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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 莊榮輝 | |
dc.contributor.author | Shin-Yu Lin | en |
dc.contributor.author | 林歆祐 | zh_TW |
dc.date.accessioned | 2021-06-13T04:18:08Z | - |
dc.date.available | 2011-08-01 | |
dc.date.copyright | 2011-08-01 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-27 | |
dc.identifier.citation | 參考文獻
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Environmental toxicology and chemistry / SETAC 24: 1731-1737 Lee S, Korban SS (2002) Transcriptional regulation of Arabidopsis thaliana phytochelatin synthase (AtPCS1) by cadmium during early stages of plant development. Planta 215: 689-693 Long X, Yang X, Ni W (2002) [Current situation and prospect on the remediation of soils contaminated by heavy metals]. Ying Yong Sheng Tai Xue Bao 13: 757-762 Maier T, Yu C, Kullertz G, Clemens S (2003) Localization and functional characterization of metal-binding sites in phytochelatin synthases. Planta 218: 300-308 Maitani T, Kubota H, Sato K, Yamada T (1996) The Composition of Metals Bound to Class III Metallothionein (Phytochelatin and Its Desglycyl Peptide) Induced by Various Metals in Root Cultures of Rubia tinctorum. Plant physiology 110: 1145-1150 Meister A (1995) Glutathione metabolism. Methods in enzymology 251: 3-7 Pal R, Rai JP (2010) Phytochelatins: peptides involved in heavy metal detoxification. 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Plant physiology 122: 793-801 Vatamaniuk OK, Mari S, Lang A, Chalasani S, Demkiv LO, Rea PA (2004) Phytochelatin synthase, a dipeptidyltransferase that undergoes multisite acylation with gamma-glutamylcysteine during catalysis: stoichiometric and site-directed mutagenic analysis of arabidopsis thaliana PCS1-catalyzed phytochelatin synthesis. The Journal of biological chemistry 279: 22449-22460 Vatamaniuk OK, Mari S, Lu YP, Rea PA (1999) AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution. Proceedings of the National Academy of Sciences of the United States of America 96: 7110-7115 Vatamaniuk OK, Mari S, Lu YP, Rea PA (2000) Mechanism of heavy metal ion activation of phytochelatin (PC) synthase: blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides. The Journal of biological chemistry 275: 31451-31459 Vestergaard M, Matsumoto S, Nishikori S, Shiraki K, Hirata K, Takagi M (2008) Chelation of cadmium ions by phytochelatin synthase: role of the cysteine-rich C-terminal. Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 24: 277-281 Vivares D, Arnoux P, Pignol D (2005) A papain-like enzyme at work: Native and acyl–enzyme intermediate structures in phytochelatin synthesis. Proceedings of the National Academy of Sciences of the United States of America 102: 18848-18853 Wang H-C, Wu J-S, Chia J-C, Yang C-C, Wu Y-J, Juang R-H (2009) Phytochelatin Synthase Is Regulated by Protein Phosphorylation at a Threonine Residue Near Its Catalytic Site. Journal of Agricultural and Food Chemistry 57: 7348-7355 Whitney SM, Andrews TJ (2001) Plastome-encoded bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco. Proceedings of the National Academy of Sciences of the United States of America 98: 14738-14743 Yang X, Feng Y, He Z, Stoffella PJ (2005) Molecular mechanisms of heavy metal hyperaccumulation and phytoremediation. J Trace Elem Med Biol 18: 339-353 Zhang H, Xu W, Guo J, He Z, Ma M (2005) Coordinated responses of phytochelatins and metallothioneins to heavy metals in garlic seedlings. Plant Science 169: 1059-1065 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32885 | - |
dc.description.abstract | 阿拉伯芥 (Arabidopsis thaliana) 中含有植物螯合素合成酶 (phytochelatin synthase, PCS, EC 2.3.2.15),會利用 glutathione (GSH) 為基質來合成植物螯合素 (phytochelatin, PC) 以結合入侵的重金屬,降低對植物的傷害。由於本實驗室以前表現出的PCS重組蛋白質N端帶有容易與金屬螫合的His-Tag,因此本論文試著將重組蛋白質上的Tag以thrombin去除。結果顯示不論是否帶有His-Tag,PCS的活性均無明顯改變。這可能是我們給予的鎘離子濃度和基質GSH超過活性催化所需,所以His-Tag的有無對PCS重組蛋白質來說沒有太大的差異。利用點突變技術將PCS的 N端保守性區域的Tyr 55進行點突變之後,測定不同突變株之表現蛋白質活性,發現同為芳香族的Y55F和Y55W突變株的活性和野生種比起來,僅稍微下降一些,而Y55H和Y55A突變株的活性則劇烈下降。進一步利用酵素動力學計算各突變株的Km和kcat,發現當側基失去芳香基團,酵素的整體催化能力也隨之下降50% 左右。由以上結果我們推測Tyr 55的苯環可以經由cation-pi 作用力,與帶著鎘的GSH複合物結合,以此將第二個基質GSH帶進催化區合成PC。另一方面,本論文以二次元電泳分析在植物體中PCS是否有磷酸化現象,發現未經過鎘逆境處理的阿拉伯芥樣本中,若加入phosphatase會使PCS蛋白質色點位移至pI較大之處,這可能是PCS被去除磷酸化所造成的結果。分別以不同濃度的鎘逆境處理後的阿拉伯芥樣本則無色點位移現象,但是各色點的比例有些不同。推測阿拉伯芥受到鎘逆境時PCS的磷酸化程度會隨之改變,所以二維電泳圖譜上才會呈現出色點比例的差異。分析阿拉伯芥粗抽取液的PCS活性,發現加以phosphatase inhibitor處理會提升PCS的活性,顯示磷酸化的確會對PCS的活性造成影響。本論文也使用免疫沉澱法來純化內生性PCS,將利用質譜儀 (LC-MS/MS) 來分析抗體辨認到的蛋白質身分,以及有無磷酸化修飾的現象。 | zh_TW |
dc.description.abstract | Phytochelatin synthase (PCS, EC 2.3.2.15) in Arabidopsis thaliana uses glutathione (GSH) as its substrate for the synthesis of phytochelatins (PCs) which could bind to heavy metals to reduce damages to cells. We have expressed AtPCS1 with His-Tag on its N-terminal sequence using E. coli expression system. In this study, His-Tag on AtPCS1 construct was removed by thrombin and the activity was then analyzed. There is no change on PCS catalytic activities whether the recombinant proteins contained His-Tag or not. This might be caused by using high concentration of GSH and Cd in the activity assay solution. To explore possible functions of the putative second substrate binding site, we tested several mutants at Tyr 55. Results showed that the activities of the mutants Y55F and Y55W were slightly lower than that of the wild-type. However, activities of Y55H and Y55A were dramatically decreased. Furthermore, changes on enzyme kinetic parameters of Tyr 55 mutants indicated that the aromatic group on Tyr 55 was important to PCS catalytic activity. These results suggested that Tyr 55 might bind GSH through cation-pi interaction. On the other hand, the two-dimensional electrophoresis (2-DE) analysis revealed that the native PCS in Arabidopsis showed several spots with different pI values. Samples treated with calf intestinal alkaline phosphatase (CIP) showed shifts of PCS spots on 2-DE, which might resulted from dephosphorylation of PCS. Samples treated with various Cd stress were also tested; the pI values of PCS spots