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學校名稱輔仁大學
系所名稱生命科學系
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學號493546211
研究生(中)游竣惟
研究生(英)Yu, Chun-Wei
論文名稱(中)Erwinia chrysanthemi產生色素性狀之應用及其基因選殖及其基因選殖
論文名稱(英)Application and gene cloning of pigmentation phenotype of Erwinia chrysanthemi
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指導教授(中)李永安
指導教授(英)Lee, Yung-An
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畢業學年度94
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關鍵字(中)軟腐病菌 藍色色素 基因選殖
關鍵字(英)Erwinia chrysanthemi blue pigment gene cloning
摘要(中)本實驗室先前研發出的NGM培養基可以用來進行軟腐病原細菌之分離,開發成檢測試紙(Ech1-檢測試紙)可對具有軟腐病徵的植物組織進行檢測,但是此檢測試紙卻無法對Erwinia chrysanthemi菌株進行檢測。當添加病葉過濾液於Ech1-檢測試紙時,可以促使E. chrysanthemi菌株藍色色素的產生,將病葉過濾液以沸水煮沸及不同蛋白質分子量大小之濾膜過濾後,仍然可以促使E. chrysanthemi菌株於Ech1-檢測試紙上產生藍色暈圈。外加不同碳源及氮源於Ech1-試紙後,發現氮源可以促進E. chrysanthemi菌株藍色色素的產生,不同的溫度及存放地點也會影響藍色色素之生成,其中以存放於28℃培養箱之處理較佳。測試了不同培養基成分及其他類似物質後,我們成功的將NGM培養基改良成為Ech2-1及Ech2-2配方,可以使E. chrysanthemi菌株在培養基上呈色更加明顯,以Ech2-2配方進一步開發成快速檢測試紙(簡稱Ech2-檢測試紙),除了可以用來進行具軟腐病徵之植物組織的快速檢測,還可以直接對E. chrysanthemi細菌進行檢測,靈敏度測試的結果中,我們發現Ech2-檢測試紙具有相當高的靈敏度,當樣本中含有低於10個colony時仍可被Ech2-檢測試紙測出。除了可以運用於次級感染來源(如發病的植物組織)之檢測外,還可以運用在初級感染來源的檢測,以土壤水溶液來配製細菌懸浮液,模擬栽培介質及灌溉水受到病原菌汙染之情形,於Ech2-2配方中添加入另一種金屬離子後,再進行檢測試紙之製作,目前已經克服了環境中其他微生物之干擾,同樣可以在診斷試紙上看到藍色暈圈的產生,而且具有相當高的靈敏度。在Ech2-檢測試紙的開發過程中,我們發現了許多培養基成分及環境因子會影響藍色色素的生成,大多數的E. chrysanthemi菌株在某一pH值中會產生最明顯的藍綠色外觀,培養基中添加某種鹽類會抑制E. chrysanthemi藍色色素的產生,另外我們發現,使用不同廠牌的agar進行培養基的配製同樣會影響藍色色素的生成。植物軟腐病組織在Ech1-檢測試紙上產生藍色暈圈之位置具有較多的菌量,不產生藍色色素的部位同樣有E. chrysanthemi菌體存在,但是菌量較少。放置抗生素紙錠於Ech1-檢測試紙上,會抑制E. chrysanthemi菌體的生長及藍色色素的生成。將蝴蝶蘭病組織放置於Ech1-檢測試紙中央,觀察其藍色暈圈隨時間點的呈色變化,可以在濾紙上發現藍色暈圈不只一個,在含有低濃度agar的Ech2-1培養基上,也同樣可以發現此現象。我們已經成功的純化出E. chrysanthemi Ly8的藍色色素,由溶解性測試中發現此藍色色素只會溶於DMSO及DMF溶劑中,經由全光譜的掃描可以在波長615 nm附近獲得最大吸光值。將藍色色素溶於DMSO溶劑後,以DMSO及DMF為移動相進行薄膜色層分析(TLC),分離的結果只會產生一種藍色色帶,另外以甲醇(methanol)為移動相進行HPLC分離,偵測波長615 nm處之吸光值,可以在大約3 min時產生一個明顯的波峰。 Erwinia chrysanthemi 12616菌株不僅可以在NGM培養基上產生深藍色的菌落外觀,也可以在LA及NA培養基上產生深藍色的外觀,因此我們選擇E. chrysanthemi 12616菌株使用fosmid pCC1FOS vector進行genomic library之建構。經過接合反應及l噬菌體之包裹之後,將l噬菌體做不同稀釋接著進行感染EPI300-T1R E. coli,總共挑出2000個colonies。並利用NGM培養基進行色素相關基因之選殖,兩星期後我們找到了38個菌株外觀變成明顯的黃色,比較黃色菌落之限制酵素(EcoRI)作用片段,發現約有18個菌株都具有3.5 kb、4.5 kb及6 kb的片段大小。以專一性引子indC(F1+R3)進行PCR擴增反應,無法擴增出644 bp之片段大小,表示這些菌株中沒有和參與藍色色素生成有關的indC基因片段,顯示應該有其他基因影響E. coli菌株的黃色外觀性狀,選擇其中四株黃色外觀較明顯的菌株之質體DNA,以EcoRI以及EcoRI+HindIII進行作用,再以DIG標定之indC基因644 bp DNA片段為探針,進行南方氏雜交反應,同樣無法產生indC基因之雜合片段。我們選取其中一個菌株E. coli 12616(1-14)內含有約44 kb之片段大小,進行基因的次選殖,已成功的將目標基因所在範圍縮短至大約22 kb之片段中,而且其中含有大約8 kb的pCC1FOS Vector,因此目前我們已經將與黃色菌落外觀有關之基因縮短至大約14 kb的片段中。經由不同培養基的測試,發現將E. coli 12616(1-14)菌株培養於1.5X NGM培養基,在特殊之pH條件下,可使E. coli 12616(1-14)菌落更快產生黃色的菌落外觀,將菌株培養於37℃兩天後置於室溫中,約7天即可觀察到黃色的菌落外觀。
