高等植物葡萄糖-6- 磷酸脱氢酶与6- 磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个关键酶。在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位。结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生。讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料。     

高等植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个酶.在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位.结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生.讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料.     

水稻质体葡萄糖-6-磷酸脱氢酶基因的克隆与表达研究

戊糖磷酸途径是高等植物中重要的代谢途径,主要生理功能是产生NADPH以及供核酸代谢的磷酸戊糖。葡萄糖-6-磷酸脱氢酶（G6PDH）是戊糖磷酸途径的关键酶,广泛存在于高等植物细胞的细胞质和质体中。木研究首次从水稻（Oryza sativa L.）幼苗中分离了核编码的质体G6PDH基因OsG6PDH2,序列分析表明OsG6PDH2编码一个具有588个氨基酸残基的多肽,等电点为8．5,分子量66kDa。OsG6PDH2的N端有1个70个氨基酸的信号肽,推测的裂解位点为Gly55和Val56,表明OsG6PDH2编码产物可能定位于质体。多序列比较的结果表明OsG6PDH2与拟南芥、烟草、马铃薯质体G6PDH的一致性分别达81％、87％、83％。进化关系说明水稻OsG6PDH2与拟南芥（AtG6PDH3）、马铃薯（StG6PDH1）处于高等植物P2型质体G6PDH分支上,暗示了OsG6PDH2可能是一个P2型的质体蛋白。Matinspector程序分析表明,OsG6PDH2在起始密码子上游含有一个bZIP转录因子识别位点、一个ABA应答元件、一个CRT／DRE元件和1个W-box元件。半定量RT-PCR分析表明,OsG6PDH2在水稻根、茎、叶和幼穗组织中都呈低丰度组成型表达,在根部表达较高,在水稻幼苗中的表达显著受暗处理的诱导。将OsG6PDH2的完整开放阅读框构建到大肠杆菌表达载体pET30a（＋）中,pET30a（＋）-OsG6PDH2在大肠杆菌中得到了有效表达。酶活性测定证明,OsG6PDH2的编码产物具有葡萄糖-6-磷酸脱氢酶的功能。     

米曲霉6-磷酸葡萄糖脱氢酶基因gsdA的克隆及生物信息学分析

6-磷酸葡萄糖脱氢酶催化6-磷酸葡萄糖生成6-磷酸葡萄糖酸,并生成NADPH,是微生物胞内磷酸戊糖途径（PPP）的关键酶。本研究以食品安全菌米曲霉CICC2012为材料,克隆获得6-磷酸葡萄糖脱氢酶基因(GenBank登录号：JN123468)。序列分析表明,该酶是由222个氨基酸组成的亲水性蛋白;128～134位氨基酸序列DHYLGKE为活性区域;170～176位氨基酸序列GTEGRGG可能为辅因子结合位点。进化树分析表明,米曲霉6-磷酸葡萄糖脱氢酶同其他丝状真菌及酵母的G6PDH较相似。     

辣椒雄性不育系葡萄糖-6-磷酸脱氢酶基因的克隆及表达分析

为了深入研究辣椒雄性不育与能量代谢之间的关系,该研究以辣椒近缘物种番茄的葡萄糖-6-磷酸脱氢酶基因(G6PDH)同源序列为基础,采用电子克隆的方法克隆出辣椒CaG6PDH基因。利用荧光定量PCR技术,对辣椒雄性不育系9704A与其保持系9704B花蕾发育的不同阶段,以及保持系9704B不同组织(茎、叶、花、果皮、胎座、种子)中CaG6PDH基因进行表达分析。结果表明:两系中获得的CaG6PDH基因的编码序列一致,全长1 533bp,编码510个氨基酸残基;辣椒CaG6PDH基因在保持系不同组织中表达量存在差异,胎座中表达量最高,茎中表达量最低;辣椒CaG6PDH基因的表达量在花蕾发育的不同阶段雄性不育系均高于保持系,此种差异在小孢子发育的单核期与成熟期尤为明显,这种差异可能使雄性不育系能量代谢供应出现异常,从而影响小孢子的正常发育而导致雄性败育。     

