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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張世宗 | |
dc.contributor.author | Ya-Ling Tseng | en |
dc.contributor.author | 曾雅伶 | zh_TW |
dc.date.accessioned | 2021-06-08T04:58:58Z | - |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23304 | - |
dc.description.abstract | SUMO E3黏合酶參與SUMO化 (sumoylation) 反應的最後一個步驟。而SUMO E3黏合酶,除了能夠加速SUMO化反應的進行及增加SUMO對於目標蛋白質的專一性外,亦可調控細胞內許多不同的生理功能。細胞中有非常多能被SUMO修飾的受質,但目前已知且較常被探討的SUMO E3黏合酶只有三種,因此相關的研究仍有很大的發展空間,尤其對單胞綠藻 (Chlamydomonas reinhardtii) 之SUMO E3黏合酶的研究的研究仍未見。
本論文先利用阿拉伯芥的SUMO E3黏合酶AtSiz1的胺基酸序列,以BLAST方法找出單胞綠藻中與其相似的序列,並找到了一個功能未知的蛋白質序列。此序列與AtSiz1擁有相似的保守性區域,如N端之SAP (scaffold attachment factor A/B/Acinus/PIAS) domain,分子中間的PHD (plant homeodomain) domain及RING (really interesting new gene) domain,因此推測這個預測的蛋白質可能為一種SUMO E3黏合酶,本論文將其暫時命名為CrSiz1。目前已成功選殖到此段序列,及表現各個保守性區域之重組蛋白質,值得一提的是CrSiz1之C端含有一段特有之胺基酸序列,並未見於其他Siz1之序列中。有研究指出阿拉伯芥於熱休克處理後,AtSiz1可以增加受質的SUMO化以抵抗逆境。然而利用CrSiz1之專一性抗體觀察單胞綠藻於熱休克處理後之CrSiz1表現情形,並未偵測出其差異性,因此目前尚無法確定CrSiz1是否具有SUMO E3黏合酶之生化性質與功能。 | zh_TW |
dc.description.abstract | The SUMO E3 ligase involves in the last step of sumoylation. In addition to faciliate sumoylation and increase specificity for the targets, SUMO E3 ligases modulate a wide range of physiological functions. There are many sumoylated proteins in the cells, but only three kinds of SUMO E3 ligases have been shown in the literatures. Notably, the study of SUMO E3 ligase in Chlamydomonas reinhardtii has not been reported.
In this study, using the Arabidopsis SUMO E3 ligase, AtSiz1, as the template for BLAST search, a gene encoding for a predicated protein with unkown function has been remarked. This predicated protein has similar conserved domains with AtSiz1, such as N-terminal SAP (scaffold attachment factor A/B/Acinus/PIAS) domain, PHD (plant homeodoamin) and RING (really interesting new gene) domain in the middle of the sequence. Based on the sequence observation, this predicated protein was herein named CrSiz1. The partial sequences of CrSiz1 have been expressed and purified from the Escherichia coli. It is noted that CrSiz1 cotains a unique C-terminal sequence which has not been found in other Siz1 proteins. It has been reported that AtSiz1 can inscrease sumoylated proteins under the heat shock condition. By using the antibodies specific to CrSiz1 for western blotting against the C. reinhardtii samples obtained from the cells cultured at 42 | en |
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dc.description.tableofcontents | 中文摘要 i
