猪NCOA3启动子甲基化对其在肌内和皮下脂肪组织差异表达的影响  

Effect of NCOA3 promotor methylation on its differential expression profile in intramuscular and subcutaneous adipose tissue in porcine

作　　者：谷淑华[1] 孙文星[1] 褚维伟[1] 韩海银 党小勇 陈杰[1] 

机构地区：[1]南京农业大学动物科技学院,江苏南京210095

出　　处：《南京农业大学学报》2016年第1期120-126,共7页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31272423)

摘　　要：[目的]以新淮猪肌内脂肪和皮下脂肪为试验材料,探讨核受体辅激活蛋白3(NCOA3)启动子甲基化对其在肌内与皮下脂肪组织中差异表达的影响。[方法]采用RT-PCR的方法检测NCOA3在肌内脂肪与皮下脂肪组织的表达水平。构建NCOA3启动子缺失报告载体,并采用双荧光报告基因系统分析启动子区域活性。预测NCOA3启动子区Cp G岛,利用亚硫酸氢盐法(BSP)检测2种脂肪组织NCOA3启动子区Cp G岛甲基化水平,并通过甲基转移酶处理,以检测甲基化对NCOA3基因启动子活性的影响。[结果]NCOA3 mRNA在肌内脂肪组织中表达水平极显著低于皮下脂肪组织(P<0.01),其中-150^-3 bp区域启动子活性最高,Cp G岛位于-930^-699 bp,且其整体甲基化水平在肌内脂肪与皮下脂肪组织中差异不显著(P>0.05),分别为90.0%,83.5%,但-904位点CG甲基化水平差异显著(P<0.05)。经甲基转移酶处理p LUC-1057(包含Cp G岛)载体荧光活性显著下降(P<0.05)。[结论]NCOA3基因在肌内脂肪与皮下脂肪组织存在着表达差异,-904位点CG甲基化水平对肌内与皮下脂肪组织表达差异存在影响。[Objectives]We explore the effect of the nuclear coactivator 3（ NCOA3） promoter methylation on its different expression patterns in intramuscular and subcutaneous adipose tissue in Xinhuai porcine. [Methods]The mRNA expression of NCOA3 in intramuscular and subcutaneous adipose tissue was determined by RT-PCR. Analysis of the activity of NCOA3 promoter of was identified by dual-luciferase reporter system. Methylation level was detected by sodium bisulfite genomic sequencing for the Cp G island in NCOA3 promoter. Effects of methylation level on promoter activity were determined through in vitro methylation analysis. [Results]The mRNA expression of NCOA3 in intramuscular adipose tissue was significantly lower than in subcutaneous adipose tissue（ P 0.01）. NCOA3 core promoter and Cp G island were located in- 150 to- 3 bp and- 930 to- 699 bp region respectively. The whole methylation level of Cp G island showed no differences between intramuscular（ 90. 0%） and subcutaneous（ 83. 5%） adipose tissue.However,the methylation level of- 904 CG site in intramuscular fat（ 55%） was higher than that in subcutaneous fat（ 20%）（ P 0.05）. Methyltransferase treatment of NCOA3 promoter region,which increased methylation level of Cp G island,led to significant depression of gene acti-vities（ P 0. 05）. [Conclusions]The expression of NCOA3 was significantly different in intramuscular and subcutaneous adipose tissue. Methylation level of- 904 CG site might have effect on NCOA3 differential expression in intramuscular and subcutaneous adipose tissue.

关 键 词：NCOA3基因 启动子 甲基化 肌内脂肪 皮下脂肪 

分 类 号：S852.23[农业科学—基础兽医学]