摘要(英)NGM medium was developed for the isolation and differentiation of the pathogenic enterobacteria causing soft rot, and NGM medium was used to make the Ech1- paper to detect E. chrysanthemi in plant soft rot tissues. However, the Ech1- paper could not be used to detect E. chrysanthemi in water. The filtrate of soft-rot Phalaenopsis leaves could induce E. chrysanthemi to develop blue color on Ech1- paper. Different carbon and nitrogen sources were added to Ech1- paper, and one nitrogen source could make E. chrysamthemi to develop color as the filtrate did. The temperature could affect the color development, and the optimal temperature was 28℃. Ech2-2 medium was developed by changing the chemical components of NGM, and used to prepare Ech2- paper. Ech2- filter paper had high sensitivity (<10 colony) to detect E. chrysanthemi in water. Ech2- paper therefore could be used to detect primary inoculum of E. chrysanthemi soft rot disease. The addition of one specific ion to Ech2- paper could increase the sensitivity and reduce the influence of saprophytic bacteria in water. On Ech2-2 agar medium, E. chrysanthemi developed deep blue color at a special pH, but did not produce color when the salt was added to the medium. In addition, different brands of agar could affect the blue pigment development of E. chrysanthemi. On Ech1- paper there was much more bacteria in the location with blue color than that without color. Antibiotics inhibited the blue pigment development in Ech1- papers. The blue pigment of E. chrysanthemi strain Ly8 had been purified, and the blue pigment could only be dissolved in DMSO and DMF solution. A maximum absorption was found at 615 nm, and the HPLC separation test showed a clear peak at 3 minute after sample injection. Erwinia chrysanthemi strain 12616 produces the dark blue color on NGM medium, and also on LA and NA medium. The fosmid pCC1FOS vector was used to construct the genomic library of E. chrysanthemi 12616, and there were 2000 colonies to store in our library. NGM medium was used to screen colonies which was could produce pigments, and there were 38 clones with yellow appearance. Most of them have the same DNA fragments after EcoRI digestion. The specific primer indC (F1+R3) could not amplify a 644 bp fragment indicating that there was no indC gene fragment in these clones. By using yellow color formation we choose 4 clones out of 38 clones and digest with EcoRI and EcoRI+HindIII. The indC fragment was labeled with Dig as probe, but we could not found the indC hybrid fragment between the 4 clones we choose. Therefore, we choose E. coli 12616 (1-14) clone for gene subcloning. Our target gene has cut down from a 44 kb fragment to a fragment about 22 kb, and inside the 22 kb fragment was a fragment about 8 kb belonging to copyControl pCC1FOS vector. Therefore, we had successfully cut down the gene fragment into a 14 kb fragment, which was related to yellow color. A medium had found to detect the yellow appearance of subclones. The medium was called 1.5X NGM and had a special pH. The yellow appearance of subclones could be found after seven days of culture.
