摘要： 为了探究骨骼肌α肌动蛋白1（ACTA1）基因在肌肉和脂肪等6个组织中的甲基化状态及mRNA表达水平，以类乌齐牦牛、麦洼牦牛为研究对象，成功克隆了牦牛ACTA1基因启动子区序列，且采用重亚硫酸氢盐测序法（BSP）检测ACTA1基因启动子区甲基化模式，并通过实时荧光定量PCR检测ACTA1基因在臀大肌、心脏、肾脏、肺脏、肝脏和脂肪组织中的mRNA表达水平。结果显示，牦牛ACTA1基因转录起始位点上游及第一外显子区部分序列总长为1 028 bp；BSP法分析发现肌肉组织DNA甲基化水平最低，脂肪组织甲基化率最高，其中，类乌齐牦牛臀大肌、心脏、肾脏、肺脏、肝脏和脂肪的甲基化概率分别为6.25%，6.88%，20.00%，16.25%，26.25%，29.38%，麦洼牦牛臀大肌、心脏、肾脏、肺脏、肝脏和脂肪的甲基化概率分别为5.00%，7.50%，18.13%，20.00%，26.25%，28.75%； ACTA1基因mRNA表达量在肾脏、肺脏、肝脏和脂肪均极显著低于臀大肌（P<0.01），且显著低于心脏（P<0.05），类乌齐牦牛和麦洼牦牛心脏mRNA表达量具有显著性差异（P<0.05），类乌齐牦牛肺脏组织甲基化率显著低于麦洼牦牛（P<0.05），其他各组织甲基化率和mRNA表达量差异均不显著（P>0.05），各组织甲基化水平与ACTA1基因mRNA表达量呈极显著负相关（r=-0.797，P=0.002）。结果表明， ACTA1基因DNA甲基化模式对肌肉发育具有一定的调控作用，可为牦牛遗传育种表观遗传标记提供部分数据支持。

关键词: 牦牛, ACTA1基因, 启动子区, DNA甲基化, 重亚硫酸氢盐测序, 组织表达

Abstract: In order to investigate the methylation status and mRNA expression levels of skeletal muscle actin alpha 1(ACTA1)gene in six tissues including muscle and adipose,et al,the experiment was conducted with Leiwuqi yak and Maiwa yak. The bisulfite-sequencing PCR(BSP)was used to detect the methylation pattern of the promoter region of ACTA1 gene,and the ACTA1 gene was detected by Real-time fluorescent quantitative PCR in the gluteus maximus,heart,kidney,lung,liver,and mRNA expression levels in adipose tissue. The results showed that the total length of the partial sequence of the transcription initiation site of the ACTA1 gene and the first exon region of yak was 1 028 bp. The BSP analysis showed that the DNA methylation level of muscle tissue was the lowest,and the methylation rate of adipose tissue was the highest. Among them,the methylation probabilities of Leiwuqi yak gluteus maximus,heart,kidney,lung,liver and adipose were 6.25%,6.88%,20.00%,16.25%,26.25% and 29.38%,methylation probabilities of Maiwa yak gluteus maximus,heart,kidney,lung,liver and adipose were 5.00%,7.50%,18.13%,20.00%,26.25%,28.75%. The expression of ACTA1 gene in kidney, lung, liver and adipose mRNA was significantly lower than that of gluteus maximus (P<0.01), which was significantly lower than that of heart (P<0.05).There was a significant difference in heart mRNA expression between Leiwuqi yak and Maiwa yak (P<0.05), but there was no significant difference in methylation rate and mRNA expression in other tissues of Leiwuqi and Maiwa yak. The methylation rate of lung tissues of Leiwuqi yak is significantly lower than that of Maiwa yak (P<0.05).There was no significant difference in methylation rate and mRNA expression between Leiwuqi and Maiwa yak (P>0.05),and the methylation level of each tissue was significantly negative correlated with the expression of ACTA1 gene mRNA(r=-0.797, P=0.002). Therefore,the results of the study indicate that the DNA methylation pattern of ACTA1 gene has a certain regulatory effect on muscle development,which can provide partial data support for the epigenetic marker of yak genetic breeding.

Key words: Yak, ACTA1 gene, Promoter region, DNA methylation, Bisulfite-sequencing PCR, Tissue expression

中图分类号: 

• S823

• S813

• Q78