title: Molecular characterization and RNA interference of three midgut aminopeptidase N isozymes from Bacillus thuringiensis-susceptible and -resistant strains of sugarcane borer, Diatraea saccharalis.
abstract: 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 5' rapid amplification of cDNA end (5' 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.
authors.affiliation: Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Yang
authors.forename: Yu Cheng
authors.initials: YC
authors.lastname: Zhu
authors.forename: James
authors.initials: J
authors.lastname: Ottea
authors.forename: Claudia
authors.initials: C
authors.lastname: Husseneder
authors.forename: B Rogers
authors.initials: BR
authors.lastname: Leonard
authors.forename: Craig
authors.initials: C
authors.lastname: Abel
authors.forename: Fangneng
authors.initials: F
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D018826:CD13 Antigens / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004731:Endotoxins / Q000494:pharmacology*; D041981:Gastrointestinal Tract / Q000737:chemistry / Q000201:enzymology; D006460:Hemolysin Proteins / Q000494:pharmacology*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007305:Insecticide Resistance; D007306:Insecticides / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths / Q000145:classification / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D034622:RNA Interference; D016415:Sequence Alignment
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D007306:Insecticides; D007527:Isoenzymes; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens