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研究生:溫孟芸
研究生(外文):WEN, MENG-YUN
論文名稱:鮑氏不動桿菌中ZraS和GacA之間的訊息傳遞
論文名稱(外文):Signal transduction between ZraS and GacA in Acinetobacter baumannii
指導教授:林光慧
指導教授(外文):LIN, GUANG-HUEY
口試委員:林光慧陳怡原蘇淑惠
口試委員(外文):LIN, GUANG-HUEYCHEN, YI-YUANSU, SHU-HUI
口試日期:2019-10-04
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學系微生物及免疫學碩士班
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2019
畢業學年度:108
語文別:中文
論文頁數:129
中文關鍵詞:鮑氏不動桿菌雙分子調控系統訊息傳遞
外文關鍵詞:Acinetobacter baumanniiTwo component systemSignal transduction
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Abstract 1
中文摘要 2
前言 3
鮑氏不動桿菌 3
雙分子調控系統 4
鮑氏不動桿菌的雙分子調控系統 4
研究源起與目的 7
材料與方法 8
一、材料 8
1. 質體、菌種以及引子 8
2. 大蠟蛾 (Galleria mellonella)生長環境以及成長史 8
3. 藥品及試劑 8
4. 儀器與器材 8
二、方法 9
1. Luria-Bertani (LB)培養基製備 9
2. 純化染色體 (Chromosome extraction) 9
3. DNA聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 10
4. 純化質體 (Plasmid extraction) 10
5. 限制酶作用 (Enzyme digestion) 11
6. DNA黏合作用 (Ligation) 11
7. Gibson assembly 11
8. 勝任細胞(Competent cell)製備 12
9. 熱休克轉形作用(Heat-shock transformation ) 12
10. 質體快速篩選 (Quick screening) 12
11. 接合生殖 (Conjugation) 13
12. 無標記基因突變法 (Marker-less mutagenesis) 13
13. 菌落聚合酶鏈鎖反應 (Colony PCR) 14
14. Phos-tagTM蛋白質樣本製備 14
15. Phos-tagTM蛋白質膠體電泳 (Phos-tag™ acrylamide gel analysis) 15
16. 西方墨點法 15
17. 鮑氏不動桿菌生長曲線 16
18. 最小抑菌濃度測試 (Minimal inhibitory concentration test) 17
19. 紙錠擴散測試 (Disk diffusion test) 17
20. 生物膜形成 (Biofilm formation) 18
21. 鮑氏不動桿菌感染大蠟蛾測試 18
22. RNA萃取 (RNA extraction) 19
23. 反轉錄作用 (Reverse transcription, RT) 19
24. 即時定量聚合酶鏈鎖反應 (Quantitative real-time PCR, qRT-PCR) 20
25. 轉錄體分析的樣本置備 20
結果 21
一、鮑氏不動桿菌的DJ41_1406以及DJ41_1407 21
1. DJ41_1406是雙分子調控系統調控蛋白質,命名為GacA 21
2. GacA蛋白質功能區域分析 21
3. DJ41_1407是雙分子調控系統感應蛋白質,命名為ZraS 22
4. ZraS蛋白質功能區域分析 22
5. gacA與zraS位於同一條mRNA 22
6. 鮑氏不動桿菌中的基因排列 23
二、ZraS/GacA在鮑氏不動桿菌的基礎代謝以及對抗環境壓力扮演重要角
色 24
1. 建構ΔzraS、ΔgacA以及ΔzraSΔgacA菌株 24
2. GacA影響鮑氏不動桿菌的生長 24
3. GacA影響鮑氏不動桿菌的醇類代謝 24
4. GacA影響鮑氏不動桿菌抵抗抗生素的能力 25
5. GacA影響鮑氏不動桿菌抵抗氧化壓力 26
6. GacA影響鮑氏不動桿菌生物膜形成 27
7. GacA影響鮑氏不動桿菌毒力 27
三、ZraS/GacA為一對雙分子調控系統,磷酸化GacA活化ZraS/GacA基因
轉錄 28
1. 在環境壓力或者營養源缺乏的環境下ZraS磷酸化GacA 28
2. 磷酸化GacA活化gacA和zraS基因表現 30
四、野生株、ΔzraS以及ΔgacA的轉錄體 (Transcriptome)分析 30
1. ΔzraS以及ΔgacA轉錄體的基因功能 (Gene ontology, GO)分類 30
2. GacA調控細菌的醇類、IAA以及胺基酸代謝 31
3. GacA活化醇類代謝、IAA代謝以及苯乙酸代謝的基因表現 33
