BticryIVB基因的克隆及表达质粒的构建

BticryIVB GENE CLONING AND CONSTRUCTION OF EXPRESSION PLASMID

采用 PCR方法 ,克隆得到 3 .5kb的开放阅读框架 ( ORF) ,其包括 Bti的杀虫蛋白基因 ,cry IVB结构基因和部分上游启动区 .将结构基因亚克隆到高表达载体 p QE52中 ,构建了高表达质粒 p QE52 B.在 E.coli SG1 3 0 0 9( p REP4 )中 ,1 3 0 k D的蛋白得到表达 ,其对韭菜迟眼蕈蚊 ( Bradysia odoriphaga)三龄幼虫具有较强的毒杀作用 .

With PCR method, a 3.5kb open reading frame (ORF) including insecticidal crystal protein(ICP) gene cryIVB was obtained from Bacillus thuringinesis var . isealensis. This ORF contains whole structural gene and partial upstream promoting region of cry IVB. The structure gene, without its starting codon, was inserted into E.coli expression vector, pQE52, and plasmid pQE52B was constructed. When transformed into E. coli SG13009(pREP4) and induced by IPTG, cry IVB expressed 130kD insecticidal protein at high level and could be checked by SDS PAGE. Bioassay result revealed that the ICP is highly toxic to the third instar larvae of Bradysia odoriphaga, a dipteran pest of Chinese chives.