DNA 序列在蕨类分子系统学研究中的应用

在分子系统学研究中, 目的基因或者基因片段的选择是最关键的一步, 由于进化速率的差异, 不同的DNA序列适用于不同分类阶元的系统发育研究。本文综述了目前蕨类分子系统发育研究中常用的DNA序列分析, 它们分别来自叶绿体基因组、核基因组和线粒体基因组, 着重阐明叶绿体基因在蕨类分子系统学研究中的应用。本文还简要介绍了分子系统学研究中常见的问题及解决方法(如内类群和外类群的选择, 适宜DNA片段的选择策略), 总结了目前蕨类植物分子系统学研究所取得的进展和研究现状, 展望了当今国际蕨类分子系统学的研究趋势。     

DNA序列在蕨类分子系统学研究中的应用

在分子系统学研究中,目的基因或者基因片段的选择是最关键的一步,由于进化速率的差异,不同的DNA序列适用于不同分类阶元的系统发育研究.本文综述了目前蕨类分子系统发育研究中常用的DNA序列分析,它们分别来自叶绿体基因组、核基因组和线粒体基因组,着重阐明叶绿体基因在蕨类分子系统学研究中的应用.本文还简要介绍了分子系统学研究中常见的问题及解决方法(如内类群和外类群的选择.适宜DNA片段的选择策略),总结了目前蕨类植物分子系统学研究所取得的进展和研究现状,展望了当今国际蕨类分子系统学的研究趋势.     

绣球属(Hydrangea L.)植物分子系统学及系统发育分析

该研究基于对绣球属(Hydrangea L.)的大尺度取样,选取国内外61种绣球属和近缘属植物,分别基于核基因片段(ITS)和叶绿体基因片段(rbcL,trnL-F,atpB)重建了绣球属及其近缘种属的系统发育关系。结果表明:(1)核基因与叶绿体基因树之间在树形上没有明显的冲突,进而基于核基因和叶绿体基因联合数据重建了绣球属及其近缘种属的系统发育关系。(2)基于联合数据构建的系统树确认了2个大分支,并得到了果实顶端截平与否这一形态学证据的强力支持;每个大分枝又分为4个类群,共确定了8个类群。部分类群也得到了广义宏观形态性状的支持,如第1类群得到了叶形、花粉以及种子形态的支持。因此,该系统发育关系的重建对于全面理解绣球属及其近缘种属的演化关系具有重要的启发。     

初探低拷贝核基因在低等分类阶元系统发育重建中的适用性——以十字花科为例

分子系统学已被广泛用于解决物种之间的亲缘关系。迄今为止,被子植物分子系统学大都采用叶绿体和线粒体基因。但叶绿体和线粒体多数采用单亲遗传方式,不能完全记录物种的进化历史。而且叶绿体和线粒体基因相对保守,可用于系统发育重建的信息有限,很难用来解决科以下类群之间的亲缘关系。相反,核基因遵守双亲遗传方式并能提供大量信息位点,但却没有得到广泛运用。本文以十字花科为例,从17种十字花科植物中得到了5个编码蛋白的单拷贝核基因序列,采用最大简约法、最大似然法和贝叶斯法重建它们之间的亲缘关系。结果表明,和目前常用的基因相比,这些基因能提供更多的信息位点,由此得到的系统发育树具有很高的支持,利用最大简约法得到的5个核基因的系统树中各分支均得到了100%的自展支持率。结果表明,我们选取的核基因可以用来进行科、属及种间的系统发育重建。因此,这5个基因可以用来解决被子植物其它科内的亲缘关系,而且也可以作为DNA条形码研究的有效分子标记。     

