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研究生:蔡榮宗
研究生(外文):Rong-Tzong Tsai
論文名稱:十字花科黑腐病菌第二型分泌系統的內胞膜蛋白XpsL,-M,-N形成一個可分合的複合體
論文名稱(外文):A reversibly dissociable ternary complex formed by XpsL, -M, -N of the Xanthomonas campestris pv. campestris type II secretion apparatus
指導教授:胡念台
指導教授(外文):Nien-Tai Hu
學位類別:博士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:59
中文關鍵詞:動態平衡複合體第二型分泌系統膜蛋白純化膜混合十字花科黑腐病菌內胞膜蛋白
外文關鍵詞:dynamic complexgeneral secretory pathwaytype II secretionXpsLXpsMXpsN
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本研究針對十字花科黑腐病菌第二型分泌系統中三個內胞膜蛋白XpsL, -M, -N間的相互作用進行分析。首先,以Ni-NTA管柱層析法部份純化包含XpsMh蛋白的複合體,免疫分析指出XpsL與XpsN因 XpsMh蛋白而被共純化。以免疫共沉澱分析共純化的XpsL與XpsN蛋白,發現此二蛋白彼此形成複合體。綜上所述,推論XpsL, -M, -N蛋白會形成一個複合體。由於在其他Xps蛋白不存在的遺傳背景之下仍可測得XpsL-M-N複合體,顯示XpsL-M-N複合體的形成可能不需要其他Xps蛋白的協助。然而將經Ni-NTA管柱層析部份純化的蛋白複合體進一步通過分子篩層析管柱後,XpsN蛋白不再與XpsL-Mh共純化,顯示XpsN蛋白易於自複合體解離。進一步將XpsL-Mh複合體及XpsMh蛋白分別固定在Ni-NTA層析管柱上,比較XpsN蛋白與它們的結合能力,結果指出XpsN與XpsL-Mh複合體的結合力明顯大於XpsMh蛋白。膜混合的實驗則顯示,XpsN蛋白與XpsL-Mh複合體在膜內可能會發生解離及再結合。比較XpsL蛋白自XpsL-Mh-N複合體或自XpsL-Mh複合體解離的速率,結果指出在XpsN蛋白存在下,XpsL蛋白的解離速率遠低於XpsN蛋白不存在時,說明XpsN蛋白有穩定XpsL-Mh複合體的功能。膜混合的實驗顯示,XpsL與XpsMh的解離及再結合也可能發生在膜內。基於以上的結果,我們對XpsL, -M, -N三者間的關係提出一個動態平衡的相互作用假說。

The cytoplasmic membrane proteins XpsL, -M, -N are components required for type II secretion in Xanthomonas campestris pv. campestris. We performed metal chelating chromatography to partially purify the XpsMh-containing complex. Immunoblot analysis revealed that both XpsL and -N co-eluted with XpsMh. The co-fractionated XpsL and -N proteins co-immune precipitated with each other, suggesting the existence of an XpsL-M-N complex. Ternary complex formation does not require other Xps protein components of the type II secretion apparatus. Further purification upon size exclusion chromatography revealed that XpsN is prone to dissociate from the complex. Reassociation of XpsN with the XpsL-Mh immobilized on a nickel column is more effective than with XpsMh. Membrane-mixing experiments suggested that the XpsL-Mh complex and XpsN probably dissociate and reassociate in the membrane vesicles. Comparison of the half-lives of XpsL-Mh-N and XpsL-Mh complexes revealed that XpsL dissociates from the latter at a faster rate than from the former. Dissociation and reassociation between XpsL and -M were also demonstrated with membrane mixing experiment. A dynamic model is proposed for the XpsL-M-N complex.

中文摘要 …………………………………… 1
英文摘要 …………………………………… 3
緒論 ………………………………………… 4
材料與方法 ………………………………… 16
結果 ………………………………………… 22
討論 ………………………………………… 30
參考文獻 …………………………………… 38
圖表 ………………………………………… 44
附錄 ………………………………………… 56

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