牛樟芝（Antrodia cinnamomea）為台灣特有的平伏型擔子菌，只生長於台灣保育類樹種牛樟樹的樹心空胴處。傳統中藥裡牛樟芝在民間被視為珍貴的藥材，被認為具有保肝、解毒、抗發炎及抗氧化等功效。而本研究即針對牛樟芝尚未被探討的具強抗氧化能力之類黃酮類生合成基因及產物進行篩選及功能分析。首先，遂由應用真菌研究室所建構及註解之牛樟芝cDNA基因資料庫，顯示三個基因和類黃酮類生合成相關，並由基因片段EST（expresstion sequence tag）設計引子對選殖出flavonol synthase（FLS）、flavonoid 3’ and 5’ hydroxylase（F3’5’H）及chalcone isomerase（CHI）三基因。此外，未被註解之其他類黃酮類生合成基因，如:flavanone 3-hydroxylase （F3H）及4-coumaroyl CoA ligase （4CL）等基因則設計簡併性引子對（degenerate primer）進行選殖，其後將此等基因探針呈雜合反應之fosmid clones進行primer walking，以求得genomic DNA全長，其後預測並進行reverse transcription以選殖cDNA全長。此等基因之生化特性亦經由相關之生物資訊網站加以分析。其後利用反轉錄牛樟芝之total RNA，將類黃酮類生合成路徑的5個基因cDNA全求得並建構在Kluvyromyces lactis之表現載體pKLAC1上，以利轉型至K. lactis乳酸酵母菌系統大量表現，並期望針對基因轉殖之K. lactis產物進行抗氧化及抗癌之活性測試，並希冀應用於臨床藥物與保健食品之領域。另一方面，針對A. cinnamomea菌絲可能產生之類黃酮進行甲醇粗萃取物分析，藉由高效液相層析儀（HPLC）及600 MHz核磁共振儀（NMR）檢測，結果顯示粗萃取物中以醣類居多，而環狀結構化合物則所佔比例不高，相對地推測類黃酮類在菌絲中含量不高，未來期望藉由基因層級於K. lactis大量表現類黃酮類產物並進行特性分析。

Antrodia cinnamomea （Neu-Chang-Ts） an unique endemic resupinate basidiomycete of Taiwan, inhabited on the heartwood cavity of Cinnamomum kanehirae, reputed for alleviating, hangover, inflammation, the hepatitis syndrome and oxidative stress, etc. Attempt to clarify the antioxidation activity of A. cinnamomea, we cloned five flavonoid biosynthesis genes: flavonol synthase （FLS）, flavonoid 3’ and 5’ hydroxylase （F3’5’H）, chalcone isomerase（CHI）, flavonone 3-hydorxylase （F3H） and 4-coumaryl CoA ligase （4CL） via the constructed cDNA and fosmid libraries and PCR approaches. Furthermore, we also worked out the full length of gDNA and cDNA of these genes and their properties characterized. Currently, these genes have been constructed on pKLAC1 shuttle vectors and transformed them into Kluyveromyces lactis, host were undergoing. The expressed secondary products will be assessed and assayed. In other aspects, to clarify wether the existence of flavonoid in A. cinnamomea, the methanolic extracts from mycelium were subjected to high performance liquid chromatography（HPLC）, nuclear magnetic resonance（NMR）analysis. The outcomes indicated carbohydrates, instead of flavonoid-like substance, dominated the most partially purified fraction. Given flavonoid processes major anti-inflammation and antioxidation capacity, perspectively, overexpress the genetically engineered flavonoid biosynthetic genes in pertinent hosts to produce adequate quantify of desired compounds for clinic trals will be the resolution.

