水稻Rubisco和RCA的日变化及其细胞定位

采用免疫胶体金标记电镜技术对水稻(Oryza satova subsp.indica cv.浙农952)叶片中的Rubisco及其活化酶(RCA)进行细胞器定位和定量,同时用免疫扩散法进行叶片含量分析,研究了这两种酶含量及活力的日变化。结果表明Rubisco主要分布于叶绿体,RCA分布于叶绿体和线粒体中;光合速率(Pn)、Rubisco初始活力和RCA活力与光合日变化密切相关;在光照最强的13时,出现光合“午休”,叶绿体中Rubisco的密度有一定程度降低,而全叶的总Rubisco保持稳定,Rubisco初始活力也有明显的“午休”,这意味着体内Rubisco的活力除受RCA调节外,可能还与叶绿体中Rubisco的分布有关。RCA活力变化与叶绿体中RCA含量变化较为一致,表明RCA在叶绿体中的分布对调节其本身活力和Rubisco活性有重要作用。     

Rubisco和RCA在青菜叶绿体中的分布及病毒侵染对其细胞定位的影响

运用免疫金标电镜术观察了青菜叶细胞中光合作用关键酶Rubisco和Rubisco活化酶(RCA)的细胞化学定位,结果显示Rubisco和RCA免疫金颗粒主要分布于薄壁组织叶绿体的间质中,在基粒片层上很少,表皮的气孔保卫细胞和维管束薄壁细胞叶绿体内也有分布,在细胞质及线粒体等细胞器中无特异性分布。同时比较观察了感染芜菁花叶病毒(TuMV)的青菜叶绿体Rubisco和RCA免疫金标记结果,发现病组织中结构尚完整的叶绿体Rubisco和RCA标记率略有下降,而结构严重破坏的叶绿体中两种酶标记率分别仅为正常叶绿体的58.44%和64.67%,表明病毒侵染可导致Rubisco和RCA含量下降,影响寄主植物的光合作用。     

小麦RCA的cDNA片断克隆和反义表达载体的构建

目的:试图分离和克隆小麦Rubisco活化酶cDNA片断并构建反义表达载体。方法:采用RT-PCR技术克隆cDNA片断,对序列用Blast等软件进行分析,并将该片断反向连接于植物表达载体pROK2的CaMV35S启动子下游,构建反义表达载体。结果:获得了小麦Rubisco活化酶(RCA)的cDNA片断(GenBank注册号:DQ984669);小麦RCA的cDNA片断推导的氨基酸序列与其它植物的RCA氨基酸序列高度同源。序列比较表明,用本实验所得的cDNA序列推导出的氨基酸序列与GenBank登录的大麦(AAA63163)、南极银须草(AAP83928)、水稻(AAX95414)、玉米(AAC97932)、拟南芥(NP 850321)和烟草(CAA78703)等的RCA序列的同源性分别为97%、95%、88%、83%、82%和82%;分析表明,该序列为新的小麦RCAα的cDNA序列;通过选择和引入合适的酶切位点进行载体构建,构建了小麦RCA的cDNA反义表达载体pR-AntiRCA。结论:构建成了小麦RCA的cDNA反义表达载体。     

大麦和玉米叶片叶绿体中Rubisco及其活化酶的免疫金标记定位

运用免疫金标记电镜技术研究了禾本科C3植物大麦(Hordeum vulgare L.)和C4植物玉米(Zea mays L.)叶片中Rubisoo及其活化酶(RCA)的细胞定位,结果表明:两种植物叶片解剖结构及叶绿体超微结构差别明显.在大麦叶细胞中,只有一种叶肉细胞叶绿体,Rubisoo和RCA主要分布于叶绿体的间质中.在玉米叶细胞中,存在着维管束鞘细胞和叶肉细胞两种类型叶绿体,Rubisco主要分布于鞘细胞叶绿体的基质中,但在叶肉细胞叶绿体中亦有少量特异性标记;RCA在鞘细胞叶绿体和叶肉细胞叶绿体的基质中都有分布.两种植物叶绿体结构及光合作用关键酶定位的不同,体现了C3植物和C4植物在光合器结构与功能上的差异.     

