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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 葉開溫 | |
dc.contributor.author | Ke-Ming Wang | en |
dc.contributor.author | 王科明 | zh_TW |
dc.date.accessioned | 2021-06-13T04:20:00Z | - |
dc.date.available | 2006-08-01 | |
dc.date.copyright | 2006-08-01 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-24 | |
dc.identifier.citation | 楊藹華. (2004) 芋頭半胱胺酸蛋白酶抑制劑(Tarocystatin)之基因選殖、表現特性及對微生物抗性的分子機制研究. 國立台灣大學植科所博士論文.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32952 | - |
dc.description.abstract | Tarocystatin屬於cysteine proteinase inhibitor中的一員,由CeCPI基因大量表現且儲藏於芋頭(Colocasia esculenta)塊莖中。Tarocystatin胺基酸序列含有一個高度保留的N端序列,與水稻OC-Ⅰ相似(~97% identity),並在C端有一段廣泛延伸的序列。本實驗利用大腸桿菌表現蛋白的系統,分別表現CeCPI全長、N端及C端的蛋白片段並進行一系列的生化及生理活性分析。全長及N端片段都有抑制papain的活性,而C端卻相反的能活化papain的活性。為了清楚其抑制特性是否不同,利用Lineweaver-Burk plot 法分析其抑制行為。全長的tarocystatin遵守混合型抑制行為(Kia=0.098 μM,Kib=0.252 μM),N端為競爭型抑制行為(Ki=0.057 μM),而C端為異位活化作用。分析其不同受質濃度下對papain的平均抑制活性,全長與N端的平均抑制活性分別為55%及39%,而C端卻具有18%活化papain的活性,約相當於全長與N端抗性之間的差距(16%)。因此不排除C端序列在第二群的phytocystatin中可能扮演調節目標蛋白構形的角色。而N端功能則類似OC-Ⅰ,提供抑制劑競爭活性區的能力。本實驗結果可推論全長對papain之抑制能力較N端片段具有較高之活性,且對papain具較高的專一性 (因其較不易對真菌產生抗性)及較長的半衰期,適合當作防禦性蛋白,且分子量較大比僅N端片段更適合當作儲藏性蛋白。 | zh_TW |
dc.description.abstract | Tarocystatin (CeCPI), a group-2 cysteine proteinase inhibitor in planta, is a defensive protein against phytopathogenic nematodes and fungi. This protein is made up of 205 amino acids, including N terminal segment (Nt peptide) of 98 amino acids and C terminal segment (Ct peptide) of 107 amino acids. The full length (FL), Nt peptide and Ct peptide segments of tarocystatin from Colocasia esculenta (Kaohsiung No.1), were separately amplified by PCR and expressed as GST fusion proteins in E. coli. Kinetic analysis of FL, Nt peptide and Ct peptide on papain activity revealed that FL exhibited mixed type inhibition (Kia =0.098 µM and Kib =0.252 µM) and Nt peptide showed competitive inhibition (Ki, 0.057 µM), whereas Ct peptide enhanced papain activity. Moreover, FL exhibited stronger antipapain activity than Nt peptide. But the antifungal activity of Nt peptide appears to be greater than that of FL, and Ct peptide did not show any antifungal activity indicating that antifungal effect is not related to proteinase inhibition. A shift in the inhibition pattern from competitive inhibition of Nt peptide segment alone to mixed type inhibition of FL implied that Ct peptide has got an influence on FL function, and Nt peptide can determine the fate of CeCPI function. Based on the inhibitory kinetics of FL and fragments of CeCPI on papain activity, a model for group-2 phytocystatin inhibitory mechanism has been proposed. The physiological significance for two varied types of proteins is also discussed. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T04:20:00Z (GMT). No. of bitstreams: 1 ntu-95-R93b42013-1.pdf: 2037494 bytes, checksum: 857109fbe3131ee249706be7ff0b3129 (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 口試委員會審定書i
誌謝ii 中文摘要3 英文摘要4 第一章 前言5 一、芋之源起5 二、Phytocystatin的分類與研究現6 三、Phytocystatin在植物體中扮演之角色8 四、抑制劑特性介紹10 五、芋頭半胱胺酸抑制因子之研究緣起與動機12 第二章 材料與方法13 一、材料13 二、方法13 (一)Phytocystatin序列分析與結構模型研究13 1.相似序列搜尋及親緣性分析13 2.二級結構預測及三級結構模擬13 (二)不同片段重組蛋白質之表現、純化及活性分析14 1.聚合酶連鎖反應放大特定片段及接合表現載體14 2.大腸桿菌勝任細胞製備程序15 3.大腸桿菌轉型 15 4.大量表現GST重組蛋白及其純化16 5.蛋白質定量分析16 6.SDS-聚丙烯醯胺膠體電泳17 7.半胱胺酸蛋白酶抑制劑之膠體活性染色分析19 8.抑制真菌活性試驗20 (三)不同片段蛋白質之活性定量分析20 1.GST重組蛋白之切離及蛋白質回收20 2.半胱胺酸蛋白酶抑制劑之活性測定21 第三章 結果22 一、Tarocystatin三級結構預測22 二、大量表現不同片段重組蛋白及其活性分析22 三、不同片段重組蛋白對真菌生長抑制活性分析23 四、不同片段之活性定量分析及抑制常數計算24 五、C端與N端蛋白之競爭試驗及C端活化特性試驗 25 第四章 討論27 一、重組蛋白之大量表現、純化及活性分析27 二、不同片段重組蛋白的抗菌性分析28 三、Tarocystatin不同片段的抑制行為判定及抑制常數計算29 四、Tarocystatin抑制木瓜酶活性機制之推衍30 結論32 圖表34 參考文獻48 附錄56 | |
dc.language.iso | zh-TW | |
dc.title | 芋頭半胱胺酸蛋白酶抑制因子上不同區域抑制特性分析 | zh_TW |
dc.title | Investigation on inhibitory characterization of different segments of tarocystatin from Colocasia esculenta | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 何國傑,王淑珍,楊藹華,廖秀娟 | |
dc.subject.keyword | 半胱胺酸蛋白酶,抑制,混合型抑制,芋頭, | zh_TW |
dc.subject.keyword | tarocystatin,cysteine proteinase inhibitor,mixed inhibition,taro, | en |
dc.relation.page | 62 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-24 | |
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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