摘 要

本論文主要在樟芝子實體減法基因庫之建立。樟芝子實體減法基因庫建立成功之後，挑選了360個separated colonies。平均長度約在500 bp左右。送至中研院sequence，最後得到246個有意義的序列。將找出來的gene依功能性的不同加以分類。發現在分類的gene組中有三個有關樟芝物種特性的代表組，依次為cancer組、cytotoxin/detoxin組、peroxidase組。cancer組佔全體3.3 %，cytotoxin/detoxin組佔全體13.4 %，peroxidase組佔全體6.1 %。三組佔全部的 22.8 %。在246個colonies之中，有些gene出現頻率較高，而這些gene又顯現出樟芝物種特徵，例如 : P450，佔總體的3.7 %。
在論文的後半部，述說由當中找出三條gene，P450、GST與prx，找出gene的全長與研究gene特性。
樟芝P450有1491 bp，可轉譯出497個胺基酸 ( 包括stop codon )。在SDS-PAGE上面觀察，約是70 kDa大小的蛋白質。經國外專門研究P450的學者David Nelson判斷，本論文的P450與Phanerochaete chrysosporium的white rot fungal 的序列相似度最高，約有46 % 的相似度，可能是CYP5014 family，屬於一個小家族，是P450家族的新成員，且尚未被官方命名。
樟芝GST有651 bp，可轉譯出217個胺基酸 (包括stop codon )，理論上約是22 kDa左右的蛋白質。由於GST是dimer structure，在SDS-PAGE上面觀察，可發現在60 kDa附近有蛋白質存在。
另外，也找出樟芝子實體中的抗氧化基因peroxiredoxin ( prx )。prx有645 bp，可轉譯出215個胺基酸 ( 包括stop codon )。在SDS-PAGE上面觀察，可發現在32.5 kDa附近有蛋白質存在。用prx的胺基酸與prx對DTT的實驗結果來判斷，樟芝子實體中的prx是屬於2-Cys prx，在蛋白質的四級結構，是monomer的構造。在prx的功用方面，本論文運用了MFO system來證明，在MFO system反應溶液內，DNA會被此system所產生的free radical切斷，某些酵素會失去活性，但是如果同時有prx存在時，可以保護DNA的完整與酵素的活性。

Abstract

The purpose of this thesis is to establish Antrodia camphorata PCR-Select cDNA Subtration Library. Approximately 360 A. camphorata cDNA clones were sequenced. The nucleotide sequences and the inferred amino acid sequences were cvncpared to the sequences in various nucleic acid and protein data banks using the FASTN and FASTP programs, respectively.
Three hundred and sixty colonies were selected and sequenced from cDNA library that was Antrodia camphorata PCR-Select cDNA Subtration Library. The inserts advantage length are 500 bp, after sequencing, we get 246 sequences meaningful. We classes these genes depend on their function and get 3 groups in 14 groups, there are belong to Antrodia camphorata's specific characters, cancer group、cytotoxin/detoxin group、peroxidase group. Cancer group has 3.3 % of the total genes，cytotoxin/detoxin group has 13.4 %，peroxidase group has 6.1 %。Three of them are 22.8 % of the total genes. In 246 colonies, some genes appear many times. For example : P450 has 3.7 % of the total genes.
We select 3 genes in this Library, P450、GST and prx. To find their cDNA full length and to study their protein function. P450 cDNA has 1491 bp and it can translated 497 amino acid residues ( add stop codon ), we observe P450 protein in SDS-PAGE, P450 maybe it‘s a kind of 70 kDa protein. David Nelson think our P450 is most like a white rot fungal sequence from Phanerochaete chrysosporium. ( 46 % ). This gene is in a small family of related genes that have not been named yet.These genes will probably be in the CYP5014 family, but that is not official yet.
GST cDNA has 651 bp and it can translated 217 amino acid residues ( add stop codon ), GST protein must be a kind of 22 kDa protein, because GST protein is dimmer structure, we find it‘s appear in 60 kDa in SDS-PAGE.
Otherwise, we find prx cDNA has 645 bp and it can translated 215 amino acid residues ( add stop codon ) , we observe prx protein appear in 32.5 kDa of SDS-PAGE. After experiments, we discover Antrodia camphorata‘s prx is 2-Cys prx, and it‘s a kind of monomer structure protein. We use MFO system to prove prx protein‘s function. DNA will be nicked and some enzymes will be destroyed and lost their function in MFO system. But prx protein can protect DNA nucleotides and glutamine synthetase‘s activity.

