Sporamin是甘藷塊根中含量非常豐富的儲存性蛋白質，過去對sporamin的研究方向著重於其運送過程及被蔗糖誘導的表現機制，直到1997年，Yeh等人才證明其具有胰蛋白抑制因子的活性，並在轉殖菸草中發現對斜紋夜盜蛾等幼蟲的啃食具有抗性。
在本篇論文中，我們期望得到具有抗蟲能力的十字花科作物，但基於CaMV 35S啟動子所驅動的基因轉殖很容易引起基因靜默的現象，故採用sporamin基因本身的prospoA啟動子來驅動sporamin基因的表現，prospoA是一具有受傷誘導的特性啟動子。經前人的研究得知，影響一基因的表現不止轉錄區的部分，在非轉錄區的地方也可能會影響基因的表現，因此我們也比較sporamin cDNA及genomic sporamin在prospoA驅動下的表現。我們首先構築了35S/sporamin、prospoA/sporamin、prospoA/sporamin —3’flanking region三種質體，利用農桿菌(Agrobacterium tumefaciens)轉殖進入阿拉伯芥及芥藍植物中。
利用阿拉伯芥及芥藍兩種植物來表現sporamin基因，經抗生素及聚合連鎖反應來初步篩選轉殖成功的植株，在阿拉伯芥的轉殖上相當有2∼5％的轉殖率，而芥藍植株也只有5％左右的成功率。轉殖成功的植株經西方點墨法分析後，證實植株均表現sporamin蛋白質，但各轉殖株之間的表現量均稍有差異。利用胰蛋白抑制因子膠體活性分析轉殖株，並沒有偵測到sporamin蛋白質表現胰蛋白抑制因子的活性，但經過胰蛋白親和性管柱純化轉殖株的粗萃取蛋白質，會得到具有活性的sporamin蛋白質。故採用敏感度較高的胰蛋白脢抑制因子生化活性分析轉殖株之間的活性，比較三種載體之間的活性，發現具有3’flanking region的sporamin基因在prospoA啟動子的驅動下表現了較高的胰蛋白抑制因子的活性。
利用芥藍轉殖株餵食二∼三齡的玉米穗蟲幼蟲，來分析轉殖株的抗蟲能力，經過2次的重複測試後，測量葉片啃食重量、蟲子平均重量級蟲子存活率，以野生型植株作為對照組，結果顯示有表現胰蛋白脢抑制因子活性的植株，都有抵抗玉米穗蟲幼蟲的啃食的能力。

Sporamin is an abundant storage protein in tuberous roots of sweet potato. It accounts for about 80% of total soluble proteins in sweet potato tubers, and no expression in leaves and a little expression in stems at the growing stage.
In 1997’s, Yeh et al., constructed a recombinant plasmid with sporamin cDNA and expressed it’s in Escherichia coli cells as fusion proteins. This protein was identified by strong inhibitory activity to trypsin in vitro and was proved to be pest-resistant in transgenic tobacco plants.
The transgenes driven by CaMV 35S promoter usually have gene silencing. To compare the gene expression driven by CcaMV 35S promoter and sporamin promoter, we selected prospoA promoter to drive it’s own cDNA sporamin and genomic sporamin, then established three different constructs: 35S::sporamin, prospoA::sporamin and prospoA::sporamin 3’-flanking region. They were introduced into Brassica oleracea var. alboglabra and Arabidopsis thaliana plants by Agrobacterium tumefaciens-mediated transformation. Genomic sporamin has longer 3’-flank region than cDNA sporamin, and contains one MAR T-box, three E-box, one SEF4 box. All transformants were screened for kanamycin resistance and confirmed by PCR. Arabidopsis thaliana transformants showed 2~5 % transformation frequency, and Brassica oleracea var. alboglabra expressed 5 % transformation ration. We used western analysis, trypsin inhibitory gel activity analysis and biochemical analysis to detect sporamin protein expression level in the transformants. Among the three-constructs, prospoA::sporamin 3’-flanking region shows better trypsin inhibitory activity than other two constructs.
We also tested insecticidal activity of these transformants by the feeding test of Helicoverpa armigera Hubner. It was demonstrated the genomic transgenic plants, with a genomic construct expressing better trypsin inhibitory activity, had better pest-resistant than wild type plants.

