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研究生:游雅琇
研究生(外文):YOU,YA-HSIU
論文名稱:自台灣淡水域中分離出六株新種黃桿菌屬 之菌株與特性鑑定
論文名稱(外文):Characterization of Six Novel Species Bacteria of The Genus Flavobacterium Isolated From Freshwater In Taiwan
指導教授:陳文明陳文明引用關係
指導教授(外文):CHEN,WEN-MING
口試委員:張淑貞許世宜陳文明
口試委員(外文):CHANG,SHU-CHENSHEU,SHIHh-YICHEN,WEN-MING
口試日期:2020-07-30
學位類別:碩士
校院名稱:國立高雄科技大學
系所名稱:水產食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:241
中文關鍵詞:特性鑑定分類學微生物黃桿菌屬
外文關鍵詞:CharacterizationTaxonomyMicroorganismFlavobacterium
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於微生物之研究中,了解其龐大的細菌族群在生物分類上極為重要。本研究即是以台灣淡水域作為樣本採集來源,進行微生物的篩選與純化,進而予以鑑定、分類與命名。六株具有成為新種潛力之菌株,分別為菌株KDG-2、KDG-16、TWA-26、CAR-11、CYK-4、CYK-51,以表現型、化學型與基因型進行分類之探究,並與系統發育密切相關之模式菌株比較。實驗結果證實六株菌株與鄰近模式菌株存在顯著差異,因此六株菌株確定成為Flavobacterium屬之新種細菌。菌株KDG-2T命名為Flavobacterium pituitosum (=BCRC 81195T=LMG 31331T);菌株KDG-16T命名為Flavobacterium difficile (=BCRC 81194T=LMG 31332T);菌株TWA-26T命名為Flavobacterium celerecrescens (=BCRC 81200T=LMG 31333T);菌株CAR-11T命名為Flavobacterium lacustre (=BCRC 81188T=LMG 31329T);菌株CYK-4T命名為Flavobacterium lotistagni (=BCRC 81192T=LMG 31330T);菌株CYK-51T命名為Flavobacterium limosum (=BCRC 81245T=LMG 31680T)。
In the study of microorganisms, it is known that bacteria of a large group are extremely important in taxonomy. This study uses Taiwan's fresh waters as a source of bacterial samples for microbial purification and screening, and then identifies, classifies and names them. Six strains with new taxonomic group, namely strains KDG-2, KDG-16, TWA-26, CAR-11, CYK-4 and CYK-51 were classified by phenotype, chemotaxonomicaltype and genotype and compared with phylogenetic closely related strains. The experimental results proved that the six strains are different from the closely related strains. Six bacterial strains were considered as novel species of the genus Flavobacterium. Strain KDG-2T named Flavobacterium pituitosum (=BCRC 81195T=LMG 31331T). Strain KDG-16T named Flavobacterium difficile (=BCRC 81194T=LMG 31332T). Strain TWA-26T named Flavobacterium celerecrescens (=BCRC 81200T=LMG 31333T). Strain CAR-11T named Flavobacterium lacustre (=BCRC 81188T=LMG 31329T). Strain CYK-4T named Flavobacterium lotistagni (=BCRC 81192T=LMG 31330T). Strain CYK-51T named Flavobacterium limosum (=BCRC 81245T=LMG 31680T).
壹、 緒論 1
1. 前言 1
2. 研究動機 2
3. 研究目的 2
貳、 文獻回顧 4
1. 微生物分類學 4
2. 表現型分類 4
3. 化學型分類 5
3.1. 極性脂肪(Polar Lipid) 5
3.2. 脂肪酸(Fatty acid) 6
3.3. 醌型(Isoprenoid quinone) 6
3.4. 多胺(Polyamine) 8
4. 基因型分類 9
4.1. DNA G+C含量(DNA G+C content) 9
4.2. DNA-DNA雜交(DNA-DNA hybridization,DDH) 9
4.3. 16S rRNA基因序列比較 10
4.4. 平均核苷酸同源性(Average nucleotid identity,ANI) 12
4.5. 系統發育樹 12
5. 新種(屬)細菌之分類標準 14
6. 基因註解 15
6.1. Rapid Annotation using Subsystem Technology(RAST) 15
7. Flavobacterium屬介紹 16
參、 材料與方法 18
1. 實驗流程與架構 18
2. 樣品採集與分離純化 20
3. 菌株保存 21
3.1. 短期保存法 21
3.2. 長期保存法 21
4. SDS-PAGE電泳圖譜分析 22
4.1. 樣品製備 22
4.2. SDS-PAGE膠體製備 24
4.3. SDS-PAGE電泳分析 25
5. 萃取菌體DNA 26
5.1. 熱破碎萃取法 26
5.2. 商業套組萃取法 26
6. 16S RRNA基因序列 26
6.1. 16S rRNA基因序列親緣性比對 29
6.2. 系統發育樹建立 29
7. 形態學(Morphology) 30
7.1. 菌落描述 30
7.2. 菌體描述 31
7.3. 其他形態描述 37
8. 生理生化特性分析 41
8.1. 生長條件測試 41
8.2. 培養基測試 44
8.3. 氧氣需求測試 47
8.4. 生化代謝 48
8.5. 色素分析 49
8.6. 抗生素感受性 50
8.7. 水解試驗 54
8.8. Analytical Profile Index(API) 60
8.9. 單一碳源試驗 66
9. 化學型(Chemotype) 68
9.1. 極性脂肪分析 68
9.2. 脂肪酸分析 73
9.3. 醌型分析 75
9.4. 多胺分析 78
10. 基因型(Genotype) 82
10.1. 16S rRNA基因定序 82
10.2. 全基因組定序 82
10.3. 序列登記 83
10.4. Overall genome relatedness index(OGRI) 83
10.5. 系統發育樹 83
11. 基因註解 84
肆、 結果與討論 85
1. 樣品採集 85
2. 菌株分離與純化 85
3. 初步鑑定 85
4. 分類與鑑定 92
4.1. 菌株KDG-2之分類鑑定 92
4.2. 菌株KDG-16之分類鑑定 114
4.3. 菌株TWA-26之分類鑑定 140
4.4. 菌株CAR-11之分類鑑定 165
4.5. 菌株CYK-4之分類鑑定 186
4.6. 菌株CYK-51之分類鑑定 211
伍、 結論 232
陸、 參考資料 237


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