Studies On The Binding Sites Of Five Cry1Toxin In Midgut And Relationship Between Hsp70and Bt Resistance Of Ostrinia Furnacalis

Asian corn borer (ACB), Ostrnia furnacalis (Guenee), is the most important insect pest on maize in China. As a new component of ACB IPM, Bt maize offers potential season long control for this insect pest. However, evolution of resistance by ACB to Bt maize will threat to use this high tech. It is necessary to understand the molecular mechanism for evolution of resistance to Bt toxins as well as the interaction of Bt toxins with larval midgut binding sites in ACB, which will play an important roll in developing a science based effective IRM strategy to ensure the sustainable use of Bt maize.The binding of biotinylated Cry1Ab, Cry1Ac, Cry1Ah, CrylF, and Crylle to brush border membrane vesicles (BBMVs) of O. furnacalis was analyzed in competition binding assays with each other of unlabelled toxins as homologous or heterologous competitors, respectively. Homologous competition assays showed that those toxins bind with high affinity to binding sites on BBMVs of O. furnacalis. Heterologous competition assays demonstrated that Cry1Ab, Cry1Ac, and Cry1Ah competed for common binding sites. Cry1F competed partially with Cry1Ab, Cry1Ac and Cry1Ah. However, Crylle did not compete to any of the other four toxins binding sites. These results indicate that Cry1Ab, Cry1Ac, and Cry1Ah binding sites are shared each other on the BBMVs of O. furnacalis, and are partially shared by Cry1F, but Cry1Ie binding sites are not recognized by Cry1Ab, Cry1Ac, Cry1Ah, and Cry1F.By designing the degenerate primers and using RT-PCR and RACE methods, a cDNA fragment Ofhsp70(GenBank assess No.:HQ434763) encoding full-length heat shock70kDa protein was cloned from larvae of the ACB-BtS. It was2169bp encoding653amino acid polypeptide, molecular weight was estimated as71.36kDa. The amino acid sequence was involving three hsp70family signatures and four typical function sites. The homologous was96%with European corn borer, O.nubilalis(Hubner).Two cDNA fragment Ofhsp70were also cloned from ACB-AbR (GenBank assess No.:JF709083) and ACB-AcR strains (GenBank assess No.:JF708084). In comparison with Ofhsp cDNA sequence in ACB-BtS strain,15and25necleotide mutations were observed in ACB-AbR and ACB-AcR strains, respectively. However, they were identy in putative protein sequences. These mutations may not associate with the resistance of ACB to CrylAb and Cry1Ac.Quantitative RT-PCT analyses showed that, in the first instar larvae midgut, expression of Ofhsp70mRNA was2.9,1.3, and1.6-fold higher in ACB-AbR and the offspring of the reciprocal crosses between ACB-AbR and ACB-AcR than in ACB-BtS. This suggests that the reduced susceptibility to Cry1Ab toxins in ACB-AbR strain may due to the increased ability of binding with Cry1Ab.