猪Nramp1基因启动子克隆及其组织表达活性分析  

Cloning of the Promoter of Porcin(Sus scrofa) Nramp1 Gene and Analysis of Its Differential Expression in Various Tissues

作　　者：顾克翠[1] 王欢欢[1] 谢周瑞[1] 徐银学[1] 陈杰[1] 

机构地区：[1]南京农业大学动物科技学院,南京210095

出　　处：《农业生物技术学报》2013年第2期165-172,共8页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育重大专项(No.2009ZX08009-143B)

摘　　要：Nramp1基因在猪肺泡巨噬细胞中具有较高的组织表达特异性,本研究的目的是克隆并分析猪(Sus scrofa)Nramp1基因在肺泡巨噬细胞中的特异性表达调控元件。本研究首先利用5'RACE技术确定了Nramp1基因转录起始位点,在此基础上构建了-1327～+86bp区域内不同长度的嵌套缺失片段,分别连接至pGL3-BASIC载体上,构建了pLUC1430、pLUC1136、pLUC840、pLUC751、pLUC487、pLUC379、pLUC274及pLUC179共8个缺失表达载体。采用脂质体转染法,与海肾荧光素酶载体共转染猪肺泡巨噬细胞、肾细胞、脂肪前体细胞和胎儿成纤维细胞,采用双荧光素酶报告系统验证了Nramp1基因不同启动子片段的表达活性,结果表明Nramp1基因核心启动子区位于-386～-173bp。进一步比较了pLUC487[-386～+86]启动子区在巨噬细胞、猪肾细胞、脂肪细胞和胎儿成纤维细胞中的表达活性,结果表明Nramp1基因在巨噬细胞中的表达活性极显著地高于肾细胞、脂肪细胞和成纤维细胞(P<0.01),表明Nramp1基因启动子具有较高的巨噬细胞特异性。本研究获得了能够在猪肺泡巨噬细胞中特异表达的调控元件,为进一步实现外源基因在猪肺泡巨噬细胞中的特异性表达提供了基础。This study aims to obtain the transcription regulatory region of Nrampl gene which is tissue-specifically expressed in porcine pulmonary alveolar macrophages. The study identified the transcription start site of pig （Sus scrofa） Nrampl gene using 5＇-RACE. Eight expression vectors, pLUC1430, pLUC1136, pLUC840, pLUC751, pLUC487, pLUC379, pLUC274 and pLUC179, with serial deleted upstream fragments were constructed by cloning different deletion fragments of-1 327 bp to ＋86 bp region into pGL3-Basic vector, respectively. The expression vectors were co-transfected with Renilla luciferase vector into porcine pulmonary alveolar macrophages, kidney ceils, preadipocytes and fetal fibroblast cells, respectively. Expression activities were analyzed using dual luciferase reporter system for different fragments in order to identify the core regulatory region. Our result showed the core -173 bp. The transcripion activities of pLUC487 regulatory region ofNrampl gene ranged from -386 bp to [-386-＋86] region were further compared among porcinepulmonary alveolar macrophages, kidney cells, preadipocytes and fetal fibroblast cells, in which Nr（unpl promoter exhibited significantly higher activity in porcine pulmonary alveolar macrophages （P〈O.O1）. We confered that Nramp I promoter was specifically expressed in porcine alveolar macropbages. Our study gained a transcription regulatory element specifically expressed in pulmonary alveolar macrophages, which facilitates the tissue-specific expression of foreign genes in porcine pulmonary alveolar macrophages.

关 键 词：猪 肺泡巨噬细胞 Nramp1 启动子 双荧光素酶检测系统 

分 类 号：S532[农业科学—作物学]