Molecular Characterization of a Dehydroascorbate Reductase from Pinus bungeana

Dehydroascorbate reductase （DHAR） plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR （PbDHAR） from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcripUon-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coil following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity （19.84 i.mnol/min per mg） and high affinity （a Krn of 0.08 mM） towards the substrates dehydroascorbate （DHA）. Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55℃. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.     

江南卷柏脱氢抗坏血酸还原酶的分子特性

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。     

普通小麦中双脱氢抗坏血酸还原酶(TaDHAR)基因的克隆与生化特性分析

利用同源克隆技术从六倍体普通小麦中获得了两个不同的双脱氢抗坏血酸还原酶(TaDHAR)基因的cDNA克隆。器官表达模式分析表明,这两个TaDHAR基因(暂时命名为TaDHAR1和TaDHAR2)在小麦根、茎、叶、幼穗以及开花后10d、20d和30d的种子中均有表达,为组成型表达基因。原生质体表达实验表明,两个基因的产物均可能定位在细胞质中。在细菌中表达并提纯了两个基因的重组蛋白。体外生化测定表明两个重组蛋白均具有将双脱氢抗坏血酸还原成抗坏血酸的能力,其最适pH为7.5,在37oC时的活性比25oC高,但25oC条件下pH6.0和7.0时,两个DHAR蛋白的活性显著不同。本研究的结果为进一步揭示TaDHAR基因在小麦抗坏血酸代谢中的生理作用奠定了基础。     

棉花GhDHAR2基因克隆、功能序列分析及原核表达

通过RT-PCR方法从棉花纤维组织中克隆得到脱氢抗坏血酸还原酶基因GhDHAR2的cDNA,该基因开放阅读框为639 bp,编码212个氨基酸的蛋白质。同源性序列对比分析显示,GhDHAR2蛋白具有较高的保守性,具有典型的功能结构域,包括GST-N家族和GST-C-DHAR家族的功能结构域;进化树分析显示GhDHAR2和拟南芥AtDHAR2在进化关系上较近。将GhDHAR2基因连接到原核表达载体pET-28a中,将重组载体pET28a-GhDHAR2转入到表达菌株BL21(DE3)中,通过IPTG诱导表达出重组GhDHAR2蛋白,SDS-PAGE凝胶电泳分析显示重组蛋白大小约为28 kD,诱导表达的重组蛋白具有较高的DHAR活性。首次克隆了棉花GhDHAR2基因,通过结构域分析其可能的作用,并成功进行蛋白体外表达及酶活性分析。     

Purification and properties of dehydroascorbate reductase from spinach leaves

Dehydroascorbate reductase was detected in the leaves of several plants and has been partially purified from spinach leaves. The enzyme has a MW of ca 25 000, a pH optimum of 7.5, a K_m for glutathione (GSH) of 4.43 ± 0.4 mM and a K_m for dehydroascorbate of 0.34 ± 0.05 mM. High concentrations of dehydroascorbate inhibit the enzyme. Cysteine cannot replace GSH as a donor. The purified dehydroascorbate reductase is extremely unstable and also inhibited by compounds which react with thiol groups. Dehydroascorbate does not protect the enzyme against such inhibition. GSH reduces dehydroascorbate non-enzymically at alkaline pH values.     

The Ascorbate System in Two Bryophytes: Brachythecium velutinum and Marchantia polymorpha

The ascorbate system, one of the major antioxidant systems, has been studied in two bryophytes; a moss, Brachythecium velutinum (Hedw.) B., S. & G., and a liverwort, Marchantia polymorpha L. The moss and liverwort gametophytes contain ascorbate both in the reduced and oxidized form; utilize ascorbate in removing hydrogen peroxide by means of ascorbate peroxidase and reconvert to ascorbate its oxidation products by means of dehydroascorbate reductase and monodehydroascorbate reductase. Ascorbate oxidase activity was measured in the cytosolic fraction suggesting a localization of the enzyme different from more evolved organisms. The ascorbate content was maintained in the moss after drought stress while it declines in the liverwort, which seems more sensitive to water stress. Since ascorbate recycling is more efficient in the moss than in the liverwort, this seems to suggest a correlation between efficiency of ascorbate recycling and water stress tolerance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase in spinach (Spinacia oleracea)

Effect of high intracellular concentrations of the antioxidants ascorbate and glutathione on the extractable activity of the reducting enzymes dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were investigated with spinach cells ( Spinacia oleracea ). An elevated ascorbate concentration was obtained by treatment with the ascorbate biosynthesis precursor L-galactono-1,4-lactone (GAL). To increase the intracellular level of glutathione, cells were treated with the 5-oxo-L-proline analog L-2-oxothiazolidin-4-carboxylate (OTC), or with the peroxidative herbicide acifluorfen (sodium 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid). Extractable monodehydroascorbate reductase activity increased in the presence of a high level of ascorbate or glutathione, and enzyme activity was at maximum when cells were treated with acifluorfen + OTC, or acifluorfen + GAL. Extractable dehydroascorbate reductase activity decreased when the intracellular concentration of glutathione was high and non-enzymatic reduction of dehydroascorbate by glutathione was the dominant reaction. Maximal decrease of enzyme activity was found in cells treated with acifluorfen + OTC. Extractable activity of glutathione reductase (GR) increased after treatment of cells with acifluorfen alone, or acifluorfen + OTC, but enzyme activity was unaffected by a high intracellular concentration of glutathione obtained by treatment of cells with OTC alone, or by treatment with acifluorfen + GAL. The degree of GR activation seemed to be controlled by several factors including inhibition by a high concentration of glutathione and possibly oxidative damage to the enzyme. Overall, the enzymes tested in this study, which provide the reduced forms of ascorbate and glutathione, were differently affected by high antioxidant levels.     

