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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭秋萍(Chiu-Ping Cheng) | |
| dc.contributor.author | Chia-Ling Yang | en |
| dc.contributor.author | 楊佳陵 | zh_TW |
| dc.date.accessioned | 2021-05-19T17:42:17Z | - |
| dc.date.available | 2029-12-31 | |
| dc.date.available | 2021-05-19T17:42:17Z | - |
| dc.date.copyright | 2019-02-20 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-02-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7367 | - |
| dc.description.abstract | 青枯病是由病原細菌 Ralstonia solanacearum (Rs) 引起之土壤傳播性病害，對眾多重要經濟作物造成嚴重危害，因此其抗病育種工作急迫且重要。在抗青枯病之番茄品系Hawaii 7996 (H7996) 中主導抗第一型 Rs 菌系之量化性狀基因座 (quantitative trait locus，簡稱QTL) 為Bwr12；我們先前研究顯示，位於此區之 12g690 可能會透過調控水楊酸 (salicylic acid, SA) 與茉莉酸 (jasmoic acid, JA) 訊息傳遞而在番茄抵抗青枯病中扮演負向調控者角色。此外， Hawaii 7998 (H7998) 為另一青枯病抗病番茄品系，但其抗病特性與防禦機制尚未闡明。本研究之第一部份為針對 12g690 在多種重要病害之角色與其基因特性進行進一步探討。以轉殖菸草驗證 12g690 在茄科植物抵抗青枯病反應中扮演負向調控者，但12g690 之轉錄表現在番茄莖基部與根部可能未受 Rs 感染調控，且在抗病 H7996 與感病 WVa700 番茄品系中之表現亦無明顯差異。此外，利用短暫基因過量表現、短暫基因靜默及基因轉殖植物等策略所得之分析結果指出 12g690 在番茄與菸草防禦 Pseudomonas syringae pv. tomato (Pst)、 Pectobacterium carotovorum subsp. carotovorum (Pcc) 及 Botrytis cinerea (Bc) 的反應中可能並非主要調控者。分析 H7996 與 WVa700 之 12g690之 genomic DNA 與 cDNA序列發現，H7996 序列中有數個非同義差異 (non-synonymous SNPs) 與一個24-bp insertion，在此兩種品系番茄之莖基部與根部中 12g690 mRNA 主要是以保留兩個內含子 (intron) 形式存在，且剪切情況並未因感染 Rs 而有明顯改變；在植體中表現重組蛋白質所得結果也一致指出，因 12g690 RNA 之第一個 intron 被保留而導致轉譯時造成 early termination 以致於產生僅有36個胺基酸之蛋白質產物 (12g6901-36)。蛋白質定位結果顯示 12g6901-36-GFP 座落於細胞膜，而全長之12g690 (12g690FL)-GFP 則無法被表現出來。本研究之第二部份分析 H7998抗病範圍所得結果顯示，H7998 不僅對 Pcc、 Pst 及 Bc 都有良好抗性，且對 Pcc與 Pst之抗性較 H7996 更佳，且其 H2O2 累積與 callose deposition等初級免疫反應也比 H7996 稍強。以上研究結果提供了番茄防禦相關之重要訊息。 | zh_TW |
| dc.description.abstract | Bacterial wilt (BW) caused by Ralstonia solanacearum (Rs) is a devastating soil-borne disease of many crops, and breeding for durable resistance is urgent and important for disease control. The major QTL associated with tomato cultivar Hawaii 7996 (H7996) resistance against Rs phylotype I strains is named Bwr12. Our previous studies suggested that 12g690 may have a negative role in tomato resistance to BW via regulating SA and JA pathways. In addition, the disease responses and defense nature of another BW-resistant tomato cultivar Hawaii 7998 (H7998) are not characterized. The first part of this study aimed to further determine the roles of 12g690 