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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志(Chien-Chih Yang) | |
| dc.contributor.author | Hsun-Chuan Kuo | en |
| dc.contributor.author | 郭巡銓 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:16:25Z | - |
| dc.date.copyright | 2010-08-04 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-03 | |
| dc.identifier.citation | Achard, P., Herr, A., Baulcombe, D.C., and Harberd, N.P. (2004). Modulation of floral development by a gibberellin-regulated microRNA. Development 131, 3357-3365.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22372 | - |
| dc.description.abstract | RNA 靜默作用對於植物的生長與發育都有重大的影響。RNA 靜默作用主要是由不同的小片段 RNA 與 ARGONAUTE (AGO) 家族蛋白質形成複合體來進行。阿拉伯芥中 AGO 蛋白質共有 10 種，AGO 複合體與不同的輔助蛋白質進行交互作用來產生不同的功能，包括對目標 mRNA 的降解、抑制轉譯作用、增加 DNA 和組蛋白質甲基化，以及對抗病蟲害等。本論文利用建立過量表現轉殖株、酵母菌雙雜合法和免疫共沉澱法等方式，尋找與 AGO1 或 AGO4 可能具有交互作用的蛋白質。由於載體上的設計錯誤，造成 frame-shift，本實驗得到兩種基因表現低下的突變株 ago1 與 ago4。藉由酵母菌雙雜合法，篩選到兩個與 AGO1 產生交互作用的 CHR 蛋白質，它們屬與需要 ATP 共同作用的染色質重組酵素。另外，以大腸桿菌表現 GST-AGO1 與 GST-AGO4 重組蛋白質作為釣餌，與阿拉伯芥葉片抽出的蛋白質粗抽液，進行免疫共沉澱測試，發現了數個候選蛋白質，進一步以 LC-MS/MS 進行分析，它們是分別是 clathrin binding protein (epsin)、F-box/FBD/LRR-repeat protein、short-chain dehydragenase/reductase、Ferredoxin-dependent glutamate synthase 1、Glutamine synthetase、ATP synthase subunit alpha和 Elongation factor 1-alpha。未來需進一步的分析，才能證實 AGO 與這些候選蛋白質間的交互作用。 | zh_TW |
| dc.description.abstract | Abstract
RNA silencing is important in the growth and development of plants. The major components of RNA silencing complex are different small RNA and ARGONAUTE (AGO) family. Ten AGOs are present in Arabidopsis. It is expected that different function, degrading the target transcript, inhibiting translation, methylating DNA or histone and defending against virus and insects, are achieved by AGOs in complex with different interacting proteins. In this study, putative interacting proteins of AGO1 or AGO4 were searched using the overexpression mutants, yeast two hybrid assay or co-immunoprecipitatin assay. Due to the presence of two extra bases in the vector pN-TAPa, two knock-down mutants, ago1 and ago4 were obtained instead of the expected overexpression. By yeast two hybrid, two CHR proteins, ATP-dependent chromatin remodeling enzymes, were found to interact with AGO1. Recombinant GST-AGO1 expressed in E.coli was used as bait to find interacting protein from crude extract of Arabidopsis using co-immunoprecipitation. The candidates found in this Co-IP experiment were analyzed by LC-MS/MS. They are clathrin binding protein (epsin), F-box/FBD/LRR-repeat protein, short-chain dehydragenase/reductase, Ferredoxin-dependent glutamate synthase 1, Glutamine synthetase, ATP synthase subunit alpha and Elongation factor 1-alpha. Further experiments are necessary to further investigate these putative interacting proteins. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:16:25Z (GMT). No. of bitstreams: 1 ntu-99-R97B47206-1.pdf: 4268744 bytes, checksum: 47257c04ca9aa4642b19af9fc3d35160 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 摘要 I
