中国医科大学学报

中国医科大学学报
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中国医科大学学报 ›› 2019, Vol. 48 ›› Issue (1): 29-33.doi: 10.12007/j.issn.0258-4646.2019.01.006

• 论著 • 上一篇    下一篇

小鼠Na+-K+-2Cl-共转运蛋白基因启动子克隆及20-HETE对其转录活性的影响

武晶晶1, 孔令慧2, 贾茹1   

  1. 1. 中国医科大学生命科学学院医学遗传学教研室, 沈阳 110122;
    2. 中国医科大学生命科学学院生物科学系, 沈阳 110122
  • 收稿日期:2018-05-23 出版日期:2019-01-30 发布日期:2019-01-05
  • 通讯作者: 武晶晶 E-mail:wujj@cmu.edu.cn
  • 作者简介:武晶晶(1983-),女,副教授,博士.
  • 基金资助:
    国家自然科学基金青年科学基金(81400709)

Cloning of the Murine Na+-K+-2Cl- Cotransporter Gene Promoter and the Effect of 20-HETE on Its Transcriptional Activity

WU Jingjing1, KONG Linghui2, JIA Ru1   

  1. 1. Department of Medical Genetics, School of Life Sciences, China Medical University, Shenyang 110122, China;
    2. Department of Biological Science, School of Life Sciences, China Medical University, Shenyang 110122, China
  • Received:2018-05-23 Online:2019-01-30 Published:2019-01-05

摘要: 目的 克隆小鼠Na+-K+-2Cl-共转运蛋白(Nkcc2)基因启动子序列,初步探讨20-HETE对该基因转录活性的调控作用。方法 应用生物信息学软件ALiBaba2.1和TRANSFAC TESS分析小鼠Nkcc2基因启动子序列;以小鼠基因组DNA为模板,利用PCR方法扩增得到小鼠Nkcc2基因启动子区片段(-1 462 bp~+40 bp),并克隆入pGL3-Basic载体,构建萤光素酶报告基因载体;脂质体法将重组报告基因载体转染到HEK293T细胞24 h后,再以20-HETE处理转染细胞2 h,利用双萤光素酶报告基因检测系统分析萤光素酶活性。结果 成功构建小鼠Nkcc2基因启动子萤光素酶报告基因载体;20-HETE可以显著下调小鼠Nkcc2基因启动子萤光素酶报告基因载体的转录活性。结论 20-HETE通过转录调控机制下调小鼠Nkcc2基因的表达。

关键词: Na+-K+-2Cl-共转运蛋白, 启动子, 20-HETE, 基因克隆

Abstract: Objective To clone the murine Na+-K+-2Cl- cotransporter (Nkcc2) gene promoter and analyze 20-HETE regulation of the murine Nkcc2 gene transcriptional activity. Methods A fragment of the murine Nkcc2 gene promoter was analyzed using bioinformatics software AliBaba and TRANSFAC TESS. The murine Nkcc2 gene promoter fragment (-1 462 bp-+40 bp) was amplified by PCR using murine genomic DNA as a template and then cloned into a pGL3-Basic vector to generate a luciferase reporter construct. The recombinant reporter construct was transiently transfected into HEK293T cells using Lipofectamine 2000 for 24 h. The transfected HEK293T cells were treated with 20-HETE for 2 h followed by measurement of the luciferase activity using the Dual-Luciferase Reporter Assay system. Results A luciferase reporter construct containing the murine Nkcc2 gene promoter was successfully generated. The results showed that 20-HETE significantly reduced the transcriptional activity of the construct. Conclusion 20-HETE may reduce the expression of the murine Nkcc2 gene through transcriptional regulation.

Key words: Na+-K+-2Cl- cotransporter, promoter, 20-HETE, gene clone

中图分类号: 

  • R394
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