苏云金芽孢杆菌WB9菌株cry2Ac4基因的克隆及表达

WB9是我国分离自武夷山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，简称Bt)菌株，经PCR-RFLP鉴定含有cry2Ac基因。根据cry2基因序列设计引物，以WB9质粒为模板扩增cry2Ac全长基因，与大肠杆菌(Escherichia coli)克隆载体pMD18-T连接获得含有cry2Ac全长基因的重组质粒pMD2Ac并测序。该基因在GenBank上登录号为DQ361267，被Bt国际命名委员会正式命名为cry2Ac4。通过亚克隆方法将cry2Ac4基因插入穿梭表达载体pHT315获得重组表达质粒pHT2Ac，将其转化大肠杆菌SCS110和Bt无晶体突变株HD73 Cry-，得到的工程菌能正常表达70 kD蛋白，形成方形晶体。生物测定结果表明，cry2Ac4基因表达产物对桔小实蝇(Bactrocera dorsalis Hendel)幼虫具有显著的毒杀作用，但对小菜蛾(Plutella xylostella)和致倦库蚊(Culex fatlgans)幼虫基本没有效果。

Cloning and Expression of cry2Ac4 Gene from Bacillus thuringiensis WB9

Bacillus thuringiensis WB9, isolated from Wuyi Mountain of China, showed high insecticidal activity against several agricultural pests and was identified for cry2Ac gene by PCR-RFLP. According to the published sequences of cry2 genes, a pair of primers was designed for full length DNA cloning of cry2Ac gene by PCR using the plasmid DNA of WB9 isolate as the template. Subsequently, the amplified fragment of cry2Ac gene was inserted into Escherichia coli cloning vector pMD18-T and sequenced. The gene had been registered in GenBank with accession number DQ361267 and designated as the novel gene cry2Ac4 by International Nomenclature Committee of Bt. An expression plasmid pHT2Ac was constructed by subcloning the cry2Ac4 gene into shuttle expression vector pHT315. pHT2Ac was transformed into Escherichia coli SCS110 and acrystalliferous Bt HD73 Cry-, respectively. SDS-PAGE analysis showed that the cry2Ac4 gene could be expressed as 70 kD peptide. In addition, square crystal was observed. The bioassay results indicated that the Cry2Ac toxic protein was distinctly insecticidal activity against Bactrocera dorsalis Hendel larvae. However, it exhibited little toxicity towards the larvae of Plutella xylostella and Culex fatlgans.