黄瓜叶片蛋白质双向电泳样品分级优化

以‘津研4号’苗期叶片为材料,采用聚乙二醇(PEG)分级沉淀法对黄瓜叶片蛋白质样品进行分级分离,将黄瓜叶片中存在的高丰度蛋白1,5-二磷酸核酮糖羧化酶/加氧酶特异性地集中于一个组分之中,以提高对黄瓜叶片蛋白质双向电泳中低丰度蛋白质的检测率.结果表明:(1)分级后各组分和全蛋白在SDS-PAGE谱带中差异显著,全蛋白得到了有效的分离.(2)二维电泳图谱上点的分辨率有很大的提高,所有组分的点数是未分级前的3倍之多.(3)浓度为24％的PEG-4000富集高丰度蛋白Rubisco的效果最好.该方法可推广应用于黄瓜叶片蛋白质组分析的样品制备.     

胺鲜酯对高产夏玉米产量及叶片光合羧化酶和保护酶活性的影响

采用田间小区试验,以高产玉米新品种登海661为材料,研究了拔节期叶面喷施10、20和40 mg·L^-1的胺鲜酯（DA-6）对玉米叶片光合羧化酶、保护酶活性和产量的影响.结果表明：喷施胺鲜酯各处理玉米分别比对照（含有表面活性剂和水）增产10.0%(10 mg·L^-1)、8.9%(20 mg·L^-1)和9.4%(40 mg·L^-1),增产效果显著,但各浓度间差异不显著.胺鲜酯处理后,花后玉米的叶面积指数、光合速率、RuBP羧化酶和PEP羧化酶活性均显著上升(P＜0.05),且对光合速率、RuBP羧化酶和PEP羧化酶活性的影响随着处理浓度的增加而提高;与对照相比,胺鲜酯处理后吐丝期、灌浆期、乳熟期和蜡熟期叶片SOD、CAT、POD和GSTs活性及可溶性蛋白质含量显著提高(P＜0.05),MDA含量显著降低(P＜0.05),其中CAT活性随着处理浓度的增加呈上升趋势,其余生理指标各浓度间无显著性差异.     

用未标记免疫酶技术在甘蔗大豆叶片内定位RUBP羧化酶

RuBP羧化酶是绿色植物同化CO_2的关键酶。研究RuBP羧化酶在C_4植物叶片内的分布,有助于阐明C_4植物的光合作用结构和功能的特点,以及RuBP羧化酶对光合作用的调控功能。这种工作具有明显的理论意义和实用价值。     

光和暗条件培养的眼虫藻(Euglena gracilis)内RuBP羧化酶的免疫定位

应用免疫金标记技术证明,在眼虫藻和其它藻类中RuBP羧化酶主要分布在蛋白核部位,这与高等植物中RuBP羧化酶分布不同,在眼虫藻叶绿体间质中有少量RuBP羧化酶存在,这与高等植物中RuBP羧化酶的分布也有相似之处。 暗中培养的眼虫藻不能形成类囊体,无RuBP羧化酶,无光合能力,只能进行异养代谢。     

适用于水稻叶片蛋白质组分析的双向电泳技术

针对水稻叶片中含有大量色素和酚等干扰物质的现象,通过对水稻叶片蛋白提取方法、上样量和聚丙烯酰胺凝胶浓度等方面做了必要改进,建立了一套适用于水稻叶片蛋白质组分析的双向电泳（2-DE）方法。     

离体稻苗叶片衰老过程中蛋白质组分的变化(简报)

离体稻苗叶片暗下衰老时,非水溶性蛋白质含量变化不大,而水溶性蛋白质含量下降迅速,其组分也同时改变。水溶性蛋白质中,Fraction Ⅰ蛋白质含量迅速下降,而Fraction Ⅱ蛋白质含量则显著增加。Fraction Ⅰ蛋白质中,RuBP羧化酶小亚基降解速率比大亚基快。叶片衰老时有两条新电泳蛋白质谱带出现。     

