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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭秋萍(Chiu-Ping Cheng) | |
dc.contributor.author | Wen-Chien Yang | en |
dc.contributor.author | 楊文潔 | zh_TW |
dc.date.accessioned | 2021-05-20T20:09:14Z | - |
dc.date.available | 2012-07-31 | |
dc.date.available | 2021-05-20T20:09:14Z | - |
dc.date.copyright | 2009-07-31 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-30 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9100 | - |
dc.description.abstract | 由Ralstonia solanacearum所引起的青枯病 (又稱為細菌性萎凋病) 係一全球重要之土壤傳播性維管束病害,但目前我們對於這個極為複雜且破壞力極強的病菌的了解仍是十分有限。先前的研究已經篩選到致病力缺失之青枯病菌突變株群,在其中兩個突變株中,跳躍子插入的基因分別為RSc0411與RSc1956,本論文之研究目標為深入探討其之確切功能。首先,LptC係一廣泛存在細菌但功能未知的DUF1239蛋白家族之一員,目前研究推測在大腸桿菌中此蛋白可能參與脂多醣(lipoplysaccharides或LPS)之生合成與運送。RSc0411之預期蛋白產物係DUF1239成員。由目前研究之結果發現,青枯病菌RSc0411突變株之粗型脂多醣(R-LPS)合成有缺失,其致病能力嚴重喪失,且游動力、生物膜(biofilm)形成能力及對番茄根部的附著力皆有明顯下降,且無法誘導菸草產生過敏反應(hypersensitive response),而進一步的結果也驗證RSc0411突變後造成青枯病菌第三型分泌系統無法被正常誘導。此外,雖然在許多革蘭氏陰性細菌中lptC相關的基因成員組合具保守性,啟動子分析結果顯示在青枯病菌中之實際操縱子組成(operon organization)與大腸桿菌並不同,且將其他革蘭氏陰性菌的DUF1239蛋白基因互補到RSc0411突變株後發現,只有親緣近序列相似度高的同源基因可以功能互補RSc0411之缺失,推測可能是因為與Ralstonia屬親緣相近之細菌的DUF1239蛋白質已經演化出具較類似之特別且新穎的功能,同時參與細菌R-LPS之生合成和第三型分泌系統。本論文第二部份的工作係研究RSc1956 (murI)之功能,其預期蛋白產物係glutamate racemase,負責將L-glutamate轉化為D-glutamate,而D-glutamate是組成細菌細胞壁肽聚醣的重要成分,可以保護細菌的細胞壁免受細胞的蛋白質酶攻擊。由目前之研究結果發現,murI突變雖然不會明顯影響生物膜形成、根部附著力和分解植物細胞壁酵素之分泌,但是會造成致病能力嚴重喪失,且游動力明顯下降,而互補試驗亦進一步證實此基因確實為青枯病之關鍵致病基因,且瞭解到其基因啟動子對於有效地表現此基因之功能十分重要。藉由這些分子層面的試驗與分析,本研究得以確認並深入探討新穎蛋白RSc0411與RSc1956確實是青枯病菌之關鍵致病基因,且對於其相關分子機制有所領悟;希望藉由深入且全面性地了解青枯病菌的致病機制、其主導基因、各訊息傳導途徑間之交互作用與調控,未來可以研擬破壞病菌感染的管道,得以研發有效控制此嚴重病害的可能策略。 | zh_TW |
dc.description.abstract | Ralstonia solanacearum is a soil-borne bacterium infecting vascular system, causing lethal wilting symptoms on many economically important crops and resulting severe crop losses. Previously, we carried out transposon (Tn5) insertional mutant screens to identify genes involved in pathogenesis of R. solanacearum. Two mutants containing a transposon insertion in RSc0411 or RSc1956 (murI) were found to be avirulent both on tomato and Arabidopsis. The aims of this study are to further elucidate roles of these genes in bacterial pathogenesis and the molecular mechanisms involved. Firstly, R. solanacearum RSc0411 