口試委員審定書---------------------------------------------------------------------------------I
中文摘要----------------------------------------------------------------------------------------- 1
Abstract ------------------------------------------------------------------------------------------ 2
前言----------------------------------------------------------------------------------------------- 3
前人研究----------------------------------------------------------------------------------------- 6
牛樟芝簡介------------------------------------------------------------------------------------- 6
分類地位------------------------------------------------------------------------------------- 6
牛樟芝型態特徵---------------------------------------------------------------------------- 7
牛樟芝粗萃取液藥理活性研究---------------------------------------------------------- 8
牛樟芝成分分析-------------------------------------------------------------------------- 10
類黃酮類化合物（Flavonoid）------------------------------------------------------------- 11
查爾酮合成酶（Chalcone synthase）-------------------------------------------------- 11
查爾酮異構酶（Chalcone isomerase）------------------------------------------------ 12
黃烷酮3-氫氧化酶（Flavonone 3-hydroxylase）------------------------------------- 12
黃酮醇合成酶（Flavonol synthase）--------------------------------------------------- 13
香豆醯輔酶A黏合酶 （4 coumaroyl-CoA ligase）--------------------------------- 13
類黃酮3’與5’氫氧化酶（Flavonoid 3’ and 5’ hydroxylase）---------------------- 14
材料與方法------------------------------------------------------------------------------------- 15
菌株及其培養----------------------------------------------------------------------------- 15
Genomic DNA萃取----------------------------------------------------------------------16
Total RNA萃取--------------------------------------------------------------------------- 16
Fosmid library類黃酮類相關基因之clone 篩選------------------------------------ 23
未註解基因之簡併性引子對設計----------------------------------------------------- 25
類黃酮類化合物甲醇萃取-------------------------------------------------------------- 25
Southern blot檢測牛樟芝類黃酮類基因copy number ----------------------------- 28
Kluyveromyces lactis 異種表現系統-------------------------------------------------- 29
序列分析----------------------------------------------------------------------------------- 31
結果---------------------------------------------------------------------------------------------- 33
類黃酮類生合成基因選殖 ------------------------------------------------------------- 33
類黃酮3’及5’氫氧化酶（Flavonoid 3’ and 5’ hydroxylase）-------------------------33
黃酮醇合成酶（Flavonol synthase）--------------------------------------------------- 35
黃烷酮3-氫氧化酶（Flavanone 3 hydroxylase）-------------------------------------- 37
香豆醯輔酶A黏合酶 （4 coumaroyl-CoA ligase, 4CL）---------------------------- 39
查爾酮異構酶(Chalcone isomerase)----------------------------------------------------- 41
類黃酮類生合成基因於牛樟芝菌絲體之copy number預測------------------------42
牛樟芝菌絲類黃酮類甲醇萃取及分析------------------------------------------------- 43
討論---------------------------------------------------------------------------------------------- 46
圖表---------------------------------------------------------------------------------------------- 52
參考文獻-------------------------------------------------------------------------------------- 102
附錄一、培養基成分------------------------------------------------------------------------ 107
附錄二、實驗試劑配方---------------------------------------------------------------------- 110
附錄三------------------------------------------------------------------------------------------113
附錄四------------------------------------------------------------------------------------------118

王柏森 (2008). 樟芝固態發酵培養產物之成分分析與抗氧化活性之研究。 國立屏東科技大學生物科技研究所碩士論文。

蔡雁暉 (2002). 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研究所碩士論文。

鄭統隆 (2008). 甘藷花青素與多酚含量之研究。 J Taiwan Aric Res 57, 33-48。

童凱鴻 (2005). 牛樟芝萜類生理活性探討及其生合成相關基因之分析。 國立台灣大學植物病理與微生物學研究所碩士論文。

黃鈴娟 (1999). 樟芝與姬松茸之抗氧化性質及其多醣組成分析。 中興大學食品科學系碩士論文。

Ao, Z., Xu, Z., Lu, Z., Xu, H., Zhang, X. & Dou, W. (2009). Niuchangchih (Antrodia camphorata) and its potential in treating liver diseases. Journal of Ethnopharmacology 121, 194-212.

