苏云金芽胞杆菌NBIC-380菌株营养期杀虫蛋白3Aa基因(vip3Aa)在大肠杆菌及芽胞杆菌中的表达  

Expression of Vegetative Insecticidal Protein 3Aa Gene(vip3Aa) from Bacillus thuringiensis NBIC-380 in Escherichia coli and Bacillus Strains

作　　者：余晓娇 刘晓艳 杨自文[1,2] 

机构地区：[1]武汉大学生命科学学院,武汉430072 [2]湖北省生物农药工程研究中心,武汉430064

出　　处：《农业生物技术学报》2015年第11期1465-1471,共7页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2011AA10A201)

摘　　要：营养期杀虫蛋白3A(vegetative insecticidal protein 3A,Vip3A)对鳞翅目(Lepidoptera)害虫具有广谱杀虫活性,具有重要研究意义。本研究以对玉米螟(Ostrinia nubilalis)具有高毒力的野生型苏云金芽胞杆菌(Bacillus thuringienesis,Bt)NBIC-380菌株总DNA为模板,PCR扩增vip3Aa(Gen Bank登录号:KT307982)全长基因约2.4 kb。将vip3Aa基因插入大肠杆菌(Escherichia coli)表达载体p GEX-6p-1和p ET-28a以及Bt表达载体p BMB1A中,构建重组表达载体p GV3、p EV3和p BV3。将p GV3和p EV3转化大肠杆菌BL21(DE3),p BV3转化Bt BMB171和枯草芽胞杆菌(Bacillus subtilis,Bs)168菌株。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gelelectrophoresis,SDS-PAGE)分析表明,vip3Aa基因在BL21(DE3)、Bt BMB171和Bs168中均有表达,蛋白质相对分子质量为88 k D。纯化后的蛋白及Bt中表达的蛋白对棉铃虫(Helicoverpa armigera)二龄幼虫进行了生物测定,在大肠杆菌表达的蛋白没有杀虫活性,而在芽胞杆菌中表达的蛋白有杀虫活性,半数致死浓度(medium lethal concentration,LC50)值分别为6.185 6(Bt BMB171)和2.984 6 mg/m L(Bs168)。生测结果表明,Bt NBIC-380菌株的vip3Aa基因表达的蛋白对棉龄虫具有一定杀虫活性。本研究为丰富Vip的基础研究和构建高效广谱的工程菌提供基础资料。Vegetative insecticidal protein 3A（Vip3A） has a broad specturm toxicity torwards Lepidoptera pests. The 2.4 kb entire coding region of vip3 Aa gene（Gen Bank 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 vip3 Aa was inserted into multiple cloning sites of the Escherichia coli expression vector p GEX-6p-1 and p ET-28 a, Bacillus expression vector p BMB1 A to generate the recombinant expression vector p GV3, p EV3 and p BV3. The p GV3 and p EV3 were transformed into E.coli BL21（DE3）, respectively, and p BV3 was electro- transformed into Bt BMB171 and Bacillus subtilis（Bs）168 strains. Sodium dodecyl sulfate polyacrylamide gel electropheresis（SDS- PAGE） showed one protein of88 k D. 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. Theproteins expressed in Bt BMB171 and Bs168 had activities against H. armigera second instar larvae with medium lethal concentration（LC50） of 6.185 6 and 2.984 6 mg/m L, respectively. Bioassay indicated that expression of vip3 Aa 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.

关 键 词：枯草芽胞杆菌 营养期杀虫蛋白(Vip) vip3Aa基因 克隆与表达 

分 类 号：S476.11[农业科学—农业昆虫与害虫防治]