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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 | Fang-Wei Liu | en |
dc.contributor.author | 劉芳瑋 | zh_TW |
dc.date.accessioned | 2021-06-08T01:41:55Z | - |
dc.date.copyright | 2016-10-05 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-17 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18997 | - |
dc.description.abstract | LsGRP1為植物第二型富含甘胺酸防禦相關蛋白,存在於百合葉片之表皮細胞及維管束組織,且位於植物細胞之表面,在百合葉片之含量會因百合灰黴病菌Botrytis elliptica感染而改變。已知在生體外處理高濃度N端區段 (LsGRP1N) 可輕微促進B. elliptica的孢子發芽,但卻抑制或不影響其他受試真菌發芽及細菌生長 (Lin et al., 2014),且LsGRP1對B. elliptica之抗菌效果較LsGRP1C為佳 (潘,2015),故推測LsGRP1N可能有助於LsGRP1C對B. elliptica之殺菌作用,於本研究擬解析LsGRP1N對B. elliptica的影響,以增進對LsGRP1抗菌功能之瞭解。研究發現LsGRP1N能促使B. elliptica孢子提早發芽及促進菌絲生長,當以SUMO-LsGRP1N融合蛋白注射於百合葉片後接種B. elliptica,可增加B. elliptica族群量與病徵嚴重度;由於LsGRP1對百合葉片不具毒殺作用,SUMO-LsGRP1N引起較為嚴重之病徵可能因其導致B. elliptica菌量增加所致。再者,胺基酸組成與LsGRP1N相同但排序不同之等長胜肽 (LsGRP1N identical amino acid composition, LsGRP1N-iaac) 對B. elliptica孢子發芽與菌絲生長的促進效果較LsGRP1N為低,因此排除LsGRP1N單純作為B. elliptica營養源的可能性。由於B. elliptica孢子在發芽階段較未發芽孢子對LsGRP1C更為敏感,且LsGRP1N與LsGRP1C共處理較單獨處理LsGRP1C之抑制B. elliptica孢子發芽的IC50為低,推測百合葉片上之LsGRP1可藉其N端區段促使B. elliptica孢子提早發芽及活躍生長而對C端毒殺區更為敏感,故能有效減少侵染百合葉片之B. elliptica菌量,抑制病斑之發展。 | zh_TW |
dc.description.abstract | LsGRP1 is a plant class II glycine-rich defense-related protein located in the cell surface of epidermis and vascular tissue of lily leaves. The LsGRP1 content appears fluctuated during Botrytis elliptica infection. Previous study demonstrated that in vitro treatment with high concentration of the N-terminal region of LsGRP1 (LsGRP1N) slightly enhanced the spore germination of B. elliptica but inhibited or did not alter the spore germination of other tested pathogenic fungi and bacterial growth (Lin et al., 2014). Moreover, LsGRP1 exhibited greater inhibitory activity on B. elliptica than LsGRP1C did (Pan, 2015), suggesting that LsGRP1N may facilitate the anti-B. elliptica effect of LsGRP1C. Thus, in this study, the effect of LsGRP1N on B. elliptica was dissected to improve the knowledge of antimicrobial activity of LsGRP1. In this research, in vitro assay showed that LsGRP1N conducted earlier spore germination and enhanced early hyphal