臺灣博碩士論文加值系統

牛樟芝，學名為Antrodia cinnamomea，為台灣特有之藥用真菌，具抗癌、抗發炎和調節免疫等療效。本試驗為了提高牛樟芝菌絲體之多醣及三萜類等活性成分，以農桿菌品系EHA105及主動誘變載體pTAG-8進行牛樟芝菌絲體基因轉殖並找出受影響之基因，試驗成功獲得16個轉殖系菌絲體(A∼P)，經由南方墨點法分析後得知有81.25％的轉殖系菌絲體為單一T-DNA嵌入，其中不僅菌絲顏色發生變化，且所有轉殖系之生長速度皆慢於未轉殖菌絲體(20％∼40％)。利用酚硫酸法進行胞外多醣含量測定，其中顯著高於未轉殖菌絲體的轉殖系為A、C、K、L、N與O。以HPLC測定同時存在牛樟芝子實體與菌絲體中之dehydrosulphurenic acid與dehydroeburicoic acid等三萜類，得知具治療攝護腺癌效果的前者在轉殖系D、H、I、J與K中的含量明顯高於未轉殖菌絲體；具抗發炎效果的後者則於轉殖系A、B、D、H與I中含量較高。同時含高量dehydrosulphurenic acid與dehydroeburicoic acid之轉殖系H的清除DPPH自由基能力為所有轉殖系中最佳，相當於子實體。在經由inverse PCR選殖得知其T-DNA嵌入基因為DIMETHYLALLYL TRYPTOPHAN SYNTHASE後，確定該基因表現量在轉殖系H中受到抑制，因此推測該基因應與轉殖系H含高量dehydrosulphurenic acid與dehydroeburicoic acid相關。
關鍵字: 牛樟芝、農桿菌、主動誘變

Antrodia cinnamomea is well known in Taiwan as a source of traditional Chinese medicine for the treatment of cancer and inflammation. In order to increase the content of active compounds, such as polysaccharides and triterpenoids in mycelia, Agrobacterium tumefaciens strain EHA105 harboring a standard binary vector pTAG-8 was used to generate the transgenic mycelia population. Sixteen transgenic mycelia (A~P) were obtained and confirmed by Southern blots(81.25％ of them with single copy insertion). Changes in mycelia color and growth rate were observed in all transgenic lines. Exopoly- ssacharides contents in transgenic lines A, C, K, L, N and O were significantly increased. Triterpenoids like dehydrosulphurenic acid (used for prostate cancer therapy) and dehydroeburicoic acid (used for anti-inflammation) in both fruiting bodies and mycelia were remarkably increased in transgenic lines D, H, I, J, K and A, B, D, H, I, respectively. Transgenic line H, containing high dehydrosulphurenic acid and dehydroeburicoic acid, was observed to have high scavenging activity. In transgenic line H, the DIMETHYLALLYL TRYPTOPHAN SYNTHASE(DTS) was as confirmed by gene expression. Mutation in DTS might cause accumulation of dehydrosulphurenic acid and dehydroeburicoic acid in transgenic line H.
Key words: Antrodia cinnamomea, Agrobacterium tumefaciens, Activation
tagging.