Austin, M. & Noel, J. (2003). The chalcone synthase superfamily of type III polyketide synthases. Natural Product Reports 20, 79-110.

Ayabe, S. & Akashi, T. (2006). Cytochrome P450s in flavonoid metabolism. Phytochemistry Reviews 5, 271-282.

Bednar, R. & Hadcock, J. (1988). Purification and characterization of chalcone isomerase from soybeans. Journal of Biological Chemistry 263, 9582.

Berente, B., Reichenbacher, M. & Danzer, K. (2001). Improvement of the HPLC analysis of anthocyanins in red wines by use of recently developed columns. Fresenius'' Journal of Analytical Chemistry 371, 68-72.

Bonekamp, F. & Oosterom, J. (1994). On the safety of Kluyveromyces lactis: a review. Applied Microbiology and Biotechnology 41, 1-3.

BRITSCH, L. & GRISEBACH, H. (1986). Purification and characterization of (2S)-flavanone 3-hydroxylase from Petunia hybrida. European Journal of Biochemistry 156, 569-577.

Cheng, J., Huang, N., Chang, T., Ling Wang, D. & Lu, M. (2005). Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells. Life sciences 76, 3029-3042.

Cherng, I., Chiang, H., Cheng, M. & Wang, Y. (1995). Three new triterpenoids from Antrodia cinnamomea. Journal of Natural products 58, 365-371.

Fukusaki, E., Kawasaki, K., Kajiyama, S., An, C., Suzuki, K., Tanaka, Y. & Kobayashi, A. (2004). Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference. Journal of Biotechnology 111, 229-240.

Geethangili, M. & Tzeng, Y. (2009). Review of pharmacological effects of Antrodia camphorata and its bioactive compounds. Evidence-based Complementary and Alternative Medicine. eCAM, 2009, Page 1 of 15

Gross, G. & Zenk, M. (1974). Isolation and properties of hydroxycinnamate: CoA ligase from lignifying tissue of Forsthia. European Journal of Biochemistry 42, 453-459.

Hahlbrock, K. & Grisebach, H. (1970). Formation of coenzyme A esters of cinnamic acids with an enzyme preparation from cell suspension cultures of parsley. FEBS letters 11, 62-64.

Hollman, P., van Trijp, J. & Buysman, M. (1997). Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man. FEBS letters 418, 152-156.

Holton, T., Brugliera, F. & Tanaka, Y. (1993). Cloning and expression of flavonol synthase from Petunia hybrida. The Plant Journal 4, 1003-1010.

Holton, T. & Cornish, E. (1995). Genetics and biochemistry of anthocyanin biosynthesis. The Plant Cell 7, 1071.

Hseu, Y., Chang, W., Hseu, Y., Lee, C., Yech, Y., Chen, P., Chen, J. & Yang, H. (2002). Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life sciences 71, 469-482.

Hseu, Y., Chen, S., Yech, Y., Wang, L. & Yang, H. (2008). Antioxidant activity of Antrodia camphorata on free radical-induced endothelial cell damage. Journal of Ethnopharmacology 118, 237-245.

Huang, S. & Mau, J. (2006). Antioxidant properties of methanolic extracts from Agaricus blazei with various doses of [gamma]-irradiation. LWT-Food Science and Technology 39, 707-716.

Jez, J., Bowman, M. & Noel, J. (2002). Expanding the biosynthetic repertoire of plant type III polyketide synthases by altering starter molecule specificity. Proceedings of the National Academy of Sciences of the USA, 99, 5319.

Katsumoto, Y., Fukuchi-Mizutani, M., Fukui, Y. & other authors (2007). Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant and Cell Physiology 48, 1589.

Lee, I., Huang, R., Chen, C., Chen, H., Hsu, W. & Lu, M. (2002). Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiology Letters 209, 63-67.

Lee, T.-H., Lee, C.-K., Tsou, W.-L., Liu, S.-Y., Kuo, M.-T. & Wen, W.-C. (2007). A new cytotoxic agent from solid-state fermented mycelium of Antrodia camphorata. Natural Product Chemistry 73, 1412-1415.

