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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉瑞芬 | |
| dc.contributor.author | Chia-Chun Chen | en |
| dc.contributor.author | 陳佳君 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:28:19Z | - |
| dc.date.copyright | 2019-09-14 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-30 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21185 | - |
| dc.description.abstract | 病原菌與植物的交互作用涉及許多機制，當面對病原菌的侵染，植物的第一線基礎防禦反應稱為pattern-triggered immunity (PTI)，藉由細胞表面的pattern recognition receptor (PRR) 辨識pathogen-associated molecular patterns (PAMPs) 或damage-associated molecular patterns (DAMPs)，引發一連串基礎防禦反應。疫病菌 (Phytophthora parasitica) 會危害多種重要經濟作物，為重要病原卵菌。OPEL是自疫病菌培養液發現的外泌蛋白，其保守性結構包含thaumatin-like domain, glycine-rich domain及glycoside hydrolase 16 (GH16) domain；先前的研究顯示將OPEL重組蛋白注射於菸草 (Nicotiana tabacum cv. Samsun-NN) 葉片後會造成細胞死亡、癒傷葡聚醣與活性氧分子累積，以及誘導水楊酸相關防禦反應基因表現等典型PTI反應，而且GH16 domain在OPEL所引發的PTI反應扮演關鍵性角色。搜尋NCBI 基因資料庫發現疫病菌共含25筆GH16-containing基因，保守性功能區分析顯示當中之PPTG_16550、PPTG_17496、PPTG_17497及PPTG_17498與OPEL有明顯保守性，很可能為OPEL同源性基因，因此本研究欲探討這些基因在植物基礎防禦反應的角色。即時定量反轉錄聚合酶連鎖反應分析結果顯示這些基因的表現情形並不相同，其中PPTG_17497 及PPTG_17498在疫病菌感染植物後第12小時的表現量顯著提升。上述四個OPEL同源性基因中，惟獨PPTG_16550缺乏thaumatin-like domain，為進一步瞭解該基因的特性，本研究以大腸桿菌表現PPTG_16550重組蛋白，並注射至菸草 (N. tabacum cv. Samsun-NN) 葉片，發現其會引發細胞死亡、活性氧分子產生及癒傷葡聚醣累積，但不會增加系統葉對Tobacco mosaic virus (TMV) 之抗性。這些研究結果顯示疫病菌PPTG_16550具有引發植物基礎防禦反應的活性，其在疫病菌感染植物時的角色仍待進一步探討。 | zh_TW |
| dc.description.abstract | To combat invasion by pathogens, plants have developed different defense mechanisms, including pattern-triggered immunity (PTI), which is elicited upon the perception of pathogen-associated molecular patterns (PAMPs) or danger/ damage-associated molecular patterns (DAMPs) by plant pattern recognition receptors (PRRs). Previously, a novel elicitor protein named OPEL was identified in the cultural fluid of Phytophthora parasitica, a notorious pathogen of a wide variety of economically important crop species. It is characterized by the presence of thaumatin-like domain, glycine-rich domain and glycoside hydrolase 16 (GH16) domain. Infiltration of OPEL recombinant protein into the leaves of tobacco (Nicotiana tabacum cv. Samsun-NN) resulted in PTI responses such as cell death, callose deposition, reactive oxygen species (ROS) production, and induced expression of salicylic acid-responsive defense genes, and the GH16 domain is indispensable for OPEL to induce PTI. Search of NCBI databases identified in total 25 GH16-containing genes in the genome of P. parasitica. Conserved sequence analysis showed that PPTG_16550, PPTG_17496, PPTG_17497, and PPTG_17498 are highly conserved with OPEL, thus they are very likely close homologs of OPEL. The purpose of this study is to investigate the role of OPEL homologs in plant basal defense response. Analysis by quantitative reverse transcriptase-PCR showed that all four genes showed different expression patterns. Especially, the expression of PPTG-17497 and PPTG_17498 was significantly induced at 12 hours post pathogen inoculation. Of the four putative OPEL homologs, PPTG_16550 is unique in that it lacks the thaumatin-like domain. To know its role in plant defense, PPTG_16550 recombinant protein was expressed in Escherichia coli, which caused cell death, reactive