目錄
中文摘要.................................................................................................... I
英文摘要...................................................................................................II
誌謝.........................................................................................................IV
目錄.........................................................................................................V
表目錄....................................................................................................VIII
圖目錄...................................................................................................IX
附錄...................................................................................................XI
壹、前言................................................................................................... 1
貳、文獻回顧........................................................................................... 2
一、牛樟芝與其主要活性成分之介紹................................................... 2
(一)牛樟芝命名與分類........................................................................ 2
(二)牛樟芝分布與特徵. ....................................................................... 3
(三)牛樟芝藥理之應用. ....................................................................... 4
(四)牛樟芝活性成分. ........................................................................... 6
二、真菌基因轉殖與主動誘變............................................................ 11
(一)真菌基因轉殖方法之介紹.......................................................... 12
(二)主動誘變之機制.......................................................................... 14
参、材料方法......................................................................................... 16
一、試驗材料........................................................................................ 16
(一)牛樟芝菌種來源與保存. ............................................................. 16
(二)農桿菌與大腸桿菌菌種來源與保存. .......................................... 16
(三)大腸桿菌勝任細胞菌製備與保存............................................... 16
二、試驗方法........................................................................................ 17
(一)牛樟芝菌絲體之基因轉殖. ......................................................... 17
(二)農桿菌與大腸桿菌質體DNA 之萃取. ....................................... 18
(三)牛樟芝菌絲體染色體DNA 之萃取. ........................................... 19
(四)轉殖系牛樟芝菌絲體之確認....................................................... 20
(五)牛樟芝菌絲體生長速率之測定................................................... 23
(六)牛樟芝菌絲體之液態培養. ......................................................... 24
(七)牛樟芝菌絲體乾重與胞外粗多醣含量之測定. .......................... 25
(八)牛樟芝菌絲體甲醇萃取物清除自由基能力之測定.................... 26
(九)牛樟芝菌絲體甲醇萃取物總多酚含量之測定. .......................... 27
(十)牛樟芝菌絲體甲醇萃取物中三萜類含量之測定. ...................... 27
(十一)T-DNA 鄰近序列之選殖. ............................................................ 28
(十二)基因定量之分析. ......................................................................... 31
肆、結果................................................................................................. 33
一、主動誘變轉殖系牛樟芝菌絲體之確認......................................... 33
(一)轉殖系牛樟芝菌絲體之抗生素篩選結果................................... 33
(二)轉殖系牛樟芝菌絲體分子鑑定之結果....................................... 33
二、轉殖系牛樟芝菌絲體外觀顏色及生長速率之改變..................... 34
三、轉殖系牛樟芝菌絲體二次代謝物之改變..................................... 35
(一)不同液態培養方式對轉殖系牛樟芝多醣體含量之影響............ 35
(二)不同培養方式之轉殖系牛樟芝菌絲體與子實體三萜含量之比較
........................................................................................................... 36
(三)轉殖系牛樟芝菌絲體與子實體甲醇萃取物抗氧化能力之比較.37
(四)轉殖系牛樟芝菌絲體與子實體甲醇萃取物總多酚含量之比較.38
四、T-DNA 鄰近序列分析之結果....................................................... 38
(一)利用inverse PCR 進行T-DNA 鄰近序列之選殖. ...................... 39
(二)選殖之基因對三萜類生合成之影響........................................... 40
伍、討論................................................................................................. 42
陸、結論與未來展望.............................................................................. 48
柒、參考文獻......................................................................................... 71
捌、附錄................................................................................................. 80
表目錄
表1、PCR 檢測轉殖基因GUS 與HPT 使用引子之一覽表................. 67
表2、Inverse PCR 反應使用引子之一覽表........................................... 67
表3、基因定量使用引子之一覽表........................................................ 67
表4、三萜類標準品移動相沖提梯度之一覽表.................................... 68
表5、高效液相層析分析使用儀器之一覽表........................................ 68
表6、轉殖系牛樟芝菌絲體甲醇萃取物中羊毛甾酸化合物含量之測定.
................................................................................................................. 69
表7、轉殖系牛樟芝菌絲體甲醇萃取物清除DPPH 自由基能力之測定.
................................................................................................................. 70
圖目錄
圖 1、牛樟芝子實體與菌絲體外觀型態............................................... 50
圖 2、多醣體含量測試之葡萄糖檢量線............................................... 51
圖 3、總多酚含量測試之catechin 檢量線............................................ 51
圖 4、三萜類標準品高效液相層析分析圖譜....................................... 52
圖 5、牛樟芝dehydrosulphurenic acid 三萜檢量線.............................. 52
圖 6、牛樟芝dehydroeburicoic acid 三萜檢量線.................................. 52
圖 7、農桿菌感染牛樟芝菌絲體疑似轉殖成功之情形....................... 53
圖 8、利用PCR 檢測農桿菌質體與轉殖系牛樟芝菌絲體.................. 54
圖 9、利用南方墨點法檢測轉殖系牛樟芝菌絲體............................... 55
圖10、依據轉殖系牛樟芝菌絲體顏色變化區分.................................. 56
圖11、轉殖系與未轉殖牛樟芝菌絲體生長曲線調查.......................... 57
圖12、轉殖系牛樟芝菌絲體胞外粗多醣體含量.................................. 58
圖13、轉殖系牛樟芝菌絲體甲醇萃取物之HPLC 分析圖譜.............. 60
圖14、轉殖系牛樟芝菌絲體甲醇萃取物總多酚類含量...................... 61
圖15、轉殖系牛樟芝菌絲體抗氧化能力與總多酚含量之關係........... 62
圖16、Inverse PCR 分析及基因選殖.................................................... 63
圖17、轉殖系牛樟芝菌絲體H 之T-DNA 鄰近序列與牛樟芝Expressed
sequence tags library 之比對........................................................ 64
圖18、轉殖系牛樟芝菌絲體H 之T-DNA 崁入之位置....................... 65
圖19、DIMETHY LALLYL TRYPTOPHAN SYNTHASE 表現量之分析66
附錄
附錄1、試驗設計流程示意圖............................................................... 80
附錄2、牛樟芝子實體中ergostane 型化合物....................................... 81
附錄3、牛樟芝子實體中lanostane 型化合物....................................... 82
附錄4、農桿菌EHA105/pTAG-8 質體之示意圖................................ 83
附錄5、AAM 培養基之組成成分......................................................... 84
附錄6、南方墨點之毛細管法轉印核酸裝置之示意圖........................ 85
附錄7、Inverse PCR 之示意圖.............................................................. 86
附錄8、pGEM®-T Easy Vector 質體之示意圖...................................... 87
附錄9、三萜類生合成相關途徑之示意圖............................................ 88

