基于基因组DNA甲基化分析的肉牛高肌间脂肪含量候选基因鉴定与功能解析

DNA methylation is one of the main epigenetic modification mechanisms in eukaryotic organisms, and plays a crucial role on the regulation of gene expression. Methylation of CpG dinucleotides in the 5' regulatory regions of genes often results in transcriptional inactivation, and some studies have shown that actively transcribed sequences are often methylated less than promoters and certain coding regions of silent genes. Significant differences in the levels or pattern of cytosine methylation have been observed in various tissues or under different functional states in the same tissue. Several studies suggested that various levels of DNA methylation may regulate tissue-specific transcription and be important for normal development or differentiation and trait. But how methylation regulates gene expression is still unclear. Therefore, the detection and analysis of levels and patterns of genome-wide methylation in various tissues or in same tissue of the different varieties is essential for understanding associations between tissue-specific or variety-specific methylation and tissue-specific or variety-specific gene expression. In recent years, the research progress in this field is going fast but there are little reports on epigenetics and its regulation patterns on meat traits in animals. The results of research in our group showed that the whole genome methylation level of muscle tissue should be correlated with meat quality traits in swine and chicken.But how DNA methylation regulated meat traits and how the related function genes expressed? Is there any relationship between genome methylation level and different meat quality characters in different breeds? In this research, whole genome methylation levels differences would be studied in different breeds and populations with different inter-muscle fat content with the fluorescence-labeled methylation-sensitive amplification polymorphism(F-MSAP) technique.Methylation mode and its relationship would be tested, biomarkers of high inter-muscle fat content would be found, condidate function genes and relationship between methylation level and expression regulation would be identified. The results of this study should make a foundation for the epi-regulation mechanism of the formation of meat quality traits and the discovery of new condidate function genes of meat quality.

DNA甲基化在真核生物中是一种重要的表观遗传学修饰方法，也是基因表达调控的重要影响因素之一。近年来该领域研究进展突飞猛进，但畜禽表观遗传对肉质性状的调控机制研究报道较少。本课题组前期工作结果表明：畜禽肌肉组织全基因组甲基化水平可能与肉质性状之间存在相关。DNA甲基化水平对肉质性状具有怎样的调控作用，与之相关的功能基因表达调控机制是怎样的，不同品种基因组DNA甲基化水平和模式与其具有的不同肉质性状特征间有无关系，均有待于进一步研究。本课题以不同肌间脂肪含量品种及群体为研究对象，利用F-MSAP技术检测不同肌间脂肪含量品种及群体背最长肌全基因组甲基化水平差异，了解甲基化水平和模式与肉质性状的相关性，寻找高肌间脂肪含量甲基化分子标记，发现和鉴定与之相关的功能基因，分析这些功能基因甲基化水平与基因表达调控的关系。研究结果将会为了解牛肉质性状形成的表观遗传调控机制，发掘新的肉质性状功能基因奠定基础。

本项目针对目前我国肉牛研究领域中功能基因较少等突出问题，以肉质性状和肌间脂肪沉积能力存在显著差异的日本和牛与草原红牛为研究对象，采用全基因组DNA甲基化测序（Whole Genome Bisulfite Sequencing，WGBS）对两个品种牛背最长肌DNA甲基化进行分析，筛选牛肉质性状相关候选基因和候选区域，并对部分候选基因和候选区域进行了功能验证和作用机制分析。甲基化分析结果显示，日本和牛与草原红牛背最长肌之间，8596个基因中存在差异甲基化区域（Differentially methylated regions，DMRs），差异甲基化基因（Differentially methylated genes，DMGs）显著富集在1046个GO terms（p<0.05），DMGs富集在276个KEGG信号通路中；亚硫酸氢钠处理测序法（BSP）检测了部分DMRs的甲基化水平，结果与测序结果趋势一致；通过不同品种间基因序列比对以及CG岛预测和遗传多态性检测，分析和预测了肉质性状候选基因SNPs位点对基因启动子序列甲基化水平，转录因子结合位点和候选基因表达可能存在的调控作用；在细胞水平上，采用基因过表达，验证了候选基因对脂肪和脂肪酸代谢的功能，全面解析候选基因对细胞内脂肪含量和脂肪酸含量的影响，以及对脂肪和脂肪酸代谢的调控机制。获得了日本和牛与草原红牛之间存在的大量DMRs和DMGs，系统筛选和挖掘决定肉牛肌肉生长、脂肪沉积等肉质性状的关键候选基因和甲基化标记，为进一步挖掘与肉质性状相关的功能基因和完善脂肪代谢调控网络奠定理论基础。此外，获得具有重要育种价值的功能基因和调控元件，将为转基因肉牛新品种培育及其产业化提供基因资源和技术支撑。