請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44479
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 曲芳華(Fang-Hua Chu) | |
dc.contributor.author | Chien-Hsiang Chao | en |
dc.contributor.author | 趙健翔 | zh_TW |
dc.date.accessioned | 2021-06-15T03:00:07Z | - |
dc.date.available | 2009-08-11 | |
dc.date.copyright | 2009-08-11 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-31 | |
dc.identifier.citation | Alvarez, M.E. (2000). Salicylic acid in the machinery of hypersensitive cell death and disease resistance. Plant Molecular Biology 44, 429-442.
Ayres, D.C., and Loike, J.D. (1990). Chemistry and pharmacology of natural products. Lignans: chemical, biological and clinical properties. Cambridge University Press, Cambridge, UK. Bar-Nun, N., and Mayer, A.M. (2008). Methyl jasmonate and methyl salicylate, but not cis-jasmone, evoke defenses against infection of Arabidopsis thaliana by Orobanche aegyptiaca. Weed Biology and Management 8, 91-96. Bretthauer, R.K., and Castellino, F.J. (1999). Glycosylation of Pichia pastoris-derived proteins. Biotechnology and Applied Biochemistry 30 ( Pt 3), 193-200. Chang, S., Puryear, J., and Cairney, J. (1993). A simple and efficient method for isolating RNA from Pine Trees. Plant Molecular Biology Reporter 11, 113-116. Charlton, J.L. (1998). Antiviral activity of lignans. Journal of Natural Products 61, 1447-1451. Chen, Y.R., Lee, Y.R., Wang, S.Y., Chang, S.T., Shaw, J.F., and Chu, F.H. (2004). Establishment of expressed sequence tags from Taiwania (Taiwania cryptomerioides Hayata) seedling cDNA. Plant Science 167, 955-957. Cragg, G.M., and Newman, D.J. (2000). Antineoplastic agents from natural sources: achievements and future directions. Expert Opinion on Investigational Drugs 9, 2783-2797. Daly, R., and Hearn, M.T. (2005). Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. Journal of Molecular Recognition 18, 119-138. Davin, L.B., and Lewis, N.G. (2003). An historical perspective on lignan biosynthesis: monolignol, allylphenol and hydroxycinnamic acid coupling and downstream metabolism. Phytochemistry Reviews 2, 257-288. Davin, L.B., and Lewis, N.G. (2005a). Dirigent phenoxy radical coupling: advances and challenges. Current Opinion in Biotechnology 16, 398-406. Davin, L.B., and Lewis, N.G. (2005b). Lignin primary structures and dirigent sites. Current Opinion in Biotechnology 16, 407-415. Davin, L.B., Bedgar, D.L., Katayama, T., and Lewis, N.G. (1992). On the stereoselective synthesis of (+)-pinoresinol in Forsythia suspensa from its achiral precursor, coniferyl alcohol. Phytochemistry 31, 3869-3874. Davin, L.B., Wang, H.B., Crowell, A.L., Bedgar, D.L., Martin, D.M., Sarkanen, S., and Lewis, N.G. (1997). Stereoselective bimolecular phenoxy radical coupling by an auxiliary (dirigent) protein without an active center. Science 275, 362-366. Gang, D.R., Costa, M.A., Fujita, M., Dinkova-Kostova, A.T., Wang, H.B., Burlat, V., Martin, W., Sarkanen, S., Davin, L.B., and Lewis, N.G. (1999). Regiochemical control of monolignol radical coupling: a now paradigm for lignin and lignan biosynthesis. Chemistry and Biology 6, 143-151. Gilbert, W. (1987). The exon theory of genes. Cold Spring Harbor Symposia on Quantitative Biology 52, 901-905. Halls, S.C., and Lewis, N.G. (2002). Secondary and quaternary structures of the (+)-pinoresinol-forming dirigent protein. Biochemistry 41, 9455-9461. Halls, S.C., Davin, L.B., Kramer, D.M., and Lewis, N.G. (2004). Kinetic study of coniferyl alcohol radical binding to the (+)-pinoresinol forming dirigent protein. Biochemistry 43, 2587-2595. Heinonen, S., Nurmi, T., Liukkonen, K., Poutanen, K., Wahala, K., Deyama, T., Nishibe, S., and Adlercreutz, H. (2001). In vitro metabolism of plant lignans: new precursors of mammalian lignans enterolactone and