|Zhu, Kun yan|
Submitted to: PLoS One
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/1/2012
Publication Date: 8/31/2012
Citation: Yao, J., Buschman, L.L., Oppert, B.S., Khajuria, C., Zhu, K. 2012. Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae. PLoS One. 7(8):e44090. http://dx.doi.org/10.1371/journal.pone.0044090. Interpretive Summary: Insect proteases affect the efficacy of insecticidal microbial toxins, such as those from the bacterium Bacillus thuringiensis (Bt). Thus, understanding the expression of protease genes in a target pest is important to more effectively design and deploy Bt toxins. Larvae of the European corn borer, a major target of Bt transgenic corn, express a suite of proteases at various locations in the gut. Our data suggests that protease genes can be grouped by genetic relatedness and expression patterns in the gut. We found that some protease genes were increased in expression when larvae were exposed to toxin. These data represent the most comprehensive study to date on larval gut proteases and the effect of Bt toxin on gene expression in the European corn borer. The data can be used to improve transgenic constructs for efficacy and durability in the field.
Technical Abstract: Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and their homologs from the European corn borer (Ostrinia nubilalis) larval gut. Our analyses of the cDNA-deduced amino acid sequences indicated that 12 were putative trypsins, 12 were putative chymotrypsins, and the remaining 10 were trypsin and chymotrypsin homologs that lack one or more conserved residues of typical trypsins and chymotrypsins. RT-PCR analysis indicated that all genes were highly expressed in gut tissues, but one group of phylogenetically-related trypsin genes, OnTry-G2, was highly expressed in larval foregut and midgut, whereas another group, OnTry-G3, was highly expressed in the midgut and hindgut. Real-time quantitative PCR analysis indicated that several trypsin genes (OnTry5 and OnTry6) were significantly up-regulated in the gut of third-instar larvae after feeding on Cry1Ab protoxin from 2 to 24 h, while one trypsin (OnTry2) was down-regulated at all timepoints. Four chymotrypsin and chymotrypsin homolog genes (OnCTP2, OnCTP5, OnCTP12 and OnCTP13) were up-regulated at least 2-fold in the gut of the larvae after feeding on Cry1Ab protoxin for 24 h. Our data represent the first in-depth study of gut transcripts encoding expanded families of protease genes in O. nubilalis larvae and demonstrate differential expression of protease genes that may be related to Cry1Ab intoxication and/or resistance.