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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭秋萍 | |
| dc.contributor.author | Feng-Chi Cheng | en |
| dc.contributor.author | 鄭峰繼 | zh_TW |
| dc.date.accessioned | 2021-06-15T16:33:18Z | - |
| dc.date.available | 2020-08-28 | |
| dc.date.copyright | 2015-08-28 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52905 | - |
| dc.description.abstract | 由Ralstonia solanacearum (Rs)引起之青枯病為土壤性維管束疾病，其寄主範圍相當廣泛且尚無有效的化學防治方法，因此抗病作物育種極為重要。番茄Hawaii 7996 (H7996)是目前抗青枯病最穩定的品系，雖然H7996抗青枯病之多個QTL已被定位，但是其抗病分子機制與具主導之抗病基因仍未明朗。本研究旨在研究pathogen-associated molecular patterns (PAMP)-trigger immunity (PTI)在H7996抗病機制中扮演之角色，並辨認在抗第一型Rs之主控QTL Bwr12中之關鍵防禦基因。為了檢測在番茄中的PTI反應，我們已經成功地建立了根部與葉部PTI反應之分析系統。實驗結果顯示抗病品系H7996之PTI反應比感病品系WVa700強，且對於多種特性不同之病原菌之抗病性也較強。另外，Bwr12之存在與較強之番茄根部PTI反應具有顯著關連性，故PTI防禦應參與在Bwr12主導之青枯病抗病機制中。此外，功能性遺傳與基因表現特性等分析所得結果顯示，12g520與12g550在番茄PTI反應與抵抗多種病原菌之抗病反應中為正向調控者，分析大量表現12g520與12g550之轉殖菸草所得結果中亦有一致的結論，而12g690則是透過調控水楊酸 (salicylic acid, SA) 與茉莉酸 (jasmoic acid, JA) 訊息傳導路徑而在植物抗病反應中扮演負調控者。此外，蛋白質定位結果顯示12g520座落於細胞膜上，12g550會被分泌至細胞外，而12g690 N端之36個胺基酸座落於細胞膜上。以上研究結果對於了解番茄之PTI訊息傳遞與番茄抗青枯病之分子機制提供了關鍵且重要訊息。 | zh_TW |
| dc.description.abstract | Bacterial wilt (BW) caused by Ralstonia solanacearum (Rs) is a complex and serious disease affecting a wide range of important crops, and breeding for durable resistance is the only effective means for disease control. Tomato cultivar Hawaii 7996 (H7996) is currently the most stable BW-resistance source. Despite BW-resistance associated quantitative trait loci (QTLs) have been mapped on H7996 chromosomes, the involved molecular mechanism and the gene identity remain undetermined. This study aims to investigate the role of pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) in H7996 disease defense and to identify genes involved in the resistance mediated by Bwr12, the major QTL associated with the H7996 resistance against Rs phylotype I strains. We have established assay systems for Rs-induced PTI responses in tomato leaf and root. We found that H7996 displays stronger PTI responses and better tolerance to multiple bacteria than BW-susceptible (BWs) cultivar WVa700. Furthermore, Bwr12 correlates with stronger root PTI responses, suggesting the involvement of PTI in Bwr12-mediated BW-defense. Results of functional genetic, gene expression analyses and disease responses