Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22443
DC FieldValueLanguage
dc.contributor廖彥銓zh_TW
dc.contributor.advisor周三和zh_TW
dc.contributor.author黃照煒zh_TW
dc.contributor.authorHuang, Chao-Weien_US
dc.contributor.other中興大學zh_TW
dc.date2007zh_TW
dc.date.accessioned2014-06-06T07:18:00Z-
dc.date.available2014-06-06T07:18:00Z-
dc.identifierU0005-2708200606223600zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/22443-
dc.description.abstract本論文研究的菌株為Xanthomonase campestris pv. campestris,屬於革蘭氏陰性菌,為兼具學術性及應用性之菌種。X. campestris能分泌多種胞外蛋白,為研究格蘭氏陰性菌蛋白分泌模式之標準系統之ㄧ,並為感染十字花科造成黑腐病之植物病原菌,屬世界性之病害。藉由基因體解碼下,此一病原菌被預測的基因約有4100個。本論文利用X-ray繞射法研究X. campestris str. 17菌株之11個目標蛋白質,其中XC1692由141個胺基酸組成,功能未知。比對已知物種基因資料庫後發現,XC1692 在不同原核生物體皆有相似序列,且同譯註為未知功能的蛋白。直至目前為止,沒有任何與XC1692序列相似(>20%)蛋白之三級結構被解析出來。我們已經得到高解析度的XC1692晶體(1.45 Å),但為利用多波長異常繞射數據解決其相位問題時, 無法獲得Se-Met 標定蛋白俱高解析度的晶體。為了解決此一問題, 我們藉由突變將Met轉成Ala, 減少Met個數,以降低XC1692晶体可能因Se-Met 標定造成的結構不穩定。藉此我們順利地得到結晶,並成功地解決相位的問題。由於XC1692為未知功能蛋白,所以我們利用結構比對得到XC1692相似結構為核醣核酸水解酶之抑制物。搜尋X. campestris基因庫發現XC1691之功能為核醣核酸水解酶,且XC1691位於XC1692上游,並由同一啟動子所調控。這暗示XC1692可能為核醣核酸水解酶抑制物。利用pull down assay實驗,證明XC1691與XC1692之間有交互作用力的存在,初步確定了XC1692的功能為核醣核酸水解酶抑制物。目前我們正在進行XC1691及XC1692複合物的結晶實驗,希望可以獲得不錯的數據,進一步從結構的角度來證實XC1692的功能的確為核醣核酸水解酶抑制物。zh_TW
dc.description.abstractThe bacterial strain studied in this thesis is Xanthomonase campestris pv. campestris, which is a gram-negative bacterium and an important pathovar both academically and industrially. X. campestris can secrete many kinds of extracellular proteins, and is a model system for studying protein secretion of gram-negative bacteria. It is phytopathogenic to cruciferous plants and can cause worldwide agricultural loss. From the sequenced genome, about 4100 genes were predicted. This thesis tries to study 11 of them by using X-ray crystallography technique. XC1692 is one of them containing 141 amino acids with no identified function. However many sequences with high similarity score were detected by a BLAST search. Until now, there is also no similar tertiary protein structure determined. However, although we have obtained high resolution crystals of native XC1692 (diffracted to 1.45 Å), we couldn't solve its phase using multiwavelength anomalous diffraction (MAD) method, due to the poor resolution data of Se-substituted crystals. To solve the problem, we have used mutagenesis method to change Met to Ala to reduce the number of Met to possibly increase the crystal stability. Using this approach, we have successfully solved the phase problem and got a preliminary structure of XC1692 using the MAD approach. XC1692 was then found to adopt a similar architecture with a ribonuclease inhibitor through a structure alignment. Also, a gene (XC1691) annotated as a ribonuclease was found to be upstream of XC1692 and controlled by the same promoter. Altogether, these data imply that XC1692 may serve as a ribonuclease inhibitor, which was further confirmed by a pull-down assay. We are currently crystallizing the XC1691/XC1692 complex to determine its functions from a structure perspective.en_US
