臺灣博碩士論文加值系統

牛樟芝，俗稱牛樟菇、牛樟芝，學名為Antrodia camphorata ( Wu, et al. 1997)。 就目前所知，牛樟芝為一台灣特有菌種，生長於牛樟樹 (Cinnamomum kanehirai) 中空樹洞中，會造成樟樹產生褐腐病 (brown rot disease)。 坊間流傳，牛樟芝具有保肝、解毒能力。 由於牛樟芝子實體生長速率緩慢，且隨寄主牛樟樹數目減少，使牛樟芝在中草藥店價格高漲。 鑑於在醫藥研究上牛樟芝逐漸熱門，但是市面上有大量容易和牛樟芝混淆的其它菌種，單就外型並不易分辨牛樟芝和它們的分別。 在進行菌種培養時，我們發現不同來源的牛樟芝其生長速率、外觀亦有差異。 因此在形態鑑定外，我們發展出一套利用分子生物技術鑑定牛樟芝的方法。利用18S 核醣體 (18S rRNA) 基因序列，internal transcribed spacer （ITS）和5.8S rRNA基因序列及超氧歧化酶（superoxide dismutase）基因序列作為研究目標，分析由台灣的苗栗和南投等地搜集到的牛樟芝子實體和菌種保存中心的牛樟芝菌絲體。 由不同牛樟芝樣品及香杉樹上的香杉芝萃取去氧核醣核酸，利用PCR擴增技術放大18S 核醣體基因序列，ITS基因序列及超氧岐化酶基因序列。 將PCR擴增技術放大的序列定序後和基因庫的序列做比對，發現牛樟芝的18S 核醣體基因序列， ITS基因序列及超氧岐化酶基因序列都具有特殊片段。 之後將牛樟芝的的序列和基因庫選取出的不同種、屬、科、目、綱序列進行演化分析。 在演化分析中，由牛樟芝的18S 核醣體基因序列，ITS基因序列及超氧岐化酶基因序列經由套裝軟體 Paup 4.0及 Phylip 3.5中的三種方法Nighbor Joning法、Parsimony法和Maximum Likelihood法進行演算，結果證實我們可以利用此三段序列來確定牛樟芝種的演化地位。 超氧岐化酶基因序列演算結果顯示牛樟芝中具有不同品系。 目前建立牛樟芝的分子鑑定系統，可以對日後牛樟芝的產品開發提供發展基礎。

Antrodia camphorata, a traditional medical fungus grows in Taiwan only, become more and more popular in recent medical research. A. camphorata grows on empty rotting trunk of Cinnamomum kanehirai only, and is well known as “niu chang ku”. Because amount of C. kanehirai decreased seriously, the price of A. camphorata rise to 162,000 NT per kilogram. Many traditional chinese herb stores sell fruiting body of A. camphorata. But there is a problem about it, none can identify A. camphorata clearly by its shape. And customer may pay a lot for a A. camphorata like mushroom.
In order to indentify A. camphorata rapidly and correctly, we use the molecular biology methods for A. camphorata species and strains analysis. First, we used 18S rRNA gene , ITS gene and intergenic spacer of rRNA , and Mn-superoxide dismutase gene. The sequencing data was submitted to PubMed for BLAST, and collected the similar sequences. When we compared the sequences, found A. camphorata have special sequences, so we can determinate the specie of A. camphorata. Then we use phylogenetic analysis methods Neighbor Joing method, Parsimony method and Maximum Likelihood method to analyze evolution relation of A. camphorata. And bootstrap analysis was used for statistical test. All the three methods can indicate A. camphorata is different from other similar sequences.
The molecular identification system we builded, could be used for product development of A. camphorata.

