莱茵衣藻中FBPaseⅠ和FBPaseⅡ的功能分析
文献类型：学位论文
| 作者 | 王文敏 |
| 学位类别 | 硕士 |
| 答辩日期 | 2014-05 |
| 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 FBPaseⅠ FBPaseⅡ amiRNA 非光合生长 |
| 其他题名 | Functional Analysis of FBPaseⅠand FBPaseⅡin Chlamydomonas reinhardtii |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 单细胞真核绿藻莱茵衣藻（Chlamydomonas reinhardtii）一直被作为研究的模式生物。其基因组序列已被测定，并在其细胞核、叶绿体基因组上已成功建立了遗传转化技术。早有研究发现该生物不仅可以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，但这一现象背后的细胞和分子机制一直不清楚。本实验室前期对莱茵衣藻光合作用的有关代谢途径进行了系统分析，惊奇地发现该生物不同于一般的光合生物具有两个Ⅰ型的果糖-1, 6-二磷酸酶（FBPase）（即参与光合卡尔文循环的“叶绿体型FBPaseⅠ”和参与糖异生的“胞质型FBPaseＩ”），而是只具有其中的“叶绿体型FBPaseⅠ”，但令人惊讶地是它具有另一种一般只存在于原核生物的Ⅱ型FBPase（FBPaseⅡ），进一步分析表明该基因是从细菌水平基因转移而来的，且该酶与其FBPaseⅠ一样都定位于衣藻独特的叶绿体中。那么该FBPaseⅡ究竟具有怎样的功能？与该生物的叶绿体型的FBPaseⅠ有着怎样的功能分工？是否参与了糖异生作用从而与该生物上述的非光合生长有关？本论文就是针对这些问题，对莱茵衣藻的FBPaseⅠ和Ⅱ的功能分工及其与非光合生长的关系进行了较系统的实验分析研究。 首先，采用人工微RNA（amiRNA）技术，分别对莱茵衣藻FBPaseⅠ和FBPaseⅡ两基因进行表达敲低实验。即通过overlapping PCR分别扩增出针对FBPaseⅠ和FBPaseⅡ的目标片段，进而构建出amiRNA载体；采用玻璃珠转化并利用选择性培养基选择出转化成功的cc-425衣藻细胞；每个实验组挑选出20个克隆，通过半定量PCR检测其各自的敲低效率，将稳定的单克隆株系（RiⅠ和RiⅡ）用于后续研究。其次，在三种光照条件（完全光照，12h光照/12h黑暗交替，完全黑暗）和两种培养基（含和不含乙酸）条件下的不同组合的培养条件下，连续培养以对比观察实验组与对照组衣藻的生长状况，即主要通过观察培养物颜色差异、衣藻细胞计数来比较各个实验组与对照组衣藻的生长状况。 主要结果如下：１）FBPaseⅠ和FBPaseⅡ基因表达分别被敲低的实验组（RiⅠ和RiⅡ），在各种培养条件下衣藻的生长速率均显著低于各自的对照组；2）无论培养基中是否含有乙酸，RiⅠ或RiⅡ实验组在完全光照、光照/黑暗交替、完全黑暗三种条件下的生长速率依次显著下降；而在同等光照条件下，有乙酸组 生长速率均快于各自对应的无乙酸组；3）在无乙酸、完全黑暗培养条件下，RiⅠ或RiⅡ实验组均无法生长；同样在完全黑暗条件下，若培养基中存在乙酸，RiⅠ或RiⅡ实验组却能缓慢生长，尽管生长速率显著低于有光照的条件下。 基于以上结果并经分析，本文认为：莱茵衣藻中FBPaseⅠ和 FBPase II尽管同处叶绿体中，但它们之间存在明显的功能分工：FBPaseⅠ主要是参与光合卡尔文循环而与该藻的光合生长密切相关，而从原核生物水平转移而来的FBPaseⅡ主要是参与了糖异生作用而与该藻能进行非光合生长有关。该研究对于进一步揭示莱茵衣藻的非光合生长机制和实际应用该藻的这一特性具有重要意义。 |
| 英文摘要 | Chlamydomonas reinhardtii is a unicellular eukaryotic green algae.It is a good model organism for studying non-photosynthetic growth. Genome sequence of C. reinhardtii has been completed and its genetic transformation of nucleus and chloroplast has been implemented successfully. C.reinhardtii not only can grow under light by photosynthesis, but also can grow under darkness using acetate as the sole carbon source through non-photosynthesis growth. Currently,the mechanism of non-photosynthesis growth of C. reinhardtii is rarely known. Our lab previously studied metabolic pathways relevant to photosynthesis system in C. reinhardtii.We surprisingly found that this organismwas quite different from common photosynthetic organisms which had two classes fructose-1, 6-bisphosphatase (FBPase) (ie involved in the Calvin cycle of photosynthesis "chloroplast FBPaseⅠ" and participated in gluconeogenesis "cytoplasmic FBPaseⅠ"), but it has only one of the "chloroplast FBPaseⅠ". Then we surprisingly found it had another classⅡFBPase (FBPaseⅡ) which generally exists in prokaryote, further analysis showed that this gene was a horizontal gene transferred from bacteria. FBPaseⅡis located in the unique Chlamydomonas chloroplasts and so as the FBPaseⅠis. So what function does the FBPaseⅡhave? Does it have division function with