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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳昭瑩 | zh_TW |
| dc.contributor.advisor | Chao-Ying Chen | en |
| dc.contributor.author | 許家偉 | zh_TW |
| dc.contributor.author | Chia-Wei Hsu | en |
| dc.date.accessioned | 2023-10-03T16:13:07Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-10-03 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-09 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90468 | - |
| dc.description.abstract | 百合灰黴病菌 (Botrytis elliptica) 為百合屬 (Lilium) 植物的專一性死體營養型病原真菌，在低溫高濕環境下感染百合組織，造成葉片焦枯、花苞畸形、花瓣枯萎等病徵，嚴重影響百合切花及種球的產量與品質。東方型葵百合 (Lilium ‘Stargazer’) 防禦蛋白LsGRP1之N端區段 (LsGRP1N)，在體外試驗標定於百合灰黴病菌之菌絲表面，推測LsGRP1N與百合灰黴病菌蛋白具有交互作用，並經免疫共沉澱法獲得百合灰黴病菌推定類枯草桿菌蛋白酶 (putative subtilisin-like proteases (subtilases), BeSerp)。本研究以雙分子螢光互補法 (bimolecular fluorescence complementation) 觀察到LsGRP1與BeSerp會在菸草細胞表面產生交互作用；以RT-qPCR分析百合灰黴病菌感染感病品種亞洲型百合Lilium ‘Tresor’之BeSerp表現時間圖譜，於感染後24小時，BeSerp表現量即提高5.5倍隨後微幅下降，並於接種後72小時增加表現量至20倍。續以噴灑誘導基因靜默 (spray-induced gene silencing) 方式降低百合灰黴病菌之BeSerp表現量，罹病面積有下降的趨勢。然而以農桿菌浸潤法於Lilium ‘Tresor’上預表現BeSerp基因三天，卻可降低百合灰黴病之罹病面積，故推測BeSerp可能具有毒力與激發子角色。進一步以大腸桿菌系統表現BeSepΔSS融合蛋白並自包涵體進行純化，所得BeSepΔSS融合蛋白在脫脂奶粉培養基分析及蛋白酶螢光檢測法均未測得酵素活性。為了解BeSerp是否經由直接或間接殺滅百合細胞以促進感染，將BeSepΔSS融合蛋白單獨或輔以能夠毒殺百合細胞的百合灰黴病菌外泌液浸潤於百合葉圓盤組織中，經由偵測細胞電解質滲漏以量化百合細胞死亡程度，發現BeSerpΔSS融合蛋白不會造成百合細胞死亡，反而能抑制由百合灰黴病菌外泌液誘發的百合細胞死亡。而以BeSerpΔSS融合蛋白預處理百合葉片時，也可減少百合灰黴病菌接種後所造成的病斑面積，並能增加百合灰黴病菌外泌液所誘發的癒傷葡聚醣沉積量，說明BeSerpΔSS融合蛋白之激發子功能。當以BeSerp基因靜默之百合灰黴病菌孢子生產外泌液並浸潤至百合葉圓盤組織時，葉組織電解質滲漏程度降低，癒傷葡聚醣沉積量則減少。綜合上述結果得知，百合灰黴病菌BeSerp在毒力及寄主防禦誘發上扮演重要角色。 | zh_TW |
| dc.description.abstract | Botrytis elliptica is a Lilium-specific necrotrophic fungal pathogen that infects lily tissues under low temperature and high humidity environments, resulting in symptom development such as leaf blight, flower bud deformation and petal blight, which seriously affects the yield and quality of lily cut flowers and bulbs. In an in vitro assay, the N-terminal region (LsGRP1N) of the defense protein LsGRP1 from Lilium ‘Stargazer’ was marked on the mycelial surface of B. elliptica. LsGRP1N was proposed to interact with the protein of B. elliptica, which was identify to be a putative subtilisin-like proteases (subtilases), BeSerp, by immune-coprecipitation