We cloned and characterized a unique and highly compartmentalized sea cucumber aromatization gene, P450cl7. The full-length sea cucumber P450cl7 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 P450cl7 cDNA had ATTTA special sequences in the 3' UTR and polyA regions. The full-length P450cl7 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 P folding in the complete protein. Sequence comparison revealed that the similarity of the P450cl7 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 P450cl7 precursor protein was clustered with the oyster and lancelet amphioxus. The P450cl7 precursor protein 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.%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l 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B-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(Anguilla japonica)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.
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