水稻葡萄糖-6-磷酸脱氢酶cDNA的电子克隆

电子克隆是基因克隆的新策略,以小麦胞质葡萄糖－6－磷酸脱氢酶cDNA(Tagpdl克隆）序列为信息探针,在GenBank水稻nr数据库中找到高度同源的水稻基因组序列,通过人工序列拼接及RT-PCR确认得到了水稻该基因的全长cDNA序列,命名为OsG6PDH,OsG6PDH与小麦Tagpdl克隆的DNA一致率为88％,推导的氨基酸序列与小麦,番茄,烟草的胞质葡萄糖－6－磷酸脱氢酶基因的一致率分别为89％,79％,80％,经RT－PCR表达谱分析,OsG6PDH在水稻幼穗,胚,根,叶中都有表达,在幼穗与根中表达略高,另外,讨论了利用水稻基因组信息的电子克隆方法克隆水稻功能基因的可行性。     

高等植物葡萄糖-6-磷酸脱氢酶的研究进展

葡萄糖-6-磷酸脱氢酶是植物戊糖磷酸途径中的一个关键性调控酶。其主要生理功能是产生供生物合成需要的NADPH及一些中间产物;此外还参与各种生物和非生物胁迫的应答反应。文中主要从葡萄糖-6-磷酸脱氢酶同工酶与调节机制等方面探讨了其生物学功能,再从胁迫耐受、基因克隆、酶的缺失和替代等方面的研究进行综述,并对已发表的高等植物中的G6PDH氨基酸序列进行聚类分析,为今后该酶的研究提供参考。     

苹果6-磷酸山梨醇脱氢酶基因启动子逆境诱导表达特性研究

为研究6-磷酸山梨醇脱氢酶(sorbitol-6-phosphate dehydrogenase,S6PDH)基因启动子(S6PDHp)的逆境诱导表达特性,利用Gateway技术构建了S6PDH基因启动子区5'端系列缺失体与GUS基因的融合表达载体,并通过农杆菌介导法转化拟南芥。对转基因拟南芥进行低温和外源ABA处理,通过GUS蛋白活性变化分析S6PDHp的逆境诱导表达特性。研究结果发现,通过Gateway技术构建了4个S6PDHp 5'端系列缺失体与β-葡萄糖苷酸酶(GUS)基因的融合表达载体(pGWB433-S6PDHp1、pGWB433-S6PDHp2、pGWB433-S6PDHp3和p GWB433-S6PDHp4)并获得了相应的转基因拟南芥。对转基因植株进行低温处理后发现,p GWB433-S6PDHp3转基因植株中的GUS活性增幅最大,达到显著水平,而其他转基因植株中的GUS活性基本保持不变。外源ABA处理后发现,除p GWB433-S6PDHp4外,其余启动子缺失体转基因拟南芥中GUS活性显著升高。以上结果表明,低温和外源ABA能够诱导S6PDHp的表达,但不同的缺失体响应程度不同,意味着在S6PDHp序列(-2 396bp至-236bp)中可能存在着响应逆境胁迫的正负调控顺式作用元件。     

发菜G6PDH基因(GND)克隆及其干旱胁迫下的差异表达分析

由GND编码的葡萄糖-6-磷酸脱氢酶(G6PDH)是戊糖磷酸途径的关键酶。为了解发菜GND分子信息以及对干旱胁迫的响应机制,该研究设计了特异性引物克隆发菜GND全长,进行序列分析、原核表达和MALDITOF-TOF/MS鉴定,并对干旱胁迫下发菜GND的差异表达水平和G6PDH活性进行分析。结果表明:(1)发菜GND全长1 431bp(GenBank登录号为KX553955),与点形念珠藻(73102)的GND核苷酸序列相似性为96%,G6PDH氨基酸相似性为98%;氨基酸序列中第26和27位的Ile疏水性最强,在第302位的Arg亲水性最强,Thr有19个磷酸化位点,Ser有18个磷酸化位点,Tys有5个磷酸化位点,二级结构和三级结构主要由α螺旋、β折叠、β转角和随机卷曲构成。(2)将GND基因进行原核表达,获得47.23kD的外源表达蛋白G6PDH。(3)随干旱胁迫程度加剧,GND在转录水平的表达量和G6PDH活性均逐渐增加。研究结果为进一步探讨干旱胁迫条件下发菜GND表达调控奠定了基础。     

