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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭貽生 | |
| dc.contributor.author | Pei-Hsuan Lin | en |
| dc.contributor.author | 林佩璇 | zh_TW |
| dc.date.accessioned | 2021-06-15T13:31:03Z | - |
| dc.date.available | 2016-03-08 | |
| dc.date.copyright | 2016-03-08 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-02-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51341 | - |
| dc.description.abstract | Ethylene response factors (ERFs)是一群在植物中參與生物與非生物逆境的特有轉錄因子，ERF96屬於其中分類第九族的成員，擁有一個會結合GCC box (AGCCGCC)的高保守性DNA binding domain名為AP2/ERF domain，前人研究ERF96在病原菌感染後，被大量誘導，以參與植物防禦反應，由於植物ERF蛋白之功能分析不多，本論文分析ERF96重組蛋白及其關鍵胺基酸與特定DNA序列結合，探討植物ERFs在對抗逆境時的可能分子機制。
本研究利用膠體過濾層析法與動態光散色儀分析，確認ERF96在溶液中為單體，且均勻穩定存在於溶液中。利用螢光電泳遷移率實驗分析ERF96與不同長度的GCC box親和性，結果顯示核心序列外的核酸長度與序列影響ERF96 R16A、W23A、R31A、W41A的結合能力。利用定點突變分析ERF96上的R16A、R19A、R21A、W23A、R31A、R39A、W41A與GCC box結合，經由螢光電泳遷移率實驗與偏極螢光實驗，發現單點突變仍具有結合能力，雙點突變(R19AR21A、R31AR39A)後之結合能力則大幅下降。在不同DNA序列的研究中，發現ERF96與其他非GCC box序列(P box、CS1 box)具有結合能力。總結以上結果，ERF96在溶液中，以單體存在，主要以Arg19、Arg21、Arg31、Arg39與GCC box 結合，除此之外，ERF96亦會與P box、CS1 box、DRE box結合，推測ERFs將可能參與調控相關基因的轉錄起始。 | zh_TW |
| dc.description.abstract | Ethylene response factors (ERFs) belong to a large family of plant-specific transcription factors which participate in biotic and abiotic stresses in plants. ERF96 has been classified as group IX and contains one conserved DNA binding domain named AP2/ERF domain, which specifically binds to GCC box (AGCCGCC). Previous study demonstrated that ERF96 involved in plant defense response. Since functional analysis for ERF proteins is still rare, we aim to study the molecular mechanism of ERF96 protein binding to GCC box or other non-GCC box sequences.
Size-exclusive chromatography and dynamic light scattering identified that full-length recombinant ERF96 is monomer and mono-disperse in the solution. Fluorescein-based electrophoretic mobility shift assay (fEMSA) showed the binding ability of ERF96 to different lengths of GCC box, and the results indicated that flanking region of GCC box influence binding affinity of R16A, W23A, R31A and W41A. Site-directed mutagenesis of ERF96 with R16A, R19A, R21A, W23A, R31A, R39A, and W41A indicated that there is no significant change of binding ability in ERF96 single mutation. However, the binding of ERF96 was abolished in double mutation with R19AR21A and R31AR39A. This results showed that Arg19, Arg21, Arg31, and Arg39 are the important residues for DNA binding. Furthermore, we examined binding ability of ERF96 to non-GCC box sequence, such as P box, CS1 box, and DRE box. The results indicated that ERF96 can bind to P box, CS1 box and DRE box. Taken together, recombinant ERF96 showed the monomer form in solution and Arg19, Arg21, Arg31, and Arg39 of ERF96 are the key residues to bind to GCC box DNA. In addition, ERF96 not only bound to GCC box, but also could bind to P box, CS1 box and DRE box. We proposed that ERFs transcription factor might involve in the transcription activation via non-GCC box binding ability. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T13:31:03Z (GMT). No. of bitstreams: 1 ntu-105-R02b42011-1.pdf: 5630874 bytes, checksum: b090cb89f16b371307707a509863b01b (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii 目錄 iii 圖目錄 vi 表目錄 vii 附錄目錄 viii 縮寫對照表 x 第一章 前言 1 1.1. 植物逆境反應 1 1.2. 乙烯反應因子 (Ethylene Response Factors, ERFs) 家族 2 1.3. 乙烯反應因子第九群(ERF Group IX) 3 1.4. 乙烯反應因子96 (Ethylene Response Factor 96, ERF96) 5 1.5. 乙烯轉錄因子之蛋白質結構 6 1.6. 研究目標 6 第二章 材料與方法 8 2.1. 實驗材料 8 2.2. 實驗方法 8 2.2.1. 蛋白質表現與純化 8 2.2.1.1. 勝任細胞(Competent cell)之製備 8 2.2.1.2. 轉型作用(Transformation) 8 2.2.1.3. 蛋白質大量表現與純化 9 2.2.1.4. SDS-聚丙烯醯胺膠體電泳(Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) 10 2.2.1.5. 蛋白質定量分析 11 2.2.2. 重組蛋白ERF96的多聚體組成分析 11 2.2.2.1. 膠體過濾層析法(Size-exclusion chromatography, SEC) 11 2.2.2.2. 動態光散射儀分析(Dynamic light scattering, DLS) 12 2.2.3. ERF96與DNA結合能力分析 12 2.2.3.1. 膠體過濾層析法(Size exclusion chromatography, SEC) 12 2.2.3.1.1. 雙股DNA製備 12 2.2.3.1.2. 膠體過濾層析實驗 13 2.2.3.2. 螢光電泳遷移率實驗(Fluorescein-based electrophoretic mobility shift assay, fEMSA) 13 2.2.3.2.1. 雙股DNA製備 14 2.2.3.2.2. 膠體製備 14 2.2.3.2.3. 原態聚丙烯醯胺膠體電泳(Native-PAGE) 14 2.2.3.2.4. 數據分析 15 2.2.3.3. 偏極螢光實驗(Fluorescence Polarization, FP) 15 2.2.3.3.1. 雙股DNA製備 15 2.2.3.3.2. 樣品製備 15 2.2.3.3.3. 螢光值測定 16 2.2.3.3.4. 數據分析 16 2.2.4. ERF96 ERF domain結構模型的建立 17 第三章 結果 18 3.1 ERF96蛋白質表現與純化 18 3.2 重組蛋白ERF96的均質性(homogeneity)分析 18 3.3 鹽濃度對ERF96結合能力的影響 20 3.4 利用fEMSA分析ERF96與其突變蛋白質對不同長度GCC-boxes的結合能力 21 3.5 ERF96及其突變蛋白質對GCC12的結合常數測定 23 3.6 ERF96序列結合特異性(binding specificity)分析 24 第四章 討論 26 4.1 ERF96以單體的形式均質存在於溶液中 26 4.2 不同長度的GCC boxes影響ERF96的結合能力 26 4.3 雙點突變蛋白質破壞ERF96與DNA結合的能力 27 4.4 ERF96具有結合P box與CS1 box等非GCC box序列的能力 29 第五章 結論 31 參考文獻 32 圖表 37 附錄 54 附圖 60 附表 75 | |
| dc.language.iso | zh-TW | |
| dc.subject | 生物逆境 | zh_TW |
| dc.subject | 轉錄因子 | zh_TW |
| dc.subject | 轉錄因子 | zh_TW |
| dc.subject | AP2/ERF domain | zh_TW |
| dc.subject | AP2/ERF domain | zh_TW |
| dc.subject | 生物逆境 | zh_TW |
| dc.subject | 偏極螢光 | zh_TW |
| dc.subject | 偏極螢光 | zh_TW |
| dc.subject | 螢光電泳遷移率實驗 | zh_TW |
| dc.subject | 螢光電泳遷移率實驗 | zh_TW |
| dc.subject | Fluorescence polarization | en |
| dc.subject | Biotic stresses | en |
| dc.subject | Transcription factor | en |
| dc.subject | AP2/ERF domain | en |
| dc.subject | fEMSA | en |
| dc.subject | Fluorescence polarization | en |
| dc.subject | Biotic stresses | en |
| dc.subject | Transcription factor | en |
| dc.subject | AP2/ERF domain | en |
| dc.subject | fEMSA | en |
| dc.title | 阿拉伯芥乙烯反應因子ERF96與GCC-boxes序列結合機制分析 | zh_TW |
| dc.title | DNA Binding Mechanism of Arabidopsis ERF96 to GCC boxes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄭石通,林讚標 | |
| dc.subject.keyword | 偏極螢光,螢光電泳遷移率實驗,AP2/ERF domain,轉錄因子,生物逆境, | zh_TW |
| dc.subject.keyword | Fluorescence polarization,fEMSA,AP2/ERF domain,Transcription factor,Biotic stresses, | en |
| dc.relation.page | 77 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-02-03 | |
| 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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