江南卷柏脱氢抗坏血酸还原酶的分子特性

Molecular characterizations of two dehydroascorbate reductases from Selaginella moellendorffii

摘要:

脱氢抗坏血酸还原酶 (DHAR) 在植物抗坏血酸?谷胱甘肽循环中发挥着重要作用。利用同源克隆技术从江南卷柏中克隆到2 个脱氢抗坏血酸还原酶基因，分别命名为SmDHAR1 和SmDHAR2。SmDHAR1 和SmDHAR2 分别编码218 和241 个氨基酸，预测分子量分别是23.97 kDa 和27.33 kDa。基因组序列分析显示这2 个基因分别含有5 和6 个内含子。器官表达模式分析发现这2 个基因在根、茎、叶中均有表达，是组成型表达基因。在大肠杆菌中表达并纯化了2 个基因的重组蛋白。酶活性分析显示SmDHAR1 和SmDHAR2 蛋白对底物DHA 的活性有显著差异，分别是19.76和0.17 μmol/(min·mg)。热力学稳定性分析显示这2 个重组蛋白的热力学稳定性具有明显差异。因此，基因结构与酶学性质的差异预示着这2 个基因可能存在功能上的分化。

Abstract:

Plant dehydroascorbate reductase (DHAR) is a physiologically important reducing enzyme in the ascorbate-glutathione recycling reaction. In this study, two DHARs genes (SmDHAR1 and SmDHAR2) were isolated from Selaginella moellendorffii. The SmDHAR1 and SmDHAR2 genes encode two proteins of 218 and 241 amino acid residues, with a calculated molecular mass of 23.97 kDa and 27.33 kDa, respectively. The genomic sequence analysis showed SmDHAR1 and SmDHAR2 contained five and six introns, respectively. Reverse transcription PCR revealed that the SmDHAR1 and SmDHAR2 were constitutive expression genes in S. moellendorffii. The recombinant SmDHAR1 and SmDHAR2 proteins were overexpressed in E. coli, and were purified by Ni-affinity chromatography. The recombinant SmDHAR1 showed 116-fold higher enzymatic activity towards the substrate dehydroascorbate than recombinant SmDHAR2. The recombinant SmDHAR1 showed higher thermal stability than recombinant SmDHAR2. These results indicated obvious functional divergence between the duplicate genes SmDHAR1 and SmDHAR2.