鸡HSP90AA1基因SNP检测及启动子区CpG岛的甲基化研究

试验旨在获得鸡热休克蛋白90α(HSP90AA1)基因序列并分析其基因结构和相关遗传变异,检测HSP90AA1基因启动子区CpG岛的甲基化状态,初步探索HSP90AA1基因在肌肉组织生长发育中的作用。以文昌鸡和北京油鸡为试验材料,利用PCR扩增鸡HSP90AA1基因组序列;通过基因测序寻找该基因中的单核苷酸多态性(SNP)位点;使用在线软件MethPrimer预测鸡HSP90AA1基因中CpG岛的位置;应用MassArray质谱法检测鸡胸肌中HSP90AA1基因启动子区CpG岛的甲基化水平,比较分析文昌鸡和北京油鸡HSP90AA1基因的甲基化差异。结果显示,在鸡HSP90AA1基因组中共发现7个SNPs位点,分别位于启动子区(A-189G,C-109T)、第1外显子(A+6G)、第2外显子(C+343T)、第2内含子(A+634G、A+836G)和第7内含子(A+3449G);鸡HSP90AA1基因包含10个外显子和9个内含子,其启动子区存在1个CpG岛,位于-1 802～-469bp处;在HSP90AA1基因启动子区共检测了42个CpG位点的甲基化水平,文昌鸡和北京油鸡中分别有9个(CpG_16.17.18、CpG_21.22.23、CpG_32.33和CpG_57)和4个CpG位点(CpG_1、CpG_5.6和CpG_57)在胸肌生长发育过程中发生甲基化改变。结果表明,文昌鸡与北京油鸡HSP90AA1基因序列信息和启动子区CpG岛的甲基化水平不同,这可能导致两种鸡对于应激反应具有不同的耐受程度。以上试验结果将为文昌鸡和北京油鸡生长发育规律、系统选育等方面的研究提供表观遗传学依据。

Detection of Single Nucleotide Polymorphism and Methylation of CpG Island in Promoter Region of Chicken HSP90AA1 Gene

This study was aimed to analyze the structure of HSP90AA1 gene,reveal the distribution of SNP in Wenchang chicken and Beijing-You chicken,investigate the promoter CpG islands methylation status of HSP90AA1 gene,and explore the role of HSP90AA1 gene in muscle tissue growth and development.HSP90AA1 gene was amplified by PCR.Then the gene mutation was identified by sequencing.CpG island was predicted using online software MethPrimer.With MassArray system,the DNA methylation levels and patterns of CpG islands in HSP90AA1 gene promoter were analyzed between the breast muscles of Wenchang chicken and Beijing-You chicken.Seven mutations were detected in the HSP90AA1 gene genomic fragment,two SNPs in the promoter region (A-189G,C-109T),one SNP in the first exon (A+6G),one SNP in the second exon (C+343T),two SNPs in the second intron (A+634G,A+836G) and one SNP in the seventh intron (A+3449G).The chicken HSP90AA1 gene sequence was determined including ten exons and nine introns.We found a CpG island,extending from -1 802 to -469 bp,in the promoter of HSP90AA1 gene.Forty-two CpG dinucleotides in HSP90AA1 promoter were tested.A total of 9 CpG loci (CpG_16.17.18,CpG_21.22.23,CpG_32.33 and CpG_57) were significantly different in different developmental stages in Wenchang chicken.A total of 4 CpG loci (CpG_1,CpG_5.6 and CpG_57) were significantly different in different developmental stages in Beijing-You chicken.This study proved that sequence information and methylation levels of HSP90AA1 gene in Wenchang chicken and Beijing-You chicken were different.The results provided some useful epigenetic basis principles for growth and development,system selection of Wenchang chicken and Beijing-You chicken.