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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭秋萍(Chiu-Ping Cheng) | |
| dc.contributor.author | Ning Wang | en |
| dc.contributor.author | 王寧 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:30:31Z | - |
| dc.date.copyright | 2019-08-19 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21297 | - |
| dc.description.abstract | 泛素化系統在許多生理功能具重要調控功能，而與目標蛋白高度專一結合的E3泛素接合酶 (E3 ligase)更扮演關鍵角色，然而目前大多數的E3 ligases尚未被研究。本研究室先前發現數個座落於微管且功能未知的植物RING-H2 type E3 ligases，將其命名為RING-type E3 ligase with an uncharacterized DAR1 domain (REDs)。本研究旨在了解阿拉伯芥RED1與RED2在有性生殖與逆境反應之功能。基因轉錄分析結果顯示RED1與RED2在花之表現量最高。酵母菌雙雜交分析結果顯示RED2形成homodimer需要DAR1 domain的存在，然而RED1可僅透過其RING domain與RED2形成heterodimer，且in vitro ubiquitination 結果顯示RED1與RED2可能會相互泛素化彼此。RED1與RED2之單基因缺失突變株並無生殖缺失，但其雙基因突變植物具明顯種子發育缺失，且雙基因突變植物與野生型植物之正反雜交試驗，結果顯示這兩個基因可能同時參與雌配子體與雄配子體之發育，雙基因突變植物之花粉管萌發率下降且胚株發育延遲。另外，利用阿拉伯芥原生質體搭配螢光觀察結果顯示RED1與RED2可能會影響葉部的微管動態。此外，RED1與RED2在乾旱與鹽害逆境反應可能未扮演重要的角色。以上結果指出RED1與RED2在阿拉伯芥有性生殖扮演關鍵之正向調控角色。為釐清其具體調控機制，目前正在製備各式互補回復株與將用於觀察微管動態之植物材料。我們期許目前所得之研究資訊與未來更進一步之研究有助於對E3 ligases有更完整的了解。 | zh_TW |
| dc.description.abstract | Protein ubiquitination is an important mechanism in regulating key cellular functions. The E3 ligases play critical roles in specific recognition of the targets; however, most plant E3 ligases are not characterized. We previously identified a group of uncharacterized plant microtubule-localized RING-H2 type E3 ligases, named RING-type E3 ligases with an uncharacterized DAR1 domain (REDs). This study aims to characterize roles of REDs in Arabidopsis reproduction and stress responses. Results of spatiotemporal transcriptional analyses by RT-qPCR assay showed that RED1 and RED2 highly express in flower. Yeast-two-hybrid (Y2H) assay revealed that the DAR1 domain is required for RED2 homodimerization, but not for RED1-RED2 interaction, and that RED1 and RED2 can form heterodimers via the RING domain of RED1. In addition, in vitro ubiquitination assay showed that RED1 and RED2 could ubiquitinate each other. red1red2 mutant plants, but not the single-gene mutants, displayed significant defects in seed production. Reciprocal cross assay revealed a critical role of RED1 and RED2 in both male and female gametogenesis, and the double mutants displayed defective pollen germination and ovule development. Microscope fluoresce assay showed that the properties of the microtubule in leaf might be affected in red1red2. Moreover, RED1 and RED2 may not play obvious roles in responses to drought and salinity stress. These results suggest that RED1 and RED2 play a vital positive role in Arabidopsis reproduction. To further elucidate the involved regulatory mechanisms, plant materials to be used for genetic complementation and microtubule dynamics analyses have being prepared. These results and the on-going studies are expected to help elucidate the regulatory role of these REDs in plant sexual reproduction. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:30:31Z (GMT). No. of bitstreams: 1 ntu-108-R06b42011-1.pdf: 8817949 bytes, checksum: e054d459fce8621f2a40927a932427cb (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 口試委員審定書 I
謝誌 II 摘要 III Abstract IV 常用縮寫與全名對照表 V 目錄 VII 圖目錄 XI 附錄目錄 XII 第一章 前言 1 1. 泛素化系統及其功能 1 1.1. 泛素化系統 1 1.2. 阿拉伯芥之E3 ligase 1 2. 細胞骨架與其功能 3 2.1. 細胞骨架 3 2.2. 微管結合蛋白 4 2.3. 阿拉伯芥細胞骨架與MAPs參與之功能 5 3. 植物之有性生殖 6 3.1. 植物配子體之發育 6 3.2. 阿拉伯芥配子體之調控因子 7 3.3. 阿拉伯芥花粉管萌發之調控因子 7 4. 前人研究與研究動機 8 4.1.前人研究 8 第二章 材料與方法 10 1. 植物材料簡介 10 2. 實驗用菌體與培養條件 10 3. 基因選殖常用實驗 10 3.1. 聚合酶連鎖反應 11 3.2. DNA 瓊脂糖凝膠電泳 11 3.3. DNA 純化 11 3.4. DNA 限制酶消化水解 12 3.5. TOPO® 質體構築 12 3.6. LR 重組互換反應 12 3.7. 大腸桿菌勝任細胞熱休克轉型作用 13 3.8. 電穿孔轉型作用之勝任細胞置備 13 3.9. 電穿孔轉型作用 13 3.10. 大腸桿菌質體萃取 14 4. 植物DNA萃取 14 4.1. 傳統genomic DNA萃取 14 4.2. genomic DNA 快速萃取法 14 5. 轉錄表現分析 15 5.1. 植物RAN萃取 15 5.2. 反轉錄聚合酶連鎖反應 15 5.3. 半定量RT-PCR 16 5.4. 即時定量PCR 16 6. 酵母菌雜合系統 17 6.1. 構築載體 17 6.2. 製備酵母菌勝任細胞 17 6.3. 酵母菌雙雜合系統 17 7. 體外泛素化試驗 18 7.1. 構築載體 18 7.2. 蛋白質表現 18 7.3. 蛋白質純化 18 7.4. 蛋白質定量 19 7.5. 蛋白質電泳 19 7.6. 西方墨點法 20 7.7. in vitro ubiquitination assay 20 8. 轉殖阿拉伯芥之製備與篩選 21 8.1. 阿拉伯芥基因轉殖 21 8.2. 基因轉殖植物篩選與檢驗 21 9. 植物生殖表現型檢測 21 9.1. 互交測試 21 9.2. 果莢與種子特性之檢測 22 9.3. 花粉特性檢測 22 9.4. 胚珠型態檢測 23 10. 蛋白質在阿拉伯芥原生質體之表現分析 23 10.1. 中量質體萃取 23 10.2. 製備阿拉伯芥原生質體與轉染作用 24 10.3. 螢光檢測計算 24 11. 根部反應表現型檢測 24 12. 非生物逆境測試 24 12.1. 葉面水分散失率測試 24 12.2. 乾旱逆境測試 25 12.3. 鹽害逆境測試 25 13. 生物逆境測試 25 13.1.細菌性軟腐病菌接種測試 25 13.2.真菌性灰黴病菌接種測試 26 14. 統計分析 26 第三章 結果 27 1. RED1與RED2之蛋白質交互作用片段 27 2. RED1與RED2互為受質 27 3. RED1與RED2之表現特性分析 28 4. RED1與RED2對果莢與種子發育之影響 28 5. red1red2作為父本與母本之生殖力分析 28 6. red1red2植株之花粉與胚珠發育 29 7. red1red2葉原生質體中之微管特性 29 8. RED1與RED2對根部反應之影響 29 9. RED1與RED2對逆境與病害反應之影響 30 第四章 討論 31 1. RED1與RED2可能有互相調控關係 31 2. RED1與RED2可能具調控微管功能 32 3. RED1與RED2為生殖功能之關鍵調控者 33 4. RED1與RED2之其他可能功能 36 5. 結語 37 參考文獻 38 | |
| dc.language.iso | zh-TW | |
| dc.title | 阿拉伯芥E3 ligase RED1與RED2 在有性生殖與逆境反應之研究 | zh_TW |
| dc.title | Studying RED1 and RED2 in reproduction and stress responses in Arabidopsis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄭貽生(Yi-Sheng Cheng),王雅筠(Ya-Yun Wang),林盈仲(Ying-Chung Lin),靳宗洛(Tsung-Luo Jinn) | |
| dc.subject.keyword | RED,泛素化系統,E3 ligase,配子體發育,微管, | zh_TW |
| dc.subject.keyword | RED,ubiquitination,E3 ligase,gametogenesis,microtubule, | en |
| dc.relation.page | 111 | |
| dc.identifier.doi | 10.6342/NTU201903530 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2019-08-14 | |
| 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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