Analyses of sequences, gene expressions, and RNAi of three Cry1Ab-resistance related aminopeptidase genes in the sugarcane borer, Diatraea saccharalis

A Cry1Ab-resistant strain of the sugarcane borer, Diatraea saccharalis (F.), capable of completing larval development on Bacillus thuringiensis (Bt) corn was developed using an F2 screening method. Midgut aminopeptidases N (APN) are considered as receptors for Bt toxins in many insects. The objective of this study is to determine if Cry1Ab resistance in D. saccharalis is associated with changes in gene structure/expression of APNs in the midgut. To achieve this goal, full length cDNAs of three APN genes were cloned and sequenced from both Cry1Ab-resistant and -susceptible strains using RT-PCR and 5’ RACE. Real-time PCR was then performed using total RNA from both strains to examine expressions of the three APN genes. Finally, RNA interference-mediated gene silencing (RNAi) was employed through dsRNA droplet feeding to assess functional linkage of the three APN genes with Cry1Ab resistance in D. saccharalis. cDNA sequences of the three APN genes were identical between the Cry1Ab-resistant and -susceptible strains. However, the Cry1Ab-resistant strain exhibited a significant reduction in gene expressions and total APN activity than the Cry1Ab-susceptible strain for all three APNs. Similarly, expression level of all three APNs and total APN activity were reduced via RNAi compared with non-RNAi treatment. Accordingly, the mortalities of neonates treated using RNAi from three APNs all decreased significantly compared to non-RNAi treated ones. The results suggest that reductions in expressions of APNs may be associated with the Cry1Ab resistance in D. saccharalis.