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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊健志 | |
dc.contributor.author | Ying-Han Lin | en |
dc.contributor.author | 林盈涵 | zh_TW |
dc.date.accessioned | 2021-06-08T04:33:24Z | - |
dc.date.copyright | 2009-08-21 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-19 | |
dc.identifier.citation | Acconcia F, Sigismund S, Polo S (2009) Ubiquitin in trafficking: the network at work. Exp Cell Res 315: 1610-1618
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Nat Cell Biol 8: 1246-1254 Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, Robert J, Rush J, Hochstrasser M, Finley D, Peng J (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137: 133-145 Yang X-H, Xu Z-H, Xue H-W (2005) Arabidopsis Membrane Steroid Binding Protein 1 Is Involved in Inhibition of Cell Elongation. Plant Cell 17: 116-131 Yang X, Song L, Xue H-W (2008) Membrane Steroid Binding Protein 1 (MSBP1) Stimulates Tropism by Regulating Vesicle Trafficking and Auxin Redistribution. Mol Plant 1: 1077-1087 高艾玲 (2002) 阿拉伯芥 MAPR 同源蛋白質基因之選殖、表現與功能分析,碩士論文,國立臺灣大學農業化學研究所 張碩修 (2003) 阿拉伯芥中與 AtMAPR 有交互作用的蛋白質之篩選,碩士論文,國立臺灣大學農業化學研究所 劉耿全 (2005) 以酵母菌雙雜合系統篩選阿拉伯芥中與 AtMAPRs 有交互作用之蛋白質,碩士論文,國立臺灣大學微生物與生化學研究所 黃勻彦 (2008) 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,碩士論文,國立臺灣大學微生物與生化學研究所 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22916 | - |
dc.description.abstract | 阿拉伯芥中的 AtMAPR 家族蛋白質由 4 個同源蛋白質所構成,分別為 AtMAPR2、AtMAPR3、AtMAPR4 與 AtMAPR5。由序列比對結果發現 AtMAPRs 具有一段與 UIM (ubiquitin-interacting motif) 相似度極高的區域。UIM 與 UBA、UEV、NZF、CUE、A20 ZnF、ZnF UBP、Ubc、GAT 等區塊皆屬於 UBDs (ubiquitin-binding domains),研究指出許多具有 UBDs 之蛋白質會進行自身泛素化修飾 (autoubiquitination)。本論文利用大腸桿菌表現胞外泛素化修飾分析方法 (in vitro ubiquitination assay) 所需要之重組蛋白質 E1 (HsUBE1)、E2 (AtUBC8)、與一個非生物性逆境相關之 RING protein (Rg, At1g74870),建立胞外泛素化修飾分析方法。透過此胞外泛素化修飾分析研究 Rg、AtMAPR3 與 AtMAPR5,發現它們具有自身泛素化修飾之活性。進一步以 LC-MS/MS 分析,發現 Rg 會催化由泛素 Lys6、Lys11 及 Lys48 相連接而形成的聚泛素鏈。另一方面,透過 LC-MS/MS 之分析,找到多個 AtMAPR3 與 AtMAPR5 上被泛素標定的 Lys 位置,也發現可能存在之聚泛素鏈。由以上結果推測 Rg 可能具有 E3 ligase 之活性,而 AtMAPR3 與 AtMAPR5 可能具有一種新穎之 E3-independent 泛素化修飾的能力。 | zh_TW |
dc.description.abstract | AtMAPR family consists of four homologs in Arabidopsis, which are AtMAPR2, AtMAPR3, AtMAPR4 and AtMAPR5. A putative ubiquitin-interacting motif (UIM) was detected from a multiple sequence alignment of AtMAPRs and UIMs. UIM is a member of UBDs (ubiquitin-binding domains) which also include UBA, UEV, NZF, CUE, A20 ZnF, ZnF UBP, Ubc, GAT domains. It has been demonstrated that UBD-containing proteins might have autoubiquitination activity. In this study, recombinant proteins required for the in vitro ubiquitination assay, E1 (HsUBE1), E2 (AtUBC8) and an abiotic stress-related RING protein (Rg, At1g74870), are produced in E.coli. In vitro ubiquitination assays indicated that Rg, AtMAPR3 and AtMAPR5 have autoubiquitination activity. By LC-MS/MS analysis, Rg was found to catalyze the formation of polyubiquitin chain via Lys6, Lys11 and Lys48 on ubiquitin. Besides, we also identified several target ubiquitination sites in AtMAPR3 and AtMAPR5. Polyubiquitination was also confirmed by detecting the presence of modified ubiquitin fragments. These results indicated that Rg may have E3 ligase activity, and AtMAPR3 and AtMAPR5 might have a novel E3-independent ubiquitination activity. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T04:33:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 | en |
dc.description.tableofcontents | 目錄..................................................I
