乙醇酸氧化酶纯化方法的改进

乙醇酸氧化酶（GO）是光呼吸的关键酶。但目前报道的GO的Mr和等电点等基本参数相差很大,重复性也不好[2,3,5～11]。本实验拟改进其纯化方法以期提高其重复性。材料与方法菠菜（Spinaciaoleracea）和菜心（Brassicacampestris）均购自市场。GO的提纯基本按文献2、3、6,只作添加FMN一项改进。具体操作如下：绿叶用100mmol·L－－‘的磷酸缓冲液（pH8．0）提取粗蛋白,过滤,4000Xg离心取上清液,加10％HAC调到PH53;然后以4000Xg离心取上清液,经15％～30％（NH;）iS04分步沉淀;高GO活性的沉淀蛋白用5mmol·L‘Tris－HCI（pH…     

菠菜叶片乙醇酸氧化酶的氧化甘油酸活性

首先从菠菜叶片中纯化了乙醇酸氧化酶（GO）。通过鉴定反应中氧的消耗以及反应产物H2O2的生成,证实菠菜GO具有氧化光呼吸途径中间代谢物甘油酸的活性。该氧化活性依赖于辅因子FMN和FAD,而不依赖核黄素和光黄素;其最适反应pH值为8．0,Km（甘油酸）值为7．14mmol／L,kcat值为1．04s^-1,活化能为17．29kJ／mol;草酸和丙酮酸对该氧化活性有明显的抑制作用,其中前者为典型的竞争性抑制。进一步通过两底物竞争作图表明：菠菜叶片GO氧化甘油酸反应和氧化乙醇酸反应为同一活性中心所催化。     

菜心乙醇酸氧化酶的纯化和催化特性分析

用改进后的方法,从菜心绿叶中分离纯化得到一个亚基分子量为42kD的乙醇酸氧化酶,用氧电极法测定该酶同时能催化乙醇酸和乙醛酸的氧化。     

菜心乙醇酸氧化酶的纯化和催化特性分析

用改进后的方法,从菜心绿叶中分离纯化得到一个亚基分子量为４２ｋＤ的乙醇酸氧化酶,用氧电极法测定该酶同时能催化乙醇酸和乙醛酸的氧化。     

植物乙醇酸氧化酶分离纯化方法的改进

通过缩短DEAE-Cellulose柱长度,加快流速并采用pH8.8的80mmol.L-1Tris-HCl为洗脱液,可在9小时内快速地从菠菜、菜心和豆角绿叶中纯化得到乙醇酸氧化酶。该酶具高活性(54.6～197.0U.mg-1)及高等电点(pI>10.0)。产率为4.1%～71.5%,纯化倍数为21.6～122.68。经SDS-PAGE检测均有40kD带,表明3种植物乙醇酸氧化酶的亚基大小无区别。     

植物乙醇酸氧化酶分离纯化方法的改进

通过缩短DEAE-Cellulose柱长度, 加快流速并采用pH8.8的80 mmol.L-1 Tris-HCl为洗脱液, 可在9小时内快速地从菠菜、菜心和豆角绿叶中纯化得到乙醇酸氧化酶。该酶具高活性(54.6～197.0 U.mg-1)及高等电点(pI >10.0)。产率为4.1%～71.5%, 纯化倍数为21.6～122.68。经SDS-PAGE检测均有40 kD带,表明3种植物乙醇酸氧化酶的亚基大小无区别。     

乙醇酸氧化酶必需精氨酸残基的化学修饰

丁二酮能使GAO迅速失活,其失活速度受介质pH和硼酸浓度的显著影响;其修饰反应具可逆性,当透析除去修饰剂和硼酸时,活性得到恢复。失活进程表现为假一级动力学。而计算表明,酶的每一活性中心单位与一分子丁二酮结合便可引起酶的失活。底物和竞争性抑制剂均能有效地保护酶免于失活。氨基酸分析表明,酶的失活是因为丁二酮修饰了精氨酸残基。丁二酮修饰GAO后使酶的K_m增大,而V_m没有变化。     

Effects of water stress on glycolate metabolism in the leaves of rice seedlings (Oryza sativa)

To investigate the effects of water stress on glycolate metabolism, seedlings of a drought-tolerant cultivar (N-22) and a susceptible cultivar (Jaya) of Oryza sativa L. were subjected to water stress for 5, 8 or 10 days. Increasing the duration of water-deficit-stress produced a proportional decrease in relative water content and leaf water potential, reduced glycolate content and catalase (EC 1.11.1.6) activity, but increased glycolate oxidase (EC 1.1.3.1) activity, hydrogen peroxide and glyoxylate contents in the leaves of both cultivars. In a radiotracer experiment, with increasing duration of water stress, the proportion of label increased in 3-phosphoglycerate, glycolate, glycine and serine. The drought-tolerant cultivar (N-22) was affected less than the susceptible cultivar (Jaya). The glycolate pathway metabolism is discussed in relation to photorespiration and the effects of water stress.     

