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研究生:吳柏宏
研究生(外文):Bohong Wu
論文名稱:芝麻再生系統及油體誘導之研究
論文名稱(外文):Studies on sesame regeneration system and oil body induction in cultured sesame cells
指導教授:曾志正曾志正引用關係蘇睿智蘇睿智引用關係
指導教授(外文):Jason T. C. TzenRuey-chih Su
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:70
中文關鍵詞:芝麻再生油體
外文關鍵詞:sesameregenerationoil body
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近年來,利用植物作為生物工廠生產外源重組蛋白質(
recombinant protein)受到相當的重視。油體膜蛋白因能專一表現在植物種子油體(oil body)上,而且可依據油體比水輕的特性,利用簡易的步驟分離及純化外源表現蛋白質,因此是一作為表達外源蛋白質的良好載體(carriers)。胡麻(Sesame),又稱芝麻,是目前世界栽種的幾種重要油料作物之一,其種子含油量超過50%,是利用油體膜蛋白表現外源蛋白質之極佳目標作物。然而,為了能夠達到實際應用的目的,建立有效的芝麻細胞外源基因表達系統是一必要條件。本研究在探討培養條件對於胡麻癒傷組織之形成、體胚之再生、以及細胞內油體含量變化之影響,以期能達到利用胡麻及其懸浮細胞作為油膜蛋白基因表現系統之目的。實驗結果發現,在不同培養條件下所誘導出之癒傷組織依其型態可分為三類。其中第三類(type III)為擬胚體(embryogenic)之癒傷組織,經挑選後進行再生條件測試發現,在含6% Maltose、1 mg/l NAA、1 mg/l BA之培養條件下,可得到較佳的再生結果。實驗中我們並以不同的鹽類、2,4-D與BA比率、以及各種不同的添加物,測試其對於胡麻癒傷組織及懸浮細胞中油體含量變化的影響,發現胡麻癒傷組織及懸浮細胞中含油量皆隨著培養基中2,4-D濃度之提高而增高。另外,在培養基中添加0.1 mg/ l 或0.2
mg/ l ABA,癒傷組織油含量亦有增加的現象。在比較多種不同胡麻品系癒傷組織細胞內油含量實驗中發現,誘導自各種不同胡麻品系之癒傷組織細胞內油含量經測試後皆在1﹪左右。

Plant-based expression systems for the production of recombinant proteins and peptides have been recently received much attention. Oleosin, an unique tissue-specific structural protein expressed in plant seed oil body, has been recognized to be a good carriers for producing foreign proteins. The recombinant proteins carried by oleosins can be easily purified using centrifugation based on the fact that oil-body has lower buoyant density than water. Sesame is one of the important oil seed crops in the world, and its seed possesses more than 50 % oil content in dry weight which makes it a very good candidate as an oil body-based expression system. However, before we can put it into practice, an efficient cell expression system has to be firstly established. This study is to investigate the effects of culture conditions on callus induction from sesame seed, regeneration of plant from somatic embryos, and also on the changes of oil-body content in the cultured cells. According to morphological appearance, the induced calli were classified into three different cell types, i.e. type I, II, and III. The type III callus, which showed embryogenic characteristic, were selected and tested for their regeneration response. The best condition was found to be with 6 % maltose, 1 mg/l NAA, and 1 mg/l BA. We also examined the effects of different salts, 2,4-D vs. BA ratios, and different additives on the oil body content in the callus and the suspension cells. It was found that the oil content in either callus or suspension cells increased with the elevated 2,4-D concentration in the culture medium. Addition of 0.1 mg/l or 0.2 mg/l ABA was found also increase the oil contents in the sesame calli. Comparison of callus induced from various sesame varieties indicated that they all have an average of about 1﹪oil content.

