Molecular Mechanisms of Bacillus thuringiensis Resistance in the Sugarcane Borer

• Main Author: Yang, Yunlong
• Other Authors: Husseneder, Claudia R
• Format: Others
• Language: en
• Published: LSU 2011
• Subjects: Entomology
• Online Access: http://etd.lsu.edu/docs/available/etd-04212011-160643/

The sugarcane borer, Diatraea saccharalis, is a major target pest of transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the U.S. mid-southern region. A Cry1Ab-resistant (Cry1Ab-RR) strain of D. saccharalis has been developed from a single two-parent family-line. To examine the molecular mechanisms of the Cry1Ab resistance in this insect strain, cDNAs of five types of potential candidate genes related to Bt resistance were sequenced using reverse transcriptase polymerase chain reaction (RT-PCR) and 5 rapid amplification of cDNA end (5 RACE). The Bt resistance candidate genes examined included three trypsins (DsTRYs), three chymotrypsins (DsCHYs), three aminopeptidases N (DsAPNs), one cadherin (DsCAD1), and three alkaline phosphatases (DsALPs). cDNA sequence of each gene and its expression levels were compared between a Cry1Ab-susceptible strain (Cry1Ab-SS) and the Cry1Ab-RR at different larval growth stages. The cDNA sequences of these genes were identical between Cry1Ab-SS and -RR strains. Gene expression levels of the trypsins, chymotrypsins, and alkaline phosphatases were similar between the two strains. There were also no significant differences in total enzymatic activity of trypsins, chymotrypsins, and alkaline phosphatases between Cry1Ab-SS and -RR. However, the gene expression levels of the three DsAPNs and the DsCAD1 in Cry1Ab-RR were significantly lower than those of the Cry1Ab-SS. RNA interference (RNAi) was employed to knock-down the three DsAPNs and the DsCAD1 in the Cry1Ab-SS strain by oral droplet feeding to neonates. Down-regulations of the expressions of these four genes by RNAi were correlated with the decrease in susceptibility to Cry1Ab toxin. Silencing each of the three DsAPNs in D. saccharalis in vivo by RNAi resulted in a decrease of total APN activity. In addition, the total specific APN activities from Cry1Ab-RR larvae were significantly lower than those of the Cry1Ab-SS strain. These results suggest that reduction in expression of the three DsAPNs and DsCAD1 is functionally associated with the Cry1Ab resistance in D. saccharalis.