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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志(Jian-Zhi Yang) | |
| dc.contributor.author | Shih-Hsin Hsueh | en |
| dc.contributor.author | 薛仕昕 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:31:38Z | - |
| dc.date.copyright | 2009-10-28 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-10-10 | |
| dc.identifier.citation | Aasland R, Gibson TJ, Stewart AF (1995) The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem Sci 20: 56-59.
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Nature 416: 703-709. Zimmermann R, Werr W (2005) Pattern formation in the monocot embryo as revealed by NAM and CUC3 orthologues from Zea mays L. Plant Mol Biol 58: 669-685. 黃勻彥. (2008) 阿拉伯芥泛素化修飾分析方法之建立與非生物逆境相關RING-型泛素黏合 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22874 | - |
| dc.description.abstract | RING蛋白質在阿拉伯芥中是一個相當大的家族，它們具有相當廣泛的功能。許多RING蛋白質與泛素/26S蛋白酶體系統有關，其中有一部分為泛素黏合酶 (E3)，能夠利用RING 區塊直接和泛素銜接酶 (E2) 作用。同時，這些RING E3 也能夠專一性地辨認可被E2 泛素化的受質。在本篇論文，我們利用微陣列資料庫找出一些阿拉伯芥中非生物逆境相關的RING E3，並利用它們當做釣餌來對cDNA庫進行篩選。使用酵母菌雙雜合系統對阿拉伯芥花序組織cDNA 庫進行篩選時，發現其中一個RING E3－R2 (at5g27420) 可能和一個NAC 家族蛋白質－NTL11(At5g04410)－具有作用性。同時，當NTL11 蛋白質融合上GAL4-DNA binding 區塊，會表現自我活化；顯示NTL11 可能如前人研究所預測為轉錄活化子。進一步的研究也顯示，R2 可以和去NAC 區塊的NTL11 具有作用性，而本實驗室先前的研究也顯示AtMAPR4 能和去NAC 區塊的NTL11 具有作用性。R2、NTL11與 AtMAPR4 在阿拉伯芥扮演的生理角色，仍須進一步確認。 | zh_TW |
| dc.description.abstract | RING proteins are defined as quite a big family in Arabidopsis, and their functions are varied. Many of RING proteins are involved in ubiquitin/26S proteasome system, some of them are ubiquitin ligases (E3s) which can interact directly with ubiquitin-conjugating enzyme (E2) by their RING domains. These RING E3s can also specifically indentify substrates that are going to be ubiquitinated by E2. In this study, we chose RING E3s related to abiotic stress in Arabidopsis based on microarray database and use them as baits to screen Arabidopsis library cDNA. Employing Yeast-two hybrid technique for cDNA (CD4-30) library screening from influoroscence meristem, floral meristem and floral buds up to stage 8 or 9, we indentified a RING E3, R2 (at5g27420), may have interaction with a NAC family protein, NTL11 (At5g04410). NTL11 also showed self-activation when it was fused to GAL4-DNA binding domain, implicating NTL11 may be a transcription factor as previous predictions from other studies. In further test, R2 can interact with NTL11 truncation without NAC domain and so does AtMAPR4 from previous study. Their roles in Arabidopsis physiological pathway need more investigations. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:31:38Z (GMT). No. of bitstreams: 1 ntu-98-R96b47213-1.pdf: 4949247 bytes, checksum: bf7775bdf5430fecdc490f1cea415d5f (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 目錄 I
縮寫表 VI 摘要 VIII Abstract IX 第一章 緒論 1 1.1 模式植物：阿拉伯芥 (Arabidopsis thaliana) 1 1.2 泛素化修飾 1 1.2.1 Ubiquitin-activating enzyme (E1) 3 1.2.2 Ubiquitin-conjugating enzyme (E2) 3 1.2.3 Ubiquitin ligase (E3) 4 1.3 RING domain (Really interesting new gene domian) 6 1.4 酵母菌雙雜合系統 (Yeast-two hyrid system) 8 1.4.1 酵母菌雙雜合系統的發展 8 1.4.2 酵母菌雙雜合系統簡介 9 1.4.2.1 一般操作：一對一轉型與營養篩選 9 1.4.2.2 由基因庫篩選有潛力的獵物基因 12 1.4.2.3 偽陽性反應的排除 12 1.5 研究動機與方向 14 第二章 材料與方法 15 2.1 儀器設備 15 2.2 實驗材料 16 2.2.1 大腸桿菌菌株 16 2.2.2 酵母菌菌株 17 2.2.3 大腸桿菌載體 17 2.2.4 Gateway cloning 使用載體 18 2.2.5 酵母菌雙雜合系統表現載體 21 2.2.6 酵母菌雙雜合篩選所使用的cDNA庫：CD4-30 23 2.3 培養基 24 2.3.1 大腸桿菌用培養基 24 2.3.2 酵母菌用培養基 25 2.4 基本DNA操作 27 2.4.1 DNA定量 27 2.4.2 瓊脂糖膠片電泳 27 2.4.3 質體DNA的製備 28 2.4.4 DNA聚合酶鏈鎖反應 (Polymerase chain reaction,PCR) 30 2.4.5 DNA 限制酶切割反應 32 2.4.6 DNA Gel Extraction 32 2.4.7 DAN A-tailing 33 2.4.8 DNA 接合反應 33 2.5 Cloning 與大腸桿菌轉型 33 2.5.1 Cloning 方法 34 2.5.1.1 一般Cloning 34 2.5.1.2 T-A Cloning 34 2.5.1.3 Gateway Cloning 35 2.5.2 大腸桿菌轉型 36 2.5.2.1 熱衝擊法 36 2.6 基本蛋白質操作 36 2.6.1 Bradford 蛋白質定量 36 2.6.2 蛋白質電泳檢定 36 2.6.3 蛋白質電泳膠片的CBR 染色與膠片乾燥保存 38 2.6.3.1 CBR 染色 38 2.6.3.2 膠片乾燥保存 39 2.6.4 蛋白質轉印 39 2.6.5 蛋白質免疫呈色 39 2.7 酵母菌雙雜合系統實驗操作 41 2.7.1 一般酵母菌雙雜合實驗流程 41 2.7. 2 酵母菌雙雜合實驗的控制組與生長狀況結果判斷 42 2.7.3 利用酵母菌雙雜合系統對花序組織cDNA 篩選流程 43 2.7.4 酵母菌轉型 45 2.7.4.1 酵母菌高效率轉型 45 2.7.4.2 酵母菌質體共轉型 (co-transformation) 與單一轉型 (sequential transformation) 46 2.7.4.3 轉型效率確認 46 2.7.5 以等張鹽水懸浮候選菌落再進行營養測試 47 2.7.6 酵母菌質體DNA 抽取 47 2.7.7 ONPG 試驗 48 2.7.7.1 ONPG 試驗流程 48 2.7.7.2 β-galactosidase units 計算 49 2.7.8 Colony Filter-lift 試驗 49 2.8 建構目標RING 基因到保存載體以及酵母菌雙雜合系統載體 50 2.8.1 本篇使用的RING 基因 50 2.8.2 建構RING 基因到保存載體 51 2.8.3 建構RING 基因到酵母菌雙雜合系統載體 51 2.8.3.1 建構RING 基因到pGBKT7 質體 51 2.8.3.2 建構RING 基因到pGADT7 質體 51 2.9 本篇酵母菌雙雜合實驗條件 52 2.9.1 RING 2 基因進行酵母菌雙雜合實驗 52 2.9.1.1 RING 2 基因進行篩cDNA 庫實驗處理 52 2.9.1.2 RING 2 候選菌落再接種處理與定序 52 2.9.1.3 RING 2 基因與候選基因進行一對一營養生長測試處理 53 2.9.2 RING 3 基因進行酵母菌雙雜合實驗處理 53 2.9.2.1 RING 3 基因進行篩cDNA 庫實驗處理 53 2.9.2.2 RING 3 候選菌落再接種處理與定序 53 2.9.2.2 RING 3 基因與其候選基因進行一對一營養生長測試處理 