菲白竹组培苗白化、绿化突变体的超微结构及15个叶绿体编码基因的表达

以菲白竹（Pleioblastus fortunei）组培苗绿化突变体及经60Coγ射线辐射获得的白化突变体为实验材料,从生长特性、超微结构及15个叶绿体编码基因的拷贝数及转录水平方面进行了研究。结果显示,白化突变体具有分化不定芽和不定根的能力,其叶肉细胞中叶绿体缺失或结构不完整,基因拷贝数与野生型无明显差异,部分基因转录水平低于野生型,psbA基因的转录水平高于野生型。绿化突变体叶色一致且能稳定遗传,与野生型绿色组织细胞的超微结构相比,其叶绿体基粒片层数量少,结构松散,基因拷贝数低或相当于野生型,多数叶绿体编码基因转录水平较野生型呈下调趋势,atpI基因转录水平高于野生型。psaA基因在两种突变体中均不表达,仅在野生型中有微弱表达。     

小麦叶绿体蛋白质编码基因RNA编辑位点的测定及与返白现象的关系

RNA编辑是一种转录后基因加工修饰现象,广泛存在于高等植物细胞器中。已有研究表明,RNA编辑与植物发生白化或者黄化有关。通过PCR、RT-PCR及测序的方法,对具有阶段性白化特性的小麦（Triticum aestivum）返白系FA85及其野生型矮变一号（Aibian 1）的叶绿体蛋白质编码基因RNA编辑位点进行了测定,在14个基因上发现了26个编辑位点。有5个编辑位点在2个株系之间存在编辑效率的差异,且这些差异的位点均位于编码叶绿体RNA聚合酶的基因上,其中3个位点编辑前后对应的蛋白质二级结构可能有差异。对2个株系叶绿体中PEP、NEP及PEP、NEP共同依赖基因转录水平的检测显示,除psbA和clpP外,其它基因在小麦返白系中的转录水平均有不同程度的下降。这种转录水平的显著下降及叶绿体RNA聚合酶基因上RNA编辑位点编辑效率的改变,可能与小麦返白系叶片的返白有关。     

叶绿素缺乏油菜突变体的LHCⅡ多肽组成、蛋白含量与cab基因转录研究

与野生型油菜相比．叶绿素缺乏油菜突变体Cr3529 LHC Ⅱ多肽组成并未发生改变,但其含量却都明显降低。RNA印迹及点杂交结果都显示出Cr3529cab基因的转录增加。这些结果表明：该叶绿素缺乏突变体仅影响LHCI多肽的蛋白含量;并未影响其组成;突变体LHC Ⅱ蛋白量的减少并非cab基因转录降低所致。可见转录水平的调节仅对LHC Ⅱ在类囊体膜上的积累起有限作用;cab核编码基因转录活性的维持和质体信号的产生并不要求有结构完整的叶绿体的存在。     

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.     

拟南芥Psrp-4基因参与光合作用机制的研究

叶绿体核糖体是植物特有的细胞器之一,其主要功能是合成质体基因编码的蛋白质。已有研究表明在叶绿体核糖体内含有6个质体特有蛋白PSRP(plastid-specific ribosomal protein),分别命名为PSRP1~PSRP6。然而,这些蛋白在叶绿体蛋白合成过程以及光合作用中的作用机制研究尚处初级阶段。在本研究中,为了阐明PSRP-4蛋白在叶绿体发育过程中的作用机制,我们利用Gateway系统构建了Psrp-4基因(At2g38140)的RNAi表达载体,转化野生型拟南芥后获得了Psrp-4基因表达量明显降低的psrp-4突变体。研究结果表明:psrp-4突变体比野生型生长略微缓慢,但叶片颜色与野生型差别不大,能够进行正常的光合作用。在高光胁迫条件下,测定psrp-4突变体光合化学效率,发现与野生型差异不明显;进一步的蛋白免疫印记实验证明Psrp-4基因表达量的降低对PSⅡ反应中心D1蛋白的周转也没有明显影响。因此,推测PSRP-4蛋白可能不是叶绿体蛋白合成以及光合作用的正常进行所必需的。     

