温度对大肠埃希菌Mn-SOD基因启动子调控条件的优化

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.

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

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.