation was confirmed by agarose gel electrophoresis and the concentration was determined spectrophotometrically. | NCBI | NM 001099823.1 | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | 10 | injection | Insect buffer saline | 10 | non_specific_dsRNA | 5 | hemolymph | For control, dsRNA specific to green fluorescent protein (GFP) was cloned into pCRII-TOPO vector(Invitrogen) and amplified with M13 forward and reverse primers. This template with flanking T7 and SP6 promoters was utilized for in vitro transcription reaction and sense and antisense RNA strands were generated with the T7 and SP6 Megascript kits (Ambion). The DNA template was removed from the transcripts by DNAse treatment and the RNA products were subsequently purified by Trizol extraction (Invitrogen) followed by isopropanol precipitation. The complementary single stranded RNAs were dissolved in DEPC treated water, combined in equimolar amounts in 1X insect buffer saline (IBS, composition: NaClâ160mM, KClâ10mM, CaCl2â4mM) and annealed by heating to 95 1C and slow cooling overnight at room temperature. The dsRNA formation was confirmed by agarose gel electrophoresis and the concentration was determined spectrophotometrically. | larva | RT-PCR (semi-quantitative) | hemolymph | not checked | 18 | high | |||||
| 169 | gloverin | RNAi | Gene duplication is a characteristic feature of eukaryotic genomes. Here we investigated the role of gene duplication in the evolution of the gloverin family of antibacterial genes (Bmglv1, Bmglv2, Bmglv3, and Bmglv4) in Bombyx mori. We observed the following two significant changes during the first duplication event: (i) loss of intronV, located in the 3-untranslated region (UTR) of the ancestral gene Bmglv1, and (ii) 12-bp deletion in exon3. We show that loss of intronV during Bmglv1 to Bmglv2 duplication was associated with embryonic expression of Bmglv2. Gel mobility shift, chromatin immunoprecipitation, and immunodepletion assays identified chorion factor 2, a zinc finger protein, as the repressor molecule that bound to a 10-bp regulatory motif in intronV of Bmglv1 and repressed its transcription. gloverin paralogs that lacked intronV were independent of chorion factor 2 regulation and expressed in embryo. These results suggest that change in cis-regulation because of intron loss resulted in embryonic expression of Bmglv2-4, a gain of function over Bmglv1. Studies on the significance of intron loss have focused on introns present within the coding sequences for their potential effect on the open reading frame, whereas introns present in the UTRs of the genes were not given due attention. This study emphasizes the regulatory function of the 3'-UTR intron. In addition, we also studied the genomic loss and show that âin-frameâ deletion of 12 nucleotides led to loss of amino acids IHDF resulting in the generation of a prepro-processing site in BmGlv2. As a result, the N-terminal pro-part of BmGlv2, but not of BmGlv1, gets processed in an infection-dependent manner suggesting that prepro-processing is an evolved feature in Gloverins. | pubmed | 18524767 | Javaregowda | Nagaraju | gloverin target | ACATTCTGTCTCGAGCAGCGAAACCTGTCACAATTCAAAATGTATTCCAAGGTGTTGTTATCCGCTGCACTCCTTGTATGCGTGAACGCTCAAGTTTCTATGCCTCCTGGTTACGCAGAGAAGTATCCGATCACCAGCCAATTTTCAAGGTCAGTCCGACACCCTCGCGATATTCACGACTTTGTCACTTGGGACAGGGAAATGGGGGGAGGGAAGGTCTTCGGGACTTTGGGAGAGAGCGACCAAGGACTTTTTGGTAAAGGTGGTTACAACAGGGAGTTCTTCAATGATGACCGCGGCAAACTGACCGGACAGGCTTACGGCACCAGAGTATTAGGACCTGGAGGCGACAGTACCAGTTACGGTGGTCGTCTAGACTGGGCCAATGAGAACGCCAAGGCTGCTATTGACTTGAACAGGCAAATTGGTGGCAGCGCTGGGATCGAAGCATCAGCTTCCGGCGTGTGGGATCTTGGTAAGAATACTCACTTGTCAGCTGGCGGAGTGGTCTCTAAGGAGTTCGGTCACAGAAGGCCTGATGTCGGTTTACAGGCCCAGATTACTCACGAGTGGTAATTGCCAACAGCATTATCAAAGCAGCTATATAAGTGCCGCTTAGACAAGCATTCTTGCCTCGTAAGCCTAGAAGTATGAATAAAATTTTGTCTAAAAACATTGTTATAAAAATCGATTGATTTGTAACTATTTATCTGCGTTCTGTAATTTCCTCTTTAAATCACCAACATTCATCTAACATTTTTTAAATATGAATAAAAATAAATTTCTTAATATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | AB190863.1 | gloverin RNAi construct | AGTGCCGCTTAGACAAGCATTCTTGCCTCGTAAGCCTAGAAGTATGAATAAAATTTTGTCTAAAAACATTGTTATAAAAATCGATTGATTTGTAACTATTTATCTGCGTTCTGTAATTTCCTCTTTAAATCACCAACATTCA | dsRNA | isopropanol precipitation | subsequent to transcription | Total RNA was isolated using TRIzol reagent (Invitrogen), dissolved in 50 µl of RNase-free water, and quantified in a spectrophotometer. 1 µg of RNA was used in 20 µl of RT reaction by using SupercriptII (Invitrogen) and oligo(dT) or gene-specific primers. For synthesizing dsRNA Bmglv1 and Bmglv2, PCR products were cloned between BamHI and KpnI sites of pB-SK_ vector, and in vitro transcription was done with linearized template using T3 and T7 RNA polymerase, separately. Single strand RNA was purified after DNase treatment to remove template plasmid and quantified in a spectrophotometer. An equal amount of sense and antisense RNA was used for annealing in annealing buffer by heating at 95 °C for 5 min followed by slow cooling to room temperature. Respective dsRNA was injected on the 1st day of 5th instar larvae. Male and female moths eclosed from dsRNA injected larvae were allowed to mate (15 experiments each for Bmglv1-dsRNA and Bmglv2-dsRNA), and egg hatching was calculated. An equivalent volume of nontarget baculoviral ie1-dsRNA was injected in control larvae. Different concentrations of dsRNA were used for standardization of RNAi, and the data presented here are from larvae injected with 10 µg of dsRNA. | InsectaCentral | gloverin RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | 10 | injection | Insect buffer saline | 10 | non_specific_dsRNA | 15 | hemolymph | An equivalent volume of nontarget baculoviral ie1-dsRNA was injected in control larvae. Different concentrations of dsRNA were used for standardization of RNAi, and the data presented here are from larvae injected with 10 µg of dsRNA. PCR product of contro gene was cloned between BamHI and KpnI sites of pB-SK_ vector, and in vitro transcription was done with linearized template using T3 and T7 RNA polymerase, separately. Single strand RNA was purified after DNase treatment to remove template plasmid and quantified in a spectrophotometer. An equal amount of sense and antisense RNA was used for annealing in annealing buffer by heating at 95 °C for 5 min followed by slow cooling to room temperature. Respective dsRNA was injected on the 1st day of 5th instar larvae. | embryo | RT-PCR (semi-quantitative) phenotype | fat body | not checked | 552 | high | ||||||
| 170 | ie-1 | RNAi | RNA interference (RNAi)-mediated viral inhibition has been used in a few organisms for eliciting viral resistance. In the present study, we report the use of RNAi in preventing baculovirus infection in a lepidopteran. We targeted the baculoviral immediate early-1(ie-1) gene in both a transformed lepidopteran cell line and in the transgenic silkworm Bombyx mori L. Constitutive expression of double-stranded RNA was achieved by piggybac-mediated transformation of Sf9 cell line with a transgene encoding double-stranded ie-1 RNA ( dsie-1). Strong viral repression was seen at early stages of infection but subsequent recovery of viral proliferation was observed. In contrast, the same transgene inserted into the chromosomes of transgenic silkworms induced long-term inhibition of B. mori nucleopolyhedrovirus infection, with nearly 40% protection compared with nontransgenic animals. Protection was efficient at larval stages after oral infection with occlusion bodiesor hemocoel injection of budded viruses. Virus injected pupae also displayed resistance. These results show that heritable RNAi can be used to protect silkworm strains from baculovirus infection. | pubmed | 17894559 | Javaregowda | Nagaraju | ie-1 target | CGATTTGCAGTTCGGGACATAAATGTTTAAATATATCAATGTCTTTGTGATGCGCGCGACATTTTGTAAGTTATTAATAAAATGCACCGACACGTTGCCCGACATTATCATTAAATCCTTGGCGTAGAATTTGTCGGGTCCGTTGTCCGTGTGCGCTAGCATGCCCGTAACGGACCTTGTGCTTTTGGCTTCAAAGGTTTTGCGCACAGACAAAATGTGCCACACTTGCAGCTCTGCTTGTGTGCGCGTTACCACAAATGCCAACGGCGCAGTGTACTTGTTGTATGTAAATAAATCTCGATAAAGGCGCGGCGCGCGAATGCAGCTGATCACGTACGCTCCTCGTGTTCCGTTCAAGGACGGTGTTATCGACCTCAGATTAATGTTTATCGGCCGACTGTTTTCGTATCCGCTCACCAAACGAGTTTTTGCATTAACATTGTATGTCGGCGGATGTTCTGTATCTAATTTGAATAAATAAACGATAACCGCATTGGTTTTAGAGGGCATAATAAAAAAAATATTATTATCGTGTTCGCCATTAGGGCAGTATAAATTGACGTTCATGTTGAATATTGTTTCAGTTGCAAGTTGACATTGGCGGGACACGATCGTGAACAACCAAACGACTATGACGCAAATTAATTTTAACGCGTCGTACACCAGTGCTCCGACGCCGTCCCGAGCGTCGTTCGACAACGGCTATTCAGAGTTTTGTGATAAACAACAGCCCAACGACTATTTGAATTATTATAACAATCCCACGCCGGATGGAGCCGACACGGTAGTATCTGACAGCAGACTGCAGGCAGCTTCAAACTTTTTGGCAAGCGTCAATTCGTTAACTGATGATAACGATATAATGGAATGTTTGCTCAAGACCACTGATAATCTCGGAGAAGCAGTTAGTTCTGCTTATAATGCGGAATCTTTTGAGCTGCCTGTTGCGGAGCAACCATCGCCCAGTTCTGCTTATAATGCGGAATCTTTTGAGCATCCTGTTGGTGTGAACCAACCATCGGCAACTGGAACTAAACGGAAGCTGGACGAATACTTGGACGATTCACAAAGTGTGGTGGGCCAATTTAACAAGAATAAATTGAAGCCTAAATACAAGAAAAGCACAATTCAAAGCTGTGCAACCCTTGAGCAGACAATTAATCACAACACGAACATTTGCACGGTTGCTTCAACTCAAGAAATTACGCATTATTTTACTAATGATTTTGCGCCGTATTTGATGCGTTTCGACGACAACGACTACAATTCCAATAGGTTCTCCGACCATATGTCCGAAACTGGTTATTACATGTTTGTGGTTAAAAAAAGTGAAGTAAAGCCGTTTGAAATTATATTTGCCAAGTACGTGAGCAATGTGGTGTACGAATATACAAACAACTATTACATGGTAGATAATCGCGTGTTTGTGGTAACTTTTGATAAAATTAGATTTATGATCTCGTACAATTTGGTTAAAGAAACCGGCATAGAAATTCCTCATTCTCAAGATGTGTGCAACGACGAGACGGCTGCACAAAATTGTAAAAAATGCCACTTTGTCGATGTGCATCACACGTTTAAAGCTGCTCTGACTTCATATTTTAATTTAGATATGTATTACGCGCAAACCACATTTGTGACTTTGTTACAATCGTTGGGCGAAAGAAAGTGTGGGTTTCTTTTGAGCAAGTTGTACGAAATGTATCAAGATAAAAATTTATTTACTTTGCCTATTATGCTTAGTCGTAAAGAGAGTAATGAAATTGAGACTGCATCTAATAATTTTTTTGTATCGCCGTATGTGAGTCAAATATTAAAGTATTCGGAAAGTATTCGGAAAGTGAAGTTTCCCGACAATCCCCCAAACAAATATGTGGTGGACAATTTAAATTTAATTGTTAACAAAAAAAGTACGCTCACGTACAAATACAGTAGTGTCGCTAATCTTTTGTTTAATAATTATAAATATCATGACAATATTGCGAGTAATAATAACGCTGAAAATTTAAAAAAGGTTAAGAAGGAGGACGGCAGCATGCACATTGTCGAACAGTATTTGACTCAGAATGTGGATAATGTAAAAGGTCACAATTTTATAGTATTGTCTTTCAAAAACGAAGAGCGGTTGACTATAGCTAAGAAAAACGAAGAGTTTTATTGGATTTCTGGCGAGATTAAAGATGTAGACGCTAGTCAAGTAATTCAAAAATATAATAGATTTAAGCATCACATGTTTGTAATCAGTAAAGTGAACCGAAGAGAGAGCACTACATTGCACAATAATTTGTTAAAATTGTTAGCTTTAATATTACAGGGTCTGGTTCCGTTGTCCGACGCTATAACGTTTGCGGAACAAAAACTAAATTGTAAATATAAAAAATTTGAATTTAATTAATTATACATATATTTTGAATTTAATTAATTATACATATATTTTATATTATTTTTGTCTTTTATTATCGACGAGGGGCCGCTGTTGATGTGGGGTGTTGCATAATAACAACAATGGGAGTTGGTGCGCCACCGCTTCCTCCTCCTCCTCCTCCTTTTGTCATGTATCTGTAGATAAAATAAAGTATTAAACCTAAAAACAAGAACGCGCCTATCATCATAATGATGAGGCATTATTTTGTTGCGGATGCTGTCACTACCGTTGGACGATTTGCCGATTAAACCTTCTCTTCCCAGTAACCAATGTAAACGCAAGTCGCCAATTAAATCACTAAACGTGTAAGGTTCGATGCACATGATTGTTTGGCCCGCAGAAGATCGCTAATATCTACGTATTGAGGCGAAGTTGGGTTAGCGGCCGGATTGCTGCCGCGACAAACTGTTTTTTCTGTTTCATAGTTAAATCCTTGGCACATGTTGGTTAGTAGGGGCGAATCGTTAGCCAACAAGGGGTCTCTTGAGCAAATGTTAACATCCGACTGAGCTAGATTGCGGTCTTGACGACAAGTGCGCTGCAATAACAAACAGGCCTCGGCGTTTTCTCCGGCGTTTCTACCTTGCACATAATAACTTCCGCCGGTTCTATTGATGGCGTTGATTATATCTTGTACTAATGTGGCGGCGGTAAACAAAAGATAACCGCCGCCGGCCCAAGAGTATGCCCACTCCTGCTACTTTCAAGGTTCTCATGTGATTATGTAAACGGGGGGGTTTTGCTGCAGTGCATTTTGAACACCTTCGGGCGTGCGCACGTTGGTCTCCGGGAAGTTTTGTCTGACTGCATTGGATCGCGTCTGTTTGGTGTGGTAATGAAAGTCTGGCACGTTGGTCCATGCGCCGCAATTGGCTCAATGAGTTTATTTGAGGGTCTGAAATGCCCTGAAATACTCCGCGTATGTTGGGGACATCGTTGTTACGAGTAATTCTGTTTATGTCTGAAGTGCTCACAAACCGGTTGTTAGATAATTGATAGCCCGGCCCATATCTGTTGTTTCCAAGGTTGCGTACACTGGGCGCGTTGAGCACATTTGTGAAACCGGCGGGAGTGCCTTGTTAAAAGACGCGTATTATCAGCAAGAAAACTGGCCTGATTAGGATACAATTTATTGACTCTGCGAAGATTTGTAAAAAAACTCATTTTAAAGCAAACTTATTTAATAAATATATCACAGTAAAGTTTTGCAAAATTGCCGTCGTCAATACAACACGGCTGCGGCGCCATGTTGGTAAAATCTAATCTTCTCCTTGCTTTAGATTTTGGGCGAAAGGGCGCATTTGTTATGTCAGTCATTTCGACGTCTGCATTATTTGTTGTGTAAGGTACTTCAACGTATGAAGCAACTTTAACATTATTATAATTTTTTTTAAATATCGACCTGCA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | NCBI | X58442 | ie-1 RNAi construct | CCAAACGACTATGACGCAAATTAATTTTAACGCGTCGTACACCAGTGCTCCGACGCCGTCCCGAGCGTCGTTCGACAACGGCTATTCAGAGTTTTGTGATAAACAACAGCCCAACGACTATTTGAATTATTATAACAATCCCACGCCGGATGGAGCCGACACGGTAGTATCTGACAGCAGACTGCAGGCAGCTTCAAACTTTTTGGCAAGCGTCAATTCGTTAACTGATGATAACGATATAATGGAATGTTTGCTCAAGACCACTGATAATCTCGGAGAAGCAGTTAGTTCTGCTTATAATGCGGAATCTTTTGAGCTGCCTGTTGCGGAGCAACCATCGCCCAGTTCTGCTTATAATGCGGAATCTTTTGAGCATCCTGTTGGTGTGAACCAACCATCGGCAACTGGAACTAAACGGAAGCTGGACGAATACTTGGACGATTCACAAAGTGTGGTGGGCCAATTTAACAA | hairpinRNA | ethanol precipitation | simultaneously with transcription | For generation of pPIGA3GFP-FF plasmid, sequential cloning strategy was adopted. The DNA of interest was PCR amplified using following primers and cloned into shuttle or intermediary vectors:ie-1 promoter-Prom F: GCCGGCCGATTTGCAGTTCGGGA,Prom R: TTGTTCACGATCGTGTCCCGCC; ie-1 F: CCAAACGACTATGACGCAAATT and ie-1 R: TTGTTAAATTGGCCCACCAC; A3 cytoplasmic actin poly(A) signal, Poly(A) F: ATCGATAAACGAACGGAATGTT, Poly(A) R: GGCCGAGGCGGCCACAC. A typical PCR reaction consisted of 10 Îĵl final volume with 5pmol each of forward and reverse primers. The PCR amplification was performed in 10 mM TrisâHCl, (pH 8.3 containing 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin and 0.01% Triton X-100), 1 mM dNTPs and 0.5 U of IMMOLASETM DNA Polymerase (Bioline, Luckenwalde, Germany) per reaction. Thermal cycling was carried out in a thermal cycler (PE9700, Applied Biosystems, Foster City, CA, USA) under the following conditions: initial denaturation of 3 min at 94 °C; 35 cycles of 30 s at 94 °C; 30 s at 52 °C (48 °C for poly(A) signal) and 2 min at 72 °C, and final extension of 10 min at 72 °C. The PCR products were quantified on 1.0% agarose gel, purified using Qiagen PCR purification columns, according to the manufacturerâs protocol (Qiagen, Hilden, Germany). For ie-1 promoter and gene fragment, pBMNPVIEG, a kind gift of R. Huybrechts (Katholieke Universiteit Leuven, Belgium), was used as the template, whereas for the polyadenylation signal, pPIGA3-GFP was used as a template. The vector pPIGA3GFP-FF was confirmed by restriction digestion and DNA sequencing. For DNA sequencing, 250 ng of plasmid was used in a sequencing reaction that contained 8 Îĵl of Ready reaction mix (big dye terminator, BDTv 3.0, Applied Biosystems) and 5 pmol of M13 primers. The cycling conditions used were as follows: 25 cycles of 96 °C for 10 s, 50 °C 5 s, 60 °C 4 min. Samples were ethanol precipitated, washed with 70% ethanol and resuspended in Hi-DiTM formamide (Applied Biosystems). The sequencing was carried out in ABI Prism 3100 Genetic Analyzer (Applied Biosystems) and the final construct pPIGA3GFP-FF was obtained. For germline transgenesis of Sf9 cell line with pPIGA3GFP-FF, exponentially growing Sf9 cells were transfected with the pPIGA3GFP-FF and helper plasmids (1 : 1 molar ratio) by using DAC-30 as the transfection reagent. Briefly, the two plasmids in the DAC-30 mixture were prepared in Grace media devoid of serum and antibiotics and layered on to cells at 50% confluence. After 4 h, the transfection medium was replaced by fresh medium containing 10% serum and antibiotics. | InsectaCentral | ie-1 RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | embryo | lab_colony | 0.5 | injection | 0.005 | non_specific_dsRNA | eggs | adult | RT-PCR (semi-quantitative) W.blot | whole organism | not checked | high | ||||||||||
| 171 | Ecdysone receptor | RNAi | 20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-response genes have not been previously characterized in the context of insect chitin biosynthesis. Here, we show that injection- based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes. Taken together, we conclude that several key genes in the chitin biosynthesis pathway are 20E late-response genes and 20E plays a key role in the regulation of chitin biosynthesis via inducing their expression. | InsectaCentral | Unpublished | Zhang | Wenqing | Ecdysone receptor target | ATGTCCATAGAGTCGCGTTTAGATAGTTTAGTGCGAGGAAAAAGTGAAGTGAAAGCCTTTGTCGGAGGATGTCCCTCGGCGCTCGTAGATACCGGAGCGTGTGACACGCTCGCAGACATGAGACGACGCTGGTATAACAACGGAGGTTTCCAGACGCTGCGAATGCTCGAGGAGAGCTCGTCTGAAGTGACGTCGTCTTCAGCGTTAGGGCTACCTCCGGCTATGGTGATGTCCCCGGAATCGCTTGCGTCGCCGGAGTACGGCGGCCTGGAGCTGTGGGGCTATGAAGATGGCCTTACAACATATACCATGGCACAGTCGCTGGGCTCTTGTACAATGGAGCAACAGCAGCAGCCGCAACAGCAACAACAGCCCCAGCAGACTCAGCCGTTACCATCAATGCCGTTGCCCATGCCGCCGACAACACCGAAATCAGAAAATGAGTCGATGTCGTCAGGTCGTGAGGAACTGTCTCCGGCTTCAAGTATTAACGGCTGCAGTACGGATGGCGAGGCGAGGCGGCAGAAGAAAGGCCCAGCACCGCGGCAACAAGAGGAGCTATGTCTCGTCTGCGGCGACAGAGCCTCCGGATATCATTACAATGCGCTCACATGTGAAGGGTGTAAAGGATTCTTCAGGCGGAGCGTAACCAAAAATGCAGTGTACATATGCAAGTTCGGGCACGCATGTGAGATGGATATGTATATGCGAAGAAAATGTCAAGAGTGTCGGTTGAAGAAATGTCTGGCGGTGGGCATGAGGCCTGAGTGTGTGGTGCCAGAAAACCAGTGTGCAATGAAAAGGGAAGAGAAAAAGGCACAAAGGGAAAAAGACAAGTCGCCAGTCAGTACAACGACAGTGGATGATCACATGCCTCCCATTATGCAGTGTGATCCACCGCCTCCAGAGGCCGCAAGAATTCACGAGGTGGTGCCACGATTCCTGAATGAGAAGCTAATGGAACAGAACAGGCTCAAGAATGTGCCCCCCCTCACTGCCAACCAGAAGTCCTTAATAGCGAGGCTGGTCTGGTACCAAGAAGGCTATGAACAGCCATCAGAAGAGGATCTAAAAAGAGTCACACAGACCTGGCAGTCGGATGAAGACGAAGAAGAGTCGGACATGCCGTTCCGTCAGATCACCGAGATGACGATCCTCACAGTGCAGCTCATTGTTGAATTCGCTAAGGGCCTACCAGGCTTCGCAAAGATCTCACAGTCGGATCAGATCACATTATTAAAGGCCTGTTCGAGTGAGGTGATGATGTTGCGAGTAGCTCGGCGGTACGACGCGGCGACAGACAGCGTGTTGTTCGCCAACAACCAGGCGTACACCCGCGACAACTACCGCAAGGCAGGCATGGCCTACGTCATCGAGGACCTGCTGCACTTCTGCCGGTGCATGTACTCCATGATGGTGGATAACGTCCACTATGCACTGCTCACTGCCATCGTCATTTTCTCAGACCGACCCGGGCTTGAGCAACCCCTGTTGGTGGAGGAGATCCAGAGATATTACCTGAACACGCTGCGGGTGTACATCCTGAACCAGAACAGTGCGTCGCCGCGCTGCCCTGTCATCCACGGCAAGATCCTCGGCATCCTGACGGAGCTGCGGACCCTGGGCATGCAGAACTCCAACATGTGCATCTCGCCCAAGCTGAAGAACAGGAAGCTGCCGCCGTTCCTCGAGGAGATCTGGGACGTGGCGGACGTGTCCACCGCGGCCCCGTCGGCAGCCGTCGTGTCGGAAACCGCGGCGCTCTAG | Lepidoptera | Noctuidae | Laphygma | exigua | cds | GenBank | Ecdysone receptor target | Ecdysone receptor RNAi construct | GTGTCGGTTGAAGAAATGTCTGGCGGTGGGCATGAGGCCTGAGTGTGTGGTGCCAGAAAACCAGTGTGCAATGAAAAGGGAAGAGAAAAAGGCACAAAGGGAAAAAGACAAGTCGCCAGTCAGTACAACGACAGTGGATGATCACATGCCTCCCATTATGCAGTGTGATCCACCGCCTCCAGAGGCCGCAAGAATTCACGAGGTGGTGCCACGATTCCTGAATGAGAAGCTAATGGAACAGAACAGGCTCAAGAATGTGCCCCCCCTCACTGCCAACCAGAAGTCCTTAATAGCGAGGCTGGTCTGGTACCAAGAAGGCTATGAACAGCCATCAGAAGAGGATCTAAAAAGAGTCACACAGACCTGGCAGTCGGATGAAGACGAAGAAGAGTCGGACATGCCGTTCCGTCAGATCACCGAGATGACGATCCTCACAGTGCAGCTCATTGTTGAATTCGCTAAGGGCCTACCAGGCTTCGCAAAGATCTCACAGTCGGATCAGATCACATTATTAAAGGCCTGTTCGAGTGAGGTGATGATGTTGCG | dsRNA | T7 RiboMAX⢠Express RNAi System Kit (Promega, USA) | ethanol precipitation | subsequent to transcription | according to the manufacturerâs instructions | InsectaCentral | Ecdysone receptor RNAi construct | Lepidoptera | Noctuidae | Laphygma | exigua | local resource | larva | lab_colony | 1 | injection | DEPC | 0.5 | buffer non_specific_dsRNA | 3 | hemolymph | larva | qPCR RT-PCR (semi-quantitative) W.blot phenotype | whole organism | not checked | 1.2243672108E10 | high | ||||||||
| 172 | Chitinase gene | RNAi | The function of insect chitinases is to digest the structural polysaccharides in the exoskeleton and gut lining during the molting process. However, it has not been determined whether insect chitinases participate in the defense against entomopathogenic fungi, which contain chitin in their cell walls. In this study, we examined the role of a chitinase (SeChi) and a bacterial type chitinase (SeChi-h) of Spodoptera exigua in defense against the entomopathogenic fungus Metarhizium anisopliae. Feeding 5th instar larvae of S. exigua with a high concentration of spores of M. anisopliae caused low survival rates and 2- to 10- fold upregulation of chitinase genes transcription in the midguts after 48 h. Additionally, we used RNA interference (RNAi) to successfully knockdown the chitinase genes through injection of double-stranded RNA (dsRNA). The anti-fungal properties of S. exigua larvae declined after injection of SeChi and SeChi-h dsRNA, leading to higher mortality rates. These results provide the first examples that chitinases of S. exigua play an important role in defense against entomopathogenic fungus. | InsectaCentral | Unpublished | Zhang | Wenqing | Chitinase gene target | ACGCGGGGACACAATCACAAGCCGGCCCGCACGCCGCAACACCGCAACTGTTCAAAATGAGAGCGATACTGGCGACGTTGGCCGTCCTAGCGGTCGTAACGACTGCAATTGAAGCGGACAGCAAAGCGCGCATAGTATGCTACTTCAGCAACTGGGCGGTGTATCGACCTGGTGTGGGTCGCTACGGCATTGAAGATATCCCCGTGGATCTCTGTACTCATATTATCTACTCTTTCATTGGAGTCACTGAGAAGTCCAATGAAGTCCTCATTATTGATCCTGAGTTGGATGTAGACAAGAATGGCTTCAAAAACTTCACAGCTCTCCGCAAATCTCATCCTGATGCCAAGTTCACGGTGGCTGTGGGAGGCTGGGCCGAGGGAGGATCAAAATACTCCCACATGGTCGCACAGAAACAAACACGAATGGCCTTCGTTAGGAGCGTTGTTGATTTTCTGAAGAAATACGACTTCGATGGTTTGGATCTGGACTGGGAGTACCCCGGCGCTGCTGACCGTGGTGGTTCCTTCTCTGACAAGGATCGATTCCTCTTCCTCGTCCAGGAGCTTAGGAGAGCATTCATCAGAGAAAAGAGAGGCTGGGAATTAACTGCTGCTGTGCCACTCGCCAACTTCAGACTGATGGAAGGTTACCACGTACCTGATCTTTGCCAGGAGCTGGATGCTATCCATGTGATGTCTTACGATCTGAGAGGAAACTGGGCTGGATTTGCTGACGTGCATTCACCATTGTTCAAACGCCCTCATGACCAATGGGCTTATGAGAAACTTAATGTTAACGATGGTCTAGCTCTCTGGGAGGAGAAGGGCTGTCCTAGCAATAAGCTGGTTGTCGGTATTCCATTCTACGGCCGTTCTTTCACTCTGTCAGCTGGTAACAACAACTACGGCCTTGGTACTTACATTAACAAGGAGGCCGGAGGCGGTGACCCTGCTCCTTACACCAACGCTACTGGATTCTGGGCTTACTATGAGATCTGTACCGAAGTTGACAAAGAAGGTTCAGGATGGACCAAGAAATGGGACGAGCATGGCAAGTGCCCCTACGCTTACAAAGGAACCCAATGGGTCGGTTACGAGGACCCTCGTAGTGTCGAGATCAAAATGAACTGGATCAAGGAGAAGGGATACCTCGGTGCCATGACCTGGGCTATCGACATGGATGACTTCAAAGGTCTTTGTGGCGACGAAAACCCTCTGATCAAGCTTTTGCACAAACATATGAGCACTTACACTGTTCCACCACCTCGTTCTGGAAATACCACTCCTACGCCTGAATGGGCGCGCCCGCCGTCAACGACGTCCGACCCGTCCGAGGGAGAGCCGATCGTGACGACTGCAAGGCCGTCCACTACCACTAAGACTAGCAGGCCTACGAAGAAGCCGACCACGACCAAGCCTCAAGTCATTATCGAAGATGATGAAAATGATATTGCCGTGAGACCTGAACCACCAAAAGCTCCCGAGACACCAGTAGCTCCGGAAGCCCCTGAAGTACCAGAATCACCTGCTGAGAATGAAATCGACGACCACGATGTCTGCAACTCTGAAGAAGATTACGTTCCTGATAAGAAGAAATGCAACATGTACTGGCGCTGCGTCAACGGTAAAGGAATGCAGTTCACATGCCAACCAGGAACAATGTTCAACACCAAACTAAACGTTTGCGATTGGCCCGACAACGCTGACCGTCAAGACTGCGAGCCCTAGACAAATCGTGCGTGAATAGGGCGCTTTACTGGTCATTGCAAAGATACCGTTTGATCGAAATCGCATTCTCCTTGCAAGACAGCTGATTAATTGTGGCGGGGCTGGGGCTTGCAAACTGATTGACACCGGTGAATGGGAATGCATGTAGAACTAAGATGGCTGTCTTTACAATTAGCAAATGATTATTCACATCCCATAAAGTTACGCTGCAAATTATTCCTTAAGGTCTGTAAATTCAACGTCTGTTTTCCAACACCAAATTTTGTTATCTTCATAAAGCTACCATTATTTACCCTATTAACTCGGTACTATAAAAATATTAGTCATCTGTATGGGAACACAAAAGGCCGCTAATAAACCTTTGTGTGGTCAGCCTGTTGGGTGGATTTTGATAAAATGGTATCTTTCCTTTACGCAATGAATCATAATAATGGTCGTATTTACGAAGTTTTAGGTTACTGAACAATATTGTCGTATTTTGTTATGAAGCAACATAATACGCCAATGTTTTCTTGTCTCTAGTTTATAAGTGGATAGGATTATTGTGATGTTAGGCTAGAAATTTACGGTTATAAATAATGTGTTTGATTTATAGATGCGCGTTTAATTGGATACACTAAAAAAAAAAACTATCAATTAAACGTTAATCTATCAAAAAGAACAATAAAAATGAGATTATTAAGCGTAACATTGCCTAAAATCGTGTACAAGACTGTATCGCATCGATTAGATTAATATAATCTATGTTTAATAAAATAAGCAACGATTTAAGTTCATTAATATAAATAAGTCTATAAATGATTCTGTATTTTTATACAAACAGAACCAGGAATAGTTTGAATAAGTCGCGAAACATGGATACTTTTATTTGTTCACTTTAGTTTGTTAAGAACTTGCAGTTTGCGTCAATAACTCCATTCAACAGTCATAAAGTTATGGCGGGAAATTGTGTTTATTTTATAAATTTTGTTTCGACGCGGAAGCCAATTAGACTTCTCCCGGCTTTGGGGATTTAAATGAATTTCCCAAGCACTTGTCGCGGCTGGTCGCTGGCTTAACCAACCTTATCTTAATTTATTTATTTTAATAATTTTAAGACAATAAATTATGATAAAAATTTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Laphygma | exigua | cds | GenBank | Chitinase gene target | Chitinase gene RNAi construct | GTCGCACAGAAACAAACACGAATGGCCTTCGTTAGGAGCGTTGTTGATTTTCTGAAGAAATACGACTTCGATGGTTTGGATCTGGACTGGGAGTACCCCGGCGCTGCTGCCCGTGGTGGTTCCTTATCTGACAAGGATCGATTCCTCTTCCTCGTCCAGGAGCTTAGGAGAGCATTCATCAGAGAAAAGAGAGGCTGGGAATTAACTGTTGCTGTGCCACTCGCCAACTTCAGACTGATGGAAGGTTACCACGTACCTGATCTTTGCCAGGAGCTGGATGCTATCCATGTGATGTCTTACGATCTGAGAGGAAACTGGGCTGGATTTGCTGACGTGCATTCACCATTGTTCAAACGCCCTCATGACCAATGGGCTTATGAGAAACTTAATGTTAACGATGGTCTAGCTCTCTGGGAGGAGAAGGGCTGTCCTAGCAATAAGCTGGTTGTCGGTATTCCATTCTACGGCCGTTCGTTCACTCTGTCGGCTGGTAA | dsRNA | T7 RiboMax Express RNAi System kit (promega) | ethanol precipitation | subsequent to transcription | Synthesis of dsRNA was performed according to the protocol from the T7 RiboMax Express RNAi System kit (Promega, USA) | InsectaCentral | Chitinase gene RNAi construct | Lepidoptera | Noctuidae | Laphygma | exigua | local resource | larva | lab_colony | 1 | injection | DEPC | 0.5 | buffer non_specific_dsRNA | 3 | hemolymph | larva | qPCR W.blot phenotype | whole organism | not checked | 24487296 | high | ||||||||