甘蓝型油菜角果特异白化种质的遗传和生理特性

本研究对在甘蓝型油菜中发现的一种新型角果特异白化种质的形态特征、遗传及生理特性进行了研究,结果表明:该种质具经济器官特异性白化的特点,即营养器官正常,生殖生长阶段所有的经济器官如花蕾、花、角果、种子均表现为白化突变。白化株结实性差,纯合白化种子未发育成熟就干瘪萎缩。通过对该角果特异白化株与野生型油菜杂交创建的分离群体分离比进行统计,表明该性状为1对核基因控制的不完全显性突变。纯合白化株系中几乎没有叶绿素和类胡萝卜素,但有少量生育酚的合成,光合速率显著低于野生型或接近于零,超微结构观察也未发现有完整叶绿体结构。结合已有研究报道,初步推断该特异白化种质为类胡萝卜素合成调控基因或决定叶绿体发育基因发生突变所引起。因角果是油菜最重要的光合器官,对油菜产量形成起着至关重要的作用,本研究为进一步揭示叶绿体发育以及角果光合作用形成的分子生理奠定了基础。     

定位于类囊体膜的PPF1在转基因拟南芥中的表达影响叶绿体的发育

PPF1是一个与植物营养生长相关的基因。它编码的产物可能是一个膜蛋白并与拟南芥叶绿体中的类囊体蛋白ALB3有很高的同源性。免疫电镜分析表明PPF1蛋白同样主要定位于类囊体膜 ,而且在短日照G2豌豆开花两周后仍发育良好的叶绿体中有很高的表达 ,在长日照豌豆同时期非正常叶绿体中丰度非常低。对转基因拟南芥和野生型植株的叶片衰老进程比较发现 ,PPF1在拟南芥中的过量表达可以延缓叶片的衰老 ,而用PPF1反义mRNA抑制拟南芥中的同源基因ALB3则明显加快叶片衰老速度。对转基因拟南芥的超微结构分析显示 ,PPF1在拟南芥中过量表达时 ,转基因植株的叶绿体比野生型植株的叶绿体大并含有更多的基粒和基质类囊体膜 ;相反 ,反义PPF1表达抑制其在拟南芥中的同源物时 ,转基因植株的叶绿体比野生型植株的叶绿体小并含有较少的基粒和发育较差的类囊体膜系统。这些数据表明叶绿体的发育状况与PPF1或拟南芥同源物ALB3的表达水平呈正相关。我们的结果提示PPF1基因可能通过控制叶绿体的发育状况来调节植物的发育。     

定位于类囊体膜的PPF1在转基因拟南芥中的表达影响叶绿体的发育

PPF1是一个与植物营养生长相关的基因.它编码的产物可能是一个膜蛋白并与拟南芥叶绿体中的类囊体蛋白ALB3有很高的同源性.免疫电镜分析表明PPF1蛋白同样主要定位于类囊体膜,而且在短日照G2豌豆开花两周后仍发育良好的叶绿体中有很高的表达,在长日照豌豆同时期非正常叶绿体中丰度非常低.对转基因拟南芥和野生型植株的叶片衰老进程比较发现, PPF1在拟南芥中的过量表达可以延缓叶片的衰老,而用PPF1反义mRNA抑制拟南芥中的同源基因ALB3则明显加快叶片衰老速度.对转基因拟南芥的超微结构分析显示,PPF1在拟南芥中过量表达时,转基因植株的叶绿体比野生型植株的叶绿体大并含有更多的基粒和基质类囊体膜;相反,反义PPF1表达抑制其在拟南芥中的同源物时,转基因植株的叶绿体比野生型植株的叶绿体小并含有较少的基粒和发育较差的类囊体膜系统.这些数据表明叶绿体的发育状况与PPF1或拟南芥同源物ALB3的表达水平呈正相关.我们的结果提示PPF1基因可能通过控制叶绿体的发育状况来调节植物的发育.     