目錄
檢索表…………………………………………………………………………..3
中文摘要………………………………………………………………………..5
英文摘要………………………………………………………………………..7
第一章 前言
第一節 植物的防禦機制…………………………………………………9
第二節 防禦性蛋白質……………………………………………………11
第三節 基因靜默…………………………………………………………15
第四節 Sporamin蛋白質………………………………………………..17
第五節 芥藍的轉殖………………………………………………………19
第二章 材料與方法
實驗材料…………………………………………………………………….21
實驗方法…………………………………………………………………….21
第一節 載體的構築………………………………………………………21
第二節 阿拉伯芥的轉殖…………………………………………………28
第三節 芥藍的轉殖………………………………………………………32
第四節 轉殖植物之鑑定與分析…………………………………………38
第五節 抗蟲能力測試……………………………………………………47
第三章 結果
第一節 構築載體…………………………………………………………48
第二節 轉型至大腸桿菌XL1-blue strain及鑑定……………………...49
第三節 阿拉伯芥植物之轉殖及篩選……………………………………50
第四節 轉殖阿拉伯芥之鑑定……………………………………………51
第五節 芥藍植物之轉殖及篩選…………………………………………51
第六節 轉殖芥藍之鑑定…………………………………………………52
第七節 西方點墨法分析sporamin蛋白質……………………………..53
第八節 分析芥藍及阿拉伯芥轉殖株胰蛋白抑制因子活性…………53
第九節 胰蛋白抑制因子生化活性分析……………………………...54
第十節 抗蟲測試…………………………………………………………55
第四章 討論
第一節 載體的構築目的……………………………………………….…56
第二節 利用農桿菌進行基因轉殖……………………………………….58
第三節 胰蛋白抑制因子活性分析…………………………………….61
第四節 未來展望………………………………………………………….61
參考文獻…………………………………………………………………………63
附圖………………………………………………………………………………70
附錄………………………………………………………………………………86

參考資料
陳淑鈺 (2001) 甘藷MAR sequence的選殖與特性之研究。台灣大學植物學研究所碩士論文
Bajaj, Y. P. S., & Nitsch, P. (1975) In vitro propagation of red cabbage (Brassica oleracea L. var. capitata). J. Exp. Bot. 26:883-890
Bergey, D. R., Hgwe, G. A., Ryan, C.A. (1996) Polypeptide signaling for plant defensive genes exhibits analogies to defence signaling in animals. Proc. Natl. Acad. Sci. USA 93:12053-12058
Borut, S., Joze, P., Pika, M., Darja, B. M., Franc, G., & Igor, K. V. T., (1992) Characterization of aspartic proteinase inhibitors from potato at the gene, cDNA and protein levels. Biol. Chem. Hoppe Seyler 373:477-482
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254
Broadway, R. M., & Duffy, S. S. (1986) Plant proteinase inhibitors: mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exihua. Journal of insect physiology 32:827-833
Buell, C. R. (1998) Arabidopsis:a weed leading the field of plant-pathogen interations. Plant Physiol. Biochem. 36:177-186
Burgess, E. P. J., Stevens, P. S., Keen, G. K., Laing, W. A. & Christeller, J. T. (1991) Effect of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus.Entomologia Experimentialis et Appl. 61:123-130
Burrows, P. R., Barker, A. D. P., Newell, C. A. & Hamilton, W. D. O. (1998) Pestic. Sci. 52:176-183
Campos, F. A. P., & Richardson, M. (1983) The complete amino acid sequence of the bifunctional α-amylase/trypsin inhinitor from seeds of ragi (Indian finger millet, Eleusine coracana Gaertn.). FEBS Lett. 152:300-304
Chen, M. S., Johnson, B., Wen, L., Muthukrishnan, S., Kramer, K. J., Morgan, T. D. & Reeck, G. R. (1992) Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity and insect growth suppressing activity of a truncated form of the protein. Protein Expression and Purification 3:41-49
Collinge, D. B., Kragh, K. M., Mikkeisen, J. D., Nielsen, K. K., Rasmussen, U. & Vad, K. (1993) Plant chitinases. Plant J. 3:31-40
Dehio, C., & Schell, J. (1994) Identification of plant genetic loci involved in a post-transcriptional mechanism for meiotically reversible transgene silencing. Proc. Natl. Acad. Sci. USA 91:5538-5542
Dunwell, J. M. (1981) In vitro regeneration from excised leaf discs of three Brassica. species. J. Exp. Bot. 32:789-799
