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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 葉開溫(Kai-Wen Yeh) | |
dc.contributor.author | Ai-hua Yang | en |
dc.contributor.author | 楊藹華 | zh_TW |
dc.date.accessioned | 2021-06-13T17:04:38Z | - |
dc.date.available | 2005-02-02 | |
dc.date.copyright | 2005-02-02 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-01-28 | |
dc.identifier.citation | 黃賢喜. 1991. 芋品種與栽培法改良之研究. 高雄區農業改良場特刊KC-004. pp.1-146.
黃賢喜. 韓青梅. 1994. 芋. 雜糧作物各論 III. 根及莖類. 台灣區雜糧發展基金會成立二十週年紀念專輯之一. 第20章. 1665-1735 Abe M. and Arai S. (1985) Purification of a cysteine proteinase inhibitor from rice, Oryza sativa L. japonica. Agric. Biol. Chem. 49(11): 3349-3350. Abe M., Arai S., Kato H., Fujimaki M. (1980) Thiol-protease inhibitor occurring in endosperm of corn. Agri. Biol. Chem. 44(3): 685-686. Abe M., Arai S. (1991) Some properties of a cysteine proteinase inhibitor from corn endosperm. Agric. Biol. Chem. 55: 1417-1418. Abe M., Emori Y., Kondo H., Suzuki K., Arai S. (1987a) Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). J. Biol. Chem. 262: 16793-16797. Abe K., Kondo H., Arai S. (1987b) Purification and characterization of a rice cysteine proteinase inhibitor. Agric. Biol. Chem. 51: 2763-2768. Abe K., Emori Y., Kondo H., Arai S., Suzuki K. (1988) The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli. J. Biol. Chem. 263: 7655-7659. Abe M., Abe K., Kuroda M., Arai S. (1992) Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin. Eur. J. Biochem. 209: 933-937. Aker C. P., Hoff, J. E. (1980) Simultaneous formation of chymopapain inhibitor activity and cubical crystals in tomato leaves. Can. J. Bot. 58: 1000-1003. Arai S., Watanabe H., Kondo H., Emori Y., Abe K. (1991) Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members. J. Biochem 109: 294-298. Barret A. J. (1981) Cathepsin B, Cathepsin H, Cathepsin L. Methods enzymol. 80: 771-778. Barrett A. J. (1987) The cystatins: a new class of peptides inhibitors. Trends Biochem Sci. 12: 193-196. de Barros E., Larkins B. A. (1990) Purification and characterization of zein-degrading protease from endosperm of germinating maize seeds. Plant Physiol. 94: 297-303. Birk Y., Applebaum S. W. (1960) Effect of soybean trypsin inhibitors on the development and midgut proteolytic activity of Tribolium castaneum larvae. Enzymologia 22: 318-326. Blechert S., Brodshelm W., Hölder S., Kammerer L., Kutchan T. M., Mueller M. J., Xia Z. Q., Zenk M. H. (1995) The octadecanoic pathway: signal molecules for the regulation of secondary pathways. Proc. Natl. Acad. Sci. USA. 92: 4099-4105. Bode W., Engh H., Musil H. (1988) The 2.0 Å X-ray crystal structure of chicken egg white cystatin and it possible mode of interaction with cysteine proteinase. EMBO J. 7: 2593-2599. Bolter C. J. (1993) Methyl jasmonate induces papain inhibitor(s) in tomato leaves. Plant Physiol. 103: 1347-1353. Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the priciple of protein-dye binding. Anal. Biochem. 72: 248-254. Brookhart G. L.,Kramer K. J. (1990) Proteinases in molting fluid of the tobacco hornworm, Manduca sexta. Insect Biochem. 20: 467-478. Brzin J., Ritonja A., Popvic T., Turk V. (1990) Low molecular mass protein inhibitor of cysteine proteinases from soybean. Biol. Chem. Hoppe-Seyler. 371:167-170. Brzin J., Popovic T., Drobnic-Kosorok M., Kotnik M., Turk V. (1988) Inhibitors of cysteine proteinases from potato. Biol. Chem. Hoppe-seyler. 