Abstract ii 縮寫表 iii 第一章 緒論 1 1.1 SUMO (small ubiquitin-like modifier) 1 1.2 SUMO化反應機制 2 1.2.1 SUMO化路徑 2 1.2.2 參與SUMO化反應之酵素 2 1.3 SUMO E3黏合酶 4 1.3.1 SIZ/PIAS 家族 5 1.3.2 NSE2/MMS21 8 1.3.3 RanBP2/Nup358 9 1.3.4 Pc2 10 1.4 SUMO化修飾之生理功能 10 1.5 逆境壓力下SUMO化修飾及SUMO E3黏合酶之調控 12 1.6 研究動機與方向 13 第二章 材料與方法 15 2.1 實驗材料 15 2.1.1 大腸桿菌菌株 15 2.1.2 單胞綠藻 15 2.2 目標基因表現載體之建構 15 2.2.1 原核表現系統載體 15 2.2.2 聚合酶鏈反應 16 2.2.3 重疊聚合酶鏈鎖反應 16 2.2.4 限制酶切反應 17 2.2.6 接合反應 17 2.3 原核宿主表現系統 17 2.3.1 化學法勝任細胞製備 17 2.3.2 大腸桿菌細胞轉形 17 2.3.3 重組蛋白質誘導表現 18 2.4 重組蛋白質之純化方法 19 2.4.1 GST-tag 重組蛋白質親和性層析法 19 2.4.2 蛋白質脫鹽與濃縮 19 2.5 核酸基本操作方法 20 2.5.1 單胞綠藻總RNA抽取 20 2.5.2 反轉錄酶-聚合連鎖反應 20 2.5.3 小量質體DNA製備 21 2.5.4 洋菜膠體電泳 21 2.5.5 核酸定量 22 2.5.6 核酸純化套組 22 2.6 蛋白質相關基本操作方法 23 2.6.1 蛋白質純化 23 2.6.2 蛋白質定量 24 2.6.3 蛋白質電泳檢定 25 2.6.4 蛋白質膠體電泳染色法 25 2.6.5 蛋白質轉印法 26 2.6.6 免疫染色法 26 第三章 結果 27 3.1 單胞綠藻之SUMO E3黏合酶CrSiz1基因選殖 27 3.1.1 CrSiz1-SAP domain基因選殖與表現質體建構 28 3.1.2 CrSiz1-SAP domain重組蛋白質表現與純化 28 3.1.3 CrSiz1-PHD domain基因選殖與表現質體建構 29 中文摘要 i Abstract ii 縮寫表 iii 第一章 緒論 1 1.1 SUMO (small ubiquitin-like modifier) 1 1.2 SUMO化反應機制 2 1.2.1 SUMO化路徑 2 1.2.2參與SUMO化反應之酵素 2 1.3 SUMO E3黏合酶 4 1.3.1 SIZ/PIAS 家族 5 1.3.2 NSE2/MMS21 8 1.3.3 RanBP2/Nup358 9 1.3.4 Pc2 10 1.4 SUMO化修飾之生理功能 10 1.5 逆境壓力下SUMO化修飾及SUMO E3黏合酶之調控 12 1.6 研究動機與方向 13 第二章 材料與方法 15 2.1 實驗材料 15 2.1.1 大腸桿菌菌株 15 2.1.2 單胞綠藻 15 2.2 目標基因表現載體之建構 15 2.2.1 原核表現系統載體 15 2.2.2聚合酶鏈反應 16 2.2.3 重疊聚合酶鏈鎖反應 16 2.2.4限制酶切反應 17 2.2.6 接合反應 17 2.3 原核宿主表現系統 17 2.3.1 化學法勝任細胞製備 17 2.3.2 大腸桿菌細胞轉形 17 2.3.3 重組蛋白質誘導表現 18 2.4 重組蛋白質之純化方法 19 2.4.1 GST-tag 重組蛋白質親和性層析法 19 2.4.2 蛋白質脫鹽與濃縮 19 2.5 核酸基本操作方法 20 2.5.1 單胞綠藻總RNA抽取 20 2.5.2 反轉錄酶-聚合連鎖反應 20 2.5.3 小量質體DNA製備 21 2.5.4 洋菜膠體電泳 21 2.5.5 核酸定量 22 2.5.6 核酸純化套組 22 2.6 蛋白質相關基本操作方法 23 2.6.1蛋白質純化 23 2.6.2 蛋白質定量 24 2.6.3 蛋白質電泳檢定 25 2.6.4 蛋白質膠體電泳染色法 25 2.6.5 蛋白質轉印法 26 2.6.6 免疫染色法 26 第三章 結果 27 3.1 單胞綠藻之SUMO E3黏合酶CrSiz1基因選殖 27 3.1.1 CrSiz1-SAP domain基因選殖與表現質體建構 28 3.1.2 CrSiz1-SAP domain重組蛋白質表現與純化 28 3.1.3 CrSiz1-PHD domain基因選殖與表現質體建構 29 3.1.4 CrSiz1-PHD domain重組蛋白質表現與純化 29 3.1.5 CrSiz1-RING domain基因選質與表現質體建構 29 3.1.6 CrSiz1-RING domain重組蛋白質表現與純化 30 3.1.7 CrSiz1 C端序列基因選殖與表現質體建構 31 3.1.8 CrSiz1 C端序列重組蛋白質表現與純化 31 3.2 SAP、PHD及C-ter多株抗體效價及專一性測試 31 3.2.1 anti-SAP多株抗體效價及專一性測試 32 3.2.2 anti-PHD多株抗體效價及專一性測試 32 3.2.3 anti-C-ter多株抗體效價及專一性測試 33 3.3利用多株抗體偵測單胞綠藻細胞中CrSiz1 34 3.4 熱逆境處理對於單胞綠藻中CrSiz1之分析 34 第四章 討論 35 4.1 單胞綠藻中確實存在CrSiz1基因序列 35 4.2 CrSiz1各保守性區域之重組蛋白質表現及純化 36 4.3 CrSiz1各保守性區域多株抗體之測試 37 4.4 多株抗體尚未找出可能為CrSiz1之蛋白質 38 參考文獻 40 圖與表 45 附錄 64 論文口試問答摘要 74 | |
dc.language.iso | zh-TW | |
dc.title | 單胞綠藻SUMO E3黏合酶Siz1之基因選殖與蛋白質表現 | zh_TW |
dc.title | Molecular cloning and protein expression of Siz1 SUMO E3 ligase in Chlamydomonas reinhardtii | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 莊榮輝,吳俊宗,張麗冠,陳威戎 | |
dc.subject.keyword | SUMO E3黏合酶,基因選殖,熱休克, | zh_TW |
dc.subject.keyword | SUMO E3 ligase,molecular coloning,heatshock, | en |
dc.relation.page | 77 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2010-08-19 | |
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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