脂联素介导绵羊肌内、内脏和皮下脂肪细胞生脂的基因表达模式与分子调控机理研究

脂联素是脂肪细胞所分泌的细胞因子中含量最高、的唯一与肥胖呈负相关的细胞因子，参与调节脂肪代谢、葡萄糖代谢、能量稳态调节及抵抗炎症反应等生理过程。脂联素及其受体基因研究主要集中在人、小鼠、猪、鸡上，绵羊脂联素及其受体的研究国内外还未见报道。本研究以兰州大尾羊为材料，首次克隆绵羊脂联素及其受体基因全长cDNA序列，qPCR研究脂联素及其受体基因时空表达规律；通过体外分离和培养绵羊肌内脂肪、皮下脂肪和内脏脂肪细胞，研究重组脂联素及其受体对不同脂肪细胞生脂及生脂相关基因表达的差异;通过siRNA技术沉默脂联素及其受体基因表达，探讨对脂联素及其受体表达信号通路关键基因PPARɑ、AMPK和p38MAPK的表达差异；分析脂联素及其受体与肌内脂肪沉积关系。初步揭示脂联素及其受体基因调控绵羊脂肪组织生脂的分子机理，为最终阐明绵羊体脂沉积和改善肉品质提供依据，也为肉羊育种中优良性状利用提供理论有益参考。

Adiponectin is the highest adipokine that secretes by adipocyte, which is also the only one that negatively correlated with obesity up until now. It plays an important role in energy supply, lipid metabolism and glucose homeostasis, as well as anti-atherogenic. Levels of adiponectin in the blood are decreased under conditions of obesity, insulin and type 2 diabetes. Studies of adiponectin and its receptors are mainly concentrated in human being, mouse, swine and chichen. Very few reports of adiponectin in ruminant species could be found, especially in sheep. .The present study, focusing on the Lanzhou fat-tailed sheep, will first clone the full length cDNA encoding adiponectin (adpn) and its receptors (adpnR). Temporal and spatial specificity of genes expression will be carried out by using quantitative real-time PCR (qPCR). Correlation analysis between gene expression of adpn and adpnR and IFM will be implement. Recombinant proteins will be induced to express in prokaryotic vectors pET-28a (+)-adpn, pET-28a (+)-adpnRs and purification process will be applied using fast protein liquid chromatography. The intramuscular fat (IMF), subcutaneous (s.c.) fat and visceral (v.c.) fat will be isolated and their adipocytes will be cultured in vitro. Mammalian ORF expression vectors of pReceiver-EU-adpn and pReceiver-EU-adpnR, eukaryotic expression vectors of pcDNA3.0-adpn and pcDNA-adpR will be constructed and transfected to cultured adipocytes. The influences of lipogenesis on above mentioned adipocytes under different concentrations of recombinant proteins will be exerted and differential expression levels of lipid metabolism genes will be analyzed by qPCR. Using technology of RNA interference (RNAi) to slience the expression of Adpn and adpnRs in cultured fat cells, the differences of gene expression of peroxisome proliferator-activate receptor ɑ (PPARɑ), AMP-activated protein kinase (AMPK) and p38 mitogen protein kinase (p38MAPK), which play key rols during signal transduction of adpn and adpnR, will be processed. Meanwhile, the lipogenesis and expression of fat-metabolismin-related genes in adpn and adpnR silent adpocytes of IMF, s.c. fat and v.c. fat will be elucdated.. These findings would initally reveal the molecular mechanism of lipogenesis in different fat dpots so as to provide some theoretical bases for finally clarifying body fat deposition and improving meat quality in sheep, as well as applying to the sheep breeding practice.