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。     

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。     

杨树葡萄糖-6-磷酸脱氢酶(G6PDH)基因启动子的克隆与分析

葡萄糖-6-磷酸脱氢酶是磷酸戊糖途径的关键性调控限速酶,其主要功能是为脂肪酸合成、氮还原和谷胱甘肽等生物分子合成提供还原力NADPH,也为核酸合成提供戊糖;此外,还参加非生物逆境胁迫应答反应.因此,G6PDH对植物的生长发育起着非常重要的作用.本文利用甜杨G6PDH基因和毛果杨基因组序列,通过PCR获得了甜杨G6PDH基因上游1 400bp的序列.序列分析结果表明,该序列具有启动子的基本元件TATA-bOX、CAAT-box.此外,还包含多个胁迫诱导元件,如低温诱导元件LTR,盐诱导元件GT-1,抗冻、缺水、脱落酸、抗寒元件MYB和MYC,以及光响应元件L-box、G-box、3AF-1、TC丰富区等.     

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。     

黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因克隆及序列分析

根据6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase,6PGDH)基因的保守氨基酸序列设计简并引物,应用RT-PCR技术从黄瓜栽培种品种'北京截头'(Cucumis sativus 'Beijingjietou')叶片中获得了640 bp的特异片段,以该序列在EST数据库进行同源检索筛选,发现甜瓜EST序列AM715537.2与之高度一致,据此设计引物经RT-PCR扩增、分子克隆和序列拼接,获得了黄瓜6-磷酸葡萄糖酸脱氢酶基因全长序列,命名为Cs6PGDH(GenBank登录号FJ610345).序列分析表明,该基因全长1 829 bp,其中开放读码框(ORF)长1 488 bp编码495个氨基酸组成的多肽,编码区内无内含子存在,5'、3'端非翻译区长度各为70 bp和271 bp.Blast同源性分析显示该基因编码的氨基酸序列与拟南芥、大豆、水稻、玉米、菠菜等物种6PGDH 基因有74%以上的一致性.由于与其他物种胞质6PGDH相类似氨基酸N端都缺少长度约为40aa的转运肽,推断Cs6PGDH为黄瓜胞质6-磷酸葡萄糖酸脱氢酶基因.     

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。     

PCR-DGGE法研究葡萄糖-6-磷酸脱氢酶缺乏症基因突变

目的：应用PCR-DGGE法和DNA测序分析云南籍G6PD缺乏症患者基因突变类型和特点、方法应用硝基四氮唑蓝（NBT）纸片法进行G6PD缺乏症定性筛查,G6PD／6PGD比值法验证,应用PCR—DGGE法和DNA测序分析46例云南籍G6PD缺乏症患者基因突变类型和特点。结果：46例云南籍G6PD缺乏症样本中有30例经PCR—DGGE法分析G6PDexon12发现有异常电泳条带,DNA测序证实26例（56、52％）为nt-1388G→A,4例（8.7％）nt-1376G→T．而PCR—DGGE法分析G6PDexon2未发现有异常电泳条带的样本出现。结论：（1）nt-1388G→A（56．52％）、nt-1376G→T（8．7％）是云南省主要的基因突变型也是中国人中最常见的两种突变型,揭示中华民族有着共同的起源;（2）所检样本中未发现nt95A→G。（3）应用PCR—DGGE法结合DNA测序检测G6PD缺乏症患者的基因型,阳性检出率高,方法简便、快捷、灵敏、结果准确可靠。     

葡萄乙醇脱氢酶基因Ⅲ的电子克隆及生物信息学分析

利用电子克隆方法获得葡萄乙醇脱氢酶基因Ⅲ(ADHⅢ),并采用生物信息学方法对该基因编码蛋白从氨基酸组成、理化性质、跨膜结构域、疏水性/亲水性、亚细胞定位、高级结构以及功能域等方面进行了预测和分析.结果表明,葡萄ADHⅢ基因全长1 602 bp,包含1 140 bp的ORF,编码379个氨基酸,该蛋白不具有明显的疏水区域,也无跨膜结构域,α-螺旋和不规则卷曲是其二级结构的主要构件.葡萄ADHⅢ包含有ADH功能域,和其他植物的ADHⅢ在序列组成、高级结构及活性位点等方面均具有高度的相似性.     

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.     

Glucose-6-phosphate dehydrogenase (G6PD) Guantánamo and G6PD Caujerí: Two new glucose-6-phosphate dehydrogenase-deficient variants found in Cuba

Glucose-6-phosphate dehydrogenase (G6PD) deficiency was identified in two children who were studied because of hemolytic episodes. The electrophoretic and kinetic properties of the mutant enzymes allowed us to conclude that both of them were new variants. They were named G6PD Guantánamo and G6PD Caujerí.     

