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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭秋萍(Chiu-Ping Cheng) | |
| dc.contributor.author | Hung-Wei Wu | en |
| dc.contributor.author | 吳紅葳 | zh_TW |
| dc.date.accessioned | 2021-06-07T23:55:31Z | - |
| dc.date.copyright | 2013-09-02 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-22 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17072 | - |
| dc.description.abstract | 由青枯病菌 (Ralstonia solanacearum, Rs) 所引發的青枯病為重要且極複雜之土壤傳播性細菌性植物病害，其寄主範圍甚廣，可造成全球嚴重作物經濟損失。屬於phylotype I的青枯病菌菌株在番茄上呈現明顯的毒力變異性，但其相關的分子機制訊息極少。源自蕃茄的Pss190菌株具由極高毒力，根據其基因體序列顯示 Pss190特有效應蛋白Pseudomonas outer protein P 3 (PopP3)，故本研究旨在探討PopP3在青枯病菌毒力之功能與其相關機制。結果發現在原本缺少PopP3的中毒力青枯病菌菌株Pss4中大量表現PopP3可提高其在番茄之毒力；在菸草中短暫表現PopP3也可使Pss4菌量提高；同時，在番茄中過量表現PopP3則會壓制植物與pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) 相關的癒傷葡聚醣之累積 (callose deposition)，也使番茄對軟腐病菌Pectobacterium carotovorum subsp. carotovorum (Pcc) 更感病。另外，啟動子分析試驗顯示popP3受到第三型分泌蛋白系統HrpG所管控，PopP3-GFP重組蛋白座落在菸草原生質體之粒線體上。以上結果顯示PopP3是會壓制植物免疫反應以提高青枯病菌的毒力之第三型分泌蛋白系統之效應蛋白，且可能干擾植物粒線體功能。 論文的第二部分針對青枯病菌噬菌體與其相關蛋白進行研究，發現可裂解中毒力青枯病菌Pss4之T7-like噬菌體RsφP29的基因組有功能未知且可能與寄主交互作用相關的蛋白。RsφP29的裂解蛋白中的L蛋白之GST重組蛋白在in vitro外加試驗下不具有裂解青枯病菌的效果，而H蛋白則無法在大腸桿菌表現，推測H蛋白可能具有裂解細菌的能力。此外，短暫表現H蛋白之菸草會造成物細胞死亡，此現象在植物感染不同病原細菌或其第三型分泌系統壞損之突變菌株後更加明顯，而全株短暫表現H蛋白之番茄對Rs與Pcc之抗性則會提升，依據目前結果推測H 蛋白對植物細胞產生的傷害可能引發類似damage-associated molecular patterns (DAMPs)-triggered immunity (DTI) 反應。此外，我們也進一步分離出本土可裂解高毒力青枯病菌的九類特性不同的噬菌體，其中噬菌體Rsφ34具有最廣的寄主範圍，而噬菌體 Rsφ4則具有最優異之殺菌效果，同時也發現高毒力青枯病菌較抗這些噬菌體。 本論文所得之重要資訊與基礎，應有助於未來對青枯病菌毒力關鍵決定因子、青枯病菌與植物/噬菌體之交互作用及病害防治更深入的研究。 | zh_TW |
| dc.description.abstract | Ralstonia solanacearum (Rs), the causal agent of bacteria wilt (BW), has an unusual wide host range, including many important crops. R. solanacearum phylotype I strains confer varied degrees of virulence/aggressiveness on tomato plants; however, genetic information on pathogen virulence-related factors is still very limited. Our comparative genome sequence analysis identified a YopJ-type effector protein Pseudomonas outer protein P 3 (popP3) unique to a high-virulence strain Pss190. The first part of this thesis aimed to study function and property of PopP3 in Rs virulence. The results showed that overexpression of PopP3 in Rs medium-virulence strain Pss4 led to increased bacterial virulence in tomato. Transient local overexpression of PopP3 in Nicotiana benthamiana caused enhanced propagation of Rs. Transient overexpression of PopP3 in tomato suppresses pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) related callose deposition and increases the susceptibility to the Pectobacterium carotovorum subsp. carotovorum (Pcc). popP3 transcription was positively regulated by the type III secretion system (T3SS) regulator HrpG. In addition, GFP-tagged PopP3 colocalized with the mitochondria of N. benthamiana protoplast. These results together suggest that PopP3 is a T3SS effector and contributes to the virulence of R. solanacearum by suppressing plant defense response which probably is related to mitochondrial functions. The second part of this thesis aimed to collect information on R. solanacearum phages and their lysis proteins. Genome annotation of a T7-like bacteriophage RsφP29 capable of lysing the medium-virulence Rs strain Pss4 revealed the existence of a group of novel proteins which may involve in host specificity. In addition, characterization of its lysis proteins showed that GST-L recombinant protein might not have in vitro anti-Rs activity and that the failure of expressing the H protein in E. coli might be due to its antibacterial activity. Interestingly, transient overexpression of RsφP29 H protein in tobacco caused cell death and this effect can be further promoted by the infection of different phytopathogenic bacteria or their T3SS-defective mutants. Furthermore, tomato plants systemically overexpressing H protein were more resistant to Rs and Pcc. These results enable us to propose an effect for RsφP29 H protein in eliciting damage-associated molecular patterns (DAMPs)-triggered immunity (DTI) in plant. In addition, nine groups of local phages with varied capability of lysing local and imported high-virulence Rs strains were isolated, and Rs strains with higher virulence generally were tolerant to these phages. The information collected from this thesis, regarding Rs virulence determinants, Rs-plant/phage interactions and related resources, would pave the way to further studies of these important aspects. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T23:55:31Z (GMT). No. of bitstreams: 1 ntu-102-R00b42004-1.pdf: 170196057 bytes, checksum: 4346c7d56244d255c41e75d5096ac88e (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書 ................................................................................................... ii 謝誌 ...................................................................................................................... iii 中文摘要 ............................................................................................................... iv 英文摘要 ............................................................................................................... v 常用縮寫與全名對照表 ....................................................................................... vii 目錄 ...................................................................................................................... viii 表目錄 .................................................................................................................. xiv 圖目錄 .................................................................................................................. xv 附錄目錄 ............................................................................................................... xvi 效應蛋白PopP3 在青枯病菌毒力之功能研究 ............................................ 1 第一章 前言 ....................................................................................................... 2 1. 第三型蛋白分泌系統 ....................................................................................... 2 1.1 調控蛋白 ......................................................................................... 2 1.2 構造蛋白 ......................................................................................... 3 1.3 效應蛋白 ......................................................................................... 3 2. 青枯病與青枯病菌 ........................................................................................... 3 3. 青枯病菌第三型分泌蛋白 ............................................................................... 5 3.1 GALA 家族 ..................................................................................... 6 3.2 AWR 家族 ....................................................................................... 7 3.3 AvrA ................................................................................................ 7 3.4 Harpin .............................................................................................. 7 3.5 YopJ superfamily ............................................................................. 8 5.植物防禦反應與細菌效應蛋白之攻擊 ............................................................ 10 5.1 植物PTI 防禦反應 ......................................................................... 10 5.2 效應蛋白對植物防禦系統的攻擊 ................................................. 11 5.2.1 作用於細胞膜之效應蛋白 ................................................ 11 5.2.2 作用於葉綠體之效應蛋白 ................................................ 12 5.2.3 作用於囊泡運輸之效應蛋白 ............................................. 13 5.2.4 作用於MAPK 訊息傳遞之效應蛋白 ............................... 13 5.2.5 作用於細胞核之效應蛋白 ................................................. 13 5.2.6 攻擊轉譯之效應蛋白 ......................................................... 14 6. 植物病原細菌毒力相關研究 ........................................................................... 14 7. 研究動機與目標 ............................................................................................... 15 第二章 材料與方法 ........................................................................................... 16 1. 供試菌株、質體及菌株培養條件 ................................................................... 16 2. 供試植物培育條件 ........................................................................................... 16 3. 生物統計分析 ................................................................................................... 16 4. 重組質體構築與popP3 過量表現菌株建構 ................................................... 16 4.1. DNA 瓊脂糖凝膠電泳 ................................................................... 16 4.1.1. 外染瓊脂糖凝膠電泳分析 ................................................ 17 4.1.2. 內染瓊脂糖凝膠電泳純化 ................................................ 17 4.2. 聚合酶連鎖反應 ............................................................................ 17 4.2.1. Taq PCR (Violet) ............................................................... 17 4.2.2. High FidelityR Taq PCR (Invitrogen) ............................... 17 4.3. 質體萃取 ....................................................................................... 18 4.3.1. 大腸桿菌質體萃取 ............................................................ 18 4.3.2.青枯病菌與農桿菌質體萃取 ............................................. 18 4.4. DNA 純化 ....................................................................................... 19 4.5. DNA 限制酶消化水解 ................................................................... 19 4.6. DNA 片段接合 ............................................................................... 19 4.7. TOPOR 質體構築 ........................................................................... 20 4.8. LR 重組互換反應 .......................................................................... 20 4.9. 大腸桿菌勝任細胞熱休克轉型作用 ............................................ 20 4.10. 電穿孔轉型作用之勝任細胞置備 ............................................. 20 4.10.1.青枯病菌、農桿菌 .......................................................... 21 4.10.2. 大腸桿菌 .......................................................................... 21 4.11. 電穿孔轉型作用 .......................................................................... 