| 成纤维细胞生长因子19转录活性及上游结合元件分析 |
| 投稿时间：2016-11-23 修订日期：2016-11-30 点此下载全文 |
| 引用本文：徐宜兰,陈淑芹,张菁,白宁宁,方启晨.成纤维细胞生长因子19转录活性及上游结合元件分析[J].医学研究杂志,2017,46(6):75-79 |
| DOI：
10.11969/j.issn.1673-548X.2017.06.020 |
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| 基金项目:上海市自然科学基金资助项目 （15ZR1431700） |
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| 中文摘要:目的 构建FGF19基因启动子区荧光素酶报告基因质粒，并研究分析其启动子区的转录活性和结合元件。方法 设计特异性引物PCR扩增基因组DNA，得到长为3323bp的FGF19基因启动子区片段，将此PCR产物插入荧光素酶报告基因载体pGL3-Basic Vector，获得全长为3323bp的FGF19基因启动子区荧光素酶报告基因质粒。在此基础上设计特异性引物，构建5'端系列缺失荧光素酶报告基因质粒。通过瞬时转染实验检测所构建质粒的相对荧光素酶活性，分析不同启动子区片段对FGF19基因转录活性的影响，并用软件预测影响启动子转录活性的关键转录因子。结果 构建了7个FGF19基因启动子区荧光素酶报告基因质粒，经双酶切和测序验证均正确。瞬时转染及荧光素酶报告基因分析实验发现启动子区-2351~-2316是调控FGF19启动子转录活性的重要序列，且在线软件预测该序列存在潜在的转录因子位点。结论 FGF19基因启动子区-2351~-2316是调控其启动子转录活性的关键位置。 |
| 中文关键词:FGF19 转录调控 启动子 质粒构建 |
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| Analysis of Fibroblast Growth Factor19 in Transcriptional Regulation and Identification of Upstream Binding Elements |
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| Abstract:Objective To investigat transcriptional regulation mechanism about FGF19, we constructed a series of plasmids containing the promoter fragments of FGF19 gene. Methods In order to get a full length of 3323bp 5'-flanking fragment of FGF19, we designed specific primer to amplify the sequence, and then it was cloned into pGL3-basic vector. Based on the constructed promoter fragment of 3323bp, several plasmids were established with deletion of 5' terminal. Subsequently, transient transfection experiments were done to localize the core fragment of FGF19 promoter. The online software gene regulation, with the function of predicting transcription factor binding sites, identify latent transcriptional factors. Results We successfully cloned 7 promoter plasmids of FGF19 gene verified by restriction endonuclease digestion and sequence analysis. In view of the transient transfection results of dual luciferase reporter plasmids, the key transcriptional regulation fragment was localized between -2351 to -2316 of FGF19 promoter. Conclusion The fragment of FGF19 promoter from -2351 to -2316 is the core transcription regulation region and may contain the pivotal binding sites of transcription factors, which act as positive response elements in the promoter of FGF19. |
| keywords:FGF19 Promoter Transcriptional regulation Plasmids construction |
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