李宛蓁，「樟芝菌絲體培養與生理活性成分生成之研究」，碩士論文，東海大學化學工程學系，台中(2003)。

肖崇厚與陳蘊如，中藥化學，科學技術出版社，上海，第323-360頁(1989)。

余淑美，「植物功能性基因體研究」，後基因體時代之生物技術，第41-50
頁(2003)。

林金鑫、曾虹萍、陳勁初、蔡明憲、楊明芬與陳巧文，「大鼠致畸測一樟芝發酵原液凍乾成品」，中華保健食品學會第二屆第一次會員大會(2001)。

高曉薇，「台灣靈芝屬新種樟芝之三萜類成份研究」碩士論文，台北醫學院天然物醫學研究所，台北(1991)。

張東柱，「牛樟之病害-牛樟生物學及育林技術研討會論文集」，林業叢刊，第72號，第127-131頁(1997)。

陳勁初、林文鑫、陳清農、許勝傑、黃仕政與陳炎鍊，「台灣特有真菌-樟芝菌絲體之開發」，中華真菌學會會刊，第16卷，第1,2期，第7-22頁(2001a)。

陳啟楨、蘇慶華與藍明煌，「樟芝固體栽培及其生物活性之研究」，Fung Science，第16期，第65-72頁(2001b)。

許勝傑、陳清農與陳勁初，「樟芝宿主專一性之探討」，台灣農業化學與食品科學，第38期，第533-539頁(2000)。

許勝傑、鍾煒惠、陳勁初、蔡明憲、王聖耀、蔡慶龍與陳巧文，「大白鼠口服高劑量樟芝菌絲體之急性毒性試驗」，中華保健食品學會第二屆第一次會員大會(2001)。

程一華，「樟芝之成份研究」，碩士論文，國立臺灣師範大學化學學系，台北(1994)。

童凱鴻，「牛樟芝萜類生理活性探討及其生合成相關基因之分析」，碩士論文，國立台灣大學植物病理與微生物學研究所，台北(2005)。
黃鈴娟，「樟芝與姬松茸之抗氧化性質及其多醣組成分析」，碩士論文，中興大學食品科學研究所，台中(2000)。

蘇毓琇，「牛樟芝利用農桿菌進行基因轉殖系統之研究」，碩士論文，朝陽科技大學生物技術研究所，台中(2006)。

水野卓與川合正允，菇類的化學-生物化學，國立編譯館(1997)。

Balhadere, P. V., Foster, A. J., and Talbot, N. J., “Identification of pathogenicity mutants of the rice blast fungus Magnaporthe grisea by insertional mutagenesis,” Mol Plant Microbe Interact, Vol. 12, pp. 129-142 (1999).

Balzergue, S., Dubreucq, B., Chauvin, S., Le-Clainche, I., Le Boulaire, F., de Rose, R., Samson, F., Biaudet, V., Lecharny, A., Cruaud, C., Weissenbach, J., Caboche, M., and Lepiniec, L., “Improved PCR-walking for large-scale isolation of plant T-DNA borders,” Biotechniques, Vol. 30, pp. 496-504 (2001).

Bradford, M. M., “A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein–dye
binding,” Anal. Chem, Vol.72, pp. 248-254 (1976).