乙醇酸氧化酶基因在巴斯德毕赤氏酵母中的表达

将菠菜乙醇酸氧化酶基因片段克隆至表达载体pPIC3．5k。提取重组质粒,进行限制性酶切鉴定。重组质粒用Sal I酶切线性化,电导入法转化毕赤酵母(Pichia pastoris),在缺乏组氨酸的RDB平板筛选重组子,提取酵母的染色体基因组进行PCR扩增鉴定整合情况,用甲醇诱导表达。结果表明,SDS-PAGE电泳显示表达蛋白的分子量约为39．8kD,与文献报道的乙醇酸氧化酶分子量接近。酶的活力达到了40．8IU／g湿菌体,比不含有目的片断的对照菌酶活提高了17倍,确认了导入的乙醇酸氧化酶基因片段在酵母中高效表达。     

Glycolate metabolism and subcellular distribution of glycolate oxidase in Spatoglossum pacificum (Phaeophyceae, Chromophyta)

Seven enzymes participating in glycolate metabolism were demonstrated to be present in crude extract of the brown alga Spatoglossum pacificum Yendo. These were phosphoglycolate phosphatase, glycolate oxidase, glutamate-glyoxylate aminotransferase, serine hydroxymethyltransferase, amino acid-hydroxy-pyruvate aminotransferase, hydroxypyruvate reductase and catalase. Malate synthase, which is involved in glycolate metabolism in the xanthophycean alga, could not be detected. On demonstration of subcellular distribution of glycolate oxidizing enzymes by linear sucrose density gradient centrifugation, glycolate oxidase was detected in the same fraction at a density of 1.23 g cm^?3 with catalase: that is, the marker enzyme of peroxisome and serine hydroxymethyltransferase was found in the same fraction at a density of 1.21 g cm^?3 with isocitrate dehydrogenase, the marker of mitochondria. From the present data, it is proposed that the brown alga Spatoglossum possesses the ability to metabolize glycolate to glycerate via the pathway which may be similar to that of higher plants.     

二倍体普通荞麦异常花朵及其结构的研究

在二倍体普通荞麦BW1 9 1的自交后代群体中有一些植株上的很多花朵形态及其结构发生变异。对这些异常花朵的形态进行了观察 ,统计了各变异花朵的被片数目 (x1)、花柱数目 (x2 )、胚珠数目 (x3 )和雄蕊数目 (x4)等参数。结果发现 ,该群体植株的花朵类型多达 2 5种以上。同一植株上花朵的被片数目 (x1)、花柱数目 (x2 )、胚珠数目 (x3 )和雄蕊数目 (x4)等参数变异广泛。大多数花朵为 5被片 3花柱 1胚珠 8雄蕊 ,但是被片数变幅为 3～ 8,雄蕊数变幅为 3～ 1 1 ,花柱数变幅为 2～ 8,胚珠数变幅为 1～ 3。当花柱数分别为 2、3、4、5、6、7或 8时 ,胚珠数分别是 1 (正常 )、1 (正常 )、1 (正常 ) +0 (未发育胚珠 )、1 (正常 ) +1 (小型胚珠 )、2 (正常 )、2 (正常) +0 (未发育胚珠 )或 2 (正常 ) +1 (小型胚珠 )。偏相关分析表明 ,被片数与花柱数存在显著相关 (r12 .3 4 =0 2 3 0 2 ) ,被片数与雄蕊数存在极显著相关 (r14 .2 3 =0 .472 7 )以及花柱数与胚珠数之间存在极显著相关(r2 3 .14 =0 .7787 ) ,这暗示被片、花柱、胚珠、雄蕊在遗传或发育上是相关的     

Anther culture and androgenetic plant regeneration in buckwheat (Fagopyrum esculentum Moench)

