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研究生:黃靜芳
論文名稱:番椒與茄科疫病菌親和性與不親和性反應之特性分析
論文名稱(外文):Characterization of compatible and incompatible interactions of pepper-phytophthora capsici leonian pathosystem
指導教授:陳隆鐘
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:茄科疫病菌番椒系統性後天抗性病程相關基因病程相關蛋白質
外文關鍵詞:Phytophthora capsicipeppersystemic acquired resistanceSARpathogenesis-related genePR genePR proteins
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茄科疫病菌 (Phytophthora capsici Leonian) 為卵菌綱 (Oomycetes) 多犯性土壤病原真菌,目前在台灣田間所分離到之菌株病原型 (pathotype) 經單孢分離純化後,可歸類為四種毒力病原型菌株,亞洲蔬菜研究發展中心 (Asian Vegetable Research and Development Center, AVRDC) 將其所收集的菌株以鑑別寄主 (differential host) 暫時區分為三個生理小種 (race) ,目前亞蔬在篩選抗疫病菌番椒品系過程中,以對具有最強毒力的 race 3 菌株的抗性反應為評估依據。經接種試驗篩選眾多番椒種原後,確定數代單株純化後的 PI201234 對台灣最高毒力分離株─ Pc17E 的抗性已達 100% 。植物遭受病原菌攻擊後,其防禦相關基因 (defence-related genes) 便活化,提供植物對病原菌系統性且廣泛的抗性,系統性後天抗性 (systemic acquired resistance, SAR) 即為植物對病原菌抗性反應相關的訊息傳遞途徑 (signal transduction pathway) 之一。與抗性表現有關的SAR蛋白質大多屬於病程相關蛋白質 (pathogenesis-related proteins, PR proteins) ,為受病原菌攻擊或以誘引物質 (elicitor) 處理後,於植物體內新生成的蛋白質,最早是在感染煙草嵌紋病毒 (Tobacco mosaic virus, TMV) 的煙草葉中發現,目前以氨基酸序列 (amino acid sequences) 、血清學關係 (serological relationship) 、酵素或生物活性 (enzymatic or biological activity) 等特性做為命名與分類依據。許多研究皆證實病程相關基因的表現或病程相關蛋白質的生成在植物對病原菌的抗性上扮演著重要的角色。目前在番椒 (Capsicum annuum L.) 上已發現之病程相關基因或病程相關蛋白質為 PR-1、β-1,3-葡聚糖 (β-1,3-glucanase) 、幾丁質分解酵素 (chitinase) 三種。為了解番椒對茄科疫病菌之抗性機制,本研究使用亞蔬所提供之抗、感茄科疫病菌番椒品種及茄科疫病菌之三種生理小種菌株,進行初步觀察研究後,再著手由番椒之病程相關基因表現差異進行了解其抗性機制。茄科疫病菌三種生理小種菌株之菌株之菌絲、孢囊型態均相似,於 V-8 培養基上的生長速率、菌落型態亦相似,但在 PDA 培養基上,Pc1E (race 1) 菌株之生長速率明顯的較其他兩者慢。接種結果顯示, PI201234 接種茄科疫病菌三種菌株後皆未有病徵產生,表示 PI201234 對茄科疫病菌確實具有抗性;而 ECW 接種三種菌株後皆呈現感病反應,且接種 race 2 (Pc33E) 與 race 3 (Pc17E) 菌株之病勢發展較接種 race 1 快,顯示 race 2 與 race 3 菌之病原性較相近,比對 RAPD 結果,亦符合接種實驗結果。依前人已發表之病程相關基因序列設計專一性引子對,以反轉錄聚合連鎖反應 (reverse-transcript polymerase chain reaction, RT-PCR) 偵測病程相關基因在不同的植物─病原菌組合之間的表現差異,結果顯示 PR-1 及幾丁質分解酵素二種病程相關基因可在已接種植株之莖部與根部表現,葉部則無表現。另與感病品系比較,同樣接種 race 1 菌株 (Pc1E) 後, PI201234 之幾丁質分解酵基因表現時間較 Early Calwonder的表現時間早;而接種race 2 或 race 3 後的表現時間則相同。以北方雜合反應分析,結果顯示信號強度在根部較強烈,表示在根部之表現量為最高;比較不同接種時間的表現,在親和性反應中, PR-1 基因在接種後第三天的表現量增加,而在不親和性反應中,反應結果的信號強度皆相近,表示 PR-1 基因的表現在不親和性反應中並無差異;而幾丁質分解酵素基因則在親和性與不親和性反應中的表現相似,且在接種後一至五天的樣本中的表現量亦無差異。綜合以上結果,得知番椒確實會因疫病菌的侵害而誘導其病程相關基因的表現,且與番椒植株對茄科疫病菌的抗性有所關聯。