外源Ca2+对PEG处理下转C4型PEPC基因水稻光合生理的调节

磷酸烯醇式丙酮酸羧化酶(PEPC)通过固定二氧化碳参与光合作用, 是关键的C₄植物光合作用酶。为了揭示高光效转C₄ PEPC基因水稻(Oryza sativa)对干旱胁迫的适应机理, 以高表达转C₄ PEPC水稻(PC)和野生型水稻Kitaake (WT)为供试材料, 在植株的4-5叶期, 使用不同浓度外源CaCl₂溶液处理, 测定在15%聚乙二醇6000 (polyethylene glycol-6000, PEG-6000)胁迫下叶片相对含水量、光合参数、内源钙总含量、叶片总蛋白激酶活性、PEPC酶活性以及相关基因表达和蛋白质含量。结果表明, 0.5 mmol∙L^-1 CaCl₂明显提高PC叶片相对含水量(P<0.05), 2 mmol∙L^-1和10 mmol∙L^-1 CaCl₂则作用不显著, 对WT则影响不显著。不同浓度钙处理对PEG处理PC的净光合速率影响不显著, 而通过维持气孔导度减少水分胁迫。内源总钙浓度的数据显示, 在PEG6000处理下, PC具有维持稳定内源Ca²⁺浓度的能力, 过高浓度(10 mmol∙L^-1 CaCl₂)钙处理反而降低了PEPC酶活性、PEPC基因表达和可溶性蛋白的含量。     

水稻剑叶取向对其光合功能的影响

水稻的水平剑叶净光合速率 (Pn)和羧化效率(CE)显著高于直立剑叶 ,其胞间CO2 浓度 (Ci)显著低于直立剑叶 ,但两者的气孔导度 (Gs)没有明显差别。这表明剑叶取向对水稻叶片的光合能力有重要影响。水平剑叶的高Pn可能同其RuBP羧化酶含量和活性高有关。这可能是水平叶生长期间吸收光量较多的结果。     

上海植物代谢研究交流座谈会简讯

上海植物生理学会曾于今夏组织一次植物代谢研究交流会。会上听取了:“PAL酶的抑制蛋白”,“羧化酶研究的近况及酶、蛋白质的磷酸化在代谢调节中的作用”;“关于国内外RuBP羧化酶研究的动态”;“硝酸还原酶的调节控制”和“硝酸还原酶的调节控制”等专题报     

水稻叶片对镉胁迫响应的蛋白质差异表达

为揭示水稻镉抗性的分子机理,以抗镉水稻品种P1312777和镉敏感水稻品种IR24为材料,在镉离子浓度为0(对照)、50和100 μmol·L-1条件下水培处理7 d,应用蛋白质组学方法分析了2种水稻叶片对镉胁迫响应的蛋白质差异表达.结果表明:镉胁迫下水稻PI312777叶片中共检测到差异表达蛋白质点31个,通过MALDI-TOF/MS分析,鉴定了其中的24个蛋白质(包括20个不同蛋白质,4个重复检出蛋白质);IR24叶片中共检测到差异表达蛋白质点19个,其中15个蛋白质得到鉴定.PI312777叶片鉴定出的20个蛋白质覆盖了IR24叶片鉴定的15个蛋白质,前者有4个与光合作用相关,11个与细胞防御代谢相关,3个与其他代谢相关,2个为功能未知蛋白.与对照相比,不同浓度镉胁迫下,抗镉水稻PI312777叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型、果糖1,6-二磷酸醛缩酶、硫氧还蛋白和DNA重组修复蛋白均上调表达;镉敏感水稻IR24叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型的表达无显著差异,果糖1,6-二磷酸醛缩酶和硫氧还蛋白则下调表达.此外,DNA重组修复蛋白仅在镉胁迫的PI312777叶片中表达.水稻PI312777比IR24具有更强的镉抗性与这些差异表达的蛋白质密切相关.     

