請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/89331
標題: 以正向遺傳學方法鑑別芒果炭疽病菌之致病因子
Identification of pathogenicity factors of Colletotrichum gloeosporioides with forward genetic approach
作者: Chia-Chi Chang
張家綺
關鍵字: 農桿菌轉殖法
芒果炭疽病
Colletotrichum gloeosporioides
致病力
Agrobacterium tumefaciens-mediated transformation (ATMT)
mango anthracnose
Colletotrichum gloeosporioides
pathogenicity
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摘要: 芒果 (Mangifera indica L.) 是世界上最重要的的熱帶水果之一,有「水果之王」之稱,而溫暖潮濕的環境利於Colletotrichum gloeosporioides所引起芒果炭疽病的發生。芒果炭疽病會在芒果的葉片、花穗、枝條、果實上造成褐色病斑,嚴重影響芒果產量及品質的下降。為了更加深入了解C. gloeosporioides致病相關機制,本研究利用農桿菌轉殖法 (Agrobacterium tumefaciens-mediated transformation, ATMT) 進行C. gloeosporioides TYC-2菌株的隨機突變以產生轉殖株,實驗中所使用的A. tumefaciens EHA105菌株分別含有pBHt2 或 p1300-CT74兩質體,在8次的轉殖實驗中總共獲得326株菌株,以PCR增幅篩選基因之片段或鏡檢綠色螢光蛋白表現篩選後,確認有181株為有T-DNA轉入之轉殖株,轉殖率約為0.0258%。以南方雜合分析T-DNA插入數量顯示,在隨機挑取的11株轉殖株中有63.6% 之轉殖株具有單一T-DNA插入點。形態觀察上,轉殖株C225-1與其二次單孢後獲得的轉殖株C225-2為白化菌株,兩菌株皆產生白色、厚實的氣生菌絲,且產孢能力都較野生型菌株低,轉殖株C225-2更在PDA及MS培養基上幾乎不產孢。118株轉殖株也於芒果幼葉上進行致病力分析,其中轉殖株48與轉殖株76則有致病力下降之情形,其病斑面積分別僅有野生型菌株所產生病斑的0.7% 與21.4%,此外,轉殖株76也在蘋果切片上有致病力下降的現象。南方雜合分析顯示轉殖株76及轉殖株C225具有T-DNA單一插入點,而轉殖株48則有具有兩個T-DNA插入點。為了分析T-DNA插入位置,利用反向聚合酵素連鎖反應 (Inverse PCR) 獲得序列,定序結果顯示,轉殖株76所插入之位置為調控pH的轉錄因子pacC,而轉殖株C225-1與C225-2則是影響與黑色素生合成有關之聚酮合成?(polyketide synthase;PKS)基因。進一步分析轉殖株C225-1與C225-2之特性,發現兩者黑色素的累積量皆比野生型菌株低,特別的是轉殖株C225-2無論於完整幼葉或是有傷口之成熟葉上,以菌絲塊接種發病情形皆較野生型菌株所產生的情形輕微,而轉殖株C225-1則沒有此現象。綜上所述,本研究已建立芒果炭疽病菌C. gloeosporioides之農桿菌轉殖系統,且獲得了181株轉殖株,其中兩株各為pacC與pks突變株,此外,也成功構築了pks互補載體,未來也將進一步分析pks在芒果炭疽病菌真正扮演的角色及功能。
Mango (Mangifera indica L.), the king of fruits, is one of the most important fruit crops in the world. Warm and humid conditions favor the occurrence of mango anthracnose, caused by Colletotrichum gloeosporioides. Mango anthracnose causes brown spots on leaves, panicles, twigs and fruits, resulting in dramatical reduction on fruit quality and yield. To identify the pathogenicity factors of Colletotrichum gloeosporioides on mango, I used Agrobacterium tumefaciens-mediated transformation (ATMT) to generate transformants of C. gloeosporioides TYC-2 by random mutagenesis. Agrobacterium tumefaciens EHA105 carrying binary vector pBHt2 or p1300-CT74 was used for ATMT in my study. Total 326 transformants were obtained from eight ATMT experiments. After PCR or GFP screening, 181 transformants had T-DNA integrations. The transformation rate is about 0.0258%. Southern blot analysis showed that 63.6% transformants carried single T-DNA insertion by analyzing 11 randomly selected transformants. Morphological examination determined that transformant C225-1 and its single spore isolate C225-2 were albino mutants. The albino mutants displayed more abundant white-aerial hyphae and lower ability of sporulation than the wild type strain. Transformant C225-1 produced few spores, whereas transformant C225-2 almost lost the ability of sporulation on PDA and MS agar medium. I also assayed the pathogenicity of 118 transformants on young mango leaf. Transformant 48 and 76 showed reduced virulence and caused 0.7% and 21.4% of the lesion area generated by the wild type strain, respectively. Besides, transformant 76 also showed reduced virulence on wounded apple fruits. Southern blot analysis indicated that transformant C225 and 76 had only one T-DNA insertion event, whereas transformant 48 carried two copies of T-DNA. In order to find out the insertion site, I used inverse PCR to obtain the T-DNA flanking regions. DNA sequence data showed that T-DNA inserted in the gene encoding a pH-response transcription factor (pacC) in transformant 76 and in the coding region of polyketide synthase (PKS) gene in transformant C225-1 and C225-2. Further analysis of transformant C225-1 and C225-2 showed that they accumulated less melanin than the wild type strain. Interestingly, transformant C225-2 showed lower virulence on both intact immature mango leaf and wounded mature mango leaf by inoculation with mycelium plugs, which was not observed in transformant C225-1. Taken together, in this study I have setup an ATMT system for mango anthracnose pathogen C. gloeosporioides and produced 181 transformants. Two of the transformants were further characterized and were pacC and pks disrupted mutants, respectively. In addition, I have generated a binary vector for pks complementation assay. The function of the PKS in C. gloeosporioides development and pathogenicity on mango will be demonstrated in the future.
URI: http://hdl.handle.net/11455/89331
文章公開時間: 10000-01-01
顯示於類別:植物病理學系

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