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研究生：	林徽鳴 Lin, Huei-Ming
論文名稱：	利用變性梯度凝膠電泳與末端螢光標定限制酵素片段長度多型性分析法監測微生物社會之分子指紋變化 Monitoring molecular fingerprinting of microbial community dynamics by denaturing gradient gel electrophoresis(DGGE) and terminal-restriction fragment length polymorphism (T-RFLP)
指導教授：	曾怡禎 Tseng, I-cheng
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生命科學系 Department of Life Sciences
論文出版年：	2005
畢業學年度：	93
語文別：	中文
論文頁數：	97
中文關鍵詞：	變性梯度凝膠電泳(DGGE) 、末端螢光標定限制片段多型性分析法(T-RFLP) 、落葉分解、16S rDNA選殖技術、微生物社會
外文關鍵詞：	DGGE, T-RFLP, microbial community, litter decomposition
相關次數：	點閱：101 下載：1
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　　土壤微生物在森林生態系的生物社會中扮演著重要的的角色，多數的微生物皆與自然環境的的養分循環息息相關。雖然絕大部分的微生物分類皆已被定義，但多數自然環境中的微生物仍屬未知。主要原因在於環境中的微生物物種僅有少數可利用現今的的培養方法加以培養與純化。在本研究中，利用16S rDNA選殖技術、變性梯度凝膠電泳(DGGE)與末端螢光標定限制片段多型性分析法(T-RFLP)等多種分子生物技術，建立南仁山當地土壤與落葉微生物社會結構資料庫與分子指紋。

　　本研究利用細菌廣泛性的引子擴增南仁山土壤中微生物之16S rDNA片段，以建立南仁山落葉袋底層土壤與黃杞落葉分解微生物的分子資料庫，並討論其親緣關係。結果顯示，南仁山落葉袋底層土壤以屬於Acidobacteria相關的菌群為優勢，佔總菌群的62.8%。而菌群之16S DNA親緣關係顯示，皆與嗜酸性的菌群相關。

　　在落葉分解的菌群中，有51.4%的菌群屬於Gammaproteobacteria，顯然落葉分解與落葉袋底層土壤的菌群有不同的微生物結構。屬於Gammaproteobacteria的菌群，其16S rDNA親緣關係顯示，菌群中微生物多為固氮作用相關菌群。將所建立之落葉袋底層土壤與落葉分解菌群之資料庫作為分子標誌，用以監測落葉分解與落葉袋土壤底層土壤之分子指紋圖譜的季節性變化。denaturing gradient gel electrophoresis (DGGE)指紋圖譜顯示，無論迎風區與背風區之落葉袋底層與其四周土壤中之微生物社會結構，均產生季節性變化，可能與東北季風有關。黃杞落葉分解DGGE指紋圖譜的變化較不顯著，但優勢菌群依然以Gammaproteobacteria為主。

　　本研究亦以terminal-restriction fragment length polymorphism (T-RFLP)的指紋圖譜監測南仁山土壤與落葉微生物社會。先行以落葉袋下土壤以及黃杞落葉樣本所建立之16S rDNA基因資料庫進行電腦限制切點模擬分析，再實際以限制酵素HhaI進行實驗。結果顯示，落葉袋底層土壤微生物社會中之菌群共可區別為八群的terminal restriction fragments (T-RFs)，同時亦顯示優勢的菌群為Acidobacteria，在樣本中約佔15.7%。而黃杞落葉樣本之微生物社會中之菌群可區分為五群的T-RFs，優勢菌群為Betaproteobacteria，在樣本中約佔38%。本研究亦分析南仁山土壤微生物以及黃杞落葉上的微生物社會之變化，並以培養方法分離纖維素分解菌，探討分離菌株之16S rDNA的親緣關係，在本文中均有詳加討論。

　Soil bacteria are essential components of the biotic community in natural forests and they are largely responsible for ecosystem function, and participate in the elements circulation. Although the main diversity of life has been proven to be microbial, the vast majority of soil bacteria still remain unknown for less than 1% of environmental microorganisms can be cultured. In this study, cloning method, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP) techniques were used to compare the prevalent microbial populations in soil and on litterfall of Nan-Jen Shan samples.

　Phylogenetic analysis based on PCR-amplified 16S rDNA revealed an effective tool to establish the microbiota development in soil and litterfall. The results from clone library showed that Acidobacteria were the dominants in soil samples (62.8%) and Gammaproteobacteria were the dominants in litterfall samples (51.4%). DGGE patterns also revealed that the microbial community of soil and litterfall were not the same. Seasonal changes in the structure of microbial community were significant in DGGE analysis. However, the DGGE patterns of dominant bacteria did not change in DGGE analysis no matter in soil and litterfall samples.

