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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李佳音 | zh_TW |
| dc.contributor.advisor | Chia-Yin Lee | en |
| dc.contributor.author | 詹睿安 | zh_TW |
| dc.contributor.author | Jui-An Chan | en |
| dc.date.accessioned | 2021-07-11T15:06:50Z | - |
| dc.date.available | 2024-08-16 | - |
| dc.date.copyright | 2019-08-22 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | Acosta, N., Pukatzki, S., and Raivio, T.L. (2015). The Cpx system regulates virulence gene expression in Vibrio cholerae. Infect Immun 83, 2396-2408.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78605 | - |
| dc.description.abstract | Cpx雙分子調控系統由感應器CpxA組胺酸激酶 (Histidine kinase)，及反應調控因子CpxR共同組成，參與細菌多種生理作用的調控。磷脂醯乙醇胺 (phosphatidylethanolamine, PE) 為腸炎弧菌細胞膜的主要組成分，負責維持細菌細胞膜的功能以及完整性。磷脂醯絲胺酸脫羧酶 (phosphatidylserine decarboxylase, Psd) 是磷脂醯乙醇胺生合成路徑中最末端的重要酵素，將磷脂醯絲胺酸 (phosphatidylserine) 脫羧反應後形成磷脂醯乙醇胺。本研究以腸炎弧菌 (Vibrio parahaemolyticus no. 93, VP93) 作為實驗菌株，建構cpxA及cpxRA刪除突變株與實驗室前人建構之cpxR刪除突變株，測試比較野生株及突變株對於環境壓力的耐受性。實驗結果顯示，CpxR促進腸炎弧菌對抗低溫、極端酸鹼值、高滲透壓及抗氧化壓力反應。接著利用生物資訊分析方法預測psd的啟動子區域，發現在腸炎弧菌psd上游之啟動子區域及其上游基因末端分別具有一段與CpxR保守性結合序列相似之核苷酸序列。在psd的啟動子區域利用ARF-TSS的方法鑑定出psd的轉錄起始點是位於其轉譯起始點上游第48個核苷酸位置。由於本研究室發現VP93之CpxR與psd有負調控相關，本論文繼續針對CpxA及CpxRA進行研究，探討cpxA及cpxRA刪除突變株對於psd的調控情形，並以CpxR蛋白質進行電泳遷移率實驗 (EMSA)，實驗結果顯示CpxR結合於cpxR上游區域，但不結合於cpxP及psd上游區域。EMSA證明psd與CpxR的調控關係並非直接由CpxR結合於psd上游區域而行使負調控關係，而是間接受CpxR的調控。進一步探討感應器CpxA組胺酸激酶的自磷酸化對於psd的調控情形，建構cpxA的自磷酸化H258A點突變株及其回補株。將CpxA自磷酸化位點為胺基酸序列258位置的Histidine (cac) 進行點突變為Alanine (gcc)，結果確認若CpxA無法自磷酸化，會使得下游CpxR無法活化，進而造成下游psd基因無法被抑制而大量表現。總結以上結果，本研究首次證實腸炎弧菌中CpxRA雙分子系統對於環境壓力的反應以及其對於磷脂質代謝途徑的探討。 | zh_TW |
| dc.description.abstract | The CpxRA two component system comprised of the histidine kinase CpxA and the response regulator, CpxR. It serves as a common stimulus response mechanism, thus allows microbes to sense and respond to diverse environments through a series of phosphorylation reactions. Phosphatidylethanolamine (PE), the major type of phospholipid which presents in V. parahaemolyticus maintaining the function and integrity of the bacterial cell membrane. Phosphatidylserine decarboxylase (Psd, EC 4.1.1.65) is an enzyme which catalyze the formation of PE. It plays a central role in the phospholipid metabolism. In this study, we investigated the role of CpxRA two component system in V. parahaemolyticus in response to different environmental stimuli by constructing the ∆cpxA single deletion mutant and ∆cpxRA double deletion mutant from V. parahaemolyticus no. 93. The CpxR was found to play essential roles in mediating tempeterature, pH stress, osmotic pressure and oxidative pressure tolerance. Moreover, we found two nucleotide sequences which similar to the CpxR conserved binding site via bioinformatics. The two nucleotide sequences located at the promoter region of the psd and the end of psd upstream gene. The transcription start site of psd was 48 nucleotides