is a homologue of E. coli lptC. LptC, a member of the novel protein family DUF1239, is suggested to be involved in E. coli lipopolysaccharides (LPS) transport into the outer membrane. Here we showed that RSc0411 mutant was defective in cell integrity and in rough LPS production. Notably, this mutant displayed defects in various pathogenesis-related properties and the induction of the type III secretion system was attenuated. The organization of DUF1239-related gene cluster is conserved among gram-negative bacteria, while sequence homology among orthologous genes in the cluster and those involved in LPS biogenesis varied according to phylogenetic relationships. Complementation tests revealed that only DUF1239 members in bacteria phylogenetically related to R. solanacearum were functional to rescue the mutant’s defects, further suggesting certain specificity in the RSc0411-involved pathogenesis mechanism and LPS biogenesis machineries may have evolved. Collectively these results imply a novel and crucial role of RSc0411 in early pathogenesis and LPS biogenesis of R. solanacearum. Secondly, R. solanacearum RSc1956 encodes glutamate racemase (MurI) protein. MurI catalyzes the conversion of L-glutamate to D-glutamate which is an essential component of peptidoglycans in bacterial cell walls. Here we showed that, although RSc1956 mutant behaved similar to the wild-type strain in biofilm formation, root attachment and production of plant cell wall degrading enzymes, it was defective in swimming motility and avirulent in tomato plants. Complementation tests further confirmed the key role of RSc1956 in R. solanacearum pathogenesis and revealed the importance of its promoter in precisely expressing MurI function. Taken together, these detailed studies on RSc0411 and RSc1956 are expected to pave the way not only for elucidating mechanisms and determinants involved in R. solanacearum pathogenesis but also potentially establishing useful disease control means. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T20:09:14Z (GMT). No. of bitstreams: 1 ntu-98-R96b42017-1.pdf: 2655604 bytes, checksum: 5fc7c6a56d9b1028b4b814260b8dc47b (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 目次
