臺灣博碩士論文加值系統

植物病原細菌利用第三型分泌系統將harpin分泌至植物細胞間隙，在非寄主植物上誘發系統性抗病反應，增加植物對抗病害的能力。本研究中利用T7 RNA polymerase dependent system在E. coli大量表現harpin蛋白後，以高溫破菌方式收取harpin蛋白HrpZ及HrpW，再以PLGA進行包裹¬，將其製作成微粒劑進行施用，並觀察其抗病效果。首先測試蛋白包裹效率，結果顯示蛋白質濃度越高、PLGA使用濃度越高，包裹效率越高，包裹後之顆粒平均直徑約280um至350um；將粒劑置於無菌水中震盪測試其釋放效率，結果發現於24小時後即可釋放約70%以上之包裹蛋白，14天後完全釋放約96%以上，釋放出的蛋白回收後，以西方墨點法偵測，確認為包裹的目標蛋白，並且施打蛋白至菸草葉片後，仍可產生過敏性反應，確認蛋白仍具有活性，將完成包裹後的微粒劑於室溫儲放1至6個月後，回溶包裹蛋白，以西方墨點法進行偵測發現並無明顯降解的情形發生；進行番茄接種試驗，於4周大番茄葉表施用harpin蛋白原液及微粒劑後4天接種病原菌Xanthomonas vesicatoria XVT45，結果顯示亦可降低病害發生，但效果並不如直接噴灑蛋白原液顯著，若施用harpin蛋白溶液及微粒劑後8天，再進行接種，則微粒劑處理皆可明顯降低病害發生，且效果較harpin蛋白溶液更好，利用RT-PCR偵測結果發現，施用包裹之蛋白於番茄葉片12小時後可誘導抗病相關基因PR-1a表現，且表現量較施用蛋白溶液來的高，而harpin蛋白溶液或是微粒劑處理誘導PR-1a基因表現皆可維持至少10天，但微粒劑處理後，長時間觀察植物的PR-1a基因表現較harpin蛋白溶液穩定，顯示harpin蛋白以微粒劑型式進行施用，可緩慢釋放於植株上，減少harpin蛋白降解，並持續誘導抗病反應發生，有效降低茄科細菌性斑點病發病情形。

The harpin proteins produced by phytobacteria are secreted into plant apoplasts via a type III secretion system (TTSS), they have been demonstrated to trigger systemic acquire resistance (SAR) and therefore to enhance the resistance of diseases in plants. In this study, hrpZ and hrpW were overexpressed in E. coli using T7 RNA-polymerase dependent system and then the proteins were partial purified by boiling bacterial cultures. We encapsulated HrpZ or HrpW proteins with PLGA into microgranules and investigated the resistance of bacterial spot disease caused by Xanthomonas vesicatoria XVT45 on tomato. In the efficiency test of protein encapsulation, the result revealed that the higher concentration of protein and PLGA solution is, the higher efficiency of encapsulation could be done. The average diameter of particles is about 280um to 350um. The test of proteins released from microgranules showed that they could be released approximately 70% after shaking microgranules in sterile water for 24 hours, and released more than 96% after 14 days. The harpin proteins released from microgranules were confirmed by using western blot analysis and still maintained activity of eliciting the hypersensitive response on tobacco. After 6 months of storaging microgranules at room temperature, the harpin proteins were not degraded and confirmed by western blot analysis, indicating harpin encapsulated into microgranule form could be preserved well in 6 months. The results of inoculation tests indicated that the encapsulated harpin microgranules also could reduce the severity of bacterial spot caused by Xvt45 on tomato plants, although it was less efficient than using harpin solutions at 4 days post treatment. When challenged inoculations were performed 8 days post harpin treatments, the treatment with harpin microgranules displayed more effective in disease resistance. According to an analysis of expression of a SAR marker gene PR-1a in tomato plants by using RT-PCR, results showed that the gene expression with microgranule treatment was higher than with protein solution treatment. The expression of PR-1a gene at both harpin solution and microgranule treatments could be sustained for at least 10 days. However, the gene expression was more stable with microgranule treatment in long term observation. Thus, the results indicate that the harpin in microgranule format could be released gradually and maintain its activity for a while, then continuously induces SAR in planta, resulting in effectively control of the bacterial spot on tomato.

中文摘要...................................................i
Abstract..................................................ii
縮寫字對照表(Abbreviation)................................iv
目錄......................................................vi
表目錄..................................................viii
圖目錄....................................................ix

壹、緒言...................................................1

貳、材料與方法.............................................7
一、供試菌株、質體及菌體生長環境...........................7
二、利用E. coli BL21(λDE3)表達P. syringae pv. averrhoi (Pav) HL1 HrpZ蛋白及A. avenae subsp. cattleyae (Aaca) OAC1 HrpW蛋白並純化目標蛋白.....................................7
三、非寄主菸草過敏性反應 (Hypersensitive response) 測定.........................................................8
四、西方墨點法 (Western blotting)..........................9
五、蛋白質濃度測定.........................................9
六、甲基氯對harpin蛋白影響之分析..........................10
七、Harpin蛋白微粒劑製備..................................10
八、微粒劑包裹效率測定 ..............................10
九、酸性環境對harpin蛋白影響之分析........................11
十、微粒劑體外釋放效率測定................................11
十一、微粒劑保存蛋白能力測定..............................11
十二、番茄接種試驗 .......................................12
十三、植物RNA萃取.........................................13
十四、半定量反轉錄聚合酶連鎖反應 (semi-quantitative RT-PCR)......................................................13
十五、聚合酶連鎖反應 (Polymerase Chain Reaction, PCR).....14

參、結果..................................................15
一、HrpZPav與HrpWAaca蛋白純化與分析.......................15
二、甲基氯對harpin蛋白活性之影響..........................16
三、Harpin微粒劑製備之包裹效率分析與其微粒劑型態觀察......16
四、酸性環境對harpin蛋白之影響............................17
五、微粒劑釋放效率測試與釋放蛋白分析......................17
六、微粒劑保存包裹蛋白能力分析............................18
七、HrpZPav與HrpWAaca蛋白液與其微粒劑誘導番茄植株抵抗細菌性斑點病之成效..............................................18
八、微粒劑於植株表面緩釋包裹蛋白與延長誘導番茄植株抗病反應發生相關性. ................................................19

肆、討論.................................................21
伍、參考文獻.............................................28
陸、圖表.................................................39
柒、附錄.................................................52

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