摘要： 以牦牛为试验对象，旨在克隆牦牛增强子结合蛋白α基因（CEBPα），预测其蛋白结构和功能，同时分离得到牦牛脂肪细胞，并检测CEBPα在牦牛不同组织中的表达及牦牛脂肪细胞诱导分化过程中的表达。采集成年牦牛（4岁）的心、肝、脾、肾、胃、小肠、皮下脂肪、内脏脂肪和肌肉组织，利用PCR技术获得CEBPα基因序列，得到其CDS序列；生物信息学的方法预测该基因的序列同源性及蛋白结构；通过实时荧光定量PCR （qRT-PCR）检测CEBPα在牦牛不同组织的表达；通过胶原酶消化法，分离得到牦牛前体脂肪细胞；qRT-PCR检测CEBPα在诱导分化0，3，6，9 d的表达模式。结果表明，牦牛CEBPα基因的CDS序列长度为645 bp，编码214个氨基酸；牦牛CEBPα基因与普通牛的同源性相对较高，核酸同源性为99.73%，氨基酸同源性为99.19%；构建系统进化树的结果显示，牦牛与普通牛和绵羊的相似性较高；CEBPα蛋白为具有跨膜结构、发生磷酸化/去磷酸化的不稳定亲水性蛋白，蛋白质的二级结构以α螺旋（27.10%）和无规则卷曲（67.29%）为主；qRT-PCR结果显示，CEBPα基因在牦牛皮下脂肪组织中表达最高，在肾、脾和内脏脂肪组织中低表达； CEBPα在牦牛脂肪细胞诱导分化过程中表达量逐渐升高。本试验克隆获得了CEBPα的CDS区序列，确定了其在牦牛各个组织中的表达量并明确了该基因在牦牛脂肪细胞诱导分化过程中的表达模式，为揭示CEBPα基因在牦牛脂肪组织和脂肪细胞中的功能提供基础数据。

关键词: 牦牛, CEBPα, 克隆, 脂肪组织, 表达模式

Abstract: Yak was used as the experimental object to clone the yak CCAAT/Enhancer binding protein alpha(CEBPα) gene, predicted its protein structure and function, isolated yak adipocyte, and detected the expression of CEBPα in different tissues of yak and yak fat expression during cell induction and differentiation. Collected the heart, liver, spleen, kidney, stomach, small intestine, subcutaneous fat, visceral fat and muscle tissue of an adult yak (4 years old), and used PCR technology to obtain the CDS sequence of the CEBPα gene;the method of bioinformatics predicted the sequence homology and protein structure of the gene;Real-time fluorescent quantitative PCR(qRT-PCR) was used to detect the expression of CEBPα in different tissues of yak;the yak preadipocytes were isolated by collagenase digestion, qRT-PCR was used to detect the expression pattern of CEBPα gene at 0, 3, 6, 9 d of induced differentiation;the results of the study showed that the CDS sequence of the yak CEBPα gene was 645 bp in length and encoded 214 amino acids. The yak CEBPα gene had relatively high homology with Bos taurus, with a nucleic acid homology of 99.73% and amino acid homology of 99.19%. The results of constructing a phylogenetic tree showed that the yak was more similar to ordinary cattle and sheep. CEBPα protein was an unstable hydrophilic protein with a transmembrane structure and phosphorylation/dephosphorylation. The secondary structure of the protein was dominated by α helix (27.10%) and random coil (67.29%). The results of qRT-PCR showed that the CEBPα gene was most highly expressed in the lower adipose tissue of the yak, and lowly expressed in the kidney, spleen and visceral adipose tissue. The expression of CEBPα gradually increased during the induction and differentiation of yak adipocytes. In this experiment, we cloned and obtained the CDS region sequence of CEBPα, determined its expression in various tissues of yak and clarified the expression pattern of this gene in the process of induced differentiation of yak adipocytes. Provide basic data to reveal the function of CEBPα gene in yak adipose tissue and adipocyte.

Key words: Yak, CEBPα, Clone, Adipose tissue, Expression pattern

中图分类号: 

• Q78

• S823