ASIP基因介导的牛脂肪和脂肪酸性状调控机制研究

Fat distribution and fatty acid composition are important economic traits in livestock and poultry which are regulated by multi-genes. In recent years, the study on function genes screening and regulation mechanism involved in regulation of lipid metabolism is one of the hotspots in animal genetics and breeding. In our previous study, the results of epigenomics and transcriptomics in the longissimus dorsi muscle between Japanese black cattle (Wagyu) and Chinese Red Steppes, and also the results of transcriptomics and micRNA analysis in mammary epithelial cells between the high milk fat and low milk fat cow showed that the ASIP gene was differentially expressed which indicated this gene may play an important role in the formation of fat and fatty acid traits in meat and milk quality. In order to analyze the regulation mechanism of ASIP gene, this research will be focus on the fat metabolism in beef and milk quality, to deeply understand the expression pattern of ASIP gene in different tissues of cattle and cow and the correlation with meat and milk quality traits. On the basis of knockdown and over-expression vector construction, the effects of ASIP on the metabolism of lipid and fatty acid will be revealed in bovine pre-adipocytes and mammary gland epithelial cells by RNA-seq and PCR array and the regulatory networks and mechanism of ASIP gene will be explored in lipogenesis and fatty acid metabolism. The results of this project will lay a foundation to the discovery of novel function genes for fat and fatty acid traits in bovine genetics and breeding.

脂肪分布和脂肪酸成分与含量是由多基因调控的畜禽重要经济性状。脂质代谢相关功能基因筛选和调控机制研究是近年来动物遗传育种领域的研究热点之一。本课题组前期的表观组学和转录组学研究结果表明，ASIP基因在和牛和草原红牛背最长肌组织中呈差异表达，在高乳脂和低乳脂奶牛乳腺上皮细胞系进行的转录组学和micRNA组学分析中也表现差异表达，表明该基因可能对牛肉质和乳品质的脂肪性状形成具有重要作用。为解析该基因对于牛肉质和乳品质中脂肪代谢的调控机制，本研究拟以ASIP基因为研究对象，深入了解该基因在肉牛和奶牛不同组织中的表达模式及与肉质和乳品质性状的相关性；在敲除和过表达载体构建的基础上，利用RNAseq和PCR array等技术，揭示ASIP表达水平对牛前体脂肪细胞和乳腺上皮细胞脂肪与脂肪酸代谢的调节作用，探索ASIP基因对脂肪生成和脂肪酸代谢相关的调控网络和作用机制，为发掘新的脂肪性状功能基因奠定基础。

肉牛和奶牛脂肪含量和脂肪酸成分与牛肉品质和乳品质形成密切相关，这些复杂性状的形成受功能基因的调控。筛选与牛脂肪代谢相关的候选基因，明确这些功能基因的作用机制，对于肉质和乳品质的分子育种具有重要的意义。基于实验室前期RNA-seq与定量验证结果表明，日本和牛与草原红牛背最长肌ASIP mRNA表达量存在显著差异，表明ASIP可能参与牛脂质代谢。通过分析ASIP基因及5’调控区的遗传变异情况，确定牛背最长肌中ASIP基因表达水平显著升高的作用机制以及ASIP基因Exon2C的重要作用；在中国西门塔尔牛群体（135头）和中国荷斯坦奶牛（88头）群体中进行了Exon2C序列的检测以及ASIP基因启动子序列SNPs位点的筛查和与肉质、乳品质性状的关联分析。以248头F2代杂交牛（夏罗莱牛×荷斯坦牛）为样本，对ASIP基因及其受体基因（MC4R，ATRN）在杂交牛的血清，不同脂肪组织，背最长肌组织，肝脏组织中的表达水平进行检测，了解了ASIP基因在牛不同组织类型中的表达模式；通过构建干扰、过表达载体以及Crispr-Cas9的方法对ASIP基因与牛胎儿成肌细胞、牛前体脂肪细胞和乳腺上皮细胞脂肪沉积、分泌和脂肪酸含量的影响进行了研究，了解了ASIP基因对牛胎儿成肌细胞、乳腺上皮细胞和牛前体脂肪细胞脂肪沉积、分泌和脂肪酸含量的影响；通过CRISPR/Cas9技术在基因组中定点整合了ASIP基因，通过与结合脂肪组织特异性表达Cre酶小鼠，获得了ASIP脂肪组织特异性表达小鼠模型。对小鼠的生长情况、对肝脏的影响、对脂肪组织脂肪代谢相关生化指标的影响、脂肪细胞大小等进行了检测。在动物水平上进一步研究了ASIP基因对脂质代谢的影响；阐明了ASIP基因对肌间脂肪含量和乳脂率的调控作用和机制，完善了牛肉质、乳品质和脂肪酸调控网络。本研究为筛选牛肉质、乳品质性状功能新基因提供了分子依据，同时，为改善牛肉质、乳品质性状和加快分子育种进程奠定了理论基础。