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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 | Yi-Ru Lai | en |
dc.contributor.author | 賴奕如 | zh_TW |
dc.date.accessioned | 2021-06-17T09:08:58Z | - |
dc.date.available | 2021-11-04 | |
dc.date.copyright | 2019-11-04 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-10-28 | |
dc.identifier.citation | 1. 林姿均。2019。百合灰黴病菌之絲胺酸蛋白酶Beserp與植物防禦相關蛋白LsGRP1之關係探討。國立臺灣大學植物病理與微生物研究所碩士論文。臺北。臺灣。
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74857 | - |
dc.description.abstract | 百合灰黴病由Botrytis elliptica (Berk.) Cooke所引起,為台灣重要的病害之一。東方型雜交品種葵百合經澆灌水楊酸後,能誘導百合系統抗病性,得以抵抗灰黴病菌的感染,伴隨著累積植物第二型富含甘胺酸蛋白LsGRP1,但關於LsGRP1各區段如何參與在防禦反應之誘發則尚未明白,因此本研究深入探討LsGRP1蛋白片段在植物中所扮演的功能及角色,本研究藉由在百合內源性地表現與綠色螢光蛋白融合之LsGRP1及其片段缺失型,發現百合過量表現LsGRP1時,除了增強表現葉的抗病性之外,亦誘導百合系統性抗病力。LsGRP1片段缺失型中,以表現LsGRP1∆N-EGFP的百合葉片相較於表現LsGRP1-EGFP失去抗性程度最多。在無LsGRP1背景的圓葉菸草中再次驗證LsGRP1N對LsGRP1誘發防禦反應的重要性,顯示LsGRP1N為LsGRP1啟動防禦反應的關鍵區段。水楊酸生合成基因LsICS1的表現,在表現LsGRP1∆N-EGFP的百合葉片有抑制作用;乙烯路徑轉錄因子基因LsEIN3的表現在灰黴病菌接種後24小時提升,推測LsGRP1N缺乏可導致ICS1水楊酸生合成路徑受到影響,並且改變百合葉的抗病性。本研究並證明表現LsGRP1N區段上泛素化潛能位點突變之LsGRP1K32, 37, 48R-EGFP與LsGRP1∆N-EGFP的百合葉片有相近的感病程度,說明泛素化潛能對位點對LsGRP1誘發抗灰黴病的重要性。另一方面,藉由綠色螢光影像觀察到於百合葉片表現LsGRP1-EGFP後接種灰黴病菌,LsGRP1蛋白分佈位置從百合葉肉細胞的細胞膜移動到細胞基質中,細胞核周圍出現少數點狀構造,推測LsGRP1的移動與防禦訊息傳導有關。LsGRP1∆N-EGFP和LsGRP1K32, 37, 48R-EGFP在百合葉細胞中有相近的分布變化,但與LsGRP1-EGFP不同,推測LsGRP1N區段與LsGRP1在百合細胞的正確移位有關。以上研究結果指出,LsGRP1N區段與百合LsICS1基因表現有關,LsGRP1N泛素化位點影響LsGRP1次細胞定位,進而能調控對灰黴病菌的防禦反應。 | zh_TW |
dc.description.abstract | Lily gray mold caused by Botrytis elliptica (Berk.) Cooke is a severe disease in Taiwan. Drench with salicylic acid could systemically enhance gray mold resistance in Lilium oriental hybrid cv. Stargazer accompanied with accumulation of a leaf-specific plant class II glycine-rich protein LsGRP1. How each region of LsGRP1 to playing role in triggering immunity is not know yet. In this research, agroinfiltration with enhanced green florescence protein (EGFP) fusions of LsGRP1 and its partial deletions showed the best local and systemic protection ability in full length-LsGRP1-overexpressed lily leaves, but lowest in the deletion of LsGRP1N. Similar results were shown in Nicotiana benthamiana, indicating that LsGRP1N is the key region of LsGRP1 for plant defense activation. In addition, expression of salicylic acid synthase gene (LsICS1) was examined and the result