MiRNAs协同调控奶山羊乳腺脂肪酸代谢的机理研究

Investigating the mechanism of fatty acid metabolism in goat mammary gland is of the vital significance on understanding the goaty flavor genesis of goat milk and dairy goat production. Based on the prediction of miRNAs regulating fatty acid metabolism of Xinong Saanen dairy goats, Gas Chromatography is used to determine the composition and contents of fatty acid in goat milk; miRNA and mRNA expression profiles of mammary gland tissues in different lactation stages will be quantified by qRT-PCR to analyze the expression pattern of target genes; luciferase report gene vectors experiment for target genes will be used to validate synergistic miRNAs targeting single or multi-genes in the epithelial cells of goat mammary gland, combining with the measurements of target genes expression level and fatty acid composition in epithelial cells to construct the network of miRNAs synergistic regulating fatty acid metabolism of goats. Furthermore, miRNAs over-expression and interference experiments will be performed by injecting oligonucleotide into mammary gland of lactating goats, the miRNA knock-out mice will be established as well, the fatty acid composition of goat and mouse mammary gland will be determined together with the correlation between miRNAs expression and fatty acid composition in goat milk to explore the molecular mechanism of synergistic miRNAs regulating the fatty acid metabolism of dairy goats, which will provide theoretical and experimental basis for manipulating goat milk fatty acids content, and understanding the goaty flavor formation etc.

乳腺脂肪酸代谢机理对探讨羊奶膻味形成原因和奶山羊产业等具有重要科学意义。本课题在预测调控山羊脂肪酸代谢基因miRNAs基础上，以西农萨能奶山羊为对象，用气相色谱分析羊奶脂肪酸含量，定量PCR分析泌乳期不同阶段乳腺组织miRNA、预测靶基因的表达规律，通过靶基因荧光素酶载体实验在奶山羊乳腺上皮细胞中验证协同调控一个或多个基因的miRNAs功能，检测靶基因表达水平和细胞脂肪酸组成及含量变化，建立miRNAs协同调控山羊乳腺脂肪酸含量的网络图。此外，利用寡核苷酸注射法在山羊乳腺组织中过表达或抑制miRNAs表达，同时构建miRNAs敲除小鼠模型，检测山羊和小鼠乳中脂肪酸含量变化，结合miRNAs表达与羊奶脂肪酸组成变化的关系，研究miRNA协同调控奶山羊乳腺脂肪酸代谢的分子机制，为调控羊奶脂肪酸成分含量和探讨羊奶膻味形成机理等提供理论和实验依据。

羊奶中蛋白质、维生素、钙、矿物质、总脂肪含量高，羊奶有益脂肪酸含量（如短、中链脂肪酸，不饱和脂肪酸和共轭酸等）均显著高于牛奶，其特殊风味与乳脂代谢有关，遗传背景及调控机理十分复杂。MiRNA具有重要的转录后调控作用，可通过靶基因影响脂肪酸代谢，是解析羊奶风味形成机理的切入点。本课题根据miRNA测序结果对不同泌乳期（泌乳前期、泌乳盛期、泌乳后期和干奶期）奶山羊乳腺组织进行miRNA表达量检测初选，利用催乳素处理乳腺上皮细胞进行第二轮筛选，发现7个差异表达的miRNAs并对其功能、调控作用以及分子机制等进行系统深入的研究。获得以下主要结果： .以泌乳前期、泌乳盛期、泌乳后期和干奶期奶山羊乳腺组织进行miRNA表达量检测，基于miRBase数据库提供的793条牛的miRNA和267条羊的miRNA进行初选，以 p 值小于0.05且差异在4倍以上为标准，筛选得到156个差异表达miRNAs,随后利用与泌乳密切相关的因子—催乳素处理乳腺上皮细胞，进行第二轮筛选，发现miR-30e-5p，miR-15a，miR-181b，miR-148a，miR-17-5p，miR-135b和miR-26等7个miRNAs在催乳素处理的乳腺上皮细胞中表达量存在显著差异。利用过表达和抑制等方法研究miRNA在乳腺上皮细胞中对脂代谢的调控功能，通过荧光素酶报告基因实验和Western Blot验证miRNAs靶基因。结果表明：MiR-181b通过IRS2抑制甘油三酯（TAG）合成并调控Hippo信号通路基因；MiR-30e-5p和miR-15a通过靶向LRP6和YAP1基因协同调控乳脂代谢；PRL通过甲基化修饰抑制miR-135b功能；MiR-148a和miR-17-5p通过PPARGC1A和PPARA基因协同调控TAG合成；山羊miR-26家族表达量与部分乳成分和乳脂肪酸含量显著相关；miR-26家族对乳腺脂肪酸代谢具有协同调控作用；山羊miR-26及宿主基因家族也具有调控乳腺脂肪酸代谢的功能；山羊miR-26a、miR-26b启动子与脂代谢关键转录因子存在网络调控关系，根据上述研究结果构建了miRNAs调控奶山羊乳腺脂代谢的网络图。 .本课题筛选的7个miRNAs及其靶基因等调控奶山羊乳脂代谢的功能研究，为解析羊奶脂肪酸代谢及羊奶风味形成机理提供了理论和实验依据，具有潜在的实际应用价值。