| 173 | bacterial type chitinase | RNAi | The function of insect chitinases is to digest the structural polysaccharides in the exoskeleton and gut lining during the molting process. However, it has not been determined whether insect chitinases participate in the defense against entomopathogenic fungi, which contain chitin in their cell walls. In this study, we examined the role of a chitinase (SeChi) and a bacterial type chitinase (SeChi-h) of Spodoptera exigua in defense against the entomopathogenic fungus Metarhizium anisopliae. Feeding 5th instar larvae of S. exigua with a high concentration of spores of M. anisopliae caused low survival rates and 2- to 10- fold upregulation of chitinase genes transcription in the midguts after 48 h. Additionally, we used RNA interference (RNAi) to successfully knockdown the chitinase genes through injection of double-stranded RNA (dsRNA). The anti-fungal properties of S. exigua larvae declined after injection of SeChi and SeChi-h dsRNA, leading to higher mortality rates. These results provide the first examples that chitinases of S. exigua play an important role in defense against entomopathogenic fungus. | InsectaCentral | Unpublished | Zhang | Wenqing | bacterial type chitinase target | ATCGGGAGGGCGTGCGCCGGCGCAGCTGTGCCGACACACAGTCAAACATAGTCAAACACACGTGAAATACAATAAAAGTGTATTTAAAAAGTGTGTGAAATACGAAGCTGTGGATTTTATACCGAAAATTTTATAATTTTTTTCGGTGTTTTTTAACTGAACCGGTTATGGGACGTCCGATGTTGGCGTTAGTCATCGGCGCGGGCCTGCTCGCCGTCGTCGCGTGCGGCCCTCCTGGCAAACCGTCCTTAGGCTGGGGAGAACGCACATTCGCCATCGTCGAAGTCAATCAAGCCGCCACAGCATACAACCAGCTGGTAAAAAAGAAAGATGCAGCCGATGTCACCGTCACATGGAACGTCTGGTCTGGCGACCCAGCTGAAAAATCGAGAGTGCTGCTCAACGACAAGGAGTTCTGGTCTGGATCTGGATCAGCTACCTCAGCCACCTTCAAAGTCAAGAAGGGTGGTAGATACCAAATGAAAGTGGAACTCTGCAACTCAGCAGGGTGCAGCTACAGTGAATCAACTGAGATAGTGGTTGCCGATACTGATGGTAGCCATCTCCCGCCTCTAGACTACTCTATCGGAGAAAGGAACAAGCCCTTCAAGCAGACTTCAGGAAAGGTTGTTGGTGCCTACTTCGTAGAATGGGGTGTCTACCCAAGGAAGTTCCCCGTTGACCGTGTGCCCATTCCTAACCTTACTCATCTGTTGTACGGTTTCATCCCAATCTGTGGAGGAGATGGCATCAACGACAGTTTGAAGGAGATCGAAGGCAGTTTCCAAGCTCTTCAAAGATCATGCAGCGGCAGGGAAGACTTCAAAGTATCAATTTACGATCCTTGGGCCGCCCTACAGAAGCCACAGAAAGGATTATCGTCCTGGAATGAGCCTTACAAGGGTAACTTCGGCCAATTGATGATGTTGAAACAAGCCAGACCTGACCTAAAGATTTTGCCTTCTGTTGGTGGATGGACCCTGGCTGATCCTTTCTTCTTCTTCACTGACGAAGTAAAGCGACACCGTTTCGTTGCTTCAGTCAAGGACTTCCTCGAAACCTGGAAGTTCTTTGATGGTGTTGACATTGATTGGGAATTCCCTGGTGGTAAAGGAGCCAACCCTGATCTTGGTGGCCCTGAAGACGGCCATATCTACGTCCAGCTAATGAAAGAACTTAGAGAGATGTTGAACGAGCTTTCTGCCAAGACTGGTAAGAAGTACGAACTGACTTCTGCCATCAGCTCCGGTTGGGACAAGATCCAAGTTGTGGACTACAAAGAGGCTCAGCAGTACATGGACCACATCTTTTTGATGAGTTATGACTTCAAGGGAGCTTGGTCTAACGACACTCTTGGTCACCAGGCTGGTTTGTACGCTCCAGCGTGGAACCCCAAGGAGACCTACACAACTGACTTTGGTGTCAAATTCCTGTTAGCCCAAGGCGTCAGTCCGAAGAAGATCGTTGTTGGTGTTGCTATGTATGGTCGAGGCTGGACTGGAGTCAATGGTTACAAAGATGGAAACCCCTTCACTGGTGTAGCTACCGGTCCCGTTAAAGGCACTTGGCAAGATGGTGTGGTGGACTACAGAGAGATTGCCAATGAAATTGCTCAAGGCAAATGGGAGTACCACTATGATAAGGTGGCTCAGGCACCTTATGTGTTCAGGAAAGAGACAGGGGATCTTATTACGTATGATGACGCGAGATCGACAATTGAGAAGGCAAAATATGTGAGAAATAATAAGTTAGGTGGATTGTTCGCTTGGGAAGTGGATGCGGACAACGGTGACATATTAAATGCCATGAATATGGGTCTCGGTAACAATGCGTGAATTGATACTAGTGCGAGAGTTGTGTGTAAATTGTGATCGTAATGATAAGTTTAGGTTTAGGCTGAATGTCTCAAATTTTTATTTACAAAAATTGATGAACGACTGAAACTGTGTAATTGTAAGTTAATATGAAAAGAAAAACTTAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Laphygma | exigua | cds | GenBank | bacterial type chitinase target | bacterial type chitinase RNAi construct | TCAGCAGTACATGGACCACATCTTTTTGATGAGTTATGACTTCAAGGGAGCTTGGTCTAACGACACTCTTGGTCACCAGGCTGGTTTGTACGCTCCAGCGTGGAACCCCAAGGAGACCTACACAACTGACTTTGGTGTCAAATTCCTGTTAGCCCAAGGCGTCAGTCCGAAGAAGATCGTTGTTGGTGTTGCTATGTATGGTCGAGGATGGACTGGAGTCAATGGTTACAAAGATGGAAACCCCTTCACTGGTGTAGCTACCGGTCCCGTTAAAGGCACTTGGCAAGATGGTGTGGTGGACTACAGAGAGATTGCCAATGAAATTGCTCAAGGCAAATGGGAGTACCACTATGATAAGGTGGCTCAGGCACCTTATGTGTTCAGGAAAGAGACAGGGGATCTTATTACGTATGATGACGCGAGATCGACAATTGAGAAGGCAAAATATGTGAGAAATAATAAGTTAGGTGGATTGTTCGCTTGG | dsRNA | T7 RiboMax Express RNAi System kit (promega) | ethanol precipitation | subsequent to transcription | Synthesis of dsRNA was performed according to the protocol from the T7 RiboMax Express RNAi System kit (Promega, USA). | InsectaCentral | bacterial type chitinase RNAi construct | Lepidoptera | Noctuidae | Laphygma | exigua | local resource | larva | lab_colony | 1 | injection | DEPC | 0.5 | buffer non_specific_dsRNA | 3 | hemolymph | larva | qPCR W.blot phenotype | whole organism | not checked | 24487296 | high | ||||||||
| 177 | trehalose synthase | RNAi | Trehalose is an important disaccharide and a key regulation factor for the development of many organisms, including plants, bacteria, fungi and insects. In order to study the trehalose synthesis pathway, a cDNA for a trehalose-6-phosphate synthase fromSpodoptera exigua (SeTPS)was clonedwhich contained an open reading frame of 2481 nucleotides encoding a protein of 826 amino acids with a predicted molecular weight of 92.65 kDa. The SeTPS genome has 12 exons and 11 introns. Northern blot and RT-PCR analyses showed that SeTPS mRNA was expressed in the fat body and in the ovary. Competitive RT-PCR revealed that SeTPSmRNA was expressed in the fat body at different developmental stages and was present at a high level in day 1 S. exigua pupae. The concentrations of trehalose and glucose in the hemolymph were determined by HPLC and showed that they varied at different developmental stages and were negatively correlated to each other. The survival rates of the insects injected with dsRNA corresponding to SeTPS gene reached 53.95%, 49.06%, 34.86% and 33.24% for 36, 48, 60 and 204 h post-injection respectivelywhichwere signiïĴcantly lower than those of the insects in three control groups. These ïĴndings provide new data on the tissue distribution, expression patterns and potential function of the trehalose-6-phosphate synthase gene. | pubmed | 20193689 | Zhang | Wenqing | trehalose synthase target | ATGAGTGGAACGGACAGCAGTGCCAGTCGATCCGCGTGCAACAGCAAGGGCAGCATGATCGTTGTGTCGAACCGATTGCCTTTTATTTTGAAGAGAAATGAAAAAACCGGCGAACTCGAGAGGAAAGCCAGCGCCGGTGGTTTGGTCACAGCTGTGGCCCCCGTAGTAATCAGGGGTAGTGGCATCTGGGTAGGATGGCCAGGAATACACCTGGACGACCCCAATGAGAAGATTCCAGAGTCCAACCCCAATGACAAGACCCCCACCGCTGGTTTGCTGTCTGAGAAGATTGTACCAATCCACGCCGAACCAAAACTTTTCGACAGTTACTACAATGGCTGCTGCAACGGTACCTTCTGGCCTTTGTTCCACTCCATGCCTGACAGAGCTACTTTCATTGCCGACCACTGGAAGGCATACATCAAGTGCAATGAGGAGTTCGCTGAGAAGACAGTCCACGCTCTCCATTTGCTCAAACAGCAGAAGGGGAAGAATGGCAACTCACCGCCAATCGTATGGGTCCATGATTATCATCTCATGTTAGCTGCTAACTGGATAAGACAGCGAGCTGAAGAAGATGAAATCAAGTGCAAGTTAGCATTCTTCTTGCACATTCCGTTCCCACCATGGGACATATTCAGACTGTTCCCATGGTCAGATGAAGTACTTCAGGGCATTCTGGGTTGTGATATGGTGGGTTTCCATATAACAGATTACTGCCTGAACTTCATTGATTGCTGCCAAAGAAACTTAGGTTGTCGTGTGGACAGGAAGAATCTCCTGGTTGAGTTGGGAGGCCGCACCATTTGTGTGCGACCTCTGCCAATTGGAGTACCCTTCGACAGATTCGTACAGTTGGCACAGAACGCGAAACCAGTGCTCTCTACCAACCAACAAGTTATATTAGGAGTCGATAGACTGGATTATACCAAAGGATTAGTACATAGACTGAAAGCCTTCGAGAGGCTGCTGGAGAAGTATCCAGAACACATCGAGAAAGTAGTACTGCTTCAAATCTCAGTGCCGTCCAGAACGGACGTCAAGGAGTACCAAGACTTGAAAGAAGGGATGGATCAATTAGTTGGAAGAATTAACGGCAGATTTACTACTCCCAACTGGTCACCTATTAGGTACATTTATGGATGCGTCGGACAGGATGAACTAGCAGCATTCTACCGTGACGCTGCAGCCGCCTTGGTAACACCTCTGCGAGACGGTATGAACCTCGTGGCGAAGGAGTTCGTCGCTTGCCAGATCAACAAGCCACCAGGAGTGCTGATAGTGTCGCCCTTCGCTGGTGCTGGAGAGATGATGCACGAGGCTCTCATCTGCAACCCATATGAGCTGGATGACGCTGCTGAAGTCATTCACAGGGCTCTGATAATGCCTGAGGATGAGCGCACAGTCCGTATGAACCATCTGAGGAGGCGAGAGCAGCTCAATGATGTCGACAGCTGGATGAAAGCGTTCCTCAAAGCTATGGACTCGTTGGAAGAGGAAGCTGATGACATCGGAGCTACATCCATGCAGCCCGTTACTATTGACGACTTCGATGAATATCTGTCAAAATACATCGGCTACACGCAAAAGCTGGCACTTTTACTGGACTACGATGGTACTCTAGCACCCATAGCGCCTCACCCCGACCTGGCGACACTGCCGCTGGAGACCAAACATACGTTGCAGAGACTCTCCAATATGTCCGATGTTTACATCGCGATTATCTCCGGCAGAAACGTCGATAACGTTAAGAATATGGTGGGCATCGAAGGCATCACTTATGCTGGTAACCACGGTCTGGAGATCCTGCACCCTGATGGCAGTAAGTTCGTGCACCCCATGCCCATGGAACTGCAAGATGCAGTTGTTAAACTGCTCAAGGCTCTGCAAGAACAGGTTTGCAAAGATGGCGCTTGGGTAGAAAACAAGGGTGCCCTGCTTACTTTCCACTACCGTGAGACACCTGTGGCGAAACGTGCTGCGCTGGCAGAACAAGCGAGGAAGCTCATCACAGAGGCTGGTTTTACACCAGCACCCGCGCATTGCGCCTTAGAAGCTCGCCCACCAGTAGAATGGGACAAAGGTCGTGCATCAATTTACATCTTGAGGACGGCCTTTGGTCTGGACTGGAGTGAAAGGATTAGGATTATCTATGCCGGTGACGACGTCACCGATGAAGACGCCATGTTGGCTCTGAAAGGTATGGCAGCTACTTTCCGTATCGCGTCGTCTAACATCACGAAGACATCAGCAGAGAGGCGATTGTCGTCAACGGACTCAGTGCTCGCCATGTTGAAGTGGGTGGAACGACACTTTTCCCGCCGCAAGCCGCGCGCCAACTCGTTGACGTACAAAAACGCGCGAAAAGCCCGCGACACCATACAGATGCACATGTCGTACCAGATGCCCGCCAAAACGTCACCACGACACACGCCGCCCCTCACGCCAGACAAAACCTCCAGCGGTTCAGAGTCCAGTTAA | Lepidoptera | Noctuidae | Laphygma | exigua | cds | GenBank | trehalose synthase target | trehalose synthase RNAi construct | CGATCCGCGTGCAACAGCAAGGGCAGCATGATCGTTGTGTCGAACCGATTGCCTTTTATTTTGAAGAGAAATGAAAAAACCGGCGAACTCGAGAGGAAAGCCAGCGCCGGTGGTTTGGTCACAGCTGTGGCCCCCGTAGTAATCAGGGGTAGTGGCATCTGGGTAGGATGGCCAGGAATACACCTGGACGACCCCAATGAGAAGATTCCAGAGTCCAACCCCAATGACAAGACCCCCACCGCTGGTTTGCTGTCTGAGAAGATTGTACCAATCCACGCCGAACCAAAACTTTTCGACAGTTACTACAATGGCTGCTGCAACGGTACCTTCTGGCCTTTGTTCCACTCCATGCCTG | dsRNA | Promega T7 express kit | ethanol precipitation | subsequent to transcription | as description | InsectaCentral | trehalose synthase RNAi construct | Lepidoptera | Noctuidae | Laphygma | exigua | local resource | larva | lab_colony | 1 | injection | DEPC | 0.5 | buffer non_specific_dsRNA | 3 | hemolymph | larva | RT-PCR (semi-quantitative) W.blot phenotype | whole organism | not checked | 1.2243648729612016E17 | high | ||||||||
| 179 | Cadherin silencing in M. sexta | RNAi | In this work we set up the conditions to efficiently deliver dsRNA by feeding M. sexta larvae.In the case of M. sexta, dsRNA was offered to starve neonates and after eating the exposed larvae were returned to artificial diet until they reached third instar. The cadherin protein levels were then determined in third instar M. sexta larvae by Western blot using specific antibody. Efficiency of silencing was estimated to be 87% Silenced larvae were exposed to different concentrations of trypsin activated Cry1Ab toxin from 0 to 10 ng/cm2 of diet. We observed no mortality of the M. sexta silenced larvae when compared with the normal M. sexta larvae, suggesting that M. sexta silenced larvae become highly tolerant to the toxin due to lost expression of cadhering protein. | InsectaCentral | Unpublished | G³mez | Isabel | caderin silencing in Msexta target | GACCAATCGGAGTGTGGTGAATTTTTGGAAAATATTTTGTGCGGTTCCTTTAGTTGTGTAATATAGTACTTTAGTTACAAATTTGGAATAATTTGGCAGCAAAACCATCTGCAGCAACAAAATCATCTGCAGCTGCGAAATCATCTGCAGCAGCAAAAGCATCTTCAGGAGCGAGAAAAGCCCCAAATAATGTGAGATGGCAGTTGACGTCCGAATCGCTGCCTTCCTGCTGGTGTTTATAGCGCCTGCAGTTTTAGCTCAAGAGAGATGTGGGTATATGACCGCCATCCCAAGGCTACCACGACCGGATAATTTGCCAGTACTAAATTTTGAAGGCCAGACATGGAGTCAGAGGCCCCTGCTCCCCGCCCCGGAGCGGGATGACCTGTGCATGGACGCCTACCACGTGATAACAGCCAACCTCGGCACGCAGGTCATCTACATGGATGAAGAGATAGAAGACGAAATCACCATCGCCATACTTAATTATAACGGACCATCAACTCCGTTCATTGAACTGCCATTTTTATCCGGTTCGTACAATCTGCTGATGCCGGTCATCAGGAGAGTTGACAACGGGGAGTGGCATCTCATCATCACGCAAAGACAGCATTACGAGTTGCCCGGCATGCAGCAGTACATGTTCAATGTGCGCGTGGACGGCCAGTCGCTGGTGGCAGGCGTGTCTCTCGCTATCGTCAACATAGATGACAACGCGCCCATCATACAAAACTTCGAGCCTTGCCGGGTTCCTGAACTGGGCGAGCCAGGGTTGACAGAATGCACATACCAAGTATCGGACGCGGACGGACGGATCAGCACAGAGTTCATGACGTTCAGGATCGACAGCGTTCGTGGCGACGAGGAGACCTTCTACATCGAACGGACGAATATCCCCAACCAATGGATGTGGCTAAATATGACCATAGGCGTTAATACCTCGCTCAACTTCGTCACCAGTCCGCTGCATATATTCAGCGTGACAGCCCTGGACTCGCTCCCGAACACCCACACGGTGACTATGATGGTGCAAGTGGCGAATGTGAACAGCCGTCCGCCGCGCTGGCTGGAGATCTTCGCTGTCCAACAGTTTGAAGAGAAATCTTACCAAAACTTCACAGTGAGGGCGATCGACGGAGACACTGAGATCAATATGCCTATCAACTACAGGCTGATCACAAATGAGGAAGACACATTCTTCAGCATTGAGGCCCTGCCTGGTGGAAAAAGCGGGGCTGTATTCCTCGTGTCGCCAATTGACCGCGACACACTGCAACGAGAGGTGTTTCCACTTACGATCGTCGCTTACAAATATGATGAGGAGGCCTTCTCCACATCAACAAACGTGGTCATCATTGTGACAGACATCAACGACCAAAGACCTGAACCTATACACAAGGAATATCGACTGGCAATCATGGAGGAGACGCCCCTGACCCTCAACTTCGATAAAGAATTCGGATTTCATGATAAGGATTTAGGTCAAAACGCTCAGTACACGGTGCGTCTAGAGAGCGTGGACCCTCCAGGCGCTGCTGAGGCATTCTACATAGCGCCTGAAGTCGGCTACCAGCGACAGACCTTCATCATGGGCACCCTCAATCACTCCATGCTGGATTACGAAGTGCCAGAGTTTCAGAGTATTACGATTCGGGTGGTAGCGACCGACAACAACGACACGAGGCACGTGGGCGTCGCGTTGGTTCACATTGACCTCATCAATTGGAACGATGAGCAGCCGATCTTCGAACACGCCGTGCAGACCGTCACCTTCGACGAGACTGAAGGCGAGGGGTTCTTCGTCGCCAAGGCGGTTGCACACGACAGAGACATCGGGGATGTCGTCGAGCATACTTTATTGGGTAACGCTGTTAACTTCCTGACCATCGACAAACTCACCGGCGACATCCGCGTCTCAGCTAACGACTCCTTCAACTACCATCGAGAAAGTGAATTATTTGTGCAGGTGCGAGCTACAGACACGCTGGGCGAACCCTTCCACACGGCGACGTCACAGCTGGTCATACGACTAAATGACATCAACAACACGCCACCCACCTTACGGCTGCCTCGAGGCAGTCCCCAAGTGGAGGAGAACGTGCCTGATGGCCACGTCATCACCCAGGAGTTACGCGCCACCGACCCCGACACCACGGCCGATCTGCGCTTCGAGATAAACTGGGACACCTCTTTCGCCACCAAGCAAGGCCGCCAGGCTAACCCCGACGAGTTTAGGAATTGCGTGGAAATCGAGACCATCTTCCCCGAGATTAACAACCGGGGACTGGCTATCGGCCGCGTTGTAGCGCGCGAAATCAGACACAACGTGACCATAGACTACGAGGAGTTTGAGGTCCTCTCCCTCACAGTGAGGGTGCGTGACCTTAACACCGTCTACGGAGACGACTACGACGAATCGATGCTCACAATAACTATAATCGATATGAACGACAACGCGCCGGTGTGGGTGGAGGGGACTCTGGAGCAGAACTTCCGAGTCCGCGAGATGTCGGCGGGCGGGCTCGTGGTGGGCTCCGTGCGCGCGGACGACATCGACGGACCGCTCTACAACCAAGTGCGATACACCATTTTCCCTCGTGAAGACACAGATAAGGACCTGATAATGATCGACTTCCTCACGGGTCAAATTTCCGTGAACACAAGCGGCGCCATCGACGCGGATACTCCTCCACGCTTCCACCTCTACTATACAGTGGTCGCTAGTGACCGATGCTCGACAGAAGATCCTGCAGATTGCCCCCCTGACCCGACTTATTGGGAAACCGAAGGAAATATCACAATCCACATCACCGACACGAACAACAAGGTCCCGCAGGCGGAAACGACTAAGTTCGATACCGTCGTGTATATTTACGAGAACGCAACCCACTTAGACGAGGTGGTCACTCTGATAGCCAGTGATCTTGACAGAGACGAAATATACCACACGGTGAGCTACGTCATCAATTATGCAGTGAACCCTCGACTGATGAACTTCTTCTCCGTGAACCGAGAGACCGGCCTGGTGTACGTGGACTATGAGACCCAGGGTAGTGGCGAGGTGCTGGACCGTGATGGTGATGAACCAACGCACCGTATCTTCTTCAACCTCATCGACAACTTCATGGGGGAAGGAGAAGGTAACAGAAATCAGAACGACACAGAAGTTCTCGTTATCTTGTTGGATGTGAATGACAATGCTCCTGAATTGCCACCGCCGAGCGAACTCTCTTGGACTATATCTGAGAACCTTAAGCAGGGCGTCCGTCTTGAACCACATATCTTCGCCCCGGACCGCGACGAGCCCGACACAGACAACTCCAGGGTCGGCTACGAGATCCTGAACCTCAGCACGGAGCGGGACATCGAAGTGCCGGAGCTGTTTGTGATGATACAGATCGCGAACGTCACGGGAGAGCTGGAGACCGCCATGGACCTCAAGGGATATTGGGGGACGTACGCTATACATATACGGGCATTCGACCACGGCATTCCGCAAATGTCCATGAACGAGACATATGAGCTGATCATCCATCCGTTCAACTACTACGCGCCTGAGTTCGTCTTCCCGACCAACGATGCCGTCATACGACTTGCGAGGGAACGAGCTGTAATCAATGGAGTTCTAGCGACAGTGAACGGAGAGTTCTTGGAGCGGATATCGGCGACTGATCCGGACGGACTCCACGCGGGCGTCGTCACCTTCCAAGTGGTAGGCGATGAGGAATCACAACGGTACTTTCAAGTAGTTAACGATGGCGAGAACCTCGGCTCGTTGAGGTTACTGCAAGCCGTTCCAGAGGAGATCAGGGAGTTCCGGATAACGATTCGCGCTACAGACCAGGGAACGGACCCAGGACCGCTGTCCACGGACATGACGTTCAGAGTTGTTTTTGTGCCCACGCAAGGAGAACCTAGATTCGCGTCCTCAGAACATGCTGTCGCTTTCATAGAAAAGAGTGCCGGCATGGAAGAGTCTCACCAACTTCCTCTAGCACAAGACATCAAGAACCATCTCTGTGAAGACGACTGTCACAGCATTTACTATCGTATTATCGATGGCAACAGCGAAGGTCATTTCGGCCTGGATCCTGTTCGCAACAGGTTGTTCCTGAAGAAAGAGCTGATAAGGGAACAAAGTGCCTCCCACACTCTGCAAGTGGCGGCTAGTAACTCGCCCGATGGTGGCATTCCACTTCCTGCTTCCATCCTTACTGTCACTGTTACCGTGAGGGAGGCAGACCCTCGTCCAGTGTTTGTGAGGGAATTGTACACCGCAGGGATATCCACAGCGGACTCCATCGGCAGAGAGCTGCTCAGATTACATGCGACCCAGTCTGAAGGCTCGGCCATTACTTATGCTATAGACTACGATACAATGGTAGTGGACCCCAGCCTGGAGGCAGTGAGACAGTCGGCTTTCGTACTGAACGCTCAAACCGGAGTGCTGACGCTTAATATCCAGCCCACGGCCACGATGCATGGACTGTTCAAATTCGAAGTCACAGCTACTGACACGGCCGGCGCTCAGGACCGCACCGACGTCACCGTGTACGTGGTATCCTCGCAGAACCGCGTCTACTTCGTGTTCGTCAACACGCTGCAACAGGTCGAAGACAACAGAGACTTTATCGCGGACACCTTCAGCGCTGGGTTCAACATGACCTGCAACATCGACCAAGTGGTGCCCGCTAACGACCCCGTCACCGGCGTGGCGCTGGAGCACAGCACGCAGATGCGCGGCCACTTCATACGGGACAACGTACCCGTACTCGCTGATGAGATAGAACAGATCCGTAGTGACCTAGTCCTCCTGAGCTCGATACAAACAACGCTGGCGGCGCGATCGCTGGTGTTGCAGGACTTGTTGACCAACTCCAGCCCGGACTCGGCGCCTGACTCGAGCCTCACGGTGTACGTGCTGGCCTCACTGTCTGCTGTGCTCGGTTTCATGTGCCTTGTGCTACTGCTTACCTTCATCATCAGGACTAGAGCGCTAAACCGACGGTTGGAAGCCCTGTCGATGACGAAGTACGGCTCACTGGACTCTGGATTGAACCGCGCCGGCATCGCCGCCCCCGGCACCAACAAACACACTGTGGAAGGCTCCAACCCTATCTTCAATGAAGCAATAAAGACGCCAGATTTAGATGCCATTAGCGAGGGTTCCAACGACTCTGATCTGATCGGCATCGAAGATCTTCCGCACTTTGGCAACGTCTTCATGGATCCTGAGGTGAACGAAAAGGCAAATGGTTATCCCGAAGTCGCAAACCACAACAACAACTTCGCTTTCAACCCGACTCCCTTCTCGCCTGAGTTCGTTAACGGACAGTTCAGAAAGATCTAGAAGATAACAACACTAGTTAAGATCATTAATTTTGGAGTTTGGAATTAAGATTTTTGAAAGGATAGTTGTGATAAGCCTGTGATTTTTAAAACTGTAATTGAAAAAAAAAATTGAGACCTCCATTTAAGCTCTTGCTCTCATCTCATCAAATTTTATAAAATGCCATTAGTCATTAAGATACTCGATTTAATTTAAGATTATTTAAGATATTATGTAAAATAAATATATTGTC | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | caderin silencing in Msexta target | caderin silencing in Msexta RNAi construct | GCTGCCTTCCTGCTGGTGTTTATAGCGCCTGCAGTTTTAGCTCAAGAGAGATGTGGGTATATGACCGCCATCCCAAGGCTACCACGACCGGATAATTTGCCAGTACTAAATTTTGAAGGCCAGACATGGAGTCAGAGGCCCCTGCTCCCCGCCCCGGAGCGGGATGACCTGTGCATGGACGCCTACCACGTGATAACAGCCAACCTCGGCACGCAGGTCATCTACATGGATGAAGAGATAGAAGACGAAATCACCATCGCCATACTTAATTATAACGGACCATCAACTCCGTTCATTGAACTGCCATTTTTATCCGGTTCGTACAATCTGCTGATGCCGGTCATCAGGAGAGTTGACAACGGGGAGTGGCATCTCATCATCACGCAAAGACAGCATTACGAGTTGCCCGGCATGCAGCAGTACATGTTCAATGTGCGCGTGGA | dsRNA | column-based | simultaneously with transcription | RT-PCR from cDNA produced from RNA samples obtained from 3rd instar larvae with primers Rev: 5â-GCTCTAGAGCTGCCTTCCTGCTGGTGTTTA-3â and For: 5â-GGAATTCCTCCACGCGCACATTGAACAT-3â that amplified a 442 bp fragment. The 442 bp PCR fragment was digested with XbaI and EcoRI and cloned into a previously digested pLITMUS 28i (HiScribeTM, New England Biolabs) vector containing two T7 promoters flanking the multi-cloning site. This enabled amplification of the cloned fragment by using a T7 oligonucleotide. The PCR product was purified with QIAquik PCR purification kit protocol (Quiagen). In vitro transcription of both DNA strands of the insert was performed with T7 RNA polymerase using the HiScribeTM transcription system (HiScribe⢠T7 In Vitro Transcription Kit, New England Biolabs) as reported by the manufacturers, yielding dsRNA. | InsectaCentral | caderin silencing in Msexta RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | feeding | buffer | 3 | Midgut | We used the same protocol that was was used for dsRNA of Cadherin | larva | W.blot | midgut | undetected | 72 | high | 87 | ||||||||
| 180 | Manduca sexta hemolymph proteins Kanost lab--Integrin alpha2 | RNAi | pubmed | 17868866 | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha2 target | AGTGCCGCGCCGACCGTTGTGAGGGGAGAACGTTTCGCCGGTCGACATTTACGACTACGAACTCGTAAAGTTTTCGAACGAATCATGAGGATGAATTATATAATAAAAAATAGTGTATGATAATTATTTTCTGTTTGTGTTGTGACGCGTAATCGACAATGGCGTTGTCTATGTGTGTATTTTTGTGCGTGTGCGCTTATGTCGCCGGGTTCAATGTGGACATCCCATCGAGGGTGGTTTACAAGGGAAATGGGAATTCTATGTTTGGATTCACTGTCCAGGCGCATGTGGAAGGCGATCAGAAAATGATCCTAGTGGGAGCGCCGGAGGATGAGCGCTATCCTCCCAAGCACTACAACATCTCACGGCCCGGCGCGGTGTACCGCTGCGAGCCTGGCCGGAGGTCCTACTCCTCTGAGGGTCATGGCCAGCGGGCGCTTGAGCGGTGCGCGGAGATGGTGTTTGATAGAACTAACATGAATCTGGTGGATAAGCAAGGTCGTCAGATCGAGGAGAAGTCCCACCAGTGGTTCGGCGCTACTCTCACCAGCACCGGGAGGAACGGGCCCATTGTGGCCTGTGCCCCTCGCTACGTCTCCTACGTGTCAGCCAAGATGAACCAGCGAGATCCTGTGGGAACGTGCTACGTCGCCAAGACATCCAACGCTGCCACCACCACAGAGTTCTCGCCTTGCAGGACTTCTAGCCACGGACAGCGCAAGACTGGTGTGTGTCAGGCTGGATTTTCCGCCTCCATATCTAAGGATGGCCAGCGTTTATTCATGGGTGCTCCGGGAAGCTTCTACTGGCAAGGTCAGCTGATTTCTCAGACCATGAATGGTCGGCCAGCACTAATCGCCACTCCTGAATCCAAAGCCATGTATGATGATTCTTACATGGGATATTCCATCGCTGTGGGAGATTTTGCCGGTCAAGGAATCCAAAGCGTCGCCGTTGGTGTACCAAGGGGAGCTAATTTAAAGGGTTTGGTTGTCCTCTACACATGGGAACTGCAGAACATAAAGAACATCAGCGGGTCCCAGATCGGCGCTTACTTCGGCTACAGTCTTGCCTCCGGAGACATCGATGGCGACGGCGCTGATGACGTCATCGTTGGAGCTCCCATGTACACCAAAACCAGGAGCGATGGTTACGAACATGGAAGGATTTATGTCATCTACCAAGGAACTGATAGGTTATTTGCCAAAAGCCACGCCAGGACCGGTGAGATCTCTCGGGGCAGATTCGGTTTGGCTGTCACCTCTTTGGGAGACATCAACTACGACGGATTTGGAGATATAGCAGTAGGCGCGCCGTACGGAGGTCAGAATGGTCGCGGTGTAGTCTACATCTACCACGGCAGCGAGCTGGGTATCCTCGAGAAGTACTCCCAGGCCATCACGGCGGAAGAGATCTCCCCGACCTCGAGCACCTTCGGCTTCTCATTATCTGGAGGCGTCGATCTGGATAATAACAACTATACTGACCTCGCTGTTGGCGCTTACAAGTCGGACAGTGTTGTGTTTTTGAAATCCCGTCCAGTGGTAAAAGTAACAGCTGACGTCAAGTTCATGGGTGACAGCAAGCTGATCTCTTTAACAGACAAGCGCTGCCACCTCTCCAACGGCACTGAGGTGGCGTGCGCAGAGCTCATGTTCTGTCTCACCTACACCGGAGTTAATGTAGACCAGCGGATCAAATTCGAGGTGACCTTAGACCTGGACTCAAGGCAGAAAACGAGCAAGCGCCTGTTCCTAGCAGAGACCCACGAGACCACGTACAAGACGCAGATACTACTGACCCAAGGCCAGCAGGAATGCAAGGACGTGATGGTGTACTTGGATGAGGAGATTAGAGACAAGCTAACGCCCATCGAGGTGAAGATGTCGTACGACTTGATCAATCAGCCGTCCGGCGACGTGGTGCCGCCCGTGTTGGACCAAACCAGGAGCAACTTCCACACCGACTCGTTGAATATTCAGAAGAACTGCGGACCTGATAATATCTGCATACCGGATCTTAGGATGTCTGCTACTACACCGACAGTAAACTACGTCCTAAGTTCCGGAGAAAACATTAACATTGATGTCAAAGTGGAAAACGCTGGCGAAGATGCCTTCGAAGCCGCCTACTACCTGAACATACCCGCTGAAGTCACATACGCGAAAATGGAGAGACTTGACAAAGATAACGCAGAGACTCCCATCTACTGCTCCATTGCTAATAGAGAAGATGGTGGAAACACTACTCTGAAATGTGATCTTGGAAACCCCATGGCTAGTGGACAGAAGGTGCATTTCCGTGTAATTCTGGAGGTGGACTCGCGAGTGATGTCGCTGGATTTCAACATGGAAGCGAACTCCACCAACCCGGAGCCGCAGGAGACCGGCTACGACAACATCAAGCACATGGTCATCGGAGTTGTTGTGAAAGCACAACTCTCCATAATTGGCACATCCGACCCCCCAGAACTTCACTACAACGCGTCTCTCTACGGTACTCAGAACCTCAAAGACGACACCAAATGGGGACCCCAAGTCCTACACAAGTACAACATCAAGAACGGAGGACCCTTCACCGTTGATGAAGCGGAGATCTATTTCATGTGGCCGAATCAGACGCTCGACGGTGAGAACATAATGTTAGTACAGCCGCAGTGGCTCGGTAACGTCCAGTGTGATGTAGCAAGACTCAAGACAGTTGAGAATTTATTTGTACAGAACCCTCACGCTTTCATGCTGGTGAAGGAAAAGGAAGCTATGCAGAGCAATGGACTTTACACCGCTTCACAATTATCCGGCGGCATAGGACACTCTGGACAGACTTTCTGGGTAGAGCACTCCAGCAGCGGTGGCGTTGTTATTTCACAAAGTGGAGGTTCTATCTCTGGTAGCCATTTCGGAGCACAAGGATCGTCAGGACATTTCAGTGGAAGCAGCTCAGGTCATGCCAGTGGAAGCTTAGGACAAACTGGGACGCAGTTCACTACCAGCCAAAGTTCGGGTGGACAGTTCAGTGGTAGCCAAAGTGGACTCAGTAACCAAGGAAGCTTCAGTGGCCAAGGCGGGCAAGGCGCCCAATCTGGTATAACATACGTTTATAATAAGACTTGGAGCAGCTCCAGTGGCGAAGGTCTGACAGCCGAAGACCAAGAGCAAATCAAGAAGAATCTAGCAGCTATGGCACAGGGTGGTGTGAACACAGGATTCACTGGACAAGGCACATACGTACAAGGAGGTTCCCAGGGTAGCTATGTACATGGTAGTGCTCAGGGATCGCAAGGCGGTTATGTTCAGGGTAGTGCTCAAGATTCACAAAGTGGATACGTCCAGGGTAGTACTCAGGGCTCTCAAGGTGGATATGTCCAGGGTAGCACGCAAGGTTCTCAGGGTACTTATGTCCAAGGTGGCTCTCAAGGAACTTTCATTCAGGGTGGCTCTCAAGGCGGTTACGTCCAGGGAGGATCTCAAGGCTTTGTACACGGAGCTGGTCAAGGAGGATATGTGAATGGAGGCAGAATTGTAAAAGTGAAAAACAGGACTATTGTCTACGATCAGAACCATAATATCATATCCCAGACGGAAACTAGCACGGAGTATGGAAAACTTGGTCATGAAGGCGAGCCTGGAAGTTCTTTCCATCTTTACGGCAACGAAGGTGGACAGCAACATAGTGCAACCTTCGCCCATGGCTCCGGAGGTAGTGTCCAGACCTCTGGCCAAGATTACTCCCAGGGAGGCCAGGGGTATGTCCATTCAGGCCAATTCGGTCAAGGAAATCAGCAATTTGCACATGGATCGGGAGGCGAGCAAGCCTTTATCCACGGGTCTTGGGGAACAACAGAAACTGTCAACCCCGATATAATGAAGGCCAGTTCCCCGACCTTCAGAGGAGCATCCACTGTGACTGTTGTCGGTGATGAAGAGGAAGACAAACTTGAAGGGTTTGGAGCGTATGCGGCCAGCAATCCAAACAATGAGTTTAGATACGGCATTGCTGACGTCAGTGGTGCTTCAGGGTCTGCTCAAAGTGGTCAACAATCTGGAAGCGGTAGCTACCAGGCCGGCGGAGGAAGCTACCATGCTAGCGGAGGCGGCATGCAAGTCGCAGGTGGCGGTATGCAGGCCGGTGGTGGTGGCAGCTATCAAGCTGGAGGTTCCAGTATGCAATCCGGAGGCGGCACCTTCCAGACCGGTGTTGGAAGCTACCAATCAGGAGGTAGTTACCAAGGTGGCGGTGGATCTTGGAGCAGCGGATACTCCTATTCATCGAGATCAGGTGGAGGTTCGTCCTATAACTCTCAGTCCAGCGGCGGCTACGGCTCAGTGGACAGCCGGCGTGAGAACACGAGGAGGAGACGGCAGACTGAACAGGTGGACCCTAAACTAAAGGAAATATTGGAGAAATGCGAAGAGAAGTACAAATGCGAAGTACTGCGGTGTACGACCGGCAGATTGCTGAAAGGACAAGAAGTTTGGGTCGCGCTTAGGTCGAGACTGAACGCGTCTGTACTTAACGAGATCTCGAAGGACCGACCGGTGGTCCTGTCAACTCTGACCGCCACGCGCGTCTCCCGGCTGCCCATGGTGGGTCGGCCGAAGGACACGACATGGCAGACGGCGGAGGCGAAAACGACGGTCACTCCTCAGCTGGAGGCTCTGGACTCGGGAACCATCCCTCTGTGGGTGGTGGTGCTGGCGGCTGTCGTCGGTGCACTGTTGCTGCTGCTACTTATCTTCGCGCTGTACAAATGCGGATTCTTCAAGCGCAACCGTCCCTCGGACCACGCCGAGCGCCAGCCGCTGAACGGGCGCGACGAGCACCTCTGATATCCGACCACCGACCTCGGCTTCACCAGCGACACGGCCGGCACCAGTGACCTGCCGCCCACCTCCCACTCCTCGTCACCATCAGACAGCTCCTCGACAGATGCCGCTAGTGATGTCACGAGTCGCGATTCCACCAGCCTCGACAGATACTGACATCATAAACTATTGGTTTATAAATTCTAAATTGCTGGTGAAAATAACATTATAATTGATGGAGTTTTGTCAAATGGTTTTTACAATGACGACGCAACTTGGGGGTGGAAATTTTTGTCCACAATAAAGTATTTCAAAGGCAATAAGTCTAATTAAGGTGCGCAAAATATTCCATATTAGCGACTATTTATTACTGAGTTTTCATCATTTCGTGACATGAAGTTAGTGTAACAGTTTTACCTCATGATATGTCGAAATCGTGGCTCCTTAATCATTTTCGTAGTAAAAATGTTAGAAGGAGCCTTTAAAAATCAGATCGTAATAACATTACTAGCCACCGTCAAACACATTTCGGTAGCCATTTAGTGATTAGTGGAAGAGTTGGAAGTGTATTATAACGTATAATGTAAAATAATTCCCGTTCTCTACTATACTTAATTCTAAATCCTAAAATCTACAGCGGGAATTATGTTACACTATAAAATAGAATGTGAGAATCAAAGTGTGATAATGTAAAAAGTGTGTCTACGTTAAGGTATGAGAGTTGTTGAGATTTGTGATTTAAGTCAATAATAATTAGATACGTTTTACTAAATGAGTTTAGGTATCATAGAATGGTTGCTATTGACGTTTTAAAACATCATTTTGGTAGAGCACGTCCGGGTAAAAACATTGGACTTAGAAATCTCGTGTAGTAAAGTTATATATTTCAGATATTACATCGTAATTCTAATTTTTACTAATAAGAACGCTTATATTAAACTAAGGAGGCATTATCTCACGTTCTTTATAAATATTAAAAACAAAACTTCACTGCCTTACCTTCAGATATATCCAAACGAATTAATTTAATTAAATTCTAAGTATCCGACAGCTAAAGTTCCAATACAGACTGATTTGCAAGATTATATAGATATATATTATAATTCGAGTAGATTTGGCAATAATTATTTTATTAGTGAATTTGATATGTAGAGTAACGCGAATATAGAAGTGCCTTATGCAATACATATCTTGGTCGTGCTAATTTAGATATGCTTGAACTACGTGCCTTCTGCCAGTAACTAATTAATGTAAATACGTTAAGAGTTTTATATTAGTATTTATGACATTTTTTCACAGAAAAAAATACGTATACTTTTTGGCCGCAAGTGTAAAAATATTTGTAGAACTATGTACTTAATATTTTTTTGAGTGAGATGTGTATTTTAAATATTTACGAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | DQ531845 | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha2 RNAi construct | ACCTCTCCAACGGCACTGAGGTGGCGTGCGCAGAGCTCATGTTCTGTCTCACCTACACCGGAGTTAATGTAGACCAGCGGATCAAATTCGAGGTGACCTTAGACCTGGACTCAAGGCAGAAAACGAGCAAGCGCCTGTTCCTAGCAGAGACCCACGAGACCACGTACAAGACGCAGATACTACTGACCCAAGGCCAGCAGGAATGCAAGGACGTGATGGTGTACTTGGATGAGGAGATTAGAGACAAGCTAACGCCCATCGAGGTGAAGATGTCGTACGACTTGATCAATCAGCCGTCCGGCGACGTGGTGCCGCCCGTGTTGGACCAAACCAGGAGCAACTTCCACACCGACTCGTTGAATATTCAGAAGAACTGCGGACCTGATAATATCTGCATACCGGATCTTAGGATGTCTGCTACTACACCGACAGTAAACTACGTCCTAAGTTCCGGA | dsRNA | column-based | simultaneously with transcription | (1) Amplify gene by PCR with forward primer (TAATACGACTCACTATAGGGAGACTCTCCAACGGCACTGAGGTG) and reverse primer (TAATACGACTCACTATAGGGAGATCCGGAACTTAGGACGTAG). (2) PCR product was recovered and used as template for preparation of dsRNA using MEGAsript RNAi Kit (Ambion). | InsectaCentral | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha2 RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | ddH2O | 0.05 | non_specific_dsRNA | 3 | abdominal hemocoels | Green fluorescent protein (GFP) dsRNA was purchased from Dharmacon and used as a control. | larva | qPCR phenotype | hemocytes | not checked | 120 | low | 50 | ||||||