FBPaseⅠin organism chloroplasts? Is it involved in gluconeogenesis and thus associated with the non-photosynthetic growth of the organisms described above?In order to understand these issues, we performed systematic experiments to validate the function of FBPaseⅡand FBPaseⅠof C. reinhardtii and its relation with non-photosynthetic growth. First, we used the artificial microRNA (amiRNA) technology, knockdown the expression of C. reinhardtii’s both FBPaseⅠandFBPaseⅡ,respectively. To do this, we amplified fragments of FBPaseⅠand FBPaseⅡgene by overlapping PCR, and then we constructed amiRNA vectors; The transform of C. reinhardtii cc-425 cells was achieved by glass beads and selective media was used to select the successful transformedclone;We screened 20 clones for each experimental group and detect their knockdown efficiency via semi-quantitative PCR.The stable monoclonal clones (RiⅠand RiⅡ) were chose for subsequent studies. Secondly, the C. reinhardtii were cultured under different conditions,combined the three light conditions（full light, 12h light / 12h dark alternation, complete darkness）and the two media（with or without acetate）, respectively, RiⅠand RiⅡ were cultured under the condition of different combinations, it was continuously cultured to juxtapose the different of experiment group and the control group which was achieved by mainlyobserving the color of the culturesandChlamydomonas cell counts. The main results showed: 1) Under various culture conditions the growth rate of RiⅠand RiⅡwhich FBPaseⅠand FBPaseⅡgene expression were been knocked down were significantly lower than the control groups,respectively; 2) Regardless the medium contains acetate or not, RiⅠor RiⅡgrowth rate are sequential decreased in three conditionswhich including light, light / dark alternation and complete darkness significantly;While at the same light conditions, acetate groups growth faster than the corresponding no acetate group, respectively; 3) RiⅠor RiⅡexperimental group can not grow in the absence of acetate and completely dark conditions; If there presence acetate in medium, RiⅠor RiⅡexperimental group were able to grow slowly in complete darkness, though the growth rate was significantly lower than under light conditions. Based on those results and analysis, the main points of this paper are: Despite the FBPaseⅠ and FBPaseⅡ ofC.reinhardtii locatedchloroplast in the same place,there has significant functional division between them. FBPaseⅠis mainly involved in the Calvin cycle and has a close relation to photosynthesis growth in the algae, while FBPaseⅡ which is orginated from prokaryotes is mainly involved in gluconeogenesis and the |
| 语种 | 中文 |
| 公开日期 | 2014-07-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7951]  |
| 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 | 王文敏. 莱茵衣藻中FBPaseⅠ和FBPaseⅡ的功能分析[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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