光/暗对高粱叶片磷酸烯醇式丙酮酸羧化酶基因表达的调节

高粱幼苗黄化叶片经照光转绿后,其PEP-Case活性提高4～15倍,mRNA含量提高了1.03倍,并测定出PEPCase mRNA的分子量为3.4kb。以等量的总RNA及mRNA进行体外翻译,发现转绿后PEPCase专一性翻译活性提高了51％～53％。这表明光照可以在转录水平上调节PEP-Case的基因表达。     

转玉米pepc基因水稻的某些光合特性

测定转玉米pepc基因水稻和未转化粳稻品种Kitaake在分蘖初期、分蘖盛期、拔节期、始穗期、齐穗期、成熟期和剑叶不同生长时期的光合特性动态变化的结果表明:转玉米pepc基因水稻的磷酸烯醇式丙酮酸羧化酶(PEPCase)活性和净光合速率(Pn)在不同时期各不相同,相对于Kitaake而言,均有不同程度的提高;剑叶完全展开时的PEPCase活性和Pn提高最明显.     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)     

Analysis of Promoter Activity for the Gene Encoding Pyruvate Orthophosphate Dikinase in Stably Transformed C4 Flaveria Species

The C₄ enzyme pyruvate orthophosphate dikinase is encoded by a single gene, Pdk, in the C₄ plant Flaveria trinervia. This gene also encodes enzyme isoforms located in the chloroplast and in the cytosol that do not have a function in C₄ photosynthesis. Our goal is to identify cis-acting DNA sequences that regulate the expression of the gene that is active in the C₄ cycle. We fused 1.5 kb of a 5′ flanking region from the Pdk gene, including the entire 5′ untranslated region, to the uidA reporter gene and stably transformed the closely related C₄ species Flaveria bidentis. β-Glucuronidase (GUS) activity was detected at high levels in leaf mesophyll cells. GUS activity was detected at lower levels in bundle-sheath cells and stems and at very low levels in roots. This lower-level GUS expression was similar to the distribution of mRNA encoding the nonphotosynthetic form of the enzyme. We conclude that cis-acting DNA sequences controlling the expression of the C₄ form in mesophyll cells and the chloroplast form in other cells and organs are co-located within the same 5′ region of the Pdk gene.     

Structure,Regulation and Biosynthesis of Phosphoenolpyruvate Carboxylase from C4 Plants

This review attempts to summarize the large body of information on the structure, regulation and biosynthesis of the enzyme phosphoenolpyruvate carboxylase in C₄ plants which has accumulated particularly since the appearance of the last review in 1987. Among the major discoveries are the involvement of protein phosphorylation-dephosphorylation cascade in the light activation of the enzyme, extraction and characteristics of PEPC-protein serine kinase, dynamic changes in oligomeric state of the enzyme in response to pH or temperature, isolation of multiple cDNAs encoding different forms of PEPC and cloning and expression of maize/sorghum PEPC in transgenic tobacco or transformed E. coli cells. Further experiments using advanced techniques of biochemistry and molecular biology would help in understanding the molecular mechanism of reaction, regulation of enzyme activity, gene expression and evolutionary pattern of C₄ PEPC.     

斑马鱼shh启动子指导绿色荧光蛋白基因在脊索中的表达

体外实验业已证明shh基因的启动子受HNF3β蛋白直接调控。为研究斑马鱼shh启动子在体内的作用模式,构建了由538bp斑马鱼shh启动子(包含两个HNF3β结合位点)与绿色荧光蛋白EGFP组成的表达载体,命名为pShh-EGFP。将pShh-EGFP注射到斑马鱼1-细胞期内的受精卵中,定期在倒置荧光显微镜下观察GFP的表达。GFP在原肠作用期就开始表达,主要发生在中轴下胚层中;在体节形成期,GFP在脊索细胞中表达,但未发现在神经底板细胞中表达。由此可见,含两个HNF3β结合区域的538bp的斑马鱼shh启动子具有脊索表达活性。     

干旱胁迫下表观遗传机制对转C4型PEPC基因水稻种子萌发的影响

为探究干旱胁迫下表观遗传机制对高表达玉米(Zea mays) C₄型PEPC转基因水稻(Oryza sativa)种子萌发的影响, 以转C₄型PEPC水稻(PC)和野生型水稻Kitaake (WT)为试材, 采用10% (m/v)聚乙二醇6000 (PEG6000)模拟干旱条件, 通过单独和联合施用PEG6000、DNA甲基化抑制剂5-氮杂胞苷(5azaC)和可变剪接抑制剂大环内酯类(PB)进行种子发芽实验, 测定种子活力、萌发过程中可溶性糖和可溶性蛋白含量、α-淀粉酶活性以及PEPC、糖信号相关基因和部分剪接因子基因的表达。结果表明, 0.25 µmol·L^-1PB处理对2种供试水稻在干旱条件下种子萌发均表现出显著抑制作用, 使干旱条件下种子萌发过程中可溶性总糖、蔗糖、葡萄糖和果糖含量以及可溶性蛋白含量均有所下降, PB也抑制糖信号-蔗糖非发酵1 (SNF1)相关蛋白激酶(SnRKs)家族和剪接因子丝氨酸/精氨酸富集蛋白家族(SR proteins)相关基因的表达以及α-淀粉酶的活性, 但对PC的抑制作用小于WT。5 µmol·L^-15azaC处理对干旱条件下种子萌发的效果与可变剪接抑制剂相反。5 µmol·L ^-1 5azaC联合PEG6000干旱处理部分减缓了干旱对水稻种子发芽率的抑制作用, 使供试材料发芽率升高, 表明DNA甲基化和可变剪接机制参与了水稻芽期干旱耐性, 其中对PC的作用更大。     

