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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李佳音(Chia-Yin Lee) | |
| dc.contributor.author | Tzu-Ling Lin | en |
| dc.contributor.author | 林慈玲 | zh_TW |
| dc.date.accessioned | 2021-06-16T08:47:43Z | - |
| dc.date.available | 2018-08-28 | |
| dc.date.copyright | 2013-08-28 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-20 | |
| dc.identifier.citation | 潘淑芬 (1999) 腸炎弧菌膠原蛋白酶基因表現及調控之研究. 博士論文, 國立臺灣大學, 台北市.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59063 | - |
| dc.description.abstract | 腸炎弧菌是台灣及亞洲國家重要的食品病原菌，人類常因為生食被汙染的海鮮引起感染導致腹瀉或腸胃炎。本研究證實腸炎弧菌Fur攝鐵調控蛋白對於腸炎弧菌胞外蛋白酶prtA(VPA0227)及prtV(VPA0459)基因的調控機制，是藉由Fur直接結合在此兩個基因的啟動子區域導致prtA及prtV基因表現受到抑制。小片段核醣核酸 RyhB參與細胞中鐵離子平衡之調控，迄今僅在霍亂弧菌和大腸桿菌中研究較透徹，腸炎弧菌目前尚未報導。本研究證實腸炎弧菌RyhB扮演調控基因的角色。以生物資訊學方法分析預測腸炎弧菌RyhB序列，與NCBI 資料庫中弧菌屬及大腸桿菌RyhB序列比對相似性。在ryhB基因的上游區域比對到一段含有19-bp保守序列可能的Fur box，利用電泳遷移率(EMSA)方法證實Fur攝鐵調控蛋白可以結合於該預測之Fur box序列，推測RyhB受到Fur及Fe2+的負向直接調控。同時建構腸炎弧菌的ryhB過量表現菌株，以即時定量聚合酶鏈鎖反應方法證實RyhB可以正向調控基因的表現，包含lafK (sigma54依賴調節因子)、flaD (鞭毛)、fliS (鞭毛蛋白)、flaK (極性鞭毛蛋白)、rpoS (RNA聚合酶-sigma38)、fhuE (ferric coprogen和ferric-rhodotorulic acid的外膜受體)；RyhB亦可以負向調控部分基因的表現，包含sodB (超氧歧化酶)、sdhCDAB (琥珀酸脫氫酶)、iscS (半胱胺酸去硫酶)、cheY (趨化性蛋白)、motB及motA (鞭毛驅動蛋白)。以北方氏雜交法亦證實RyhB可以調控上述基因表現，除了flaD、fliS、flaK 結果訊號微弱較難判讀。本研究結果闡明腸炎弧菌RyhB具有調控趨化性、移動性及與鐵離子相關基因的功能。 | zh_TW |
| dc.description.abstract | Vibrio paraheamolyticus is a major food-borne pathogen in many countries that causes diarrhea through the consumption of raw or undercooked seafood. These results indicated that PrtA (VPA0227) and PrtV (VPA0459) are repressed directly by Fur and iron in V. parahaemolyticus No.93. A small untranslated RNA, RyhB, was discovered in Escherichia coli and Vibrio cholerae that were involved in the regulation of iron metabolism. In this study, we identify and characterize a small RNA RyhB ( gene code VP0105-VP0107 intergenic ) in V. parahaemolyticus No.93 to clarify its regulation roles. Bioinformatics analysis appeared a putative RyhB sequence in V. paraheamolyticus that has the similarity with other small non-coding RNA RyhB in NCBI database. Interestedly, a putative fur box containing 19-bp
conserved sequence was found in the upstream of ryhB gene from V. parahaemolyticus. We used EMSA to demonstrate the interaction of Fur protein and ryhB promoter. We show that RyhB is functional and is down regulated by Fur. The transcription level of twelve RyhB-regulated target genes were examined in RyhB overexpression strain by using RT-PCR and Northern Blot hybridization. LafK (sigma54-dependent regulator), FlaD (flagellin), FlaK (polar flagellar protein), RpoS (RNA polymerase-sigma38) and FhuE (Outer membrane receptor for ferric coprogen and ferric-rhodotorulic acid) were positively regulated by RyhB, while SodB (superoxide dismutase), SdhC (succinate dehydrogenase), IscS (cysteine desulfurase), CheY (chemotaxis vi protein), MotB and MotA (flagellar motor protein) were negatively regulated by RyhB. In this report, we identify and characterize a small RNA RyhB in V. paraheamolyticus and show that it is similar in sequence to that of V. cholerae and is functional to regulate the chemotaxis, motility, metabolism and iron-related genes. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T08:47:43Z (GMT). No. of bitstreams: 1 ntu-102-R00623025-1.pdf: 2925478 bytes, checksum: 86cdb608e3b31b93b8ef623132808633 (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 口試委員會審定書
誌謝 ii 中文摘要 iv 英文摘要 v 目錄 vii 表次 x 圖次 xi 附錄圖次 xii 縮寫表 xiii 壹、前言 1 一、腸炎弧菌(Vibrio parahaemolyticus) 1 1. 概述 1 2. 性狀 2 3. 毒性因子(virulence factors) 3 4. 腸炎弧菌疾病流行情形 4 二、胞外蛋白酶(extracellular protease) 5 1. 概述 5 2. 腸炎弧菌胞外蛋白酶相關研究 5 3. 膠原蛋白酶 6 4. 絲胺酸蛋白酶 7 5. 腸炎弧菌胞外膠原蛋白酶PrtV 7 6. 腸炎弧菌胞外絲胺酸蛋白酶PrtA 8 三、攝鐵機制 9 1. 概述 9 2. 攝鐵調控蛋白 9 3. 攝鐵調控蛋白調控之基因及相關研究 10 4. 弧菌屬的攝鐵調控蛋白 12 四、小片段核糖核酸(small RNA) 13 1. 概述 13 2. 小片段RNA功能和相關研究 14 3. RyhB調控之機制 16 4. RyhB主要調控之範圍和基因 16 5. RyhB於弧菌屬之研究 17 6. RyhB於其他細菌之研究 18 五、研究目的與動機 19 貳、實驗材料與方法 20 I.實驗材料 20 一、實驗菌株、質體與引子 20 二、培養基 20 三、藥品與試劑 21 四、各種溶液及緩衝溶液 21 五、儀器設備 24 六、實驗使用套組 25 II.實驗方法 25 一、DNA技術 25 二、RNA技術 29 三、蛋白質技術 32 四、生物資訊庫軟體分析 34 五、電泳遷移率實驗(Electrophoretic mobility shift assay, EMSA) 35 六、北方墨點法(Northern blot) 37 七、細菌生長曲線之測定 38 八、腸炎弧菌運動能力之測定 39 九、統計分析 39 參、實驗結果 40 一、Fur蛋白質會與prtV、prtA上游Fur box片段結合 40 二、prtA及prtV的轉錄表現會受到Fur蛋白質的負向調控 41 三、Fur攝鐵調控蛋白會抑制prtV的蛋白質表現，但不影響其分泌 41 四、Fur攝鐵調控蛋白會抑制prtA的蛋白質表現，但不影響其分泌 42 五、弧菌屬及大腸桿菌中RyhB 序列相似性的比較 43 六、大腸桿菌、霍亂弧菌及腸炎弧菌No.93 RyhB 的二級結構預測 43 七、RyhB的轉錄表現會受到Fur蛋白質的負向調控 43 八、腸炎弧菌與霍亂弧菌超氧歧化酶相似性之比較 44 九、可能受腸炎弧菌No.93 RyhB調控的目標基因互補序列的配對 44 十、比較WT/p、WT/pryhB菌株及鐵離子對於菌體生長速率及ryhB基因轉錄之影響 45 十一、以qRT-PCR分析受RyhB調控之目標基因的表現 46 十二、以北方墨點法分析各菌株間受RyhB調控之目標基因的表現 47 十三、RyhB的過量表現會抑制腸炎弧菌遊走和泳動能力 48 肆、討論 49 一、Fur調控機制與胞外蛋白酶之關係 49 二、Fur與RyhB調控之關係 50 三、腸炎弧菌可受RyhB調控的目標基因 50 四、在固態培養基中觀察菌體遊走和泳動能力 52 伍、結論 53 陸、參考文獻 54 | |
| dc.language.iso | zh-TW | |
| dc.subject | Vibrio parahaemolyticus | zh_TW |
| dc.subject | RyhB | zh_TW |
| dc.subject | Fur | zh_TW |
| dc.subject | 攝鐵調控蛋白 | en |
| dc.subject | 小核醣核酸 | en |
| dc.subject | 腸炎弧菌 | en |
| dc.title | 腸炎弧菌胞外蛋白酶受Fur調控及RyhB特性分析 | zh_TW |
| dc.title | Regulation of extracellular protease by Fur and characterization of RyhB in Vibrio parahaemolyticus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 彭慧玲(Hwei-Ling Peng),陳建先(chien-hsien chen) | |
| dc.subject.keyword | Fur,RyhB,Vibrio parahaemolyticus, | zh_TW |
| dc.subject.keyword | 攝鐵調控蛋白,小核醣核酸,腸炎弧菌, | en |
| dc.relation.page | 102 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-08-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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