几种因子对离体豌豆叶绿体蛋白质合成的影响

利用制备的豌豆完整叶绿体研究了离体条件下蛋白质合成的条件。结果表明：叶绿体蛋白质合成的饱和光强为４５０μｍｏｌ ｍ＾－２ｓ＾－１,合成的速率在最初５ｍｉｎ内最大,此后随时间延长而合成速率下降;Ｋ＾＋对蛋白质合成有促进作用,其最适浓度为３０￣４０ｍｍｏｌ／Ｌ,进一步增加浓度其促进使用反而降低;Ｍｇ＾２＋在１ｍｍｏｌ／Ｌ以下对蛋白质合成有轻微的促进作用,当浓度超过１．５ｍｍｏｌ／Ｌ则开始产生明显的抑制     

高温对小麦叶绿体核糖体和叶绿体蛋白质生物合成的影响

本实验用蔗糖密度梯度离心分离小麦叶片的核糖体,用 SDS-聚丙烯酰胺凝胶电泳分离叶绿体蛋白质。对在高温和常温条件下生长的小麦分析比较表明:在34℃高温下,小麦叶片能正常地形成细胞质的80S 核糖体,而影响了叶绿体的70S 核糖体的形成,从而使由叶绿体基因组控制的蛋白质的生物合成受阻。由 SDS-凝胶电泳分析表明:高温处理的小麦,其叶绿体蛋白质的电泳条带少于常温下生长的小麦。在这些消失的多肽中,主要是叶绿体基因组的翻译产物,如二磷酸核酮糖羧化酶大亚基。由于叶绿体内这些具有光合生理功能的蛋白质的合成受阻,从而导致小麦叶片光合强度的降低。     

利用CHAPS分离豌豆叶绿体偶联因子复合物

近年来不少工作者对叶绿体偶联因子复合物的结构、功能和发生的问题颇为关注(程秋琛等 1986,Merchant等 1985,Nelson 1982,Pick等1979,Strontman等1983,Suss等 1983)。分离生物膜蛋白质的     

渗调物质和fluridone对离体花生胚蛋白质合成的调节

离体花生胚发育过程中,渗透调节物质提高了胚内源ＡＢＡ含量,促进了贮藏蛋白质花生球蛋白和伴花生球蛋白Ⅱ两个组分的合成,诱导和促进ＬＥＡ蛋白的合成,内源ＡＢＡ合成抑制剂ｆｌｕｒｉｄｏｎｅ抑制了渗调物质的作用。     

高粱离体叶绿体蛋白质合成的研究

高粱叶绿体蛋白质在SDS凝胶电泳上至少可分离出染色程度不同的30多个条带,而具有放射性的条带只有13条。它们大部分属于叶绿体膜结构蛋白。可溶性蛋白质中只有两个具有放射性的条带。其中一个57000道尔顿的多肽是二磷酸核酮塘羧化酶大亚基。该酶的小亚基只在染色图谱中显示而无放射性。说明小亚基是由核基因编码合成的。该酶是受叶绿体和核基因联合控制的产物。在膜蛋白中32000道尔顿的多肽只在成熟叶绿体中出现,在黄化苗的质体中未发现这个多肽。它可能是叶绿体DNA上“光基因32”的产物。     

叶绿体蛋白质组研究进展

亚细胞蛋白质组学是近年来蛋白组学研究中的一个热点。通过细胞器的纯化和亚细胞组分的分离,降低了样品的复杂性,增大了相应蛋白质组分的富集,有利于由此分离获得的蛋白质的序列分析及功能鉴定。叶绿体蛋白质组为植物亚细胞蛋白质组学研究中相对全面的一部分,利用亚细胞分离结合双向电泳技术系统地鉴定叶绿体中蛋白质组分是获取叶绿体蛋白质信息、确定其功能的重要技术手段。本文就近年来植物叶绿体蛋白质组涵盖的叶绿体内、外被膜、叶绿体基质、类囊体膜和类囊体腔蛋白的研究进行综述,以全面认识叶绿体蛋白的组成、特点及其在叶绿体生理生化代谢网络中的作用。     