| 181 | Cadherin silencing in adult M.sexta | RNAi | In this work we injected M. sexta adults using a Brinkmann micromanipulator coupled to a 10 µl VWR Digital Microdispensor (VWR Scientific, San Francisco CA). Each adult was injected with 0.5 µg of M.sexta cadherin dsRNA in 10 µl of final volume directly into the hemolymph using 10 µl replacement capillaries for microdispensors (Thoman Scientific,Swedesboro, NJ). The needle was inserted at the dorsal side in the last segment of the abdomen, parallel to the longitudinal axis of the insect, minimizing damage of internal organs. The dsRNA was injected and the needle was left inside the insects for a few seconds before removal. Control adults were injected with water only. After injections of cadherin dsRNA or water only, all adults were kept in the cages until laid eggs. Eggs were recovered and first instar larvae were treated with different concentrations of trypsin activated Cry1Ab toxin from 0 to 10 ng/cm2 of diet. We observed more than 60% mortality of the M. sexta silenced larvae while the control M. sexta injected with water showed 100 % mortality. Survival larvae were feed with artificial diet until they reached third instar. The cadherin protein levels were then determined in third instar M. sexta larvae by Western blot using specific antibody. Efficiency of silencing was estimated to be 28% | InsectaCentral | Unpublished | Bravo | Alejandra | Cadherin silencing in adult M.sexta target | GACCAATCGGAGTGTGGTGAATTTTTGGAAAATATTTTGTGCGGTTCCTTTAGTTGTGTAATATAGTACTTTAGTTACAAATTTGGAATAATTTGGCAGCAAAACCATCTGCAGCAACAAAATCATCTGCAGCTGCGAAATCATCTGCAGCAGCAAAAGCATCTTCAGGAGCGAGAAAAGCCCCAAATAATGTGAGATGGCAGTTGACGTCCGAATCGCTGCCTTCCTGCTGGTGTTTATAGCGCCTGCAGTTTTAGCTCAAGAGAGATGTGGGTATATGACCGCCATCCCAAGGCTACCACGACCGGATAATTTGCCAGTACTAAATTTTGAAGGCCAGACATGGAGTCAGAGGCCCCTGCTCCCCGCCCCGGAGCGGGATGACCTGTGCATGGACGCCTACCACGTGATAACAGCCAACCTCGGCACGCAGGTCATCTACATGGATGAAGAGATAGAAGACGAAATCACCATCGCCATACTTAATTATAACGGACCATCAACTCCGTTCATTGAACTGCCATTTTTATCCGGTTCGTACAATCTGCTGATGCCGGTCATCAGGAGAGTTGACAACGGGGAGTGGCATCTCATCATCACGCAAAGACAGCATTACGAGTTGCCCGGCATGCAGCAGTACATGTTCAATGTGCGCGTGGACGGCCAGTCGCTGGTGGCAGGCGTGTCTCTCGCTATCGTCAACATAGATGACAACGCGCCCATCATACAAAACTTCGAGCCTTGCCGGGTTCCTGAACTGGGCGAGCCAGGGTTGACAGAATGCACATACCAAGTATCGGACGCGGACGGACGGATCAGCACAGAGTTCATGACGTTCAGGATCGACAGCGTTCGTGGCGACGAGGAGACCTTCTACATCGAACGGACGAATATCCCCAACCAATGGATGTGGCTAAATATGACCATAGGCGTTAATACCTCGCTCAACTTCGTCACCAGTCCGCTGCATATATTCAGCGTGACAGCCCTGGACTCGCTCCCGAACACCCACACGGTGACTATGATGGTGCAAGTGGCGAATGTGAACAGCCGTCCGCCGCGCTGGCTGGAGATCTTCGCTGTCCAACAGTTTGAAGAGAAATCTTACCAAAACTTCACAGTGAGGGCGATCGACGGAGACACTGAGATCAATATGCCTATCAACTACAGGCTGATCACAAATGAGGAAGACACATTCTTCAGCATTGAGGCCCTGCCTGGTGGAAAAAGCGGGGCTGTATTCCTCGTGTCGCCAATTGACCGCGACACACTGCAACGAGAGGTGTTTCCACTTACGATCGTCGCTTACAAATATGATGAGGAGGCCTTCTCCACATCAACAAACGTGGTCATCATTGTGACAGACATCAACGACCAAAGACCTGAACCTATACACAAGGAATATCGACTGGCAATCATGGAGGAGACGCCCCTGACCCTCAACTTCGATAAAGAATTCGGATTTCATGATAAGGATTTAGGTCAAAACGCTCAGTACACGGTGCGTCTAGAGAGCGTGGACCCTCCAGGCGCTGCTGAGGCATTCTACATAGCGCCTGAAGTCGGCTACCAGCGACAGACCTTCATCATGGGCACCCTCAATCACTCCATGCTGGATTACGAAGTGCCAGAGTTTCAGAGTATTACGATTCGGGTGGTAGCGACCGACAACAACGACACGAGGCACGTGGGCGTCGCGTTGGTTCACATTGACCTCATCAATTGGAACGATGAGCAGCCGATCTTCGAACACGCCGTGCAGACCGTCACCTTCGACGAGACTGAAGGCGAGGGGTTCTTCGTCGCCAAGGCGGTTGCACACGACAGAGACATCGGGGATGTCGTCGAGCATACTTTATTGGGTAACGCTGTTAACTTCCTGACCATCGACAAACTCACCGGCGACATCCGCGTCTCAGCTAACGACTCCTTCAACTACCATCGAGAAAGTGAATTATTTGTGCAGGTGCGAGCTACAGACACGCTGGGCGAACCCTTCCACACGGCGACGTCACAGCTGGTCATACGACTAAATGACATCAACAACACGCCACCCACCTTACGGCTGCCTCGAGGCAGTCCCCAAGTGGAGGAGAACGTGCCTGATGGCCACGTCATCACCCAGGAGTTACGCGCCACCGACCCCGACACCACGGCCGATCTGCGCTTCGAGATAAACTGGGACACCTCTTTCGCCACCAAGCAAGGCCGCCAGGCTAACCCCGACGAGTTTAGGAATTGCGTGGAAATCGAGACCATCTTCCCCGAGATTAACAACCGGGGACTGGCTATCGGCCGCGTTGTAGCGCGCGAAATCAGACACAACGTGACCATAGACTACGAGGAGTTTGAGGTCCTCTCCCTCACAGTGAGGGTGCGTGACCTTAACACCGTCTACGGAGACGACTACGACGAATCGATGCTCACAATAACTATAATCGATATGAACGACAACGCGCCGGTGTGGGTGGAGGGGACTCTGGAGCAGAACTTCCGAGTCCGCGAGATGTCGGCGGGCGGGCTCGTGGTGGGCTCCGTGCGCGCGGACGACATCGACGGACCGCTCTACAACCAAGTGCGATACACCATTTTCCCTCGTGAAGACACAGATAAGGACCTGATAATGATCGACTTCCTCACGGGTCAAATTTCCGTGAACACAAGCGGCGCCATCGACGCGGATACTCCTCCACGCTTCCACCTCTACTATACAGTGGTCGCTAGTGACCGATGCTCGACAGAAGATCCTGCAGATTGCCCCCCTGACCCGACTTATTGGGAAACCGAAGGAAATATCACAATCCACATCACCGACACGAACAACAAGGTCCCGCAGGCGGAAACGACTAAGTTCGATACCGTCGTGTATATTTACGAGAACGCAACCCACTTAGACGAGGTGGTCACTCTGATAGCCAGTGATCTTGACAGAGACGAAATATACCACACGGTGAGCTACGTCATCAATTATGCAGTGAACCCTCGACTGATGAACTTCTTCTCCGTGAACCGAGAGACCGGCCTGGTGTACGTGGACTATGAGACCCAGGGTAGTGGCGAGGTGCTGGACCGTGATGGTGATGAACCAACGCACCGTATCTTCTTCAACCTCATCGACAACTTCATGGGGGAAGGAGAAGGTAACAGAAATCAGAACGACACAGAAGTTCTCGTTATCTTGTTGGATGTGAATGACAATGCTCCTGAATTGCCACCGCCGAGCGAACTCTCTTGGACTATATCTGAGAACCTTAAGCAGGGCGTCCGTCTTGAACCACATATCTTCGCCCCGGACCGCGACGAGCCCGACACAGACAACTCCAGGGTCGGCTACGAGATCCTGAACCTCAGCACGGAGCGGGACATCGAAGTGCCGGAGCTGTTTGTGATGATACAGATCGCGAACGTCACGGGAGAGCTGGAGACCGCCATGGACCTCAAGGGATATTGGGGGACGTACGCTATACATATACGGGCATTCGACCACGGCATTCCGCAAATGTCCATGAACGAGACATATGAGCTGATCATCCATCCGTTCAACTACTACGCGCCTGAGTTCGTCTTCCCGACCAACGATGCCGTCATACGACTTGCGAGGGAACGAGCTGTAATCAATGGAGTTCTAGCGACAGTGAACGGAGAGTTCTTGGAGCGGATATCGGCGACTGATCCGGACGGACTCCACGCGGGCGTCGTCACCTTCCAAGTGGTAGGCGATGAGGAATCACAACGGTACTTTCAAGTAGTTAACGATGGCGAGAACCTCGGCTCGTTGAGGTTACTGCAAGCCGTTCCAGAGGAGATCAGGGAGTTCCGGATAACGATTCGCGCTACAGACCAGGGAACGGACCCAGGACCGCTGTCCACGGACATGACGTTCAGAGTTGTTTTTGTGCCCACGCAAGGAGAACCTAGATTCGCGTCCTCAGAACATGCTGTCGCTTTCATAGAAAAGAGTGCCGGCATGGAAGAGTCTCACCAACTTCCTCTAGCACAAGACATCAAGAACCATCTCTGTGAAGACGACTGTCACAGCATTTACTATCGTATTATCGATGGCAACAGCGAAGGTCATTTCGGCCTGGATCCTGTTCGCAACAGGTTGTTCCTGAAGAAAGAGCTGATAAGGGAACAAAGTGCCTCCCACACTCTGCAAGTGGCGGCTAGTAACTCGCCCGATGGTGGCATTCCACTTCCTGCTTCCATCCTTACTGTCACTGTTACCGTGAGGGAGGCAGACCCTCGTCCAGTGTTTGTGAGGGAATTGTACACCGCAGGGATATCCACAGCGGACTCCATCGGCAGAGAGCTGCTCAGATTACATGCGACCCAGTCTGAAGGCTCGGCCATTACTTATGCTATAGACTACGATACAATGGTAGTGGACCCCAGCCTGGAGGCAGTGAGACAGTCGGCTTTCGTACTGAACGCTCAAACCGGAGTGCTGACGCTTAATATCCAGCCCACGGCCACGATGCATGGACTGTTCAAATTCGAAGTCACAGCTACTGACACGGCCGGCGCTCAGGACCGCACCGACGTCACCGTGTACGTGGTATCCTCGCAGAACCGCGTCTACTTCGTGTTCGTCAACACGCTGCAACAGGTCGAAGACAACAGAGACTTTATCGCGGACACCTTCAGCGCTGGGTTCAACATGACCTGCAACATCGACCAAGTGGTGCCCGCTAACGACCCCGTCACCGGCGTGGCGCTGGAGCACAGCACGCAGATGCGCGGCCACTTCATACGGGACAACGTACCCGTACTCGCTGATGAGATAGAACAGATCCGTAGTGACCTAGTCCTCCTGAGCTCGATACAAACAACGCTGGCGGCGCGATCGCTGGTGTTGCAGGACTTGTTGACCAACTCCAGCCCGGACTCGGCGCCTGACTCGAGCCTCACGGTGTACGTGCTGGCCTCACTGTCTGCTGTGCTCGGTTTCATGTGCCTTGTGCTACTGCTTACCTTCATCATCAGGACTAGAGCGCTAAACCGACGGTTGGAAGCCCTGTCGATGACGAAGTACGGCTCACTGGACTCTGGATTGAACCGCGCCGGCATCGCCGCCCCCGGCACCAACAAACACACTGTGGAAGGCTCCAACCCTATCTTCAATGAAGCAATAAAGACGCCAGATTTAGATGCCATTAGCGAGGGTTCCAACGACTCTGATCTGATCGGCATCGAAGATCTTCCGCACTTTGGCAACGTCTTCATGGATCCTGAGGTGAACGAAAAGGCAAATGGTTATCCCGAAGTCGCAAACCACAACAACAACTTCGCTTTCAACCCGACTCCCTTCTCGCCTGAGTTCGTTAACGGACAGTTCAGAAAGATCTAGAAGATAACAACACTAGTTAAGATCATTAATTTTGGAGTTTGGAATTAAGATTTTTGAAAGGATAGTTGTGATAAGCCTGTGATTTTTAAAACTGTAATTGAAAAAAAAAATTGAGACCTCCATTTAAGCTCTTGCTCTCATCTCATCAAATTTTATAAAATGCCATTAGTCATTAAGATACTCGATTTAATTTAAGATTATTTAAGATATTATGTAAAATAAATATATTGTC | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | Cadherin silencing in adult M.sexta target | Cadherin silencing in adult M.sexta RNAi construct | GCTGCCTTCCTGCTGGTGTTTATAGCGCCTGCAGTTTTAGCTCAAGAGAGATGTGGGTATATGACCGCCATCCCAAGGCTACCACGACCGGATAATTTGCCAGTACTAAATTTTGAAGGCCAGACATGGAGTCAGAGGCCCCTGCTCCCCGCCCCGGAGCGGGATGACCTGTGCATGGACGCCTACCACGTGATAACAGCCAACCTCGGCACGCAGGTCATCTACATGGATGAAGAGATAGAAGACGAAATCACCATCGCCATACTTAATTATAACGGACCATCAACTCCGTTCATTGAACTGCCATTTTTATCCGGTTCGTACAATCTGCTGATGCCGGTCATCAGGAGAGTTGACAACGGGGAGTGGCATCTCATCATCACGCAAAGACAGCATTACGAGTTGCCCGGCATGCAGCAGTACATGTTCAATGTGCGCGTGGA | dsRNA | HiScribe T7 In Vitro Transcription Kit, New England Biolabs | column-based | simultaneously with transcription | RT-PCR from cDNA produced from RNA samples obtained from 5th instar larvae with primers Rev: 5â-GCTCTAGAGCTGCCTTCCTGCTGGTGTTTA-3â and For: 5â- GGAATTCCTCCACGCGCACATTGAACAT-3â that amplified a 442 bp fragment. The 442 bp PCR fragment was digested with XbaI and EcoRI and cloned into a previously digested pLITMUS 28i (HiScribeTM, New England Biolabs) vector containing two T7 promoters flanking the multi-cloning site. This enabled amplification of the cloned fragment by using a T7 oligonucleotide. The PCR product was purified with QIAquik PCR purification kit protocol (Quiagen). In vitro transcription of both DNA strands of the insert was performed with T7 RNA polymerase using the HiScribeTM transcription system (HiScribe⢠T7 In Vitro Transcription Kit, New England Biolabs) as reported by the manufacturers, yielding dsRNA. | InsectaCentral | Cadherin silencing in adult M.sexta RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | adult | lab_colony | 1 | injection | 0.1 | buffer | 2 | hemolymph | We used the same as used for dsRNA Cadherin | larva | W.blot | midgut | undetected | 72 | low | 30 | |||||
| 185 | Attacin | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | Attacin target | ACGACAACGTCAATGGTCACGGCGCTACTCTTACAAAAACGCATATACCCAGCTTCGGTGACAAGCTGACGGCGGCCGGCAAGCTGAACGTGTTCCACAACGACAACCACAACCTGGACGTGAAGGCGTTGGCCACCAGGACCATGCCGGATATTCCACACGCGCCCGACTTCAACACCTTCGGCGGCGGCGTTGACTACATGTTCAAGGACAAGGTGGGCGCGTCGGCGAGCGCTGCGCACACGCCTCTCTTCGACCGCAGCGACTACTCCGTGGGCGGCAAGCTGAACCTGTTCCGCGACAAGACCACCTCGCTCGACTTCAACGCCGACTACAAGAAGTTCGAGATGCCCAACTTCAAGTCCGACTGGACACCCAACATCGGCTTCTCATTCAGCAAGTTTTGGTAGTTCGGTATTAAGACGGAGTTTTAACAACAGACACTTAAACCATTTCTGTGATATTTCGTGTTTGTAAATAGGAGGTTTTGAATGTCAAGTATTTATATTAAGGTATTTTATTACAAAAATAAATGATATAATATTGTCCC | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | Attacin target | Attacin RNAi construct | ACGACAACGTCAATGGTCACGGCGCTACTCTTACAAAAACGCATATACCCAGCTTCGGTGACAAGCTGACGGCGGCCGGCAAGCTGAACGTGTTCCACAACGACAACCACAACCTGGACGTGAAGGCGTTGGCCACCAGGACCATGCCGGATATTCCACACGCGCCCGACTTCAACACCTTCGGCGGCGGCGTTGACTACATGTTCAAGGACAAGGTGGGCGCGTCGGCGAGCGCTGCGCACACGCCTCTCTTCGACCGCAGCGACTACTCCGTGGGCGGCAAGCTGAACCTGTTCCGCGACAAGACCACCTCGCTCGACTTCAACGCCGACTACAAGAAGTTCGAGATGCCCAA | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | Attacin cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: ATT_F: 5'-GGTCACGGCGCTACTCTTAC-3' and ATT_R: 5'-TTGGGCATCTCGAACTTCTT-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for attacin. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | Attacin RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body | not checked | 18 | high | ||||||
| 186 | Cecropin RNAi | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | Cecropin RNAi target | CAACAACACAATATAACAAAATGAAATTCTCCCGTGTTTTATTCTTCGTCTTCGCTTGCTTCGCCGCATTTACAGTAACTGCGGCCAAGCCATGGGACTTCTTAAAGGAGCTGGAGGGTGCAGGTCAAAGGATTCGTGACGCTATCATCAGCGCGCAGCCGGCGGTGGAAACCATCGCGCAGGCAACCGCCATTTTCAAAGGACAATCAAAAGAAGAAGATTAATTGTGTCATTACAGTATTACATATTTAAGATATAATTTATTTTGACAATATATTCATTTAATTC | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | Cecropin RNAi target | Cecropin RNAi RNAi construct | TTCTTCGTCTTCGCTTGCTTCGCCGCATTTACAGTAACTGCGGCCAAGCCATGGGACTTCTTAAAGGAGCTGGAGGGTGCAGGTCAAAGGATTCGTGACGCTATCATCAGCGCGCAGCCGGCGGTGGAAACCATCGCGCAGGCAACCGCCATTTTCAAAGG | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | Cecropin cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: CEC_F: 5'-TTCTTCGTCTTCGCTTGCTT-3' and CEC_R: 5'-CCTTTGAAAATGGCGGTTG-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for cecropin. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | Cecropin RNAi RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body | not checked | 18 | high | ||||||
| 187 | Lebocin RNAi | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | Lebocin RNAi target | ACGTGCGTAGTGTGAACGAGCCGTCGTCACAGGAGCATCACGAACGCTTTGTGAGGAGCTTCGACTCCCGCAGCAGCAGGCATCACGGCGGCAGCCACTCTACGTCCAGCGGCAGCCGCGACACTGGAGCTACTCATCCGGGATACAATCGTCGTAACTCATAATCTGCGGTTTAATCCATTAGAAATTTGTGTTTGTATTTTGATAAAAACAATGAAACATACATAAAATTGCCTTTTTGAATAAATAAGT | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | Lebocin RNAi target | Lebocin RNAi RNAi construct | ACGTGCGTAGTGTGAACGAGCCGTCGTCACAGGAGCATCACGAACGCTTTGTGAGGAGCTTCGACTCCCGCAGCAGCAGGCATCACGGCGGCAGCCACTCTACGTCCAGCGGCAGCCGCGACACTGGAGCTACTCATCCGGGATACAATCGTCGTAACTCATAATCTGCG | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | Lebocin cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: LEB_F: 5'-ACGTGCGTAGTGTGAACGAG-3' and LEB_R: 5'-CGCAGATTATGAGTTACGACGA-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for lebocin. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | Lebocin RNAi RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body | not checked | 18 | high | ||||||
| 188 | Lysozyme RNAi | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | Lysozyme RNAi target | ACTCCCGGCAAAGACTGCAATGTCAAATGTAGTGACTTGCTGATTGACGACATCACCAAGGCATCGACTTGCGCCAAGAAGATATACAAACGCCACAAGTTCCAAGCGTGGTACGGATGGCGCAACCACTGCCAGGGCTCCCTGCCTGACATCAGCTCCTGTTAGATATCTCGTCAGTGAAGATCAGAGACAGTCCTGAAACCCATCGTAAATATTTCCACCTCCTCAGTATCTTCTATTCCTTTAATTTCGCAACAGTTTAAAATTAATTCGCGGATCTACACCACTTGGTCTTCTAAAATGATTGCCGCTGTTTGGCCTGGTTTACAGAAGTTGCAGTTGTGAAAGAGGGATAATTCCACCTTTAAGCATGTCGTCCTTGGTTACAAGCCTTTGGCGATATGGCTCATTTAAATTCAAATCTCGCGCGCACTAAACCTGTAAAACTGTTTTTACACTCATCTTACCTAGAACCGGCTCTGATCTTATTAATATATCATGTGCTATATTTTAGAAGGTGGGTATACAAACACCTGTTTATTCTCGAAACGCTTATTTATCGAATAAATATTTTTTTTTNCGA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | Lysozyme RNAi target | Lysozyme RNAi RNAi construct | TACAAACGCCACAAGTTCCAAGCGTGGTACGGATGGCGCAACCACTGCCAGGGCTCCCTGCCTGACATCAGCTCCTGTTAGATATCTCGTCAGTGAAGATCAGAGACAGTCCTGAAACCCATCGTAAATATTTCCACCTCCTCAGTATCTTCTATTCCTTTAATTTCGCAACAGTTTAAAATTAATTCGCGGATCTACACCACTTGGTCTTCTAAAATGATTGCCGCTGTTTGGCCTGGTTTACAGAAGTTGCAGTTGTGAAAGAGGGATAATTCCACCTTTAAGCATGTCGTCCTTGGTTACAAGCCT | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | Lysozyme cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: LYS_F: 5'-TACAAACGCCACAAGTTCCA-3' and LYS_R: 5'-AGGCTTGTAACCAAGGACGA-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for lysozyme. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | Lysozyme RNAi RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body | not checked | 18 | high | ||||||
| 189 | Moricin RNAi | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | Moricin RNAi target | AATTGCAAGAGTCGTCNGNAAAATATTGTTAAANTGAAGTTAACAAGTTTATTTATTTTTGTTATTGTCGCGTTGTCGCTATTATTTTCGAGTACCGACGCAGCACCGGGTAAAATTCCCGTGAAGGCTATAAAGCAGGCTGGCAAGGTCATTGGAAAAGGTCTACGAGCAATAAATATTGCTGGCACCACACACGATGTTGTTAGTTTCTTCAGGCCTAAGAAGAAGAAGCACTAGACGTGTAATGTTTAAACGATAACTTTACAAATTGATATTATATAGTAATTACTTAATAGTGTTTTGTAATGGTATTTATTTTATGTACGAAAATTGCAACTAATTTTATGATGTTTTGACTAAGTTATGCCTGCCGTTCTCCCTTCGGAATCTTCTGTGAATTCTCAGTTAAACAATGTAAAATAATTACCTTTTGCATAAAATATTCAACAAGACATGGAATATTATTTTAACGAAAACTGCTTTCTTTAACCTTTGGTCCTCATTTCAAAATAACTTTTGTTTTCACATCAAAGTTNATGGTCCCCGAACTTTATACAGTGCATTCGGTTTATTTTTCATCTTTCAATATAGATTGA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | Moricin RNAi target | Moricin RNAi RNAi construct | TCGCGTTGTCGCTATTATTTTCGAGTACCGACGCAGCACCGGGTAAAATTCCCGTGAAGGCTATAAAGCAGGCTGGCAAGGTCATTGGAAAAGGTCTACGAGCAATAAATATTGCTGGCACCACACACGATGTTGTTAGTTTCTTCAGGCCTAAGAAGAAGAAGCACTAGACGTGTAATGTTTAAACGATAACTTTACAAATTGATATTATATAGTAATTACTTAATAGTGTTTTGTAATGGTATTTATTTTATGTACGAAAATTGCAACTAATTTTATGATGTTTTGACTAAGTTATGCCTGCCGTTCTCCCTTCGGAATCTTCTG | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | Moricin cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: MOR_F: 5'-TCGCGTTGTCGCTATTATTTT-3' and MOR_R: 5'-CAGAAGATTCCGAAGGGAGA-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for moricin. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | Moricin RNAi RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body | not checked | 18 | high | ||||||
| 190 | PPO RNAi | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | PPO RNAi target | GGAACTGTGAAGTGGGTATACTAAAAAGTTTTTTTTTTATTTATTTAAAAATACGTTTATCAGACACCATGGCTGATATTTTTGATAGTTTCGAACTCCTCTACGACCGTCCAGGAGAACCTATGATTAACACCAAAGGGGAAGACAAAGTTCTATTCGAACTCACTGAACAATTTCTGACCCCGGAATACGCCAACAATGGTCTGGAGTTGAATAACCGCTTCGGTGATGAGGAGGAGGTGTCTCGTAAAATAATATTGAAGAATCTTGACAAGATTCCAGAGTTTCCTAAAGCTAAACAACTCCCAAACGATGCCGATTTCTCCCTTTTCCTGCCCAGCCATCAAGAAATGGCTAATGAAGTCATTGATGTCCTAATGAGTGTAACTGAGAACCAACTACAAGAACTCCTGTCTACTTGTGTGTATGCCCGAATCAATCTCAACCCGCAGCTGTTCAACTACTGCTACACTGTTGCCATTATGCACAGACGTGACACGGGTAAGGTTCGTGTACAGAACTATGCAGAAATTTTCCCTGCAAAGTTTTTGGACTCTCAAGTATTCACCCAGGCCCGTGAAGCTGCAGCAGTCATCCCGAAAACTATTCCTCGAACTCCTATCATCATTCCACGAGACTACACTGCTACCGACTTGGAAGAAGAGCATCGCCTGGCGTACTGGCGTGAAGACCTCGGTATCAATCTTCACCATTGGCATTGGCATCTCGTTTACCCGTTCTCTGCTAGCGACGAGAAAATTGTAGCCAAAGACCGTCGCGGGGAGCTGTTCTTCTACATGCACCAGCAAATCATTGCCAGATACAACTGCGAGCGTTTGTGCAATTCTCTGAAGAGGGTAAAGAAATTCAGCGACTGGCGCGAGCCTATCCCGGAGGCATATTACCCTAAACTAGACAGCTTGACTTCAGCCCGCGGCTGGCCGCCACGCCAAGCTGGTATGCGTTGGCAAGACCTGAAGAGACCTGTGGACGGTCTAAACGTTACAATTGATGACATGGAACGTTACAGGAGGAACATTGAAGAGGCTATTGCTACTGGTAACGTTATATTGCCAGACAAATCTACCAAAAAGTTGGATATCGATATGCTCGGCAACATGATGGAAGCAAGCGTCCTGTCACCCAACCGCGATTTGTATGGCTCCATCCACAACAACATGCACAGCTTCAGCGCGTACATGCACGACCCCGAACATCGATACCTCGAATCATTCGGTGTGATCGCTGATGAAGCGACGACGATGCGCGATCCGTTCTTCTACCGCGTGCATGCGTGGGTCGATGACATCTTCCAGAGTTTCAAAGAGGCCCCTCATAACGTGCGCCCATACAGTCGCTCTCAGCTCGAGAACCCTGGCGTTCAAGTGACATCCGTCGCGGTGGAGTCTGCTGGCGGCCAACAGAACGTGCTGAACACTTTCTGGATGCAGAGCGATGTGAACCTCTCTAAAGGTTTAGATTTCTCGGACCGCGGGCCGGTGTACGCGCGCTTCACTCATCTCAACCACAGACCTTTCCGTTACGTTATAAAGGCGAACAACACTGCGTCCGCTCGCCGCACGACCGTGCGCATCTTCATAGCCCCGAAGACAGACGAGCGCAACCTGCCGTGGGCTCTGTCCGACCAACGCAAGATGTTCATTGAGATGGATAGATTCGTAGTACCCTTGAGCGCTGGCGAAAACACAATTACTCGTCAGTCCACAGAATCGTCACTGACAATTCCGTTCGAACAGACGTTCCGCGACCTTTCCATCCAGGGAAGTGACCCTAGGCGTTCGGAACTCGCCGCATTTAATTACTGCGGGTGTGGCTGGCCGCAGCACATGCTCGTGCCTAAGGGAACGGTCGGCGGTGTTGCCTATCAACTCTTTGTAATGCTTTCCAACTACGAACTGGATAAGATTGAGCAACCAGACGGCAGGGAACTGAGTTGCGTCGAAGCTTCCATGTTCTGTGGCTTGAAGGATAAGAAGTACCCAGACGCACGTCCGATGGGCTATCCGTTCGACCGGCCATCCAACTCTGCTACCAACATCGAAGACTTCAGCGCGATGTCCAACATGGGACTGCAAGACATAGTCATCAAACTATCCGATGTAACCGAACCAAACCCTAGAAACCCACCCGCTTAAACTACCAAGCTTGCTAAATCATTGTGAATTTACTATCACAAAGGATTTTTGTTTATAATGATACTTTATGATACTTAAGTATTACATTTATAATTGAATTTTAAGCTTGAATTATCCTCATAGTGTTTTTCTTTTAATTTTACTTTTCTTTAAAAATAAAAAATGAAAGGCTGATAGTGTTTATCTTGTAAATTCTTAAGTGTAAATATGTTATATATGTTTGTAGAGTTATGTAACTATTATTGACCCATTTTATAAGGAATAAGACAATAAATAAAACAATGCATTTAT | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | PPO RNAi target | PPO RNAi RNAi construct | AAACAACTCCCAAACGATGCCGATTTCTCCCTTTTCCTGCCCAGCCATCAAGAAATGGCTAATGAAGTCATTGATGTCCTAATGAGTGTAACTGAGAACCAACTACAAGAACTCCTGTCTACTTGTGTGTATGCCCGAATCAATCTCAACCCGCAGCTGTTCAACTACTGCTACACTGTTGCCATTATGCACAGACGTGACACGGGTAAGGTTCGTGTACAGAACTATGCAGAAATTTTCCCTGCAAAGTTTTTGGACTCTCAAGTATTCACCCAGGCCCGTGAAGCTGCAGCAGTCATCCCGAAAACTATTCCTCGAACTCCTATCATCATTCCACGAGACTACACTGCTACCGACTTGGAAGAAGAGCATCGCCTGGCGTACTGGCGTGAAGACCTCGGTATCAATCTTCACCATTGGCATTGGCATCTCGTTTACCCGTTCTCTGCTAGCGACGAGAAAATTGTAGCCAAAGACCGTCGCGGGGAGCTGTTCTTCTACATGCACCAGCAAATCATTGCCAGATACAACTGCGAGCGTTTGTGCAATTCTCTGAAGAGGGTAAAGAAATTCAGCGACTGGCGCGAGCCTATCCCGGAGGCATATTACCCTAAACTAGACAGCTTGACTTCAGCCCGCGGCTGGCCGCCACGCCAAGCTGGTATGCGTTGGCAAGACCTGAAGAGACCTGTGGACGGTCTAAACGTTACAATTGATGACATGGAACGTTACAGGAGGAACATTGAAGAGGCTATTGCTACTGGTAACGTTATATTGCCAGACAAATCTACCAAAAAGTTGGATATCGATATGCTCGGCAACATGATGGAAGCAAGCGTCCTGTCACCCAACCGCGATTTGTATGGCTCCATCCACAACAACATGCACA | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | PPO cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: PPO_F: 5'-AAACAACTCCCAAACGATGC-3' and PPO_R: 5'-TGTGCATGTTGTTGTGGATG-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for PPO. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | PPO RNAi RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body, hemocytes, hemolymph | not checked | 18 | high | ||||||
| 191 | PSP RNAi | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | PSP RNAi target | TGCGCGTATAGTTTTTCCAGATGACGATCAAGATGTTGCGCCAAGGCGTGAGCAACCTAAAATAACCACAAAGGCACCTACGACAGAGGCACCGACCACGACAACCACTGCTGCAAACACTGCTGAGACAACGACCACAAAAGAAGGAAGAGAAAATTATTTATTAGGGTAAGCGCATAGAATACGCCGTCAAAATGAAGTTATTTTTTATAGTTTCGTGCGTTGCAATATTAGCAATTAGCTGTTCTAGTCCGGTTAATGGAGATTTGAATGGATTTTTTGGTGATATTCACAAGAAGGTGCATCATGCCGGGCGTGGTGTGGGAAAGTTATTTAAATTGGAAAATTCGGATACGCGCAATGAAGCGAGGATAGTTTTTCCAGATGACGATCAAGATGTTGCGCCAAGGCGTGAGCAACCTAAAATAACCACAAAGGCACCTACGACAGAGGCACCGACCACGACAACCACTGCTGCAAACACTGCTGAGACAACGACCACAAAAGAAGGAAGAGAAAACTTTGCAGGGGGTTGTGCCACGGGCTTCTTGCGTACTGCGGATGGTAGATGCAAACCTACATTTTGACTAAATAATTAAAAACAAAACATGATTAATAAATTCAACATAACTATTTTTTATTTGATTTTTAGTAAAATATTAAAATATTAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | PSP RNAi target | PSP RNAi RNAi construct | ATGAAGTTATTTTTTATAGTTTCGTGCGTTGCAATATTAGCAATTAGCTGTTCTAGTCCGGTTAATGGAGATTTGAATGGATTTTTTGGTGATATTCACAAGAAGGTGCATCATGCCGGGCGTGGTGTGGGAAAGTTATTTAAATTGGAAAATTCGGATACGCGCAATGAAGCGAGGATAGTTTTTCCAGATGACGATCAAGATGTTGCGCCAAGGCGTGAGCAACCTAAAATAACCACAAAGGCACCTACGACAGAGGCACCGACCACGACAACCACTGCTGCAAACACTGCTGAGACAACGACCACAAAAGAAGGAAGAGAAAACTTTGCAGGGGGTTGTGCCACGGGCTTCTTGCGTACTGCGGATGGTAGATGCAAACCTACATTTTGA | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | Pro-PSP cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: proPSP_F: 5'--3' and proPSP_R: 5'--3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for proPSP. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | PSP RNAi RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body, hemocytes, hemolymph | not checked | 1 | high | ||||||
| 192 | NOS RNAi | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | NOS RNAi target | GAACTCTATCGGTATGCATATATATTATGCTCCGAGACTCGCTCCCGTCAGTCAAGCAGTCTCGCACGCAAGCGACATTACAGTGCGGTCAGAAAGCAAATGTCAAATATATTTAAAATTTGTTGCATGAAGGATCCCAGCGGATCGGTTAAGGACACTCTTGGAAAAATGGAACACGTTAACGGTCATTTCGTTCCCTCAAAATGTCCTTTTAGTGGTGAAAGTGATTTCAAAGTTGACAATCAGAAAACAGTGAAGCCGAACCTTCGTATAAAAGTTCCTCAACCGATAAGACTGAAGAATCATCTCGTGAACGAAGAGAATTTCGACACGCTACATTCTCGAATTGATGAAGTCACTAGTTTTAACACAAAATGCACAGAGAAAGTATGCCAGACCAGCCTCATGGACATTCCGAACCGAGGAGACACGCCGCGAACTGCCGAAGAAGTATTCCAAGACGCTCAGACTTTCCTTAGACAATACTTCGCCTCTATACGCAGAGAGAATTCCGAGGCCCACACTGCCCGACTGGAGGAGGTAAAAAGGGAATTGAAAGATAAAGGCTCATACCAACTTAAAACTTCGGAATTGGTGTTTGGGGCGAAATTGGCATGGCGAAATGCGACGCGGTGCATCGGCCGAATACAGTGGAAGAAACTTCAGACATTCGATTGCAGAGAAGTTACGACTGCAAGTGGGATGTTTGAGGCGTTATGCAATCATATCAAATATGCAACGAATAAAGGCAATATAAGGTCAGCAATAACCATATTTCCACAACGTACAGACGGCAAGCATGATTATAGAATATGGAATCCCCAATTAATCGGTTACGCAGGATATCAAGAGCCGGATGGAAGTATTTTGGGTGATCCAGCACGCGTTGAGTTCACTGAGGTATGTTTGAAATTGGGATGGAAGCCTGCTCGAACAGCATGGGATATACTACCGTTAGTACTATCAGCAGATGGTAAAGATCCGGAGTACTTTGAAATACCTCGCGAAATTGTCATGGAAGTGCAGATCGTGCATCCCAAATACGACTGGTTCAAAGAGCTAGGACTGCAATGGTACGCTTTGCCAGCTGTTTCCAATATGAGGTTGGATTGTGGAGGGCTGGAGTTCACAGCAACTGCCTTCAACGGCTGGTACATGGGCACCGAGATCGGCTGCAGGAATTTCTGCGATAGCAATCGTCTCAATGTTGTTGAGAACGTAGCAAGGCAGATGGGTTTGGACACGAACTCTTTCGTTTCACTGTGGAAAGACAAAGCGTTAGTGGAGGTGAACATAGCGGTACTCCACAGTTATCTTAGAGATAATGTCTCCATCGTAGATCATCATTCCGCCTCGGAACAGTTCCTCAAGCATCTTGACAATGAGAATAAAAGCAGGGGTGGCTGTCCGGCCGATTGGATATGGATTGTTCCACCGATGTCGTCTTCCTTAACATCAGTGTTCCACCAAGAAATGGCGTTATATTACATAAGACCATCCTATGATTATCAGGAGCCCGCGTGGAAGACGCATCAGTGGACGAAGTCCGACGGCACTAAAGCGGTGACCAGAAAATACCATTTCAAACAGATAGCCCGCGCTGTTAAGTTCACTTCCAAACTCTTCGGACGAGCCCTCTCTAAGCGCATCAAGGCGACCATTCTCTACGCCACGGAGACAGGCAAATCTGAACAGTATGCCAAAGAGCTAGGAACCATTTTCGGCCACGCATTTAATGCTCAGGTCCATTGCATGTCTGAATACGACATGTTCTCTATAGAACACGAGACATTGCTTTTGATTGTTACATCTACCTTCGGGAACGGAGAACCCCCTGCAAACGGAGTCGACTTTACTGAACATCTGTTCCAAATGTTATACAACGAGTCTAAAAATCAAGGAGACCAGACGGGTGATTTAGGTTCAGGAAATTTCAAGACACAAACTCCGAAGTCCCTGATGAGAACTAATAGTATGATGACACCAAGTTTTGAATATAAGAGACAGTTAACGAGACTGGAGTCAAACAAAAGCAGTATAGCTGGAACATCAACAGCAGAACAAATAGGACCTCTAAGCAACGTCTGTTTTGCGGTCTTCGCTTTAGGGTCTAGTGCTTATCCAAAATTCTGCAATTTTGGAAAGACAGTCGACAAAGTACTGGGTGATTTGGGAGGCGAAAGGATTTTGGAATTAGCATGCGGCGATGAATTGTACGGGCAGGAGCAACAATTTAGGACTTGGTCGTCGAATATCTTCCATGTGGCCTGTGAGACCTTCTGCTTGGACGAAAACGATATGGTCAAAGATGCAAAGAAAGCATTAGGCACAGTTCCATTAACAGAAGAAACCGTACGATTCGGTAAACCAACTACAAACCCGACCCTCAAAACAGCTTTGGAAGCAGGGTTTAGAAAACAACTTATCGTTTGTAAAGTTAAAGAGAACAAGCATTTGGGAGATTATTCAGTAGATCGAGCAACCATATTCGTTGATATGGAACCACAAAGTGAATTCAAGTACGACCCAGGTGACCACGTGGGCGTGATGGCGTGCAATCGCAAGGAGATTGTTGACGCTGTACTGAGCCGGACGAAGGATGATGACAATTACGATAAACAAGTGCAATTGCAGGTCATGAAGGAAACTCTCACGCCTACTGGCGCAGTTAAGACATGGGAACGACACGAAAGAATCCCAGCTGTGACAGTACGAGAAATATTCACTCGTTTCCTGGATATAACAACACCGCCGTCGACGACTGTTCTCAAATATCTCGCCAATTCGTGCACCGATCAACAAGACGCTGAGAAACTTCTAGAATTGGCCACGGATTCCAACAAGTACGATGACTGGAGGCATTTTCATTATCCAAATTTAGCTGAAGTGCTCGCGCAATTTCCTTCGTGCAAACCGCAGGCGTCTCTGTTGGCCGCGCTACTACCGCCCTTACAGCCGAGGTTCTATTCAATATCGTCCTCACCAGTCGCACATCCGGAGAGAATCCATGTCACTGTTGCTATAGTCGTGTACAACACACAGAATGGAAAAGGCCCCACACATTACGGAGTCTGCTCTACGTACCTACAAAGCCTCAAACCAGATGACGAAGTTTTCGTGTTTATTAGACGTGCACCAAGCTTCCACATGCCTAAAGACGTGTCAGCACCGCTTATCCTAGTCGGACCTGGTTCAGGAGTCGCTCCGTTCCGAGGGTTCTGGCATCATAGACGGCACCAGATGAAAAACCTAGTGCCAAACAATAAGAAAGCTGGTCATATGTGGTTGTTCTTCGGATGCCGACACAGTGGCATGGATTTATATAAAGACGAGAAAGAGGCAGCTGTGAATGAAGGCGTTCTTACTAAGACTAGATTGGCGCTTTCAAGGGAAAAGGGAATTGAGAAGAAACATGTGCAAGCTCTTCTAGAAGAAGAAGGAGCCGAAGTAACAAGAATGTTGTTAGACGAAGAAGGACATTTCTACGTTTGCGGAGATTGCAAAATGGCGGAGGAAGTTCAACAAAAACTAAAATTCATCATTAAAAAACACGCAAAAATGACTGAGCAGGAAGTTGAAGAATTTATTTTCAGTTTAATGGATGAAAATAGATATCACGAAGACATCTTTGGCATCACCCTGCGTACTGCAGAGGTCCACAGTGCATCTAGGGAATTCGCGAAACGGACACGACAAGAGTCTTTGCAATCGCAAGCTTGAAGCATTTCGTTTAAATAATATAACAAGGACTGATAATATTAACTGAAGAAAATTTTATCACAAGCCATTTAATTAATCTAGATATTTGCTTACTGAATAAGTCTTCAAATGATAGAAGCGTTGTGTTGTAATATAAGCATCTAGATAATTGGTAATACTTCAATCGTTCATTAAATATAAACGGTAATGACCACATTATAAGGTACATTAATTTGGAAATAATTAATTATATAACGCCTGTCAAAATGTAAATTTTACTGTCCTAATACTTATTTGATCTGTGAAAATCAGCCCCACACAATAAAATACACCTAATTATAACAGATCTAATCGCTTATATTCTATAAGACGCGTTATTTTATTTATGAATCAGTAATCACAAATTTTAGTGCTATTTTCTACATGATAATAAGAACATATATACACTTTAAAAGTTTTGGCATACCAAGCATCGACGCAAGTCAATAAGACTAAATTATTTGGCCGACTGCCGTGTTATAGAGATATAATATGATACCGTTAAAATAATAAAATTATTGCATTATTTCACTAATTTTATTATAATTGTATTTACATTATAGTACTATAATTTTAATTATACGCATTTAATAGCTTAGTTTACACCCGTAAGTTAAAAGAACAGAACTTTTGGTTAGGAATTTACATGCCTAACCAAAACAAAGACAAAGTAAAGTAAAGTAACAAAGTAATTACATAAAAATTTTATATTGTGCGTTAATTTTGAAATATTTTCATCGATTCGATTAGTATGTAATACTTGATTATTTATAACTAAAAGCATCACAAAATAATTGCCTAAATACATTTATGATGAAGATTGTCTTTGATTCGTAGTTCATCCAGAGATTAATTTACGAAAACTTAGAGCAGTTATTATTTAGAAGTTAAATAATATTATATGCGACCAATGGCTTAATATTTAAGTATAATGTATGTTAATTATAAGGTTCAAAACCATTTTGAACCTTGAGAACAATATTATAAACGTCATTTTTATTAATATAGATACATGAGAATATTATCTAAACTAATCTAAGTATTTTAATTCTACCTAAGTTTAATTGTTAAGGCTATTAGTTTATATTTTTTATTGATACATTATTTAAATTTGTTTGCATAAATTCAAGCAGCTTGCGTCAAGTATACCACAATTATTTATTAATAAATCATGTATCGAGGATTTCATAACAAAGAAGTGTATGGACAAAACATCGGTTTTGGAGGAGCGACAGAAATGTGACAATAGCAAAACTAATTGATTGTATATCAAATATGTTATGTTTAGAAAGATGCGTGTACCAAATTATAGCCTTATACAATTAACATCGTATTACTTTCGACTTCGCCCGCGTGAACAGTCTTTTCTGCTACAAAATAGCCATTTTTACCACCATTTACGCCATCTATACCACTATTTAAAATCCATCATTTTCCATTTAAGCAAATTACCGAAAAAAAAAGCCTACGTGTTAATCTAGGACATGGTTTGCCCCTACGCTAAATTCCATCTAAATCAGTTTAGGTTTCTGTGTTTAGTTCTAACAAGCATCCAAATATGTATAGCGAAGTATATGGGCGCCACTGTAGAGGAACAGTTGGTTGCGTTGCTCCTTGGAAGGGGAAGATCTCCAGTCAGACAGATATTTCGATTGATCGGCCGCGGCCTACGGTTTCTATTCGGAGGTGGTATAAATATTGCGCGTCGCGTTAATGTTGCTATCAATTTAGTATTGACTAGTGCCATCTGTTCTTAACATTGGGAGTTATTTCCTAATTGTCAATATCATTGACAGATGAGCAGTTATGTGTTCTAATAATTATCTATATCATCTTTTAGTTCCAGTGATATGATCAGATGGCGCTGTATGTTAATAGTGCCGCCCCACGTCCAAACCTCACAATCTTTCATATTTACGATATTAGTAAAAAGCATATTTTTTTTCATATCTTTGTCGCTCCCACAAACACTTTTATTTGTTATGAAGCCCTCGATATAACTTCCAACCAGTATTACTACAATGTTTAAGTCCTCCACGACATTTGAATTATCTAAGAAATTTAATAAAACAAAATAATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | NOS RNAi target | NOS RNAi RNAi construct | ACGCAAGCGACATTACAGTGCGGTCAGAAAGCAAATGTCAAATATATTTAAAATTTGTTGCATGAAGGATCCCAGCGGATCGGTTAAGGACACTCTTGGAAAAATGGAACACGTTAACGGTCATTTCGTTCCCTCAAAATGTCCTTTTAGTGGTGAAAGTGATTTCAAAGTTGACAATCAGAAAACAGTGAAGCCGAACCTTCGTATAAAAGTTCCTCAACCGATAAGACTGAAGAATCATCTCGTGAACGAAGAGAATTTCGACACGCTACATTCTCGAATTGATGAAGTCACTAGTTTTAACACAAAATGCACAGAGAAAGTATGCCAGACCAGCCTCATGGACATTCCGAACCGAGGAGACACGCCGCGAACTGCCGAAGAAGTATTCCAAGACGCTCAGACTTTCCTTAGACAATACTTCGCCTCTATACGCAGAGAGAATTCCGAGGCCCACACTGCCCGACTGGAGGAGGTAAAAAGGGAATTGAAAGATAAAGGCTCATACCAACTTAAAACTTCGGAATTGGTGTTTGGGGCGAAATTGGCATGGCGAAATGCGACGCGGTGCATCGGCCGAATACAGTGGAAGAAACTTCAGACATTCGATTGCAGAGAAGTTACGACTGCAAGTGGGATGTTTGAGGCGTTATGCAATCATATCAAATATGCAACGAATAAAGGCAATATAAGGTCAGCAATAACCATATTTCCACAACGTACAGACGGCAAGCATGATTATAGAATATGGAATCCCCAATTAATCGGTTACGCAGGATATCAAGAGCCGGATGGAAGTATTTTGGGTGATCCAGCACGCGTTGAGTTCACTGAGGTATGTTTGAAATTGGGATGGAAGCCTGCTCGAACAGCATGGGATATACTACCGTTAGTACTATCAGCAGATGGTAAAGATCCGGAGTACTTTGAAATACCTCGCGAAATTGTCATGGAAGTGCAGATCGTGCATCCCAAATACGACTGGTTCAAAGAGCTAGGACTGCAATGGTACGCTTTGCCAGCTGTTTCCAATATGAGGTTGGATTGTGGAGGGCTGGAGTTCACAGCAACTGCCTTCAACGGCTGGTACATGGGCACCGAGATCGGCTGCAGGAATTTCTGCGATAGCAATCGTCTCAATGTTGTTGAGAACGTAGCAAGGCAGATGGGTTTGGACACGAACTCTTTCGTTTCACTGTGGAAAGACAAAGCGTTAGTGGAGGTGAACATAGCGGT | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | NOS cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: NOS_F: 5'-ACGCAAGCGACATTACAGTG-3' and NOS_R: 5'-ACCGCTATGTTCACCTCCAC-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for NOS. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | NOS RNAi RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) W.blot phenotype | Fat body, hemocytes, gut | not checked | 24 | high | ||||||