369: 233-238. Carneiro M., Coutinho M. V., Rodrigues C. A., de Castro L. A. B. (1990) Isolation and characterization of the major albumin from Colocasia esculenta corms. Plant Sci. 67: 39-46. Castro L. A. B., Carneiro M., Neshich D. C. M., Paiva. G. R. (1992) Spatial and temporal gene expression patterns occur during corm development. Plant Cell. 4: 1549-1559. Chen M. S., Johson 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 Expr. Purify. 3: 41-49. Chenchik A., Zhu Y., Diatchenko L. R., Hill J., Siebert P. 1998. Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR. In RT-PCR methods for gene cloning and analysis. Eds. Siebert P. and Larrick J. (Bio Techniquws Books, MA). pp 305-319. Darvill A. G., Albersheim P. (1984) Phytoalexins and their elictors: a defence against microbial infection in plants. Annu. Rev. Plant Physiol. 35: 243-276. Ding L. C., Hu C. Y., Yeh K. W., Wang P. J. (1998) Development of insect-resistant transgenic cauliflower plants expressing the trypsin inhibitor gene isolated from local sweet potato. Plant Cell Rep. 17: 854-860. Diop N. N., Kidrič M., Repellin A., Garell M., ď Arcy-Lameta A., Thi A. T. P., Zuily-Fodail Y. 2004. A multicystatin is induced by drought-stress in cowpea (Vigna unguiculata (L.) Walp.) leaves. FEBS Lett. 577: 545-550. Drenth J., Jansonius J. N., Koekoek R., Swen H. M., Wolthers B G., (1968) Structure of papain. Nature. 218: 929-32. Fernandes K. V. S., Sabelli P. A., Barratt D. H. P., Richardson M., Xavier-Filho J., Shewry P. R., (1993) The resistance of cowpea seeds to byuchid is not related to levels of cysyeine proteinase inhibitors. Plant Mol. Biol. 23: 215-219. Fujinaga M., Read R. J., Sielecki A., Ardelt W., Laskowski M. Jr., James M. N. G. (1982) Refined crystal structure of the molecular complex of Streptomyces griseus protease B, A serine protease, with the third domain of the ovomucoid inhibitor from turkey. Proc. Natl. Acad. Sci. USA 79: 4868-4872. Green T. R. Ryan C. A. (1972) Wound induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects. Science 175: 776-777. Guerrero C., de la Calle M., Reid M. S., Valpuesta V. (1998) Analysis of the expression of two thiolprotease genes from daylity (Hemerocallis spp.) during flower senescence. Plant Mol. Biol. 36: 565-571. Gutierrez-Campos R., Torres-Acosta J. A., Saucedo-Arias L. J., Gomez-Lim M. A. 1999. The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. Nature Biotech. 17: 1223-1226. Hilder V. A., Gatehouse A. M. R., Sheerman S. E., Barker R. F., Boulte D. (1987) A novel mechanism of insect resistance engineered into tobacco. Nature 300: 160-163. Hines M. E., Osuala C. I., Nielsen S. S. (1991) Isolation and partial characterization of a soybean cystatin cysteine proteinase inhibitor of coleopteran digestive proteolytic activity. J. Agri. Food Chem. 39: 1515-1520. Hirai M., Nakamura K., Imai T., Sato T. (1993) cDNAs encoding for storage proteins in the tubers of taro ( Colocasia esculenta Schott ). Jpn. J. Genet. 68(3): 229-36. Huber R., Bode W. (1978) Structure basis of the activation and action of trypsion. Acc. Chem. Res. 11: 114-122. Isabel C. B., Castro L. A. B., Neshich G., de Almeida E. R. P., de Sá M. F. G., Mello L. V., Monte-Neshich D. C. (1995) A corm-specific gene encodes tarin, a major globulin of taro ( Colocasia esculenta L. Schott ). Plant Mol. Biol. 28: 137-144. Jacinto T., Fernandes K. V. S., Machado O. L. T., Siwueira C. L. Jr. (1998) Leaves of transgenic tomato plants overexpressing prosystemin asscumulate high levels of cystatin. Plant Sci. 138: 3542. John T. C., Wasmann C. C., Echt C., Dunn R. L., Bohnert H. J., McCoy T. J. (1994) Introduction and expression of an insect proteinase inhibitor in alfalfa (Medicago sativa L.). Plant Cell Rep. 14: 31-36. Johnson R., Narvaez J., An G., Ryan C. (1989) Expression of proteinase inhibitors I and II in transgenic tobacco plant: Effects on natural defense against Manduca sexta larvae. Proc. Natl. Acad. Sci. USA 86: 9871-9875. Joshi B. N., Sainani M. N., Bastawade K. B., Deshpande V. V., Gupta V. S., Ranjekar P. K. (1998) Pearl millet cysteine protease inhibitor. Evidence for the presence of two distinct sites responsible for anti-fungal and anti-feedent activities. Eur. J. Biochem. 