蛋白质向叶绿体的转运

对近年来叶绿体蛋白质前导肽序列、叶绿体被膜中的蛋白质转运器、监护蛋白在蛋白转运过程中的作用、蛋白质导入叶绿体的途径、前体蛋白的加工的研究进展进行了介绍和评述     

铈对黄瓜叶绿体叶绿素蛋白质复合物形成的影响

黄瓜（Ｃｕｃｕｍ ｉｓｓａｔｉｖｕｓＬ．）叶片叶绿体中铈（Ｃｅ）含量随Ｈｏａｇｌａｎｄ 培养液中ＣｅＣｌ３ 浓度的增加而增加。Ｃｅ 对黄瓜叶片Ｃｈｌａ／ｂ 比值的影响与光强度有关,当植株生长在强光下,对照和处理叶片的Ｃｈｌａ／ｂ 比值均为３．０７;但在弱光下对照叶片的Ｃｈｌａ／ｂ 比值为２．７２,而处理叶片为２．８６。这说明只有在弱光下Ｃｅ才对叶片色素的组分有影响,Ｃｅ 使叶片中的Ｃｈｌｂ 略有下降。Ｃｅ能促进叶绿体光系统Ⅰ叶绿素蛋白质复合物及１１０ ｋＤ多肽的形成,并使捕光叶绿素ａ／ｂ 蛋白质复合物及其２７ ｋＤ多肽的含量减少。     

低渗膨胀对菠菜完整叶绿体光合作用的影响

菠菜离体完整叶绿体需要合适的介质渗透压（约０．９ＭＰａ）以保持其较高的光合作用速率。当渗透压因降低介质中山梨醇浓度（从０．３３ｍｏｌ／Ｌ至０．１７ｍｏｌ／Ｌ）而降低时,叶绿体的完整率保持不变。低于临界渗透压（约０．５ＭＰａ）,叶绿体被膜就发生破裂．并丧失ＣＯ２同化能力。在轻度低渗条件下,虽然叶绿体被膜未破,但依赖ＣＯ２的放氧速率已受抑制。渗透压在０．９ＭＰａ与０．５ＭＰａ之间,叶绿体依赖ＰＧＡ的放氧抑制,可由加入山梨醇至正常浓度（０．３３ｍｏｌ／Ｌ）而解除。膨涨叶绿体的ＡＴＰ合成水平与正常叶绿体相同,而ＮＡＤＰＨ形成速率则明显降低。利用能透过被膜的不同电子受体ＮＣ２、ＰＧＡ和ＯＡＡ发现,在膨胀叶绿体中,ＮＯ２的还原不受形响,而ＰＧＡ及ＯＡＡ的还原明显被抑制。我们推测,低渗膨胀叶绿体中光合作用的抑制,至少有一个原因是Ｆｄ－ＮＡＤＰ氧化还原酶作用的受阻。     

光周期敏感核不育水稻叶绿体的特异性蛋白质

用双向聚丙烯酰胺凝胶电泳技术,将光周期敏感核不育水稻“农垦58S”和其对照品种“农垦58”苗期及育性转换光周期敏感期的叶绿体蛋白质分离为大约90个蛋白质点。“农垦58S”的叶绿体内有一个45kD(pI_(6.7))和一个61kD(pI_(6.0))的特异性蛋白质点,而“农垦58”没有。“农垦58S”的另一个61kD(pI_(6.2))蛋白质点的含量明显高于“农垦58”。不同光周期(长日照和短日照)处理不影响光敏感期的这种叶绿体蛋白质的差异。     

Circadian Rhythm of Total Protein Synthesis in the Cytoplasm and Chloroplasts of Gonyaulaxpolyedra

Protein synthesis of Gonyaulax polyedra was analyzed by means of electron microscopic autoradiographs under constant conditions at different times of the 24-hr cycle. Circadian rhythmic changes in the synthesis rate of total protein were determined in the cytoplasm and chloroplasts of growing cells. Three independent series of experiments in constant light showed a maximum of grains per unit area during the 'subjective' dark phase (=phase that corresponds to the dark phase during a 12:12 hr LD cycle) in both compartments. Minimum and maximum grain number are different by a factor of 5-10. The maximum of total protein synthesis coincided with the maximum phase shift by cycloheximide pulses (1) suggesting protein species within the total pool involved in the mechanism of the circadian clock. A similar rhythm of lower amplitude was observed in the mitochondria, but this rhythm cannot with certainty be attributed to these organelles. In a slowly growing culture a rhythm of total protein synthesis was observed that showed a smaller amplitude and a different phasing.     

