山羊DCT基因启动子区甲基化水平、SNP与毛色特征关系研究

doi:10.11843/j.issn.0366-6964.2019.02.005

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (2): 271-279.doi: 10.11843/j.issn.0366-6964.2019.02.005

山羊DCT基因启动子区甲基化水平、SNP与毛色特征关系研究

杜小龙¹, 王麒¹, 张乐超¹, 葛琳涵¹, 刘小辉¹, 王涵¹, 李兰会^1,3*, 李祥龙^2*

1. 河北农业大学动物科技学院, 保定 071000;
2. 河北科技师范学院, 秦皇岛 066004;
3. 河北省牛羊胚胎工程技术研究中心, 保定 071000

收稿日期:2018-05-14 出版日期:2019-02-23 发布日期:2019-02-23
通讯作者: 李兰会,主要从事动物遗传育种研究,E-mail:lanhuili13@163.com;李祥龙,主要从事动物遗传育种研究,E-mail:lixianglongcn@yahoo.com
作者简介:杜小龙(1993-),男,内蒙古呼和浩特人,硕士,主要从事动物遗传育种与繁殖研究,E-mail:1464902509@qq.com;王麒(1993-),男,河北井陉人,硕士,主要从事动物遗传育种研究,E-mail:15369307136@qq.com。
基金资助:
河北省应用基础研究计划重点基础研究项目（15962901d）；河北省自然科学基金项目（C2015204176）

Study on the Relationship between Methylation Level, SNP in Promoter Region of DCT Gene and Hair Color in Goat

DU Xiaolong¹, WANG Qi¹, ZHANG Lechao¹, GE Linhan¹, LIU Xiaohui¹, WANG Han¹, LI Lanhui^1,3*, LI Xianglong^2*

1. College of Animal Science and Technology, Hebei Agricultural University, Baoding 071000, China;
2. Hebei Normal University of Science & Technology, Qinhuangdao 066004, China;
3. Research Center of Cattle and Sheep Embryo Engineering Technique of Hebei, Baoding 071000, China

Received:2018-05-14 Online:2019-02-23 Published:2019-02-23

摘要/Abstract

摘要：

旨在探究DCT基因启动子区甲基化水平和SNP突变对山羊毛色的影响，为探索DCT基因调控山羊毛色变化的机理提供理论依据。本研究以山羊为试验动物，对DCT基因启动子区进行CpG岛预测，设计引物对预测的2个CpG岛富集区域进行亚硫酸氢盐甲基化测序，使用甲基化水平分析软件BISMA统计甲基化位点，比较唐山奶山羊（白色）和南江黄羊（黑色品系）两种不同毛色山羊群体DCT基因启动子区甲基化水平差异。克隆DCT基因核心启动子区，筛选不同毛色山羊群体的SNPs，使用JASPAR和Nsite预测SNPs位点突变前后转录因子的改变，并检测比较突变前后DCT基因启动子活性变化。结果，成功克隆了山羊DCT基因启动子区甲基化序列及核心启动子区（g.-1045~-318）。在g.-348~-150区域和g.+222~+502区域分别发现6个和23个甲基化位点，其中g.+312、g.+352和g.+400位点与g.+389和g.+404位点白色山羊甲基化水平分别显著和极显著高于黑色山羊（P<0.05和P<0.01），并且g.+222~+502区域白色山羊甲基化平均水平极显著高于黑色山羊（P<0.01）。在DCT基因核心启动子区的g.-804 T > G、g.-705 C > T和g.-679 G > A，3个SNPs位点的基因型构成在白色山羊和3个有色山羊群体中存在差异，g.-804 T > G突变导致该区域的SOX10转录因子结合位点缺失，DCT基因启动子活性显著下降（P<0.05）。结果显示，白色山羊DCT基因g.+222~+502区域的高甲基化水平，g.-804、g.-714和g.-679 3个位点的突变，尤其是g.-804 T > G造成SOX10转录因子结合位点的缺失，突变的G型DCT基因启动子活性显著降低。因此，DCT基因启动子区SNP突变和高甲基化水平可能抑制了基因的表达从而形成山羊白色被毛。

Abstract:

The purpose of this study was to investigate the effects of the methylation and SNPs in DCT gene promoter region on the coat color of goats, and to provide a theoretical basis for exploring the mechanism of DCT gene regulating the coat color of goats. Goats were used as experimental animals. The CpG island in the promoter region of goat DCT gene was predicted and two pairs of primers were designed for bisulfite methylation sequencing of the two CpG island enriched regions. Methylation level online analysis software, BISMA, was used to count the number of methylation sites and to compare the differences of methylation levels between Tangshan dairy goats(with white coat) and Nanjiang Huang goats(with black coat). Core promoter region sequence of DCT was cloned and SNPs were screened for goats with different coat colors. JASPAR and Nsite, the transcription factor online prediction software, were used to predict the transcription factor alterations with the mutation of SNPs, and promoter activity variation with the mutation of SNPs was detected. The methylation sequence and the core promoter region sequence(g.-1045--318) of goat DCT gene were successfully cloned. Six and 23 methylation sites were found in the g.-348--150 region and the g.+222-+502 region, respectively. The methylation levels of white goats at the g.+312, g.+352 and g.+400 sites and at the g.+389 and g.+404 sites were significantly and extremely significantly higher than those of black goats (P<0.05 and P<0.01), respectively. And the average methylation level of white goats in g.+222-+502 region was extremely significantly higher than that of black goats (P<0.01). The genotype compositions of the 3 SNPs in the core promoter region, g.-804 T > G, g.-705 C > T and g. -679 G > A, was different between white goats and the 3 colorful goats. SOX10 transcription factor binding site disappeared with the g.-804T > G mutation, and the promoter activity of the DCT gene was significantly decreased (P<0.05). The higher methylation level in the g.+222 -+502 region of DCT gene in white goats, and the 3 mutations at g.-804, g.-714 and g.-679 sites, especially the g.-804 T > G mutation resulted in the loss of the SOX10 transcription factor binding site and the promoter activity of DCT with G genotype was significantly decreased.In conclusion, the SNP mutation and the higher methylation level in promoter region may suppress the expression of DCT gene and form the white coat color of goat.

中图分类号:

S827.2

杜小龙, 王麒, 张乐超, 葛琳涵, 刘小辉, 王涵, 李兰会, 李祥龙. 山羊DCT基因启动子区甲基化水平、SNP与毛色特征关系研究[J]. 畜牧兽医学报, 2019, 50(2): 271-279.

DU Xiaolong, WANG Qi, ZHANG Lechao, GE Linhan, LIU Xiaohui, WANG Han, LI Lanhui, LI Xianglong. Study on the Relationship between Methylation Level, SNP in Promoter Region of DCT Gene and Hair Color in Goat[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(2): 271-279.

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山羊DCT基因启动子区甲基化水平、SNP与毛色特征关系研究

Study on the Relationship between Methylation Level, SNP in Promoter Region of DCT Gene and Hair Color in Goat

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