苏云金芽胞杆菌HD-73菌株sigE基因敲除突变株的构建与特点  

Construction and Characteristics of the sigE Mutation in Bacillus thuringiensis HD-73

作　　者：魏娟[1,2] 孙长坡[2,3] 宋福平[2] 张杰[2] 高继国[1] 黄大昉[3] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193 [3]中国农业科学院生物技术研究所,北京100081

出　　处：《微生物学通报》2008年第10期1581-1586,共6页Microbiology China

基　　金：国家“973项目”(No.2003CB114201)资助

摘　　要：SigE因子是芽胞杆菌芽胞形成过程中起重要作用的sigma因子,它控制着众多芽胞形成相关基因的表达。在苏云金芽胞杆菌中,Cry1等杀虫晶体蛋白的表达也受SigE因子的控制。本研究利用同源重组技术构建了苏云金芽胞杆菌库斯塔克亚种标准菌株HD-73sigE缺失突变株。研究表明:突变株衰亡期提前,丧失了形成芽胞和晶体的能力;cry1Aa指导的β-半乳糖苷酶活性分析表明sigE基因对cry1Aa基因的转录有较大影响。利用载体pHT315携带sigE所在操纵子spoIIG及其启动子序列在突变株中表达,使突变株恢复了产生芽胞和形成杀虫晶体的能力,生长基本恢复正常,这些结果表明SigE因子是Bt库斯塔克亚种菌株产生芽胞和形成晶体所必需的。SigE, encoded by sigE gene is an important sigma factor in the sporulation process. Many known genes and operons are controlled by this sporulation-specific factor, as well as most of cry genes in Bacillus thuringiensis. In this study, a sigE deletion mutant HD-73 （AsigE） was constructed by homologous recombination from Bacillus thuringiensis subsp, kurstaki HD-73 strain. The result showed that the mutant strain lost the ability of sporulation and crystallization of Cry protein. At the same time, the expression of insecticidal crystal proteins was severely reduced in HD-73 （AsigE） mutant, and growth speed of the mutant was affected intensively. The lacZ gene was fused with the promoter of the crylAa gene, and expressed in HD-73 （△sigE） mutant and HD-73 acrystalliferous mutant respectively. The activity of β-galactosidase expressed by lacZ gene in HD-73 （AsigE） mutant was much lower than that in the HD-73 acrystalliferous mutant. The ability of sporulation and crystallization of Cry protein was normally complemented by the expression of spollG operon, which contained the sigE gene after transformation with recombinant plasmid, and growth speed of the complemented strain also recovered. All the results indicated that for the B. thuringien- sis subsp. Kurstaki strain, sigE gene is essential to sporulate and crystallize. Acquirement of HD-73 （△sigE） mutant will redound to understanding mechanism of Cry protein expression and regulation, crystallization and its relationship with sporulation.

关 键 词：苏云金芽胞杆菌 CRY基因 sigE基因 突变 

分 类 号：S476[农业科学—农业昆虫与害虫防治] Q78[农业科学—植物保护]