【作者】 郭文波；

【导师】 包振民； 宋微波；

【作者基本信息】 中国海洋大学 ， 遗传学， 2009， 博士

【摘要】 纤毛虫分子系统学工作的开展将在很大程度上解决和完善某些缺乏发生学资料的关键类群在系统进化位置上的争议。近年来,由于引入分子系统学导致曾经被视为多膜纲重要类群的腹毛类和异毛类被置于不同进化分枝的旋唇纲和异毛纲。异毛类和腹毛类内大部分物种被分子数据所支持并保留在阶元内,但同时部分物种被移出本阶元。但多膜纲某些关键类群的分子信息,如异毛类突口虫属Condylostoma以及广义腹毛类重要类群的缺失,以至影响到系统进化树中异毛类及腹毛类各目的位置。深入分析纤毛虫基因信息,有利于研究真核生物(细胞)分裂过程中核质关系及遗传信息表达提供发生学模型,并可为阐述真核生物细胞器在分化与反分化、细胞分裂周期中新老结构的相承关系、亚细胞水平的信息传导机制及动力学等细胞生物学基本现象。本工作第一部分拟获得异毛类和腹毛类纤毛虫中重要类群中相对保守的SSU rRNA、α-tubulin基因和相对变异较大的ITS1-5.8S-ITS2基因序列,并构建系统发生树;同时结合形态学和细胞发生学的信息,进而探讨多膜纲中异毛类重要类群和腹毛类各目的系统地位。本工作第二部分利用海洋纤毛虫肉色伪角毛虫为材料,完成了对其cDNA文库的构建和部分基因表达序列标签的分析,以期为下一步功能基因的探讨铺就基础。本论文主要成果如下:1.游仆目纤毛虫系统关系:游仆目不是一个单源发生系;盘头虫类从游仆目中较早分化出来,呈现与排毛类、寡毛类平行的进化关系;腹棘虫科内的腹棘虫和拟游仆虫体现了密切的亲缘关系;游仆虫科、舍太虫科、楯纤虫科在所有分子树中均表现了十分稳定的拓扑结构;游仆虫属内可以细分为7个亚类群;尾刺虫科在游仆目内分化较早,支持前人有关其在系统演化时序中处于原始阶段的论断。2.尾柱目纤毛虫系统关系:基本上支持尾柱目和排毛类、寡毛类的姊妹群关系;尾柱目的Pseudourstylidae(伪尾柱虫科)、Pseudokeronopsidae(伪角毛虫科)聚类在一起,与尾柱虫科呈现姊妹群关系;全列虫为多源发生;曾经被视为尾柱类纤毛虫的伪小双虫,移出本阶元;Epiclintidae(额斜虫科)与寡毛类、散毛类的亲缘关系更为紧密,不支持Lynn(2008)在尾柱目下设立该科。根据分子系统树研究结果,尾柱目不再是个单源发生系。3.散毛目纤毛虫系统关系:尖颈虫、半腹柱虫和殖口虫从散毛目中较早分化出来;尖毛科除尖毛已有亚科、棘毛亚科外,应新建一亚科;尖毛虫属分类混淆,部分物种应该被移出本属;建议原腹柱虫属移出尖毛科,置于排毛亚纲某一新建的阶元内;弹跳虫科与尖毛虫关系紧密,但弹跳虫科置于散毛目需要分子和形态数据的进一步支持。4.测定了突口虫属的三个海洋种的SSU rRNA和ITS1-5.8S-ITS2基因序列,并在此基础上构建了突口虫属的系统发生树,讨论了该属在异毛纲(Heterotrichea)内的系统位置及其与纲内其它属的关系。5.利用α-tubulin基因对伪角毛虫属、突口虫属纤毛虫的分子系统学系统研究。基于α-tubulin蛋白并与SSU rRNA构建系统发生树的结果进行了比较,由于不同基因进化速率的差异,由特定基因构建系统树并对其相应物种进行系统进化地位讨论的结果可能会有较大不同。6.以海洋腹毛类纤毛虫--肉色伪角毛虫为材料开展了cDNA文库的构建和部分基因表达序列标签(EST)的分析。其中,利用Smart技术构建cDNA文库,质量检测表明该文库容量为1×106 pfu/mL,重组率为91%,表明所建文库能够满足后续的EST文库构建及从文库中分离筛选重要基因等研究工作。从该库中随机抽取了237个克隆进行5′端测序,拼接后得到189条非冗余基因(UniGene),Blast X比对分析后得到57条有功能注释的序列,这些功能基因片段多为首次发现。更多还原