and mass spectrometry. In this study, the bimolecular fluorescence complementation assay showed that LsGRP1 and BeSerp colocalized to the surface of tobacco cells. RT-qPCR analysis showed the BeSerp expression pattern in B. elliptica-infected susceptible Asiatic lily cultivar ‘Tresor’ increasing 5.5-fold at 24 hours post inoculation, then slightly reduced, followed by an increase to 20-fold at 72 hours post inoculation. By spray-induced gene silencing (SIGS) assay, reduced BeSerp gene expression and lesion area caused by B. elliptica were observed. However, pre-expression of BeSerp gene in Lilium ‘Tresor’ by Agrobacterium tumefaciens-mediated plant transformation reduced the lesion area caused by B. elliptica. Thus, BeSerp might have dual functions as virulence factor and elicitor. To further investigate the function of BeSerp, the BeSepΔSS fusion protein was expressed in Escherichia coli system and purified from inclusion bodies. However, BeSepΔSS fusion protein did not show enzyme activity in skim milk agar assay and protease fluorescent detection method. To investigate whether BeSerp could promote B. elliptica infection via directly or indirectly killing lily cells, the BeSepΔSS fusion protein was infiltrated into lily leaf disc tissues alone or with B. elliptica secretion which could kill lily cells, and the cell electrolyte leakage was detected to quantify the level of lily cell death. The results showed that BeSerpΔSS fusion protein did not casue lily cell death but inhibit the cell death induced by B. elliptica secretion. Moreover, infiltration of BeSerpΔSS fusion protein in lily leaf disc tissues reduced lesion area caused by B. elliptica and enhanced callose deposition, indicating the elicitor function of BeSerpΔSS fusion protein. Further examination by infiltrating lily leaf disc tissues with the secretion of BeSerp gene-silenced B. elliptica spore culture showed that the reduction of BeSerp expression not only reduced the lethality of B. elliptica secretion but also decreased the callose deposition. In summary, BeSerp of B. elliptica plays important roles in the virulence and plant defense induction. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-10-03T16:13:07Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-10-03T16:13:07Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 致謝 i