Leonard, E., Lim, K., Saw, P. & Koffas, M. (2007). Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Applied and Environmental Mmicrobiology 73, 3877–3886.

Liu, D., Liang, H., Chen, C. & other authors (2007). Comparative anti-inflammatory characterization of wild fruiting body, liquid-state fermentation, and solid-state culture of Taiwanofungus camphoratus in microglia and the mechanism of its action. Journal of Ethnopharmacology 113, 45-53.

Mau, J., Huang, P., Huang, S. & Chen, C. (2004). Antioxidant properties of methanolic extracts from two kinds of Antrodia camphorata mycelia. Food Chemistry 86, 25-31.

Muir, S., Collins, G., Robinson, S., Hughes, S., Bovy, A., De Vos, C., van Tunen, A. & Verhoeyen, M. (2001). Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols. Nature Biotechnology 19, 470-474.

Ono, E., Fukuchi-Mizutani, M., Nakamura, N. & other authors (2006). Yellow flowers generated by expression of the aurone biosynthetic pathway. Proceedings of the National Academy of Sciences USA 103, 11075.

Rani, A., Singh, K., Sood, P., Kumar, S. & Ahuja, P. (2009). p-Coumarate: CoA ligase as a key gene in the yield of catechins in tea [Camellia sinensis (L.) O. Kuntze]. Functional & Integrative Genomics 9, 271-275.

Rao, Y., Fang, S. & Tzeng, Y. (2007). Evaluation of the anti-inflammatory and anti-proliferation tumoral cells activities of Antrodia camphorata, Cordyceps sinensis, and Cinnamomum osmophloeum bark extracts. Journal of Ethnopharmacology 114, 78-85.

Read, J., Colussi, P., Ganatra, M. & Taron, C. (2007). Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains. Applied and Environmental Microbiology 73, 5088–5096.

Shen, B. (2003). Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Current Opinion in Chemical Biology 7, 285-295.

Song, T. & Yen, G. (2002). Antioxidant properties of Antrodia camphorata in submerged culture. Ariculture and general of Food Chemistry 50, 3322-3327.

Teffo, L. S., Aderogba, M. A. & Eloff, J. N. (2010). Antibacterial and antioxidant activities of four kaempferol methyl ethers isolated from Dodonaea viscosa Jacq. var. angustifolia leaf extracts. South African Journal of Botany 76, 25-29.

Terao, J., Yamaguchi, S., Shirai, M., Miyoshi, M., Moon, J., Oshima, S., Inakuma, T., Tsushida, T. & Kato, Y. (2001). Protection by quercetin and quercetin 3-O-￡]-D-glucuronide of peroxynitrite-induced antioxidant consumption in human plasma low-density lipoprotein. Free Radical Research 35, 925-931.

Trantas, E., Panoulos, N. & Ververidis, F. (2009). Metabolis engneering of the complete pathway leading to heterologous bisynthesis of various flavonoids and stilbenoids in Sacchramyces cerevisiae. Metabolic Engineering 11, 355-366.

Wong, E. (1967). Flavanone biosynthesis. Tetrahedron Letters 7, 3021-3022.

Wu, S. H., Ryvarden, L. & Chang, T. T. (1997). Antrodia camphoratum (“niu-chang-chih”), new combination of a medicinal fungus in Taiwan. Bot Bull Acad Sin 38, 273-275.

Yan, X., Murphy, B., Hammond, G., Vinson, J. & Neto, C. (2002). Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon). Ariculture and general of Food Chemistry 50, 5844-5849.

Yang, H., Hseu, Y., Chen, J., Yech, Y., Lu, F., Wang, H., Lin, P. & Wang, B. (2006). Antrodia camphorata in submerged culture protects low density lipoproteins against oxidative modification. American Journal of Chinese Medicine 34, 217.