目 錄

中文摘要

英文摘要

縮寫表

第壹章 續論

1-1 樟芝物種介紹
1-2 樟芝菌種發表經過
1-3 樟芝的形態
1-4 樟芝引起之病徵
1-5 樟芝的發生生態
1-6 樟芝成份
1-7 樟芝的功效與價值

第貳章: 樟芝子實體與菌絲體的PCR Select Subtraction cDNA
Library

2-1 PCR Select Subtraction cDNA Library

2-2 PCR Select Subtraction cDNA Library實驗材料與方法

2-3 PCR Select Subtraction cDNA Library結果與討論


第參章 : 樟芝Cytochrome P450 (P450)之研究

3-1 前言
3-2 P450文獻回顧
3-3 P450 and GST實驗材料與方法
3-4 P450結果與討論

第肆章: 樟芝Glutathione s transferase (GST)之研究

4-1 GST文獻回顧
4-2 GST kinetic study實驗材料與方法
4-3 GST結果與討論

第伍章: 樟芝Peroxiredoxin (Prx)之研究

5-1 前言
5-2 prx文獻回顧
5-3 prx實驗材料與方法
5-4 prx結果與討論

文獻索引

附錄

文獻索引

張東柱、謝煥儒、張瑞璋、傅春旭, 台灣常見樹木病害， 1990

高曉薇，”台灣靈芝屬新種樟芝之三帖類成份研究”，台灣醫學院天然物醫學研究所碩士論文，1991

程一華，”樟芝之成份研究”，台灣師範大學化學研究所碩士論文，1994

水野卓 川合正允原著，菇類的化學、生物化學，國立編譯館，1997

陳勁初、林文鑫、陳勁農、許勝傑、黃仕政、陳炎鍊…台灣特有真菌-樟芝菌絲體之開發，
Fungal Science, 2001, 16 (1,2):7-12.

蔡雁暉，”樟芝深層培養液及其多醣體之抗氧化特性”，國立中興大學食品科學系碩士論文，2002

郭淑卿，”樟芝發酵液對大鼠肝臟纖維化及胃腸功能之改善作用”，中國醫藥學院中國藥學研究所碩士論文，2002

葉怡真，”樟芝對血管內皮細胞之影響集保心血管疾病之機制探討”，中國醫藥學院營養研究所碩士論文，2002

陳欣怡，”牛樟芝發酵過濾液對大白鼠肝臟生理機能之影響”，中國醫藥學院營養研究所碩士論文，2002

藏穆、蘇慶華，”我國台灣產靈芝屬新種樟芝”，雲南植物研究，1990，12，305-396

Boyland E, Chasseaud LF. Glutathione S-aralkyltransferase. The Biochemical journal. 1969, 115(5): 985-91.

B. Bieseler, C. Fedtke, T. Neuefeind, W. Etzel, L. Prade and P. Reinemer.
Maize selectivity of FOE 5043: Degradation of active ingredient by glutathione-S-transferases. Pflanzenschutz-Nachrichten Bayer. 1997, 2 : 117-140.

Barr SD, Gedamu L. Cloning and characterization of three differentially expressed peroxidoxin genes from Leishmania chagasi. The Journal of Biological Chemistry. 2001, 276(36): 34279–34287.

Brehelin C, Meyer EH, de Souris JP, Bonnard G, Meyer Y. Resemblance anddissemblance of Arabidopsis type II peroxiredoxins: similar sequences for divergent gene expression, protein localization, and activity. Plant Physiology. 2003, 132: 2045–2057.

Chang, T. T. & Chou, W. N, Antrodia cinnamomea sp. nov.on Cinnamomea kanehirai in Taiwan. Mycological research. 1995, 99: 756-758.