中文摘要 ii 英文摘要 iii 壹、前言 1 貳、前人研究 3 一、百合 3 二、百合灰黴病 3 三、葵百合之富含甘胺酸防禦相關蛋白LsGRP1 4 四、絲胺酸蛋白酶 6 五、類枯草桿菌蛋白酶 7 六、基因靜默 8 七、農桿菌介導之基因暫時性表現 9 八、細胞電解質滲漏 10 九、包涵體蛋白純化 10 十、病原激發子 11 參、材料與方法 13 一、供試植物之栽培 13 1. 栽培介質調配 13 2. 百合栽培 13 3. 菸草栽培 13 二、供試真菌之培養、保存與接種源製備 13 1. 供試真菌培養與保存 13 2. 供試真菌接種源製備 13 三、反轉錄-即時定量聚合酶連鎖反應 (RT-qPCR) 14 四、十二烷基硫酸鈉聚丙烯醯胺凝膠電泳偵測目標蛋白 14 1. 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 14 2. 考馬斯亮藍染色 15 3. 西方墨點法 15 五、細菌勝任細胞之製備與轉形 16 1. 大腸桿菌熱休克勝任細胞製備 16 2. 大腸桿菌熱休克轉形法 16 3. 鹼性溶解法抽取大腸桿菌質體 17 4. 農桿菌電轉形勝任細胞製備 17 5. 農桿菌電穿孔轉形法 18 六、農桿菌浸潤法 18 七、百合灰黴病菌外泌液製備 18 八、BeSerp基因序列選殖 19 九、以雙分子螢光互補 (bimolecular fluorescence complementation, BiFC) 檢測LsGRP1與BeSerp於圓葉菸草中的交互作用 19 1. 構築BeSerp農桿菌轉形株 20 2. 以雷射掃描共軛焦顯微鏡觀察目標蛋白於植物中的交互作用 20 十、分析BeSerp基因在B. elliptica侵染百合過程中的表現趨勢 21 十一、分析靜默BeSerp基因對B. elliptica致病性之影響 21 1. 合成dsRNA 21 2. 評估葉面施用dsRNA對病原菌侵染之影響 22 十二、在百合中暫時性表現BeSerp對B. elliptica侵染之影響 22 1. 構築帶有pBI121-BeSerp載體之農桿菌轉形株 22 2. 農桿菌介導之BeSerp暫時性表現對B. elliptica侵染的影響 23 十三、製備大腸桿菌表現BeSerpΔSS融合蛋白 24 1. 大腸桿菌蛋白表現株構築與保存 24 2. 融合蛋白生產條件測試 24 3. 包涵體蛋白生產之誘導時間優化 25 4. 包涵體蛋白純化 26 5. 蛋白透析與定量保存 27 十四、BeSerpΔSS融合蛋白之酵素活性測試 27 1. 使用脫脂奶粉培養基檢測酵素活性 27 2. 以蛋白酶螢光檢測試劑檢測酵素活性 27 十五、BeSerpΔSS融合蛋白對Lilium ‘Tresor’葉圓盤細胞電解質滲漏之影響 28 十六、B. elliptica孢子與BeSerpΔSS融合蛋白混合接種對真菌侵染之影響 29 十七、BeSerpΔSS融合蛋白浸潤百合葉片對B. elliptica侵染之影響 29 1. Lilium ‘Tresor’ 29 2. Lilium ‘Starfigter’ 29 十八、BeSerpΔSS融合蛋白處理百合葉片對植物癒傷葡聚醣沉積之影響 30 1. Lilium ‘Tresor’ 30 2. Lilium ‘Starfighter’ 30 十九、降低百合灰黴病菌外泌液中BeSerp含量對Lilium ‘Tresor’葉圓盤細胞死亡及癒傷葡聚醣沉積量之影響 30 1. 以基因靜默方式降低BeSerp於百合灰黴病菌外泌液中之表現量 30 2. 評估百合灰黴病菌外泌液對Lilium ‘Tresor’葉圓盤細胞死亡的影響 31 3. 評估百合灰黴病菌外泌液對Lilium ‘Tresor’癒傷葡聚醣沉積量的影響 31 二十、統計分析 31 肆、結果 32 一、BeSerp核酸與胺基酸序列 32 二、雙分子螢光互補顯示LsGRP1與BeSerp於菸草細胞表面有交互作用 32 三、BeSerp於百合灰黴病菌感染Lilium ‘Tresor’後期大量表現 32 四、BeSerp基因靜默使百合灰黴病菌對Lilium ‘Tresor’感染能力呈現減弱趨勢 32 五、Lilium ‘Tresor’中暫時性表現BeSerp能減輕百合灰黴病菌感染的病斑面積 33 六、BeSerpΔSS融合蛋白生產條件測試與純化 33 七、由大腸桿菌包涵體純化之BeSerpΔSS融合蛋白未測得酵素活性 34 八、BeSerpΔSS融合蛋白共處理能減緩百合灰黴病菌外泌液於Lilium ‘Tresor’葉圓盤所誘發之細胞死亡 35 九、百合灰黴病菌孢子與BeSerpΔSS融合蛋白混合接種於Lilium ‘Tresor’葉片可抑制病斑發展 35 十、百合葉片預處理BeSerpΔSS融合蛋白能減緩百合灰黴病菌之感染 36 十一、百合葉片預處理BeSerpΔSS融合蛋白能增加癒傷葡聚醣沉積 36 十二、減少百合灰黴病菌外泌液之BeSerp含量會降低外泌液對Lilium ‘Tresor’葉圓盤細胞致死能力且會降低癒傷葡聚醣沉積量 36 伍、討論 38 陸、參考文獻 45 柒、圖表集 