enterodiol. Journal of Agricultural and Food Chemistry 49, 3178-3186. Hirano, T., Gotoh, M., and Oka, K. (1994). Natural flavonoids and lignans are potent cytostatic agents against human leukemic HL-60 cells. Life Sciences 55, 1061-1069. Hitzeman, R.A., Chen, C.Y., Dowbenko, D.J., Renz, M.E., Liu, C., Pai, R., Simpson, N.J., Kohr, W.J., Singh, A., Chisholm, V., and et al. (1990). Use of heterologous and homologous signal sequences for secretion of heterologous proteins from yeast. Methods in Enzymology 185, 421-440. Huylebroeck, D., Van Nimmen, K., Waheed, A., von Figura, K., Marmenout, A., Fransen, L., De Waele, P., Jaspar, J.M., Franchimont, P., Stunnenberg, H., and et al. (1990). Expression and processing of the activin-A/erythroid differentiation factor precursor: a member of the transforming growth factor-beta superfamily. Molecular Endocrinology (Baltimore, Md.) 4, 1153-1165. Kangas, L., Saarinen, N., Mutanen, M., Ahotupa, M., Hirsinummi, R., Unkila, M., Perala, M., Soininen, P., Laatikainen, R., Korte, H., and Santti, R. (2002). Antioxidant and antitumor effects of hydroxymatairesinol (HM-3000, HMR), a lignan isolated from the knots of spruce. European Journal of Cancer Prevention : The Official Journal of the European Cancer Prevention Organisation (ECP) 11 Suppl 2, S48-57. Kim, M.K., Jeon, J.H., Fujita, M., Davin, L.B., and Lewis, N.G. (2002). The western red cedar (Thuja plicata) 8-8 ' DIRIGENT family displays diverse expression patterns and conserved monolignol coupling specificity. Plant Molecular Biology 49, 199-214. Kitts, D.D., Yuan, Y.V., Wijewickreme, A.N., and Thompson, L.U. (1999). Antioxidant activity of the flaxseed lignan secoisolariciresinol diglycoside and its mammalian lignan metabolites enterodiol and enterolactone. Molecular and Cellular Biochemistry 202, 91-100. Koulman, A., Bos, R., Medarde, M., Pras, N., and Quax, W.J. (2001). A fast and simple GC MS method for lignan profiling in Anthriscus sylvestris and biosynthetically related Plant species. Planta Medica 67, 858-862. Laemmli, U.K. (1970). Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227, 680-&. Lambert, J.D., Sang, S.M., Dougherty, A., Caldwell, C.G., Meyers, R.O., Dorr, R.T., and Timmermann, B.N. (2005). Cytotoxic lignans from Larrea tridentata. Phytochemistry 66, 811-815. Lee, C.H., Chan, M.H., Wang, Y.N., and Chu, F.H. (2006). Gene Investigation into the Inner bark of Taiwania (Taiwania cryptomerioides). Botanical Studies 47, 111-118. Loake, G., and Grant, M. (2007). Salicylic acid in plant defence--the players and protagonists. Current Opinion in Plant Biology 10, 466-472. Lourith, N., Katayama, T., Ishikawa, K., and Suzuki, T. (2005). Biosynthesis of a syringyl 8-O-4 ' neolignan in Eucommia ulmoides: formation of syringylglycerol-8-O-4 '-(sinapyl alcohol) ether from sinapyl alcohol. Journal of Wood Science 51, 379-386. Lu, H., and Liu, G.T. (1992). Anti-oxidant activity of dibenzocyclooctene lignans isolated from Schisandraceae. Planta Medica 58, 311-313. Metraux, J.P., Signer, H., Ryals, J., Ward, E., Wyss-Benz, M., Gaudin, J., Raschdorf, K., Schmid, E., Blum, W., and Inverardi, B. (1990). Increase in Salicylic Acid at the Onset of Systemic Acquired Resistance in Cucumber. Science 250, 1004-1006. Moinuddin, S.G., Hishiyama, S., Cho, M.H., Davin, L.B., and Lewis, N.G. (2003). Synthesis and chiral HPLC analysis of the dibenzyltetrahydrofuran lignans, larreatricins, 8'-epi-larreatricins, 3,3'-didemethoxyverrucosins and meso-3,3'-didemethoxynectandrin B in the creosote bush (Larrea tridentata): evidence for regiospecific control of coupling. Organic and Biomolecular Chemistry 1, 2307-2313. Moss, G.P. (2000). Nomenclature of lignans and neolignans (IUPAC Recommendations 2000). Pure and Applied Chemistry 72, 1493-1523. Murashige, T., and