博士 / 國立中山大學 / 海洋生物研究所 / 101 / The highly productive coral reef ecosystem is based on the cnidarian-dinoflagellate endosymbiosis. However, the complex biological processes and interactions between the host and symbionts were still unclarified. Here, I firstly investigated the roles of Aprab11, Aprab3 and Aprab4 to describe the interaction between the symbiosomes and vesicles in host cell, which based on a sea anemone model organism, Aiptasia pulchella. Additionally, I also presented two approaches to dissect the molecular regulatory mechanism of the endosymbiosis. One was the characterization of host cathepsin family proteases, and the other was the identification of differentially expressed Symbiodinium genes during the symbiosis/free-living stages by surppressive subtractive hybridization (SSH). The results showed most of zooxanthellae-harbored symbiosomes were absent of Aprab11 but were abundant of Aprab3 and Aprab4 that indicated the TGN-derived vesicles and fast recycling routes were interacted with symbiosomes, but slow recycling routes were not. Lysotracker red staining and bafilomycin A1 treatment with phagosomes/symbisosomes in host cells indicated that the acidification of phagosomes was mainly mediated by ApV-ATPase and the symbiosomal lumen was mildly acidic. That’s consistent with the findings of immunostaining which the symbiosome were filled with many Apcathepsins except Apcathepsin H and ApV-ATPase. Additionally, the intracellular Apcathepsin H protease also accumulated in the early phagosmes and part of Aprab5-positive early endosomes and its proteolytic acivity could be activated at pH 6.8. But DCMU treatment of healthy symbiosomes could simultaneously induce the acidification and re-contribution of Apcathepsin H and ApV-ATPase to symbiosomes. Consequently, these results revealed that the Apcathepsin H and ApV-ATPase were actively excluded from symbiosome by the zooxanthellae to maintain the pH of symbiosomal lumen which were able to prevent damages from Apcathepsin proteases. On the other hand, the application of SSH technique produced 216 symbiosis and 175 nonsymbiosis differentially expressed zooxanthellae genes. These differentially-expressed genes were classified with different biological processes into cell division, cell signaling/communication, cell structure/motility, cell/organism defense, RNA synthesis, proten synthesis, metabolism and unclassified. Unfortunately, ~ 30 % differentially-expressed genes were belonged to unknown genes whether symbiosis or nonsymbiosis that implicated the understanding to zooxanthellae was still less than clear. Quantitative PCR assay revealed that the zooxanthellal genes related with vesicle trafficking, cytoskeleton, protease inhibitor, stress response and photosynthesis were more active during symbiotic phase. Conversely, the zooxanthellal genes highly expressed at the nonsymbiotic phase were involved with flagellate formation and motility, nitrogen transporter, cell division, cell wall-associated process… and so on. Together, the current investigation of host cathepsin family and symbiosis differentially-expressed zooxanthellal genes provided new knowledge and insights of endosymbiosis regulatory mechanism.
| Identifer | oai:union.ndltd.org:TW/101NSYS5270019 |
| Date | January 2013 |
| Creators | Ming-Chang Hong, 洪明昌 |
| Contributors | Keryea Soong, Ming-Chyuan Chen, 宋克義, 陳鳴泉 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 255 |
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