in several important diseases and its molecular characteristics. Characterization of transgenic tobacco plants confirmed the negative role of 12g690 in BW-defense. However, 12g690 transcription in tomato root and stem-base might not be regulated by Rs infection nor differentially regulated in H7996 and susceptible cultivar WVa700. In addition, results of transient and transgenic functional genetic analyses suggested that 12g690 may not have a major role in tomato and tobacco against Pseudomonas syringae pv. tomato (Pst), Pectobacterium carotovorum subsp. carotovorum (Pcc) and Botrytis cinerea (Bc). Furthermore, sequence analysis of 12g690 genomic DNA in H7996 and WVa700 revealed non-synonymous SNPs and a 24-bp insertion are present in H7996. Analyses of 12g690 transcripts showed that most transcripts retain both introns in both cultivars and this intron-retention phenomenon is not altered by Rs infection. Intron retention in 12g690 transcripts would lead to early termination with a predicted protein product of 36 amino acids (12g6901-36) and in planta expression of 12g690 recombinant protein showed consistent results. Protein subcellular localization assay further showed that 12g6901-36-GFP localizes on plasma membrane, while 12g690FL-GFP was not detectable. The second part of this study aimed to determine the disease responses and defense nature. The results showed that H7998 confers similar or higher degrees of resistance to Pcc, Pst and Bc compared to H7996. Moreover, H7998 confers slightly higher levels in H2O2 accumulation and callose deposition, two key characteristics of innate immunity. Together, these results provide important information regarding the nature of tomato defense. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T17:42:17Z (GMT). No. of bitstreams: 1 ntu-108-R05b42006-1.pdf: 10008172 bytes, checksum: 4b61cfb175f51dab1ae0d43d0010be3a (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目錄
口試委員會審定書 i 謝誌 ii 中文摘要 iii Abstract iv 常用縮寫與全名對照表 v 目錄 vii 表目錄 xi 圖目錄 xii 附錄目錄 xii 第一章 前言 1 1. 植物病害防禦機制 1 1.1植物抗病反應 1 1.2植物抗病訊息傳遞 2 1.3 PTI訊息傳遞與其下游相關反應 3 1.4植物信使 RNA (mRNA)前驅物之剪切與免疫反應 4 2. 植物白胺酸重覆 (Leucine rich repeat, LRR)蛋白質的特性 5 2.1 LRR receptors結構與功能 5 2.2 LRR receptors與病原菌之交互作用 6 3.青枯病 (Bacterial wilt, BW) 7 3.1番茄之青枯病反應相關研究 7 3.2阿拉伯芥之青枯病反應相關研究 8 3.3其他作物之青枯病反應相關研究 9 4. 前人研究與研究動機 10 第二章材料與方法 12 1. 植物材料與栽培條件 12 2. 微生物材料與培養條件 12 3. 序列分析與統計分析 12 4. 