A spectrophotometric assay for dehydroascorbate reductase

A simple spectrophotometric assay for dehydroascorbate reductase based on the change in absorbance associated with the formation of ascorbic acid is described. Using a partially purified preparation from spinach leaves, the reaction was found to be linear with time and enzyme concentration. The reaction rate determined by this assay correlated well with that obtained by a high-performance liquid chromatography method. Possible advantages over currently available assays as well as potential applications are discussed.     

Hydrogen peroxide-scavenging enzymes and antioxidants in Echinochloa frumentacea as affected by triazole growth regulators

Paclobutrazol (PBZ)- and uniconazole (UCZ)-treated plants of Echinochloa frumentacea were shorter but had much wider leaves than untreated controls 10 days after treatment. Leaves of treated plants had a slightly higher concentration of soluble protein than the controls and exhibited enhanced activities of ascorbate peroxidase, monodehydroascorbate (MDHA) reductase, and glutathione (GSH) reductase. The triazoles did not influence the activity of dehydroascorbate (DHA) reductase. The leaves of treated plants had increased concentrations of water-soluble sulfhydryls and ascorbic acid. In contrast, the concentration of malondialdehyde (MDA), a by-product of lipid peroxidation, was lower in the leaves of treated plants than in controls. These results suggest that triazole growth regulators increased the activity of the endogenous H₂O₂-scavenging system in E. frumentacea.     

星星草脱氢抗坏血酸还原酶（PtDHAR）cDNA的克隆及表达分析

脱氢抗坏血酸还原酶是抗坏血酸代谢循环中的关键酶,在多种植物中与抗胁迫相关。为了获得抗盐碱植物星星草中该基因序列,利用RACE技术,从星星草中克隆出脱氢抗坏血酸还原酶基因（PtDHAR）的cDNA全长序列,其GenBank登录号为HM125046。PtDHAR cDNA核苷酸序列长度为987bp,开放阅读框为639bp,编码213个氨基酸。该基因编码的氨基酸序列与水稻、小麦等禾本科作物具有很高的同源性。Northern杂交分析表明,该基因在盐碱胁迫下表达量显著升高。     

Antioxidative defense system in lupin roots exposed to increasing concentrations of lead

Yellow lupin (Lupinus luteus) cv. Juno seedlings exposed to increasing concentrations of Pb²⁺ (50–350 mg l⁻¹) were analysed in respect to its effect on the degradation of lipids, the content of antioxidants (ascorbate, α-tocopherol) and the activity of the ascorbate glutathione cycle enzymes (dehydroascorbate reductase DAR; EC 1.8.5.1 and glutathione reductase GR; EC 1.6.4.2). Lipid peroxidation, expressed as the content of TBArm (thiobarbituric acid reactive metabolites), increased only at 50 and 100 mg l⁻¹ Pb²⁺, whereas at higher lead concentrations it decreased as compared with the control. The level of free fatty acids was not significantly affected as a result of Pb²⁺ exposure, except for 300 mg l⁻¹ Pb²⁺. The content of α-tocopherol increased significantly at the range of concentrations between 150 and 300 mg l⁻¹ and at these concentrations lipid peroxidation was inhibited. Ascorbic acid (AA) and dehydroascorbic acid (DHA) content increased considerably in roots treated with 100 and 150 mg l⁻¹ Pb²⁺. In general the activity of DHAR and GR was stimulated by lead, however at higher Pb²⁺ concentrations (300 and 350 mg l⁻¹) GR revealed lower enzymatic activity. Our results suggest that in lead-treated roots lipids might be protected against reactive oxygen species (ROS) by lipid-soluble molecules of α-tocopherol and this direct defense seems to be supported by AA as well as the enzymes: DHAR and GR.     

大肠杆菌trpBA基因的克隆表达

目的:提高大肠杆菌中色氨酸合成酶的表达量和表达活性。方法:利用PCR方法从大肠杆菌K-12的基因组中直接克隆出紧密连锁trpB和trpA基因(简称trpBA),并将其连接到原核表达载体pet22b( )中,得到重组质粒pet22b( )-trp-BA,转化大肠杆菌BL21,IPTG诱导重组蛋白表达,表达产物经SDS-PAGE分析并用比色法测定其活性。结果:凝胶电泳可见PCR扩增产物大小约为2kb,SDS-PAGE鉴定目的蛋白的Mr分别约为29000和44000,色氨酸合成酶α、β亚基分别得到了高效表达,色氨酸合成酶活性提高到对照菌的3.7倍。结论:成功构建了重组质粒pet22b( )-trpBA,色氨酸合成酶的表达量和表达活性在大肠杆菌中得到了提高,为高产色氨酸基因工程菌的构建奠定基础。