Skoog, F. (1962). A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum 15, 473-&. Petcher, T.J., Weber, H.P., Kuhn, M., and Vonwartb.A. (1973). Crystal-structure and absolute configuration of 2'-bromopodophyllotoxin-0,5 ethyl acetate. Journal of the Chemical Society-Perkin Transactions 2, 288-292. Ralph, S.G., Jancsik, S., and Bohlmann, J. (2007). Dirigent proteins in conifer defense II: Extended gene discovery, phylogeny, and constitutive and stress-induced gene expression in spruce (Picea spp.). Phytochemistry 68, 1975-1991. Saleem, M., Kim, H.J., Ali, M.S., and Lee, Y.S. (2005). An update on bioactive plant lignans. Natural Product Reports 22, 696-716. Schultz, L.D., Tanner, J., Hofmann, K.J., Emini, E.A., Condra, J.H., Jones, R.E., Kieff, E., and Ellis, R.W. (1987). Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus. Gene 54, 113-123. Scorer, C.A., Buckholz, R.G., Clare, J.J., and Romanos, M.A. (1993). The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene 136, 111-119. Sinclair, G., and Choy, F.Y. (2002). Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris. Protein Expression and Purification 26, 96-105. Thompson, L.U., Robb, P., Serraino, M., and Cheung, F. (1991). Mammalian lignan production from various foods. Nutrition and Cancer 16, 43-52. Turner, J.G., Ellis, C., and Devoto, A. (2002). The jasmonate signal pathway. Plant Cell 14, S153-S164. Villatte, F., Hussein, A.S., Bachmann, T.T., and Schmid, R.D. (2001). Expression level of heterologous proteins in Pichia pastoris is influenced by flask design. Applied Microbiology and Biotechnology 55, 463-465. Walling, L.L. (2008). Avoiding effective defenses: strategies employed by phloem-feeding insects. Plant Physiology 146, 859-866. Willfor, S., Hemming, J., Reunanen, M., Eckerman, C., and Holmbom, B. (2003). Lignans and lipophilic extractives in Norway spruce knots and stemwood. Holzforschung 57, 27-36. Willfor, S.M., Smeds, A.I., and Holmbom, B.R. (2006). Chromatographic analysis of lignans. Journal of Chromatography A 1112, 64-77. Yamauchi, S., Ina, T., Kirikihira, T., and Masuda, T. (2004). Synthesis and antioxidant activity of oxygenated furofuran lignans. Bioscience, Biotechnology, and Biochemistry 68, 183-192. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44479 | - |
dc.description.abstract | 木酚素(Lignan)是重要的植物酚類化合物,就生合成角度來看,其歸屬於植物二次代謝產物,具抗癌、抗微生物、抗真菌、抗病毒、抗發炎、抗氧化及免疫調節等等的生物活性,同時在植物對抗多樣性的病原或害蟲扮演了重要的防禦角色。已有研究指出木酚素生合成需要經過Dirigent protein(DIR)的參與,協助基質進行專一性的結合。
臺灣杉(Taiwania cryptomerioides Hayata)是地質時代第三紀之孑遺植物,為臺灣原產珍貴的針葉樹種。本研究自2年生臺灣杉之葉部組織cDNA以反轉錄聚合酶連鎖反應(RT-PCR)技術獲得DIR部分片段,續以Rapid Amplification of cDNA Ends(RACE)技術獲得基因頭尾序列,以及Genome Walking技術釣取promoter序列,共得到2563 bp,包括Promoter序列1570 bp、5’端73 bp、轉錄區(ORF)576 bp、3’端341 bp,並命名為TcDIR。經分析預測其蛋白質大小約為21.6 kDa。將所得基因構築於大腸桿菌(Escherichia coli BL21)用的載體pET-28a(+)與酵母菌(Pichia pastoris SMD1168)表現用載體pGAPZαA。由大腸桿菌得到可溶部重組蛋白約22 kDa;而由酵母菌表現之重組蛋白約27 kDa,且經分析具有醣基化現象。以反轉錄聚合酶連鎖反應分析不同組織,發現樹皮、雄毬花、雌毬花、毬果、老葉與幼葉均有TcDIR基因表現。以茉莉酸(Jasmonic acid, JA)600 μM與水楊酸(Salicylic acid, SA)400 μM處理小苗6小時後發現TcDIR表現量均較對照組為多。並且無論老枝葉或幼枝葉在創傷處理過後TcDIR之表現量均有上升之趨勢。另外,將TcDIR轉殖至阿拉伯芥(Arabidopsis thaliana)發現生長較野生型為快且葉部有較大的現象。 本研究結果將可提供未來透過不同的基因表現層級,來推測TcDIR在臺灣杉面臨生物及物理逆境上所扮演的角色之重要參考。 | zh_TW |
dc.description.abstract | Lignan is one of important phenolics compounds, which is belonging to plant secondary metabolites. Numbers of literatures show that lignan possess anticancer, antioxidant, antimicrobial, antifungal, anti-inflammatory and immunosuppressive activities. In the meantime, it might contribute to defense and resistant mechanism against insects and pathogens in plant species. The previous reports have shown that the dirigent protein (DIR) is involved in biosynthesis of lignans in vivo, which contribute to the stereoselective linkage of radical-radical couplings.
