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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳昭瑩(Chao-Ying Chen) | |
dc.contributor.author | Ching-Hsuan Huang | en |
dc.contributor.author | 黃靖軒 | zh_TW |
dc.date.accessioned | 2023-03-19T22:15:40Z | - |
dc.date.copyright | 2022-09-30 | |
dc.date.issued | 2022 | |
dc.date.submitted | 2022-09-21 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84560 | - |
dc.description.abstract | 百合灰黴病菌Botrytis elliptica為死體營養型病原真菌,藉由外泌性蛋白質成分誘導植物組織發生程序性細胞死亡,並自死亡之寄主組織獲得養份,造成壞疽病徵,嚴重危害百合之栽培與生產,然導致此程序性細胞死亡的蛋白質身分與特性仍待鑑定與分析。實驗室前人透過電解質滲漏分析發現百合灰黴病菌之果膠甲基酯酶蛋白(pectin methyl esterase 1, BePME1) 會引起百合細胞死亡,並可透過外源添加促進病原菌的感染;本研究進一步闡明BePME1對百合灰黴病菌之毒力與引起寄主程序性細胞死亡的貢獻。首先建立百合灰黴病菌之聚乙二醇介導原生質體轉型法以建構BePME1剔除突變株,獲得兩個突變株bepme1-1及bepme1-2,所造成之病徵明顯地較野生型菌株弱,但外源添加BePME1蛋白可挽救之,證實BePME1為一毒力因子。以BePME1蛋白處理百合會引起PCD典型徵狀DNA片段化,處理程序性細胞死亡抑制物會抵消BePME1對百合細胞的致死效應,而BePME1突變株之外泌液引起百合細胞死亡及DNA片段化程度較野生型菌株為低,故認為BePME1為百合灰黴病菌引起寄主程序性細胞死亡之因子。已知百合防禦蛋白LsGRP1可抑制百合灰黴病菌外泌液引起之寄主細胞死亡,而BePME1在LsGRP1基因靜默的百合葉組織會造成更嚴重的寄主細胞死亡,說明BePME1是百合防禦系統的干擾目標。此外,BePME1也能引發阿拉伯芥葉組織的死亡,顯示BePME1的致死能力應無寄主選擇性。另一方面,以源自不同植物的果膠作為基質均無法測得BePME1的酵素活性。本研究首次證明百合灰黴病菌之分泌蛋白BePME1對病原菌毒力的貢獻以及誘發寄主程序性細胞死亡的功能。 | zh_TW |
dc.description.abstract | Botrytis elliptica, a necrotrophic fungal pathogen causing lily gray mold disease, induces host programmed cell death (PCD) via secretory proteinaceous compounds to obtain nutrients from dead host cells, resulting in necrosis symptom and severely damaging lily cultivation and production. However, the host PCD-eliciting compounds of B. elliptica have not been identified and characterized. A secretory pectin methyl esterase protein of B. elliptica, named BePME1, had been previously demonstrated capable of causing lily leaf cell death and promoting B. elliptica infection via exogenous application. In this study, the contributions of BePME1 in the virulence and PCD-eliciting activity of B. elliptica were clarified. Firstly, two BePME1-deleted mutant lines, bepme1-1 and bepme1-2, were generated using a polyethylene glycol-mediated B. elliptica protoplast transformation procedure established in this study. The two mutant lines caused smaller necrotic lesions as compared to wild-type B. elliptica, but this phenomenon could be rescued by exogenous application of BePME1, revealing BePME1 is a virulence factor. BePME1 treatment triggered a PCD-specific characteristic DNA fragmentation of lily cells, which could be rescued by the application of PCD inhibitors. The secretions of BePME1-deleted mutant lines conducted weaker DNA fragmentations of the host plant. Accordingly, BePME1 is a host PCD inducer. Since plant defense protein LsGRP1 had been known to prevent lily cell death caused by B. elliptica secretion, more severe host cell death in LsGRP1-silenced lily caused by BePME1 indicated that BePME1 is the interfering target of lily defense system. Besides, BePME1 caused the cell death of Arabidopsis leaves, revealing the cell death-inducing ability of BePME1 is non-host selective. Besides, BePME1 did not show detectable enzymatic activity as assayed using pectin substrates from different plants. This study firstly proves that the secretory protein BePME1 contributes to the virulence and functions as a host PCD inducer of B. elliptica. | en |