Cherng, I. H. & Chiang, H. C. Three new triterpenoids from Antrodia cinnamomea. Journal of Natural Products, 1995, 58: 365-371.

Chen, C. H., Yang, S. W. & Shen, Y. G. New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum. Journal of Natural Products, 1995, 58: 1655-1661.

Chiang, H. C., Wu, D. P., Cherng, I. H. & Ueng, C. H. A sesquiterpene lactone,phenyl and biphenyl compounds from Antrodia cinnamomea. Phytochemistry, 1995, 39: 613-616..

Chapple C. Molecular-genetic analysis of plant cytochrome P450-dependendmonooxygenases. Annu. Rev. Plant Physiol. Plant Molecular Biology. 1998, 49: 311-343.

Cheong NE, Choi YO, Lee KO, Kim WY, Jung BG, Chi YH, Jeong JS, Kim K, Cho MJ, Lee SY. Molecular cloning, expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage. Plant Molecular Biology. 1999, 40: 825–834.

Choi YO, Cheong NE, Lee KO, Jung BG, Hong CH, Jeong JH, Chi YH, Kim K, ChoMJ, Lee SY. Cloning and expression of a new isotype of the peroxiredoxin gene of chinese cabbage and its comparison to 2Cys-peroxiredoxin isolated from the same plant. Biochemical and Biophysical Research Communications . 1999, 258: 768–771.



Curran JE, Weinstein SR, Griffiths LR. Polymorphisms of glutathione S-transferase genes (GSTM1, GSTP1 and GSTT1) and breast cancer susceptibility. Cancer Letters 2000, 153: 113-120.

Castro H, Sousa C, Santos M, Cordeiro-da-Silva A, Flohe L, Tomas AM. Complementary antioxidant defense by cytoplasmic and mitochondrial peroxiredoxins in Leishmania Infantum. Free Radical Biology & Medicine. 2002, 33(11) :1552–1562.

Coon MJ. Multiple oxidants and multiple mechanisms in cytochrome P450 catalysis. Biochemical and Biophysical Research Communications. 2003, 312: 163–168.

Declercq JP, Evrard C, Clippe A, Stricht DV, Bernard A, Knoops B. Crystal structure of human peroxiredoxin 5, a novel type of mammalian peroxiredoxin at 1.5 a resolution. Journal of Molecular Biology. 2001, 311: 751-759.

Dixon DP, Lapthorn A, Edwards R. Plant glutathione transferases. Genome Biology. 2002, 3(3): reviews 3004.1–reviews 3004.10.

Farnden, K. J. F. & Robertson, J. G. Methods for studying enzymes involved in metabolism related to nitrogenase. Nitrogen Fixation. 1980, 265-314.

Günther Regelsberger, Christa Jakopitsch, Lisa Plasser, Hansjörg Schwaiger, Paul Georg Furtmüller, Günter A. Peschek, Marcel Zámocký and Christian Obinger Occurrence and biochemistry of hydroperoxidases in oxygenic phototrophic prokaryotes (cyanobacteria) Plant Physiology and Biochemistry. 2002, 479-490.

Graham SE, Peterson JA. How similar are P450s and what can their differences teach us? Biochemistry and Biophysics. 1999, 369(1) : 24–29.

Habig WH, Pabst MJ, Jakoby WB. Glutathione s-transferases. The first enzymatic step in mercapturic acid foemation. The Journal of Biological Chemistry. 1974, 249(22): 7130-7139.




Hseu, Y. C., Chang, W. C., Hseu, Y. T., Lee, C. Y., Yech, Y. J., Chen, P. C., Chen, J. Y., Yang, H. L., Protection of oxidative damage by aqueous extract from Antrodia cinnamomea mycelia in normal human erythrocytes. Life Science, 2002, 71:469-482.

Hillebrand H, Schmidt A, Krauth-Siegel RL. A second class of peroxidases linked to the trypanothione metabolism. The Journal of Biological Chemistry. 2003, 278(9): 6809–6815.