dc.description.tableofcontents中文摘要 I 英文摘要 II 縮寫檢索表 III 實驗儀器設備 IV 本文目錄 VI 第一章 前言 1 第二章 材料與方法 4 I. 蛋白質表現載體之構築 4 A. 染色體DNA 之抽取 4 B. 引子之設計與合成 4 C. 聚合酶連鎖反應(PCR, polymerase chain reaction) 5 D. 膠體電泳 6 E. 表現載體 6 F. 質體DNA之抽取 7 G. 質體DNA之限制酶作用 7 H. E. coli 勝任細胞之製備 8 I. 接合反應(Ligation) 8 J. 轉殖作用(Transformation) 9 K. DNA 定序 9 II. 蛋白質之大量表現與純化 9 A. SDS-PAGE 9 B. 蛋白質之大量表現 11 C. 蛋白質的純化 12 D. 蛋白質濃度測定 14 E. 蛋白質結晶實驗所需之蛋白質樣品製備 14 III. 利用X-ray 晶體繞射技術解析蛋白質之結構 15 A. 蛋白結晶 15 B. 結晶條件篩選 15 C. 大量結晶 15 D. 保護晶體的方法 16 E. cryo-protectant 的選擇 16 F. Native蛋白晶體X-ray繞射數據收集及分析實驗 17 G. 晶體結構的建立 17 H. 相位(phase)判定 17 I. 相位決定方法 18 J. 電子密度圖 (electron density map) 19 K. 分子模型的建立 (model building and refinement) 20 第三章 結果與討論 21 A. XC1692的基因註解及分析 21 B. XC1692表現載體 22 C. XC1692蛋白的大量表現及純化 22 D. XC1692蛋白結晶及X-ray繞射實驗 23 E. 點突變之XC1692蛋白大量表現、純化 24 F. XC1692的功能討論 25 第四章 參考文獻 28 圖表 31 圖一 引子設計原理 31 圖二 pMCsG7 32 圖三 蒸氣擴散法(Vapor diffusion) 33 圖四 XC1692之基因預測結果 34 圖五 XC1692 BLASTp 35 圖六 XC1692純化結果 36 圖七 XC1692初步結晶圖 37 圖八 XC1692手動微調結晶圖 37 圖九 XC1692 Native晶體繞射圖 38 圖十 點突變XC1692 39 圖十一 點突變XC1692純化結果 40 圖十二 點突變XC1692經Se標定後,利用Mass Spectrum測定的結果 41 圖十三 XC1692之初步結構圖 42 圖十四 XC1692與 1AY7的superposition 43 圖十五 XC1692二級結構之topology與不同物種相似序列之比較 44 圖十六 XC1691基因預測結果 45 圖十七 XC1691 BLASTp 46 圖十八 XC1691蛋白序列與其他物種的比對結果 47 圖十九 Δ1-95XC 1691純化結果 48 圖二十 Pull-down assay 49 圖二十一 XC1692/Δ1-95XC 1691 complex結晶圖 50 圖二十二 XC1692定序結果 51 圖二十三 點突變XC1692定序結果 52 圖二十四 Δ1-95XC 1691定序結果 53 表一 挑選之目標蛋白整理表 54 表二 XC1692胺基酸組成及特性分析 55 表三 點突變XC1692胺基酸組成及特性分析 56 表四 Δ1-95XC 1691胺基酸組成及特性分析 57 表五 XC1692 Native晶體繞射數據 58 表六 引子設計 59zh_TW
dc.language.isoen_USzh_TW
dc.publisher生命科學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2708200606223600en_US
dc.subjectXanthomonase campestris pv. campestrisen_US
dc.subject十字花科黑腐病病原菌zh_TW
dc.subjectcomplexen_US
dc.subjectunknown function proteinen_US
dc.subject複合物zh_TW
dc.subject未知功能蛋白zh_TW
dc.titleXanthomonas campestris pv.campestris 未知功能蛋白XC1692/XC1691複合物的晶體結構及功能分析zh_TW
dc.titleStructural determination and functional analysis of unknown function protein complex XC1692/XC1691 in Xanthomonas campestris pv. campestrisen_US
dc.typeThesis and Dissertationzh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
item.grantfulltextnone-
item.cerifentitytypePublications-
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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