臺灣博碩士論文加值系統

第三型分泌系統（Type III secretion system ， T3SS）是多數革蘭氏陰性菌中經由多種蛋白複合體形成的跨膜通道，可經由分泌蛋白或把具有致病活性的蛋白直接注入宿主細胞中發揮致病作用。柑橘潰瘍病是黃桿菌所引起的細菌疾病，經由第三型分泌系統進行感染，雖然對人體無害，但潰瘍嚴重影響柑橘樹的生長能力，由黃單胞菌（Xanthomonas axonopodis pv. Citri，Xac）引起，會導致植物葉子脫掉落，柑橘植物的果實早熟而掉落。XopAI蛋白引起植物細菌性潰瘍的機制目前尚不清楚，我們希望經由以下實驗確定XopAI蛋白的致病機制，首先經由重組XopAI蛋白在大腸桿菌中表達，用Ni2+柱純化並結晶進行結構分析。經由序列比對我們發現XopAI C-末端區域與假單胞菌的效應物HopU1預測的ADP-核糖基轉移酶結構區段相似，利用iGEMDOCKv2.1軟體模擬分子對接預測，我們篩選出NAD +，NADP，ADP三種cofactors進行螢光分析實驗。我們利用螢光光譜法確定XopAI蛋白會與NAD +結合，這結果類似於先前研究的效應蛋白HopU1的ADP-核糖基轉移酶結構區段，進而影響植物生長和代謝。

Type III secretion system is a protein appendage found in several Gram-negative bacteria. In pathogenic bacteria, the needle-like structure is used as a sensor probe to detect the presence of eukaryotic organisms and secrete proteins that help the bacteria infect them. Citrus canker is a disease affecting the bacterium species caused by the bacterium Xanthomonas axonopodis. While not harmful to humans, canker serious affects the vitality of citrus trees, causing leaves and fruit to drop prematurely. Although a fruit infected with canker is safe to eat, it will be too unsightly to be sold. The disease is caused by Xanthomonas axonopodis pv. citri (Xac) and leads to defoliation, and premature fruit drop of the citrus plant. The mechanism by which XopAI proteins cause plant bacterial ulcers is not yet known. The recombinant XopAI protein was expressed in E.coli, purified by using Ni2+ column and crystallized. The crystals belonged to space group P41212 and diffracted resolution to 1.63 Å. Because the C-terminal region of XopAI has similarity to the ADP-ribosyl transferase domains of the effector HopU1 of Pseudomonas syringae, we performed fluorescent spectroscopic analyses using three potential cofactors (NAD+, NADP+, and ADP) in order to determine the binding preference of XopAI.

第一章 前言 1
一、 細菌第三型分泌系統(T3SS, Type III secretion system) 1
(一) 第三型分泌系統概述 1
(二) T3SS之結構特性 1
(三) T3SS蛋白組成 2
(四) T3SS的感染途徑 2
二、 柑橘潰瘍 3
(一) 柑橘潰瘍病概述 3
(二) 柑橘潰瘍病病徵 3
(三) 柑橘潰瘍病分類 4
(四) 柑橘潰瘍病寄主 4
(五) 柑橘潰瘍病形態 4
三、 黃單胞菌屬(Xanthomonas) 4
(一) 黃單孢菌屬(Xanthomonas)概述 4
(二) 黃單孢菌形態和生長 5
四、 羅斯曼摺疊（Rossmann fold） 5
五、 丁香假單胞菌(Pseudomonas syringae) 5
六、 ADP-核醣基化（ADP-Ribosylation） 6
(一) 催化機制 6
七、 研究動機 7
第二章 材料與方法 8
一、 實驗流程 8
二、 實驗材料與方法 8
(一) 蛋白質可溶性測試 8
(二) SDS-PAGE分析 11
(三) 蛋白質大量表現 14
(四) 鎳親和性管柱純化 15
(五) 蛋白質濃縮 20
(六) 蛋白質透析 21
(七) 蛋白質定量 22
(八) 蛋白質結晶實驗 23
(九) X-ray晶體繞射實驗數據收集與處理 24
(十) 電腦模擬分子對接實驗 27
(十一) 螢光光譜分析法 (Fluorescence spectroscopy) 28
(十二) 蛋白質熱穩定性分析 (Thermostability measurements) 30
第三章 結果 32
一、 XopAI蛋白大量表現與純化 32
(一) XopAI蛋白可溶性測試與大量表現 32
(二) XopAI蛋白藉由親和性管柱進行純化 32
(三) XopAI蛋白透析與濃縮 33
二、 XopAI蛋白晶體結構 33
(一) XopAI蛋白結晶條件篩選 33
(二) XopAI蛋白結晶 33
(三) 經由X-ray繞射數據分析與晶格判斷 34
(四) 利用分子取代法 (molecular replacement) 進行XopAI蛋白結構解析 34
三、 蛋白質結構比較分析 35
(一) XopAI蛋白與相似蛋白序列比對 35
四、 電腦模擬分子對接尋找輔因子(cofactor) 36
五、 XopAI螢光光譜分析 36
六、 XopAI蛋白與輔因子(cofactor)之熱穩定性分析 37
第四章 討論 38
一、 蛋白質的表現與純化 38
二、 蛋白質透析 38
三、 XopAI蛋白結晶實驗 39
四、 XopAI蛋白結構分析 39
五、 蛋白質序列結構比較分析 40
六、 電腦模擬分子對接分析 41
七、 XopAI螢光光譜分析 42
八、 XopAI蛋白與輔因子(cofactor)之熱穩定性分析 43
第五章 結論 44
表次 45
圖次 51
附錄 78
參考文獻 91

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