广灵驴FTO基因克隆、序列分析及组织差异表达

目的] 对广灵驴脂肪和肥胖相关基因（fat mass and obesity-associated gene，FTO）进行克隆及序列分析，同时检测其在广灵驴各组织中的表达差异，以探究广灵驴FTO基因结构对其生理代谢功能的影响。方法] 运用RT-PCR技术扩增并克隆广灵驴FTO基因CDS序列，进行基因及蛋白质功能分析，同时运用实时荧光定量PCR方法检测FTO基因在广灵驴7种组织（心脏、肝脏、脾脏、肺脏、肾脏、背最长肌及皮下脂肪）中的差异表达。结果] 广灵驴FTO基因CDS区序列长1 518 bp，编码505个氨基酸，序列提交至NCBI，登录号：MZ169553。广灵驴FTO基因与马、猪、牛、人、羊驼、绵羊和山羊的相似性为99.3%、90.3%、89.5%、90.8%、90.7%、89.2%和89.2%；系统进化树分析发现，广灵驴与马的亲缘关系最近。FTO蛋白分子质量为58.35 ku，理论等电点为5.07，脂肪系数为80.36，不稳定系数为48.82，平均疏水指数为-0.550，为不稳定的酸性亲水蛋白。FTO蛋白无信号肽和跨膜区，主要定位于细胞质中，具有34个磷酸化位点与5个糖基化位点。FTO蛋白二级结构主要以α-螺旋（43.96%）和无规则卷曲（37.82%）为主。实时荧光定量PCR分析发现，FTO基因在广灵驴7个组织中均有表达，其中在肺脏和皮下脂肪中的表达量极显著高于其他组织（P<0.01），在背最长肌中表达量最低。结论] 本试验结果可为下一步基因表达与调控脂肪沉积机制及改善驴肉品质的研究奠定基础。

Cloning,Sequence Analysis and Tissue Differential Expression of FTO Gene in Guangling Donkey

Objective] The purpose of this study was to clone and analyze the sequence of fat mass and obesity-associated gene(FTO), detect the expression difference of FTO gene in various tissues of Guangling donkey, and explore the effect of the structure of FTO gene related to fat on the physiological and metabolic functions of Guangling donkey. Method] RT-PCR method was used to amplify and clone the CDS sequence of FTO gene, and analyze the gene and protein function. Meanwhile, Real-time quantitative PCR was used to detect the differential expression of FTO gene in seven tissues (heart, liver, spleen, lung, kidney, longissimus dorsi muscle and subcutaneous fat) of Guangling donkey. Result] The results showed that the CDS sequence of FTO gene in Guangling donkey was 1 518 bp and could encode 505 amino acids. The sequence was uploaded to NCBI and obtained the accession No.:MZ169553. The similarity of FTO gene in Guangling donkey were 99.3%, 90.3%, 89.5%, 90.8%, 90.7%, 89.2% and 89.2% with Equus caballus, Sus scrofa, Bos taurus, Homo sapients, Vicugna pacos, Ovis aries and Capra hircus, respectively. Phylogenetic tree analysis result showed that FTO gene of Guangling donkey was closely to Equus caballus. The molecular weight of FTO protein was 58.35 ku, the theoretical isoelectric point was 5.07, the fat index was 80.36, the instability coefficient was 48.82, and the average hydrophobic index was -0.550, the FTO protein of Guangling donkey was an unstable acidic hydrophilic protein. There was no signal peptide and transmembrane region of FTO protein, which were mainly located in the cytoplasm, and had 34 phosphorylation sites and 5 glycosylation sites. The secondary structure prediction of FTO protein showed that α-helix (43.96%) and random coil (37.82%) were the main structures. Real-time quantitative PCR analysis showed that FTO gene was all expressed in 7 kinds of tissues, the expression of FTO gene in lung and subcutaneous fat were extremely significantly higher than other tissues (P<0.01), and the expression was the lowest in longissimus dorsi muscle. Conclusion] The results of this study provided a solid theoretical basis for the further research on gene expression, mechanism of fat deposition, and improved the meat quality of Guangling donkey.