臺灣博碩士論文加值系統

分子農場可生產具有高價值的醫藥保健用重組蛋白質或工業應用之酵素。建立菇類的基因轉形系統，有助於分子育種和分子農場的發展。本研究使用農桿菌媒介轉形法，經由感染宿主的過程，將 Ti (tumor-inducing) 質體上的目標基因 T-DNA 隨機鑲嵌入宿主染色體，可使目標基因較穩定且有利於後續蛋白質之分析。實驗中建構帶有同源美白菇甘油醛-3-磷酸脫氫酶 (GPD) 啟動子、篩選標記潮黴素抗藥性基因 (hygromycin phosphotransferase, hph) 和報導子綠色螢光蛋白質 (enhanced green fluorescent protein, egfp) 基因的表現載體，將帶有此表現載體之農桿菌與轉形材料美白菇菌絲、菌絲塊進行共培養，皆可順利得到表現 EGFP 之轉形株。在潮黴素選擇性培養基上得到表現 EGFP 之轉形株。篩選標記 hph 轉形效率：菌絲塊具有較佳的轉形效率，轉形效率約 30-40 %，而菌絲體每毫克可獲得 1 個轉形株。帶有異源洋菇、金針菇甘油醛-3-磷酸脫氫酶 (GPD) 啟動子之農桿菌皆可順利感染美白菇，在潮黴素選擇性培養基上得到表現 EGFP 之轉形株。在酵素免疫分析進行 EGFP定量後，美白菇同源啟動子所測得最高 EGFP 含量為每克菌粉乾重中達 27.4 ng，高於洋菇和金針菇啟動子所測得之 EGFP 含量，顯示美白菇啟動子較適合作為美白菇表現 egfp 之啟動子。在研究中選殖出琥珀酸去氫酶 (succinate dehydrogenase, sdi1) 基因序列，進行特定序列的點突變後，利用農桿菌媒介轉形法將此質體鑲嵌入美白菇染色體，可得到具有同源性篩選標記萎�觖F (carboxin) 之轉形株。

The molecular farming may produce pharmaceutically valuable protein or enzymes of the industrial application. The establishment of gene transformation contribute to develop molecular breeding and molecular farming.
In this study, the expression system using Agrobacterium tumefaciens-mediated transformation (ATMT) was developed. A. tumefaciens inserts the T-DNA which is a portion of Ti-plasmid into the genome DNA randomly during infecting host. It could make the target gene more stable and enhance protein analysis. The expression plasmids were constructed to harbor homologous glyceraldehyde-3-phosphate (gpd) promoter from Hypsizygus marmoreus variant (Hmv), Escherichia coli hygromycin B phosphotransferase gene (hph) as the selectable marker and enhanced green fluorescent protein gene (egfp) as reporter gene. A. tumefaciens carrying these plasmids were cocultivated with vegetative mycelia, modified mycelia pellets (MMP) of Hmv for 3~6 days, respectively. The transformation efficiency was 1 transformant per mg dry weight of mycelia and about 30~40% with MMP. The heterologous gpd promoters from Agaricus bisporous and Flammulina velutipes expressed egfp gene successfully in Hmv. The western blotting analysis revealed the EGFP was expressed indeed. From the ELISA quantification analysis, the egfp expressed by Hmv promoter could reach to 27.4 ng per gram dry weight of mycelia, which was higher than by A. bisporus and F. velutipes. This study show the homologous Hmv gpd promoter was more efficient for egfp expression in Hmv than heterologous promoter. In this study, we cloned the succinate dehydrogenase gene (sdi1) from Hmv and introduced a point mutation that confers carboxin resistance into it. After transfer the recombinant plasmid into Hmv by ATMT, we could get the transformants which confers homologous selectable resistance.