Bundock, P. den., Dulk-Ras A., and Beijersbergen A., “Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cervisiae,” EMBO, Vol. 14, pp. 3206-3214 (1995).

Caroline, B., Michielse, A., Paul, J. J., Hooykaas, Cees A. M. J. J. van den Hondel, A., and Arthur F. J. Ram., “Agrobacterium-mediated transformation as a tool or functional genomics in fungi,” Curr. Genet., Vol. 48, pp. 1-17 (2005).

Chakraborty, B. N., Patterson, N. A., and Kapoor, M., “An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi,” Canadian Journal of Microbiology, Vol. 37, pp. 858-863 (1991).

Chang, T. T., and Chou, W. N., “Antrodia cinnamomea reconsidered and A. salmonea sp.nov. on Cunninghamia konishii in Taiwan,” Bot. Bull. Acad. Sin. Vol. 45, pp. 347-352 (2004).

Chang, T. T., and Chou, W. N., “ Antrodia cinnamomea sp. nov. on Cinnamomum kanehirai in Taiwan,” Mycol. Res, Vol. 99, pp. 756-758 (1995).


Chen, C. H., Yang, S. W., and Shen, Y. C., “New steroid acids from antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum,” J. Nat. Prod, Vol. 58, pp. 1655-1661 (1995).

Cherng, I. H., and Chiang, H. C., “Three new triterpenoids from antrodia
cinnamomea,” J. Nat. Prod, Vol. 58, pp. 365-371 (1995).

Cherng, I. H., Wu, D. P., and Chiang, H. C., “Triterpenoids from antrodia
cinnamomea,” Phytochemistry, Vol. 41, pp. 263-267 (1996).

Chiang, H. C., Wu, D., Cherng, I. W., and Ueng, C. H., “A sesquiterpene
lactone, phenyl and biphenyl compounds from Antrodia cinnamomea,”
Phytochemistry, Vol. 39, pp. 613-616 (1995).

Combier, J. P., Melayah, D., Raffier, C., Gay, G., and Marmeisse, R.,
“ Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum,” FEMS Microbiol Lett, Vol. 220, pp. 41-148 (2003).

Covert, S. F., Kapoor, P., Lee, M. H., Briley, A., and Nairn, C. J., “Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum,” Mycol Res, Vol. 105, pp. 259-264 (2001).

Cvitanich, C., and Judelson, H. S., “Stable transformation of the oomycete,
Phytophthora infestans, using microprojectile bombardment,” Curr. Genet, Vol. 42, pp. 228-235 (2003).

Dobinson, K. F., Grant, S. J., and Kang, S., “Cloning and targeted disruption, via Agrobacterium tumefaciens-mediated transformation, of a trypsin protease gene from the vascular wilt fungus Verticillium dahliae ,” Curr Genet, Vol. 45, pp. 104-110 (2004).

Dogasaki, C., Murakami, H., Nishijima, M. N. Oohno., Yadomae, T., and Miyazaki, T., “Biological activity and structural characterization of alkaline-soluble polysaccharides from the kernels of Prunus mume sieb et Zacc,” Bio Pharm Bull, Vol. 17, pp. 386-390 (1994).

Eo, S. K., Kim, Y. S., Lee, C. K., and Han, S. S., “Antiherpetic activities of various protein bound polysaccharide isolated from Ganoderma lucidum,” J Ethnopharma, Vol. 72, pp. 475-81 (1999).


Filley, T. R., Cody, G. D., Goodell, B., Jellison, J., Noser, C., Ostrofsky, A., Adam, G., and Duncan, H., “Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi,” Org. Geochem, Vol. 33, pp. 111-124 (2002).

Fits, L., Hilliou, F., and Memelink, J., “T-DNA activation tagging as a tool to isolate regulators of a metabolic pathway from a genetically non-tractable plant species,” Transgenic Res, Vol. 10, pp. 513-521 (2001).

Ge, X. and Wu, J., “Tanshinone production and isoprenoid pathways in Salvia miltiorrhiza hairy roots induced by Ag+ and yeast elicitor,” Plant Sci, Vol. 168, pp. 487-491 (2005).

Grant, J. J., Chini, A., Basu, D., and Loake, G. J., “Targeted activation tagging of ADR1, a NBS-LRR gene, conveys resistance to virulent pathogens,” Mol Plant Microbe Interact, Vol. 16, pp. 669-680 (2003).

