苏云金芽孢杆菌cry2Aa基因的克隆、表达与活性

B-8-G和Ly30是我国自行分离的对多种重要农业害虫具有高毒力的苏云金芽孢杆菌（Bacillus thuringiensis）菌株,经PCR-RFLP鉴定均含有cry2Aa基因.根据cry2Aa全长基因序列设计特异引物,以B-8-G总DNA为模板扩增其中的cry2Aa全长基因,与大肠杆菌（Escherichia coli）表达载体pET-21b相连接,获得含有cry2Aa全长基因的重组质粒pET2Aa,该基因在大肠杆菌BL21菌株能够正常表达65 kD蛋白.通过构建Ly30总DNA文库方法从中筛选获得cry2Aa基因,将其连接至Bt-E.coli穿梭表达载体pHT315上,转化Bt无晶体突变株HD-73中,该基因能正常表达65 kD蛋白,并形成立方体状晶体.这两种基因序列已被国际Bt基因命名委员会分别正式命名为cry2Aa9和cry2Aa10.杀虫生物活性测定结果表明cry2Aa基因表达产物对黄胫小车蝗（Oedaleusinfernlis）、尖音库蚊（Culex pipiens）、黑翅伊蚊（Aedes melanopterus）、水稻二化螟（Chilo suppressalis）和小菜蛾（Plutellaxylostella）幼虫均具有显著的毒杀作用.首次报道cry2Aa10基因表达蛋白对蝗虫、库蚊具有杀虫活性.这些基因的获得,将为高效工程菌和抗虫转基因植物的研制提供了新的基因资源.

Cloning and Expression of cry2Aa Genes from Isolates of Bacillus thuringiensis and Their Bioactivity

Two wild-type isolates of Bacillus thuringiensis, B-8-G and Ly30, were isolated from China, which showed high insecticidal activity against several primary agricultural pests, and identified for cry2Aa genes by PCR-RFLP. According to the published sequence of cry2Aa gene, a pair of special primers was designed for full length DNA cloning of cry2Aa gene by PCR amplification using total DNA of the B-8-G isolate as the template. Subsequently, the amplified fragment of cry2Aa gene was inserted into the pro- caryotic vector pET-21b and a 65 kD peptide was expressed in Escherichi coli by IPTG induction at room temperature. Alterably, another cry2Aa gene was obtained by screening of a DNA library of Ly30 isolate and was transformed into Bt acrystalliferous mutant HD73 by using Bt-E. coil shuttle vector pHT315. The result of SDS-PAGE analysis showed that the cry2Aa gene could be expressed as 65 kD peptide. These two genes had been nominated as the novel cry genes of cry2Aa9 and cry2Aa10 by International Nomenclature Committee of Bt, respectively. The bioassay results indicated that the Cry2Aa toxic protein was distinctly insecticidal activity against Oedaleus infernlis, Culex pipiens, Aedes melanopterus, Chilo suppressalis and Plutella xylostella, respectively. It is first report that Cry2Aa10 protein has pesticidal activity against Oedaleus infemlis and Culex pipiens larva. This study result will facilitate the construction of genetically engineered bacterium and transgenic plant for biocontrol of agricultural and sanitary insect pests.