Identification And Cloning Of Vip Genes From Bacillus Thuringiensis

Using the temperature screening method,73 Bacillus thuringiensis isolates were obtained from 202 soil samples collected from some locations of Hebei and Guizhou provinces.The microscope examination and SDS-PAGE result indicated that most of them expressed 130kDa and 60kDa insecticidal crystal proteins.With the universal primers of Svip5/Svip3,the vip3A-gene types of these isolates were identified by PCR—RFLP(polymerase chain reaction—restriction fragment length polymorphism)method.1.45kb of expected PCR products were identified from 63 isolates and the distribution rate was 36.8%,RFLP results showed that three bands of 881bp,255bp, 186bp which marked as vip3Aal type gene.TF9 and Bt16 isolates showed high and moderate toxicity to Spodoptera exigua larvae and chosen for vip3A gene cloning.Two novel vip3Aa genes from TF9 and Bt16 were cloned by full-length PCR method, respectively.GenBank accession number are EU332167(Vip3Aa27)and EU294496 (Vip3Aa26).The amino acid sequence similarity showed 97.34%～99.49 and 96.70%～99.11%with other known Vip3A proteins.vip3A-16 and vip3A-TF9 were inserted into a pQE30 expression vector respectively, and transformed into E.coli M15.The E.coli harbouring the cloned vip3A genes were induced with lmmol/L IPTG,and expressed the proper recombinant proteins.The bioassay results indicated that Vip3Aa27 was highly toxic to Trichoplusia ni,Spodoptera exigua and Helicoverpa arrnigera,The LC₅₀values were 0.125μg/mL,0.238μtg/mL and 9.238μg/mL,respectively.However,Vip3Aa26 only possessed toxicity to Trichoplusia ni.A PCR-RFLP method was used for identification of vip1A/vip2A genes and cryl-type genes from Bt isolates,respectively.The result showed that vip1A/vip2A genes appeared in 20 of 117 Bt isolates.A vip1A/vip2A gene fragment from Bt16 was cloned and sequenced. GenBank accession number is EU594327.The common cryl-type gene was identified as crylFa,which indicated that some linkage occurred between the vip1A/vip2A and crylFa.