五、建構pACYC184/TCSR報導基因系統 34
討論 35
一、如何研究雙分子調控系統 35
二、ZraS/GacA並非典型雙分子調控系統,GacA能夠接收來自不同感應蛋白
質的磷酸根 36
1. 在其他細菌中GacA如何調控下游基因 37
2. ZraS在其他細菌中的調控機制 38
3. 其他細菌中雙分子調控系統的交互訊息傳遞(cross-talk) 39
三、ZraS/GacA調控鮑氏不動桿菌的胺基酸、醇類以及IAA代謝 40
表一、本篇論文使用的質體 42
表二、本篇論文使用的菌種 43
表三、本篇論文使用的引子 45
表四、ZraS正調控的基因列表 48
表五、ZraS負調控的基因列表 49
表六、GacA正調控的基因列表 50
表七、GacA負調控的基因列表 59
圖一、雙分子調控系統調控蛋白質DJ41_1406演化樹 63
圖二、GacA蛋白質功能區域 64
圖三、雙分子調控系統感應蛋白質DJ41_1407演化樹 65
圖四、ZraS蛋白質功能區域 67
圖五、確認gacA與zraS位於同一條mRNA 68
圖六、鮑氏不動桿菌、克雷伯氏肺炎菌以及小束噬脯氨酸菌的基因排列示意
圖 69
圖七、確認ΔzraS、ΔgacA以及ΔgacAΔzraS菌株 70
圖八、鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA培養在LB培養
液中的生長曲線 72
圖九、鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA培養在以1%乙
醇為單一碳源的生長曲線 73
圖十、鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA培養在以1%
1-丙醇為單一碳源的生長曲線 74
圖十一、鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA培養在以
0.5%1-丁醇為單一碳源的生長曲線 75
圖十二、即時定量聚合酶鏈鎖反應分析野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA
在含有1%乙醇的LB培養液中adh4基因表現量 76
圖十三、鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA培養在不同
抗生素中的最小抑菌濃度 77
圖十四、即時定量聚合酶鏈鎖反應分析鮑氏不動桿菌野生株在含有抗生素的環
境中gacA以及zraS的基因表現量 78
圖十五、紙錠擴散測試鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA
對於H2O2的抵抗能力 79
圖十六、紙錠擴散測試鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA
對於tBHP的抵抗能力 80
圖十七、即時定量聚合酶鏈鎖反應分析鮑氏不動桿菌野生株、ΔzraS、ΔgacA
以及ΔzraSΔgacA中ohr基因表現量 81
圖十八、測試鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA的生物
模形成能力 82
圖十九、鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA感染後大蠟
蛾幼蟲的存活率 83
圖二十、鮑氏不動桿菌野生株、ΔzraS、ΔgacA以及ΔzraSΔgacA感染後大蠟
蛾幼蟲的黑化程度 84
圖二十一、使用phos-tag™西方墨點法分析GacA磷酸化情形 85
圖二十二、使用phos-tag™西方墨點法分析磷酸化以及未磷酸化GacA 86
圖二十三、即時定量聚合酶鏈鎖反應分析野生株以及ΔzraS在不同培養狀況下
gacA基因表現量 87
圖二十四、以Gene ontology將野生株和ΔzraS轉錄體分析中基因表現量不同
的基因分類 88
圖二十五、以Gene ontology將野生株和ΔgacA轉錄體分析中基因表現量不同
的基因分類 90
圖二十六、野生株和ΔgacA的轉錄體分析中基因表現量差異大於2倍之分析
圖 92
圖二十七、野生株和ΔgacA的轉錄體分析結果中基因叢 DJ41_2041-DJ4_2054
的基因表現量差異分析圖 93
圖二十八、野生株和ΔgacA的轉錄體分析結果中基因叢 DJ41_332-DJ4_338、
DJ41_2892-DJ4_2896以及DJ41_3071-DJ4_3075的基因表現量差異分析圖
94
圖二十九、即時定量聚合酶鏈鎖反應分析野生株、ΔzraS、ΔgacA以及
ΔzraSΔgacA中adh4、ipdC、iacH以及paa基因表現量 95
圖三十、預測GacA可能結合的啟動子區域 96
圖三十一、雙分子調控系統ZraS/GacA調控的細菌基礎代謝路徑圖 97
圖三十二、pACYC184/TCSR報導基因系統示意圖 99
圖三十三、建構pACYC184/TCSR示意圖 100
圖三十四、建構pACYC184/TCSR確認圖 101
附錄一、pK18mobsacB示意圖 102
附錄二、pACYC184示意圖 103
參考文獻 104

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