Elmayan, T., & Vaucheret, H. (1996) Expression of single copies of a strongly expressed 35S transgene can be silenced post-transcriptionally. Plant J. 9:787-797
English, J. J., Mueller, E., & Baulcombe, D. C. (1996) suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8:178-188
Flick, C. E., Evans, D. A., & Sharp, W. R. (1983) Organogenesis. In: Evans, D. A., Sharp, W. R., Ammirato, P. V., Yamada, Y. (Eds) Handbook of Plant Cell Culture. 1:13-81
Green, T. R., & Ryan, C. A. (1972) Wound-induced proteinase inhibitor in plant leaves:a possible defence mechanism against insects. Science (Washington) 175:776-777
Garcia-Olmedo, F., Salcedo, G., Sanchez-Monge, R., Gomez, L., Royo, J., & Carbonero, P., (1987) Plant proteinaceous inhibitors of proteinase and α-amylase. Oxf. Surv. Plant Mol. Cell Biol. 4:345-350
Gatehouse, A. M. R., & Boulter, D., (1983) Assessment of the anti-metabolic effects of trypsin inhibitors from cowpea (Vigna unguiculata) and other legumes on development of the bruchid bettle Callosobruchus maculatus. Journal of the Science of Food and Agriculture. 34:345-350
Gatehouse, A. M. R., Davison, G. M., Newell, C. A., Merryyweather, A., Hamilton, W. D. O., Burgess, E. P. J., Gilbert, R. J. C., & Gatehouse, J. A., (1997) Transgenic potato plants with enhznced resistance to the tomato month, Lacanobia oleracea: growth room trails. Molecular Breeding 31:49-63
Hammerschmidt, R., Schultz, J. C. (1996) Multiple defenses and signals in plant defense against pathogens and herbivores. Rec. Adv. Phytochem. 30:121-154
Hammond-Kosack, K. E., Jones, J. D. G. (1996) Risistance gene-dependent plant defense response. Plant Cell 8:1773-1791
Hattori, T., Nakagawa, T., Maeshima, M., Nakamura, K., & Asahi, T. (1985) Molecular clonging and nucleotide sequence of cDNA for sporamin, the major soluble protein of sweet potato tuberous roots. Plant Mol. Biol. 5:313-320
Hattori, T., Yoshide, N., & Nakamura, K. (1989) Structural relationship among the members of a multigene family coding for the sweet potato tuberous root storage protein. Plant Mol. Biol. 13:563-572
Hilder, V. A., Gatehouse, A. M. R., Sheerman, S. E., Barker, R. F., & Boulter, D., (1987) A novel mechanism of insect resistance engineered into tobacco. Nature 330:160-163
Iizuka, M., Hla-Bo S., & Madrigal, R. (1978) Floral organ culture and organogenesis of cruciferous plants. Japan J. Breed. 28:151-158
Ingelbrecht, I., Van, H. H., Van, M. M., & Depicker, A. (1994) Posttranscriptional silencing of reporter transgengs in tobacco correlates with DNA methylation. Proc. Natl. Acad. Sci. USA 91:10502-10506
Jongsma, M. A., Bakker, p. l., Peters, J., Bosch, D., & Stiekema, W. J., (1995) Adaptation of Spodoptera exigua larval to plant proteinase inhibitors by induction of proteinase activity insensitive of inhibition. Proc. Natl. Acad. Sci. USA 92:8041-8045
Jouanin , L., Michel, B. B., Cecile, G., Gil, M. and Marc, G. (1998)Transgenic plants for insect resistance. Plant Science 131:1-11
Kartha, K. K., Gamborg, O. L., & Constabel, F. (1974) In vitro plant formation from stem explants of rape (Brassica napus cv. Zephyr). Physiol Plant 31:217-220
Keller, W. A., Rajathy, T., & Lacapra, J. (1975) In vitro production of plants from pollen in Brassica campestris. Can. J. Genet. Cytol. 17:655-666
Kim, Y. K., & Kim, K. S., (1994) Expression of GRP78 enhancer-CAT fusion constructs microinjected into Xenopus laevis oocytes. Korean Journal of Zoology. 37:137-143.