| 193 | Arylphorin-alpha | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | Arylphorin-alpha target | ATGAAAACTGTCGTAATCCTAGCGGGGCTGGTGGCCCTGGCACTCAGCAGTGCTGTACCACCTCCCAAGTATCAGCACCATTATAAGACCAGTCCGGTGGACGCCATATTTGTAGAAAAACAAAAGAAAGTTTTTTCTCTTTTCAAAAATGTAAACCAACTCGACTACGAGGCTGAGTATTACAAAATCGGCAAGGACTATGACGTTGAAGCTAACATTGACAACTACTCGAACAAGAAAGTAGTCGAAGACTTCTTGCTGCTATACAGAACTGGCTTCATGCCGAAGGGCTTTGAATTCTCTATTTTCTACGAAAGAATGAGGGAGGAAGCCATCGCTCTTTTCGAGTTGTTCTACTACGCTAAGGACTTCGAAACTTTCTACAAGACAGCTAGCTTCGCCCGTGTTCACGTTAACGAGGGCATGTTCTTGTATGCATACTACATTGCGGTCATCCAACGTATGGACACTAATGGTTTAGTTCTCCCAGCGCCTTACGAAGTCTACCCACAATACTTCACCAACATGGAAGTCCTATTCAAGGTCGACCGTATAAAGATGCAGGACGGTTTCCTTAACAAGGATCTCGCCGCGTACTACGGCATGTATCATGAAAATGACAACTATGTATTTTATGCCAATTACTCCAACTCTCTCAGCTACCCCAACGAGGAGGAAAGGATTGCATACTTCTACGAGGACATTGGTCTAAACTCGTACTACTACTACTTCCACATGCACTTGCCTTTCTGGTGGAACTCGGAGAAATACGGCCCATTCAAGGAACGTCGTGGTGAAATCTACTACTACTTCTATCAGCAATTGATCGCCCGTTACTACCTGGAGCGTCTTACTAACGGCTTGGGTGAAATTCCTGAATTCTCCTGGTACTCTCCAGTGAAGACCGGCTACTACCCCATGCTCTACGGTTCCTACTATCCATTTGCTCAAAGGCCCAACTATTACGATATTCACAATGACAAGAACTACGAGCAAATCCGTTTCTTAGACATGTTTGAAATGACCTTCTTGCAATACCTGCAGAAGGGACACTTCAAGGCTTTCGACAAAGAAATTAACTTCCATGACGTAAAGGCTGTTAACTTCGTGGGCAACTACTGGCAGGCTAATGCTGACTTATACAACGAAGAAGTTACCAAACTCTACCAACGTTCCTATGAAATCAATGCTCGTCATGTACTTGGTGCTGCTCCTAAACCTTTCAACAAGTATAGCTTCATTCCAAGTGCACTTGACTTCTACCAAACTTCGCTGCGTGACCCTGTCTTCTACCAGCTCTACGACAGGATCATTAACTACATCAACGAATTCAAACAATACTTACAGCCCTACAACCAAAACGATTTGCACTTCGTTGGTGTAAAGATTAGCGACGTCAAAGTAGACAAATTGGCCACATACTTCGAATATTACGACTTTGACGTCAGCAACAGCGTATTTGTAAGCAAGAAGGACATCAAGAACTTCCCCTACGGTTACAAGGTCCGTCAGCCCCGCCTTAATCACAAACCATTCTCTGTCTCCATCGGCGTCAAATCTGATGTTGCTGTTGATGCAGTTTTCAAGATTTTCTTAGGTCCTAAATACGACAGCAATGGCTTCCCAATTCCATTGGCTAAGAACTGGAATAAATTCTACGAACTGGATTGGTTCGTGCACAAGGTCATGCCTGGGCAGAACCACATCGTCCGCCAGTCCAGTGACTTCCTATTCTTTAAGGAGGACTCTCTGCCCATGTCTGAGATCTACAAACTGCTTGACGAAGGCAAAATACCCTCTGACATGTCAAACTCATCCGACACGCTGCCTCAGAGATTGATGCTGCCAAGAGGTACCAAGGACGGTTATCCATTCCAATTGTTCGTGTTCGTGTACCCGTACCAGGCTGTGCCTAAGGAGATGGAGCCATTCAAGTCGATTGTGCCAGACAGCAAGCCTTTCGGATATCCGTTCGACCGCCCCGTCCACCCTGAATACTTCAAGCAGCCCAACATGCACTTCGAGGATGTGCACGTTTACCACGAGGGAGAACAGTTCCCGTACAAGTTCAACGTTCCTTTTTACGTTCCGCAAAAGGTTGAGGTTTGAACGTTAATTTAGAATAGCTTGAAAGGTATCGTGTCTTTTAATTACAACTTGGATATGTATATAATTTAAATGGACAAAGAAATATATGTATAATTTAT | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | Arylphorin-alpha target | Arylphorin-alpha RNAi construct | TGGCAAGGAATACAACATCGAGGCTAACATCGACAATTACTCGAACAAGAAGGCCGTCGAAGAATTCTTGCAGTTATACAGGACAGGTTTCTTGCCTAAGTACTATGAATTTTCACCCTTCTATGACAGACTAAGGGACGAGGCCATTGGTGTTTTCCACCTCTTTTACTACGCTAAAGATTTTGATACGTTCTACAAATCTGCCGCATGGGCGCGTGTGTACCTCAACGAAGGACAGTTCTTATACGCCTACTACATTGCTGTGATTCAGCGTAAAGATACTCAGGGCTTCGTTGTACCAGCACCGTATGAAGTCTACCCTCAATTCTTCGCAAACTTGAACACTATGCTCAAAGTCTACCGTACCAAAATGCAGGATGGAGTTGTTAGTGCCGATTTAGCTGCACAACACGGCATCGTAAAGGAGAAAAACTACTACGTATACTATGCCAATTACTCCAACTCATTAGTGTACAACAACGAGGAACAGAGACTGTCGTACTTCACTGAGGACATCGGCTTGAATTCGTACTACTACTACTTCCACTCTCACTTGCCTTTCTGGTGG | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | Arylphorin alpha subunit cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: ARYL_A_F: 5'-tggcaaggaatacaacatcg-3' and ARYL_B_R: 5'-ccaccagaaaggcaagtgag-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for arylphorin-alpha. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | Arylphorin-alpha RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body | not checked | 18 | none | ||||||
| 194 | Arylphorin-beta | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | Arylphorin-beta target | ATGAAGACTGTCATAATCCTAGCGGGGTTGGTGGCCCTGGCCCTCGGCAGCGAAGTGCCTGTCAAGCACTCCTTCAAAGTTAAGGATGTTGATGCGGCTTTCGTCGAACGTCAAAAGAAGGTCTTAGATCTTTTCCAAGATGTCGACCAAGTAAATCCTAACGATGAGTACTACAAGATTGGCAAGGAATACAACATCGAGGCTAACATCGACAATTACTCGAACAAGAAGGCCGTCGAAGAATTCTTGCAGTTATACAGGACAGGTTTCTTGCCTAAGTACTATGAATTTTCACCCTTCTATGACAGACTAAGGGACGAGGCCATTGGTGTTTTCCACCTCTTTTACTACGCTAAAGATTTTGATACGTTCTACAAATCTGCCGCATGGGCGCGTGTGTACCTCAACGAAGGACAGTTCTTATACGCCTACTACATTGCTGTGATTCAGCGTAAAGATACTCAGGGCTTCGTTGTACCAGCACCGTATGAAGTCTACCCTCAATTCTTCGCAAACTTGAACACTATGCTCAAAGTCTACCGTACCAAAATGCAGGATGGAGTTGTTAGTGCCGATTTAGCTGCACAACACGGCATCGTAAAGGAGAAAAACTACTACGTATACTATGCCAATTACTCCAACTCATTAGTGTACAACAACGAGGAACAGAGACTGTCGTACTTCACTGAGGACATCGGCTTGAATTCGTACTACTACTACTTCCACTCTCACTTGCCTTTCTGGTGGAATTCTGAGAGATACGGAGCACTAAAATCGCGCCGTGGTGAAATCTACTATTACTTCTATCAGCAATTAATTGCACGTTATTACTTTGAACGTCTCTCGAACGGCCTGGGTGACATTCCCGAATTCTCATGGTACTCACCAGTCAAGTCTGGCTACTATCCACTGATGTCTTCTTATTACTACCCCTTCGCTCAAAGGCCCAACTACTGGAACGTGCACAGCGAAGAAAACTACGAGAAAGTACGATTCTTGGACACGTATGAAATGTCATTCCTTCAGTTCCTCCAAAACGGACACTTCAAAGCGTTTGACCAGAAGATTGACTTCCACGATTTCAAAGCTATCAACTTTGTTGGAAACTACTGGCAAGATAATGCTGACCTGTACGGTGAGGAAGTTACTAAGGACTACCAACGTTCATATGAAATTATAGCCCGCCAAGTGCTTGGTGCTGCACCTAAACCATTCGACAAGTACACATTCATGCCCAGCGCTTTAGACTTCTACCAGACGTCTCTGCGTGACCCAATGTTCTACCAACTTTACAACAGAATTCTGAAGTACATATATGAGTACAAGCAGTACCTGCAACCGTACTCTTCAGAAAAACTGGCATTCAAGGGTGTCAAGGTGGTCGATGTTGTAGTAGACAAACTGGTTACCTTCTTCGAGTACTACGACTTTGATGCGTCCAACAGCGTTTTCTGGAGCAAAGAGGAGGTTAAATCTAGCTACCCCCATGATTTCAAGATCCGTCAGCCACGCCTTAACCACAAGCCATTCTCTGTCTCTATCGACATCAAATCTGAAGCTGCCGTTGATGCCGTTGTCAAGATATTCATGGCACCTAAATACGACGATAATGGATTCCCTCTGAAATTAGAAAACAACTGGAACAAATTCTTCGAGCTGGACTGGTTCACATACAAATTTGTTGCTGGTGACAACAAAATCGTGAGGAACTCAAACGACTTCTTGATCTTCAAGGACGACTCTGTTCCCATGACTGAGTTGTACAAATTATTAGAACAAAATAAGGTTCCACACGACATGTCCGAGGATTACGGCTACCTGCCTAAAAGACTGATGCTGCCAAGAGGTACTGAGGGTGGTTTCCCATTCCAGTTCTTCGTTTTCGTATATCCATTCAACGCTGACAGCAAAGATCTTGCACCGTTCGAGGCCTTCATCCAGGACAACAAACCTTTGGGCTATCCATTCGACCGTCCCGTTGTTGACGCTTACTTCAAGCAACACAACATGTTCTTCAAGGACGTCTTCGTATACCATGACGGCGAGTACTTCCCGTACAAGTTCAATGTTCCTTCCCATGTGATGCACTCAAACGTTGTTCCTAAACACTGAAAAGGAATTTGCTTAAATAGCATAGATCACATTATATTGTATTGAAATGTGTGCAAAGTAACTTAAATAAAACAGATTCATATTAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | Arylphorin-beta target | Arylphorin-beta RNAi construct | TGGCAAGGAATACAACATCGAGGCTAACATCGACAATTACTCGAACAAGAAGGCCGTCGAAGAATTCTTGCAGTTATACAGGACAGGTTTCTTGCCTAAGTACTATGAATTTTCACCCTTCTATGACAGACTAAGGGACGAGGCCATTGGTGTTTTCCACCTCTTTTACTACGCTAAAGATTTTGATACGTTCTACAAATCTGCCGCATGGGCGCGTGTGTACCTCAACGAAGGACAGTTCTTATACGCCTACTACATTGCTGTGATTCAGCGTAAAGATACTCAGGGCTTCGTTGTACCAGCACCGTATGAAGTCTACCCTCAATTCTTCGCAAACTTGAACACTATGCTCAAAGTCTACCGTACCAAAATGCAGGATGGAGTTGTTAGTGCCGATTTAGCTGCACAACACGGCATCGTAAAGGAGAAAAACTACTACGTATACTATGCCAATTACTCCAACTCATTAGTGTACAACAACGAGGAACAGAGACTGTCGTACTTCACTGAGGACATCGGCTTGAATTCGTACTACTACTACTTCCACTCTCACTTGCCTTTCTGGTGG | dsRNA | salt precipitation | subsequent to transcription | Arylphorin-beta subunit cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: ARYL-B_F: 5'-tggcaaggaatacaacatcg-3' and ARYL_B_R: 5'-ccaccagaaaggcaagtgag-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for arylphorin-beta. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | Arylphorin-beta RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body | not checked | 18 | none | |||||||
| 195 | SPARC RNAi | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | SPARC RNAi target | ATGAAGTCGCATCTTCTGCTGCTGGCTCTTGTATTTTGCCTTTTGGTTTCTGATGCTTTTGCAGATAAAGTGAGGAAACACAAGAAATACCGGCGAGTCCGCACTACAACTACTACCACAACAGAAGCCATTCAAGGAGCAGAGGATGGTGATGTTTTTTCAGTACTAGCACAGGATGAAGAAGAAGAAGAAAAAGCACAGGAGAATGAAGATTTAAAAAAGACACCAGAAGAGGAAGAAGAGGAACCACAGCAACCAGACGATAATGAAGTCATAGCCATTGATCCTTGTGCAGAGAAGCACTGTGGAGCTGGTCAAGTGTGTGAAGTGAATGAACAAGGAGAAGGGCACTGTGTGTGTATTCCTGACTGTCCTGTCGAAGTGGATCCTCGCAGGAAGGTGTGTTCCAATCACAATGAGACTTGGGGCTCAGACTGTGAGGTCTACCAGATGCGTTGCTGGTGCAAGCATGGCATGGATGGCTGCCGGGGTGATGAATACTCTCATGTCCACATTGAATATTATGGTGAATGCAGAGACATGCCTGAATGCACACCTGATGAAATGGCTGACTTCCCACGCAGAATGCGTGACTGGCTTTTCAACATAATGCGTGATTTGGCTGACCGTCGTGAACTGACACCTCACTACCTGGAAATGGAACGTGAAGCTGAAAATAATATGACCCGTCGGTGGACCAATGCTGCCATCTGGAAATGGTGTGACTTGGATGGACACCCACATGACAGGAGTGTGTCAAGGCATGAGCTGTTCCCAATCCGTGCTCCACTTATGGCTCTAGAGCACTGTATAGCACCTTTCCTTGACAGCTGTGATGTTAACGACGATCACACCATCTCCCTGGTAGAATGGGGGAAATGTCTTGAATTGGATCAGGATGATCTGGATGAAAGGTGCGATGAACTGAGAGAAGGCTAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | SPARC RNAi target | SPARC RNAi RNAi construct | AGAAGAGGAAGAAGAGGAACCACAGCAACCAGACGATAATGAAGTCATAGCCATTGATCCTTGTGCAGAGAAGCACTGTGGAGCTGGTCAAGTGTGTGAAGTGAATGAACAAGGAGAAGGGCACTGTGTGTGTATTCCTGACTGTCCTGTCGAAGTGGATCCTCGCAGGAAGGTGTGTTCCAATCACAATGAGACTTGGGGCTCAGACTGTGAGGTCTACCAGATGCGTTGCTGGTGCAAGCATGGCATGGATGGCTGCCGGGGTGATGAATACTCTCATGTCCACATTGAATATTATGGTGAATGCAGAGACATGCCTGAATGCACACCTGATGAAATGGCTGACTTCCCACGCAGAATGCGTGACTGGCTTTTCAACATAATGCGTGATTTGGCTGACCGTCGTGAACTGACACCTCACTACCTGGAAATGGAACGTGAAGCTGAAAATAATATGACCCGTCGGTGGACCAATGCTGCCATCTGGAAATGGTGTGACTTGGATGGACACCCACATGACAGGAGTGTGTCAAGGCATGAGCTGTTCCCAATCCGTGCTCCACTTATGGCTCTAGAGCACTGTATAGCACCTTTCCTTGAC | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | SPARC cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: SPARC_F: 5'-agaagaggaagaagaggaa-3' and SPARC_R: 5'-gtcaaggaaaggtgctat-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for SPARC. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | SPARC RNAi RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) phenotype | Fat body | not checked | 18 | none | ||||||
| 196 | PGRP-1A RNAi | RNAi | InsectaCentral | Unpublished | Eleftherianos | Ioannis | PGRP-1A RNAi target | GCAAAATGAAGTTATTTTTGTGTGCATTTTTAGTGCTCGTCGCAAAAACAAGATTCCTTAATGCTGACTGCAACGTGGTCAGTAAAGATGACTGGGACGGTATCACTTCCGTCCACATTGAGTACCTTACCCGTCCAATCAAACTGGTCATCATTCAACACACTGACACACCTGGCTGCGATACCGACGACGCATGCGCAGCGAGGGTTCGCAGCATTCAGGACTATCACTTGGACACTTTAAATTACTGGGACATCGGATCTTCGTTCCTGATTGGCGGTAATGGTAAAGTTTACGAAGGCTCCGGGTGGCTTCACGTGGGCGTGCCCAACTATGCTTACAACCGAAAAGCTATCAAAATCACGTTCATCGGAAGCTATAATAGTAAAGAGCCAAACTCCCAACAACTAAATGCTATCAAAGCCCTGCTGAAGTGTGGCGTTGACAATGGACATCTATCTTCGGATTACAAAGTCGTGGGCCATCGCCAGCTCTTGGACACCGACAGCCCTGGACGGAAATTATACAACATCATCAGGAGATGGCCAGAATGGACTAACGATGTGTCCGAATATAAAGACTAATTCTTATTGATTGTTGTTCAGCTACTTTTGGAGAAGACCATACGTATAAGTTGGTAAACAAATACCAAAGTTCAAATCTGTTATACAATTTTGTCTCAGCAATTTGTATTTCTTTTTTAAGGCAAATTATATATTTACGATTAAAAAAAAATATTTTATTACTTATTGTATCATTATAATGTATATGATTTAAAATAGCGTCTATTGCACTTGATTTCAATTAATTTAATTATTAGCAATAAATAATTGTTAACTATGCAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | PGRP-1A RNAi target | PGRP-1A RNAi RNAi construct | ACGGTATCACTTCCGTCCACATTGAGTACCTTACCCGTCCAATCAAACTGGTCATCATTCAACACACTGACACACCTGGCTGCGATACCGACGACGCATGCGCAGCGAGGGTTCGCAGCATTCAGGACTATCACTTGGACACTTTAAATTACTGGGACATCGGATCTTCGTTCCTGATTGGCGGTAATGGTAAAGTTTACGAAGGCTCCGGGTGGCTTCACGTGGGCGTGCCCAACTATGCTTACAACCGAAAAGCTATCAAAATCACGTTCATCGGAAGCTATAATAGTAAAGAGCCAAACTCCCAACAACTAAATGCTATCAAAGCCCTGCTGAAGTGTGGCGTTGACAATGGACATCTATCTTCGGATTACAAAGTCGTGGGCCATCGCCAGCTCTTGGACACCGACAGCCCTGGACGGAAATTATACAACATCATCAGGAGATGGCCAGAATG | dsRNA | T3 and T7 MegaScript kit / Ambion | salt precipitation | subsequent to transcription | PGRP-1A cDNA was amplified from fat body total RNA extracted from insects previously injected with E. coli to elicit an immune response. The primers used were as follows: PGRP_F: 5'-ACGGTATCACTTCCGTCCAC-3' and PGRP_R: 5'-CATTCTGGCCATCTCCTGAT-3'. Semi-quantitative single-step reverse transcription (RT)-PCR was performed with the âOneStepâ RT-PCR kit (Qiagen, UK). Each reaction was carried out in a 50 ul volume containing 0.6 uM of forward and reverse gene primers and 2 ug of RNA template. Amplifications were performed on a PTC-100 thermal controller (MJ Research, USA) under the following cycling conditions: room temperature (approx. 20C) at 50C for 30 min, 95C for 15min, followed by 35 cycles of 94C for 30 sec, 45C for 30 sec and 72C for 1 min with a final extension of 72C for 10 min. The resulting PCR product was cloned into pCR4-TOPO vector (Invitrogen, UK) and checked for the correct nucleotide sequence. It was then used as a template to generate double-stranded RNAs (dsRNA) for PGRP-1A. Sense and antisense RNAs were synthesized using the T3 and T7 âMegascriptâ kits (Ambion, UK), respectively, annealed in DMPC-treated water, and stored as dsRNA reagent at -20C until required. | InsectaCentral | PGRP-1A RNAi RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | DMPC-treated water | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | Hemolymph | As a negative dsRNA control (dsCON) I used a gene from a plant, Manihot esculenta catalase CAT1 (GenBank accession no.AF170272). The template for synthesis of dsCON was a pBluescript II KS+ plasmid containing the cloned gene, which was amplified by PCR using T7 (5'-TAATACGACTCACTATAGGG-3') and T3 (5'-ATTAACCCTCACTAAAGGGA-3') sequencing primers and dsCON was then synthesized as described for dsRNA of Gloverin. Controls involving injection of DMPC-treated water without dsRNA were also used. | larva | RT-PCR (semi-quantitative) W.blot phenotype | Fat body, hemocytes | not checked | 18 | low | ||||||
| 198 | Manduca sexta hemolymph proteins Kanost lab | RNAi | pubmed | 17868866 | Kanost | Michael | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha2 target | AGTGCCGCGCCGACCGTTGTGAGGGGAGAACGTTTCGCCGGTCGACATTTACGACTACGAACTCGTAAAGTTTTCGAACGAATCATGAGGATGAATTATATAATAAAAAATAGTGTATGATAATTATTTTCTGTTTGTGTTGTGACGCGTAATCGACAATGGCGTTGTCTATGTGTGTATTTTTGTGCGTGTGCGCTTATGTCGCCGGGTTCAATGTGGACATCCCATCGAGGGTGGTTTACAAGGGAAATGGGAATTCTATGTTTGGATTCACTGTCCAGGCGCATGTGGAAGGCGATCAGAAAATGATCCTAGTGGGAGCGCCGGAGGATGAGCGCTATCCTCCCAAGCACTACAACATCTCACGGCCCGGCGCGGTGTACCGCTGCGAGCCTGGCCGGAGGTCCTACTCCTCTGAGGGTCATGGCCAGCGGGCGCTTGAGCGGTGCGCGGAGATGGTGTTTGATAGAACTAACATGAATCTGGTGGATAAGCAAGGTCGTCAGATCGAGGAGAAGTCCCACCAGTGGTTCGGCGCTACTCTCACCAGCACCGGGAGGAACGGGCCCATTGTGGCCTGTGCCCCTCGCTACGTCTCCTACGTGTCAGCCAAGATGAACCAGCGAGATCCTGTGGGAACGTGCTACGTCGCCAAGACATCCAACGCTGCCACCACCACAGAGTTCTCGCCTTGCAGGACTTCTAGCCACGGACAGCGCAAGACTGGTGTGTGTCAGGCTGGATTTTCCGCCTCCATATCTAAGGATGGCCAGCGTTTATTCATGGGTGCTCCGGGAAGCTTCTACTGGCAAGGTCAGCTGATTTCTCAGACCATGAATGGTCGGCCAGCACTAATCGCCACTCCTGAATCCAAAGCCATGTATGATGATTCTTACATGGGATATTCCATCGCTGTGGGAGATTTTGCCGGTCAAGGAATCCAAAGCGTCGCCGTTGGTGTACCAAGGGGAGCTAATTTAAAGGGTTTGGTTGTCCTCTACACATGGGAACTGCAGAACATAAAGAACATCAGCGGGTCCCAGATCGGCGCTTACTTCGGCTACAGTCTTGCCTCCGGAGACATCGATGGCGACGGCGCTGATGACGTCATCGTTGGAGCTCCCATGTACACCAAAACCAGGAGCGATGGTTACGAACATGGAAGGATTTATGTCATCTACCAAGGAACTGATAGGTTATTTGCCAAAAGCCACGCCAGGACCGGTGAGATCTCTCGGGGCAGATTCGGTTTGGCTGTCACCTCTTTGGGAGACATCAACTACGACGGATTTGGAGATATAGCAGTAGGCGCGCCGTACGGAGGTCAGAATGGTCGCGGTGTAGTCTACATCTACCACGGCAGCGAGCTGGGTATCCTCGAGAAGTACTCCCAGGCCATCACGGCGGAAGAGATCTCCCCGACCTCGAGCACCTTCGGCTTCTCATTATCTGGAGGCGTCGATCTGGATAATAACAACTATACTGACCTCGCTGTTGGCGCTTACAAGTCGGACAGTGTTGTGTTTTTGAAATCCCGTCCAGTGGTAAAAGTAACAGCTGACGTCAAGTTCATGGGTGACAGCAAGCTGATCTCTTTAACAGACAAGCGCTGCCACCTCTCCAACGGCACTGAGGTGGCGTGCGCAGAGCTCATGTTCTGTCTCACCTACACCGGAGTTAATGTAGACCAGCGGATCAAATTCGAGGTGACCTTAGACCTGGACTCAAGGCAGAAAACGAGCAAGCGCCTGTTCCTAGCAGAGACCCACGAGACCACGTACAAGACGCAGATACTACTGACCCAAGGCCAGCAGGAATGCAAGGACGTGATGGTGTACTTGGATGAGGAGATTAGAGACAAGCTAACGCCCATCGAGGTGAAGATGTCGTACGACTTGATCAATCAGCCGTCCGGCGACGTGGTGCCGCCCGTGTTGGACCAAACCAGGAGCAACTTCCACACCGACTCGTTGAATATTCAGAAGAACTGCGGACCTGATAATATCTGCATACCGGATCTTAGGATGTCTGCTACTACACCGACAGTAAACTACGTCCTAAGTTCCGGAGAAAACATTAACATTGATGTCAAAGTGGAAAACGCTGGCGAAGATGCCTTCGAAGCCGCCTACTACCTGAACATACCCGCTGAAGTCACATACGCGAAAATGGAGAGACTTGACAAAGATAACGCAGAGACTCCCATCTACTGCTCCATTGCTAATAGAGAAGATGGTGGAAACACTACTCTGAAATGTGATCTTGGAAACCCCATGGCTAGTGGACAGAAGGTGCATTTCCGTGTAATTCTGGAGGTGGACTCGCGAGTGATGTCGCTGGATTTCAACATGGAAGCGAACTCCACCAACCCGGAGCCGCAGGAGACCGGCTACGACAACATCAAGCACATGGTCATCGGAGTTGTTGTGAAAGCACAACTCTCCATAATTGGCACATCCGACCCCCCAGAACTTCACTACAACGCGTCTCTCTACGGTACTCAGAACCTCAAAGACGACACCAAATGGGGACCCCAAGTCCTACACAAGTACAACATCAAGAACGGAGGACCCTTCACCGTTGATGAAGCGGAGATCTATTTCATGTGGCCGAATCAGACGCTCGACGGTGAGAACATAATGTTAGTACAGCCGCAGTGGCTCGGTAACGTCCAGTGTGATGTAGCAAGACTCAAGACAGTTGAGAATTTATTTGTACAGAACCCTCACGCTTTCATGCTGGTGAAGGAAAAGGAAGCTATGCAGAGCAATGGACTTTACACCGCTTCACAATTATCCGGCGGCATAGGACACTCTGGACAGACTTTCTGGGTAGAGCACTCCAGCAGCGGTGGCGTTGTTATTTCACAAAGTGGAGGTTCTATCTCTGGTAGCCATTTCGGAGCACAAGGATCGTCAGGACATTTCAGTGGAAGCAGCTCAGGTCATGCCAGTGGAAGCTTAGGACAAACTGGGACGCAGTTCACTACCAGCCAAAGTTCGGGTGGACAGTTCAGTGGTAGCCAAAGTGGACTCAGTAACCAAGGAAGCTTCAGTGGCCAAGGCGGGCAAGGCGCCCAATCTGGTATAACATACGTTTATAATAAGACTTGGAGCAGCTCCAGTGGCGAAGGTCTGACAGCCGAAGACCAAGAGCAAATCAAGAAGAATCTAGCAGCTATGGCACAGGGTGGTGTGAACACAGGATTCACTGGACAAGGCACATACGTACAAGGAGGTTCCCAGGGTAGCTATGTACATGGTAGTGCTCAGGGATCGCAAGGCGGTTATGTTCAGGGTAGTGCTCAAGATTCACAAAGTGGATACGTCCAGGGTAGTACTCAGGGCTCTCAAGGTGGATATGTCCAGGGTAGCACGCAAGGTTCTCAGGGTACTTATGTCCAAGGTGGCTCTCAAGGAACTTTCATTCAGGGTGGCTCTCAAGGCGGTTACGTCCAGGGAGGATCTCAAGGCTTTGTACACGGAGCTGGTCAAGGAGGATATGTGAATGGAGGCAGAATTGTAAAAGTGAAAAACAGGACTATTGTCTACGATCAGAACCATAATATCATATCCCAGACGGAAACTAGCACGGAGTATGGAAAACTTGGTCATGAAGGCGAGCCTGGAAGTTCTTTCCATCTTTACGGCAACGAAGGTGGACAGCAACATAGTGCAACCTTCGCCCATGGCTCCGGAGGTAGTGTCCAGACCTCTGGCCAAGATTACTCCCAGGGAGGCCAGGGGTATGTCCATTCAGGCCAATTCGGTCAAGGAAATCAGCAATTTGCACATGGATCGGGAGGCGAGCAAGCCTTTATCCACGGGTCTTGGGGAACAACAGAAACTGTCAACCCCGATATAATGAAGGCCAGTTCCCCGACCTTCAGAGGAGCATCCACTGTGACTGTTGTCGGTGATGAAGAGGAAGACAAACTTGAAGGGTTTGGAGCGTATGCGGCCAGCAATCCAAACAATGAGTTTAGATACGGCATTGCTGACGTCAGTGGTGCTTCAGGGTCTGCTCAAAGTGGTCAACAATCTGGAAGCGGTAGCTACCAGGCCGGCGGAGGAAGCTACCATGCTAGCGGAGGCGGCATGCAAGTCGCAGGTGGCGGTATGCAGGCCGGTGGTGGTGGCAGCTATCAAGCTGGAGGTTCCAGTATGCAATCCGGAGGCGGCACCTTCCAGACCGGTGTTGGAAGCTACCAATCAGGAGGTAGTTACCAAGGTGGCGGTGGATCTTGGAGCAGCGGATACTCCTATTCATCGAGATCAGGTGGAGGTTCGTCCTATAACTCTCAGTCCAGCGGCGGCTACGGCTCAGTGGACAGCCGGCGTGAGAACACGAGGAGGAGACGGCAGACTGAACAGGTGGACCCTAAACTAAAGGAAATATTGGAGAAATGCGAAGAGAAGTACAAATGCGAAGTACTGCGGTGTACGACCGGCAGATTGCTGAAAGGACAAGAAGTTTGGGTCGCGCTTAGGTCGAGACTGAACGCGTCTGTACTTAACGAGATCTCGAAGGACCGACCGGTGGTCCTGTCAACTCTGACCGCCACGCGCGTCTCCCGGCTGCCCATGGTGGGTCGGCCGAAGGACACGACATGGCAGACGGCGGAGGCGAAAACGACGGTCACTCCTCAGCTGGAGGCTCTGGACTCGGGAACCATCCCTCTGTGGGTGGTGGTGCTGGCGGCTGTCGTCGGTGCACTGTTGCTGCTGCTACTTATCTTCGCGCTGTACAAATGCGGATTCTTCAAGCGCAACCGTCCCTCGGACCACGCCGAGCGCCAGCCGCTGAACGGGCGCGACGAGCACCTCTGATATCCGACCACCGACCTCGGCTTCACCAGCGACACGGCCGGCACCAGTGACCTGCCGCCCACCTCCCACTCCTCGTCACCATCAGACAGCTCCTCGACAGATGCCGCTAGTGATGTCACGAGTCGCGATTCCACCAGCCTCGACAGATACTGACATCATAAACTATTGGTTTATAAATTCTAAATTGCTGGTGAAAATAACATTATAATTGATGGAGTTTTGTCAAATGGTTTTTACAATGACGACGCAACTTGGGGGTGGAAATTTTTGTCCACAATAAAGTATTTCAAAGGCAATAAGTCTAATTAAGGTGCGCAAAATATTCCATATTAGCGACTATTTATTACTGAGTTTTCATCATTTCGTGACATGAAGTTAGTGTAACAGTTTTACCTCATGATATGTCGAAATCGTGGCTCCTTAATCATTTTCGTAGTAAAAATGTTAGAAGGAGCCTTTAAAAATCAGATCGTAATAACATTACTAGCCACCGTCAAACACATTTCGGTAGCCATTTAGTGATTAGTGGAAGAGTTGGAAGTGTATTATAACGTATAATGTAAAATAATTCCCGTTCTCTACTATACTTAATTCTAAATCCTAAAATCTACAGCGGGAATTATGTTACACTATAAAATAGAATGTGAGAATCAAAGTGTGATAATGTAAAAAGTGTGTCTACGTTAAGGTATGAGAGTTGTTGAGATTTGTGATTTAAGTCAATAATAATTAGATACGTTTTACTAAATGAGTTTAGGTATCATAGAATGGTTGCTATTGACGTTTTAAAACATCATTTTGGTAGAGCACGTCCGGGTAAAAACATTGGACTTAGAAATCTCGTGTAGTAAAGTTATATATTTCAGATATTACATCGTAATTCTAATTTTTACTAATAAGAACGCTTATATTAAACTAAGGAGGCATTATCTCACGTTCTTTATAAATATTAAAAACAAAACTTCACTGCCTTACCTTCAGATATATCCAAACGAATTAATTTAATTAAATTCTAAGTATCCGACAGCTAAAGTTCCAATACAGACTGATTTGCAAGATTATATAGATATATATTATAATTCGAGTAGATTTGGCAATAATTATTTTATTAGTGAATTTGATATGTAGAGTAACGCGAATATAGAAGTGCCTTATGCAATACATATCTTGGTCGTGCTAATTTAGATATGCTTGAACTACGTGCCTTCTGCCAGTAACTAATTAATGTAAATACGTTAAGAGTTTTATATTAGTATTTATGACATTTTTTCACAGAAAAAAATACGTATACTTTTTGGCCGCAAGTGTAAAAATATTTGTAGAACTATGTACTTAATATTTTTTTGAGTGAGATGTGTATTTTAAATATTTACGAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | NCBI | DQ531845 | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha2 RNAi construct | ACCTCTCCAACGGCACTGAGGTGGCGTGCGCAGAGCTCATGTTCTGTCTCACCTACACCGGAGTTAATGTAGACCAGCGGATCAAATTCGAGGTGACCTTAGACCTGGACTCAAGGCAGAAAACGAGCAAGCGCCTGTTCCTAGCAGAGACCCACGAGACCACGTACAAGACGCAGATACTACTGACCCAAGGCCAGCAGGAATGCAAGGACGTGATGGTGTACTTGGATGAGGAGATTAGAGACAAGCTAACGCCCATCGAGGTGAAGATGTCGTACGACTTGATCAATCAGCCGTCCGGCGACGTGGTGCCGCCCGTGTTGGACCAAACCAGGAGCAACTTCCACACCGACTCGTTGAATATTCAGAAGAACTGCGGACCTGATAATATCTGCATACCGGATCTTAGGATGTCTGCTACTACACCGACAGTAAACTACGTCCTAAGTTCCGGA | dsRNA | column-based | simultaneously with transcription | (1) Amplify gene by PCR with forward primer (TAATACGACTCACTATAGGGAGACTCTCCAACGGCACTGAGGTG) and reverse primer (TAATACGACTCACTATAGGGAGATCCGGAACTTAGGACGTAG). (2) PCR product was recovered and used as template for preparation of dsRNA using MEGAsript RNAi Kit (Ambion). | InsectaCentral | Manduca sexta hemolymph proteins Kanost lab-Integrin alpha2 RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 0.1 | injection | ddH2O | 0.05 | non_specific_dsRNA | 3 | abdominal hemocoels | Green fluorescent protein (GFP) dsRNA was purchased from Dharmacon and used as a control. | larva | qPCR phenotype | hemocytes | not checked | 120 | low | 50 | ||||||
| 210 | bombykol biosynthetic pathway (pgACBP) | RNAi | pubmed | 16537410 | Ohnishi | Atsushi | bombykol biosynthetic pathway (pgACBP) target | ATGTCTCTCCAAGAAAAATTTGACCAAGCCGCAGCCAACGTGAAGAACCTGAAAGCTCTTCCAACTTATGCCCAACTCCTTAACTTGTATGCCCATTTCAAACAGGCCACAGTTGGAGATGCCGATCCAGCCAATAGACCTGGTCTTCTAGACTTGAAGGGTAAAGCCAAATTTGACGCTTGGCACAAACTGGCCGGCACTTCGAAGGAGGATGCCCAGAAAGCCTACATCGAGATCGTCGAAGGTCTCATAGCTTCCATCGGCCTCAAAGAAtAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AF246695 | bombykol biosynthetic pathway (pgACBP) RNAi construct | ATGTCTCTCCAAGAAAAATTTGACCAAGCCGCAGCCAACGTGAAGAACCTGAAAGCTCTTCCAACTTATGCCCAACTCCTTAACTTGTATGCCCATTTCAAACAGGCCACAGTTGGAGATGCCGATCCAGCCAATAGACCTGGTCTTCTAGACTTGAAGGGTAAAGCCAAATTTGACGCTTGGCACAAACTGGCCGGCACTTCGAAGGAGGATGCCCAGAAAGCCTACATCGAGATCGTCGAAGGTCTCATAGCTTCCAT | dsRNA | phenol_chloroform | subsequent to transcription | The templates for synthesis of dsRNAs corresponding to pgACBP was prepared by using gene-specific primers containing T7 polymerase sites and plasmids constructed previously. PCR was performed by using KOD-Plus (Toyobo, Osaka) with the resulting products purified (Promega SV PCR purification kit) and used as templates to generate dsRNAs using the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | GenBank | AF246695 | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 5 | injection | DEPC-treated water | 0.02 | buffer non_specific_dsRNA | 10 | pheromone gland (abdominal tip) | Control pupae were injected with 2ul of DEPC-treated H2O alone or dsRNA corresponding to the enhanced GFP sequence (Clontech). After injection, pupae were maintained under normal conditions until adult emergence. | adult | RT-PCR (semi-quantitative) phenotype | pheromone gland | not checked | 192 | high | 90 | ||||||
| 211 | bombykol biosynthetic pathway(mgACBP) | RNAi | pubmed | 16537410 | Ohnishi | Atsushi | bombykol biosynthetic pathway(mgACBP) target | ATGTCTCTCGACGAGCAATTCAAACAGGTCGCCGATAAGGTTAGGAACTGGAAGACCAAGCCCAGTGACGATGAGAACCTTGCGCTGTACTCCCTGTACAAGCAGGCTACCATAGGTGATGTTAACATTGCCCAGCCCAGCGGTCTTGTGGAGAGCGCCAAGTGGAAGGCATGGAACGGTCGCAAAGGCATCTCCCAAGACGATGCCAAGAAGCAATACATCGAAAATGCGGAGAAACTCCACTCCAAATACGCAtAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AF246696 | bombykol biosynthetic pathway(mgACBP) RNAi construct | ATGTCTCTCGACGAGCAATTCAAACAGGTCGCCGATAAGGTTAGGAACTGGAAGACCAAGCCCAGTGACGATGAGAACCTTGCGCTGTACTCCCTGTACAAGCAGGCTACCATAGGTGATGTTAACATTGCCCAGCCCAGCGGTCTTGTGGAGAGCGCCAAGTGGAAGGCATGGAACGGTCGCAAAGGCATCTCCCAAGACGATGCCAAGAAGCAATACATCGAAAATGCGGAGAAACTCCACTCCAAATACG | dsRNA | AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI) | phenol_chloroform | subsequent to transcription | The templates for synthesis of dsRNAs corresponding to mgACBP was prepared by using gene-specific primers containing T7 polymerase sites and plasmids constructed previously. PCR was performed by using KOD-Plus (Toyobo, Osaka) with the resulting products purified (Promega SV PCR purification kit) and used as templates to generate dsRNAs using the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | GenBank | AF246696 | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 5 | injection | DEPC-treated water | 0.02 | buffer non_specific_dsRNA | 10 | pheromone gland (abdominal tip) | Control pupae were injected with 2ul of DEPC-treated H2O alone or dsRNA corresponding to the enhanced GFP sequence (Clontech). After injection, pupae were maintained under normal conditions until adult emergence. | adult | RT-PCR (semi-quantitative) phenotype | pheromone gland | not checked | 192 | high | 50 | |||||
| 212 | bombykol biosynthetic pathway (Bmpgdesat1) | RNAi | pubmed | 16537410 | Ohnishi | Atsushi | bombykol biosynthetic pathway (Bmpgdesat1) target | ATGCCTCCTAATTCAGTGGATAAAACAAATGAAACAGAATACCTCAAGGATAATCATGTTGATTATGAAAAACTCATTGCACCCCAGGCCTCGCCCATCAAACACAAAATTGTAGTGATGAACGTGATACGTTTTAGCTATTTACATATTGCTGGTCTATATGGACTTTATCTCTGTTTCACCTCAGCAAAATTGGCTACATCGGTTTTTGCTATTGTGTTATTCTTCCTTGGGAACTTTGGTATTACAGCTGGAGCTCATCGTCTGTGGTCTCATAATGGTTACAAAGTCAAACTGCCCCTCGAGATCCTGTTGATGGTCTTCAACAGTATTGCTTTTCAGAACACCATTTTCACATGGGTGAGAGATCACAGGCTACATCACAAGTATACGGACACTGATGCAGACCCTCATAATGCTACGAGAGGTTTTTTTTTCTCACACATCGGCTGGTTGTTGGTGCGAAAACATCCAATGGTGAAAATTGCTGGAAAGAGCCTTGACATGTCAGATATTTATTGTAATCCTCTTTTAAGATTTCAGAAAAAGTACGCAATACCATTTATAGGAACAATATGTTTTATCATACCGACATTAGCTCCAATGTATTTTTGGGGTGAAAGCTTAAACAACGCTTGGCATATTACAGTTTTAAGATACATTTTCAGTCTCAATGGCACTTTTCTTGTAAATAGCGCTGCTCATTTGTGGGGATACAAACCGTACGATAAGAGCCTGAAAGCTACTCAAAGTGGCATGGCAAATGCATTTACTTTTGGTGAAGGATTTCACAATTACCATCACGTTTTTCCGTGGGATTATCGGGCAGACGAATTGGGGGACAGGTACATAAATTTGACAACAAGGTTCATAGATTTCTTTGCATGGATGGGATGGGCTTATGATCTGAAAACTGCTTCCACCAATATTATTGAGAAGAGGGCACTAAGAACAGGGGATGGGACTTACAAACGACCAAATGGAATGAATtGA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AB166851 | bombykol biosynthetic pathway (Bmpgdesat1) RNAi construct | ATGCCTCCTAATTCAGTGGATAAAACAAATGAAACAGAATACCTCAAGGATAATCATGTTGATTATGAAAAACTCATTGCACCCCAGGCCTCGCCCATCAAACACAAAATTGTAGTGATGAACGTGATACGTTTTAGCTATTTACATATTGCTGGTCTATATGGACTTTATCTCTGTTTCACCTCAGCAAAATTGGCTACATCGGTTTTTGCTATTGTGTTATTCTTCCTTGGGAACTTTGGTATTACAGCTGGAGCTCATCGTCTGTGGTCTCATAATGGTTACAAAGTCAAACTGCCCCTCGAGATCCTGTTGATGGTCTTCAACAGTATTGCTTTTCAGAACACCATTTTCACATGGGTGAGAGATCACAGGCTACATCACAAGTATACGGACACTGATGCAGACCCTCATAATGCTACGAGAGGTTTTTTTTTCTCACACATCGGCTGGTTGTTGGTGCGAAAACATCCAATGGTGAAAATTGCTGGAAAGAGCCTTGACATGTCAGATATTTATTGTAATCCTCTTTTAAGATTTCAGAAAAAGTACGCAATACCATTTATAGGAACAATATGTTTTATCATACCGACATTAGCTCCAATGTATTTTTGGGGTGAAAGCTTAAACAACGCTTGGCATATTACAGTTTTAAGATACATTTTCAGTCTCAATGGCACTTTTCTTGTAAATAGCGCTGCTCATTTGTGGGGATACAAACCGTACGATAAGAGCCTGAAAGCTACTCAAAGTGGCATGGCAAATGCATTTACTTTTGGTGAAGGATTTCACAATTACCATCACGTTTTTCCGTGGGATTATCGGGCAGACGAATTGGGGGACAGGTACATAAATTTGACAACAAGGTTCATAGATTTCTTTGCATGGATGGGATGGGCTTATGATCTGAAAACTGCTTCCACCAATATTATTGAGAAGAGGGCACTAAGAACAGGGGATGGGACTTACAAACGACCAAATGGAATGAAT | dsRNA | AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI) | phenol_chloroform | subsequent to transcription | The templates for synthesis of dsRNAs corresponding to Bmpgdesat1 was prepared by using gene-specific primers containing T7 polymerase sites and plasmids constructed previously. PCR was performed by using KOD-Plus (Toyobo, Osaka) with the resulting products purified (Promega SV PCR purification kit) and used as templates to generate dsRNAs using the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | GenBank | AB166851 | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 5 | injection | DEPC-treated water | 0.02 | buffer non_specific_dsRNA | 10 | pheromone gland (abdominal tip) | Control pupae were injected with 2ul of DEPC-treated H2O alone or dsRNA corresponding to the enhanced GFP sequence (Clontech). After injection, pupae were maintained under normal conditions until adult emergence. | adult | RT-PCR (semi-quantitative) phenotype | pheromone gland | not checked | 192 | high | 85 | |||||