Taiwania (Taiwania cryptomerioides Hayata) is a relic plant from Tertiary period of geological age. It is an economical species and an endemic conifer in Taiwan. In our current study, a DIR gene in Taiwania cryptomerioides, TcDIR was cloned. By using reverse transcription-polymerase chain reaction from Taiwania 2-year-old leaf cDNA, 576 base pairs (bp) of coding region were obtained. Then the 5’ and 3’ RACE (Rapid Amplify of cDNA End) and Genome Walking were used to clone the full length of TcDIR. The promoter region is 1570 bp. The uncoding region on 5’-end and 3’-end is 73 bp and 341bp, respectively. The TcDIR gene was constructed into BL21 and SMD1168 to generate a great quantity of TcDIR recombinant protein. It’s found that TcDIR expresses in SMD1168 might be glycosylated. We found that TcDIR expresses in bark, male cone, female cone, cone, old leaf and young leaf. The TcDIR were up-regulated by 600 μM JA (Jasmonic acid) and 400 μM SA (Salicylic acid) after 6 hr treatments of 22-day-old seedings. The TcDIR were up-regulated by wounding in either old leaf or young leaf. We also found that transgenic Arabidopsis plants were growing faster and have bigger leaf than wildtypes. These results obtained in this study might provide a useful reference for the future studies in more gene expression level experiments, which are correlated with the rule of TcDIR with defense system and/or physiology in Taiwania. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T03:00:07Z (GMT). No. of bitstreams: 1 ntu-98-R96625005-1.pdf: 8770852 bytes, checksum: 1ee6dfdec099905534f7c42ed3d556e6 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 口試委員審定書 i
誌謝 ii 摘要 iii Abstract iv 目錄 vi 圖目錄 xi 表目錄 xiv 第一章、 前言 1 第1節、 臺灣杉dirigent protein基因研究之重要性 1 第2節、 文獻回顧 1 2.1 Lignan在自然界物中扮演的角色 1 2.2 Dirigent protein之功能 3 第二章、 材料與方法 8 第1節、 植物材料 8 1.1 臺灣杉 8 1.2 基因轉殖用之阿拉伯芥 8 第2節、 載體(Vector) 8 2.1 pGEM-T Easy 8 2.2 pET-21a(+) 8 2.3 pET-28a(+) 10 2.4 pGAPZαA 10 2.5 pBI121 10 第3節、 實驗所使用菌株 10 3.1 Escherichia coli DH5α 10 3.2 Escherichia coli BL21(DE3) 10 3.3 Pichia pastoris SMD1168 10 3.4 Agrobacterium tumefaciens GV3101 10 第4節、 TcDIR基因之選殖 10 4.1 臺灣杉TcDIR專一性序列片段之選殖 10 4.2 TcDIR基因之5’端與3’端序列之釣取 11 4.3 Genome Walking 11 第5節、 核酸與蛋白質之序列分析 12 第6節、 TcDIR基因序列之親緣關係比較 12 第7節、 蛋白質表現質體之構築 12 7.1 大腸桿菌之蛋白質表現質體構築 12 7.2 酵母菌之蛋白質表現質體構築 