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United States Department of Agriculture

Agricultural Research Service

Research Project: Insecticide Resistance Management and New Control Strategies for Pests of Corn, Cotton, Sorghum, Soybean, and Sweet Potato

Location: Southern Insect Management Research Unit

Title: Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and -resistant strains of sugarcane borer Diatraea saccharalis

Authors
item Yang, Yunlong -
item Zhu, Yu Cheng
item Ottea, James -
item Husseneder, Claudia -
item Leonard, Rogers -
item Abel, Craig
item Luttrell, Randall
item Huang, Fangeneng -

Submitted to: Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 16, 2012
Publication Date: October 10, 2012
Citation: Yang, Y., Zhu, Y., Ottea, J., Husseneder, C., Leonard, R., Abel, C.A., Luttrell, R.G., Huang, F. 2012. Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and -resistant strains of sugarcane borer Diatraea saccharalis. Insect Science. 00:1-12.

Interpretive Summary: Sugarcane borer is a major corn borer pest and a target of transgenic Bt corn in South America and the U.S. mid-southern region. With a major role in dietary protein digestion, midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is also responsible for Bt resistance development in some insect species. To clone and characterize midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in the sugarcane borer is associated with changes in midgut proteinases, enzymatic activities of midgut trypsins and chymotrypsins, cDNA sequences and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Bt-susceptible and -resistant strains. Results showed that midgut trypsins and chymotrypsins of the sugarcane borer efficiently hydrolyzed corresponding substrates, with higher chymotrypsin activity compared to the trypsin activity. No significant differences in both trypsin and chymotrypsin activities were observed between Bt-susceptible and -resistant strains. Full-length cDNAs encoding the three trypsin- and three chymotrypsin-like proteinases were identical in both strains. All deduced proteins have typical secretion signal peptide and activation peptide, conserved amino acids for the catalytic active sites, three pairs of cysteine residues for disulfide bridge configurations, and conserved substrate specificity determination residues for trypsins and chymotrypsins. Transcriptional levels of the three trypsin and three chymotrypsin genes were similar between Bt-susceptible and -resistant strains. Results of this study showed that three trypsin cDNAs and three chymotrypsin cDNAs code for active forms of midgut serine proteinases with all functional motifs. The data of similar cDNA sequences and gene expression levels between Bt-susceptible and –resistant strains suggest that the development of Cry1Ab resistance in the sugarcane borer was not associated with these trypsins and chymotrypsins.

Technical Abstract: Sugarcane borer, Diatraea saccharalis, is a major corn borer pest and a target of transgenic Bacillus thuringiensis (Bt) corn in South America and the U.S. mid-southern region. With a major role in dietary protein digestion, midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is also responsible for Bt resistance development in some insect species. To clone and characterize midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, enzymatic activities of midgut trypsins and chymotrypsins, cDNA sequences and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab-RR) strains. Results showed that midgut trypsins and chymotrypsins of D. saccharalis efficiently hydrolyzed corresponding substrates in vitro, with higher chymotrypsin activity compared to the trypsin activity. No significant differences in both trypsin and chymotrypsin activities were observed between Cry1Ab-SS and -RR strains. Full-length cDNAs encoding the three trypsin- and three chymotrypsin-like proteinases were identical in Cry1Ab-SS and -RR larvae. All deduced proteins have typical secretion signal peptide and activation peptide, conserved residues (His, Asp, and Ser) for the catalytic triad, three pairs of cysteine residues for disulfide bridge configurations, and conserved substrate specificity determination residues for trypsins and chymotrypsins. Transcriptional levels of the three trypsin and three chymotrypsin genes were similar between Cry1Ab-SS and -RR strains. Results of this study showed that three trypsin cDNAs and three chymotrypsin cDNAs code for active forms of midgut serine proteinases with all functional motifs. The data of similar cDNA sequences and gene expression levels between Cry1Ab-SS and Cry1Ab-RR suggest that the development of Cry1Ab resistance in D. saccharalis was not associated with these trypsins and chymotrypsins.

Last Modified: 10/31/2014
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