| 213 | bombykol biosynthetic pathway(pgFAR) | RNAi | pubmed | 16537410 | Ohnishi | Atsushi | bombykol biosynthetic pathway(pgFAR) target | ATGTCACACAATGGAACTTTGGATGAACATTATCAGACCGTGAGGGAATTTTACGATGGTAAATCCGTTTTTATTACAGGAGCGACCGGATTCCTTGGAAAGGCGTACGTTGAGAAACTTGCGTATTCGTGTCCGGGCATAGTAAGCATTTACATTTTGATTCGAGACAAGAAAGGGTCGAACACTGAAGAGCGAATGAGAAAATATCTTGATCAGCCCATTTTTTCACGAATAAAATACGAACATCCAGAATATTTCAAAAAAATCATTCCCATCTCTGGTGATATCACCGCACCCAAACTAGGATTGTGCGATGAAGAAAGAAATATATTAATCAACGAGGTGTCGATAGTGATTCACTCAGCCGCTTCGGTCAAACTAAACGATCATTTAAAATTTACTCTGAACACGAATGTTGGAGGTACGATGAAAGTGTTGGAACTCGTTAAAGAAATGAAAAACTTAGCGATGTTCGTATATGTGTCTACAGCTTATTCTAACACAAGTCAAAGAATTTTAGAAGAAAAACTTTATCCTCAGTCCCTAAATCTAAACGAAATACAGAAGTTCGCTGAAGAACATTATATATTAGGTAAAGACAACGATGAAATGATAAAATTTATAGGTAACCACCCAAACACATATGCATACACAAAAGCGCTGGCAGAAAACTTAGTTGCTGAAGAACACGGTGAAATACCAACAATTATCATTAGGCCTTCGATTATTACAGCGAGTGCTGAAGAGCCAGTGCGAGGGTTCGTGGATAGTTGGAGTGGTGCAACTGCTATGGCAGCATTTGCTTTGAAGGGATGGAACAACATAATGTACAGCACAGGCGAGGAGAATATTGATCTGATACCTCTAGACTATGTTGTCAACCTTACATTAGTAGCGATTGCTAAATATAAGCCTACCAAAGAGGTCACAGTATATCACGTCACAACCAGCGATCTTAATCCCATTTCAATCCGAAGAATATTTATAAAGCTCTCCGAATTCGCATCAAAAAATCCAACGAGTAATGCCGCACCATTCGCAGCAACGACTTTATTGACTAAACAGAAGCCATTAATAAAACTGGTCACGTTTCTCATGCAGACCACGCCGGCTTTCCTAGCTGACTTATGGATGAAAACCCAGAGGAAAGAAGCCAAATTCGTCAAGCAACATAATCTAGTAGTGAGAAGTCGTGATCAACTGGAATTCTTCACGTCTCAGTCGTGGTTGCTTCGTTGCGAGCGAGCCCGTGTGCTTAGTGCGGCACTGAGCGATTCAGACCGCGCTGTCTTCCGCTGCGATCCTTCCACCATAGACTGGGACCAATATTTGCCAATATACTTCGAAGGAATCAACAAGCATCTGTTCAAAAATAAATTAtAG | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | 3utr 5utr cds | GenBank | AB104896 | bombykol biosynthetic pathway(pgFAR) RNAi construct | GGCACGAGGCGACGAAACAATACTTGCCTGATTTAAACGATTATAAGTTTTGTTAAGGAAAAACTGTGATCAGCTTCAAATTATTTTCAAATTCCGCGTAATCTCCAAGATGTCACACAATGGAACTTTGGATGAACATTATCAGACCGTGAGGGAATTTTACGATGGTAAATCCGTTTTTATTACAGGAGCGACCGGATTCCTTGGAAAGGCGTACGTTGAGAAACTTGCGTATTCGTGTCCGGGCATAGTAAGCATTTACATTTTGATTCGAGACAAGAAAGGGTCGAACACTGAAGAGCGAATGAGAAAATATCTTGATCAGCCCATTTTTTCACGAATAAAATACGAACATCCAGAATATTTCAAAAAAATCATTCCCATCTCTGGTGATATCACCGCACCCAAACTAGGATTGTGCGATGAAGAAAGAAATATATTAATCAACGAGGTGTCGATAGTGATTCACTCAGCCGCTTCGGTCAAACTAAACGATCATTTAAAATTTACTCTGAACACGAATGTTGGAGGTACGATGAAAGTGTTGGAACTCGTTAAAGAAATGAAAAACTTAGCGATGTTCGTATATGTGTCTACAGCTTATTCTAACACAAGTCAAAGAATTTTAGAAGAAAAACTTTATCCTCAGTCCCTAAATCTAAACGAAATACAGAAGTTCGCTGAAGAACATTATATATTAGGTAAAGACAACGATGAAATGATAAAATTTATAGGTAACCACCCAAACACATATGCATACACAAAAGCGCTGGCAGAAAACTTAGTTGCTGAAGAACACGGTGAAATACCAACAATTATCATTAGGCCTTCGATTATTACAGCGAGTGCTGAAGAGCCAGTGCGAGGGTTCGTGGATAGTTGGAGTGGTGCAACTGCTATGGCAGCATTTGCTTTGAAGGGATGGAACAACATAATGTACAGCACAGGCGAGGAGAATATTGATCTGATACCTCTAGACTATGTTGTCAACCTTACATTAGTAGCGATTGCTAAATATAAGCCTACCAAAGAGGTCACAGTATATCACGTCACAACCAGCGATCTTAATCCCATTTCAATCCGAAGAATATTTATAAAGCTCTCCGAATTCGCATCAAAAAATCCAACGAGTAATGCCGCACCATTCGCAGCAACGACTTTATTGACTAAACAGAAGCCATTAATAAAACTGGTCACGTTTCTCATGCAGACCACGCCGGCTTTCCTAGCTGACTTATGGATGAAAACCCAGAGGAAAGAAGCCAAATTCGTCAAGCAACATAATCTAGTAGTGAGAAGTCGTGATCAACTGGAATTCTTCACGTCTCAGTCGTGGTTGCTTCGTTGCGAGCGAGCCCGTGTGCTTAGTGCGGCACTGAGCGATTCAGACCGCGCTGTCTTCCGCTGCGATCCTTCCACCATAGACTGGGACCAATATTTGCCAATATACTTCGAAGGAATCAACAAGCATCTGTTCAAAAATAAATTATAGTCGATTCCTAATAGGCTTGTAATTGATTCCAAAAATACTTAGATAACAATAACTTTAATCAACTACAATGAAATAAATGTAAAGTCGTCGTGACCTATAGGATAAGACGTACGGTGCATTCGTATATAGCGATGCACCGGTGTTCGAATCCCGCAGGCGGATACCAATTTTTCTAATGAAATACGTACTTAACAAATGTTCACGATTGACTTCCGCGGTGAAGGAACAACATCGTGTAATAAAAATCAAACCCGCAAAATTATAATTTGCGTAATTACTGGTGGTAGGACCTCTTGTGAGTCCGCACGGGTAGGTACCACCACTCCGTCTATTTCTGCCCTGAAGCAGCATTGCGTTTCGGTTTGAAAGGTGGGGTAGCCGTGGTAACTATACTATACTATAGTAATTCCAAAATTCGCTGATTTATGTTCTATTAGTCTGATTTAGTTAAACCATTTCTATAGTTA | dsRNA | AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI) | phenol_chloroform | subsequent to transcription | The templates for synthesis of dsRNAs corresponding to pgFAR was prepared by using gene-specific primers containing T7 polymerase sites and plasmids constructed previously. PCR was performed by using KOD-Plus (Toyobo, Osaka) with the resulting products purified (Promega SV PCR purification kit) and used as templates to generate dsRNAs using the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | GenBank | AB104896 | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 5 | injection | DEPC-treated water | 0.02 | buffer non_specific_dsRNA | 10 | pheromone gland (abdominal tip) | Control pupae were injected with 2ul of DEPC-treated H2O alone or dsRNA corresponding to the enhanced GFP sequence (Clontech). After injection, pupae were maintained under normal conditions until adult emergence. | adult | RT-PCR (semi-quantitative) phenotype | pheromone gland | not checked | 192 | high | 85 | |||||
| 215 | bombykol biosynthetic pathway(PBANR) | RNAi | pubmed | 16537410 | Ohnishi | Atsushi | bombykol biosynthetic pathway(PBANR) target | ATGATGGCAGATGAAACCGTCAACATGGAAATGCTCGAGAACAATTTGCTGAATGTCACGAACGTGACAGACCAATCTTCTGCGTACAGTGAATCCTACCCATTACATCTGCTAGTACCATTAAGTGTTACGTACGCTGTCATATTCATAGTCGGGATCTTAGGGAATACAAGCACATGCGTGGTCATAGCGCGTAACCGCTCCATGCACACCGCCACTAACTTCTACCTCTTCAGTCTTGCTATCTCCGATATAATACTCCTTGTGTGCGGTCTGCCCTTAGAATTGTATCGCTTGTGGAATCCGTTTACGTACCCCCTCGGTGAGGCGCAATGTATCACGATAGGTCTGGCGTCTGAAACGTCTGCGAACGCAACCGTATTGACTATTACGGCATTCACTATGGAGCGATACATTGCTATATGTAGACCGTTCATGTCGCATACCATGTCGAAGCTTTCACGGGCTGTGCGATTTATTATTGCTATATGGGTGTTCGCGCTGTGTACGGCGGTACCTCAAGCGATGCAATTCGGTATAGTGTCTTACGTTGAAAACGGACAGAGCATGAGTGCTTGTACTGTGAAAGGACCTGGTGTGCACCAAGTGTTTGTCATTTCGAGTTTTGTTTTCTTCGTTGTACCTATGTCGGTTATATCGGTGTTGTACGCGTTGATAGGGCTCAAGTTACGTACGTCTCGTATTTTGCATCCCGTTAAGAAGCTGTCATTAGATAGCAATGAGAGGCCTGGCGCGCACACACCGTACAGAAACGGAAGCTCACAGAGGAGGGTCATCAGAATGCTCGTTGCTGTGGCGCTGTCGTTTTTCATCTGTTGGGCTCCATTTCACGTGCAGAGGCTTCTCGCGATTTATGGGAAGAGTTTGGAACATCCGTCTGATACATTTTATCTGGTGTACATAGTGTTGACCTTTTTATCTGGAGTCCTGTATTTCCTTTCGACGGCTATTAATCCATTTCTCTACAACATAATGTCGAACAAATTCAGGAACGCTTTCAAGATGACATTAGCGGCTTGGTGTGGAAGGCGAGGTGGGCCTCGCATGGGTCGGTCATACAGTGCACTGCTGGCTTCGCAACGGCAGAGGGCTGCCAATGGCTTGACAGATCCAGTTCGAGGGCCTCGAAGACTTCGGCGACTGTCGACCGCTACTACGCACCTTTGCGATGCGCCTCCTAGAGCGCAGGTGAGTGCTACCAAAATCGCGATCTCTCCATAGtGA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AB181298 | bombykol biosynthetic pathway(PBANR) RNAi construct | ATTGACTATTACGGCATTCACTATGGAGCGATACATTGCTATATGTAGACCGTTCATGTCGCATACCATGTCGAAGCTTTCACGGGCTGTGCGATTTATTATTGCTATATGGGTGTTCGCGCTGTGTACGGCGGTACCTCAAGCGATGCAATTCGGTATAGTGTCTTACGTTGAAAACGGACAGAGCATGAGTGCTTGTACTGTGAAAGGACCTGGTGTGCACCAAGTGTTTGTCATTTCGAGTTTTGTTTTCTTCGTTGTACCTATGTCGGTTATATCGGTGTTGTACGCGTTGATAGGGCTCAAGTTACGTACGTCTCGTATTTTGCATCCCGTTAAGAAGCTGTCATTAGATAGCAATGAGAGGCCTGGCGCGCACACACCGTACAGAAACGGAAGCTCACAGAGGAGGGTCAT | dsRNA | AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI) | phenol_chloroform | subsequent to transcription | The templates for synthesis of dsRNAs corresponding to PBANR was prepared by using gene-specific primers containing T7 polymerase sites and plasmids constructed previously. PCR was performed by using KOD-Plus (Toyobo, Osaka) with the resulting products purified (Promega SV PCR purification kit) and used as templates to generate dsRNAs using the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | GenBank | AB181298 | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 5 | injection | DEPC-treated water | 0.02 | buffer non_specific_dsRNA | 10 | pheromone gland (abdominal tip) | Control pupae were injected with 2ul of DEPC-treated H2O alone or dsRNA corresponding to the enhanced GFP sequence (Clontech). After injection, pupae were maintained under normal conditions until adult emergence. | adult | RT-PCR (semi-quantitative) W.blot phenotype | pheromone gland | not checked | 192 | high | 75 | |||||
| 216 | bombykol biosynthetic pathway(BmSTIM1) | RNAi | pubmed | 19740753 | Ohnishi | Atsushi | bombykol biosynthetic pathway(BmSTIM1) target | ATGCGTATCGGTTTCATTTTGTATTGGTGCGTATTTATTCTTCATCTGTGTAAAGCTGACGATGCCGAGAGGACAGAAAAAGATGGTGGAAGGCGGGAGAAGTGGGGTCCAGTGGTCCGAAGTGGTTCTTTCGTAACAGATCCAGCTTTTACAGCCTCAAATGATCAGATATTACTGGAGGCGTGTCAGAATGATGCGTCGTGCCTACAAGACCATGCCGGATTGGAGGCCATCACACTTTTGCACCGGCAGCTCGATGATGACGCCAACGGAAACGTGGACTTGAGCGAGAGCGATGATTTCCTCCGCGAAGAGTTGCAGTACGACAGCGGCTACGAGAAGCGACAGAGGGCGTTCCACCACAATGACGATATGCACATATCTGTCAAGGAACTATGGGAGGCGTGGCTTAGATCGGAGGTTCACAATTGGACGGTGGAGCAAACGGTCGAATGGTTATCAGAATCCGTGGATCTTCCGCAGTACACAACATTATTTTTACAGCACAAAGTCACTGGCGCTACGTTACCAAGACTCGCCGTAAACAACATGCAGTATCTGAGCAATGTGCTCGGCATCAAAGATCCCATACACAAACAGAAGCTGGCGCTTAAAGCTATGGACGTTGTGCTTTTTGGACCACCCAAAGGTAGCAGATGGAAGGACTGGCTCCTCGCTTCGTTGCTGCTCGGCGCTGTTGTGGGGGGTTGGGCCGCGTTGCGCGCGGGACGTGCGAGCAGGACACAAGTGCAACGGATGCTGCGCGACATGGAACAATTACGGAGAGCCGAGATGGCATTGGACGAGATGCAGAAGGAACTCGAGAAGGCACGACTAGAACAGGAAAGTGCAACAACAGAGAAGAAAAATCTAGAAAAGAAACTTAAAGAGACAAGTGACACGCCAGTCCTGAACACGACCACGTCGGATCTCGAAGTGTCTAAACTCAAAGCCGAAGTCGAGATGTTGCGAGCTGAACTCCGTAGGGCGGAGGGTGAGCTCGAGGACCGGTGCTGGGCCCCTCCTCCCGGGCTCCAGCAGTGGCTCCAGCTGACGCACGAGGTCGAGAACAGATCCTACCTCAGGAAGAAGCAGCAGGCCGACCTACAGCTGCAACAGGCTAGGGACGCGTGCGAAAAATTACGCAAGAAGCGTTCTAGTTTGGTCGGCGCCTTCGTGTCGACGCACGGCAAGTCCATAGACGACGTGGATCGGTCCATTGTGGAAGCGCGCACTGCACTCAACGAGGTCACTCAAGAGCTACAGGAACGCATGCACCGATGGAAGCAGATCGAACGCCTGTGCGGGTTCAACATCATCAACAACAACGGGCTGCAGTACCTGGAGACCACGCTGTACAGGAACATGAACGGTCGACCGACGGGAAGACCCCGCATGAGCAGTTCCCAGGACGACCTGAGCGTCGGCGACGACACATCCCTGTGCGGCTCAGTGGTGGATAACTTTGGATGGCGCGAGGACTCATCGGGAAGCGAAGAGCACGATTCTCCACACTATCCTCTAGATTTAAGACTCGCCGAGGCTAGGTTACAATTGGAAGAGCGTCTCGCTCAAGAAGCCCGCGAGAGACGTCAGGACGAGCGACGTTTAGAGGACAAGAAACTGTCGAGCGAGGAACTGCGCATGCGGAACTCGCACAACGACGTTCGCAAGAGTCCCGTTGACGACAGACGCAAGGACCAAGTTCAGTTCGTGCTGGGAGGAGACAAGTGA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AB425069 | bombykol biosynthetic pathway(BmSTIM1) RNAi construct | TTTCCTCCGCGAAGAGTTGCAGTACGACAGCGGCTACGAGAAGCGACAGAGGGCGTTCCACCACAATGACGATATGCACATATCTGTCAAGGAACTATGGGAGGCGTGGCTTAGATCGGAGGTTCACAATTGGACGGTGGAGCAAACGGTCGAATGGTTATCAGAATCCGTGGATCTTCCGCAGTACACAACATTATTTTTACAGCACAAAGTCACTGGCGCTACGTTACCAAGACTCGCCGTAAACAACATGCAGTATCTGAGCAATGTGCTCGGCATCAAAGATCCCATACACAAACAGAAGCTGGCGCTTAAAGCTATGGACGTTGTGCTTTTTGGACCACCCAAAGGTAGCAGATGGAAGGACTGGCTCCTCGCTTCGTTGCTGCTCGGCGCTGTTGTGGGGGGTTGGGCCGCGTTGCGCGCGGGACGTGCGAGCAGGACACAAGTGCAACGGATGCTGCGCGACATGGAACAATTACGGAGAGCCGAGATGGCATTGGACGAGATGCAGAAGGAACTCGAGAAGGCACGACTAGAACAGGAAAGTGCAACAACAGAGAAGAAAAATCTAGAAAAGAAACTTAAAGAGACAAGTGACACGCCAGTCCTGAACACGACCACGTCGGATCTCGAAGTGTCTAAACTCAAAGCCGAAGTCGAGATGTTGCGAGCTGAACTCCGTAGGGCGGAGGGTGAGCTCGAGGACCGGTGCTGGGCCCCTCCTCCCGGGCTCCAGCAGTGGCTCCAGCTGACGCACGAGGTCGAGAACAGATCCTACCTCAGGAAGAAGCAGCAGGCCGACCTACAGCTGCAACAGGCTAGGGACGCGTGCGAAAAATTACGCAAGAAGCGTTCTAGTTTGGTCGGCGCCTTCGTGTCGACGCACGGCAAGTCCATAGACGACGTGGATCGGTCCATTGTGGAAGCGCGCACTGCACTCAACGAGGTCACTCAAGAGCTACA | dsRNA | AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI) | phenol_chloroform | subsequent to transcription | The templates for the synthesis of dsRNAs corresponding to BmSTIM1 was prepared using KOD-Plus- with gene-specific primers containing T7 polymerase sites. Portions of each gene were amplified from plasmid DNAs using the BmSTIM1 sense primer (5-TAATACGACTCACTATAGGGCGTTCCTCC- GCGAAGAGTTG-3) with the antisense primer (5-TAATACG-ACTCACTATAGGGCGTGTAGCTCTTGAGTGACC-3). PCR products were purified using a SV PCR purification kit (Promega) and used as templates for in vitro transcription using the AmpliScribe T7 High Yield Transcription kit (Epicenter) according to the manufacturerâs recommended protocol. | GenBank | AB425069 | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | DEPC-treated water | 0.02 | buffer non_specific_dsRNA | 10 | pheromone gland | Control females were injected with dsRNA corresponding to the EGFP ORF (Clontech). After injection, adults were maintained overnight under normal conditions. | adult | RT-PCR (semi-quantitative) phenotype | pheromone gland | not checked | 48 | high | 50 | |||||
| 217 | bombykol biosynthetic pathway(BmOrai1) | RNAi | pubmed | 19740753 | Ohnishi | Atsushi | bombykol biosynthetic pathway(BmOrai1) target | ATGTCGGGTGAGACGCCGATACAGTCGGGGGACGGGCTTCATACGCCGGCGTACCTCTCGTGGAGGAAGTTGCAGCTTAGTAGGGCGAAACTGAAGGCTTCCAGCAAAACATCTGCCCTACTTTCCGGTTTCGCTATGGTGGCTATGGTCGAAGTACAATTAAATCCAGCCCCCACTGCAGTACCAAAGGAGATGCTCGTCGCTTTCACGGTGTGCACAACACTCTTAGTCGCAGTCCACATGCTCGCGCTCATGATTAGCACCTGCATTTTGCCAAACATCGAAGCAGTCGGCAACTTGCATAGTATAGCGCTCGTCCATGAGTCGCCACACGAGAGACTCCACTGGTACATCGAAGTGGCTTGGGCTTTTTCGACCCTCTTAGGCTTAATCTTATTCTTGATAGAGATAGCTATACTATGTTGGGTTAAATTCTACGATTTGAGTCCGACGGCAGCTTGGTCGGCCTGTGTGGTCCTTATACCTGTTATGATTGTATTTTTGGCGTTCGCAATACATTTTTACATGTCCCTGGCGACGCACAAGTACGAAGTCACGGTGACAGGTATCAAAGAATTAGAATTGCTCAAAGAACAGATCGAAATGGGCGACCACGACGCCAGAATGAACAGCTTAACGTTGCTGGACCAAAGTCGGGTCGTCtGA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AB425231 | bombykol biosynthetic pathway(BmOrai1) RNAi construct | ATGTCGGGTGAGACGCCGATACAGTCGGGGGACGGGCTTCATACGCCGGCGTACCTCTCGTGGAGGAAGTTGCAGCTTAGTAGGGCGAAACTGAAGGCTTCCAGCAAAACATCTGCCCTACTTTCCGGTTTCGCTATGGTGGCTATGGTCGAAGTACAATTAAATCCAGCCCCCACTGCAGTACCAAAGGAGATGCTCGTCGCTTTCACGGTGTGCACAACACTCTTAGTCGCAGTCCACATGCTCGCGCTCATGATTAGCACCTGCATTTTGCCAAACATCGAAGCAGTCGGCAACTTGCATAGTATAGCGCTCGTCCATGAGTCGCCACACGAGAGACTCCACTGGTACATCGAAGTGGCTTGGGCTTTTTCGACCCTCTTAGGCTTAATCTTATTCTTGATAGAGATAGCTATACTATGTTGGGTTAAATTCTACGATTTGAGTCCGACGGCAGCTTGGTCGGCCTGTGTGGTCCTTATACCTGTTATGATTGTATTTTTGGCGTTCGCAATACATTTTTACATGTCCCTGGCGACGCACAAGTACGAAGTCACGGTGACAGGTATCAAAGAATTAGAATTGCTCAAAGAACAGATCGAAATGGGCGACCACGACGCCAGAATGAACAGCTTAACGTTGCTGGACCAAAGTCGGGTCGTC | dsRNA | AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI) | phenol_chloroform | subsequent to transcription | The templates for the synthesis of dsRNAs corresponding to BmOrai1B was prepared using KOD-Plus- with gene-specific primers containing T7 polymerase sites. Portions of each gene were amplified from plasmid DNAs using the BmOrai1B sense primer (5-TAATACGACTCACTATA- GGGCGATGTCGGGTGAGACGCCG-3) with the antisense primer (5-TAATACGACTCACTATAGGGCGGACGACCCGACTTTGGTC-3). PCR products were purified using a SV PCR purification kit (Promega) and used as templates for in vitro transcription using the AmpliScribe T7 High Yield Transcription kit (Epicenter) according to the manufacturerâs recommended protocol. | GenBank | AB425231 | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | DEPC-treated water | 0.02 | buffer non_specific_dsRNA | 10 | pheromone gland | Control females were injected with dsRNA corresponding to the EGFP ORF (Clontech). After injection, adults were maintained overnight under normal conditions. | adult | RT-PCR (semi-quantitative) phenotype | pheromone gland | not checked | 48 | high | 50 | |||||
| 218 | bombykol biosynthetic pathway | RNAi | pubmed | 19112106 | Ohnishi | Atsushi | bombykol biosynthetic pathway target | ATGGTGAACGTTGTTACGACGGTAGATAGCAACATGAATAACACAAATAATAATCATAAAGAGATGAAACCCGGTGCTGTTGACATCGAAAAAAGCAAACAGAACAGTGATTCGGATGGAGTGTCTTGGGGAAAAATATTTTACGTGTTGCTGTCGCTAGCAGTTATCGTGATAGCCTGTGGCGTCGCCTGGGTGTTCCAGGACTGGCTCACCAGTCTTATCATCTTCGTTGTACTTTTGGTGGTATTCACCGTTGGCTATTTCTGGAAATGGCTGTACATCGCGGCCAGAACTGCTCCTAGAGATTTTTCGGCACTGTGGTGTTATGTTAAAATCCTGAGACTTTCGGGTAATTTTGGTAAGAAGAACTGGTCAATGCCCGATATATTCCATGAAAATGTAAAGAGACATCCGAACAAAGCATGCTTCCTTTACGAGAACGAGTCATGGTCCTTTAAACAGGTCGAAGAATTCAGTCTTCGTGTTACGGCAGTGCTTAAGAATCATGGTGTGAAACGTGGAGATGTTGTCGGCGTAATGATGAATAACTGTCCGGAATTGCCGGCGACGTGGCTAGGCGTAGCTCGCATGGGAGGCGTGTCGCCTCTCATAAACACGAACCAAACCGGAAACGCTCTTCTGCATTCGGTTAACGTTGCGAAATGTAACGTTGTGATATACGGAAGCGAGTTTCAGAGTGCATTCGATGAAATATCAAATGAAATAAATCCCGCAATAAAACTGTATAGATACAACCGGAGACCCCTAAATGCATCAGGTGATGCTGTTAGGGTTGTAGAATCGGAAAACGATTTCACTCATATGTTGGAGACTACACCTCCTGCCCCGTGGAGTCTTTCCGACGGAGAAGGCTTCACCGGAAAACTTCTATATATATACACGTCAGGGACTACTGGATTGCCCAAAGCAGCAGTTATATCTCCGTCAAGGATGGTGTTCATGGCATCGGGCGTACATTATTTAGGTGGATTGAGAAAAAACGATATAATGTACTGTCCAATGCCTTTGTATCATTCAGCTGGAGGGTGTATTTCAGTTGGTCAGGCCTTCATATTCGGGTGCACTGTAGCTTTGAGAGCGAAATTTTCGGCGTCTGCATATTTCCCGGATTGCATTAAATTCAAAGCAACGGCCGCTCACTACATTGGGGAGATGTGTCGGTATATCCTAGCAACACCTCCGTCCGCAACTGACAGGCAACACAAAGTTCGAACCGTATACGGCAATGGAATGAGGCCAACCATCTGGACAGAATTCGTCAAACGCTTCAACATCAAACGTGTCGTCGAGTTTTATGGCGCAACAGAAGGCAACGCAAATATCGTAAACATCGACAACAAGACAGGTGCAATAGGATTTGTATCGAGAATCATCCCCGCAGTTTACCCGATAGCGATTCTCAAGGTTGATCAAGAAACTGGCGAACCAATCAGGAATTCCAAAGGACTTTGCCAGCTAGCAAAGCCGTATGAGCCAGGAGTATTTATTGGCAAGATTAAACCTAACAATCCATCAAGGGCGTTCCTGGGTTACGTGGACAAAGAAGCATCTGAGAAGAAAATAGTTAGAGACGTATTTAACATCGGCGATTCTGCGTTTATATCAGGTGACATCCTGGTGGCGGACGAATTAGGTTATCTGTACTTCCGCGACCGCACCGGAGACACGTTCCGGTGGCGCGGAGAGAACGTTTCCACCACAGAGGTCGAGGCGGCCGTGTCTAGATGTGCCAATCAAAGGGACGCTGTAGTCTACGGTGTAGAGATTCCGAATACGGAAGGTCGGGCTGGCATGTGCGGCATAGTGGACATTGAAGGCACATTGGACCTGGATAAACTCGCCAAAGATATTGCGAAAGACCTGCCTAAATACGCGAGGCCAATATTCATTAGAATAATGACCAGCGTTGATATGACAGGAACATTCAAAATGAAGAAAGTGGATTTACAAAAAGAAGGTTATAATCCATCAACTGTGAGCGACAAAATGTTCTTCTTTGAACCAAAACAGAATAAGTACGTGCCCTTAGGAGTTGAAGAATACGAAAAGATTATATCTGGTGAAATTCGGCTAtGA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AB451529 | bombykol biosynthetic pathway RNAi construct | TATACACGTCAGGGACTACTGGATTGCCCAAAGCAGCAGTTATATCTCCGTCAAGGATGGTGTTCATGGCATCGGGCGTACATTATTTAGGTGGATTGAGAAAAAACGATATAATGTACTGTCCAATGCCTTTGTATCATTCAGCTGGAGGGTGTATTTCAGTTGGTCAGGCCTTCATATTCGGGTGCACTGTAGCTTTGAGAGCGAAATTTTCGGCGTCTGCATATTTCCCGGATTGCATTAAATTCAAAGCAACGGCCGCTCACTACATTGGGGAGATGTGTCGGTATATCCTAGCAACACCTCCGTCCGCAACTGACAGGCAACACAAAGTTCGAACCGTATACGGCAATGGAATGAGGCCAACCATCTGGACAGAATTCGTCAAACGCTTCAACATCAAACGTGTCGTCGAGTTTTATGGCGCAACAGAAGGCAACGCAAATATCGTAAACATCGACAACAAGACAGGTGCAATAGGATTTGTATCGAGAATCATCCCCGCAGTTTACCCGATAGCGATTCTCAAGGTTGATCAAGAAACTGGCGAACCAATCAGGAATTCCAAAGGACTTTGCCAGCTAGCAAAGCCGTATGAGCCAGGAGTATTTATTGGCAAGATTAAACCTAACAATCCATCAAGGGCGTTCCTGGGTTACGTGGACAAAGAAGCATCTGAGAAGAAAATAGTTAGAGACGTATTTAACATCGGCGATTCTGCGTTTATATCAGGTGACATCCTGGTGGCGGACGAATTAGGTTATCTGTACTTCCGCGACCGCACCGGAGACACGTTCCGGTGGCGCGGAGAGAACGTTTCCACCACAGAGGTCGAGGCGGCCGTGTCTAGATGTGCCAATCAAAGGGACGCTGTAGTCTACGGTGTAGAGATTCCGAATACGGAAGGTCGGGCTGGCATGTGCGGCATAGTGGACATTGAAGGCACATTGGACCTGGATAAACTCGCCAAAGATATTGCGAAAGACCTGCCTAAATACGCGAGGCCAATATTCATTAGAATAATGACCAGCGTTGATATGACAGGAACATTCAAAATGAAGAAAGTGGATTTACAAAAAGAAGGTTATAATCCATCAACTGTGAGCGACAAAATGTTCTTCTTTGAACCAAAACAGAATAAGTACGTGCCCTTAGGAGTTGAA | dsRNA | phenol_chloroform | subsequent to transcription | The templates for synthesis of dsRNAs corresponding to BmFATP were prepared using gene-specific primers containing T7 polymerase sites (NRPG1860T7-F1 [sense primer]: 5-CCTAATACGACTCACTATAGGGCGGTATACACGTCAGGGA-3 and NRPG1860T7-R1 [antisense primer]: 5-CCTAATACGACTCACTATAGGGCGGTTCAACTCCTAAGGG-3; nucleotide sequences corresponding to the T7 promoter region are underlined). PCR was performed under the conditions of 6 cycles of 94 °C for 30s, 56.5 °C for 30s, 68 °C for 90s followed by 30 cycles of 94°C for 30 s, 66°C for 30 s, 68°C for 90 s using KOD-Plus- (Toyobo, Osaka, Japan) with the resulting products purified (Wizard SV Gel and PCR Clean-Up kit, Promega, Madison, WI) and used as templates to generate dsRNAs using the AmpliScribeTM T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI) according to the manufacturerâs instructions. | GenBank | AB451529 | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | pupa | lab_colony | 5 | injection | DEPC-treated water | 0.02 | buffer | 10 | pheromone gland (abdominal tip) | Control pupae were injected with 2ul of DEPC-treated H2O alone. After injection, pupae were maintained under normal conditions until adult emergence. | adult | RT-PCR (semi-quantitative) W.blot phenotype enzymatic_activity | pheromone gland | not checked | 192 | high | 50 | ||||||
| 220 | soluble trehalase | RNAi | Background: Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1) and membrane-bound (Tre-2) trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. Principal Findings: The membrane-bound trehalase of Spodoptera exigua (SeTre-2) was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1) and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi) of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA) and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB) expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. Conclusions: SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2 has an important role in CHSB expression and chitin synthesis in the midgut. | pubmed | 20405036 | Zhang | Wenqing | soluble trehalase target | ACGCGGGGAGTCGCGTTCGTGTTTGCGACTCGAGCGCATGTAATCGATTAACTTATCCCAACGACATGTCAAATCGATAAAATCAATCGATTGTAAAGAAATGATATGATAATTTAATTTTGTTTTATCTGTTACCAGTGTAAATCCTAGTGTAGTTGCGATTAGTTACGTGTATCGATATTTCGATTTTAAATTCAACTTGTGTTCGGTTGCCCGTGAAGTGAATGGTGTCCTTTTGTAAGATGCGGGGATACCTGATTGTCTTGGCGGCGGCCGTTGCGCTGGCCGGTGCTGCCGACATGCGACCCACTTGTAGCAAGCCTGTCTACTGTAATAGCAAACTACTCCATAAAGTTCAAATGGCCAGGCTCTACAGTGATTCTAAAACCTTCGTCGATCTTCAAATGAATTACGATCAAAACACAACATTAAATGCTTTTGATACTTTACTTAACGATACGGACCAGGAACCGTCAGTAGAACAATTAAGAGAATTTGTTGAAAAACACTTCTCTAATAATAGCGAACTCATGCCATGGAGACCACCAGATTTTAATACTGATCCTTACTCATTAAATACTATACAAGACGATGATTTAAGAGAATTCGCCAGAAACATCACCAACATCTGGCCACTTCTTGCCCGAAAAGTCAAAGATGAGGTTATTCAAAATCCTGATCGTTATAGTTTGATCCCTATCACTAATGGATTCATAATCCCTGGAGGAAGGTTCACGGAGATTTATTACTGGGATACTTACTGGATTATCGGAGGTCTGCTGATAAGTGGAATGCAAGAAATCGCGAAAGGAATTATTGGAAATCTCATTGAACTATTAAATATGCTCGGTCATATTCCCAATGGAAGCAGGTGGTATTATCAAGAGCGCAGTCTGCCACCAATGTTGACAGCTATGGTTGCTATTTACTACCAATACACCAATGACACGGAGTTCTTGAGAAACAACATTGCATATTTAGAAAAGGGAATGGACTTCTGGTTTGATGAAAGATCAGTGACTGTTGAAAAAGAAGGTAGTAACCACACTCTGCTTAGGTATTTTGCTATCAGCTCAGGACCAAGACCTGAATCTTATTATGAAGACTACGAAAACGCGGCAGAATTTAGTGAACAAGCTCGCACAGACTTCTTTATCGATATTAAGAGCGCAGCTGAGAGTGGTTGGGACTTCTCAACGCGCTGGTTCGTTAACCCTGATGGCAGCAACACAGGAACTTTAAAAGACATTCACACCAGATACATCATACCTGTCGATTTAAACGCCATATTTGCGGGTGCTCTTCAGAATATCGCAAACTTCCATGTTATTTTGGGGAATCACCGGGATGCTGTCTCATTTAGCCAATCAGCTCAACAGTGGAGAGATAGCATTCAGTCAATTCTGTGGAATGAGGAAGAGGGAATGTGGTTTGATTATGACATCAGAGATCAGATACATCGCAAATACTTCTACCCATCTAACCTGGCTCCTTTGTGGCAAGGTGCTGTCGATCCGAATATAGTGAAAGCGAATGCACTCAGAATTCTGAATAATCTTAAACAATCTGGTGCTTTTGACTTCCCTGGAGGGGTGCCTACATCCCTCAGTAGGAGTGGAGAGCAGTGGGACTTCCCTAACGTATGGCCACCAGAAATGAGCATCGCAGTGAACGCCATTGAAAACATTGGAACACCAGAGGCGAGTGTGCTGGCTTTTGAAACTGCGCAAACATTCGTTAGGGCTTGTCATTCGGGCTTCTCAGAATACCACCAGATGTTCGAGAAATATGATGCAGAGAATCCTGGCAAGTTTGGAGGAGGGGGTGAATACAACGTGCAATATGGTTTTGGATGGACCAATGGTGTAGTCTTAGAGTTTATGAAGAAATATGGAGAAGGCATGACTGCAAACGACTCTAATGAGTTAGATACTACTGCATCTCCCTCCAACAGTAGCGATACATCGAATAATGCCGCATGACTAGGCGTGGGTACAATAAATAACTTTTTAATTTTTCTTTCAGCATTCAAAATATTTATTGGTGTTCATAATAACTAAAGGTTGTGACTACATAAATAATAATTAGCTAAAACAGTGCCATAAGATCATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Laphygma | exigua | cds | InsectaCentral | soluble trehalase target | soluble trehalase RNAi construct | ACCAGGAACCGTCAGTAGAACAATTAAGAGAATTTGTTGAAAAACACTTCTCTAATAATAGCGAACTCATGCCATGGAGACCACCAGATTTTAATACTGATCCTTACTCATTAAATACTATACAAGACGATGATTTAAGAGAATTCGCCAGAAACATCACCAACATCTGGCCACTTCTTGCCCGAAAAGTCAAAGATGAGGTTATTCAAAATCCTGATCGTTATAGTTTGATCCCTATCACTAATGGATTCATAATCCCTGGAGGAAGGTTCACGGAGATTTATTACTGGGATACTTACTGGATTATCGAAGGGCTGCTGATAAGTGGAATGCAAGAAACTGCGAAAGGAATTATTGAAAATCTCATTGAACTATTAAATATGTTTGGTCATATTCCCAATGGAAGCAGATGGTATTATCAAGAGCGCAGTCAGCCACCAATGTTGACAGCTATGGTTGC | dsRNA | T7 RiboMAXTM Express RNAi System Kit (Promega) | ethanol precipitation | subsequent to transcription | according to the manufacturerâs instructions | GenBank | soluble trehalase RNAi construct | Lepidoptera | Noctuidae | Laphygma | exigua | local resource | larva | lab_colony | 1 | injection | DEPC | 0.5 | buffer non_specific_dsRNA | 3 | hemolymph | larva | qPCR W.blot phenotype enzymatic_activity | whole organism | not checked | 12244872 | high | ||||||||
