臺灣博碩士論文加值系統

十字花科蔬菜，例如青花菜、甘藍、花椰菜，含有高量的蘿蔔硫素(sulforaphane)，其具有抑制癌細胞繁殖、防止癌細胞的發展、抗癌細胞轉移等作用。蘿蔔硫素是一種異硫氰酸鹽(isothiocyanate)，由硫代葡萄糖苷(glucosinolates)經存在於植物體液泡內的黑芥子酶(myrosinase, MYR)水解而得。研究顯示ESP (epithiospecifier protein, 上皮硫特異蛋白)會抑制蘿蔔硫素的合成，ESM (epithiospecifier modifier, 表皮特異硫蛋白修飾子)則促進蘿蔔硫素的生成。由於蘿蔔硫素的萃取效率極低及高價格，限制其在醫藥的應用。
甘藍是世界重要經濟蔬菜，是台灣栽培面積最廣的葉菜類蔬菜，青花菜是十字花科作物中最具有抗癌效果的蔬菜，二者有其重要的民生及經濟地位。利用基因轉殖作物為生物反應器以生產工業、食品、飼料及醫藥方面的產品，在高附加價值、低成本及環保因素考量之下，是台灣極適合積極研究發展的重要項目。
研究理念是將自青花菜分離之MYR及ESM基因構築到蕓苔屬葉綠體基因轉殖載體系統，ESP基因構築到RNAi的之農桿菌介導的基因轉殖系統，將此三個基因各別或共同轉移至青花菜或甘藍之葉綠體/細胞核中，達到抑制ESP基因表現或/及葉綠體大量合成MYR及ESM，且區格MYR及ESM在葉綠體中，如此當植物器官組織受到傷害時，即可大量合成蘿蔔硫素。
本研究分別構築帶有青花菜Myr及Esm基因為目標基因之蕓苔屬葉綠體轉殖載體與帶有青花菜Esp基因為目標基因之RNAi抑制方式(ESPi)的農桿菌基因轉殖載體。總共完成六種蕓苔屬葉綠體轉殖載體：pMT91t-Esm-A、pMT91t-Myr-A、pMT91t-Esm-Myr-A、pMT91t-Esm-GA、pMT91t-Myr-GA及pMT91t-Esm-Myr-GA，以aadA為篩選基因或/且以gus為報導基因。完成二種RNAi抑制方式的農桿菌基因轉殖載體﹕p1304-Esp-IN-GH及p1304-Esp-IN-GD，以mgfp-gusA為報導基因，以hptII或daao為篩選基因。
本研究將pMT91t-Esm-Myr-GA載體，利用基因槍法將其轉移至'初秋'甘藍及'綠王'青花菜下胚軸中。再生培植體經10 ppm spectinomycin持續篩選，已獲得再生殖株。轉殖再生植株葉片之PCR、RT-PCR及qrt-RT-PCR分析之結果顯示，轉殖之Esm、Myr基因已存在於轉殖植株之葉綠體基因組，並表現其mRNA。

Cruciferous vegetables, such as broccoli, cabbage, and cauliflower, are rich sources of sulforaphane. Sulforaphane have been found to inhibit proliferation of cancer cells, prevent cancer cells to develop, and anti-metastasis of cancer cells. Sulforaphane is an isothiocyanate which is produced from glucosinolates in myrosinase-catalyzed hydrolysis. Myrosinase (MYR) is physically separated from glucosinolates in intact plant cells. Production of sulforaphane is upregulated by the MYR and ESM (epithiospecifier modifier) and downregulated by ESP (epithiospecifier protein). The complicated separation and purification procedure result in low extraction efficiency and extremely high price for the sulforaphane, which hampers a wide application in pharmaceutical science.
Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetable crops grown worldwide, and also has been the most widely cultivated leafy vegetables in Taiwan. Broccoli (Brassica oleracea var. italica Planck) is well established as the best anti-cancer vegetable among the cruciferous vegetables. Using transgenic crops as bioreactors to produce industrial, feed and fodder additives, and pharmaceutical proteins become a new way for increasing economic values of crops and may solve the problems we are facing.
