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Title: Expressed sequence tags from larval gut of the european corn borer (Ostrinia nubilalis): exploring candidate genes potenially involved in Bacillus thuringiensis toxicity and resistance

item Zhu, Yu Cheng
item Chen, Ming-Shun
item CRESPO, ANDRE - Kansas State University
item SIEGFRIED, BLAIR - University Of Nebraska
item MUTHUKRISHANAN, SUBBARATNAM - Kansas State University
item ZHU, KUN YAN - Kansas State University

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/29/2009
Publication Date: 6/29/2009
Citation: Khajuria, C., Zhu, Y., Chen, M., Buschman, L.L., Higgins, R.A., Yao, J., Crespo, A.L., Siegfried, B.D., Muthukrishanan, S., Zhu, K. 2009. Expressed sequence tags from larval gut of the european corn borer (Ostrinia nubilalis): exploring candidate genes potenially involved in Bacillus thuringiensis toxicity and resistance. Biomed Central (BMC) Genomics. 10:286.

Interpretive Summary: The main target for Bt toxins is the insect midgut, where Bt protoxins are activated by gut proteases to produce activated Bt toxins. The activated toxins then bind to specific receptor(s) to confer the Bt toxicity. This means that insect resistance to Bt toxins could be conferred by protease-mediated and receptor-mediated mechanisms. Because Bt toxin and insect gut interactions are determined by many gene products in the insect gut, including many proteins/enzymes involved in Bt protoxin activation, toxin binding to receptors and toxin degradation, any change in these systems has the potential to affect a particular Bt’s specificity and efficacy, and could lead to Bt resistance in insects. Our goals are to develop a gut-specific EST library from ECB larvae and identify candidate genes that are potentially involved in insect-Bt interactions and Bt resistance. We report the analysis and annotations of 15,000 sequences derived from the gut of ECB larvae. We discuss the putative identities of the sequences, their potential biological and molecular functions, and present comparative analyses of our sequences with sequences from other insects. Furthermore, we identified 55 candidate genes that could be involved in Bt toxicity and resistance. This work provides the opportunity for developing an ECB gut-specific microarray that can be used to study insect-Bt interactions and genetic basis of Bt resistance in ECB. In addition, the information generated from this study may serve as a model for studying Bt resistance mechanisms and for developing bio-pesticides for all closely related corn stalk borers.

Technical Abstract: Background: Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt) toxin and for discovering new targets for novel toxins for use in pest management. This study analyzed the ESTs generated from the larval gut of the European corn borer (ECB, Ostrinia nubilalis), one of the most destructive pests of corn in North America and the western world. Our goals were to develop a gut-specific EST library from ECB larvae and to identify candidate genes potentially involved in insect-Bt toxicity and resistance. Results: Two cDNA libraries were constructed from the guts of the fifth-instar larvae of ECB. A total of 15,000 ESTs were derived from these libraries. The ESTs represented 2,895 unique sequences, including 1,738 singletons and 1,157 contigs. Among the unique sequences, 62.7% encoded putative proteins that shared significant sequence similarities (E-value =10-3) with the sequences available in GenBank. Furthermore, we identified 55 genes that are potentially involved in Bt toxicity and resistance. These genes encode 18 trypsin-like proteases, 21 chymotrypsin-like proteases, 13 aminopeptidases, 1 cadherin-like protein, and 2 alkaline phosphatases. Certain trypsin-like and chymotrypsin-like proteases have previously been found to activate or degrade Bt protoxins and toxins, whereas several aminopeptidases, cadherin-like proteins and alkaline phosphatases have been demonstrated to serve as Bt receptor proteins in other insect species. Conclusions: A relatively large EST database containing 15,000 sequences from ECB larval gut was developed. The putative functions of the expressed gut genes were analyzed according to the sequence similarity to known genes. Fifty five candidate genes that could be potentially involved in Bt toxicity and resistance were identified. Our work provides a foundation for future research to develop an ECB gut-specific DNA microarray for the analysis of the global changes of gene expression in response to Bt protoxins/toxins as well as the genetic difference between Bt- resistant and susceptible strains of ECB.