Studies On Cross-resistance Of Ostrinia Furnacalis To Other Bt Toxins And Bt Binding Sites In The Midgut

The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is the most important insect pest of maize in China. Insect resistant biotech Bt maize, with efficient, user-friendly advantages has provide a new way for the maize pest control. However the evolution of resistance in target pest is a major concern to threaten the use of Bt maize. Although exotic Cry1Ab- and Cry1F-expressing maize derived from genetic transformation events MON810, Bt11, and TC1507 as well as domestic Cry1Ah-, Cry1A-, and Cry1Ac-Cry1Ie-expressing maize were evaluated to suppress O. furnacalis in field trials, the patten of cross-resistance among these toxin proteins has not been investigated in the Asian corn borer, which would provide valuable information for considering the priority event of Bt maize adaptation in China where Cry1Ac expressing cotton, one of the important hosts of O. furnacalis, is a widespread Bt crop. In this study, the cross-resistant petten of Cry1Ac-selected Asian corn borer to other Bt toxins were studied. In addition, the binding patten among three different Bt proteins to Asian corn borer midgut BBMV were tested. The cadherin-like gene, potential Cry protein receptor gene in the BBMV of Asian corn borer was expressed by parts in E.coli. Then, binding abilities of these expressed peptides were assayed with Cry1Ab protein. The results are as follows:A resistant strain of Asian corn borer, ACB-AcR, selected with Cry1Ac protein incorporated into artificial diet developed significant levels of resistance (14-fold) after 27 generations. Additionally, ACB-AcR maintained on a constant concentration (8μg/g) was developed 48.9-fold resistance at generation 82. LC50s of Cry1Ab, Cry1Ah, and Cry1Ie were measured to ACB-AcR and ACB-BtS stains. The susceptibility of ACB-BtS strain to four Cry proteins ranked as Cry1Ah> Cry1Ab>Cry1Ac>Cry1Ie. Significantly, the highest cross-resistance was observed to Cry1Ah with resistance ration up to 14.9-fold. Low levels (6.3-fold) of cross-resistance were detected with Cry1Ab. In contrast, Cry1Ie susceptibility was unaffected by selection with Cry1Ac (0.9-fold). Dose-growth inhibition curves showed that for Cry proteins significantly inhibited the growth of the Asian corn borer larvae. Growth inhibition rate got 70%-95% at the concentration of Cry1Ac,Cry1Ab and Cry1Ah less than 0.5μg/g.The Asian corn borer BBMV was separated from the midgut of ACB-BtS fifth instar larvae. Binding studies were conducted with the biotinylated Cry1Ab, Cry1Ac, Cry1Ie to the BBMV. Results indicated that three Bt proteins could bind with the BBMV. Heterologous competition results showed that Cry1Ab, Cry1Ac, and Cry1Ie had same bingding sites on the BBMV. Cry1Ab performed the highes bingding ability, Cry1Ac was the second, and Cry1Ie had the less ability than Cry1Ab and Cry1Ac. These results could explain the why cry1Ab selected strain ACB-AcR was cross-resistant to Cry1Ab toxin. However, it could not explain why it did not cross-resistant to Cry1Ie toxin.Four different legth of cadherin-like gene was expressed in E. coli. The molecular weight of the expressed peptides is 25.7, 32.8, 40.3 and 83.9kDa. Ligand blot showed that 40.3 kDa peptides could bind with Cry1Ab. This toxin-binding region was located 1003-1317 peptide chain of cadherin-like protein, as 3007-3951 bases in the cadherin gene open reading frame sequence. Through this study, cross-resistance patterns of Cry1Ac-selected Ostrinia furnacalis to other Bt toxins were preliminary analyzed. The toxicity of Cry1Ab, Cry1Ac and Cry1Ie to O. furnacalis and binding capacity between three Bt toxins and BBMV was revealed. At the same time cadherin-like protein was proved to be the receptor of Cry1Ab in the midgut of asian corn borer. This study laid the foundation for the research of Bt resistance mechanism, and provides some reference basis for the new Bt maize.