The Investigation Of Resistance Risk On Bt-Cry1Ac-resistant Helicoverpa Armigera (Lepidoptera: Noctuidae) To Cry2Ab

The wide spread of the first generation transgenic cotton which expresses Cry1 Ac has played an important role in the integrated pest management of cotton. However, there is a risk for resistance evolution of the cotton bollworm (Helicoverpa armigera). Therefore, a strategy of pyramiding different Bt genes in cotton is much valuable for managing the resistance risk from the target pest. One of requirements for the dual (pyramided) gene strategy to work is that the two toxins must have different modes of action, which means that there must be a low probability of cross-resistance between the two toxins. Here, we reports the investigation on the cross-resistance of CrylAc-resistant H. armigera strains (LFR₁₀ and BtR) to Cry2Ab by bioassay and other methods for understanding the molecular basis of resistance to Cry1 Ac and cross-resistance to Cry2Ab, the binding kinetics between the BBMV from Cry1 Ac-susceptible and -resistant strains of H. armigera and Cry toxins (Cry1A and Cry2Ab). In addition, the interaction analysis between the esterases from Cry1 Ac-susceptible and -resistant strains of H. armigera were conducted. The results were as follows:1,Bioassays between Cry toxins (Cry1Aa, Cry1 Ab, Cry1 Ac and Cry2Ab) and larvae of a Cry1 Acsusceptible (96S) and two -resistant strains (BtR and LFR₁₀) were conducted for investigating cross-resistance of Cry1 Ac-resistant strains to Cry2Ab in H. armigera. The resistance ratios (RR) of the BtR and LFR₁₀ strains to Cry1Ac and Cry2Ab were 2971.3- and 1.1-fold, and 253.0- and 1.0-fold, respectively, indicating that the Cry1Ac-resisrant strains of H. armigera had no cross-resistance to Cry2Ab.2,Binding in vitro and surface plasma resonance were conducted for binding kinetics between the toxins (Cry1A and Cry2Ab) and BBMV, the results suggested that Cry1A could not compete with Cry2Ab either in the Cry1 Ac-susceptible strain of H. armigera or in -resistant strains, it also indicated that Cry1A could not share the binding site with Cry2Ab in H. armigera. In a word, the difference of binding sites between the -susceptible and -resistant strains may be contributed to no cross-resistant to Cry2Ab in H. armigera.The data from the binding kinetics implied that the binding ability of Cry1 Ab to the binding site 1 and Cry1Ac to site 2 decreased, and the decrease of binding ability corresponding to the increase of resistant ratio. At the same time, we observed that the binding ability of Cry1Ab did not decrease anymore until it decreased to some degree, but the Cry1Ac did not stop decreasing until it lost the binding ability to binding site 2. To our surprise, there was no difference in the binding affinities of Cry1Ac and BBMV between the Cry1 Ac-susceptible strain and -resistant strain of H. armigera, but the binding site concentration of Cry1Ac and the binding ability of Cry1Ac in -resistant strain were lower than in the -susceptible, the results from Surface Plasma Resonance (SPR) suggested that the velocity of association in Cry1 Ac-susceptible strain was higher than in -resistant strain during the binding procession may be answered for this conflict. All these results suggested that the changes of binding ability of the Cry1Ac to BBMV is the main reason for its resistance, but the binding ability was affected by the binding affinity, binding site concentration and the velocity of association during the binding procession.3,Esterase and alkaline phosphatase may be also contributed to Cry1Ac resistance in H. armigera. We surveyed the activity of the total esterase, alkaline phosphatase and acid phosphatase in the Cry1Ac- susceptible and -resistant stain of H. armigera by microplate reader 550. The results indicated that the activity of total esterase in -susceptible was lower than that in -resistant strain of H. armigera, but the activity of alkaline phosphatase in Cry1Ac-resistant strain was significantly higher than that in -susceptible strain of H. armigera. The interaction analysis between esterase and Cry1Ac protoxin and toxin were also investigated, the results suggested that the esterase of Cry1Ac-resistant strain could bind with Cry1Ac protoxin and toxin, but the -susceptible strain could not. At the same time, the ability of activation of Cry1Ac protoxin in Cry1Ac-suscptible was stronger than that in -resistant strain. According to these results, we proposed that once H. armigera resistant to Cry1Ac, the reduction of alkaline phosphatase would affect the activation of Cry1Ac protoxin, at the same time, more esterase could bind with Cry1Ac protoxin and toxin, and this binding would reduce or lose the chance of Cry1Ac toxin binding to the receptor on the midgut of epithelium, and these procession would cause H. armigera reduce its susceptiblity and resistance to Cry1Ac.In a conclusion, these results demonstrated that Cry1 A and Cry2Ab had different binding sites in H. armigera and provided a potential mechanism for the lack of cross-resistance between Cry1A and Cry2Ab toxins, and it also suggested that Cry2Ab could be used to delay the evolution of H. armigera resistance to Cry1Ac.