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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭秋萍(Chiu-Ping Cheng) | |
| dc.contributor.author | Chi Huang | en |
| dc.contributor.author | 黃琪 | zh_TW |
| dc.date.accessioned | 2021-06-16T03:41:38Z | - |
| dc.date.available | 2020-03-02 | |
| dc.date.copyright | 2015-03-02 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-02-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54922 | - |
| dc.description.abstract | 植物經常會受到各種不同病原菌攻擊，導致嚴重作物生產損失，例如：由青枯病菌所引起的青枯病，即是全世界最嚴重的植物病害之一，影響全球許多重要經濟作物，造成極大損失。本研究包含兩個子題。在第一子題中，我們先前的研究顯示，在菸草中表現源自青枯病菌之噬菌體的一個裂解蛋白質H會造成被細菌感染誘導的植物細胞死亡，並使植物對數種病原細菌之抗性增加。本研究進一步證實，在番茄植株系統性表現H蛋白質可加強植物抵抗多種特性不同之細菌病害，亦會造成細菌誘發性細胞死亡與增強pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) 反應，故表現H蛋白質可在植物體內誘導廣泛性防禦反應而促使植物得以抵抗不同的病原菌。在第二子題中，我們先前的研究顯示，青枯病菌參與糖解作用之酵素基因gpsA經破壞後，會導致病菌在番茄抗病品系中之毒力下降。本研究進一步證明gpsA確實是決定青枯病菌致病力的關鍵基因，並參與病菌之糖利用作用；同時，定量分析顯示番茄感病番茄品系L390之葡萄糖、果糖及蔗糖含量較抗病品系H7996高，且在L390根部能測到低量甘油 (glycerol) 而在H7996之各部位中均無法測得甘油；此外，L390在感染gpsA突變株後，其根部中之甘油含量下降趨勢比感染野生型菌株快速。這些結果顯示，在甘油含量低至無測得之抗病番茄品系H7996中，gpsA在青枯病菌感染的初期扮演重要角色，使病菌得以在甘油含量極低之H7996根部增殖，以達成後續的系統性感染，同時，這些發現也暗指番茄之含糖量可能與其青枯病抗性具一定相關性。另外，從本研究之番茄與馬鈴薯糖類傳輸蛋白 (sugar transporters) SWEETs序列分析結果發現，茄科植物之SWEETs基因可能比阿拉伯芥與水稻略多，且短暫基因靜默分析初步發現番茄SWEET2a (SISWEET2a) 參與植株生長發育。預期本研究所獲資訊將有助於瞭解植物病害發生之具體機制，並研擬防治病害之可能措施。 | zh_TW |
| dc.description.abstract | Plants often suffer from attacks of different pathogens, causing serious losses in crop production. For example, bacterial wilt (BW), caused by Ralstonia solanacearum (Rs), is a serious disease affecting a wide range of economically important crops worldwide. This work contains two studies. Firstly, our previous studies revealed that expression of a lysis protein (H) derived from a bacteriophage of Rs promoted bacteria-induced cell death and resistance to several bacteria in Nicotiana benthamiana. Results from this study further revealed that systemic overexpression of H or signal peptide (sp)-fused H proteins enhanced tomato resistance against three distinct bacteria, bacteria-triggered cell death and pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) responses. Therefore, expression of H protein can prime plants for enhanced defense responses, leading to boosted resistance to different pathogens. Secondly, our previous studies revealed that the disruption of Rs gpsA, which encodes an enzyme involved in glycolysis, led to differential bacterial virulence in BW-resistant and -susceptible tomato cultivars. Data from this study further evidence that gpsA is a general virulence determinant of Rs and is involved in sugar utilization. In addition, contents of sugars (glucose, fructose, sucrose) in BW-susceptible tomato cultivar L390 are higher than that in the resistant cultivar Hawaii7996 (H7996). The content of glycerol was detected at a very low level in the root of L390 but not in other tissues of L390, and glycerol was undetectable in various tissues of H7996. Moreover, the glycerol content in L390 root after the infection of Rs gpsA mutant decreased more dramatically than that after the infection of the wild-type strain. These results suggest that, to achieve a successful infection in H7996 with very limited glycerol, if there is any, functional gpsA is required for Rs sugary utilization and thus proliferation at the initial infection stage. In addition, these data imply an effect of sugar contents on tomato BW response. Furthermore, sequence analyses reveal a higher number of sugar transporters SWEET homologs in tomato and potato than that in Arabidopsis and rice. Moreover, transient gene silencing assay suggest a key role tomato SWEET2a (SISWEET2a) in plant growth. This work together with the proposed further studies are anticipated to provide important information on plant defense mechanisms and explore useful defense genes, potentially paving the way to assemble effective approaches for BW control. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T03:41:38Z (GMT). No. of bitstreams: 1 ntu-104-R01b42019-1.pdf: 6490787 bytes, checksum: 6046b09bb65d38688a00c6bcf7cedbee (MD5) Previous issue date: 2015 | 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 .................................................................................................................. ix 表目錄 .............................................................................................................. xiv 圖目錄 .............................................................................................................. xv 附錄目錄 ........................................................................................................... xvii 青枯病菌噬菌體裂解蛋白之分析 .................................................................... 1 第一章 前言 ................................................................................................ 2 1. 植物病害防禦機制 .................................................................................... 2 1.1. 植物PTI防禦反應 .................................................................... 2 1.2. 植物ETI防禦反應 ..................................................................... 3 1.3. 植物DTI防禦反應 ..................................................................... 3 2. 本研究使用之作物病原菌 ........................................................................... 3 2.1. 青枯病菌 ………………………………………………………. 4 2.2. 細菌性細斑病菌 ………………………………………………. 4 2.3. 細菌性軟腐病菌 ……………………………………………….. 4 2.4. 灰黴病菌 ………………………………………………………. 5 3. 噬菌體之裂解蛋白 ...................................................................................... 5 3.1. 噬菌體λ的holin S105 ............................................................... 6 3.2. 噬菌體P21 的holin S21 ……………………………………… 6 3.3. 噬菌體T4 的holin T …………………………………………… 7 4. 青枯病菌噬菌體 ........................................................................................... 7 5. 前人研究與研究動機 ……………………................................................... 7 第二章 材料與方法 ................................................................................... 9 1. 供試菌株、質體及菌株培養條件 .............................................................. 9 2. 供試噬菌體培養件 ...................................................................................... 9 3. 供試植物培育條件 ...................................................................................... 9 4. 生物統計分析 .............................................................................................. 9 5. 番茄短暫性病毒誘導基因過量表現 .............................................………. 10 6. 病原菌處理 ................................................................................................. 10 6.1. 細菌性細斑病菌接種番茄試驗 ................................................ 10 6.2. 軟腐病菌接種番茄試驗 ............................................................ 10 6.3. 灰黴病菌接種番茄試驗 ........................................................... 11 7. 真空抽氣法接種第三型分泌蛋白損壞之突變菌株 ................................... 11 8. 錐藍染色法.................................................................................................... 