By using the art of genetic engineering, we attempt to modify the glucosinolate-myrosinase substrate-enzyme system. Increasing MYR and ESM expression and knockdown the expression of ESP are achieved by chloroplast gene transformation and RNAi manipulation, respectively. Thus, overexpression of MYR and ESM in chloroplast and suppression of ESP expression, thorough breakdown of modified broccoli and cabbage will enhance the production of sulforaphane.
In this study, Brassica chloroplast transformation vectors harboring the MYR and ESM genes were constructed. Vectors harboring antisense--oriented ESP gene (ESPi) were also constructed for Agrobacterium-mediated transformation. Six Brassica chloroplast transformation vectors had been constructed, namely pMT91t-Esm-A, pMT91t-Myr-A, pMT91t-Esm-Myr-A, pMT91t-Esm-GA, pMT91t-Myr-GA and pMT91t-Esm-Myr-GA. Two RNAi constructs for ESP gene has been obtained, namely, p1304-Esp-IN-GH and p1304-Esp-IN-GD.
Brassica chloroplast transformation vectors, pMT91t-Esm-Myr-GA (harboring Myr and Esm gene), was transferred into the hypocotyls of 'K-Y cross' cabbage and 'Green King' broccoli via biolistic bombardment. Transformed plantlets are selected by 10 ppm spectinomycin. The results of PCR, RT-PCR, and qrt-RT-RCR analysis indicated that the transformed genes are present in the chloroplast genome of transplastomic plants, and expressed its mRNA.

中文摘要…………………………………………………………………………………i
Abstract………………………………………………………………………………ii
目次……………………………………………………………………………………iv
圖目次…………………………………………………………………………………v
表目次…………………………………………………………………………………viii
前言……………………………………………………………………………………1
前人研究………………………………………………………………………………3
一、葉綠體基因轉殖………………………………………………………………3
二、蘿蔔硫素、合成、分佈與功效………………………………………………4
三、蘿蔔硫素生合成及調控………………………………………………………6
四、外在環境對蘿蔔硫素生成之影響……………………………………………7
五、黑芥子酶 (myrosinase, MYR, EC3.2.3.1)……………………………………9
六、表皮特異硫蛋白修飾子(epithiospecifier modifier, ESM)…………………10
七、上皮硫特異蛋白(epithiospecifier protein，ESP)……………………………11
材料與方法……………………………………………………………………………12
結果……………………………………………………………………………………24
一、 蘿蔔硫素生合成相關基因Esm、Myr及Esp基因轉殖載體之構築………24
二、Spectinomycin對未轉殖之甘藍及青花菜下胚軸培養再生之影響………30
三、甘藍及青花菜的葉綠體基因轉殖、培植體篩選及誘導再生………………31
四、轉殖植株之基因及表現分析………………………………………………32
討論……………………………………………………………………………………97
參考文獻……………………………………………………………………………103
附錄…………………………………………………………………………………108

林經偉。青花菜栽培之土壤及施肥管理。2013。臺南區農業專訊85: 13-17。
薛?、李勝、馬紹英、劉浩、羅麗媛、方艷。2010。不同光質對西蘭花癒傷組織及蘿蔔硫素含量的影響。甘肅農業大學學報 45(4): 95-99。
Andreasson, E., J. Taipalensuu, L. Rask, and J. Meijer, 1999. Age-dependent wound induction of a myrosinase-associated protein from oilseed rape (Brassica napus). Plant Molecular Biology 41: 171-180.
Andréasson, E., L. B. Jörgensen, A. Höglund, L. Rask, and J. Meijer. 2001. Different Myrosinase and Idioblast Distribution in Arabidopsis and Brassica napus. Plant Physiol. 127: 1750-1763.
Boddupalli, S., J. R. Mein, S. Lakkanna, and D. R. James. 2012. Induction of phase 2 antioxidant enzymes by broccoli sulforaphane: perspectives in maintaining the antioxidant activity of vitamins A, C, and E. Front. Genet. 3:7.