【Abstract】 Systematics and phylogenetics on the ciliated protozoa have been carried out for about 20 years. However, its phylogeny still remains confusing as regards the evolutionary relationships and systematic positions of many well-known groups. This is due to the high diversity of their morphology, the difficulty in recognizing which similarities are due to convergent evolution, the loss of intermediate forms during the long period of time and insufficient molecular information.Molecular marker gene were chosen e.g. SSU rRNA gene, ITS1-5.8S-ITS2 gene andα-tubulin gene as to study systemetics, phylogenetic position or relationships and evolutionary histories of some key and / or ambiguous taxa of ciliated protozoa. Based on the gene sequenceing and analyses, this work resolved some confusions of morphological taxonomy and phylogenetic positions of some key taxa within three calsses at the genetical level. We used multiple computer-assisitent algorithms inferred from combinations of molecular sequences and morphological and morphogenetic characterizations to supply important basis and parameters for molecular systematics, a reconstruction of phylogenetic positions and evolutionary relationships of key taxa ciliated protozoa.The technology of expressed tag (EST) was broadly applied in analysis of gene function and expression pattern. In this study, total rRNA of cDNA library of Marine Ciliate P. carnea was constructed using the Smart technique for the further studies, such as special nuclear dualism, complicated morphogenetic process and unique sexual reproduction (conjugation).The conclusions were confirmed:1. The order Euplotida:The typical euplotids belong to a paraphyletic group composed of three diverged clades (Uronychiidae, Euplotidae-Certesiidae-Aspidiscidae and Gastrocirrhidae); both clades formed by Euplotidae-Certesiidae-Aspidiscidae and Gastrocirrhidae were firmly supported with high bootstrap; the family Discocephalidae forms a sister group to the large clade containing Euplotidae-Certesiidae-Aspidiscidae and Gastrocirrhidae, though its position remains ambiguous due to low supports; Uronychiidae is clearly separated from the most other euplotids.2. The order Urostylida:The order Urostylida should be a paraphyletic group. (1) Pseudoamphisiella always branch firstly off the clade followed by Licnophorea which indicates that they might represent an ancestral form for urostylid species. (2) Pseudokeronopsis and Thigmokeronopsis may not share a recent common ancestor, casting doubt on the monophyly of the family Pseudokeronopsidae. (3) Our data justify the redefinition of Holosticha and confirm that Anteholosticha should be apart from Holosticha. (4) DNA sequence data can be harmonized with the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis which suggests that the urostylid midventral pattern evolved from an oxytrichine ancestor, developing a second time within the Oxytrichidae. (5) Epiclintidae clusters with Oligotrichida and Sporadotrichida, which against its establishment of the family in the order Urostylida.3. The order Sporadotrichida:(1) the oligotrichine family Halteriidae which has been transferred to the order Sporadotrichida Lynn (2008) invariably clusters within the oxytrichid clade. This contradicts the current ontogenetic findings; possibly, it is an artifact caused by taxa undersampling and/or special molecular evolutionary events. (2) The systematic position of one of the two key genera could be clarified with the SSU rRNA sequences: Gastrostyla is a stylonychine oxytrichid. (3) sequence analyses of several Oxytricha species resulted in a high molecular diversity, which does not support monophyly of this genus. (4) the molecular phylogenetic tree basically supports the establishment of the family Trachelostylidae Small & Lynn, 1985. 4. Condylostoma species and other related Heterotrich ciliates:The systematically poorly-known ciliate genus Condylostoma was erected by Vincent in 1826. (1) all the trees have similar topologies with high supports; (2) Condylostoma is mostly related to the genus Condylostentor; and (3) three Condylostoma species as well as Condylostentor auriculatus cluster together and form a sister group with other heterotrichs. This is moderately consistent with the assessment of phylogenetic relationships of Condylostoma-related heterotrichs due to morphological information. The phylogenetic relationship of some other related heterotrichs, Peritromus, Folliculina, Stentor and Blepharisma, has been also discussed.5. The cDNA library of marine ciliate, Pseudokeronopsis carnea:The cDNA library was constructed using the SMART construction kit, from which totally 237 expressed sequence tags (ESTs) were randomly selected for analyses. The capacity of the library was 1×106 pfu/mL, in which more than 90% clones are recombinant. 189 unabridged cDNA sequences lacking X or N were made homology comparison using the Blast X search; 57 of which were nearly identical to known genes in the NCBI database. These data show that the full length cDNA library of P. carnea constructed using the Smart technique is of reasonably good quality for the further studies.更多还原