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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 | Shuen-Huang Tsai | en |
dc.contributor.author | 蔡蕣隍 | zh_TW |
dc.date.accessioned | 2021-06-15T13:25:19Z | - |
dc.date.available | 2018-07-06 | |
dc.date.copyright | 2016-07-06 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-05-23 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51107 | - |
dc.description.abstract | 草莓灰黴病菌(Botrytis cinerea)是草莓栽培生產過程中的重要病害之一,由於草莓是連續採收作物,在這期間必須噴施化學農藥以減少果實因灰黴病而造成的損失,很難有安全採收期,一方面對環境造成汙染,及導致抗藥性菌株的產生。基於永續農業的發展,生物防治可以做為減少化學農藥使用的替代方案。生物防治的其中一個機制是利用微生物所產生之二次代謝物殺死或抑制病原菌,本實驗室自草莓植株葉圈分離到多黏類芽孢桿菌菌株(Paenibacillus polymyxa TP3),可以有效抑制灰黴病菌生長,於溫室及田間試驗均能有效降低草莓灰黴病的罹病嚴度;另外也以本菌株防治草莓炭疽病,能在溫室試驗減緩炭疽病菌的感染,並能誘發草莓對炭疽病的抗性,說明菌株TP3 具有生物防治潛力。本研究主要分析菌株TP3 產生之有效抗灰黴病菌物質及其組成結構。利用生化分析之方法萃取菌株TP3 之抗菌物質,其粗萃物能有效抑制灰黴病菌之生長,進一步以高效液相層析儀並配合基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF MS)偵測抗灰黴病菌分液之抗菌物質,推斷其為殺鐮孢菌素(fusaricidins)。此外,影像質譜儀(MALDI-imaging MS)顯示殺鐮孢菌素主要分佈在菌體周圍,同時在抑菌圈也有明顯的多黏菌素E 質譜訊號,於體外試驗發現其具有抑制灰黴病菌絲生長能力。最後以液相層析串聯質譜儀分析菌株TP3 殺鐮孢菌素之組成及結構,發現大部分與已知者相同,但其中質荷比(m/z) 869胺基酸組成之排列與相同質荷比之已知殺鐮孢菌素不同,而質荷比(m/z) 917 與已知(m/z) 931 在第一個胺基酸組成不同,推測為新穎的殺鐮孢菌素。 | zh_TW |
dc.description.abstract | Botrytis cinerea is one of the major pathogens in the cultivation of strawberry.Strawberry is a kind of continual harvest crop; spraying pesticides is necessary to reduce the loss caused by gray mold and difficult to have safety harvest period. Moreover,pesticides cause pollution in enviroments and emergence of drug-resistant strains. Based on the development of sustainable agriculture, biological control is proposed as an alternative to reduce the use of chemical pesticides. One of the mechanisms of biological
control is the production of secondary metabolites to kill or inhibit the pathogens.Paenibacillus polymyxa strain TP3 isolated previously from the phyllosphere of strawberry could effectively inhibit the growth of B. cinerea, and in greenhouse and field trials, strain TP3 could effectively reduce the disease severity of strawberry gray mold. Furthermore, strain TP3 could retard the infection of Colletotrichum gloeosporioides in greenhouse experiment and induce strawberry resistance against anthracnose, illustrating the potential of strain TP3 as a biocontrol agent. In this research, the anti-B. cinerea compounds produced by strain TP3 and its composition and structure were investigated. By a biochemical approach, antifungal compounds which could effectively inhibit the growth of B. cinerea were extracted and analyzed. High performance liquid chromatography and matrix-asisted laser desorption ionization-time of flight mass spectrometry were applied to identify the composition of the anti-B. cinerea compounds, which was presumed to be fusaricidins. Moreover, matrix-asisted laser desorption ionization-time of flight-imaging mass spectrometry showed that fusaricidins mainly distributed around the bacterial colony. Simultaneously, it also found that polymyxin E appeared at inhibition zone with apparent mass signals and could inhibit B. cinerea growth in vitro. Finally, composition and structure of fusaricidins produced by strain TP3 were analysed by liquid chromatography-tandem mass spectrometry and the results indicated an identity of fusaricidins as previously reported; however, the amino acid sequence of m/z 869 was different from the known fusaricidin of same m/z and the first amino acid of m/z 917 was different from that of known m/z 931, suggesting to be a novel fusaricidin member. | en |
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dc.description.tableofcontents | 壹、中文摘要...................................................1