Rubisco活化酶及其对Rubisco的调节作用

介绍了Robisco活化酶的发现和分子生物学特性，以及活化酶对光合作用中的关键酶──Ru－bisco的调节机制，这种调节主要是通过减轻磷酸糖的抑制作用实现的。     

低温弱光对黄瓜幼苗Rubisco与Rubisco活化酶的影响

以‘津优3号'黄瓜幼苗为试材,研究弱光(100 μmol·m-2·s-1)下适温(WL:25℃/18℃)、亚适温(ST+WL:18℃/12℃)和低温(LT+WL:10℃/5℃)对黄瓜幼苗光合速率(Pn)、核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)、Rubisco活化酶(RCA)活性及其基因表达量的影响.结果表明:与对照(25℃/18℃,400 μmol·m-2·s-1)相比,WL、ST+WL和LT+WL处理的单株叶面积和干物质量均明显减小.处理初期,Pn、Rubisco活性及其大亚基基因(rbcL)、小亚基基因(rbcS)表达、RCA活性与基因(CsRCA)表达量大幅度降低,5～7 d后,WL处理趋于平稳,ST+WL处理缓慢回升,而LT+WL处理持续下降,表明黄瓜光合机构对适温弱光和亚适温弱光环境有逐步适应机制.Rubisco和RCA活性及其基因表达对低温弱光的响应与Pn基本一致,表明低温弱光下RCA和Rubisco活性及其基因表达量下降是黄瓜幼苗Pn降低的重要原因.     

水稻叶片Rubisco 活化酶表达的昼夜变化

在 48h的自然光照、连续光照和连续黑暗处理下 ,水稻幼苗rcamRNA的含量均表现出昼夜节奏特性 ,其中以自然光周期中变化最为明显。在光暗交替的条件下 ,RCA含量虽也表现出明显的昼夜变化 ,但连续黑暗处理其含量持续下降 ,连续光照则其含量先上升然后下降 ,说明它不发生节昼现象。这些结果表明RCA表达在转录和翻译水平上的调控机制不同 ,转录既由光暗交替控制又受内生节奏调节 ,而翻译则更大程度上由光调节     

光和糖对水稻Rubisco活化酶基因表达的影响

水稻黄化苗在光照２ｈ内其Ｒｕｂｉｓｃｏ活化酶的ｍＲＮＡ物收白量明显增加,然后维持在相对 水平。光对水稻Ｒｕｂｉｓｃｏ活化酶的基因表达的诱导作用主要在转录水平上。Ｒｕｂｉｓｃｏ活化酶主要在绿叶中表达,这与Ｒｕｂｉｓｃｏ基因表达的器官特异性完全一致。用等渗葡萄糖喂养成熟的水稻叶片１ｈ,促使水稻Ｒｏｕｂｉｓｃｏｄｄ 、ｉｈ ｇｏｇ ａｄ ｔ Ｒｕｂｉｓｃｏ活化酶可ｍＲＮＡ含量下降同样蔗糖对Ｒ７ｕｂｉｓｃ     

水稻转绿型白化突变系W25转绿过程中Rubisco、Rubisco活化酶活性与光合速率的变化

水稻 (OryzasativaL .)转绿型白化突变系W2 5在转绿过程中叶绿素、可溶性蛋白质和Rubisco含量的动态变化过程表明 ,白化突变体内叶绿素、可溶性蛋白质和Rubisco含量极低 ,随着转绿过程各组分含量迅速提高 ,转绿至第 30天时超过野生种 2 177s;Rubisco初始活力与Rubisco活化酶含量呈极显著正相关。Rubisco活化酶基因表达的研究结果表明 ,突变体的Rubisco活化酶表达高于野生种 2 177s。在转绿过程中 ,Rubisco活化酶含量的提高要先于Rubisco和光合速率     

Heat sensitivity of Rubisco,Rubisco activase and Rubisco binding protein in higher plants

During the past few years the investigations concerning Rubisco and the changes of its activity and properties at elevated temperature were reconsidered with special reference to the important role of Rubisco activase and Rubisco binding protein. The major changes in Rubisco, Rubisco activase and Rubisco binding protein reported recently are presented in this review. New information on these proteins, including their changes under heat stress conditions, is discussed together with open questions.     

小麦Rubisco活化酶基因的克隆和表达特性

Rubisco活化酶是广泛存在于光合生物中调节Rubisco活性的酶,我们利用PCR技术,从小麦(Triticum aestivum)叶片cDNA文库中克隆得到Rubisco活化酶基因cDNA片段,该片段长度为850 bp,编码201个氨基酸.Northern blot表明,小麦叶片在暗诱导衰老的条件下,叶片中活化酶基因表达水平逐渐下降;同时,小麦叶片的光合特性、叶绿素含量和Rubisco活性呈现下降趋势.这些结果表明,衰老时小麦叶片Rubisco活化酶基因表达水平下降与光合速率下降密切相关.     