黄瓜矮生突变体C1056的茎解剖结构及其生理特性分析

该研究以黄瓜矮生突变体C1056和野生型CCMC为材料,对其主要生理特性、叶绿体超微结构以及茎显微结构进行了观察、测定和比较分析,以探讨黄瓜株高调控机理并挖掘新的矮化种质,为黄瓜的矮化育种提供依据。结果显示:(1)突变体C1056的株高较野生型极显著变矮,且叶色加深、叶脉加粗、叶尖内卷、叶片皱缩,但茎粗、节间数与野生型无显著差异,而节间长度极显著低于野生型。(2)茎横切显微结构显示,突变体的维管束数量与野生型无显著差异,但导管直径缩小;纵切结果显示,突变体茎节间细胞长度变短,细胞变小,细胞数目略有补偿。(3)与野生型相比,突变体的叶绿素和类胡萝卜素含量均有不同程度的下降,叶绿素/类胡萝卜素和叶绿素a/b的比值明显增高。(4)突变体叶绿素荧光各参数与野生型相比无明显变化;突变体的净光合速率较野生型降低8%,气孔导度、蒸腾速率较野生型分别提高15%和10%,但差异均不显著,而胞间CO2浓度显著高于野生型。(5)透射电镜观察结果发现,与野生型相比,突变体的叶肉细胞比较小,叶绿体所占细胞面积明增大,且叶绿体形状为半圆形和纺锤形,部分非正常结构的叶绿体的大部分基质、基粒片层未完全分化且不清晰,垛叠不整齐。研究表明,黄瓜矮生突变体C1056的矮化主要因其节间长度缩短以及细胞变小所致,且突变体的叶绿体结构受到一定程度的影响,但并未明显影响其光合能力。     

小麦黄化突变体叶绿体超微结构研究

利用透射电镜对小麦自然黄化突变体及其突变亲本(西农1718)叶片细胞叶绿体的数目、形态及超微结构进行比较分析。结果发现:(1)3种不同黄化程度突变体的叶绿体分布、数目、形状及大小与突变亲本无明显差异;(2)突变体叶绿素含量为野生型58%的黄绿植株与其突变亲本叶绿体超微结构无明显差异,基质类囊体与基粒类囊体高度分化,基粒数目以及基粒片层数目较多;(3)突变体金黄和绿黄植株的叶绿素含量分别为野生型的17%、24%,其叶绿体超微结构与突变亲本明显不同,突变体的叶绿体发育存在明显缺陷,其中突变体金黄植株的叶绿体内无基粒、基质片层清晰可见,有淀粉粒,嗜锇颗粒较多,而突变体绿黄植株的叶绿体内有基粒,但明显少于突变亲本,且基粒片层较少,基质类囊体较发达。结果表明该黄化突变体叶绿体超微结构的改变,是由于叶绿素含量降低造成,推测,该黄化突变是由于叶绿素合成受阻导致的。     

Efficient translation in chloroplasts requires element(s) upstream of the putative ribosome binding site from atpI

Thousands of proteins make up a chloroplast, but fewer than 100 are encoded by the chloroplast genome. Despite this low number, expression of chloroplast-encoded genes is essential for plant survival. Every chloroplast has its own gene expression system with a major regulatory point at the initiation of protein synthesis (translation). In chloroplasts, most protein-encoding genes contain elements resembling the ribosome binding sites (RBS) found in prokaryotes. In vitro, these putative chloroplast ribosome binding sequences vary in their ability to support translation. Here we report results from an investigation into effects of the predicted RBS for the tobacco chloroplast atpI gene on translation in vivo. Two reporter constructs, differing only in their 5'-untranslated regions (5'UTRs) were stably incorporated into tobacco chloroplast genomes and their expression analyzed. One 5'UTR was derived from the wild-type (WT) atpI gene. The second, Holo-substitution (Holo-sub), had nonchloroplast sequence replacing all wild-type nucleotides, except for the putative RBS. The abundance of reporter RNA was the same for both 5'UTRs. However, translation controlled by Holo-sub was less than 4% that controlled by WT. These in vivo experiments support the idea that translation initiation in land plant chloroplasts depends on 5'UTR elements outside the putative RBS.     

