不同浓度的NO^—3和NH^＋4对小麦根谷氨酰胺合成酶及其相关酶的影响

利用酶活性测定和 Northern分子杂交等技术 ,研究了小麦幼苗根在不同浓度的 Na NO3 和(NH4) 2 SO4的供应下 ,其谷氨酰胺合成酶 (GS)、天冬酰胺合成酶 (AS)、谷氨酸脱氢酶 (GDH)、硝酸还原酶 (NR)以及 GS- m RNA的变化。结果表明 :NH 4 处理的小麦 ,其根部 GS活性比 NO-3 处理的高 ;高浓度处理的比低浓度处理的高 ;Northern杂交结果说明 GS- m RNA转录量与 GS活性一致 ;3mmol/ L NO-3促进了 AS的活性。AS酶活性变化与 GS酶活性变化无明显依赖关系。在实验的条件下 ,没能测出 GDH的活性 ,不同浓度的 NO-3 和 NH 4 处理对 NR活性没有明显的规律。     

硝态氮对黄瓜子叶谷氨酰胺合成酶和谷氨酸脱氢酶活性的影响

在硝态氮存在或缺乏的条件下,测定了黄瓜(Cucumis sativus L．)种子萌发和子叶发育过程中子叶可溶性蛋白质含量以及谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(NAD(H)-GDH)活性的变化。在子叶发育初期,无论外源氮存在与否,每对子叶可溶性蛋白质含量和GS、NADH—GDH、NAD^ -GDH活性随发育上升。在外源氮存在下,第4d后,可溶性蛋白质含量虽有所下降,但基本保持恒定;第6d后,GS和NADH—GDH活性逐渐降低,NAD^ -GDH却相反增高。但在无外源氮条件下,于第4d后,可溶性蛋白质水平以及GS、NADH—GDH和NAD^ -GDH活性都逐渐降低。在子叶发育的整个过程中,外源氮对GS和NAD^ -GDH活性有促进作用,尤其是在子叶发育的后期对NAD^ -GDH活性的促进更为明显。     

氮素水平对花生氮素代谢及相关酶活性的影响

 在大田高产条件下研究了氮素水平对花生(Arachis hypogaea)可溶性蛋白质、游离氨基酸含量及氮代谢相关酶活性的影响, 结果表明, 适当提高氮素水平既能增加花生各器官中可溶性蛋白质和游离氨基酸的含量, 又能提高硝酸还原酶、谷氨酰胺合成酶和谷氨酸脱氢酶等氮素同化酶的活性, 使其达到同步增加; 氮素水平过高虽能提高硝酸还原酶和籽仁蛋白质含量, 但谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)的活性下降; N素施肥水平不改变花生植株各器官中可溶性蛋白质、游离氨基酸含量以及硝酸还原酶(NR)、谷氨酰胺合成酶、谷氨酸脱氢酶活性的变化趋势, 但适量施N (A2和A3处理)使花生各营养器官中GS、GDH活性提高; 氮素水平对花生各叶片和籽仁中GS、GDH活性的高低影响较大, 但对茎和根中GDH活性大小的影响较小。     

不同生育期花生叶片蛋白质含量及氮代谢相关酶活性分析

以5个珍珠豆型花生(Arachis hypogaea Linn.)品种(系)‘汕E’(‘Shan E’)、‘汕G’(‘Shan G’)、‘TH’、‘TJ’和‘泉花7号’(‘Quanhua No.7’)为研究对象,分析了花针期、结荚期和饱果期花生叶片中可溶性蛋白质含量及硝酸还原酶(NR)、谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)活性的变化趋势,并比较了5个品种(系)荚果和秆产量的差异。结果表明:在3个生育期内,5个花生品种(系)叶片可溶性蛋白质含量和GDH活性的变化趋势基本一致,而NR和GS活性的变化趋势则有差异。其中,可溶性蛋白质含量均呈"低—高—低"的变化趋势,在结荚期最高;GDH活性均逐渐升高,至饱果期达最高;‘泉花7号’叶片NR活性呈"高—低—高"的变化趋势,而其他4个品种(系)叶片NR活性均逐渐降低;‘汕E’、‘TJ’和‘泉花7号’叶片GS活性呈逐渐降低趋势,而‘汕G’和‘TH’叶片GS活性呈"低—高—低"的变化趋势。总体上看,5个品种(系)中,‘汕G’和‘泉花7号’叶片的可溶性蛋白质含量及NR和GDH活性、‘汕E’叶片的NR和GS活性以及‘TH’叶片的GDH活性均较高。5个品种(系)的2个产量指标(单株荚果鲜质量和单株秆鲜质量)均有明显差异,总体上看,‘汕G’、‘泉花7号’和‘TH’的2个产量指标均较高,而‘汕E’和‘TJ’的2个产量指标均较低。综合分析结果显示:‘汕G’和‘泉花7号’叶片可溶性蛋白质含量及NR和GDH活性均相对较高,其荚果和秆产量也均较高,表明花生荚果和秆产量与不同生育期叶片氮代谢水平有一定关系。     

