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。     

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.     

还原型谷胱甘肽治疗儿童葡萄糖-6-磷酸脱氢酶缺乏症中的临床效果研究

目的:研究还原型谷胱甘肽治疗儿童葡萄糖-6-磷酸脱氢酶(G-6-P-D)缺乏症并发急性溶血的临床疗效,为临床治疗提供参考。方法:选取我院2015年6月-2017年6月因葡萄糖-6-磷酸脱氢酶(G-6-P-D)缺乏症并发急性溶血的患儿78例并将其随机分为两组,每组39例。对照组予以停用氧化类药物,卧床休息,水化、碱化尿液,贫血严重者输注去白红细胞治疗;观察组在对照组基础上加用还原型谷胱甘肽治疗。观察和比较两组患儿第1天、第2天、第3天小便恢复率以及平均恢复时间,血清总胆红素第3天、第5天恢复率、平均恢复时间及平均住院时间。结果:治疗后,观察组第1天、第2天、第3天小便恢复率分别为51.3%、92.3%、100%,对照组分别为25.6%、64.1%、89.7%,观察组第1天、第2天、第3天小便恢复率均显著高于对照组(P0.05);观察组及对照组小便恢复正常平均时间分别为1.8±0.7天、2.6±0.9天,观察组明显短于对照组(P0.05);观察组第3天、第5天血清总胆红素恢复率分别为71.8%、100%,对照组为46.2%、97.4%;观察组和对照组血清总胆红素恢复正常平均时间分别为3.6±0.9天、4.1±1.0天;平均住院时间分别为2.3±0.6天、2.8±0.6天;观察组小便及血清总胆红素平均恢复时间(P0.05)、平均住院时间均显著短于对照组(P0.05)。结论:在儿童葡萄糖-6-磷酸脱氢酶缺乏并发急性溶血中应用还原型谷胱甘肽可增强其治疗疗效,缩短治疗疗程。     

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。     

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.     

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。     

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.     

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)     

Glucose-6-Phosphate Dehydrogenase Deficiency in Nigerian Children

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy and in Sub-Saharan Africa, is a significant cause of infection- and drug-induced hemolysis and neonatal jaundice. Our goals were to determine the prevalence of G6PD deficiency among Nigerian children of different ethnic backgrounds and to identify predictors of G6PD deficiency by analyzing vital signs and hematocrit and by asking screening questions about symptoms of hemolysis. We studied 1,122 children (561 males and 561 females) aged 1 month to 15 years. The mean age was 7.4±3.2 years. Children of Yoruba ethnicity made up the largest group (77.5%) followed by those Igbo descent (10.6%) and those of Igede (10.2%) and Tiv (1.8%) ethnicity. G6PD status was determined using the fluorescent spot method. We found that the overall prevalence of G6PD deficiency was 15.3% (24.1% in males, 6.6% in females). Yoruba children had a higher prevalence (16.9%) than Igede (10.5%), Igbo (10.1%) and Tiv (5.0%) children. The odds of G6PD deficiency were 0.38 times as high in Igbo children compared to Yoruba children (p = 0.0500). The odds for Igede and Tiv children were not significantly different from Yoruba children (p = 0.7528 and 0.9789 respectively). Mean oxygen saturation, heart rate and hematocrit were not significantly different in G6PD deficient and G6PD sufficient children. The odds of being G6PD deficient were 2.1 times higher in children with scleral icterus than those without (p = 0.0351). In conclusion, we determined the prevalence of G6PD deficiency in Nigerian sub-populations. The odds of G6PD deficiency were decreased in Igbo children compared to Yoruba children. There was no association between vital parameters or hematocrit and G6PD deficiency. We found that a history of scleral icterus may increase the odds of G6PD deficiency, but we did not exclude other common causes of icterus such as sickle cell disease or malarial infection.     

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.     

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.     

Light Modulation of Glyceraldehyde-3-phosphate Dehydrogenase and Glucose-6-phosphate Dehydrogenase by Photosynthetic Electron Flow in Pea Chloroplasts

Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) and light inactivation of glucose-6-P dehydrogenase (EC 1.1.1.49) appear to be modulated within pea leaf chloroplasts by mediators which are reduced by photosynthetic electron flow from the photosystem I reaction center. Dichlorophenyl-1, 1-dimethylurea inhibition of this modulation can be completely reversed by ascorbate plus 2,6-dichlorophenolindophenol in broken chloroplasts, but not in intact chloroplasts. Intact chloroplasts are impermeable to 2,6-dichlorophenolindophenol at pH 7.5. Studies on the effect of light in reconstituted chloroplasts with photosystem I-enriched particles in the place of whole thylakoids revealed that photosystem I participates in the light modulation of NADP-linked glyceraldehyde-3-P dehydrogenase and of glucose-6-P dehydrogenase.