請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/30981
標題: 桃褐腐病菌抗生物質之分離及聚酮化合物生合成基因之選殖
Isolation of antibiotic compounds and cloning of polyketide synthase genes from the peach brown rot pathogen Monilinia fructicola
作者: 余芳儀
Yu, Fang-Yi
關鍵字: Monilinia fructicola
抗生物質
antibiotic compounds
polyketide synthase
聚酮化合物生成酶
出版社: 植物病理學系所
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摘要: 桃褐腐病菌是世界性分布的病原真菌,可感染核果類作物,引起果腐和花腐,造成採收前、後嚴重的經濟損失。先前研究報導指出褐腐病菌產生聚酮化合物類 (polyketides) 的抗生物質拮抗人類病原真菌、細菌。本實驗室自台灣桃栽培區採集桃褐腐病菌,其中一分離株TW5-4生長緩慢、在培養基中菌落呈黑褐色、具有拮抗其他植物病原真菌、細菌的能力,培養過程並分離到 TW5-4 自發性突變之白化菌株,命名TW5-4WM。TW5-4WM 生長正常,但喪失對所測試的植物病原菌的拮抗能力。實驗以M1菌株作為對照菌株,探討TW5-4與TW5-4WM之生長和培養特性、產孢能力、附著器形成能力、黑色素生成量、致病能力及抗生表現能力,萃取抗生物質並以薄層色層分析和液相層析串聯質譜分析抗生物質結構,以及選殖可能參與抗生物質和黑色素生成的聚酮化合物合成酶 (polyketide synthase, PKS) 基因,利用real time-rt PCR 和 rt-PCR 分析 PKS 基因表現量。實驗結果顯示TW5-4 菌株的菌株生長緩慢,產孢能力、附著器形成能力和致病能力均低於 M1 和 TW5-4WM,但會累積大量黑色素及拮抗植物病原真菌之抗生物質。乙酸乙酯萃取TW5-4 菌體所獲得的萃取液可抑制綠黴病菌和灰黴病菌的菌絲生長和孢子發芽,以薄層色層分析法結合抗性測試和定性分析顯示抗生物質可能是酚化物或/及具有胺基。薄層色層法純化後的萃取液在液相層析串聯質譜分析下顯示萃取液內含有兩物質,分子量分別是350 和 346。聚酮化合物生合成基因之選殖,本研究共獲得一條全長的及十一條不完整的 PKS 基因,這些PKS 基因皆與合成色素和植物毒素的PKS 基因有高相似度,其中兩條基因 (LC1 和 LC2) 與合成黑色素的 PKS 基因有高相似度並且在TW5-4 菌株的表現量高於 TW5-4WM,其餘十條PKS基因之表現量與菌株之抗生能力和黑色素累積能力並未呈現相關性。
Monilinia fructicola is a fungal pathogen which causes blossom blight and fruit rot of Prunnus species. In several early studies, M. fructicola was reported that could produce phenolic polyketides as antimicrobial compounds against bacterial and fungal pathogens of human. Among our M. fructicola collections, strain TW5-4 grew slowly, forms dark colony in agar medium and has strong antimicrobial activity against phytopathogenic fungi and bacteria. A spontaneous albino mutant of TW5-4 (TW5-4WM) with normal growth and less antimicrobial activity is also identified during subculturing in the lab. In this study, the differences of the two strains (TW5-4 and TW5-4MW) on fungal growth, sporulation, appressorium formation, melanin accumulation, pathogenicity and antimicrobial activity were investigated. Antimicrobial compounds were extracted by ethyl acetate and analyzed by thin layer chromatography (TLC) and LC-MS-MS. Furthermore, polyketide synthase genes, which are potentially involved in the biosynthesis of melanin and antimicrobial compounds of M. fructicola, were cloned by degenerate PCR and inverse PCR. Their expressions were detected by rt-PCR and real time-rt-PCR. Our data showed that compared with strain TW5-4MW and M1, a typical strain of M. fructicola, strain TW5-4 has less ability on growth, sporulation, appressorium formation and pathogenicity but has stronger ability on melanin accumulation and anti-fungal activity. The ethyl acetate extracts from mycelia of TW5-4 showed inhibitory activity against mycelial growth and spore germination of two target plant pathogens Penicillium digitatum and Botrytis cinerea. Antimicrobial compounds were localized with Rf=0.45 by bioassay after separated by TLC, which could react with FeCl3 and ninhydrin solution, indicating that the antimicrobial compound might be phenolic and/or contain amine(s). Two compounds which may contribute to antifungal activity were further determined by LC-MS-MS. Their molecular weights are 350 and 346. One full-length and 11 partial PKS genes of M. fructicola were cloned and analyzed. Some PKSs have high sequence similarity to fungal PKS involved in pigment formation and phytotoxin production. PKS gene LC1 and LC2, which are closely related to the PKS genes involved in melanin biosynthesis, expressed at higher levels in TW5-4 than TW5-4WM. No significant correlation was found between the expression levels of the other 10 genes and antimicrobial activity or melanin accumulation.
URI: http://hdl.handle.net/11455/30981
其他識別: U0005-1808201113004000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1808201113004000
顯示於類別:植物病理學系

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