苏云金芽孢杆菌cry1Ac15基因的克隆及原核表达

目的]为了分析苏云金芽孢杆菌cry1Ac15基因序列。方法]以全长基因PCR产物的黏端定向克隆的方法,设计1对特异引物,分别引入SalⅠ和BamHⅠ酶切位点。以Ly30质粒DNA为模板扩增cry1Ac全长基因,与表达载体pUCM-T相应的酶切产物连接,转化大肠杆菌,获得含有cry1Ac基因的重组质粒pUCLy1Ac。结果]cry1Ac基因编码区为3 564 bp,编码蛋白分子量为134 kD,含1 184个氨基酸,与cry1Ac3同源性最高,该基因经诱导获得高效表达,SDS-PAGE电泳检测到明显的134 kD蛋白带。结论]诱导表达的cry1Ac蛋白对棉铃虫、甜菜夜蛾等鳞翅目害虫幼虫均有较高的杀虫活性。

Cloning and Expression of crylAcl5 Gene from Bacillus thuringiensis

Objective] The research aimed to analyze the nucleotide sequence of cry1Ac15 Gene from Bacillus thuringiensis.Method] According to cry1A gene sequences of 5′-terminal and 3′-terminal,a pair of primers for full-length cry1Ac gene was designed.And PCR was performed to produce a full-length cry1Ac fragment by using plasmid DNA from Bt Ly30 as the template.Result] Sequence analysis results showed that the open reading frame of the cry1Ac gene was 3 564 bp which coded for 1 184 amino acids.The deduced molecular weight of the predicted protein was about 134 kD.The cry1Ac15 gene was registered in GenBank as novel gene expressed by inserting into an expressing vector pUCM-T.The 134 kD protein could be identified obviously by SDS-PAGE.Conclusion] The results of bioassay proved that the expression products of cry1Ac15 gene had high toxicity against Helicoverpa armigera and Spodotera exigua.