臺灣博碩士論文加值系統

處理蔬果加工廢棄物，將其所產生的堆肥，直接應用在小白菜之生長上，發現堆肥除提供養分外，尚有其它作用可增進小白菜產量。因此本研究探討在小白菜栽培時，添加處理蔬果加工廢棄物生產之堆肥、化肥對土壤微生物相的影響；實驗以三種樣品，分別為無施肥、添加堆肥、添加化肥土壤，了解三種土壤微生物多樣性之差異。
利用增殖16S rRNA基因來比較微生物族群的技術，在今日已廣泛應用在微生物生態上。本實驗之策略在分離出所有族群DNA，再利用此DNA來當模板，以專一性引子增殖出16S rRNA基因(rDNA)。隨後建立16S rDNA之基因庫後，藉由16S rDNA之限制片段長度多型性(RFLP)來快速篩選。
本實驗過程中針對原核微生物來分析，首先，我們針對16S rDNA專一性的寡核統計出其個別與RDP(Ribosomal Database Project)中完整的3000條以上SSU rRNA基因序列之穩合機率，最終結果我們選用了一組能夠選殖出1.5 kb左右長度的引子。之後我們也對選用的限制做了一系列的評估，實驗以電腦模擬的方式，選出能將上述引子選殖的16S rDNA基因中間序列切割成片段長度範圍集中的一組酵素，最終我們選用了AciI、BstUI、RsaI三種限制。
本研究我們總共分析了165條從土壤DNA增殖的16S rDNA，然後以AciI，RsaI和BstUI三種限制來鑑定”phylotype”。最終的75個”phylotype”之種類與數量分佈情形等調查皆以多樣性指標來表示。所有微生物共可分為18個群落，最終結果無施肥土壤與添加堆肥土壤較接近。無施肥、添加堆肥、添加化肥土壤三者之歧異度分別為0.990、0.986、0.962；均勻度為0.979，0.977，0.931。

To deal with vegetable/fruit waste material, we used its compost on Brassica chinesis cultivation。We appear compost not only provide nutrients, but also have other effect increase the production of Brassica chinesis cultivation.So the study focus on the microbial diversity during the Brassica chinesis cultivation, and we compared the microbial diversity in blank, compost, Chemical fertileizer soil.
Techniques based on amplification of 16S rRNA genes for comparing bacteria community are now widely used in microbial ecology. The stepwise strategy in this study is to isolate total community DNA and use this DNA as a template for PCR amplification of 16S rRNA genes(rDNA) with domain-specific primers. Followed by construction of a clone library for genes encoding 16S rDNA and rapid screening of the library by determining restriction fragment length polymorphisms (RFLPs) of the 16S rDNAs.
In this study only prokaryotes have been analysised. First, we matched 16S rDNA specific primer with over 3000 SSU rRNA sequences, at last we choose the primer pair that amplify around 1.5kb in length.Then we also compare the restriction enzyme, which could digest the sequence amplified with the primer pair into appropriate fragment length, Lastly we used AciI、BstUI、RsaI.
We analyzed 165 16S rDNAs amplified from extracted soil DNA, then defied phylotypes with the restriction enzymes AciI，RsaI and BstUI. At last, a total of 75 phylotypes richness, frequency distribution of the three clone libraries were investigated by using diversity indices. All phylotypes were divided into 18groups, the similarity is higher between blank soil and compost soil. The indices of blank, compost, chemical fertileizer soil are 0.990、0.986、0.962 for D value；and 0.979，0.977，0.931 for Evenness.

