延边牛PPARγ基因的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of PPAR γ Gene of Yanbian Cattle

研究旨在克隆和分析延边牛过氧化物酶体增殖物激活受体γ(PPARγ)基因,并探讨其在延边牛不同组织中的表达规律。参考GenBank数据库中牛PPARγ的序列(登录号:NM_181024.2)设计引物,以18月龄延边牛为试验对象,利用RT-PCR克隆得到延边牛PPARγ基因,利用NCBI中的BLAST程序与其他物种进行相似性分析并构建系统进化树;用生物信息学软件分析其核苷酸序列及其编码蛋白的理化性质、疏水性、信号肽、跨膜结构域、亚细胞定位、磷酸化位点、糖基化位点、二级结构、三级结构等;用实时荧光定量PCR检测延边牛心脏、肝脏、脾脏、肺脏、肾脏、小肠、皮下脂肪、肌肉8个组织中PPARγ基因的相对表达水平。结果显示,延边牛PPARγ基因CDS区序列为1620 bp,相似性比对和系统进化树结果显示,与黄牛的亲缘关系最近,相似性为99.9%,与鸡的亲缘关系最远,相似性为82.1%;该基因编码505个氨基酸,无信号肽和跨膜结构域,为亲水性蛋白,主要分布在细胞核和细胞质中;延边牛PPARγ蛋白二级结构主要为α-螺旋和无规则卷曲,存在53个潜在的磷酸化位点,24个O-糖基化位点;实时荧光定量PCR结果显示,延边牛PPARγ基因在脾脏、皮下脂肪、肝脏中表达量显著高于心脏(P<0.05),肺脏、肾脏、肌肉和小肠中表达量显著低于心脏(P<0.05)。以上试验结果可为进一步研究PPARγ基因的功能提供借鉴。

The purpose of this study was to clone and analyze the peroxisome proliferator-activated receptorγ(PPARγ)gene of Yanbian cattle,and explore its expression patterns in different tissues of Yanbian cattle.Primers were designed based on the sequence of bovine PPARγgene in the GenBank database(accession No.:NM_181024.2).Taking 18-month-old Yanbian cattle as the experimental object,the PPARγgene of Yanbian cattle was cloned by RT-PCR,and BLAST in NCBI was used to carry out homology analysis and construct a phylogenetic tree with other species.Bioinformatics softwares were used to analyze the nucleotide sequence and the physicochemical properties,hydrophobicity,signal peptides,transmembrane domains,subcellular localization,phosphorylation sites,glycosylation sites,secondary structure and tertiary structure of PPARγprotein.Real-time quantitative PCR technology was used to detect the expression level of PPARγgene in heart,liver,spleen,lung,kidney,small intestine,subcutaneous fat,muscle of Yanbian cattle.The results showed that the CDS region sequence of Yanbian cattle PPARγgene was 1620 bp,the sequence and phylogenetic tree analysis showed that it had 99.9%homology with that of yellow cattle and 82.1%homology with chicken.PPARγgene encoded a protein of 505 amino acids,and the protein had no signal peptide and transmembrane domain,which was hydrophilic protein and mainly distributed in the nucleus and cytoplasm.The main secondary structure of Yanbian cattle were alpha helix and random coil,and there were 53 potential phosphorylation sites and 24 O-glycosylation sites.Real-time quantitative PCR results showed that the expression of Yanbian cattle PPARγgene in spleen,subcutaneous fat and liver was significantly higher than that in heart(P<0.05),the expression of it in lung,kidney,muscle and small intestine was significantly lower than that in heart(P<0.05).The above results could provide reference for further study of the function of PPARγgene.