| 雷学华,吴风瑞,刘勇,丁彪,王荣,李文雍.2013.小鼠组蛋白H3K4甲基化酶Smyd3启动子荧光素酶报告载体的构建与活性分析.动物学杂志,48(1):22-27. |
| 小鼠组蛋白H3K4甲基化酶Smyd3启动子荧光素酶报告载体的构建与活性分析 |
| Construction and Analysis of Luciferase Reporter Vector for Promoter ofSmyd3, A Gene Encoding Mouse H3K4 Methylase |
| 投稿时间：2012-07-02 修订日期：2012-10-22 |
| DOI： |
| 中文关键词: 组蛋白H3K4甲基化 Smyd3启动子 活性分析 小鼠 |
| 英文关键词:Histone H3K4 methylase Smyd3 promoter Activity analysis Mouse |
| 基金项目:国家自然科学基金项目(No. 31071310,31201789);安徽省教育厅自然科学基金项目(No. KJ2012B132) |
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| 中文摘要: |
| 为研究小鼠(Mus musculus)组蛋白H3K4甲基化酶基因Smyd3转录调控的分子机制,本研究首先通过PCR的方法克隆了5条不同长度的Smyd3启动子5'端缺失片段,与pMD19-T载体连接后,双酶切克隆入pGL3-Basic荧光素酶报告基因载体,构建Smyd3启动子-pGL3-Basic报告基因重组质粒,瞬时转染HEK293细胞48 h后采用双报告基因检测试剂盒检测Smyd3启动子各缺失片段的相对荧光活性。结果表明,本研究成功构建Smyd3启动子5'端缺失片段-pGL3-Basic荧光报告基因重组质粒,所构建的启动子重组子转染组与阳性对照组相比表现出荧光活性,并且pGL3-Smyd3-4的荧光活性最强,是其他的2至4倍左右,pGL3-Smyd3-5的荧光活性最弱。本研究初步确定Smyd3基因的启动子核心区域可能位于-533~-42 bp之间,在-2 026~-533 bp之间可能存在启动子负调控序列。 |
| 英文摘要: |
| To further study the molecular mechanism of transcription regulation of the mouse(Mus musculus)histone H3K4 methylase gene Smyd3, different 5'-flanking regions of Smyd3 promoter were cloned by PCR and inserted into pMD19-T vector. The pMD19-T vector was digested by two enzymes, and then inserted into pGL3-Basic luciferase reporter vector. The recombination plasmids were transiently transfected into HEK293 cells, and then its fluorescence activity was measured with the dual-luciferase reporter assay after 48 hours. The results showed that pGL3-Basic-Smyd3-1-pGL3-Basic-Smyd3-5 luciferase reporter gene vectors were constructed successfully. Compared with the positive control, construction of promoter recombinants transfected group showed fluorescence activity, and the pGL3-Smyd3-4 fluorescence was most active, about 2-4 times of the others, while the pGL3-Smyd3-5 fluorescence activity was the weakest. This study suggests that the core promoter region of the Smyd3 gene may be located on the up-stream between -533 bp to -42 bp and that the area between -2 026 bp to -533 bp is a transcriptional negative regulation region. |
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