upstream of the translation start site. We have previously reported that ∆cpxR from V. parahaemolyticus no. 93 significantly downregulated the psd. A comparative transcriptomic analysis of VP93 WT, ∆cpxR, ∆cpxA, ∆cpxRA by real-time quantitative PCR and luminescence assay were subsequently employed to understand how Cpx system affects the psd expression. EMSA analysis was used to determine the interaction between cpxRA, cpxP, psd I as well as psd II promoter region and CpxR. The results indicated that the CpxR was able to specificity bind to the cpxRA promoter region. However, no shift band from EMSA results was observed in cpxP, psd I as well as psd II promoter region. These results indicated that psd is indirectly regulated by CpxR. Furthermore, we investigated the role of autophosphorylation of CpxA in regulation of psd by constructing the CpxA H258A point mutation mutant. The results confirmed that CpxA autophosphorylate, and activate the downstream CpxR, resulting in repression of psd gene expression. In this thesis, we firstly analyzed the CpxRA two component system in V. parahaemolyticus and found that CpxR mediating tempeterature, pH stress, osmotic pressure and oxidative pressure tolerance. Furthermore, real-time quantitative PCR and luminescence assay were used and indicated that psd gene expression is downregulated by CpxRA two component system. However, EMSA results indicated that psd is indirectly regulated by CpxR. At last, the autophosphorylatation of CpxA play an important role in CpxRA two component system. It can activate the CpxR, resulting in repression of psd gene expression. Taken together, these findings first characterize the role of the CpxRA two component system in V. parahaemolyticus conferring stress response tolerance and in phospholipid metabolism, which will deepen our understanding in V. parahaemolyticus. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:06:50Z (GMT). No. of bitstreams: 1 ntu-108-R04623023-1.pdf: 4705808 bytes, checksum: ec770bfc36fafddcc13790cd10226f24 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目錄
中文摘要……………………………………………………………………….i 英文摘要……………………………………………………………………….ii 目錄…. iv 附錄圖次 xii 縮寫表.. xiii 前言…. 1 一、 腸炎弧菌 (Vibrio parahaemolyticus) 1 1. 生理特性及分類 1 2. 分布及感染途徑 1 3. 致病因子 2 4. 腸炎弧菌引起之疾病 2 二、 Cpx雙分子調控系統 (Two component regulatory system) 3 1. 雙分子調控系統 3 2. Cpx雙分子調控系統 4 3. Cpx雙分子調控系統與輔助調節蛋白CpxP及NlpE 5 4. Cpx雙分子系統在大腸桿菌中的調控作用 6 5. Cpx雙分子系統在杜氏嗜血桿菌中的調控作用 7 6. Cpx雙分子系統在霍亂弧菌中的調控作用 7 三、 磷脂醯乙醇胺 (phosphatidylethanolamine, PE) 8 1. 原核的磷脂醯乙醇胺合成途徑 8 2. 酵母菌、植物、哺乳動物的磷脂醯乙醇胺合成途徑 10 3. 磷脂醯乙醇胺影響大腸桿菌的附著力 10 4. 磷脂醯乙醇胺影響自噬作用及延緩老化 11 5. 酵母菌中磷脂醯乙醇胺調控路徑的競爭抑制 12 四、 磷脂醯絲胺酸 (Phosphatidylserine, PS) 與磷脂醯絲胺酸合成酶 (Phosphatidylserine systhase, Pss) 13 1. 磷脂醯絲胺酸 13 2. 磷脂醯絲胺酸合成酶 13 五、 磷脂醯絲胺酸脫羧酶 (Phosphatidylserine decarboxylase, Psd) 14 1. 磷脂醯絲胺酸脫羧酶 14 2. 細菌的磷脂醯絲胺酸脫羧酶 15 3. 酵母菌的磷脂醯絲胺酸脫羧酶 16 4. 瘧原蟲的磷脂醯絲胺酸脫羧酶 17 5. 