延边牛PLIN2基因CDS区克隆、生物信息学分析及其表达规律研究

Cloning,Bioinformatics Analysis and Expression of PLIN2 Gene CDS Region in Yanbian Cattle

本研究旨在克隆延边牛脂肪分化相关蛋白PLIN2基因完整CDS区,通过生物信息学分析PLIN2基因CDS区序列和蛋白质基本特性,探讨其在延边牛不同组织及前体脂肪细胞成脂过程中的表达规律。选取18月龄去势的延边牛为研究对象,利用RT-PCR和基因克隆获得延边牛PLIN2基因,与其他物种进行同源性比对及系统进化树构建,利用生物信息学方法预测PLIN2编码蛋白质的理化性质、潜在磷酸化位点及糖基化位点、二硫键分析、信号肽、亚细胞定位、蛋白高级结构,利用实时荧光定量PCR检测PLIN2基因在延边牛不同组织中的表达水平。结果显示,延边牛PLIN2基因CDS长1353 bp,编码450个氨基酸;同源性比对发现延边牛PLIN2基因与黄牛、水牛、绵羊、山羊、野猪、人、小鼠和野鸡的同源性分别为100%、98.7%、96.7%、97.1%、85.5%、86.3%、47.3%和49.1%;系统进化树表明,延边牛与黄牛、水牛的亲缘关系最近,与野鸡的亲缘关系最远。PLIN2蛋白为不稳定蛋白,具有一定亲水性,存在61个潜在的磷酸化位点、16个O-糖基化潜在位点、12个N-糖基化潜在位点,存在2个二硫键,不存在信号肽,主要分布于细胞核中,细胞质和线粒体中有少量分布。PLIN2蛋白高级结构预测该蛋白是由α-螺旋、延伸链、无规则卷曲和β-转角组成,为混合型蛋白,通过无规则卷曲连接,以α-螺旋为主。实时荧光定量PCR结果显示,PLIN2基因在延边牛小肠组织中表达量最高,其次为背最长肌和肾脏组织。本研究结果可为进一步研究PLIN2基因的功能提供借鉴。

The present study was aimed to clone the complete CDS region of Yanbian cattle adipose differentiation-related protein PLIN2 gene,analyze the sequence and basic protein characteristics of PLIN2 gene CDS region through bioinformatics,and explore its presence in different tissues of Yanbian cattle.The 18-month-old castrated Yanbian cattle were selected as the research object.PLIN2 gene of Yanbian cattle was obtained by RT-PCR and gene cloning,and the homology comparison with other species was carried out and the phylogenetic tree was constructed.The PLIN2 encoded protein was predicted by bioinformatics methods the physicochemical properties,potential phosphorylation sites,glycosylation sites,disulfide bond analysis,signal peptides,subcellular localization and high-level structure of the protein,Real-time quantitative PCR was used to detect the expression level of PLIN2 gene in different tissues of Yanbian cattle.The results showed that the CDS of PLIN2 gene in Yanbian cattle was 1353 bp long and encoded 450 amino acids.The homology comparison showed that Yanbian cattle PLIN2 gene had 100%homology with cattle,98.7%for buffalo,96.7%for sheep,97.1%for goat,85.5%for wild boar,86.3%for human,47.3%for mouse,and 49.1%for pheasant.The phylogenetic tree showed that Yanbian cattle had the closest genetic relationship with Yellow cattle and buffalo,and the farthest genetic relationship with pheasants.PLIN2 protein was an unstable protein with a certain degree of hydrophilicity.There were 61 potential phosphorylation sites,16 potential O-glycosylation sites,12 potential N-glycosylation sites,and 2 disulfide sites,there was no signal peptide,mainly distributed in the nucleus,a small amount in the cytoplasm and mitochondria.The high-level structure of PLIN2 protein predicted that the protein mainly contains a mixed protein composed ofα-helix,extended chain,random coils andβ-turns,which were connected by random coils,mainlyα-helix.Real-time quantitative PCR results showed that PLIN2 gene was expressed the highest in small intestine tissue of Yanbian cattle,followed by longissimus dorsi muscle and kidney tissue.The results of this study could provide a reference for further research on the function of PLIN2 gene.