growth of B. elliptica. When SUMO-LsGRP1N fusion protein was infiltrated into lily leaves before inoculation with B. elliptica, higher fungal biomass and more severe host cell death occurred as compared with the control treatment of SUMO partner of fusion protein. Further assay revealed that LsGRP1 did not conduct plant cell death, the enhanced growth of B. elliptica triggered by SUMO-LsGRP1N was likely a cause of higher extent of plant cell death. In addition, in vitro treatment of equal-length peptide with identical amino acid composition of LsGRP1N (LsGRP1N-iaac) did not affect spore germination and early hyphal growth of B. elliptica, the possibility of LsGRP1N simply as a nutrient supplement was excluded. On the other hand, spores of B. elliptica in germinating stage were more sensitive to LsGRP1C as compared with that of un-germinated ones, and a combination of LsGRP1N and LsGRP1C conducted stronger inhibitory effect than the sole treatment of LsGRP1C did. Thus, the N-terminal region of LsGRP1 on cell surface of lily leaf triggering earlier spore germination of B. elliptica and conducting germinating spores vulnerable to LsGRP1C, which then capable of reducing the population of B. elliptica and diminishing symptom development were persumed. | en |
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dc.description.tableofcontents | 目錄
壹、中文摘要 I 貳、英文摘要 II 參、前言 1 肆、前人研究 3 一、百合與百合灰黴病 3 二、百合灰黴病菌 4 三、植物抗菌胜肽 (Antimicrobial peptides, AMPs) 5 四、富含甘胺酸蛋白 (Glycine-rich proteins, GRPs) 6 五、葵百合防禦相關蛋白LsGRP1 7 伍、材料與方法 9 一、植物栽培 9 二、真菌材料製備與保存 9 1. 培養真菌 9 2. 收取分生孢子與製備孢子懸浮液 10 3. 保存真菌 10 三、表現與生產LsGRP1及其區段之融合蛋白 10 1. 建構表現載體與大腸桿菌轉形菌株 10 2. 大腸桿菌轉形菌株保存 11 3. 以大腸桿菌表現重組蛋白 11 4. 胞內蛋白質粗萃取 11 5. 純化與透析蛋白 12 6. 蛋白質定量 12 四、Tricine-SDS-聚丙烯醯胺膠體電泳 13 五、以考馬斯藍染色法、銀染法與西方墨點法偵測電泳膠體中的蛋白 13 六、偵測百合葉組織的LsGRP1 15 七、胜肽結構預測、設計與合成 15 1. 胺基酸序列比較與親緣關係樹分析 15 2. 預測coiled-coil二級結構 15 3. 設計與LsGRP1N胺基酸組成相同但序列不同之等長胜肽 16 4. 合成胜肽 16 八、LsGRP1區段胜肽生體外影響孢子發芽試驗 16 1. 以胜肽處理真菌 16 2. B. elliptica孢子發芽率 17 3. B. elliptica半數孢子發芽時間點 17 4. B. elliptica發芽後菌絲每小時平均增長量 17 5. 對真菌的半抑制濃度 17 九、植體內LsGRP1區段蛋白抑制灰黴病的試驗 17 1. 組織切片與免疫組織化學 17 2. 葉盤處理LsGRP1區段蛋白與接種病原菌 18 3. 抽取百合葉組織及B. elliptica全基因核酸 18 4. 定量百合葉組織中的真菌族群 19 十、評估LsGRP1區段融合蛋白對百合葉片細胞滲漏之影響 19 1. 