苹果和葡萄果实发育过程中蛋白激酶特性的比较研究

在苹果 (MalusdomesticaL .Borkh .cv .RedStarking)和葡萄 (VitisviniferaL .×V .lubruscaL .cv .Kyoho)的不同发育阶段均检测到蛋白激酶活性 ,它们可被Ca2 强烈激活 ,表明依赖钙的蛋白激酶是苹果和葡萄果实中一类非常重要的蛋白激酶。钙调素 (CaM)对苹果和葡萄果实蛋白激酶没有激活作用 ,同时CaM拮抗剂calmidazolium、W7及三氟乙酸 (TFA)均可不同程度地抑制其活性 ,表明依赖钙的蛋白激酶中可能有含类似钙调素结构域的蛋白激酶(CDPK)的存在。在苹果和葡萄果实发育中 ,依赖钙的蛋白激酶无论在其活性变化还是特性上都存在着显著差异 :苹果果实发育的不同阶段依赖钙的蛋白激酶活性无明显变化 ,而葡萄果实在发育的第Ⅱ期蛋白激酶活性大大高于第Ⅰ、Ⅲ期 ;苹果果实依赖钙的蛋白激酶可被Mn2 强烈激活并对热敏感 ,而葡萄果实依赖钙的蛋白激酶却不受Mn2 及热处理的影响 ;此外 ,葡萄果实蛋白激酶活性不受磷脂酰丝氨酸 (PS)的影响 ,而苹果果实蛋白激酶可被PS激活 ,表明苹果果实中还可能有钙和磷脂激活的蛋白激酶即PKC的存在。苹果和葡萄果实发育中蛋白激酶种类、特性及其活性变化的不同 ,暗示了这两种果实发育调控的差异 ,也意味着苹果和葡萄果实发育调控可能与蛋白激酶密切相关。     

苹果和葡萄果实蛋白激酶特性分析

以组蛋白Ⅲ S作苹果和葡萄果肉蛋白激酶制剂底物时 ,反应体系中加EGTA可抑制蛋白激酶活性 ,而加Ca2 可激活蛋白激酶的活性 ,表明苹果和葡萄果实中有依赖钙的蛋白激酶存在。而且 ,葡萄果实微粒体蛋白激酶呈热稳定性 ,苹果果实微粒体蛋白激酶对热敏感。以髓鞘碱性蛋白 (MBP)作底物 ,在苹果和葡萄果实微粒体中都检测出很高的蛋白激酶活性 ,并且不依赖于钙 ,说明苹果和葡萄果实中可能有分裂原激活的蛋白激酶 (MAP激酶 )的存在。苹果和葡萄果实MAP激酶的活性都表现出对二价阳离子Mg2 或Mn2 的依赖 ,并对高温处理表现出了激活效应     

苹果果实发育期间细胞壁组分变化特性

以 '富士'、'国光'、'红星'、'金冠'和'嘎拉'5个苹果品种为试材,分析了果实发育成熟过程细胞壁物质(CWM)、水溶性果胶(WSP)、共价结合果胶(CSP)、离子结合果胶(ISP)、纤维素及半纤维素各组分变化.结果表明:在苹果果实发育过程中,5个品种果实CWM含量变化均呈先升后降的变化规律,均以果实膨大期为其含量下降的转折点;果实总果胶含量均呈不断降低的趋势,其中CSP为主导成分,'富士'和'国光'果实CSP含量最高,WSP含量最低,'嘎拉'与'红星'果实3种果胶含量变化居中,'金冠'果实总果胶含量最低且变化小,但在近成熟期'红星'和'金冠'果实WSP呈明显的上升趋势.果实半纤维素含量也具相似的变化规律,'国光'、'富士'和'金冠'等3个品种的高峰值显著高于'嘎拉'和'红星';比较5个品种纤维素含量,'国光'果实在成熟期之前显著高于其他4品种,而其他4品种的纤维素含量变化比较平稳.     