Klimaszewska, K., & Keller, W. A. (1985) High frequency plant regeneration from thin cell layer explantsof Brassica napus. Plant Cell Tiss. Org. Cult. 4:183-197
Knowles, B. H. and Ellar, D. J. (1987) Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus thuringiensis δ-endotoxins with differenet insect specificity. Biochem. Biophy. Acta. 924:509-518
Koncz C. Martini N. Mayerhofer R. Koncz Kalman Z. Koerber H. Redei G P. Schell J. (1989) High-frequency T DNA-medisted gene tagging in plants. Proceedings of the National Academy of Sciences of the United States of America 86(21): 8467-8471
Kuroda, M., Ishmto, M., Suzuki, K., Kondo, H., Abe, K., Kitamura, K., & Arai, S. (1996) oryzacystatins exhibit growth-inhibitory and lethal effects on different species of bean insect pest, Ccallosobruchus chinensis(Coleoptera)and Riptortus clavatus(Hemiptera). Bioscience Biotechnology and Biochemistry 60:209-212
Laskowski, M. J., & Kato, I., (1980) Protein inhibitors of proteinases. Ann. Rev. Biochem. 49:593-626
Lazzeri, P. A., & Dunwell, J. M. (1984) In vitro shoot regeneration from seeding root segments of Brassica oleracea and Brassica napus cultivars. Ann. Bot. 54:341-350
Lichter, R. (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z. Pflanzenphysiol 105:427-434
Lin, J. Y., Chu, S. C., Wu, H. C., & Hsieh, Y. S. (1991) Trypsin inhibitor from the seeds of Acacia confusa. J. Biochem. 110:879-883
Lyccett, G. W., (1990) Genetic engineering of insect control agents and production of insect-resistance plants. Genetic Engineering of Crop Plants 49-58
Maeshima, M., Sasaki, T. A., & Asahi, T. (1985) Characterization of major proteins in sweet potato tuberous roots. Pytochemistry 24:1899-1920
Mascarenhas, D., Mettler, I. J., Puerce, D. A., & Lowe, H. W. (1990) Intron-mediated enhancement of heterologous gene expression in maize. Plant Molecular Biology 15(6): 913-920
Matsuoka, K., Matsumoto, S., Hattori, T., Machida, Y., & Nakamura, K. (1990) Vaculor targeting and posttranslational processing of the precusor to the sweet potato tuberous root storage protein in heterogous plant cell. J. Biol. Chem. 265:19750-19757
Mcelroy, D., Blowers, A. D., Henes, B., & Wu, R. (1991) Construction of expression vectors based on the rice actin 1 act1 5’region for use in monocot transformation. Molecular & General Genetics 231(1):150-160.
Murakami, S., Hattori, T., & Nakamura, K. (1986) Structural dufferences in full-length cDNAs for two classes of sporamin, the major soluble protein of sweet potato tuberous roots. Plant Mol. Biol. 7:343-355
Murphy D J. Cummins I. Ryan A J. (1989) Immunocytochemical and biochemical study of the biosynthesis and mobilization of the major seed storage protein of Brassica napus. Plant Physiology & Biochemistry (Paris) 27 (5): 647-658
Nakamura, K., Matsuoka, K., Mukumoto, F., & Watanabe, N. (1993) Processing and transport to the vacuole of a precursor to sweet potato sporamin in transformed tobocco cell line BY-2. J. Exp. Botany 44:331-338
Narasimhulu, S. B., Kirti, P. B., Mohapatra, T., Prakash, S., & Chopra, V. L. (1992) Soot regeneration in stem explants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica Carinata. Plant Cell Report 11: 359-362
Norris, D. M. & Kogan, M.(1980) Biochemical and morphological bases of resistance. In Breeding Plants Resistant to insect, ed. F. G. Maxwell and P. R. Jennings. John Wiley & Sons, London, 23-60
Park, Y. D., Papp, I., Moscone, E. A., Iglesias, V. A., Vaucheret, H., Matzje, A. J. M., & Matzke, M. A., (1996) Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity. Plant J. 9:183-194
Pius, J., George, L., Eapen, L., & Rao, P. S. (1994) Influence of genotype and phytohormones on somatic embryogenesis and plant regeneration in finger millet. Proceeding of the Indian National Science Academy Part B Biological Science. 60(1): 53-56
Pua, E. C., & Chi, G. L. (1993) De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene. Physiologia Plantarum. 88(3): 467-474.
Puddephat, I. J., Riggs, T. J., & Fenning, T. M. (1996) Transformation of Brassica oleracea L.: A critical review. Molecular Breeding 2(3):185-210
Ryals, J. A., Neuenschwander, U. H., Willits, M. G., Molina, A., Steiner, H. Y., Hunt, M. D. (1996) Systemic cquired resistance. Plant Cell 8:1809-1819
Ryan, C. A., (1990) Protease inhibitors in plants: genes for improving defence against insects and pathogens. Ann. Rev. Phytopathol., 28:425-449
Sambrook, J., Firtxch, E. F., & Maniatis, T. (1989) Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, N. Y.
