Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89361
標題: 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
Functional analysis of two transcription factors containing homeodomain-like motif in Colletotrichum higginsianum
作者: Shang-Wei Yang
楊尚唯
關鍵字: 十字花科炭疽病菌
homeodomain
轉錄因子
形態發育
病原性
逆境反應
Colletotrichum higginsianum
crucifer anthracnose
homeodomain
transcription factor
morphological development
pathogenicity
stress response
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摘要: 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。
URI: http://hdl.handle.net/11455/89361
文章公開時間: 2018-08-20
Appears in Collections:植物病理學系

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