were not affected by CIP, but the propotion of spots were slightly changed. These results suggested that phosphorylation of PCS might be enhanced in plants under Cd stress. In addition, PCS activity in Arabidopsis was decreased when sample was treated with CIP, and the activity could be recovered by adding phosphatase inhibitors. The above observations showed that the PCS activity might be regulated by phosphorylation. We also purified the endogenous PCS from Arabidopsis plants by immunoprecipitation and will identify the protein spots and its phosphorylation by LC-MS/MS. | 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 環境中重金屬的衝擊 1 1.1.1 重金屬於現今社會的現況 1 1.1.2 重金屬的定義 1 1.1.3 重金屬對於生物體的危害 1 1.1.4 如何清除重金屬汙染 2 1.2 植物體遇到重金屬逆境該如何自處 3 1.2.1 植物體抵抗重金屬逆境的機制 3 1.2.2 植物螯合素之結構與在重金屬累積時所扮演的角色 3 1.2.3 植物螯合素之特性與其和金屬之間的關係 4 1.3 植物螯合素經由植物螯合素合成酶 (phytochelatin synthase, PCS) 催化產生 5 1.3.1 植物螯合素合成酶之特性與純化 5 1.3.2 植物螯合素合成酶之基因研究 5 1.3.3 植物螯合素合成酶基因於自然界之分布 7 1.3.4 植物螯合素合成酶之催化機制 8 1.3.5 植物螯合素合成酶在生理上之功能 10 1.4 植物螯合素合成酶催化機制研究 10 1.4.1 植物螯合素合成酶可能受到蛋白質磷酸化的調控 11 1.4.2 植物螯合素合成酶上重要胺基酸Tyr 55的角色 12 1.5 研究動機 12 第二章 材料與方法 14 2.1 實驗材料 14 2.1.1 植物材料 14 2.1.2 表現載體 (vectors) 14 2.1.3 大腸桿菌 (Escherichia coli) 菌株 14 2.2 阿拉伯芥植物螯合素合成酶重組蛋白質製備 14 2.2.1 質體之轉形 (Transformation) 14 2.2.2 AtPCS1重組蛋白質之表現及純化 15 2.3 阿拉伯芥植物螯合素合成酶重組蛋白質之催化機制探討 16 2.3.1 決定最適基質和酵素反應時間 16 2.3.2 阿拉伯芥植物螯合素合成酶重組蛋白質之活性分析 16 2.3.3 阿拉伯芥植物螯合素合成酶重組蛋白質之酵素動力學 16 2.4 阿拉伯芥粗抽取液中AtPCS1有無磷酸化之電泳圖譜和活性分析差異 17 2.4.1 種植阿拉伯芥植株 17 2.4.2 阿拉伯芥植株以鎘處理之方法 17 2.4.3 添加磷酸酶抑制劑與蛋白質去磷酸化分析 17 2.4.4 蛋白質樣本萃取方法 17 2.4.5 蛋白質二維電泳 (2-dimensional electrophoresis) 18 2.4.6 阿拉伯芥粗抽取液中AtPCS1活性分析 18 2.5 以Ca2+/phytate分劃阿拉伯芥總蛋白質 19 2.6 免疫沉澱法 (immunoprecipitation, IP) 19 2.6.1 Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) 多株抗體之製備 19 2.6.2 免疫沉澱法 20 2.7 阿拉伯芥白化苗處理 21 2.7.1 無菌播種 21 2.7.2 阿拉伯芥白化苗以鎘處理之方法 21 2.7.3 蛋白質分析方法 21 第三章 結果與討論 22 3.1 PCS表現蛋白質反應條件測試 22 3.2 點突變型AtPCS1之活性分析和酵素動力學 24 3.2.1 His-Tag對於重組蛋白質AtPCS1之影響 24 3.2.2 Tyr 55點突變型AtPCS1之活性分析及酵素動力學 25 3.3 觀察植物內生性PCS是否有磷酸化現象 34 3.3.1 以二維電泳圖譜觀察阿拉伯芥內生性PCS是否有磷酸化現象 34 3.3.2 磷酸化可能會影響阿拉伯芥PCS活性 35 3.3.3 利用抗體檢測阿拉伯芥內生性PCS是否有磷酸化 37 3.4 去除阿拉伯芥中的Rubisco以利於分析含量較低的內生性PCS 42 3.4.1 去除Rubisco來進行電泳分析低量蛋白質 42 3.4.2 利用阿拉伯芥白化苗去除Rubisco 43 3.4.3 使用anti-Rubisco多株抗體以免疫沉澱法去除Rubisco 43 3.5 純化阿拉伯芥內生性PCS 49 3.5.1 鎘誘導阿拉伯芥內生性PCS表現量增加 49 3.5.2 以免疫沉澱法純化阿拉伯芥內生性PCS 49 第四章 未來研究方向 55 參考文獻 56 附錄 60 附錄一. 實驗藥品 60 1.1.1 一般化學試劑 60 1.1.2 酵素 60 1.1.3 培養基 60 附錄二. 儀器設備 61 問答錄 63 | |
dc.language.iso | zh-TW | |
dc.title | 磷酸化及Tyr 55突變對阿拉伯芥植物螯合素合成酶之催化活性影響 | zh_TW |
dc.title | The effect of phosphorylation and Tyr 55 mutation on the catalytic activity of phytochelatin synthase from Arabidopsis thaliana | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張世宗,楊健志,常怡雍,陳翰民 | |
dc.subject.keyword | 磷酸化,Tyr 55突變,阿拉伯芥植物螯合素合成酶, | zh_TW |
dc.subject.keyword | phosphorylation,Tyr 55 mutation,phytochelatin synthase, | en |
dc.relation.page | 64 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-28 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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