縮寫表...............................................IV 摘要.................................................VI Abstract............................................VII 第一章 緒論..........................................1 1.1 模式植物:阿拉伯芥 (Arabidopsis thaliana)......1 1.2 泛素化修飾作用.................................1 1.2.1 The Ubiquitin Protein......................2 1.2.2 ubiquitin-activating enzyme (E1)...........3 1.2.3 ubiquitin-conjugating enzyme (E2)..........3 1.2.4 ubiquitin ligase (E3)......................3 1.2.5 ubiquitin-binding domains (UBDs)...........5 1.2.6 泛素化修飾的蛋白質體研究...................9 1.3 AtMAPR家族.....................................9 1.3.1 AtMAPR5 之功能.............................10 1.4 研究動機與方向.................................12 第二章 材料與方法....................................13 2.1 實驗材料.......................................13 2.1.1 載體 (Vectors..............................13 2.1.2 菌種.......................................15 2.2 實驗藥品.......................................16 2.2.1 一般化學藥劑...............................16 2.2.2 酵素.......................................16 2.2.3 大腸桿菌培養基.............................16 2.3 儀器設備.......................................17 2.4 實驗方法.......................................19 2.4.1 DNA 相關之基本操作方法.....................19 2.4.1.1 質體 DNA 之抽取........................19 2.4.1.2 小量 DNA 限制酶切割反應 (Restriction enzyme digestion)............................................20 2.4.1.3 DNA 瓊脂糖膠體電泳 (Agarose gel electrophoresis)......................................21 2.4.1.4 DNA 之定量.............................21 2.4.1.5 化學法勝任細胞 (competent cell) 製備及質體轉形....................................................22 2.4.1.6 Gateway cloning........................22 2.4.1.7 大腸桿菌菌種保存.......................25 2.4.2 目標基因之選殖及保存.......................25 2.4.2.1 At1g74870 (Rg) 保存質體與表現質體之建構.25 2.4.3 蛋白質相關之基本操作方法...................26 2.4.3.1 蛋白質電泳檢定.........................26 2.4.3.1.1 SDS 膠體電泳.......................26 2.4.3.1.2 梯度膠體電泳.......................27 2.4.3.1.3 膠片染色法..........................31 2.4.3.1.4 膠片乾燥法.........................32 2.4.3.2 蛋白質轉印.............................32 2.4.3.3 免疫呈色...............................33 2.4.3.4 Bradford 蛋白質定量法..................34 2.4.4 各目標基因重組蛋白質表現與純化.............34 2.4.4.1 HsUBE1 表現與純化......................34 2.4.4.2 6×His-AtUBC8 表現與純化................36 2.4.4.3 GST-Rg表現與純化.......................37 2.4.4.4 GST-AtMAPR3 及GST-AtMAPR5 表現與純化...38 2.4.5 胞外泛素化修飾 (In vitro ubiquitination assay)................................................39 2.4.6 蛋白質身份鑑定 - LC-MS/MS..................39 2.4.6.1 In-gel digestion.......................41 2.4.6.2 LC-MS/MS 分析..........................42 第三章 結果與討論....................................44 3.1 AtMAPRs 胺基酸序列與 ubiquitin-binding domain 之比對分析..................................................44 3.2 胞外泛素化修飾分析 (In vitro ubiquitination assay)................................................44 3.2.1 胞外泛素化修飾分析材料之選擇...............44 3.2.2 重組蛋白質表現與純化結果之分析.............45 3.2.2.1 HsUBE1 蛋白質表現與純化之分析..........45 3.2.2.2 6×His-AtUBC8 重組蛋白質表現與純化之分析.46 3.2.2.3 GST-AtMAPR3 及GST-AtMAPR5 重組蛋白質表現與純化之分析..............................................46 3.2.2.4 GST-Rg 表現質體確認及重組蛋白質表現與純化之分析....................................................47 3.2.3 分析 GST-Rg 之 E3 ligase 活性..............47 3.2.4 分析 GST-AtMAPR3 及 GST-AtMAPR5 之自身泛素化修飾活性..................................................48 3.3 LC-MS/MS 蛋白質身分鑑定結果....................49 3.3.1 分析 in vitro ubiquitination assays 電泳膠片上的色帶....................................................49 3.3.2 分析泛素在目標蛋白質標定之位置..............49 3.4 AtMAPR3 與 AtMAPR5 自身泛素化修飾之討論........50 第四章 結論與未來展望.................................52 4.1 結論...........................................52 4.2 未來展望.......................................52 4.2.1 探討 AtMAPR3 與 AtMAPR5 標定泛素的位置及類似 UIM 之區塊對其活性或功能的影響............................52 4.2.2 AtMAPR2 與 AtMAPR4 自身泛素化修飾的研究....53 4.2.3 AtMAPRs 與泛素結合的研究...................53 4.2.4 探討 AtMAPRs 與不同 E2之間的專一性.........53 4.2.5 尋找 AtMAPR3 與 AtMAPR5 可能的受質蛋白質...54 參考文獻..............................................55 圖與表................................................59 附錄..................................................96 | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥中 AtMAPR 家族蛋白質與其潛在自身泛素化修飾功能之研究 | zh_TW |
dc.title | Potential Autoubiquitination of AtMAPRs in Arabidopsis | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇仲卿,王愛玉,陳佩燁,李平篤 | |
dc.subject.keyword | 自身泛素化修飾,AtMAPR家族蛋白質,泛素結合區塊, | zh_TW |
dc.subject.keyword | autoubiquitination,AtMAPR,UBDs, | en |
dc.relation.page | 119 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2009-08-20 | |
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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