oxygen species (ROS) production, and callose deposition upon infiltration on the leaves of tobacco. Nevertheless, this protein shows no activity to induce systemic acquired resistance against Tobacco mosaic virus (TMV). These results indicate that PPTG-16550 is an elicitor, which elicits PTI response in tobacco. Its role in the process of plant infection by P. parasitica awaits further investigation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:28:19Z (GMT). No. of bitstreams: 1 ntu-108-R04633016-1.pdf: 6119506 bytes, checksum: a2089b3f89c572a3b788c92120c5114f (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 口試委員會審定書 ..... i
中文摘要 ..... ii 英文摘要 ..... iii 目錄 ..... I 壹、 前言 ..... 1 一、 植物的先天性免疫 (plant innate immunity) ..... 1 1. PAMP-triggered imunity (PTI) ..... 2 2. Effector-triggered imunity (ETI) ..... 3 二、 疫病菌Phytophthora parasitica ..... 4 三、 醣苷水解酶在疫病菌的研究 ..... 5 1. XEG1 ..... 6 2. ppxyn1與ppxyn2 ..... .6 研究動機 ..... 8 貳、 材料與方法 ..... 9 一、 供試植株與菌種來源 ..... 9 1. 植物材料與生長條件 ..... 9 2. 菌種來源及培養條件 ..... 9 二、 疫病菌GH16蛋白親緣演化樹及胺基酸序列功能性區域分析 ..... 9 三、 即時定量聚合酶連鎖反應 (Quantitative real time polymerase chain reaction, qPCR) 分析OPEL同源性基因於疫病菌不同生長階段表現情形 ..... 10 1. 疫病菌不同生長階段樣本收集 ..... 10 (1) 菌絲 (mycelia) 樣本收集 ..... 10 (2) 孢囊 (sporangia) 樣本收集 ..... 10 (3) 游走子 (zoospores) 樣本收集 ..... 11 (4) 靜止子 (cysts) 樣本收集 ..... 11 (5) 發芽靜止子 (germinating cysts) 樣本收集 ..... 11 (6) 罹病圓葉菸草葉片樣本收集 ..... 12 2. 總量RNA抽取 ..... 12 3. 製備cDNA ..... 13 4. 即時定量聚合酶連鎖反應 (Quantitative real time polymerase chain reaction, qPCR) ..... 13 四、 農桿菌注入法 (Agroinfiltration) ..... 14 五、 以大腸桿菌表現重組蛋白 ..... 14 1. PPTG_16550 重組蛋白可溶性測試 ..... 15 2. PPTG_16550及OPEL-GH domain重組蛋白表現及純化 ..... 15 3. 膠體過濾法 (Gel filtration) ..... 16 4. 蛋白質定量 ..... 16 六、 植物抗病反應分析 ..... 16 1. 細胞死亡染色 ..... 7 2. 活性氧分子 (Reactive Oxygen Species, ROS) 染色 ..... 17 3. 癒傷葡聚醣堆積分析 (Callose deposition assay) ..... 18 4. Tobacco mosaic virus (TMV) 接種 ..... 18 參、 結果… ..... 20 一、 OPEL同源性基因胺基酸序列功能性區域分析 ..... 20 二、 疫病菌GH16蛋白親緣演化樹及胺基酸序列功能性區域分析 ..... 21 三、 OPEL同源性基因於疫病菌不同生長階段表現情形 ..... 21 四、 以農桿菌注入法於菸草 (N. tabacum cv. Samsun-NN) 葉片短暫表現OPEL同源性基因會引起葉片黃化 ..... 22 五、 重組蛋白的表現及純化 ..... 23 六、 PPTG_16550會導致菸草 (N. tabacum cv. Samsun-NN) 細胞死亡 ..... 24 七、 PPTG_16550會引發菸草 (N. tabacum cv. Samsun-NN) 活性氧分子累積 ..... 24 八、 PPTG_16550會引發菸草 (N. tabacum cv. Samsun-NN) 累積癒傷葡聚醣 ..... 25 九、 PPTG_16550不會引發菸草 (N. tabacum cv. Samsun-NN) 對Tobacco mosaic virus (TMV)的抗性 ..... 25 肆、 討論 ..... 27 一、 疫病菌GH16保守性功能區分析 ..... 27 二、 OPEL同源性基因在疫病菌各個階段表現情形 ..... 28 三、 PPTG_16550會引發菸草PTI，但不會引發系統性抗性 ..... 29 四、 PPTG_16550在疫病菌與菸草交互作用所扮演的角色 ..... 31 五、 結語 ..... 32 伍、 參考文獻 ..... 34 陸、 附表 ..... 41 柒、 附圖 ..... 42 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討疫病菌OPEL同源性基因在植物基礎防禦反應的角色 | zh_TW |
| dc.title | To investigate the roles of OPEL homologs from Phytophthora parasitica in plant basal defense response | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張雅君,陳穎練 | |
| dc.subject.keyword | 疫病菌,植物基礎防禦反應,OPEL同源性基因,糖?水解?, | zh_TW |
| dc.subject.keyword | glycoside hydrolase (GH),Phytophthora parasitica,pattern-triggered immunity (PTI),OPEL homologs, | en |
| dc.relation.page | 51 | |
| dc.identifier.doi | 10.6342/NTU201904107 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2019-08-30 | |
| 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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