Antisense inhibition of Rubisco activase increases Rubisco content and alters the proportion of Rubisco activase in stroma and thylakoids in chloroplasts of rice leaves

BACKGROUND AND AIMS: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) is a nuclear-encoded chloroplast protein that modifies the conformation of Rubisco, releases inhibitors from active sites, and increases enzymatic activity. It appears to have other functions, e.g. in gibberellin signalling and as a molecular chaperone, which are related to its distribution within the chloroplast. The aim of this research was to resolve uncertainty about the localization of RCA, and to determine whether the distributions of Rubisco and RCA were altered when RCA content was reduced. The monocotyledon, Oryza sativa was used as a model species. METHODS: Gas exchange and Rubisco were measured, and the sub-cellular locations of Rubisco and RCA were determined using immunogold-labelling electron microscopy, in wild-type and antisense rca rice plants. KEY RESULTS: In antisense rca plants, net photosynthetic rate and the initial Rubisco activity decreased much less than RCA content. Immunocytolocalization showed that Rubisco in wild-type and antisense plants was localized in the stroma of chloroplasts. However, the amount of Rubisco in the antisense rca plants was greater than in the wild-type plants. RCA was detected in both the chloroplast stroma and in the thylakoid membranes of wild-type plants. The percentage of RCA labelling in the thylakoid membrane was shown to be substantially decreased, while the fraction in the stroma was increased, by the antisense rca treatment. CONCLUSIONS: From the changes in RCA distribution and alterations in Rubisco activity, RCA in the stroma of the chloroplast probably contributes to the activation of Rubisco, and RCA in thylakoids compensates for the reduction of RCA in the stroma, allowing steady-state photosynthesis to be maintained when RCA is depleted. RCA may also have a second role in protecting membranes against environmental stresses as a chaperone.     

Changes in protein quantities of phosphoenolpyruvate carboxylase and Rubisco activase in various wheat genotypes

In early seedlings of wheat genotypes two isoforms of Rubisco activase with molecular weights of 42 and 46 kDa are expressed. Amounts of both isoforms significantly increase in early seedlings of the durum wheat genotype Barakatli-95 exposed to salt stress. But at the beginning of the tillering stage, the changes in quantities of both RCA isoforms are different in durum and bread wheat genotypes subjected to a 3-day drought stress. In the leaves of the early seedlings of the studied wheat genotypes exposed to drought stress quantities of PEPC subunits increase compared to the control but they remain relatively stable in early roots and germinating seeds. However, quantities of its subunits decrease sharply in roots and germinating seeds of early seedlings under the influence of 100 mM NaCl. In flag leaves and ear elements of the Barakatli-95 genotype grown under normal water supply conditions protein quantities of PEPC subunits change differently depending on time. Changes in protein quantities of RCA, PEPC and Rubisco enzymes have been studied comparatively in ear elements and flag leaves after the fourth day of anthesis.     

光和糖对水稻Rubisco活化酶基因表达的影响

水稻黄化苗在光照2h内其Rubisco。活化酶的mRNA和蛋白量明显增加,然后维持在相对稳定的水平。光对水稻Rubisco活化酶的基因表达的诱导作用主要在转录水平上。Rubisco活化酶主要在绿叶中表达,这与Rubisco基因表达的器官特异性完全一致。用等渗葡萄糖喂养成熟的水稻叶片1h,促使水稻Rubisco大、小亚基和Rubisco活化酶可翻译mRNA含量下降。同样蔗糖对Rubisco小亚基和Rubisco活化酶的表达也有抑制,其作用弱于葡萄糖。     

烟草Rubisco活化酶的纯化及其特性

利用35％饱和硫酸铵分部、DEAE－Sephacel和FPIC－MonoQ柱层析等步骤从烟草叶片中纯化了Rubisco活化酶,并制备了其专一性抗体。此法不仅快速,而且比活力高。以往认为菠菜和拟南芥Rubisco活化酶由两种亚基组成。通过快速制备的粗提液分析．发现烟草Rubisco活化酶由一种42kD的亚基组成。即使在有多种蛋白酶抑制剂存在的情况下,此亚基仍很易降解为39kD的亚基。ATP不仅对酶的活性所必需,而且也有利于维持酶的稳定性。该酶的热稳定性远比Rubisco差。