牛DSCAM基因的基因组印记和DNA甲基化状态分析

Analysis of DNA methylation on DSCAM gene in different bovine tissues

为鉴定牛DSCAM基因的印记状态以及DNA甲基化修饰在调控印记表达中的作用,本研究以牛胎盘和成年牛组织(心、肝、脾、肺、肾、肌肉、脂肪和大脑)为试验材料,利用基于单核苷酸多态(SNP)的RT-PCR直接测序法分析DSCAM基因的印记状态,采用亚硫酸氢盐测序法分析基因启动子区的甲基化状态。结果发现,在DSCAM基因的第32个外显子上存在1个A/G杂合的SNP位点(rs136908595),利用该SNP位点区分亲本等位基因发现,在被检测的成年牛组织中,DSCAM基因为双等位基因表达;而在胎盘中DSCAM基因为母源等位基因表达。对牛DSCAM基因启动子区及第一个外显子处402 bp的CpG岛甲基化进行分析,发现在单等位基因表达的胎盘中,2条亲本链间存在差异甲基化区,而在双等位基因表达的组织中,未发现差异甲基化区。结果表明,DNA甲基化修饰参与调控牛DSCAM基因的胎盘特异性父源印记,可为深入探讨牛DSCAM基因功能和调控机制提供参考依据。

The DNA methylation status of DSCAM gene in cattle were analyzed to determine the role of DNA methylation on gene expression.The allelic expression of DSCAM gene was analyzed in bovine placenta and somatic tissues including heart,liver,spleen,lung,kindy,muscle,fat and brain by a SNP-based RT-PCR products direct sequencing method.The DNA methylation status of CpG island in promoter region of DSCAM gene was analyzed by bisulfite sequencing.An A/G SNP(rs136908595)was found on the exon 32 of bovine DSCAM gene and used to distinguish the parental alleles.The results showed that DSCAM gene was biallelically expressed in all detected bovine somatic tissues,including heart,liver,spleen,lung,kidney,muscle,fat and brain.In bovine placenta,DSCAM gene was maternally expressed based on the SNP(rs136908595).The DNA methylation was analyzed on the CpG island(402 bp)in the promoter region and the first exon of DSCAM gene.A differentially methylated region(DMR)was detected in placenta.However,DMR was not found in spleen,kidney and brain tissues.The results indicated that DNA methylation played a role in the placenta-specific paternal imprinting of bovine DSCAM gene,which provides a reference for further study of the function and regulation mechanism of bovine DSCAM gene,which provides a reference for further study of the function and regulation mechanism of bovine DSCAM gene and regulation mechanism of bovine DSCAM gene.