Chloroplast genome aberration in micropropagation-derived albino <Emphasis Type="Italic">Bambusa edulis</Emphasis> mutants, <Emphasis Type="Italic">ab1</Emphasis> and <Emphasis Type="Italic">ab2</Emphasis>

Two albino mutants (ab1 and ab2) have been derived from long-term shoot proliferation of Bambusa edulis. Based on transmission electronic microscopy data, the chloroplasts of these mutants were abnormal. To study the mutation of gene regulation in the aberrant chloroplasts, we designed 19 pairs of chloroplast-encoded gene primers for genomic and RT-PCR. Only putative NAD(P)H-quinone oxidoreductase chain 4L (ndhE; DQ908943) and ribosomal protein S7 (rps7; DQ908931) were conserved in both the mutant and wild-type plants. The deletions in the chloroplast genome of these two mutants were different: nine genes were deleted in the chloroplast genomic aberration in ab1 and 11 genes in ab2. The chloroplast genes, NAD(P)H-quinone oxidoreductase chain 4 (ndhD; DQ908944), chloroplast 50S ribosomal protein L14 (rpl14; DQ908934), and ATP synthase beta chain (atpB; DQ908948) were abnormal in both mutants. The gene expressions of 18 of these 20 genes were correlated with their DNA copy number. The two exceptions were: ATP synthase CF0 A chain (atpI; DQ908946), whose expression in both mutants was not reduced even though the copy number was reduced; ribosomal protein S19 (rps19; DQ908949), whose expression was reduced or it was not expressed at all even though there was no difference in genomic copy number between the wild-type and mutant plants. The genomic PCR results showed that chloroplast genome aberrations do occur in multiple shoot proliferation, and this phenomenon may be involved in the generation of albino mutants.     

An Arabidopsis cotyledon-specific albino locus: a possible role in 16S rRNA maturation

We report here the isolation and characterization of a cotyledon-specific albino locus of Arabidopsis, WHITE COTYLEDONS (WCO). This recessive mutation in the WCO locus, located on the top of Chromosome 1, results in albino cotyledons but green true leaves. An accumulation profile of chlorophylls and ultrastructure of chloroplasts indicate that WCO is necessary for development of functional chloroplasts in cotyledons but is dispensable in true leaves. This was further supported by the fact that the mutants request feeding of sucrose for their survival at the early seedling stage where true leaves have not emerged, but the mutants which have developed true leaves are able to grow autotrophically without sucrose supplementation. The wco mutants accumulate low levels of chloroplast mRNA encoding photosynthesis-related proteins and have a specific defect in 16S rRNA maturation in a cotyledon-specific manner. Although wco mutants exhibited abnormal chloroplasts and chloroplast gene expression in cotyledons, nuclear genes for photosynthetic components are expressed at similar levels to those found in wild-type siblings. This lack of suppression of the nuclear genes is not due to a defect in the signaling of the so-called "plastid factor" to the nucleus since normal suppression of the nuclear genes was observed in response to the photo-oxidative damage due to norflurazon application.     

Characterization of Arabidopsis glutamine phosphoribosyl pyrophosphate amidotransferase-deficient mutants

Using a transgene-based screening, we previously isolated several Arabidopsis mutants defective in protein import into chloroplasts. Positional cloning of one of the loci, CIA1, revealed that CIA1 encodes Gln phosphoribosyl pyrophosphate amidotransferase 2 (ATase2), one of the three ATase isozymes responsible for the first committed step of de novo purine biosynthesis. The cia1 mutant had normal green cotyledons but small and albino/pale-green mosaic leaves. Adding AMP, but not cytokinin or NADH, to plant liquid cultures partially complemented the mutant phenotypes. Both ATase1 and ATase2 were localized to chloroplasts. Overexpression of ATase1 fully complemented the ATase2-deficient phenotypes. A T-DNA insertion knockout mutant of the ATase1 gene was also obtained. The mutant was indistinguishable from the wild type. A double mutant of cia1/ATase1-knockout had the same phenotype as cia1, suggesting at least partial gene redundancy between ATase1 and ATase2. Characterizations of the cia1 mutant revealed that mutant leaves had slightly smaller cell size but only half the cell number of wild-type leaves. This phenotype confirms the role of de novo purine biosynthesis in cell division. Chloroplasts isolated from the cia1 mutant imported proteins at an efficiency less than 50% that of wild-type chloroplasts. Adding ATP and GTP to isolated mutant chloroplasts could not restore the import efficiency. We conclude that de novo purine biosynthesis is not only important for cell division, but also for chloroplast biogenesis.