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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 曾顯雄(Shean-Shong Tzean) | |
dc.contributor.author | Tzu-Wei Huang | en |
dc.contributor.author | 黃子葳 | zh_TW |
dc.date.accessioned | 2021-06-16T10:29:30Z | - |
dc.date.available | 2018-08-20 | |
dc.date.copyright | 2013-08-20 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-14 | |
dc.identifier.citation | 王淞民,2009。基轉超寄生菌Trichoderma spp.黑色素生合成基因以提昇其逆境抗性與致病力。國立台灣大學生農學院植物病理與微生物所學研究所碩士論文。
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60771 | - |
dc.description.abstract | 褐根病菌(Phellinus noxius)為白腐病,其所造成之褐根病,再近幾年漸趨嚴重。自1980年起,在世界各地都有報導過此一病害,如馬來西亞、印度、非洲等熱帶、亞熱帶國家。褐根腐病主要侵染闊葉喬木,在經濟或觀賞作物上造成相當的損害,如果樹、橡膠樹及常見之行道樹或建材用樹種,皆是其侵染之目標。褐根腐病最明顯之病徵,即是在受到侵染的樹木,其樹勢會急速衰退,末端枝葉黃化或小葉化,其根部或近基部之表面,會有褐色或深褐色之菌絲覆層。在樹皮下之木質部,會有褐色之網狀紋路。褐根腐病在長時間受到侵染的樹木上,則會發育出不規則層疊狀之子實體。為了侵染植物組織,褐根病菌等白腐菌會分泌一群可分解木質素之酵素,如木質分解素(lignin peroxides, LiP)或是錳過氧化酵素(Manganese peroxidase, MnP)等。在本研究中,將探討錳過氧化酵素與褐根腐病在病原性上之關係。研究中以基因靜默(RNA silencing)之技術,並以農桿菌轉殖Agrobacterium-mediated transformant)之方式,導入一段與過氧化酵素互補之DNA片段,以誘發其基因靜默,降低錳過氧化酵素之表現量。在轉殖成功的六個轉型株中,其錳過氧化酵素之表現量降低50%~90%,其中有兩個轉型株之錳過氧化酵素表現量有更明顯之減少。之後將轉型株接種於枇杷樹(Eriobotrya japonica)上,以探討其錳過氧化酵素與病原性之關係。結果顯示其基因靜默效果越好之轉型株,其病原性會越差,換言之錳過氧化酵素與其病原性確實有一正向之相關性,而錳過氧化酵素也可作為設計防治褐根腐病之系統性殺菌劑目標之一。 | zh_TW |
dc.description.abstract | Brown root rot disease, caused by basidiomycetous fungus Phellinus noxius, becomes more noticeable from 1980s. The pathogen primarily attacks board-leave trees, fruit trees, and ornamental plants, such as rubber, coffee, Ficus and Cinnamomum, etc. The most pronounced symptom and sign of the infection are the massive yellowing and defoliating of the leaves, retarding the twig growth, and in the later infection stage, the butt and root near the ground covered with brown to dark mycelial mat, accompanied with internal wood decay and occasional formation of resupinate or sessile pileate fruiting body. The dense mycelium and deep brown to black rhizomorphs under the bark also are the indication of infection. In order to penetrate and colonize the host to acquire the essential nutrients, rationally, the brown root rot pathogen has to produce an array of tissue degrading enzymes, i.e., cellulase, hemicellulase, and lignin peroxidase, etc. Therefore, the magnitude of such enzymic activity might relate to its virulence. In this study, we explored the relevance of MnP to virulence. A gene silencing strategy is initiated to down regulate the expression of manganese peroxidase which is usually encoded by multiple copy gene family. The RNA silencing cassette was constructed based on the skeleton of pCAMBIA plasmid, and introduced into P. noxious genomic DNA by Agrobacterium tumefaciens-medicated transformation (ATMT) to obtain stable transformants. Subsequently, bioassay and pathogenicity test of the transformants have been performed in vitro and in vivo, to determine the relatedness between lignin-degrading manganese peroxidase and the virulence of P. noxious. The results reveal that the virulence of P. noxius declined considerably on loquat (Eriobotrya japonica) seedlings relevant to the down regulation of MnP gene expression by gene silencing. Prospectively, the MnP can be envisaged as the selection target for designing systemic fungicide in control P. noxius brown rot. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:29:30Z (GMT). No. of bitstreams: 1 ntu-102-R99633016-1.pdf: 11928452 bytes, checksum: 9f052239853f1dfb3db09a164865a1e6 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | CONTENTS
