Doctor of Philosophy / Department of Entomology / Larry L. Buschman / Kun Yan Zhu / European corn borer, Ostrinia nubilalis, is one of the most destructive insect pests of corn in the Midwest corn belt of the United States. The crystal protein toxin (Cry1Ab) expressed by the bacterium, Bacillus thuriginesis (Bt), specifically targets O. nubilalis gut and functions as “stomach poison”. Transgenic corn expressing Cry1Ab can effectively control O. nubilalis larval infestation. However, O. nubilalis has the potential to develop resistance to Bt toxins which prompts concerns that transgenic corn will lose its control efficacy. Previous studies found that O. nubilalis gut serine proteases and membrane proteins were involved in Bt toxicity and resistance. Therefore, this study was to identify and characterize gut transcripts potentially involved in Bt toxicity and resistance, and to compare their transcriptional responses to the ingestion of Cry1Ab protoxin and transgenic corn leaves expressing Cry1Ab toxin. We identified and characterized 34 cDNAs encoding putative trypsins, chymotrypsins, and trypsin- and chymotrypsin-like protease homologs from O. nubilalis gut-specific expressed sequence tags (ESTs). Blast and phylogenetic analysis of their deduced amino acid sequences indicated that 15 were putative trypsins belonging to Try-G2 and Try-G3 groups (none of them was grouped in Try-G1), another 15 were putative chymotrypsins in one large group (CTP-G1), and the remaining four were serine protease homologs in Try-G4 and CTP-G2 groups, respectively. The existence of diverse trypsins, chymotrypsins and serine protease homologs in O. nubilalis could be an adaptation to different food sources and also a defense mechanism against plant-specific protease inhibitors and Cry toxins from transgenic corn. The expressions of four putative trypsins (OnTry4, OnTry5, OnTry6 and OnTry14) were up-regulated in O. nubilalis larvae after the ingestion of Cry1Ab protoxin. The differential expressions of these protease transcripts may implicate a link to Cry1Ab intoxication. To better understand the basic physiology of insect gut and Bt toxin interactions, we developed a high-resolution 8×15K cDNA microarray chips based on the larval gut specific ESTs. Each microarray contains 12,797 probes representing 2,895 unique larval gut transcripts. The expressions of 174 transcripts were differentially regulated at least 2-fold (P-value ≤0.05) after the larvae fed Cry1Ab protoxin for 6 hours. Among them, 13 transcripts, putatively encoding eight serine protease, three aminopeptidase, one alkaline phosphatase, and one cadherin-like protein, were identified and further validated their expression ratios by quantitative PCR (qPCR). Three trypsin transcripts were up-regulated by more than 5-fold in larvae fed Cry1Ab protoxin. Sequence analysis suggests that they may have role in protoxin activation and toxin degradation. The transcriptional responses of laboratory-selected Cry1Ab resistant (R) and susceptible (S) strains of O. nubilalis to the ingestion of transgenic corn (MON811) leaves expressing Cry1Ab toxin were also examined. Even though R-strain larvae showed 200-fold resistance to Cry1Ab protoxin as compared with S strain, the larvae from both strains eventually died after fed transgenic corn leaves. However, the survival time of R-strain larvae was significantly different from that of S-strain larvae. The median lethal time (LT50) for the early third-instar larvae of R- and S-strains were 5.4 and 3.6 days, respectively. Furthermore, we identified 398 and 264 transcripts from the larvae of the S and R strains, respectively, with a significantly increased or decreased expression (expression ratio cut off ≥2.0 fold with p-value ≤0.05) as compared with those in the larvae fed on non-transgenic corn leaves. The number of transcripts and their expression ratios of S-strain larvae are larger than these of R-strain larvae. These significantly differentially expressed transcripts may play important roles in influencing Cry1Ab toxicity from toxin degradation, toxin binding, to intracellular defense. Seventeen transcripts including serine protease and aminopeptdiase in S strain and nine in R strain were further analyzed by qPCR to validate their expression ratios. This study not only revealed information about the difference in the transcriptional responses of these genes to Cry1Ab between Bt-resistant and susceptible strains of O. nubilalis, but also provided new insights into potential interactions of the protoxin, toxin from transgenic corn with important proteins in the gut of O. nubilalis larvae.
| Identifer | oai:union.ndltd.org:KSU/oai:krex.k-state.edu:2097/12131 |
| Date | January 1900 |
| Creators | Yao, Jianxiu |
| Publisher | Kansas State University |
| Source Sets | K-State Research Exchange |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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