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研究生:鄭英敏
研究生(外文):Ying-Min Cheng
論文名稱:共生藻存活於腔腸動物消化細胞內的機制-ApRab5及ApRab7之關鍵角色
論文名稱(外文):Intracellular Survival Mechanisms of Zooxanthellae in Cnidarian Digestive Cells—The Critical Role of ApRab5 and ApRab7
指導教授:李澤民李澤民引用關係陳鳴泉
指導教授(外文):Tse-Min LeeMing-Chyuan Chen
學位類別:碩士
校院名稱:國立中山大學
系所名稱:海洋生物研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:58
中文關鍵詞:腔腸動物胞內共生
外文關鍵詞:Rab7cnidarianRab5endosymbiosis
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在海洋中腔腸動物(cnidarian)與共生藻(zooxanthellae)所形成的胞內共生現象,是珊瑚礁生態系中十分重要的基礎生產力貢獻者。這樣的共生關係是如何建立?相關的機制又為何?目前已有相關研究顯示在endocytosis過程的主要調控蛋白-Rab5(early endocytic phase regulating protein)和Rab7 (late endocytic phase regulating protein),可能扮演相當重要的角色。本研究主要是從細胞分子生物學的角度來探討細胞中的Rab proteins-Rab5和Rab7在Aiptasia-Symbiodinium共生現象之關連性。Rab蛋白質在氨基酸序列上具演化保留性,實驗中利用退化反轉錄聚合連鎖反應(degenerate RT-PCR)和cDNA端點反應(RACE)找到Aiptasia Rab5和Rab7(ApRab5和ApRab7)蛋白質序列,其氨基酸序列與人類Rab5C和Rab7十分相近,在以微珠(latex bead)所進行之phagocytosis,顯示ApRab5主要分佈在前30分鐘的phagosomes上;ApRab7要分佈在30-120分鐘的phagosomes上。過去已知光合作用抑制劑-DCMU會破壞共生關係,因此本研究以免疫染色法比較有無DCMU處理,其包著共生藻的phagosomes (又稱為共生小體)上ApRab5和ApRab7之分佈比率。結果發現未經DCMU處理之大部份共生小體上有ApRab5分佈(~65 %),但少有ApRab7之存在(~35 %);在經DCMU處理後一小時,有ApRab5分佈的共生小體數量銳減(~30 %),而有ApRab7分佈的共生小體數目卻等比例增多(~70 %)。餵食共生藻實驗中顯示-餵食活的共生藻二小時後,共生小體上約有65 %的ApRab5分佈;ApRab7的分佈僅約36 %。餵食死的共生藻,二小時後共生小體上ApRab5分佈降至約30 %;ApRab7的分佈遽升到約70 %。由上述種種實驗數據顯示,我們推測共生藻成功生存在宿主細胞中可能是藉由使Rab5停留在共生小體上及排除Rab7的作用,使共生小體停留在類似early phagosome stage階段。
Marine cnidarian-microalgal endosymbiosis is an ecologically important intracellular association. However, its underlying molecular mechanisms are essentially unknown. In light of the critical roles of host phagocytosis in intracellular fates of a variety of microbes, and the Rab small GTPases as key mediators of host-symbiont interaction, we set out to investigate the potential involvement of Aiptasia Rab proteins in the model photosynthetic endosymbiosis between the sea anemone, Aiptasia pulchella and the symbiotic dinoflagellate (commonly called zooxanthellae), Symbiodinium spp. Many Aiptasia Rab homologue-encoding cDNA fragments were first cloned through our degenerate RT-PCR and RACE reactions. Significantly, Aiptasia homologues of Rab5 and Rab7 (ApRab5 and ApRab7), two Rabs known to be critical regulators of phagosome maturation were also identified in the screen. The overall sequence identities of ApRab5 and ApRab7 to those of human Rab5C and Rab7 were very extensive, and EGFP reporter, protein fractionation, and immuno-fluorescence studies all suggested that the similarity of the Aiptasia Rabs to their human counterparts extended to the functional levels. Finally, although the phagosomes enclosing latex beads stained positive for ApRab5 and ApRab7 with kinetics characteristics of normal phagosomal maturation, the phagosomes housing zooxanthellae only stained positive for ApRab5. Furthermore, the association of ApRab5 with and the exclusion of ApRab7 from the zooxanthellae-containing phagosomes could be reversed by the heat-killed or photosynthesis-impaired symbionts. Overall, our present study has identified ApRab5 and ApRab7 as potential key regulators of the Aiptasia-Symbiodinium endosymbiosis
一、緒論
(一)細胞藉由胞飲作用擷取細胞外物質
(二)Rab蛋白調控細胞中囊泡運輸
(三)Rab5和Rab7調控胞飲作用的囊泡運送過程及胞噬泡成熟過程
(四)細胞中胞內微生物生存機制
(五)胞內共生藻生存在一個類似胞噬泡的胞器中,與溶小體沒有融合現象
(六)Mycobacterium和Salmonella藉由干擾胞噬泡成熟,以達到胞內寄生目的
(七)研究構想與方向
二、材料與方法
(一)細胞與動物
(二)ApRab5和ApRab7 cDNA選殖與序列分析
(三)ApRab5和ApRab7重組蛋白與純化
(四)蛋白質免疫分析
(五)EGFP-ApRab5和EGFP- ApRab7於哺乳類細胞內之定位
(六)胞噬作用
(七)免疫螢光分析
三、結果
(一)ApRab5和ApRab7全長cDNA選殖與分析
(二)ApRab5和ApRab7蛋白質序列分析
(三)ApRab5位於早期endocytic compartments,會促進early endosomes融合; ApRab7位於晚期、酸性endocytic compartments
(四)ApRab5和ApRab7同時存在海葵細胞細胞質(cytosolic form)及膜器(intracellular membrane)中
(五)ApRab5主要分佈在early phagosomes;
ApRab7主要分佈在late phagosomes
(六)不同控制下ApRab5和ApRab7在phagosomes上的分佈
四、討論與結論
(一)ApRab5和ApRab7與脊推動物Rab5和Rab7蛋白質序列具高度相似性
(二)在哺乳類細胞中EGFP-ApRab5分佈在EE;EGFP-ApRab7分佈在LE
(三)美麗海葵細胞質與細胞膜器皆有ApRab5和ApRab7分佈ApRab5和ApRab7參與phagocytic pathway
(四)ApRab5和ApRab7參與phagocytic pathway(五)ApRab5和ApRab7參與共生藻在腔腸生物消化細胞胞內生存機制調控
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