牛Ascl2 基因印记及DNA甲基化状态分析

doi:10.11843/j.issn.0366-6964.2014.12.006

摘要/Abstract

摘要：

为探讨Ascl2基因在成年牛不同组织中的印记状态，并揭示DNA甲基化在调控Ascl2基因印记中的可能作用。本研究首先应用基于核苷酸多态的RT-PCR产物直接测序法对Ascl2基因在牛7个组织（心、肝、脾、肺、肾、肌肉和脂肪）中的表达及印记状态进行了分析，进一步应用亚硫酸氢盐测序法分析牛肺、肝和肾组织中Ascl2基因启动子区的等位基因特异的甲基化状态。RT-PCR产物测序结果显示，Ascl2基因印记表现出组织特异性，在肺和肝中为单等位基因表达，而在心、脾、肾、肌肉和脂肪5个组织中为双等位基因表达。亚硫酸氢盐测序发现，在Ascl2单等位基因表达的肺和肝中，2条亲本链的甲基化水平存在极显著差异（P<0.01）， A链的甲基化水平（61.21%、87.28%）显著高于G链（24.70%、25.61%）；而在Ascl2双等位基因表达的肾组织中，A链（54.70%）与G链（49.55%）的平均甲基化水平差异不显著（P >0.05）。以上结果表明，Ascl2基因启动子区DNA的甲基化修饰调控Ascl2基因的印记表达。

关键词: Ascl2, 印记状态, DNA甲基化, 差异甲基化

Abstract:

In order to determine the imprinting status of Ascl2 in bovines different tissues，and to reveal the possible role of DNA methylation in regulating Ascl2 gene imprinting，we analyzed the expression of Ascl2 in 7 organs (heart，liver，spleen，lung，kidney，muscle and subcutaneous fat) of calves.Single nucleotide polymorphisms were used to identify imprinting by direct sequencing the RT-PCR products，then the DNA methylation status in promoter region of Ascl2 gene were analyzed in lung，liver and kidey tissues of cattle using bisulfite sequencing analysis.The results of RT-PCR showed that Ascl2 gene monoallelic expressed in lung and liver tissues while biallelic expressed in heart，spleen，kidney，muscle and subcutaneous fat tissues.The results of bisulfite sequencing showed that the methylation level of 2 parental strands exhibited extremely significant difference (P<0.01) in lung and liver tissues where Ascl2 was monoallelic expression.The mean methylation levels of A-strands (61.21%，87.28%) showed seriously hypermethylated compared with G-strands (24.70%，25.61%).However，in kidey tissues where Ascl2 was biallelic expression，A-strand (54.70%) and G-strand (49.55%) had no significant difference（P>0.05）.Our results suggest that the differential methylation of CpG island in promoter of Ascl2 regulate the imprinting expression of Ascl2 in lung and liver tissues.

Key words: Ascl2, imprinting status, DNA methylation, differential methylation

中图分类号:

S823
S813.3

王梦楠，崔亚丽，吴国江，李冬杰，李世杰. 牛Ascl2 基因印记及DNA甲基化状态分析[J]. 畜牧兽医学报, 2014, 45(12): 1949-1956.

WANG Meng-nan，CUI Ya-li,WU Guo-jiang，LI Dong-jie,LI Shi-jie. Genomic Imprinting and DNA Methylation Status of Ascl2 Gene in Cattle[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(12): 1949-1956.

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