論文目次第一章 軟腐病菌Erwinia chrysanthemi藍色色素之應用 中文摘要-------------------------------------------------------- I 英文摘要------------------------------------------------------- III 前言----------------------------------------------------------- 1-1 材料與方法----------------------------------------------------- 1-8 菌種的取得及培養條件----------------------------------------- 1-8 植物接種材料的取得方式--------------------------------------- 1-8 E. chrysanthemi細菌懸浮液的配製方式---------------------------- 1-8 軟腐病菌E. chrysanthemi的接種方式----------------------------- 1-8 1. E. chrysanthemi鑑別性培養基及Ech1-檢測試紙之研發 不同菌株之E. chrysanthemi或E. carotovora在鑑別性培養基之呈色- 1-9 Ech1-檢測試紙之製作-------------------------------------- 1-9 Ech1-檢測試紙對具軟腐病徵之植物組織之檢測---------------- 1-9 Ech1-檢測試紙之製作方式差異對呈色之影響------------------ 1-10 Ech1-檢測試紙對E. chrysanthemi細菌之檢測------------------ 1-10 Ech1-檢測試紙添加病葉過濾液對E. chrysanthemi 細菌之檢測--- 1-11 病葉過濾液中促使E. chrysanthemi可在Ech1-檢測試紙呈色之成分探討 ------------------------------------------------------------- 1-11 2. E. chrysanthemi鑑別性培養基及Ech2-檢測試紙之改良 鑑別性培養基成分分析------------------------------------ 1-11 Ech1-檢測試紙額外添加碳源、氮源之方法------------------- 1-11 改良後的E. chrysanthemi鑑別性培養基配方及使用方法-------- 1-12 以改良後的鑑別性培養基進行軟腐病菌(E. chrysanthemi)之分離-- 1-12 Ech2-檢測試紙之製作方式-------------------------------- 1-12 Ech2-檢測試紙之使用方法--------------------------------- 1-13 Ech2-檢測試紙之靈敏度測試------------------------------- 1-13 Ech2-檢測試紙運用於初級感染來源之檢測------------------- 1-13 Ech2-檢測試紙之保存方法--------------------------------- 1-14 3.鑑別性培養基中各成分及環境因子對E. chrysanthemi呈色之影響 Pectin及PGA對藍色色素呈色之影響----------------------- 1-14 不同廠牌Agar對藍色色素呈色之影響---------------------- 1-15 4. 植物軟腐病組織在Ech1-檢測試紙上藍色暈圈之變化與菌體生長之關係 Ech1-檢測試紙上藍色暈圈與菌量之關係探討---------------- 1-15 抗生素紙錠對菌落生長及藍色色素呈色之影響--------------- 1-15 Ech1-檢測試紙上藍色暈圈隨時間點之變化情形-------------- 1-16 E. chrysanthemi PB1細菌懸浮液在含低濃度agar之PGM培養基上 的色圈變化--------------------------------------------- 1-16 5. E. chrysanthemi藍色色素之純化及相關化學性質分析 E. chrysanthemi藍色色素之純化方式----------------------- 1-16 E. chrysanthemi藍色色素之溶解性測試--------------------- 1-17 E. chrysanthemi藍色色素之全光譜掃描(波長200~900 nm)----- 1-17 藍色色素以DMSO及DMF為移動相之TLC實驗----------------- 1-18 藍色色素之HPLC分離條件測試-------------------------- 1-18 結果--------------------------------------------------------- 1-19 1. E. chrysanthemi鑑別性培養基及Ech1-檢測試紙之研發 E. chrysanthemi及E. carotovora菌株在鑑別性培養基上的呈色差異 ------------------------------------------------------- 1-19 受到E. chrysanthemi感染的蝴蝶蘭組織在Ech1-試紙上之呈色-- 1-19 不同植物寄主之軟腐病徵之檢測--------------------------- 1-19 Ech1-試紙之製作方式差異對藍色圈產生的影響-------------- 1-20 E. chrysanthemi細菌懸浮液在Ech1-試紙上之呈色情形-------- 1-20 病葉過濾液對E. chrysanthemi菌體懸浮液於Ech1-試紙上之呈色影響 ------------------------------------------------------- 1-20 2. E. chrysanthemi鑑別性培養基及Ech2-檢測試紙之改良 外加不同碳源及氮源對藍色暈圈產生之影響----------------- 1-21 不同成分配方搭配對藍色色素生成之影響------------------- 1-21 E. chrysanthemi細菌懸浮液在Ech1-試紙及改良後新開發之Ech1-試紙上 之呈色情形--------------------------------------------------- 1-21 不同稀釋倍數之E. chrysanthemi在改良後之Ech1-檢測試紙之靈敏度 ------------------------------------------------------- 1-21 改良後的E. chrysanthemi鑑別性培養基配方及病原菌分離效果- 1-22 Ech2-檢測試紙之呈色結果比較---------------------------- 1-22 Ech2-檢測試紙之靈敏度測試------------------------------ 1-23 Ech2-檢測運用於初級感染來源之檢測---------------------- 1-23 Ech2-檢測試紙之保存方法-------------------------------- 1-24 3.