猪MKRN3基因的印记表达和DNA甲基化状态分析

doi:10.11843/j.issn.0366-6964.2024.09.011

摘要/Abstract

摘要：

旨在探讨MKRN3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MKRN3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MKRN3-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MKRN3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，MKRN3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。MKRN3-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MKRN3-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，MKRN3表达父本来源等位基因，MKRN3-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，MKRN3不再维持印记状态，提示克隆猪脾脏组织中MKRN3印记异常。综上所述，MKRN3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，MKRN3印记表达和甲基化状态均为异常；启动子的DMR调控MKRN3表达。

关键词: DNA甲基化, 猪, DMR, 基因组印记

Abstract:

The aim of this study was to investigate the imprinting status of the MKRN3 gene and its DNA methylation level in wild type and cloned pigs. The imprinting status of the MKRN3 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 MKRN3 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 MKRN3-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 MKRN3 gene between Western (Duroc) and local (Bama and Rongchang) pig breeds: A/G; RT-PCR sequencing showed that MKRN3 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. MKRN3-DMR showed hypomethylation (0%) in spermatozoa and showed hypermethylation (70.9%) in oocytes. The average methylation levels of MKRN3-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, MKRN3 was expressed as the allele of paternal origin, and the level of MKRN3-DMR methylation was similar to that of the wild-type crossbred pigs. While MKRN3-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 MKRN3 imprinting status was abnormal in cloned pig spleen tissues. In summary, MKRN3 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; MKRN3 imprinted expression and methylation status were abnormal in some tissues of the cloned pigs. The expression of MKRN3 was regulated by the DMR of the promoter.

Key words: DNA methylation, porcine, DMR, genomic imprinting

中图分类号:

S828.2

陈南珠, 李俊良, 余大为, 周心仪, 王晶, 邹惠影, 杜卫华. 猪MKRN3基因的印记表达和DNA甲基化状态分析[J]. 畜牧兽医学报, 2024, 55(9): 3853-3863.

Nanzhu CHEN, Junliang LI, Dawei YU, Xinyi ZHOU, Jing WANG, Huiying ZOU, Weihua DU. Analysis of Imprinted Expression and DNA Methylation Status of the Porcine MKRN3 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 3853-3863.

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	退火温度/℃ Tm	用途 Application	片段长度/bp Length
esm-MKRN3-snp1F	CAGTGTCGGAAGGAACAATGGA	58	SNP检测	1 131
esm-MKRN3-snp1R	ACGAAAGCATTGCCCCATGATA	58	SNP检测	1 131
esm-MKRN3-snp2F	GGAGCATGCCATTGAGTTTGTTC	59	SNP检测	1 067
esm-MKRN3-snp2R	GGATGTTTTGTGACTTTTTAGCACAGTG	59	SNP检测	1 067
esm-MKRN3-snp3F	AGAAGCTGCTGAAACCATTTGT	58	SNP检测	929
esm-MKRN3-snp3R	CAAAGAATGTGTATCTGAGTCCAGG	58	SNP检测	929
cDNA-SNP1F	CTGTCGATACTCCCATGACCT	58	MKRN3表达	169
cDNA-SNP1R	CAGAGGAAAAGAGCGTGAGGA	58	MKRN3表达	169
MKRN3-WF1	GTTAGTAGGGAAAGTTAGGA	50	DMR甲基化	692
MKRN3-WR1	AAATAAACCAAAACTTAATCTCC	50	DMR甲基化	692
MKRN3-NF1	GATATAAAATGGAGTGAATAAATAAAG	55	DMR甲基化	465
MKRN3-NR1	TCCATAACAACTTCTACTCCCC	55	DMR甲基化	465

步骤 Procedure	时间/min Time	温度/℃ Temperature
变性 Denaturation	5	95
转化 Conversion	25	60
变性 Denaturation	5	95
转化 Conversion	85	60
变性 Denaturation	5	95
转化 Conversion	175	60
保存 Preservation	+∞	20

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