卵形鲳鲹PPARα基因cDNA序列的克隆、组织表达及生物信息学分析

Molecular Cloning and Expression Distribution and Bioinformatics Analysis of Peroxisome Proliferator Activated Receptors-α in Trachinotus ovatus

采用RACE-PCR 克隆卵形鲳鲹（Trachinotus ovatus）过氧化物酶体增殖物激活受体（peroxisome proliferator activated receptors-α,PPAR α）基因的cDNA 序列全长,并应用生物信息学方法分析其编码蛋白质的理化性质和结构特征.结果表明：卵形鲳鲹PPAR α 基因（GenBank 登录号KP893147） cDNA 全长1 930 bp,开放阅读框（ORF）为1 425 bp,共编码474 个氨基酸,其编码的蛋白质为不稳定蛋白,无信号肽和跨膜结构,二级结构由α 螺旋、β转角、伸展片段和无规则卷曲组成,且α 螺旋占较大比例;预测显示,该蛋白有PPARs 基因家族典型的DNA 结合区（DBD）和配体结合区（LBD）;序列对比表明,卵形鲳鲹PPAR α 基因与尼罗罗非鱼（Oreochromis niloticus）、花鲈（Lateolabrax japonicas）、大黄鱼（Larimichthys crocea）、金头鲷（Sparus aurata）、军曹鱼（Rachycentroncanadum）等有较高的同源性（81% - 89%）;蛋白系统进化树分析显示,在人（Homo sapiens）、鼠（Mus musculus）、鸭（Gallus gallus）、花鲈、大黄鱼、军曹鱼等动物中,卵形鲳鲹的PPAR α 蛋白与军曹鱼的进化关系最为密切（94%）,与人（68%）、鼠（68%）、鸭（67%）等的同源性较低.荧光定量分析显示,卵形鲳鲹PPAR α mRNA 在脑、肾脏、肠、脾脏等组织表达水平较高,其次是皮肤、肌肉,在心脏、肝脏中表达量较低.

The full-length c DNA of PPAR α in Trachinotus ovatus was cloned by RACE(Rapid Amplification of c DNA Ends), and the protein structures, physical, chemical properties and their functional domain structures which encoded by PPAR α gene were analyzed by bioinformatics method. The results showed that c DNA sequences of PPAR α in T. ovatus were 1 930 bp, and the open reading frame was 1 425 bp, encoding 474 amino acids(Gen Bank accession No. KP893147). The protein was unstable protein without signal peptide and trans-membrane. The secondary structure of PPAR α contains alpha helixes, beta turns, and extended strands, random coils. At the same time, alpha helixes were 45.15%. There were some typical structures in PPAR α, including a DNA-binding domain with zinc-finger structures, and a ligand binding domain which can integrate ligands and activate PPAR α. That the sequences of T. ovatus PPAR α were highly homologous with those cloned from Japanese sea perch, cobia, large yellow croaker and gilthead sea-bream(81%- 89%), phylogenetic tree analysis of PPAR α protein showed that the nearest relationship existed between T. ovatus and cobia among all the species mentioned above, and the lowest homology was human. Using quantitative real-time RT-PCR, we observed that PPAR α m RNA was expressed predominantly in brain-lipid, spleen, intestine and head-kidney tissues of T. ovatus.