壹、前言……………………………………………………………………………1
貳、材料與方法…………………………………………………………………..7
一、供試植物之栽種與病原菌之培養保存………………………………….7
二、供試植物接種試驗………………………………………………………..7
1.接種源製備………………………………………………………………...7
2.植物接種與接種樣本之收取……………………………………………..8
三、利用隨機增幅核酸多型性 (random amplified polymorphic DNA, RAPD) 分析兩種番椒品系之差異……………………………………...8
1.植物之去氧核糖核酸(deoxyribonucleic acid, DNA)萃取……………8
2.隨機增幅核酸多型性分析兩種番椒品系之差異……………………….9
四、以隨機增幅核酸多型性 (RAPD) 進行茄科疫病菌三種生理小種菌株之差異分析………………………………………………………………10
1.茄科疫病菌之總量去氧核糖核酸(total DNA)的萃取………………10
2.隨機增幅核酸多型性分析茄科疫病菌三種生理小種菌株之差異…..11
五、以反轉錄聚合連鎖反應 (reverse-transcript polymerase chain reaction, RT-PCR) 偵測接種茄科疫病菌之植物病程相關基因的表現……….11
1.植物總量核糖核酸 (total ribonucleic acid, total RNA) 萃取…………11
2.反轉錄聚合連鎖反應 (RT-PCR)……………………………………..12
六、以北方雜合 (Northern hybridization) 反應偵測病程相關基因於接種之植株體內的表現………………………………………………………12
1.電泳分析 (formaldehyde gel electrophoresis)…………………………..13
2.RNA 轉漬 (transfer)……………………………………………………..13
3.核酸探針製備與標識 (labeling)………………………………………...13
4.雜合反應 (hybridization)………………………………………………...13
5.偵測反應 (detection)…………………………………………………….14
參、結果………………………………………………………………………….15
一、茄科疫病菌三種生理小種菌株之基本型態比較……………………..15
二、兩種番椒品系接種茄科疫病菌三種生理小種後之抗、感病病徵反應差異………………………………………………………………………….15
三、兩種番椒品系 (Early Calwonder、PI201234) 之核酸差異分析結果…16
四、茄科疫病菌三種生理小種菌株 (Pc1E、Pc33E、Pc17E) 之隨機增幅核酸多型性結果之分析…………………………………………………..17
五、PR-1 、幾丁質分解酵素基因在親和性與不親和性反應之番椒植株根、莖、葉組織中的表現…………………………………………………17
六、PR-1 、幾丁質分解酵素基因在親和性與不親和性番椒植株莖部的表現差異………………………………………………………………………18
七、利用北方雜合反應分析 PR-1 與幾丁質分解酵素基因於接種後之親和性與不親和性番椒植株根、莖、葉的表現………………………….19
八、利用北方雜合反應分析 PR-1 、幾丁質分解酵素基因於親和性反應與不親和性反應之表現差異……………………………………………..19
肆、討論…………………………………………………………………………….21
伍、中文摘要………………………………………………………………………25
陸、英文摘要………………………………………………………………………27
柒、圖表說明……………………………………………………………………….29
捌、引用文獻………………………………………………………………………49
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