Proteolytic processing of Bacillus thuringiensis(Bt)crystal toxins by insect midgut proteases play an essential role in their insecticidal toxicities against target insects.In present study,proteolysis of Bt crystal toxin Cry2Ab by Plutella xylostella L.midgut proteases(PxMJ)was evaluated.Both trypsin and chymotrypsin were identified involving in the proteolytic activation of Cry2Ab,and cleaving Cry2Ab at Arg139 and Leu144,respectively.Three Cry2Ab mutants(R139A,L144A and R139AL144A)were constructed by replacing residues Arg139,Leu144 and Arg139-Leu144 with alanine.Proteolysis assays revealed that mutants R139A and L144A but not R139A-L144A could be cleaved into 50 kDa activated-toxins by PxMJ.Bioassays showed that mutants R139A and L144A were high toxic against P.xylostella larvae,while mutant R139AL144A was almost non-insecticidal.Those results demonstrated that proteolysis by PxMJ was associated with Cry2Abs toxicity against P.xylostella.It also revealed that either trypsin or chymotrypsin was enough to activate Cry2Ab protoxin.This characteristic was regarded as a belt-and-braces approach and might contribute to control of resistances development in target insects.Our studies characterized the proteolytic processing of Cry2Ab and provided a new insight into the activation of this Bt toxin.