Dppa2基因启动子区Oct4结合位点突变对其活性的影响  

The Mutation of Oct4 Binding Site in Promoter Region of Dppa2 Gene and its Impacts on the Dppa2 Gene Expression

作　　者：马娜[1] 陈天姬[1] 李珑[1] 陈婧[1] 杜娟[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,湖南长沙410005

出　　处：《现代生物医学进展》2015年第21期4001-4004,共4页Progress in Modern Biomedicine

基　　金：湖南省科技厅科技计划项目(2009FJ3058);中南大学中央高校基本科研业务费专项资金项目(2013zzts273);中南大学教师研究基金(905010042)

摘　　要：目的:探讨Dppa2基因5'端启动子区Oct4结合位点突变对Dppa2基因启动子活性的影响。方法:PCR扩增包括Oct4结合位点的Dppa2基因5'端转录起始点上游-2439^+293 bp的启动子序列,片段长度为2732 bp。将该片段连接到p GL3-Basic载体,构建野生型p GL3-2439表达载体。采用定点突变法,将-1959^-1957位碱基的GCA突变成TAG,构建Oct4结合位点突变型p GL3-mo2439表达载体。用上述两种表达载体、PGL3-basic载体和Oct4表达载体分别瞬时转染HEK 293细胞。细胞培养48 h后,利用双荧光素酶报告系统测定各组细胞表达的荧光素酶的相对活性。结果:经琼脂糖凝胶电泳及测序鉴定,证实野生型(p GL3-2439)和突变型(p GL3-mo2439)载体构建成功。荧光素酶活性测定结果显示,转染Dapp2基因启动子野生型p GL3-2439表达载体的细胞组荧光素酶的相对活性为16.307,突变型p GL3-mo2439表达载体的细胞组荧光素酶的相对活性为10.634。Oct4结合位点突变后,Dppa2基因启动子区转录活性较野生型降低了35%。结论:Dppa2基因5'端启动子区-1959^-1957位的Oct4结合位点突变可能导致Dppa2基因启动子活性下降。Objective： To investigate the mutation of Oct4 binding site in promoter region of Dppa2 gene and its impacts on the Dppa2 gene expression. Methods： The promoter sequence, -2439～＋293 bp region upstream of Dppa2 which contain the recognition site of Oct4, was amplified through PCR method. The mutation of GCA→TAG （-2439～＋293 bp） in the conserved recognition site of Oct4 . was obtained using site directed mutagenesis method. The wild type and mutant were subeloned into pGL3 lueiferase reporter vectors to construct recombinant eukaryotic expression vector, named as pGL3-2439 and pGL3-mo2439 respectively. These two recombinant plas- mids, pGL3-basic plasmids or control vector and Oct4 plasmids were transient transfected into HEK 293 cells using lipofectamin 2000. After cultured 48 hours, the activity of promoters was determined by examine the relative activity of luciferase by dual Luciferase Re- porter Assay System. Results： Wild-type and mutant promoter sequence of Dppa2 was successfully cloned into recombinant vectors, which was vertified by Gel electrophoresis and DNA sequencing methods. The expression activity of human wild-type and mutant was also confirmed in the transfected HEK 293 cell. The relative activity of luciferase was 16.307 in the cells transfected with wild-type pro- moter, and 10.643 in the cells transfected with mutant promoter. When compared with wild type, the promoter activity of Dppa2 gene de- creased 35 % in the promoter with mutation of Oct4 binding site. Conclusion： The mutation of Oct4 binding site in the promoter region may down-regulates the transciption activity of Dppa2 gene.

关 键 词：Dppa2启动子 Oct4结合位点 荧光素酶 

分 类 号：Q-291[生物学]