猪MKRN 3基因的印记表达和DNA甲基化状态分析

Analysis of Imprinted Expression and DNA Methylation Status of the Porcine MKRN 3 Gene

旨在探讨MKRN 3基因在野生型猪和克隆猪中的印记状态及其DNA甲基化水平。本研究利用西方猪种(杜洛克猪)与本地猪种(巴马猪或荣昌猪)中存在的种间单核苷酸多态性(single nucleotide polymorphism,SNP),检测MKRN 3基因在3只足月出生0天的野生型杂交猪中的印记状态。利用MethPrimer网站预测MKRN3启动子区附近的CpG岛,并在卵母细胞和精子基因组中验证CpG岛是否为差异甲基化区域(differentially methylated region,DMR)。对3只野生型杂交猪和两只克隆猪基因组DNA进行亚硫酸盐转化后测序,检测MKRN 3-DMR在野生型猪和克隆猪的甲基化状态,并分析其甲基化状态与基因表达的关系。结果表明,在MKRN 3基因的编码区域,西方猪种(杜洛克猪)与本地猪种(巴马猪和荣昌猪)之间存在一个SNP:A/G;RT-PCR测序结果显示,MKRN 3在其中1只野生型个体(杜洛克猪与巴马猪杂交后代:DB2)的心脏组织呈双等位基因表达,在肝脏、脾脏、肺等器官和脑组织中均为单等位基因表达,且均表达父本来源的等位基因。MKRN 3-DMR在精子中表现为低甲基化(0%),在卵母细胞中表现为高甲基化(70.9%)。杂交猪6个组织中的MKRN 3-DMR平均甲基化水平分别为:心脏(43.1%)、肝脏(46.2%)、脾脏(48.4%)、肺(45.6%)、肾脏(48.5%)、脑(44.3%);在新生克隆猪的大部分组织中,MKRN 3表达父本来源等位基因,MKRN 3-DMR甲基化水平与野生型杂交猪相近。而在NT201和NT207脾脏中,其甲基化水平分别为79.0%和80.1%,SNP测序结果也表明在两个克隆猪脾脏中,MKRN 3不再维持印记状态,提示克隆猪脾脏组织中MKRN 3印记异常。综上所述,MKRN 3在野生型猪肝脏、脾脏、肺、肾和脑组织中呈母本印记、父本表达,在心脏为非印记状态;克隆猪部分组织中,MKRN 3印记表达和甲基化状态均为异常;启动子的DMR调控MKRN 3表达。

The aim of this study was to investigate the imprinting status of the MKRN 3 gene and its DNA methylation level in wild type and cloned pigs.The imprinting status of the MKRN 3 gene in the 3 wild-type crossbred pigs,which were born at full term,were examined using interspecies single nucleotide polymorphism(SNP)present in Western(Duroc)and local(Bama or Rongchang)pig breeds.CpG islands near the MKRN 3 promoter region were predicted using the MethPrimer website,and CpGs were verified as differentially methylated region(DMR)in oocyte and sperm genomes.The genomes of the 3 wild-type hybrid and 2 cloned pigs were sequenced after bisulfite transformation to detect the methylation status of MKRN 3-DMR in wild-type and cloned pigs,and to analyze the relationship between methylation status and expression.The results showed that a single nucleotide polymorphism(SNP)existed in the coding region of the MKRN 3 gene between Western(Duroc)and local(Bama and Rongchang)pig breeds:A/G;RT-PCR sequencing showed that MKRN 3 was bi-allelic expressed in the heart tissue of one of the wild-type individual(offspring of a cross between Duroc pig and Bama pig:DB2),and monoallelic expressed in organs such as liver,spleen,lungs,and brain tissues,and all of them expressed the allele from paternal origin.MKRN 3-DMR showed hypomethylation(0%)in spermatozoa and showed hypermethylation(70.9%)in oocytes.The average methylation levels of MKRN 3-DMR in the six tissues of hybrid pigs were:heart(43.1%),liver(46.2%),spleen(48.4%),lung(45.6%),kidney(48.5%),and brain(44.3%),respectively.In most tissues of the newborn cloned pigs,MKRN 3 was expressed as the allele of paternal origin,and the level of MKRN 3-DMR methylation was similar to that of the wild-type crossbred pigs.While MKRN 3-DMR methylation assay results showed that the methylation levels were 79.0%and 80.1%in spleen and spleen of NT201 and NT207,respectively,SNP sequencing results also indicated that the imprinted state was no longer maintained in the two cloned pig spleens.It is suggested that MKRN 3 imprinting status was abnormal in cloned pig spleen tissues.In summary,MKRN 3 was maternally imprinted and paternally expressed in liver,spleen,lung,kidney and brain tissues of wild-type pigs,and was in a non-imprinted state in the heart;MKRN 3 imprinted expression and methylation status were abnormal in some tissues of the cloned pigs.The expression of MKRN 3 was regulated by the DMR of the promoter.