Study On The Formation Mechanism Of Modified Vip3A Protein For Stem Borer Resistance In Rice

Bacillus thuringiensis(Bt) is a bacteria that would be the most widely used as biological insecticides. VIP is a soluble protein secreted during the whole vegetative phase without evolutional homology with ICP. The pesticidal activities of VIP could be up to nanogram level which would be considered as new generation of biopesticides. No doubt, VIP will be more and more applied in the breeding program of GM crop, in fact the genetically modified crop with vip gene have been constructed in rice, corn, cotton in China. The vip gene with stem borer resistance has nearly not been found from the existing studies. We found a new vip gene named as SBR gene that causes stem borer resistance. According to the Vip3 A protein structure and predecessors’ research report, Vip3 A protein sequence can be divided into the N-terminal(1-199 amino acids), the core region(200-533 amino acids) and C-termianl(534-789 amino acids). The SBR gene is a chimeric gene from vip3Aa1 and vip3Ad2 gene, which including the N-terminal and core region of Vip3Ad2 and the C-termianl of Vip3Aa1. This study through different chimeric vip3Aa1 and vip3Ad2 in accordance with the functional domains into three different vip3 A genes including SBR, vip3 Aa Ad1, vip3 Aa Ad2. Transformation of rice and prokaryotic expression the five vip3 A genes including vip3Aa1, vip3Ad2, SBR, vip3AaAd1 and vip3AaAd2 to identify the insecticidal activity. Sensitivity analysis of the five insecticidal proteins under trypsin process and analysis of digestion mechanism in the target pests. Meanwhile, Transformation of rice and prokaryotic expression the five Vip3 A two-color fluorescent proteins including GAa1 R, GAd2 R, GSR, GV1 R and GV2 R to feed striped stem borer(SSB), and analysis of vip3 A gene stem borer resistance formation mechanism. The main results are as follows,1. The vip3Ad2, vip3 Aa Ad1 and vip3 Aa Ad2 were obtained by overlapping PCR. The p1300-Vip3Aa1, p1300-Vip3Ad2, p1300-SBR, p1300-Vip3 Aa Ad1 and p1300-Vip3AaAd2 plant expression vector were constructed. The constructed vector were introduced into Nipponbare via Agrobacterium-mediated transformation. The T0 transgnic rice with five vip3 A genes were screened by PCR, RT-PCR and Vip3 A strip. Positive T0 generation of transgenic plants with vip3Aa1, SBR, vip3AaAd1 and vip3AaAd2 genes have a high resistance to SSB through detached-leaf bioassay while transgenic plants with vip3Ad2 gene has mild resistance to SSB.2. The pET28-Vip3Aa1, pET28-Vip3Ad2, pET28-SBR, pET28-Vip3 Aa Ad1 and pET28-Vip3AaAd2 prokaryotic expression vector were constructed, and the five vip3 A genes expressed from E.coli. When the concentration of trypsin is 0.1μg/μ L, the five Vip3 A proteins were digested to produce 62 KDa core peptide. Further analysis revealed that the C-terminal of Vip3 A may play a key role in the process of trypsin digestion. Meanwhile, the five toxic proteins were displayed insecticidal activity by feeding SSB.3. Five vip3 A genes fusion gfp and rfp genes plant expression vector and prokaryotic expression vector were constructed. So far, the T0 transgenic rice with five vip3 A genes fusion gfp and rfp genes were obtained and the five vip3 A genes fusion gfp and rfp genes expressed from E.coli.