初探低拷贝核基因在低等分类阶元系统发育重建中的适用性——以十字花科为例

分子系统学已被广泛用于解决物种之间的亲缘关系.迄今为止,被子植物分子系统学大都采用叶绿体和线粒体基因.但叶绿体和线粒体多数采用单亲遗传方式,不能完全记录物种的进化历史.而且叶绿体和线粒体基因相对保守,可用于系统发育重建的信息有限,很难用来解决科以下类群之间的亲缘关系.相反,核基因遵守双亲遗传方式并能提供大量信息位点,但却没有得到广泛运用.本文以十字花科为例,从17种十字花科植物中得到了5个编码蛋白的单拷贝核基因序列,采用最大简约法、最大似然法和贝叶斯法重建它们之间的亲缘关系.结果表明,和目前常用的基因相比,这些基因能提供更多的信息位点,由此得到的系统发育树具有很高的支持,利用最大简约法得到的5个核基因的系统树中各分支均得到了100％的自展支持率.结果表明,我们选取的核基因可以用来进行科、属及种间的系统发育重建.因此,这5个基因可以用来解决被子植物其它科内的亲缘关系,而且也可以作为DNA条形码研究的有效分子标记.     

叉蕨科是一个多系类群: 基于叶绿体rbcL和atpB基因的分析

叉蕨科植物为泛热带分布, 全世界约有20余属, 中国产8属. 本研究以属为单位进行类群取样, 利用来自叶绿体基因组的两个基因(rbcL和atpB)对叉蕨科中国产全部8属植物进行系统发育重建, 用以探讨叉蕨科及其科下系统发育关系. 研究结果显示, 传统(秦仁昌系统)的叉蕨科是一个多系类群, 肋毛蕨属Ctenitis, 轴鳞蕨属Dryopsis和节毛蕨属Lastreopsis应该从叉蕨科分出而作为鳞毛蕨科成员, 黄腺羽蕨属Pleocnemia也暂时置于鳞毛蕨科. 轴脉蕨属Ctenitopsis, 沙皮蕨属Hemigramma, 牙蕨属Pteridrys, 地耳蕨属Quercifilix和叉蕨属Tectaria在系统发育树上聚为一支, 形成一个得到强烈支持的单系类群. 在rbcL单基因分析中, 爬树蕨属Arthropteris同上述单系类群聚在一起. 本研究基于叶绿体基因的证据对叉蕨科进行了重新定义.     

高等植物叶绿体和线粒体免疫亲近性的研究

以火箭免疫电泳分析表明:大豆叶绿体抗体与大豆线粒体有免疫交叉反应,同时大豆线粒体抗体与大豆叶绿体也有免疫交叉反应,但是大豆线粒体的抗体与鼠肝线粒体之间无免疫交叉反应。这说明高等植物线粒体对叶绿体比之对动物线粒体在免疫特性上有更大的亲近性,亦即高等植物线粒体和高等植物的叶绿体有更大的同源性。经火箭免疫电泳、交叉免疫电泳和线状免疫电泳进一步分析表明:菠菜偶联因子抗体(AbCF_1)和大豆线粒体、大豆叶绿体间,大豆线粒体抗体与CF_1和大豆叶绿体之间,以及大豆叶绿体的抗体(AbC)与CF_1和大豆线粒体间有免疫交叉反应,说明两种换能器之间有免疫亲近性,并分别与CF_1存在免疫亲近性。这揭示两种换能器免疫亲近性的表现是由于存在共同物质基础所致,这内在共同物质基础是偶联因子。这个结果有力地支持高等植物叶绿体和线粒体在结构和功能上以及发生上存在同源性的观点,在理论上也为两种换能器的起源和演化上存在同源性提供了一些依据。     

