Study On BBMV Specific Binding Receptor Proteins Of Bt Vip3Aa Protein In Spodoptera Exigua

Bacillus thuringiensis(Bt)is one of the most widely used environmental insecticides in the world.Bacillus thuringiensis has good feasible conditions as a biological insecticide.At present,the research mainly explores the insecticidal activity and insecticidal mechanism of Bacillus thuringiensis.In this paper,the insecticidal proteins in vegetative period were studied.Vegetative Insecticidal Protein(Vip)is a new class of representative Insecticidal Proteins,which have been discovered after the Insecticidal Crystal Proteins(ICPs).Vip3Aa11 and Vip3Aa39 were isolated from Bt strains C9 and T03B001,respectively.There were 39 a mino acid difference sites between the two proteins,and the a mino acid sequence similarity was 95.06%.These protein shave high insecticidal activity against Lepidoptera insects.Spodoptera exigua is a serious agricultural pest in China.However,in recent years,with the global warming and the change of crop distribution,the beet exigua has increased from a secondary pest to a major agricultural pest.They not only seriously harm corn sorghum and other food crops,but alsoharm vegetables,flowers,fruit trees,tobacco,cotton,sugar beet and other cash crops,causing great losses.Relevant studies show that Bacillus thuringiensis has a good killing effect on beet exigua.At present,only CRY proteins have been confirmed as insecticidal mechanism and corresponding receptors for S.exigua,while the vegetative stage insecticidal protein(Vip)for S.exigua remains unclear.In order to improve the insecticidal mechanism of Vip protein,we searched for the specific receptor of Vip protein for the vegetative stage of S.exigua.In this study,recombinant proteins Vip3Aa11 and Vip3Aa39 with His protein tags were constructed by molecular biology methods,and purified by using affinity chromatography.At the same time,the midgut tissue was extracted using liquid nitrogen with the 5-10 age S.exigua as the experimental material to gain its BBMV.Vip3Aa11 and Vip3Aa39 proteins were used as materials,and p ET28 a vector was used as negative control to conduct protein pull-down assay for midgut proteins of S.exigua,and the analysis was carried out by nanoscale reverse phase chromatography.The result showed that 26 and 30 potential receptors for Vip3Aa11 and Vip3Aa39 of midgut proteins were obtained,respectively.According to the corresponding data,the potential receptor proteins of Vip3Aa11 and Vip3Aa39 were obtained by proteomic analysis.According to the relevant research progress,alkaline phosphatase has been found to be related to the toxicological action of Vip protein,but ithas not been directly proved to be the direct receptor in the insecticidal mechanism of Vip.In this study,four proteins including alkaline phosphatase,apolipoprotein,LL1 and Repat 33 were identified as the first batch of verification objects based on the results of previous experiments and related studies.Referring to Gen Bank,We obtained nucleic acid sequences of alkaline phosphatase,apolipoprotein,LL1 and Repat33,and designed corresponding primers to obtain gene fragments of four potential receptor proteins by PCR using c DNA of S.exigua.The genes were constructed on protein expression vectors to express prokaryotic proteins.The optimal induction conditions of the four potential receptor proteins were deter mined by gradient concentration and gradient inducer concentration,respectively.The results showed that the optimal induction condition of ALP is 12℃ 0.5 mmol/L IPTG,while Apotripoprotein Ⅲ,LL1 and Repat 33 is 16℃ 1 mmol/L IPTG.Subsequently,the four potential receptor proteins induced under the optimal induction conditions were verified by protein interactions with Vip3Aa11 and Vip3Aa39.Western Blot and BLI results did not prove that alkaline phosphatase,apolipoprotein,LL1 and Repat33 are the direct receptors of Vip3Aa11 and Vip3Aa39.The above results can be further verified by yeast two-hybrid experimentsIn summary,this study successfully obtained the list of potential receptor proteins for Vip3Aa11 and Vip3Aa39 in midgut proteins of S.exigua,and preli minarily verified the four proteins with high probability,which provided an experimental basis and technical ideas for the deter mination of Vip3Aa11 and Vip3Aa39 receptor proteins in the future.