ITGB2基因在苏博美利奴羊不同细度皮肤组织中的DNA甲基化及mRNA表达水平分析

doi:10.16431/j.cnki.1671-7236.2020.04.005

摘要/Abstract

摘要： 本研究旨在分析ITGB2基因在苏博美利奴羊不同细度皮肤组织中的DNA甲基化和mRNA表达水平。以苏博美利奴羊周岁母羊为试验动物，以不同细度的皮肤组织样为试验样本，对ITGB2基因（GenBank登录号：NC_040252.1）启动子区CpG岛进行预测并设计BSP引物，并对ITGB2基因（GenBank登录号：NM_001009485.1）、GAPDH基因（GenBank登录号：NM_001190390.1）mRNA序列设计引物，采用重亚硫酸盐测序法（BSP法）进行扩增纯化后将其连接pMD19-T载体，转化JM109细胞过夜培养，形成单菌落，筛选阳性克隆菌进行测序，对所获序列进行分析，分析ITGB2基因启动子区CpG岛在周岁母羊皮肤组织的甲基化模式，并运用实时荧光定量PCR检测ITGB2基因在苏博美利奴羊不同细度皮肤组织中的mRNA表达水平。结果显示，极细组苏博美利奴羊CpG岛甲基化率（94.29%）高于极粗组苏博美利奴羊的CpG岛甲基化率（87.62%），其中，极细组苏博美利奴羊CpG2、CpG3、CpG4、CpG7甲基化率（100%、100%、100%和80.00%）均高于极粗组（86.67%、93.33%、80.00%和73.33%）；ITGB2基因在苏博美利奴羊极粗皮肤组织中的表达量极显著高于极细皮肤组织的表达量（P < 0.01），且ITGB2基因的DNA甲基化水平与mRNA表达量呈明显负相关。研究表明，DNA甲基化对皮肤生长发育有一定作用，可作为一个候选的表观遗传标记用于苏博美利奴羊。

关键词: 苏博美利奴羊; ITGB2基因; DNA甲基化

Abstract: This study was aimed to analyze the DNA methylation and mRNA expression levels of ITGB2 gene in different fine skin tissues of Subo Merino sheep.Using Subo Merino sheep aged ewe as experimental animal and skin tissue samples of different fineness as experimental samples,the promoter CpG island of ITGB2 gene (GenBank accession No.:NC_040252.1) were predicted,the BSP primer,the primers of the mRNA sequence of ITGB2 gene (GenBank accession No.:NM_001009485.1) and GAPDH gene (GenBank accession No.:NM_001190390.1) were designed,respectively.The bisulfite sequencing method (BSP method) was used to amplify and purify,and then inserted into pMD19-T vector and transferred to JM109 cells for overnight culture to form a single colony.The positive clones were screened for sequencing and the obtained sequences were analyzed.The methylation pattern of CpG island in the promoter region of ITGB2 gene in skin tissue of aged ewe,and the mRNA expression level of ITGB2 gene in different fineness skin tissues of Subo Merino sheep was detected by Real-time quantitative PCR.The results showed that the methylation rate of CpG island of extremely fine group in Subo Merino sheep (94.29%) was higher than that of extremely thick group (87.62%).Among them,the methylation rate of CpG2,CpG3,CpG4 and CpG7 island in extremely fine group of Subo Merino sheep (100%,100%,100% and 80.00%) were higher than that in extremely thick group (86.67%,93.33%,80.00% and 73.33%).The expression of ITGB2 gene in extremely thick skin tissues of Subo Merino sheep was extremely significantly higher than that in extremely fine skin tissues(P < 0.01),and there was a significant negative correlation between the level of DNA methylation and the mRNA expression of ITGB2 gene.This results indicated that DNA methylation had a role in skin growth and development,and could be used as a candidate epigenetic marker for Subo Merino sheep.

Key words: Subo Merino sheep; ITGB2 gene; DNA methylation

中图分类号:

S813.3

杜建文, 杨雪梅, 王丹, 朱桦, 何军敏, 阿布来提·苏来曼, 田月珍, 赵冰茹, 徐新明, 付雪峰, 哈尼克孜·吐拉甫, 吴伟伟, 田可川, 黄锡霞. ITGB2基因在苏博美利奴羊不同细度皮肤组织中的DNA甲基化及mRNA表达水平分析[J]. 中国畜牧兽医, 2020, 47(4): 1009-1017.

DU Jianwen, YANG Xuemei, WANG Dan, ZHU Hua, HE Junmin, ABLAT Sulayman, TIAN Yuezhen, ZHAO Bingru, XU Xinming, FU Xuefeng, HANIKEZI Tulafu, WU Weiwei, TIAN Kechuan, HUANG Xixia. Analysis of DNA Methylation and mRNA Expression Levels of ITGB2 Gene in Different Fineness of Subo Merino Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(4): 1009-1017.

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