猪肌肉组织中ApoE基因5′调控区甲基化的研究

DNA methylation in the 5′regulatory region of porcine ApoE in muscle tissues

【目的】研究猪肌肉组织中猪载脂蛋白E(Apolipoprotein E,ApoE)基因5′调控区的DNA甲基化状况。【方法】以180日龄长白母猪为试验动物,采集其肌肉组织,提取其DNA,经亚硫酸氢盐修饰后用作PCR反应模板;通过生物信息学预测猪ApoE5′调控区潜在的CpG岛,根据预测结果选用甲基化特异性引物对CpG岛进行PCR扩增,对回收、纯化的PCR产物进行克隆和测序,并与GenBank中的ApoE5′调控区序列进行比对,分析CpG岛的甲基化水平。【结果】生物信息学预测发现,猪ApoE基因5′调控区有2个CpG岛,第1个CpG岛有10个CpG位点,第2个CpG岛有12个CpG位点;PCR扩增获得长度分别为233bp的CpG岛1和288bp的CpG岛2序列,这2个CpG岛均存在着9个甲基化CpG位点。【结论】猪ApoE基因5′调控区是CpG位点的富集区域,2个CpG岛存在不同程度的甲基化。

【Objective】 This study focused on DNA methylation levels of the 5′regulatory region of porcine ApoE gene in muscle tissues of Landrace sows.【Method】 Landrace sows at the age of 180 days were used as experimental animals,and muscle tissues were collected for subsequent DNA extraction.Modified DNA using sodium bisulfite was employed as the PCR reaction templates.The potential CpG islands in the porcine ApoE 5′regulatory region were predicted using bioinformatics and methylation-specific primers obtained from the prediction were chosen for amplifying the potential CpG islands.PCR amplicons were recycled,purified,and subsequently cloned into pMD-18T vectors for sequencing and then the sequences were aligned with that of the porcine ApoE 5′regulatory region from GenBank for analyzing the methylation levels of CpG islands in this region.【Result】 The results showed that two CpG islands within the porcine ApoE gene 5′regulatory region were found using bioinformatics and there were ten CpG sites within CpG island 1 and twelve within CpG island 2.Two sequences including the 233 bp length of CpG island 1 and the 288 bp of CpG island 2 were obtained.There were nine methylated sites within the two CpG islands.【Conclusion】 The CpG sites were rich in the 5′regulatory region of porcine ApoE gene and the two CpG islands were methylated in different degrees.