| 222 | Chitin Synthase A | RNAi | RNA interference (RNAi) is a powerful tool for rapidly analyzing gene functions. However, little is known about the possible use of dsRNA/siRNA as a pest control method. Here we demonstrate that dsRNA/siRNA can induce the silence of chitin synthase gene A (CHSA) which is an important gene for the growth and development of cuticles and trachea in beet armyworm Spodoptera exigua. Based on the in vitro RNAi experiments in an insect cell line (Trichoplusia ni High 5), in vivo RNAi was performed by injecting synthesized dsRNA/siRNA into the 4th instar larvae of S. exigua. Significantly lower levels of CHSA transcripts were detected. In addition, the cuticle of these insects was disordered and the epithelial walls of larval trachea did not expand uniformly in injected individuals. Moreover, Injections significantly increased abnormalities relative to control larvae.. These results highlighted the possibility of dsRNA/siRNA for gene function studies in lepidopteran insects and future pest control. | pubmed | 18662430 | Zhang | Wenqing | Chitin Synthase A target | GAGTGAGCGCGCGGCCTACCGACCGAACGCACGCGCAAACCATGCGCAGCGACGCTGTCCCGCGAGTGCACTCCTCGTTACATTGTGAAGTGATATACAGTATAAACACGGATTACAACAACGCTTATCTCCACAACATACTGAACTAATATTAATTAAAAATGGCGACGTCAGGAGGGAAACGGCGGGAAGAGGGCAGCGATAACTCCGATGATGAGCTTACACCGCTCGCTAACGATATTTATGGCGGAAGCCAAAGGACAGTACAAGAAACGAAAGGATGGGACGTGTTCCGGGAGTTTCCACCGAAGCAGGACAGTGGGTCTATGGAGACTCAGAAATGTTTGGAGTTCACAGTGCGGTTGCTGAAGGTGACGGCATATCTAGTCGTCTTCATTGCGGTCCTCGGGTCCGGAGTCGTAGCAAAGGGCAGCACGCTCTTTATGACATCACAGCTAAAAAAGGACAGGCGGATTGCGTATTGTAATAGGAATTTAGGTAGGGATAAACAATTTATAGTAAGTCTTCCAGACGAGGAACGAGTGGCTTGGATGTGGGCTATTTTGGCTGCGTTCGCCATACCAGAAATAGGAACACTCATTAGATCAGTGAGGATATGCTTTTTCAAAACTTCTAGACGACCGACTAGCACACAATTCGCTGTGATTTTCATAGCGGAGACGTTGCATACGATAGGAATGGGTCTCCTATTCTTTTTGATCCTACCAGAACTGGACGTGGTCAAAGGAGCAATGATTACGAACTGCCTTTGCATAATTCCTGCCGTTTTGGGCTTGCTCTCACGCAACTCCAGAGATTCGAAACGGTTCATAAAAGTGATTGTGGACATGGCGGCAATAGTTGCGCAAGTCACTGGATTCATTGTATGGCCACTATTAGAGAATAAACCTGTACTATGGTTGATACCAGTCGCATCACTATGCATATCCCTTGGATGGTGGGAAAACTATGTCACACGTCAGAGTCCGATAGGTATAATCAAAAGCCTTGGCAGATTAAAAGATGAATTAACCTTCACTCGCTACTACACGTACCGTTTTATATCTGTCTGGAAAATCTTGGTCTTCCTCATGTGCATTCTCTTCAGCATTTGGCTCGAAGGTGACGAGCCTGCCATGTTTTTCCAACTGTTCAATGCCGGTTTTGGACCACACAGTATCGTTGTCGAAGAGGTACAAATTCAATTAGGCGGGACCGTCATTCCTGATTTAGCTAATGTTACTTTAACCGGAGACTCAGTTGAGGTCGCAGCTGCTTACAAATCCGCATTCTACGTGATGCTTATCCAAATATTTGCAGCGTATATCTGCTACATATTTGGAAAGTTCGCTTGTAAGATCCTCATCCAAGGCTTCAGTTACGCGTTCCCCATCAATCTCGTCATTCCATTGGTGGTCAACTTGTTGATTGCCGCGTGTGGTATCAGAAATGGTGACAATTGCTATTTCCATGGGACAGTTCCCGATTATCTTTACTTCGAGAGTCCACCAGTGTTTACGCTAAGCGATTTCATATCTCGTCAAATGGCATGGATATGGCTACTATGGCTATTGTCGCAAACATGGATCACCATACACATCTGGACACCAAAAGCTGAACGTTTGGCCTCTACGGAGAAGTTATTCGTGATGCCAATGTACAACGGTTTACTTATTGATCAGAGTATGGCCTTAAACAGAAAGAGGAATGATCAAAGAGATGTTAAGACTGAGGACCTCGCAGAAATAGAAAAAGAAAAAGGCGACGAATACTATGAAACTATATCAGTTCACACGGATAACACTGGGTCTTCTCCAAAAGCTATTAAGTCATCAGATCAGATCACCAGGATATATGCATGCGCTACTATGTGGCACGAAACTAAAGACGAGATGATGGAGTTCTTGAAGTCCATTCTTCGGTTAGACGAGGATCAGTGCGCTCGGCGTGTAGCTCAAAAGTATTTACGAGTCGTTGACCCTGATTACTATGAATTCGAAACACATATCTTCTTAGACGACGCCTTCGAAATATCAGATCACAGTGACGATGATTCTCAGGTGAATCGATTCGTAAAACTGCTTGTTGACACTATCGATGAAGCGGCTTCCGAGGTACATCAGACGAACATTCGTATTCGACCACCTAAGAAGTATCCCGCGCCTTACGGAGGACGATTGACGTGGGTACTGCCAGGAAAGACGAAGATGATTTGTCACTTGAAGGATAAGGCAAAGATTCGTCACAGGAAACGTTGGTCTCAGGTGATGTACATGTACTACCTACTCGGTCACCGACTAATGGAGCTGCCAATATCTGTGGATCGTAAAGAAGTTATGGCTGAGAACACCTATCTGCTGACCCTGGACGGAGACATCGATTTCCAACCTCATGCTGTACGTTTGCTTATCGATTTGATGAAGAAGAACAAGAATCTGGGAGCTGCTTGCGGTCGTATTCATCCCGTAGGCTCTGGCCCTATGGTGTGGTATCAAATGTTCGAGTATGCCATTGGTCATTGGCTGCAAAAGGCAACTGAACACATGATTGGCTGTGTACTGTGTAGCCCTGGCTGCTTCTCCCTCTTCAGAGGAAAGGCTTTGATGGACGACAACGTAATGAAGAAGTATACATTGAGGTCTGATGAAGCTCGGCATTACGTACAGTACGATCAAGGGGAAGATCGATGGTTATGTACGCTGTTACTTCAACGAGGTTACCGTGTAGAGTACTCAGCTGCCTCCGACGCCTACACTCACTGTCCCGAAGGTTTTAACGAGTTCTACAACCAACGTCGTCGTTGGGTGCCTTCCACCATCGCCAACATTATGGACTTGCTTGCCGATTGCAAACACACCATCAAGATTAACGATAACATCTCCAGTCCTTATATCGCATACCAGATGATGTTGATGGGTGGTACAATCTTGGGTCCCGGAACTATATTCCTTATGTTGGTGGGTGCCTTCGTGGCCGCTTTCCGTATTGACAACTGGACTTCTTTCGAATATAACTTGTATCCCATTTTGATCTTCATGTTTGTATGTTTTACGATGAAATCCGAAATTCAATTGCTCGTGGCTCAGATATTATCGACGGCATACGCCATGATTATGATGGCTTTTATAGTCGGTACCGCGCTTCAGTTAGGCGAGGACGGTATCGGATCTCCTTCGGCTATATTTTTGATATCACTTTCGAGTTCGTTCTTCATAGCCGCTTGCTTGCATCCGCAGGAGTTCTGGTGTATTGTACCGGGAATTATTTATCTTTTATCTATACCCTCTATGTACTTGCTTTTGATTTTATATTCGATTATAAATCTTAACGTAGTATCTTGGGGTACTCGAGAAGTAGCTGTTAAGAAGACGAAGAAGGAAATCGAAGCAGAAAAGAAAGAAGCAGAATTAGCAAAAAAATCGGCAAAACAGAAGTCTTTGTTAGGTTTCCTTCAAGGAGTAAACAGCAATGAAGAAGAAGGATCTATAGAATTCTCGTTCGCCGGTCTATTCAAGTGTCTGTTATGCACGCATCCAAAAGGAAACGAAGAGAAAGTGCAACTCTTGCATATTGCATCTACTCTAGAGAAGTTGGAAAAGAAATTAGAAACTGTTGAGAGGGCTGTTGATCCTCACGGCATTAGCAGAGGACGTAAACTGTCGGTTGGACCAAGAGGTAGCACCACTGGAGATCATGGTTTGGACGCTCTAGCTGAAGGACCAGAAGAGGATAACGACTCAGATTCTGAAACTGACACACTTTCTACTGTGCCAAGAGAAAAGAGAGATGATCTCATAAACCCATACTGGATTGAGGATCCTGAGTTGAAAAAGGGTGAAGTAGACTTTTTGAGTCCCGCCGAATTATCTTTCTGGAAAGATCTCATTGACAAATATTTATACCCTATTGATGCTAACAAGGAGGAGCAGGCCCGTATATCCAAGGATCTGAAAGAATTGAGAGACTCGTCTGTTTTTTCTTTCTTTATGGTCAATGCTCTCTTTGTATTGATTGTATTCTTGCTACAACTGAACAAGGACAACCTTCACATTAAGTGGCCCTTCGGAGTTAAAACTAACATAACATACGATGAGGTTACTCAAGAGGTGTTAATATCAAAGGAATATCTGCAACTGGAGCCTATTGGTTTAGTGTTTGTATTCTTCTTCGCCTTGATCCTGGTGATACAGTTCTCCGCCATGTTGTTCCATAGATTTGGAACCCTTTCACACATATTAGCGTCGACAGAACTGAACTGGTTCTGTTCTAAGAAGTCCGACGACTTGTCTCAAGACGCACTATTAGATAAGAATGCAATAGCAATAGTAAAAGACCTGCAGAAATTGAATGGTCTGGACGACGATTATGACAATGACTCGGGCTCGGGTCCTCATAACGTCGGCAGAAGAAAGACTATTCACAATTTGGAGAAGGCGAGACAGAAGAAGAGGAATATAGGCACACTGGATGTGGCCTTCAAGAAGAGATTCTTCAATATGAACGCCAACGATGGACCAGGTACACCAGTTCTAAATCGTAAGATGACGTTGAGAAGGGAAACGCTAAAGGCTTTAGAGACGAGAAGGAATTCAGTGATGGCGGAAAGAAGAAAATCCCAGATGCAAACACTTGGTGCCAATAATGAATATGGAGTGACAGGAATGCTCAATAATAACTTAGGTGTCGGGCCGCGGCACAGGACATCTAATGCCAACATATCAGTGAAAGATGTTTTCGGCGAGCCAAACGGAGGTCAAGTCAACCGAGGCTACGAAACCACCATAGGCGACGAAGATGACACAAACTCAATGAGATTACAACCTAGACAAAACCAAGTTTCCTTCCAGGGTAGATTCTAAAAGAGTTGCCAAACTAAGTGCCTAGGTTAAACAAATGTAGATACTAGAAATATAGACTGTAAAATTTTTTACAATGAAGAATCTCAGCAAATGTTTGCGGGAATCATGTATTGCTTGTGATATATACTTTTTTTTAGGTCGTGACAGCAATGTGCTGTCATTAAATACCAACATCGGACAATTTACAGCTTTATTGTAAGATAAAACATACGAATTTACACTGCCACTTAACTTTACATTATTGATCTTTTTATACTTATTTAAAATAATATCTTTTGTATTAAAAGATTTTTGTTTTAGTTTTTATGAAAGTAGACATCGAATTAGTGTTGTATGTTTTTAATGGGG | Lepidoptera | Noctuidae | Laphygma | exigua | cds | GenBank | Chitin Synthase A target | Chitin Synthase A RNAi construct | GGTGAATCGATTCGTAAAACTGCTTGTTGACACTATCGATGAAGCGGCTTCCGAGGTACATCAGACGAACATTCGTATTCGACCACCTAAGAAGTATCCCGCGCCTTACGGAGGACGATTGACGTGGGTACTGCCAGGAAAGACGAAGATGATTTGTCACTTGAAGGATAAGGCAAAGATTCGTCACAGGAAACGTTGGTCTCAGGTGATGTACATGTACTACCTACTCGGTCACCGACTAATGGAGCTGCCAATATCTGTGGATCGTAAAGAAGTTATGGCTGAGAACACCTATCTGCTGACCCTGGACGGAGACATCGATTTCCAACCTCATGCTGTACGTTTGCTTATCGATTTGATGAAGAAGAACAAGAATCTGGGAGCTGCTTGCGGTCGTATTCATC | dsRNA | T7 RiboMAXTM Express RNAi System Kit (Promega) | ethanol precipitation | subsequent to transcription | according to the manufacturerâs instructions | InsectaCentral | Chitin Synthase A RNAi construct | Lepidoptera | Noctuidae | Laphygma | exigua | local resource | larva | lab_colony | 1 | injection | DEPC | 0.30000000000000004 | buffer non_specific_dsRNA | 3 | hemolymph | larva | RT-PCR (semi-quantitative) phenotype | whole organism | not checked | 72 | high | ||||||||
| 227 | Manduca sexta MAPK p38 | RNAi | The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. In this work we analyzed the response of two different insect orders, the Lepidopteran Manduca sexta and Dipteran Aedes aegypti to highly specific Cry toxins, Cry1Ab and Cry11Aa, respectively. One pathway activated in different organisms in response to a variety of poreforming toxins is the mitogen-activated protein kinase p38 pathway (MAPK p38) that activates a complex defense response. We analyzed the MAPK p38 activation by immunodetection of its phosphorylated isoform, and the induction of p38 by RT-PCR, real-time PCR quantitative assays and immunodetection. We show that MAPK p38 is activated at postraductional level after Cry toxin intoxication in both insect orders. We detected the p38 induction at the transcriptional and traductional level, and observed a different response. In these three levels, we found that both insects respond to Cry toxin action but M. sexta responses more strongly than A. aegypti. Gene silencing of MAPK p38 in vivo, resulted in both insect species becoming hypersensitive to Cry toxin action, suggesting that the MAPK p38 pathway is involved in insect defense against Bt Cry toxins. This finding may have biotechnological applications for enhancing the activity of some Bt Cry toxins against specific insect pests. | pubmed | 20040372 | Garbutt | Jennie | Manduca sexta MAPK p38 target | CGGCCATAGACACTTTGCATAATATGAAAGTGGCCATTAAGAAATTAGCAAGACCATTTCAATCTGCTGTACATGCTAAGAGAACGTACCGCGAACTTCGGATGCTTAAACACATGAACCATGAAAATGTAATCGG | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | cds | InsectaCentral | Manduca sexta MAPK p38 target | Manduca sexta MAPK p38 RNAi construct | ATAATATGAAAGTGGCCATTAAGAAATTAGCAAGACCATTTCAATCTGCTGTACATGCTAAGAGAACGTACCGCGAACTTCGGATGCTTAAACACATGAACCATGAAAATGTAATCGG | dsRNA | HiScribe RNai transcription kit | unknown | simultaneously with transcription | In vitro transcription of both DNA strands of the insert was performed with T7 RNA polymerase using the HiScribe RNAi Transcription Kit (New England Biolabs) as reported by the manufacturer, yielding dsRNA. | InsectaCentral | Manduca sexta MAPK p38 RNAi construct | Lepidoptera | Sphingidae | Manduca | sexta | 7130 | local resource | larva | lab_colony | 5 | feeding | 123456789 | buffer | Feeding | larva | RT-PCR (semi-quantitative) W.blot | midgut | not checked | 170 | high | 80 | |||||||
| 228 | Ostrinia nubilalis chitinase | RNAi | Chitinases belong to a large and diverse family of hydrolytic enzymes that break down glycosidic bonds of chitin. However, very little is known about the function of chitinase genes in regulating the chitin content in peritrophic matrix (PM) of the midgut in insects. We identified a cDNA putatively encoding a chitinase (OnCht) in European corn borer (ECB; Ostrinia nubilalis). The OnCht transcript was predominately found in larval midgut but undetectable in eggs, pupae, or adults. When the larvae were fed on an artificial diet, the OnCht transcript level increased by 4.4-fold but the transcript level of a gut-specific chitin synthase (OnCHS2) gene decreased by 2.5-fold as compared with those of unfed larvae. In contrast, when the larvae were fed with the food and then starved for 24 h, the OnCht transcript level decreased by 1.8-fold but the transcript level of OnCHS2 increased by 1.8-fold. Furthermore, there was a negative relationship between OnCht transcript level and chitin content in the midgut. By using a feeding-based RNAi technique, we were able to reduce the OnCht transcript level by 63-64% in the larval midgut. Consequently, these larvae showed significantly increased chitin content (26%) in the PM but decreased larval body weight (54%) as compared with the control larvae fed on the diet containing GFP dsRNA. Therefore, for the first time, we provide strong evidence that OnCht plays an important role in regulating chitin content of the PM and subsequently affecting the growth and development of the ECB larvae. | pubmed | 20542114 | Garbutt | Jennie | Ostrinia nubilalis chitinase target | GGGCTTCACTATGGGATTGATTTTATTATTTGTGTTGGGTTTTTGTGCCAGTGCGGTGTTCGCGAATGACGATAAAATAGTAGTCTGCTACTATGGCACATGGGCGACATACCGGACTGGCTTGGGCAAGTTCGACGTAGACGACATCGACCCATTCCTCTGCACGCATCTGGTATATGCCTTCATCGGCATTAATGCTGAAGGAACAGCTCTGGCGCTTGACCCTGAGCTTGATGTTGAGAGAGGTAACTTCAAGAACTTCACTTCGTTGAAAGAAAAGAACCCGAACCTCAAAACGCTTGTAGCTGTCGGAGGATGGAGCGAAGGATCAGCTAATTACTCTATTATGGCAGCAGAACCAGAATATCGGCAGAACTTCATCAACACCTCTCTGGCAATGATACTGGAATACAACTTCGATGGTCTGGACGTAGACTGGGAGTACCCCAATCGCAGAGACACGGTGCATGGTGAAGACGACATTGAGAACTTCAGCACTCTCCTTAAGGAGTTGAGGGAGGAATTCGATAATTACGGTCTACTGCTGACAGTCGCTGTGGCTGCTGTGGAGGAAGCTGCTGTGCAGTCCTATGACGTTCCAAGTGTTGCCAAGTACGTAGACTACATTGGCGTCATGACGTATGACATGCACGGCGCCTGGGACTCCGTGACCGGCCACAACGCGCCTCTGTTCATAAGTGAAGGCGAGAGTGCTGAAAACGAAAGCACTCTGTACAACGTCAATAACGCTGTCCAGTATTGGCTTAGTGCAGGATGTCCTCCCGAGAAGTTAGTTATGGGGGTTCCATTCTATGGGCGCACTTTCAACCTGAGTGACCCTTCAGTTAACGCTCCAAATTCACCTTCCAACGGAGCTGGTCTCGCCGGTCCTTACACTGCTGAAAGTGGATTTATCGGGTATAATGAGTTCTGCTATATTCTCCAGAACGAGTCTTCTTGGACCGTCCAGACCGACAACCTTGCCAAAGTGCCTTACGCCTTCCTCGACTACAACTGGGTATCTTTCGATAACGTTGAGTCCATGACCGCCAAAGTGGAGTACGCTAACAGCTTCAACCTTCGCGGCATCATGCTCTGGAGTATCGAGACTGATGACTTCCACGGCCTCTGTGGAGAGGGAACATTCCCTCTGCTGAACACCATCAACACAGTTCTAGCTGAAGGCTCAACAGAAGCCAGACACAACAACCCTCCTCATCATCACCATCATTAAATATGGAAACCTACTTCCAGGTCCTTAAAAGAAGAATTAAAGACAAATCCATAGTACAAGTATTATGAATCTTCCAGAGTGCCAGGAGACGGCACTCTTTTCATAATTAACACCAAAAACGAGGCAAAGATAATAAAAAATATATTTCTGCAAAAAAAAAAAAAAAAAAA | Lepidoptera | Crambidae | Ostrinia | nubilalis | 29057 | cds | GenBank | GU329524.1 | Ostrinia nubilalis chitinase RNAi construct | CGGAGGATGGAGCGAAGGATCAGCTAATTACTCTATTATGGCAGCAGAACCAGAATATCGGCAGAACTTCATCAACACCTCTCTGGCAATGATACTGGAATACAACTTCGATGGTCTGGACGTAGACTGGGAGTACCCCAATCGCAGAGACACGGTGCATGGTGAAGACGACATTGAGAACTTCAGCACTCTCCTTAAGGAGTTGAGGGAGGAATTCGATAATTACGGTCTACTGCTGACAGTCGCTGTGGCTGCTGTGGAGGAAGCTGCTGTGCAGTCCTATGACGTTCCAAGTGTTGCCAAGTACGTAGACTACATTGGCGTCATGACGTATGACATGCACGGCGCCTGGGACTCCGTGACCGGCCACAACGCGCCTCTGTTCATAAGTGAAGGCGAGAGT | dsRNA | AmpliScribe T7-Flash Kit (Epicentre Technologies, Madison, WI) | phenol_chloroform | simultaneously with transcription | Each dsRNA was prepared using plasmid DNA as template by in vitro transcription for RNAi. The primers were designed by using Beacon Designer software (version 7) and T7 primer sequence was placed in front of both forward and reverse primers. The primer sequences for generating dsRNA were 50- TAATACGACTCACTATAG GCGGAGGATGGAGCGAAG and 30- TAATACGACTCACTATAGGACTC TCGCCTTCACTTAT for the OnCht gene, and 50- TAATACGACTCA CTATAGGCCATTCTTTTGTTTGTCTGC and 30- TAATACGACTCACTA TAGGGGCCAACACTTGTCAC for the green fluorescent protein (GFP) gene. The expected dsRNA sizes are 404 and 309 bp for OnCht- and GFP-dsRNA, respectively. The dsRNA was transcribed by using AmpliScribe T7-Flash Kit (Epicentre Technologies, Madison, WI) according to the manufacturerâs protocol. The dsRNA was purified by phenol/chloroform extraction followed by ammonium acetate precipitation. | InsectaCentral | Ostrinia nubilalis chitinase RNAi construct | Lepidoptera | Crambidae | Ostrinia | nubilalis | 29057 | local resource | larva | lab_colony | feeding | non_specific_dsRNA | 3 | Feeding | Control larvae were fed with the same amount of the artificial diet containing the same amount of GFP dsRNA as that of OnCht dsRNA. | larva | qPCR | midgut | not checked | 192 | high | 64 | |||||||
| 229 | Diatraea saccharalis | RNAi | Aminopeptidase N (APN) proteins located at the midgut epithelium of some lepidopteran species have been implicated as receptors for insecticidal proteins from Bacillus thuringiensis. cDNAs of three APN isoforms, DsAPN1, DsAPN2, and DsAPN3, from Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab- RR) strains of the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), were identified and sequenced using reverse transcriptase polymerase chain reaction (RT-PCR) and 50 rapid amplification of cDNA end (50 RACE). The characteristic APN sequence features were derived from deduced amino acid sequences of the cloned cDNAs. cDNA sequences of the three APN genes were identical between the Cry1Ab-SS and -RR strains. However, total APN proteolytic activity and gene expression of the three APNs from Cry1Ab-RR larvae were significantly lower than those of the Cry1Ab-SS strain. RNA interference (RNAi) was employed using an oral droplet feeding technique for the three APNs of the Cry1Ab-SS strain. Down-regulating expressions of the three APN genes by RNAi were corresponding to the reductions in the specific APN activity. In addition, silencing of all three APNs in D. saccharalis in vivo by RNAi resulted in a decrease in Cry1Ab susceptibility. Our results showed that reduction in expression of the three APNs is functionally associated with the Cry1Ab resistance in D. saccharalis. | InsectaCentral | Unpublished | Garbutt | Jennie | Diatraea saccharalis target | GGGGGGGGGGGGGGGGGATCATTTGCTAAAGTGACATGATGGCGTACCGATGGCTATCGTTGTCAGTGGTAGTCACTTTACTCCAGGGCGCATTATTTCTAAGCCCTTTACCAGTACCTGATAAACAATGGGATGATTTTGATGTACACCCTTTACAAACAGCAGAAGAAGAATGGGAACAATATAACGAATTGTTAAGAAACTCCGATTATAGACTACCGAGAACAACGGTACCAGATCATTATGTTCTTTCCTTGACTCCATATTTTGAACATACGGATGTAAACCGAGCATTCACATTCGATGGAAAGGTTAAAATTAACATTAGAGCAACTACAGAAGGTGTAAATGAAATTCTCATGCACTGCAATGATCTCACCGTGAAAAGTGTCACTGTTCAATACACGGACAGTAATAACGAAACTAAGAATATTGCCTCATCTGAACAGAACTTGCAGTGTCAAATGCCTTATAGCTTCTTAAGGATATCTACAACAGAATATTTGCAACAAGTTGTGACGTATGAAGTAGAAATGGAATTTACTGGTCATCTTCAATCCAATATGAGAGGTTTTTATCGGAGTTGGTACTCTGACCATAATACCACTAGGAGATGGATGGCTACTACTCAATTCCAACCAGGTCATGCGCGTCAAGCATTCCCCTGCTATGACGAACCTGGTTTCAAGGCTACTTTCGACATCACCATCAATAGGGAACCTGATTTCAGCCCTACGATTTCGAATATGCCTATTAAAGATACTTCAAATGAGCTAGTACCCGGTAGAGTCTCTGAAACATTCCACACAACCCCACGGACTTCTACATACCTGCTTGCGTTTATCGTTTCTCATTACGAGGTAGTTGCATCAAAAAATGATGAAGAACGGCCGTTTAGAATCTACGCTAGAAATAATGCTGGCACGACTGGAGACTGGTCTCTAAAAATTGGTATAGACTTACTAAGAGCGATGGAAGAATACACACAAATACCTTACTACACAATGGCTGATAACATGGATATGAAACAAGCCGCTATACCTGATTTTTCAGCTGGTGCTATGGAAAATTGGGGACTCTTAACCTACAGAGAGGCTTTGATACTTTACGATCCTCAAAACTCGAACCACTTTTATAAGCAACGTGTTGCTAATATTGTGTCCCATGAAGTCGCCCATATGTGGTTTGGAAACCTAGTCACGTGCGCCTGGTGGGATAACTTGTGGCTAAATGAAGGCTTTGCTAGATTCTACCAATATTATTTGACTGCCTCGGTGGCACCTGAATTGGGTTATGAAACTCGATTCATAGTTGAGCAGTTTGAGCAAGCCATGAGTGCAGATTCCGTAGACACTGCGCACGCCCTCACAAATCAGGCCGTTAGTGATCCAATAACAGTCAGTGCCCACTTTTCATCTATCACTTACGCGAGAGGAGCATGCATATTGAGAATGACAGAGCATCTTCTATCTCACACAACTTTTGTTAAAGGCCTCCGAAAATATTTACAAGACAGGAGCTACGATGTAGCCGAACCTCATGATTTATTCAAAGCACTTGATGACGCTGCTCTAGAAGACGGTGTTCTGAATGATTATGATGGCATTACCATTGATCAATACTTTAGAAGCTGGTCAGAGAAAGCAGGACATCCATTGCTCACAGTTAACATTGATCAAGCTACAGGGGAAATGACAATAAAACAGGCTCGTTGGGAACGTTCGTCGGGTGAGTCAATTCATTTTAGTTTGTGGGATATTCCTATAACATGGACTCGAGCCGATGATCCCGATTTCTTAAATCTGAAGCCTTCCCAATTCATGAGAACCGAAAGTACAGTCATCCAACGTGGTACCACAGGAAATGAATGGGTCGTTTTCAACAAGCAAGCATCAGGTTTCTACAGAATTAACTATGACAACACCAACTGGGCTCTTATTACACAAGCTTTGAGGTCGGATGTTCATGTTATCCATGAATACAATCGGGCACAGATTATTGACGATTTGTTCAACTTCGGTAGAGCAACTGTGATGCCTTATGATAGAGTGTTCAACATTATATCATTCCTCGAGTTTGAGGATCAATATGCACCATGGATCAATGCAATTACTGGATTTAACTTCCTTATCAGGAGGCTTGCTCATGATACACCCAACTTGAAAAGATTACAGGAAGTGATTATTGCATTCAGTAAAACAATAACAGCGCGATTAGGGTATGGGGAAATTGAGAATGAGCCTTTCATGGATGGATTATTGAGAATGTATGTCATGCAGTTCCTCTGTAATATTGGAGACCAGCAATGTATAAACGTTGGCAAGGAGAGGTTCACGATGTGGAGGAGCGGAAATGTTCATATTCCTGCAAATATGCGGCCTTGGGTTTACTGCGTTGGTCTTCGTGAAGGAACAGCTGAAGATTTCAACTTTTTCTGGGGAAAATATTTGAATGAAGACCTTGCTAGTGAACAAGTCGTTATGCTTCAAGCAGCTGGATGCACTACTGACCAAAACAGTCTTACAGTGTTCTTGGACGCTATTGTTGCGGAAGAGGAAATTGTTAGACCACAAGATCTCACTACTGCTCTCAACTCAGCAGTGACCAGAAACGAAGTTAATACGCTCAGAGTTTTTGAATGGCTGAAAAATAATATTGATAAAACTGCAGCCAAATTTGGAAGTATCACTACACCTCTCAGCTATATTGCACCACGACTTTTGACTCAAAGTGACATAGACCGGTTTGAAAGTTGGTTGCAAGAAAATGAAGATAGAATTGGTCCAACGGCTTTCGCAACGGGCAGCAGTGGTGTAAATAACGTCAGAAACGCTCTCATTTGGTCAGATCTACGAATTCCAGAAATTGTTAAATTCCTTGAAAATGGTTATGTAGAAGATGATATTCCCAATAATACTACAACTACTGAAACTACTACCACTGAAACTACTACCACTGAAACTACTACCACTGAAACTACTACCACTGAAACTACTACTACTGAAACTACCACTACTGATACTACTACTACAGAAGCACCCACAGAAGCACCCACAGAAGCACCCACAGAAGCACCCACAGAAGCCACTGAAACTACTACCACTGAAACTACTACCACTGAAACTACTACCACTGAAACTACTACCACTGAAACTACTACCACTGAAACTACTACTACTGAAACTACCACTACTGATACTACTACTACAGAAGCACCCACAGAAGCACCCACAGAAGCACCCACAGAAGCACCCACAGAAGCACCCACAGAAGCACCCACAGAAGCACCTACAGAAGAACCTACTACGCCTGGCGACAATGGTGCGGCCACTATTGTATTAAGTTTTGCAGCCCTTGCAGTATCATTTTTTATAACATTCTTTAATTAGAGTTTTTATTAATAAAGCTTAATGTTTTAGTATTTTGAAAAATAAATAAGTATATTTTTCCAAAAAAAAAAAAAAAAAAAAAAAAAAA | Lepidoptera | Crambidae | Diatraea | saccharalis | 40085 | cds | GenBank | HM231316.1 | Diatraea saccharalis RNAi construct | GTCATCTTCAATCCAATATGAGAGGTTTTTATCGGAGTTGGTACTCTGACCATAATACCACTAGGAGATGGATGGCTACTACTCAATTCCAACCAGGTCATGCGCGTCAAGCATTCCCCTGCTATGACGAACCTGGTTTCAAGGCTACTTTCGACATCACCATCAATAGGGAACCTGATTTCAGCCCTACGATTTCGAATATGCCTATTAAAGATACTTCAAATGAGCTAGTACCCGGTAGAGTCTCTGAAACATTCCACACAACCCCACGGACTTCTACATACCTGCTTGCGTTTATCGTTTCTCATTACGAGGTAGTTGCATCAAAAAATGATGAAGAACGGCCGTTTAGAATCTACGCTAGAAATAATGCTGGCACGACTGGAGACTGGTCTCTAAAAATTGGTATAGACTTACTAAGAGCGATGGAAGAATACACACAAATACCTTACTACACAATGGCTGATAACATGGATATGAAACAAGCCGCTATACCTGATTTTTCAGCTGGTGCTATGGAAAATTGGGGACTCTTAACCTACAGAGAGGCTTTGATACTTTACGATCCTCAAAA | dsRNA | MEGAScript RNAi kit (Ambion, Austin, TX). | other | simultaneously with transcription | dsRNA was produced following the protocol described in the MEGAScript RNAi kit (Ambion, Austin, TX). After incubation at 37 C for 4 h, dsRNA was prepared in an elution solution (ES) (10 mMTriseCl, pH 7,1 mMEDTA) provided in the RNAi kit. | InsectaCentral | Diatraea saccharalis RNAi construct | Lepidoptera | Crambidae | Diatraea | saccharalis | 40085 | local resource | larva | lab_colony | feeding | elution solution (ES) (10 mMTriseCl, pH 7,1 mMEDTA) | buffer | feeding | larva | qPCR | gut | not checked | 24 | low | 37 | ||||||||
| 231 | Bombyx mori sex pheromone biosynthetic pathway - PLCb1 RNAi | RNAi | pubmed | 20546038 | Hull | Joe | Bombyx mori sex pheromone biosynthetic pathway - PLCb1 RNAi target | ATGAGTGCTAGCACGAGATCAGCCAAAGTTAGCTTAAAGTCCATCGAAGTGCCGAAAGCTTTGCAGGATGGAGAAAAATTTATTAAATGGGATGAGGATTCCGGGACAGGCCTTCCAGTTACCTTGAGGGTTGATCCTAATGGCTTCTACCTGTACTGGACTGATCAGAACATGGAGGTGGAGTTGCTAGACATTGTCACCATCAGAGATGTCAGAACTGGAGTTCATGCTAAAGTGCCAAAAGATCCGAAAATCCGCAACGTCGTCCTCATCGGGAGCCAAGGGTCGCTCGAGGAGAAGACGGTCACGGTTTGTCACGGCACGGACTTCGTCAACGTGAATTTCATTAACTTCTGCTGCACTAGGAAGGAAATTGCACAGTTATGGACCGAAGAGTTGTTTGCGTTGGCTTACAACTTGAACCAGTTCAACAATCCAACGGTGAAGTTTCTAGAGAAATTGCACACGAAGATAACGTTAAAGGCTGACAAATCGGGGAAAATTCTCGTCAAGAACATTGTCCGCCAATTCGCGCAGAACAAGGAAGACAAGAAGCGTGTGGAGAAAGCGCTCAGTGAGTCTGGTCTCCCGACGGGCAAAAACGATACCATCAGCCAGTCCAAGTTCCAGTTCGAAGACTTCTTCGCTTTCTACAAGAGTCTCACGCAGAGAACCGAAGTACAGAAGATATTTAACGGACTAACGGACGGAAAACCTTATCTATCAGCGACTCAATTAGTAGACTTTTTAAACGACGTGCAAAGGGACCCACGTCTCAATGAGATATTGCATCCGTACGCTGATCTCCAGCGAGCGAAAGACCTCATAAAAGCTTACGAACACAATAAGTATCATCAGCAAAGATCGCAGCTTACCTTCGATGGATTCCTCAGGTTCCTAATGTCTGAAGACAATCCTATAGTGGCACCAAACAAGTTGGATTTGTGTGACGATATGGATCAGCCATTAGCTCATTATTTCATCAACAGCTCACACAACACGTATCTAACCGGCCATCAGATAACTGGCAAGTCTAGCGTCGAGATATACAGGCAGAGCTTGCTAGCCGGGTGCAGATGCGTGGAGTTGGACTTCTGGAATGGTCGAACCGAAGAGCCGGTCATCGTTCACGGTTACACGTTCGTGCCGGAGATAAGCGCCAGGGAAGTGTTGGAGGCGATAGCGGAAAGCGCTTTCAAGACTTCCGATTTTCCGGTTATTTTGAGTTTCGAGAACCACTGCAACCCGCGCCAGCAGGCGAAGATAGCCAACTACTGTAGAGAGATTTTCGGGGACATGCTGCTCGATAAGCCTCTGGACTCGCACCAGTTGGAACCAGGAGGTGAACTTCCACCGCCTTCGTTGCTACGGCACAAAATAATAATTAAGAACAAAAAGAAGCACCATCACCATCACAAGAAGGAGGACACACCGCCGATAGAGGAGTGCGAGCAGAGAGCCGAGCTGACAACGCAGGGGAACGGCGAGATGACCCACGCCACATTAGGCAGACAAGGCTCGAAGGATTCGTCCGAGTCGTCGGAGTCGGAGAGCTCGTCTGGCGAGGAGGAGGCGGGGGGCGCGGAGGGCGGCGCGGCGGGGGGCGAGGGCGAGCCGCGCGAGTGTCACGCCGCCGCCGAGATCTCCGCGCTCGTCAACTACGTGCAGCCCGTGCACTTTAGCTCGTTCGAGAGCGCCGAGAAAAAGAATCGTTTCTACGAGATGTCATCGTTCGACGAGAAACAAGCGACCACGCTGCTGAAGGAACGTCCCATCGAGTTCGTGAACTACAACAAGCACCAGCTGTCCCGGGTGTACCCGGCCGGGACCAGGTTCGACTCGTCCAACTTCATGCCACAGGTCTTCTGGAACGCGGGTTGTCAATTAGTCGCTCTCAACTATCAAACTTTGGACCTCGCCATGCAGCTCAACCTAGGCACGTTCGAGTACAACAAGCGATGCGGATTCCTATTGAAACCTGAATTTATGAGGAGAAAGGATCGACGTTTGGATCCATTCGCGGAGAGCACCGTGGACGGCATCATAGCCGGCAGTCTCACCGTGACGGTTCTGTCCGGGCAGCTACTGACCGACAAGCGTTGCGGCACGTACGTGGAGGTGGACATGTTCGGTCTGCCGGCCGACACCGTCAGGAAGAAGTTCCGCACCAGGGTCGTGCCCAATAACGGAATCAATCCGATTTACGGAGACGAACCCTTCGAATTTAAGAAGGTGGTGCTGCCGGAGCTGGCGATGCTGCGGGTGGCGGCGCACGAGGAGGGCGGGCGGCTGCTGGGGCACCGCGTGGTGCCGGTGCGCGGGCTGCGGCCCGGCCTGCGCGCGCTGCCGCTGCGCACGGAGCGGGGCCTGCCGCTGCACGCCGCGCTGCTGCTGCACGTGCAGGTCAAGGACTACGTGCCGGACAAGCTGTCGGAGCTGGCGGAGGCCCTGGCCAACCCCATAAAGTACCAGAGCGAGCTGGACAAGCGCGAGCACCAGCTGGCCGTGCTCACCGACGGCACCGACGAGCCCGAGCAGGAGCCGCGCCGCTCCGTCGCGCAGCCGGTAAGACACGAGGCGGTGCCGGAGAGCAGTCCCAGTAAGTTGGCTTTGGAGACTAAGAAGTTAATAGAGACCGAGTCCATAGTGACCCTGCCGATACCGCCGCCGATTGATACTAAAGAGCAAGAGAAGAAAGCGAACGGCACGGACGAGAGCCTGGCCGAGACCCTAGAAACGTTGCTGAACTCGAAGCTGGTGCGGTTAAAAGAAGCCGAGCTGGCCCGCAAGCTGGACACGCTGCGTCGCAAGTTCGACAAGGACAAGAAGCCCTTCTCGGCCAAGTTCTACGTCAGCAACAAGCTCGTCAAGAAGCTCTCGTCCAATAACATGTGCGGTGAGGCGGGGGGCAGCGGCGCGGCGGAGGAGGGCGCGGCGGAGGAGCGCGCGCTGCGGCTGCGCTACCACCACGCCATCTACGCCGCGCTCGAGAAGCACGTGCGCGCCGACCACTCGCAGCAGATGAAGACGCTCAGCGATCTGTTAGAAGTCGAGAAGAAGGAAATATTAAATCGACTAGCAAACTCGCGGAAGGAAGACGTCAAAGCGCTCGCCAAAAAGACAAATCGAGATAAAGACGAGATACAGAGGATAAAGAGAGAGATCCACTCGCAGAGCGTGGAGCGCGGCGTGTCGGAATGTTGCCGGCTAGAGCAAGCCTACACCAAGCGGCGCGAGCAGCTCGCCCGCGACCACGAGAAGCTGCTGCAGATGCTGCTCAAACATCGAGATCAGGAGCTCGTGAAGCTGGACCGTCTGTGCGGGGGCACGCCGGAGGGCTAG | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | GU266210 | Bombyx mori sex pheromone biosynthetic pathway - PLCb1 RNAi RNAi construct | ATGAGTGCTAGCACGAGATCAGCCAAAGTTAGCTTAAAGTCCATCGAAGTGCCGAAAGCTTTGCAGGATGGAGAAAAATTTATTAAATGGGATGAGGATTCCGGGACAGGCCTTCCAGTTACCTTGAGGGTTGATCCTAATGGCTTCTACCTGTACTGGACTGATCAGAACATGGAGGTGGAGTTGCTAGACATTGTCACCATCAGAGATGTCAGAACTGGAGTTCATGCTAAAGTGCCAAAAGATCCGAAAATCCGCAACGTCGTCCTCATCGGGAGCCAAGGGTCGCTCGAGGAGAAGACGGTCACGGTTTGTCACGGCACGGACTTCGTCAACGTGAATTTCATTAACTTCTGCTGCACTAGGAAGGAAATTGCACAGTTATGGACCGAAGAGTTGTTTGCGTTGGCTTACAACTTGAACCAGTTCAACAATCCAACGGTGAAGTTTCTAGAGAAATTGCACACGAAGATAACGTTAAAGGCTGACAAATCGGGGAAAATTCTCGTCAAGAACATTGTCCGCCAATTCGCGCAGAACAAGGAAGACAAGAAGCGTGTGGAGAAAGCGCTCAGTGAGTCTGGTCTCCCGACGGGCAAAAACGATACCATCAGCCAGTCCAAGTTCCAGTTCG | dsRNA | phenol_chloroform | subsequent to transcription | In vitro transcription templates were generated via PCR using plasmid DNA containing the gene of interest in conjunction with gene-specific primers containing T7 polymerase promoter sites at the 5â ends of the sense and antisense primers. PCR was performed with KOD-Plus (Toyobo, Osaka) and the resulting product purified (Promega SV PCR purification kit) was used as a template to generate dsRNAs with the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | InsectaCentral | Bombyx mori sex pheromone biosynthetic pathway - PLCb1 RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | DEPC-H2O | 0.02 | buffer non_specific_dsRNA | 12 | pheromone gland | Pheromone glands of newly emerged adults were injected either with 2 uL DEPC-H2O or 2 uL (ie 10 ug) dsRNA corresponding to a portion of the green fluorescent protein (GFP) coding sequence using a 10 uL Hamilton syringe fitted with a 35 gauge needle. Moths were then maintained under normal conditions for 48 hr prior to assay. | adult | phenotype | pheromone gland | not checked | 48 | high | 60 | ||||||
