博碩士論文 103821009 詳細資訊




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姓名 羅凱泓(Kai-Hong Luo)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 Pseudomonas sp. A46全基因組分析與重金屬復育基因工程菌開發
(Genomic Characterization of Pseudomonas sp. A46 and Establishment of Genetic Bacteria for Enhancing Bioremediation of Heavy Metals)
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摘要(中) 汞是一種自然存在的元素,常溫下唯一的液態金屬,帶有強烈的生物毒性。環境中有許多細菌具有汞抗性,能抵抗汞汙染環境並將高毒性型態汞轉化成低毒性型態。抗汞菌株Pseudomonas sp. A46能培養於含60 ppm Hg2+ LB培養基中,並於24小時內移除培養基中89.24%中汞離子,推測可為汞汙染生物復育之優勢菌株。本論文利用次世代定序技術分析Pseudomonas sp. A46基因體組成,分析結果顯示除mer operon外可針對汞進行生物復育外,菌株還帶有鎘抗性基因czcABC、鉻抗性與還原基因chr operon、銅抗性和氧化基因copB與cueO、鉛抗性基因ZntA與砷抗性和還原基因acr3與arsC,因此此菌株除汞外可能帶有上述重金屬之抗性與生物復育潛力可供外來進一步研究。有研究指出重金屬會對細胞造成氧化壓力,qPCR分析結果顯示菌株培養在含汞環境下,除汞操作組外其抗氧化相關基因如SOD、GPx、CAT等表現量皆有上升。由於利用汞操作組還原揮發汞,經過汞循環路徑,最終仍可能於其它地區沉降,無法完全解決汞汙染問題,本論文參考前人文獻在Pseudomonas sp. A46菌株中表現能結合重金屬之金屬硫蛋白,將重金屬固定於菌株內以便後續回收,此外為解決蛋白質表現問題,本論文開發Pseudomonas sp. A46用pJBR持續表現型蛋白質表現系統,並將綠螢光蛋白與金屬硫蛋白融合,使金屬硫蛋白能於菌株中穩定表現並能簡易觀察蛋白質表現量,汞固定實驗顯示轉殖金屬硫蛋白之Pseudomonas sp. A46菌體內汞濃度較野生型高3.7倍,顯示利用金屬硫蛋白增加汞吸收量是可行的。
摘要(英) Mercury is a nature liquid element which has strong toxicity. In the environment, many mercury-resistant bacteria can transform the high-toxic state mercury into low-toxic state mercury. One of the mercury-resistant bacteria, Pseudomonas sp. A46, is able to growth in the LB medium with 60ppm mercuric ion, to remove 89.24% mercuric ion from the medium in 24 hours. We suggested that P. spp A46 can be the potential strain for mercury-bioremediation. Using the Next Generation Sequencing (NGS) technique to analyze whole genome of P. spp A46, our results indicated that the strain not only carries mer operon for mercury-bioremediation but also cadmium resistant gene czcABC、chromium resistant and reduction gene chr operon、copper resistant and oxidation gene copB and cueO、lead resistant gene zntA and arsenic resistant and reduction gene acr3 and arsC. As mercury was reported to induce the oxidative stresss, our results also confirmed that the expression levels of superoxide dismutase, glutathione peroxidase and catalase were increased under mercury stress. Although mercury was potentially removed by Pseudomonas sp. A46, part of mercury could be vaporized into air. To avoid the vaporization of mercury when bioremediation was operating, we developed one genetic bacteria harbouring metallothionein, a heavy metal-binding protein. Our results found out that the gentic bacteria, P. spp A46, could bind to mercury with 3.7 fold concentration of mercury than wild type strain.
