基于β-半乳糖苷酶为报告基因的耐辐射异常球菌启动子检测载体构建  

Construction of Promoter Function Analysis Vector Using β-galactosidase Report System in Deinococcus radiodurans

作　　者：李杰[1] 张陈[1] 张维[1] 周正富[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081

出　　处：《生物技术进展》2016年第4期265-270,共6页Current Biotechnology

基　　金：国家973计划项目(2015CB755700);国家转基因新品种培育重大专项(2014ZX0800301B);国家自然科学基金项目(31370126)资助

摘　　要：耐辐射异常球菌是一种超强电离辐射抗性的微生物,其含有大量的胁迫反应相关蛋白及其转录调控因子,在适应环境变化和各种胁迫反应中发挥着重要作用。为深入分析该菌的抗逆基因在不同胁迫下的功能,以β-半乳糖苷酶为报告基因构建了耐辐射异常球菌启动子检测体系。以耐辐射异常球菌基因组为模板扩增gro EL、hsp20、relA和relQ启动子片段,将其连接到到大肠杆菌-耐辐射异常球菌穿梭载体pRADZ1上,构建成具有启动子活性检测功能的重组质粒pRADZ-P_(groE)、pRADZ-P_(hsp20)、pRADZ-P_(relA)和pRADZ-P_(relQ),并将其转化至耐辐射异常球菌,测定β-半乳糖苷酶酶活。结果显示,这些启动子能在耐辐射异常球菌中启动Lac-Z报告基因的表达,并受到胁迫条件的诱导调控。耐辐射异常球菌启动子活性检测方法构建为该菌株特异性胁迫应答系统的研究提供了重要的实验基础。Deinococcus radiodurans is a robust bacterium which shows extreme ionizing-radiation tolerance. Abundant proteins and transcriptional factors are important for the various stress response in D. radiodurans. In order to construct the promoter function analysis system that use β-galactosidase gene as report gene in D. radiodurans,we used the genome of D. radiodurans as template to amplificate the promoters of gro EL,hsp20,rel A and rel Q by PCR,then connected them to E. coli-D. radiodurans shuttle vector pRADZ1,obtained the recombinant plasmids pRADZ-PgroEL,pRADZ-Phsp20,pRADZ-PrelA and pRADZ-PrelQ,which could analyze the activity of promoter. Then we transformed those recombinant plasmids into D. radiodurans,and measured the activity of β-galactosidase,the result showed that these promoters can start expression of Lac-Z,and could be regulated under stress. This study provided theoretical basis for the further work of stress response mechanism in Deinococcus radiodurans.

关 键 词：Β-半乳糖苷酶 启动子活性检测 耐辐射异常球菌 穿梭质粒pRADZ1 

分 类 号：Q78[生物学—分子生物学]