Bones, A. M. and J. T. Rossiter. 1996. The myrosinase-glucosinolate system: its organization and biochemistry. Physiologia Plantarum 97: 194-208.
Campas-Baypolia, O. N., D. I. Sánchez-Machadoa, C. Bueno-Solanoa, B. Ramírez-Wongb, and J. López-Cervantesa. 2010. HPLC method validation for measurement of sulforaphane level in broccoli by-products. Biomed. Chromatogr. 24: 387-392.
Chen, S. and E. Andreasson. 2001. Update on glucosinolate metabolism and transport. Plant Physiol. Biochem. 39(9): 743-758.
Esfandiari, A., A. Saei, M. J. McKenzie, A. J. Matich, M. Babalar, and D. A. Hunter. 2017. Preferentially enhancing anti-cancer isothiocyanates over glucosinolates in broccoli sprouts: How NaCl and salicylic acid affect their formation. Plant Physiol. Biochem. 115: 343-353.
Fahey, J. W., A. T. Zalcmann, and P. Talalay. 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochem. 56(1): 5-51.
Fahey, J. W., S. L. Wehage, W. D. Holtzclaw, T. W. Kensler, P. A. Egner, T. A. Shapiro, and P. Talalay. 2012. Protection of humans by plant glucosinolates: Efficiency of conversion of glucosinolates to isothiocyanates by the gastrointestinal microflora. Cancer Prev. Res. 5(4): 603-611.
Falk, A., B. Ek, and L. Rask. 1995. Characterization of a new myrosinase in Brassica napus. Plant Molecular Biology 27: 863-874.
Falk, K. L. J. G. Tokuhisa, and J. Gershenzon. 2007. The effect of sulfur nutrition on plant glucosinolate content: physiology and molecular mechanisms. Plant Biol. 9(5): 573–581.
Foo, H. L., L. M. Gronning, L. Goodenough, A. M. Bones, B. Danielsen, D. A. Whiting, and J. T. Rossiter. 2000. Purification andcharacterisationof epithiospecifier proteinfrom Brassica napus: enzymic intramolecular sulphur addition within alkenyl thiohydroximates derived from alkenyl glucosinolate hydrolysis. FEBS Lett. 468: 243-246.
Guo, L., R. Yang, Y. Zhou, and Z. Gu. 2016. Heat and hypoxia stresses enhance the accumulation of aliphatic glucosinolates and sulforaphane in broccoli sprouts. Eur. Food Res. Technol. 242(1): 107-116.
Guo, R., G. Yuan, and Q. Wang. 2013. Effect of NaCl treatments on glucosinolate metabolism in broccoli sprouts. J. Univ-Sci. B. 14(2): 124-131.
Iamtham, S. and A. Day. 2000. Removal of antibiotic resistance genes from transgenic tobacco plastids. Nat. Biotechnol. 18: 1172-1176.
Jones, R. B., C. L. Frisina, S. Winkler, M. Imsic, and R. B. Tomkins. 2010. Cooking method significantly effects glucosinolate content and sulforaphane production in broccoli florets. Food Chem. 123(2): 237-242.
Jones, R. B., J. D. Faragher, and S. Winkler. 2006. A review of the influence of postharvest treatments on quality and glucosinolate content in broccoli (Brassica oleracea var. italica) heads. Postharvest Biol. Technol. 41(1):1-8.
Khan, M. A. M., C. Ulrichs, and I. Mewis. 2011. Water stress alters aphid-induced glucosinolate response in Brassica oleracea var. italica differently. Chemoecology 21(4): 235-242.
Kissen, R., J. T. Rossiter, and A. M. Bones. 2009. The ‘mustard oil bomb’: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system. Phytochemistry Reviews 8: 69-86.
Kliebenstein, D. J., J. Kroymann, and T. Mitchell-Olds. 2005. The glucosinolate-myrosinase system in an ecological and evolutionary context. Curr. Opin. Plant Biol. 8: 264-271.
Kobayashi, A., M. I. Kang, H. Okawa, M. Ohtsuji, Y. Zenke, T. Chiba, K. Igarashi, and M. Yamamoto. 2004. Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase To Regulate Proteasomal Degradation of Nrf2. Mol. Cell Biol. 24:7130-7139.