13 第8節、 TcDIR基因之蛋白質表現與純化 13 8.1 大腸桿菌之蛋白質表現與純化 13 8.2 酵母菌之蛋白質表現與純化 14 第9節、 蛋白質鑑定 15 第10節、 TcDIR基因於臺灣杉各組織中之基因表現量 15 10.1 不同組織之RNA抽取 15 10.2 RT-PCR 15 10.3 Real-time PCR 15 第11節、 JA及SA處理後之TcDIR基因表現 16 11.1 濾紙苗 16 11.2 幼枝葉 16 11.3 老枝葉 16 第12節、 創傷處理後之TcDIR基因表現 17 第13節、 TcDIR之基因轉殖與阿拉伯芥種子之篩選 17 13.1 植物轉殖質體之構築與轉型 17 13.2 轉殖阿拉伯芥種子之篩選 17 第14節、 以GC/MS檢測轉殖阿拉伯芥葉子 18 14.1 材料準備 18 14.2 GC/MS條件 19 第三章、 結果 20 第1節、 臺灣杉TcDIR基因序列之選殖與序列分析 20 1.1 臺灣杉TcDIR基因體核酸序列之選殖 20 1.2 啟動子(Promoter)序列預測 23 1.3 蛋白質序列預測 25 1.4 醣基化位置(N-glycosylation sites)預測 25 1.5 磷酸化位置(Phosphorylation site)預測 25 1.6 Signal peptide切位(Signal peptide cleavage site) 26 第2節、 臺灣杉TcDIR之序列親緣關係 27 第3節、 TcDIR重組蛋白質之表現與純化 30 3.1 大腸桿菌之蛋白質表現與純化 30 3.2 酵母菌之蛋白質表現與純化 31 3.3 不同時間梯度之重組蛋白表現 31 3.4 去醣基分析 32 3.5 不同廠牌抗體測試 34 第4節、 蛋白質鑑定 35 第5節、 TcDIR基因於臺灣杉各組織中之基因表現量 36 第6節、 不同組織於JA及SA處理後之TcDIR基因表現 36 6.1 濾紙苗 36 6.2 幼枝葉 37 6.3 老枝葉 39 第7節、 創傷處理後之TcDIR基因表現 40 7.1 幼枝葉 40 7.2 老枝葉 41 第8節、 TcDIR基因轉殖之阿拉伯芥 42 8.1 轉殖株之表現型 42 8.2 轉殖株基因表現鑑定 42 8.3 T2轉殖株進行GC/MS分析 44 第四章、 討論 46 第五章、 結論 51 第六章、 參考文獻 52 第七章、 附錄 56 第1節、 實驗引子(Primer)之設計 56 第2節、 實驗藥品配方 57 2.1 抗生素Stock 57 2.2 其他 57 第3節、 基本實驗方法 59 3.1 抽取總體RNA(Total RNA) 59 3.2 反轉錄(Reverse transcription, RT)反應 60 3.3 聚合酶連鎖反應(Polymerase chain reaction, PCR) 60 3.4 洋菜膠電泳分析 60 3.5 PCR產物純化 60 3.6 接合反應(Ligation) 60 3.7 E. coli轉型(Transformation)及篩選 61 3.8 抽取質體DNA(Plasmid) 62 3.9 DNA定序 62 3.10 抽取基因體核酸(Genomic DNA) 62 3.11 SDS-聚丙醯胺膠體進行電泳分析 64 3.12 免疫雜合分析 64 3.13 蛋白質純化與濃縮 65 3.14 酵母菌轉型 65 第4節、 縮寫表 67 | |
dc.language.iso | zh-TW | |
dc.title | 臺灣杉 Dirigent protein之基因選殖及基因表現情形 | zh_TW |
dc.title | Molecular Cloning and Characterization of
Dirigent Protein Gene from Taiwania cryptomerioides Hayata | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 孟孟孝(Meng-hsiao Meng),陳振榮(Zenn-Zong Chen),張淑華(Shu-Hwa Chang),王升陽(Sheng-Yang Wang) | |
dc.subject.keyword | Dirigent protein,木酚素,抗性,酚類化合物,醣基化,臺灣杉, | zh_TW |
dc.subject.keyword | Dirigent protein,Glycosylation,Lignan,Phenoic compound,Resistance,Taiwania cryptomerioides Hayata, | en |
dc.relation.page | 68 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-31 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 森林環境暨資源學研究所 | zh_TW |
顯示於系所單位: | 森林環境暨資源學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-98-1.pdf 目前未授權公開取用 | 8.57 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。