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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭貽生(Yi-Sheng Cheng) | |
| dc.contributor.author | Wei Chen | en |
| dc.contributor.author | 陳葳 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:17:21Z | - |
| dc.date.available | 2016-09-16 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-19 | |
| dc.identifier.citation | 簡嘉佑 (2014) 阿拉伯芥PIF3與G-box序列結合的分子機制之研究。國立台灣大學植物科學研究所碩士論文。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49146 | - |
| dc.description.abstract | PHYTOCHROME INTERACTING FACTOR3(PIF3)是植物體內和光訊息傳導有關的重要轉錄因子，透過負調控光訊息傳遞，使植物在黑暗下抑制葉綠素生合成、光合作用及光型態發生等反應。PIF3參與在負調控光訊息傳遞路徑，惟其與上游啟動子的結合機制尚不清楚，本研究透過前人模擬結構及生物物理測定，探究PIF3 DNA結合區與DNA之結合機制。
以單點突變實驗分析各胺基酸位點對二聚體形成的影響，將疏水性胺基酸Leu392、Tyr388、Leu366及Met363個別突變後以膠體過濾層析結果顯示，bHLH會凝集成一巨大分子，失去原有二聚體構型；Met399及Lys362突變後仍能維持原有二聚體狀態。利用螢光電泳遷移實驗(Fluorescein-based electrophoretic mobility shift assay; fEMSA)及等溫滴定微熱量法(Isothermal titration calorimetry; ITC)測量bHLH與G-box DNA(CACGTG)專一性結合能力，將bHLH與DNA鍵結的胺基酸His348、Glu352、Arg355及Arg356，分別突變成Alanine後使得與DNA結合常數上升約1.5至3倍。為測試G-box (CACGTG) 與E-box (CANNTG) 結合差異由R356負責，因此以R356A與四種不同序列之E-box (CANNTG)進行螢光標定電泳遷移實驗，結果四種E-box DNA與R356A的結合常數皆相近，顯示R356作為負責辨認G box核心CG序列的重要功能。最後結構生物學的部分以蛋白質共結晶的方式，篩選出兩種結晶試劑再進行溶液微調，希望透過X-ray繞射分析能解出蛋白質複合體之結構。 | zh_TW |
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| dc.description.tableofcontents | 中文摘要 v
Abstract vi 縮寫對照表 vii 第一章 前言 - 1 一、光敏素(Phytochromes)訊息傳遞路徑 - 2 二、PHYTOCHROME INTERACTING FACTOR3(PIF3)簡介 - 4 三、Basic helix-loop-helix (bHLH)轉錄因子家族 - 5 四、植物之Basic helix-loop-helix (bHLH)轉錄因子家族 - 6 五、各物種相關Basic helix-loop-helix (bHLH)蛋白質之結構 - 7 六、研究目標 - 8 第二章 材料與方法 - 9 一、 材料 - 9 二、 方法 - 9 (一) bHLH點突變基因之構築 - 9 1. bHLH domain 點突變H348A、E352A、R356A、R356A、K362A、M363A、L366F、Y388A、L392H、M399A 之DNA構築 - 9 (二) bHLH蛋白質之表現與純化 - 9 1. 大腸桿菌勝任細胞之製備 - 9 2. 大腸桿菌之轉形作用(Transformation) - 10 3. 重組蛋白質之大量表現(Over-expression)及純化 - 10 4. 蛋白質濃縮以及蛋白質緩衝液置換 - 11 5. 陽離子交換法 - 12 6. 蛋白質定量分析 - 12 7. SDS-聚丙烯醯胺膠體電泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) - 12 (三) bHLH蛋白質形成二聚體之重要胺基酸分析 - 13 1. 膠體過濾層析法(Gel filtration chromatography) - 13 (四) bHLH蛋白質與突變蛋白質之熱穩定性測定 - 14 1. 差異螢光掃描法(Differential scanning fluorimetry, DSF) - 14 (五) bHLH domain對 DNA結合能力之分析 - 14 1. 螢光電泳遷移實驗(Fluorescein-based electrophoretic mobility shift assay) - 14 1.1 雙股DNA製備 - 14 1.2 蛋白質樣品製備 - 15 1.3 原態聚丙烯醯胺膠體電泳(Native-PAGE) - 15 2. 等溫滴定微熱量法(Isothermal titration calorimetry; ITC) - 15 2.1 雙股DNA製備 - 16 2.2 蛋白質樣品製備 - 16 2.3 ITC200儀器操作 - 17 2.4 數據分析方式 - 17 (六) PIF3 bHLH domain蛋白質結晶實驗 - 18 1. Pre-crystallization test (PCT測試) - 18 2. bHLH蛋白質與雙股G-box DNA複合體之結晶條件篩選 - 18 3. X-ray繞射晶體數據搜集 - 19 4. 