　Terminal-restriction fragment length polymorphism (T-RFLP) analysis is also used in this study for rapid comparison of the complex bacterial communities. By T-RFLP analysis of soil and litterfall samples, we got eight groups Terminal-restriction fragments (T-RFs) in soil samples and five groups T-RFs in litterfall samples. T-RFLP analysis also revealed that Acidobacteria were the dominant bacteria in soil and were not affected by season. However, the results of T-RFLP showed that Betaproteobacteria are the dominant in litterfall. Otherwise, the dominant bacteria were changing with season on litterfall.

　Cultivation-approached method was also applied in isolation of cellulose degrading bacteria. Five strains were isolated from soil samples, all of them can use α-cellulose as carbon source. It showed that the soil bacteria in Nan-Jen Shan might play an important role in litter decomposition mechanism.

中文摘要                                                                I
英文摘要                                                                II
誌謝                                                                    III
目錄                                                                    IV
表目錄                                                                  VI
圖目錄                                                                  VII
第一章 前言                                                             1
1-1研究緣起                                                             1
1-2研究目的                                                             1
1-3研究架構                                                             2
第二章 文獻回顧                                                         3
2-1森林中的落葉分解                                                     3
2-2分子生物方法應用於微生物生態的研究                                   3
  2-2-1聚合酵素鏈鎖反應                                                 4
  2-2-2變性梯度凝膠電泳在微生物生態研究的應用                           5
  2-2-3末端螢光標定限制酵素片段長度多型性分析法在微生物生態研究的應用   6    
  2-2-4其他的分子檢測技術                                               7
  2-2-5 DNA 萃取技術與PCR反應的改良                                     8
第三章 材料與方法                                                       10
3-1樣區與樣本                                                           10
3-2優厚培養與分離                                                       10
3-3樣本DNA萃取                                                          13
  3-3-1 土壤DNA萃取                                                     13
  3-3-2落葉DNA萃取                                                      13
  3-3-3純菌DNA萃取                                                      13
3-4分析方法                                                             13
  3-4-1 DNA萃取方法                                                     13
  3-4-2聚合酵素鏈鎖反應                                                 15
  3-4-3瓊脂膠體電泳                                                     16
  3-4-4 16S rDNA 分子選殖                                               18
  3-4-5變性梯度凝膠電泳                                                 18
  3-4-6變性梯度凝膠電泳上之DNA片段萃取                                  21
  3-4-7限制酵素片段長度多型性分析法                                     21
  3-4-8末端螢光標定限制酵素片段長度多型性分析法                         21
  3-4-9定序                                                             22
  3-4-10 親源關係分析                                                   22
3-5主要儀器                                                             22
第四章 實驗結果                                                         24
4-1 DNA萃取與環境中之PCR反應抑制物                                      24
  4-1-1土壤DNA萃取方法比較                                              28
4-2分子生物技術分析菌群結構                                             28
  4-2-1落葉袋底部之表層土壤樣本之選殖分析                               28
  4-2-2落葉袋底部之表層土壤樣本選殖資料庫親緣關係分析                   28
  4-2-3落葉樣本之選殖分析                                               36
  4-2-4落葉樣本選殖資料庫親緣演化分析                                   36
4-3微生物社會變動之監測                                                 46
  4-3-1落葉袋底部土壤樣本微生物社會變動之監測                           46
  4-3-2落葉袋四周土壤樣本微生物社會變動之監測                           50
  4-3-3落葉分解微生物社會變動之監測                                     54
4-4利用末端螢光標定限制酵素片段長度多型性分析法分析南仁山微生物菌群結構 61
  4-4-1南仁山微生物菌群結構分析                                         61
  4-4-2利用T-RFLP監測菌群季節性變動                                     69
  4-4-3優勢物種的檢測                                                   75
4-5纖維素分解菌的純菌分離                                               76
  4-5-1厭氧纖維素分解菌之純菌分離                                       76
  4-5-2耗氧纖維素分解菌之純菌分離                                       77
  4-5-3厭氧發酵產氫菌株分離                                             77
  4-5-4分離菌株親緣關係分析                                             79
第五章 討論                                                             82
第六章 結論與建議                                                       85
6-1結論                                                                 85
6-2建議                                                                 86
第七章 參考文獻                                                         87
自述                                                                    97

