摘要
本研究旨在通过克隆鸭慢速骨骼肌型肌钙蛋白I 1(Slow skeletal muscle troponin I 1,TNNI1)基因5'侧翼区序列,检测鸭骨骼肌组织TNNI1基因的mRNA表达水平和启动子CpG岛区甲基化状态,初步探索TNNI1基因转录调控机制。采用染色体步移方法克隆测序获得鸭TNNI1基因5'侧翼区序列,进行生物信息学分析,采用荧光定量PCR(Real-time quantitative PCR,RT-PCR)检测鸭胸肌和腿肌TNNI1基因mRNA表达水平,采用亚硫酸氢盐测序法(Bisulfite sequencing PCR,BSP)检测核心启动子区CpG岛在鸭肌肉组织中的甲基化水平。结果表明,克隆获得鸭TNNI1基因5'侧翼区序列2 078 bp,预测存在2个CpG岛,其中CpG岛(-2 032～-1 833 bp)位于预测的核心启动子区内,并存在多个真核生物结构元件和转录因子结合位点;甲基化检测发现,其总体甲基化水平在胸肌和腿肌组织中分别为52. 66%、57. 04%,差异不显著(P>0. 05);荧光定量检测结果表明,鸭胸肌和腿肌TNNI1基因表达量差异显著(P<0. 05);相关性分析结果表明,CpG4位点甲基化程度与胸肌TNNI1基因表达量呈极显著负相关(P<0. 01)。鸭胸肌和腿肌TNNI1基因的mRNA表达量存在显著差异,其总体甲基化水平无显著差异。在胸肌中,启动子区CpG4位点可能通过甲基化修饰影响TNNI1基因的转录调控。
The experiment aims to clone the 5′flanking sequences of duck slow skeletal muscle troponin I 1(TNNI1)gene,investigate the mRNA expression levels and promoter CpG island methylation status of TNNI1 gene in the skeletal muscle of duck,and explore the mechanism of TNNI1 gene transcriptional regulation.The 5′flanking sequence of duck TNNI1 gene was cloned by genome walking method,and analyzed using online bioinformatics software.The mRNA expression levels of TNNI1 gene in duck breast muscle and leg muscle were detected by real-time quantitative PCR(RT-PCR),and the methylation level of CpG island in duck muscle tissue was detected by bisulfite sequencing PCR(BSP).The 5′flanking sequence of 2 078 bp duck TNNI1 gene was obtained and existed two CpG islands.The CpG island(-2 032--1 833 bp)located in the deduced core promoter region,and existed multiple eukaryotic structural elements and transcription factor binding sites.The overall methylation level showed that no differences existed between breast muscles(52.66%)and leg muscles(57.04%),but there were significant differences between breast muscles and leg muscles on TNNI1 mRNA expression levels.The correlation analysis results showed that the methylation degree of CpG4 site was significantly negatively correlated with the expression of the TNNI1 gene in the breast muscle(P<0.01).The expression of TNNI1 gene was significantly different in duck breast muscle and leg muscle,but there was no significant difference on total methylation level.The CpG4 locus in the promoter region might affect the transcriptional regulation of the TNNI1 gene in the pectoral muscles by methylation modification.
作者
姬改革
束婧婷
单艳菊
章明
屠云洁
巨晓军
刘一帆
JI Gai-ge;SHU Jing-ting;SHAN Yan-ju;ZHANG Ming;TU Yun-jie;JU Xiao-jun;LIU Yi-fan(Jiangsu Institute of Poultry Science,Yangzhou 225125,China)
出处
《江苏农业学报》
CSCD
北大核心
2018年第6期1312-1318,共7页
Jiangsu Journal of Agricultural Sciences
基金
江苏省自然科学基金项目(BK20151316)
江苏省六大人才高峰项目(NY-026)
扬州市现代农业项目(YZ2015039)