Hsiao, G., Shen, M. Y., Lin, K. H., Lan, M. H., Wu, L. Y., Chou, D. S., Su, C. H., and Sheu, J,. R. Antioxidative and Hepatoprotective effect of Antrodia cinnamomea extract. Journal of Agricultural and Food Chemistry. 2003, 51:3302-3308.

Irina F. Sevrioukova, Huiying li, Hong zhang, Julian A. Peterson, Thomas L. Poulos Biochemistry structure of a cytochrome P450–redox partner electron-transfer complex. Proceedings of the national academy of sciences of the United States of America. 1999,.96: 1863–1868.

Jakoby WB, Ziegler DM. The enzymes of detoxication. The Journal of Biological Chemistry. 1990, 265(34):20715-20718.

Joaquin Albenisio Gomes da Silveira, José Luis Condato, Jorge Luiz Mazza Rodrigues, José Tadeu Abreu de Oliveira. Phosphoenolpyruvate carboxy1ase and glutamine synthetase activities in relation to nitrogen fixation in cowpen nodules. Revista Brasileira de Fisiologia Vegetal. 1998, 10(1): 19-23.

Kim K, Kim IH, Lee KY, Rhee SG, Stadtman ER..
The isolation and purification of a specific〝Protector〞protein which inhibits enzyme inactivation by a Thiol/Fe(Ⅲ)/O2 mixed-funtion oxidation system. The Journal of Biological Chemistry. 1988, 263 : 47044-4711.

Kong W, Shiota S, Shi Y, Nakayama H, Nakayama K.
A novel peroxiredoxin of the plant Sedum lineare is a homologue of Escherichia coli bacterioferritin co-migratory protein (Bcp). The Biochemical journal. 2000, 351: 107-114.


Kawazu S, Tsuji N, Hatabu T, Kawai S, Matsumoto Y, Kano S. Molecular cloning and characterization of a peroxiredoxin from the human malaria parasite Plasmodium falciparum. Molecular and Biochemical Parasitology. 2000, 109 :165–169.

Kawazu S, Komaki K, Tsuji N, Kawai S, Ikenoue N, Hatabu T, Ishikawa H, Matsumoto Y, Himeno K, Kano S. Molecular characterization of a 2-Cys peroxiredoxin from the human malaria parasite Plasmodium falciparum. Molecular & Biochemical Parasitology . 2001, 116 :73–79.

Konig J, Lotte K, Plessow R, Brockhinke A, Baier M, Dietz KJ. Reaction mechanism of plant 2-Cys peroxiredoxin. The Journal of Biological Chemistry. 2003, 278( 27): 24409–24420.

Lee AJ, Huntley J, Van den Broek A, Coates D, Isaac RE Expression and characterisation of a Psoroptes ovis glutathione s-transferase. Veterinary Parasitology. 2002, 105 : 49–63.

Mannervik B, Danielson UH. Glutathione transferases--structure and catalytic activity. Critical reviews in biochemistry and molecular biology . 1988;23(3):283-337.

Meister A. Glutathione metabolism and its selective modification. The Journal of Biological Chemistry. 1988, 263(33): 17205-17208.

Mizuno, T. Biomolecules of mushroom: Food function and medicinal effect of mushroom fungi. Food Research International. 1995, 11: 7-21

Nelson DR, Koymans L, Kamataki T,Stegeman JJ, Feyereisen R, Waxman DJ,Waterman MR,Gotoh O,Coon MJ, Estabrook RW,et al,. P450 superfamily: update on new sequences,gene mapping accession numbers and nomenclature. Pharmacogenetics. 1996, 6:1-42.

Nishiyama Y, Massey V, Takeda K, Kawasaki S, Sato J, Watanabe T, Niimura Y. Hydrogen peroxide-forming NADH oxidase belonging to the peroxiredoxin oxidoreductase family: existence and physiological role in bacteria. Journalof Bacteriolog, 2001, 183(8): 2431–2438.