中文摘要 I
Abstract II
目錄 III
表目錄 V
圖目錄 VI
第一章 前言 1
一、 基因工程與分子農場 1
1. 基因工程 1
2. 分子農場 1
3. 異源表達系統 2
二、 微生物異源表達系統 4
1. 原核表達系統 4
2. 真核表達系統 4
三、 食用菇類 7
1. 營養成分 7
2. 嗜好特性 7
3. 藥理價值 8
四、 美白菇 10
1. 起源 10
2. 形態特性 10
3. 栽培優勢 10
4. 營養價值 10
5. 藥理潛力 11
五、 菇類表達系統之建構 12
1. 載體 12
3. 報導基因 12
4. 分生策略 13
5. 篩選標記 13
6. 轉形策略 16
六、 研究動機與目的 21
第二章 材料與方法 24
一. 實驗材料 24
1. 實驗菌株 24
2. 質體 24
二. 實驗方法 28
1. 美白菇 sdi1 基因之選殖 28
2. 轉形質體之建構 36
3. 農桿菌媒介轉形法41
4. 轉形株分析 44
5. 提高異源基因 egfp表現 47
第三章 結果 48
一. 美白菇 sdi1 基因之選殖 48
1. 部分保守性 DNA 序列 48
2. 5’ genome walking 48
3. 3’ genome walking 48
4. 完整 sdi1 基因之釣取 48
5. 點突變之sdi1 基因 49
6. sdi1 基因啟動子之選殖 49
二. 轉形質體之建構 56
三. 農桿菌媒介轉形 57
1. 表現載體電轉入農桿菌 57
2. 美白菇與農桿菌共培養 57
3. 轉形效率 57
四. 轉形株分析 61
1. 轉形株 DNA 分析 61
2. 轉形株 EGFP 分析 63
3. 轉形株異源基因穩定度測試 69
五. 提高異源基因 egfp表現 71
第四章 討論 74
一. 美白菇農桿菌媒介轉形法探討 74
二. 異源蛋白質以同源及異源啟動子表現之差異 76
三. GMO管理 77
第五章 結論 78
第六章 未來工作 79
第七章 參考文獻 80

1.Ackrell, B. A. C. 2000. Progress in understanding structure-function relationships in respiratory chain complex II. FEBS Letters 466:1-5.
2.Akihisa, T., S. G. Franzblau, H. Tokuda, M. Tagata, M. Ukiya, T. Matsuzawa, K. Metori, Y. Kimura, T. Suzuki, and K. Yasukawa. 2005. Antitubercular activity and inhibitory effect on Epstein-Barr virus activation of sterols and polyisoprenepolyols from an edible mushroom, Hypsizigus marmoreus. Biol Pharm Bull 28:1117-9.
3.Arakawa, T., D. K. Chong, J. L. Merritt, and W. H. Langridge. 1997. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res 6:403-13.
4.Armaleo, D., G. N. Ye, T. M. Klein, K. B. Shark, J. C. Sanford, and S. A. Johnston. 1990. Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi. Curr Genet 17:97-103.
5.Beckett, G. J., and J. R. Arthur. 2005. Selenium and endocrine systems. J Endocrinol 184:455-65.
6.Binninger, D. M., C. Skrzynia, P. J. Pukkila, and L. A. Casselton. 1987. DNA-mediated transformation of the basidiomycete Coprinus cinereus. Embo J 6:835-40.
7.Burns, C., K. E. Gregory, M. Kirby, M. K. Cheung, M. Riquelme, T. J. Elliott, M. P. Challen, A. Bailey, and G. D. Foster. 2005. Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns. Fungal Genet Biol 42:191-9.
8.Cabanas, M. J., D. Vazquez, and J. Modolell. 1978. Dual interference of hygromycin B with ribosomal translocation and with aminoacyl-tRNA recognition. Eur J Biochem 87:21-7.
9.Casas-Flores, S., T. Rosales-Saavedra, and A. Herrera-Estrella. 2004. Three decades of fungal transformation: novel technologies. Methods Mol Biol 267:315-25.
10.Chang, J. S., J. K. Son, G. Li, E. J. Oh, J. Y. Kim, S. H. Park, J. T. Bae, H. J. Kim, I. S. Lee, O. M. Kim, N. Kozukue, J. S. Han, M. Hirose, and K. R. Lee. 2004. Inhibition of cell cycle progression on HepG2 cells by hypsiziprenol A9, isolated from Hypsizigus marmoreus. Cancer Lett 212:7-14.
11.Chang, R. 1996. Functional properties of edible mushrooms. Nutr Rev 54:S91-3.
12.Chen, X., M. Stone, C. Schlagnhaufer, and C. P. Romaine. 2000. A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ Microbiol 66:4510-3.