關鍵字(中) ★ 汞
★ 重金屬
★ 生物復育
★ 次世代定序
★ 金屬硫蛋白
★ 基因工程菌
關鍵字(英) ★ mercury
★ heavy metal
★ bioremediation
★ next generation sequencing
★ metallothionein
★ genetic bacteria
論文目次 致謝………………………………………………………………………………..…V
中文摘要………………………………………………………………………..…...VI
英文摘要……………………………………………………………………………VII
第壹章 緒論 (Introduction)…………………………………………………………..1
1.1 研究緣起…………………………………………………………………….1
1.2汙染物「汞」之特性…………………………………………………………..1
1.2.1汞的型態、污染來源及傳輸 1
1.2.2 汞在環境中的危害途徑…………………………………………………..2
1.2.3 汞對人體的危害與影響…………………………………………………..3
1.2.3.1 元素汞…………………………………………………………………...3
1.2.3.2 無機汞…………………………………………………………………...5
1.2.3.3 有機汞…………………………………………………………………...6
1.3 汞汙染生物復育法………………………………………………………….8
1.4 次世代定序技術 (Next generation sequencing, NGS) 10
1.5 基因工程細菌 (Genetic engineering bacteria)與生物復育 12
1.5.1 金屬硫蛋白 (Metallothionein) 12
第貳章 實驗目的和實驗架構 14
實驗目的………………………………………………………………………..14
第參章 實驗材料與方法 (Materials and Methods) 15
3.1.2 常用藥品與試劑…………………………………………………………17
第二節 實驗方法………………………………………………………………22
一、菌株來源、保存與繼代培養 (Bacterial culture) 22
二、菌株揮發汞離子能力測試…………………………………………………23
三、汞消化萃取與檢測方法……………………………………………………24
三、聚合?鏈鎖反應 (Polymerase chain reaction) 25
四、細菌RNA萃取 (Total RNA isolation) 26
五、反轉錄作用 (Reverse transcription) 27
六、DNA/RNA電泳……………………………………………………………28
七、即時定量聚合?連鎖反應 (qRT-PCR) 29
八、大腸桿菌電穿孔勝任細胞製備 (E. coli competent cell) 30
九、大腸桿菌電穿孔轉型法 (E. coli electroporation) 31
十、傳統法質體抽取與純化 (Plasmid extraction and purification) 32
十一、質體小量抽取試劑組……………………………………………………33
十二、DNA純化………………………………………………………………..34
十三、TSS competent cell………………………………………………………35
十四、限制酵素處理 (Digestion) 36
十五、DNA膠體純化 (Gel extraction) 36
十六、接合反應 (Ligation)…………………………………………………….37
十七、pJBME持續表現型載體建構 38
十八、蛋白質萃取 (Protein extraction) 40
十九、蛋白質濃度測定 (RC.DC protein assay) 41
二十、蛋白質電泳 (SDS Poly-acrylamide-gel-electrophoresis) 42
二十一、蛋白質轉漬……………………………………………………………43
二十二、西方免疫墨點法 (Western immune blotting) 44
二十三、細菌genomic DNA萃取 45
二十四、NGS菌種全基因組定序 (Whole genome sequencing) 46
二十五、生長細胞 (growth cell)汞固定實驗 47
第肆章 實驗結果 (Result) 49
4.1 評估Pseudomonas sp. A46於含60 ppm Hg2+ LB培養基生長情況與Hg2+移除效率………………………………………………………………………..49
4.2 Pseudomonas sp. A46 draft genome屬性分析 49
4.3 以Pseudomonas sp. A46 16S rDNA序列建立菌株親緣關係圖 50
4.4 Pseudomonas sp. A46 gene COGs功能性分析 50
4.5 Pseudomonas sp. A46 draft genome汞生物復育相關基因分析 50
4.6 Pseudomonas sp. A46 draft genome常見重金屬生物復育相關基因分析………………………………………………………………………………..50
4.7 以qRT-PCR分析汞生物復育相關基因表現量 52
4.8 以qRT-PCR分析抗氧化壓力相關基因表現量 52
4.9 以綠螢光蛋白評估持續表現型載體於Pseudomonas sp. A46目標蛋白質表現能力………………………………………………………………………..52
4.10 以西方墨點法評估MT1-EGFP-His蛋白質於Pseudomonas sp. A46內表現量與穩定度…………………………………………………………………..53
4.11 以生長細胞汞固定法測試基因工程菌株固定汞能力 53
第伍章 討論 ( Discussion ) 54
5.1 菌株Pseudomonas sp. A46汞抗性與還原揮發汞離子能力 54
5.2 Pseudomonas sp. A46 draft genome屬性分析 55
5.3 Pseudomonas sp. A46 16S rDNA親緣關係圖 55
5.4 Pseudomonas sp. A46 gene COGs功能性分析 56
5.5 Pseudomonas sp. A46重金屬生物復育相關基因分析 56
5.6.1 Pseudomonas sp. A46汞生物復育相關基因分析 56
5.6.2 重金屬鎘生物復育相關基因分析 57
5.6.3 重金屬鉻生物復育相關基因分析 58
5.6.4 重金屬銅生物復育相關基因分析 59
5.6.5 重金屬鉛生物復育相關基因分析 59
5.6.6 重金屬砷生物復育相關基因分析 60
5.7.1 以即時定量聚合?鏈鎖反應 (Quantitative real-time PCR, qPCR)分析抗汞相關基因表現量變化…………………………………..……………………61
5.7.2 重金屬產生之氧化壓力相關基因變化 61
5.8 金屬硫蛋白 (metallothionein)與基因工程菌 62
5.9 持續表現型載體開發……………………………………………………...63
5.10 金屬硫蛋白穩定度分析………………………………………………….65
5.11 基因工程菌株汞生物固定分析 65
結論……………………………………………………………………………..66
參考文獻 (Reference) 67
圖表 78
補充說明 95

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指導教授 陳師慶(Ssu-Ching Chen) 審核日期 2017-1-6
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