文章摘要
乔春敏,乔笑芹,周国平.人FAT1基因启动子的克隆鉴定及其功能浅析[J].南京医科大学学报,2019,(6):862~866
人FAT1基因启动子的克隆鉴定及其功能浅析
Identification and primary function of human FAT1 gene promoter
投稿时间:2018-12-07  
DOI:10.7655/NYDXBNS20190614
中文关键词: 脂肪非典型钙黏蛋白1  启动子  转录调控
英文关键词: FAT1  promoter  transcriptional regulation
基金项目:江苏省“科教强卫”工程创新团队(领军人才)项目(CXTDA2017018)
作者单位
乔春敏 南京医科大学第一附属医院儿科江苏 南京 210029 
乔笑芹 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 
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中文摘要:
      目的:克隆人脂肪非典型钙黏蛋白1(FAT atypical cadherin 1,FAT1)基因启动子,找到核心启动子区域并对其转录调控机制进行初步分析。方法:通过PCR方法获得人FAT1基因5′上游1 163 bp(-1 029~+134 bp)的片段,亚克隆至pGL3?basic载体;通过步移缺失构建不同缺失片段的重组质粒。双荧光素酶报告活性分析检测各重组质粒在A549细胞和HEK293T细胞中的活性,找到核心启动子区域。利用生物信息学方法预测核心区域的转录因子结合位点。结果:经测序、酶切鉴定,成功构建了人FAT1启动子荧光素酶活性报告基因重组质粒。与pGL3?basic质粒相比,人FAT1启动子重组质粒的相对荧光素酶活性增加(P < 0.05)。通过生物信息学软件预测人FAT1启动子区域(-233~-110 bp)可能含有TFAP2C、KLF5等转录因子结合位点。结论:成功构建人FAT1启动子不同缺失片段的荧光素酶报告基因重组质粒。通过荧光素酶活性比较,推测人FAT1的核心启动子区域位于-233~+134 bp区域,其中可能含有若干转录因子结合位点。
英文摘要:
      Objective:To clone the promoter sequences of fat atypical cadherin 1(FAT1),and to preliminarily analyze the transcriptional regulatory mechanism of the promoter. Methods:Promoter region was consructed by bioinformatic methods,and the application of 1 163 bp(-1 029~+134 bp)fragment of 5′upstream sequence of FAT1 gene by PCR was conducted,followed by cloning to pGL3?basic vector to establish the luciferase report gene recombinant plasmid. Another four recombinant plasmids of different lengths were obtained through walking deletion and then cloned to pGL3?basic plasmid as before. The resultant plasmids were transfected into A549 cells and HEK293T cells respectively together with pGL3?basic vector. After this their activities were detected via dual?luciferase reporter assay. Bioinformatic methods was performed to predict the sequences of the potential transcriptional factor binding sites of the core region found in the promoter. Results:The lusiferase reporter gene recombinant plasmids of human FAT1 promoter were successfully conducted. The relative luciferase activities of recombinant promoters,in contrast to pGL3?basic vector,were much higher(P < 0.05). Note the sequences of the binding sites such as TFAP2C、KLF5 were possibly included in the promoter region(-233~-110 bp)of FAT1 gene,which could bepredicted through bioinformatic means. Conclusion:The construction of the luciferase report recombinant plasmids of FAT1 gene were successfully done,and four of these plasmids had strong luciferase activities in A549 cells and HEK293T cells,comparing to the activities of pGL3?basic plasmid. Through this the core region was probably found. It is concluded the core promoter region of FAT1 gene was possibly located in the(-233~+134 bp)region,in which may preserve potential important transcriptional binding sites.
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