中文摘要 IV
ABSTRACT V
第一章 緒論 8
1-1. 牛樟芝簡介 2
1-1-1. 牛樟芝命名 2
1-1-2. 牛樟芝特點 2
1-1-3. 牛樟樹簡介 4
1-1-4. 牛樟芝效用 4
1-1-5. 牛樟芝分類地位 4
1-1-6. 子實體特徵 5
1-1-7. 菌絲體特徵 5
1-1-8. 牛樟芝成分分析 7
1-1-9. 牛樟芝的液態醱酵培養 7
1-2. 薄層菌屬 8
第二章 牛樟芝形態、生理性質 9
2-1. 前言 9
2-2. 材料與方法 9
2-2-1. 實驗標本及菌株 9
2-2-2. 牛樟芝菌株保存 12
2-2-4. 牛樟芝菌絲體玻片培養觀察 12
2-2-5. 牛樟芝生長速率觀察 12
2-2-6. 牛樟芝生質量觀察 12
2-3. 結果與討論 13
2-3-1. 牛樟芝子實體標本和菌株 13
2-3-2. 牛樟芝菌絲體觀察 14
2-3-3 牛樟芝生長速率 16
2-3-4. 牛樟芝不同菌株的生質量 18
第三章 利用18S RRNA基因序列進行牛樟芝分類研究 20
3-1. 前言 20
3-2. 材料與方法 22
3-2-1. 實驗標本及菌株 22
3-2-2. 菌種培養 22
3-2-3. 去氧核糖核酸萃取 22
3-2-4. 聚合酶連鎖反應（PCR，polymerase chain reaction） 23
3-2-5. 基因選殖 26
3-2-6. 基因序列處理 27
3-2-7. 和薄層孔菌屬序列之18S rRNA序列比較 27
3-2-8. 和基因庫得到的相似序列做比對 27
3-3. 結果與討論 27
3-3-1. 聚合酶連鎖反應結果 27
3-3-2. 牛樟芝序列和薄層菌屬序列的差異 31
3-3-3. 牛樟芝18S rRNA序列和基因庫中相近序列的比對 34
3-3-4. 牛樟芝18S 核醣體基因序列和高相同度的序列計算之演化樹 34
第四章 利用INTERNAL TRANSCRIBED SPACER（ITS）和5.8S核醣體基因序列進行牛樟芝分類研究 41
4-1. 前言 41
4-2. 材料與方法 41
4-2-1. 實驗標本及菌株 41
4-2-2. 菌種培養 41
4-2-3. 去氧核糖核酸萃取 41
4-2-4. ITS聚合酶連鎖反應（PCR，polymerase chain reaction） 42
4-2-5. 基因選殖 42
4-2-6. 基因序列處理 43
4-2-7. 和薄層孔菌屬序列之比較 44
4-2-8. 和基因庫得到的相似序列做比對 44
4-3. 結果與討論 44
4-3-1. 聚合酶連鎖反應結果 44
4-3-2. 牛樟芝ITS及5.8S核醣體基因序列和薄層孔菌屬ITS序列及5.8S核醣體基因的差異 45
4-3-3. 牛樟芝序列和基因庫中相近序列的差異 45
4-3-4. 牛樟芝ITS及5.8S核醣體基因序列和相似序列的演化樹分析 50
第五章 利用含錳超氧歧化酶基因序列進行牛樟芝分類研究 55
5-1. 前言 55
5-2. 材料與方法 55
5-2-1. 實驗標本及菌株 55
5-2-2. 菌種培養 56
5-2-3. 去氧核糖核酸萃取 56
5-2-4. 超氧歧化酶聚合酶連鎖反應（PCR，polymerase chain reaction） 56
5-2-5. 基因選殖 58
5-2-6. 基因序列處理 58
5-3. 結果與討論 58
5-3-1. 基因序列之定序結果 58
5-3-2. 牛樟芝種內和薄層孔菌屬超氧歧化酶基因序列的差異 58
5-3-3. 牛樟芝超氧歧化酶基因序列和基因庫中的相近序列 59
5-3-4. 牛樟芝超氧歧化酶基因序列之組成分析 65
5-3-5. 利用演化樹圖譜分析牛樟芝超氧歧化酶基因序列的演化地位 69
第六章 總討論及未來展望 74
參考文獻 76

許瑞祥，1984。人工培養冬蟲夏草屬之性質及其代謝物之探討，碩士論文，國立台灣大學農業化學研究所。
許瑞祥，1990。靈芝屬菌株鑑定系統之研究，博士論文，國立台灣大學農業化學研究所。
王伯徹和陳啟楨，1994。常見食藥用菇類介紹，食品工業發展研究所，新竹市，中華民國。
王伯徹、陳啟楨和華傑，1998。食藥用菇類的培養與應用，食品工業發展研究所，新竹市，中華民國。
王伯徹，2000。食藥用菇保健食品之開發，食品工業32（5）：18-27。
王惠芳，1996。靈芝屬含錳超氧歧化酶基因之研究，碩士論文，國立台灣大學農業化學研究所。
吳德鵬，1995。牛樟芝微量成分的研究，碩士論文，台灣師範大學化學研究所。
徐唯哲，1998。藉由分析18S rDNA序列探討牛樟芝在多孔菌的親緣關係，碩士論文，東海大學生物研究所。
高曉薇，1991。台灣靈芝屬新種牛樟芝之三萜類成分研究，碩士論文，台北醫學院天然物醫學研究所。
陳勁初、林文鑫、陳清農、許勝傑、黃仕政和陳炎鍊，2001。台灣特有真菌-牛樟芝菌絲體之開發. Fung. Sci. 16（1,2）；7-22.
