ACSL1调控牛肌内前体脂肪细胞不饱和脂肪酸合成作用及表观遗传学机制

Long-chain acyl-CoA synthetase 1 (ACSL1) has been identified as a plausible functional and positional candidate gene for manipulation of fatty acid composition in bovine skeletal muscle. However, the regulation and epigenetic on the synthesis of unsaturated fatty acids in bovine intramuscular preadipocytes remain elusive. In this study, in order to clarify the regulation and epigenetic mechanism of ACSL1 on regulation unsaturated fatty acid synthesis, which is vitally significant for promoting the nutritive value of beef, the bovine intramuscular preadipocytes are used as a study model. 1) overexpression of ACSL1 and RNA small interference are performed to verify the functions for the synthesis of unsaturated fatty acids. 2) To screen the specific lncRNAs from siRNA small interference and control in intramuscular preadipocytes using lncRNA sequencing technology, and to analyze the effects of specific lncRNAs on methylation of ACSL1 promoter; 3) The target genes of the specific lncRNAs will be verified by dual-luciferase reporter assay；4) To screen the key protein binding to lncRNAs by ChIRP-MS technology. Furthermore, the target protein binding specific lncRNAs will be verified by western blotting、RIP and EMSA. The synthesis mechanism of unsaturated fatty acids will be elaborated from the aspects of lncRNA regulation, which will reveal the mechanism unsaturated fatty acid synthesis in bovine intramuscular preadipocytes and provide theoretical basis for promoting the content of healthy fatty acids and improving the quality of beef.

ACSL1作为最重要位置和功能候选基因，直接影响牛肉骨骼肌不饱和脂肪酸的组成，但目前该基因调控肌内脂肪细胞不饱和脂肪酸合成的作用及表观遗传学机制尚不清楚，明确ACSL1基因作用及其表观遗传学机制对提高牛肉的营养价值具有重要科学意义。本课题以牛肌内前体脂肪细胞为模型；1）利用超表达和siRNA干扰验证ACSL1基因调控不饱和脂肪合成的作用；2）拟筛选IncRNA-seq获得干扰ACSL1和对照组特异lncRNAs，分析验证特异lncRNAs对ACSL1启动子甲基化的影响；3）双荧光素酶系统验证特异lncRNAs调控的靶基因；4）通过ChIRP-MS筛选与特异lncRNAs结合的蛋白，利用Western blotting、RIP和EMSA验证特异lncRNAs与靶蛋白的结合；从表观遗传学角度阐明lncRNAs对不饱和脂肪酸合成的调控机制，为提高有益脂肪酸含量、改善牛肉品质提供理论依据。

ACSL1作为最重要位置和功能上的候选基因，直接影响牛肉骨骼肌中不饱和脂肪酸的组成，且ACSL1基因表达可能受到表观遗传学调控。lncRNAs最重要的功能是参与编码基因表达调控的表观遗传学机制，其中许多lncRNAs仅在特定的发育阶段出现或具有细胞组织特性。因此，本研究以牛肌内前体脂肪细胞为模型，发现过表达或干扰ACSL1基因对牛脂肪细胞中不饱和脂肪酸合成相关基因的表达、不饱和脂肪酸组成及脂滴形成具有重要的调控作用。通过RNA-seq对干扰ACSL1的牛前脂肪细胞和对照组进行了测序，筛选出差异表达的107个mRNAs、75个lncRNAs、28个miRNAs和39个circRNAs，差异表达mRNAs、lncRNAs以及miRNAs的靶基因在多个UFAs合成相关通路中显著富集。其中差异表达lncRNA-mRNA共表达网络、组织和时序表达分析结果表明，TCON_00069661等16个lncRNAs及ACSL1等23个mRNAs可能是调控牛脂肪细胞UFAs合成的关键lncRNAs和mRNAs。此外，构建了circRNA-miRNA-mRNA的ceRNA调控网络，生物信息预测发现novel_circ_0004855、novel_circ_0001507、novel_circ_0001731、novel_circ_0005276、novel_circ_0002060、novel_circ_0005405和novel_-circ_0004254可能通过与相关miRNAs相互作用调控牛脂肪细胞不饱和脂肪酸合成。研究结果为揭示牛脂肪细胞不饱和脂肪酸合成及分子调控机制，丰富不饱和脂肪酸形成的分子机理，提高有益脂肪酸含量以及改善牛肉品质提供理论依据。