仿刺参性别相关基因P450c17的克隆与序列分析

Cloning and bioinformatics analysis of aromatization gene P450c17 in sea cucumber Apostichopus japonicus(Selenka)

本研究克隆了仿刺参(Apostichopus japonicus)P450c17基因的全长cDNA,结果表明其cDNA的5′端UTR为256 bp,3′端UTR为250 bp,ORF为1 530 bp。在3′端有特殊序列ATTTA、终止密码子TAA和polyA加尾。其全长cDNA编码509个氨基酸的前体蛋白,预测蛋白分子量57.6 kD,理论等电点5.5。P450c17的氨基酸序列存在长度为25个氨基酸的信号肽,1个长度为22个氨基酸的跨膜区,1个糖基结合位点NHS,1个P450特有的结构域,其氨基酸序列具有明显的细胞色素P450基因家族的特征。结构域中亚铁血红素结合域中的精氨酸R和发挥活性所必须的半胱氨酸C都是保守的。这些结构组成了血红素结合环、质子传递槽和K螺旋及绝对保守EXXR基序。蛋白前体二级结构中无规则卷曲占41.22%,延伸链占12.77%,α–螺旋占46.01%,不含–折叠。氨基酸序列与其他物种氨基酸序列的同源性在35%～41%之间。系统进化树表明仿刺参与长牡蛎(Crassostrea gigas)及文昌鱼(Branchiostoma belcheri)聚为一支,黄颡鱼(Pelteobagrus fulvidraco)、鲇(Clarias gariepinus)、日本鳗鲡(Anguillajaponica)及白斑角鲨(Squalus acanthias)聚为一支,三刺鱼(Gasterosteus aculeatus)、金眼门齿鲷(Stenotomus chrysops)、斑马鱼(Danio rerio)、鲤(Cyprinus carpio)、虹鳟(Oncorhynchus mykiss)、牙鲆及青鳉(Fundulus heteroclitus)聚为一支。研究结果为进一步探讨仿刺参P450c17在性激素调控性别分化方面的功能提供参考。

We cloned and characterized a unique and highly compartmentalized sea cucumber aromatization gene,P450c17.The full-length sea cucumber P450c17 cDNA was 2 036 bp in length,and contained a 1 530 bp open reading frame（ORF）,a 250 bp 3′UTR,and a 256 bp 5′UTR.Based on the effective length of gene translation,the full-length P450c17 cDNA had ATTTA special sequences in the 3′ UTR and polyA regions.The full-length P450c17 cDNA encoded 509 amino acids constituting a 57.6 kD protein molecule with an isoelectric point of 5.5.The precursor protein was hydrophilic.The protein sequence had one signal peptide,consisting of a 25 amino acid residue.The signal peptide consisted primarily of an alpha helix.The sequence of amino acids contained one transmembrane region,involving in electronic transfer catalysis between the internal and external membrane.We found a single N-glycosylation site,suggesting the precursor protein belonged to the family of secretion trans-membrane glycoproteins.The secondary structure of the precursor protein consisted primarily of an alpha helix,with irregular coiling and a strand chain,but without β folding in the complete protein.Sequence comparison re-vealed that the similarity of the P450c17 precursor protein ranged from 35% to 41% with other aquatic animals.We conducted a phylogenetic analysis using the neighbor joining（NJ） method.The evolutionary tree indicated that the P450c17 precursor protein was clustered with the oyster and lancelet amphioxus.The P450c17 precursor pro-tein contained a P450 specific structure domain.The conserved core was composed of a coil termed the ＂meander＂,a four-helix bundle,helices J and K,and two sets of beta-sheets.These constitute the haem-binding loop（with an absolutely conserved cysteine that serves as the 5th ligand for the haem iron）,the proton-transfer groove,and the absolutely conserved EXXR motif in helix K.Our results provide a reference for investigating the function of this gene.