選殖技術 (Cloning) 13 4.1聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 13 4.2 DNA瓊脂糖凝膠電泳（agarose gel electrophoresis） 13 4.3 DNA片段純化 (DNA purification) 14 4.4 DNA限制酶消化水解 (DNA digestion) 14 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) 15 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 6.1植物RNA萃取 (Plant RNA extraction) 17 6.2 RNA 樣本 genomic DNA殘留檢測 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) 19 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 20 10.3 細菌性斑點病接種試驗 21 10.4灰黴病接種試驗 21 11. 番茄葉部 PTI 反應 22 11.1 病原菌處理 22 11.2 癒傷葡聚醣累積分析 (Callose deposition assay) 22 11.3 H2O2累積檢測 (H2O2 accumulation assay) 22 12. 接種青枯病菌後番茄基因轉錄之檢測 22 13. 蛋白質在菸草葉片細胞之定位分析 23 13.1 基因短暫表現載體構築 23 13.2 蛋白質定位分析與細胞質壁分離 23 14. 培育基因轉殖植物 (Generation of transgenic plants) 23 14.1 基因過量表現構築 23 14.2 菸草基因轉殖 24 14.3 番茄基因轉殖 24 14.4 基因轉殖植物之檢驗 25 15. 蛋白質轉譯表現 (Transgenic tobacco protein translation) 25 15.1 植物膜蛋白質萃取 (Plant protein extraction) 25 15.2 蛋白質定量 (Protein quantification) 25 15.3 蛋白質電泳 (SDS-PAGE) 26 15.4 西方墨點法 (Western blot) 26 第三章結果 27 1. 接種青枯病菌後番茄 12g690之基因轉錄表現 27 2. 抗病與感病番茄品系中 12g690 之 DNA序列分析 27 3. 抗病與感病番茄品系中 12g690 RNA 之剪切與蛋白質表現 27 4. 培育大量表現 12g690FL與 12g6901-36 之轉殖菸草 28 5. 12g690蛋白質在植物細胞中之座落位置 29 6. 12g690 在茄科青枯病之角色 29 7. 12g690 在其他茄科重要病害反應之角色 30 8. 接種青枯病菌後12g520之基因轉錄表現 30 9. 12g550蛋白質在植物細胞之座落位置 31 10. 不同番茄品系對病害反應與 PTI反應之比較 31 第四章 討論 33 1. 番茄基因 12g690 在青枯病防禦反應中為負向調控者 33 2. Rs 感染可能並未影響 H7996與 WVa700 中 12g690 之轉錄表現 33 3. 12g690 在茄科植物對 Pcc、 Pst 及 Bc 之病害反應中可能並非主要調控者 34 4. 12g690 genomic DNA序列 在 H7996、H1706 及 WVa700 具一定之差異性 35 5. 番茄基因 12g690 之 mRNA 可能保留其 intron 36 6. 番茄品系 H7998 具強 PTI 反應與廣效抗病力 38 7. 結語 39 第五章 參考文獻 40 第六章 附錄 53 表目錄 表一、統整大量表現 12g520與 12g550轉殖菸草之培育、PTI反應及病害反應 53 表二、大量表現 12g690FL轉殖菸草培育 54 表三、大量表現 12g6901-36轉殖菸草培育 55 圖目錄 圖一、接種青枯病菌後番茄莖基部之12g690 轉錄表現 56 圖二、接種青枯病菌後番茄根部之12g690 轉錄表現 58 圖三、12g690之genomic DNA序列分析 60 圖四、12g690之cDNA基因序列分析 62 圖五、以西方點墨法檢測12g690剪切狀況 64 圖六、大量表現 12g690FL與 12g6901-36之轉殖菸草的培育 65 圖七、12g690FL-GFP與 12g6901-36重組蛋白質在菸草葉片之座落位置 66 圖八、大量表現 12g690FL之轉殖菸草接種中毒力青枯病菌 Pss4之反應 67 圖九、大量表現 12g6901-36之轉殖菸草接種中毒力青枯病菌 Pss4之反應 68 圖十、番茄H7996中短暫過量表現12g690FL與12g6901-36後接種 Pst之反應 69 圖十一、番茄WVa700中靜默12g690後接種接種 Pst之反應 70 圖十二、番茄 H7996中短暫過量表現 12g690FL與 12g6901-36後接種 Pcc 之反應 71 圖十三、番茄 WVa700中靜默 12g690後接種 Pcc 之反應 72 圖十四、大量表現 35S::12g690FL-GFP之轉殖菸草接種 Pcc 之反應 73 圖十五、番茄H7996中短暫過量表現 12g690FL與 12g6901-36後接種 Bc 之反應 74 圖十六、番茄WVa700中靜默 12g690後接種灰黴病菌 Bc 後之反應 75 圖十七、大量表現 35S::12g690FL-GFP轉殖菸草接種灰黴病菌 