Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23945
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dc.contributor詹迺立zh_TW
dc.contributor廖彥銓zh_TW
dc.contributor.advisor周三和zh_TW
dc.contributor.author邱國城zh_TW
dc.contributor.authorChiu, Kuo-Chengen_US
dc.contributor.other中興大學zh_TW
dc.date2007zh_TW
dc.date.accessioned2014-06-06T07:21:35Z-
dc.date.available2014-06-06T07:21:35Z-
dc.identifierU0005-2608200615283200zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/23945-
dc.description.abstractXanthomonas campestris pv. campestris為格蘭氏陰性菌,主要感染十字花科植物,造成農業損失,因此研究這株菌的蛋白功能將有助於減少農業上的損失。其中XC2113利用BLAST 預測為YaeQ 蛋白。而在其它菌株中,例如在Salmonella enterica sv. Typhimurium 菌株中,YaeQ 蛋白調控hemolysin 致病毒素的表現。而hemolysin 主要的功能是分解紅血球,造成溶血的作用。XC2113在XCC的作用機制並不清楚,但可能會調控Xcc 其它的致病毒素,進而感染十字花科植物,造成植物腐壞。目前已經成功得到XC2113的可溶性蛋白,並且找到合適的結晶條件。經過X-ray 繞射技術分析後,其解析度達到1.28Å。未來將利用結構分析,了解該蛋白的功能。另外還分別得到XC2712及XC5630的蛋白結晶。而利用序列比對的方法,XC2712被預測具有glycine cleavage system (GCS) transcriptional repressor的功能。不論在原核生物或是真核生物中,GCS 都是很重要的調節機制,藉由此系統可以將甘胺酸分解成NH3、CO2和一個單一碳單元。目前XC2712雖可得到很多不同晶形的晶體,但由於結晶速度太快,以致於得到的晶體都太小。X-ray 繞射技術分析後,也無法得到很好的解析度。未來嘗試以晶種結晶的方式,試圖得到較大的晶體。利用BLAST 作分析,判斷XC5630 的功能可能為酮固醇異構酶(ketosteroid isomerase)。酮固醇異構酶可以改變類固醇環上雙鍵的位置。這種酮固醇調控養份分解,產生生物體所需的能量。然而此調控機制還不清楚,因此研究XC5630將有助於了解生命體如何產生這種類固醇,進而了解整個能量代謝的機制。XC5630得到一個晶形不錯的結晶,晶體大小為0.3×0.2×0.1 mm 。但以X-ray 繞射技術分析後,卻沒有辨法得到很好的解析度。zh_TW
dc.description.abstractXanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium. It is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. To reduce this loss, it is necessary to find Xcc pathogenic factors and to block their functions. Among them, XC2113 was predicted to belong to the YaeQ family by a BLAST search, and proposed to regulate the expression of toxins such as hemolysin in other bacterial strains. For example, the YaeQ in S. enterica sv. Typhimurium play the function of hemolysin. The chief function of hemolysin is presumably to decompose red blood cell. Although the function of XC2113 in Xcc is not clear yet, it may infect cruciferous plants, causing rot disease through the release of hemolysin. The XC2113 protein has been successfully expressed and purified, and crystals suitable for X-ray diffraction (reached to 1.28 Å) have been obtained. By analyzing its tertiary structure, it may be possible to decipher its function. Besides XC2113, the XC2712 and XC5630 proteins were also successfully crystallized. XC2712 is predicted as a glycine cleavage system (GCS) transcriptional repressor by sequence alignment. GCS is a very important mechanism in every organism to decompose glycine into NH3, CO2 and C1 unit, which is an important source of methyl donor in many biosynthesis reactions. At present, many different kinds of small crystals were observed for XC2712. But no good data could be collected yet, possibly due to the fast crystallization. Seeding method can be tried to improve the crystal size. XC5630 is predicted as a ketosteroid isomerase by a BLAST serch, which can change the position of double bond of ketosteroids. This ketosteroid can control the decomposition of nutrient to energy. Good-liking crystals of XC5630 of 0.30.20.1 mm size have been obtained. However, no good data could be derived from this crystal, possibly due to their high mosacity values.en_US
