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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭秋萍(Chiu-Ping Cheng) | |
dc.contributor.author | Yi-Syue Ho | en |
dc.contributor.author | 何宜學 | zh_TW |
dc.date.accessioned | 2021-06-16T05:11:54Z | - |
dc.date.available | 2019-09-02 | |
dc.date.copyright | 2014-09-02 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-18 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55968 | - |
dc.description.abstract | 泛素化系統在許多植物功能之調控路徑中具關鍵功能,而其中與目標蛋白質結合之E3泛素接合酶 (E3 ligase) 更扮演極重要之決定角色。我們先前在番茄及阿拉伯芥中發現一群功能未知之RING-H2 type E3 ubiquitin ligase,將之命名為RING-type E3 ubiquitin ligases with an uncharacterized DAR1 domain (REDs),並確認番茄Sl_RED1是一坐落於細胞骨架微管上的泛素接合酶。本研究旨在探討番茄SlRED1及其同源基因之蛋白質特性及功能,並尋找REDs之交互作用蛋白質。結果顯示,SlRED1、SlRED2、AtRED1、AtRED2皆坐落於微管上,而SlRED1之胺基酸片段1-60為其坐落於微管之必要片段。此外,位於SlRED1 C端的RING domain與DAR1 domain則為與E2進行直接結合之關鍵片段。另外,過量表現SlRED1的菸草(Nicotiana benthamiana)轉殖株對青枯病菌(Ralstonia solanacearum)與細菌性軟腐病原細菌(Pectobacterium carotovorum subsp. carotovorum)的抗性提高,但不影響植物葉部氣孔密度與水份散失率。以上結果顯示,REDs為一群坐落於微管之新穎E3泛素接合酶,且SlRED1在植物對抗病原細菌具正向功能。目前正積極搜尋REDs之交互作用蛋白質,以期釐清RED之作用機制。 | zh_TW |
dc.description.abstract | Ubiquitin E3 ligases are highly complex regulators and play a pivotal role in plant signaling transduction pathways switching extracellular signals into various cellular responses. In previous studies, we identified a group of plant RING-H2 type E3 ubiquitin ligases with an uncharacterized DAR1 domain (REDs) and our data indicated that SlRED1 is a microtubule-localized E3 ligase. The objective of this study was to characterize these REDs and identify their interacting proteins. The results show that SlRED1, SlRED2, AtRED1 and AtRED2 are co-localize on the microtubules and the N-terminal region (amino acid 1~60) is required for the microtubular localization of SlRED1. In addition, the RING and DAR1 domains are both important for the interaction between SlRED1 and E2 conjugating enzyme. Furthermore, the overexpression of SlRED1 in transgenic Nicotiana benthamiana plants led to enhanced resistance to the infection of R. solanacearum and Pectobacterium carotovorum pv. carotovorum, but had no effect on leaf stomata number and transpiration. Collectively, these results indicate that REDs define a new group of plant microtubule-localized E3 ligases and that SlRED1 plays a positive role in resistance to pathogenic bacteria. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:11:54Z (GMT). No. of bitstreams: 1 ntu-103-R01b42009-1.pdf: 3993070 bytes, checksum: da32299b60afa94c969151f69523d93b (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員審定書 ii
謝誌 iii 縮寫與全名對照表 v 中文摘要 1 Abstract 2 第一章 前言 3 1. 泛素化系統與參與功能 3 2. 細胞骨架 4 3. 植物抗病機制 5 4. 青枯病(Bacterial wilt) 6 5. 植物對青枯病害之研究 7 6. 細菌性軟腐病及相關抗病研究 8 7. 植物病害與非生物逆境之交互作用 8 8. 研究動機 10 第二章 材料與方法 11 1. 實驗菌株、質體及菌株培養條件 11 2. 試驗植物培育條件 11 3. 生物統計分析 11 4. 基因選殖常用實驗 重組質體構築與過量表現菌株建構 11 4.1. DNA 瓊脂糖凝膠電泳 (Agarose Gel Electrophoresis) 12 4.1.1. 外染瓊脂糖凝膠電泳分析: 12 4.1.2. 內染瓊脂糖凝膠電泳純化: 12 4.2. 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 12 4.3微量抽取質體DNA(Minipreparation of plasmid DNA) 13 4.4限制酶剪切作用(Digestion) 13 4.5 DNA片段純化 14 4.6載體與目標片段接合(Ligation) 14 4.7 TOPOR cloning (Invitrogen) 14 4.8大腸桿菌勝任細胞之轉型作用(Transformation of E. Coli by heat-shock)15 4.9農桿菌勝任細胞製備 15 4.10農桿菌電穿孔法(Transformation of A. tumefaciens by electroporation15 5. 螢光重組蛋白質之定位分析(sub-cellular localization) 15 5.1構築載體 16 5.2 阿拉伯芥原生質體製備 16 5.3 菸草原生質體製備 16 5.4細胞骨架抑制劑相關處理 17 6. 酵母體單雜交(Yeast one-hybrid) 17 6.1製備實驗所需質體 17 6.2製備酵母菌勝任細胞 18 6.3酵母菌雜合系統(Yeast two-hybrid system) 18 7. 植物RNA萃取 18 7.1 RNeasy Mini Kit (Qiagen, Hilden, Germany) 19 7.2 DNA 消化水解作用 (DNase treatment) 19 8. 反轉錄聚合酶連鎖反應 (Reverse transcription PCR, RT-PCR) 20 8.1 半定量RT-PCR (Semi-quantitative RT-PCR) 20 9. 生物逆境測試 21 9.1 R.Solanacerum對煙草之毒力測試(Virulence assay) 21 9.2菸草植株青枯病菌量濃度檢測 21 9.3煙草接種細菌性軟腐病菌測試 22 10. 非生物性逆境測試 22 10.1菸草水分散失率測試 22 10.2菸草葉片 (leaf-disc) 之氧化逆境測試 22 11. 植物生理檢測 22 11.1葉綠素含量測量 23 11.2 離子流失測試(electron leakage assay) 23 第三章 結果 24 1. REDs在植物細胞中之蛋白質座落位置之分析 24 2. 探討影響SlRED1微管坐落特性之蛋白質區域 24 3. SlRED1與E2之交互作用關係 25 4. SlRED1在植物病害反應之功能分析 26 5. SlRED1與氣孔調節及生長發育性狀 26 6. 氧化逆境測試 27 7. 植物賀爾蒙及相關基因檢測 27 8. RED作用蛋白對象之釣取 27 第四章 討論 29 1. REDs蛋白坐落於細胞骨架之微管 29 2. SlRED1以其C端與E2接合 30 3. SlRED1基因在不同病害扮演不同角色 31 4. SlRED1與植物非生物性逆境相關反應 33 5. REDs之調控與蛋白質特性 34 6. 總結 34 第五章 未來展望 35 第六章 參考文獻 37 | |
dc.language.iso | zh-TW | |
dc.title | 新穎微管E3 ligase RED 基因群之功能分析 | zh_TW |
dc.title | Characterization of microtubule-associated RING E3 Ligase with DAR1 Domain (REDs) | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 謝旭亮(Hsu-Liang Hsieh),邱子珍(Tzyy-Jen Chiou),葉國楨(Kuo-Chen Yeh),符宏勇(Hong-yong Fu) | |
dc.subject.keyword | 泛素化系統,E3 ligase,微管,青枯病, | zh_TW |
dc.subject.keyword | ubiquitination,E3 ligase,microtubule,bacterial wilt, | en |
dc.relation.page | 87 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-19 | |
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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