目 錄
頁次
中文摘要………………………………………………………… 1
英文摘要………………………………………………………… 3
前言………………………………………………………………… 5
前人研究………………………………………………………….. 6
植物蛋白質表達系統………………………………………….. 6
油體與油體膜蛋白………………………………………….…. 8
油體重組蛋白質表達系統………………………………….…. 10
胡麻簡介……………………………………………………….. 10
芝麻細胞表達系統…………………………………………….. 11
芝麻癒傷組織的誘導與再生系統的建立…………………….. 12
芝麻培養細胞油體的導……………………………………….. 12
材料與方法……………………………………………………….. 14
植物材料……………………………………………………….. 14
植物培養基…………………………………………………….. 14
多株抗體……………………………………………………….. 15
芝麻植株栽培………………………………………………….. 15
芝麻種子表面消毒…………………………………………….. 15
芝麻癒傷組織誘導…………………………………..………… 15
芝麻癒傷組織生長速率測試……………………………..…… 16
芝麻植株再生………………………………………………….. 16
芝麻懸浮細胞培養…………………………………………..… 17
芝麻細胞內油含量測定………………………………..……… 17
培養基成分對芝麻細胞內含油量的影響………………..…… 18
不同添加物對芝麻癒傷組織內含油量的影響……………..… 18
不同添加物對芝麻懸浮細胞細胞內含油量的影響………….. 19
不同品系芝麻癒傷組織細胞內含油量比較………………….. 19
油體之純化…………………………………………………….. 19
芝麻種子與癒傷組織油體組成分比較……………………….. 21
蛋白質樣品處理……………………………………………….. 21
硫酸十二酯納聚丙烯醯胺膠體電泳………………………….. 21
西方墨點法之分析…………………………………………….. 23
結果………………………………………………………………… 25
芝麻植株再生…………………………………………………… 25
芝麻癒傷組織誘導……………………………………………. 25
不同基礎培養基及維他命對Type III癒傷組織誘導形成率
的影響………………………………………………………. 25
芝麻植株栽培…………………………………………………. 26
芝麻癒傷組織植株再生……………………………………….. 27
不同auxin/cytokinin濃度對芝麻癒傷組織再生之影響….. 27
7天黑暗處理,對芝麻再生率的影響……………………….. 27
不同醣類對台南一號癒傷組織再生的影響………………….. 28
不同植物賀爾蒙對台南一號癒傷組織再生的影響………….. 28
芝麻細胞油體誘導……………………………………………… 29
不同2,4-D / BA 濃度比例,對台南一號癒傷組織生長速
率的影響………………………………………………….…. 29
不同基礎培養基,對台南一號癒傷組織細胞內含油量的
影響………………………………………………………….. 29
不同2,4-D / BA 濃度比例,對台南一號癒傷組織細胞內
含油量的影響…………………………………………….…. 30
癒傷組織誘導過程中,加入不同添加物對台南一號癒傷
組織細胞內含油量及細胞乾重的影響…………………….. 30
台南一號懸浮細胞培養……………………………………….. 31
懸浮細胞培養過程中,加入不同添加物對台南一號懸浮細
胞,細胞內含油量的影響… …………………………..…... 31
懸浮細胞培養過程中,加入不同濃度ABA對台南一號懸
浮細胞,細胞內含油量的影響… ………………………..... 32
不同芝麻品系癒傷組織細胞內由含量比較………………..… 33
討論………………………………………………………………… 34
芝麻植株再生………………………………………………….. 34
芝麻細胞油體誘導…………………………………………….. 35
參考文獻…………………………………………………………… 37
圖表………………………………………………………………… 45

台灣省政府農林廳 (1997) 台灣雜糧作物品種圖說第三輯,台灣省政府農林廳編印。
李文輝 (1995) 胡麻。台灣農家要覽農作篇(一),財團法人豐年社附設出版部,117-122。
董大成 (1961) 台灣產常見食品之營養成分,台灣醫學會。
Abell, B. M., Holbrook L. A., Abenes, M., Murphy, D. J., Hills, M. J., and Moloney, M. M. (1997) Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil boday targeting. Plant Cell 9, 1481-1493.
Alonzo, M. N. (1955) Photometric determination of fatty acid ester grounds in phospholipids. J. Biol. Chem. 217, 193-198.
Avjioglu, A. and Knox, R. B. (1989) Storage lipid accumulation by zygotic and somatic embryos in culture. Ann. Bot. 63, 409-420.
Baker, C. M., Durham, R. E., Burns, J. A., Parrott, W. A. and Wetzstein, H. Y. (1995) High frequency somatic embryogenesis in peanut (Arachis Hypogaea L.) using mature, dry seed. Plant Cell Rep. 15, 38-42.
Burnette, W. N. (1981) “Western blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radioiodinated protein A. Anal. Biochem. 112, 195-203.
Chengalrayan, K., Mhaske, V. B. and Hazra, S. (1997) High-frequency conversion of abnormal peanut somatic embryos. Plant Cell Rep. 16, 783-786.
Chuang, R. L. C., Chen, J. C. F., Chu, J. and Tzen, J. T. C. (1996) Char-acterization of seed oil bodies and their surface oleosin isoforms from rice embryos. J. Biochem. 120, 74-81.
Dittmer, J. C. and Wells, M. A. (1969) Quantitative and qualitative analysis of lipids and lipid components. Mothods in Enzymol. 14, 482-531.
