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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 劉瑞芬 | |
dc.contributor.author | Yi-Hsuan Chang | en |
dc.contributor.author | 張怡萱 | zh_TW |
dc.date.accessioned | 2021-06-08T00:21:15Z | - |
dc.date.copyright | 2013-07-26 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-07-23 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17561 | - |
dc.description.abstract | 疫病菌屬 (Phytophthora) 中有許多種類 (species) 皆屬於重要經濟作物的病原菌,往往造成農業重大損失。Phytophthora與植物交互作用過程所涉及的分子機制非常複雜,近年來的研究發現Phytophthora含有可引發植物免疫反應的分子如pathogen-associated molecular patterns (PAMPs) 及avirulence effectors等,這些分子被植物辨識到後所引發之植物免疫反應分別為PAMP-triggered immunity (PTI) 以及effector-triggered immunity (ETI),但仍有許多putative effectors的功能尚待研究釐清,本研究的目的即是探討一未知功能的疫病菌 (Phytophthora parasitica)外泌性蛋白OPEL的功能與特性。在OPEL N端具有一段訊號肽,並有三個保守性區域分別為thaumatin-like domain (Thau)、glycine-rich domain (GR) 及glycosyl hydrolase domain (GH) 。OPEL為卵菌特有之基因,其同源性序列僅在Phytophthora spp.、Albugo、Hyaloperonospora arabidopsidis及Pythium被發現。即時定量聚合酶鏈鎖反應 (real-time quantitative reverse transcriptase-PCR) 分析顯示,OPEL在疫病菌不同生長階段皆有表現,但在感染植物後表現量顯著提升。為了瞭解其在疫病菌與植物交互作用所扮演的角色,我們利用E. coli表現OPEL重組蛋白以進行後續分析,結果顯示將OPEL注射入菸草葉片會引發癒傷葡聚醣堆積 (callose deposition)、產生活性氧分子 (reactive oxygen species, ROS) 以及誘導水楊酸 (salicylic acid, SA) 和PTI防禦反應相關基因表現。除此之外,經由OPEL處理之菸草可增加系統葉對Tobacco mosaic virus (TMV) 之抗性。而為了進一步了解是OPEL中哪個功能性區域可引發植物抗病反應,我們同樣利用E. coli表現Thau、GR、GH及GR+GH重組蛋白,研究結果顯示GH及GR+GH與OPEL結果相似,皆可引發植物累積癒傷葡聚醣及產生H2O2。綜合上述結果顯示OPEL具有既可引發植物基礎防禦反應PTI,也可誘導植物產生後天系統性抗性 (systemic acquired resistance, SAR) 之獨特性質,而glycosyl hydrolase domain則是引發抗病反應所必須。 | zh_TW |
dc.description.abstract | The genus Phytophthora includes more than 100 species, with many of them notorious pathogens of economically important crops. Interaction between Phytophthora pathogens and plant hosts involves the exchange of complex molecular signals from both sides. Recent studies in Phytophthora have led to the identification of some pathogen-associated molecular patterns (PAMPs) as well as many avirulence effectors, which are known to elicit PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. However, there are still numerous putative effectors with unknown function. In this study, we report the functional characterization of OPEL, a secretory protein of Phytophthora parasitica, which contains a signal peptide in the N-terminus and three other conserved domains including a thaumatin-like domain (Thau), a glycine-rich domain (GR), and a glycosyl hydrolase domain (GH). Homologs of OPEL were identified only in Oomycetes, including Phytophthora spp., Albugo, Hyaloperonospora arabidopsidis, and Pythium but not fungi and other organisms. Analysis by quantitative reverse transcriptase-PCR indicated that OPEL is expressed throughout development of P. parasitica, but is especially highly induced following the infection on plant. To characterize its function, we obtained OPEL recombinant proteins from E. coli. Infiltration of the recombinant protein into leaves of tobacco (Nicotiana tabacum) resulted in callose deposition, production of reactive oxygen species (ROS), and induced expression of salicylic acid-responsive defense genes. Moreover, when the systemic leaves of the OPEL-treated tobacco plants were challenged with Tobacco mosaic virus (TMV), the number of local lesions caused by TMV