牛ATP10A基因组织特异性印记研究  

Tissue-specific Imprinting of Bovine(Bos taurus)ATP10A Gene

作　　者：刘晓倩 董艳秋 靳兰杰 谷书凯 李冬杰[2] 李世杰[1] LIU Xiao-Qian;DONG Yan-Qiu;JIN Lan-Jie;GU Shu-Kai;LI Dong-Jie;LI Shi-Jie(College of Life Science,Hebei Agricultural University,Baoding 071001,China;College of Bioscience and Bioengineering,Hebei University of Science and Technology,Shijiazhuang 050018,China)

机构地区：[1]河北农业大学生命科学学院,保定071001 [2]河北科技大学生物科学与工程学院,石家庄050018

出　　处：《农业生物技术学报》2021年第11期2109-2117,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31372312);河北省自然科学基金(C2020204004)。

摘　　要：基因组印记是存在于哺乳动物中的一种重要的表观遗传修饰机制,与生物体的生长发育密切相关。ATP10A(ATPase phospholipid transporting 10A)基因位于PWS/AS印记区域的一端,该印记区域与人类(Homo sapiens)的PWS(Prader-Willi syndrome)综合征和AS(Angelman syndrome)综合征发生有关。ATP10A基因在人中表现为大脑特异性的印记基因,而在牛(Bos taurus)中的印记状态未见报道。本研究首先对32头牛心脏组织和15个牛胎盘基因组DNA进行PCR扩增,通过产物直接测序鉴定基因组中的单核苷酸多态位点,在牛ATP10A基因外显子20上鉴定出A/G杂合的SNP位点(rs136134264)。然后对杂合子牛各组织及胎盘总RNA进行RT-PCR,产物测序结果与上述杂合SNP位点进行对比,确定了ATP10A基因在牛大脑组织中为单等位基因表达,而在其他组织和胎盘中为双等位基因表达。进一步利用亚硫酸氢盐测序法,分析ATP10A基因启动子区在牛组织和胎盘中的DNA甲基化状态,发现所检测区域的46个CpG位点在牛组织和胎盘中均呈现轻甲基化状态,说明ATP10A基因启动子区的DNA甲基化修饰不参与调控ATP10A基因在牛中的印记表达。本研究可为深入探讨牛ATP10A基因功能和印记的分子机制提供参考依据。Genomic imprinting is an important epigenetic modification mechanism in mammals,which is closely related to the growth and development of organisms.The ATP10 A(ATPase phospholipid transporting10 A)located at one end of the PWS/AS imprinted region,which is related to the development of human(Homo sapiens)PWS(Prader-Willi syndrome)and AS(Angelman syndrome)syndromes.ATP10 A is a brainspecific imprinted gene in human,but its imprinting status in cattle(Bos taurus)is still unknown.In this study,genomic DNA from 32 bovine heart tissues and 15 placentas were amplified by PCR first,and the single nucleotide polymorphic sites were found on ATP10 A gene by direct sequencing of PCR products,the A/G heterozygous SNP locus(rs136134264)was identified on exon 20 of bovine ATP10 A gene.Then total RNA in tissues and placenta of heterozygous cattle were used for RT-PCR to determine the allele expression status of ATP10 A gene.The sequencing results of PCR products were compared with above heterozygous SNP locus,it was determined that ATP10 A gene was monoallelic expression in bovine brain tissues and biallelic expression in other tissues and placenta.The DNA methylation status of the promoter region of ATP10 A gene in bovine placenta and adult bovine tissues was further analyzed by bisulfite sequencing.It was found that the 46 CpG loci in the detected region showed hypomethylation in bovine tissues and placenta,indicating that the DNA methylation modification in the promoter region of ATP10 A gene might not be involved in regulating imprinted gene expression of ATP10 A.The present study provides a reference for further exploring of bovine ATP10 A gene function and imprinting mechanism.

关 键 词：牛 PWS/AS印记区域 ATP10A(ATPase phospholipid transporting 10A) 印记状态 DNA甲基化 

分 类 号：S823[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]