Acclimation of rice to changing atmospheric carbon dioxide concentration

Abstract. The effects were studied of season-long (75 and 88d) exposure of rice (Oryza sativa L. cv. IR-30) to a range of atmospheric CO₂ concentrations in outdoor, computer-controlled, environment chambers under natural solar radiation. The CO₂ concentrations were maintained at 160, 250, 330, 500, 660 and 900μmol mol^-1 air. Photosynthesis increased with increasing growth CO₂ concentrations up to 500u.mol moP1, but levelled off at higher CO₂ values. Specific leaf area also increased significantly with increasing CO₂. Although leaf dry weight and leaf area index increased, the overall response was not statistically significant. Leaf nitrogen content dropped slightly with elevated CO₂, but the response was not statistically significant. The specific activity of ribulose bisphosphate carboxylase/oxygenase (rubisco) declined significantly over the CO₂ concentration range 160 to 900μmol mol^-1. When expressed on a leaf area basis, rubisco activity decreased by 66%. This was accompanied by a 32% decrease in the amount of rubisco protein as a fraction of the total soluble leaf protein, and by 60% on a leaf area basis. For leaves in the dark, the total rubisco activity (CO₂/Mg²⁺-activated) was reduced by more than 60%. This indicates that rice accumulated an inhibitor in the dark, probably 2-car-boxyarabinitol 1-phosphate (CA-1-P). However, the inhibitor did not seem to be involved in the acclimation response. The degree of carbamylation of the rubisco enzyme was unchanged by the CO2 growth regime, except at 900 [μmol mol^-1 where it was reduced by 24%. The acclimation of rice to different atmospheric CO₂ conditions involved the modulation of both the activity and amount of rubisco protein in the leaf.     

非生物胁迫下水稻OsLRR基因的表达分析

植物中含有多种富含亮氨酸重复（leucine-rich repeats,LRRs）的蛋白质,这类蛋白质在植物生长、发育和抗病反应等方面发挥着重要作用。本研究在水稻中克隆到一个编码LRRs结构的基因OsLRR,以半定量RT-PCR检测了OsLRR在水稻不同组织和不同非生物胁迫的表达情况,并进一步分析了铝毒胁迫下OsLRR在抗铝和铝敏感水稻品种之间的表达差异。结果表明OsLRR在水稻根、叶鞘和叶中都有较高表达。铝、砷、PEG6000和ABA可诱导水稻根中OsLRR的表达,而镉、硝普钠和铁则抑制其表达。只有盐胁迫能诱导叶片中OsLRR的表达。铝毒可以诱导抗铝和铝敏感水稻品种根中OsLRR的表达,但随着处理时间的延长,抗铝品种中OsLRR的表达逐渐加强,而铝敏感品种中OsLRR的表达则逐渐减弱。     

The proteomics of senescence in leaves of white clover,Trifolium repens (L.)

A proteomics approach has been used to study changes in protein abundance during leaf senescence in white clover. Changes in cell ultrastructure were also examined using transmission electron microscopy. The most obvious ultrastructural changes during senescence occurred in chloroplasts, with progressive loss of thylakoid integrity and accumulation of osmiophilic globules in the stroma. Quantitative analysis of 590 leaf protein spots separated by two-dimensional electrophoresis indicated that approximately 40% of the spots showed significant senescence related changes in abundance. Approximately one-third of the protein spots present in mature green leaves were also visible by two-dimensional electrophoresis of an isolated chloroplast fraction, and these spots represented a major proportion of the proteins showing senescence related declines in abundance. Chloroplast proteins that were identified by matrix-assisted laser desorption/ionization-time of flight mass fingerprinting included rubisco large and small subunits, a rubisco activase and the 33 kDa protein of the photosystem II oxygen-evolving complex. These proteins declined in abundance late in senescence, indicating that the photosynthetic apparatus was being degraded. A chloroplast glutamine synthetase showed partial decline in abundance during late senescence but was maintained at levels that may support provision of glutamine for export to other tissues. The results emphasise the importance of proteolysis, chloroplast degradation and remobilisation of nitrogen in leaf senescence.     

