根据本实验室构建的三角帆蚌cDNA文库中已标注的EST序列, 利用cDNA末端快速扩增法(RACE)克隆了三角帆蚌(Hyriopsis cumingii)谷胱甘肽过氧化物酶(Glutathione peroxidase, GPX)基因cDNA全序列。序列分析表明, 该基因cDNA序列全长1286 bp, 包括5′端非翻译区(Untranslated Region) 39 bp、3′端非翻译区659 bp和开放阅读框(Open reading frame, ORF)588bp, 共编码195个氨基酸, 分子量约为22.2 kDa, 理论等电点为8.44, 属于含硒类GPX。该氨基酸序列具有GPX所有亚型中均高度保守的3个环状结构, 对酶的三级结构起稳定作用。在线分析结果表明: GPX的氨基酸序列不存在明显的疏水区, 也不存在信号肽序列。氨基酸相似性对比结果显示, 三角帆蚌GPX氨基酸序列与脊椎动物GPX-2及GPX-1的序列相似度较高, 为73.1%-80.8%, 与其他型GPX相似度较小, 相似度低于60%。构建的系统进化树显示三角帆蚌GPX与其他几种鱼类GPX聚为一类, 与其他已发表的几种软体动物GPX相距较远, 推测本实验克隆的三角帆蚌GPX基因和已发表的软体动物不属于同一种GPX类型。
Based on the EST sequence of mantle cDNA library of Hyriopsis cumingii, the complete cDNA of Hyriopsis cumingii GPX gene was cloned by rapid amplification of cDNA ends (RACE) in this paper. The GPX full-length cDNA sequence was 1286 bp including a 39 bp 5′-untranslated region, a 659 bp 3′-untranslated region and an open reading frame (ORF) 588 bp in length, which encodes for 195 amino acids with the predicted molecular weight of 22.2 kDa and the isoelectric point of 8.44. Molecular structure analysis revealed that GPX was Se-GPX. The amino acid sequences have three highly conserved loop structures that have a stabilizing effect on the tertiary structure of the enzyme. The amino acid sequence did not contain obvious hydrophobic domain and signal peptide. Homologous analysis of amino acid sequences indicated that the GPX of H. cumingii shared a high similarity with GPX-2 and GPX-1 of vertebrates (73.1%-80.8%), and low similarity with other types (< 60%). Phylogenetic trees showed that GPX of Hyriopsis cumingii was clustered together with GPX of some fishes and far away from GPX of several other published Molluscas. These results indicated GPX gene of H. cumingii cloned in this study was different from the GPX genes from other Molluscas.
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