圖目錄………………………………………………………………………vii
表目錄………………………………………………………………………viii
壹、緒論………………………………………………………………………1
一、生物多樣性………………………………………………………………1
(一)生物多樣性之定義…………………………………………………1
(二)生物多樣性之指標…………………………………………………1
二、堆肥之簡介………………………………………………………………3
三、微生物之重要性…………………………………………………………4
(一)微生物之貢獻………………………………………………………4
(二)土壤微生物的功能…………………………………………………4
四、微生物分類及鑑定………………………………………………………5
(一)傳統分類方法………………………………………………………10
(二)近代分類方法………………………………………………………10
(三)rRNA鑑定的意義……………………………………………………11
(四)RFLP的介紹…………………………………………………………13
(五)系統分類及樹狀分析圖……………………………………………14
五、土壤中DNA之萃取………………………………………………………19
(一)不同土壤抽出之DNA產量…………………………………………19
(二)土壤之DNA抽取方法………………………………………………19
(三)腐植酸的影響及純化方法…………………………………………20
貳、研究目的…………………………………………………………………23
參、材料與方法………………………………………………………………25
一、材料……………………………………………………………………25
二、儀器設備………………………………………………………………27
三、實驗方法………………………………………………………………29
肆、結果與討論………………………………………………………………37
一、土壤DNA之確認…………………………………………………………37
(一)土壤抽取之total DNA……………………………………………37
(二)土壤DNA之PCR選殖…………………………………………………37
二、引子對之選擇…………………………………………………………40
三、16S rDNA的轉形………………………………………………………40
四、限制之選擇…………………………………………………………40
五、生物多樣性……………………………………………………………52
(一)各樣品土RFLP分群結果……………………………………………52
(二)各樣品土菌相之生態指標…………………………………………53
(三)各樣品土細菌族群相似性…………………………………………54
(四)各樣品土菌相的分類鑑定…………………………………………62
伍、結論………………………………………………………………………64
陸、參考文獻…………………………………………………………………65

吳秋曄 1995. 臺灣地區三種土壤之脫氮作用及以核酸探針定量土壤脫氮菌, 國立台灣大學農業化學研究所博士論文.
林良平 1993 土壤微生物學 南山堂出版社 台北
施盈如 1995 施用尿素與豬糞堆肥對土壤之肥力及水稻生長之影響 國立台灣大學農業化學研究所碩士論文.
陳建霖 1993. 生態目標與最適林分結構之研究. 國立台灣大學森林學研究所碩士論文.
陳麗鈴 1999 以演化類源分類的方式來探討細菌分類 CCRC News. pp.6-9.
許振宏 1999 豬糞堆肥之重金屬及有機物化學特性與其於土壤之應用研究 國立台灣大學環境工程學研究所博士論文.
張承晉 2001 外分泌纖維水解酵素之枯草桿菌轉植株在蔬菜加工廢棄物應用之研究 國立台灣大學園藝學研究所博士論文.
鍾國芳 1996. 以限制酵素片段長度多型性研究菸草屬之親緣關係 國立臺灣大學植物學研究所碩士論文.
Alexander, M. 1977. Introduction to soil microbiology, p.472. John Wiley&Sons, New York.
Amann, W. Ludwig, and K. Schleifer. 1995. Phylogenetic identification and in situ detection of indivisual microbial cells without cultivation. Microbiol. Rev. 59：143-169.
Arian, D. S., and J. H. Bell. 1980 Resource recovery through composting-a sleeping giant in Proc. 1980. Am. Soc. Mech. Eng., New York, NY, 121-129.
Beckmann, J. S., and M. Soller. 1983. Restriction fragment length polymorphism in genetic improvement：methodologies, mapping and costs. Theor. Appl. Genet. 67：35-43.
Bryant, J. E. 1995. Detection of Cryptosporidium parvum oocysts in soil using the polymerase chain reaction. M. S. thesis. Cornell University, Ithaca, N. Y.
Chen, M. Y., K. Y. Chen, S. S. Tsay, and Z. C. Chen. 1997 Use of random amplified polymorfic DNAs markers to distinquish temperature isolates of Aspergillus fumigatus of Taiwan. Taiwania. 42：201-206.
Chen, Y., Y. Inbar, and R. L. Malcolm. 1989 Chemical properties and solid-state CPMAS 13C-NMR of composted organic matter. Sci. Total Environ. 81/82, 201-208.
Erwin, T. L. 1983 Tropical forests：Their richness in coleoptera and arthropod species. Coleopt. Bull. 36：74-75.
Fink, W. L. 1986. Microcomputers and phylogenetic analysis. Science 234：1135-1139.
Goodfellow, M. and O’Donnell, A. G. 1993. Handbook of new Bacterial Systematics. London：Academic Press.
Hayashi, K. 1991. PCR-SSCP：a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Appl.1：34-38.
Hennig, W. 1966. Phylogenetic Systematics. Univ. Illinois Press: Urbana.
Hillis, D. M., Allard, M. W. and M. M. Miyamoto. (1993) Analysis of DNA sequence data: phylogenetic inference. pp. 456-487 in Zimmer, E.A., White, T.J., Cann, R.L. & Wilson, A.C. (eds.) Molecular Evolution: Producing the Biochemical Data (Academic Press: San Diego) .