21 4.12. 青枯病菌染色體DNA 萃取 ........................................................ 22 5. 青枯病菌PopP3 功能研究 ............................................................................... 22 5.1. 啟動子活性測試 ............................................................................ 22 5.2. 效應蛋白外泌試驗 ........................................................................ 23 5.2.1. 培養基中之效應蛋白外泌檢測 ......................................... 23 5.2.2. 效應蛋白外泌試驗之西方墨點法 ..................................... 23 5.3. 菸草原生質體萃取與蛋白座落位置檢測 ..................................... 24 5.4. PopP3 重組蛋白質表現及酵素活性分析 ...................................... 25 5.4.1. 重組蛋白質表現 ................................................................ 25 5.4.2. 蛋白質電泳 ........................................................................ 25 5.4.3. 重組蛋白表現之西方墨點法 ............................................ 26 5.4.4. 重組蛋白質純化與定量 ..................................................... 26 5.4.5. 乙醯化轉移酶活性試驗 ..................................................... 26 5.5. 青枯病菌毒力測試 ......................................................................... 27 6. 植物體短暫表現popP3 與其功能性研究 ........................................................ 27 6.1. 菸草葉部基因短暫表現 ................................................................. 27 6.2. 青枯病菌植物體內增殖能力分析 ................................................. 27 6.3. 番茄短暫性病毒誘導基因過量表現 ............................................. 28 6.4. 軟腐病菌接種試驗 ......................................................................... 28 6.5. 植物PTI 防禦反應指標檢測 ......................................................... 29 6.5.1 癒傷葡聚糖堆積分析 ......................................................... 29 6.5.2. 植物PTI 防禦反應之ROS 檢測 ...................................... 29 6.6. 植物體短暫表現基因檢測 ............................................................. 29 6.6.1 植物RNA 萃取 ................................................................... 30 6.6.2 反轉錄聚合酶連鎖反應 ...................................................... 30 6.6.3.半定量RT-PCR ................................................................... 30 第三章 結果 ........................................................................................................ 32 1. 青枯病菌染色體定序與毒力相關基因篩選 .................................................... 32 2. 青枯病菌PopP 家族成員存在與胺基酸序列分析 .......................................... 32 3. 在高毒力菌株popP3 之分佈 ............................................................................ 33 4. PopP3 對青枯病菌毒力之影響 ....................................................................... 33 5. PopP3 對菸草病徵產生與過敏性反應之影響 ................................................. 34 6. PopP3 對於青枯病菌增殖能力影響 ............................................................... 34 7. PopP3 短暫表現對番茄病害反應之影響 ....................................................... 34 8. PopP3 對番茄PTI 防禦反應之影響 ............................................................... 35 9. popP3 操縱組結構與第三型分泌蛋白系統調控分析 ................................... 35 10. PopP3 胞外分泌試驗測試 ............................................................................ 36 11. PopP3 在植物細胞中之蛋白質座落位置分析 ............................................ 36 12. PopP3 乙醯化轉移酶活性測試 .................................................................... 36 第四章 討論 ........................................................................................................ 38 1. PopP3 為青枯病菌毒力關鍵因子 .................................................................. 38 2. PopP3 應可分泌到青枯病菌胞外 .................................................................. 39 3. PopP3 重組蛋白不具有乙醯轉移酵素之活性 .............................................. 39 4. PopP3 座落在植物粒線體可能與其功能有關 .............................................. 40 5. PopP3 抑制植物PTI 防禦反應 ....................................................................... 41 6. PopP 家族成員可能為青枯病菌Pss190 之高毒力成因 ............................... 42 第五章 結語 ........................................................................................................ 44 參考文獻 ................................................................................................................ 45 青枯病菌噬菌體裂解蛋白之分析 ................................................................... 92 第一章 前言 ........................................................................................................ 93 1. 噬菌體 ................................................................................................................ 93 2. 噬菌體裂解細胞 ............................................................................................... 94 2.1 細胞壁分解酵素特性 ...................................................................... 94 2.2 穿孔素 .............................................................................................. 95 2.2.1 噬菌體λ的holin S105 ....................................................... 95 2.2.2 噬菌體P21 之holin S21 .................................................... 96 2.2.3 噬菌體T4 之holin T .......................................................... 97 3. T7-like 噬菌體 ................................................................................................... 