COL1A1基因启动子区突变（-106C〉T）对其表达的影响  

The mutation in promoter region of COL1A1 gene and its impacts on the COL1A1 gene expression

作　　者：李连永[1] 赵群[1] 高红[2] 王莉莉[2] 

机构地区：[1]中国医科大学附属盛京医院小儿骨科,沈阳110104 [2]卫生部小儿先天畸形重点实验室

出　　处：《中华小儿外科杂志》2010年第7期541-544,共4页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金资助项目（编号：30600654）;辽宁省科学技术计划项目（编号;20052250131）

摘　　要：目的 探讨COL1A1基因启动子区突变（-106C〉T）对其表达的影响。方法PCR扩增正常人COL1A1基因转录起始点上游-1024＋36bp的启动子序列，片段长度为1060bp。经T／A克隆，连接酶切位点，再连接到PGL3-basic载体，得到野生型的PGL3-COL1A1表达载体。再采用定点突变的方法，把-106位碱基的C突变成T，再连接到PGL3-basic载体，构建突变型的PGL3-COL1A1表达载体。用上述两种表达载体及PGL3-basic分别瞬时转染NIH3T3细胞。细胞培养48h后，测定各组细胞表达的荧光素酶的相对活性，以明确COL1A1启动子区-106位碱基C〉T突变对基因转录调控的影响。结果经琼脂糖凝胶及基因测序鉴定，证实载体构建成功。荧光素酶活性测定结果显示，在转染野生型PGL3-COL1A1表达载体的细胞组，荧光素酶的相对活性为3．8665，为阴性对照组（PGL3-basic）的4．8倍；转染突变型PGL3-COL1A1表达载体的细胞组，荧光素酶的相对活性为1．3917，为阴性对照组的1．7倍。COL1A1基因启动子-106位碱基C〉T突变后，其转录活性较野生型降低了64％。结论COL1A1基因启动子区-106C〉T突变可以在转录水平抑制该基因的表达。Objective To investigate the mutation in promoter region of COL1A1 gene and its impacts on the COL1A1 gene expression. Methods The wild-type promoter sequence （ - 1024 bp-＋ 36 bp） of human COL1A1 gene was amplified. The mutations in the promoter region were obtained using site directed mutagenesis method. The wild-type and mutant promoter were subcloned into pGL3 luciferase reporter vectors to construct recombinant eukaryotic expression vector （PGL3-COL1A1）. The recombinant plasmids were transfected into NIH 3T3 cells using lipofectamin method, and the activity of promoters was determined by examine the relative activity of lueiferase. Results Enzyme digestion and sequencing analysis showed the target sequences were cloned into recombinant vectors successfully. The expression activity of human wild-type and mutant promoter was also confirmed in the transfected NIH 3T3 cell. The relative activity of luciferase was 3, 8665 in the cells transfected with wild-type promoter, and 1. 3917 in the cells transfected with mutant promoter, The mutation of - 106C〉T in promoter region of COL1A1 gene decreased the promoter activity by 64%. Conclusions The mutation （-106C〉T） in the promoter region down-regulates the transcription activity of COL1/A1 gene.

关 键 词：髋发育不良 先天性 COL1A1基因 基因表达调控 

分 类 号：R726.8[医药卫生—儿科]