Kopsell, D. A. and C. E. Sams. 2013. Increases in shoot tissue pigments, glucosinolates, and mineral elements in sprouting broccoli after exposure to short-duration blue light from light emitting diodes. J. Amer. Soci. Horti. Sci. 138(1): 31-37.
Ku, K. M., J. H. Choi, H. S. Kim, M. M. Kushad, E. H. Jeffery, et al. 2013. Methyl jasmonate and 1-methylcyclopropene treatment effects on quinone reductase inducing activity and post-harvest quality of broccoli. PLoS ONE 8(10): e77127. doi:10.1371/journal.pone.0077127.
Lambrix, V., M. Reichelt, T. Mitchell-Olds, D. J. Kliebenstein, and J. Gershenzon. 2001. The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory. Plant Cell 13(12): 2793-2807.
Liang, H., Q. Yuan, and Q. Xiao. 2006. Effects of metal ions on myrosinase activity and the formation of sulforaphane in broccoli seed. J. Mol. Catal. B Enzym. 43: 19-22.
Maliga, P. 2003. Progress towards commercialization of plastid transformation technology. Trends in Biotech. 21(1): 20-28.
Maliga, P. 2014. Chloroplast Biotechnology: Methods and Protocols. Methods in Molecular Biology. Vol. 1132. Springer, New York York, Humana Press.
Matusheski, N. V., R. Swarup, J. A. Juvik, R. Mithen, M. Bennett, and E. H. Jeffery. 2006. Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. Italic) inhibits formation of the anticancer agent sulforaphane. J. Agri. Food Chem. 54: 2069-2076.
Matusheski, N. V., J. A. Juvik, and E. H. Jeffery. 2004. Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli. Phytochem. 65: 1273-1281.
Padilla, G., M. E. Cartea, P.Velasco, A. Haro, and A. Ordás. 2007. Variation of glucosinolates in vegetable crops of Brassica rapa. Phytochem. 68(4): 536-545.
Park, J.-H., S.-J. Lee, B.-R. Kim, E.-T. Woo, J.-S. Lee, E.-H. Han, Y.-H. Lee, and Y.-D.Park. 2011. Isolation of myrosinase and glutathione S-transferase genes and gransformation of ghese genes to develop phenylethylisothiocyanate enriching Chinese cabbage. Kor. J. Hort. Sci. Technol. 29(6): 623-632.
Pérez-Balibrea, S, D. A. Moreno, and C. García-Viguera. 2008. Influence of light on health-promoting phytochemicals of broccoli sprouts. J. Sci. Food Agri. 88 (5): 904-910.
Pérez-Balibrea, S, D. A. Moreno, and C. García-Viguera. 2011. Improving the phytochemical composition of broccoli sprouts by elicitation. Food Chem. 129 (1): 35-44.
Prakash, O., A. K. Rai, J. Singh, and P.M. Singh. 2013. Effect of heavy metal ions and carbohydrates on the activity of cauliflower (Brassica oleracea Var. botrytis) myrosinase. J. Stress Physiol. Biochem. 9: 108-117.
Rangkadilok, N., M. E. Nicolas, R. N. Bennett, R. R. Premier, D. R. Eagling, and P. W. J. Taylor. 2002. Developmental changes of sinigrin and glucoraphanin in three Brassica species (Brassica nigra, Brassica juncea and Brassica oleracea var. italica). Sci. Hort. 96: 11-26.
Rask, L., E. Andréasson, B. Ekbom, S. Eriksson, B. Pontoppidan, J. Meijer. 2000. Myrosinase: gene family evolution and herbivore defense in Brassicaceae. Plant Mole. Biol. 42: 93-113.
Razin, A. and J. Friedman. 1982. DNA Methylation and its Possible Biological Roles. Prog. Nucletic Acids Res. Mol. Biol. 25: 33-52.
Royston, K. J. and T. O. Tollefsbol. 2015. The epigenetic impact of cruciferous vegetables on cancer prevention. Curr. Pharmacol. Rep. 1(1): 46-51.
