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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志 | |
| dc.contributor.author | Yun-Yen Huang | en |
| dc.contributor.author | 黃勻彥 | zh_TW |
| dc.date.accessioned | 2021-06-08T07:24:37Z | - |
| dc.date.copyright | 2008-07-26 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-17 | |
| dc.identifier.citation | Aaron Ciechanover, A.O.A.L.S. (2000). Ubiquitin-mediated proteolysis: biological regulation via destruction. BioEssays 22, 442-451.
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Dharmasiri, N., Dharmasiri, S., Weijers, D., Lechner, E., Yamada, M., Hobbie, L., Ehrismann, J.S., JAaron Ciechanover, A.O.A.L.S. (2000). Ubiquitin-mediated proteolysis: biological regulation via destruction. BioEssays 22, 442-451. Aguilar, R.C., and Wendland, B. (2003). Ubiquitin: not just for proteasomes anymore. Current Opinion in Cell Biology 15, 184-190. Andersen, P., Kragelund, B.B., Olsen, A.N., Larsen, F.H., Chua, N.-H., Poulsen, F.M., and Skriver, K. (2004). Structure and Biochemical Function of a Prototypical Arabidopsis U-box Domain. J. Biol. Chem. 279, 40053-40061. Bachmair, A., Novatchkova, M., Potuschak, T., and Eisenhaber, F. (2001). Ubiquitylation in plants: a post-genomic look at a post-translational modification. Trends in plant science 6, 463-470. Bernard, P., and Couturier, M. (1992). Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. Journal of Molecular Biology 226, 735-745. Bishop, G.J., and Koncz, C. (2002). Brassinosteroids and Plant Steroid Hormone Signaling. Plant cell 14, S97-110. Borden, K.L.B. (2000). RING domains: master builders of molecular scaffolds? Journal of Molecular Biology 295, 1103-1112. Chau, V., Tobias, J.W., Bachmair, A., Marriott, D., Ecker, D.J., Gonda, D.K., and Varshavsky, A. (1989). A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science (New York, N.Y) 243, 1576-1583. Chen, Z.J. (2005). Ubiquitin signalling in the NF-[kappa]B pathway. Nat Cell Biol 7, 758-765. Creelman, R.A., and Mullet, J.E. (1997). BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS. Annual Review of Plant Physiology and Plant Molecular Biology 48, 355-381. Devoto, A., Nieto-Rostro, M., Xie, D., Ellis, C., Harmston, R., Patrick, E., Davis, J., Sherratt, L., Coleman, M., and Turner, J.G. (2002). COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis. Plant J 32, 457-466. Dharmasiri, N., Dharmasiri, S., and Estelle, M. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26766 | - |
