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

Abstract

Background: Lepidoptera represents more than 160,000 insect species which include some of the most devastating pests of crops, forests, and stored products. However, the genomic information on lepidopteran insects is very limited. Only a few studies have focused on developing expressed sequence tag (EST) libraries from the guts of lepidopteran larvae. 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) toxins, 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 establish an ECB larval gut-specific EST database as a genomic resource for future research and to explore candidate genes potentially involved in insect-Bt interactions and Bt resistance in ECB.

Results: We constructed two cDNA libraries from the guts of the fifth-instar larvae of ECB and sequenced a total of 15,000 ESTs from these libraries. A total of 12,519 ESTs (83.4%) appeared to be high quality with an average length of 656 bp. These 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 <or= 10-3)with the sequences available in GenBank. Our EST analysis revealed 52 candidate genes that potentially have roles in Bt toxicity and resistance. These genes encode 18 trypsin-like proteases, 18 chymotrypsin-like proteases, 13 aminopeptidases, 2 alkaline phosphatases and 1 cadherin-like protein. Comparisons of expression profiles of 41 selected candidate genes between Cry1Ab-susceptible and resistant strains of ECB by RT-PCR showed apparently decreased expressions in 2 trypsin-like and 2 chymotrypsin-like protease genes, and 1 aminopeptidase genes in the resistant strain as compared with the susceptible strain. In contrast, the expression of 3 trypsin- like and 3 chymotrypsin-like protease genes, 2 aminopeptidase genes, and 2 alkaline phosphatase genes were increased in the resistant strain. Such differential expressions of the candidate genes may suggest their involvement in Cry1Ab resistance. Indeed, 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.

Conclusion: We developed a relatively large EST database consisting of 12,519 high-quality sequences from a total of 15,000 cDNAs from the larval gut of ECB. To our knowledge, this database represents the largest gut-specific EST database from a lepidopteran pest. Our work provides a foundation for future research to develop an ECB gut-specific DNA microarray which can be used to analyze the global changes of gene expression in response to Bt protoxins/toxins and the genetic difference(s) between Bt- resistant and susceptible strains. Furthermore, we identified 52 candidate genes that may potentially be involved in Bt toxicity and resistance. Differential expressions of 15 out of the 41 selected candidate genes examined by RT-PCR, including 5 genes with apparently decreased expression and 10 with increased expression in Cry1Ab-resistant strain, may help us conclusively identify the candidate genes involved in Bt resistance and provide us with new insights into the mechanism of Cry1Ab resistance in ECB.

Figure 1

(A) Distribution of ECB gut-specific ESTs in each contig. (B) Distribution of the 2,895 ECB gut-specific contigs and singletons with or without match in NCBI database using BLASTx. Sequences were defined as identical or similar cDNA sequences when they had E-values ≤ 10^-3.

Figure 2

Open reading frame (ORF), secretory protein, and BLASTx results. (A) The proportion of the unique ESTs from ECB gut cDNA library with or without 450 bp of ORF region along with their matches in BLASTx using NCBI database. (B) Proportion of the unique ESTs with or without signal peptide along with their match in BLASTx using NCBI database.

Figure 3

Similarity of ECB gut-specific ESTs with other insects. The first hit sequence (highest score) was used to determine the most similar organism.

Figure 4

(A) Percentage of the 2,895 ECB gut-specific unique ESTs which shared matches with O. nubilalis and B. mori sequences with E-value ranging from E-150 to E-5 using BLASTN. (B) Comparative analyses of the 2,895 ECB gut-specific unique ESTs to B. mori sequences and other O. nubilalis sequences available in NCBI database using BLASTN.

Figure 5

Distribution of the ECB gut-specific unique ESTs annotated at GO level 2. The Y-axis shows the percentage of the sequences. The x-axis shows 3 areas of annotation and with each area the sequences were further divided into subgroups at GO level 2.

Figure 6

Expression profiles of 41 candidate genes relevant to Bt toxicity and resistance, which include 15 trypsin-like serine protease (TP), 13 chymotrypsin-like serine protease (CP), 2 alkaline phosphatase (AK), 10 aminopeptidase (AP), and 1 cadherin-like protein (CA) genes in the midgut of one-day-old fifth-instar larvae in Cry1Ab-susceptible (S) and resistant (R) strains of ECB as determined by RT-PCR. The arrows next to the gel pictures indicate the apparently decreased (↓) or increased (↑) expression of a particular gene in Cry1Ab-resistant strain as compared with the susceptible strain of ECB. The ribosomal S3 (RPS3) gene was used as a reference gene. At least two biological replications were used for each primer pair. The sequences of PCR primers used in this study were given in Appendix A.