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研究生:楊尚唯
研究生(外文):Shang-Wei Yang
論文名稱:十字花科炭疽病菌之兩個含有homeodomain-like motif轉錄因子的功能分析
論文名稱(外文):Functional analysis of two transcription factors containing homeodomain-like motif in Colletotrichum higginsianum
指導教授:王智立王智立引用關係
指導教授(外文):Chih-Li Wang
口試委員:李敏惠沈偉強
口試日期:2015-07-15
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系所
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:2015
畢業學年度:103
語文別:中文
論文頁數:93
中文關鍵詞:十字花科炭疽病菌homeodomain轉錄因子形態發育病原性逆境反應
外文關鍵詞:Colletotrichum higginsianumcrucifer anthracnosehomeodomaintranscription factormorphological developmentpathogenicitystress response
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植物炭疽病造成全球許多重要經濟作物的生產損失,主要由Colletotrichum 屬的真菌所引起,其寄主範圍廣泛,包括蔬菜、花卉及果樹等,而Colletotrichum higginsianum 為引起蕓苔屬 (Brassica) 及蘿蔔屬 (Raphanus) 等重要蔬菜之十字花科炭疽病 (crucifer anthracnose) 的病原菌,在台灣普遍發生於有機種植的菜園, 另外C. higginsianum 也感染模式植物阿拉伯芥 (Arabidopsis thaliana) ,被推薦為研究炭疽病菌與雙子葉植物交互作用的模式病理系統 (model pathosystem) 。過去曾報導植物病原真菌含有 homeodomain 之轉錄因子,該類轉錄因子在Magnaporthe oryzae 與Botrytis cinerea可藉由影響植物病原真菌之形態構造發育而影響病原菌侵染植物的能力。於前人研究十字花科炭疽病菌侵染阿拉伯芥的transcriptome 分析,顯示C. higginsianum CH063_09099 和CH063_11689 在已知具有homeodomain-like motif 之轉錄因子中有較高的表現量,本實驗室早先的研究中也發現此2個基因在侵染白菜的死體營養期 (necrotrophic stage) 具有較高的表現量,因此本研究針對此2個基因探討其功能。本研究使用農桿菌媒介轉殖 [Agrobacterium tumefaciens-mediated transformation (ATMT) ] 獲得基因剔除菌株 (gene knockout strain) ,並利用PCR 與南方墨點法 (Southern blot) 進行基因剔除菌株的基因型確認,獲得正確的基因剔除菌株後,進一步進行表現型分析 (phenotype analysis) 。CH063_09099 基因剔除菌株形態構造與野生型菌株有明顯的差異,其分生孢子較細長、菌落生長緩慢、在Czapek- Dox培養基上菌絲缺乏螺旋生長的趨勢,當菌絲生長於載玻片上其側生菌絲常自分岔點過度分岔形成束狀菌絲團。CH063_09099 基因剔除菌株在生體外 (in vitro) 與生體內 (in vivo) 可正常地形成附著器 (appressorium) ,有趣的是在阿拉伯芥之病原性測試顯示其與野生型菌株有相似的毒力表現,但接種於白菜上僅顯現輕微病徵或是沒有病斑形成,檢視在白菜上的侵染構造,大多數基因剔除菌株的分生孢子之侵染構造多停留在附著器時期或活體營養時期,而野生型菌株則多數發展至死體營養時期。在逆境反應的測試中顯示,CH063_09099基因剔除株對滲透壓逆境、高濃度之氧化逆境與細胞壁逆境均較野生型菌株有耐受性。另一方面 CH063_11689 基因剔除菌株亦可在生體外與生體內產生正常的附著器,其菌落生長速度及形態構造與野生型菌株相似,唯具有較短的分生孢子。而在逆境反應的測試中顯示,CH063_11689基因剔除株對離子型的滲透壓逆境及氧化逆境與野生型菌株相比呈現相類似之耐受趨勢,但在細胞壁逆境中則較野生型菌株敏感,且在非離子型的滲透壓逆境中耐受性較高。值得注意的是,CH063_11689基因剔除株在阿拉伯芥與白菜的病原性測試,顯示其僅造成輕微的點狀初期病斑,在白菜上大多數基因剔除株的分生孢子之侵染構造多停留在活體營養時期,唯有在輕微病斑形成處觀察到少部分發展到死體營養時期的侵染構造。本研究結果顯示推測,CH063_09099 為可影響真菌形態發育及感知多種環境逆境訊號傳遞路徑的轉錄因子,而此兩方面的功能改變造成病原菌在不同寄主植物上顯現不同的毒力表現;CH063_11689 則可能影響致病過程感染初期的基因表現。

中文摘要 i
Abstract iii
目次 v
表次索引 vii
圖次索引 viii
附圖索引 ix
前言 1
材料與方法 8
菌株、載體來源與保存及聚合酶鏈鎖反應引子對序列 8
生物資訊分析 8
基因剔除載體之構築 9
細菌質體DNA萃取 10
農桿菌基因轉殖技術 10
真菌基因體DNA萃取 11
轉殖株之基因型確認 12
南方墨點法 12
(一)南方墨點法核酸探針製備 12
(二)南方墨點法分析 13
基因恢復載體之構築與基因片段增幅 14
PEG媒介共轉殖基因技術獲得基因恢復菌株 15
(一)原生質體之製備 15
(二)PEG媒介原生質體基因轉殖 16
基因剔除菌株表現型分析 16
(一)菌落形態觀察 16
(二)菌絲形態觀察 17
(三)產孢與孢子形態觀察 17
(四)環境逆境耐受性測試 18
(五)附著器與侵入孔之形成觀察 18
(六)病原性測試 19
結果 20
目標基因之胺基酸序列分析 20
基因剔除載體之構築 20
基因剔除菌株之驗證 22
基因剔除菌株對菌落形態之影響 23
基因剔除菌株菌絲形態特徵分析 25
基因剔除菌株對產孢的影響 26
基因剔除菌株之病原性測試及侵染過程觀察 27
環境逆境耐受性測試分析 30
討論 32
參考文獻 39


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