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。
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 HEK293 T 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 TFAP2 C、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 HEK293 T 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.
作者
乔春敏
乔笑芹
周国平
Qiao Chunmin;Qiao Xiaoqin;Zhou Guoping(Department of Pediatrics,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2019年第6期862-866,共5页
Journal of Nanjing Medical University(Natural Sciences)
基金
江苏省“科教强卫”工程创新团队(领军人才)项目(CXTDA2017018)