发育过程中苹果果皮和果肉细胞的超微结构

用透射电镜对发育过程中苹果（Ｍａｌｕｓ ｄｏｍｅｓｔｉｃａ Ｂｏｒｋｈ ｃｖ．Ｒｅｄ Ｆｕｊｉ）果皮和果肉细胞的超微结构进行了观察。结果表明,不同发育期果皮细胞的超微结构发生了变化,其中最引人注目的是,内质网自始至终密布于整个细胞质中,而且大多为槽库膨大的、合成功能旺盛的管状粗面内质网,并分泌出大量的具运输功能的小泡;观察到这些小泡与液泡融合的景象;细胞中也存在活跃的高尔基体。超微结构上的这些现象     

丝裂原活化蛋白激酶激活蛋白激酶(MK)研究进展

丝裂原活化蛋白激酶(MAPK)信号通路介导多种重要的细胞生理反应.对下游蛋白激酶的磷酸化是MAPK家族成员发挥生理作用的重要方式.在MAPK的下游存在3个结构上相关的MAPK激活蛋白激酶(MAPKAPKorMK),即MK2,MK3和MK5.在被MAPK激活后,MK可将信号传递至细胞内不同靶标,从而在转录和翻译水平调节基因表达,调控细胞骨架和细胞周期,介导细胞迁移和胚胎发育.最近,在基因敲除研究的基础上,不同MK亚族成员之间的功能区分已经逐渐明晰,使我们对于MK的认识有了长足的进步.     

套袋苹果果实质地及其显微结构与果面裂纹的关系北大核心CSCD

以套白色单层纸袋的着色富士系‘天红2号’和短枝富士后代优系4-1-103,以及不着色姊妹系‘冀苹4号’和‘冀苹5号’4个苹果品种(系)为试验材料,利用质构仪整果穿刺和质地多面分析法(texture profile analysis,TPA)测定果实质地,光学显微镜观察果实显微结构,探讨苹果果实质地及其显微结构与果面裂纹的关系,为抗裂纹品种的选育和潜在裂纹机制提供理论依据。结果表明:(1)盛花后132~185 d,‘天红2号’和‘冀苹4号’裂纹率和裂纹指数始终高于4-1-103和‘冀苹5号’,其裂纹主要部位为果肩部。(2)‘天红2号’和‘冀苹4号’的果肉脆裂性、果肉硬度分别显著小于4-1-103和‘冀苹5号’。(3)‘天红2号’和‘冀苹4号’的表皮细胞密度显著小于4-1-103和‘冀苹5号’,其果肉细胞间隙率显著大于4-1-103和‘冀苹5号’,且其角质膜有“V”型凹陷,并发生龟裂,而4-1-103和‘冀苹5号’角质膜完整均一。(4)主成分和灰色关联度分析表明,果肉硬度、果皮果肉硬度比和果肉细胞间隙率是解释裂纹的主要指标。研究发现,果实角质膜完整均一、果肉硬度大、果肉细胞间隙率小的苹果品种不易发生果面裂纹。     

葡萄浆果发育过程中激素水平的变化

对三个葡萄品种浆果发育过程中可溶性糖累积和内源IAA、GA3 、ABA含量的动态变化等进行比较研究。结果表明 ,巨峰和木纳格葡萄浆果生长呈双S型曲线 ,无核白葡萄果实生长呈S型曲线 ;浆果内可溶性糖的累积主要发生在果实发育的后期。浆果发育初期 ,无核白葡萄IAA含量远高于两个有核品种的含量 ,有利于幼果坐果 ;浆果发育后期 ,无核白葡萄IAA和GA3 的含量低于两个有核品种 ,而ABA的含量比两个有核品种高。GA3 与ABA比值的变化对浆果的发育起着关键的作用。     

植物抗逆相关蛋白激酶的结构与功能

植物在遭受外界逆境胁迫时,体内的信号传导系统能够感知、传递逆境胁迫信号,并引起各种生理生化反应以适应环境。植物蛋白激酶在信号感知、传导以及基因的表达调控中起重要的作用。蛋白激酶在信号传导过程的功能是磷酸化修饰目的蛋白,而磷酸化的实现需要蛋白质之间相互作用。本文从植物蛋白激酶的结构、分类、与激素信号传导之间的关系等方面进行了系统的阐述,对蛋白激酶介导的植物抗性与发育的最新研究进展进行了系统的总结,为解析蛋白激酶在植物生长发育中的抗逆机理提供依据。     

Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases

The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies.     

