摘要： 旨在克隆延边牛解偶联蛋白-1（UCP1） CDS区序列，利用RT-PCR技术和基因克隆获得延边牛UCP1基因，与其他物种进行同源性比对及构建系统进化树，利用生物信息学方法预测UCP1编码蛋白质的理化性质、潜在的磷酸化位点等性质，利用实时荧光定量PCR （Quantitative Real-time PCR，qPCR）方法检测UCP1基因在延边牛不同组织的表达丰度。结果显示，延边牛UCP1基因的CDS区全长749 bp，编码249个氨基酸。同源性比对发现延边牛UCP1基因与印度牛同源性为99.47%，系统进化树表明，延边牛与印度牛的亲缘关系最近，和南美羊驼亲缘关系最远。UCP1蛋白缺乏稳定性，脂溶系数是91.53，网状红细胞于体外的半衰期是30 h，总平均亲水性等于0.212，预测其具备一定亲水性。二级结构由α-螺旋（54.03%）、延伸链（12.90%）、无规卷曲（27.42%）和β-转角（5.65%）构成的混合型蛋白。磷酸化位点和糖基化位点分析结果表明，UCP1共有21个磷酸位点，4个潜在的O-糖基化位点，3个N-糖基化潜在位点。UCP1基因的编码产物无跨膜螺旋（TMHs）结构，跨膜螺旋氨基酸残基数量的预测值为19.008 57，蛋白质前60个氨基酸的跨膜螺旋数预测值为10.980 09，位于膜细胞质侧的总概率为22.995%且属于非分泌蛋白。经实时荧光定量PCR （RT-PCR）可知，延边牛脂肪与小肠内UCP1基因表达水平相对较高，而在肌肉、心脏中的表达水平较低。该试验结果可为进一步研究该基因的功能提供借鉴。

关键词: 延边牛, UCP1 基因, 克隆, 生物信息学, 组织mRNA表达

Abstract: The aim was to clone the CDS region sequence of Yanbian cattle UCP1 (Uncoupling protein-1), use RT-PCR technology and gene cloning to obtain Yanbian cattle UCP1 gene, conduct homology comparison with other species and construct a phylogenetic tree, using bioinformatics methods to predict the physical and chemical properties of UCP1 encoded protein, potential phosphorylation sites and other properties, and use Quantitative Real-time PCR (qPCR) method to detect the expression abundance of UCP1 gene in different tissues of Yanbian cattle. The results showed that the CDS region of Yanbian cattle UCP1 gene was 749 bp in length, encoding 249 amino acids. The homology comparison revealed that the UCP1 gene of Yanbian cattle had 99.47% homology with Indian cattle. The phylogenetic tree showed that Yanbian cattle and Indian cattle had the closest genetic relationship and the farthest related to South American alpaca. The UCP1 protein lacked stability, the fat solubility coefficient was 91.53, the half-life of reticulocytes in vitro was 30 h, and the total average hydrophilicity was equal to 0.212, which was predicted to have a certain degree of hydrophilicity. The secondary structure was a mixed protein composed of α-helix (54.03%), extended chain (12.90%), random coil (27.42%) and β-turn (5.65%). The analysis of phosphorylation sites and glycosylation sites showed that UCP1 had a total of 21 phosphate sites, 4 potential O-glycosylation sites, and 3 N-glycosylation potential sites. The encoded product of UCP1 gene had 0 transmembrane helix (TMHs) structure, the predicted value of the amino acid residues of the transmembrane helix was 19.008 57, and the predicted value of the transmembrane helix of the first 60 amino acids of the protein was 10.980 09, which was the total probability of being located on the cytoplasmic side of the membrane 22.995% and was a non-secreted protein. Real-time fluorescent quantitative PCR (RT-PCR) showed that the expression level of UCP1 gene in Yanbian beef fat and small intestine was relatively high, while the expression level in muscle and heart was low. The experimental results could provide a reference for further research on the function of this gene.

Key words: Yanbian cattle, UCP1 gene, Cloning, Bioinformatics, Tissue mRNA expression

中图分类号: 

• Q78

• S826