13.Combs, G. F., Jr., and W. P. Gray. 1998. Chemopreventive agents: selenium. Pharmacol Ther 79:179-92.
14.Cregg, J. M., T. S. Vedvick, and W. C. Raschke. 1993. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (N Y) 11:905-10.
15.Daniell, H., S. J. Streatfield, and K. Wycoff. 2001. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in Plant Science 6:219-226.
16.Davies, S. J., P. Golby, D. Omrani, S. A. Broad, V. L. Harrington, J. R. Guest, D. J. Kelly, and S. C. Andrews. 1999. Inactivation and regulation of the aerobic C(4)-dicarboxylate transport (dctA) gene of Escherichia coli. J Bacteriol 181:5624-35.
17.Derkx, P. M., and S. M. Madrid. 2001. The foldase CYPB is a component of the secretory pathway of Aspergillus niger and contains the endoplasmic reticulum retention signal HEEL. Mol Genet Genomics 266:537-45.
18.Desiderio, A., B. Aracri, F. Leckie, B. Mattei, G. Salvi, H. Tigelaar, J. S. Van Roekel, D. C. Baulcombe, L. S. Melchers, G. De Lorenzo, and F. Cervone. 1997. Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris. Mol Plant Microbe Interact 10:852-60.
19.Driscoll, D. M., and P. R. Copeland. 2003. Mechanism and regulation of selenoprotein synthesis. Annu Rev Nutr 23:17-40.
20.Fan, Y. M., M. Y. Xu, L. Y. Wang, Y. Zhang, L. Zhang, H. Yang, P. Wang, and P. Cui. 1989. The effect of edible black tree fungus (Auricuaria auricula) on experimental atherosclerosis in rabbits. Chin Med J (Engl) 102:100-5.
21.Fischer, R., and N. Emans. 2000. Molecular farming of pharmaceutical proteins. Transgenic Res 9:279-99; discussion 277.
22.Fullner, K. J., and E. W. Nester. 1996. Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J Bacteriol 178:1498-504.
23.Gaertig, J., L. Gu, B. Hai, and M. A. Gorovsky. 1994. High frequency vector-mediated transformation and gene replacement in Tetrahymena. Nucleic Acids Res 22:5391-8.
24.Gellissen, G., and K. Melber. 1996. Methylotrophic yeast hansenula polymorpha as production organism for recombinant pharmaceuticals. Arzneimittelforschung 46:943-8.
25.Guan, G. P., H. X. Wang, and T. B. Ng. 2007. A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus. Biochim Biophys Acta 1770:1593-7.
26.Helber, N., and N. Requena. 2008. Expression of the fluorescence markers DsRed and GFP fused to a nuclear localization signal in the arbuscular mycorrhizal fungus Glomus intraradices. New Phytol 177:537-48.
27.Hiatt, A. 1990. Antibodies produced in plants. Nature 344:369-70.
28.Hockney, R. C. 1994. Recent developments in heterologous protein production in Escherichia coli. Trends Biotechnol 12:456-63.
29.Hodgkins, M., D. Mead, D. J. Ballance, A. Goodey, and P. Sudbery. 1993. Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations. Yeast 9:625-35.
30.Ikekawa, T., H. Saitoh, W. Feng, H. Zhang, L. Li, and T. Matsuzawa. 1992. Antitumor activity of Hypsizigus marmoreus. I. Antitumor activity of extracts and polysaccharides. Chem Pharm Bull (Tokyo) 40:1954-7.
31.Inglis, P. W., P. R. Queiroz, and M. C. Valadares-Inglis. 1999. Transformation with green fluorescent protein of Trichoderma harzianum 1051, a strain with biocontrol activity against Crinipellis perniciosa, the agent of witches''-broom disease of cocoa. J Gen Appl Microbiol 45:63-67.
32.Irie, T., Y. Honda, T. Hirano, T. Sato, H. Enei, T. Watanabe, and M. Kuwahara. 2001. Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals. Appl Microbiol Biotechnol 56:707-9.
33.Irie, T., T. Sato, K. Saito, Y. Honda, T. Watanabe, M. Kuwahara, and H. Enei. 2003. Construction of a homologous selectable marker gene for Lentinula edodes transformation. Biosci Biotechnol Biochem 67:2006-9.