Anther culture for haploid induction of buckwheat was studied over a period of five years. Approximately 24,000 anthers were isolated and cultured on different culture media. The regeneration capacity was generally very low. Data are presented for experiments that included 7278 anthers on which 99 calluses were formed and 20 buds regenerations were noted. Regeneration occurred most readily on gellan-gum solidified media, with 90 g l^-1 maltose, 2.5 mg l^-1 BA, 0.5 mg l^-1 IAA, and preferably in darkness. Haploid cells, as established by chromosome counts, were observed in eight regenerants. Several abnormalities of pollen development in vitro were detected. Starch presence in pollen as a possible sign of androgenic capacity was studied. Microspores in uninucleate and early binucleate stages contained only proplastids, while in adult pollen grains a number of amyloplasts were present.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - 2,4 D-2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - 2iP 6- c,c-dimethylallylaminol-purine - NAA -naphthalene acetic acid     

Unusual regulatory properties of sucrose-phosphate synthase purified from soybean (Glycine max) leaves

Sucrose-phosphate (SPS) from source leaves of soybean ( Glycine max (L.) Merr. cv. Ransom II) was purified 74-fold to a final specific activity of 1.8 U (mg protein)¹. The partially purified preparation was free from phosphoglucoseisomerase (EC 5.3.1.9), pyrophosphatase (EC 3.6.1.1), phosphoenolpyruvate-phosphatase (EC 3.1.3.-), phosphofructokinase (EC 2.7.1.11), and uridine diphosphatase (EC 3.6.1.6), and was used for characterization of the kinetic and regulatory properties of the enzyme. The enzyme showed hyperbolic saturation kinetics for both fructose-6-phosphate (K_m=0.57 m M ) and UDPGlucose (UDPG) (K_m=4.8 m M ). The activity of SPS was inhibited by the product UDP. In vitro this inhibition could be partially overcome by the presence of Mg²⁺. Inorganic orthophosphate was only slightly inhibitory (35% inhibition at 25 m M phosphate). Glucose-6-phosphate (up to 20 m M ) had no effect on activity, and did not show any significant interaction with phosphate inhibition. A range of potential effectors was tested and had no effect on SPS activity: Glucose-1-phosphate, fructose-1, 6-bisphosphate, α-glycero-phosphate, dihydroxyacetone-phosphate, 3-phosphoglyceric acid, (all at 5 m M ), sucrose at 100 m M and pyrophosphate at 0.1 m M . The apparent lack of allosteric regulation of soybean SPS makes this enzyme markedly different from SPS previously characterized from spinach and maize.     

Plant regeneration from cotyledon tissues of common buckwheat (Fagopyrum esculentum Moench)

Summary Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l⁻¹) and kinetin (KIN) (0.2 mg l⁻¹) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l⁻¹ KIN, 2.0 mg l⁻¹ BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.     

Identification and characterization of a rhamnosyltransferase involved in rutin biosynthesis in Fagopyrum esculentum (common buckwheat)

Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6″-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6″RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6″RhaT enzyme expressed in Escherichia coli exhibited 6″-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6″RhaT converted the administered quercetin into rutin, suggesting that FeF3G6″RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6″RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6″RhaT is involved in rutin biosynthesis in common buckwheat.     

Induction of glycolate oxidase by SO2 in Nicotiana tabacum

In contrast to the inhibitory action of sulfite on glycolate oxidase, the specific activity of the enzyme in tobacco leaves exposed to SO₂ for 18 hr increases in proportion to the SO₂ concentration. This increase is strongly reduced by pretreatment with cycloheximide. As a consequence of induced de novo synthesis of glycolate oxidase the glycolate content of the leaves is markedly reduced after 18 hr exposure to SO₂.     

Expression analysis of buckwheat (Fagopyrum esculentum Moench) metallothionein-like gene (MT3) under different stress and physiological conditions

The buckwheat metallothionein-like (MT3) gene expression was studied throughout seed and leaf development, as well as under the influence of different external stimuli. MT3 mRNAs were detected from the early stage of seed development to the end of maturation, reaching the highest level during the mid-maturation stage. High MT3 mRNA level was noticed for both green and senescent leaves. The influence of raising Cu ion concentrations on MT3 gene expression was studied only in leaves, while the effect of Zn ions was analyzed through seed development as well. It was found that Cu and Zn ions had stimulatory effects on expression in leaves. MT3 expression was significantly enhanced in the early stage of seed development in response to Zn ions, while after this stage, influence of Zn ions was not detected. After H2O2/NaCl treatment, MT3 mRNA level was decreased in green leaves, contrary to senescent leaves where expression levels remained unchanged. H2O2 treatment caused the increase of MT3 mRNA levels in the mid-maturation stage of seed development. NaCl had no effect on expression levels in seeds. According to obtained results, proposed functions in different plant organs regarding oxidative stress and metal homeostasis are discussed.