牦牛的分类学地位及起源研究:mtDNA D-loop序列的分析

牦牛的起源与属级分类学地位至今仍然存在一定的争议。我们测定了家养牦牛和野生牦牛线粒体控制区(D-loop)序列,并以此构建牦牛和牛属、野牛属、水牛属以及非洲水牛属相关种的系统发育树。研究结果表明线粒体D-loop区与Cytb基因序列在构建牛族的系统发育具有同样重要的价值。系统发育关系显示野牛属的灭绝种草原野牛与现存种美洲野牛先聚合为一单系群,然后再和牦牛形成一单系分支,表明牦牛与野牛属的草原野牛、美洲野牛亲缘关系最近,具有最近的共同祖先,而与牛属的其它亚洲物种亲缘关系较远。因此,本研究不支持将牦牛独立为牦牛属———Poephagus,牛属与野牛属在分类上也应合并为一个属。基于上述研究结果和化石证据,我们进一步对牦牛起源的历史背景进行了讨论,认为牦牛与野牛属的分化是由于第四纪气候变化在欧亚大陆发生的,野牛通过白令陆桥进入北美;冰期结束后,由于欧亚大陆其它地区温度升高,牦牛只能局限分布在较为寒冷的青藏高原;而野牛属在北美先后分化为草原野牛和美洲野牛,前者可能是后者的直接祖先。     

核基因在鸟类分子系统发育学研究中的应用

核基因作为一种新的遗传标记,近年来被广泛应用于鸟类分子系统发育研究中.核基因与线粒体基因位于不同的遗传载体上,因此被引入到系统发育学研究中为物种树的重建提供独立的证据.常用的外显子标记为重组激活基因1(RAG-1),重组激活基因2(RAG-2),癌基因c-myc,原癌基因c-mos,它们由于缓慢的进化速率而被用于鸟类高级分类阶元的系统学研究中.常用的内含子标记是β纤维蛋白原基因内含子7(β-fibrinogen intron7,β-fibint7),肌红蛋白基因内含子Ⅱ(myoglobin intionⅡ).内含子标记通常与线粒体序列联合使用,形成具有互补系统发育信号的数据集,应用于各种分类阶元的系统学研究中.     

叶绿体的遗传进化

叶绿体是半自主性细胞器,其生长和增殖受核基因组和自身的基因组2套遗传系统的控制、关于叶绿体的起源有2种学说,近年来．大量叶绿体基因组全序列被测定,以及分子生物学的研究结果为内共生起源学说提供了更多证据。相对于线粒体,叶绿体DNA的结构更趋于保守一,叶绿体与核基因组所编码的蛋白质互相协调来维持叶绿体的正常功能。在进化过程中,基因可能从叶绿体大量转移到细胞核中。叶绿体基因组的信息常常表现出“母性遗传”特征．因而,使之更具生物反应器的优势。     

Tetraploid somatic hybrids of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) obtained from diploid breeding lines

Intraspecific somatic hybrids between 16 different diploid breeding lines of Solanum tuberosum L. were produced by PEG-induced fusion. Manually selected heterokaryons were cultured in a Millicells-CM using a post-fusion protoplast mixture. Plants were regenerated from calli derived from heterokaryons obtained from 10 out of 38 combinations of diploid lines. Of the tested putative somatic hybrids, 14.2% were diploid, 72.8% were tetraploid and 13% pentaploid. The DNA amplification pattern obtained with RAPD or semi-random primers confirmed that 6 fusion combinations were hybrids. In most cases, the morphological traits were intermediate to those of the diploid fusion partners. About 23.0% of the tested somatic hybrids showed variation in their morphology. Of the tested somatic hybrids, 78.0% flowered and 86.0% tuberized. The cytoplasm of 9 diploid lines and 6 somatic hybrid combinations was analysed. Two of the diploid lines had W/S chloroplasts and α or ε mitochondria; the remainder contained T chloroplasts and β mitochondria. All the analysed somatic hybrids carried T chloroplasts and β mitochondria.     

Evolution of the chaperonin families (HSP60, HSP 10 and TCP-1) of proteins and the origin of eukaryotic cells