Gomes-Barcellos, F., Pelegrinelli-Fungaro, M. H., Furlaneto, M . C., Lejeune, B., Pizzirani-Kleiner, A. A., and Azevedo, J. L., “Genetic analysis of Aspergillus nidulans unstable transformants obtained by the biolistic process,” Can. J. Microbiol, Vol. 44, pp. 1137-1141 (1998).

Hawksworth, D. L., Kirk, P. M., Sutton, B. C., and Pegler, D. N., “Dictionary of the fungi,” International Mycological Institute (1996).

Hseu, Y. C., Yang, H. L., Lai, Y. C., Lin, J. G., Chen, G. W., and Chang, Y. H., “Induction of apoptosis by Antrodia camphorata in human premyelocytic leukemia HL-60 cells,” Nutr Cancer, Vol. 48, pp. 189-97 (2004).

Hsu, Y. C., Chang, W. C., Hseu, Y. T., Lee, C. Y., Chen, P. C., Chen, J. Y., and Yang, H. L., “Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes,” Life Sci, Vol. 71, pp. 469-482 (2002).

Huang, L. C., “Antioxidant properties and polysaccharide composition analysis of Antrodia camphorata and Agaricus blazei,” Master Thesis, NationalChung-Hsing University, Taichung, Taiwan, pp. 63-76 (2000).

Huang, L. C., Huang, S. J., Chen, C. C., and Mau, J. L., ”Antioxidant
properties of Antrodia camphorata.” 3rd International conference on
Mushroom Biology and Mushroom Product & AMGA’s 26th National
Mushroom Industry conference, pp. 12-16, Sydeny, Australia (1999).
Hutchinson, H. T., and Hartwell, L. H., “Macromolecule synthesis in yeast spheroplasts,” J. Bacteriol, Vol. 94, pp. 1697-1705 (1967).

Jeon, J. S., Lee, S., Jung, K. H., Jun, S. H., Jeong, D. H., Lee, J., Kim, C., Jang, S., Lee, S., Yang, K., Nam, J., An, K., Han, M. J., Sung, R. J., Choi, H. S., Yu, J. H., Choi, J. H., Cho, S. Y., Cha, S. S., Kim, S. I., and An, G., “T-DNA insertional mutagenesis for functional genomics in rice,” Plant J , Vol. 22, pp. 561-570 (2000).

Jeong, D. H., An. S., Kang, H. G., Moon, S., Han, J. J., Park, S., Lee, H. S., An, K., and An, G., “T-DNA Insertional Mutagenesis for Activation Tagging in Rice1,” Plant Physiology, Vol. 130, pp. 1636-1644 (2002).

Komari, T., Hiei, Y., Ishida, Y., Kumashiro, T., and Kubo, T., “Advances in cereal gene transfer,” Plant Biotechnol. Vol. 1, pp. 161-165 (1998).

Kwon-Chung, K. J., Goldman, W. E., Klein, B., and Szaniszlo, P. J., “Fate of transforming DNA in pathogenic fungi,” Medical Mycology, Vol. 36, pp. 38-44 (1998).

Lee, C. Y., Agrawal, D. C., Chou, T. M., Wang, C. S., Yu, S. M., Chen, J. W., and Tsay, H. S., “T-DNA activation tagging as a tool to isolate Salvia miltiorrhiza Bunge transgenic lines for higher yields of tanshinones,” Planta Med, Vol. 74, pp. 1-7 (2008) .

Lieber C. S., “Prevention and treatment of liver fibrosis based on
pathogenesis,” Alcohol Clin Exp Res. Vol.23, No.5, pp. 944-949. Review (1999).

Liu,Y. G., and Whittier, R., “Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking,” Genomics, Vol. 25, pp. 674-681 (1995).

Lu, S., Lyngholm., Yang, G., Bronson, B., Yoder, O. C., and Turgeon, B. G., “Tagged mutations at the Tox1 locus of Cochliobolus heterostrophus by restriction enzyme-mediated integration,” Proc Natl Acad Sci USA, Vol. 91, pp. 12649-12653 (1994).

McClelland, C. M., Chang, Y. C., and Kwon-Chung, K. J., “High frequency transformation of Cryptococcus neoformans and Cryptococcus gattii by Agrobacterium tumefaciens,” Fungal Genet Biol, Vol. 42, pp. 904-913 (2005).