条形柄锈菌小麦专化型葡萄糖-6-磷酸脱氢酶基因PsG6PDH1的表达特征及功能分析

葡萄糖-6-磷酸脱氢酶是磷酸戊糖途径中的关键限速酶。基于已测序的条形柄锈菌小麦专化型基因组序列,利用RT-PCR方法克隆了该病菌葡萄糖-6-磷酸脱氢酶PsG6PDH1的全长cDNA序列（1 497bp）,编码498个氨基酸的蛋白。编码蛋白含有葡萄糖-6-磷酸脱氢酶的保守功能域。系统进化分析发现,PsG6PDH1与禾柄锈菌小麦专化型的G6PDH聚为一簇。qRT-PCR分析表明,PsG6PDH1在病菌侵染早期的表达明显上调,其中侵染24h时表达量最高,为对照夏孢子的30倍。将PsG6PDH1导入酿酒酵母G6PDH缺失突变体中成功表达,并表现出较强的葡萄糖-6-磷酸脱氢酶活性,明显酵母增强了菌株对过氧化氢的耐受性。由此推测,PsG6PDH1可能参与了条形柄锈菌小麦专化型在侵染寄主时抵御寄主的氧化胁迫反应。研究结果为进一步研究该病菌基础代谢及侵染机理奠定一定的理论基础。     

Thessaly variant of Glucose-6-phosphate Dehydrogenase

Summary A new variant of the erythrocytic enzyme Glucose-6-phosphate Dehydrogenase was detected in two unrelated Greek individuals. The variant was designated G6PD Thessaly. It is characterized by normal levels of G6PD activity in the red cells and electrophoretic migration slower than G6PD B on phosphate and T.E.B. buffers while faster than G6PD B on Tris-HCl buffer. In addition, the Thessaly variant has distinctly decreased affinity for NADP.This study was supported by National Institutes of Health Grant GM 15253.     

Isolation and Expression Analysis of Plastidic Glucose-6-phosphate Dehydrogenase Gene from Rice (Oryza sativa L.)

Glucose-6-phosphate dehydrogenase is a rate-limiting enzyme of pentose phosphate pathway, existing in cytosolic and plastidic compartments of higher plants. A novel gene encoding plastidic glucose-6-phosphate dehydrogenase was isolated from rice (Oryza sativa L.) and designated OsG6PDH2 in this article. Through semiquantitative RT-PCR approach it was found that OsG6PDH2 mRNA was weakly expressed in rice leaves, stems, immature spikes or flowered spikes, and a little higher in roots. However, the expression of OsG6PDH2 in rice seedlings was significantly induced by dark treatment. The complete opening reading frame (ORF) of OsG6PDH2 was inserted into pET30a (+), and expressed in Escherichia coli strain BL21 (DE3). The enzyme activity assay of transformed bacterial cells indicated that OsG6PDH2 encoding product had a typical function of glucose-6-phosphate dehydrogenase.     

Rapid Purification of Glucose-6-phosphate Dehydrogenase from Mammal's Erythrocytes

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6pD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362–3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2′5′ ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2′5′ ADP-Sepharose with KC1 and NADP.

By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2′5′ ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5–4 ml of rabbit blood, which can be performed in about 8 hours and a macro-scale purification starting from 180–200 ml of human blood, which takes a day and a half.     

Purification and Kinetic Characterization of Glucose-6-phosphate Dehydrogenase from Dictyostelium discoideum

Reimers, J. M., Huang, Q., Albe, K. R., and Wright, B. E. 1993. Purification and kinetic characterization of glucose-6-phosphate dehydrogenase from Dictyostelium discoideum. Experimental Mycology 17, 1-6. Glucose-6-phosphate dehydrogenase from Dictyostelium discoideum was purified 650-fold and kinetically characterized. The enzyme catalyzed the conversion of G6P + NADP to 6PG + NADPH stoichiometrically and irreversibly in vitro . The purified enzyme is specific for NADP. Michaelis constants for G6P and NADP were 0.040 and 0.011 mM, respectively. NADPH was found to be a competitive inhibitor with respect to NADP with a K_i of 0.006 mM and a noncompetitive inhibitor with respect to G6P. The data from initial velocity and product inhibition studies were consistent with a sequential mechanism.     