植物的磷脂醯絲胺酸脫羧酶 17 六、 研究目的 18 貳、 實驗材料與方法 20 一、 菌株、質體、引子 20 二、 培養條件 20 三、 藥品與試劑 20 四、 溶液與緩衝溶劑 21 五、 儀器設備 23 六、 實驗使用套組 24 七、 DNA技術 25 八、 RNA技術 29 九、 蛋白質技術 30 十、 建構基因缺損突變株 32 十一、 建構點突變株 34 十二、 建構腸炎弧菌cpxA回補株 34 十三、 生長曲線測定方法 36 十四、 環境壓力測試 36 十五、 LuxAB螢光活性分析 37 十六、 ARF-TSS分析轉錄起始點 (Adaptor and radioactivity free identification of TSS) 38 十七、 電泳遷移率實驗 (Electrophoretic mobility shift assay, EMSA) 38 十八、 生物資訊分析 41 十九、 統計分析 41 參、 實驗結果 42 一、建構腸炎弧菌no. 93之∆cpxA、∆cpxRA突變株 42 二、腸炎弧菌no. 93野生株與cpx突變株於不同條件下 (滲透壓、溫度、pH值及氧化壓力測試) 之生長情形 42 三、腸炎弧菌no. 93之psd啟動子序列以及轉錄起始點分析 43 四、以qRT-PCR分析腸炎弧菌CpxR及CpxA抑制cpxP、psd表現 44 五、以LuxAB報導基因系統確認腸炎弧菌野生株cpxP及psd的啟動子具有表現…………. 44 六、LuxAB分析腸炎弧菌cpxR、cpxA基因對cpxP及psd的啟動子活性的影響…………. 45 七、 CpxR的表現及其純化 45 八、凝膠電泳遷移率實驗分析CpxR對cpxP、cpxRA、psd基因表現的影響…………. 46 九、建構CpxAH258A點突變株 46 十、qRT-PCR分析腸炎弧菌點突變株∆cpxA*H258A之cpxP及psd基因表現…………. 46 肆、 討論 48 伍、 結論 51 陸、 未來展望 52 柒、 參考文獻 53 表次 表一、本研究中所使用之菌株 Table 1. Strains used in this study. 63 表二、本研究中所使用之質體 Table 2. Plasmids used in this study. 66 表三、本研究中所使用之引子 Table 3. Primers used in this study. 67 表四、腸炎弧菌No. 93野生株與突變株於不同條件下 (滲透壓、溫度、pH值及氧化壓力測試) 之生長情形……………………………………………………70 表五、腸炎弧菌及大腸桿菌之CpxR保守結合位…………………………71 圖次 圖一AB、腸炎弧菌cpxA鄰近基因與引子位置，並利用同源重組建構∆cpxA突變株之示意圖 72 圖一CD、經由基因定序結果確認∆cpxA突變株之序列 73 圖三A、分析VP93 psd基因轉錄起始點及預測psd基因之啟動子、-10 box、-35 box及保守性CpxR結合區 75 圖三BC、ARF-TSS分析psd轉錄起始點示意圖及定序結果確認psd之轉錄起始點…….. 76 圖四、腸炎弧菌野生株及其突變株之生長曲線 77 圖五、qRT-PCR分析腸炎弧菌野生株cpxP、psd、cpxR及cpxA基因表現時間………… 78 圖六、qRT-PCR分析腸炎弧菌野生株及其突變株之cpxP、psd基因表現……………… 79 圖七、qRT-PCR分析腸炎弧菌野生株、突變株及其回補株之cpxP、psd基因表現…….. 80 圖八、以LuxAB報導基因系統確認腸炎弧菌野生株cpxP及psd的啟動子具有表現……. 81 圖九、LuxAB分析腸炎弧菌cpxP及psd的啟動子活性在野生株及其突變株受調控情形…. 82 圖十、限制酵素截切確認pET28a-cpxR 83 圖十一、His6-CpxR小量表現結果 83 圖十二、凝膠電泳位移CpxR與(A) cpxRA (B) cpxP (C) psdI (D) psdII啟動子的交互作用…. 84 圖十三、建構點突變株以∆cpxA*H258A為示意圖 (A) 及經由基因定序結果(B) 確認∆cpxA*H258A點突變株之序列 85 圖十四、腸炎弧菌點突變株之生長曲線 86 圖十五、qRT-PCR分析腸炎弧菌點突變株之cpxP、psd基因表現 …….87 圖十六、腸炎弧菌中Cpx系統調控機制…………………………………...88 附錄圖次 附錄圖一、原核細菌之磷脂質生合成途徑…………...……………………89 附錄圖二、大腸桿菌Psd之丙酮醯基形成途徑……………………………90 附錄圖三、腸炎弧菌cpxR 鄰近基因……………………………….………91 附錄圖四、腸炎弧菌psd 鄰近基因………………………………...………92 附錄圖五、大腸桿菌cpxR 鄰近基因……………………………….………93 附錄圖六、大腸桿菌psd 鄰近基因……………………………...………….94 | - |
| dc.language.iso | zh_TW | - |
| dc.title | 腸炎弧菌Cpx雙分子系統與調控 磷脂醯絲胺酸脫羧酶基因之研究 | zh_TW |
| dc.title | The Cpx two component system and regulation of phosphatidylserine decarboxylase by Cpx in Vibrio parahaemolyticus | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 謝陸盛;廖淑貞 | zh_TW |
| dc.contributor.oralexamcommittee | Lu-Sheng Hsieh;Shwu-Jen Liaw | en |
| dc.subject.keyword | 腸炎弧菌,Cpx系統,磷脂醯絲胺酸脫羧?,基因調控,磷酸化, | zh_TW |
| dc.subject.keyword | V. parahaemolyticus,CpxRA two component system,phosphatidylserine decarboxylase,phosphatidylethanolamine,gene regulation, | en |
| dc.relation.page | 94 | - |
| dc.identifier.doi | 10.6342/NTU201903547 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-14 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 農業化學系 | - |
| dc.date.embargo-lift | 2024-08-22 | - |
| 顯示於系所單位： | 農業化學系 | |
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