Isolation And Purification Of BBMV From Susceptible And Resistant Cotton Bollworm And Binding Models Between CrylA And BBMVs

The Bt transgenic cotton(Bt cotton) is high effective tool for control of cotton bollworm Helicoverpa armigera (Hubner), an important agricultural insect pest in the world. As the growing area of Bt cotton increases rapidly, the resistant development of H. armigera to Bt has become a critical problem that can threaten deployment of Bt cotton. In this article, the brush border membrane vesicles (BBMV) from Bt resistance strain of H. armigera systemically selected for more than 52 generations in the laboratory, were isolated and purified. We put an emphasis for the study on the binding kinetics between Cryl A toxin and BBMVs of both in resistant and susceptible strains, and binding models between Cryl A and BBMVs. The results were as follows:The BBMV was isolated from midgut of H. armigera by means of differential centrifugation method. The results of SDS-PAGE analysis showed that protein bands in susceptible strain were much more than those in resistant strain. The BBMV in susceptible strain has a protein band about 110kDa, which was absent in resistant cotton bollworm. In addition, a protein band, about 175 kDa, was purified by using affinity chromatography, this protein can bind with Cryl Ac.Three Cry1A toxins, namely Cry1Aa, Cry1Ab and Cryl Ac, labeled with Na125I, were bound to the BBMVs obtained from Cryl Ac resistant or susceptible strains of H armigera. The results of homologous competition test showed that there was no significant difference in binding affinity, although the concentration of binding site dramatically decreased in resistant strain. The heterologous competition test showed that there were three Cryl Ac binding sites in the susceptible strain. Among them, site 1 could bind with all the three toxins, site 2 bound with both CrylAb and Cry1Ac, and site 3 just for Cry1Ac. While in the Cry1Ac resistant strain, site 1 could bind with all these toxins, but the binding capability with Cry1Ab was decreased drastically. Especially, site 2 had lost the binding capability with Cry1Ac and only slightly bound with CrylAb. On the other hand, Western blot verified that there were three proteins (110, 120 and 175kDa) could bind with CrylAc in susceptible strain, but only twoproteins (120 and 175kDa) bound with Cry 1 Ac in resistant one. According to these results, two binding models between Cry1A and BBMV from two strains were constructed respectively. It is suggested that the absence of one binding site may be responsible for the evolution of H. armigera Cry 1 Ac resistance.