猪血清白蛋白基因(alb)启动子克隆及活性分析  

Clone and Activity Analysis of Pig(Sus scorfa) Serum Albumin Gene(alb) Promoter

作　　者：程英[1,2] 黄雷[2] 吴添文[2] 樊俊华[2] 牟玉莲[2] 赵书红[1] 李奎[2] 

机构地区：[1]华中农业大学,农业动物遗传育种与繁殖教育部重点实验室,武汉430070 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《农业生物技术学报》2013年第9期1076-1084,共9页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育重大专项(No.2011ZX08010-004)

摘　　要：血清白蛋白(albumin,alb)是血清中重要的运输蛋白,主要由肝脏合成。本研究旨在确定猪血清alb启动子活性区域及其主要调控元件,探索alb基因的表达调控机制。首先通过qRT-PCR方法检测猪(Sus scorfa)不同组织及肝脏细胞系(hepa1-6)、和胎儿成纤维细胞系(PEF)中内源性alb基因表达情况;采用基因克隆,DNA测序等技术手段,构建含有5个不同长度猪alb基因启动子区域的双荧光素酶报告基因载体,分别转染hepa1-6以及PEF,检测不同长度启动子活性。结果表明,猪alb基因为肝脏特异性表达基因;其启动子能启动体外荧光素酶报告基因表达;其核心启动子区域位于-184～-31 bp,该段序列的缺失造成启动子启动活性丧失;-184～-2 034 bp间存在参与猪alb基因表达调控的潜在正负调控元件。本研究构建了含有5个五指山猪alb基因启动子片段的双荧光素酶报告载体,确定了该基因的启动子核心区域,预测了alb基因起始密码子上游约2 kb内主要调控元件,有助于进一步研究该基因表达调控分子机制以及构建外源基因组织特异性表达载体。Serum albumin（alb） is the majority transport protein in serum, which mainly is synthesized by the liver. The objective of this study was to identify porcine serum albumin gene（alb） promoter region and its main regulation elements, to quest the alb gene＇ s expression and regulation mechanisms. Firstly, endogenous expression level of alb from different tissues of pig（Sus scorfa） and different cell lines of hepatic（hepal-6） and porcine embryo fibroblkast （PEF） were analyzed by quantitative RT-PCR（qRT-PCR）; Luciferase reporter vectors containing five different lengths of pig alb promoter region were constructed by cloning, DNA sequencing and other methods. Hepal-6 and non hepatic PEF cell lines were transfected with these 5 vectors above-mentioned, then functions of different alb promoter region were validated through the observation of relative activities of luciferase reporter genes. Our results demonstrated that the pig alb gene showed a unique liver-specific expression pattern; the dual luciferase reporter assays revealed core region of alb promoter waslocated between -184 to -31 bp, deletion of this area would lead to loss of function. Between -184 - -2034 bp there existed some potential positive and/or negative regulatory elements involved in the regulation of pig alb gene expression. In this study five dual luciferase reporter vectors containing different fragments of Wuzhishan pig alb gene promoter were successfully constructed, core region of the promoter of the Wuzhishan pig alb gene were identified and the main regulatory elements within 2 kb upstream of the start codon were predicted. These studies may contribute to our understanding of the effective mechanisms of pig alb gene promoter, as well as construction of liver-specific expression vectors for further exogenous gene studies.

关 键 词：猪 血清白蛋白 核心启动子 调控元件 

分 类 号：S532[农业科学—作物学]