Reinemer P, Prade L, Hof P, Neuefeind T, Huber R, Zettl R, Palme K, Schell J, Koelln I, Bartunik HD, Bieseler B. Three-dimensional structure of glutathione s-transferase from arabidopsis thaliana at 2.2 Å resolution: structural characterization of herbicide-conjugating plant glutathione s-transferases and a novel active site architecture. Journal of Molecular Biology. 1996, 255: 289–309.

Rhee SG, Kang SW, Netto LE, Seo MS, Stadtman ER. A family of novel peroxidase, peroxiredoxins. BioFactors. 1999, 10: 207-209.

Robertson L, Robertson WM, Sobczak M, Helder J, Tetaud E, Ariyanayagam MR, Ferguson MA, Fairlamb A, Jones JT. Cloning, expression and functional characterisation of a peroxiredoxin from the potato cyst nematode Globodera rostochiensis. Molecular and Biochemical Parasitology. 2000, 111: 41–49.

Radyuk SN, Klichko VI, Spinola B, Sohal RS, Orr WC. The peroxiredoxin gene family in Drosophila Melanogaster. Free Radical Biology & Medicine. 2001, 31( 9): 1090–1100.

Roberts GA, Grogan G, Greter A, Flitsch SL, Turner NJ. Identification of a new class of cytochrome P450 from a Rhodococcus sp. Journal of Bacteriology. 2002, 184(14): 3898–3908.

Rauch N, Nauen R. Characterization and molecular cloning of a glutathione S-transferase from the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae). Insect Biochemistry and Molecular Biology. 2004, 34(4): 321-329.

Seo MS, Kang SW, Kim K, Baines IC, Lee TH, Rhee SG. Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate. The Journal of Biological Chemistry. 2000, 275(27): 20346–20354.

Son ES, Song KJ, Shin JC, Nam HW Molecular cloning and characterization of peroxiredoxin from Toxoplasma gondii. The Korean Journal of Parasitol. 2001 , 39(2):133-141.

Sheehan D, Meade G, Foley VM, Dowd CA. Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. The Biochemical journal. 2001, 360: 1–16.
Sakamoto A, Tsukamoto S, Yamamoto H, Ueda-Hashimoto M, Takahashi M, Suzuki H, Morikawa H. Functional complementation in yeast reveals a protective role of chloroplast 2-Cys peroxiredoxin against reactive nitrogen species. The Plant Journal. 2003, 33: 841–851.

Tsuji N, Kasuga-Aoki H, Isobe T, Yoshihara S. Cloning and characterisation of a peroxiredoxin from the swine roundworm Ascaris suum . International Journal for Parasitology. 2000, 30: 125-128.

The Worldwide Physiologist: cytochrome P450 www.anaesthetist.com/physiol/basics/metabol/cyp/cyp.htm

Urlacher V, Schmid RD. Biotransformations using prokaryotic P450 monooxygenases. Biotechnology. 2002, 13:557–564.

Werck-Reichhart D, Feyereisen R. Cytochromes P450: a success story.
Genome Biology. 2000, 1(6): reviews 3003.1–reviews 3003.9.

Wood ZA, Poole LB, Hantgan RR, Karplus PA. Dimers to doughnuts: redox-sensitive oligomerization of 2-Cysteine peroxiredoxins. Biochemistry. 2002, 41: 5493-5504.

Woo HA, Chae HZ, Hwang SC, Yang KS, Kang SW, Kim K, Rhee SG. Reversing the inactivation of peroxiredoxins caused by cysteine sul.nic acid formation. The Journal of Biological Chemistry. 2002, 277: 38029.

Wiseman A. Limitations of in silico predictability of specificity of co-immobilised cytochromes P450 and mimics in food-bioprocessing. Biotechnology Letters. 2003, 25: 515–519.

Wood ZA, Schroder E, Robin Harris J, Poole LB. Structure, mechanism and regulation of peroxiredoxins. Biochemical Sciences. 2003, 28(1):32-40.

Yang S. W., Shen Y. C., Chen C. H., Steroids and triterpenoids from Antrodia cinnamomea-a fungus parasitic on Cinnamomum micranthum. Phytochemistry. 1996, 41: 1389-1392.