| dc.description.abstract | 在阿拉伯芥中參與泛素化修飾作用的 E3 ligase 約有 1000 種。許多重要的植物訊息傳遞作用直接或間接與 E3 ligase 及其受質蛋白質之間的辨識作用有關。本實驗室先前酵母菌雙雜合實驗結果顯示，阿拉伯芥中 AtMAPR5 可能與一個含有 RING 區塊的蛋白質 At3g58030 (RING3g) 有交互作用。經序列比對發現另一個與 RING3g 具有高度相似性的蛋白質 At2g42030 (RING2g)。本論文建立本實驗室之胞外泛素化修飾分析方法，試圖研究 AtMAPR5 是否為 RING3g 之生理基質。胞外泛素化修飾分析顯示 RING2g 及 RING3g 具有 E3 ligase 的活性，但是進一步研究顯示 RING3g 無法有效將 AtMAPR5 進行泛素化修飾。推測 AtMAPR5 可能不是 RING3g E3 ligase 之受質蛋白質。將泛素化修飾分析方法擴展，利用微陣列分析資料庫挑選並選殖出 8 個與非生物逆境相關的 RING 蛋白質 (R1 ~ R8)，以胞外泛素化修飾分析發現其中 7 個具有 E3 ligase 之活性。這些 RING-type E3 ligase 生理功能仍有待進一步研究。此外，進行蛋白質體之胞外泛素化修飾，以 2D 電泳分析全蛋白質，尋找參與高鹽非生物逆境之 RING-type E3 ligase 的方法也在本論文中討論。 | zh_TW |
| dc.description.abstract | There are about 1000 E3 ligases in Arabidopsis, which recognize specific protein substrate and catalyze the ubiquitination reaction to these proteins. The recognition between E3 ligases and their substrate proteins occurs in response to many plant signaling pathways directly or indirectly. Previous yeast two-hybrid experiments indicated that AtMAPR5 might interact with a RING protein, RING3g, which is encoded by At3g58030. In vitro ubiquitination assay was established to find out whether AtMAPR5 is the substrate protein of RING3g. RING3g and RING2g (encoded by At2g42030), a close homolog of RING3g, showed the E3 ligase activity in the in vitro ubiquitination assay. However, RING3g did not show the ability to modify AtMAPR5 in vitro and might not be the E3 ligase of AtMAPR5. To expand our studies to find out novel E3 ligases involved in the abiotic stress signaling, eight RING proteins (R1 ~ R8) were cloned based on microarray database. Seven recombinant proteins among them possessed the activity of autoubiquination using in vitro ubiquitination assay. The physiological roles of the RING proteins still await to determine. Finally, the possibility of how to employ in vitro ubiquitination assay in the discovery of novel RING-type E3 ligases involved in salt stress signaling is also discussed. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T07:24:37Z (GMT). No. of bitstreams: 1 ntu-97-R95b47201-1.pdf: 3331784 bytes, checksum: 43beccfb1eee4c0eb47a0c4e193617d6 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 目錄 I
縮寫表 VI 摘要 VIII Abstract IX 第一章 緒論 1 1.1 阿拉伯芥 1 1.2 蛋白質品質管制 2 1.3 泛素-蛋白酶體系統 2 1.3.1 蛋白質泛素化修飾作用原理 3 1.3.2 蛋白質泛素化修飾作用與體內蛋白質調控 7 1.4 蛋白質泛素化修飾與植物荷爾蒙及植物逆境之關係 8 1.4.1 泛素化修飾與植物生長素 9 1.4.2 泛素化修飾與吉貝素 9 1.4.3 泛素化修飾與離層酸 10 1.4.4 泛素化修飾與乙烯 10 1.4.5 泛素化修飾與蕓薹素內酯 11 1.4.6 泛素化修飾與茉莉酸 11 1.5 AtMAPR5 與 AtMAPRIPs 11 1.6 Gateway Cloning 介紹 12 1.7 研究動機與方向 14 第二章 實驗材料與方法 15 2.1 儀器設備 15 2.2 實驗材料 17 2.2.2 植物材料 17 2.2.3 載體 (Vectors) 17 2.2.4 大腸桿菌菌株 20 2.3 基本實驗藥品 21 2.3.1 一般化學藥劑 21 2.3.2 大腸桿菌培養基 21 2.3.3 阿拉伯芥固態培養基 22 2.4 DNA 相關之基本操作方法 23 2.4.1 質體 DNA 小量製備 (Mini-prep) 23 2.4.2 DNA 限制酶切割反應 (Restriction enzyme digestion, RE) 24 2.4.3 聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 24 2.4.4 BP cloning 25 2.4.5 LR cloning 26 2.4.6 洋菜膠體電泳 (agarose gel electrophoresis) 26 2.4.7 大腸桿菌轉形 27 2.4.8 DNA 濃度估算 29 2.4.9 Quikchange 點突變 29 2.4.10 大腸桿菌菌種保存 