烤烟叶片衰老期氨气挥发特征及其生理调控研究

以烤烟品种K326为试验材料,利用氨气收集装置测定烟叶的氨气挥发量,并利用谷氨酰胺合成酶(GS)抑制剂(Glufosinate)处理叶片和质外体提取等方法,研究了叶片氨气挥发及其与氮代谢相关生理指标的关系。结果表明:(1)随着叶片的衰老,氨气挥发量在叶龄70d时最大(10.96μg.m-2.h-1),与衰老初期(叶龄40d)相比增加了2.15倍;质外体NH4+浓度和pH、氨气补偿点逐渐上升,GS和硝酸还原酶(NR)活性下降,谷氨酸脱氢酶(GDH)活性升高,可溶性蛋白和总氮降解,叶片NH4+浓度升高。(2)GS抑制剂处理后,叶片组织NH4+浓度和氨气补偿点升高,氨气挥发量增大,与对照相比差异显著。(3)氨气挥发量与质外体NH4+浓度、质外体pH和氨气补偿点呈极显著或显著正相关,与GS活性呈显著负相关,与GDH活性呈显著正相关,与叶片组织NH4+浓度等其他指标相关性不显著。研究认为,烤烟叶片衰老期间氨挥发量大幅上升,挥发量的大小受气孔氨气补偿点、GS和GDH活性的直接调控,以及其他氮素代谢相关指标的间接调控,其中GS起主导作用。     

浑球红假单胞菌氨同化途径的研究

本文测定了浑球红假单胞菌(Rhodobacter sphaeroides)菌株601谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)和丙氨酸脱氢酶(ADH)的活性。低氨时,GS/GOGAT活力高,GDH活力低,高氨时,GS/GOGAT活力低,GDH活力高。在以分子氮或低浓度氨为氮源的培养条件下,加入GS抑制刑MSX(L—methionine—DL—sulphoximine),细菌生长受到抑制。但是,生长在以谷氨酸为氮源的细菌则不受影响。上述结果表明,浑球红假单胞菌菌株601氨同化是通过GS/GOGAT途径和GDH途径。     

灌浆期不同水分处理对玉米籽粒蛋白质及其组分和相关酶活性的影响

蛋白质作为氮素代谢的终极产物,与玉米(Zea mays)籽粒品质呈正相关关系,其生物合成主要在硝酸还原酶(NRase)、谷氨酰胺合成酶(GS)、谷氨酸脱氢酶(GDH)等一系列酶催化下完成,受制于品种自身遗传特性及环境因素,栽培管理措施和生态环境条件对玉米品质具有十分重要的影响。关于土壤水分供应状况对玉米籽粒主要品质成分的分布、积累动态和相关酶活性的影响的研究尚少见报道。以两种不同类型玉米:普通玉米‘掖单22’和高油玉米‘高油115’为研究对象,采用防雨棚池栽试验。水分处理以开花期为界线,设置3种水分处理,花后不浇水(W0)、花后浇1水(灌浆期,W1)、花后浇3水(灌浆期、乳熟期、蜡熟期,W2)。结果表明:两种类型玉米籽粒蛋白质及其组分含量的积累动态基本一致,且不受土壤水分供应状况的影响。玉米籽粒蛋白质及清蛋白、谷蛋白含量,均为‘高油115’较高,球蛋白含量为‘掖单22’较高,醇溶蛋白两类型玉米含量相近。在不同水分供应条件下,两种类型玉米叶片中NRase、GS酶活性和籽粒中GS、GDH酶活性的变化动态一致,NRase酶活性自灌浆初期至成熟期一直下降,GS、GDH酶活性呈单峰曲线,在授粉后20~40 d达到高峰,充足的水分供应有利于酶的活性维持较高水平;玉米叶片中NRase酶活性,‘掖单22’高于‘高油115’,叶片GS和籽粒GDH酶活性显著低于‘高油115’。研究表明:用玉米叶片中NRase和GS活性的高低表征籽粒蛋白质含量的高低不确切,土壤水分条件与不同类型玉米穗位叶和籽粒中GS 和GDH活性关系密切。     

