普通小球藻八氢番茄红素合成酶基因psy的cDNA克隆及生物信息学分析

通过RACE-PCR首次克隆获得了普通小球藻(Chlorella vulgaris)中八氢番茄红素合成酶编码基因psy 的cDNA全长序列.该序列全长1 605 bp,包括1 245 bp开放阅读框,编码415个氨基酸.氨基酸序列对比及系统发生分析表明其与其他物种的PSY蛋白具有同源性,与绿藻聚为一支,与小球藻(Chlorella variabilis)、原壳小球藻(Auxenochlorella protothecoides)和佐夫色绿藻(Chromnochloris zofingiensis的遗传距离最近(分别为0.173、0.188和0.239),并具有较高的相似性(81％ ～ 84％)和一致性(69％ ～ 76％).亚细胞定位预测表明普通小球藻PSY前45个氨基酸残基为叶绿体转运肽,可能引导翻译后的肽链进入叶绿体.保守区块、特征基序分析及三维建模显示,序列中具有多个潜在的蛋白质修饰位点,以及PSY蛋白的保守区块和特征基序,包括两个保守的底物-镁离子结合位点和两个活性位点遮蔽残基,用于结合底物及保护催化反应中间产物;普通小球藻PSY蛋白中还具有一个特异的底物-镁离子结合位点,其活性和功能还未知.总之,研究结果揭示了普通小球藻psy基因的cDNA全长序列及可能的蛋白质序列结构特性,结果可为构建过表达载体,提高类胡萝卜素产量提供相关信息.

cDNA cloning and bioinformatics analysis of the phytoene synthase encoding gene psy in Chlorella vulgaris

The full-length cDNA sequence of phytoene synthase (PSY) encoding gene psy in Chlorella vulgaris(C.vulgaris)was isolated by RACE-PCR.The 1605 bp sequence contained a 1245 bp open reading frame (ORF),which encoded a protein composed of 415 amino acids residues.Results of multiple sequence alignment and phylogeny showed that the deduced protein sequence of the C.vulgaris PSY was homologous with other PSY proteins,and clustered into the green algae group.The genetic distances of PSY between C.vulgaris and Chlorella variabilis,Auxenochlorella protothecoides,Chromochloris zofingiensis was 0.173,0.188and 0.239 respectively.And those PSYs also kept the high similarity(81％-84％) and identity(69％-76％).The prediction result of subcellular location showed that the amino acids residues from 1 st to 45th might perform as a chloroplast transit peptide,suggesting that PSY must be translated outside before entering the chloroplast and being active.Analyses of conserved blocks/motifs and tertiary structure modelling indicated that this PSY sequence contained several potential modification sites,with the conserved blocks and characteristic motifs of PSY protein,including two substrate-Mg2+ binding sites and two active site lid residues which might mediate binding of substrate and shield highly reactive intermediates from solvent.A specific substrate-Mg2+ binding site (the third) was found in the C.vulgaris PSY,and its activity and function was still unconfirmed.Taken together,these results uncovered the full-length cDNA sequence of psy,and predicted the putative structure of PSY protein in C.vulgaris,and thus provided information for over-expression of psy and for enhancing the productivity of carotenoids.