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研究生:阮子軒
研究生(外文):Tzu-Hsuan Juan
論文名稱:百合LMADS1同質雙倍體形成的結構研究
論文名稱(外文):Structural studies on lily LMADS1 homo-dimerization
指導教授:劉俊宏劉俊宏引用關係
指導教授(外文):Jyung-Hurng Liu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:78
中文關鍵詞:LMADS1重組蛋白重新摺疊純化
外文關鍵詞:LMADS1recombinant proteinrefoldprufication
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中文摘要


植物花器的形成是由 MADS box 蛋白質所控制,這類轉錄因子具有能與特定 DNA 序列結合的 MADS box domain,以及與蛋白質相互作用有關的 Keratin-like domain (K domain)。LMADS1 是百合 (Lilium longiflorum) 中的 MADS box 轉錄因子,全長為228個胺基酸。不同於擬南芥的同源基因 AP3 會形成異質二聚體 (Hetrodimer),LMADS1 形成同質二聚體 (Homodimer) 來調控花器的發育。為了更加了解 LMADS1 二聚體形成的機制,本研究嘗試解析出 LMADS1 之晶體結構。我們以大腸桿菌作為宿主大量表現 LMADS1 蛋白。因 LMADS1 無法以可溶型式存在於大腸桿菌內,我們將 LMADS1 重組蛋白重新摺疊再進行純化,而後著手 LMADS1 的結晶條件篩選。因先前研究指出 K domain 的突變會影響 LMADS1 雙倍體的形成,本研究同時構築 LMADS1 的 K domain 重組蛋白基因,並送入大腸桿菌內進行大量表現。此 K domain 重組蛋白在宿主內為可溶蛋白。目前我們已將純化後的 K domain 蛋白進行結晶條件的篩選。同時我們也利用分析級超高速離心技術證實 K domain 在in vitro 的環境中是以二聚體的形式存在。
前言 1
開花誘導 1
花器形成分子機制 1
MIKC-type MADS-box 蛋白質 3
LMAD1 蛋白質 4
研究目的 5
材料與方法 6
材料 6
實驗方法 6
大腸桿菌的轉型 6
液態培養 8
質體 DNA 萃取 8
蛋白質表現量測試 9
蛋白質可溶性測試 9
SDS 凝膠電泳分析 10
蛋白質濃度測定 13
蛋白質透析 13
蛋白質濃縮 13
分析級超高速離心技術 (analytical ultracentrifugation, AUC) 14
電泳流動性轉移分析 (eletrophoretic mobility shift assay, EMSA) 15
西方墨跡法 (Western Blotting) 16
TA cloning 19
瓊脂凝膠膠片之配製 19
DNA 之回收與純化 20
PCR 產物的純化回收 20
重組DNA之構築 20
定點突變 22
實驗結果 23
LMADS1 全長蛋白質之表現與可溶性分析 23
LMADS1 全長蛋白質之純化 23
利用 AUC 分析 LMADS 1 全長蛋白質形成二聚體的能力 24
利用 Cross-linking 分析 LMADS1 全長蛋白質形成二聚體的能力 25
利用 EMSA 分析 LMADS1 全長蛋白質的 DNA 結合能力 26
LMADS1 全長蛋白質之結晶條件篩選 27
LMADS1 K domain 之構築 27
LMADS1 K domain 之表現及可溶性分析 28
LMADS1 K domain 之純化 28
利用 AUC 分析 LMADS1 K domain 形成二聚體的能力 29
LMADS1 K domain之結晶條件篩選 30
構築LMADS1 K domain的突變株 30
討論 32
LMADS1 全長蛋白質的表現及純化 32
LMADS1 全長蛋白質形成二聚體的能力 33
LMADS1 全長蛋白質的 DNA 結合能力 34
LMADS1 全長蛋白質之結晶條件篩選 35
LMADS1 K domain 蛋白質的表現及純化 35
LMADS1 K domain 蛋白質形成二聚體的能力 35
LMADS1 K domain 之結晶條件篩選 36
結論 37
參考文獻 38
實驗圖表 41
附圖 72
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