34.Iwashita, K. 2002. Recent studies of protein secretion by filamentous fungi. J Biosci Bioeng 94:530-5.
35.Johnston, S. A. 1990. Biolistic transformation: microbes to mice. Nature 346:776-7.
36.Jung, E., and K. L. Williams. 1997. The production of recombinant glycoproteins with special reference to simple eukaryotes including Dictyostelium discoideum. Biotechnol Appl Biochem 25 ( Pt 1):3-8.
37.Kemp, A., J. Parker, and C. Grierson. 2001. Biolistic transformation of Arabidopsis root hairs: a novel technique to facilitate map-based cloning. Plant J 27:367-71.
38.Kiho, T., J. Hui, A. Yamane, and S. Ukai. 1993. Polysaccharides in fungi. XXXII. Hypoglycemic activity and chemical properties of a polysaccharide from the cultural mycelium of Cordyceps sinensis. Biol Pharm Bull 16:1291-3.
39.Kilaru, S., P. J. Hoegger, A. Majcherczyk, C. Burns, K. Shishido, A. Bailey, G. D. Foster, and U. Kues. 2006. Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters. Appl Microbiol Biotechnol 71:200-10.
40.Kilstrup, M., and K. N. Kristiansen. 2000. Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer. Nucleic Acids Res 28:E55.
41.Kuo, C. Y., S. Y. Chou, and C. T. Huang. 2004. Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes. Appl Microbiol Biotechnol 65:593-9.
42.Kuo, C. Y., and C. T. Huang. 2008. A reliable transformation method and heterologous expression of beta-glucuronidase in Lentinula edodes. J Microbiol Methods 72:111-5.
43.Lam, S. K., and T. B. Ng. 2001. Hypsin, a novel thermostable ribosome-inactivating protein with antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hypsizigus marmoreus. Biochem Biophys Res Commun 285:1071-5.
44.Meyer, V., D. Mueller, T. Strowig, and U. Stahl. 2003. Comparison of different transformation methods for Aspergillus giganteus. Curr Genet 43:371-7.
45.Michielse, C. B., P. J. Hooykaas, C. A. van den Hondel, and A. F. Ram. 2005. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet 48:1-17.
46.Michielse, C. B., P. J. J. Hooykaas, C. A. M. J. J. van den Hondel, and A. F. J. Ram. 2005. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Current Genetics 48:1-17.
47.Miller, E. M., and J. A. Nickoloff. 1995. Escherichia coli electrotransformation. Methods Mol Biol 47:105-13.
48.Minetoki, T., C. Kumagai, K. Gomi, K. Kitamoto, and K. Takahashi. 1998. Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efficient expression of Aspergillus oryzae amylase-encoding genes. Appl Microbiol Biotechnol 50:459-67.
49.Mori, K., C. Kobayashi, T. Tomita, S. Inatomi, and M. Ikeda. 2008. Antiatherosclerotic effect of the edible mushrooms Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Hypsizygus marmoreus (Bunashimeji) in apolipoprotein E-deficient mice. Nutr Res 28:335-42.
50.Morigiwa, A., K. Kitabatake, Y. Fujimoto, and N. Ikekawa. 1986. Angiotensin converting enzyme-inhibitory triterpenes from Ganoderma lucidum. Chem Pharm Bull (Tokyo) 34:3025-8.
51.Nakai, R., H. Masui, H. Horio, and M. Ohtsuru. 1999. Effect of maitake (Grifola frondosa) water extract on inhibition of adipocyte conversion of C3H10T1/2B2C1 cells. J Nutr Sci Vitaminol (Tokyo) 45:385-9.
52.Nevalainen, K. M., V. S. Te''o, and P. L. Bergquist. 2005. Heterologous protein expression in filamentous fungi. Trends Biotechnol 23:468-74.
53.Ooi, V. E., and F. Liu. 2000. Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Curr Med Chem 7:715-29.
54.Peng, M., N. K. Singh, and P. A. Lemke. 1992. Recovery of recombinant plasmids from Pleurotus ostreatus transformants. Curr Genet 22:53-9.
55.Piers, K. L., J. D. Heath, X. Liang, K. M. Stephens, and E. W. Nester. 1996. Agrobacterium tumefaciens-mediated transformation of yeast. Proc Natl Acad Sci U S A 93:1613-8.
