山羊PPARα基因克隆、分子特性及组织差异表达分析

doi:10.16431/j.cnki.1671-7236.2018.03.001

摘要/Abstract

摘要：

试验旨在克隆山羊过氧化物酶体增殖物激活受体α（peroxisome proliferators-activated receptors-alpha，PPARα）基因的CDS区序列，分析其编码蛋白的结构与功能，并探讨其在山羊不同组织中的表达模式。采用RT-PCR方法扩增并克隆PPARα基因CDS编码区；通过在线软件对其一级结构、二级结构和三级结构进行生物信息学分析；利用基因序列和编码蛋白构建系统发育树，进行系统发育进化分析；采用实时荧光定量PCR方法检测PPARα基因在简州大耳羊心脏、肺脏、肝脏、肾脏、脾脏和网膜6个组织中的相对表达情况。结果表明，山羊PPARα基因CDS区全长1 413 bp，结构稳定，共编码470个氨基酸。生物信息学分析发现，PPARα蛋白是一种结构较为稳定的带负电的亲水性蛋白，以α-螺旋和无规则卷曲为主，无信号肽和跨膜蛋白，属于膜内蛋白。蛋白序列中总共有49个磷酸化位点，9个糖基化位点。保守结构域中含有明显的DBD区域和LBD区域，蛋白结构高度保守。三级结构预测发现其蛋白结构主要为通过长链卷曲连接的2个明显不同的结构区域，结构均以helix螺旋为主。序列比对结果表明，山羊PPARα氨基酸序列与绵羊和牛的同源性最高。实时荧光定量检测结果表明，PPARα基因在肾脏和肝脏中表达量较高，显著高于其他组织（P < 0.05）；在网膜组织和心脏中中度表达，显著高于肺脏和脾脏（P < 0.05）；在肺脏和脾脏中相对低表达；说明山羊PPARα基因可能与体内脂肪氧化、脂质代谢和抗氧化应激等调控功能有关。试验结果为深入研究PPARα基因在山羊中的生理功能和调控机制奠定了理论基础。

关键词: 山羊; PPARα 基因; 克隆; 生物信息学; 差异表达

Abstract:

This study was aimed to clone the CDS of goat peroxisome proliferators-activated receptors-alpha (PPARα) gene,analyze the structure and function of its encoding protein,and discuss its expression pattern in different tissues.In this study,the CDS of PPARα gene was amplified and cloned by RT-PCR,its primary,second and tertiary structures were analyzed by online bioinformatics softwares.Phylogenetic tree was constructed and then used to perform the phylogenetic analysis.Six tissues,including heart,lung,liver,kidney,spleen and omentum,were collected from Jianzhou Da’er goat to determine the relative expression pattern of PPARα by Real-time PCR.The results showed that the CDS of PPARα gene was 1 413 bp in length encoding 470 amino acids,which was a kind of stable hydrophilic protein.Bioinformatics analysis suggested that the second structures of PPARα protein were mainly α-helix and random coil,and 9 potential glycosylation sites and 49 phosphorylation sites were also revealed.The signal peptide and transmembrane domain were not existed in PPARα protein,however,there were obviously conserved DNA-binding and ligand-binding domains.Two different structure enrichment domains were connected by a long chain crimp link and generally consisted of helix structure in the tertiary structure.PPARα amino acid phylogenetic trees indicated that goat had a highly homology with sheep and cattle.PPARα gene mRNA were expressed in all the collected tissues and the highest expression was found in kidney and liver,which were significantly higher than those in the other tissues (P < 0.05).There were moderately relative expression in heart and omentum,while there were the lowest relative expression in lung and spleen.The results showed that the differential expression pattern of PPARα gene in goat might associate with the physiologic function and regulation mechanism,such as fat oxidation,lipid metabolism and anti-oxidative stress.This study would provide a theoretical foundation for further research on physiologic function and regulation mechanism of PPARα gene in goats.

Key words: goat; PPARα gene; clone; bioinformatics; differential expression

中图分类号:

饶辽源, 周靖宣, 王林杰, 李利, 张红平, 仲涛. 山羊PPARα基因克隆、分子特性及组织差异表达分析[J]. 《中国畜牧兽医》, 2018, 45(3): 559-570.

RAO Liaoyuan, ZHOU Jingxuan, WANG Linjie, LI Li, ZHANG Hongping, ZHONG Tao. Cloning, Molecular Characteristics and Differential Expression Pattern of PPARα Gene in Goats (Capra hircus)[J]. , 2018, 45(3): 559-570.

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