| 其他摘要 | 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 |
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