貳、英文摘要...................................................2 參、前言.......................................................3 肆、前人研究...................................................5 一、草莓與草莓灰黴病............................................5 二、多黏類芽孢桿菌 (Paenibacillus polymyxa).....................7 三、殺鐮孢菌素(fusaricidins)生合成機制..........................9 四、殺鐮孢菌素殺真菌及革蘭氏陽性菌的機制.........................10 五、多黏類芽孢桿菌以及殺鐮孢菌素相關之專利.......................11 伍、材料與方法................................................12 一、供試菌株的培養及保存.......................................12 1. 供試菌株之培養 .............................................12 2. 供試菌株之保存 .............................................12 二、萃取菌株TP3抗菌物質有機溶劑之選定...........................12 三、菌株TP3抗菌物質小規模粗萃取.................................13 四、菌株TP3抗灰黴病菌物質分析(流程如圖一).......................13 1. 以基質輔助雷射脫附游離飛行時間質譜儀分析菌株TP3粗萃物可能之抗菌物質成分........................................................13 2. 以逆相高效液相層析儀分離並以MALDI-TOF質譜儀解析抗灰黴病菌有效成分 .............................................................14 3. 液相層析串聯質譜儀(LC/MS/MS) 解析抗菌物質結構................16 陸、結果 .....................................................18 一、含0.1%甲酸之乙腈萃取菌株TP3抗菌物質之效率較乙酸乙酯高.........18 二、菌株TP3能產生殺鐮孢菌素等抗菌物質...........................18 三、菌株TP3粗萃物之高效液相層析部份分液具有抑制灰黴病菌菌絲生長之活性 .............................................................19 四、菌株TP3透過殺鐮孢菌素抑制灰黴病菌菌絲生長....................19 五、殺鐮孢菌素以及多黏菌素E為菌株TP3外泌性抗菌物質...............20 六、多黏菌素E可抑制灰黴病菌菌絲生長 .............................20 七、殺鐮孢菌素類抗菌物質間之結構具有一定關聯性...................20 八、菌株TP3殺鐮孢菌素類抗菌物質之組成及結構 .....................21 柒、討論 .....................................................22 捌、參考文獻..................................................27 玖、圖表集....................................................36 表一、Paenibacillus polymyxa 產生已命名抗菌物質一覽表(I)........37 表二、Paenibacillus polymyxa 產生已命名抗菌物質一覽表 (II) .....38 表三、菌株TP3產生之殺鐮孢菌素胺基酸組成.........................39 圖一、菌株TP3抗菌物質分析之流程圖...............................40 圖二、比較不同有機溶劑之菌株TP3萃取物抑制灰黴病菌菌絲生長之效果 .............................................................41 圖三、以MALDI-TOF分析菌株TP3粗萃物可能的抗菌物質成分.............42 圖四、菌株TP3粗萃物之C18 逆相高效液相層析圖 .....................43 圖五、菌株TP3經C18逆相高效液相層析分離之分液抗菌分析 .............44 圖六、以MALDI-TOF分析菌株TP3抗灰黴病菌分液所含之抗菌物質.........45 圖七、菌株TP3與灰黴病菌對峙培養之基質輔助雷射脫附游離影像質譜 .....47 圖八、多黏菌素E之基質輔助雷射脫附游離影像質譜....................47 圖九、多黏菌素E對灰黴病菌菌絲生長之影響..........................48 圖十、菌株TP3產生之殺鐮孢菌素分子關聯網絡........................50 圖十一、由液相層析串聯質譜碰撞誘發鍵解反應產生之碎片離子(b or y ions)推衍之fusaricidin A (m/z 883.56)胜肽序列 ([M+H]+ group) .............................................................52 圖十二、由液相層析串聯質譜碰撞誘發鍵解反應產生之碎片離子(b or y ions)推衍之fusaricidin B (m/z 897.58)胜肽序列及其同分異構物胜肽序列 ([M+H]+ group)............................................55 圖十三、由液相層析串聯質譜碰撞誘發鍵解反應產生之碎片離子(b or y ions)推衍之[M+H]+=931.561胜肽序列 ([M+H]+ group) .............57 圖十四、由液相層析串聯質譜碰撞誘發鍵解反應產生之碎片離子(b or y ions)推衍之[M+H]+=947.556胜肽序列 ([M+H]+ group) .............59 圖十五、由液相層析串聯質譜碰撞誘發鍵解反應產生之碎片離子(b or y ions)推衍之[M+H]+=869.546胜肽序列 ([M+H]+ group) .............61 圖十六、由液相層析串聯質譜碰撞誘發鍵解反應產生之碎片離子(b or y ions)推衍之[M+H]+=911.592及其同分異構物胜肽序列 ([M+2H]2+ group) .............................................................64 圖十七、由液相層析串聯質譜碰撞誘發鍵解反應產生之碎片離子(b or y ions)推衍之[M+H]+=917.544胜肽序列 ([M+2H]2+ group)............65 圖十八、由液相層析串聯質譜碰撞誘發鍵解反應產生之碎片離子(b or y ions)推衍之[M+H]+=945.576胜肽序列 ([M+2H]2+ group)............66 圖十九、由液相層析串聯質譜碰撞誘發鍵解反應產生之碎片離子(b , y ions)推衍之[M+H]+=959.592胜肽序列 ([M+2H]2+ group).................67 | |
dc.language.iso | zh-TW | |
dc.title | 多黏類芽孢桿菌TP3產生之有效抗菌物質分析 | zh_TW |
dc.title | Analysis of active antifungal compounds produced by Paenibacillus polymyxa TP3 | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 劉世東(Shih-Tung Liu),楊玉良(Yu-Liang Yang),黃健瑞(Chien-Jui Huang),吳蕙芬(Whei-Fen Wu) | |
dc.subject.keyword | 草莓灰黴病菌,多黏類芽孢桿菌,基質輔助雷射脫附游離飛行時間質譜儀,殺鐮孢菌素, | zh_TW |
dc.subject.keyword | Botrytis cinerea,Paenibacillus polymyxa,matrix-asisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS),fusaricidins, | en |
dc.relation.page | 67 | |
dc.identifier.doi | 10.6342/NTU201600252 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-05-24 | |
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 |
顯示於系所單位: | 植物病理與微生物學系 |
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