臺灣博碩士論文加值系統

近年來由於環境改變，土壤微生物多受到了干擾，故探討土壤菌相改變為重要的課題。早期相關技術如：RAPD、RFLP、DGGE、RISA…等已被運用在菌相分析上，上述技術多以16S rDNA為主，近年以16S-23S（900bp~1100bp） rDNA(ITS，0.5-1.4Kb)進行之相關研究也有增加的趨勢。本研究利用培養法(選擇性培養基LB，LB＋Kan＋Cyc)與化學法(CE和DGGE)進行種植基因改造木瓜隔離試驗田對土壤中一般細菌與KanR菌相分佈的分析。結果顯示利用DGGE技術配合16S rDNA中V3區域進行土壤中總DNA之一般細菌菌相分析，發現不同土壤樣本總DNA呈現不同的DGGE圖譜，經叢聚分析（Cluster analysis）顯示可得到基因改造作物對一般細菌菌相分佈的差異性。利用16S rDNA（1.4Kb）及ITS配合配合BLAST比對序列也可得到不同土壤樣本中KanR細菌分佈。

In recent years due to environmental changes, and more by the disruption of soil microbes, the soil bacteria to explore the importance of changing the subject. Early-related technologies such as: RAPD, RFLP, DGGE, RISA ... and have been used for analysis of bacteria, many of these technologies to 16S rDNA-based in recent years to 16S-23S (900bp ~ 1100bp) rDNA (ITS, 0.5~1.4 Kb ) carried out research is also increasing. In this study, culture method (selective medium LB, LB + Kan + Cyc) and chemical methods (CE and DGGE) for the cultivation of genetically modified papaya in isolated experimental field of soil bacteria with the general distribution of bacteria KanR analysis. The results showed that with the use of DGGE technology V3 region of 16S rDNA of total DNA of soil bacteria of the general analysis of bacteria and found that the total DNA of different soil samples showed different DGGE patterns, the clustering analysis (Cluster analysis) shows available on genetically modified crops general bacterial strain differences in distribution. The use of 16S rDNA (1.4Kb) and with the ITS sequence comparison with BLAST is also available in the soil samples of different bacteria KanR distribution.

目錄
中文摘要 I
Abstract II
誌謝 III
目錄 IV
表目錄 VII
圖目錄 VIII
附錄 XI
中英文對照表 XII
第一章 前言 1
第二章 文獻回顧 3
2-1分子生物技術的介紹 3
2-2變性梯度凝膠電泳 4
2-3毛細管電泳分析 6
2-3-1電滲流原理 7
2-3-2 DNA於CE中之分離機制 8
2-3-3毛細管凝膠電泳分析之應用 11
2-4 16S rDNA和16S-23S rDNA介紹 12
2-4-1 16S rDNA 12
2-4-2 16S-23S rDNA(ITS) 13
2-5分子演化系統樹介紹 14
2-5-1演化關係推導的方法 15
2-5-2 Bootstrap分析方法 21
第三章 材料和方法 23
3-1 試驗流程 23
3-2 試驗材料 24
3-2-1 土壤採樣來源和方法 24
3-2-2 土壤KanR細菌之篩選 25
3-3 試驗方法 26
3-3-1 DNA萃取和純化 26
3-3-2 聚合酶連鎖反應(PCR)條件 29
3-3-3 變性凝膠梯度電泳法 30
3-3-4 毛細管分析法 32
3-3-5 基因選殖(Gene Cloning) 33
3-3-6 統計分析 34
第四章 結果與討論 38
4-1 16S rDNA和ITS gene分析 38
4-1-1 16S rDNA 38
4-1-2 ITS gene分析 43
4-2 毛細管電泳法(CE)分析 46
4-3 變性梯度凝膠電泳(DGGE) 49
第五章 結論 56
第六章 參考文獻 95
6-1中文 95
6-2英文 96

表目錄
表1. 24株KanR菌株比對16S rDNA序列之結果 57
表2. 24株KanR菌株比對V3 gene序列之結果 58
表3. 14株KanR菌株比對16S-23S rDNA序列之結果 59
表4. 毛細管電泳儀器計算準確度之結果 60
表5. CE分析儀對土壤樣品產生之波峰訊號值 61
表6. 相同土壤不同時間菌相之相似係數矩陣 62
表7. 相同時間不同土壤菌相相似係數矩陣 63