Hoitink, H. A. J., and P. C. Fahy. 1986. Basis for the control of soilborn plant pathogens with composts. Ann. Rev. Phytopathol. 24：93-114.
Holben, W. E., J. K. Jansson, B. K. Chelm, and J. M. Tiedje. 1988. DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl. Environ. Microbial. 54：703-711.
Jackson, C. R., J. P. Harper, D. Willoughby, E. E. Roden, and P. F. Churchill. 1997. A simple, efficient method for the seperation of humic substances and DNA from environmental samples. Appl. Environ. Microbial. 63：4993-4995.
Laguerre, G., M. Allard, F. Revoy, and N. Amarger. 1994. Rapid identification of rhizobia by restriction fragment length polymorphisms analysis of PCR-amplified 16S rRNA genes. Appl. Environ. Microbiol. 60：56-63.
Lane , D. J.. 1983. thesis. University of Colorado.
Lin, R. F. 1995. Production and identification of alien chromosome addition lines of Nicotiana plumbaginifolia. Dr. Dissertation, Institute of Botany, National Taiwan University, Taipei, Taiwan.(in Chinese)
Liu, W. T., T. L. Marsh, H. Cheng, and L. J. Forney. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63：4516-4522.
Lovelock, J. E. 1987 Gaia-A New Look at Life on Earth. Oxford, UK.
Ludwig, J. A., and J. F. Reynold. 1988. Statistical Ecology. Wiley N. Y. p.85-103.
Ludwig, W., R. Amann, E. M. Romero, W. Schonhuber, S. Bauer, A. Neef; and K. H. Schleifer 1998 rRNA based identification and detection systems for rhizobia and other bacteria. Plant soil. 204：1-19.
McCouch, S. R., G. Kochert, Z. H. Yu, Z. Y. Wang, W. R. Coffman, and S.D.Tanksley.1988.Molecular mapping of rice chromosomes. Theor. Appl. Genet. 76：815-829.
Maidak, B. L., N. Larsen, N. J. McCaughey, R. Overbeek, G. J. Olsen, K. Gogel, J. Blandy, and C. R. Woese. 1994. The Ribosomal Database Project. Nucleic Acids Res. 22：3485-3487.
Miller, D. N., J. E. Bryant, E. L. Madsen, and W. C. Ghiorse. 1999. Evaluation and Optmization of DNA Extraction and Purification Procedures for Soil and Sediment Sample. Appl. Environ. Microbiol. 65：4715-4724.
Moeseneder, J. M. Arrieta, G. Muyzer, C. Winter, and G. J. Herndl. 1999. Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis. Appl. Environ. Microbiol. 65：3518-3525.
More, M. I., J. B. Herrick, M. C. Silva, W. C. Ghiorse, and E. L. Madsen. 1994. Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment. Appl. Environ. Microbiol.60：1572-1580.
Moyer, C. L., F. C. Dobbs, and D. M. Karl. 1994. Estimation of diversity and community structure through restriction fragment length distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi seamount, Hawaii. Appl. Environ. Microbiol. 60：871-879.
Moyer, C. L., J. M. TIEDJE, F. C. DOBBS, AND D. M. KARL. 1996. A Computer-Simulated Restriction Fragment Length Polymorphism Analysis of Bacterial Small-Subunit rRNA Genes: Efficacy of Selected Tetrameric Restriction Enzymes for Studies of Microbial Diversity in Nature. Appl. Environ. Microbiol.62：2501-2507.
Murry, A. E., J. T. Hollibaugh, and C. Orrego. 1996. Phylogenetic compositions of bacterioplankton from two California estuaries compared by denaturing gradient gel electrophoresis of 16S rDNA fragments. Appl. Environ. Microbiol.62：2676-2680.
Muyzer, G., E. C. DeWaal, and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59：695-700.
Ogram, A., G. S. Sayler, and T. Barkay. 1987. The extraction and purification of microbial DNA from sediments. J. Microbiol. Methods 7：57-66.
Olsen, D. J. Lane, S. J. Giovannoni, and N. R. Pace. 1986. Microbial Ecology and Evolution：A Ribosomal RNA Approach. Ann. Rev. Microbiol. 40：337-65.
