鸡ApoB基因g.-112A>G单核苷酸多态性的功能性鉴定与分析  

Functional Identification and Analysis of ApoB gene SNP g.-112A>G in Chickens(Gallus gallus)

作　　者：陈耀峰[1] 谢可嘉 张长超 王宁[1] 李辉[1] 王守志[1] CHEN Yao-Feng;XIE Ke-Jia;ZHANG Chang-Chao;WANG Ning;LI Hui;WANG Shou-Zhi(Key Laboratory of Chicken Genetics and Breeding,Ministry of Agriculture/Key Laboratory of Animal Genetics,Breeding and Reproduction,Education Department of Heilongjiang Province/College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]农业部鸡遗传育种重点实验室/黑龙江省普通高等学校动物遗传育种与繁殖重点实验室/东北农业大学动物科学技术学院,哈尔滨150030

出　　处：《农业生物技术学报》2018年第11期1919-1927,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31572394);东农学者计划学术骨干项目(No.15XG13);现代农业产业技术体系建设专项资金(CARS-41);国家高技术研究发展计划(863)项目(No.2011AA100301)

摘　　要：载脂蛋白B (apolipoprotein B, ApoB)在极低密度脂蛋白(very low density lipoproteins, VLDL)组装、转运以及脂类代谢中都具有重要作用。本课题组前期研究发现鸡(Gallus gallus) ApoB基因g.-112A>G单核苷酸多态性(single nucleotide polymorphism, SNP)与鸡腹脂重和腹脂率显著相关。生物信息学分析显示,该SNP位于鸡ApoB基因的启动子区,可能是功能性SNP。为确定该SNP的功能性,本研究开展了该位点不同基因型与血清生化指标的相关分析;比较了ApoB基因3种基因型在鸡肝脏和小肠中的表达水平;构建了含该SNP位点A和G等位基因的启动子报告基因载体,并利用双荧光素酶报告系统分析候选转录因子TATA-box binding protein (TBP)和CCAAT/enhancer binding protein alpha (C/EBPα)对该SNP位点不同等位基因荧光素酶活性的影响。关联分析结果显示,携带GG基因型的公鸡高密度脂蛋白(highdensity lipoprotein, HDL)/低密度脂蛋白(low-density lipoprotein, LDL)显著高于AA型个体(P<0.05);qRTPCR结果显示,在鸡肝脏和小肠组织,AG基因型的ApoB基因表达量显著低于AA和GG基因型(P<0.05);报告基因检测结果显示,A等位基因荧光素酶活性显著高于G等位基因(P<0.05)。此外,转录因子TBP和C/EBPα对A等位基因荧光素酶活性的促进作用强于G等位基因(P<0.05)。上述结果提示,ApoB基因g.-112A>G可能是功能性SNP位点,可初步确定其为优质鸡育种的功能性分子标记。本研究为深入探讨ApoB基因在脂肪生长发育中的功能提供了实验数据。The apolipoprotein family plays an important role in the transport and metabolism of lipids and cholesterol in the body. The apolipoprotein B （ApoB） plays an important role in lipid metabolism. Previously it was found that the genotypes of chicken （Gallus gallus） ApoB g.-112A〉G （single nucleotide polymorphism, SNP） were significantly associated with abdominal fat weight and abdominal fat percentage in chickens. To determine whether the ApoB gene g.-112A〉G is a functional SNP, we conducted the following research work： Association analysis between different genotypes and plasma biochemical markers, body weight and abdominal fat traits; The mRNA expression levels of the 3 ApoB genotypes in the liver and small intestine of chickens were detected and compared; The luciferase reporter gene vector containing different alleles in the promoter region of chicken ApoB gene was constructed and transfected into chicken fibroblast cell （DF1） and human hepatoma （HepG2） cells respectively; The possible mechanism of action of the ApoB gene g.-112A〉G was predicted and analyzed by on-line bioinformatics software. It was found that the A allele could change the TBP and C/EBPα transcription factor binding sites in the chicken ApoB gene promoter region. It could be assumed that the g.-112A〉G could influence the promoter activity of ApoB gene, which in turn affected gene expression. To confirm this hypothesis, the effect of pCMV-TBP and pCMV-C/EBPα on the activity of the dual-luciferase reporter gene of different alleles was analyzed in DF1 and HepG2 cells. The major results as follows： （1） Association analysis showed that genotypes of g.-112A〉G in ApoB gene were significantly associated with high-density lipoprotein （HDL） （P〈0.01）, and significantly associated with HDL/low-density lipoprotein （LDL） and Body Weight （BW） （P〈0.05）, and closely associated with Triglyceride （TG）（P=0.0549）, Total Cholesterol （TC）（P=0.0926） and Abdominal Fat Weight （AFW） （P

关 键 词：鸡(Callus gallus) 载脂蛋白B基因(ApoB) 启动子 功能性SNP 

分 类 号：S831[农业科学—畜牧学]