Molecular characterization and RNA interference of three midgut aminopeptidase N isozymes from Bacillus thuringiensis-susceptible and -resistant strains of sugarcane borer, Diatraea saccharalis

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-R...

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Bibliographic Details
Published in:Insect biochemistry and molecular biology 2010-08, Vol.40 (8), p.592-603
Main Authors: Yang, Yunlong, Zhu, Yu Cheng, Ottea, James, Husseneder, Claudia, Rogers Leonard, B., Abel, Craig, Huang, Fangneng
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Aminopeptidase N
Animals
Bacillus
Bacillus thuringiensis
Bacterial Proteins - pharmacology
CD13 Antigens - chemistry
CD13 Antigens - genetics
CD13 Antigens - metabolism
Crambidae
Cry1Ab resistance
Diatraea saccharalis
Endotoxins - pharmacology
Gastrointestinal Tract - chemistry
Gastrointestinal Tract - enzymology
Hemolysin Proteins - pharmacology
Insect Proteins - chemistry
Insect Proteins - genetics
Insect Proteins - metabolism
Insecticide Resistance
Insecticides - pharmacology
Isoenzymes - chemistry
Isoenzymes - genetics
Isoenzymes - metabolism
Lepidoptera
Molecular cloning
Molecular Sequence Data
Moths - classification
Moths - drug effects
Moths - enzymology
Moths - genetics
Phylogeny
Quantitative RT-PCR
RNA Interference
RNAi
Sequence Alignment
Total aminopeptidase N activity
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Summary: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.
ISSN:0965-1748
1879-0240
DOI:10.1016/j.ibmb.2010.05.006