Abstract：Vegetative insecticidal protein 3A (Vip3A) has a broad specturm toxicity torwards Lepidoptera pests. The 2.4 kb entire coding region of vip3Aa gene (GenBank No. KT307982) was amplified by PCR with total DNA extracted from Bacillus thuringienesis (Bt) NBIC-380 which had high insecticidal activity against corn borer (Ostrinia nubilalis) as template. The vip3Aa was inserted into multiple cloning sites of the Escherichia coli expression vector pGEX-6p-1 and pET-28a, Bacillus expression vector pBMB1A to generate the recombinant expression vector pGV3, pEV3 and pBV3. The pGV3 and pEV3 were transformed into E. coli BL21 (DE3), respectively, and pBV3 was electro- transformed into Bt BMB171 and Bacillus subtilis (Bs) 168 strains. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) showed one protein of 88 kD. The expressed protein in E. coli was purified by GST affinity chromatography and Ni affinity chromatography, which demonstrated no toxicity towards Helicoverpa armigera second instar larvae. The proteins expressed in BtBMB171 and Bs168 had activities against H. armigera second instar larvae with medium lethal concentration (LC50) of 6.185 6 and 2.984 6 mg/mL, respectively. Bioassay indicated that expression of vip3Aa gene from Bt NBIC-380 strain had certain toxicity torwards H. armigera. This work enriches the fundamental research of Vip and provides basic data for construction of high toxicity and broad spectrum engineering strains.
[1]Adang M J.Bacillus thuringiensis insecticidal crystal protein：Gene structure， action and utilization[M].CRC Press，London, 1991, :3-24
[2]Beard C E，Court L，Boets A，et al.Unusually high frequency of genes encoding vegetative insecticidal proteins in all Australian Bacillus thutingiensis collection[J].Current Microbiology, 2008, 57(3):159-199
[3]蔡启良，刘子铎，孙明，等.苏云金芽胞杆菌营养期杀虫基因的克隆及表达分析[J].生物工程学报, 2009, 18(5):578-582
[4]Estruch J J，Warren G W，Mullins M A，et a.Vip3A，a novel Bacillus thuringienis vegetative insecticdal protein with a wide spectrum of activities against lepidopteran insectsProceedings of the National Academy of Sciences of the United States of America，93(11)：5389-5394.[J].Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(11):5389-5394
[5]Heimpel A M.A critical review of Bacillus thuringiensis var.thuringiensis Berliner and other crystalliferous bacteria[J].Annual Review of Entomolgy, 1967, 12:287-
[6]Hofte Whiteley R.Insecticidal crystal proteins of Bacillus thuringiensis[J].Microbiolory Review, 1989, 53:242-255
[7]刘荣梅，张杰，高继国，等.苏云金芽胞杆菌营养期杀虫蛋白基因的研究[J].高技术通讯, 2004, 14(9):39-24
[8]PENG D H，LUO Y，GUO S X，et al.Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis[J].Applied Microbiology, 2009, 106(6):1849-1858
[9]Ramasamy B，Nadarajah V D，et a.A preliminary study of the bioactivity of vegetative proteins extracted from Malaysian Bacillus thutingiensis isolates[J].Trop Biomed, 2008, 25(1):64-74
[10]Schnepf E，Crickmore N，Van R J，et al.Bacillus thuringiensis and its pesticidal proteins[J].Microbiology Molecular Biology Review, 1998, 66:775-806
[11]Selvapandiyan A，Arora N，et at.Toxicity analysis of N-and C-terminus deleted vegetative insecticidal protein from Bacillus thuringiens[J].Applied Enviromental Microbiology, 2001, 67(12):5855-5858
[12]孙明，喻子牛，刘子铎.苏云金芽孢杆菌 基因改造[J].中国生物防治, 1996, 12(3):127-129
[13]Warren G W，Koziel M G，Mullins M A.Pesticidal proteins and strains[P].World Intellectual Property Organization Patent, 1998, 5846870:
[14]巫益鸣，阮丽芳，彭东海，等.苏云金芽胞杆菌菌株- 的插入突变体库的构建及芽胞萌发突变体的筛选[J].华中农业大学学报自然科学版, 2010, 29(5):577-581
[15]YU Z N，ed.Bacillus thuringiensis[M].Science Publication Press，Bejing, 1990, :306-307
[16]YU C G，Mullins M A，Warren G W，Koziel M G and Estruch J J.The Bacillus thuringiensis vegetative insecticidal proteinVip3A lyses midgut epithelium cells of susceptible insects[J].Applied and Environmental Microbiology, 1997, 63:532-536
[17]张志刚、杨自文、王开梅，等.新型新杀虫剂的高通量筛选方法研究[J].湖北农业科学, 2010, 49(12):3045-3048