The members of the 10 kDa and 60 kDa heat-shock chaperonin proteins (Hsp10 and Hsp60 or Cpn10 and Cpn60), which form an operon in bacteria, are present in all eubacteria and eukaryotic ceil organelles such as mitochondria and chloroplasts. In archaebacteria and eukaryotic cell cytosol, no close homologues of Hsp10 or Hsp60 have been identified. However, these species (or ceil compartments) contain the Tcp-1 family of proteins (distant homologues of Hsp60). Phylogenetic analysis based on global alignments of Hsp60 and Hsp10 sequences presented here provide some evidence regarding the evolution of mitochondria from a member of the α-subdivision of Gram-negative bacteria and chloroplasts from cyanobacterial species, respectively. This inference is strengthened by the presence of sequence signatures that are uniquely shared between Hsp60 homologues from α-purple bacteria and mitochondria on one hand, and the chloroplasts and cyanobacterial hsp60s on the other. Within the α-purple subdivision, species such as Rickettsia and Ehrlichia, which live intracellularly within eukaryotic cells, are indicated to be the closest relatives of mitochondrial Homologues, In the Hsp60 evolutionary tree, rooted using the Tcp-1 homologue, the order of branching of the major groups was as follows: Gram-positive bacteria — cyanobacteria and chloroplasts — chlamydiae and spirochaetes —β and γ-Gram-negative purple bacteria —α-purple bacteria — mitochondria. A similar branching order was observed independently in the Hsp10 tree. Multiple Hsp60 homologues, when present in a group of species, were found to be clustered together in the trees, indicating that they evolved by independent gene-duplication events. This review also considers in detail the evolutionary relationship between Hsp50 and Tcp-1 families of proteins based on two different models (viz. archaebacterial and chimeric) for the origin of eukaryotic cell nucleus. Some predictions of the chimeric model are also discussed.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.     

Substitution of the gene for chloroplast RPS16 was assisted by generation of a dual targeting signal

Organelle (mitochondria and chloroplasts in plants) genomes lost a large number of genes after endosymbiosis occurred. Even after this major gene loss, organelle genomes still lose their own genes, even those that are essential, via gene transfer to the nucleus and gene substitution of either different organelle origin or de novo genes. Gene transfer and substitution events are important processes in the evolution of the eukaryotic cell. Gene loss is an ongoing process in the mitochondria and chloroplasts of higher plants. The gene for ribosomal protein S16 (rps16) is encoded in the chloroplast genome of most higher plants but not in Medicago truncatula and Populus alba. Here, we show that these 2 species have compensated for loss of the rps16 from the chloroplast genome by having a mitochondrial rps16 that can target the chloroplasts as well as mitochondria. Furthermore, in Arabidopsis thaliana, Lycopersicon esculentum, and Oryza sativa, whose chloroplast genomes encode the rps16, we show that the product of the mitochondrial rps16 has dual targeting ability. These results suggest that the dual targeting of RPS16 to the mitochondria and chloroplasts emerged before the divergence of monocots and dicots (140-150 MYA). The gene substitution of the chloroplast rps16 by the nuclear-encoded rps16 in higher plants is the first report about ongoing gene substitution by dual targeting and provides evidence for an intermediate stage in the formation of this heterogeneous organelle.     

A novel ubiquitous protein 'chaperonin' supports the endosymbiotic origin of mitochondrion and plant chloroplast

The deduced amino acid sequences for a major mitochondrial protein (designated P1, related to the 'chaperonin' family of proteins) from human and Chinese hamster cells show extensive similarity (greater than 60% identity observed over the entire length) with a related protein present in evolutionarily as divergent organisms as Escherichia coli, Coxiella burnetii, Mycobacterium species, cyanobacteria as well as in yeast mitochondria and higher plant chloroplasts. Of the different groups of bacteria for which sequence data is available, maximum similarity of the mammalian/yeast P1 protein is observed with the corresponding protein from purple bacteria (especially C. burnetii) while the protein from plant chloroplasts exhibited highest similarity with the corresponding protein from cyanobacteria. The sequence data for this protein thus support the contention that the endosymbiont that gave rise to mitochondrion was a member of purple bacteria, while plant chloroplast originated from a member of the cyanobacterial lineage.     