鑑別性培養基中各成分及環境因子對E. chrysanthemi呈色之影響 pH值對E. chrysanthemi藍色色素生成之影響---------------- 1-24 Pectin及PGA對藍色色素呈色之影響----------------------- 1-24 不同廠牌的Agar對藍色色素呈色之影響--------------------- 1-25 4. Ech1-檢測試紙上藍色暈圈之變化與菌體生長之關係 植物軟腐病組織在Ech1-檢測試紙上藍色暈圈與菌量之關係探討- --- 1-25 抗生素紙錠對E. chrysanthemi菌落生長及藍色色素呈色之影響-- 1-25 Ech1-檢測試紙上藍色暈圈隨時間點之變化情形--------------- 1-26 E. chrysanthemi PB1細菌懸浮液在含低濃度agar之鑑別性培養基 上的色圈變化-------------------------------------------- 1-26 5. E. chrysanthemi藍色色素之純化及相關化學性質分析 E. chrysanthemi藍色色素之純化與溶解性測試---------------- 1-26 E. chrysanthemi藍色色素之全光譜掃描(波長200~900 nm) ----- 1-27 藍色色素以DMSO及DMF為移動相之TLC測試------------ 1-27 藍色色素之HPLC分離條件測試--------------------------- 1-27 討論---------------------------------------------------------- 1-28 參考文獻------------------------------------------------------ 1-34 表------------------------------------------------------------ 1-40 圖------------------------------------------------------------ 1-45 第二章 軟腐病菌Erwinia chrysanthemi色素相關基因之選殖 中文摘要-------------------------------------------------------- I 英文摘要------------------------------------------------------- II 前言----------------------------------------------------------- 2-1 材料與方法----------------------------------------------------- 2-6 菌種的取得及培養條件----------------------------------------- 2-6 1.Genomic library之建構 Genomic library建構所使用菌株之選擇------------------------- 2-6 基因組DNA大量分離法(CsCl centrifugation) ------------------- 2-6 DNA濃度測定--------------------------------------------- 2-8 菌種之確認------------------------------------------------ 2-8 Genomic library建構系統之選擇-------------------------------- 2-9 DNA濃度之確定及DNA片段之剪切--------------------------- 2-9 將DNA兩端補成齊頭端(end-repaired DNA) -------------------- 2-10 適當大小DNA片段之選擇----------------------------------- 2-10 電泳膠體中DNA之回收與DNA濃度測定---------------------- 2-11 Insert DNA的接合反應(ligation) ------------------------------ 2-11 l噬菌體之包裹(Lambda Packaging) --------------------------- 2-12 計算Titer-------------------------------------------------- 2-12 塗盤挑選所需clones數目------------------------------------ 2-13 2.藍色色素相關基因之選殖 以NGM培養基進行藍色色素相關基因之選殖------------------ 2-13 誘導質體產生多個複本及質體DNA的分離-------------------- 2-13 限制酵素圖譜(pattern)比對---------------------------------- 2-14 藍色色素相關基因的次選殖(Subcloning) ---------------------- 2-14 基礎分生技術--------------------------------------------- 2-16 結果--------------------------------------------------------- 2-24 1. Genomic library之建構 Genomic library建構所使用菌株之選擇----------------------- 2-24 E. chrysanthemi 12616基因組DNA大量分離與定量------------ 2-24 菌種之確認---------------------------------------------- 2-25 Library建構時DNA所使用濃度之確定及DNA片段的剪切----- 2-25 適當大小DNA片段之選擇與回收--------------------------- 2-26 計算Titer------------------------------------------------ 2-26 2.藍色色素相關基因之選殖 藍色色素相關基因之選殖---------------------------------- 2-26 誘導質體產生多個複本及質體DNA的分離------------------- 2-27 黃色菌落之限制酵素作用片段(pattern)比對---------------- 2-27 以indC基因片段之專一性引子對進行聚合?連鎖反應(PCR) --- 2-27 indC基因片段的雜合反應------------------------------- 2-27 藍色色素相關基因的次選殖(Subcloning) ----------------- 2-28 加快黃色菌落外觀表現之培養基篩選----------------------- 2-28 討論---------------------------------------------------- 2-30 參考文獻------------------------------------------------ 2-32 表------------------------------------------------------ 2-35 圖------------------------------------------------------ 2-38
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