Identification Of The Cry1Ac/Cry2Aa Binding Proteins And Their Functional Analysis In Spodoptera Exigua

The bacterium Bacillus thuringiensis produces Crystal (Cry) proteins that are toxic to a diverse range of insects, and cry genes have been transformed into transgenic plants for resistance to insect. Transgenic cotton "Bollgard ?" that express both Cry 1 Ac and Cry2Ab proteins are predicted to be resistant to S. exigua.Nonetheless, large-scale plant Bt crops have led to field-evolved resistance in several pests. It has been reported that "Bollgard ?", cotton have field-evolved resistance in Helicoverpa armigera, the Beet armyworm also exist the risk of becoming resistant to the transgenic cotton. However, little is known about the receptor proteins that mediate the toxicity of the Cry subfamily of proteins in the Lepidoptera. It seriously restricts its resistance management and hreatents the future sustainable utilization of Bt transgenic crops. In this study, we used the methods of proteomics, molecular biology, insect toxicology and biochemistry to identify the Bt binding proteins and analyze the function of receptor in S. exigua. The results of our study are of great theoretical significance for understanding the molecular bases of Bt mode of action,and integrated management of insect resistance to Bt crops.The main research contents and conclusions are as follows:1. Cry1Ac binding proteins in S. exigua BBMVsIn the current work, we used a combined one-dimernsional (1D) gel electrophoresis and immunoblotting approach to identify Aminopeptidase N (APN1 and APN3) act as CrylAc binding proteins in the midgut of S. exigua larvae, these results will provide theoretical basis for subsequent study of CrylAc receptor.Meanwhile, we performed heterologous competitive binding assays with BBMV from S. exigua. The results showed that CrylAc and Cry2Aa did not share binding sites on S. exigua Brush Border Membrane Vesicles (BBMVs). Our results suggest that the potential cross-resistance of this insect to CrylAc and Cry2Aa is unlikely.2. Functional CrylAc receptor assays of APNs in S. exiguaFunctional analyses by gene silencing of six different APN genes of S. exigua(SeAPNl, SeAPN2, SeAPN3, SeAPN4, SeAPN5 and SeAPN6), the results showed that larval ingestion of dsRNAs specific for the SeAPNs are significantly reduced transcript levels of these genes. The results of bioassay showed that only suppression of SeAPN1 resulted in decreased larval susceptibility to Cry1Ac protein. The results suggest that SeAPNl act as the potentially functional receptor for Cry1Ac in S.exigua larvae.3. Binding of Cry2Aa to S. exigua BBMVsThe goal of this section was to identify Cry2Aa binding proteins in S. exigua BBMVs using two-dimension gel electrophoresis (2DE) and LC-MS (liquid chromatography-mass spectrometry)/MS techniques,we identified several proteins that bind Cry2Aa (Polycalin, V-ATPase subunits A and B, actin, 4-hydroxybutyrate CoA-transferase [4-HB-CoAT]) and a receptor for activated protein kinase C (Rack)in S. exigua. Our study is the first Cry2Aa receptor identified in S. exigua brush border membrane vesicles.4. Functional CrylAc receptor assays of APNs in S. exiguarBasing on the type of Cry1Ac binding proteins, Recombinant, expressed versions of these proteins were able to bind the Cry2Aa protein performed by competitive ELISA assays. The binding affinities with Cry2Aa are different from each of the recombinant peptide. Moreover, RNA interference knockdown of the Se-V-ATPase subunit B significantly decreased the susceptibility of S. exigua larvae to Cry2Aa, but not to other target genes. Finally, an in vitro homologous competition assay demonstrated that the Se-V-ATPase subunit B binds specifically to the Cry2Aa. The results suggest that Se-V-ATPase subunit B act as the potentially functional receptor for Cry2Aa in S.exigua larvae.