Baseline Toxicity Of Cry2Ab And Identificaiton Of Midgut Cry2Ab-Binding Proteins And Midgut Juice Proteins Of Helicoverpa Armigera

It has already been15years since the first generation transgenic Bt cotton commercialized in1996. Evolution of resistance by pests is the primary threat to the continued efficacy of Bt crops. To delay the pest resistance, some second-generation Bt crops producing two distinct Bt toxins (such as Cry1Ac and Cry2Ab) were developed and commercialized in2003. This approach, which is called a "pyramid", extremely enhanced the efficacy of Bt cotton to some lepidopteran pests, but dural-Bt cotton has not been adopted in China. Currently, researches on Bt resistance focus on CrylA family toxins, little is known about the mode of action and resistance mechanism of Cry2A toxins.In the present study, susceptibility of Bt toxin Cry2Ab in14field populations collected from northern and northwestern China in2010was measured by laboratory bioassay to establish the toxicity baseline of Cry2Ab. Binding patterns of Cry2Ab to the midgut brush border membrane vesicles (BBMV) in the susceptible strain and the Cry2Ab-resistant strain were compared. A proteomic approach was used to identify Cry2Ab binding proteins from midgut BBMV of H. armigera, proteins in the midgut juice were also identified. Our results will provide an important basis for Cry2Ab susceptibilty monitoring and resistance mechanism investigating in H. armigera.1. Baseline susceptibility to Cry2Ab in field populations of H. armigera from ChinaSusceptibility to Cry2Ab in14field populations of H. armigera collected in2010from China was surveyed, and the results demonstrated that the LC50of Cry2Ab ranged from22to170ng/cm2, and less than10-fold variations occurred among populations. Although field populations showed decreaded susceptibility to Cry1Ac in some populatiosn from northern China, there was no significant difference in Cry2Ab susceptibility among field populations from northern China and northwestern China, it suggests that the second generation Bt cotton (expressing CrylAc+Cry2Ab) can achieve good control efficacy against field populations of H. armigera in China. 2. Comparative analyses of the binding patterns of Cry2Ab with the resistant and susceptible H. armigera BBMVs by ligand blottingSix binding bands to Cry2Ab toxin (76、62、49、38、33kDa respectively) were detected by using ligand blotting from BBMV in both the resistant strain (An2Ab) and the susceptible strain (An) of H. armigera. The bands of79kDa and49kDa were weaker. In the resistant strain An2Ab than in the susceptible strain An. When add200times unlabeled Cry2Ab, all binding bands became weaker, which suggest these binding proteins were specific to Cry2Ab, and the decreased binding of79kDa and49kDa proteins may be related to Cry2Ab reisistance in An2Ab strain. At least9Cry1Ac-binding bands (between30kDa and200kDa) were detected. The band around200kDa (putative cadherin) was present in Cry1Ac blotting, but not present when blotted with Cry2Ab toxin. In heterologous competition assays, Cry1Ac binding bands were almost not affected by200times unlabeled Cry2Ab, while all the Cry2Ab bands were disappeared under the presence of200times Cry1Ac competition. The present results showed there are significant differences in the binding proteins of Cry2Ab and Cryl Ac in the BBMVs of H. armigera.3. Separation and identification of Cry2Ab-binding proteins from larval midgut BBMVs and of proteins from larval midgut juice from H. armigeraWith combined2-DE, ligand blotting and MALDI-TOF mass spectrometry approaches, thirty-two Cry2Ab-binding proteins (with MWs between30-to120-kDa) were identified from midgut BBMVs of H. armigera. Among these Cry2Ab-binding proteins, there were3membrane-associated proteins, APN5, ATP/ADP translocase and annexin Ⅸ. APN and ATP/ADP translocase were previously identified as Cry1A-binding proteins. Annexin Ⅸ as a novel membrane protein binding with Cry2Ab deserves further investigating. In addition, a large amount of binding proteins are related to metabolism, cell signals, and their involvement in binding and mode of action is not clear. Meanwhile, the midgut lumen proteome of H. armigera were identified in order to know more about the activiation and digestion of Bt toxins. We identified a total of thirty-three proteins, including10proteases,16digestive enzymes other than proteases, and7other proteins.