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研究生:廖俊博
研究生(外文):Chun-Po Liao
論文名稱:南仁山古湖底泥甲烷氧化菌社會結構之研究
論文名稱(外文):Community Structure of Methane Oxidizing Bacteria in Nan-Jen Lake Sediment
指導教授:曾怡禎曾怡禎引用關係
指導教授(外文):I-Cheng Tseng
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:84
中文關鍵詞:pmoA基因甲烷氧化菌變性梯度電泳親緣關係
外文關鍵詞:methane oxidizing bacteria (MOB)pmoA geneDGGEphylogenic analysis
相關次數:
  • 被引用被引用:14
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  • 下載下載:57
  • 收藏至我的研究室書目清單書目收藏:2
本研究利用分子生物的方法來建立南仁山古湖底泥甲烷氧化菌的社會結構。針對甲烷氧化菌的功能性基因pmoA設計專一性的引子對A189Cf和mb661AC,選殖分析後共獲得120 clones,再經由變性梯度電泳比對分析,共獲得14種不同的OTU。親緣關係分析的結果顯示,此14條pmoA序列與Methylobacter和Methylomonas的關係較為相近。南仁山古湖底泥甲烷氧化菌之豐富度高於湖水,底泥層之種豐富度隨深度遞減。不同深度所出現的甲烷氧化菌的種類略有差異,但屬於Methylobacter之NJ M2、46、88和屬於Methylomonas之NJ M77在不同的深度均有出現,應屬於古湖底泥優勢的甲烷氧化菌。本研究亦結合MPN的方法和利用甲烷氧化菌功能性基因的引子,分析底泥表層甲烷氧化菌的含量,其含量為5 X 107 MPN/g soil,顯示底泥層甲烷氧化菌的含量相當豐富。將底泥分別以5%和10%的甲烷培養之後,分別抽取DNA和RNA,經DGGE分析之後比對甲烷氧化菌族群的變動,結果顯示以5%甲烷培養的呈現較大的微生物社會結構的改變。
Methane is an important greenhouse gas that significantly contributes to global warming. Methane-oxidizing bacteria ( MOB ) are considered to play a major role in regulating the fluxes of methane from important methane sources such as forest, wetlands and rice paddies. The MOB are obligately aerobic bacteria that can utilize methane as the sole source of carbon and energy for growth. They are important biological regulators of methane in nature. Very limited information is presently available on the levels of populations of MOB in Taiwan. The object of this study was to construct the community structure of methane-oxidizing bacteria in Nan-Jen lake. The methane oxidizing population was sampled by PCR amplification and cloning of particulate methane monooxygenase A ( pmoA ) gene from the total soil population. Primer set A189Cf and mb661AC were designed specific for pmoA gene of MOB. A total of 120 pmoA clones was screened by DGGE and fourteen OTUs were found. The similarity of the fourteen pmoA gene sequences with those of Methylobacter and Methylomonas were 85.6% and 91.3%. The diversity of MOB in lake sediment were higher than lake water. The MOB content in the surface layer of soil was about 5 X 107 MPN/g-soil determined by MPN-PCR method. The dynamic of MOB population was analyzed by DGGE cultivated with 5% and 20% methane. ARDRA also applied in this study for analysis MOB community in Nan-Jen lake.
中文摘要..................................................I
英文摘要.................................................II
致謝....................................................III
目錄.....................................................IV
表目錄..................................................VII
圖目錄...................................................IX
第一章 前言...............................................1
第二章 文獻回顧...........................................2
2.1 碳循環............................................3
2.2 甲烷氧化菌........................................4
2.3 甲烷氧化菌分解甲烷的機制..........................5
2.4 甲烷氧化菌的研究..................................7
2.5 甲烷氧化菌16S rDNA序列之核酸探針和引子...........9
2.6 甲烷氧化菌單氧氧化酵素序列之核酸探針和引子.......10
2.7 ARDRA在環境微生物生態研究的應用..................11
2.8 研究目的.........................................12
第三章 材料與方法........................................13
3.1 實驗材料.........................................13
3.1.1 採樣地點....................................13
3.1.2 甲烷氧化菌菌種來源..........................13
3.1.3 培養基......................................15
3.1.3.1 液體培養基.............................15
3.1.3.2 瓊脂培養基.............................15
3.1.4 化學藥品....................................15
3.1.5 主要儀器....................................17
3.2 實驗方法與步驟...................................19
3.2.1 底泥土壤的前處理............................19
3.2.2 甲烷氧化菌菌量計數..........................19
3.2.3 DNA萃取.....................................19
3.2.4 RNA萃取.....................................22
3.2.5 引子的設計..................................22
3.2.6 聚合酵素連鎖反應(PCR).......................23
3.2.7 反轉錄(RT)..................................24
3.2.8 瓊脂膠體電泳................................24
3.2.9 變性梯度電泳................................25
3.2.10 選殖分析...................................27
3.2.10.1 選殖步驟..............................27
3.2.10.2 選殖樣品的製備........................28
3.2.11 定序.......................................30
3.2.12 親緣關係分析...............................30
3.2.13 利用ARDRA分析甲烷氧化菌pmoA序列..........30
第四章 結果..............................................31
4.1 PCR鍊合的溫度....................................31
4.1.1 甲烷氧化菌pmoA序列PCR引子.................31
4.1.2甲烷氧化菌pmoA序列DGGE之PCR引子...........31
4.2 DNA萃取方法的比較................................34
4.2.1 環境樣本未經過培養直接萃取DNA...............34
4.2.2 環境樣本經過培養之後萃取DNA.................34
4.3 RNA的萃取........................................37
4.4 甲烷氧化菌pmoA序列選殖分析(cloning).............39
4.5 親緣分析.........................................41
4.5.1 甲烷氧化菌pmoA序列親緣關係.................41
4.5.2甲烷氧化菌pmoA胺基酸序列親緣關係............44
4.6 ARDRA結果比較....................................48
4.7 甲烷氧化菌的計數.................................54
4.8 南仁山古湖樣區不同深度樣本之比較.................55
4.8.1 10公分之南仁山古湖底泥......................55
4.8.2 20公分之南仁山古湖底泥......................55
4.8.3 30公分之南仁山古湖底泥......................56
4.8.4 南仁山古湖湖水樣本..........................56
第五章 討論..............................................65
第六章 結論..............................................73
第七章 參考文獻..........................................74
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