Effects of Epigenetic Mechanisms on C4 Phosphoenolpyruvate Carboxylase Transgenic Rice (Oryza sativa) Seed Germination Under Drought Stress

为探究干旱胁迫下表观遗传机制对高表达玉米(Zea mays) C₄型PEPC转基因水稻(Oryza sativa)种子萌发的影响, 以转C₄型PEPC水稻(PC)和野生型水稻Kitaake (WT)为试材, 采用10% (m/v)聚乙二醇6000 (PEG6000)模拟干旱条件, 通过单独和联合施用PEG6000、DNA甲基化抑制剂5-氮杂胞苷(5azaC)和可变剪接抑制剂大环内酯类(PB)进行种子发芽实验, 测定种子活力、萌发过程中可溶性糖和可溶性蛋白含量、α-淀粉酶活性以及PEPC、糖信号相关基因和部分剪接因子基因的表达。结果表明, 0.25 μmol·L^-1PB处理对2种供试水稻在干旱条件下种子萌发均表现出显著抑制作用, 使干旱条件下种子萌发过程中可溶性总糖、蔗糖、葡萄糖和果糖含量以及可溶性蛋白含量均有所下降, PB也抑制糖信号-蔗糖非发酵1 (SNF1)相关蛋白激酶(SnRKs)家族和剪接因子丝氨酸/精氨酸富集蛋白家族(SR proteins)相关基因的表达以及α-淀粉酶的活性, 但对PC的抑制作用小于WT。5 μmol·L^-15azaC处理对干旱条件下种子萌发的效果与可变剪接抑制剂相反。5 μmol·L ^-1 5azaC联合PEG6000干旱处理部分减缓了干旱对水稻种子发芽率的抑制作用, 使供试材料发芽率升高, 表明DNA甲基化和可变剪接机制参与了水稻芽期干旱耐性, 其中对PC的作用更大。     

Multiple Inter-Kingdom Horizontal Gene Transfers in the Evolution of the Phosphoenolpyruvate Carboxylase Gene Family

Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.     

Phosphate Activates the Phosphoenolpyruvate Carboxylase from the C4 Plant Amaranthus viridis L.

Phosphoenolpyruvate carboxylase from Amaranthus viridis leaves was activated by inorganic orthophosphate in a concentration- and pH-dependent manner. Maximal activation at pH 7.0 was achieved at phosphate concentrations above 20 mM, and a positive cooperativity was observed for the binding of the anion at this pH. At pH 8.0 the maximum of activity was achieved at 10 mM phosphate; higher concentrations reduced the activation. K_M for phosphoenolpyruvate-Mg at pH 7.0 was lowered by phosphate in all concentrations tested up to 30 mM. While at pH 8.0 the K_M values were lower than that of the control up to 10 mM phosphate; higher anion concentrations raised the minimum value of K_M at this pH. V_MAX increased at pH 7.0, and remained unchanged at pH 8.0. A K_A value of 0.41 mM was calculated for phosphate at the alkaline pH. The phosphate analogue arsenate also behaved as an activating agent, while other anions (e.g. nitrate, nitrite, sulfate, tetraborate) were ineffective. The phosphate-activated enzyme was shown to be insensitive to glucose-6-phosphate, but was inhibited by l -malate to the same extent as the control.     

The Light-Dependent Transduction Pathway Controlling the Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis

Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent protein-serine kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent protein kinase and a cytosolic protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.     

甘露碱合成酶基因启动子调控的萤光素酶基因在转化烟草中的表达

利用我们自己分离的甘露碱含成酶基因启动子与萤光素酶结构基因、胭脂碱合成酶基因’3末端结构相拼构成一融合基因,并在带有此融合基因的中间载体PBZ7610插入Ti质粒T区的tmr基因,构成中间载体pBZ7621。利用改建的Ti质粒载体PGV3850,将萤光素酶融合基因引入了烟草植株,结果表明,萤光素酶融合基因在转化烟草中能表达。中间载体pBZ7610还带有PstⅠ,HindⅢ,XbaⅠ等多个单一的酶切位点,外源基因极易插入。利用中间载体pBZ7621,还可研究启动子在高等植物不同发育阶段中的功能特征。     

Calcium-Dependent and -Independent Phosphoenolpyruvate Carboxylase Kinases in Sorghum Leaves: Further Evidence for the Involvement of the Calcium-Independent Protein Kinase in the in situ Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase

Calcium-dependent phosphoenolpyruvate carboxylase protein kinasewas copurified with C₄ phosphoenolpyruvate carboxylase (C₄ PEPC)from illuminated Sorghum leaves during purification by variousprocedures. Isolated mesophyll cell protoplasts contained bothcalcium-dependent and -independent protein kinases. The latterwas induced by light and weak bases and was found to be themajor protein kinase phosphorylating C₄ PEPC in the mesophyll. (Received July 29, 1997; Accepted November 28, 1997)     

Phosphate and Sulfate Activate the Phosphoenolpyruvate Carboxylase from the C4 Plant Cynodon dactylon L.

The effect of phosphate, sulfate and other inorganic ions on the activity of phosphoenolpyruvate carboxylase (PEPC) from the C₄ plant Cynodon dactylon were investigated for the first time, as well as their interaction with Clc-6-P, AMP and ma-late. Activation of PEPC by phosphate and sulfate ions was demonstrated and it was not dependent on the accompanying cations, something that was not clarified for PEPCs from other plant sources. No activation of this enzyme was observed by nitrate. PEPC activation was found to be competitive with glucoses-phosphate (Clc-6-P) and AMP stimulation and less sensitive to malate inhibition. This work showed that PEPC from C₄plants could exhibit similar activation properties with the enzyme from CAM plants and different activation properties in plants of the same type, rendering the study of this enzyme from different plant sources necessary.