Transformation Of Bt Insecticidal Gence, Vip3A(a), Into A TB2 Strain(Bacillus Amyloliquefaciens)

Abstract: A pair of primers was designed according to the sequence of the strain BS168 (Bacillus subtilis subsp. subtilis str.168, GenBank accession number NC000964) , then to PCR to obtain the promoter of rpsD, designated as rpsP,using a modle of the chromosome DNA of BS 168．Another pair of primers was designed according to the vegetative insecticidal protein (VIPs) gene (GenBank accession number AF442384) , then to PCR to obtain vip3A(a) gene using a modle of total DNA of the strain WB7( Bacillus thuringiensis )which had high insecticidal activity. The rpsP and vip3A(a) sequences were cut by the endoenzyme Nde I and purified by the DNA pulification kit respectively, then the two genes were ligated by T4 DNA Ligas, and to ampify using the ligated genes as a modle to obtain the p-vip gene with RpsF/vip-R as primers. The p-vip gene was cloned into the strain JM109(Escherichia coli) using pMD-18T vector. Then the recombinant plasmid, pMD-p-vip, was extracted and further amplified and analyzed with restrictionendoenzymes. After that the p-vip gene was cut down from the recombinant plasmidpMD-p-vip and ligated with expression vector Pgfp4412 to yield the recombinant plasmid pVipgfp.The recombinant plasmid pVipgfp was transferred into the strain TB2(Bacillus amyloliquefaciens), then transformants were verified by PCR. Therefore, 6 engineering bacterial strains of B. amyloliquefaciens with Bt insecticidal gene were obtained. Bioassays showed that the engineering strain, TB2-16, had high toxicity against the second instar larva of Spodoptera litura. The inhibiting activities of TB2-16 to Colletotrichum truncatum, C.gloeosprioides , Fusarium oxyporum f.sp. cucumerium, F. oxysporum f.sp. niveum, Alternaria solani were as the same as that of the TB2 strain. The endophytic and plant growth promoting tests showed that TB2-16 had no difference from that of TB2．...