Michielse, C. B., Arentshorst, M., Ram, A. F. J., and Hondel, C. A. M. J. J. v. d., “Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori,” Fungal Genet Biol, Vol. 42, pp. 9-19 (2005).

Michielse, C. B., Ram, A. F. J., Hooykaas, P. J. J., and Hondel, C. A. M. J. J. v. d., “Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full length VirD2, VirC2 or VirE2 leads to insertion of aberrant T-DNA structures,” J. Bacteriol, Vol. 186, pp. 2038-2045 (2004a).

Michielse, C. B., Ram, A. F. J., Hooykaas, P. J. J., Hondel., and Hondel, C. A. M. J. J. v. d., “Role of bacterial virulence proteins in Agrobacterium mediated transformation of Aspergillus awamori,” Fungal Genet Biol, Vol. 45, pp. 571-578 (2004b).

Mizuno, T., “Bioactive biomolecules of mushrooms: food function and medicinal effect of mushroom fungi,” Food Rev.int, Vol. 11, pp. 7-21 (1995).

Mizuno, T., Kinoshita T., Zhuang C., Ito, H., and Mayuzumi, Y.,
“Antitumor-active heteroglycans from niohshimeji mushroom, Tricholoma giganteum,” Biosci., Biotechnol., Biochem, Vol. 59, No.4, pp. 568-571 (1995).

Mohacek-Grosev. V., Bozac. R., Puppels., and G. J., “Vibrational spectroscopic Characterization of wild growing mushrooms and toadstools,” Spectrochimica Acta Part A, Vol. 57, pp. 2815-2829 (2001).

Mullins, E. D., Chen, X., Romaine, P., RAINA, R., GEISER, D. M., and KANG, S., “Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer,” Phytopathology, Vol. 91, pp. 173-180 (2001).

Nakazawa1, M., Ichikawa1, T., Ishikawa1, A., Kobayashi1, H., Tsuhara1, Y., Kawashima1, M., Suzuki1, K., Muto, S., and Minami M., “Activation tagging, a novel tool to dissect the functions of a gene family, ” Plant J, Vol. 34, pp. 741-750 (2003).

Osman, E., Owen, J. S., and Burroughs, A. K., “S-adenosyl-L-methionine - a new therapeutic agent in liver disease,” Aliment Pharmacol Ther, Vol. 7, No.1, pp. 21-28. Review (1993).

Rho, H. S., Kang, S., and Lee, Y. H., “Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea,” J. Mol Cells, Vol. 12, pp. 407-411 (2001).

Riach, M. B. R. and Kinghorn, J. R., “Genetic transformation and vector
developments in filamentous fung,” In Fungal Genetics: Principles and
Practiced, Bos, C.J. pp. 209-233 (1996).

Rice-Evans, C. A., Miller, N. J., and Paganga, G., “Antioxidant properties of phenolic compounds,” Trends Plant Sci. Vol. 2, pp. 152-159 (1997).

Ryhanen, L., Stenback, F., Ala-Kokko, L., and Savolainen, E. R., “The effect of malotilate on type III and type IV collagen, laminin and fibronectin metabolism in dimethylnitrosamine-induced liver fibrosis in the rat,” J. Hepatol, Vol. 24, No. 2, pp. 238-245 (1996).

Schneider A., Kirch, T., Gigolashvili, T., Mock, H. P., Sonnewald, U., Simon, R., Flugge, U. I., and Werr, W., “A transposon-based activation-tagging
population in Arabidopsis thaliana (TAMARA) and its application in the
identification of dominant developmental and metabolic mutations,” FEMS Lett, Vol. 579, pp. 4622-4628 (2005).

Shen, C. C., Kuo, Y. C., Huang, R. L., Lin, L. C., Don, M. J., Chang, T. T., and Chou, C. J., “New ergostane and lanostane from Antrodia camphorata,” J. Chin. Med, Vol. 14, pp. 247-258 (2003).

Shen, Y. C., Yang, S. W., Lin, C. S., Chen, C. H., Kuo, Y. H., and Chen, C. F.,“Zhankuic acid F: a new metabolite from a formosan fungus Antrodia cinnamomea,” Planta Med, Vol. 63, pp. 86-88 (1997).

Shuster, J. R., and Bindel-Connelley, M., “Promoter-tagged restriction enzyme-mediated insertion (PT-REMI) mutagenesis in Aspergillus niger,” Mol Gen Genet, Vol. 262, pp. 27-34 (1999).

