大肠杆菌yigP基因转录调控序列的鉴定

Identification of transcriptional regulatory sequences of yigP gene in Escherichia coli

【目的】调查yigP基因启动子的活性,并对该转录调控序列进行分析。【方法】以lacZ为报告基因,克隆启动子片段至启动子探针质粒中,通过检测β-半乳糖苷酶活性判断启动子活性,并通过克隆一系列逐步缩短的启动子片段来确定启动子所在区域。利用定点突变技术,对启动子的重要序列进行定点突变,调查其对启动子活性的影响。【结果】确定了yigP基因启动子的区域,鉴定了启动子的-10区和-35区,并发现了启动子上游存在一个负调控序列,对该序列进行了初步的研究显示其中部分序列是这种负调控作用的核心序列。【结论】对yigP基因的转录调控序列进行了鉴定,丰富了我们对基因转录调控的认识。

[Objective] We investigated the promoter activity of yigP gene and analyzed its transcriptional regulatory sequence.[Methods] We cloned promoter fragment into promoter probe plasmid which has a reporter gene lacZ,thus the promoter activity could be measured by detecting β-galactosidase activity.Then,the promoter region was minimized by cloning different truncated promoter fragments into promoter probe plasmid.Using site-directed mutagenesis technology,we introduced site mutations in some key sequences and investigated their effects on promoter activity.[Results] The promoter region of yigP gene was determined,and also the-10 region and-35 region were identified.Meanwhile,a negative regulatory sequence just upstream of yigP promoter was discovered,and our results show that these sequences have important influence on the transcriptional regulation.[Conclusion] We identified the transcriptional regulatory sequences of yigP gene,and this facilitated our understanding of gene transcription.