口試委員會審定書 i 致謝 ii 中文摘要 iii 英文摘要 v 常用名詞之縮寫與全名對照表 vii 目 次 viii 表目次 xiii 圖目次 xiv 附錄目次 xvi 第一章 前言 1 Ⅰ. 植物病原菌的致病策略 1 Ⅱ. 植物病原菌的第三型分泌系統 1 Ⅲ. 青枯病菌 (細菌萎凋病) 3 Ⅳ. 青枯病菌之致病機制 4 1.胞外多醣體 (exopolysaccharides, EPS) 4 2.游動力、趨化性及趨氧性 5 3.分解植物細胞壁的酵素 (plant cell wall degrading enzymes, CWDEs) 5 4.螯鐵蛋白 (siderophores) 6 5.第三型分泌系統 6 6.細菌脂多醣 (lipopolysaccharides或簡稱LPSs) 7 7. PhcA調控青枯病菌致病網 7 Ⅴ. LPS生合成與運送機制及DUF1239相關研究 8 Ⅵ. murI基因的相關研究 9 Ⅶ 研究目標 11 第二章 實驗材料與方法 13 1.萃取青枯病菌genomic DNA 13 2.製備plasmid DNA 13 3.選殖技術與程序 (Cloning and transformation in E. coli) 14 3-1.聚合酶連鎖反應 (polymerase chain reaction, PCR) 14 3-2.DNA純化 15 3-3.限制酶 (restriction enzyme) 的消化水解 (digestion) 15 3-4.載體與基因之接合 (ligation) 15 3-5.大腸桿菌勝任細胞的轉型作用 15 4. 構築質體 15 4-1.構築互補 15 4-2.構築 lacZ transcriptional fusions 16 5. 青枯病菌勝任細胞的製備與轉型作用 16 5-1.青枯病菌勝任細胞的製備 16 5-2.青枯病菌勝任細胞的轉型作用 16 6. 製備 allelic mutants 17 7. RNA表現的測定 17 7-1.萃取青枯病菌的RNA 17 7-2.甲醛電泳膠體製備 18 7-3.反轉錄聚合酶連鎖反應 (RT-PCR) 18 7-4.即時定量RT-RCR 18 8. 生理特性分析 18 8-1.生長曲線 18 8-2.點盤分析 19 8-3.青枯病菌在植物體內增生能力分析 19 8-4.逆境反應分析 19 8-5.細菌碳源、氮源 (BiologTM) 和API-Zym酵素的分析 19 8-6.穿透式電子顯微鏡 20 9. 青枯病菌致病力相關能力之分析 20 9-1.游動能力的測試 20 9-2.番茄根部的附著力測試 20 9-3.生物膜形成能力之分析 21 9-4.胞外酵素 (Exoenzyme activity) 的分析 21 9-5.菸草的反應測試 21 9-6.番茄病原性測試 22 10. 青枯病菌脂多醣的測定 22 10-1.青枯病菌脂多醣的萃取 22 10-2.一維電泳 (SDS-PAGE) 的製備 23 10-3.青枯病菌脂多醣銀染分析 23 11. β-galactosidase activity assay 23 第三章 結果 25 Ⅰ. RSc0411基因之功能分析 25 1. 生物資訊分析 (bioinformatics) 25 2. RSc0411突變株之生理與生化特性分析 25 2-1.生長曲線 25 2-2.逆境反應分析 25 2-3.API-Zym酵素的分析 26 2-4.細胞膜形態的觀察 26 3. RSc0411突變株致病力相關能力之分析 26 3-1.細菌游動能力的測試 26 3-2.番茄根部的附著力測試 26 3-3.生物膜形成能力之分析 27 3-4.胞外酵素的分析 27 3-5.菸草的反應測試 27 3-6.番茄病原性測試 27 4. 驗證RSc0411基因功能 28 4-1.構築isogenic突變株 28 4-2.互補株的構築與分析 28 5. 判定青枯病菌RSc0409~RSc0413 之操縱子 (operon) 結構 28 6. 青枯病菌脂多醣的測定 29 7. 以比較生物資訊學探討DUF1239基因群及協助LPS運送相關蛋白群 29 8. 同源基因互補株的構築與分析 30 9. RSc0411參與第三型分泌系統 31 10. 青枯病菌其他突變株的型態與致病能力分析 32 Ⅱ. murI基因之功能分析 33 1. murI突變株之生理與生化特性分析 33 1-1.生長曲線 33 1-2.逆境反應分析 33 1-3.API-Zym酵素與細菌碳、氮源 (BiologTM) 的的分析 33 1-4.細胞膜形態的觀察 33 2. murI突變株致病力相關能力之分析 33 2-1.青枯病菌早期致病因子之分析 33 2-2.菸草的反應測試 34 2-3.番茄病原性測試 34 3. 驗證murI基因功能 34 3-1.構築isogenic 突變株 34 3-2.互補株的構築與分析 35 第四章 討論 36 I. RSc0411基因之功能探討 36 1.DUF1239蛋白是否為生長必需蛋白 36 2.RSc0411參與LPS生合成 36 3.青枯病菌脂多醣對致病力相關因子之影響 37 4.RSc0411參與青枯病菌第三型分泌系統之調控 39 5.Rastonia親緣相近之DUF1239家族新穎功能探討 40 6.青枯病菌第三型分泌系統與其它致病因子的關係 42 7.青枯病菌與菸草之交互作用 42 Ⅱ. murI基因之功能探討 44 1.MurI蛋白是否為生長必需蛋白 44 2.青枯病菌MurI在細胞型態與生理特性之功能 45 3.