Table 3-1 α-cellulose medium                                            12
Table 3-2 TYG medium                                                    12
Table 3-3 Solution M                                                    12
Table 3-4 3-4 Primers used in this study                                17
Table 3-5 PCR conditions of different primer pairs                      17
Table 3-6 Procedure of cloning.                                         19
Table 3-7 Procedure of DGGE analysis.                                   20
Table 4-1 The comparison of DNA extraction methods.                     25
Table 4-2 Phylogenetic affiliations of the 16S rDNA clone library of 
the soil sample beneath litterfall.                                     30
Table 4-3 Phylogenetic affiliations of the 16S rDNA clone library of 
the litterfall sample.                                                  39
Table 4-4 The comparison of soil beneath litterfall and litterfall
clone library.                                                          45
Table 4-5 (A) Analysis of species richness in the soil sample beneath 
litterfall by DGGE method.                                              47
Table 4-5 (B) Analysis of species richness in the soil sample 
by DGGE method.                                                         51
Table 4-6 (A) Analysis of species richness in litterfall sample 
by DGGE method.                                                         57
Table 4-6 (B) Analysis of species richness in litterfall sample 
by DGGE method.                                                         59
Table 4-7 Simulation cutting site in clone library of soil sample 
beneath litterfall by HhaI.                                             62
Table 4-8 Simulation cutting site in clone library of litterfall 
by HhaI.                                                                64
Table 4-9 The T-RFLP result of soil sample beneath litterfall cut 
by HhaI.                                                                65
Table 4-10 Comparison of soil sample beneath litterfall and simulation 
cutting site of clone library by HhaI.                                  66
Table 4-11 The T-RFLP results of litterfall sample cut by HhaI.         67
Table 4-12 Comparison of litterfall sample and simulation cutting 
site of clone library by HhaI.                                          68
Table 4-13 Comparison of RFLP selection clones and soil beneath 
litterfall clone library.                                               75

Fig.1-1 Schematic representation of the steps for the study of 
Nan-Jen Shan bacterial community by cultivation-dependent and 
-independent approaches.                                                2
Fig.3-1 Sample site of Nan-Jen Shan                                     11
Fig.4-1 Electrophoresis profile of different DNA extraction methods.    26
Fig.4-2 Electrophoresis profile of 11F-1512R PCR product by different 
DNA extraction methods.                                                 27
Fig.4-3 The relationship of the number of clones and operational 
taxonomic unit (OTU) from the 16S rDNA clone library of the soil sample 
beneath litterfall. (screened by DGGE)                                  32
Fig.4-4 DGGE profile of the 16S rDNA clone library of soil sample 
beneath litterfall.                                                     33
Fig.4-5 The illustration of DGGE fingerprinting of the bacterial 
16S rDNA clone library from soil sample beneath litterfall.             34
Fig.4-6 Phylogenetic dendrogram of bacterial 16S rDNA sequence from 
the soil sample beneath litterfall.                                     35
Fig.4-7 The relationship of the number of clones and operational 
taxonomic unit (OTU) from the bacterial 16S rDNA clone library of 
the litterfall sample. (screened by DGGE)                               41
Fig.4-8 DGGE profile of the 16S rDNA clone library of litterfall sample.42
Fig.4-9 The illustration of DGGE fingerprinting of the bacterial 
16S rDNA clone library from litterfall sample.                          43
Fig.4-10 Phylogenetic dendrogram of 16S rDNA bacterial sequence 
from the litterfall sample.                                             44
Fig.4-11 (A) DEEG profiles of 16S rDNA gene fragments for PCR from 
seasonal soil samples beneath litterfall.                               48
Fig.4-11(B) The illustration of DGGE fingerprinting of different 
seasonal soil samples beneath litterfall.                               49
Fig.4-12 (A) DEEG profiles of 16S rDNA gene fragments for PCR 
from seasonal soil samples.                                             52
Fig.4-12(B) The illustration of DGGE fingerprinting of different 
seasonal soil samples.                                                  53
Fig.4-13(A) DEEG profiles of 16S rDNA gene fragments for PCR from 
seasonal litterfall samples.                                            56
Fig.4-13(B) The illustration of DGGE fingerprinting of different 
seasonal litterfall samples.                                            58
Fig.4-13(C) The illustration of DGGE fingerprinting of different 
seasonal litterfall samples.                                            60
Fig.4-14 T-RFLP electropherograms of PCR-amplified 16S rDNA gene from 
soil sample beneath litterfall. (sample were digested with 
restriction enzyme HhaI)                                                65
Fig.4-15 T-RFLP electropherograms of PCR-amplified 16S rDNA gene 
from litterfall sample. (sample were digested with 
restriction enzyme HhaI)                                                67
Fig.4-16 Representive T-RFLP profile of bacteria community from 
different seasonal soil samples.                                        72
Fig.4-17 Representive T-RFLP profile of bacteria community from 
different seasonal soil samples beneath litterfall.                     73
Fig.4-18 Representive T-RFLP profile of bacteria community from 
different seasonal litterfall samples.                                  74
Fig.4-19 Restriction fragment length polymorphism (RFLP) banding 
patterns of soil sample beneath litterfall analyzed by DGGE. 
(digested by endonuclease HhaI and MspI)                                76
Fig.4-20 DEEG profile representing the bacteria community of 
MPN cultivation.                                                        78
Fig.4-21 Phylogenetic tree of 16S rDNA of isolated strain.              80  
Fig.4-22 Growth of isolated strain ISO 05 onα-cellulose agar plate.     81
Fig.4-23 Growth of isolated strain ISO GB01 on TYG agar plate.          81
                                    

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