56.Rolland, S., C. Jobic, M. Fevre, and C. Bruel. 2003. Agrobacterium-mediated transformation of Botrytis cinerea, simple purification of monokaryotic transformants and rapid conidia-based identification of the transfer-DNA host genomic DNA flanking sequences. Curr Genet 44:164-71.
57.Rose, A. B., and J. A. Beliakoff. 2000. Intron-mediated enhancement of gene expression independent of unique intron sequences and splicing. Plant Physiol 122:535-42.
58.Saunders, J. A., and B. F. Matthews. 1995. Pollen electrotransformation in tobacco. Methods Mol Biol 55:81-8.
59.Shark, K. B., F. D. Smith, P. R. Harpending, J. L. Rasmussen, and J. C. Sanford. 1991. Biolistic transformation of a procaryote, Bacillus megaterium. Appl Environ Microbiol 57:480-5.
60.Sharma, K. K., S. Gupta, and R. C. Kuhad. 2006. Agrobacterium-mediated delivery of marker genes to Phanerochaete chrysosporium mycelial pellets: a model transformation system for white-rot fungi. Biotechnol Appl Biochem 43:181-6.
61.Siebert, P. D., A. Chenchik, D. E. Kellogg, K. A. Lukyanov, and S. A. Lukyanov. 1995. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087-8.
62.Sugui, J. A., Y. C. Chang, and K. J. Kwon-Chung. 2005. Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Appl Environ Microbiol 71:1798-802.
63.Sun, J., H. He, and B. J. Xie. 2004. Novel antioxidant peptides from fermented mushroom Ganoderma lucidum. J Agric Food Chem 52:6646-52.
64.Sunagawa, M., and Y. Magae. 2002. Transformation of the edible mushroom Pleurotus ostreatus by particle bombardment. FEMS Microbiol Lett 211:143-6.
65.Tsuchida, K., Y. Aoyagi, S. Odani, T. Mita, and M. Isemura. 1995. Isolation of a novel collagen-binding protein from the mushroom, Hypsizigus marmoreus, which inhibits the Lewis lung carcinoma cell adhesion to type IV collagen. J Biol Chem 270:1481-4.
66.van de Rhee, M. D., P. M. Graca, H. J. Huizing, and H. Mooibroek. 1996. Transformation of the cultivated mushroom, Agaricus bisporus, to hygromycin B resistance. Mol Gen Genet 250:252-8.
67.Vitaly Citovsky, S. V. K., Beno Lacroix, Adi Zaltsman, Mery Dafny-Yelin, Shachi Vyas, Andriy Tovkach, Tzvi Tzfira,. 2007. Biological systems of the host cell involved in Agrobacterium infection. Cellular Microbiology 9:9-20.
68.Ward, P. P., C. S. Piddington, G. A. Cunningham, X. Zhou, R. D. Wyatt, and O. M. Conneely. 1995. A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic. Biotechnology (N Y) 13:498-503.
69.Wong, J. H., H. X. Wang, and T. B. Ng. 2008. Marmorin, a new ribosome inactivating protein with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from the mushroom Hypsizigus marmoreus. Appl Microbiol Biotechnol 81:669-74.
70.Zeilinger, S. 2004. Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation. Curr Genet 45:54-60.
71.Zeng, Q., A. J. Morales, and G. Cottarel. 2001. Fungi and humans: closer than you think. Trends in Genetics 17:682-684.
72.Zhang, G., V. Gurtu, and S. R. Kain. 1996. An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells. Biochem Biophys Res Commun 227:707-11.
73.范�嫄�. 2007. 以甘油醛-3-磷酸脫氫酶基因啟動子研究美白菇轉形系統，國立臺灣大學微生物與生化學研究所碩士論文。.
74.徐韻涵. 2008. 以農桿菌媒介轉形法進行金針菇表達系統之研究，國立臺灣大學微生物與生化學研究所碩士論文。.
75.郭俊毅. 2008. 食用菇異源基因表現系統之建立及其應用，國立臺灣大學微生物與生化學研究所博士論文。
76.蔡媛禎. 2009. 以農桿菌媒介轉形法作為鮑魚菇異源表達之工具，國立臺灣大學微生物與生化學研究所碩士論文。.