of overexpressing transgenic tobacco plants further reveal a positive role of 12g520 and 12g550 in PTI against distinct pathogens and a negative role of 12g690 in defense via modulating SA and JA pathways. In addition, 12g520-GFP localizes at the plasma membrane, 12g550-GFP could be secreted to extracellular space, and 12g6901-36-GFP localizes at the plasma membrane. These results shed light on the mechanism of PTI signaling and the disease-defense mechanisms in tomato. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T16:33:18Z (GMT). No. of bitstreams: 1 ntu-104-R02b42003-1.pdf: 10098055 bytes, checksum: b64fffd1dbc756b73525930cffb7a5cd (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 目錄 口試委員審定書 I 謝誌 II 中文摘要 IV Abstract V 常用縮寫與全名對照表 VI 目錄 VIIIIII 圖目錄 XIIII 附錄目錄 XIVV 第一章 前言 1 1. 青枯病 (Bacterial wilt, BW) 1 1.1 番茄之青枯病反應相關研究 1 1.2 其他作物之青枯病反應相關研究 2 1.3 阿拉伯芥之青枯病反應相關研究 3 2. 植物病害防禦機制 4 2.1 植物抗病反應 4 2.2 植物抗病訊息傳遞 5 2.3 PTI訊息傳遞與其下游相關反應 6 3. 植物白胺酸重覆 (Leucine rich repeat, LRR)蛋白質的特性 8 3.1 LRR receptors結構與功能 8 3.2 LRR receptors與病原菌之交互作用 8 4. 前人研究與研究動機 9 第二章材料與方法 11 1. 植物材料與栽培條件 11 2. 微生物材料與培養條件 11 3. 序列分析與統計分析 12 4. 選殖技術 (Cloning) 12 4.1聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 12 4.2 DNA瓊脂糖凝膠電泳（agarose gel electrophoresis） 13 4.3 DNA片段純化 (DNA purification) 13 4.4 DNA限制酶消化水解 (DNA digestion) 13 4.5 DNA片段結合 (DNA ligation) 14 4.6 TOPO ®質體構築 (TOPO® cloning) 14 4.7 LR重組互換反應 (LR recombination) 14 4.8大腸桿菌勝任細胞熱休克轉型作用 (Heat shock transformation) 15 4.9電穿孔轉型作用之勝任細胞製備 (Competent cell preparation for electroporation) 15 4.10 電穿孔轉型作用 (Electroporation transformation) 16 4.11 質體萃取 (Plasmid purification) 16 5. 植物 DNA 萃取 (Plant DNA extraction) 17 6. 植物 RNA 萃取 (Plant RNA extraction) 17 7. 反轉錄反應 (Reverse transcription) 18 8. 定量聚合酶連鎖反應 (Quantitative polymerase chain reaction) 18 8.1 半定量PCR (Semi-quantitative PCR, sqPCR) 18 8.2 定量 PCR (Quantitative PCR, qPCR) 18 9. 病毒誘導短暫性基因靜默與大量表現 (Virus-induced gene silencing [VIGS] and virus-mediated gene overexpression [VMGO]) 19 9.1病毒誘導短暫性基因靜默 (Virus-induced gene silencing, VIGS) 19 9.2病毒誘導短暫性大量表現 (Virus mediated gene overexpression, VMGO) 20 10. 植物病原菌處理與病害反應分析 20 10.1 青枯病接種試驗 20 10.2 細菌性軟腐病菌接種試驗 21 10.3 細菌性斑點病接種試驗 21 10.4 番茄疫病接種試驗 22 10.5 灰黴病接種試驗 22 11. 番茄 PTI 反應分析 23 11.1 番茄根部 PTI 反應 23 11.2 番茄葉部 PTI 反應 24 12. 接種青枯病菌後番茄基因轉錄之檢測 25 13. 酵母體雜交 (Yeast two-hybrid) 25 13.1 質體構築 25 13.2 製備酵母菌勝任細胞 26 13.3 酵母菌雙雜合系統 26 14. 