蛋白質結晶溶液之微調 - 19 (七) bHLH蛋白質之序列比對及結構模擬 - 19 1. PIF3 bHLH domain與其他bHLH家族成員之胺基酸序列比對- 19 2. PIF3 bHLH domain與DNA之複合體模型建立 - 19 第三章 結果 - 20 一、bHLH蛋白質進行陽離子交換實驗 - 20 二、PIF3 bHLH domain形成二聚體之重要胺基酸分析 - 20 (ㄧ)模擬bHLH蛋白質形成二聚體之重要胺基酸結構 - 20 (二)膠體過濾層析法分析不同突變bHLH蛋白質在溶液中多聚體形式 - 21 三、bHLH蛋白質與突變蛋白質之熱穩定性測定 - 21 四、 PIF3 bHLH domain之不同突變蛋白質與G-box DNA之結合能力分析 - 22 (ㄧ)模擬bHLH蛋白質與DNA之重要胺基酸結構 - 22 (二)利用螢光電泳遷移率實驗分析不同蛋白質與G-box DNA之親和性 - 22 (三)利用等溫滴定微熱量法(ITC)分析不同蛋白質和DNA之親和性 -23 五、 PIF3 bHLH 之突變蛋白質R356A與不同E-box DNA結合能力分析 - 23 六、bHLH蛋白質之晶體篩選 - 24 第四章 討論 - 25 一、 PIF3 bHLH蛋白質進行陽離子交換實驗 - 25 二、PIF3之bHLH domain序列分析及蛋白質結構模擬探討- 26 三、 以膠體過濾層析法(GF)分析突變蛋白質多聚體組成之形式 -26 四、 bHLH蛋白質與突變蛋白質之熱穩定性探討 - 26 五、 以不同結合力試驗分析bHLH蛋白質與DNA之結合能力- 27 六、 以fEMSA及ITC分析G-box DNA與不同突變bHLH蛋白質之結合能力 - 28 七、PIF3 bHLH domain之突變蛋白質R356A與E-box DNA結合能力 -29 第五章 結論 - 30 參考文獻 - 31 圖表 - 36 表一、PIF3之bHLH domain以DSF法測得之變性溫度(Tm) - 36 表二、不同bHLH蛋白質以ITC分析對G-box DNA結合能力之熱力學參數 - 37 表三、ITC分析R356A蛋白質與不同E-box DNA之結合常數- 38 圖一、bHLH蛋白質大量表現及純化結果 - 39 圖二、陽離子交換法分離出具有功能的bHLH蛋白質 - 40 圖三、bHLH蛋白質形成二聚體形式之重要胺基酸分析 - 42 圖四、標準分子量之蛋白質膠體過濾色層分析 - 43 圖五-1、膠體過濾層析法分析bHLH蛋白質在溶液中的多聚體形式 -44 圖五-2、膠體過濾層析法分析不同突變bHLH蛋白質在溶液中的多聚體形式 - 45 圖六、bHLH蛋白質與G-box DNA結合之重要胺基酸分析 - 46 圖七、fEMSA測定bHLH-WT蛋白質與G-box DNA結合能力 - 48 圖八、ITC測定bHLH蛋白質與G-box DNA結合能力 - 50 圖九、PIF3 bHLH domain之突變蛋白質R356A與E-box DNA結合能力分析 - 52 圖十、ITC測定bHLH-R356A蛋白質與不同E-box DNA結合分析- 53 圖十一、篩晶實驗得到之bHLH與雙股DNA複合體結晶 - 54 附錄 - 55 附錄一、bHLH domain單點突變所使用之引子(primer) - 55 附錄二、單點突變之聚合酶連鎖反應參數設定 - 56 附錄三、PIF3之bHLH domain重組蛋白純化之試劑 - 56 附錄四、陽離子交換法之試劑 - 57 附錄五、SDS 膠體電泳之試劑 - 58 附錄六、fEMSA之試劑 - 59 附錄七、探針序列列表 - 60 附錄八、ITC之試劑 - 60 附錄九、蛋白質結晶濃度之PCT試劑 - 61 附錄十、蛋白質養晶實驗母液成分微調整理 - 62 附圖一、阿拉伯芥中PIFs家族成員 - 63 附圖二、阿拉伯芥PIF3 之bHLH domain構築於表現載體pET21b- 64 附圖三、bHLH蛋白質之多聚體組成分析 - 65 附圖四、差異性螢光掃描法 (Differentail scanning fluorimetry, DSF) - 66 附圖五、坐式蒸氣擴散法示意圖 - 67 附圖六、利用序列分析探討bHLH domain之保守性胺基酸- 68 附圖七、植物bHLH domain序列相似性分析 - 69 | |
| dc.language.iso | zh-TW | |
| dc.title | 阿拉伯芥 PIF3 之bHLH 功能區結構與功能分析 | zh_TW |
| dc.title | Strucutral and Functional Analyses of bHLH Domain of
Arabidopsis PIF3 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張孟基(Men-Chi Chang),張世宗(Shih-Chung Chang),詹明才(Ming-Tsair Chan),吳克強(Keqiang Wu) | |
| dc.subject.keyword | 光敏素調控因子, | zh_TW |
| dc.subject.keyword | PIF3,bHLH domain, | en |
| dc.relation.page | 69 | |
| dc.identifier.doi | 10.6342/NTU201602954 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-20 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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