牛ATP5B基因启动子双荧光素酶报告基因载体构建与活性检测

被引：4

作者：

段美艳 ^{[1
]}

李安宁 ^{[1
]}

赵志东 ^{[1
]}

王明明 ^{[1
]}

昝林森 ^{[1
,2
]}

机构：

[1] 西北农林科技大学动物科技学院

[2] 国家肉牛改良中心

来源：

西北农林科技大学学报(自然科学版) | 2015年 / 43卷 / 08期

关键词：

牛; ATP5B基因启动子; 双荧光素酶报告基因载体;

D O I：

10.13207/j.cnki.jnwafu.2015.08.026

中图分类号：

S823 [牛]; Q78 [基因工程（遗传工程）];

学科分类号：

0905 ; 071007 ; 0836 ; 090102 ;

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 898bp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-763-85bp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-763-230bp为牛ATP5B基因的核心启动子区域。

引用

页码：39 / 45

页数：7

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