对粉纹夜蛾高毒力cry9Ea基因的克隆及表达  

Cloning and characterization of a novel gene cry9Ea7 encoding crystal protein with high toxicity against Trichoplusia ni

作　　者：孙永祥[1] 刘廷辉[2] 郭巍[1,2] 王立光[2] 孙伟明[1] 

机构地区：[1]河北农业大学生命科学学院,保定071001 [2]河北农业大学植物保护学院,河北省农作物病虫害生物防治工程技术中心,保定071001

出　　处：《微生物学报》2010年第5期601-605,共5页Acta Microbiologica Sinica

基　　金：国家"973项目"(2009C13118900);国家自然科学基金(30771447);农业厅科技项目~~

摘　　要：【目的】从本实验室分离的Bt4菌株中克隆cry9Ea基因,并研究其表达和杀虫活性。【方法】以PCR-RFLP方法鉴定Bt4菌株含有cry9基因,然后以菌株Bt4的质粒为模板,利用全长引物F9EA/R9EA进行PCR扩增全长基因。【结果】将目的片段插入到表达载体pET21b,得到大肠杆菌重组表达质粒pETcry9Ea。转化E.coli BL21(DE3),诱导后表达130kDa的蛋白,再将cry9Ea7基因连接到穿梭载体pSXY422b,电激转化HD73-(cry-),得到工程菌BioHD9Ea7,提取Cry9Ea7晶体蛋白,并进行生物活性测定。生物活性测定结果显示Cry9Ea7蛋白对粉纹夜蛾(Trichoplusia ni)初孵幼虫具有高毒力,LC50为0.044μg/mL,而对甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)初孵幼虫未显示活性。【结论】克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Ea7,并成功构建了工程菌BioHD9Ea7。[Objective]We cloned a novel cry9Ea gene encoding a Cry9Ea protein with a high insecticidal activity against Trichoplusia ni.[Methods]We identified a cry9-type gene from Bt strain （Bt4） by PCR-RFLP.The full length cry9Ea gene was amplified by PCR with a pair of primers F9EA/R9EA.[Results]We cloned the complete cry9Ea7 gene into pET21b.The SDS-PAGE results showed that the 130 kDa Cry9Ea7 protein was expressed in E.coli BL21（DE3）.We also constructed an engineering strain BioHD9Ea7 by transforming a shuttle vector pSXY422b containing the cry9Ea7 gene into Bt acrystalliferous mutant HD73-（cry-）.The bioassay results indicated that the Cry9Ea7 protein was highly toxic against Trichoplusia ni neonates,and the LC50 value was 0.044 μg/mL.However,the Cry9Ea7 protein showed no activity against Spodoptera exigua and Helicoverpa armigera neonates.[Conclusion]The novel cry9Ea7 gene encoding Cry9Ea7 is highly toxic against Trichoplusia ni neonates.

关 键 词：苏云金杆菌 cry9Ea7基因 基因克隆 基因表达 杀虫活性 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]