三种酰胺类新农药对水稻孕穗期稻纵卷叶螟的防效试验

单季杂交晚稻孕穗期稻纵卷叶螟Cnaphalocrocis medinalis Guenée发生不整齐,为害重,在主治药剂氟虫腈即将禁用之际,急需防治高龄幼虫的长效药剂,酰胺类农药是满足这一条件的新一代农药。3种酰胺类农药试验结果,在单季稻孕穗期防治稻纵卷叶螟,20%氟虫双酰胺(WDG)150g/hm2处理速效性与持效性表现最好,药后3d防效达90.3%,药后15d防效高达96.2%;20%氯虫苯甲酰胺(SC)150mL/hm2处理速效性略低于氟虫双酰胺,持效接近,药后3d、15d防效分别达75.2%、91.2%;40%氯虫·噻虫嗪(WDG)120mL/hm2处理速效介于氟虫双酰胺和氯虫苯甲酰胺之间,持效略低于前二者,药后3d、15d防效分别为83.2%、88.3%。对照药剂5%氟虫腈(SC)750mL/hm2和90%杀虫单3000g/hm2处理药后3d、15d的防效均低于上述3种酰胺类新药剂。保叶效果以氯虫苯甲酰胺、氟虫双酰胺处理最高,药后15d保叶率分别为83.6%和85.0%,二者无显著差异,其次为氯虫·噻虫嗪处理为59.6%,3种酰胺类新药剂保叶效果均显著高于对照药剂氟虫腈和杀虫单。结果表明,3种酰胺类农药孕穗期防治稻纵卷叶螟的药效、持效性和保叶效果均高于当前主治药剂氟虫腈、杀虫单,可在生产上推广应用。     

稻纵卷叶螟和白背飞虱序列危害及间隔天数对水稻生理生化的影响

【目的】为了解稻纵卷叶螟Cnaphalocrocis medinalis和白背飞虱Sogatella furcifera的复合危害对水稻产量相关因子和相关酶类的影响。【方法】本文设定两种害虫的先后危害顺序,并通过调整两者开始危害的时间,研究了受害后水稻在灌浆期根、茎和叶片中淀粉和蔗糖含量的变化以及蔗糖合成酶(Sucrose synthase,SS)和蔗糖磷酸合成酶(Sucrose phosphate synthase,SPS)活性的变化。【结果】随着接虫量的增加,水稻不同组织内各生理指标与对照相比均显著下降。随着间隔天数的增加,先接白背飞虱为害要重于先接稻纵卷叶螟。比如叶片中蔗糖含量在先接稻纵卷叶螟的处理中随着间隔天数的增加显著增加,相应的SPS活性显著增加,间隔24 d的处理显著高于间隔6 d和12 d;而先接白背飞虱的处理中蔗糖含量与SPS活性均显著降低,间隔24 d的处理显著低于间隔6 d和12 d的处理。茎部淀粉含量在先接稻纵卷叶螟的处理中随着间隔天数的增加逐渐增加,SS活性显著增加,而先接白背飞虱的处理中淀粉含量和SS活性均显著降低;叶片中淀粉含量均随着间隔天数的增加逐渐降低,而相应的SS活性在先接稻纵卷叶螟的处理中显著增加,在先接白背飞虱的处理中相反。另外,接虫量和间隔天数间有显著的交互作用。【结论】本研究对指导水稻生产中的"两迁害虫"防治具有潜在应用价值。     