程一華，1993。牛樟芝之成份研究，碩士論文，台灣師範大學化學研究所。
徐唯哲，1998。藉由分析18S rDNA序列探討牛樟芝在多孔菌的親緣關係，碩士論文，東海大學生物研究所。
黃惟敏，2000。牛樟芝微量成分的研究(Ⅱ)，碩士論文，靜宜大學應用化學系。
黃鈴娟，2000。牛樟芝與姬松茸之抗氧化性質及其多醣組成分析，碩士論文，中興大學食品科學系。
黃惠琴，2001。牛樟芝菌絲體深層培養之研究，碩士論文，東海大學化學工程學系。
陳宏文，2001。以回應曲面法探討牛樟芝高密度發酵並分析相關的成份與機能性，碩士論文，交通大學生物科技研究所。
戴宇昀，2001。牛樟芝菌絲體與子實體對四氯化碳及酒精誘導之慢性及急性肝損傷之保肝功能評估，碩士論文，中興大學食品科學系。
Ames, B. N., Shigenaga, M. K. & Hagen, T. M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A 90, 7915-22. (1993).
Andersson, B. E., Tornberg, K., Henrysson, T. & Olsson, S. Three-dimensional outgrowth of a wood-rotting fungus added to a contaminated soil from a former gasworks site. Bioresour Technol 78, 37-45. (2001).
Baldrian, P., Gabriel, J. & Pospisek, M. Improved isolation of nucleic acids from basidiomycete fungi. Biotechniques 27, 458-60, 462. (1999).
Bialek, R., Ibricevic, A., Fothergill, A. & Begerow, D. Small subunit ribosomal DNA sequence shows Paracoccidioides brasiliensis closely related to Blastomyces dermatitidis. J Clin Microbiol 38, 3190-3. (2000).
Bidochka, M. J., St Leger, R. J., Stuart, A. & Gowanlock, K. Nuclear rDNA phylogeny in the fungal genus Verticillium and its relationship to insect and plant virulence, extracellular proteases and carbohydrases. Microbiology 145, 955-63. (1999).
Binder, M. & Hibbett, D. S. Higher-Level Phylogenetic Relationships of Homobasidiomycetes (Mushroom- Forming Fungi) Inferred from Four rDNA Regions. Mol Phylogenet Evol 22, 76-90. (2002).
Buzina, W., Lang-Loidolt, D., Braun, H., Freudenschuss, K. & Stammberger, H. Development of Molecular Methods for Identification of Schizophyllum commune from Clinical Samples. J Clin Microbiol 39, 2391-6. (2001).
Castle, A. et al. Morphological and molecular identification of Trichoderma isolates on North American mushroom farms. Appl Environ Microbiol 64, 133-7. (1998).
Chang, T. T. & Chou, W. N. Antrodia cinnamomea sp. nov. on Cinnamomea kanehirai in Taiwan. Mycol. Res. 99, 756-758 (1995).
Chaturvedi, S. et al. Molecular cloning, phylogenetic analysis and three-dimensional modeling of Cu,Zn superoxide dismutase (CnSOD1) from three varieties of Cryptococcus neoformans. Gene 268, 41-51. (2001).
Chen, C. H., Yang, S. W. & Shen, Y. C. New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum. J Nat Prod 58, 1655-61. (1995).
Cherng, I. H. & Chiang, H. C. Three new triterpenoids from Antrodia cinnamomea. J. Natural Products 58, 365-371 (1995).
Cherng, I. H., Wu, D. P. & Chiang, H. C. Triterpenoids from Antrodia cinnamomea. Phytochemistry 41, 263-267 (1996).
Chiang, H. C., Wu, D. P., Cherng, I. H. & Ueng, C. H. A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. Phytochemistry 39, 613-616 (1995).
Culotta, V. C. Superoxide dismutase, oxidative stress, and cell metabolism. Curr Top Cell Regul 36, 117-32 (2000).
Dams, E. et al. Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res 16, r87-173. (1988).
Denis, C. M. et al. Genetic divergence at the SODA locus of six different formae speciales of Pneumocystis carinii. Med Mycol 38, 289-300. (2000).
Dolashka-Angelova, P. et al. Isolation and characterization of a novel superoxide dismutase from fungal strain Humicola lutea 110. J Pept Res 54, 279-89. (1999).
Eisenga, B. H., Trestrail, J. H., 3rd & Emerson, T. S. Identification of unknown mushrooms: the good, the bad, and the ugly. J Toxicol Clin Toxicol 36, 635-6 (1998).