口試委員會審定書 # 誌謝 i 中文摘要 ii ABSTRACT iii CONTENTS iv 表目錄 viii 圖目錄 ix Chapter 1 前人研究 1 1.1 分類地位 1 1.2 褐根腐病於世界之分布 1 1.3 褐根腐病之病徵 1 1.4 褐根腐病之侵染方式 2 1.5 褐根腐病在台灣的分布 2 1.6 褐根腐病之型態特徵 3 1.7 褐根腐病的寄主範圍 4 1.8 木材腐朽菌之分類 5 1.9 褐腐菌(Brown-rot fungi) 5 1.10 白腐菌(white-rot fungi) 5 1.11 木質素與木質素分解酵素(ligninolytic enzyme) 6 1.12 第二類過氧化酵素(Class II peroxidase) 7 1.13 細胞壁分解酵素(cell wall degrading enzyme, CWDE)與病原性 8 1.14 農桿菌轉型(Agrobacterium tumefaciens mediated transformation) 9 1.15 RNA interference (RNAi, RNA干擾) 10 Chapter 2 材料與方法 13 2.1 真菌菌種分離、培養與保存 13 單倍體(haploid)褐根腐菌株分離 13 菌種培養 13 RNA萃取與cDNA製備 13 孢子懸浮液製備 14 菌種保存 14 2.2 DNA/RNA/cDNA 材料之製備 14 基因體DNA之萃取 14 大分子基因體DNA (Large molecular weight DNA, LMW DNA)之萃取 15 基因體RNA之萃取 15 反轉錄(reverse transcription) 16 製備RACE (Rapid Amplification of cDNA Ends)所需之cDNA 17 2.3 褐根腐病錳離子過氧化酵素(Manganese Peroxidase, PnMnP)基因之選殖 18 2.4 RNA silencing hairpin cassette construction 18 部分互補之MnP DNA序列之取得 18 構築RNA silencing hairpin cassette 19 2.5 農桿菌轉型 (Agrobacterium mediated transformation) 19 電穿孔勝任細胞(competent cell)製備 19 電穿孔勝任細胞 20 農桿菌轉型 (Agrobacterium mediated transformation) 20 2.6 南方式雜合(Southern blot) 21 DIG標定探針 21 基因體DNA酵解與電泳 21 轉印 22 漂洗 22 雜和反應 22 壓片 22 移除探針 23 2.7 Real-time quantitative PCR (qPCR) analysis 23 qRT-PCR 23 2.8 MnP and Laccase activity assay 24 Guaiacol:木質素分解酵素初步篩選 25 MnP:2,6 dimethoxyphenol (2,6 DMP) 25 Laccase : 2,2'-azino-bis(3-ethylbenzothiazoline-6-sμlphonic acid) (ABTS) 25 2.9 病原性測試接種實驗 26 Chapter 3 結果 28 3.1 萃取P. noxius wildtype A42大分子DNA 28 3.2 P. noxius wildtype A42單核株全基因體解序 (Full genome sequencing and de novo assembly) 28 3.3 選殖受錳離子刺激表現之錳過氧化酵素 29 3.4 設計建構基因靜默(RNA silencing)所需之silencing cassette shRNA 29 3.5 架構農桿菌轉殖所需之質體系統pCAMBIA1300 30 3.6 農桿菌A. tumefaciens EHA105之轉型 30 3.7 P. noxius wildtype A42與農桿菌A. tumefaciens EHA105共培養轉型 31 3.8 轉型之P. noxius之菌落形態 31 3.9 初步篩選轉型株之木質素分解酵素活性 32 3.10 測定轉型株MnP活性 32 3.11 測定轉型株Laccase活性 33 3.12 以qRT-PCR測定MnP基因之相對表現量 33 3.13 病原性接種試驗 34 Chapter 4 討論 36 4.1 P. noxius wildtype A42單核株全基因體解序 36 4.2 農桿菌轉型之優缺點 37 4.3 P. noxius wildtype A42與轉型株之性狀 39 4.4 RNA silencing與生理活性之變化 41 4.5 RNA silencing與MnP的表現量 41 4.6 MnP與病原性之關係 43 4.7 未來展望 44 Chapter 5 Reference 46 Chapter 6 表 54 Chapter 7 圖 58 Chapter 8 附錄 91 Chapter 9 附圖 95 表目錄 Table. 1 使用HiSeq 2000定序儀進行P. noxius A42全基因解序之結果。 54 Table. 2 P. noxius A42 全基因解序組裝之結果(de novo assembly)。 56 Table. 3 於本研究中所使用之引子對列表。 57 | |
dc.language.iso | zh-TW | |
dc.title | 選殖褐根腐病菌 Phellinus noxius 之錳過氧化酵素(PnMnP)及其
與其病原性之相關性 | zh_TW |
dc.title | Cloning and characterization of Phellinus noxius manganese peroxidase (PnMnP) gene and its relatedness with virulence | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 劉瑞芬(Ruey-Fen Liou),葉信宏(Hsin-Hung Yeh),鍾嘉綾(Chia-Lin Chung) | |
dc.subject.keyword | 褐根腐病,錳過氧化酵素,木質素分解酵素,木質素, | zh_TW |
dc.subject.keyword | phellinus noxius,Brown root rot,Manganese peroxidase,lingin, | en |
dc.relation.page | 99 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-15 | |
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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