製備B. elliptica外泌液 19 2. 將LsGRP1區段融合蛋白導入葉盤 19 3. 將B. elliptica外泌液導入葉盤 20 4. 測量導電度 20 十一、分析LsGRP1C對B. elliptica孢子或於發芽階段的毒性 20 1. 真菌樣品處理 20 2. PI與DAPI複合染色 21 十二、LsGRP1N於B. elliptica菌絲分布之免疫螢光定位 21 十三、收取B. elliptica 非細胞質蛋白及免疫共沉澱 22 十四、高效液相層析電灑法串聯式質譜鑑定 22 十五、統計分析 23 陸、結果 24 一、LsGRP1N促使B. elliptica孢子提早發芽與早期菌絲延長 24 二、建構SUMO CK蛋白與表現大腸桿菌LsGRP1區段融合蛋白 25 三、植體上處理SUMO-LsGRP1N可增加B. elliptica族群量與植體細胞死亡 程度 25 四、初步鑑定LsGRP1N不具毒殺百合葉片細胞的能力 26 五、LsGRP1N對B. elliptica的作用有序列特殊性 27 六、發芽階段之B. elliptica對LsGRP1C更為敏感 28 七、共處理LsGRP1N與LsGRP1C之抗菌功效較單獨處理LsGRP1C為佳 29 八、處理B. elliptica之百合葉部LsGRP1含量變化 31 九、LsGRP1N分布於B. elliptica菌體外側 31 十、與LsGRP1N交互作用之B. elliptica蛋白預測 32 柒、討論 33 捌、參考文獻 39 玖、圖表集 48 表一、百合灰黴病菌孢子經LsGRP1區段合成胜肽處理後之半數發芽時間 49 表二、LsGRP1區段合成胜肽對真菌孢子發芽之半抑制濃度 50 表三、本研究所使用引子對之序列 51 圖一、於不同時間點觀察經LsGRP1N或LsGRP1C處理之百合灰黴病菌孢子 發芽及早期菌絲生長 52 圖二、經LsGRP1N或LsGRP1C處理之百合灰黴病菌孢子發芽時間模式 54 圖三、LsGRP1N對百合灰黴病菌發芽孢子之早期菌絲平均延長速率的影響 55 圖四、經LsGRP1N處理16小時後的百合灰黴病菌菌絲長度與孢子發芽率 56 圖五、以不同大腸桿菌菌株表現SUMO CK蛋白 57 圖六、LsGRP1區段融合蛋白於百合葉組織之分布 58 圖七、LsGRP1N融合蛋白對百合灰黴病發展之影響 59 圖八、LsGRP1區段融合蛋白對百合葉組織電解質滲漏之影響 61 圖九、LsGRP1區段融合蛋白與百合灰黴病菌培養濾液依序處理對百合葉組 織電解質滲漏之影響 63 圖十、處理LsGRP1N-iaac對百合灰黴病菌孢子發芽速率之影響 65 圖十一、LsGRP1N-iaac對百合灰黴病菌發芽孢子之早期菌絲平均延長速率 的影響 66 圖十二、植物第二型富含甘胺酸蛋白之序列分析與coiled-coil結構預測 69 圖十三、LsGRP1、LsGRP1N與 LsGRP1N-iaac之coiled-coil結構預測 70 圖十四、LsGRP1C對發芽或未發芽之百合灰黴病菌孢子致死率比較 72 圖十五、LsGRP1N共處理對LsGRP1C抑制百合灰黴病菌孢子發芽之影響 73 圖十六、LsGRP1N共處理對LsGRP1C防治百合灰黴病的影響 74 圖十七、LsGRP1於百合灰黴病菌接種葉與未接種系統葉中之動態變化 76 圖十八、以免疫螢光法檢測LsGRP1N於百合灰黴病菌菌絲之分布 77 圖十九、B. elliptica之LsGRP1N交互作用假定蛋白釣取 78 壹拾、附錄 79 圖一、本篇研究所使用之LsGRP1區段融合蛋白 80 | |
dc.language.iso | zh-TW | |
dc.title | 抗菌蛋白LsGRP1之N端區段對百合灰黴病菌之孢子發芽促進作用 | zh_TW |
dc.title | Spore germination-enhancing effect on Botrytis elliptica
driven by the N-terminal region of anti-fungal protein LsGRP1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄭秋萍(Chiu-Ping Cheng),沈偉強(Wei-Chiang Shen),王智立(Chih-Li Wang),路幼妍(Yu-Yen Lu) | |
dc.subject.keyword | LsGRP1,LsGRP1N,LsGRP1C,百合灰黴病菌, | zh_TW |
dc.subject.keyword | LsGRP1,LsGRP1N,LsGRP1C,B. elliptica, | en |
dc.relation.page | 80 | |
dc.identifier.doi | 10.6342/NTU201603114 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-19 | |
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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