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研究生:廖經裕
研究生(外文):Chin-Yu Liao
論文名稱:以分子生物方法與培養方式分析台灣兩處溫泉之嗜熱菌族群
論文名稱(外文):Microbial community analysis of thermophilic bacteria in two hot springs of Taiwan using molecular biology method and cultivation method
指導教授:黃啓裕
指導教授(外文):Chi-Yu Huang
學位類別:碩士
校院名稱:東海大學
系所名稱:環境科學系
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:139
中文關鍵詞:變性梯度電泳凝膠分類運算單位多樣性指數限制酵素片段長度多型性
外文關鍵詞:DGGEOTUsdiversity indexRFLP
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本研究選定轆轆溫泉及十三坑溫泉,進行分析嗜熱箘群的結構,本實驗中利用聚合酶鍊鎖反應,擴增箘群中的16S rRNA gene。利用16S rRNA gene的差異,分析兩處樣區的嗜熱箘群結構。
本研究中利用兩種分析方式:以變性梯度電泳凝膠法分析16S rDNA中V3-V5的變異區段,利用亮帶擷取、核醣核酸萃取、PCR擴增及定序的步驟,進行菌群結構分析。而另外一種方式為分子選殖分析法,進行菌群結構分析。在兩溫泉樣區中定義以核酸序列相似度大於98%視為一個分類運算單位(OTUs),在親源關係及核酸序列相似度的比較結果中,變性梯度電泳凝膠法分析得到24個OTUs,而分子選殖法中的所取的80個clone中有28個OTUs。以兩樣區所取得的OTUs進行相似性、多樣性及變異性的計算結果中發現,十三坑溫泉的菌群生物多樣性及變異性均高於轆轆溫泉。以親源關係分析兩溫泉樣區菌群發現相同的group包括:Clostridia, Chroococcales, β-proteobacteria, Spirochaetes 及 CFB (Cytophaga- Flavobacterium- Bacteroides ).
本研究中並嘗試以75℃的溫度下進行純菌培養、分離及純化,以限制酵素片段長度多型性的測試中,將100株純菌分類為22個phylotype。
The thermophilic community structure was investigated in this study at Lulu hot spring and Shihsankeng hot spring. For this investigation, it based on the analysis of PCR amplification containing the 16S ribosomal RNA gene.
Two types of libraries were generated in this study: DGGE libraries analysis of the variable region (V3-V5), which were further analysed by excising, reamplification, sequencing, and random shotgun sequence libraries. Phylogenetic and sequence similarity analyses of the resultant in DGGE libraries revealed the presence of 24 operational taxonomic units (OTUs) or phylotypes, which were defined as having more than 98% of sequence similarity. 80 clones (40 clones from each sample site) sequences in shotgun libraries revealed 28 OTUs.
The similarity, diversity, and evenness in these libraries were estimated using a variety of diversity indices. Shinsankeng exhibited more diverse and variegated bacterial populations. Phylogenetic analysis confirmed the diversity of thermophilic divisions. The common group in two communities appeared to include Clostridia, Chroococcales, β-proteobacteria, Spirochaetes and CFB (Cytophaga- Flavobacterium- Bacteroides ).
Cultivation was used to isolate pure cultures under 75℃ from 2 sample sites. Among 100 pure strains, 22 phylotypes were distinguished by RFLP (Restriction Fragment Length Polymorphism).
第一章 前 言……………………………………………………. 1
第二章 文獻回顧……………………………………………………. 3
2.1 生物多樣性(Biodiversity)…………………………………. 3
2.2 原核生物(The Prokaryotes)……………………………… 9
2.3 嗜熱菌概述及應用(Introduction and application of thermophilicbacteria)……………………………………….. 11
2.3.1 嗜熱菌的特性…….………………………………………… 11
2.3.2 高溫環境生態………………………………………………. 12
2.3.3 嗜熱菌的應用………………………………………………. 13
2.4 台灣溫泉(Taiwan hot spring)……………………………… 20
2.5 分子分類學(Molecular Taxonomy)……………………….. 21
2.5.1 原核生物分子演化………………………………………… 21
2.5.2 核醣體核醣核酸(ribosomal RNA, rRNA)…………….. 22
2.5.3 分子分類學方法(Methodology of molecular taxonomy) 25
2.5.3.1 限制酵素片段多型性(Restriction Fragment Length Polymorphism, RFLP)………………………………… 25
2.5.3.2 變性梯度電泳(Denature Gradient Gel Electrophoresis,DGGE)……………………………… 26
2.6 鑑定與分類 (Identification and taxonomy)……………… 28
2.7 生物多樣性指標(Diversity indices)………………………. 32
2.8 研究目的……………………………………………………… 33
第三章 實驗材料與方法……………………………………………. 35
3.1 實驗流程設計…………………………………………………. 35
3.2 環境樣本採集…………………………………………………. 35
3.3 溫泉菌群之分析……………………………………………… 37
3.3.1 分子生物技術分析………………………………………… 37
3.3.1.1 溫泉樣品之前處理……………………………………… 37
3.3.1.2 溫泉菌群Chromosomal DNA萃取…………………….. 37
3.3.1.3 DNA的純度及定量檢測……………………………….. 41
3.3.1.4 溫泉菌群16S rDNA引子選定…………………………. 42
3.3.1.5 聚合連鎖反應(Polymerase Chain Reaction, PCR)…... 46
3.3.1.6 分子選殖(Melecular Cloning)……………………….. 48
3.3.1.7 限制酵素多型性(Restriction fragment length polymorphism, RFLP)…………………………………. 53
