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题名	几类处在关键进化地位藻类的TIM基因和糖酵解途径的研究
作者	孙桂玲
学位类别	博士
答辩日期	2007-08
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	TIM 糖酵解 眼虫 衣藻 进化
其他题名	Studies on TIM genes and glycolytic pathway in several algae of important evolutionary status
学位专业	动物学
中文摘要	糖酵解作为细胞的重要的基本代谢途径广泛存在于各类生物中。但参与该途径的tim基因／酶和整个途径在细胞中的区室定位情况在一些处在关键进化地位的藻类中还存在许多未知或争议。本文首先对参与该途径的一个重要酶――磷酸丙糖异构酶（TIM）在两种不同营养方式的眼虫上进行了鉴定和序列结构分析，并结合了包括绿藻、红藻和动基体类等在内的其它生物的数据进行了分子系统分析；其次，对该途径在绿藻类的衣藻细胞中的区室定位情况进行了研究，并对该特殊的区室定位途径的进化进行了探讨。得到了如下结果和结论： 1）通过3’与5’-RACE实验，在光合型眼虫Euglena gracilis和Euglena intermedia和腐生型眼虫Astasia longa中各获得了一长一短两个TIM的全长cDNA序列，同时通过基因组DNA-PCR和序列搜索，还获得了两种绿藻和红藻的tim基因序列。前导序列分析显示眼虫的两个cDNA分别编码定位到胞质的cTIM和定位到叶绿体的cpTIM或质体的pTIM。腐生眼虫A. longa的pTIM与光合型眼虫E. gracilis的cpTIM高度相似，其前导序列也具有眼虫典型的核编码叶绿体蛋白质的典型特征；两类不同营养型眼虫的成熟TIM间的同源性达91.6％，并且具有其它TIM所共有的活性中心氨基酸和保守的序列motifs。这表明A. longa的pTIM确实是定位于质体、具有生物活性的cpTIM的同工酶，并提示A. longa的质体还具有与TIM相关的代谢功能（例如脂肪酸的合成），而不仅仅是一个叶绿体的“残迹”。 TIM的序列比对和分子系统分析结果显示：眼虫类和红藻类的TIM共有一个由两个氨基酸组成的插入；更重要的是，眼虫类TIM既没有与被认为与它共祖的动基体类的TIM聚在一起，又没有与被认为为其提供叶绿体（经二次内共生）的绿藻的TIM聚在一起，而是与红藻TIM聚为一枝。这提示眼虫与红藻间在进化历史上可能曾经有过基因交流。 2）采用生物信息学手段结合分子生物学实验，对衣藻基因组和转录组中参与糖酵解相关基因／酶进行了鉴定、定位预测和表达水平分析，结果表明：与其它极大多数的真核生物不同，衣藻细胞质中不具有完整的糖酵解途径，尽管该途径的后三步主要发生在胞质中，但前七步则是发生在叶绿体中的。 分子系统分析表明衣藻叶绿体中参与前6步和细胞质中参与最后2步的糖酵解酶都是胞质型起源，其中参与第4步的FBA更像是由很早时期的胞质型基因重复而来的；而第7步的PGK是由内共生形成叶绿体的蓝细菌的水平基因转移而来。这表明衣藻胞质中所缺少的糖酵解酶是一种次生性丢失的结果，而非原始的特征；其叶绿体中的糖酵解步骤应该是由于原有胞质型糖酵解酶的基因重复之后重新定位或者直接重新定位到叶绿体中，以及内共生产生叶绿体时由蓝细菌的水平基因转移所致。
英文摘要	As an important basic metabolic pathway, glycolysis is ubiquitous in all kinds of organisms. But the genes/enzymes involved in this pathway and the compartmentation of the pathway in some algae of important evolutionary status remains unknown or controversial. In the present work, firstly, triosephosphate isomerase (TIM), one of the important enzymes of the pathway, was identified in two trophic modes of euglenoids, and phylogenetic analysis was carried out combined with sequences from other organisms including chlorophytes, rhodophytes and kinetoplastids. Secondly, the compartmentation of the pathway was investigated in Chlamydomonas, and furthermore phylogenetic analyses of each classic glycolytic enzyme were conducted to trace the evolutionary history of the pathway in this species. The results and conclusions are as follows: 1) By 3’-RACE and 5’-RACE, two types of cDNA sequences were obtained in phototrophic Euglena gracilis and Euglena intermedia, and saprotrophic Astasia longa. Meanwhile tim gene sequences of two chlorophytes and rhodophytes were acquired by genome DNA-PCR and database searches. Sequence analyses and presequence prediction indicated that the shorter one encodes a cytosol TIM (cTIM) and the longer one encodes a chloroplast TIM (cpTIM) or a plastid TIM (pTIM). The typical presequence of the putative A. longa pTIM and the high sequence similarity between A. longa pTIM and E. gracilis cpTIM imply that A. longa pTIM is targeted to plastids, and therefore that A. longa plastids still have function(s) involving in TIM (e.g. probably glycolysis and fatty acid synthesis). Sequence alignment and phylogenetic analyses of TIM indicated I) euglenoid and rhodophyte TIMs share a unique 2-aa insertion within their loop-4 areas; II) most important, euglenoid TIMs neither group with TIMs of kinetoplastids, which share the nearest common ancestor with euglenoids, nor are closely related to TIMs of III chlorophytes, which are considered to offer euglenoids chloroplasts through secondary endosymbiosis, but group with TIMs of rhodophytes. These suggest that tim lateral gene transfer might have occurred between euglenoids and rhodophytes after the divergence of euglenoids with kinetoplastids. 2) By using the methods of bioinformatics and molecular biology experiments, glycolyticsis-associated genes/enzymes were identified and analyzed for their sub-cellular targeting and expression level based on Chlamydomonas reinhardtii genomic and transcriptomic databases. The results indicated that different from the majority of eukaryotes, Chlamydomonas cytosol does not possess a complete glycolytic pathway. Although the last three steps of the pathway occur in cytosol, the forward seven steps take place in the chloroplast. Molecular phylogenetic analyses indicated all the enzymes involved in the forward six steps in Chlamydomonas chloroplast and the last two steps in Chlamydomonas cytosol derived from the original cytosolic genes, and chloroplast FBAs involved in the fourth step were most probably from the duplication of the endogenous cytosolic gene at the very early stage; chloroplast PGK involved in the seventh step originated from cyanobacterial endosymbionts by lateral gene transfer. These suggest the lack of glycolytic enzymes in Chlamydomonas cytosol was not a primary character but due to a secondary lost. The occurrence of the forward seven glycolytic steps in chloroplast should result from the original cytosolic enzymes by relocalization to the chloroplast either directly or after gene duplication, or derive from cyanobacterial endosymbionts by lateral gene transfer.
语种	中文
公开日期	2010-10-14
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6121]
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	孙桂玲. 几类处在关键进化地位藻类的TIM基因和糖酵解途径的研究[D]. 北京. 中国科学院研究生院. 2007.

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