牛Nramp1基因启动子的克隆及其活性分析  

Cloning and Activity Analysis of Bovine Natural Resistance Associated Macrophage Protein 1(Nramp1) Gene Promoter

作　　者：王洪梅[1] 张利博[1] 侯明海[1] 王长法[1] 王玲玲[1] 孙涛[1] 何洪彬[1] 仲跻峰[1] 

机构地区：[1]山东省农业科学院奶牛研究中心,济南250100

出　　处：《中国农业科学》2011年第5期1022-1028,共7页Scientia Agricultura Sinica

基　　金：国家"863"计划项目(2006AA10Z1D9;2007AA10Z169);山东省自然基金(ZR2010CM012);公益性行业科技专项(nyhyzx07-036-09);泰山学者海外特聘专家(何洪彬);转基因专项(2009ZX08007-006B)

摘　　要：【目的】牛Nramp1基因是主要的抗病候选基因,但其转录调控的分子机制尚不清楚。本研究欲确定牛Nramp1基因的启动子区域,找到启动子核心序列和主要的调控区,探索Nramp1基因表达机制。【方法】采用基因克隆、DNA测序、半定量RT-PCR和荧光素酶报告基因系统等技术手段,构建牛Nramp1基因5′侧翼区长片段及固定3′端的不同节段的pEGFP-N1和/或pGL3重组质粒,分别转染293T和RAW264.7细胞,并进行脂多糖(LPS)诱导,对不同片段的启动子活性进行定性和定量测定。【结果】牛Nramp1基因5′侧翼区长片段具有较强的启动子活性,+58—-89区域具有基本的启动子功能,+58—-1 748启动子活性最强。进一步研究表明,-89—-205 bp区域、-278—-1 495 bp区域存在着正调控元件,在-205—-278 bp区域内存在着负调控元件;另外,LPS能显著增强启动子活性,其诱导牛Nramp1基因的表达具有细胞特异性和剂量依赖性。【结论】成功构建了含推测的牛Nramp1基因启动子片段的重组报告基因载体,确定了启动子核心区域和主要的调控区域。【Objective】Bovine natural resistance associated macrophage protein 1 gene（Nramp1） is a major anti-disease candidate gene,but its mechanism of transcriptional regulation remains unclear.To discover the Nramp1 gene expressional mechanism,its promoter region was determined,and the core sequences and major domains were found.【Method】The 5′flanking region and different fixed 3′ terminal fragments of bovine Nramp1 were cloned and recombined into EGFP-N1 and/or pGL3-basic plasmids.After DNA sequencing confirmation,the recombined vectors were transfected into 293T and murine mammary epithelial cells RAW264.7,respectively,and treated with lipopolysaccharide（LPS）,and then the promoter activities of different fragments of 5′flanking region of Nramp1 gene were determined by qualitative and quantitative assays including semi-quantitative RT-PCR and luciferase reporter gene analysis assay.【Result】The studies showed that the long DNA fragment,sequence from ＋58 to-89,and sequence up to-1 748 of 5′flanking region,had the stronger,basal,or maximal promoter activities,respectively.Further studies showed that there were positive（-89/-205 and-278/-1 495） and negative（-205/-278） regulatory domains,respectively.In addition,Escherichia coli LPS significantly enhanced the activity of Nramp1 promoter,and induced its expression in a dose-dependent and cell specific way.【Conclusion】The recombinant reporter plasmids containing different deduced fragments of Nramp1 promoter were successfully constructed,and its core and major regulatory regions were found.

关 键 词：牛Nramp1基因 启动子活性 调控区 LPS诱导 

分 类 号：S823[农业科学—畜牧学]