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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭貽生(Yi-Sheng Cheng) | |
dc.contributor.author | Chun-Yen Chen | en |
dc.contributor.author | 陳俊彥 | zh_TW |
dc.date.accessioned | 2021-06-16T17:49:30Z | - |
dc.date.available | 2012-08-20 | |
dc.date.copyright | 2012-08-20 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64477 | - |
dc.description.abstract | 光訊息對於植物生長與發育佔有重要的地位,光型態發生與暗型態發生是植物為了適應光照環境產生的生理機制,包含了植物感應光的強度、週期、向光性、避蔭反應以及概日韻律等現象。茉莉酸是植物重要的荷爾蒙,於植物受到創傷或是病原菌感染時大量產生並產生防禦反應,近年來的研究證明茉莉酸會影響根部的形成、花器的發育以及果實的成熟,對於生殖生理有重要的影響。
FIN219 (Far-red insensitive 219)又稱為JAR1 (Jasmonate Resistant 1),參與植物遠紅外光訊息路徑,同時具有催化茉莉酸與異白胺酸形成JA-Ile的酵素活性分子的功能。此外,利用酵母菌雙雜合系統篩選到的FIP1 (FIN219-Interacting Protein 1)同樣也參與在遠紅外光訊息路徑,為阿拉伯芥AtGSTU的成員,擁有GST酵素活性,由先前研究已知FIN219能與FIP1產生交互作用,而且FIP1能穩定FIN219並調控FIN219的酵素活性。 本論文為探討FIN219與FIP1的蛋白質結構、功能與酵素活性,利用大腸桿菌系統表現蛋白質以及利用膠體過濾層析法純化FIN219與FIP1。純化過程發現穩定性提升的FIN219能夠與表現載體pGEX 4T-1表達的SjGST蛋白質結合,並影響GST結合至親和性管柱的能力,並在膠體過濾層析中發現FIN219能與SjGST產生蛋白質複合體。利用石英晶體微天平分析證實FIN219與FIP1以及FIN219與SjGST的交互作用,分析FIN219酵素活性發現FIP1可以提升FIN219的最大速率以及催化常數。最後我們篩選出FIN219-GST、FIN219-FIP1以及FIP1晶體的結晶條件,並解出FIP1結構為對稱性的蛋白質雙體並帶有兩個活性區域,活性區中G-site帶有高度保守性的Glu以及Ser殘基,而H-site擁有比GmGSTU4-4更大的結合位,顯示GST tau類型對於植物代謝毒素以及殺草劑類化合物的重要性。 | zh_TW |
dc.description.abstract | Light signal plays an important role in plant growth and development. Photomorphogenesis and skotomorphogenesis are plant physiological mechanisms developed for adapting to light, including light fluence, photoperiodism, phototropism, shade avoidance and circadian rhythms. Jasmonate is a crucial phytohormone. It can be accumulated in the wounded or infected tissue and triggered defense response. Recent study confirms that jasmonate regulates root formation, flower development and fruit maturation, it affects deeply the reproductive physiology.