3.3.1.8 變性梯度凝膠電泳法(Denaturing Gradient Gel Electrophoresis, DGGE)………………………………. 54
3.3.1.9 變性梯度凝膠膠片萃取(QIAGEN gel extraction kit) 57
3.4 溫泉菌株之培養、純化、選取與保存……………………… 59
3.4.1 極端嗜熱厭氧菌株之培養基………………………………. 60
3.4.2 菌株之培養、純化與分離…………………………………. 65
3.4.3 菌種保存……………………………………………………. 65
3.5 菌種鑑定……………………………………………………… 66
3.5.1 API ZYM酵素測試………………………………………… 66
3.5.2 API20A厭氧菌鑑定套件………………………………….. 66
3.6 核酸序列之分析……………………………………………… 67
3.6.1 核酸序列之定序……………………………………………. 67
3.6.2 核酸序列之比對..………………………….………………. 67
3.6.3 親源樹之製作………………………………………………. 68
3.7 數據整理與生物多樣性指標估算…………………………… 68
第四章 結果與討論…………………………………………………. 69
4.1 採樣地點位置及背景…………………………………………. 69
4.2 不同核酸萃取方法的比較與選擇……………………………. 70
4.3 PCR擴增16S rDNA片段…………………………………… 72
4.4 以分子選殖法(Molecular Cloning)進行菌群結構分析…. 76
4.4.1 16S rDNA 純化……………………………………………. 76
4.4.2 菌群結構分析………...…...………………………………... 77
4.5 以變性梯度電泳凝膠(DGGE)進行菌群結構分析………. 90
4.5.1 以Nested PCR擴增V3-V5變異區段……………………. 90
4.5.2 DGGE變性梯度範圍選定………........................................ 92
4.6 Clone及DGGE之生物多樣性評估…………………………. 98
4.7 以培養方式分離純菌及菌株之篩選………………………… 102
4.7.1 培養期間內菌群的變化…………………………………… 102
4.7.2 極端嗜熱菌株之篩選與測試……………………………… 110
4.7.2.1 以RFLP進行純箘篩選………………………………… 110
4.7.2.2 API20A基質利用測試…………………………………. 115
4.7.2.3 API-ZYM酵素分析…………………………………….. 118
第五章 結論與建議…………………………………………………. 121
5.1 結論……………………………………………………………. 121
5.2 建議……………………………………………………………. 124
參考文獻…………..…………………………………………………. 125
表 目 錄
Table 2-1 Known and estimated numbers of biological species in biosphere of the Earth………………………………………. 8
Table 2-2 Number and biomass of prokaryotes in the world………….. 10
Table 2-3 The classification of bacteria by the different optimal growth temperatures............................................................................ 12
Table 2-4 The properties of thermostable enzymes produced from thermohpiles………………………………………………… 15
Table 2-5 Bioconversion reactions and application of thermostable enzymes……………………………………………………… 19
Table 2-6 Characteristics used to describe thermophiles………………. 30
Table 3-1 The components and concentrations of TAE buffer............... 42
Table 3-2 The components of agarose electrophoresis…….......... ……. 42
Table 3-3 Broad specificity PCR priners for amplification and sequencing of SSU rDNAs………………………………….. 44
Table 3-4 The PCR primers of variable regions in 16S rRNA….……... 45
Table 3-5 The components and their concentrations in PCR and nested PCR.………………………………………………………… 47
Table 3-6 The heating program of PCR……………………………….. 48
Table 3-7 The reagents for ligation.…………………………………... 50
Table 3-8 The components for transformation during cloning in this study………………………………………………………… 52
Table 3-9 The heating program of colony PCR for amplifyication of cloned DNA.………………………………………………… 52
Table 3-10 The components used in RFLP……………………………… 54
Table 3-11 The protocol of SYBR……..………………………………. 54
Table 3-12 The relationship of DNA fragment and polyacrylamide percentage…………………………………………………… 56
Table 3-13 The reagent and concentration of DGGE……….................... 56
Table 3-14 The component of diffusion buffer……………………….. 59
Table 3-15 The component of TA medium……………………………… 62
Table 3-16 The component and concentration of TA medium………….. 62
Table 3-17 The component of trace element……………………………. 63
Table 3-18 The component of vitamin solution…..…………………….. 64
Table 4-1 The information of hot spring Lulu and Shihsankeng………. 69
Table 4-2 The ratio of DNA/ protein(A260/A280) with different extraction methods.................................................................. 71
Table 4-3 The condition of amplification 16S rDNA by PCR…............ 75
Table 4-4 The purity of DNA during purification process…………….. 76
Table 4-5 The condition for PCR amplifyication of cloned 16S rDNA 77
Table 4-6 Sequence length and phylogenetic affiliation of the clone in this study…………………………………………………….. 88
Table 4-7 The condition for amplifying V3-V5 variable regions of 16S rDNA by nested PCR………………………………………... 91