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dc.description.tableofcontents | 致謝 i 摘要 ii Abstract iii 壹、前言 1 貳、前人研究 2 一、 百合品種與病害抗性 2 二、 百合灰黴病菌 2 三、百合灰黴病菌果膠甲基酯酶(BePME1) 4 四、程序性細胞死亡(PCD) 4 五、細胞電解質滲漏 5 六、聚乙二醇(polyethylene glycol,PEG)介導基因轉型 6 七、植物果膠組成與果膠酶活性 7 參、材料與方法 11 ㄧ、病原菌製備 11 二、植物栽培 11 三、病毒介導百合LsGRP1靜默 11 四、製備BePME1缺失突變株 12 五、分析BePME1對百合灰黴病菌生理與致病性的影響 16 六、BePME1蛋白生產以及純化 17 七、蛋白質電泳與定量 18 八、植物葉盤與BePME1蛋白致死特性分析 20 九、BePME1蛋白果膠甲基酯酶活性測試 22 肆、結果 24 一、建構百合灰黴病菌之最適化PEG介導基因轉型條件 24 二、以PEG介導基因轉型同源置換BePME1基因取得突變菌株 25 三、突變株之BePME1基因表現情形 25 四、BePME1突變株生理特性測試 26 五、突變株致病力與BePME1回補之補救能力測試 27 六、突變株外泌液致死能力檢驗 27 七、BePME1蛋白與程序性細胞死亡(PCD)關聯性測試 28 八、BePME1蛋白致死特性與其濃度之關係 30 九、葵百合LsGRP1基因與BePME1蛋白致死性之關聯 30 十、BePME1蛋白酵素活性分析 31 十一、BePME1蛋白對阿拉伯芥之致死性分析 32 伍、討論 33 陸、參考文獻 36 柒、圖表集 45 表一、本研究使用之引子對 46 圖一、不同濃度之葡萄糖溶液對細胞壁分解酵素作用生成百合灰黴病菌原生質體之影響 47 圖二、百合灰黴病菌原生質體之外觀與細胞活性染色結果 48 圖三、以同源置換法刪除百合灰黴病菌的BePME1基因 49 圖四、百合灰黴病菌抗生素抗性菌株之觀察 50 圖五、以反轉錄聚合酶連鎖反應測試檢驗百合灰黴病菌轉型株BePME1及hygromycin抗性基因表現情形 51 圖六、百合灰黴病菌野生型菌株與BePME1突變株bepme1-1及bepme1-2之菌落外觀及分生孢子與產孢構造觀察 52 圖七、百合灰黴病菌野生型菌株與突變株菌落進展之比較 53 圖八、百合灰黴病菌野生型菌株和突變株產孢量比較 54 圖九、百合灰黴病菌野生型菌株和突變株孢子發芽率比較 55 圖十、百合灰黴病菌突變株bepme1-1在葵百合上引起較小的病斑並可被外源添加的BePME1蛋白補救 56 圖十一、百合灰黴病菌突變株bepme1-2在葵百合上引起較小的病斑並可被外源添加的BePME1蛋白補救 57 圖十二、百合灰黴病菌野生型菌株和突變株外泌液處理葵百合葉盤之電解質滲漏數值比較 58 圖十三、百合灰黴病菌野生型菌株及突變株外泌液處理穿梭百合葉盤之電解質滲漏數值比較 59 圖十四、經百合灰黴病菌野生型菌株及BePME1刪除突變株外泌液處理的葵百合與穿梭百合葉組織死亡細胞定量 60 圖十五、比較百合灰黴病菌野生型菌株及BePME1刪除突變株外泌液引發葵百合離葉壞疽之能力比較 61 圖十六、檢測百合灰黴病菌野生型菌株及BePME1刪除突變株外泌液處理誘發葵百合離葉之DNA片段化能力 62 圖十七、BePME1蛋白對葵百合離葉之致死性檢測 63 圖十八、BePME1蛋白誘導葵百合離葉DNA片段化檢視 64 圖十九、處理程序性細胞死亡抑制物影響BePME1蛋白誘導葵百合細胞死亡能力之電解質滲漏分析 65 圖二十、處理程序性細胞死亡抑制物影響BePME1蛋白誘導葵百合細胞死亡之伊凡氏藍染色分析 66 圖二十一、BePME1蛋白對百合的致死性與施用濃度之關聯性 67 圖二十二、BePME1引起葵百合與穿梭百合細胞死亡之伊凡氏藍檢測 68 圖二十三、以電解質滲漏分析檢測BePME1蛋白對LsGRP1基因靜默葵百合的致死程度改變 69 圖二十四、以伊凡氏藍染色量化LsGRP1基因靜默對葵百合抵抗BePME1致死性的影響 70 圖二十五、以蘋果果膠培養基測試BePME1果膠甲基酯酶活性 71 圖二十六、以柑橘果膠培養基測試BePME1果膠甲基酯酶活性 72 圖二十七、以葵百合果膠培養基測試BePME1果膠甲基酯酶活性 73 圖二十八、以吸光值數值變化檢測BePME1果膠甲基酯酶活性 74 圖二十九、BePME1對阿拉伯芥的致死分析 75 圖三十、百合灰黴病菌BePME1蛋白誘導植物細胞死亡之特性總結 76 | |
dc.language.iso | zh-TW | |
dc.title | 百合灰黴病菌分泌蛋白BePME1為引發寄主程序性細胞死亡之毒力因子 | zh_TW |
dc.title | The secreted protein BePME1 of Botrytis elliptica is a virulence factor inducing host programmed cell death | en |
dc.type | Thesis | |
dc.date.schoolyear | 110-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李敏惠(Miin-Huey Lee),沈偉強(Wei-Chiang Shen),葉信宏(Hsin-Hung Yeh),黃健瑞(Chien-Jui Huang) | |
dc.subject.keyword | 死體營養型真菌,百合灰黴病菌,果膠甲基酯酶,分泌蛋白BePME1,百合,程序性細胞死亡, | zh_TW |
dc.subject.keyword | Necrotrophic fungus,Botrytis elliptica,pectin methyl esterase,secretory protein BePME1,lily,programmed cell death, | en |
dc.relation.page | 76 | |
dc.identifier.doi | 10.6342/NTU202201488 | |
dc.rights.note | 同意授權(限校園內公開) | |
dc.date.accepted | 2022-09-22 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
dc.date.embargo-lift | 2022-09-30 | - |
顯示於系所單位: | 植物病理與微生物學系 |
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