植物氮代谢硝酸还原酶水平调控机制的研究进展

氮代谢是植株体内最基本的物质代谢之一,硝酸还原酶是植物氮代谢的关键酶。主要对植物氮代谢在硝酸还原酶水平上调控的研究新进展,尤其是其合成／降解及活性调控机制进行了较为系统的综述。硝酸还原酶合成的调控主要发生在转录水平和翻译水平上,硝酸还原酶降解的调控主要发生在翻译后水平上,同时NO3^-及光在硝酸还原酶转录水平调控上的作用重大,硝酸还原酶编码基因转录的mRNA的稳定性强弱影响植物的氮代谢,而影响mRNA稳定性的因素很多,机理复杂;磷酸化／去磷酸化在硝酸还原酶活性调控中占举足轻重的地位,研究也比较深入。钝化蛋白也能够影响硝酸还原酶活性,许多小分子物质对硝酸还原酶活性有影响。     

低温和外源不饱和脂肪酸对高山被孢霉脂肪酸脱氢酶基因表达的影响

[目的]在转录水平上研究低温和外源不饱和脂肪酸对于高山被孢霉脂肪酸脱氢酶基因的表达调控机制.[方法]通过实时定量PCR技术和启动子报告基因融合载体的方法,研究低温和外源不饱和脂肪酸对于高山被孢霉3种脂肪酸脱氢酶基因表达随时间进程的影响.[结果]实时定量PCR的结果表明:低温对于3种脂肪酸脱氢酶基因的转录具有激活作用,外源不饱和脂肪酸对基因转录起抑制作用,而且这两种作用都是快速响应的,随时间延长逐渐减弱并消失.脂肪酸组成测定结果证明了基因转录水平变化与对应产物变化之间没有相关性.低温能够在短时间内诱导pFAD6启动子活性增加,并随时间延长而持续增强 ;外源不饱和脂肪酸对pFAD6启动子活性起抑制作用,其不饱和度和浓度越高,抑制作用越强,而且抑制作用是快速且持续的.[结论]低温和外源不饱和脂肪酸除了在转录水平上调控高山被孢霉脂肪酸脱氢酶基因表达发生变化之外,可能主要在转录后水平上介导了胞内脂肪酸组成的变化.而且,脂肪酸脱氢酶基因的表达可能受到胞内脂肪酸组成变化的反馈调节作用.本文首次在转录水平上对高山被孢霉脂肪酸脱氢酶基因的表达调控机制进行了探索,为深入了解脂肪酸脱氢酶基因表达及多不饱和脂肪酸合成对外界信号的应答机制提供了有用信息,也对应用微生物发酵和转基因技术生产不饱和脂肪酸具有指导意义.     

慢性氨暴露对中华鳖幼鳖血浆总氨氮、皮质酮浓度及组织氨代谢酶活性的影响

本文观测了慢性氨暴露对中华鳖(Pelodiscussinensis)幼鳖血浆总氨氮、皮质酮浓度及与氨代谢有关的酶活性的影响。将中华鳖暴露在总氨氮(TAN)浓度为32.4、57.6、83.5mg/L(分别记为1、2、3组)的水环境中饲养42d,以自然晾晒脱氯自来水饲养组为对照(记为0组),pH值控制在7.80-7.85。检测氨暴露2、4、8、24、48h、42d后血浆TAN与8、24、48h、42d后皮质酮浓度,42d后肝、肌肉与脑中谷氨酰胺合成酶(Glutaminesynthetase,GS)、谷氨酸脱氢酶(Glutamatedehydrogenase,GDH)的活性以及特定生长率(Specificgrowthrate,SGR)的变化。结果表明:0、1组血浆TAN随时间没有明显变化,2、3组血浆TAN随时间呈现先增加后降低的趋势,分别在8h和48h达到峰值,42d时各组间血浆TAN没有显著差异。氨暴露显著影响暴露初期血浆皮质酮水平,暴露后24h,1、3组血浆皮质酮水平显著高于对照组;42d后除2组外其他组间无显著差异。4组之间肝、肌肉和脑中GS活性均没有显著差异,肝和肌肉GDH(氨化和去氨化方向)活性也没有显著差异。各暴露组脑GDH活性和对照组相比差异不明显,但各处理组间氨化和去氨化方向脑GDH酶活差异显著。42d饲养期间各处理组SGR没有显著差异。     

Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. I. Cellular levels of glutamine synthetase and NADPH-dependent glutamate dehydrogenase

Cell-free extracts of nitrate-grown as well as of ammonium-grown cells of the filamentous non-nitrogen-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate-grown cells was higher than that of ammonium-grown cells, whereas the GDH level was higher in ammonium-grown cells and depended on the external ammonium concentration. When nitrate-grown cells were transferred to an ammonium-containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia-grown cells were transferred to a nitrate-containing medium, an increase of GS and a decrease of GDH-specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate-grown cells were transferred to nitrate medium with L-methionine-DL-sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.     