Table 4-8 Comparison of the diversity indices of rDNA libraies with other microbecal comm.unities found in different environments……………………………………………….. 99
Table 4-9 Distribution of the 16S rRNA gene clone and OTUs obtained Lulu and Shihsankeng hot spring with clone and DGGE method………………………………………………………. 101
Table 4-10 The condition of amplification 16S rDNA by PCR………… 104
Table 4-11 The condition of amplification 16S rDNA by PCR………… 104
Table 4-12 The condition of amplification V3-V5 Variable region by PCR…………………………………………………………. 105
Table 4-13 The condition of amplification V3-V5 Variable region by PCR…………………………………………………………. 106
Table 4-14 The similarity of each pure cultural isolated from Lulu hot spring……………………………………………………….. 112
Table 4-15 The similarity of each pure cultural isolated from Shihsankeng hot spring……………………………………… 114
Table 4-16 API20A results of each isolates form Lulu hot spring………. 116
Table 4-17 API20A results of each s isolates form Shihsankeng hot spring………………………………………………………… 117
Table 4-18 API-ZYM results of each strains isolated form Lulu hot spring………………………………………………………… 119
Table 4-19 API-ZYM results of each strains isolated form Shihsankeng hot spring……………………………………………………. 120
圖 目 錄
Fig. 2.1 Presentation of the universal and variable region in SSU rRNAs……………………………………………………….. 24
Fig. 2.2 The principle of denaturing gradient gel electrophoresis…… 27
Fig. 2-3 The classification of Bacteria super kingdom by nucleotide... 31
Fig. 3.1 Flow chart of experimental design in this study……………. 36
Fig. 3.2 The processes of isolation, purification and quantitation DNA………………………………………………………… 38
Fig. 3.3 The composition of gas station………….…………………... 64
Fig. 4.1 Agarose gel electrophoresis of chromosomal DNA extracted from Lulu hot spring(L)and Shihsankeng hot spring(S). 72
Fig. 4.2 The 16S rDNA electrophoresis of Lulu hot spring(L)and Shihsankeng hot spring(S)by PCR amplification……….. 75
Fig. 4.3 The prediction line of clone for Lulu and Shihsankeng hot springs………………………………………………………. 78
Fig. 4-4 Phylogenetic tree showing the relationship of cloned 16S rDNA partial sequence from Lulu hot spring ( TA clones)…. 80
Fig. 4.5 The unrooted phylogenetic tree showing the position of the Lulu hot spring……………………………………………… 81
Fig. 4.6 Phylogenetic tree showing the relationship of cloned 16S rDNA partial sequence from Shihsankeng hot spring spring ( TA clones)…………………………………………………. 83
Fig. 4.7 The unrooted phylogenetic tree showing the position of the Shihsankeng hot spring……………………………………… 84
Fig. 4.8 The V3-V5 variable regions electrophoresis of Lulu hot spring(L)and Shihsankeng hot spring(S)by nested PCR 91
Fig. 4.9 The profiles of DNA distribution with different percentage of denaturant in DGGE……………………………………… 93
Fig. 4.10 The position of each single band….………………………… 94
Fig. 4.11 The phylogenetic tree of Lulu hot spring by using DGGE….. 96
Fig. 4.12 The phylogenetic tree of Shihsankeng hot spring by using DGGE……………………………………………………….. 97
Fig. 4.13 The growth curve of extreme thermophilic anaerobes in Lulu 102
Fig. 4.14 The growth curve of extreme thermophilic anaerobes in Shihsankeng hot spring……………………………………… 103
Fig. 4.15 Analysis the culturable community by DGGE……………… 107
Fig. 4.16 The cluster tree shows the change community of Lulu hot spring………………………………………………………… 107
Fig. 4.17 Analysis the culturable community by DGGE……………… 109
Fig. 4.18 The cluster tree shows the change community of Shihsankeng hot spring……………………………………… 109
Fig. 4.19 RFLP profile of 16S rRNA genes of pure cultures isolated from LuLu hot spring……………………………………….. 111
Fig. 4.20 RFLP profile of 16S rRNA genes of pure cultures isolated from Shihsankeng hot spring………………………………... 113
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