| 摘要: |
| 【目的】构建牛ATP5B基因启动子双荧光素酶报告基因重组质粒，并检测其在C2C12细胞系中的表达活性。【方法】从牛外周血中提取基因组DNA，通过PCR方法从牛基因组DNA中克隆获得牛ATP5B基因的5′端转录调控区的 1 898 bp目的片段，通过设计引物逐段缺失后获得7个亚克隆，将其纯化后经SmaⅠ和KpnⅠ双酶切与pGL3-Basic载体连接，连接产物转化感受态细胞DH5α，得到牛ATP5B基因启动子双荧光素酶报告基因重组质粒，经脂质体基因转染法转染C2C12细胞系后，检测7个重组质粒的荧光素酶活性；运用在线软件Gen-omatix和TFSEARCH对ATP5B启动子区序列进行分析。【结果】成功克隆获得7个系列缺失的牛ATP5B基因启动子双荧光素酶报告基因重组质粒pATP5B-1898、pATP5B-1607、pATP5B-1293、pATP5B-992、pATP5B-678、pATP5B-462和pATP5B-145；通过转染细胞和荧光素酶活性分析，可知构建的重组质粒均有启动子活性，且重组质粒pATP5B-678和pATP5B-462与空载体pGL3-Basic的荧光素酶活性差异极显著。软件分析结果显示，ATP5B基因启动子区域 -763～-85 bp存在多个重要转录调控元件。【结论】成功构建了7个系列缺失的牛ATP5B基因启动子双荧光素酶报告基因重组质粒，且证实 -763～-230 bp为牛ATP5B基因的核心启动子区域。 |
| 关键词: 牛 ATP5B基因启动子 双荧光素酶报告基因载体 |
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| 基金项目:国家自然科学基金项目（31272411）；国家“863”计划项目（2013AA102505，2011AA100307-02）；国家科技支撑计划项目（2011BAD28B04-03）；国家转基因育种专项（2011ZX08007-002）；国家肉牛牦牛产业技术体系项目（CARS-38）；陕西省科技统筹创新工程计划项目（2014KTZB02-02） |
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| Construction and identification of bovine ATP5B gene promoter dual luciferase report gene vector |
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DUAN Mei-yan,LI An-ning,ZHAO Zhi-dong,et al
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| Abstract: |
| 【Objective】The research was conducted to construct the dual luciferase reporter gene plasmid of ATP5B gene promoter from bovine and detect the expression activities by transfecting into the C2C12 cells.【Method】Bovine genome DNA was extracted from blood cells,and from which a 1 898 bp fragment of the 5′ flanking region of ATP5B gene was isolated by PCR.Then 7 sub-clones were obtained by the design of primers.The PCR products were purified and double digested by SmaⅠ and KpnⅠ before being inserted into pGL3-Basic vectors by homologous recombination and the vectors were transferred into DH5α cells.At last,dual luciferase report gene vectors of bovine ATP5B gene promoter were constructed and transfected into the C2C12 cells by LPS before the activities of 7 recombined plasmids were measured and the sequence of ATP5B gene 5′ end was analyzed by online software.【Result】A total of 7 recombined plasmids (pATP5B 1898,pATP5B-1607,pATP5B-1293,pATP5B-992,pATP5B-678,pATP5B-462,and pATP5B-145) of bovine ATP5B gene promoter dual luciferase report gene vectors were successfully cloned.Luciferase activity assay indicated that all the recombined plasmids had significant activity.pATP5B-678 and pATP5B-462 had extremely significant difference compared to pGL3 Basic.Sequence analysis by Gen-omatix and TFSEARCH indicated that there were many putative binding sites for transcription factors in the region of -763－-85 bp of ATP5B gene promoter.【Conclusion】A total of 7 dual luciferase report gene recombined plasmids of bovine ATP5B gene promoter were successfully constructed,activity analysis in C2C12 cells indicated that the regionsof －763--230 bp contained key transcription regulatory elements. |
| Key words: bovine ATP5B promoter dual-luciferase reporter |