Orita, M., H. Iwahana, H. Kanazawa, K. Hayashi, and T. Sekyia. 1989. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Nat. Acad. Sci. USA 86：2766-2770.
Penny, D., Hendy, M.D., Zimmer, E.A. & Hamby, R.K. (1990) Trees from sequences: panacea or Pandora''s box? Aust. Syst. Bot. 3: 21-38.
Peters, S. K., F. Schwieger, and C. C. Tebbe. 2000. Succession of microbial communities during hot composting as detection by PCR-Single-Strand-Conformation-Polymorphism- Based genetic profiles of small-subunit rRNA genes. Appl. Environ. Microbiol. 66：930-936.
Picard, C., C. Ponsonnet, E. Paget, X. Nesme, and P. Simonet. 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl. Environ. Microbiol. 58：2717-2722.
Porteous, L. A., and J. L. Armstrong. 1991. Recovery of bulk DNA from soil by a rapid, small-scale extraction method. Curr. Microbiol. 22：345-348.
Reilly, P. J. 1985. chap.2 in Starch Conversion Technology edited by G.M.A. van Beynum, J. A. Roels. New York：M. Dekker.
Song, K. M., T. C. Osborn, and P. H. Williams. 1988. Brassica taxonomy based on nuclear restriction fragment length polymorphisms(RFLPs). 1. Genome evolution of diploid and amphidiploid species. Theor. Appl. Genet. 75：784-794.
Stackebrandt, E. 1992. Unifying phylogeny and phenotypic properties. In Balows, A. et al.,(eds) The Prokaryotes, A Handbook on the biology of Bacteria：Ecophysiology, Isolation, Identification, Applications, 2nd edn New York：Springer.
Stahl, D. J. Lane, G. J. Olsen, and N. R. Pace. 1984. Analysisof hydrothermal vent-associated symbionts by ribosomal RNA sequences. Science 224：409-411.
Stahl, D. J. Lane, G. J. Olsen, and N. R. Pace. 1985. Characterization of a Yellowstone Hot Spring Microbial Community by 5S rRNA Sequences. Appl. Environ. Microbiol. 49：1379-1384.
Steffan, R. J., J. Goksoyr, A. K.1988. Recovery of DNA from soils and sediments. Appl. Environ. Microbiol. 54：2908-2915.
Steffan, R. J. and R. M. Atlas. 1988. DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Appl. Environ. Microbiol. 54：2185-2191.
Swofford, D. and G. Olsen, 1990. Phylogeny reconstruction. Pp. 411-501 in Hillis, D.M. & Moritz, C. (eds.) Molecular Systematics (Sinauer: Sunderland, Mass.)
Torsvik, V., J. Coksoyr, and F. L. Daae. 1990. High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56：782-787.
Tebbe, C. C., and W. Vahjen. 1993. Interference of humic acid and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and yeast. Appl. Environ. Microbiol.59：2657-2665.
Tsai, Y., and B. H. Olson. 1991. Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57：1071-1074.
Tsai, Y., and B. H. Olson. 1992. Rapid method for seperation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl. Environ. Microbiol. 58：2292-2295.
Van de Peer Y, S. Chapelle, R. De Wachter, 1996. A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucleic Acids Res. 24:3381-3391
Volossiouk, E., E. J. Robb, and R. N. Nazar.1995. Direct DNA extraction for PCR-mediated assay of soil organism. Appl. Environ. Microbiol.61：3972-3976.
Weston, P. H. (1994) Methods for rooting cladistic trees. Pp. 125-155 in Scotland, R. W., Siebert, D. J. & Williams, D. M. (eds.) Models in Phylogeny Reconstruction (Oxford Univ. Press: Oxford) .
Wilson, E. O. 1984 The current state of biological diversity. p3-18 in E. O. Wilson, (ed) Biodiversity, National Academy Press, Washington, DC.
Woese, C. R., Gutell, R., Gupta, R., and H. Noller. 1983. Detailed analysis of the higher order structure of 16S like ribosomal ribonucleic acids. Microbiological Reviews. 47：621-669.
Woese, C. R. 1987. Bacterial evolution. Microbiological Reviews. 51：221-227.
Zhou, J., M. A. Bruns, and J. M. Tiedje. 1996. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62：316-322.
Zuckerkandl, E. and L. Pauling. 1965. Molecules as documents of evolutionary history. Journal of Theoretical Biology. 8：357-366.