蛋白質在菸草葉片細胞之定位分析 26 14.1 基因短暫表現載體構築 26 14.2 蛋白質定位分析與細胞質壁分離 27 15. LUC互補影像系統 (Luciferase complementation imaging assay, LCI) 27 15.1 基因大量表現載體構築 27 15.2 LUC活性偵測 28 16. 培育基因轉殖植物 (Generation of transgenic plants) 28 16.1 基因過量表現構築 28 16.2 菸草基因轉殖 29 16.3 番茄基因轉殖 29 16.4 基因轉殖植物之檢驗 30 第三章結果 33 1. 番茄根部 PTI 反應分析系統之建立 33 2. 青枯病抗病與感病番茄品系之 PTI 反應比較 34 3. 青枯病抗病與感病番茄品系對其他病害反應之比較 34 4. Bwr12 與 Bwr6 對番茄根部 PTI 表現之影響 35 5. 12g520 與 12g550 在其他病害反應之功能 35 6. 12g520 與 12g550 蛋白質在植物細胞中之座落位置 36 7. 12g520 與 12g550 之蛋白質交互作用 37 8. 以短暫基因靜默與過量表現檢視12g690在番茄青枯病抗性之角色 37 9. 接種青枯病菌後之 12g690 表現 38 10. 12g690 序列分析與 12g690 1-36之功能分析 38 11. 12g690 蛋白質之座落位置 39 12. 12g690 在 PTI 反應與其他病害反應中之角色 39 13. 12g690 與防禦荷爾蒙訊息傳遞之關係 40 14. 12g690 與 12g690 1-36轉殖植物之培育 40 第四章 討論 41 1. 番茄 PTI 反應對廣效抗病力之重要性 41 2. Bwr12 區段之多個 RLK 基因共同參與 H7996 抗病反應 42 3. 番茄基因 12g520 參與抗病之可能機制 43 4. 番茄基因 12g550 參與抗病之可能機制 45 5. 番茄基因 12g690 調控抗病反應之可能機制 46 6. WVa700 抗細菌性軟腐病之可能機制 47 7. 結語 49 第五章 參考文獻 50 圖目錄 圖一、青枯病抗病與感病番茄品系之葉部callose deposition反應 60 圖二、青枯病抗病與感病番茄品系之葉部過氧化氫(H2O2)累積反應 61 圖三、青枯病抗病與感病番茄品系之根部PTI反應callose deposition分析 62 圖四、青枯病抗病與感病番茄品系之根部PTI反應標誌基因PTI5表現 63 圖五、青枯病抗病與感病番茄品系之根部PTI反應生長抑制分析 64 圖六、不同番茄品種接種細菌性斑點病菌後之反應 65 圖七、不同番茄品種接種軟腐病菌13~16小時後之反應 66 圖八、不同番茄品種接種灰黴病菌後之反應 67 圖九、不同番茄品種接種番茄疫病菌後之反應 68 圖十、番茄RILs之根部PTI反應callose deposition分析 69 圖十一、番茄RILs之根部PTI反應標誌基因PTI5表現 71 圖十二、番茄H7996靜默12g520與12g550後接種Pst後之反應 72 圖十三、番茄H7996靜默12g520與12g550後接種Pcc後之反應 73 圖十四、番茄H7996靜默12g520與12g550後接種灰黴病菌後之反應 74 圖十五、番茄H7996靜默12g520與12g550後接種番茄疫病菌後之反應 75 圖十六、大量表現12g520或12g550之基因轉殖菸草的培育 76 圖十七、大量表現12g520與12g550之轉殖菸草接種Pcc後之反應 77 圖十八、大量表現12g520與12g550之轉殖菸草接種灰黴病菌後之反應 78 圖十九、大量表現12g520與12g550之轉殖菸草接種疫病菌後之反應 79 圖二十、12g520-GFP重組蛋白質在轉殖菸草葉片之座落位置 80 圖二十一、12g550-GFP重組蛋白質在轉殖菸草葉片之座落位置 81 圖二十二、以酵母菌雙雜合檢測12g520與12g550之蛋白質交互作用 82 圖二十三、在H7996中短暫過量表現12g690後之青枯病反應 83 圖二十四、在WVa700中短暫過量表現或靜默12g690後之青枯病反應 84 圖二十五、接種青枯病菌後之12g690 轉錄表現 85 圖二十六、不同番茄品系之12g690 genomic DNA 序列比較 86 圖二十七、番茄品系H7996之12g690 genomic DNA 與 cDNA 之部份片段序列與其胺基酸序列比較 87 圖二十八、在WVa700中短暫過量表現12g6901-36後之青枯病反應 88 圖二十九、12g690與12g690 1-36重組蛋白質在菸草葉片之座落位置 89 圖三十、在H7996中短暫過量表現12g690對PTI反應callose deposition之影響 90 圖三十一、在H7996中短暫過量表現12g690對PTI反應H2O2累積之影響 91 圖三十二、在WVa700中短暫靜默12g690對PTI反應callose deposition之影響 92 圖三十三、在WVa700中短暫靜默12g690對PTI反應H2O2累積之影響 93 圖三十四、在H7996中短暫過量表現12g690後接種Pcc 13~16小時後之反應 94 圖三十五、在H7996中短暫過量表現12g690對防禦相關荷爾蒙之標誌基因的影響 95 圖三十六、番茄H7996 Bwr12抵抗青枯病之模式圖 96 附錄目錄 附表一、番茄 Bwr12相關 RLKs基因之VIGS 分析中使用之構築 