| 232 | Bombyx mori sex pheromone biosynthetic pathway - PLCg RNAi | RNAi | pubmed | 20546038 | Hull | Joe | Bombyx mori sex pheromone biosynthetic pathway - PLCg RNAi target | ATGAAGTCGGTCTGCTTCAACGGGGCCAAAGATAGGCTCATACACGAGGCAGACCAGCTTATCTCCTACATAGAAAGAGGCTCTACCGTTTTAAAATTCTTCCCTCGTAAGAGACCCGAAAGGCGTGCACTTTGCGTGAGAAGAGAGACCCATCAAGTTCTTTGGTCTCGAATCAATGCCCCTCAGAGACAGGGCTACGAAGGGGCTCTCGATATCCGTGATGTCAAAGAAGTTCGCACTGGAAAATCATCCAAAGACTTTGAGCGTTGGCCTGAAGAAACCAAACGTCTTGAAAGTTCAAAATGCTTTACCATTTATTATGGAACTGAATTTAAACTTCGCTGTGCATCATTTGTTGCTCAAGCAGATAAAGAGTGTGAAGCTTGGGTGAAAGGTGTACTGTATCTAATAGCAGAAGCTGTCAGTGCATCATGTCCTCTACAGATTGAAGGATGGCTCCGGAAAGAATTCTATTCTATTGAAAACTCCCATGAAAAAATAACATTAAAAGAGGTCAAAGCTTTTCTACCAAAAATTAATTGTAAAATTTCAACAAGTAAGCTTCGAGATGTATTCCAAGATGTTGACACTGAAAAAAGAGGGGAGATTGGCTTTGATGATTTTGCTGTTCTTTATCAGAAGCTAATTTTTGATGAGAATAATGTTCAGGACATATTTGATAAATACTCAGTCTATTGTTCTAATGGTACTACTATTACTCTTAGAGAGTTTCAAAATTTCCTTAGAGATGAACAGCATGATAACCTTGGTGATGATGAAGTACAAGCCTCACAGTTCATCCGTGATTACTTAAGAGATCCACAAAGAGACATCTATGAACCTTATTTCACTTTAAGCGAGTTTGTCGAGATGCTGTACTCCAAGCAGAATTCCATCTATGACAGTCAGTATGATAAGGTTACACAAGATATGACCAGGCCTTTGTCACACTATTGGATATCTTCTTCTCATAACACGTATCTGACAGGTGACCAGTTTTCAAGTGAATCATCACTGGAGGCCTACGTGCGGTGCTTGAGGTCAGGCTGTCGTTGTATTGAGCTAGATTGTTGGGATGGTCCTGACGGCACACCTTTTATTTATCACGGGCACACACTGACAACGAAAATAAGGTTTATGGCTGTACTGCGAACAATAAAAGAACATGCATTCATCACATCCGAATATCCATTAATTTTGTCTATCGAAGACAACTGCTCACTCCCACAGCAAAGACGAATGGCGAGCGCATTTCAAGACGTATTCGGTGACCTTCTTCTTGTTCATCCAATGGATAAGAATGAAACATCACTTCCCTCTCCTCATGACTTACGCAGAAAAATAATCCTGAAACATAAAAAATTACCTGAAGGTGCTGAAGAAAGTTCTTTCGCAGTACGTCAAGAAGAAGGAAAAGATTTAGACCTACGAAATTCGATCAAAAATGGTATATTGCATTTGGAAGATCCTGTCGACAAAGAATGGAAGCCTCACGCCTTCGTGCTCACCGAGAACAAACTTTACTATACAGAGAGCTACAACAGTCAAGAAGAAACCGACACTGAAAGTGACGGAGACTCAGATGCGGAAAACGCTATTGTACCACAAGATGAATTACATTTCGCAGAATGTTGGTTCCACGGGAAGCTGGCAGGGAATCGACAAGAAGCCGAAGATTTGCTGAGAGCCCACGCCCATCTTGGCGATGGAACCTTTCTGGTGAGGGAGAGCGTTACTTTTGTCGGTGACTACTGTCTATCATTCTGGCGGCAAGGGAAAGTAAATCACTGCCGCATCAAGCTCAAACAGGAACGGGGGGTCACCAAGTTCTATCTGATAGATAGTATGTGTTTCGACAGCCTGTACAACCTCATCACCCATTATAGGTCACATCCTCTGAGGAGTCAAGAGTTCCTGATAACGTTAAAGGAGCCAGTGCCTCAGCCTAACAAGCATGAAGGCAAGGAATGGTTCCACGCGCACTGCACGCGTACTCAAGCCGAAGAATTACTGCGGAAGACCAACACCGACGGCGCTTTCCTAGTACGCCCCAGCGAAAAGGAACAAGGGAGCTTCGCAATTAGTTTCAGGACTGAGAAGGAGATAAAGCATTGCAGGATTAAACAAGAGGGTCGCCTGTACACAATCGGTACAGTCAAGTTCGAGAGCCTCATTGAATTAGTCTCGTACTACGAGAACCATCCGCTGTATAAAAAAGTAAAACTCTGGTATCCGATCAGCGAAGAAACCGTCAGAAGACTTGTCTCTGAACCAGACGATAATACTGTCTATGGGACACCGGGATACATGGATCCCACGTCATTCACGTCCAAGGTGACCGTTAAAGCGCTTTACGATTATCGGGCTCGTCAAGGTGACGAGTTGTCCTTCTGCAAGCACGCCATCATTACAAACGTCGACAAGCCGGACGAGGGCTGGTGGCGGGGCGACTACGGCGGCAAACGTCATCACTGGTTCCCGGCCAACTACGTGCTTGAGATTGAGGTTCCACACACACCCGACGTGACCAGCGGTCTAGAAAACGAATCAGCCGCCCTGGGATCGTTGCAGAAAGGCGTGCTGGACGTGCTCGGCGCTATCGTTGAATTGGTGGTGGGCGACGGGGGAGGCGCGGCGCGGTGGCTGGTGCGTGTCCAGAGCCCCGCCATGTACTCGCCGTTCGACATGGCAGCCGCCACCCGCGAGTCCGCGCTCGAATGGCTCTCGGCCATCGAAGAGGCCGCGCACAGTGCTAGCGCTCGCTCGCTCCACCACAGGAAAATGGAACGAACATGGCGCATCGCCAAAGAGATATCGGACCTCATCATCTACTGCCGCTCAGTCACCTTCAACATCGAACGTCTACGCATCAAGGGCTTTGTTTACAACGAGATGTCCTCGTTTCCCGAGACCAAAGCCGAACGCCTTATGTCTCAGCAAGAGAACACGTTCTTCGTCAAGTACCACACGACGCAACTTAGCCGTATCTACCCTAAAGGCCAGAGAATCGATTCGTCGAACTACAATCCGGTGCCGTTCTGGAACTGCGGCTCTCAAATGGTCGCTTTGAATTACCAAACGCCCGACAAACCGATGCAGGTCAACATGGGCAAGTTTAAACAGAACGGCGGCTGTGGCTTTATCTTGAAGCCACAGTTTATGTTCGAGGAAGGATACAATCCGTGCGATAAAAAATCGATCGAGGGCAAAGTGAAGCCGGTAACTGTGATGTTGCGTGTTATCGGGGCGAGGCATCTGTGCAAGACGGGTCGCGGCACGGCCAGTCCCTTTGTAGAAGTCGAGATCATCGGGGCGGACTACGACAGCGGCGTCAAACTCGTCACTAAAACAGTGTCTGATAACGGACTGAACCCTTTGTGGAACGACATCTGCGAGTTCGAAGTTGCGAACCCAGAACTGGCCTTGATCCGTTTCGTGGTACAAGACGAAGATATATTTGGCGAGCCGAACTTCATAGGTCAAGCGACTTTCCCGCTGCTGTGCCTGCGTAAAGGATACCGAAGCGTGCCTCTCACTAACGCATTCTCCGAAGAACTGGAACTATCCACACTGATGGTTCACTTGTCAATTATAACAGACGGTTGA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | GU266212 | Bombyx mori sex pheromone biosynthetic pathway - PLCg RNAi RNAi construct | ATGAAGTCGGTCTGCTTCAACGGGGCCAAAGATAGGCTCATACACGAGGCAGACCAGCTTATCTCCTACATAGAAAGAGGCTCTACCGTTTTAAAATTCTTCCCTCGTAAGAGACCCGAAAGGCGTGCACTTTGCGTGAGAAGAGAGACCCATCAAGTTCTTTGGTCTCGAATCAATGCCCCTCAGAGACAGGGCTACGAAGGGGCTCTCGATATCCGTGATGTCAAAGAAGTTCGCACTGGAAAATCATCCAAAGACTTTGAGCGTTGGCCTGAAGAAACCAAACGTCTTGAAAGTTCAAAATGCTTTACCATTTATTATGGAACTGAATTTAAACTTCGCTGTGCATCATTTGTTGCTCAAGCAGATAAAGAGTGTGAAGCTTGGGTGAAAGGTGTACTGTATCTAATAGCAGAAGCTGTCAGTGCATCATGTCCTCTACAGATTGAAGGATGGCTCCGGAAAGAATTCTATTCTATTGAAAACTCCCATGAAAAAATAACATTAAAAGAGGTCAAAGCTTTTCTACCAAAAATTAATTGTAAAATTTCAACAAGTAAGCTTCGAGATGTATTCCAAGATGTTGACACTGAAAAAAGAGGGGAGATTGGCTTTGATGATTTTGCTGTTCTTTATCAGAAGCTAATTTTTGATGAGAATAATGTTCAGGACATATTTGATAAATACTCAGTCTATTGTTCTAATGGTACTACTATTACTCTTAGAGAGTTTCAAAATTTCCTTAGAGATGAACAGCATGATAACCTTGGTGATGATGAAGTACAAGCCTCACAGTTCATCCGTGATTACTTAAGAGATCCACAAAGAGACATCTATGAACCTTATTTCACTTTAAGCGAGTTTGTCGAGATGCTGTACTCCAAGCA | dsRNA | Ampliscribe T7 High Yield Transcription kit; Epicentre Technologies | phenol_chloroform | subsequent to transcription | In vitro transcription templates were generated via PCR using plasmid DNA containing the gene of interest in conjunction with gene-specific primers containing T7 polymerase promoter sites at the 5â ends of the sense and antisense primers. PCR was performed with KOD-Plus (Toyobo, Osaka) and the resulting product purified (Promega SV PCR purification kit) was used as a template to generate dsRNAs with the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | InsectaCentral | Bombyx mori sex pheromone biosynthetic pathway - PLCg RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | D | 0.02 | buffer non_specific_dsRNA | pheromone gland | Pheromone glands of newly emerged adults were injected either with 2 uL DEPC-H2O or 2 uL (ie 10 ug) dsRNA corresponding to a portion of the green fluorescent protein (GFP) coding sequence using a 10 uL Hamilton syringe fitted with a 35 gauge needle. Moths were then maintained under normal conditions for 48 hr prior to assay. | adult | phenotype | pheromone gland | not checked | 48 | high | 60 | ||||||
| 233 | RNAi experiment Bombyx | RNAi | InsectaCentral | Unpublished | Swevers | Luc | RNAi experiment Bombyx target | CTCCGTTGACGACGGTCGCGCGTGCGGTACGTCCGTTTTTACGGCTCAAACGTACACGGTAACCTCCGTCTCTGCATCATCGGCGGAACTCGTGAAATTCGCGTGCTTTTCTCACCTGTTGAACGAGTTGTGTTGTGACTGAAAAACATCATCACAAATATCAAGCTTCAAAACTAATAAGTGAATGAGAGTCGAGAACGTGGATAACGTATCGTTTGCTTTGAACGGACGCGCTGACGAGTGGTGTATGTCTGTAGAGACGCGTTTAGATAGTTTAGTGCGAGAAAAAAGTGAAGTGAAAGCCTACGTCGGAGGATGTCCCTCGGTAATCACGGATGCTGGAGCGTATGACGCGCTCTTCGACATGAGACGCCGCTGGTCTAATAACGGTGGCTTCCCGCTGCGAATGCTTGAAGAGAGCTCTTCAGAAGTGACATCGTCTTCGGCACTGGGTTTGCCACCGGCCATGGTTATGTCGCCGGAATCCTTGGCGTCGCCCGAGTATGGAGCCCTCGAGCTATGGAGCTACGATGACGGAATCACTTATAATACAGCCCAGTCTCTGCTGGGTGCATGCAATATGCAACAGCAACAGCTACAACCTCAGCAACCACATCCAGCACCACCGACGCTCCCCACGATGCCTTTACCAATGCCTCCCACAACACCGAAATCAGAAAATGAATCGATGTCATCAGGTCGAGAGGAACTTTCGCCGGCTTCAAGCATAAATGGCTGCAGTGCTGATGCTGACGCCAGACGGCAGAAGAAAGGTCCTGCACCTCGACAGCAAGAGGAGCTATGTCTTGTCTGCGGCGACAGAGCCTCCGGATACCACTACAACGCACTGACGTGTGAAGGATGCAAAGGATTCTTCAGGCGGAGTGTCACCAAAAACGCAGTATATATTTGTAAATTTGGACATGCCTGTGAAATGGATATGTACATGAGGAGGAAATGTCAAGAGTGTCGATTAAAGAAATGTCTAGCGGTAGGAATGAGGCCTGAATGTGTCATACAGGAGCCCAGTAAAAATAAAGACAGGCAAAGACAAAAGAAAGACAAAGGAATATTATTACCTGTTAGTACGACCACAGTCGAAGACCACATGCCCCCGATCATGCAATGTGATCCACCTCCGCCCGAGGCCGCCAGGATTCACGAAGTCGTCCCGAGGTATCTTTCGGAGAAGCTGATGGAGCAGAACAGGCAGAAGAACATACCACCATTGTCGGCGAATCAGAAGTCTCTGATCGCGAGGCTCGTGTGGTACCAGGAGGGATATGAGCAGCCCTCCGACGAGGATCTCAAAAGAGTAACGCAGACTTGGCAGTCGGATGAAGAGGACGAGGAATCCGATCTACCCTTCCGCCAGATCACGGAGATGACGATCTTAACGGTCCAGTTGATCGTCGAGTTCGCCAAGGGTCTACCGGGCTTTTCGAAGATATCACAGTCTGATCAAATCACCTTATTAAAAGCCTCGTCCAGCGAGGTGATGATGCTGCGGGTGGCGAGGCGATACGACGCCGCGTCCGACAGCGTGCTGTTCGCCAACAACAAGGCGTACACGCGCGACAACTACCGCCAAGGCGGCATGGCCTACGTCATCGAAGACCTCCTACACTTCTGCCGGTGCATGTTCGCGATGGGCATGGACAATGTGCACTTTGCACTGCTCACGGCCATCGTTATATTCTCAGATCGGCCCGGGCTCGAGCAGCCGTCGCTGGTAGAAGAGATCCAGAGATACTACCTGAACACGTTGCGAATTTACATCATCAACCAGAACAGCGCGTCGTCGCGCTGCGCCGTGATCTACGGCAGGATCCTGAGCGTGCTGACCGAGCTACGCACGCTCGGCACGCAAAACTCCAACATGTGCATCTCGCTGAAGCTGAAGAACAGGAAGCTGCCGCCGTTCCTCGAGGAGATCTGGGACGTGGCGGAGGTGGCCACGACGCATCCCACGGTGCTGCCGCCCACCAACCCGGTGGTGCTATAGCCTCCGCCCGCCCCAGGAGAGAACGCTCATAGACTGGCTAGTTTTAGTGAACGTGCGCTGATCCGTATTCGGTGACAGATTAGTGATTATATGTGTTGTTGAACGTTTGGAGAGTATATATATAGTGTTGACGGCGAGGCCCGTCCGGCCCCGTACTTGTTTCGTTTCTGACCGGATGCTGCGTCGGTCGCGCCCTTGCGACCACGATAAGACTACTTTCTATAAGTACGTCTCTAAATTGAGGCCCCAAGAACCGGAAGCCTAACTAAGATACGACGGTTTATTTTATCACAGAGGAAACTGAAGTAATTAATATATTAACAGTTAACACAGGTTCAAAGCAGTTAGGCCGCTCCGGACTTCTCATGGTCTCAATAAGGCGGACGTATAAGAGTTACATAGAGAATAAAATAATAATATATGAAGAGATGTTTCTATTGGATGGAATGCGTGATGTAAAGTTGATAGTTATTTTTATTTACCAAATTAATGAAGCGTCGGGTGTAGACCTTTTGTATGTGATGTGGCGAGGAGTGGATCGCAGTGTCGGCCGCGTGCTCTCACCAAAAGCGTGCGGTCGACGCTAATAGTGCGATGGTTTTGGAATATGTTTGTTTATATATAGTTTATGTGTGAGGTGTTATCGTGTCCCGTCAATTTTAATTTCGATTCGCGTTCATTCGTCCTGTGCTCGCTACTCAGATTTAAAAAAAAAAAAAAAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | RNAi experiment Bombyx target | RNAi experiment Bombyx RNAi construct | GATATCACAGTCTGATCAAATCACCTTATTAAAAGCCTCGTCCAGCGAGGTGATGATGCTGCGGGTGGCGAGGCGATACGACGCCGCGTCCGACAGCGTGCTGTTCGCCAACAACAAGGCGTACACGCGCGACAACTACCGCCAAGGCGGCATGGCCTACGTCATCGAAGACCTCCTACACTTCTGCCGGTGCATGTTCGCGATGGGCATGGACAATGTGCACTTTGCACTGCTCACGGCCATCGTTATATTCTCAGATCGGCCCGGGCTCGAGCAGCCGTCGCTGGTAGAAGAGATCCAGAGATACTACCTGAACACGTTGCGAATTTACATCATCAACCAGAACAGCGCGTCGTCGCGCTGCGCCGTGATCTACGGCAGGATCC | dsRNA | pLitmus 28i & 38i vectors (New England Biolabs) | phenol_chloroform | subsequent to transcription | Preparation of dsRNA. pLitmus28i and 38i vectors containing respectively the complete ORF of GFP (EcoRI-HindIII, 0.7 kb) or a fragment of the ligand-binding domain of BmEcR (EcoRV-BamHI, 0.35 kb) were linearized using appropriate restriction enzymes and used for RNA synthesis by T7 RNA polymerase (Fermentas). Sense and antisense strand annealing occurred in 0.3 M NaCl, 1 mM EDTA after heating at 95°C for 5 min followed by slow cooling to room temperature (> 2 hrs). | InsectaCentral | RNAi experiment Bombyx RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | larva | lab_colony | 0.5 | injection | 0.3 M NaCL, 1mM EDTA | 0.005 | non_specific_dsRNA | whole organism | pupa | RT-PCR (semi-quantitative) phenotype | fat body | not checked | 168 | none | ||||||||
| 234 | Bombyx mori sex pheromone biosynthetic pathway - PLCb4 RNAi | RNAi | pubmed | 20546038 | Hull | Joe | Bombyx mori sex pheromone biosynthetic pathway - PLCb4 RNAi target | ATGACCAAGAAATTTGAATTCAACTGGCAGATACCAGTCCCCGAGCCGCTGTTGCAGGGCGCAGTCTTCGACAGATGGACGGAAGAGAAGGACAATACAGAGTTTGAACAAAATTGCTTATTCAAGGTCGACGAATATGGCTTCTTCATCTACTGGAAGAGTGATGGAAAGGACGGTGACGTGATAGAATTGTGTCAAGTAAGCGACGTCAGATCCGGAGGTGTTCCCAAGGACTCCAAGCTCAACTCGAATCTGGTGAACAAACACGGGGACACGTTGGAAGACAAATCGCTAATCATATGCAGTGGAACTGACTATATTAACATCAACTACCAACACGTGGTGTGTCCTGACGCCGCCACAGCGAAGGTTTGGAAGGAAGGACTACGAGCTATCACGCACAACAACAAAATCAACAATGTCTGCCCGAGAACCAATCTAATGAAGCACTGGATGCGCCTCTGTTTCCTGACCGACCCTCGGGGCAAAGTTCCCGTCAAGGTGGTGGCCAGGACATTTGCTTCTGGTAAAACTGAGAAGCTGGTCTACCAGTGCCTGTCCGAACTGGGTCTGCCGTCAGGGAAGAATGACGTCATGGAAAAAGAAGCCTTCACGTTTGACAAGTTTTACGCTCTGTATCATAAGATTTGCCCGAGAAACGATATTGAAGAGTTGTTTCGTTCTATAACTCAAGGCAAATCCGATCGCATCAATTTGGACCAGTTCGTGAACTTCCTGAATGAGAAGCAACGTGATCCTCGACTCAACGAGATCCTGTACCCGCTGTACGATGAGAAGAGAGCAGCTGAGATCATCACGACTTATGAACAGAATGAGGAAGCCAAAAATGCCAAATGCTTGACAAAAGACGGTCTGATCCGCTACTTGATGTCGGACGAGAATGCTCCTGTGTTCCTCGACCGTCTCGACAACTATATGGACATGGACCAGCCACTCGCTCACTACTACATCAACTCATCGCACAACACGTACCTAAGCGGACGACAGTTCGGAGGGAAAAGCTCCGTGGAGATGTACCGACAGGTCTTGTTGGCTGGTTGCCGATGCGTAGAGCTAGACTGTTGGGATGGCAAAGGCGAGGATGAAGAGCCAATCATAACGCACGGCATGGCAATGTGCACTGACATACTGTTCAAAGATGTCATATACGCCCTGAGGGATACCGCCTTCGTGACTTCAGACTACCCAGTCATCCTATCATTTGAGAACCACTGTTGCAAGGCCCAGCAATACAAGCTGGCCAAATACTGTGACGAGATCTTGGGTGATTTGCTGCTTAAGGAACCGCTGCCTGATCATCCGTTGGACCCAGGAGTGCCATTACCTCCGCCTTCAGCCCTCAAACGCAAGATCATGATTAAGAACAAACGTCTAAAGCCAGAAGTTGAGAAGCAAGAATTAGAGCTGTTCCGTCAAGGACAGTTTGTCATCGAAGAAGAAGAGAAAGAAGATGCTTCTGCAGTCGCCATTCTGGATAAAAAGCCTGAAGAGATCGTAGCCGAAGCAGCATCGGCTGGCGATGACGCACCTCCTCCCGTTGCGTACACAGGATCTACAACCAACGTTCATCCATGGCTCTCGTCCATGGTCAACTACGCGCAGCCCATCAAGTTCCAAGGCTTCGAAGAGGCAGATAAGCGCAACATTTACCACAACATGTCCTCGTTCGCTGAGACGACCGGCATGAACTACCTAAAGAGCCAAGCCATCGACTTCGTGAATTACAACAAGCGACAGATGTCCCGTATTTATCCTAAAGGAACCCGTGCTGACTCCTCTAATTACATGCCTCAGGTATTCTGGAACGCCGGATGTCAAATGGTGTCCCTAAACTTCCAAACGTCAGACTTGCCAATGCAACTGAACCAAGGCAAGTTCGAGTACAACGGGAACTGCGGATACCTGTTGAAGCCGGACTTCATGCGGCGCGCGGACCGTAGCTTCGATCCTTTTGCCGACGCGCCAGTCGATGGCGTCATCGCGGCTCAGTGCAGTGTCCAGGTCATAGCTGGTCAGTTTCTGTCAGACAAGAAGATCGGGACCTACGTTGAAGTGGATATGTACGGTCTACCGTCAGACACGATCCGCAAGGAATTCCGCACGAGAATGGTGCCTGCGAACGGATTGAACCCAGTTTACAACGAGGAGCCTTTCTTGTTTAGGAAGGTCGTGCTTCCTGACCTAGCCGTCCTCCGTTTCGGTGTTTACGACGAGAACGGCAAGCTCCTGGGCCAGAGGATCCTGCCGCTGGACGGGCTCCAAGCCGGCTACAGGCACATCTCGCTCAGGACCGAAGCAAACTTCCCTATGTCACTGCCCATGTTGTTCTGCAACATCGAACTCAAGATTTACGTCCCGGACGGTTTTGCTGATTTCATGGACGCTCTCTCAGACCCGCGTGCGTTCAAGAAACGTTCCGAAGACATGCAGCAGATGGGCATTGAAGCTGGTGGACCTGAAAAAAGAGTGCAGCTCGAAGAGAAGAAAGAAGAGCCTCCCATTTTTTTTCGACCTATAACAGTGGAGTCCTTGCGCCAAGAGAAGGCTTCCTTAAAGACCGGCAGGAAACAGCAGAAAGACCTGGACGCAATGAGGAAGCGACATGCCAAAGAGAAGATGGCATTGCAAAAGCAACAATGCACTGCACTTGAGAAGATGATAAAGGGAAAGAACAAAGAGCAGTTGAGCACTGATCCGGCCTTCCGCAAGATGGTCAACGAACAGTCGACGGCGTGGAGTGAGCTGGTAGCCAAGCACCGAGTGGAAGAGTGGACCAACACCAGGAACCGCCTCAACGAACAACGGGAACTGCTGAAGAAAATGATGGAGCAGACACAACAGGCTCAAGTCAAACGCCTCGAAGCCAAGCATGAGAGGGAGCTGAAAGAGATGAACGCTCGTCAGGCCAGGATCAGTATGGAGACCAGCAAAGAGGTCGCCAACGACAAAACTTTGAAGACCAAACAAGAAAAAGATAGACGACTCAGGGAGAAAAAACAAAACAACACAAAGAAGTTTATGGACGAAAGACTGAGTCACACTAAGAAACAAAACCGCGAGAAGGATAACTTGAAGGCGACACACGAGAAACAGATGGAAGAGCTGTCAAAAGATGTTGATAATCTAATCGAGATGTACAAGATGGAGGAATCCGAATTCGACATGGCGAGTAAAACGGAATTTTTTGCATGA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | GU266211 | Bombyx mori sex pheromone biosynthetic pathway - PLCb4 RNAi RNAi construct | AACTGGCAGATACCAGTCCCCGAGCCGCTGTTGCAGGGCGCAGTCTTCGACAGATGGACGGAAGAGAAGGACAATACAGAGTTTGAACAAAATTGCTTATTCAAGGTCGACGAATATGGCTTCTTCATCTACTGGAAGAGTGATGGAAAGGACGGTGACGTGATAGAATTGTGTCAAGTAAGCGACGTCAGATCCGGAGGTGTTCCCAAGGACTCCAAGCTCAACTCGAATCTGGTGAACAAACACGGGGACACGTTGGAAGACAAATCGCTAATCATATGCAGTGGAACTGACTATATTAACATCAACTACCAACACGTGGTGTGTCCTGACGCCGCCACAGCGAAGGTTTGGAAGGAAGGACTACGAGCTATCACGCACAACAACAAAATCAACAATGTCTGCCCGAGAACCAATCTAATGAAGCACTGGATGCGCCTCTGTTTCCTGACCGACCCTCGGGGCAAAGTTCCCGTCAAGGTGGTGGCCAGGACATTTGCTTCTGGTAAAACTGAGAAGCTGGTCTACCAGTGCCTGTCCGAACTGGGTCTGCCGTCAGGGAAGAATGACGTCATGGAAAAAGAAGCCTTCACGTTTGACAAGTTTTACGCTCTGTATCATAAGATTTGCCCGAGAAACGATATTGAAGAGTTGTTTCGTTCTATAACTCAAGGCAAATCCGATCGCATCAATTTGGACCAGTTCGTGAACTTCCTGAATGAGAAGCAACGTGATCCTCGACTCAACGAGATCCTGTACCCGCTGTACGATGAGAAGAGAGCAGCTGAGATCATCACGACTTATGAACAGAATGAGGAAGCCAAAAATGCCAAATGCTTGACAAAAGACGGTCTGATCCGCTACTTGATGTCGGACGAGAATGCTCCTGTGTTCCTCGACCGTCTCGACAACTATATGGACATGGACCAGCCACTCGCTCACTACTACATCAACTCATCGCACAACACGTACCTAAGCGGACGACAGTTCGGAGGGAAAAGCTCCGTGGAGATGTACCGACA | dsRNA | Ampliscribe T7 High Yield Transcription kit; Epicentre Technologies | phenol_chloroform | subsequent to transcription | In vitro transcription templates were generated via PCR using plasmid DNA containing the gene of interest in conjunction with gene-specific primers containing T7 polymerase promoter sites at the 5â ends of the sense and antisense primers. PCR was performed with KOD-Plus (Toyobo, Osaka) and the resulting product purified (Promega SV PCR purification kit) was used as a template to generate dsRNAs with the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | InsectaCentral | Bombyx mori sex pheromone biosynthetic pathway - PLCb4 RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | DEPC-H2O | 0.02 | buffer non_specific_dsRNA | 18 | pheromone gland | Pheromone glands of newly emerged adults were injected either with 2 uL DEPC-H2O or 2 uL (ie 10 ug) dsRNA corresponding to a portion of the green fluorescent protein (GFP) coding sequence using a 10 uL Hamilton syringe fitted with a 35 gauge needle. Moths were then maintained under normal conditions for 48 hr prior to assay. | adult | phenotype | pheromone gland | not checked | 48 | none | ||||||
| 235 | Bombyx mori sex pheromone biosynthetic pathway - BmIP3R RNAi | RNAi | pubmed | 20546038 | Hull | Joe | Bombyx mori sex pheromone biosynthetic pathway - BmIP3R RNAi target | ATCCTACAGCGCATGATCAAGTACTGCACGCACGGCAACAGCGGCGCCGGCGACTGCGGCCGCCCGCGCCGCCACGAGCAGCGCCTGCTGCGCAACATCGGCGTGCACAACATCGTGCTGGACTTGCTGCAGGTGCCGCACGACGAGGACGACGCAGCTATGGACGAGCTGCTGTCGCTAGCTCACGAGTTCCTCCAGCACTTCTGCCACGGAAACCAGCAGAACCAGACGATCCTCCACAAACATCTCGACTTGTTCCTCAACGCGGGAATCCGAGAGGCGCAGACGGTCTGCGCGATCTTCAAGGACAACGCGTCGCTGTGCGGGCACGAGGGCAACGAGAAGGTGGTGGCGCACTTCGTGCACTGCGTGGAGTCCCGCGGCAGGAAGCCCGACTACCTGCGCTTCCTGCAGACCATCGTCCACGCCGACAAGCAGTACATACGGAAGAGCCAGGACTTGGTCATGCAAGAGATGGTGAACGCCGGCGAGGATGTCTTGGTCTTCTACAATGACAAAGTTTCGTTCAATTACTTCATACAGATGATGAAACAGTACAAAGAGAAAGGGGAAATGCCCGAAGCTCTTACATACCACATCCAGCTCGTGAAGCTGCTGACGTGCTGCACTATGGGGAAGAACGTGTACACGGAGATCAAGTGCCACTCGCTGCTGCCGCTGGACGACATCGTGTCCGCCATCACCAACCCGCACTGCATCCCCGAGGTGAAAGAAGCGTACGTGGGCTTCCTGAACCACTGCTACATCGACACGGAGGTGGAGATGAAGGAGATCTACAGCAGCAACTACATGTGGGACCTGTTCGAGCGCTCCTTCCTCCAGGACATGCACGCCATCCTGCAGAGCGGCGCCGCCTGGTCGGACGTGCCGTCGTCGTCGCACAAGCCGTCCACCACCAGCACGCACCGCGCCTGGGTCAACTACGTCACCGACGTGCTCATGCACACCATATGCACGTTCTTCAACTCGCCCTTCAGCGACCAAAGCACAACCGTGCAGACCCGTCAGTCGATCTTCGTGAAGCTACTCCAGACGACGTTCAGTATATACCAGTGTCCGTGGCTCACGCCGCCGCAGAAGCTGAACGTGGAGAAGTGTATACGGACGCTCAGCGAGGTCGCGAAATCTCGCAGTATCGCGATCCCGCTGGAGCTCGAGGCTAAAATACAGACCGTGTTCGAGAAGGCTGCCGCGCTGTCCCGCCAGACCAGCAAGTGGCTGCAGGCCTCGAAGACTTCGAAACTTGAGAAGATGGCTTCGCAGTTGAACTTGCAGAACGATAGGTCGGTGATGGAGGGTCTACAGGAGATCGTGTCCCTACTGGAGGAACAGCTCCGGCCGCTGCTGCAGGCGGAGCAGTCGCTCCTGGTGGACATCCTGTACAAGCCGCACCTCCTGTTCACCAGCCCCGCCCAGCCCGACGCCAGCGGGATCTTTATATCACGATTAATACGTCACACCGAGAAGCTTCTAGAAGAAAAAGAAGAAAAACTGTGCGTGAAAGTCTTACGCACGTTGAGAGAGATGATGGC | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | InsectaCentral | Bombyx mori sex pheromone biosynthetic pathway - BmIP3R RNAi target | Bombyx mori sex pheromone biosynthetic pathway - BmIP3R RNAi RNAi construct | TCCTACAGCGCATGATCAAGTACTGCACGCACGGCAACAGCGGCGCCGGCGACTGCGGCCGCCCGCGCCGCCACGAGCAGCGCCTGCTGCGCAACATCGGCGTGCACAACATCGTGCTGGACTTGCTGCAGGTGCCGCACGACGAGGACGACGCAGCTATGGACGAGCTGCTGTCGCTAGCTCACGAGTTCCTCCAGCACTTCTGCCACGGAAACCAGCAGAACCAGACGATCCTCCACAAACATCTCGACTTGTTCCTCAACGCGGGAATCCGAGAGGCGCAGACGGTCTGCGCGATCTTCAAGGACAACGCGTCGCTGTGCGGGCACGAGGGCAACGAGAAGGTGGTGGCGCACTTCGTGCACTGCGTGGAGTCCCGCGGCAGGAAGCCCGACTACCTGCGCTTCCTGCAGACCATCGTCCACGCCGACAAGCAGTACATACGGAAGAGCCAGGACTTGGTCATGCAAGAGATGGTGAACGCCGGCGAGGATGTCTTGGTCTTCTACAATGACAAAGTTTCGTTCAATTACTTCATACAGATGATGAAACAGTACAAAGAGAAAGGGGAAATGCCCGAAGCTCTTACATACCACATCCAGCTCGTGAAGCTGCTGACGTGCTGCACTATGGGGAAGAACGTGTACACGGAGATCAAGTGCCACTCGCTGCTGCCGCTGGACGACATCGTGTCCGCCATCACCAACCCGCACTGCATCCCCGAGGTGAAAGAAGCGTACGT | dsRNA | Ampliscribe T7 High Yield Transcription kit; Epicentre Technologies | phenol_chloroform | subsequent to transcription | In vitro transcription templates were generated via PCR using plasmid DNA containing the gene of interest in conjunction with gene-specific primers containing T7 polymerase promoter sites at the 5â ends of the sense and antisense primers. PCR was performed with KOD-Plus (Toyobo, Osaka) and the resulting product purified (Promega SV PCR purification kit) was used as a template to generate dsRNAs with the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | InsectaCentral | Bombyx mori sex pheromone biosynthetic pathway - BmIP3R RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | DEPC-H2O | 0.02 | buffer non_specific_dsRNA | 12 | pheromone gland | Pheromone glands of newly emerged adults were injected either with 2 uL DEPC-H2O or 2 uL (ie 10 ug) dsRNA corresponding to a portion of the green fluorescent protein (GFP) coding sequence using a 10 uL Hamilton syringe fitted with a 35 gauge needle. Moths were then maintained under normal conditions for 48 hr prior to assay. | adult | phenotype | pheromone gland | not checked | 48 | high | 60 | |||||
| 236 | Bombyx mori sex pheromone biosynthetic pathway - BmGq1 RNAi | RNAi | pubmed | 20546038 | Hull | Joe | Bombyx mori sex pheromone biosynthetic pathway - BmGq1 RNAi target | ATGGAGTGCTGCATGTCAGAAGAAGCAAAGGAACAAAAAAGGATCAATCAAGAAATAGAGAGGCAACTCAGAAAGGACAAACGAGATGCCAGAAGAGAACTCAAACTGCTCCTACTTGGAACTGGAGAGTCGGGCAAATCTACTTTTATCAAGCAGATGAGAATCATCCACGGCTCGGGTTATAGCGACGAAGACAAGCGCGGCTTCATCAAACTCGTTTACCAGAACATCTTCATGGCGATGCAGAGTATGATACGCGCCATGGACCTACTAACAATACAATATGGGAACCCGTCCAATGTAGAAAAGGCGGAATTGATATCATCGATCGACTTTGAGAGTGTGACAACGTTCGAGAGTCCGTACGTGGAAGCTATCAAGGGCTTATGGGCCGATTCCGGCATCCAGGAATGTTATGATCGCAGGCGAGAATACCAACTTACTGACTCGGCCAAATACTACTTGCAGGAGATCGACCGCGTCGCAGCGCCAAATTACCTACCAACCGAACAGGATATCCTCCGTGTGAGAGTTCCCACAACGGGCATCATAGAGTACCCATTTGACTTGGAGGAAATACGATTTCGAATGGTGGACGTCGGGGGCCAGAGATCCGAAAGAAGGAAGTGGATCCATTGTTTCGAAAACGTCACATCCATCATATTCTTAGTAGCTCTTAGTGAATATGACCAAATTTTATTCGAATCAGAGAACGAGAATCGAATGGAAGAATCGAAAGCATTGTTCAAGACCATCATAACGTACCCGTGGTTCCAGCACTCCTCGGTCATTCTGTTTCTCAACAAAAAGGATTTGCTCGAGGAGAAGATTATGTATTCTCACCTTGTTGACTACTTCCCTGAATACGACGGCCCGCAACGCGACGCCAATGCGGCGCGCGAGTTCATTCTGCGGATGTTCGTCGACTTGAACCCGGACGCGGAGAAAATCATCTATTCGCATTTCACATGTGCGACAGATACCGAAAACATCCGGTTCGTATTCGCCGCCGTAAAGGACACAATCCTACAGTCCAACCTGAAAGAGTACAATCTCGTTTAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | AB425234 | Bombyx mori sex pheromone biosynthetic pathway - BmGq1 RNAi RNAi construct | ATGGAGTGCTGCATGTCAGAAGAAGCAAAGGAACAAAAAAGGATCAATCAAGAAATAGAGAGGCAACTCAGAAAGGACAAACGAGATGCCAGAAGAGAACTCAAACTGCTCCTACTTGGAACTGGAGAGTCGGGCAAATCTACTTTTATCAAGCAGATGAGAATCATCCACGGCTCGGGTTATAGCGACGAAGACAAGCGCGGCTTCATCAAACTCGTTTACCAGAACATCTTCATGGCGATGCAGAGTATGATACGCGCCATGGACCTACTAACAATACAATATGGGAACCCGTCCAATGTAGAAAAGGCGGAATTGATATCATCGATCGACTTTGAGAGTGTGACAACGTTCGAGAGTCCGTACGTGGAAGCTATCAAGGGCTTATGGGCCGATTCCGGCATCCAGGAATGTTATGATCGCAGGCGAGAATACCAACTTACTGACTCGGCCAAATACTACTTGCAGGAGATCGACCGCGTCGCAGCGCCAAATTACCTACCAACCGAACAGGATATCCTCCGTGTGAGAGTTCCCACAACGGGCATCATAGAGTACCCATTTGACTTGGAGGAAATACGATTTCGAATGGTGGACGTCGGGGGCCAGAGATCCGAAAGAAGGAAGTGGATCCATTGTTTCGAAAACGTCACATCCATCATATTCTTAGTAGCTCTTAGTGAATATGACCAAATTTTATTCGAATCAGAGAACGAGAATCGAATGGAAGAATCGAAAGCATTGTTCAAGACCATCATAACGTACCCGTGGTTCCAGCACTCCTCGGTCATTCTGTTTCTCAACAAAAAGGATTTGCTCGAGGAGAAGATTATGTATTCTCACCTTGTTGACTACTTCCCTGAATACGACGGCCCGCAACGCGACGCCAATGCGGCGCGCGAGTTCATTCTGCGGATGTTCGTCGACTTGAACCCGGACGCGGAGAAAATCATCTATTCGCATTTCACATGTGCGACAGATACCGAAAACATCCGGTTCGTATTCGCCGCCGTAAAGGACACAATCCTACAGTCCAACCTGAAAGAGTA | dsRNA | Ampliscribe T7 High Yield Transcription kit; Epicentre Technologies | phenol_chloroform | subsequent to transcription | In vitro transcription templates were generated via PCR using plasmid DNA containing the gene of interest in conjunction with gene-specific primers containing T7 polymerase promoter sites at the 5â ends of the sense and antisense primers. PCR was performed with KOD-Plus (Toyobo, Osaka) and the resulting product purified (Promega SV PCR purification kit) was used as a template to generate dsRNAs with the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | InsectaCentral | Bombyx mori sex pheromone biosynthetic pathway - BmGq1 RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | DEPC-H2O | 0.02 | buffer non_specific_dsRNA | 28 | pheromone gland | Pheromone glands of newly emerged adults were injected either with 2 uL DEPC-H2O or 2 uL (ie 10 ug) dsRNA corresponding to a portion of the green fluorescent protein (GFP) coding sequence using a 10 uL Hamilton syringe fitted with a 35 gauge needle. Moths were then maintained under normal conditions for 48 hr prior to assay. | adult | phenotype | pheromone gland | not checked | 48 | high | 60 | |||||
| 237 | Bombyx mori sex pheromone biosynthetic pathway - BmGo1 RNAi | RNAi | pubmed | 20546038 | Hull | Joe | Bombyx mori sex pheromone biosynthetic pathway - BmGo1 RNAi target | ATGGGGTGCGCCCAGTCGGCCGAAGAGCGTGCAGCGGCTGCTCGAAGCAAATTGATCGATAAGAACTTGAAAGAGAATGGTATTCAGGCATCAAAAGACATCAAGCTATTGTTGTTAGGTGCTGGCGAATCGGGCAAGAGTACTATAGTCAAACAGATGAAAATTATCCATGAAAGTGGATTCACAAATGAAGACTTCAAACAGTACCGGCCCGTGGTCTACAGCAATGCGATCCAGTCGCTCGTCGCCATCTTGCGTGCCATGCCCAACTTGGGCATCATCTATGGCAATAGAGACCGAGAAAGCGATGGCAAGATGGTGTTCGACGTGATTCAACGGATGGAGGACACGGAACCGTTCAGTGAGGAGCTTCTCGCCGCGATGAAAAGGCTCTGGGCCGACTCTGGCGTTCAGGAGTGCTTTGGTCGTTCCAACGAGTACCAACTCAACGACTCCGCTAAATATTTCCTGGACGACCTCGACCGGTTAGGAGCAAAGGATTACCAGCCCACCGAACAAGATATCCTAAGGACTAGAGTCAAAACGACTGGAATCGTCGAAGTACATTTCTCCTTCAAAAACCTCAATTTCAAATTGTTCGACGTGGGCGGGCAGCGATCGGAGAGAAAGAAATGGATCCATTGCTTCGAGGACGTGACCGCGATCATATTCTGTGTTGCCATGTCTGAATATGATCAGGTGTTGCACGAGGATGAAACAACGAACCGTATGCAGGAGAGTCTAAAGTTGTTCGATTCTATCTGCAACAACAAATGGTTCACCGACACCAGCATCATCTTGTTCCTGAACAAGAAGGATCTCTTCGAGGAAAAAATTCGCAGATCGCCGCTCACCATATGCTTTCCAGAATATACAGGCGCGCAAGAGTACGGCGAGGCGGCCGCGTACATCCAGGCGCAGTTCGAAGCGAAGAACAAGTCCACCACGAAGGAGATCTACTGCCACATGACGTGCGCCACCGACACGAACAACATCCAGTTTGTGTTCGACGCGGTGACCGACGTGATCATCGCCAACAACCTGCGCGGCTGCGGCCTGTAC | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | GU266213 | Bombyx mori sex pheromone biosynthetic pathway - BmGo1 RNAi RNAi construct | ATGGGGTGCGCCCAGTCGGCCGAAGAGCGTGCAGCGGCTGCTCGAAGCAAATTGATCGATAAGAACTTGAAAGAGAATGGTATTCAGGCATCAAAAGACATCAAGCTATTGTTGTTAGGTGCTGGCGAATCGGGCAAGAGTACTATAGTCAAACAGATGAAAATTATCCATGAAAGTGGATTCACAAATGAAGACTTCAAACAGTACCGGCCCGTGGTCTACAGCAATGCGATCCAGTCGCTCGTCGCCATCTTGCGTGCCATGCCCAACTTGGGCATCATCTATGGCAATAGAGACCGAGAAAGCGATGGCAAGATGGTGTTCGACGTGATTCAACGGATGGAGGACACGGAACCGTTCAGTGAGGAGCTTCTCGCCGCGATGAAAAGGCTCTGGGCCGACTCTGGCGTTCAGGAGTGCTTTGGTCGTTCCAACGAGTACCAACTCAACGACTCCGCTAAATATTTCCTGGACGACCTCGACCGGTTAGGAGCAAAGGATTACCAGCCCACCGAACAAGATATCCTAAGGACTAGAGTCAAAACGACTGGAATCGTCGAAGTACATTTCTCCTTCAAAAACCTCAATTTCAAATTGTTCGACGTGGGCGGGCAGCGATCGGAGAGAAAGAAATGGATCCATTGCTTCGAGGACGTGACCGCGATCATATTCTGTGTTGCCATGTCTGAATATGATCAGGTGTTGCACGAGGATGAAACAACGAACCGTATGCAGGAGAGTCTAAAGTTGTTCGATTCTATCTGCAACAACAAATGGTTCACCGACACCAGCATCATCTTGTTCCTGAACAAGAAGGATCTCTTCGAGGAAAAAATTCGCAGATCGCCGCTCACCATATGCTTTCCAGAATATACAGGCGCGCAAGAGTACGGCGAGGCGGCCGCGTACATCCAGGCGCAGTTCGAAGCGAAGAACAAGTCCACCACGAAGGAGATCTACTGCCACATGACGTGCGCCACCGACACGAACAACATCCAGTTTGTGTTCGACGCGGTGACCGACGTGATCATCGCCAACAACCTGCG | dsRNA | Ampliscribe T7 High Yield Transcription kit; Epicentre Technologies | phenol_chloroform | subsequent to transcription | In vitro transcription templates were generated via PCR using plasmid DNA containing the gene of interest in conjunction with gene-specific primers containing T7 polymerase promoter sites at the 5â ends of the sense and antisense primers. PCR was performed with KOD-Plus (Toyobo, Osaka) and the resulting product purified (Promega SV PCR purification kit) was used as a template to generate dsRNAs with the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | InsectaCentral | Bombyx mori sex pheromone biosynthetic pathway - BmGo1 RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | DEPC-H2O | 0.02 | buffer non_specific_dsRNA | 25 | pheromone gland | Pheromone glands of newly emerged adults were injected either with 2 uL DEPC-H2O or 2 uL (ie 10 ug) dsRNA corresponding to a portion of the green fluorescent protein (GFP) coding sequence using a 10 uL Hamilton syringe fitted with a 35 gauge needle. Moths were then maintained under normal conditions for 48 hr prior to assay. | adult | phenotype | pheromone gland | not checked | 48 | none | ||||||