Regulation of ammonia assimilation in ammonia-limited chemostat cultures of Escherichia coli ML 30: evidence of bistability

In a preceding paper evidence of two stationary stable states (bistability) in the specific activity of glutamine synthetase (GS) in ammonia-limited steady-state cultures of Escherichia coli ML 30 at dilution rates (D) about 0.15 h-1 was described (Müller et al. 1977). For better understanding of the regulation mechanisms leading to GS bistability chemostat experiments were performed over a wide range of dilution rates up to D = 0.8 h-1. For each steady state the specific activities of GS and glutamate dehydrogenase (GDH)--the other key enzyme of the two NH3 assimilation routes in E. coli--and in addition the remaining NH3 concentration in the culture liquid were determined. Parallel to GS bistability two states of GDH activity and NH3 concentration are found. The higher state of GS is connected with a lower GDH activity and NH3 concentration. With rising D the GS activities decrease whereas GDH activities and NH3 concentrations increase. Since no adenylation of the GS is detectable GS bistability seems to be regulated on the level of enzyme synthesis like GDH bistability. From the experimental findings a mathematical model is derived based on the bottle neck enzyme theory of growth. It describes the dependence between the specific growth rates on the one hand and the specific enzyme activities and NH3 concentration on the other. It is shown that the specific uptake rate of the limiting NH3 and the specific growth rates, respectively, depend on the simultaneous action of two bottle neck enzymes which are connected by a regulative link.     

NaCl对水稻谷氨酸合酶和谷氨酸脱氢酶的胁迫作用

在ＮａＣｌ的胁迫下,水稻幼苗根和叶的谷氨酸合酶和谷氨酸脱氢酶的活性随着营养液中的ＮａＣｌ浓度的升高而降低;游离ＮＨ４＾＋在叶中积累,在根中未见明显变化。与根相比,叶对ＮａＣｌ的胁迫作用更为敏感。叶的ＮＡＤＨ－ＧＯＧＡＴ和ＮＡＤＨ－ＧＤＨ活性在ＮａＣｌ胁迫降低的程度明显大于根。无论是否有ＮａＣｌ存在,根的ＮＡＤＨ－ＧＤＨ活性明显高于叶。ＧＳ／ＧＤＨ比值分析提示,对对照下,根中的ＮＨ４＾存在,根的ＮＡ     

The influence of exogenously supplied sucrose on glutamine synthetase and glutamate dehydrogenase levels in excisedPisum sativum roots

Glutamine synthetase (GS) level is positively influenced by exogenously supplied sucrose in isolated pea roots (similarly as nitrate reductase - NR), glutamate dehydrogenase (GDH) level negatively. Comparison with previous results shows that GS level decreases more slowly than NR level when sucrose is omitted from the medium; the rate of changes in GS level corresponds rather to that in GDH level. The increase in GDH level in the tips of isolated roots cultivated in the medium lacking sucrose stops after approx. 24 h, but continues for at least 72 h in more mature root parts. GS level decreases during the first 24 h in the tips of isolated roots (compared with roots of intact seedlings) cultivated both with sucrose and without it (without sucrose more), however it again rises in the course of further cultivation with sucrose. The differences in GS and GDH levels caused by omission of sucrose are small in isolated roots from which root tips were removed, the difference in NR level in decapitated roots is similar to that found in isolated roots with root tips left. Decapitated isolated roots cultivated without sucrose contain higher amounts of soluble sugars than corresponding roots with root tips left. These facts are dismissed with regard to sugar consumption, transport, and compartmentalisation, and with respect to production in root tips and other plant parts of unknown compounds involved in GS and GDH regulation. The results obtained suggest that GDH functions in pea roots in the deaminating direction.     

