小鼠PGC-1a基因启动子转录调控体系载体的构建及活性鉴定
Cloning and Analyzing the Promoter of Mouse PGC-1a Gene
投稿时间:2013-08-27  修订日期:2013-11-05
DOI:
中文关键词:  细胞生物学  PGC-1a  启动子  转录调控
英文关键词:
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作者单位E-mail
冉莉 南华大学附属第一医院内分泌科 22236347@qq.com 
涂晶 南华大学附属第一医院临床研究所  
杨靖 南华大学附属第一医院内分泌科  
钟警 南华大学附属第一医院临床研究所  
洪涛 南华大学附属第一医院内分泌科  
文格波 南华大学附属第一医院临床研究所  
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中文摘要:
      目的:克隆小鼠PGC-1a基因的启动子并分析其转录调控模式。方法:设计引物,以小鼠肝脏组织DNA 为模板, PCR 扩增小鼠 PGC-1a 基因启动子区并克隆入荧光素酶报告基因表达载体pGL3-Basic 中,构建具有PGC-1a 基因启动子区转录活性的报告质粒pGL3-PGC-1a-Luc。在此基础上,通过设计特异性引物,引入替换碱基突变相关转录因子调控模序,构建点突变融合载体。报告质粒与内参质粒共转染1c1c7 及c2c12 细胞系,48 h 后收获细胞检测荧光素酶的表达情况。结果:构建的pGL3- PGC-1a -Luc 系列报告质粒经过酶切鉴定及DNA 测序分析都显示正确;转染2 种细胞系后进行荧光素酶活性检测的结果表明,PGC-1a基因启动子区域(-2533- 78)具有较强的转录活性;突变失活PGC-1a基因启动子区域中的CRE反应原件导致转录活性明显降低。结论:成功构建小鼠PGC-1a基因启动子报告质粒,证实PGC-1a 基因上游区域(-2533- 78)具有较强的启动子活性;启动子区域中的CRE和IRE反应原件为主要转录调控位点。
英文摘要:
      In this paper, we cloned mouse PGC-1a promotor (-2533- 78)and constructed the pGL3-PGC-1a-Luc reporter vector and its mutants. The transcription activity was analyzed by Luciferase assay. PCR was used to obtain DNA fragments that contain the promoter of PGC-1a gene by using genome DNA prepared from mouse liver as the templates. Reporter plasmids pGL3-PGC-1a-Luc which contain wild-type or mutant-type promoter were constructed. After the reporter plasmids were transiently transfected into 1c1c7 and c2c12 cells, the Luciferase activity were tested. Result showed that mouse PGC-1a promotor fragments were correctly amplified and the reporter plasmids were successfully constructed ;Luciferase assays of the constructed reporter plasmids in two cell lines showed that the promoter region(-2533- 78)had transcriptional activity, the CRE and IRE sites were major transcriptional regulatory regions in the promoter.
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