UCP3基因在巴马猪和藏猪脂肪组织中的表达和甲基化分析

Expression and Methylation Analysis of UCP3 Gene in Adipose Tissue of Bama and Tibetan Pigs

为探究解偶联蛋白3(uncoupling protein 3,UCP3)基因在巴马猪和藏猪皮下脂肪组织中的表达和甲基化水平,试验采用实时荧光定量PCR技术检测UCP 3基因在巴马猪和藏猪皮下脂肪组织中的mRNA表达水平;针对猪UCP 3基因启动子区域(-3580^+920 bp),利用在线软件MethPrimer对该区域进行CpG岛预测,并采用亚硫酸氢盐测序法(bisulfite sequencing PCR,BSP)检测其甲基化水平,探究UCP 3基因甲基化水平在巴马猪和藏猪中的差异。结果显示,巴马猪皮下脂肪组织UCP 3基因表达量显著高于藏猪(P<0.05);在UCP 3基因启动子区预测到3个CpG甲基化岛,分别是CpG island1(-3171^-2928 bp)、CpG island2(-154^-2 bp)和CpG island3(+648^+806 bp),其中CpG island1和CpG island3的甲基化水平在巴马猪和藏猪中差异较小,而藏猪CpG island2的甲基化水平(42.61%)高于巴马猪(24.49%)。本研究绘制了2个猪种CpG island2甲基化水平的黑白点图,其中CpG位点为4、8、9、10、11、12、15,藏猪甲基化频率分别比巴马猪高28.26%、17.39%、26.09%、26.09%、26.09%、23.91%和34.78%。在CpG island2处预测到3个转录因子结合位点(SP2、PPARγ和EGR1)。结果表明,巴马猪和藏猪皮下脂肪组织中UCP 3基因mRNA水平的表达差异可能是由于CpG island2的甲基化水平不同所导致,藏猪DNA甲基化水平在一定程度上阻碍了转录因子与启动子调控区域的结合,从而抑制了UCP 3基因的表达。

The aim of this study was to investigate the expression and methylation levels of the uncoupling protein 3(UCP3)in subcutaneous adipose tissue of Bama and Tibetan pigs.The expression of UCP 3 gene at mRNA level was detected by Real-time quantitative PCR method.CpG islands were predicted for the sequence in pig UCP 3 gene promoter region(-3580 to+920 bp)using online software MethPrimer.The difference methylation level of UCP 3 gene in Bama and Tibetan pigs was detected by bisulfite sequencing PCR(BSP).The results showed that the expression of UCP 3 gene in subcutaneous adipose tissue of Bama pigs was significantly higher than that of Tibetan pigs(P<0.05).Three CpG methylation islands were predicted in the promoter region of UCP 3 gene,which were recorded as CpG island1(-3171 to-2928 bp),CpG island2(-154 to-2 bp)and CpG island3(+648 to+806 bp).The methylation levels of CpG island1 and CpG island3 were less different in Bama and Tibetan pigs,while the methylation level of CpG island2(42.61%)was significantly higher in Tibetan pigs than that in Bama pigs(24.49%).A black and white dot maps were built based on the CpG island2 methylation of Bama and Tibetan pigs,the methylation frequency of CpG sites 4,8,9,10,11,12 and 15 in Tibetan pigs were 28.26%,17.39%,26.09%,26.09%,26.09%,23.91%and 34.78%higher than that in Bama pigs,respectively.In addition,three transcription factor binding sites(SP2,PPARγand EGR1)were predicted at CpG island2.The results indicated that the differential expression of UCP 3 gene in subcutaneous adipose tissue of Bama and Tibetan pigs might be caused by the different methylation levels of CpG island2.The higher DNA methylation of adipose tissue in Tibetan pigs might inhibit the expression of UCP 3 gene at mRNA level by inhibiting the binding of transcription factors to promoter regulatory regions.