Purification and assay of rubisco activase from leaves

Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase protein was purified from spinach leaves by ammonium sulfate precipitation and ion exchange fast protein liquid chromatography. This resulted in 48-fold purification with 70% recovery of activity and yielded up to 18 milligrams of rubisco activase protein from 100 grams of leaves. Based on these figures, the protein comprised approximately 2% by weight of soluble protein in spinach (Spinacia oleracea L.) leaves. The preparations were at least 95% pure and were stable when frozen in liquid nitrogen. Addition of ATP during purification and storage was necessary to maintain activity. Assay of rubisco activase was based on its ability to promote activation of rubisco in the presence of ribulose-1,5-bisphosphate. There was an absolute requirement for ATP which could not be replaced by other nucleoside phosphates. The initial rate of increase of rubisco activity and the final rubisco specific activity achieved were both dependent on the concentration of rubisco activase. The initial rate was directly proportional to the rubisco activase concentration and was used as the basis of activity. The rate of activation of rubisco was also dependent on the rubisco concentration, suggesting that the activation process is a second order reaction dependent on the concentrations of both rubisco and rubisco activase. It is suggested that deactivation of rubisco occurs simultaneously with rubisco activase-mediated activation, and that rubisco activation state represents a dynamic equilibrium between these two processes.     

Rubisco Activase Activity in Spinach Leaf Extracts

Rubisco activase is a chloroplast stromal protein that catalyzesthe activation of ribulose-1,5- bisphosphate carboxylase/oxygenase(rubisco) in vivo. Activation must occur before rubisco cancatalyze the photosynthetic assimilation of CO₂. In leaves,photosynthesis and rubisco activation increase with increasinglight intensity. Techniques are described that allow the activityof rubisco activase to be measured in extracts of spinach (Spinaceaoleracea L.) leaf tissue. In this context, rubisco activaseactivity is defined as the ability to promote activation ofthe inactive ribulose-1,5- bisphosphate-bound rubisco in anATP-dependent reaction. Determination of rubisco activase activityin extracts of dark and light treated leaf tissue revealed thatthe activation state of rubisco activase was independent oflight intensity. ¹Present address: Department of Biological Sciences, 213 Carson-TaylorHall, Louisiana Tech University, Ruston, Louisiana 71272, U.S.A.     

A new form of crystalline rubisco and the conversion to its common dodecahedral form

In this paper, we present a new purification procedure that yields a new crystalline form of rubisco and has enabled us to completely remove this most abundant protein from tobacco leaf extract. The crystals formed within 48 h after refrigeration at 4 degrees C at pH 5.6. However, these crystals were not well-ordered crystals and lacked well-defined facets or edges. The remaining leaf extract (fraction 2 protein) was void of rubisco. Conversion of this new crystalline form of rubisco to its common dodecahedral form was achieved by dialysing the protein solution in Tris buffer at pH 8.0 or purified water. Since the molecular size of its large subunit of rubisco (55 kD) is similar to that of the papillomavirus capsid protein, L1 (57 kD), its complete removal from fraction 2-protein may facilitate the detection, purification, and recovery of the Li protein.     

吉林省47年育成的水稻品种根系伤流液重量变化及其与剑叶光合速率的关系

为了解不同年代水稻品种根系活力的变化及其与叶片光合的关系,以根系伤流液重量作为根系活力指标,研究了吉林省1958-2005年间育成的33个水稻品种抽穗后根系伤流液重量变化及其与剑叶净光合速率的关系。2--~-的研究结果表明,47年的遗传改良导致了水稻品种根系伤流液重量增加,根系伤流液重量与品种的育成年份呈显著正相关,与剑叶光合速率也呈显著正相关。抽穗后根系伤流液重量可以作为剑叶光合能力和高产的参考指标。     

植物蛋白质组学研究若干重要进展

植物蛋白质组学近年来正从定性向精确定量蛋白质组学的方向发展。国际上近两年发表的约160篇研究论文报道了利用不断改进的双向电泳结合生物质谱技术、多维蛋白质鉴定技术,以及包括双向荧光差异凝胶电泳、幅N体内代谢标记、同位素标记的亲和标签、同位素标记相对和绝对定量等在内的第2代蛋白质组学技术,对植物组织（器官）与细胞器、植物发育过程和植物响应环境胁迫的蛋白质组特征,以及植物蛋白质翻译后修饰和蛋白质相互作用等方面的研究成果。该文对上述报道进行总结,综述了2007年以来植物蛋白质组学若干重要问题研究的新进展。