dc.description.tableofcontents致謝 I 中文摘要 Ⅱ Abstract Ⅲ 圖目錄 Ⅵ 表目錄 Ⅶ 第一章 前言 1 1-1、Xanthomonas campestris pv.campestris之結構基因體計畫簡介 1 1-2、利用X-ray 繞射技術解析蛋白質結構 4 1-3、研究動機及目的 4 第二章 材料與方法 6 2-1、目標蛋白質之選定 6 2-2、蛋白質表現載體之構築 6 2-2-1、染色體DNA之抽取 6 2-2-2、引子之設計與合成 7 2-2-3、聚合酶連鎖反應(PCR, polymerase chain reaction) 8 2-2-4、膠體電泳 9 2-2-5、PCR product之純化 9 2-2-6、蛋白質表現系統 9 2-2-7、質體DNA之抽取 10 2-2-8、PCR產物與質體DNA之限制酶作用 11 2-2-9、DNA 之接合反應 11 2-2-10、E.coli 勝任細胞之製備 11 2-2-11、轉殖作用(Transformation) 12 2-2-12、DNA 定序 12 2-3、蛋白質之大量表現與純化 12 2-3-1、蛋白質大量表現之誘發條件 12 2-3-2、SDS-PAGE 13 2-3-3、蛋白質之大量表現 14 2-3-4、蛋白質之純化 16 2-3-5、蛋白質濃度測定 18 2-3-6、蛋白質結晶實驗所需之蛋白質樣品製備 18 2-4、利用X-ray晶體繞射技術解析蛋白質之結構 19 2-4-1、蛋白質結晶實驗 19 2-4-2、結晶條件篩選 19 2-4-3、大量結晶 19 2-4-4、篩選合適之抗凍劑(Cryo-protectant) 20 2-4-5、Native 蛋白晶體X-ray晶體繞射數據收集及分析實驗 20 2-4-6、相位(Phase)判定 21 2-4-7、以X-ray決定蛋白質之結構 21 第三章 結果與討論 23 3-1、目標蛋白質之選定 23 3-2、XC2113 23 3-2-1、XC2113功能註解分析 23 3-2-2、XC2113蛋白質表現載體之構築 24 3-2-3、XC2113蛋白質之大量表現與純化 24 3-2-4、XC2113蛋白質之結晶與X-ray 晶體繞射實驗測試 25 3-2-5、XC2113之X-ray 實驗結果討論 26 3-2-6、探討XC2113功能 26 3-3、XC2712 27 3-3-1、XC2712之功能註解 27 3-3-2、XC2712蛋白質表現載體之構築、蛋白表現及純化 27 3-3-3、XC2712蛋白質之結晶 28 3-3-4、XC2712的功能探討 29 3-4、XC5630 30 3-4-1、XC5630之功能註解 30 3-4-2、XC5630蛋白質表現載體之構築、蛋白表現及純化 31 3-4-3、XC5630蛋白質之結晶 32 3-4-4、XC5630的功能探討 32 參考文獻 34 附錄 38 縮寫檢索表 38 實驗儀器設備 39 圖目錄 圖2-1、以X-ray晶體繞射與NMR決定蛋白質結構之實驗流程圖 41 圖2-2、Sticky-end PCR 之原理與方法 42 圖2-3、pMCSG7之map 43 圖2-4、蒸氣擴散法 44 圖2-5、以X-ray 晶體繞射技術解析蛋白質之結構流程 45 圖3-1、XC2113之基因預測結果 46 圖3-2、XC2113 之domain searching 及BLAST之結果 49 圖3-3、XC2113與序列相似的幾個蛋白作序列比對 50 圖3-4、XC2113之定序結果 51 圖3-5、XC2113之純化結果 52 圖3-6、XC2113的蛋白晶體 53 圖3-7、XC2113 Native 晶體繞射圖及數據 54 圖3-8、XC2113的胺基酸序列與PDB 資料庫作BLAST 55 圖3-9、利用Threading modeling 預測XC2113的蛋白結構 56 圖3-10、RfaH 蛋白促進致病毒菌產生的示意圖 56 圖3-11、XC2712之基因預測結果 57 圖3-12、XC2712 之BLAST 結果 58 圖3-13、XC2712與其序列相似的幾個蛋白作序列比對 59 圖3-14、XC2712之定序結果 60 圖3-15、XC2712純化結果及未標定之晶體 61 圖3-16、selenomethionine 標定之XC2712蛋白晶體 62 圖3-17、 Gcs 的調控機制 63 圖3-18、GcsA 與GcsR 的調控機制 64 圖3-19、XC2712的胺基酸序列與PDB 資料庫作BLAST 65 圖3-20、XC2712與1U8S 作序列比對 65 圖3-21、利用Threading modeling預測XC2712的蛋白結構 66 圖3-22、1U8S 的結構 66 圖 3-23、XC5630之基因預測結果 67 圖 3-24、XC5630 之BLAST 結果 68 圖 3-25、XC5630與其序列相似蛋白作序列比對 69 圖3-26、XC5630的定序結果 70 圖 3-27、XC5630純化結果及未標定蛋白之結晶 71 圖3-28、XC5630的胺基酸序列與PDB 資料庫作BLAST 72 圖3-29、利用Threading modeling 預測XC5630的蛋白結構 72 圖3-30、NTF2-like domain 的基本原理 73 圖3-31、KSI 催化酮固醇異構化之反應機構 74 表目錄 表3-1、挑選之目標蛋白質整理列表 75 表3-2、引子設計 76zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物化學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2608200615283200en_US
dc.subjectX-ray 繞射技術zh_TW
dc.subjectX-ray diffraction techniqueen_US
dc.subjectXanthomonas campestrisen_US
dc.subjectglycine cleavage systemen_US
dc.subjectYaeQen_US
dc.subjectketosteroid isomeraseen_US
dc.title利用X-ray 繞射技術研究植物病原菌Xanthomonas campestris 的XC2113, XC2712及XC5630 蛋白zh_TW
dc.titleThe studies of XC2113, XC2712 and XC5630 proteins from a plant pathogen Xanthomonas campestris by X-ray diffraction techniqueen_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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