几类“无线粒体”原生生物的DNA拓扑异构酶II基因及进化地位研究
文献类型：学位论文
| 作者 | 何德 |
| 学位类别 | 博士 |
| 答辩日期 | 2004-07 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 文建凡 |
| 关键词 | 无线粒体原生生物 DNA拓扑异构酶II 鉴定分子系统分析 长枝吸引 进化地位 |
| 其他题名 | DNA Topoisomerase II Genes and Evolutionary Status of Several Amitochondriate Protists |
| 中文摘要 | 以贾第虫、毛滴虫、内变形虫和微抱子虫等为代表的几类原生生物，不仅因为他们的寄生致病性而在医学上长期备受关注，它们的进化地位也是一个十分令人注目的问题。因为曾认为它们不具线粒体等细胞器，再加上一些分子系统学研究表明它们处在真核生物的最基部，因此不少人认为它们是在线粒体产生之前即已分化的极原始真核生物，其进化地位是处在原核生物向真核生物的过渡阶段，并有人称之为achezoa。这一发现一度被认为对探讨真核细胞（生物）的起源进化极为重要，是进化生物学上的重要突破。然而，近年来不断有新的证据对此提出质疑，其进化地位也就存在较大争议。本文首先利用PCR扩增、测序和基因组数据库搜索等技术方法鉴定了蓝氏贾第虫（Giardialamblia）、阴道毛滴虫（Trichomonasvaginalis）和痢疾内变形虫（entamoebahistolytica）的II型DNA拓扑异构酶基因序列。RT-PCR和序列分析表明它们均不具内含子。蛋白质序列搜索的结果表明它们与其它真核生物的DNA拓扑异构酶H是高度同源的。用生物信息学的方法，我们还对这些酶的性质进行了初步分析。分析还表明蓝氏贾第虫的DNA拓扑异构酶H具有一些不同于其．宿主的特征，如在ATPase区和中间区有六个插入，中间区要长大约100个氨基酸，而C端区又短大约200个氨基酸且富含带电荷的氨基酸残基。这些结果对研制以该酶为靶分子的专一性抗贾第虫药物具有指导意义。其次，将上述获得的序列数据结合GenBank数据库中已有的脑炎微抱子虫（Encephalitozooncuniculi）和其它一系列处在不同进化地位的真核生物的相应序列数据，用多种方法构建出分子系统树，对这些"无线粒体"原生生物的进化地位进行了探讨，并对"长枝吸引"对系统树的影响进行了分析。结果表明，由于DNA拓扑异构酶H的特点和可以克服"长枝吸引"等以往分子系统分析中的不足，所构建的系统树不仅能有效地反映出已普遍接受的真核生物各主要类群的系统关系，而且显示出这些"无线粒体"原生动物不同于以前系统树所反映的进化地位：它们并非是最早分支出来的真核生物，而是在具有线粒体的生物如动基体类或菌虫类等之后才分化的、分别属于不同进化地位的类群。结合近来它们中发现了类似线粒体细胞器等证据，我们认为这些所谓"无线粒体"的原生生物虽然其中有些种类（如以贾第虫为代表的双滴虫类）进化地位很低等，对探讨真核细胞的早期进化具有一定意义，但总体上它们并非过去所认为的那么极端原始，它们应该是线粒体产生之后才分别分化出来的不同生物类群 |
| 英文摘要 | Several groups of protists (such as Microsporidia, Giardia, Trichomonas and, Entamoeba, and so on) have attracted long attention not only in medicine for their parasitical nosogenesis, but also in evolutionary biology for their unique evolutionary-status. It was once thought that they lacked mitochondria (amitochondriate), in addition to that some molecular phylogenetic analyses showed that they branched at the base of eukaryotes, thus not a few people considered that they were the most primitive eukaryotes which diverged before the endosymbiotic origin of mitochondria, and that they were the relicts of transformation between prokaryotes and eukaryotes. A hypothesis was formalized by calling them 'Archezoa' to denote the primitively amitochondriate condition. It was thought that they were very important for the study of the origin and evolution of eukaryotic cells. However, recently, a growing body of evidence challenged their evolutionary status. In the present paper, firstly, the genes encoding type II DNA topoisomerases were identified from Giardia lamblia, Trichomonas vaginalis and Entamoeba histolytica by PCR-amplifying, sequencing and database searching. The results of RT-PCR and sequence analysis showed that there exist no introns in all the genes. Sequence blast indicated that their sequences showed strong homology to known type II DNA topoisomerases, especially to eukaryotic DNA topoisomerase II. Using bioinformational methods, we have analyzed the characters of their encoded enzymes. Our analysis showed that G lamblia topoisomerase II was different from that of its human host, for example, six insertions in the ATPase domain and the central domain; a ~100aa longer central domain; a ~200aa shorter C-terminal domain containing rich charged residues. These detail features might be helpful to exploit drug selectivity for antigiardial therapy whose target enzyme is DNA topoisomerase II. Secondly, besides the above obtained sequences, the sequences of Encephalitozoon cuniculi and a series of eukaryotes of different evolutionary positions were obtained from GenBank, and then phylogenetic trees were reconstructed by various methods to study the evolutionary status of these amitochondrate protists. Simultaneously, the affection of "Long Branch Attraction (LBA)" to the phyologenetic trees was also investigated. Our results showed that owing to the unique characters of DNA topoisomerase II, LBA had little influence on the trees based on the enzyme. Our phylogenetic trees not only reflected the widely accepted phylogenetic relationships among the main eukaeyotic groups, but also showed that the evolutionary positions of those amitochondriate protists were different from those in previous reported trees: they were not the earliest branching eukaryotes, but emerged later than mitochondriate protozoa (e.g. Kinetoplastid and Mycetozoa); they belonged to different groups of eukaryotes with different evolutionary positions. In addition to the other recent evidence such as the discovery of mitochondria-like organelle, our results suggest that all these amitochondriate protists are not extremely primitive as was considered before, and they might be different groups of eukaryotes and diverged after the endosymbiotic origin of mitochondria, though some of them, such as GiardiUj are primitive eukaryotes and are of significance to the study of early evolution of eukaryotes. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6196]  |
| 专题 | 昆明动物研究所_真核细胞进化基因组 |
| 推荐引用方式 GB/T 7714 | 何德. 几类“无线粒体”原生生物的DNA拓扑异构酶II基因及进化地位研究[D]. 北京. 中国科学院研究生院. 2004. |
入库方式： OAI收割
来源：昆明动物研究所
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