54 2.9.3 R2 對NLT11 Truncation 進行一對一轉型相關實驗 54 2.9.3.1 NLT11 Truncation 54 2.9.3.2 NTL11 truncations 與R2 進行一對一營養生長測試 54 2.9.3.2 NTL11 truncations 與R2 進行ONPG assay 55 2.9.3.3 NTL11 truncations 與R2 進行filter-lift assay 55 2.10 GST-R2 與6x His- NTL11 融合蛋白質的表現與純化 55 2.10.1 GST-R2 的表現與純化 55 2.10.2 6x His- NTL11 的表現與純化 56 第三章 結果與討論 58 3.1 RING 基因在T vactor 保存載體的建構與確認 58 3.2 RING 基因在pGBKT7/pGADT7 酵母菌表現載體的建構與確認 59 3.2.1 RING 基因在pGBKT7 載體的建構與確認 59 3.2.2 RING 基因在pGADT7 載體的建構與確認 59 3.3 RING 基因在Gateway 系統載體的建構與確認 59 3.3.1 pDONR221-R2 的確認 59 3.3.2 pDEST15-R2 的確認 59 3.3.3 pDEST17-R2 的建構 60 3.3.4 pDONR221-NTL11 的建構 60 3.3.5 pDEST15-NTL11 的建構 60 3.3.6 pDEST17-NTL11 的建構 60 3.4 以R2 對CD4-30 基因庫進行酵母菌雙雜合系統篩選結果 61 3.4.1 R2 篩選實驗的轉型效率 61 3.4.2 R2 篩選所得之候選菌落以及定序後基因確認 61 3.4.3 候選基因與R2 進行一對一生長測試 61 3.5 以R3 對CD4-30 基因庫進行篩選結果 62 3.5.1 R3 篩選實驗的轉型效率 62 3.5.2 R3 篩選所得之候選菌落以及定序後基因確認 63 3.5.3 候選基因與R3 進行一對一生長測試 63 3.6 R2 與NTL11 在篩選實驗後的domain 預測 64 3.6.1 NTL11 65 3.6.2 設計不同長度的NTL11 片段 65 3.6.3 R2 66 3.6.4 R2 的Domian 預測 66 3.7 R2 對ANAC078 片段進行一對一轉型及測試 66 3.7.1 R2 對ANAC078 片段進行一對一生長測試 66 3.7.2 R2 對ANAC078 片段進行β-galactosidase 活性測試 67 3.7.2.1 ONPG assay 67 3.7.2.2 Filter-lift assay 68 3.8 NTL11 與R2 在酵母菌雙雜合系統的交互作用討論 68 3.8.1 NTL11 可能的domain 分析 68 3.8.2 NTL11 可能為轉錄因子 (Transcription factor) 69 3.8.3 NTL11 與R2 可能存在交互作用 69 3.8.4 NTL11 片段與R2 間交互作用消失的可能原因 70 3.8.5 NTL11 可能的生理功能 71 3.9 GST-R2 與6x His- NTL11 蛋白質的表現與純化 71 3.9.1 GST-R2 蛋白質的表現與純化 71 3.9.2 6x His- NTL11 蛋白質的表現與純化 72 3.7.3 GST-R2 與6x His- NTL11 蛋白質的表現與純化討論 72 第四章 結論與未來展望 73 4.1 結論 73 4.2 未來展望 74 4.2.1 R2 與NTL11 間作用區域的探討 74 4.2.2 以其他實驗驗證R2 與NTL11 的交互作用性 74 4.2.3 以其他方式檢驗NTL11 是否為轉錄活化子 74 4.2.4 探討NTL11 與R2、AtMAPR4 之間的關係 75 4.2.5 建立NTL11 與R2、AtMAPR4 的突變株觀察生理變化 75 4.2.6 R2 與其他正反應株間作用性的研究 75 4.2.7 R3 與其正反應株間作用性的研究 75 參考文獻 77 圖與表 82 | |
| dc.language.iso | zh-TW | |
| dc.subject | RING | zh_TW |
| dc.subject | 泛素銜接 | zh_TW |
| dc.subject | E3 | en |
| dc.subject | RING | en |
| dc.title | 以RING E3蛋白質作為誘餌進行酵母菌雙雜合系統分析 | zh_TW |
| dc.title | Using RING E3 as bait to perform yeast two-hybrid screening | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李平篤(Ping-Du Li),王愛玉(Ai-Yu Wang) | |
| dc.subject.keyword | 泛素銜接,RING, | zh_TW |
| dc.subject.keyword | E3,RING, | en |
| dc.relation.page | 103 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2009-10-12 | |
| 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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