| Bt crops harboring insecticidal genes of Bacillus thuringiensis (Bt) has developed a new strategy for pest control. In our country, planting transgenic cotton (Bt-Cry1Ac) has evidently reduced the costs and increased yields of cotton by controlling the damage of cotton bollworm. But as the planting scope became wider and wider, the risk of resistance development has been increased, and outmost threatened the long-term effectiveness of Bt cotton. To prevent and control the pest resistance, we employed some methods, such as: multiple genes strategy, new toxin strategy and shelters strategy. As one of the multiple genes strategy, Cry2Ab toxin is the new Cry toxin used in transgenic cotton (expressing Cry2Ab and Cry1Ac) to against the resistance development. It was proved that there was no cross-resistance between Cry2Ab and Cry1Ac against the cotton bollworm. Hence, the pest resistence to Cry1Ac could be avoided by using this new Bt cotton. Until now, it still remains unclear for receptors bound to Cry2Ab toxin and toxin-binding sites in the midgut of Helicoverpa armigera. It is essential for toxicity of toxins to bind with its specific receptors in the larval midgut of the insect. Finding the receptors in the midgut and its binding sites became very crucial to understand the molecular mechanisms of Cry2Ab toxin, and develop the management of pest insistence. In our research, the epitope of Cry2Ab toxin have been mapped by phage display, in vitro, and demonstrated their biological functions of the selected peptides.After activation and purification of Cry2Ab toxin, we carried out four rounds panning and then sequenced the insert DNA fragment of eluted phage. After that, we got two peptide sequences: NQFPSGAVYEHS and ATNEFPNPLHAP. ELISA analysis showed that these two peptides could specifically bind to activated Cry2Ab toxin and it has been detected that they could competitively inhibit the binding between Cry2Ab and BBMV by Western-bloting. After incubating the peptides with Cry2Ab toxin respectively, bioassay analysis showed that the two peptides can reduce the toxicity to target insect larvae (H. armigera). By Ligand-blot, it has been found that Cry2Ab toxin could identify two proteins on BBMV and the molecular weight of their binding proteins are about 42 kDa and 100 kDa, respectively.The results of these experiments which are showed above demonstrated that the selected peptides have biological functions, moreover have very close similarity with the receptors in the midgut of insect in amino acid sequences and dimensional structure. Meanwhile, the related factors to the two binding proteins found by Ligand-blot could regulate Cry2Ab action. Using phage display, our research not only provide us a new efftective thecnique to study the mode of Bt action, but also helps us to know the differences among different Bt toxins, and further more provides important theoretical foundation for the mechanism of Bt resistance. |