Song, T. Y., and Yen, G. C., “Antioxidant properties of Antrodia camphorate in submerged culture,” J. Agri. Food Chem, Vol. 50, pp. 3322-3327 (2002).

Sone, Y., Okuda, R., Wada, N., Kishida, E., and Misaki, A., “Structures and antitumor activaties of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum,” Agric. Biol. Chem, Vol. 49, pp. 2641-2653 (1985).

Tai, T., Akahori, A., and Shingu, T., “Triterpenes of Poria cocos,” Phytochem, Vol.32, No. 5, pp. 1239-1244 (1993).

Tani, H., Xinwei, C., Pedro, N., John, J. G., Marjorie, S., Andrea, C., Eleanor, G., Paul, R. J. B., and Gary, J. L., “Activation tagging in plants: a tool for gene discovery,” Funct. Integr. Genomics, Vol. 4, pp. 258-266 (2004).

Thon, M. R., Nuckles, E. M., and Vaillancourt, L. J., “ Restriction
enzyme-mediated integration used to produce pathogenicity mutants of
Colletotrichum graminicola,” Mo. Plant Microbe Interact, Vol. 13, pp. 1356-1365 (2000).

Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang, M., Thornton, S., and Brettell, R., “ Agrobacterium tumefaciens-mediated barley transformation,” Plant J. Vol. 11, pp. 1369-1375 (1997).

Triglia, T. M., Peterson, G., and Kemp, D. J., “A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences,” Nucleic Acids Res, Vol. 16, pp. 81-86 (1988).

Toroser, D., and Huber, S. C., “3-Hydroxy-3-methylglutaryl-coenzyme A
reductase kinase and sucrose–phosphate synthase kinase activities
in cauliflower florets: Ca2+ dependence and substrate specificities,
Arch,” Biochem. Biophys. Vol. 355, pp. 291-300 (1998).

Wang, S. Y., Hsu, M. L., Hsu, H. C., Tzeng, C. H., Lee, S. S., Shiao, M. S., and Ho, C. K., “The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages,” Int. J. Cancer, Vol.70, pp. 699-705 (1997).

Wasser, S. P., and Weis, A. L., “Medicinal properties of substances occurring in higher basidomycetes mushrooms,” Intern. J. Med. Mushrooms, Vol. 1, pp. 31-62 (1999a).

Weigel, D., Ahn, J. H., Blazquez, M. A., Borevitz, J. O., Christensen, S. K., Fankhauser, C., Ferrandiz, C., Kardailsky, I., Malancharuvil, E. J., Neff, M. M., Nguyen, J. T., Sato, S., Wang, Z., Xia, Y., Dixon, R. A., Harrison, M. J., Lamb, C. J., Yanofsky, M. F., and Chory, J., “Activation Tagging in Arabidopsis,” Plant Physiol, Vol. 122, pp. 1003-1013 (2000).

Wu, D. P., and Chiang, H. C., “Constituents of Antrodia cinnamomea,” J.
Chin.Chem. Soc, Vol. 42, pp. 797-800 (1995).
Wu, S. H., Ryvarden, L., and Chang, T. T., “Antrodia camphorate (“niu-chang-chih”), new combination of a medicinal fungus
in Taiwan,” Bot. Bull. Acad. Sin, Vol. 38, pp. 273-275 (1997).

Wu, S. H., Yu, Z. H., Dai, Y. C., Chen, C. T., Su, C. H., Chen, L. C., Hsu, W. C., and Hwang, G. Y., “Taiwanofungus, a polypore new genus,” Fung. Sci, Vol. 19, pp. 109-16 (2004).

Yang, S. W., Shen, Y. C., and Chen, C. H., “Steroids and triterpenoids of Antrodia cinnamomea-fungus parasitic on Cinnamomum micranthum,” Phytochemistry, Vol. 41, pp. 1389-1392 (1996).

Young, D. S., Chiang, H. C., and Liu, L. K., “Identification of bioactive
components in Antrodia cinnamomea by MS/MS via EI ionization,” J.
Chin.Chem. Soc, Vol. 45, pp. 123-129 (1998).

Zang, M and Su, C. H., “Ganoderma camphoratum, a new taxon in genus Ganoderma from Taiwan,” China. Acta Bot. Yunnanica, Vol. 12, pp. 395-396 (1990).