山羊PHLDA2启动子克隆和甲基化分析

Cloning and Methylation Analysis of PHLDA2 Promoter in Goat

为了研究山羊血小板白细胞C激酶底物同源性样结构域蛋白家族A成员2 (pleckstrin homology-like domain family A member 2, PHLDA2)基因表达水平与其启动子甲基化状态相关性,试验采用RT-PCR方法检测PHLDA2在各组织间的表达水平(心脏、肝脏、脾脏、肺脏、肾脏、肌肉、脂肪、舌、大脑、胎盘),以及扩增了山羊PHLDA2基因启动子区域序列,并利用Methylation specific PCR技术检测CpG岛在山羊各组织间的甲基化水平。结果表明:克隆得到的山羊PHLDA2 1842 bp的启动子序列与绵羊、牛PHLDA2启动子同源性分别达93.2%和87.9%;启动区CpG岛内第三位点CpG的甲基化频率和胎盘第一外显子区域甲基化水平显著低于其他组织(P 【0.05)。说明PHLDA2的mRNA相对表达量水平与启动子区CpG岛(−302~−88 bp)内第三个CpG位点甲基化频率和第一外显子CpG岛(49~333 bp)甲基化水平呈负相关。

In order to investigate the correlation between gene expression level of pleckstrin homology-like domain family A member 2 (PHLDA2) and its promoter methylation status of goat, RT-PCR was applied to detect the expression level of PHLDA2 in various tissues (heart, liver, spleen, lung, kidney, muscle, fat, tongue, brain and placenta) and clone the sequence of PHLDA2 gene promoter region of goat. Methylation specific PCR was used to detect the methylation level of CpG island in goat tissues. The results showed that the cloned goat PHLDA2 1842 bp promoter sequence had 93.2% homology with sheep and 87.9% homology with cattle PHLDA2 promoter, respectively, and the methylation frequency of CpG at the third site of CpG in the promoter region and the methylation level of the first exon of placenta were significantly lower than those of other tissues (P <0.05). These results suggest that the mRNA relative expression level of PHLDA2 was negatively correlated with the methylation frequency of the third CpG site and the methylation level of the first exon CpG island (49~333 bp) in the promoter region CpG island (−302~−88 bp).