圖目錄
圖1. 24株抗KanR菌菌株16S rDNA之電泳圖， 64
圖2. KanR菌之16S rDNA分析的親緣樹狀圖 65
圖3. KanR菌之16S rDNA分析的親緣樹狀圖 66
圖4. 24株抗KanR菌菌株V3基因之電泳圖 67
圖5. KanR菌依據V3基因分析的親緣樹狀圖 68
圖6. KanR菌依據V3基因分析的親緣樹狀圖 69
圖7. 24株抗KanR菌菌株ITS基因之電泳圖 70
圖8. KanR菌依據ITS基因分析的親緣樹狀圖 71
圖9. KanR菌依據ITS基因分析的親緣樹狀圖 72
圖10. 24株KanR菌之毛細管電泳分析結果 73
圖11. 24株抗KanR菌之毛細管電泳分析層析圖 79
圖12. Kan0481之電泳層析圖譜 80
圖13. Kan0506之電泳層析圖譜 80
圖14. 毛細管分析儀器之檢量線圖 80
圖15. Kan0468和Kan0501之電泳層析圖譜 81
圖16. Kan0457和Kan0513之電泳層析圖譜 81
圖17. Kan0483和Kan0502之電泳層析圖譜 81
圖18. Kan0501和Kan0502的之電泳層析圖譜 82
圖19. Kan0455和Kan0502的之電泳層析圖譜 82
圖20. Kan0455和Kan0468之電泳層析圖譜 82
圖21. Kan0457和Kan0501之電泳層析圖譜 83
圖22. Kan0455、Kan0457、Kan0501之電泳層析圖譜 83
圖23. Kan0457、Kan0501、Kan0502之電泳層析圖譜 83
圖24. Kan0455、Kan0457、Kan0501、Kan0502之電泳層析圖譜 84
圖25. Kan0455、Kan0456、Kan0457、Kan0501、Kan0502之電泳層析圖譜 84
圖26. 毛細管電泳分析相同土壤品系不同時間之電泳圖譜 85
圖27. 毛細管電泳分析相同時間不同土壤品系之電泳圖譜 85
圖28. 毛細管電泳分析土壤樣品10-4之層析圖 86
圖29. 毛細管電泳分析土壤樣品12-4之層析圖 86
圖30. 毛細管電泳分析土壤樣品14-3之層析圖 87
圖31. 毛細管電泳分析土壤樣品TCK之層析圖 87
圖32. 毛細管電泳分析土壤樣品空白組之層析圖 88
圖33. DGGE分析不同bp長度和GC%排列之電泳圖譜 89
圖34. DGGE分析菌株Kan0515和Kan0516之電泳圖譜 90
圖35. 以PCR-DGGE分析KanR菌菌株的PCR產物混合之圖譜 91
圖36. 不同時間和品系土壤之電泳圖 92
圖37. DGGE分析不同時間和品系土壤之電泳圖 93
圖38. 以AlphaEaseFC分析不同時間和品系土壤之電泳圖 93
圖39. 不同土壤品系和時間菌相分佈之相似度樹狀圖 94

附錄
附錄1.毛細管電泳分離模式及其分類 106
附錄2.電滲流示意 107
附錄3. 有根樹及無根樹表示法 108
附錄4. UPGMA公式推導、距離矩陣、演化樹 109
附錄5. NJ公式推導、距離矩陣、演化樹 110
附錄6. MP公式推導、距離矩陣、演化樹 111
附錄7. ML公式推導、距離矩陣、演化樹 112
附錄8. Bootstrap分析方法 113
附錄9. 實驗室編碼和採樣樣品說明 114
附錄10. 研究中使用引子對說明 115
附錄11. 培養基和變性膠配置 116
附錄12. DGGE製膠藥品配製 117
附錄13. 主控程式之狀態列 118
附錄14. 樣本分析實例和選殖藥品配置 119
附錄15. 基因選殖藥品配置 120

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