|
| 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: |
| 基金项目: |
|
| 摘要点击次数: 707 |
| 全文下载次数: 0 |
| 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. |
| 查看/发表评论 下载PDF阅读器 |
| 关闭 |