97 4. 青枯病菌噬菌體相關研究 ................................................................................ 98 5. 研究動機與目標 ................................................................................................ 99 第二章 材料與方法 ............................................................................................ 100 1. 供試菌株、質體及菌株培養條件 ................................................................... 100 2. 供試噬菌體培養條件 ....................................................................................... 100 3. 供試植物培育條件 ........................................................................................... 100 4. RsφP29 裂解蛋白實驗 ...................................................................................... 101 4.1. lysozyme 與holin 重組蛋白表現與細菌裂解試驗 ..................... 101 4.1.1. lysozyme 重組蛋白質表現S21 ....................................... 101 4.1.2. lysozyme 重組蛋白裂解青枯病菌測試S21 ................... 101 4.1.3. holin 重組蛋白質小量表現 ............................................. 102 4.2. 菸草葉部基因短暫表現 ............................................................... 102 4.3. 菸草病原菌接種 ........................................................................... 102 4.4. 青枯病菌植物體內增殖能力分析 ............................................... 103 4.5. 錐藍染色法 ................................................................................... 103 4.6. 離子流失試驗 ............................................................................... 104 4.7. RsφP29 對植物病原菌宿主分析 .................................................. 104 4.8. 番茄短暫性病毒誘導基因過量表現 ........................................... 104 4.9. 青枯病菌接種番茄試驗 ............................................................... 105 4.10. 軟腐病菌接種番茄試驗 ............................................................. 105 5. 本土可裂解高毒力青枯病菌之噬菌體分離檢測 ........................................... 105 5.1. 自土壤中分離噬菌體 .................................................................... 106 5.2. 檢測濾液是否含有可裂解青枯病菌之噬菌體 ............................ 106 5.3. 單一溶菌斑分離與噬菌體濃度測量 ............................................ 106 5.4. 噬菌體濃度測量 ............................................................................ 107 5.5. 噬菌體放大 .................................................................................... 107 5.6. 噬菌體宿主範圍分析 .................................................................... 107 5.7. 噬菌體殺菌效果分析 .................................................................... 108 第三章 結果 ...................................................................................................... 109 1. RsφP29 轉譯蛋白分析比較 ............................................................................ 109 2. RsφP29裂解蛋白表現與殺菌力試驗 ............................................................ 109 3. 大量表現RsφP29裂解蛋白對菸草葉部反應之影響 ................................... 110 4. RsφP29sp-holin 誘導細胞死亡之分析 ............................................................ 111 5. 短暫大量表現RsφP29holin 與sp-holin 之菸草對植物病原細菌感染之反應 111 6. 系統性表現RsφP29holin 與sp-holin 之番茄的病害反應 ............................ 112 7. 本土可裂解高毒力青枯病菌之噬菌體分離篩選 .......................................... 112 第四章 討論 ....................................................................................................... 114 1. RsφP29基因組分析 ......................................................................................... 114 2. RsφP29之lysozyme 與holin 之in vitro 裂解細菌效果 ................................ 114 3. holin 可能造成植物細胞傷害 ......................................................................... 116 4. holin 的存在會增加植物產生抗病性 ............................................................. 116 5. 自本土分離得9 株可裂解高毒力青枯病菌之噬菌體 ................................... 118 第五章 結語 ....................................................................................................... 120 參考文獻 ............................................................................................................... 121 | |
| dc.language.iso | zh-TW | |
| dc.subject | 毒力 | zh_TW |
| dc.subject | PopP3 | zh_TW |
| dc.subject | 效應蛋白 | zh_TW |
| dc.subject | 青枯病菌 | zh_TW |
| dc.subject | 裂解蛋白 | zh_TW |
| dc.subject | DAMPs-triggered immunity | zh_TW |
| dc.subject | lysis protein | en |
| dc.subject | virulence | en |
| dc.subject | PopP3 | en |
| dc.subject | effector | en |
| dc.subject | Ralstonia solanacearum | en |
| dc.subject | DAMPs-triggered immunity | en |
| dc.title | 效應蛋白PopP3 在青枯病菌毒力之功能研究與青枯病菌噬菌體裂解蛋白之分析 | zh_TW |
| dc.title | Role of Effector PopP3 in Virulence of Ralstonia solanacearum and Characterization of Phage Lysis Proteins | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王肇芬(Jaw-Fen Wang),劉瑞芬(Ruey-Fen Liou),葉信宏(Hsin-Hung Yeh),林乃君(Nai-Chun Lin) | |
| dc.subject.keyword | 青枯病菌,效應蛋白,PopP3,毒力,裂解蛋白,DAMPs-triggered immunity, | zh_TW |
| dc.subject.keyword | Ralstonia solanacearum,effector,PopP3,virulence,lysis protein,DAMPs-triggered immunity, | en |
| dc.relation.page | 156 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2013-08-22 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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