Mechanism Of Synergism Between Cry9Aa And Vip3Aa Protein Against Chilo Suppressalis

Bacillus thuringiensis (Bt), a gram-positive bacterium,can produce crystalline protein during its sporulation phase of growth. Due to its non-toxic to non-target pests, safe to the environment, it has been widely used to control insect pests, including lepidoptera, diptera, and coleoptera. As more and more applications of transgenic crops, the risk of insect resistance to Cry proteins is increasing. In this research, the Cry9Aa protein soluble modification, the minimal active fragment, the screening of purification condition, the influence factors of oligomerization, the N-terminal breaking point analysis of Cry9Aa and Vip3Aa protein digested by trypsin, and synergistic mechanism of Cry9Aa and Vip3 Aa protein against Chilo suppressalis were investigated.cry9Aa3 gene, a novel gene with highly toxic to lepidopteran pests was isolated in this laboratory, Cry9Aa protein can not be expressed in the crystal mutant, and mainly be in the form of inclusion bodies in E. coli with low expression in the supernatant. In this paper, we modified Cry9Aa protein through two ways:(1) 4.6kb cry9AaAc fusion gene was constructed by BP and RecA recombinant, transferred into HD73 crystals mutant. We found no crystal, and no target protein was detected by SDS-PAGE analysis of total protein of recombinant strain; (2) According to bioinformatics analysis results of Cry9Aa protein, eleven different fragments of cry9Aa3 gene were amplified by PCR designed, and then they were expressed in E. coli Rosetta (DE3) respectively. We found that the quantity of Cry9Aa3-1-654, Cry9Aa3-1-655 protein expressed in soluble polypeptide increase significantly by SDS-PAGE analysis, boosting the researches of the mechanism of the Cry9Aa protein against pests.Bioassay results against Plutella xylostella and Ostrinia furnacalis showed that the minimal active fragment of Cry9Aa toxin was located between amino acid residues 45’and 654V. This result is helpful for construction of transgenic plant, and for promoting commercial application of gene.By studying influence of imidazole and NaCl concentration in the Binding buffer to Cry9Aa protein purification, we found that the optimal condition of purification is that 50 mmol/L imidazol and 500 mmol/L NaCl were included in Binding buffer. By studying effect of pH, DTT and the NaCl concentration to oligomerization of Cry9Aa protein, we concluded that the high pH and NaCl concentration could inhibit oligomerization of the protein, and the oligomerization is mediated by intermolecular disulfide bond, so 1 mmol/L DTT must exist in the storage buffer. This would solve the problem of the protein precipitation in the process of dialysis.Full-length Vip3Aa could be digested to activated Vip3Aa peptide with the molecular weight about 62 kDa and truncated Cry9Aa peptide could be digested to activated Cry9Aa peptide with the molecular weight about 60 kDa by trypsin, chymotrypsin and larval midgut juice. Through N-term AA sequencing and mass spectrometry, the breaking point of the N terminal of activated Cry9Aa peptide lies between 23K and 24Y, the breaking point of the N terminal of activated Vip3Aa peptide lies between 39K and 40T.Through bioassay against Chilo suppressalis, synergistic activity between Cry9Aa and Vip3Aa protein was verified, and the synergistic factor is 35.7. Analyzing their receptors by the receptor pull-down platform, we found that they had the same receptor aminopeptidase family. In addition, there were also different receptors.