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。     

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。     

玉米脱氢抗坏血酸还原酶基因的克隆和特性研究

通过电子克隆方法得到玉米脱氢抗坏血酸还原酶基因,命名为ZmDHAR1。结果显示,ZmDHAR1基因全长723bp,开放阅读框642bp,编码214个氨基酸。ZmDHAR1基因与高粱、水稻及小麦等植物的脱氢抗坏血酸还原酶基因高度相似,其中与高粱的DHAR基因相似性为85%,与水稻的DHAR1基因相似性为83%。基因表达分析显示,ZmDHAR1基因在根以外的其他部位都可以检测到表达,而且ZmDHAR1基因可以在4℃低温、100μmol/L ABA、100mmol/L PEG、250mmol/L NaCl和机械损伤等多种逆境胁迫下均可应答表达。研究表明,ZmDHAR1基因可能在玉米抗逆境胁迫反应中发挥重要的作用。     

24-脱氢胆固醇还原酶基因转基因小鼠血生化指标分析

目的建立全身表达24-脱氢胆固醇还原酶基因（Dhcr24）转基因小鼠动物模型,研究该基因过表达对小鼠代谢的影响。方法RT-PCR法克隆小鼠Dhcr24基因,把该基因插入CMV启动子下游,构建转基因表达载体,通过显微注射法建立Dhcr24转基因小鼠。PCR鉴定Dhcr24转基因小鼠的基因型,RT-PCR和Western Blot检测基因表达水平,血生化检测仪检测转基因小鼠血生化指标的改变。结果建立了2个不同表达水平的Dhcr24转基因小鼠品系,转入的Dhcr24基因在肝和脾组织中的表达高于内源的Dhcr24。血生化检测证实：乳酸脱氢酶（LDH）、总蛋白（TP）、白蛋白（Alb）和血肌酐（SCr）较野生型小鼠明显降低,而高密度脂蛋白胆固醇（HDL-c）和碱性磷酸酶（ALP）较野生型小鼠明显增加,并且Dhcr24转基因雌鼠的体重比野生型小鼠明显降低,均有显著差异。但Dhcr24转基因雄鼠各项指标与野生型小鼠相比没有显著差异。结论成功建立了全身表达Dhcr24转基因小鼠,并证实Dhcr24基因对雌性小鼠的体重和血生化指标,包括LDH,TP,Alb,SCr,HDL-c and ALP具有明显的影响。     

甘蔗类异黄酮还原酶(IRL)基因的克隆与表达分析

采用RT-PCR技术从甘蔗中克隆So IRL基因,用生物信息学方法对获得的氨基酸序列进行分析,利用荧光定量PCR技术研究So IRL基因在甘蔗不同组织和不同胁迫条件下的表达特性。结果表明,克隆获得甘蔗So IRL,Gen Bank登录号为KF808324。该c DNA全长1 169 bp,含有1个927 bp的完整开放阅读框(ORF),编码309个氨基酸。系统进化树分析显示,甘蔗So IRL与玉米的IRL蛋白亲缘关系较近。q RT-PCR分析表明So IRL在甘蔗根、茎、叶中均有表达;在RSD病菌及低温(4℃)、聚乙二醇(PEG)、Na Cl和脱落酸(ABA)4种非生物胁迫下均被诱导表达,但表达模式不同。说明该基因可能参与甘蔗应答RSD过程,并可能在非生物胁迫中也发挥了作用。     

毛白杨细胞质抗坏血酸过氧化物酶基因的克隆及表达分析

利用同源克隆技术得到1个毛白杨细胞质抗坏血酸过氧化物酶基因,命名为PcAPX。该基因编码249个氨基酸残基,预测分子量为33.01kD。采用原核表达技术在大肠杆菌中表达并纯化该蛋白并进行酶活性分析,结果表明:重组PcAPX蛋白对抗坏血酸(AsA)和过氧化氢(H2O2)有很高的活性,其对抗坏血酸的米氏常数(Km)和最大反应速度(Vmax)分别为(0.71±0.03)mmol·L-1和(0.41±0.02)mmol·L-1·min-1·mg-1;对过氧化氢的Km和Vmax分别为(0.60±0.21)mmol·L-1和(0.35±0.12)mmol·L-1·min-1·mg-1,表明PcAPX对AsA和H2O2拥有较高的催化底物的能力和催化效率。利用实时定量RT-PCR分析毛白杨PcAPX基因的表达模式,结果表明其在老叶中表达量高于新叶、韧皮部、形成层和根部。该研究结果将进一步促进毛白杨APX基因家族成员参与植物生长调控的研究。     

