枯草芽孢杆菌sacB基因的功能验证及应用  

FUNCTIONAL IDENTIFICATION OF Bacillus subtilis sacb GENE AND ITS APPLICATION

作　　者：吴菁[1] 刘秀敏[1,2] 张维[1] 陈明[1] 林敏[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]河北交通职业技术学院,河北石家庄050091

出　　处：《核农学报》2008年第5期590-594,共5页Journal of Nuclear Agricultural Sciences

基　　金：国家高技术研究发展计划项目(2004AA214170)课题;国家自然科学基金项目(206700050)

摘　　要：本研究将枯草芽孢杆菌(Bacillus subtilis)基因组DNA中克隆的1.4 kbsacB基因片段连接到表达载体pET-28a(+),构建了sacB基因表达载体pSacB。通过测定蔗糖水解产生的葡萄糖具有的还原性,确定sacB基因产物具有蔗糖果聚糖酶活性。该基因表达后,导致大肠杆菌(Escherichia coli)细胞不能在含5%蔗糖的培养基中生存。同时发现sacB基因也能赋予耐辐射菌株Deinococcussp.BR501蔗糖敏感性。利用条件致死sacB基因的这一特性,我们分析了D.sp.BR501的突变率,结果表明DNA错配修复缺失突变株(ΔmutS1)的突变频率明显高于野生型。因此枯草芽孢杆菌sacB基因可作为验证DNA损伤修复能力的一种筛选标记。In this study, the 1.4 kb sacB gene from Bacillus subtilis genomic DNA was cloned into the expression vector pET- 28a（ ＋ ）. The product of gene sacB had the levansucrase activity determined by glucose release from sucrose hydrolysis. The expression of sacB gene in Escherichia coli could cause cell death when exposed to 5 % sucrose in medium. And sacB gene also confered a sucrose-sensitive phenotype for the extremely radiation-resistant bacterium, Deinococcus sp. BR501. Using the conditional-lethal gene sacB to analyze the mutation rates of Deinococcus sp. BR501, we found that the rate of the DNA mismatch repair deficient strain （△ mutS 1 ） was obviously higher than that of wild type strain. Our result suggested that B. subtilis sacB gene could be used as a negative-selection marker to identify and characterize the genes involved in DNA demage repair.

关 键 词：枯草芽孢杆菌 sacB基因 蔗糖果聚糖酶 突变率 

分 类 号：S182[农业科学—农业基础科学]