11 9. 離子流失試驗................................................................................................ 12 10. 植物PTI防禦反應指標檢測...................................................................... 12 10.1. H2O2檢測…………………………………………..………….. 12 10.2. 癒傷葡聚糖堆積分析………………………………..………... 12 10.3. PTI反應相關標誌基因表現之檢測 ........................................ 13 10.3.1. 植物RNA萃取 .............................................................. 13 10.3.2. 去除DNA殘留 .............................................................. 13 10.3.3. 反轉錄聚合酶連鎖反應 ................................................ 14 10.3.4. 定量PCR ...................................................................... 14 第三章 結果 .............................................................................................. 15 1. 表現 RsφP29 holin或sp-holin對番茄病害反應之影響 ......................... 15 2. 表現 RsφP29 holin或sp-holin對病菌誘導之番茄細胞死亡的影響 ..... 15 3. 表現 RsφP29 holin或sp-holin對番茄PTI防禦反應之影響 ................ 16 第四章 討論 ............................................................................................... 18 1. 表現RsφP29 holin或sp-holin可提升植物之廣效抗病力 ....................... 18 2. 表現RsφP29 holin或sp-holin會造成病菌誘導性植物細胞死亡 ............. 18 3. 表現RsφP29 holin 或sp-holin會加強植物PTI反應 ............................... 19 4. 表現RsφP29 sp-holin或許也會誘發植物DTI反應 …………………… 20 5. 表現RsφP29 holin 或sp-holin使植物發生priming現象 ………………. 20 6. 結語 …………………………………………………………………….….. 21 參考文獻 ........................................................................................................... 22 青枯病菌gpsA對番茄青枯病反應之功能研究 .................................................. 48 第一章 前言 .................................................................................................. 49 1. 植物SWEETs功能之研究 ............................................................................. 49 2. 細菌碳水化合物的代謝途徑 .................................................................... 50 3. 細菌secB與gpsA之研究 .......................................................................... 51 4. 青枯病與青枯病菌 ..................................................................................... 51 5. 青枯病菌之致病過程與分子機制………………………………………….. 6. 前人研究與研究動機 ..................................................................................... 52 53 第二章 材料與方法 .................................................................................... 54 1. 供試菌株、質體及菌株培養條件 ............................................................... 54 2. 供試植物培育條件 ....................................................................................... 54 3. 生物資訊分析 .............................................................................................. 54 4. 生物統計分析 ............................................................................................... 54 5. 重組質體構築 ............................................................................................... 55 5.1. 聚合酶連鎖反應 ……………………………………………….. 55 5.2. DNA瓊脂糖凝膠電泳 …………………………………….….. 55 5.3. 質體萃取 ……………………………………………………….. 55 5.3.1. 大腸桿菌質體萃取 ……………………………………… 55 5.3.2. 青枯病菌質體萃取 ……………………………………… 56 5.4. DNA 純化 …………………………………………………….… 56 5.5. DNA限制酶反應 …………………………………………….… 56 5.6. DNA 片段接合 ……………………………………………….… 57 5.7. TOPOR 質體構築 ……………………………………………….. 57 5.8. LR 重組互換反應 ………………………………………………. 57 5.9. 大腸桿菌勝任細胞熱休克轉型作用 ……………………….….. 57 5.10. 電穿孔轉型作用之勝任細胞置備 ……………………………. 58 5.11. 電穿孔轉型作用 ………………………………………………. 58 5.12. 青枯病菌染色體DNA 萃取 ……………………………….…. 58 6. 青枯病菌GpsA功能研究 ………………………………………………… 59 6.1. 啟動子活性測試 ……………………………………………….. 59 6.2. 青枯病菌基因表現量 …………………………………………... 60 6.2.1. 