Fluckiger, S. et al. Comparison of the crystal structures of the human manganese superoxide dismutase and the homologous Aspergillus fumigatus allergen at 2-A resolution. J Immunol 168, 1267-72. (2002).
Flynn, T. M. & Niehaus, W. G. Improved method for isolation of DNA from slow-growing basidiomycetes such as Armillaria mellea. Biotechniques 22, 47, 50-2. (1997).
Gilbertson, R. L. and L. Ryvarden. 1986-1987. North American Polyporaceae vols. 1-2. Fungiflora A/S, Oslo.
Goel, S., Raina, S. N. & Ogihara, Y. Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of nuclear ribosomal DNA in the phaseolus- vigna complex. Mol Phylogenet Evol 22, 1-19. (2002).
Halliwell, B., Murcia, M. A., Chirico, S. & Aruoma, O. I. Free radicals and antioxidants in food and in vivo: what they do and how they work. Crit Rev Food Sci Nutr 35, 7-20. (1995).
Henry, T., Iwen, P. C. & Hinrichs, S. H. Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. J Clin Microbiol 38, 1510-5. (2000).
Hseu, Y. C. et al. Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life Sci 71, 469-82. (2002).
Hwang, S. K. & Kim, J. G. Secondary structural and phylogenetic implications of nuclear large subunit ribosomal RNA in the ectomycorrhizal fungus Tricholoma matsutake. Curr Microbiol 40, 250-6. (2000).
Jackson, C. J. Molecular identification and strain typing of dermatophyte fungi. Nippon Ishinkin Gakkai Zasshi 42, 7-10 (2001).
Jacob, C. et al. Molecular cloning, characterization and regulation by cadmium of a superoxide dismutase from the ectomycorrhizal fungus Paxillus involutus. Eur J Biochem 268, 3223-32. (2001).
James, T. Y., Moncalvo, J. M., Li, S. & Vilgalys, R. Polymorphism at the ribosomal DNA spacers and its relation to breeding structure of the widespread mushroom Schizophyllum commune. Genetics 157, 149-61. (2001).
Jorgensen, F., Petruson, B. & Hansson, H. A. Extensive variations in nasal mucosa in infants with and without recurrent acute otitis media. A scanning electron-microscopic study. Arch Otolaryngol Head Neck Surg 115, 571-80. (1989).
Joshi, P. & Dennis, P. P. Structure, function, and evolution of the family of superoxide dismutase proteins from halophilic archaebacteria. J Bacteriol 175, 1572-9. (1993).
Kelly, J. M. & Cox, R. A. The nucleotide sequence at the 3''-end of Neurospora crassa 18S-rRNA and studies on the interaction with 5S-rRNA. Nucleic Acids Res 10, 6733-45. (1982).
Kevei, F., Hamari, Z., Varga, J., Kozakiewicz, Z. & Croft, J. H. Molecular polymorphism and phenotypic variation in Aspergillus carbonarius. Antonie Van Leeuwenhoek 70, 59-66. (1996).
Kwiatowski, J., Skarecky, D. & Ayala, F. J. Structure and sequence of the Cu,Zn Sod gene in the Mediterranean fruit fly, Ceratitis capitata: intron insertion/deletion and evolution of the gene. Mol Phylogenet Evol 1, 72-82. (1992).
Kwiatowski, J., Skarecky, D., Bailey, K. & Ayala, F. J. Phylogeny of Drosophila and related genera inferred from the nucleotide sequence of the Cu,Zn Sod gene. J Mol Evol 38, 443-54. (1994).
Lee, I. et al. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiol Lett 209, 61-65. (2002).
Lee, J. H. et al. Protective role of superoxide dismutases against ionizing radiation in yeast. Biochim Biophys Acta 1526, 191-8. (2001).
Lee, S. B. & Taylor, J. W. Isolation of DNA from fungal mycelia and single spores. In: PCR Protocols. A Guide to Methods and Applications., 315-322 (1990).
Lott, T. J., Burns, B. M., Zancope-Oliveira, R., Elie, C. M. & Reiss, E. Sequence analysis of the internal transcribed spacer 2 (ITS2) from yeast species within the genus Candida. Curr Microbiol 36, 63-9. (1998).
Martin, F., Selosse, M. A. & Le Tacon, F. The nuclear rDNA intergenic spacer of the ectomycorrhizal basidiomycete Laccaria bicolor: structural analysis and allelic polymorphism. Microbiology 145, 1605-11. (1999).
McCord, J. M. & Fridovich, I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244, 6049-55. (1969).
Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71, 491-9. (1988).
Michiels, C., Raes, M., Toussaint, O. & Remacle, J. Importance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radic Biol Med 17, 235-48. (1994).
Mizushina, Y. et al. A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases. Biochem Biophys Res Commun 249, 17-22. (1998).
Nagy, A., Vagvolgyi, C. & Ferenczy, L. Simple method for obtaining chromosomal DNA for orthogonal field alternation gel electrophoresis analysis from Phaffia rhodozyma. Biotechniques 18, 63, 65. (1995).
Parenicova, L. et al. Combined molecular and biochemical approach identifies Aspergillus japonicus and Aspergillus aculeatus as two species. Appl Environ Microbiol 67, 521-7. (2001).
Poyart, C., Berche, P. & Trieu-Cuot, P. Characterization of superoxide dismutase genes from gram-positive bacteria by polymerase chain reaction using degenerate primers. FEMS Microbiol Lett 131, 41-5. (1995).
Revill, P. A., Davidson, A. D. & Wright, P. J. Identification of a subgenomic mRNA encoding the capsid protein of mushroom bacilliform virus, a single-stranded RNA mycovirus. Virology 260, 273-6. (1999).
Schroeder, S., Kim, S. H., Cheung, W. T., Sterflinger, K. & Breuil, C. Phylogenetic relationship of Ophiostoma piliferum to other sapstain fungi based on the nuclear rRNA gene. FEMS Microbiol Lett 195, 163-7. (2001).
Sies, H. Strategies of antioxidant defense. Eur J Biochem 215, 213-9. (1993).
Smith, M. W. & Doolittle, R. F. A comparison of evolutionary rates of the two major kinds of superoxide dismutase. J Mol Evol 34, 175-84. (1992).
Song, T. Y. & Yen, G. C. Antioxidant Properties of Antrodia camphorata in Submerged Culture. J Agric Food Chem 50, 3322-7. (2002).
Sonnenberg, A. S. et al. Isolation of expressed sequence tags of Agaricus bisporus and their assignment to chromosomes. Appl Environ Microbiol 62, 4542-7. (1996).
Specht, C. A., DiRusso, C. C., Novotny, C. P. & Ullrich, R. C. A method for extracting high-molecular-weight deoxyribonucleic acid from fungi. Anal Biochem 119, 158-63. (1982).
Suguimoto, H. H., Barbosa, A. M., Dekker, R. F. & Castro-Gomez, R. J. Veratryl alcohol stimulates fruiting body formation in the oyster mushroom, Pleurotus ostreatus. FEMS Microbiol Lett 194, 235-8. (2001).
Turenne, C. Y., Sanche, S. E., Hoban, D. J., Karlowsky, J. A. & Kabani, A. M. Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system. J Clin Microbiol 37, 1846-51. (1999).
Varga, J. et al. Phylogenetic analysis of Aspergillus section Circumdati based on sequences of the internal transcribed spacer regions and the 5.8 S rRNA gene. Fungal Genet Biol 30, 71-80. (2000).
Velegraki, A., Kambouris, M., Kostourou, A., Chalevelakis, G. & Legakis, N. J. Rapid extraction of fungal DNA from clinical samples for PCR amplification. Med Mycol 37, 69-73. (1999).
Wang, S. X., Hunter, W. & Plant, A. Isolation and purification of functional total RNA from woody branches and needles of Sitka and white spruce. Biotechniques 28, 292-6. (2000).
White, T. J., Bruns, T., Lee, S. & Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genesfor phylogenetics .In: PCR protocols. A Guide to Methods and Applications., 315-322 (1990).
Woese, C. R. & Olsen, G. J. Archaebacterial phylogeny: perspectives on the urkingdoms. Syst Appl Microbiol 7, 161-77 (1986).
Wu, S. H., Leif, R. & Chang, T. T. Antrodia camphorata ("niu-chang-chih"), new combination of a medicinal fungus in Taiwan. Bot. Bull. Acad. Sin. 38, 273-275 (1997).
Zang, M. & Su, Q. Ganoderma camphoratum, a new taxon in genus Ganoderma from Taiwan, China. Acta Bot. Yunnanica 12, 395-396 (1990).
Zhang, D., Yang, Y., Castlebury, L. A. & Cerniglia, C. E. A method for the large scale isolation of high transformation efficiency fungal genomic DNA. FEMS Microbiol Lett 145, 261-5. (1996).
Zolg, J. W. & Philippi-Schulz, S. The superoxide dismutase gene, a target for detection and identification of mycobacteria by PCR. J Clin Microbiol 32, 2801-12. (1994).