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研究生:許博堯
研究生(外文):Po-Yao Shu
論文名稱:寡核苷酸微陣列同時快速診斷鑑定多種重要防檢疫森林病原真菌之研發
論文名稱(外文):Development of oligonucleotide array for simultaneous identification of multiple crucial forest fungal pathogens
指導教授:曾顯雄曾顯雄引用關係
指導教授(外文):Shean-Shong Tzean
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:121
中文關鍵詞:細胞核內轉錄區真菌病原木層孔菌屬蜜環菌寡核苷酸探針反轉式點膜雜合生物晶片
外文關鍵詞:fungal pathogensAntrodiaArmillariaHeterobasidiumGanodermaPhellinusOligonucleotide probereverse dot hybridizationmicrochip
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植物病原真菌可引起農藝、園藝或森林等植物各種不同類型之病害,危害嚴重時可引起糧食飢荒、林木大量死亡,故如何快速、精確、靈敏診斷植物真菌性病原,以提早採取適當有效之防制管理措施,以減少生命財產之損失,並提升全民之福址,實刻不容緩。近年來由於氣候變化,許多社區之綠化樹木、公園行道樹、造林樹以及海岸防風林普遍發生褐根病造成嚴重損失與公共危害,樹木病害才逐漸受到重視,而在樹木病害上木材腐朽菌最為常見,因此在本研究中收集了國內最常見的腐朽病菌,以及國外報導最嚴重之樹木病原真菌,共9科22屬67種,包含木層孔菌屬(Phellinus)之多種成員,如Phellinus pini及P. werii、隸屬於假蜜環屬Armillariella之A. mellea(假蜜環菌)、蜜環菌屬Armillaria之A. ostyae以及靈芝屬(Ganoderma spp.)成員等重要林木真菌病原,這些病原菌也曾報導於美加、紐澳、歐洲等地侵染許多針葉林木或濶葉樹,造成生長不良、弱化、落葉、提早開花、腐朽、萎凋、倒伏等病徵,成為林業經營莫大限制因子和潛在嚴重威脅。本研究開發寡核苷酸生物晶片,係利用細胞核內轉錄區(Internal transcribed spacer, ITS)基因序列上具有保守性區域經增幅、定序、並比分析多種目標病原菌之基因,設計寡核苷酸探針(oligonucleotide probe),目標基因經毛地黃素(DIG)或生物素(Biotin)標定後再進行反轉式點膜雜合(reverse dot blot hybridization)。此檢測技術可同時、快速且精確鑑定重要林木病原真菌。實驗結果顯示本生物晶片具專一性、精準性,於尼龍膜晶片上其靈敏度可達1 pg/μl,於塑膠晶片可達100 fg/μl。此晶片可於7小時內完成精確診斷鑑定受病原感染之林間罹病組織或病原證據標本,可應用於果樹、林木、苗木之植物病原真菌早期檢測認證,以及在生態、防治、造林決策等多方面。
Plant fungal pathogens can cause various types of diseases in agronomical, horticultural and forestry plants, which can lead to widespread plant mortality and famine. Therefore, use of time-saving, accurate, and sensitive diagnosis methods coupled with appropriate and effective control measures, is essential in reducing economic loss and enhancing public well-being. In recent years, due to climate change, an outbreak of brown root disease among community greenery street trees, artificial forests, and windbreak plants has caused serious losses, but has also forced the public to take a serious view of fungal pathogens. Attempt to rapid and accurate diagnosis, totally 67 species of wood rot fungal pathogens were collected, including many crucial pathogens, i.e. Phellinus noxius, P. pini and P. werii, Armillaria mellea, Armillaria ostyae, and several Ganoderma species. These pathogens, which have also been reported in the United States, Canada, New Zealand, Australia and Europe, have infected many coniferous and broadleaf forests, resulting in the growth decline; defoliating, early flowering, decay, wilting, lodging and other symptoms, and become a great limiting factor and a potentially serious threat in forest management. The oligonucleotide microchip developed for simultaneous rapid identification of the 67 crucial forest pathogens was based on the DIG or biotin-labeled specific probes derived forms ribosomal DNA gens (ITS1-5.8S-ITS2) by using reverse-dot hybridization. These chips can precisely and accurately identify and diagnose many Armillaria, Antrodia, Antrodiella, Heterobasidium, and Phellinus species, including A. mellea, A. ostoyae, H. annosum, P. noxius, and P. weirii, etc. with a sensitivity of 1 pg DNA/μl on nylon membrane chip, and 100 fg DNA/μl on plastic chip, respectively. And the verification and identification of forest Phellinus pathogens in authentic samples or voucher specimens can be accomplished within 7 hrs. The chip can be applied in inspection and quarantine, also for healthy free, seedling and nursery certification, and in ecology and silviculture management, as well.
中文摘要 1
Abstract 2
前 言 4
壹、前人研究 7
一、重要森林病原真菌及生態、為害習性 7
(一) Phenillus spp. 7
(二) 木材腐朽菌(Wood-rot fungi) 8
(三) 其他重要森林病原真菌 11
二、傳統鑑定病原真菌之診斷、鑑定方法 13
(一) 組織病理學 13
(二) 分離培養鑑定 13
三、利用分子生物學檢測病原真菌 14
(一) 實驗原理 14
(二) 分子生物學檢測技術種類 16
四、核糖體核酸基因內轉錄區(rDNA internal transcribed spacer, ITS) 序列在真菌分生技術之應用 23
貳、材料與方法 25
一、菌株及培養 25
二、菌體核苷酸(DNA)之萃取 26
三、DNA內轉錄區(ITS)之增幅(PCR反應) 27
四、PCR產物電泳分析 27
五、PCR產物純化 28
六、不同林木病原真菌ITS片段之選殖、定序 29
七、探針設計(Probe design) 30
八、晶片製備 31
九、雜合反應(hybridization) 33
參、結 果 37
一、多層孔菌屬(Phellinus)晶片之研發 37
二、薄層菌屬(Antrodia spp.)之晶片之研發 41
三、重要森林病原真菌之晶片之研發 44
肆、討 論 48
伍、結 論 52
陸、圖 表 53
柒、參考文獻 100
捌、附錄圖 110
玖、附錄表 118
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