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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 broad spectrum insecticidal activity against Lepidoptera pests, which has important research significance. In this study, the total DNA of wild-type Bacillus thuringiensis (Bt) NBIC-380 strain with high virulence to Ostrinia nubilalis was used as a template to amplify the full-length gene of vip3Aa (Gen Bank accession number: KT307982) About 2.4 kb. The vip3Aa gene was inserted into Escherichia coli expression vectors pGEX-6p-1 and pET-28a and Bt expression vector pBMB1A to construct recombinant expression vectors pGV3, pVEG3 and pBV3. PGV3 and p EV3 were transformed into E. coli BL21 (DE3) and p BV3 was transformed into Bt BMB171 and Bacillus subtilis (Bs) 168 strains. SDS-PAGE analysis showed that the protein of vip3Aa was expressed in BL21 (DE3), Bt BMB171 and Bs168, the relative molecular mass of the protein was 88 k D. The purified protein and the protein expressed in Bt were used to bioassay the second-instar larvae of Helicoverpa armigera. The proteins expressed in Escherichia coli had no insecticidal activity. The proteins expressed in Bacillus sp. Had insecticidal activity and half The LC50 values were 6.1856 (Bt BMB171) and 2.984 6 mg / mL (Bs168), respectively. The results of bioassay indicated that the protein expressed by vip3Aa gene of Bt NBIC-380 strain had certain insecticidal activity against cotton instars. This study provides the basic information for enriching basic research of Vip and constructing efficient and broad-spectrum engineering bacteria.