苏云金芽孢杆菌杀虫晶体蛋白基因cry1 Ab22的克隆与表达  

Cloning and expression of ICP cry1Ab22 gene of Bacillus thuringiens

作　　者：伍金元[1] 冯纪年[1] 张兴[2] 

机构地区：[1]西北农林科技大学植保资源与病虫害防治教育部重点实验室,陕西杨凌712100 [2]西北农林科技大学无公害农药研究服务中心,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2009年第1期151-154,共4页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家"十五"科技攻关项目(2002BA516A04);西北农林科技大学研究生教育创新计划项目(05YCH007)

摘　　要：【目的】克隆并表达苏云金芽胞杆菌(Bacillus thuringiens,Bt)杀虫晶体蛋白cry1Ab22基因,为解决昆虫抗性问题提供有效途径,并为构建新型转基因工程菌和转基因植物提供基因材料。【方法】根据GenBank中cry1Ab型基因序列设计1对引物,并加入BamHⅠ和PstⅠ酶切位点。用全长基因PCR产物粘端定向克隆的方法,从苏云金芽孢杆菌S249菌株中扩增到1个苏云金芽胞杆菌杀虫晶体蛋白cry1Ab型基因cry1Ab22,将其克隆到表达载体pMAL-c2X中,转化大肠杆菌TB1,进行诱导表达,并对表达产物的杀虫活性进行检测。【结果】cry1Ab22基因长度约为3.5 kb;其与已知的cry1Ab3型基因同源性最高,所编码的蛋白质存在5个氨基酸差异。cry1Ab22基因已在GenBank中登录,登录号为EU220269,并被Bt毒素基因国际命名委员会正式命名为cry1Ab22。SDS-PAGE电泳结果表明,cry1Ab22基因表达了170 ku左右的融合蛋白。cry1Ab22蛋白对小菜蛾(Plutella xylostella)的LC50为225.1μg/mL。【结论】S249菌株含有新型的cry1Ab基因,且该基因表达的蛋白具有较强的杀虫活性。[Objective] Cloning and expression of ICP cry1Ab22 gene of Bacillus thuringiens not only give a way to resolve insect resistance,but also provide the genetic material for constructing the new transgenie project fungus and transgenic plant. [Method] According to cry1Ab gene sequences of 5′-terminal and 3′-terminal, a pair of primers for full-length cry1Ab gene on the GenBank was designed. The cry1Ab type gene was cloned into the E. coli expression vector pMAL-c2X,which was then transformed into E. coli TB1 ,induction expression for Bacillus thuringiens and detected the activity for expression product. [Result] PCR was performed to produce a cry1Ab type gene which was designated cry1Ab22 in GenBank （Accession No. EU220269）. The length of gene was about 3.5 kb,and the comparison showed the highest homology with the cry1Ab3 and 5 amino acid differences existed in Encoded protein of cry1Ab22. The 170 ku fusion protein could be identified obviously by SDS-PAGE. Bioassay showed that the LC50 of cry1Ab22 against Plutella xylostella larve with a spread method was 225.1 μg/mL. [Conclusion] There is a novel cry1Ab type in strain S249 and cry1Ab22 protein has a stronger biological insecticidal activity.

关 键 词：苏云金芽胞杆菌 杀虫晶体蛋白基因cry1Ab22 生物杀虫活性 

分 类 号：S482.398[农业科学—农药学]