论文部分内容阅读
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(Chromochloris 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。
The full-length cDNA sequence of phytoene synthase encoding gene psy in Chlorella vulgaris was obtained by RACE-PCR for the first time. The full length of this sequence is 1 605 bp, including 1 245 bp open reading frame and encodes 415 amino acids. Amino acid sequence comparison and phylogenetic analysis showed that it shared homology with other species of PSY protein, and was associated with Chlorella together with Chlorella variabilis, Auxenochlorella protothecoides and Zov green Chromochloris zofingiensis had the closest genetic distance (0.173, 0.188 and 0.239, respectively), and had high similarity (81% -84%) and consistency (69% -76%). Subcellular localization prediction showed that the first 45 amino acid residues in PSY of Chlorella vulgaris were chloroplast transit peptides, which may guide the post-translational peptide chains into the chloroplast. The conserved blocks, signature motif analysis and three-dimensional modeling show that there are several potential protein modification sites in the sequence, as well as the conserved blocks and characteristic motifs of PSY proteins, including two conserved substrates, the Mg ion binding site Point and two active sites to mask the residues for binding to the substrate and to protect the catalytic reaction intermediates; Chlorella vulgaris PSY protein also has a specific substrate - magnesium ion binding sites, its activity and function is unknown . In conclusion, the results revealed the full-length cDNA sequence of psy gene of Chlorella vulgaris and possible structural characteristics of the protein sequence. The results may provide relevant information for the construction of overexpression vector and improvement of carotenoid yield.