Dunstan, D. I., Bethune, T. D. and Abrams S. R. (1991) Racemic abscisic acid and abscisyl alcohol promote maturation of white spruce (Picea glauca) somatic embryos. Plant Sci. 76, 219-228.
Dutta, P. C., Appelqvist, L. A. (1989) The effects of different cultural conditions on the accumulation of depot lipids notably petroselinic acid during somatic embryogenesis in Daucus carota L. Plant Sci. 78, 259-267.
Eapen, S. and George, L. (1993) Somatic embryogenesis in peanut: Influence of growth regulators and sugars. Plant Cell Tiss. Org. Cult 4, 151-156.
Etienne, H., Montorro, P., Michaux-Ferriere, N. and Carron, M. P. (1993) Effect of desiccation, medium osmolarity and abscisic acid on the muturation of Hevea brasilensis somatic embryos. J. Exp. Bot. 44, 1613-1619.
Ewins A. (1992) Oilseed prices still going up on the Winnipwg exchange. Westerm Producer 70, 11.
George, L., Bapat, V. A. and Rao, P. S. (1987) In vitro multiplication of sesame (Sesamun indicum) through tissue culture. Ann. Bot. 60, 17-21.
Goldberg, R. B., Barker, S. J. and Perez-Grau L. (1989) Regulation of gene expression during plant embryogenesis. Cell 56, 149-160.
Guo, Y. D. and Pulli, S. (1996) High-frequency embryogenesis in Brassica campestris microspore culture. Plant Cell Tiss. Org. Cult 46, 219-225.
Herman, E. M. (1995) Cell and molecular biology of seed oil development in Seed Development and Germination (Kigel, J. and Galili, G., eds.), pp. 195-214, Marcel Dekker, New York
Hiatt, A., Cafferkey, R. and Bowdish, K. (1989) Production of antibodies in transgenic plant . Nature 342, 76-78.
Hoffman, L. M., Donaldson, D. D. and Herman, E. M. (1988) A modified storage protein is synthesized, processed and degraded in the seeds of transgenic plant. Plant Mol. Biol. 11, 717-729.
Horsch, R. B., Fry, J. E., Hoffman, N. L., Wallroth, M., Eichholtz, D., Roger, S. G. and Fraley, R. T. (1985) A simple and general method for transferring genes into plant. Science 227, 1229-1231.
Huang, A. H. C. (1992) Oil bodies and oleosins in seeds. Annu. Rev. Plant Physiol. 43, 177-200.
Huang, A. H. C. (1996) Oleosin and oil bodies in seeds and other organs. Plant Physiol. 110, 1055-1061.
Jacks, T. J., Hensaring, T. P., Neucere, J. N. Yatsu, L. Y. and Barker, R. H. (1990) Isolation and physicochemical characterization of the half-unit membranes of oilseed lipid bodies. J. Am. Oil chem. Soc. 67, 353-361.
Janick, J. (1991) Production of seed lipids via culture of somatic embryos. In:Rattray, J. (ed) Biotechnology of plant fats and oils, American Oil Chemists Society, Champaign, III, pp 76-103.
Krebbers, E. and Vandekerckhove, J. (1990) Production of peptide in plant seeds. Trends Biotechnol 8, 1-3.
Kyswly, W. and Jacobsen, H. J. (1990) Somatic embryogenesis from pea embryos and shoot apices. Plant Cell Tiss. Org. Cult. 20, 7-14.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Maquoi, E., Hanke, D. E. and Deltour, R. (1993) The effects of abscissic acid on the maturation of Brassica napus somatic embryos: an ultrastructural study. Protoplasma 174, 147-157.
Mary, R. J. and Jayabalan, N. (1997) Influence of growth regulators on somatic embryogenesis in sesame. Plant Cell Tiss. Org. Cult 49, 60-70.
Mhaske, V. B., Chengalrayan, K. and Hazra, S. (1998) Influence of osmotica and abscisic acid on triglyceride accumulation in peanut somatic embryos. Plant Cell Rep. 17, 742-746.
Murphy, D. J. (1994) Biogenesis, function and biotechnology of plant storage lipids. Prog. Lipid Res. 33, 71-85.
Murphy, D. J., and Au, D. M. Y. (1989) A new class of highly abundant apolipoproteins involved in lipid storage in oilseeds. Biochem. Soc. Trans. 117, 682-683.
Murphy, D. J., Rawsthorne, S. and Hills, M. J. (1993) Storage lipid formation in seeds. Seed Sci. Res. 3, 79-95.
Osborne, T. B. (1924) The vegetable proteins. Longmans, Green & Co., London.