青枯病菌MurI影響致病力分析 46 4. murI為青枯病菌之關鍵致病基因 47 參考文獻 49 表目次 表一、青枯病菌RSc0411研究所使用的菌株與質體 59 表二、青枯病菌野生株與RSc0411及rfaF突變株的特性比較 62 表三、參與脂多醣生合成相關基因之序列比較 64 表四、青枯病菌murI研究所使用的菌株與質體 68 表五、青枯病菌野生株與murI突變株的特性比較總表 69 圖目次 I、RSc0411基因之功能分析 71 圖一、青枯病菌野生型菌株Pss190和其RSc0411突變株的生長曲線 71 圖二、青枯病菌各個菌株在不同固體培養基下的生長情形 72 圖三、野生型菌株和RSc0411突變株在菸草N. benthamiana內的增生 73 圖四、穿透式電子顯微鏡觀察青枯病菌野生型菌株和RSc0411突變株細胞膜 型態 74 圖五、青枯病菌野生型菌株和RSc0411突變株游動力 (swimming) 測試 75 圖六、青枯病菌野生型菌株和RSc0411突變株游動力 (twitching) 測試 76 圖七、青枯病菌野生型菌株和其各式互補株對番茄根部著附著力分析 77 圖八、青枯病菌野生型菌株、RSc0411突變株和互補株生物膜形成能力之 分析 78 圖九、青枯病菌野生型菌株、RSc0411突變株和互補株對菸草的反應分析 79 圖十、青枯病菌野生型菌株、RSc0411突變株及互補株番茄病原性測試 80 圖十一、青枯病菌RSc0409~RSc0413之操縱子 (operon) 結構 81 圖十二、青枯病菌RSc0411與大腸桿菌LptC胺基酸序列比對 82 圖十三、利用銀染分析各式青枯病菌株之脂多醣生合成 83 圖十四、比較各細菌中DUF1239相關基因群之操縱子結構及基因成員 84 圖十五、青枯病菌野生株與RSc0411突變株第三型分泌系統相關基因表現量 的測定 85 圖十六、RSc0411啟動子在不同菌株中的表現分析 86 圖十七、青枯病菌野生株、RSc0411突變株及其他膜完整性缺失突變株特性 分析 87 Ⅱ、murI基因之功能分析 88 圖十八、青枯病菌野生型菌株和murI突變株生長速度比較 88 圖十九、青枯病菌野生菌株和murI突變株在不同固體培養基下的生長情形 89 圖二十、青枯病菌野生型菌株和其murI突變株碳源 (BiologTM) 利用分析 90 圖二十一、穿透式電子顯微鏡觀察青枯病菌野生菌株和murI突變株細胞態 91 圖二十二、青枯病菌野生型菌株和murI突變株游動能力的測試 92 圖二十三、青枯病菌野生型菌株和murI突變株游動力 (twitching) 測試 93 圖二十四、青枯病菌野生型菌株和murI突變株番茄根部附著力與生物膜形 成能力分析 94 圖二十五、青枯病菌野生菌株和murI突變株於菸草的反應 95 圖二十六、青枯病菌野生菌株和murI突變株番茄病原性測試 96 圖二十七、青枯病菌野生菌株、murI突變株和其互補株游動力 (swimming) 測試 97 附錄目次 附錄一、青枯病菌RSc0411研究所使用之引子 98 附錄二、青枯病菌murI研究所使用之引子 101 附錄三、利用生物資訊軟體分析DUF1239蛋白家族 102 附錄四、RSc0411膜蛋白的預測 103 附錄五、大腸桿菌LPS的合成與運送及LPS運送突變株之表現型態分析 104 附錄六、青枯病菌複雜的致病調控系統 105 附錄七、實驗室前人測試野生型菌株Pss190及其其突變株在番茄及阿拉伯芥 病原性 106 附錄八、實驗室前人在Pss190背景下互補murI突變株的病原性分析 107 附錄九、本研究Pss4背景下murI突變株進行不同長度的互補實驗 108 附錄十、肽聚醣生合成路徑圖 109 附錄十一、生物體內glutamate的代謝路徑 110 附錄十二、常用培養基配方 111 | |
dc.language.iso | zh-TW | |
dc.title | 植物青枯病菌致病基因RSc0411及murI之功能性研究 | zh_TW |
dc.title | Functional studies of Ralstonia solanacearum novel pathogenicity determinants RSc0411 and murI | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 翁淑芬(Shu-Fen Weng),王肇芬(jaw-fen wang),賴爾?(Erh-Min Lai) | |
dc.subject.keyword | 青枯病菌,脂多醣,DUF1239蛋白,LptC,第三型分泌系統,MurI, | zh_TW |
dc.subject.keyword | Ralstonia solanacearum,DUF1239,LptC,lipopolysaccharide,type III secretion system,MurI, | en |
dc.relation.page | 116 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2009-07-30 | |
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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