| 题名 | 莱茵衣藻基于乙酸的非光合生长机制 及FBPase II 在其中的作用 |
| 作者 | 邹勇 |
| 学位类别 | 硕士 |
| 答辩日期 | 2012-05 |
| 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 | 莱茵衣藻 果糖-1 乙酸碳源 6-二磷酸酶 非光合生长 进化 |
| 其他题名 | The mechanism of non-photosynthetic growth with acetate as carbon source in Chlamydomonas reinhardtii and the role of FBPase II in it |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 衣藻是一种单细胞绿藻，而绿藻被认为是陆地植物的祖先。早有研究表明莱 茵衣藻不仅能进行一般的光合生长，还可以在无光条件下利用乙酸作为唯一碳源 进行非光合生长。但其背后的机制并不清楚。本文首先基于基因组数据库和EST 库等对莱茵衣藻及与其近缘种中与利用乙酸进行非光合生长的相关代谢途径的 所有基因/酶进行了系统的调查分析，以探讨这一有趣并可能有重要应用前景的 生长现象的分子机制。其次，对其中具有特殊来源且起关键作用的II 型FBPase （果糖-1，6-二磷酸酶）进行了较为详尽的生物信息学分析和实验验证，以探讨 这一生长现象的起源形成机制。主要研究结果和结论如下： I．通过莱茵衣藻及其近缘种从乙酸到淀粉这一系列合成代谢通路（包括乙 醛酸循环、糖异生等）的基因/酶的调查，发现：莱茵衣藻与其亲缘关系非常近 但不能进行这一非光合生长的近缘种团藻中所有的相关基因/酶都高度一致，这 可能暗示着是其它原因（如细胞外基质阻挡了乙酸进入细胞等）导致了多细胞的 团藻不能进行该非光合生长。Prasinophceae 纲的绿藻不能进行上述非光合生长， 我们发现其原因是由于它们没有乙醛酸途径的关键酶异柠檬酸（裂合）酶和苹果 酸合酶。这些结果表明不同的绿藻之所以有的能有的不能进行乙酸作为唯一碳源 进行非光合生长其原因各不相同。而具备该能力的绿藻在乙酸的转运、乙醛酸循 环和糖异生等代谢途径必须完整。 II．我们发现在绿藻中存在着一个一般真核生物均不具有FBPase。Pfam 分 析表明此FBPase 具有典型的原核型II 类FBPase 的FBPase_glpX 结构域，且莱 茵衣藻的叶绿体蛋白质组含有此酶，因此我们称它为叶绿体型的II 类FBPase --cpFBPase II。系统发生分析表明绿藻中的此酶很可能是通过水平基因转移从放 线菌亚纲的共同祖先转移到绿藻的共同祖先中的。Real-time PCR 结果显示， cpFBPase II 基因在整个光周期中表达基本平稳，不受光的调节，而参与Calvin 循环的cpFBPase I 基因的表达是受光调节的。莱茵衣藻可以利用乙酸为唯一碳源 进行非光合生长，这一过程需要乙醛酸循环，糖异生途径和淀粉合成途径的参与。 II 但是cpFBPase I 具有三个保守的半胱氨酸，因此在蛋白水平也是受光调节的，也 就是说在无光条件下没有催化活性；而已有研究表明莱茵衣藻的糖异生途径大部 分定位于叶绿体，我们所发现的该cpFBPase II 也正好定位于叶绿体，因而在无 光条件下，应该是该酶在起作用从而保证了糖异生途径的进行。因此，cpFBPase II 的获得是莱茵衣藻进化出以乙酸作为唯一碳源进行非光合生长这一能力的前 提条件。 |
| 英文摘要 | Chlamydomonas are unicellular green algae that are considered as the ancestor of land plants. Besides thriving through photosynthesis, C. reinhardtii also possesses the ability to grow nonphotosynthetically with acetate as the sole carbon source in total darkness. In this work, based on the genome and EST data from C. reinhardtii and its close relatives, we performed comprehensive investigation and analysis of the genes/enzymes of metabolic pathways relative to the ability of growing nonphotosynthetically with acetate as the sole carbon source to philosophize the molecular mechanism of this amazing phenomenon which probably has applicable prospect. Furthermore, we also conducted detailed bioinformatic and experimental analysis of fructose-1,6-bisphosphatase (FBPase) II which has a special origin and plays a crucial role in nonphotosynthetical growth, to understand the origin of this phenomenon. The main results and conclusions are listed as follows: I. After the investigation of the whole pathway from acetate to starch (including glyoxylate cycle, gluconeogenesis and starch synthesis) in C. reinhardtii and its close relatives, we found that the relative genes/enzymes in C. reinhardtii are nearly the same as that in its closest relative Volvox carteri which do not have the ability to utilize acetate under darkness, suggesting other mechanisms, such as massive members of extracellular matrix that presents as a obstacle to prevent the acetate entrance, lead to the inability of using acetate in V. carteri. Besides, the green algae in Prasinophceae are not reported with forementioned ability of utilizing acetate. Our results indicated that is just because of the lack of isocitrate lyase and malate synthase which play the pivotal role in glyoxylate cycle. All these results demonstrate that the IV reason why these different algae can or can’t grow nonphotosythetically with acetate as the sole carbon source are all different and the algae with this ablity must contain the integral pathway of acetate transport, glyoxylate cycle and gluconeogenesis. II. We found a novel FBPase harbored in green algae which is never reported in eukaryotes. This enzyme contains a classic FBPase_glpX domain, a symbol indicated that is a class II FBPase. For being included in the chloroplast proteome of C. reinhardtii, we called it chloroplastic FBPase II (cpFBPase II). Phylogeny analysis showed that the cpFBPase II of green algae orginated from the ancestor of actinobacteridae through horizontal gene transfer (HGT). Real-time PCR results showed that the expression level of cpFBPase II is smooth, while that of cpFBPase I participating in Calvin Cycle is substantially regulated by light, and meanwhile no conserved cysteine regulated by light was found in cpFBPase II, demonstrating that cpFBPase II was not light-regulated. C. reinhardtii can grow with acetate as the sole carbon source in dark, which need the combination of glyoxylate cycle, gluconeogenesis and starch synthesis. However, cpFBPase I possesses three conserved cysteines for light regulation and does not work due to lack of light activation in the dark. It has been reported that most steps of the gluconeogenesis occurred in chloroplast in C. reinhardtii, and cpFBPase II is a chloroplastic enzyme, all of which lead to the conclusion that cpFBPase II plays the role when acetate was the sole carbon source in total dark. Therefore, it is the acquisition of cpFBPase II in green algae that endows C. reinhardtii with the ability to thrive with acetate as the sole carbon source under darkness. |
| 语种 | 中文 |
| 公开日期 | 2012-06-06 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6961]  |
| 专题 | 昆明动物研究所_结构生物信息学 |
| 推荐引用方式 GB/T 7714 | 邹勇. 莱茵衣藻基于乙酸的非光合生长机制 及FBPase II 在其中的作用[D]. 北京. 中国科学院研究生院. 2012. |
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