高温胁迫下葡萄叶片蛋白激酶的诱导形成与活性变化

以"京秀"葡萄(Vitis vinifera L.cv.Jingxiu)幼苗为试材,研究了高温胁迫激活的蛋白激酶的类型和活性.结果表明,高温胁迫10～60min明显地激活了一个分子量约为52 kD的蛋白激酶,该蛋白激酶能将凝胶中所嵌入的髓鞘碱性蛋白(MBP)磷酸化,在放射自显影中表现出很高的放射活性,而对凝胶中的组蛋白-Ⅲ(histone-Ⅲ)则没有这样的作用.在溶液反应体系中该蛋白激酶对MBP也表现出很高的磷酸化活性,而对histone-Ⅲ却无作用.Ca2 对其活性变化无显著影响.酪氨酸特异性蛋白磷酸酶(YOP)对该激酶的活性有显著的钝化作用.结果表明该52 kD蛋白激酶是MAPK家族中的一种.     

Overexpression of The Calcium-Dependent Protein Kinase OsCDPK2 in Transgenic Rice is Repressed by Light in Leaves and Disrupts Seed Development

Independent transgenic rice lines overexpressing the rice CDPK isoform OsCDPK2 were generated by particle bombardment. High levels of OsCDPK2 were detected in leaves removed from etiolated plants, as well as in stems and flowers. However, there was no overexpression in green leaves that had been exposed to light, confirming that OsCDPK2 protein stability was subject to light regulation. The morphological phenotype of transgenic plants producing high levels of recombinant OsCDPK2 was normal until the onset of seed development. Flowers developed normally, producing well-shaped ovaries and stigmas, and mature anthers filled with pollen grains. However, seed formation in these plants was strongly inhibited, with only 3–7% of the flowers producing seeds. Seed development was arrested at an early stage. We discuss these data with respect to the possible requirement for specific CDPK isoforms during rice seed 4.4ptdevelopment.     

苹果果实韧皮部及其周围薄壁细胞的超微结构观察和功能分析

利用透射电镜技术,对发育过程中的苹果（Ｍａｌｕｓ ｄｏｍｅｓｔｉｃａ Ｂｏｒｋｈ）果实韧皮部及其周围薄壁细胞的超微结构进行了观察研究。结果表明,在主脉和细脉的筛分子（ＳＥ）和伴胞（ＣＣ）之间存在胞间连丝,胞间连丝在筛分子一侧是单通道,在伴胞一侧呈多分枝通道。在细脉中筛分子小,伴胞大,在主脉中则是筛分子大,伴胞小。伴胞内胞质和核质稠密,富含线粒体、内质网和高尔基体,液泡内往往呈现多膜包被的囊泡结构,     

葡萄早熟芽变与其亲本果实发育特征比较分析

‘峰早’和‘洛浦早生’葡萄分别是‘巨峰’和‘京亚’的早熟芽变,本研究对比分析了早熟芽变与其亲本间在果实发育过程中与成熟有关的生理指标动态变化的差异。结果表明,与亲本相比,两个早熟芽变品种在果实横径、纵径、单粒重的增长速率,叶绿素、类黄酮和总酚含量变化趋势,以及β-半乳糖苷酶活性等方面的变化趋势差别不大。可溶性固形物和可溶性糖的变化趋势、类胡萝卜素的含量及脂氧合酶（LOX）活性的变化趋势在两对芽变与亲本间无明显一致的趋势,表现出的情形比较复杂。但两芽变品种花色素苷的增长速率在花后50 d均显著快于亲本,且在成熟时的绝对含量均大于对应时期亲本,这可能是早熟芽变品种果实先着色的原因之一。两芽变与亲本间果胶甲酯酶（PE）活性和多聚半乳糖醛酸酶（PG）活性变化模式相一致,且两芽变的PG酶活性增长速率均明显大于其亲本,这种变化幅度也与早熟芽变性状变化的幅度相关,所以PG酶活性增加加快了芽变品种的成熟软化,这可能是芽变比亲本早熟的原因之一。     

Effect of Down-Regulation of Ethylene Biosynthesis on Fruit Flavor Complex in Apple Fruit

The role of ethylene in regulating sugar, acid, texture and volatile components of fruit quality was investigated in transgenic apple fruit modified in their capacity to synthesize endogenous ethylene. Fruit obtained from plants silenced for either ACS (ACC synthase; ACC-1-aminocyclopropane-1-carboxylic acid) or ACO (ACC oxidase), key enzymes responsible for ethylene biosynthesis, expectedly showed reduced autocatalytic ethylene production. Ethylene suppressed fruits were significantly firmer than controls and displayed an increased shelf-life. No significant difference was observed in sugar or acid accumulation suggesting that sugar and acid composition and accumulation is not directly under ethylene control. Interestingly, a significant and dramatic suppression of the synthesis of volatile esters was observed in fruit silenced for ethylene. However, no significant suppression was observed for the aldehyde and alcohol precursors of these esters. Our results indicate that ethylene differentially regulates fruit quality components and the availability of these transgenic apple trees provides a unique resource to define the role of ethylene and other factors that regulate fruit development.     