| 238 | Bombyx mori sex pheromone biosynthetic pathway - BmGs1 RNAi | RNAi | pubmed | 20546038 | Hull | Joe | Bombyx mori sex pheromone biosynthetic pathway - BmGs1 RNAi target | ATGGGTTGCTTCGGGTCACCGGGCGGCAAGAGCAGCGAGGGCGACGACATCAAGAAACAGAAGAGACGCAGCGATGCTATCACCAGGCAGCTGCAGAAGGATAAACAGTTGTATCGGGCGACGCACCGGCTTCTCCTGCTGGGCGCCGGCGAGTCCGGGAAGTCGACGATCGTGAAGCAGATGCGCATCTTGCACGTCAACGGTTTCTCCGACAAGGAGCGCAAGGAGAAGATTGAGGACATCAAGAAGAACATCCGCGATGCTATACTCACAATCACGGGCGCAATGAGTACCCTGACTCCTCCGATTCCACTCGAGAAGCAGGAGAACAAAGTACGCGTCGATTACATTCAGGACGTGGCGTCGCAGCCGGACTTCGACTACCCTTCGGAGTTCTACGAGCACACGGAGGCGCTGTGGAAGGACCAGGGCGTGCAGCGCACCTACGAGCGCAGCAACGAGTACCAGCTCATCGACTGCGCGAAATACTTCTTGGATAAAGTTCACATAATAAAGCAGGCGGACTACACGCCCACCGAGCAGGACATCCTGCGCTGCCGAGTCCTCACCTCCGGGATATTCGAGACGCAGTTCGTCGTGGACAAAGTCAATTTCCACATGTTCGACGTGGGCGGGCAACGTGACGAGCGCCGTAAGTGGATCCAGTGCTTCAATGACGTGACGGCCATCATCTTCGTGACGGCCTGCTCCTCCTACAACATGGTGCTGCGCGAGGACCCCACGCAAAACCGGCTGCGCGAGTCCCTAGAGCTGTTCAAGAGCATCTGGAACAACCGGTGGCTGCGGACTATCTCCGTCATCCTGTTCCTCAACAAGCAGGACCTGCTCGCCGAGAAGGTGCTGGCGGGGAAGTCCCGGCTCGAGGATTACTTCCAGGAGTTCGCCCGTTACCAGACCCCGCCTGATGCTGTGCCCGACGCCAAGGACCATCCTGAGGTGTCCCGCGCCAAGTACTTCATACGCGATGAGTTTCTGCGCATAAGTACGGCCAGCGGCGACGGTAAGCACTACTGCTACCCGCACTTCACGTGCGCCGTCGACACGGAGAACATCAAGCGCGTGTTCAACGACTGCCGCGACATCATCCAGCGCATGCACCTGCGCCAGTACGAGCTGCTCTAA | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | NM_001099822.1 | Bombyx mori sex pheromone biosynthetic pathway - BmGs1 RNAi RNAi construct | ATGGGTTGCTTCGGGTCACCGGGCGGCAAGAGCAGCGAGGGCGACGACATCAAGAAACAGAAGAGACGCAGCGATGCTATCACCAGGCAGCTGCAGAAGGATAAACAGTTGTATCGGGCGACGCACCGGCTTCTCCTGCTGGGCGCCGGCGAGTCCGGGAAGTCGACGATCGTGAAGCAGATGCGCATCTTGCACGTCAACGGTTTCTCCGACAAGGAGCGCAAGGAGAAGATTGAGGACATCAAGAAGAACATCCGCGATGCTATACTCACAATCACGGGCGCAATGAGTACCCTGACTCCTCCGATTCCACTCGAGAAGCAGGAGAACAAAGTACGCGTCGATTACATTCAGGACGTGGCGTCGCAGCCGGACTTCGACTACCCTTCGGAGTTCTACGAGCACACGGAGGCGCTGTGGAAGGACCAGGGCGTGCAGCGCACCTACGAGCGCAGCAACGAGTACCAGCTCATCGACTGCGCGAAATACTTCTTGGATAAAGTTCACATAATAAAGCAGGCGGACTACACGCCCACCGAGCAGGACATCCTGCGCTGCCGAGTCCTCACCTCCGGGATATTCGAGACGCAGTTCGTCGTGGACAAAGTCAATTTCCACATGTTCGACGTGGGCGGGCAACGTGACGAGCGCCGTAAGTGGATCCAGTGCTTCAATGACGTGACGGCCATCATCTTCGTGACGGCCTGCTCCTCCTACAACATGGTGCTGCGCGAGGACCCCACGCAAAACCGGCTGCGCGAGTCCCTAGAGCTGTTCAAGAGCATCTGGAACAACCGGTGGCTGCGGACTATCTCCGTCATCCTGTTCCTCAACAAGCAGGACCTGCTCGCCGAGAAGGTGCTGGCGGGGAAGTCCCGGCTCGAGGATTACTTCCAGGAGTTCGCCCGTTACCAGACCCCGCCTGATGCTGTGCCCGACGCCAAGGACCATCCTGAGGTGTCCCGCGCCAAGTACTTCATACGCGATGAGTTTCTGCGCATAAGTACGGCCAGCGGCGACGGTAAGCACTACTGCTACCCGCACTTCACGTGCGCCGTCGACACGGAGAACATCAAGCGCGTGTTCAACGACTGCCGCGACATCATCCAGCGCATGCACCTGCGCCAGTACGAGCTGCT | dsRNA | Ampliscribe T7 High Yield Transcription kit; Epicentre Technologies | phenol_chloroform | subsequent to transcription | In vitro transcription templates were generated via PCR using plasmid DNA containing the gene of interest in conjunction with gene-specific primers containing T7 polymerase promoter sites at the 5â ends of the sense and antisense primers. PCR was performed with KOD-Plus (Toyobo, Osaka) and the resulting product purified (Promega SV PCR purification kit) was used as a template to generate dsRNAs with the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | InsectaCentral | Bombyx mori sex pheromone biosynthetic pathway - BmGs1 RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | DEPC-H2O | 0.02 | buffer non_specific_dsRNA | 26 | pheromone gland | Pheromone glands of newly emerged adults were injected either with 2 uL DEPC-H2O or 2 uL (ie 10 ug) dsRNA corresponding to a portion of the green fluorescent protein (GFP) coding sequence using a 10 uL Hamilton syringe fitted with a 35 gauge needle. Moths were then maintained under normal conditions for 48 hr prior to assay. | adult | phenotype | pheromone gland | not checked | 48 | none | ||||||
| 239 | Bombyx mori sex pheromone biosynthetic pathway - BmGs2 RNAi | RNAi | pubmed | 20546038 | Hull | Joe | Bombyx mori sex pheromone biosynthetic pathway - BmGs2 RNAi target | ATGGGCTGTTTCAAGTCGTTAGGAGGTCACAGAAACGAAAGCGAAGACGCGAAGGAGCAGAGAAGACGGAACCATGCCATAAATCAACAGCTCAAACAAGATAGAGAGCTGTACCAAGCGACGCACCGGCTTCTCCTGCTGGGCGCCGGCGAGTCCGGGAAGTCGACGATCGTGAAGCAGATGCGCATCCTGCACGTCAACGGCTTCTCCGACAAGGAGCGCAGGGAGAAGATCGAGGACATCAAGAAGAACGTTCGAGATGCCATCATTACGATCACCGGTGCCATGAGTACCTTAACTCCTCCGATTCCTCTCGAGAAACCGGAGAATAAACCACGCGTGGATTACATTCAGGACGTGGCGTCGCAGCCGGACTTCGACTATCCGTCGGAGTTCTACGAGCACACGGAGGCGCTGTGGAAGGACCAGGGCGTGCAGCGCACCTACGAGCGCAGCAACGAGTACCAGCTCATCGACAGTGCCAAATACTTCCTGGACAAGATCCACGTGATCAGACAGCCGGGCTACACGCCCACCGAGCAGGACGTCCTCCGCTGCCGGGTCCTCACCTCCGGGATCTACCAGACGCAGTTCGTCGTGGACAAAGTTCACTTCCATATATTCGATGTCGGCGGACAACGCGACGAAAGACGCAAATGGATCCAGTGCTTCAACGATGTTACCGCGATCATATTCGTAGCGGCCTGCTCCGCCTACAACATGGTGCTGCGCGAGGACCCCACGCAGAACCGGCTGCGCGAGTCCCTGGATCTGTTCAAGAGCATCTGGAACAACCGGTGGCTGCGGACCATCTCCGTCATCCTGTTCCTCAACAAGCAGGACCTGCTCGCCGAGAAGGTGCTGGCGGGCAAGTCCCGGCTCGAGGACTACTTCCACGAGTTCGCCCAGTACCAGACCCCGACCGACATGAAGCCAGACCCCAAAGATCACCCTGAAGTGACCCGCGCCAAGTTCTTCATTCGTGAGGAGTTTCTGCGCATCAGCACCGCCACGGGCGACGGCAAGCACGACTGCTACGCTCACTTCACGTGCGCCGTCGACACCGACAACATCCAACGAGTGTTCAACGGCTGCCGGAACATCATCCAGCGGTGGCATTTGAAGCAATACGAACTACTATAG | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | cds | GenBank | NM_001109932.1 | Bombyx mori sex pheromone biosynthetic pathway - BmGs2 RNAi RNAi construct | ATGGGCTGTTTCAAGTCGTTAGGAGGTCACAGAAACGAAAGCGAAGACGCGAAGGAGCAGAGAAGACGGAACCATGCCATAAATCAACAGCTCAAACAAGATAGAGAGCTGTACCAAGCGACGCACCGGCTTCTCCTGCTGGGCGCCGGCGAGTCCGGGAAGTCGACGATCGTGAAGCAGATGCGCATCCTGCACGTCAACGGCTTCTCCGACAAGGAGCGCAGGGAGAAGATCGAGGACATCAAGAAGAACGTTCGAGATGCCATCATTACGATCACCGGTGCCATGAGTACCTTAACTCCTCCGATTCCTCTCGAGAAACCGGAGAATAAACCACGCGTGGATTACATTCAGGACGTGGCGTCGCAGCCGGACTTCGACTATCCGTCGGAGTTCTACGAGCACACGGAGGCGCTGTGGAAGGACCAGGGCGTGCAGCGCACCTACGAGCGCAGCAACGAGTACCAGCTCATCGACAGTGCCAAATACTTCCTGGACAAGATCCACGTGATCAGACAGCCGGGCTACACGCCCACCGAGCAGGACGTCCTCCGCTGCCGGGTCCTCACCTCCGGGATCTACCAGACGCAGTTCGTCGTGGACAAAGTTCACTTCCATATATTCGATGTCGGCGGACAACGCGACGAAAGACGCAAATGGATCCAGTGCTTCAACGATGTTACCGCGATCATATTCGTAGCGGCCTGCTCCGCCTACAACATGGTGCTGCGCGAGGACCCCACGCAGAACCGGCTGCGCGAGTCCCTGGATCTGTTCAAGAGCATCTGGAACAACCGGTGGCTGCGGACCATCTCCGTCATCCTGTTCCTCAACAAGCAGGACCTGCTCGCCGAGAAGGTGCTGGCGGGCAAGTCCCGGCTCGAGGACTACTTCCACGAGTTCGCCCAGTACCAGACCCCGACCGACATGAAGCCAGACCCCAAAGATCACCCTGAAGTGACCCGCGCCAAGTTCTTCATTCGTGAGGAGTTTCTGCGCATCAGCACCGCCACGGGCGACGGCAAGCACGACTGCTACGCTCACTTCACGTGCGCCGTCGACACCGACAACATCCAACGAGTGTTCAACGGCTGCCGGAACATCATCCAGCGGTGGCATTTGAAGCAATACGA | dsRNA | Ampliscribe T7 High Yield Transcription kit; Epicentre Technologies | phenol_chloroform | subsequent to transcription | In vitro transcription templates were generated via PCR using plasmid DNA containing the gene of interest in conjunction with gene-specific primers containing T7 polymerase promoter sites at the 5â ends of the sense and antisense primers. PCR was performed with KOD-Plus (Toyobo, Osaka) and the resulting product purified (Promega SV PCR purification kit) was used as a template to generate dsRNAs with the AmpliScribe T7 High Yield Transcription kit (Epicentre Technologies, Madison, WI), according to the manufacturerâs instructions. After synthesis, the dsRNAs were diluted with DEPC-treated H2O, the RNA concentrations measured (Abs260), and the products analyzed by gel electrophoresis to confirm annealing. | InsectaCentral | Bombyx mori sex pheromone biosynthetic pathway - BmGs2 RNAi RNAi construct | Lepidoptera | Bombycidae | Bombyx | mori | 7091 | local resource | adult | lab_colony | 5 | injection | DEPC-H2O | 0.02 | buffer non_specific_dsRNA | 17 | pheromone gland | Pheromone glands of newly emerged adults were injected either with 2 uL DEPC-H2O or 2 uL (ie 10 ug) dsRNA corresponding to a portion of the green fluorescent protein (GFP) coding sequence using a 10 uL Hamilton syringe fitted with a 35 gauge needle. Moths were then maintained under normal conditions for 48 hr prior to assay. | adult | phenotype | pheromone gland | not checked | 48 | none | ||||||
| 241 | RNAi of IIM gene expression in Trichoplusia ni | RNAi | Only a small percentage (~5%) of RNAi treated larvae showed significantly reduced IIM. | InsectaCentral | Unpublished | Wang | Ping | RNAi of IIM gene expression in Trichoplusia ni target | GAAAAGAATAACCAGCGAACAAGTTATGATAAAGACCCTCCTATTCCTGACGGCCCTCGGGCTCGTCGCCGCGCGTCCTGAAGTCAGCGACGCGGAGAAGAACCCCGCTCTCCACGAGCCGCACCCAGACTGCCCTCCCGCTGAGCAGCACTGGCTCCTGCCTCACGAATACGACTGCACCAAGTTCTACTACTGTGAATATGGTCTCAAGTTCATCGCACCGAGAGACTGTGCTCCTGGTACCGAATTCAAGTTCTCCGCTCAGACTTGTGTTCACGCCGCTTTAGCCGGATGCACCCTGCCAGGACCTCCAGCTGAGACAACCCAGGCCCCAGCAACAACTCAGGCCCCAACAACCACCCAGGCCCCAACCACAACTACTCAGGCCCCTACTACAACCACCCAGGCCCCAACCACAACCACCCAGGCCCCAACCACCACCCAGGCCCCAACCACCACCCAGGCCCCAACTACCACTCAGGCCCCTACTACTACCACTCAGGCCCCAACCACAACCACTCAGGCCCCTACCACAACCACCCAGGCCCCAACCACCACCCAGGCCCCAACTACCACCCAGGCCCCAACTACCACTCAGGCCCCAACTACAATCACCCAGGCTGCAACTACCCCGGCCGCAACTACCCCGGCCGCAACTACCCCGGCCGCAACTACCCCTGCCGCGACAACCCCCGCTGCAACTACCCCAGGTGTTCCTGCACCCACTTCAGCCCCAGTCTGGCCCCCGATCTGTGAACTGTTGCCCAATGGTTGCCCAGCTGACTTCGACATCCACTTGTTGATTCCCCACGACAAGTACTGCAACCTCTTCTACCAGTGCTCCAACGGTTACACCTTCGAACAGAGGTGCCCTGAGGGACTCTACTTCAACCCCTACGTCCAGCGCTGCGACTCTCCTGCTAACGTTGAATGCGACGGCGAAATCAGCCCCGCACCCCCAGTCACAGAAGGCAACGAAGACGAAGACATTGACATCGGAGACCTCCTCGACAATGGATGCCCAGCTAACTTCGAAATCGACTGGCTCTTGCCCCACGGAAACCGTTGCGACAAGTATTACCAGTGCGTCCACGGTAACTTGGTAGAGAGGCGTTGTGGAGCCGGCACCCACTTCAGTTTTGAACTTCAGCAATGTGACCACATCGAGCTCGTTGGCTGCACCCTCCCCGGCGGCGAGAGCGAAGAAGTTGACGTCGACGAGGATGCCTGCACCGGCTGGTACTGCCCCACGGAACCCATTGAATGGGAGCCCCTCCCCAACGGCTGCCCTGCCGACTTCAGCATCGACCACCTCCTCCCCCACGAGAGCGACTGCGGCCAGTATCTACAGTGTGTCCATGGACAGACTATCGCAAGACCTTGCCCTGGAAACCTGCACTTCAGTCCTGCCACACAGTCCTGTGAGTCTCCTGTGACCGCTGGTTGCCAAGTTTTCGAGTGCGATTCTGACAACCAGTGCACATCGACTGCTGCCCCGACAGCTGCTCCAACGGCTGCCCCAACGGCTGCCCCAACGGCTGCCCCAACTGCCGCACCCTCCACCGTGGTCCCACCTGCAACGCCACCCGCAACTGCAGCCCCAGTCCCACCTACAACCGCAATTCCTACTCCGGCCCCCACCGCTGCCCCCACCGCAGCTCCTACTACTGCTGCCCCTGAATCCCCAACCACTGTCACAGTACCACCTACTGCTGCTCCCACCGCAGCCCCTACTACTGCTGTCCCTGAAATCCCAATCACTGTCACATCAGCGCCTACCGCTGCCCCCACCGCTGCCCCCACCGCTGCCCCCACCGCAGCCCCTACTACTGCTGTCCCAGAAATCCCAACTACTGTCACATCACCACCTACTGCTGCCCCCACTACCGCAGCACCTGCCCCCAACACCACAGTCACTGTACCACCCACTGCTGCCCCTACTACCGCAGCACCTGCTCCCAACACCACAGTGACTGCACCACCCACCGCAGCCCCTACTACCGCAGCACCTGCCCCCAACACCACAGTCACTGTACCACCCACTGCTGCCCCCACTGCAGCTCCCCCTACCGTCGCACCTGCACCCAACACCACAGCTGCCCCGGTAACTACAACCAGCGCACCAGCTACCACACCTGAAGATGATGACATCGACCCCCCTCTCCCCAACGACCCCATCAACCCTTGCGTTGAAGAATGCAACGTTTTGCCCTGGGCTCACGCTGACTGCGACAAATACTGGGTCTGTGACGGCAACAACCAAGTCCTGGTGGTTTGCTCTGAGGGTCTCCAGTTCAACCCCACTACTAAGACCTGTGACTTCGCTTGCAACGTCGGTTGCGTGAGGAGCAACATTCAGATGTCTGAAAGCTACGAAGGAGTCCAGGTCTTCATCCCATGGAACAAACTAGATGAAGACATCAGACAGGCGCTGAACTTTGAGTTGTAAACCTACTTAAATTAATGAAGGTTTTGTTTTATTTTTGAGTTATTATTCCAATGGGCGGGAAAGTCCGCCATTATTGGGTCTTGCCAGTTTTGAGGAAACCTTTTTTTTTACTACCAACATTCTTGTGAACCCATATTTATTACGTATTAAACATCGTGATTTGAAAAACGTTACATGATTTTTTCATTAATTTGAAACAATTTATGTTGTTTTTGTTCTCATTAAATATCAAATATCATTTTCGAAACTGGCAATTTTGGATTGGAATAATCAACAAATGGTTAAGAAAAAAAACGATTTCTTAAAAATGTATTTATTATAAAATGTGTAAATAAATATACAAATTAGCATTTAAAAAAAAAAAAAAAAAAA | Lepidoptera | Noctuidae | Trichoplusia | ni | 7111 | cds | GenBank | AF000606.1 | RNAi of IIM gene expression in Trichoplusia ni RNAi construct | GAAAAGAATAACCAGCGAACAAGTTATGATAAAGACCCTCCTATTCCTGACGGCCCTCGGGCTCGTCGCCGCGCGTCCTGAAGTCAGCGACGCGGAGAAGAACCCCGCTCTCCACGAGCCGCACCCAGACTGCCCTCCCGCTGAGCAGCACTGGCTCCTGCCTCACGAATACGACTGCACCAAGTTCTACTACTGTGAATATGGTCTCAAGTTCATCGCACCGAGAGACTGTGCTCCTGGTACCGAATTCAAGTTCTCCGCTCAGACTTGTGTTCACGCCGCTTTAGCCGGATGCACCCTGCCAGGACCTCCAGCTGAGACAACCCAGGCCCCAGCAACAACTCAGGCCCCAACAACCACCCAGGCCCCAACCACAACTACTCAGGCCCCTACTACAACCACCCAGGCCCCAACCACAACCACCCAGGCCCCAACCACCACCCAGGCCCCAACCACCACCCAGGCCCCAACTACCACTCAGGCCCCTACTACTACCACTCAGGCCCCAACCACAACCACTCAGGCCCCTACCACAACCACCCAGGCCCCAACCACCACCCAGGCCCCAACTACCACCCAGGCCCCAACTACCACTCAGGCCCCAACTACAATCACCCAGGCTGCAACTACCCCGGCCGCAACTACCCCGGCCGCAACTACCCCGGCCGCAACTACCCCTGCCGCGACAACCCCCGCTGCAACTACCCCAGGTGTTCCTGCACCCACTTCAGCCCCAGTCTGGCCCCCGATCTGTGAACTGTTGCCCAATGGTTGCCCAGCTGACTTCGACATCCACTTGTTGATTCCCCACGACAAGTACTGCAACCTCTTCTACCAGTGCTCCAACGGTTACACCTTCGAACAGAGGTGCCCTGAGGGACTCTACTTCAACCCCTACGTCCAGCGCTGCGACTCTCCTGCTAACGTTGAATGCGACGGCGAAATCAGCCCCGCACCCCCAGTCACAGAAGGCAACGAAGACGAAGACATTGACATCGGAGACCTCCTCGACAATGGATGCCCAGCTAACTTCGAAATCGACTGGCTCTTGCCCCACGGAAACCGTTGCGACAAGTATTACCAGTGCGTCCACGGTAACTTGGTAGAGAGGCGTTGTGGAGCCGGCACCCACTTCAGTTTTGAACTTCAGCAATGTGACCACATCGAGCTCGTTGGCTGCACCCTCCCCGGCGGCGAGAGCGAAGAAGTTGACGTCGACGAGGATGCCTGCACCGGCTGGTACTGCCCCACGGAACCCATTGAATGGGAGCCCCTCCCCAACGGCTGCCCTGCCGACTTCAGCATCGACCACCTCCTCCCCCACGAGAGCGACTGCGGCCAGTATCTACAGTGTGTCCATGGACAGACTATCGCAAGACCTTGCCCTGGAAACCTGCACTTCAGTCCTGCCACACAGTCCTGTGAGTCTCCTGTGACCGCTGGTTGCCAAGTTTTCGAGTGCGATTCTGACAACCAGTGCACATCGACTGCTGCCCCGACAGCTGCTCCAACGGCTGCCCCAACGGCTGCCCCAACGGCTGCCCCAACTGCCGCACCCTCCACCGTGGTCCCACCTGCAACGCCACCCGCAACTGCAGCCCCAGTCCCACCTACAACCGCAATTCCTACTCCGGCCCCCACCGCTGCCCCCACCGCAGCTCCTACTACTGCTGCCCCTGAATCCCCAACCACTGTCACAGTACCACCTACTGCTGCTCCCACCGCAGCCCCTACTACTGCTGTCCCTGAAATCCCAATCACTGTCACATCAGCGCCTACCGCTGCCCCCACCGCTGCCCCCACCGCTGCCCCCACCGCAGCCCCTACTACTGCTGTCCCAGAAATCCCAACTACTGTCACATCACCACCTACTGCTGCCCCCACTACCGCAGCACCTGCCCCCAACACCACAGTCACTGTACCACCCACTGCTGCCCCTACTACCGCAGCACCTGCTCCCAACACCACAGTGACTGCACCACCCACCGCAGCCCCTACTACCGCAGCACCTGCCCCCAACACCACAGTCACTGTACCACCCACTGCTGCCCCCACTGCAGCTCCCCCTACCGTCGCACCTGCACCCAACACCACAGCTGCCCCGGTAACTACAACCAGCGCACCAGCTACCACACCTGAAGATGATGACATCGACCCCCCTCTCCCCAACGACCCCATCAACCCTTGCGTTGAAGAATGCAACGTTTTGCCCTGGGCTCACGCTGACTGCGACAAATACTGGGTCTGTGACGGCAACAACCAAGTCCTGGTGGTTTGCTCTGAGGGTCTCCAGTTCAACCCCACTACTAAGACCTGTGACTTCGCTTGCAACGTCGGTTGCGTGAGGAGCAACATTCAGATGTCTGAAAGCTACGAAGGAGTCCAGGTCTTCATCCCATGGAACAAACTAGATGAAGACATCAGACAGGCGCTGAACTTTGAGTTGTAAACCTACTTAAATTAATGAAGGTTTTGTTTTATTTTTGAGTTATTATTCCAATGGGCGGGAAAGTCCGCCATTATTGGGTCTTGCCAGTTTTGAGGAAACCTTTTTTTTTACTACCAACATTCTTGTGAACCCATATTTATTACGTATTAAACATCGTGATTTGAAAAACGTTACATGATTTTTTCATTAATTTGAAACAATTTATGTTGTTTTTGTTCTCATTAAATATCAAATATCATTTTCGAAACTGGCAATTTTGGATTGGAATAATCAACAAATGGTTAAGAAAAAAAACGATTTCTTAAAAATGTATTTATTATAAAATGTGTAAATAAATATACAAATTAGCATTTAAAAAAAAAAAAAAAAAAA | dsRNA | MEGAscript RNAi Kit/Ambion | phenol_chloroform | subsequent to transcription | Method 1: Target DNA fragments flanked with T3 and T7 promoter sequences were amplified from the constructs, pIIM-763, pIIM-845 and pIIM-1214, by PCR and were used as templates after purification for single strain RNA synthesis using the MEGAscript T3 and MEGAscript T7 RNA synthesis kits (Ambion), respectively. To prepare dsRNA preparations, the single strain RNA preparations made with T3 and T7 polymerases were combined and heated at 95°C for 3 min and then let slowly cooled down to room temperature. The dsRNA preparations were cleaned by extraction with phenol/chloroform (1:1) and the dsRNA in aqueous phase was precipitated with ethanol. Method 2: Target DNA fragments were amplified by PCR using primers containing T7 promoter sequence at 5â ends to add the T7 promoter sequence at the ends of the PCR products. The PCR products were used as templates after purification for dsRNA synthesis using the MEGAscript T7 or MEGAscript RNAi kit. Methods 3: The target DNA fragments were cloned into the plasmid L4440. dsRNAs were produced in E. coli strain HT115 (Timmons et al., 2001. Gene 263: 103-112) and isolated using the TRI Reagent from Ambion. | InsectaCentral | RNAi of IIM gene expression in Trichoplusia ni RNAi construct | Lepidoptera | Noctuidae | Trichoplusia | ni | 7111 | local resource | larva | lab_colony | 5 | injection feeding | 20 | buffer non_specific_dsRNA | 10 | hemocoel and midgut | larva | W.blot | peritrophic membrane, feces | not checked | low | ||||||||
| 243 | RNAi of SfT6 in Spodoptera frugiperda feeding | RNAi | Entered by Olle Terenius. SfT6 has been identified in a subtracted cDNA library of Spodoptera frugiperda larval midgut transcripts as a serine-protease gene downregulated within 24 h of intoxication with Bacillus thuringiensis Cry1Ca1 protein. In the present study, the specific role of SfT6 during Cry1Ca1 intoxication was investigated by RT-PCR and in vivo RNA interference. Quantitative real-time RT-PCR analysis showed SfT6 mRNA levels in the midgut tissue were significantly reduced after injecting or feeding 4th-instar larvae with specific long-size dsRNA. Gut juice-mediated in vitro protoxin activation and susceptibility for Cry1Ca1 were investigated in Sft6-knockdown larvae and compared with control treated with nonspecific dsRNA. Our results demonstrate SfT6 plays a determinant role in Cry1Ca1 toxicity against S. frugiperda since a decreased expression caused a reduced protoxin activation by larval gut juice and reduced susceptibility of insects to toxin in bioassays. We propose SfT6 downregulation occurring at the early stages of Cry1Ca1 intoxication is part of a complex and multifaceted defensive mechanism triggered in the insect gut to withstand B. thuringiensis pathogenesis. | pubmed | 20545748 | AyraâPardo | Camilo | RNAi of SfT6 in Spodoptera frugiperda target | ATGCGTGTCCTCGCTTGCTTGGCCCTTCTCTTAGCTGTGGTAGCAGCCGTCCCCTCCAATCCCCAGAGGATTGTGGGTGGTTCGGTCACCACCATTGACCGGTACCCCACCATTGCATCTCTGCTGTACTCGTGGAACTTGAGCTCCTACTGGCAGGCATGCGGTGGTTCCATCTTGAACAACCGTGCTATCCTTACTGCTGCCCACTGCACAGTTGGTGACGCCGCTAACAGATGGAGAATCCGTCTCGGTTCCACCTGGGCCAACAGCGGTGGTGTCGTTCACAACGTCAACACCAACATCGTCCACCCCTCATACAATTCCGCAACTTTGAACAACGACATCGCTATCCTCCGCTCCGCCACCACTTTCTCCTTCAACAACAATGTTCAGGCTGCCTCCATCGCTGGCGCCAACTACTTGCCCGGTGACAACACCGCCGCTTGGGCCGCTGGATGGGGAACTACCTCTGCTGGTGGCTCTAGCTCTGAGCAGCTCCGTCACGTTGAGCTGCGCATCATCAACCAAGCTACTTGCAGAAACAACTACGCTACCCGCGGTATCACCATTACCGACAACATGTTGTGCTCCGGCTGGCCCACTGGTGGTCGCGACCAGTGCCAGGGTGACTCTGGTGGTCCTCTCTACCACAACGGCATCGTTGTTGGTGTCTGCTCTTTCGGTATTGGCTGTGCTCAGGCTGCTTTCCCCGGTGTCAACGCTCGTGTATCCCGCTACACTGCCTGGATCTCTTCCAATGCATAA | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | GenBank | FJ940726 | RNAi of SfT6 in Spodoptera frugiperda RNAi construct | ATGCGTGTCCTCGCTTGCTTGGCCCTTCTCTTAGCTGTGGTAGCAGCCGTCCCCTCCAATCCCCAGAGGATTGTGGGTGGTTCGGTCACCACCATTGACCGGTACCCCACCATTGCATCTCTGCTGTACTCGTGGAACTTGAGCTCCTACTGGCAGGCATGCGGTGGTTCCATCTTGAACAACCGTGCTATCCTTACTGCTGCCCACTGCACAGTTGGTGACGCCGCTAACAGATGGAGAATCCGTCTCGGTTCCACCTGGGCCAACAGCGGTGGTGTCGTTCACAACGTCAACACCAACATCGTCCACCCCTCATACAATTCCGCAACTTTGAACAACGACATCGCTATCCTCCGCTCCGCCACCACTTTCTCCTTCAACAACAATGTTCAGGCTGCCTCCATCGCTGGCGCCAACTACTTGCCCGGTGACAACACCGCCGCTTGGGCCGCTGGATGGGGAACTACCTCTGCTGGTGGCTCTAGCTCTGAGCAGCTCCGTCACGTTGAGCTGCGCATCATCAACCAAGCTACTTGCAGAAACAACTACGCTACCCGCGGTATCACC | dsRNA | T7 RiboMAX Express RNAi System (Promega) | ethanol precipitation | subsequent to transcription | To generate a SfT6 template for dsRNA synthesis, a fragment comprising the first 567 bp of the ORF was PCR amplified from pGEM-SfT6 (section 2.2) with primers (F) 5â²-ATGC GTGTCCTCGCTTGCTTGG-3â²ï and (R) 5â²-GGTGATACC GCGGGTAGCGTAG-3â², and subcloned into the unique EcoRV restriction site of the multiple cloning region of Litmus 29 plasmid (New England BioLabs, Beverly, MA, USA). For RNAi control experiments with nonspecific dsRNA, a fragment from the E. coli bâDâglucuronidase (uidA) gene (GenBank accession no. CP001509) spanning 58â669 nucleotides was produced by PCR with primers 5â²-GCATTCAGTCTGGATCGCGAAAAC-3â²ï (forward) and 5â²-CACCTGTTGATCCGCATCACGCAG-3â²ï (reverse) and subcloned into the EcoRV-digested Litmus 29 vector. The source for uidA gene was the pPIC-GUS plasmid (Ayra- Pardo et al., 1998). Since the Litmus 29 polylinker region is flanked by two T7 promoters in opposite orientations, singlestrand sense RNA (sRNA) and antisense RNA (asRNA) of cloned fragments were generated in vitro using the T7 RiboMAX Express RNAi System (Promega). Two different T7 RNA Pol templates, for each Litmus 29-SfT6 and Litmus 29-Gus, were prepared by linearizing with XhoI and XbaI restriction endonucleases that do not cut SfT6 or uidA inserts, purified using the PCR Clean-Up System (Promega) and transcribed in separated reactions. To produce dsRNA, equal amounts of corresponding sRNA and asRNA were mixed, heated to 65°C, and annealed by slow cooling over 4 h followed by DNase treatment for 15 min at 37°C. The dsRNA were then precipitated with ice-cold ethanol in the presence of 0.3 M sodium acetate, pH 5.4. The dsRNA pellets were washed with 75% ethanol and resuspended in DEPC-treated water to a final concentration of 5 mg ml-1. The dsRNA was quantified at 260 nm. The dsRNA were stored at â20°C until use. | InsectaCentral | RNAi of SfT6 in Spodoptera frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | local resource | larva | lab_colony | 3 | feeding | DB, 10 mM Tris-Cl, pH 7.5; 10 mM EDTA | buffer non_specific_dsRNA | 48 | feeding | Two groups of larvae were then fed with a 5 ml drop of 3 mg dsRNA from uidA, in delivery buffer (DB, 10 mM Tris-Cl, pH 7.5; 10 mM EDTA). The third group of larvae were fed with DB as a control. | larva | qPCR phenotype | qPCR midguts pools, Phenotype toxin resistance protein or survival | not checked | 48 | high | 90 | |||||
| 244 | RNAi of SfT6 in Spodoptera frugiperda injection | RNAi | Entered by Olle Terenius. SfT6 has been identified in a subtracted cDNA library of Spodoptera frugiperda larval midgut transcripts as a serine-protease gene downregulated within 24 h of intoxication with Bacillus thuringiensis Cry1Ca1 protein. In the present study, the specific role of SfT6 during Cry1Ca1 intoxication was investigated by RT-PCR and in vivo RNA interference. Quantitative real-time RT-PCR analysis showed SfT6 mRNA levels in the midgut tissue were significantly reduced after injecting or feeding 4th-instar larvae with specific long-size dsRNA. Gut juice-mediated in vitro protoxin activation and susceptibility for Cry1Ca1 were investigated in Sft6-knockdown larvae and compared with control treated with nonspecific dsRNA. Our results demonstrate SfT6 plays a determinant role in Cry1Ca1 toxicity against S. frugiperda since a decreased expression caused a reduced protoxin activation by larval gut juice and reduced susceptibility of insects to toxin in bioassays. We propose SfT6 downregulation occurring at the early stages of Cry1Ca1 intoxication is part of a complex and multifaceted defensive mechanism triggered in the insect gut to withstand B. thuringiensis pathogenesis. | pubmed | 20545748 | AyraâPardo | Camilo | RNAi of SfT6 in Spodoptera frugiperda target | ATGCGTGTCCTCGCTTGCTTGGCCCTTCTCTTAGCTGTGGTAGCAGCCGTCCCCTCCAATCCCCAGAGGATTGTGGGTGGTTCGGTCACCACCATTGACCGGTACCCCACCATTGCATCTCTGCTGTACTCGTGGAACTTGAGCTCCTACTGGCAGGCATGCGGTGGTTCCATCTTGAACAACCGTGCTATCCTTACTGCTGCCCACTGCACAGTTGGTGACGCCGCTAACAGATGGAGAATCCGTCTCGGTTCCACCTGGGCCAACAGCGGTGGTGTCGTTCACAACGTCAACACCAACATCGTCCACCCCTCATACAATTCCGCAACTTTGAACAACGACATCGCTATCCTCCGCTCCGCCACCACTTTCTCCTTCAACAACAATGTTCAGGCTGCCTCCATCGCTGGCGCCAACTACTTGCCCGGTGACAACACCGCCGCTTGGGCCGCTGGATGGGGAACTACCTCTGCTGGTGGCTCTAGCTCTGAGCAGCTCCGTCACGTTGAGCTGCGCATCATCAACCAAGCTACTTGCAGAAACAACTACGCTACCCGCGGTATCACCATTACCGACAACATGTTGTGCTCCGGCTGGCCCACTGGTGGTCGCGACCAGTGCCAGGGTGACTCTGGTGGTCCTCTCTACCACAACGGCATCGTTGTTGGTGTCTGCTCTTTCGGTATTGGCTGTGCTCAGGCTGCTTTCCCCGGTGTCAACGCTCGTGTATCCCGCTACACTGCCTGGATCTCTTCCAATGCATAA | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | cds | GenBank | FJ940726 | RNAi of SfT6 in Spodoptera frugiperda RNAi construct | ATGCGTGTCCTCGCTTGCTTGGCCCTTCTCTTAGCTGTGGTAGCAGCCGTCCCCTCCAATCCCCAGAGGATTGTGGGTGGTTCGGTCACCACCATTGACCGGTACCCCACCATTGCATCTCTGCTGTACTCGTGGAACTTGAGCTCCTACTGGCAGGCATGCGGTGGTTCCATCTTGAACAACCGTGCTATCCTTACTGCTGCCCACTGCACAGTTGGTGACGCCGCTAACAGATGGAGAATCCGTCTCGGTTCCACCTGGGCCAACAGCGGTGGTGTCGTTCACAACGTCAACACCAACATCGTCCACCCCTCATACAATTCCGCAACTTTGAACAACGACATCGCTATCCTCCGCTCCGCCACCACTTTCTCCTTCAACAACAATGTTCAGGCTGCCTCCATCGCTGGCGCCAACTACTTGCCCGGTGACAACACCGCCGCTTGGGCCGCTGGATGGGGAACTACCTCTGCTGGTGGCTCTAGCTCTGAGCAGCTCCGTCACGTTGAGCTGCGCATCATCAACCAAGCTACTTGCAGAAACAACTACGCTACCCGCGGTATCACC | dsRNA | T7 RiboMAX Express RNAi System (Promega) | ethanol precipitation | subsequent to transcription | To generate a SfT6 template for dsRNA synthesis, a fragment comprising the first 567 bp of the ORF was PCR amplified from pGEM-SfT6 (section 2.2) with primers (F) 5â²-ATGC GTGTCCTCGCTTGCTTGG-3â²ï and (R) 5â²-GGTGATACC GCGGGTAGCGTAG-3â², and subcloned into the unique EcoRV restriction site of the multiple cloning region of Litmus 29 plasmid (New England BioLabs, Beverly, MA, USA). For RNAi control experiments with nonspecific dsRNA, a fragment from the E. coli bâDâglucuronidase (uidA) gene (GenBank accession no. CP001509) spanning 58â669 nucleotides was produced by PCR with primers 5â²-GCATTCAGTCTGGATCGCGAAAAC-3â²ï (forward) and 5â²-CACCTGTTGATCCGCATCACGCAG-3â²ï (reverse) and subcloned into the EcoRV-digested Litmus 29 vector. The source for uidA gene was the pPIC-GUS plasmid (Ayra- Pardo et al., 1998). Since the Litmus 29 polylinker region is flanked by two T7 promoters in opposite orientations, singlestrand sense RNA (sRNA) and antisense RNA (asRNA) of cloned fragments were generated in vitro using the T7 RiboMAX Express RNAi System (Promega). Two different T7 RNA Pol templates, for each Litmus 29-SfT6 and Litmus 29-Gus, were prepared by linearizing with XhoI and XbaI restriction endonucleases that do not cut SfT6 or uidA inserts, purified using the PCR Clean-Up System (Promega) and transcribed in separated reactions. To produce dsRNA, equal amounts of corresponding sRNA and asRNA were mixed, heated to 65°C, and annealed by slow cooling over 4 h followed by DNase treatment for 15 min at 37°C. The dsRNA were then precipitated with ice-cold ethanol in the presence of 0.3 M sodium acetate, pH 5.4. The dsRNA pellets were washed with 75% ethanol and resuspended in DEPC-treated water to a final concentration of 5 mg ml-1. The dsRNA was quantified at 260 nm. The dsRNA were stored at â20°C until use. | InsectaCentral | RNAi of SfT6 in Spodoptera frugiperda RNAi construct | Lepidoptera | Noctuidae | Spodoptera | frugiperda | 7108 | local resource | larva | lab_colony | 5 | injection | DEPC-treated water | buffer non_specific_dsRNA | 144 | the base of a pseudopodium | larvae were injected with 1 ml of nonspecific dsRNA solution (5 mg ml-1, N = 24 each) or 1 ml nuclease free water (N = 20) for the control. | larva | qPCR | midguts pools | not checked | 48 | high | 90 | |||||
