温度对大肠埃希菌Mn-SOD基因启动子调控条件的优化  

Regulation on the Mn-SOD gene promoter in Escherichia coli through temperature

作　　者：邵海刚[1] 侯亚文[1] 张莹[1] 刘奕彦[1] 许海峰[1] 张洪勤[2] 应俊[2] 

机构地区：[1]温州医学院检验医学院与生命科学学院,浙江温州325035 [2]温州医学院生物学实验教学中心,浙江温州325035

出　　处：《中国微生态学杂志》2012年第10期886-888,892,共4页Chinese Journal of Microecology

基　　金：温州医学院2011年学生科研立项课题(wyx201101077)

摘　　要：目的构建大肠埃希菌BL21 Mn-SOD-RFP报告基因载体,并探讨温度对大肠埃希菌Mn-SOD基因启动子的调控。方法利用重组PCR技术,构建以Mn-SOD启动子调控的红色荧光蛋白(RFP)报告基因载体,将融合基因与T载体连接导入大肠埃希菌中,在不同温度(20、37、40和45℃)培养不同时间(13、20、30、37、44和54 h)后,利用荧光显微镜和荧光光度计观察大肠埃希菌表达RFP的情况。结果正确构建Mn-SOD-RFP融合基因,重组PCR结果与测序结果完全一致;不同温度不同时间诱导后,Mn-SOD启动子在37℃,培养30～37 h表达的红色荧光蛋白最多。结论成功构建该报告基因载体,并完成温度、时间对其调控的优化,为更进一步研究其他因素对SOD基因启动子的调控机制奠定基础。Objective To construct a vector of Mn-SOD-RFP as a reporter gene in E.coli and investigate how the temperature regulate Mn-SOD gene promoter in BL21 E.coli.Methods Recombinant PCR was used to construct Red Fluorescent Protein（RFP） reporter gene vector regulated by Mn-SOD gene promoter;the fusion gene was ligated with Vector T and transformed into E.coli.Then the expression of REP in BL21 E.coli was observed using fluorescenece microscope and microfluorophtometer after cultivating E.coli at different temperatures（20 ℃,37 ℃,40 ℃,45 ℃） for different times（13 h,20 h,30 h,37 h,44 h,54 h）.Results The Mn-SOD-RFP fusion gene was correctly constructed;the result of Recombinant PCR was in accordance with that of sequencing completely;the condition for the optimal RFP expression in E.coli inducted by Mn-SOD gene promoter was at 37 ℃,after 30-37 h of cultivation.Conclusion The reporter gene vector was successfully constructed and the regulation on its promoter was optimized by temperature and time,which provides a foundation and tool further studies on how other factors influence the mechanism of regulation of SOD gene promoter.

关 键 词：超氧化物歧化酶 启动子 重组PCR 红色荧光蛋白 报告基因 温度 

分 类 号：Q78[生物学—分子生物学]