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研究生:林怡萱
研究生(外文):I-Hsuan Lin
論文名稱:GGDEF區域蛋白Xcc1294與Xcc2731對於Xanthomonascampestris表型之影響
論文名稱(外文):The effects of GGDEF domain proteins Xcc1294 and Xcc2731 on Xanthomonas campestris phenotypes
指導教授:蕭懿民
指導教授(外文):Yi-Min Hsiao
學位類別:碩士
校院名稱:中臺科技大學
系所名稱:醫學生物科技研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:65
中文關鍵詞:GGDEF 區域蛋白第二訊息分子黑腐病菌
外文關鍵詞:Cyclin-di GMPGGDEF domain proteinXanthomonas
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中文摘要
Cyclic di-GMP (c-di-GMP) 是ㄧ個廣泛存在的第二訊息傳遞因子,參與許多細胞功能的調控。c-di-GMP 經由 diguanylate cyclases 催化合成,此酵素之特性為具有一個GGDEF 區域;然而,c-di-GMP 的水解是藉由 phosphodiesterases,此酵素被註解為含有 EAL 或是 HD-GYP 區域。
Xanthomonas campestris pv. campestris (Xcc) 為一植物病原菌,會導致十字花科的黑腐病,此菌基因體編碼有 37 個具有 GGDEF、EAL 或 HD-GYP 區域之蛋白質。在本篇研究,我們顯示 GGDEF 區域蛋白基因 Xcc1294 和 Xcc2731 的過度表現,或是他們的刪除突變與野生株相比,導致不同的表型改變。當 Xcc1294 被過度表現或刪除,沒有發現有顯著性差異,包括胞外酵素、胞外多醣、運動能力以及貼附能力。而在野生株 Xcc 中過度表現 Xcc2731,則顯示: (1) 毒力因子胞外酵素和胞外多醣的產生下降;(2) 運動能力降低;以及 (3) 細胞貼附力減少。
Abstract
Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger with a role in the regulation of a range of cellular function. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases, the enzymes characterized by the presence of a GGDEF domain, whereas the hydrolysis of c-di-GMP is catalyzed by phosphodiesterases, the enzymes noted for possessing an EAL or a HD-GYP domain. Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The bacterial genome encodes 37 proteins with GGDEF, EAL or HD-GYP domain. In this study, we showed that over-expression of GGDEF domain protein genes Xcc1294 and Xcc2731 or their deletion mutants caused different phenotypic changes when compared with wild type strain. When Xcc1294 was over-expressed or deleted, no significant differences were found including extracellular enzymes, exopolysaccharides, motility, and adhesion. While over-expression of Xcc2731 in wild-type Xcc caused (i) decrease in production of virulence factor extracellular enzymes and exopolysaccharides, (ii) reduction in motility, and (iii) reduction in cell attachment.
目錄
中文摘要............................................................................................................. 1
英文摘要............................................................................................................. 2
第一章 前言....................................................................................................... 3
一、Xanthomonas 的特性.......................................................................... 3
二、Xcc 之致病機轉................................................................................... 4
三、Xcc 中 DSF (diffusible signal factor) 訊息調控網........................... 4
四、Cyclic-di-GMP...................................................................................... 5
第二章 文獻回顧............................................................................................... 6
一、第二訊息傳遞因子cyclic-di-GMP...................................................... 6
二、C-di-GMP 之合成與降解.................................................................. 6
三、Xanthomonas 之毒力因子相關研究................................................. 8
四、Xanthomonas 中,含 GGDEF/EAL/HD-GYP 區域蛋白之相關研究.................................................................................................................. 10
第三章 材料與方法........................................................................................... 14
ㄧ、菌種及質體......................................................................................... 14
二、ㄧ般藥品............................................................................................... 14
三、培養基.................................................................................................... 14
(ㄧ)、液體培養基.................................................................................... 14
1. LB 培養基........................................................................................ 14
2. SOC 培養基..................................................................................... 14
3. XOLN 培養基.................................................................................. 15
(二)、固體培養基................................................................................... 15
四、儀器..................................................................................................... 15
(ㄧ)、光譜儀........................................................................................ 15
(二)、桌上型微量離心機.................................................................... 15
(三)、桌上型低溫冷凍離心機............................................................ 15
(四)、電孔儀........................................................................................ 15
(五)、酵素連結免疫吸附分析儀........................................................ 15
(六)、低溫迴轉式振盪培養箱............................................................ 15
(七)、紫外光照相系統........................................................................ 15
(八)、洋菜凝膠電泳............................................................................ 16
(九)、其他............................................................................................ 16
1. 振盪器....................................................................................... 16
2. 攪拌器....................................................................................... 16
3. 電子天秤................................................................................... 16
4. 烘箱........................................................................................... 16
五、方法...................................................................................................... 17
(ㄧ)、細菌之培養................................................................................ 17
(二)、小量質體 DNA 抽取法............................................................ 17
(三)、勝任細胞的製備........................................................................ 18
(四)、電孔法........................................................................................ 18
(五)、胞外酵素之平板測試................................................................ 19
1. 胞外蛋白酶定性分析............................................................... 19
2. 胞外纖維酶定性分析............................................................... 19
3. 胞外聚甘露醣酶定性分析....................................................... 19
(六)、胞外多醣定量分析..................................................................... 20
(七)、運動能力測試............................................................................. 20
(八)、吸附能力分析............................................................................. 21
第四章 結果與討論........................................................................................... 22
ㄧ、Xcc1294 及 Xcc2731 過度表現菌株之取得.................................... 22
二、XC17、1294M 與 XC17(pRK1294) 之表型分析…………...……… 22
(ㄧ)、胞外酵素之定性分析................................................................ 23
1. 胞外蛋白酶定性分析............................................................... 24
2. 胞外纖維酶定性分析............................................................... 24
3. 胞外聚甘露醣酶定性分析....................................................... 24
(二)、胞外多醣之定量分析................................................................ 24
(三)、運動能力測試............................................................................ 24
(四)、吸附能力分析............................................................................ 25
三、XC17、2731M 與 XC17(pRK2731) 之表型分析…......................... 26
(ㄧ)、胞外酵素之定性分析................................................................. 27
1. 胞外蛋白酶定性分析............................................................... 27
2. 胞外纖維酶定性分析............................................................... 27
3. 胞外聚甘露醣酶定性分析....................................................... 28
(二)、胞外多醣之定量分析................................................................. 28
(三)、運動能力測試............................................................................ 28
(四)、吸附能力分析............................................................................ 29
四、討論...................................................................................................... 30
第五章 結論....................................................................................................... 32
參考文獻............................................................................................................. 35
表......................................................................................................................... 41
圖......................................................................................................................... 42
附錄 縮寫字對照表........................................................................................... 56
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