Modulation of key nitrogen assimilating enzymes by NAA and in vitro culture in Cuscuta reflexa

Nitrogen assimilation in the callus of an angiosperm holoparasitic plant, Cuscuta reflexa, has been investigated by studying the level of key enzymes of the nitrogen assimilation pathway, namely nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH), during its growth in the absence and presence of NAA. The activity of all these enzymes in culture exhibited a developmental profile of an initial increase followed by a decrease. The presence of NAA increased the activity of all the enzymes throughout the culture period without altering their developmental profiles. Isozyme profiles of GS and GDH in the callus of C. reflexa were analyzed by PAGE and direct in gel activity staining. In the absence of NAA, the callus exhibited one isozyme of GS and two isozymes of GDH. NAA treatment led to the development of one additional isozyme of GS. Further stimulating effect of NAA on the activity of each of these enzymes was also evident by in gel activity staining of the isozymes. A comparison of the levels of NR, GS, GOGAT and GDH in field vines of C. reflexa, leaves of its host plant, Catheranthus with those of Cuscuta callus, led to the observation that all the nitrogen assimilating enzymes except GDH, were absent in the field vines of C. reflexa. Callus and field vines revealed a preponderance of GDH as compared to GS activity, while a reverse trend was observed in the host plant. The data are suggestive of ammonia assimilation through GDH pathway in this parasite.     

Regulation of Glutamate Dehydrogenase and Glutamine Synthetase in Avocado Fruit during Development and Ripening

The activity, protein, and isoenzymic profiles of glutamate de-hydrogenase (GDH) and glutamine synthetase (GS) were studied during development and ripening of avocado (Percea americana Mill. cv Hass) fruit. During fruit development, the activity and protein content of both GDH and GS remained relatively constant. In contrast, considerable changes in these enzymes were observed during ripening of avocado fruit. The specific activity of GDH increased about 4-fold, coincident with a similar increase in GDH protein content and mRNA levels. On the other hand, GS specific activity showed a decline at the end of the ripening process. On the isoenzymic profile of GDH, changes in the prevalence of the seven isoenzymes were found, with a predominance of the more cathodal isoenzymes in the unripe and of the most anodal isoenzymes in the ripe fruit. Two-dimensional electrophoresis revealed that avocado fruit GDH consists of two subunits whose association gives rise to seven isoenzymes. The results support the view that the predominance of the more anodal isoenzymes in the overripe fruit was due to the accumulation of the [alpha]-polypeptide.     

Response of Ammonia Assimilation in Cucumber Seedlings to Nitrate Stress

The influence of increased nitrate concentration—14 (control) and 140 mmol L⁻¹ (T)—in hydroponic culture on ammonia assimilation in cucumber (Cucumis sativus L. cv. Xintaimici) seedlings was investigated. The results showed that NH₃ accumulation in the roots and leaves of T seedlings increased significantly, indicating that NH₃ toxicity might be involved in nitrate stress. Under control conditions, GS and GOGAT activity were much higher in the leaves than in the roots, whereas GDH activity was much higher in the roots than in the leaves. Correlation analysis showed that NH₃ concentration had a strong negative linear relationship with GDH activity in the roots but had a strong negative linear relationship with GS and GOGAT activity in the leaves. These results indicate that NH₃ might be assimilated primarily via GDH reaction in the roots and via GS/GOGAT cycle in the leaves. Short-term nitrate stress resulted in the increase of GS and GOGAT activity in the roots and GDH activity in the leaves of T seedlings, indicating possible shifts in ammonia assimilation from the normal GDH pathway to GS/GOGAT pathway in the roots and from the normal GS/GOGAT pathway to the GDH pathway in the leaves under nitrate stress, but with the increase of treatment time, GS, GOGAT, and GDH activity in the roots and leaves of T seedlings decreased possibly due to low water potential and NH₃ toxicity.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

南瓜种子萌发及子叶发育时谷氨酰胺合成酶和其它氨同化酶的变化

在发育的新生组织中 ,来自种子胚乳储存蛋白的降解和氨基酸分解代谢产生的氨由谷氨酰胺合成酶 ( Glutamine synthetase,GS)重新同化 ,生成的谷氨酰胺 ( Gln)被转运到正在生长着的部分。GS是高等植物氮素代谢的关键酶 [1] ,这个酶能同化不同来源的氨。 GS有多种同工酶 ,存在于植物的各种组织和器官中。它们是由一小的同源但分离的核基因家族编码的 [2 3 ] ,这些不同的 GS在植物氮素同化中起着非重叠的作用 [4] ,它们的表达受到环境、发育进程以及组织或细胞类型等许多因素的影响。在大多数已研究过的植物叶片中存在两种 GS,即胞液型GS(…