拟南芥DG1参与叶绿体早期光合蛋白合成功能

拟南芥中已有466个PPR蛋白,已有研究证实许多PPR蛋白参与细胞器基因表达的转录后调节,但大部分PPR蛋白分子作用机制尚不清楚.Delayed greening 1(DG1)是定位于叶绿体中的的PPR蛋白,研究结果证实该蛋白是通过与SIG6因子相互作用降低PEP转录活性从而影响叶绿体早期发育.本研究利用拟南芥Dg1基因功能缺陷型突变体研究了DG1蛋白对光系统蛋白复合体组成及其光转化效率的影响.77K荧光发射光谱分析发现dg1突变体幼叶PSII中电子传递速度明显低于野生型,而成熟叶片与野生型基本一致;蓝绿温和胶分析结果表明:相对于野生型在dg1突变体新生叶中PSII、PS玉及其超聚复合物含量均有不同程度降低;进一步温和胶二向电泳及蛋白免疫印迹分析显示,在dg1突变体新生叶中,由叶绿体编码的光系统蛋白复合物组成亚基含量显著降低,而核编码复合物组成亚基含量与野生型相比没有明显区别.上述实验结果进一步确定了DG1蛋白是通过调控叶绿体编码基因的表达进而调节光系统复合物的生物合成与组装,最终影响拟南芥叶绿体早期发育.因此,我们认为DG1蛋白对于叶绿体发育早期光合蛋白的合成是必需的.     

Protein Synthesis in Isolated Plastids during Chloroplast Development in Wheat

Light-driven protein synthesis in isolated plastids was studiedduring the greening of etiolated wheat (Triticum aestivum L.)seedlings. The process was divided into five phases (I to V)according to the recovery of plastids from the leaf tissue.The activity was not detected in the etioplasts, but rapidlyincreased to the maximum level in phase I and remained at thislevel through phase II. During the transition from phase IIto III, the activity rapidly decreased to one-third and thencontinued to decrease slowly. The plastid polypeptides synthesizedduring the greening were analyzed by SDS-polyacrylamide gelelectrophoresis. In phase I, membrane polypeptides having molecularweights of about 21k were synthesized, while 23 k membrane polypeptidewas synthesized in phases III, IV and V. Synthesis of solublepolypeptides of 50–60 k and membrane polypeptides of 15k and 30–35 k was active in phases I and II, but decreasedbetween phases II and III. (Received October 31, 1983; Accepted May 14, 1984)     

In vivo Studies on the Roles of Tic55-Related Proteins in Chloroplast Protein Import in Arabidopsis thaliana

The TicS5 （Translocon at the inner envelope membrane of chloroplasts, 55 kDa） protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthetic machinery. Two Tic55 homologs have been proposed to exist in Arabidopsis： atTic55-11 and AtPTC52 （Protochlorophyllide-dependent Trans- Iocon Component, 52 kDa; has also been called atTic55-1V）. Our phylogenetic analysis shows that attic55-11 is an ortholog of psTic55 from pea （Pisum sativurn）, and that AtPTC52 is a more distant homolog of the two. AtPTC52 was included in this study to rule out possible functional links between the proteins in Arabidopsis. No detectable mutant phenotypes were found in two independent T-DNA knockout mutant plant lines for each Arabidopsis protein, when compared with wild- type： visible appearance, chlorophyll content, photosynthetic performance, and chloroplast protein import, for example, were all normal. Both wild-type and tic55-11 mutant chloroplasts exhibited deficient protein import when treated with diethylpyrocarbonate, indicating that Tic55 is not the sole target of this reagent in relation to protein import. Furthermore, ptc52 mutant chloroplasts were not defective with respect to pPORA import, which was previously reported to involve PTC52 in barley. Thus, we conclude that atTic55-11 and AtPTC52 are not strictly required for functional protein import in Arabidopsis.     

AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery

AtToc159 is a GTP-binding chloroplast protein import receptor. In vivo, atToc159 is required for massive accumulation of photosynthetic proteins during chloroplast biogenesis. Yet, in mutants lacking atToc159 photosynthetic proteins still accumulate, but at strongly reduced levels whereas non-photosynthetic proteins are imported normally: This suggests a role for the homologues of atToc159 (atToc132, -120 and -90). Here, we show that atToc90 supports accumulation of photosynthetic proteins in plastids, but is not required for import of several constitutive proteins. Part of atToc90 associates with the chloroplast surface in vivo and with the Toc-complex core components (atToc75 and atToc33) in vitro suggesting a function in chloroplast protein import similar to that of atToc159. As both proteins specifically contribute to the accumulation of photosynthetic proteins in chloroplasts they may be components of the same import pathway.     