猪JARID1C基因的cDNA克隆、生物信息学及组织表达谱分析

JARID1C是高度保守的ARID蛋白家族的成员,该家族的蛋白参与并引起一系列生物学效应,如染色质重塑、细胞增殖与分裂、个体发育以及基因转录调控。JARID1C在人脑中表达丰富,对脑的发育和维持正常功能具有重要作用,突变可引起智力迟钝。本研究采用电子克隆（insilicocloning）的方法并结合5′末端快速扩增技术（RACE）,从猪卵巢中克隆到JARID1C的全长cDNA序列（GenBank登录号：EF139241）。猪JARID1C基因的cDNA全长5,908bp,包括4,551bp的开放阅读框（ORF）、522bp的5′非翻译区（5′UTR）和835bp的3′非翻译区（3′UTR）,polyA加尾信号序列AATAAA位于5,881bp和5,886bp之间。生物信息学分析揭示JARID1C蛋白含有1517个氨基酸残基,定位于细胞核中,该蛋白含有5个保守的结构域：JmjN结构域、ARID结构域、JmjC结构域、C5HC2锌指结构域和PHD锌指结构域。应用ClusterW程序分别对猪、狗、小鼠、大鼠、人和猿的JARID1C核苷酸序列和氨基酸序列进行多重序列比对,发现猪的JARID1C与其他哺乳动物具有很高的相似性。借助Mega3.1软件,采用N-J算法构建JARID1亚家族蛋白的系统进化树,揭示不同物种的进化关系。应用实时荧光定量PCR技术分析该基因在不同组织的表达差异,结果表明该基因在各组织均不同程度地表达,其中在肺和骨骼肌表达水平最低,而在脑和性腺表达水平最高。     

米曲霉木糖还原酶基因的克隆及序列分析

木糖还原酶（XR, EC 1.1.1.21）是真菌微生物代谢木糖的关键酶之一。本文以米曲霉基因组DNA为模板,克隆木糖还原酶基因（xr,GenBank登录号：FJ957890.1）,并对XR的序列、系统进化树、理化性质及蛋白结构等进行生物信息学分析。结果表明： xr基因序列长1449 bp,其中开放阅读框长960 bp,编码319个氨基酸,蛋白质分子质量35.9 kDa,等电点为5.78;米曲霉XR与其他菌种XR有较高的同一性,含有醛酮还原酶家族的两个指纹结构和一个参与辅酶结合活性位点指纹结构,以及醛酮还原酶家族典型的（β/α）8 TIM桶结构,说明米曲霉XR属于醛酮还原酶家族。     

Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.     

Glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase of wild oat seeds

The presence of the initial enzymes of the pentose phosphate pathway, namely glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase, has been demonstrated in dormant seed of wild oat. Before a partial characterization of these enzymes was made, an inherent NADP-reducing activity and an enzyme deactivating component, both present in the crude extract, were removed by ammonium sulphate precipitation and subsequent desalting. Both enzymes were then shown to be NADP-specific. Typical Michaelis-Menten kinetics were shown by each enzyme towards NADP and their respective substrates. Soluble cytoplasmic dehydrogenase enzymes were present in both embryo and endosperm extracts.     

G6PD缺陷通过Caspase-3和PARP-1诱导人黑色素瘤细胞凋亡

葡糖-6-磷酸脱氢酶(G6PD)在许多肿瘤细胞中高表达,但其发生的作用机理目前仍然不明确.以正常人表皮黑色素细胞(HEM)、野生型人黑色素瘤A375细胞(A375-WT)和G6PD缺陷的A375细胞(A375-G6PDΔ)为对象,经real-time PCR、Western印迹和紫外分光光度法分析显示,A375-WT细胞的mRNA、G6PD蛋白和G6PD活性分别是HEM细胞的1.89倍(P0.05)、6.86倍(P0.01)和2.30倍(P0.05).Annexin V/PI流式细胞仪和Western印迹测定表明,A375-G6PDΔ的凋亡率是A375-WT的5.10倍(P0.01),活化半胱氨酸蛋白酶3(caspase-3)增高1.84倍(P0.01)以及89 kD多聚二磷酸腺苷核糖聚合酶-1(PARP-1)生成增加2.87倍(P0.01).分光光度法分析显示,A375-G6PDΔ的NADPH和GSH分别降低了72.30%(P0.01)和27.39%(P0.05),并伴有75.43%的H2O2增高(P0.01).结果提示,G6PD在黑色素瘤细胞中高表达和高活性,而敲减G6PD表达通过caspase-3和PARP-1信号诱发人黑色素瘤细胞凋亡,这为深入揭示黑色素瘤的发生机理提供了新思路。     

Glucose-6-phosphatase proteins of the endoplasmic reticulum (Review)

Summary

Hepatic glucose-6-phosphatase (G-6-Pase) catalyses the terminal step of hepatic glucose production and it plays a key role in the maintenance of blood glucose homeostasis. Hepatic G-6-Pase is an integral resident endoplasmic reticulum (ER) protein and it is part of a multicomponent system. Its active site is situated inside the lumen of the ER and transport proteins are needed to allow its substrates, glucose-6-phosphate (G-6-P) (and pyrophosphate), and its products, phosphate and glucose, to cross the ER membrane. In addition, a calcium-binding protein is also associated with the G-6-Pase enzyme. Recent immunological studies have shown that G-6-Pase (which has conventionally been thought to be present only in the gluconeogenic organs) is present in minor cell types in a variety of human tissues and that its distribution changes dramatically during human development. In all the tissues, enzymatic analysis, direct transport assays and/or immunological detection of the ER glucose and phosphate transport proteins have been used to demonstrate the presence and activity of the whole G-6-Pase system. The G-6-Pase protein is very hydrophobic and has proved difficult to purify to homogeneity. Four proteins of the system have now been isolated and polyclonal antibodies have been raised against them; two have also been cloned. The available sequences, together with topologicai studies, have given some information about both the topology of the proteins in the ER and the probable mechanisms by which the proteins are retained in the ER.