CD44基因对牛前体脂肪细胞分化和脂肪沉积的作用及调控机制

Cattle industry is an important part of the modern agricultural production system in our country. Although many cattle breeds with excellent meat quality traits have been obtained through continuous breeding for many years, the ability of intramuscular fat deposition still has gaps compared with the foreign beef breeds. Therefore, study of genes involved in lipid metabolism and intramuscular fat deposition is particularly important. Previously, comparative genome-wide methylation and RNA-seq analysis of longissimus dorsi muscles between Japanese black (Wagyu) and Chinese Red Steppes cattle, the meat quality traits candidate gene CD44 were obtained, and overexpressing in the bovine fetal fibroblasts could increase intracellular triglyceride content. However, at present, the role and molecular mechanisms of CD44 on preadipocyte differentiation or fat deposition are still not clear. The aim of this study is to reveal the molecular mechanism of CD44 in regulating of bovine preadipocyte differentiation and adipogenesis by investigating the function of CD44 on fat metabolism in beef cattle with detecting the expression and location of CD44 gene in adipose tissue and preadipocytes, analyzing the effect of CD44 expression levels on preadipocyte differentiation and fat synthesis, constructiing the regulation network of CD44 involved in fat metabolism and verifying the proteins that interact with the CD44 gene. It will provide a theoretical basis for study new genes of bovine fat metabolism.

养牛业是我国现代农业生产体系的重要组成部分。经过多年持续选育，我国已培育了多个肉质性状优良的新品种，但其肌内脂肪沉积能力与国外优良肉牛品种相比依然存在差距。因此，脂肪代谢和肌内脂肪沉积功能基因的挖掘和鉴定依然是肉牛分子育种的关键问题。申请人前期转录组和全基因组DNA甲基化联合分析表明，CD44可能对肉质性状具有决定作用，在牛成纤维细胞初步验证结果表明，CD44过表达可以增加细胞内甘油三酯含量，但目前对于CD44在前体脂肪细胞分化和脂肪沉积的作用及分子机制尚不清楚。本项目通过检测CD44在牛脂肪组织和细胞中的表达水平和蛋白质定位，分析CD44表达水平对前体脂肪细胞分化和脂肪合成的影响，构建CD44参与脂肪代谢的基因调控网络以及验证与CD44存在相互作用的蛋白质，系统研究CD44对牛脂肪代谢的作用，揭示CD44调控牛前体脂肪细胞分化和脂肪合成的分子机制，为挖掘牛脂肪代谢功能基因提供理论依据。

本研究针对我国已培育的肉牛品种肌内脂肪沉积能力与国外优良肉牛品种相比依然存在差距，以及肉牛新品种培育和改良过程中脂肪代谢和脂肪沉积功能基因的挖掘和鉴定的突出问题，以申请人所在团队前期筛选的CD44基因作为影响牛脂肪性状的候选功能基因，采用慢病毒介导的RNA干扰技术、转录组与代谢组联合分析、Seahorse细胞代谢分析，PCR array等，结合常规分子遗传技术手段，分析了CD44基因在牛的各组织中的表达水平，牛前体脂肪细胞分化过程中的表达规律，以及前体脂肪细胞、乳腺上皮细胞中的亚细胞定位，并首次将CD44蛋白质与脂肪细胞内脂滴共定位；解析CD44表达水平对前体脂肪细胞增殖、分化，以及细胞中甘油三酯含量和其它代谢产物成分的影响，证明了CD44表达水平降低促进前体脂肪细胞分化和脂肪细胞内甘油三酯合成；同时预测蛋白质结构、功能和可能的蛋白与分子间互作模式结果显示，CD44部分结构域能与甘油和脂肪酸类代谢物结合。综上所述，系统的解析CD44基因对于牛前体脂肪细胞分化和脂肪代谢的影响，并初步揭示了其在此过程中发挥作用的分子机制。为我国肉牛分子育种提供了新的功能基因，为牛品种培育和改良奠定了分子遗传基础。