Rungapamestry, V., A. J. Duncan, Z. Fuller, and B. Ratckiffe. 2006. Changes in Glucosinolate Concentrations, Myrosinase Activity, and Production of Metabolites of Glucosinolates in Cabbage ( Brassica oleracea Var. capitata ) Cooked for Different Durations. J. Agric. Food Chem. 54: 7628-7634.
Scholl, C., B. D. Eshelman, D. M. Barnes, and P. R. Hanlon. 2011. Raphasatin is a more potent inducer of the detoxification enzymes than its degradation products. J. Food Sci. 76(3): 504-511.
Sivakumar, G., A. Aliboni, and L. Bacchetta. 2007. HPLC screening of anti-cancer sulforaphane from important European Brassica species. Food Chem. 104(4): 1761-1764.
Song, L. and P. J. Thornalley. 2007. Effect of storage, processing and cooking on glucosinolate content of Brassica vegetables. Food Chem. Toxicol. 45(2): 216-224.
Svab, Z., and P. Maliga. 1993. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. P. Natl. Acad. Sci. USA. 90: 913-917.
Taipalensuu, J., E. Andreasson, S. Eriksson, and L. Rask L. 1997. Regulation of the wound-induced myrosinase-associated protein transcript in Brassica napus plants. Eur. J. Biochem. 247(3): 963-971.
Tilaar, W., S. Ashari, B. Yanuwiadi, and J. Polii-Mandang. 2012. Synthesis of sulforaphane during the formation of plantlets from broccoli (Brassica oleracea L. var. italica) in vitro. IJET-IJENS. 12(3):1-5.
Vaughn, S. F. and M. A Berhow. 2005. Glucosinolate hydrolysis products from various plant sources: pH effects, isolation, and purification. Ind. Crops Prod. 21(2): 193-202.
Wang, G. C., M. Farnham, and E. H. Jeffery. 2012. Impact of Thermal Processing on Sulforaphane Yield from Broccoli (Brassica oleracea L. ssp. italica). J. Agric. Food Chem. 60(27): 6743-6748.
Williams, D. J., C. Critchley, S. Pun, S. Nottingham, and T. J. O’Har. 2008. Epithiospecifier proteinactivity in broccoli: The link between terminal alkenyl glucosinolates and sulphoraphane nitrile. Phytochemistry 69: 2765-2773.
Williams, D. J., C. Critchley, S. Pun, M. Chaliha, and T. J. O’Har. 2010. Key Role of Fe2+ in Epithiospecifier Protein Activity. J. Agric. Food Chem. 58(15): 8512–8521.
Wittstock, U. and B. A. Halkier. 2002. Glucosinolate research in the Arabidopsis era. Trends Plant Sci. 7(6):263-270.
Wu, Q., J. Lin, K. Huang and M. Liu. 2013. Characterization and expression analysis of myrosinase for sulforaphane synthesis in broccoli. Int. J. Agric. Biol. 15: 83-89.
Xue, J., M. Jorgensen, U. Pihlgren, and L. Rask. 1995. The myrosinase gene family in Arabidopsis thaliana: gene organization, expression and evolution. Plant Mol. Biol. 27: 911-922.
Yuan, G. and Q. Wang. 2012. Function of epithiospecifier protein gene from broccoli. J. Zhejiang University Agric. & Life Sci. 38 (5): 529-534.
Yang, R., L. Guo, X. Jin, C. Shen, Y. Zhou, and Z. Gu. 2015. Enhancement of glucosinolate and sulforaphane formation of broccoli sprouts by zinc sulphate via its stress effect. J. Funct. Foods 13: 345-349.
Zhang, C., Z. Y. Su, T. O. Khor, L. Shu, and A. T. Kong. 2013. Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation. Biochem. Pharmacol. 85(9): 1398-1404.
Zhang, Z. Y., J, A, Ober, and D. J. Kliebenstein. 2006, The gene controlling the quantitative trait locus EPITHIOSPECIFIER MODIFIER1 alters glucosinolate hydrolysis and insect resistance in Arabidopsis. Plant Cell 18: 1524-1536.