showed that LsICS1 expression was supressed in LsGRP1∆N-EGFP-expressed lily leaves as compared with that in LsGRP1-EGFP-expressed leaves. Expression of LsEIN3 encoding for a transcription factor of ethylene signaling pathway increased at 24 hours post inoculation with B. elliptica. Howener, lack of LsGRP1N interfering ICS1 salicylic acid synthesis pathway and altering disease resistance ability of lily leaves was presumed. Lily leaves expressing LsGRP1K32, 37, 48R with triple point mutation in LsGRP1N showed similar disease level as that expressing LsGRP1∆N, indicating that ubiquitination is important for LsGRP1 to trigger resistance against gray mold disease. On the other hand, subcellular localization of LsGRP1-EGFP showed moving from the cell membrane to cytoplasm and around nucleus with particles in lily leaf cells after inoculation with B. elliptica inoculation, suggesting that LsGRP1 is related to defense signaling transduction. The alternation of subcellular location of LsGRP1∆N-EGFP and LsGRP1K32, 37, 48R-EGFP was similar but different from that of LsGRP1-EGFP; thus LsGRP1N related to accurate translocation of LsGRP1 in lily cells was presumed. According to these results, LsGRP1N would be involved in the expression of salicylic acid synthase gene LsICS1 and playing a role in the subcellular localization of LsGRP1 via the function of ubiquitnation site of LsGRP1N to trigger defense against gray mold disease. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T09:08:58Z (GMT). No. of bitstreams: 1 ntu-108-R06633002-1.pdf: 12866263 bytes, checksum: a7a53573f28cd4bb282cb31c95d1750b (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 目 錄
口試委員審定書 誌謝 i 壹、中文摘要 I 貳、英文摘要 III 參、前言 1 肆、前人研究 3 一、百合病害 3 二、百合灰黴病 4 三、植物富含甘胺酸蛋白 5 四、葵百合LsGRP1蛋白之發現及其功能 6 五、植物免疫反應 8 伍、材料方法 10 一、 構築表現LsGRP1片段缺失-EGFP型之重組載體 10 2. 抽取大腸桿菌質體 10 3. 增幅LsGRP1片段缺失-EGFP重組片段 11 4. 重組含目標基因之pBluescript載體 11 5. 大腸桿菌熱休克轉形法 12 6. 過量表現之T-DNA載體構築 12 7. 農桿菌勝任細胞製備 13 8. 農桿菌電轉型法 13 二、 農桿菌浸潤法 (Agroinfiltration) 13 三、 葵百合適應性農桿菌菌株之馴化 14 四、植物栽培 14 五、 病原菌培養與接種 15 1. 百合灰黴病菌培養與接種 15 2. 灰黴病菌培養與接種 15 3. 