Inheritance of Glucose-6-phosphate Dehydrogenase Variation in Kangaroos

THE production of glucose-6-phosphate dehydrogenase (EC 11149, G6PD) in human¹, horse and donkey² and brown and blue hare³ cells is governed by genes carried by the X chromosome. Two electrophoretic forms of G6PD have been found in wallaroos and euros (Macropus robustus Gould) and one in red kangaroos (Macropus rufus (Desm.)); members of the marsupial family Macropodidae (kangaroos). This analysis used electrophoresis of red blood cell haemolysates on cellulose acetate⁴. No polymorphic populations were found⁵ but electrophoretic phenotypes of euros (Macropus robustus erubescens Sclater) were characterized by a single slow moving band (G6PD-S) while those of wallaroos (Macropus r. robustus Gould) had a single fast moving band (G6PD-F). Red kangaroo populations were uniformly G6PD-S.     

Aggregated Forms of Glucose-6-phosphate Dehydrogenase from Cultured Rice Plant Cells

Multiple molecular forms of glucose-6-phosphate dehydrogenaseincluding aggregated ones were observed in rice plant suspensioncultures by disc electrophoresis. By sucrose density gradientcentrifugation, the enzyme was resolved into three componentswith the sedimentation coefficients of 42.5, 23 and 6, respectively.The first two components were dissociated into the third componentin the presence of KC1, NADP⁺ and NADPH. These three componentswere interconvertible molecular species. (Received September 16, 1980; Accepted December 19, 1980)     

Glucose-6-phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase of Candida tropicalis Partial Purification and Some Properties

Glucose-6-phosphate (G6P) dehydrogenase and 6-phosphogluconate (6PG) dehydrogenase were partially purified about 53-fold and 47-fold, respectively, from the cell-free extract of glucose-grown Candida tropicalis by means of ammonium sulfate fractionation and DEAE-cellulose column chromatography. AMP acted as the competitive inhibitor against G6P and NADP in the G6P dehydrogenase reaction. This inhibition was remarkable at low concentrations of NADP, increasing the sigmoidicity of the NADP-saturation curve. On the other hand, 6PG dehydrogenase was not affected by AMP. Fructose-1,6-bisphosphate (FDP) and phosphoenolpyruvate (PEP) inhibited slightly G6P dehydrogenase. 6PG dehydrogenase was also weakly inhibited by FDP. Apparent Km values of G6P dehydrogenase were calculated as 1.8 × 10^?4 m for G6P and 3.1 × 10^?5 m for NADP. Those of 6PG dehydrogenase were 9.4 × 10^?5 m for 6PG and 2.8 × 10^?5 m for NADP.     

Glucose-6-phosphate Dehydrogenase Deficiency and Malaria: Cytochemical Detection of Heterozygous G6PD Deficiency in Women

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a X-chromosomally transmitted disorder of the erythrocyte that affects 400 million people worldwide. Diagnosis of heterozygously-deficient women is complicated: as a result of lyonization, these women have a normal and a G6PD-deficient population of erythrocytes. The cytochemical assay is the only reliable assay to discriminate between heterozygously-deficient women and non-deficient women or homozygously-deficient women. G6PD deficiency is mainly found in areas where malaria is or has been endemic. In these areas, malaria is treated with drugs that can cause (severe) hemolysis in G6PD-deficient individuals. A cheap and reliable test is necessary for diagnosing the deficiency to prevent hemolytic disorders when treating malaria. In this review, it is concluded that the use of two different tests for diagnosing men and women is the ideal approach to detect G6PD deficiency. The fluorescent spot test is inexpensive and easy to perform but only reliable for discriminating hemizygous G6PD-deficient men from non-deficient men. For women, the cytochemical assay is recommended. However, this assay is more expensive and difficult to perform and should be simplified into a kit for use in developing countries. (J Histochem Cytochem 57:1003–1011, 2009)