调控山羊奶脂肪酸代谢的miRNA功能分析与验证

Studies on the mechanism of fatty acid metabolism in goat milk is of the vital scientific significance on investigating the goaty flavor genesis and imporving the goat milk quality. Based on our preliminary research results which miRNA regulate the fatty acid metabolisms in goat mammary epithelial cells (GMEC), 14 miRNAs conducted previously the function analysis were selected for the further precision function verification, as the experimental animal, the milk samples of Xinong Saanen dairy goats were collected in the different stages of lactation to measure the fatty acid composition, the biopsy samples of mammary gland were taken to determine the expression levels of selected miRNAs, and the 2-3 key miRNAs that significantly related with milk fatty acid content will be identified by the correlation analysis of milk fatty acid composition and miRNAs expressions; the epigenetic analysis of miRNAs and its target genes were analysed using GMEC to elucidate the molecular mechanisms of miRNAs epigenetically regulating the fatty acid metabolism; analysis on the target genes and signal pathway of miRNAs investigate the regulation of signal pathway on fatty acid metabolism of GMEC; Furthermore, the miRNAs knockout GMEC and knockout goats were produced using CRISPR/Cas9 technique, determining the fatty acid composition in GMEC and milk, which the regulation of miRNAs on fatty acid metabolism will be verified to reveal the molecular mechanisms of precision regulation of milk fatty acid metabolism by single or multiple miRNAs, the results will be beneficial to decipher the regulation mechanism of milk fatty acids and to provide the evidence for cause analysis of goaty flavor in goat milk.

山羊奶脂肪酸代谢机理研究对探讨羊奶膻味成因和改良羊奶品质等具有重要科学意义。在miRNAs调控奶山羊乳腺脂肪酸代谢研究基础上，本项目选择功能已知的14个miRNAs为研究对象，以西农萨能奶山羊为实验动物，采集泌乳期不同阶段奶样和活体乳腺组织样，通过羊奶脂肪酸含量与miRNA表达量的关联分析，筛选2-3个与羊奶脂肪酸显著相关的miRNAs，在奶山羊乳腺上皮细胞上分析miRNAs及其靶基因的表观遗传作用规律，阐明miRNAs表观遗传调控乳腺脂肪酸代谢的特点；通过miRNAs靶基因及信号通路分析，探讨信号通路对乳腺上皮细胞脂肪酸代谢的作用；利用CRISPR/Cas9技术制备miRNAs敲除乳腺上皮细胞和敲除奶山羊，检测细胞和羊奶脂肪酸含量，验证miRNAs对脂肪酸代谢的调控作用；揭示单个或多个miRNAs精细调控羊奶脂肪酸代谢的分子机理，有助于解析羊奶脂肪酸调控机制，为羊奶膻味成因探究提供依据。

羊奶的独特风味和营养保健功效与羊奶中含有的大量短、中链脂肪酸及不饱和脂肪酸等密切相关，羊奶脂肪酸代谢调控机理研究对探究羊奶膻味成因和改善羊奶品质等具有重要的理论意义和实际应用价值。本研究选择了在奶山羊乳腺上皮细胞前期研究中发现的14个已知调控山羊乳腺乳脂代谢的miRNAs，采集西农萨能奶山羊泌乳中期的奶样和乳腺组织样，通过羊奶脂肪酸含量与乳腺miRNAs表达量的关联分析以及干扰过表达研究，筛选出6个与羊奶中短链和长链脂肪酸含量相关的miRNAs。本项目利用CRISPR/Cas9基因编辑技术在奶山羊乳腺上皮细胞和小鼠活体中敲除miRNAs并深入分析验证miRNAs通过靶基因及信号通路调控脂肪酸代谢的分子机理，为miRNAs调控羊奶品质提供理论和实验依据。主要研究结果如下：. 本研究利用气相色谱测定了西农萨能奶山羊泌乳中期（产后第100天）奶样中的脂肪酸组成，手术法活体采集乳腺组织并检测了14个前期验证过的miRNAs的表达量。关联分析表明，miR-145、miR-24、miR-130b、miR-26a、miR-26b和miR-103等6个miRNAs与羊奶中短链及长链脂肪酸含量相关。利用CRISPR/Cas9技术在山羊乳腺上皮细胞中敲除miRNAs，研究miRNAs在胞内脂质及脂肪酸代谢中的作用；通过双荧光素酶、RT-qPCR、Western Blot和rescue实验证实靶基因在miRNA调控脂质及脂肪酸代谢的功能。结果发现：miR-145通过INSIG1基因调控山羊乳腺上皮细胞的脂肪酸代谢；miR-24靶向INSIG1与FASN基因调控乳腺上皮细胞的胆固醇代谢与单不饱和脂肪酸成分；miR-130b靶向PPARG-PGC1α 信号轴调控乳腺上皮细胞脂肪酸代谢；敲除miR-26a通过靶向INSIG1基因抑制乳腺上皮细胞中脂质及不饱和脂肪酸合成；miR-26b敲除通过靶向INSIG1基因调控乳腺上皮细胞的脂质及脂肪酸合成；山羊乳腺上皮细胞中敲除miR-103抑制脂质合成且影响脂肪酸组成；miR-204-5p与miR-211协同下调αS1-酪蛋白表达进而影响羊奶的低致敏性。通过制作基因编辑小鼠验证了miR-26a2与miR-130b调控功能。本项目为利用CRISPR/Cas9技术编辑miRNAs进行动物基因编辑育种和乳品质调控等提供理论依据和技术手段。