FIN219 (Far-red insensitive 219), also known as JAR1 (Jasmonate Resistant 1), involved in far-red light signaling pathway and possessed catalytic activity in conjugating jasmonic acid to isoleucine. In addition, FIP1 (FIN219-interacting protein 1) screened from yeast two-hybrid system also participated in far-red light signaling pathway. It belongs to the member of Arabidopsis thaliana AtGSTU family and possesses GST enzyme activity, previous study identified that FIN219 interacts with FIP1, then FIP1 improves FIN219 stability and regulates FIN219 enzyme activity. This study aimed to resolve FIN219 and FIP1 protein structure, function and enzyme activity. FIN219 and FIP1 proteins were expressed in Escherichia coli system and further purified using gel filtration chromatography. Interestingly, FIN219 could interacts with SjGST, a GST protein expressed from vector pGEX 4T-1, and interfered the ability of SjGST binding to GST affinity column. The FIN219-SjGST complex could be observed in gel filtration chromatography. The binding affinities and enzymatic kinetics between FIN219 and FIP1, and between FIN219 and SjGST were determined by Quartz Crystal microbalance (QCM). The results of kinetics of FIP1 demonstrated that FIP1 would promote maxmium velocity (Vmax) and catalytic constant (Kcat) of FIN219. Finally we screened FIN219-GST, FIN219-FIP1 and FIP1 crystallization condition. Only the crystals of FIP1 were obtained. The resolved structure of FIP1 is a dimer and possesses two active sites, with highly conserved residues Glu and Ser in G-site. In addition, FIP1 contained a larger H-site in comparison to that of GmGSTU4-4. It exhibited the importance of GST tau class in detoxification and in metabolism of herbicide compounds. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:49:30Z (GMT). No. of bitstreams: 1 ntu-101-R99B42034-1.pdf: 3172137 bytes, checksum: bfe5bf16e1a7bc086e1d2c38500cccfc (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 目錄 I
中文摘要 III Abstract IV 縮寫對照表 VI 前言 1 一、緒論 1 二、光訊息對植物之影響 1 三、茉莉酸訊息對植物之影響 2 四、光與茉莉酸訊息之關聯性 3 五、FIN219(AR-RED INSENSITIVE 219)在遠紅外光與茉莉酸訊息之角色 5 六、FIP1(FIN219-interacting protein 1)之功能 6 七、研究目標 7 材料與方法 8 一、勝任細胞(competent cell)之製備 8 二、質體DNA轉形作用(transformation) 8 三、錐形瓶大量表現FIP1蛋白質 8 四、醱酵槽(fermentor)大量表現FIN219蛋白質 9 五、GST融合FIN219重組蛋白萃取與純化 10 六、His融合FIP1重組蛋白萃取與純化 10 七、去除GST-FIN219融合蛋白之GST 11 八、蛋白質濃度定量 11 九、膠體過濾層析(Gel filtration chromatography)純化FIN219蛋白質單體 11 十、聚丙烯醯胺膠體電泳(SDS-polyacrylamide gel electrophoresis) 12 十一、石英晶體微天平(QCM)分析分子交互作用與酵素動力學 12 十¬二、蛋白質結晶實驗 13 十三、X-ray繞射晶體數據之收集與處理 14 十四、FIP1蛋白質與穀胱甘肽結構之解析與精調 15 十五、FIP1蛋白質結構驗證與繪製 16 結果 17 一、FIN219蛋白質與FIP1蛋白質的表現與純化 17 二、利用膠體過濾法篩選抑制FIN219聚集體形成之緩衝液條件 19 三、膠體過濾法顯示FIN219與GST產生交互作用 19 四、QCM分析FIN219與FIP1以及FIN219與GST之交互作用 20 五、QCM分析並比較FIN219以及FIN219-FIP1複合體之酵素動力學變化 21 六、FIN219-GST、FIN219-FIP1與FIP1蛋白質結晶實驗與X光繞射結果 22 七、FIP1蛋白質與穀胱甘肽受質複合體之結構 24 討論 26 一、FIN219與FIP1以及FIN219與SjGST之交互作用 26 二、FIP1對於FIN219酵素活性之影響 27 三、FIP1與其他GST Tau類型蛋白質結構比較 28 四、FIP1結構活性區域與其他GST tau類型蛋白質之比較 29 結論 31 圖表 33 參考文獻 57 附錄 68 | |
dc.language.iso | zh-TW | |
dc.title | FIP1晶體結構及其與FIN219/JAR1協同活性之研究 | zh_TW |
dc.title | Crystal structure of FIP1 and its cooperative activity with FIN219/JAR1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 謝旭亮(Hsu-Liang Hsieh),林讚標(Tsan-Piao Lin),徐駿森(Chun-Hua Hsu) | |
dc.subject.keyword | FIN219,JAR1,茉莉酸,JA-Ile,FIP1, | zh_TW |
dc.subject.keyword | FIN219,JAR1,Jasmonate,JA-Ile,FIP1, | en |
dc.relation.page | 76 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-14 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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