Schenvk, H. R., & Hoffmann, F. (1983) Isolation and culture of protoplasts: Brassica. In: Vasil IK (Ed) Cell Culture and Somatic Cell Genetics of Plants 1:356-369
Schroeder, M. R., Borkhsenious, O. N., Matsuoka, K., Nakamura, K., & Raikhel, N. V. (1993) Colocalization of barley lectin and sporamin in vacuoles of transgenic tobacco plants. Plant Physiol. 101:451-458
Sheng, J., & Citovsky, V. Agrobacterium-plant cell DNA transport: Have virulence proteins, will travel. Plant Cell. 8(10): 1699-1710
Stam, M., Mol, J. N. M., & Kooter, J. M., (1997) The silence of genes un transgenic plants. Ann. Bot. 79:63-72
Stotz, H. U., Pittendrigh, B. R., Kroymann, J., Weniger, K., Fritsche, J., Bauke, a., Mitchell, T. (2000) Induced plant defense responses against chewing insects. Ethylene signaling reduces resistance of Arabidopsis against Egyptian Cotton worm but not Diamondback Moth. Plant Physiology 124:1007-1017
Thomas, J. C., Adams, D. G., Keppenne, V. D., Wasmann, C. C., Brown, J. K., Kanost, M. R., & Bohnert, H. J., (1995) Protease inhibitors of Manduca sexta expressed in transgenic cotton. Plant Cell Reports. 14:758-762
Turlings, T. C. J., Loughrin, J. H., McCall, P. J., Rose, USR.,Lewis, W. J., Tumlinson, J. H. (1995) How caterpillar-damaged plants protect themselves by attracting parasitic. Proc. Natl. Acad. Sci. USA 92:4169-4174
Uriel, J., & Berges, J. (1968) Characterization of nutral inhibitors of trypsin and chymotrypsin by elrctrophoresis in acrylamide-agarose gels. Nature 218: 578-580
Urwin, P. E., Atkinson, H. J., Waller, D. A., & McPherson, M. J., (1995) Engineered oryxacystatin-I expressed in transgenic hairy roots confers resistance to Giobodera pallida. Plant Journal 8:121-131
Vain, P., Finer, K. R., & Dean, E. (1996) Intron-mediated enhancement of gene expression in maize(Zea mays L.) and bluegrass(Poa pratensis L.) Plant Cell Reports 15(7): 489-494
Vaucheret, H., (1993) Identification of a general silencer for 19S and 35S promoters in a transgenic tobacco plant: 90 bp of homology in the promoter sequences are sufficient for transinactivation. C. R. Acad. Sci. Paris, 316:1471-1483
Wang, S. J., Lan, Y. C., Chen, S. F., Chen, Y. M., & Yeh, K. W. (2002) Wond-response regulation of the sweet potato sporamin gene promoter region. Plant Molecular Biology 48:223-231
Wesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M., Rouse, D. T., Liu, Q., Gooding, P. S., Singh, S. P., Abbort, D., Stoutjesdijk, P. A., Robinson, S. P., Gleave, A. P., Green, A. G., & Waterhouse, P. M. (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27(6): 581-590
Wong, K. W., & Loh, C. S. (1987) In vitro regeneration of plantlets in Brassica alboglabra. Plant Cell Tiss. Org. Cult. 10:143-148
Xu, D., Xue, Q., McElroy, D., Mawal, Y., Hilder, V. A., & Wu, R., (1996) Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests. Molecular Breeding 2:167-173
Yeh K. W., Chen J. C., Lin M.I., Chen Y.M., & Lin C.Y. (1997) Functional activity of sporamin from sweet potato (Ipomoea bataras Lam.): A tuber storage protein with trypsin inhibitory activity. Plant Molecular Biology 33(3):565-570
Yeh K. W., Lin M.I., Tuan S.J., Chen Y.M., Lin C.Y., & Kao S. S. (1997) Sweet potato (Ipomoea bataras) trypsin inhibitors expressed in transgenic tobacco plants con R., (1996) Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests. Molecular Breeding 2:167-173
Yeh K. W., Chen J. C., Lin M.I., Chen Y.M., & Lin C.Y. (1997) Functional activity of sporamin from sweet potato (Ipomoea bataras Lam.): A tuber storage protein with trypsin inhibitory activity. Plant Molecular Biology 33(3):565-570
Yeh K. W., Lin M.I., Tuan S.J., Chen Y.M., Lin C.Y., & Kao S. S. (1997) Sweet potato (Ipomoea bataras) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura. Plant Cell reports 16:696-699