青枯病菌RNA萃取 …………………………..……….… 60 6.2.2. 去除DNA殘留 ………………………………………...… 60 6.2.3. 反轉錄聚合酶連鎖反應 …………………………………. 61 6.2.4. 定量PCR ……………………………………………….… 61 6.3. 青枯病菌毒力測試 ………………………………………….….. 61 6.4. 青枯病菌植物體內增殖能力分析 ……………………………… 62 6.5. 青枯病菌的自然轉型作用 ……………………………………... 62 6.6. 青枯病菌使用碳源能力 ………………………………………... 62 6.7. 青枯病菌的生長情形 ……………………………………….….. 62 7. 檢測植物中各部位之糖類含量 ………………………………..………….. 63 8. 番茄SWEETs功能研究 …………………………………………………….. 63 8.1. 番茄短暫性病毒誘導基因靜默 ………………………………… 63 第三章 結果 1. secB與gpsA基因在不同毒力青枯病菌株中之序列比較 ………….…… 64 2. gpsA相關操縱組結構與其表現調控分析 …………………………….…. 64 3. 青枯病菌gpsA基因在跳躍子插入突變株中之轉錄表現分析 ……….…. 65 4. 青枯病菌突變株gpsA-在抗病番茄品系H7996中之增殖能力分析 ….… 65 5. 青枯病菌突變株gpsA-在不同番茄品系之致病力分析 ……………….…. 65 6. 中毒力與低毒力青枯病菌之gpsA突變菌株之致病力分析 ………….…. 66 7. 青枯病菌突變株gpsA-之營養使用分析 ………………………………..… 66 8. 抗病與感病番茄品系中主要糖類之含量分析 …………………………… 66 9. 青枯病抗病與感病番茄品系經青枯病菌感染後之糖類含量分析 …….... 67 10. 番茄SWEETs序列比對分析 ……………………………………………. 68 11. 以短暫基因靜默策略檢測番茄SWEETs對植株生長發育之影響 ……. 68 12. 以短暫基因靜默策略檢測番茄SWEETs對植株青枯病反應之影響 …. 69 第四章 討論 ............................................................................................... 70 1. 青枯病菌侵染造成番茄全株之糖含量快速減少 ......................................... 70 2. 番茄含糖量與抗青枯病能力可能有關聯 ................................................... 71 3. gpsA是具不同毒力之青枯病菌菌株共有的致病基因 ............................... 71 4. gpsA參與青枯病菌之糖利用功能 …………….. ......................................... 72 5. gpsA在青枯病菌感染番茄之初期扮演關鍵角色 ....................................... 73 6. 茄科植物SWEETs基因數比阿拉伯芥與水稻多且集中分佈在特定染色體 73 7. 番茄SWEET2a參與植株生長發育 ………………………………………. 74 8. 結語 …………………………………………………………………..…..… 74 參考文獻 ............................................................................................................. 76 表目錄 青枯病菌gpsA對番茄青枯病反應之功能研究 表一、secB 與gpsA在已完全解序之青枯病菌菌株基因組中之分析 ............. 81 表二、BiologTM分析青枯病菌Pss190野生型與gpsA突變株 ………........... 82 表三、番茄SWEETs蛋白質成員搜尋 ….............................…………............ 85 表四、不同植物中SWEET蛋白質之數目 ……….…....................................... 87 表五、不同植物中SWEET基因位在各染色體位置之數目 …….….............. 88 表六、本研究使用之番茄SWEETs基因之VIGS構築 …………………….. 89 圖目錄 青枯病菌噬菌體裂解蛋白之分析 圖一、短暫大量表現RsφP29 holin或sp-holin之番茄在接種細菌性細斑病菌 後之反應 ............................................................................................ 27 圖二、短暫大量表現RsφP29 holin或sp-holin之番茄在接種軟腐病菌後之反應 ......................................................................................................... 28 圖三、短暫大量表現RsφP29 holin或sp-holin之番茄在接種灰黴病菌後之反應 ......................................................................................................... 29 圖四、短暫大量表現RsφP29 holin或sp-holin之番茄在接種植物病菌後之細 胞死亡分析 ............................................................................................. 31 圖五、短暫大量表現RsφP29 holin或sp-holin之番茄在接種植物病菌後之細 胞染色死亡分析 .................................................................................... 31 圖六、短暫大量表現RsφP29 holin或sp-holin之番茄在接種植物病菌後之離 子流失檢測 ........................................................................................... 32 圖七、短暫大量表現RsφP29 holin或sp-holin之番茄接種植物病菌後之過氧 化氫含量檢測 ..................................................................................... 33 圖八、短暫大量表現RsφP29 holin或sp-holin之番茄接種植物病菌後之癒傷 葡聚糖含量檢測 ............................................................................... 34 圖九、短暫大量表現RsφP29 holin或sp-holin之番茄對PTI反應相關標誌基 因表現之影響 ...................................................................................... 35 青枯病菌gpsA對番茄青枯病反應之功能研究 圖一、不同毒力之青枯病菌株與GMI1000之SecB與GpsA胺基酸序列比對 ........................................................................................................... 