草莓MDHAR及AO基因的克隆及序列分析

从新鲜幼嫩‘丰香’草莓(Fragaria×ananassa cv.Toyonaka)果实中提取分离总RNA,反转录成cDNA,根据已报道的其他植物单脱氢抗坏血酸还原酶(MDHAR)及抗坏血酸氧化酶(AO)基因的保守区分别设计 一对引物,通过PCR扩增均得到目的条带.序列分析发现:mdhar基因片段长372bp,与刺梨同源性最高达96％,该片段编码123个氨基酸,推导的氨基酸序列与苹果属植物同源性为91％,与其他植物该基因编码的氨基酸序列也有较高相似性;a基因片段长842 bp,编码280个氨基酸,该片段与其他多种植物的ao基因均具有较高同源性,与甜瓜和黄瓜ao基因的同源性最高,均为70％,编码的氨基酸序列与其他多种植物均具有70％左右的相似性.     

普通小麦中一氧化氮相关因子(TaNOA)编码基因的克隆和分子生物学分析

一氧化氮是动植物体内重要的信号分子。本研究利用同源克隆技术从六倍体普通小麦中获得一个一氧化氮相关因子(TaNOA)编码基因的全长基因组和cDNA克隆。该基因具有13个外显子和12个内含子,与拟南芥以及水稻中同源基因结构相似。根据cDNA推导的氨基酸序列与拟南芥AtNOA1的序列一致性达60%以上,具备P-环GTPaseG4-G5-G1-G2-G3的排列特征和保守的序列。对其中2个内含子的测序分析表明在六倍体小麦中TaNOA至少有3个成员。进一步用中国春小麦缺体-四体材料将这3个TaNOA基因成员分别定位在第六同源群的6A、6B和6D染色体上,本研究中获得的成员定位于6B染色体上,因此将其命名为TaNOA-B1。原生质体表达实验表明,TaNOA-B1可能定位在线粒体中。TaNOA基因在小麦根、叶片中表达较高,在幼穗和小花中有少量表达,茎中几乎检测不到表达。TaNOA的转录本水平还因脱落酸或盐处理而上升,表明它可能参与小麦对非生物胁迫的反应。本研究为进一步克隆六倍体小麦中TaNOA的其他成员及研究该基因在小麦中的功能奠定了基础。     

Trichilia dregeana胚轴的脱水敏感性与抗坏血酸的抗氧化作用

以顽拗性Trichilia dregeana Sond.种子为材料,研究其胚轴的脱水敏感性与抗坏血酸的抗氧化作用.T.dregeana胚轴的脱水耐性随着脱水进程逐渐下降,50%的胚轴被脱水致死的含水量(W50)大约为0.16 g H2O/g DW.在脱水过程中,胚轴的电解质渗漏速率逐渐增加,超氧物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶 (CAT)、谷胱苷肽还原酶(GR)和脱氢抗坏血酸还原酶(DHAR)的活性下降,硫代巴比妥酸(TBA)-活性产物的含量增加.2.5～10.0 mmol/L抗坏血酸处理能有效地增加胚轴的脱水耐性和SOD、APX、CAT和GR的活性,降低电解质渗漏速率和TBA活性产物的含量.结果表明,T.dregeana胚轴的脱水耐性与抗氧化酶的活性增加和脂质过氧化作用的降低密切相关.     

Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene

To analyze the physiological role of dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, T1 transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants expressing a human DHAR gene in chloroplasts were biochemically characterized and tested for responses to various stresses. Fully expanded leaves of transgenic plants had about 2.29 times higher DHAR activity (units/g fresh wt) than non-transgenic (NT) plants. Interestingly, transgenic plants also showed a 1.43 times higher glutathione reductase activity than NT plants. As a result, the ratio of AsA/DHA was changed from 0.21 to 0.48, even though total ascorbate content was not significantly changed. When tobacco leaf discs were subjected to methyl viologen (MV) at 5 mumol/L and hydrogen peroxide (H2O2) at 200 mmol/L, transgenic plants showed about a 40% and 25% reduction in membrane damage relative to NT plants, respectively. Furthermore, transgenic seedlings showed enhanced tolerance to low temperature (15 degrees C) and NaCl (100 mmol/L) compared to NT plants. These results suggest that a human derived DHAR properly works for the protection against oxidative stress in plants.     

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.     

Co-expression of monodehydroascorbate reductase and dehydroascorbate reductase from Brassica rapa effectively confers tolerance to freezing-induced oxidative stress

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing.     

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.     

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.