SIRT1调控牦牛肌内脂肪代谢的分子机制研究

Studies on the mechanism of intramuscular fat metabolism in yak is of the vital scientific significance on improving the meat quality. Our preliminary research results which comparative analyses of the differentially mRNA expression profiles of the longissimus dorsi muscle and adipose tissue of different ages in yak, and the correlation between differential gene expression abundance and intramuscular fat content in longissimus dorsi tissue, showed that the expression of SIRT1 is closely related to intramuscular fat content. However, the regulation mechanism was still unclear. Therefore, SIRT1 is selected as the research point, the adenovirus-mediated overexpression and lentivirus-mediated interference technologies will be used to clarify the biological function of SIRT1 in intramuscular fat metabolism. Cloning and analyzing the SIRT1 promoter, using site-directed mutagenesis and ChIP to explore the transcriptional regulation mechanism of SIRT1 gene by key transcription factors of lipid metabolism, and elucidate the upstream regulation mechanism of SIRT1. Furthermore, the downstream epigenetic regulatory pathway of SIRT1 will be analyzed using ChIP-Seq combined with ChIP, Western blot and Co-IP technologies to reveal the molecular mechanisms of SIRT1 epigenetically regulating the intramuscular fat metabolism. These results will be beneficial to reveal the molecular mechanisms of SIRT1 regulating intramuscular fat metabolism in yak, and provide a theoretical basis for improving the meat quality, which is of great scientific significance for promoting the development of yak industry.

牦牛肌内脂肪代谢机理研究对改善牦牛肉品质具有重要科学意义。申请人前期通过建立不同月龄育肥牦牛背最长肌和脂肪组织差异基因表达谱、差异基因表达丰度与肌内脂肪含量相关性的两次筛选，证实SIRT1与肌内脂肪含量密切相关，但调控机制尚不清楚。因此，本项目以SIRT1为切入点，利用病毒介导的超表达和干扰技术，明确SIRT1在牦牛肌内脂肪代谢中的生物学功能；克隆分析SIRT1启动子，利用定点突变和ChIP等技术探索脂代谢关键转录因子对SIRT1基因的转录调控机制，阐明SIRT1上游调控方式；利用ChIP-Seq结合ChIP、Western blot、Co-IP等技术，筛选受SIRT1去乙酰化调控的关键靶基因并进行功能验证，明析SIRT1下游表观遗传调控通路。本项目旨在揭示SIRT1调控牦牛肌内脂肪代谢的分子机理，为提高牦牛肉品质提供理论依据，对于促进牦牛产业发展具有重要的科学意义。

牦牛肌内脂肪代谢机理研究对改善牦牛肉品质具有重要科学意义，SIRT1是一种依赖NAD+的去乙酰化酶，通过从多种蛋白质脱去乙酰基从而参与到多种生物学功能的调控。本研究以牦牛肌内脂肪细胞为研究对象，探讨SIRT1调控牦牛肌内脂肪代谢的机制。结果表明，牦牛SIRT1基因CDS区长1,674bp，在牦牛各组织中呈广泛性表达，且在肌内前体脂肪细胞分化过程中其表达量呈现下降的趋势，并与IMF含量呈显著负相关；超表达SIRT1后，脂质分化标志基因（PPARγ、C/EBPα和AP2）及脂合成相关基因（FASN、ACACA、FABP3、SCD、SREBP1、SCAP、PLIN2）显著下调，而脂解（ATGL）及脂肪酸氧化相关基因(CPT1A、PGC-1α)的表达量显著上调；最终导致脂滴沉积及甘油三酯含量降低。干扰后得到相反的结果。进一步研究发现SREBP1干扰后下调了SIRT1的启动子活性，定点突变及CHIP实验结果表明，SREBP1蛋白可直接与SIRT1启动子上的SRE元件结合，通过直接调控SIRT1基因表达进而参与脂肪酸合成的调控。H3K4ac和H3K9ac抗体进行CHIP-seq并结合mRNA-seq分析结果表明，SIRT1可能靶向TRIM23、HSD17B11、TRIM21、HADH等基因参与肌内脂肪沉积调控；靶基因TRIM23的功能研究进一步表明SIRT1可通过TRIM23降低肌内脂肪沉积。这些结果揭示了SIRT1一方面受到脂代谢关键转录因子SREBP1的转录调控；另一方面，SIRT1通过对脂代谢相关因子进行去乙酰化修饰，进而在肌内脂肪沉积中发挥重要调控作用。本项目研究结果对通过分子遗传改良提升牦牛肉品质奠定了坚实的理论基础。