文章摘要
陈海燕,徐妍妍,周国平,徐华国.人cGAS 基因启动子的克隆鉴定及功能初探[J].南京医科大学学报,2019,(6):856~861
人cGAS 基因启动子的克隆鉴定及功能初探
Cloning and characterization of human cGAS gene promoter
投稿时间:2019-01-13  
DOI:10.7655/NYDXBNS20190613
中文关键词: 环鸟苷酸⁃腺苷酸合成酶  启动子  转录调控
英文关键词: cGAS  promoter  transcriptional regulation
基金项目:江苏省自然科学基金面上项目(SBK 2018022334);江苏省研究生科研创新项目(KYCX17_1287)
作者单位
陈海燕 南京医科大学第一附属医院检验学部江苏 南京 210029 
徐妍妍 南京医科大学第一附属医院儿科江苏 南京 210029 
周国平 南京医科大学第一附属医院儿科江苏 南京 210029 
徐华国 南京医科大学第一附属医院检验学部江苏 南京 210029 
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中文摘要:
      目的:构建人环鸟苷酸?腺苷酸合成酶(cyclic guanosine monophosphate?adenosine monophosphate synthase,cGAS)基因启动子,确认其活性,进而对转录调控机制进行初步探究。方法:采用 PCR 方法扩增人cGAS 基因 5′上游 1 254 bp(-1 178~+76 bp)的片段,酶切后连接到pGL3?basic 质粒,以该质粒为模板合成不同长度的启动子质粒。通过双荧光素酶报告基因实验验证不同质粒在Hela细胞中的活性,利用生物信息学方法预测启动子区存在的转录因子结合位点。通过点突变实验验证潜在的转录因子结合位点。结果:人cGAS启动子质粒初步构建成功。转染后,cGAS 启动子重组质粒的相对荧光素酶活性增加(P<0.05)。人cGAS近端启动子区域(-414~+76 bp)经生物信息学软件分析可能含有 Sp1、CREB、USF1、RAP1、C?JUN、OCT?1等转录因子的结合位点。点突变实验证实Sp1、CREB正向调控该启动子区域。结论:人cGAS近端启动子区域(-414~+76 bp)存在较强的启动子活性,该区域含有多个潜在的转录因子结合位点。转录因子Sp1、CREB调控人cGAS启动子区。
英文摘要:
      Objective:This study aims to construct the promoter of human cyclic guanosine monophosphate?adenosine monophosphate synthase(cGAS)gene,and explore its promoter activity and transcriptional regulation mechanism. Methods:The 1 254 bp(-1 178~+76 bp)fragment of 5′ upstream of human cGAS gene was amplified by PCR and subcloned into pGL3?basic plasmid. By a series of 5′ deletion and promoter constructions,the core region of cGAS promoter was founded. Luciferase assays were used to detect the activity of recombinant plasmids in Hela cells. Then,the transcription factor binding sites in promoter region were predicted by bioinformatics. Finally,potential transcription factor binding sites were verified by point mutation experiments. Resluts:The luciferase reporter gene recombinant plasmids of cGAS gene promoter were successfully constructed. In comparison with the pGL3?basic plasmid,the relative luciferase activities of recombinant palsmids of cGAS promoter were much higher(P < 0.05). What’s more,bioinformatics software predicts that the proximal promoter region of human cGAS(-414~+76 bp) may contain binding sites of transcription factors such as Sp1,CREB,USF1,RAP1,C?JUN and OCT?1. Sp1 and CREB positively regulated promoter region,which was confirmed by point mutation experiment. Conclusion:It is concluded that the proximal promoter core region of human cGAS has strong promoter activity,which contains many potential transcription factors binding sites. The transcription factor Sp1 and CREB regulates the human cGAS promoter region.
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