55 表一、本研究使用之引子 56 圖一、BeSerp核酸序列及胺基酸序列 58 圖二、以雙分子螢光互補分析LsGRP1與BeSerp交互作用之載體構築 60 圖三、雙分子螢光互補檢分析LsGRP1與BeSerp於菸草細胞表面產生交互作用 61 圖四、百合灰黴病菌感染Lilium ‘Tresor’後之BeSerp基因表現時間圖譜 62 圖五、BeSerp基因靜默有減弱百合灰黴病菌對Lilium ‘Tresor’感染毒力之趨勢 63 圖六、Lilium ‘Tresor’暫時性表現BeSerp不影響百合灰黴病菌之侵染 64 圖七、Lilium ‘Tresor’暫時性表現BeSerp能減輕百合灰黴病菌的感染面積 65 圖八、BeSerpΔSS融合蛋白生產條件優化 67 圖九、由菌體之可溶性粗萃產物純化帶有His-tag標定的BeSerpΔSS融合蛋白 68 圖十、以固定金屬離子親合性層析法純化BeSerpΔSS融合蛋白 69 圖十一、由大腸桿菌包涵體純化之BeSerpΔSS融合蛋白未呈現酵素活性 71 圖十二、BeSerpΔSS融合蛋白的單獨浸潤處理不會誘發Lilium ‘Tresor’的細胞死亡 72 圖十三、BeSerpΔSS融合蛋白共處理能減緩百合灰黴病菌外泌液於Lilium ‘Tresor’誘發細胞死亡 73 圖十四、百合灰黴病菌與BeSerpΔSS融合蛋白混合接種於Lilium ‘Tresor’葉片可抑制病斑發展 74 圖十五、Lilium ‘Tresor’葉片預處理BeSerpΔSS融合蛋白能減輕百合灰黴病菌感染之病徵 75 圖十六、將Lilium ‘Starfighter’葉片預處理BeSerpΔSS融合蛋白能減輕百合灰黴病菌感染之病徵 76 圖十七、Lilium ‘Tresor’葉片預處理BeSerpΔSS融合蛋白能強化癒傷葡聚醣沉積 77 圖十八、Lilium ‘Starfighter’葉片預處理BeSerpΔSS融合蛋白能強化癒傷葡聚醣沉積 78 圖十九、以dsRNA-BeSerp處理百合灰黴病菌孢子並生產BeSerp含量較低之外泌液流程及BeSerp基因靜默分析 79 圖二十、減少百合灰黴病菌外泌液中BeSerp含量會降低外泌液對Lilium ‘Tresor’葉圓盤的致死能力 80 圖二十一、減少百合灰黴病菌外泌液中BeSerp含量會降低Lilium ‘Tresor’葉圓盤癒傷葡聚醣沉積量 82 捌、附錄 83 附圖一、Lilium ‘Starfighter’及Lilium ‘Tresor’之LsGRP1同源基因 85 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 百合灰黴病菌 | zh_TW |
| dc.subject | 毒力 | zh_TW |
| dc.subject | 類枯草桿菌蛋白酶 | zh_TW |
| dc.subject | 植物防禦 | zh_TW |
| dc.subject | 激發子 | zh_TW |
| dc.subject | subtilisin-like proteases | en |
| dc.subject | virulence | en |
| dc.subject | plant defense | en |
| dc.subject | Botrytis elliptica | en |
| dc.subject | elicitor | en |
| dc.title | 百合灰黴病菌BeSerp參與致病及植物防禦之功能探討 | zh_TW |
| dc.title | The functional study of Botrytis elliptica BeSerp involved in virulence and plant defense | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 王智立;黃祥恩;林玉儒 | zh_TW |
| dc.contributor.oralexamcommittee | Chih-Li Wang;Hsiang-En Huang;Yu-Ju Lin | en |
| dc.subject.keyword | 百合灰黴病菌,類枯草桿菌蛋白酶,毒力,植物防禦,激發子, | zh_TW |
| dc.subject.keyword | Botrytis elliptica,subtilisin-like proteases,virulence,plant defense,elicitor, | en |
| dc.relation.page | 85 | - |
| dc.identifier.doi | 10.6342/NTU202303916 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2023-08-11 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 植物病理與微生物學系 | - |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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