罹病嚴重程度分析 15 六、 以苯胺藍染色法偵測癒傷葡聚醣 16 七、 以錐藍蟲染色法偵測植物細胞死亡 16 九、 植物RNA萃取 16 十一、反轉錄連鎖聚合酶反應 (reverse transcription-polymerase chain reaction, RT-PCR) 17 十一、百合基因表現分析 17 十五、統計方法 19 陸、結果 20 一、 百合葉適應性農桿菌菌株可於百合葉部介導更高量的基因表現 20 二、 以農桿菌LsC5菌株於百合葉片介導目標基因表現 20 三、 表現LsGRP1-EGFP提升百合對灰黴病的抗性 21 四、表現LsGRP1-EGFP增強百合防禦灰黴病菌的癒傷葡聚醣累積 21 五、表現LsGRP1-EGFP誘發百合對灰黴病的系統性 21 六、表現LsGRP1-EGFP誘發圓葉菸草對灰黴病菌之系統抗性 22 七、剔除LsGRP1N嚴重降低在百合葉片表現LsGRP1-EGFP所觸發的灰黴病抗性 23 八、LsGRP1對B. elliptica接種早期荷爾蒙相關基因之表現影響與LsGRP1N區段在其中參與 23 九、百合接種灰黴病菌後的LsMAPK6基因表現的波動與LsGRP1N相關 24 十、表現LsGRP1-EGPF與LsGRP1∆N-EGFP菌可促進接種灰黴病菌後的百合葉部氣孔關閉 25 十一、表現LsGRP1∆N-EGFP與LsGRP1K32, 37,48R-EGFP的百合葉片對灰黴病具相近的感病程度 25 十二、LsGRP1-EGFP和LsGRP1∆N-EGFP之次細胞定位比較 26 柒、討論 27 一、LsGRP1對百合抗灰黴病的重要性 27 二、適應LsGRP1之農桿菌於百合之表現效率提升 27 三、表現LsGRP1於百合葉片促進防禦反應提升 28 四、百合灰黴病菌與LsGRP1N之密切交互作用 28 五、LsGRP1所影響的防禦訊號傳導 29 六、位於LsGRP1N的泛素化區位點影響百合抵抗灰黴病 30 七、水楊酸與LsGRP1之間的關係 31 捌、參考文獻 32 玖、圖表集 41 表一、用以增幅基因之引子對列表 42 表二、即時定量連鎖反應引子對列表 44 圖二、百合適應性農桿菌菌株在百合暫時性表現GUS的效率比較 47 圖三、Agrobacterium tumefaciens LsC5於百合葉片表現LsGRP1-EGFP和其他片段缺失型之訊息核酸分析 48 圖四、 表現完整LsGRP1-EGFP的葉片相較其他缺失型片段提供百合葉對灰黴病菌最佳的防禦力 50 圖五、表現LsGRP1-EGFP的百合葉片增強灰黴病菌感染之植物細胞周圍癒傷葡聚醣的累積 51 圖六、百合表現LsGRP1-EGFP及其他缺失型片段之系統葉在感染灰黴病菌後3天之罹病嚴重程度 53 圖七、表現完整的LsGRP1-EGFP與其他缺失型片段之百合系統葉,灰黴病菌感染後的植物細胞死亡情形 54 圖八、表現LsGRP1-EGFP明顯增強百合系統葉抗灰黴病及累積癒傷葡聚醣 56 圖九、圓葉菸草中LsGRP1-EGPF及其片段缺失型基因及蛋白表現偵測 57 圖十、表現LsGRP1-EGFP及其他缺失型片段之圓葉菸草的系統葉罹病嚴重程度 59 圖十一、LsGRP1-EGFP及LsGRP1∆N-EGFP表現之百合葉片菌量、壞疽死亡病斑及癒傷葡聚醣累積情形 61 圖十二、表現LsGRP1-EGFP及LsGRP1∆N-EGFP之百合葉片,LsICS1基因在接種灰黴病菌後的表現情形 62 圖十三、表現LsGRP1-EGFP及LsGRP1∆N-EGFP之百合葉片,LsEIN3基因在接種灰黴病菌後的表現情形 63 圖十四、表現LsGRP1-EGFP及LsGRP1∆-EGFP之百合葉片,LsMAPK6基因在接種灰黴病菌後的表現情形 64 圖十五、表現LsGRP1∆N-EGFP相較於表現LsGRP1-EGFP的百合葉片對灰黴病菌的感受性提升 66 圖十六、表現LsGRP1-EGFP及LsGRP1∆N-EGFP 之百合葉片接種灰黴病菌後的氣孔關閉比例 67 圖十七、LsGRP1的泛素化位點突變與失去LsGRP1N同樣影響百合對灰黴病的抗性 69 圖十八、以綠色螢光影像觀察LsGRP1-EGFP與LsGRP1∆N-EGFP蛋白在圓葉菸草中的次細胞定位 71 圖十九、灰黴病菌接種引起LsGRP1-EGFP及LsGRP1∆N-EGFP和LsGRP1K32,37,48R-EGFP蛋白在百合葉片中改變次細胞定位 74 | |
dc.language.iso | zh-TW | |
dc.title | 百合防禦蛋白LsGRP1抗灰黴病之關鍵區段探討 | zh_TW |
dc.title | Investigation of the key region of Lilium defense protein LsGRP1 against gray mold | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄭秋萍(Chiu-Ping Cheng),葉信宏,黃祥恩,鍾嘉綾 | |
dc.subject.keyword | LsGRP1,百合灰黴病菌,植物第二型富含甘胺酸蛋白,水楊酸生合成基因,泛素化,蛋白次細胞定位, | zh_TW |
dc.subject.keyword | LsGRP1,Botrytis elliptica,plant class II glycine-rich protein,salicylic acid synthase,ubiquitination,protein sub cellular localization, | en |
dc.relation.page | 74 | |
dc.identifier.doi | 10.6342/NTU201904224 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-10-29 | |
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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