基于单细胞RNA组学分析的肉牛肌内脂肪沉积分子调控机理解析

The intramuscular fat, one of the main target traits in beef cattle breeding, is an important factor in determining the beef quality. As an complex traits, the intramuscular fat deposition of beef cattle should be controlled by multi-gene and many regulation networks. The process involving networks contains the multiple signaling factors, interactions of multiple pathways and fine regulation of genetically modified. The timing and heterogeneity of functional gene expression during adipocyte differentiation in different breeds of cattle have caused great difficulties in the regulatory mechanisms research during fat deposition. Single-cell RNomics technology could accurately analyze gene expression profiles and heterogeneity of single cells and cell subsets of adipose tissue. In this research, whole genome single-base resolution levels differences would be studied in different development stages of two cattle breeds with different intramuscular fat deposition with single-cell RNA sequencing technique. By the interaction model of gene expression networks analysis, spatiotemporal specificity, manipulation of genetic modification and the distribution characteristics of lncRNA/mRNA in functional domains of key genes should be elucidated. The function genes of intramuscular fat deposition, relationship between genetic modification level and expression regulation networks would be identified. The results of this study should make a foundation for the molecular mechanism of gene expression regulation in adipocyte differentiation and fat deposition, the new condidate function genes and regulatory networks of meat quality should be discovered.

肌内脂肪是决定牛肉品质的重要因素，也是培育优良肉牛品种的主要目标性状之一。而肉牛肌内脂肪沉积涉及多信号因子与多种脂肪代谢通路的互作与精细的遗传修饰，其调控机制非常复杂。深入了解不同品种肉牛肌内脂肪细胞分化时期功能基因表达的时序性和异质性及在脂肪沉积过程中的精确调控具有重要科学意义。单细胞RNA组学技术可以全面、精确分析脂肪组织中单个细胞和细胞亚群基因表达谱及其异质性特征。本项目在前期工作基础上，拟以不同肌内脂肪沉积的和牛和延边黄牛为研究对象，利用单细胞RNA组学技术对不同发育阶段肉牛肌内脂肪沉积过程中的基因表达进行单细胞、单碱基、全基因组范围的精准分析。通过研究此过程中关键基因和特异lncRNA表达时空特异性、互作组合因子遗传修饰变化及功能域的分布特点，结合对脂代谢通路候选基因的功能验证分析，明确肉牛肌内脂肪沉积的基因表达调控分子机制，为发掘新的肉质性状功能基因及调控网络奠定理论基础。

肌内脂肪沉积对于肉质性状的形成，尤其对肉牛的大理石花纹、嫩度、风味和多汁性具有决定性的影响，是牛肉品质和优良肉牛品种的重要评价指标。为了揭示肉牛肌内脂肪沉积的分子调控机制，本项目基于10xGenomics组学、lncRNA组学及miRNA测序及单细胞PCR Arrays多组学联合筛查获得了一批与肉牛肌内沉脂性状及脂肪细胞分化相关的候选基因和调控元件（miRNA、lncRNA、可变剪接体）。于细胞水平、活体水平和群体水平利用基因分子生物学、细胞工程学、基因修饰等技术解析了脂肪沉积和脂肪细胞分化相关基因的功能和遗传修饰元件的调控作用，分析了lncRNA/miRNA-靶基因在脂代谢通路中的互作方式及功能域特点，并验证了GPAM、ACSL5、DLK1、CPT1B等候选基因功能位点在肉牛资源群体中的遗传效应。本项目结果有助于深入解析肉牛肌内脂肪沉积的分子调控机制，为发掘肉牛肉质性状新候选基因和遗传修饰元件及肉牛分子育种提供理技术依据。