香猪卵巢RARRES1基因第1外显子CG位点的甲基化及其与基因差异表达的关联

doi:10.16431/j.cnki.1671-7236.2018.06.006

摘要/Abstract

摘要：

为验证和探索香猪卵巢转录组RNA测序检测到的视黄酸受体应答1（retinoic acid receptor responder 1，RARRES1）基因在香猪高、低产仔组之间差异表达的原因，本试验针对RARRES1基因第1外显子ATG下游富含CpG位点区段设计特异性引物，采用亚硫酸氢盐测序法（bisulfite sequencing PCR，BSP）研究卵巢RARRES1基因中的甲基化修饰水平；利用实时荧光定量PCR方法检测高、低产仔组香猪卵巢RARRES1基因的表达量，并探究其与基因甲基化水平之间的相关性。结果表明，与低产仔组相比，香猪高产仔组的甲基化水平较高；4个CpG位点均未被甲基化（CpG_7、CpG_11、CpG_12和CpG_15），另外3个CpG位点（CpG_8、CpG_17和CpG_18）基本上全部发生了甲基化。此外，与低产仔组相比，香猪高产仔组中CpG_4（P>0.05）、CpG_9（P<0.05）和CpG_16（P<0.05）位点的甲基化占比较高，而CpG_6位点在香猪低产仔组中的甲基化比例较高，两组差异极显著（P<0.01）。实时荧光定量PCR结果显示，高产仔组香猪RARRES1基因的表达水平较高（P<0.05）。Spearman相关分析结果显示，CpG_9和CpG_16两个位点的甲基化比例与RARRES1基因的表达水平高度正相关（R²=0.896，P<0.01），提示这两个位点的甲基化可能是香猪高产仔组RARRES1基因表达量较高的原因。

关键词: 香猪; 卵巢; RARRES1基因; 甲基化; 表达量

Abstract:

From the transcriptome data of ovary using the RNA-seq technology, retinoic acid receptor responder 1 (RARRES1) gene was screened out from the differential expression profile of Xiang pigs.To further reveal the underlying reason for the gene differential expression,the ovarian tissues samples were collected from two groups of Xiang pig with high and low litter sizes.Based on bioinformatics analysis which predicted that the downstream of ATG start codon in exon 1 of RARRES1 gene was rich with CpG sites,specific primers for this region were designed,and the methylation status of RARRES1 gene was tested by bisulfite sequencing PCR method (BSP).The expression level of RARRES1 gene was detected by quantitative Real-time PCR method,and its relationship with methylation level was analyzed.The results showed that the methylation level of RARRES1 gene in high litter size group was higher,and in both of the two groups, there were four CpG sites (CpG_7,CpG_11,CpG_12 and CpG_15) keeping unmethylated state,while the other three sites (CpG_8,CpG_17 and CpG_18) were all modified by methylation.There were four CpG sites were differently modified between the two groups with sites,CpG_4(P>0.05),CpG_9(P<0.05) and CpG_16(P<0.05) were higher and site CpG_6 (P<0.01) to be lower in high litter group than that in low litter size group.And the results of quantitative Real-time PCR showed that the mRNA levels of RARRES1 gene in high litter size group were much higher than that in another group (P<0.05).Besides,Spearman correlation analysis estimated there was a strong positive relationship between the methylation ratio of CpG_9 and CpG_16 and the expression level of RARRES1 gene (R²=0.896,P<0.01).It suggested that the methylation of exon 1 region might have a certain effect on promoting the expression of RARRES1 gene in Xiang pig ovary.

Key words: Xiang pig; ovary; RARRES1 gene; methylation; expression level

中图分类号:

S828

阮亦麒, 李大鹏, 冉雪琴, 毛宁, 易凡利, 张福平, 王嘉福. 香猪卵巢RARRES1基因第1外显子CG位点的甲基化及其与基因差异表达的关联[J]. 《中国畜牧兽医》, 2018, 45(6): 1471-1478.

RUAN Yiqi, LI Dapeng, RAN Xueqin, MAO Ning, YI Fanli, ZHANG Fuping, WANG Jiafu. The Methylation in the Exon 1 of RARRES1 Gene and its Association with Gene Differential Expression of Xiang Pig Ovary[J]. , 2018, 45(6): 1471-1478.

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