Parmenter, D. L., Boothe, J. G., van Rooijen, G. J. H., Yeung, E. C. and Moloney, M. M. (1995) Production of biologically active hirudin in plant seeds using oleosin partitioning. Plant Mol. Bio. 29, 1167-1180.
Pen, J., Verwoerd, T. C.,Van Paridon, P. A., Beudeker, R. F., Van den Elzen, P. J. M., Gearse, K.., Van der Klis, J. D., Versteegh, H. A. J.,Van Ooyen, A. J. J. and Hoekema, A. (1993) Phytase-containing transgenic seeds as a novel feed additive for improved phosphorous utilization. Bio/technology 11, 811-814.
Pence, V. C., Hasegawa, P. M. and Janick, J. (1981) Sucrose mediated regulation of fatty acid composition in asexual embryos of Theobroma cacao. Physiol. Plant 53, 378-384.
Piqueras, A., Olmos, E. and Hellin, E. (1994) Cytological changes related with salt tolerance in embryogenic callus of Citrus limon. Plant Cell Tiss. Org. Cult 39, 13-18.
Qu, R., Wang, S. M., Lin, Y. H., Vance, V. B. and Huang, A. H. C. (1986) Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (Zea mays L.). Biochem. J. 234, 57-65.
Radetzky, R. and Langheinrich U. (1994) Induction of accumulation and degradation of the 18.4-kDa oleosin in a triacylglycerol-storing cell culture of anse (Pimpinella anisum L.). Planta 194, 1-8.
Shewry, P. R., Napier, J.A. and Tatham A. S. (1995) Seed storage proteins: structure and biosynthesis. Plant Cell. 7, 945-956.
Sijmons, P. C., Dekker, B. M., Schrammeijer, B.,Verwoerd, T. C., van den Elzen, P. J. M. and Hoekema, H. (1990) Production of correctly processed human serum albumin in transgenic plant. Bio/technology 8, 217-221.
Stymne, S. and Stobart, A. K. (1987) Triacylglycerol biosynthesis in The Biochemistry of Plants (Stumpf, P. K. and Conn, E. E., eds.) Vol. 10, pp. 175-214, Academic Press, New York
Tzen, J. T. C., Cao, Y. Z., Laurent, P., Ratnayake, C. and Huang, A. H. C. (1993) Lipids, proteins, and structure of seed oil bodies from diverse species. Plant Physiol. 101, 267-276.
Tzen, J. T. C., Chuang, R. L. C., Chen, J. C. F. and Wu, L. S. H. (1998) Coexistence of both oleosin isoforms on the surface of seed oil bodies and their individual stabilization to the organelles. J. Biochem. 123, 319-324.
Tzen, J. T. C. and Huang, A. H. C. (1992) Surface structure and properties of plant seed oil bodies. J. Cell Biol. 117, 327-335.
Tzen, J. T. C., Lai, Y. K., Chan, K. L. and Huang, A. H. C. (1990) Oleosin isoforms of high and low molecular weights are present in the oil bodies of diverse seed species. Plant Physiol. 94, 1282-1289.
Tzen, J. T. C., Lie, G. C. and Huang, A. H. C. (1992) Characterization of the charged components and their topology on the surface of plant seed oil bodies. J. Biol. Chem. 267, 15626-15634.
Tzen, J. T. C., Peng, C. C., Cheng, D. J., Chen, E. C. F. and Chiu, J. M. H. (1997) A new method for seed oil body purification and examination of oil body integrity following germination. J. Biochem. 121, 762-768.
Vandekerckhove, J., Damme, J. V., Lijsebettens, M. V., Botterman, J., DeBlock, M., Van Montagu, M. and Krebber, E. (1989) Enkephalins produced in transgenic plant using modified 2S seed storage proteins. Bio/technology 7, 929-932.
Vance, V. B. and Huang, A. H. C. (1987) The major protein from lipid
bodies of maize. Characterization and structure based on cDNA clon-ing. J. Biol. Chem. 262, 11275-11279.
van Rooijen, G. J. H. and Moloney, M. M. (1995) Plant seed oil-bodies as carriers for foreign proteins. Bio/Technology 13, 72-77.
Wilson, K. A. (1986) In:Plant Proteolytic Enzymes, vol. II, pp. 19-47. CRC Press, London.
Wilson, M. A. and Nakane P. K. (1978) Recent development in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. Knapp W, Holubar K, Wick G, eds, Immunofluorescence and Related Staining Techniques. Elsevier/North Holland Biomedical Press, Amsterdam, 215-224
Yatsu, L. Y. and Jacks, T. J. (1972) Spherosome membranes. Half unit-
membranes. Plant Physiol. 49, 937-943.

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