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研究生: 賴昱龍
論文名稱: CIA2與HAP蛋白之交互作用
指導教授: 孫智雯
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2009
畢業學年度: 97
語文別: 中文
論文頁數: 42
中文關鍵詞: CIA2HAP
論文種類: 學術論文
相關次數: 點閱:157下載:5
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  • 葉綠體是植物細胞進行光合作用重要的胞器。為了維持葉綠體的正常功能,從細胞核基因合成的葉綠體蛋白必須正確地被葉綠體膜上的轉運蛋白辨識及輸入。我們之前的研究顯示,CIA2蛋白是CCT-class的轉錄因子,可正向調控葉綠體運組基因Toc33和Toc75。本研究發現CIA2的CCT區域和HAP2序列具有相似性,且其保守胺基酸與HAP蛋白質交互作用及DNA結合有關。利用酵母菌雙雜交實驗證實CIA2和HAP5a、HAP5b及HAP5c有交互作用。檢測不同長度CIA2片段蛋白與HAP5進行酵母菌雙雜交實驗,結果顯示其N端序列和C端的CCT motif,可能與蛋白質交互作用有關。比對植物的CIA2同源蛋白發現N端序列和CCT motif皆具高度保守性,顯示CIA2的N端和CCT motif具有重要蛋白質功能。

    第一章、研究背景…………………………………………………………………0 1.1細胞核基因與葉綠體發育之關聯性 1.2葉綠體轉運蛋白 1.3 CIA2影響葉綠體蛋白之運輸 1.4 CIA2為可調控Toc33和Toc75的基因表現之轉錄因子 1.5 HAP蛋白複合體 1.6 CCT-class蛋白與HAP蛋白的交互作用 1.7 研究動機與目的 第二章、材料與方法………………………………………………………………5 1. CIA2和HAP基因選殖 1-1. 植物材料 1-2. 阿拉伯芥核醣核酸之萃取 1-3. 阿拉伯芥cDNA之合成 1-4. 聚合酶連鎖反應 1-5. 基因選殖及定序 2. 酵母菌雙雜交系統 2-1. 建構CIA2與HAP基因表現質體 2-2. 勝任細胞之製備 2-3. 酵母菌轉型 2-4. 以營養缺失培養基及β-galatosidase活性測試方法篩選蛋白質之交互作用 2-5. β-gal活性分析 3. 親緣分析及其他生物資訊軟體 第三章、結果………………………………………………………………………12 3-1. 阿拉伯芥之HAP基因家族 3-2. CIA2的CCT-motif和HAP2蛋白成員之DNA結合區域有較高的保守度 3-3. 在酵母菌中HAP5a、HAP5b、HAP5c與CIA2有蛋白質間交互作用 3-4. 在酵母菌中CIA2蛋白間有交互作用 3-5. CIA2蛋白N端區域或CCT-motif對於CIA2/HAP5之交互作用扮演重 要的角色 3-6. 植物的CIA2同源蛋白之親緣分析 第四章、討論………………………………………………………………………19 第五章、參考文獻…………………………………………………………………23表目錄 表一、本論文所使用的阿拉伯芥HAP基因專一性引子…………………………28 表二、阿拉伯芥HAP基因家族……………………………………………………29 表三、HAP與不同CIA2片段之交互作用……………………………………….30 表四、Y2H之對照組結果……………………………………………………...31 圖目錄 圖一、Y2H系統載體建構策略示意圖.............................32 圖二、阿拉伯芥CIA2 CCT-motif與HAP2成員胺基酸比對分析示意圖....33 圖三、HAP5a、HAP5b和HAP5c與CIA2蛋白交互作用檢測結果.........34 圖四、CIA2蛋白間交互作用檢測結果............................35 圖五、HAP5a與不同CIA2片段檢測組別之β-gal定量................36 圖六、HAP5b與不同CIA2片段檢測組別之β-gal定量................37 圖七、HAP5c與不同CIA2片段檢測組別之β-gal定量................38 圖八、阿拉伯芥CIA2與其他同源基因之N端區域比對分析結果.........39 圖九、CIA2以及同源蛋白親緣關係樹............................40 圖十、HAP及CIA2之突變株外表型比較. ……………………………………41 附圖目錄 附圖一、阿拉伯芥HAP2、HAP3及HAP5蛋白之親緣關係樹(取自Sifer et al., 2009)………………………………………………………42

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