Cytoplasmic inheritance of organelles in brown algae

Brown algae, together with diatoms and chrysophytes, are a member of the heterokonts. They have either a characteristic life cycle of diplohaplontic alternation of gametophytic and sporophytic generations that are isomorphic or heteromorphic, or a diplontic life cycle. Isogamy, anisogamy and oogamy have been recognized as the mode of sexual reproduction. Brown algae are the characteristic group having elaborated multicellular organization within the heterokonts. In this study, cytoplasmic inheritance of chloroplasts, mitochondria and centrioles was examined, with special focus on sexual reproduction and subsequent zygote development. In oogamy, chloroplasts and mitochondria are inherited maternally. In isogamy, chloroplasts in sporophyte cells are inherited biparentally (maternal or paternal); however, mitochondria (or mitochondrial DNA) derived from the female gamete only remained during zygote development after fertilization. Centrioles in zygotes are definitely derived from the male gamete, irrespective of the sexual reproduction pattern. Female centrioles in zygotes are selectively broken down within 1–2 h after fertilization. The remaining male centrioles play a crucial role as a part of the centrosome for microtubule organization, mitosis, determination of the cytokinetic plane and cytokinesis, as well as for maintaining multicellularity and regular morphogenesis in brown algae.     

A pathway of protein translocation in mitochondria mediated by the AAA-ATPase Bcs1

The AAA+ family in eukaryotes has many members in various cellular compartments with a role in protein unfolding and degradation. We show that the mitochondrial AAA-ATPase Bcs1 has an unusual function in protein translocation. Bcs1 mediates topogenesis of the Rieske protein, Rip1, a component of respiratory chains in bacteria, mitochondria, and chloroplasts. The oligomeric AAA-ATPase Bcs1 is involved in export of the folded Fe-S domain of Rip1 across the inner membrane and insertion of its transmembrane segment into an assembly intermediate of the cytochrome bc(1) complex, thus revealing an unexpected mechanistical concept of protein translocation across membranes. Furthermore, we describe structural elements of Rip1 required for recognition and export by as well as ATP-dependent lateral release from the AAA-ATPase. In bacteria and chloroplasts Rip1 uses the Tat machinery for topogenesis; however, mitochondria have lost this machinery during evolution and a member of the AAA-ATPase family has taken over its function.     

A mutation causing variegation and abnormal development in tobacco is associated with an altered mitochondrial DNA

A variegated mutation appeared in the leaves of a tobacco cybrid plant resulting from fusion of protoplasts from tobacco with Petunia . The mutation was inherited maternally. The light green coloration of leaf sectors resulted from a substitution of spongy parenchyma for palisade parenchyma. No defects were detected in the chloroplasts of the plants, which were derived from Petunia . The mitochondria, as judged by the electrophoretic pattern of their DNA after digestion with restriction endonucleases, were very similar to mitochondria of tobacco, although with some unique cybrid-specific fragments. A second round of fusions was performed to confirm that mitochondria, rather than chloroplasts, were associated with the variegated phenotype. In these fusions, the Petunia chloroplasts of the variegated plants were replaced by tobacco chloroplasts. The mitochondria, according to the DNA restriction pattern, retained all or some of the unique cybrid-specific fragments found in the original variegated tobacco cybrid. Since the variegated phenotype remained after the chloroplast exchange, the chloroplast DNA cannot be the site of the mutation which is responsible for the mutant phenotype. This result eliminates the chloroplast and confirms that the mitochondrial genome is associated with the mutant phenotype.     

A mitochondrial presequence can transport a chloroplast-encoded protein into yeast mitochondria

Ribulose-1,5-bisphosphate carboxylase/oxygenase of chloroplasts contains eight large and eight small subunits. The small subunit is encoded by nuclear DNA, synthesized in the cytoplasm, and imported into chloroplasts. The large subunit is encoded by chloroplast DNA and synthesized within chloroplasts. We show in this communication that the large subunit of Chlamydomonas chloroplasts could be efficiently imported into isolated yeast mitochondria if it was attached to the presequence of a protein transported into the yeast mitochondrial matrix. Thus, synthesis of the large subunit within chloroplasts does not reflect the inability of this subunit to cross membranes. The same mitochondrial presequence could also transport the nuclear-encoded small subunit into yeast mitochondria. However, when the two types of subunits were coimported into mitochondria, they did not assemble with each other inside the heterologous organelle.