Comparative study on enzyme activities and cDNA sequences of Bacillus thuringiensis-resistance related genes between Cry1Ab -susceptible and -resistant strains of Diatraea saccharalis

A Bacillus thuringiensis (Bt)-resistant strain (Bt-RR) of the sugarcane borer, Diatraea saccharalis (F.), capable of completing larval development on commercial Bt corn has been selected using a novel F₂ screening technique. The objective of this study is to determine the physiological and molecular mechanisms of Bt resistance in D. saccharalis. Midgut enzymatic activities of trypsins, chymotrypsins, N-aminopeptidases, and alkaline phosphatases from Bt-susceptible (Bt-SS) and Bt-RR larvae were assayed in vitro using Na-Benzoyl-DL-arginine-p-nitroanilide, N-succinyl-Ala-Ala-Pro-phenylalanine-p-nitroanilide, leucine-p-nitroanilide, and p-nitrophenyl phosphate disodium as the substrates, respectively. Trypsin activity of late 4th instar larvae of the Bt-SS strain is significantly greater than that of Bt-RR strain, while the chymotrypsin activity of the Bt-SS strain is lower than that of the Bt-RR strain. Full lengths of three aminopeptidase (AP-1, AP-2, AP-3), three alkaline phosphatase (AL-1, AL-2, AL-3), and one cadherin (CAD-1) cDNAs were cloned and sequenced. Expression levels of these genes were examined for the 3rd and 5th instars of the two strains using realtime PCR. 3′ mRNA level for the AP-1 gene from Bt-RR strain is lower in both 3rd and 5th instars, while mRNA level for AL-3 gene from Bt-RR strain is about 7 times higher than that from Bt-SS strain in 5th instar. mRNA level for CAD-1 gene from Bt-RR strain is lower in both 3rd and 5th instars. The Bt resistance in this D. saccharalis strain may be associated with the changes in proteinase activity and/or expression of the Bt binding genes in the midgut.