Abstract II 縮寫表 III 第一章 緒論 1 1.1 RNA 靜默作用 (RNA silencing) 1 1.1.1 上位基因調控作用 (epigenetic modification) 2 1.1.2 小片段 RNA (small RNA) 2 1.1.3 Argonaute 家族 (AGO family) 4 1.1.3.1 Argonaute1 蛋白質 5 1.1.3.2 Argonaute4 蛋白質 6 1.2 RNA 靜默作用路徑 6 1.2.1 轉錄後基因靜默作用 (Post-transcriptional gene silencing, PTGS) 7 1.2.2 轉錄基因靜默作用 (Transcriptional gene silencing, TGS) 7 1.3 對植物生長發展的影響 8 1.4 生物性逆境反應 9 1.5 輔助蛋白質 11 1.6 模式植物：阿拉伯芥 (Arabidopsis thaliana) 17 第二章 材料與方法 19 2.1 實驗材料 19 2.1.1 植物材料 19 2.1.2 載體 (vectors) (各圖譜見附錄二) 19 2.1.3 菌種 22 2.2 實驗藥品 23 2.2.1 一般化學藥劑 23 2.2.2 酵素 23 2.2.3 大腸桿菌培養基 23 2.2.4 酵母菌培養基 24 2.3 儀器設備 26 2.4 實驗方法 27 2.4.1 DNA 相關之基本操作 27 2.4.1.1 質體 DNA 之抽取 27 2.4.1.2 阿拉伯芥染色體 DNA 之抽取 28 2.4.1.3 小量限制酶切反應 (Restriction enzyme reaction) 28 2.4.1.4 DNA 瓊脂糖膠體電泳 28 2.4.1.5 DNA 之定量 29 2.4.1.6 Gateway cloning 29 2.4.2 RNA 之抽取與分析 30 2.4.2.1 Total RNA 之抽取 (REzol reagent method) 30 2.4.2.2 RNA 之定量 30 2.4.2.3 RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) 31 2.4.3 阿拉伯芥種植 32 2.4.3.1 培養基之製備 32 2.4.3.2 種子表面消毒及低溫處理 33 2.4.3.3 種子之無菌培養 34 2.4.3.4 種子之土壤培養 34 2.4.3.5 種子之收集 34 2.4.4 蛋白質相關之基本操作 34 2.4.4.1 蛋白質電泳檢定 34 2.4.4.1.1 SDS 膠體電泳 34 2.4.4.1.2 梯度膠體電泳 36 2.4.4.1.3 膠片染色法 37 2.4.4.1.4 膠片乾燥法 38 2.4.4.2 蛋白質轉印 38 2.4.4.3 免疫呈色 39 2.4.4.4 Bradford 蛋白質定量法 39 2.4.5 各目標基因重組蛋白質表現與純化 40 2.4.6 ARGONAUTE 轉殖株之建立 41 2.4.6.1 電穿孔轉形法 41 2.4.6.2 農桿菌感染-花序沾潤法 (Floral dip method) 41 2.4.7 ARGONAUTE1 和 ARGONAUTE 4 交互作用蛋白質之測定 42 2.4.7.1 阿拉伯芥全蛋白質之抽取 42 2.4.7.2 蛋白質免疫共沉澱法 42 2.4.7.3 LC-MS/MS 分析 43 2.4.8 酵母菌雙雜合法 44 2.4.8.1 酵母菌轉型 44 2.4.8.1.1 勝任細胞 (competent cell)的製備 44 2.4.8.1.2 質體轉形-熱衝擊法 44 2.4.8.2 配對雜合 (Mating) 45 2.4.8.3 酵母菌質體抽取 46 2.4.9 基因槍轉殖 (Particle bombardment) 47 2.4.9.1 製備洋蔥表皮細胞 47 2.4.9.2 製備 DNA 包覆鎢粒子 47 2.4.9.3 基因槍操作 48 2.4.9.4 螢光顯微鏡觀察 48 第三章 結果與討論 49 3.1 酵母菌雙雜合法篩選 49 3.1.1 AGO1 和 AGO4 基因序列選殖 49 3.1.2 獵物質體 (prey) 與釣餌 (bait) 質體的建構 50 3.1.3 對 CD4-30 cDNA 庫進行酵母菌雙雜合篩選結果 50 3.1.4 候選者身分鑑定 50 3.1.5 以 AGO1 對篩選之候選者進行一對一轉形篩選結果 51 3.1.6 雙分子螢光互補實驗 (BiFC) 分析 52 3.1.6.1 BiFC 短暫表現質體之建構 52 3.1.6.1.1 CHR1 基因序列選殖 52 3.1.6.1.2 表現用質體之建構 52 3.1.6.2 AGO1 與 CHR1 之 BiFC assay 53 3.1.7 CHR 家族蛋白質 53 3.1.7.1 AGO1 與 CHR1 交互作用的可能機制 54 3.2 阿拉伯芥轉殖株建立 54 3.2.1 轉殖用表現質體建構 54 3.2.2 篩選轉殖株 55 3.2.3 轉殖株鑑定 55 3.2.3.1 ago1-1~ago1-4轉殖株鑑定 55 3.2.3.2 ago4-1~ago4-3轉殖株鑑定 56 3.2.4 轉殖株結果與討論 56 3.3 免疫共沉澱法篩選 57 3.3.1 大腸桿菌表現質體的建構 57 3.3.2 重組蛋白質表現與純化結果分析 57 3.3.2.1 GST-AGO1 和 GST-AGO4 蛋白質表現與純化分析 58 3.3.3 蛋白質身分鑑定結果 58 3.3.3.1 分析 AGO1 電泳色帶 58 3.3.3.2 LC-MS/MS 鑑定結果 60 3.3.3.3 AGO1 候選者的功能及可能參與的機制 60 3.3.3.4 AGO4 候選者的功能及可能參與的機制 61 3.3.4 免疫共沉澱法結果討論 63 第四章 未來研究重點 65 4.1 酵母菌雙雜合法展望 65 4.2 轉殖株展望 65 4.3 免疫共沉澱法展望 66 第五章 圖與表 67 附錄 87 附錄一 AGO1 和 AGO4 之 T-DNA 同型合子突變株篩選 87 篩選結果 89 附錄二 各載體之圖譜 91 附錄三 引子序列 95 附錄四 LC-MS/MS 鑑定 AGO1 和 AGO4 電泳膠體色帶 97 參考文獻 104 問答集 113 | |
| dc.language.iso | zh-TW | |
| dc.subject | 蛋白質交互作用 | zh_TW |
| dc.subject | 阿拉伯芥 | zh_TW |
| dc.subject | ARGONAUTE | en |
| dc.subject | interactiing protein | en |
| dc.subject | Arabidopsis | en |
| dc.title | 阿拉伯芥中與 ARGONAUTE 有潛在交互作用之蛋白質研究 | zh_TW |
| dc.title | Putative interacting proteins of ARGONAUTE in Arabidopsis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇仲卿,王愛玉,陳佩燁,章為皓 | |
| dc.subject.keyword | 阿拉伯芥,蛋白質交互作用, | zh_TW |
| dc.subject.keyword | ARGONAUTE,Arabidopsis,interactiing protein, | en |
| dc.relation.page | 114 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2010-08-03 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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