29 2.5 蛋白質相關基本操作方法 30 2.5.1 蛋白質電泳檢定 30 2.5.3 蛋白質轉印 37 2.5.3 免疫呈色 37 2.5.4 蛋白質 N 端定序 37 2.5.5 膠體過濾法 38 2.5.6 陰離子交換法 39 2.5.7 Bradford 蛋白質定量法 39 2.5.8 BCA 蛋白質定量法 39 2.6 各目標基因表現載體建構及序列確認 40 2.6.1 HsUBE1、AtUBC8、pDEST15-RING+ 序列確認 42 2.6.2 Ubiquitin 表現載體建構 42 2.6.3 pDEST15-RING2g、pDEST15-RING3g 表現載體建構 43 2.6.4 與植物逆境相關之 pDEST15-RINGs 表現載體建構 44 2.6.5 pDEST15-AtHOS1 表現載體建構 44 2.7 各目標基因重組蛋白質表現方法 45 2.7.1 HsUBE1 表現及純化 45 2.7.2 6x-His-AtUBC8 表現及純化 46 2.7.3 10x-His-RING3g 表現及純化 47 2.7.4 GST-RINGs 表現及純化 48 2.7.5 GST-AtMAPR5 表現及純化 48 2.7.6 Ubiquitin-6x-His 及 Ubiquitin 表現及純化 49 2.8 Ubiquitin 多株抗體製備 50 2.9 阿拉伯芥培養及 300 mM NaCl 處理 51 2.10 In vitro ubiquitination assay (胞外泛素化修飾) 52 第三章 結果與討論 53 3.1 胞外泛素化修飾分析 (in vitro ubiquitination assay) 之建立 53 3.1.1 胞外泛素化修飾分析材料之選擇 53 3.1.2 HsUBE1 表現質體確認及蛋白質表現 54 3.1.3 AtUBC8 表現質體確認及 6x-His-AtUBC8 重組蛋白質表現 56 3.1.4 RING+ 表現質體確認及 GST-RING+ 重組蛋白質表現 57 3.1.5 human ubiquitin 及 human ubiquitin-6x-His表現質體確認及重組蛋白質表現 57 3.1.6 Ubiquitin 多株抗體製備 58 3.1.7 以 GST-RING+ 建立胞外泛素化修飾分析 59 3.1.7 HsUBE1、6x-His-AtUBC8、GST-RING+ 活性探討 59 3.1.8 以 human ubiquitin 或 human ubiquitin-6x-His 作為胞外泛素化修飾分析中泛素來源之比較 61 3.1.9 以自製泛素多株抗體偵測胞外泛素化修飾 61 3.2 分析 At2g42030 (RING2g)、At3g58030 (RING3g) 之 E3 ligase 活性 62 3.2.1 RING2g、RING3g 表現質體建構 62 3.2.2 GST-RING2g、GST-RING3g 重組蛋白質表現 64 3.2.3 以胞外泛素化修飾分析 GST-RING2g、GST-RING3g 之 E3 ligase 活性 65 3.3 分析 At3g58030 (RING3g) 是否為 AtMAPR5 之 E3 ligase 65 3.3.1 pET-16b-RING3g 表現質體確認及重組蛋白質表現 66 3.3.2 以胞外泛素化修飾分析 10x-His-RING3g 之 E3 ligase 活性 66 3.3.3 pGEX4T-1-AtMAPR5 表現質體確認及重組蛋白質表現 67 3.3.4 以胞外泛素化修飾發現 RING3g 不是 AtMAPR5 之 E3 ligase 67 3.4 與植物逆境相關之 RING-type E3 ligase 篩選 68 3.4.1 以 Microarray挑選 8 個與植物逆境相關之 RING protein 基因 69 3.4.2 R1 ~ R8 可能具有之植物生理角色 69 3.4.3 R1 ~ R8 表現質體建構 70 3.4.4 GST-R1 ~ GST-R8 重組蛋白質表現 72 3.4.5 以胞外泛素化修飾分析 GST-R1 ~ GST-R8 之 E3 ligase 活性 74 3.4.6 高鹽處理之阿拉伯芥蛋白質體泛素化修飾 74 3.4.7 全蛋白質胞外泛素化修飾之討論 75 第四章 結論與未來展望 78 4.1 結論 78 4.2 未來展望 79 4.2.1 RING2g、RING3g、RING+ 及 7 個與非生物逆境相關 RING-type E3 ligase 之植物生理功能探討 79 4.2.2 在全蛋白質胞外泛素化修飾分析中以不同的阿拉伯芥 E2來釣取不同類型的 E3 ligase 80 4.2.3 以全蛋白質胞外泛素化修飾來釣取 E3 ligase 之受質蛋白質 80 4.2.4 AtFMPR5 與 E3 ligase 之訊息傳遞探討 81 參考文獻 82 圖與表 87 | |
| dc.language.iso | zh-TW | |
| dc.subject | 泛素化修飾 | zh_TW |
| dc.subject | ubiquitination | en |
| dc.title | 阿拉伯芥泛素化修飾分析方法之建立與非生物性逆境相關 RING-型泛素黏合酶選殖 | zh_TW |
| dc.title | Establishing in vitro ubiquitination assay and the cloning of RING-type E3 ligases induced by abiotic stress in Arabidopsis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇仲卿,陳佩燁,章為皓,常怡雍,李平篤 | |
| dc.subject.keyword | 泛素化修飾, | zh_TW |
| dc.subject.keyword | ubiquitination, | en |
| dc.relation.page | 86 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2008-07-17 | |
| 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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