Effects of Polyhydroxyl Alcohols on Protein Synthesis in Brain Slices

Stressors such as tissue slicing, toxic chemicals, and heat shock applied to cultured cells, organ tissues, or whole animals in vivo induce the synthesis of a 71,000-kilodalton stress protein (SP71) that is not normally present in most organ tissues. In the present experiment, an attempt was made to inhibit selectively the synthesis of SP71 in rat brain tissue slices. Of several manipulations to the brain slice incubation medium that were examined, only addition of very high concentrations of certain polyhydroxyl alcohols, i.e., 1.0 M glycerol, selectively inhibited SP71 synthesis. Glycerol also selectively inhibited SP71 synthesis in heat-shocked cerebral microvascular cells in culture but failed to inhibit SP71 synthesis in anesthetized rats in vivo in response to heat shock. The effects of glycerol on SP71 synthesis are discussed in relationship to current hypotheses concerning the function of SP71.     

Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution

In bacteria, membrane proteins are targeted cotranslationally via a signal recognition particle (SRP). During the evolution of higher plant chloroplasts from cyanobacteria, the SRP pathway underwent striking adaptations that enable the posttranslational transport of the abundant light-harvesting chlorophyll-a/b-binding proteins (LHCPs). The conserved 54-kDa SRP subunit in higher plant chloroplasts (cpSRP54) is not bound to an SRP RNA, an essential SRP component in bacteria, but forms a stable heterodimer with the chloroplast-specific cpSRP43. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane whereby cpSRP43 plays a central role. This study shows that the cpSRP system in the green alga Chlamydomonas reinhardtii differs significantly from that of higher plants as cpSRP43 is not complexed to cpSRP54 in Chlamydomonas and cpSRP54 is not involved in LHCP recognition. This divergence is attributed to altered residues within the cpSRP54 tail and the second chromodomain of cpSRP43 that are crucial for the formation of the binding interface in Arabidopsis. These changes are highly conserved among chlorophytes, whereas all land plants contain cpSRP proteins with typical interaction motifs. These data demonstrate that the coevolution of LHCPs and cpSRP43 occurred independently of complex formation with cpSRP54 and that the interaction between cpSRP54 and cpSRP43 evolved later during the transition from chlorophytes to land plants. Furthermore, our data show that in higher plants a heterodimeric form of cpSRP is required for the formation of a low molecular weight transit complex with LHCP.     

Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.     

拟南芥未知功能基因At3g61870编码蛋白的叶绿体定位研究

利用反向遗传学研究方法对1个预测的拟南芥叶绿体未知功能基因At3g61870编码蛋白进行了亚细胞定位研究.通过克隆At3g61870基因5′端长229 bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-CP-TP-GFP,经农杆菌介导转化拟南芥.转基因植株的叶肉细胞经激光共聚焦显微镜观察,叶绿素自发荧光与GFP荧光共定位于叶绿体中.结果表明,未知功能基因At3g61870编码的蛋白质为叶绿体蛋白质.     

Effects of Protein Synthesis on the Maturation of Mouse Oocytes in vitro

卵母细胞成熟过程中伴随有多种蛋白质的合成与磷酸化,蛋白质的合成对卵细胞的成熟具有重要作用。本实验较系统地阐述小鼠卵母细胞体外成熟培养的不同阶段蛋白质合成对卵母细胞成熟的影响。放线菌酮是肽链延伸的抑制因子。将生发泡（ＧＶ）期的卵母细胞分别于Ｔ６成熟培养液中培养０、４、６、９小时后,转至含有１０ｍｇ／ｍｌ放线菌酮的Ｔ６成熟培养液继续培养１２１５小时。固定、染色、观察卵母细胞。结果如Ｔａｂｌｅ１。０小时实验组：抑制处理４小时,其生发泡破裂（ＧＶＢＤ）发生率与对照组无明显差异。表明：卵母细胞ＧＶＢＤ所需蛋白质（如：成熟促进因子ＭＰＦ等）是在卵巢的卵泡卵母细胞生长过程中完成的。４、６小时实验组：笫一极体的释放被完全抑制,卵母细胞不能达到ＭＩ期,染色质处于凝集状态（Ｆｉｇ．３＆４）。表明：ＧＶＢＤＭＩ期间所需蛋白质的合成对卵母细胞ＭＩ期中期纺缍体的形成与维持具有重要作用。９小时实验组：可能由于卵母细胞发育速度存在个体间的差异。没有进入ＭＩＩ期的便停滞于ＭＩ期以前。进入ＭＩ期的则能排出笫一极体。因此,笫一极体的释放总体上呈不完全抑制状态,其释放率低于对照组。但是,后者虽然弪过恢复培养至１５小时,可能由于微管蛋白的合成