97 附表二、本研究所使用之質體與菌株特性 98 附表三、培養基與藥品配方 101 附表四、抗生素濃度與配方 109 附表五、本研究中所使用之引子 110 附圖一、pCR®II/GW/TOPO®與pCR®8⁄GW⁄TOPO®載體 (Invitrogen) 113 附圖二、TRV-based VIGS vectors 與 PVX-based VMGO vector 114 附圖三、綠色螢光重組蛋白所使用之載體 115 附圖四、酵母菌雙雜合系統使用之載體 116 附圖五、LCI重組蛋白所使用之載體 117 附圖六、不同番茄品種接種Pcc 16~20 小時後之反應 118 附圖七、番茄根部 PTI 反應 callose deposition 之測試 119 附圖八、番茄幼苗接種病原細菌後之根部 PTI 反應 callose deposition 分析 120 附圖九、番茄根部 PTI 反應H2O2累積之測試 121 附圖十、番茄根部 PTI 反應標誌基因 PTI5 表現之建立 122 附圖十一、番茄根部 PTI 反應生長抑制之測試 123 附圖十二、Recombinant inbreeding lines (RILs) 之分析 124 附圖十三、位於Bwr12區段中之基因分類與胺基酸序列分析 125 附圖十四、以VIGS與VMGO在番茄中短暫靜默與大量表現 RLKs 與 RLPs後之青枯病測試 126 附圖十五、在不同番茄品系中 12g520 與 12g550 胺基酸序列比較 127 附圖十六、接種青枯病菌後 12g520 與 12g550 之表現 128 附圖十七、在H7996中靜默12g520與12g550對 PTI 反應- callose deposition的影響 129 附圖十八、在H7996中靜默12g520與12g550對 PTI 反應-過氧化氫H2O2的累積與 PTI 反應相關標識基因 PTI5 現的影響 130 附圖十九、本研究使用之 VIGS 構築所對應之標靶基因與檢測其基因靜默效力時使用之引子 131 附圖二十、番茄進行 VIGS 後之基因靜默效力檢測 132 附圖二十一、番茄進行 VMGO 後之基因大量表現效力檢測 133 附圖二十二、以西方墨點法偵測大量表現之轉殖菸草蛋白質表現 134 附圖二十三、12g520 蛋白質表現位置預測分析 135 附圖二十四、12g550 蛋白質表現位置預測分析 136 附圖二十五、以 LUC互補 (LCI) 分析蛋白質交互作用 137 附圖二十六、在 H7996中大量表現 12g690對防禦相關荷爾蒙之標誌基因的影響 138 附圖二十七、12g690 蛋白質表現位置預測分析 139 附圖二十八、12g690 1-36蛋白質表現位置預測分析 140 附圖二十九、12g720蛋白質表現位置預測分析 141 附圖三十、12g740蛋白質表現位置預測分析 142 附圖三十一、、以西方墨點法偵測短暫大量表現 12g690 與 12g6901-36之菸草蛋白質表現 143 | |
| dc.language.iso | zh-TW | |
| dc.subject | H7996 | zh_TW |
| dc.subject | PTI | zh_TW |
| dc.subject | 數量性狀位點 | zh_TW |
| dc.subject | 青枯病菌 | zh_TW |
| dc.subject | 青枯病 | zh_TW |
| dc.subject | RLK | zh_TW |
| dc.subject | LRR | zh_TW |
| dc.subject | PTI | en |
| dc.subject | Ralstonia solanacearum | en |
| dc.subject | QTL | en |
| dc.subject | Hawaii 7996 | en |
| dc.subject | LRR | en |
| dc.subject | RLK | en |
| dc.subject | Bacterial wilt | en |
| dc.title | 初級免疫反應在番茄Hawaii 7996數量性狀位點Bwr12抗青枯病能力扮演重要角色 | zh_TW |
| dc.title | Innate immunity plays an important role in the resistance of tomato Hawaii 7996 QTL Bwr12 against Ralstonia solanacearum | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳克強,王肇芬,葉國楨,詹明才 | |
| dc.subject.keyword | 青枯病,青枯病菌,數量性狀位點,H7996,LRR,RLK,PTI, | zh_TW |
| dc.subject.keyword | Bacterial wilt,Ralstonia solanacearum,QTL,Hawaii 7996,LRR,RLK,PTI, | en |
| dc.relation.page | 143 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-13 | |
| 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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