Activity, but not Expression, of Soluble and Cell Wall-Bound Acid Invertases Is Induced by Abscisic Acid in Developing Apple Fruit

The present experiment, involving both the in vivo injection of abscislc acid （ABA） Into apple （Malus domestica Brohk.） fruits and the in vivo Incubation of fruit tissues in ABA-contalnlng medium, revealed that ABA activates both soluble and cell wall-bound acid invertases. Immunoblottlng and enzyme-linked Immunosorbent assays showed that this ABA-induced acid invertase activation is Independent of the amount of enzyme present. The acid Invertase activation induced by ABA is dependent on medium pH, time course, ABA dose, living tissue and developmental stage. Two isomers of cls-（＋）-ABA, （-）-ABA and trans- ABA, had no effect on acid invertases, showing that ABA-induced acid invertase activation is specific to physiologically active cis-（＋）ABA. Protein kinase inhlbltors K252a and H7 as well as acid phosphatase Increased the ABA-Induced effects. These data indicate that ABA specifically activates both soluble and cell wall-bound acid Invertases by a posttranslational mechanism probably Involving reversible protein phosphorylatlon, and this may be one of the mechanisms by which ABA Is Involved In regulating fruit development.     

Role of Extracellular Signal-Regulated Protein Kinases 1 and 2 in Oligodendroglial Process Extension

Abstract: The relationship between extracellular signal-regulated protein kinase (ERK) activation and process extension in cultured bovine oligodendrocytes (OLGs) was investigated. Process extension was induced through the exposure of cultured OLGs to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), for various intervals. During the isolation of these OLGs from bovine brain, the original processes were lost. Therefore, any reinitiation of process extension via PMA stimulation was easily discernible through morphological monitoring. It was found that exposure of OLGs to PMA for 10 min was enough to induce OLG process extension 24–72 h later. Furthermore, this extension was still evident at least 1 week after the initial PMA stimulation, indicating that OLGs do not need continuous PKC activation to sustain process extension. Control and PMA-stimulated OLGs were also subjected to immunocytochemistry using an anti-ERK antibody selective for the mitogen-activated protein kinases p42 Erk2 (ERK2) and p44 Erk1 (ERK1) isoforms. ERK immunoreactivity in the nucleus was evident after PMA stimulation of OLGs but not in control OLGs. In parallel experiments, the control and PMA-stimulated OLGs were purified by Mono Q fractionation and subjected to ERK phosphotransferase assays using [γ-³²P]ATP and either myelin basic protein (MBP) or a synthetic peptide substrate based on the Thr⁹⁷ phosphorylation site in MBP. These assays indicated that in PMA-treated OLGs, ERK activation was at least 12-fold higher than in control OLGs. Anti-ERK and anti-phosphotyrosine western blots of the assay fractions verified an enhanced phosphorylation of ERK1 and ERK2 in PMA-treated fractions relative to control fractions. When OLGs were pretreated for 15 min with the ERK kinase (MEK) inhibitor PD 098059 before PMA stimulation, they exhibited a 67% decrease in ERK activation as compared with cells treated with PMA alone. Furthermore, these MEK inhibitor-pretreated cells were still viable but showed no process extensions up to 1 week later. Therefore, we propose that a threshold level of ERK activity is required for the initiation of OLG process extension.     

植物类受体蛋白激酶的研究进展

植物类受体蛋白激酶(receptor-like protein kinase,RLKs)通过胞外结构域识别病原信号分子,发生磷酸化、去磷酸化反应而开启或关闭下游靶蛋白,调节植物固有免疫反应,诱导抗病防御反应.目前对植物类受体蛋白激酶的功能、信号传导和配体识别等方面的研究已成为该领域的重点.本文对近年来国内外有关植物类受体蛋白激酶的结构、功能及其在植物抗病防御反应中的作用研究进行综述,为今后进一步深入研究植物类受体蛋白激酶的生理生化功能及应用提供参考.