Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89360
標題: 利用農桿菌媒介轉殖法探討辣椒炭疽病菌感染寄主之相關致病基因
Identification of pathogenicity genes of Colletotrichum acutatum with Agrobacterium tumefaciens mediated transformation
作者: Yung-Chu Lin
林詠筑
關鍵字: 辣椒
炭疽病
Colletotrichum acutatum
農桿菌媒介轉殖法
chili pepper
anthracnose
Colletotrichum acutatum
Agrobacterium tumefaciens-mediated transformation (ATMT)
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摘要: 辣椒 (Capsicum spp.) 為世界重要辛香料之一,在其栽培過程及採收後之儲藏期皆可能遭受植物病原真菌Colletotrichum spp.造成之炭疽病 (anthracnose) 危害,使辣椒產業損失嚴重,辣椒炭疽病菌在辣椒果實上造成之典型病徵為水浸狀凹陷並伴隨黑色輪紋及橘紅色分生孢子堆的產生;在台灣則是以Colletotrichum acutatum為造成辣椒炭疽病的主要病原菌,並已嚴重威脅辣椒產業。農桿菌媒介轉殖 (Agrobacterium tumefaciens mediated transformation, ATMT)系統目前已廣泛運用於真菌基因功能的研究,許多研究團隊在Colletotrichum gloeosporioides、Colletotrichum higginsianum等Colletotrichum species皆已建立高轉殖效率之ATMT系統,並成功找到病原菌相關致病基因。雖然C. acutatum對辣椒具有嚴重之摧毀性,但目前對C. acutatum相關基因研究發表相對仍較少,因此本實驗目的即為藉由ATMT對C. acutatum Coll-153進行隨機突變,並從獲得之轉殖株中篩選出可深入研究的致病相關基因。實驗中以載體pBHt2與p1300-CT74F轉殖並分別獲得40及275株轉殖株,經南方墨點法 (southern blot) 確認獲得之轉殖株70%以上為單一T-DNA插入。在96孔盤篩選試驗中,測試轉殖株對不同營養及滲透壓與溫度敏感性,並成功從189株轉殖株中發現一株在Czapek's medium上生長不良之轉殖株B7;在辣椒接種試驗中,則成功於39株轉殖株中篩選出兩株毒力下降之轉殖株B42與B84。以南方墨點法確認三株轉殖株B7、B42及B84皆為單一T-DNA插入,並以Inverse-PCR與Nested-PCR獲得三株轉殖株之T-DNA插入點序列。經序列比對與semi-quantitative RT-PCR分析結果,推測B7受影響的基因可能為T-DNA插入點下游的GPI-anchored serine-threonine rich protein gene,目前已構築split marker進行基因剔除(knock out),並已獲得1株GPI-anchoredprotein 基因剔除之轉殖株可供將來深入研究此基因之功能。
Chili pepper (Capsicum spp.) is an important spice in the world. Anthracnose, which causes by Colletotrichum species, is one of the most important fungal diseases on chili pepper and can reduce yield marketablely. All growth stages of chili pepper fruit could be affected by anthracnose. The typical symptom on chili pepper fruit is sunken necrotic tissues with concentric rings of acervuli. In Taiwan, Colletotrichum acutatum is the major species causing chili anthracnose, resulting in severe yield losses. Agrobacterium tumefaciens-mediated transformation (ATMT) is a useful method to study gene function in fungi. It has been successfully used to identify pathogenicity genes from Colletotrichum gloeosporioides, Colletotrichum higginsianum and other Colletotrichum species. Although anthracnose caused by C. acutatum is the most destructive disease on chili pepper, almost none athogenicity gene has been analyzed in this pathogen. To discover novel genes involved in pathogenicity or fungal development in C. acutatum Coll-153, I used A. tumefaciens strain EHA105, carrying binary vector pBHt2 or p1300-CT74F, to generate T-DNA insertional mutants and have obtained 40 and 275 transformants, respectively. Southern blot showed that more than 70% of the Coll-153 transformants had single T-DNA insertion. After 96-well plate screening and pathogenicity assay, transformant B7 showed nutritional deficiencies with poor growth on Czapek's medium and transformants, B42 and B84, showed reproducible pathogenicity defect on chili pepper. T-DNA left- and right-border sequences were recovered from the three transformants with Inverse-PCR and Nested-PCR. Sequence analysis and semi-quantitative RT-PCR showed that the transformant B7 may affect the expression of gene encoding GPI-anchored serine-threonine rich protein. I have constructed split markers for gene disruption and generated one mutant for gene knockout on GPI-anchored protein. The function of this GPI-anchored protein in transformant B7 can be studied by the knockout mutants in the future.
URI: http://hdl.handle.net/11455/89360
文章公開時間: 10000-01-01
Appears in Collections:植物病理學系

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