infection was significantly reduced compared to the control plants. To identify conserved domain(s) which is involved in elicitation of plant immunity, we obtained recombinant proteins representing Thau, GR, GH, or GR+GH, respectively, from E. coli and analyze their activity. The results showed that GH and GR+GH could induce callose deposition and ROS production in a level similar to those induced by OPEL recombinant protein. These results demonstrate the unique characteristics of OPEL which can induce plant defense response and systemic resistance in tobacco. Moreover, the glycosyl hydrolase domain plays an essential role for OPEL to elicit plant immunity. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T00:21:15Z (GMT). No. of bitstreams: 1 ntu-102-R00633010-1.pdf: 1995886 bytes, checksum: 30c34bbd8775312afad7c3f32526b0d4 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 中文摘要 I
Abstract III 目 錄 V 壹、前 言 1 1. 植物防禦反應 1 1.1 植物免疫系統 1 1.2 植物防禦反應機制 1 1.3 全株性系統性抗病(systemic acquired resistance, SAR) 2 1.4 植物防禦反應相關標記基因 (marker genes) 3 2. 疫病菌Phytophthora parasitica 5 3. 疫病菌之外泌蛋白 6 3.1 Cytoplastic effectors 7 3.2 Apoplastic effectors 7 研究動機 10 貳、材料與方法 11 1. 供試植株及菌種來源 11 1.1 植物材料及生長條件 11 1.2 病原菌來源及保存 11 2. 多序列比對分析及功能性區域預測 11 2.1 蛋白功能性區域預測以及親緣演化樹分析 11 3. 以簡併式引子對增幅不同種疫病菌之OPEL基因 12 3.1 Genomic DNA抽取 12 3.2 聚合酶鏈鎖反應增幅不同種疫病菌之OPEL基因 13 4. OPEL對於Phytophthora parasitica 致病力之影響分析 13 4.1 表現載體構築 13 4.2 農桿菌轉殖法 (Agrobacterium transformation) 14 4.3 農桿菌注入法 (Agroinfiltration) 14 4.4 抽取植物葉片全蛋白 15 4.5 西方轉漬法 (Western blot analysis) 15 4.6 疫病菌接種 15 5. OPEL於Phytophthora parasitica 不同生長階段之RNA表現量分析 16 5.1 疫病菌total RNA抽取 16 6. OPEL在植物細胞的表現位置分析 18 6.1 OPEL-GFP表現載體構築 18 6.2 以雷射掃描式共軛焦顯微鏡 (Laser Scanning Confocal Microscope) 觀察蛋白分布位置 19 7. 以大腸桿菌表現OPEL蛋白 19 7.1 OPEL全長蛋白表現載體之構築 19 7.2 OPEL不同功能性區域蛋白表現載體構築 20 7.3 大腸桿菌轉型試驗 20 7.4 OPEL全長蛋白表現及可溶性測試 20 7.5 OPEL不同功能性區域蛋白表現及可溶性測試 21 7.6 OPEL重組蛋白純化 21 7.7 OPEL不同功能性區域蛋白純化 22 7.8 膠體過濾法 (Gel filtration) 22 8. 植物抗病反應分析 22 8.1 抗病反應相關基因表現分析 22 8.2 癒傷葡聚醣堆積分析 (Callose deposition assay) 24 8.3 Reactive Oxygen Species (ROS) 染色 24 8.4 Tobacco mosaic virus (TMV)接種 25 叁、結 果 26 1. 序列分析及功能性區域預測 26 2. 疫病菌 (Phytophthora spp.)外泌蛋白OPEL胺基酸序列與其他物種同源性序列之親源演化樹分析 26 3. OPEL基因普遍存在於不同種之疫病菌中 27 4. OPEL在疫病菌感染菸草後會大量誘導表現 27 5. OPEL-GFP位於植物細胞間隙 28 6. 以agroinfiltration表現OPEL蛋白可提升菸草對於疫病菌之抗病性 28 7. OPEL重組蛋白之表現與純化 29 8. OPEL重組蛋白可導致Nicotiana tacabum葉部細胞死亡 30 9. OPEL重組蛋白可引發菸草癒傷葡聚糖堆積 30 10. OPEL重組蛋白可引發H2O2累積 30 11. 免疫反應相關基因表現分析 31 12. Tobacco mosaic virus (TMV) 接種 31 13. 表現及純化OPEL不同功能性區域之重組蛋白 32 14. Glycosyl hydrolase domain可引發菸草癒傷葡聚醣累積及產生活性氧分子 32 肆、討 論 33 1 Phytophthora parasitica將OPEL外泌至植物細胞間隙 33 2 OPEL在疫病菌與菸草交互作用所扮演的角色 33 3 OPEL引發菸草PTI及SAR 34 4 Glycosyl hydrolase domain為引發植物抗病反應所必需 36 5 結語…………. 38 伍、參考文獻 39 陸、附 表 49 柒、附 圖 52 | |
dc.language.iso | zh-TW | |
dc.title | 疫病菌外泌蛋白OPEL在植物反應所扮演的角色 | zh_TW |
dc.title | The role of OPEL, a secretory protein of Phytophthora parasitica, involved in induction of plant immunity | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李敏惠,鄭秋萍,賴爾?,林乃君 | |
dc.subject.keyword | OPEL,PAMP-triggered immunity (PTI),Phytophthora parasitica,Systemic acquired resistance (SAR), | zh_TW |
dc.relation.page | 67 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2013-07-23 | |
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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