成子硕，兰婷，李迪，杨海灵，曾庆银

1 中国科学院植物研究所 系统与进化植物学国家重点实验室，北京 100093

2 北京林业大学生物科学与技术学院，北京 100083

3 中国科学院研究生院，北京 100049

江南卷柏脱氢抗坏血酸还原酶的分子特性

成子硕1,3，兰婷1,3，李迪2，杨海灵2，曾庆银1

1 中国科学院植物研究所 系统与进化植物学国家重点实验室，北京 100093

2 北京林业大学生物科学与技术学院，北京 100083

3 中国科学院研究生院，北京 100049

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。

江南卷柏，脱氢抗坏血酸还原酶，克隆，蛋白表达，酶活

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 byNi-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.

Keywords:Selaginella moellendorffii, dehydroascorbate reductase (DHAR), cloning, protein expression, enzyme activity

还原型抗坏血酸 (Ascorbic acid，AsA) 是植物体内普遍存在的一种小分子抗氧化物质，可清除由光合作用、呼吸作用等氧化反应产生的超氧自由基、过氧化氢、单线态氧、羟自由基等活性氧自由基[1]，并被氧化成单脱氢抗坏血酸 (MDA)。……