Bc 之反應 76 圖十八、接種青枯病菌後番茄 12g520之轉錄表現 78 圖十九、12g550-GFP重組蛋白質在菸草葉片之座落位置 80 圖二十、不同番茄品種接種細菌性軟腐病菌 Pcc 之反應 81 圖二十一、不同番茄品種接種細菌性斑點病菌 Pst 之反應 82 圖二十二、不同番茄品種接種灰黴病菌 Bc 之反應 83 圖二十三、青枯病抗病番茄品系之葉部過氧化氫累積反應 84 圖二十四、青枯病抗病番茄品系之葉部PTI反應callose deposition分析 85 圖二十五、番茄 H7996 Bwr12之 12g520、 12g550及 12g690參與抵抗青枯病之模式圖 86 附錄目錄 附表一、統整大量表現 12g690FL與 12g6901-36轉殖菸草之病害反應 87 附表二、整合 Bwr12基因在植物防禦之研究 88 附表三、番茄 Bwr12相關 RLKs 與訊息傳遞相關基因之 VIGS 分析中使用之構築 89 附表四、本研究所使用之質體與菌株特性 90 附表五、培養基與藥品配方 92 附表六、抗生素濃度與配方 99 附表七、本研究中所使用之引子 100 附圖一、位於Bwr12區段LRR (Leucine rich repeat)蛋白質之序列分析 102 附圖二、在H7996中短暫過量表現12g690後之青枯病反應 103 附圖三、在WVa700中短暫過量表現或靜默12g690後之青枯病反應 104 附圖四、接種青枯病菌後之12g690 轉錄表現 105 附圖五、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 106 附圖六、12g690與12g6901-36重組蛋白質在菸草葉片之座落位置 107 附圖七、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 108 附圖八、12g550 蛋白質在菸草葉片中之表現位置 109 附圖九、12g550-GFP重組蛋白質在轉殖菸草葉片之座落位置 110 附圖十、12g690蛋白質功能性區段預測分析 111 附圖十一、12g690蛋白質表現位置預測分析 112 附圖十二、12g6901-36蛋白質表現位置預測分析 113 附圖十三、12g690FL-GFP與 12g6901-36重組蛋白質在轉殖菸草葉片之座落位置 114 附圖十四、12g690FL-GFP重組蛋白質在轉殖菸草葉片之座落位置 115 附圖十五、以西方點墨法檢測大量表現12g690FL 轉殖菸草之蛋白表現 116 附圖十六、接種青枯病菌後12g520與12g550之表現 117 附圖十七、接種青枯病菌後12g520與12g550之表現 118 附圖十八、12g5501-93蛋白質表現位置預測分析 119 附圖十九、12g550-GFP重組蛋白質在轉殖菸草葉片之座落位置 120 附圖二十、12g690 基因轉錄表現引子設計示意圖 121 附圖二十一、在H7996中短暫過量表現12g690後接種 Pcc 之反應 122 附圖二十二、番茄進行 VMGO 後之基因大量表現效力檢測 123 附圖二十三、番茄進行VIGS後之基因靜默效力檢測 124 附圖二十四、在WVa700中短暫過量表現 12g6901-36 後之青枯病反應 125 附圖二十五、青枯病抗病與感病番茄品系之葉部過氧化氫累積反應 126 附圖二十六、pCR®II/GW/TOPO®與pCR®8⁄GW⁄TOPO®載體 127 附圖二十七、TRV-based VIGS vectors 與 PVX-based VMGO vector 128 附圖二十八、綠色螢光GFP重組蛋白所使用之載體 129 附圖二十九、黃色螢光YFP重組蛋白所使用之載體 130 附圖三十、菸草葉片表現N端 YFP 重組蛋白所使用之載體 131 附圖三十一、菸草葉片表現N端 GFP 重組蛋白所使用之載體 132 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討番茄 12g690 調控之病害反應與抗青枯病番茄 Hawaii 7998 之特性 | zh_TW |
| dc.title | Study on tomato disease responses mediated by 12g690 and Hawaii 7998 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 謝旭亮,王肇芬,陳逸然,林盈仲 | |
| dc.subject.keyword | 青枯病,青枯病菌,H7996,內含子保留,H7998,初級免疫, | zh_TW |
| dc.subject.keyword | Bacterial wilt,Ralstonia solanacearum,H7996,intron retention,H7998,innate immunity, | en |
| dc.relation.page | 133 | |
| dc.identifier.doi | 10.6342/NTU201900610 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2019-02-15 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| dc.date.embargo-lift | 2029-12-31 | - |
| 顯示於系所單位： | 植物科學研究所 | |
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