90 圖二、不同毒力之青枯病菌株之secB與gpsA轉譯起始密碼子上游序列比對 ……………………………………………….................................... 91 圖三、gpsA基因組結構分析 ………………..…………………………..….... 92 圖四、gpsA啟動子受關鍵轉錄因子之調控分析 …………………………... 93 圖五、青枯病菌gpsA基因經跳躍子插入後之轉錄表現分析 ……….......... 94 圖六、青枯病菌gpsA-在抗病番茄品系H7996中之增殖情形 ………….…. 95 圖七、青枯病菌gpsA-在不同番茄品系之致病力測試 ………………….…. 96 圖八、中毒力與低毒力青枯病菌之gpsA突變菌株之致病力測試 ………... 97 圖九、青枯病菌gpsA-之營養使用特性 ………………………………….…. 98 圖十、青枯病抗病與感病番茄品系之glycerol、glucose、sucrose及fructose 含量檢測 ………………………………………………………………. 99 圖十一、青枯病抗病與感病番茄品系經青枯病菌感染後之糖類含量分析 ... 101 圖十二、番茄SWEETs之氨基酸序列親緣性分析 …………………………. 103 圖十三、感病番茄品系 L390 SWEET2a基因靜默後之植株生長情形 …..… 圖十四、抗病番茄品系H7996 SWEET2a基因靜默後之植株生長情形 ……. 104 105 附錄目錄 青枯病菌噬菌體裂解蛋白之分析 附表一、本研究所使用之質體與菌株特性 .................................................... 36 附表二、培養基與藥品配方 ........................................................................... 38 附表三、抗生素濃度與配方 ........................................................................... 41 附表四、本研究中所使用之引子 .................................................................... 42 附圖一、短暫表現RsφP29之裂解蛋白之菸草在接種青枯病菌後之反應 … 43 附圖二、短暫表現RsφP29 holin或sp-holin之菸草在接種青枯病菌後之細胞 死亡分析 ............................................................................................. 44 附圖三、短暫大量表現RsφP29 holin或sp-holin之菸草葉片的離子流失檢測 ........................................................................................................ 45 附圖四、短暫表現RsφP29 holin或sp-holin之菸草接種植物病原細菌後之應 ........................................................................................................ 46 附圖五、短暫大量表現RsφP29 holin或sp-holin之番茄接種青枯病菌後之反應 ........................................................................................................ 47 青枯病菌gpsA對番茄青枯病反應之功能研究 附表一、青枯病菌毒力數據 .......................................................................... 106 附表二、本研究團隊定序之青枯病菌菌株特性 .......................................... 107 附表三、本研究所使用之質體與菌株特性 ..................................................... 108 附表四、培養基與藥品配方 ............................................................................. 110 附表五、抗生素濃度與配方 ............................................................................. 114 附表六、本研究中所使用之引子 .................................................................... 115 圖一、青枯病菌Pss190 gpsA突變株之致病力與增殖分析 ………………… 117 圖二、青枯病菌gpsA突變株與基因互補株之致病力與增殖測試 ................. 118 圖三、qRT-PCR檢測青枯病菌gpsA基因在跳躍子插入突變株中之表現量 119 圖四、GpsA與其相關代謝途徑 …………………………………………….. 120 圖五、可被病菌誘導表現之阿拉伯芥SWEETs基因 ………………………. 121 圖六、番茄SWEET1之核苷酸與胺基酸序列比對 ………………………… 112 圖七、番茄SWEET2之核苷酸與胺基酸序列比對 ………………………… 124 圖八、番茄SWEET10之核苷酸與胺基酸序列比對 ………………………. 125 圖九、番茄SWEET17之核苷酸與胺基酸序列 ……………………………. 126 圖十、番茄SWEET11 之核苷酸與胺基酸序列比對 …………………….… 127 圖十一、VIGS番茄SWEET1cd基因靜默構築之比對 ……………………… 129 圖十二、VIGS番茄SWEET10/11/12基因靜默構築之比對 ………………… 130 圖十三、在感病番茄品系L390將SWEET基因靜默後之青枯病測試 …….. 131 圖十四、在感病番茄品系L390 SWEET2a基因靜默後之效率檢測 .……….. 132 | |
| dc.language.iso | zh-TW | |
| dc.title | 噬菌體裂解蛋白與青枯病菌gpsA對番茄青枯病反應之影響 | zh_TW |
| dc.title | Effects of a phage lysis protein and Ralstonia solanacearum gpsA on tomato bacterial wilt response | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林乃君(Nai-Chun Lin),柯淳涵(Chun-Han Ko),王肇芬(Jaw-fen Wang),張英?(Ing-Feng Chang) | |
| dc.subject.keyword | 番茄,青枯病菌,噬菌體,裂解蛋白質,gpsA,糖類,SWEET, | zh_TW |
| dc.subject.keyword | tomato,Ralstonia solanacearum,bacteriophage,lysis protein,gpsA,sugars,SWEET, | en |
| dc.relation.page | 134 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-02-12 | |
| 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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