265: 556-63. Kimura M., Ikeda T., Fukumoto D., Yamasaki N., Yonekura M. (1995) Primary structure of a cysteine proteinase inhibitor from the fruit of avocado (Persea Americana Mill). Biosci. Biotech. Biochem. 59: 2328-2329. Kondo H., Abe K., Nishimura I., Watanabe H., Emori Y., Arai S. (1990). Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. J. Biol Chem. 265: 15832-15837. Kondo H., Abe K., Emori Y., Arai S. (1991) Gene organization of oryzacystatin-II, a new cystatin superfamily member of palnt from those animal cystatins. FEBS Lett. 278: 87-90. Kouzuma Y., Kawano K., Kimura M., Yamasaki N., Kadowaki T., Yamamoto K. (1996) Purification, characterization, and sequencing of two cysteine proteinase inhibitor, Sca and Scb, from sunflower (Helianthus annuus) seeds. J. Biochem. 119: 1106-1113. Krizaj I., Drobnic-Kosorok M., Brzin J., Jerala R., Turk V. (1993) The primary structure of inhibitor of cysteine proteinases from potato. FEBS Lett. 333:15-20. Kuroda M., Ishimoto M., Suzuki K., Kondo H., Abe K., Kitamura K., Abai S. (1996) Oryzacystatins exhibit growth-inhibitory and lethal effects on different species of bean insect pests, Callosobruchus chinensis (Coleoptera) and Riptortus clavatus (Hemiptera). Biosci. Biotech. Biochem. 60: 209-212. Laemmli U. K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680-685. Laskowski M. Jr., Kato I. (1980) Protein inhibitors of proteinases. Annu. Rev. Biochem. 49: 593-626. Leplé J. C., Bonadé-Bottino M., Augustin S., Pilate G., Dumanois Lê Tân V., Delplanque A., Cornu D., Jouanin L. (1995) Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidase) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol. Breed. 1: 319-328. Li Z. Y., Sommer A., Dingermann J., Noe C. R. (1996) Molecular cloning and sequence analysis of a cDNA encoding a cysteine proteinase inhibitor from Sorghum bicolor seedings. Mol. Gen. Genet. 251: 499-502. Liang C., Brookhart G., Feng G. H., Reeck G. R., Kramer K. J. (1991) Inhibition of digestive proteinases of stored grain coleoptera by oryzacystatin, a cysteinases inhiitor from rice seed. FEBS Lett. 278: 139-142. Light A., Greenberg J. (1965) The sequence of 26 amino acid residues at the amino terminus of papain. J. Biol. Chem. 204: 258-265. Lim C. O., Lee S. I., Chung W. S., Park S. H., Hwang I., Cho M. J. (1996) Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp pekinensis) flower buds. Plant Mol. Biol. 30: 373-379. Machleidt W., Thiele U., Laber B., Assfalg-Machleidt I. A., esterl A., Wiegang G., Kos J., Turk V., Bode W. (1989) Mechanism of inhibition of papain by chicken egg white cystatin. FEBS Lett. 243: 234-238. Margis R., Reis E. M., Villeret V. (1998) Structural and phylogenetic relationships among plant and animal cystatins. Arch. Biochem. Biophys. 359: 24-30. Martin J. R., Craven C. J., Jerala R., Kroon-Zitko L., Zerovnik E., Turk V., Waltho J. P. (1995) The three-dimensional solution structure of human stefin. J. Mol. Biol. 246:331-343. Michaud D., Cantin L., Raworth D. A., Vrain T. C. (1996) Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin- polyacrylamide gel electrophoresis. Electrophoresis 17: 74-79. Michaud D., Faye L., Yelle S. (1993) Electrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrylamide gels and class-specific proteinase inhibitors. Electrophoresis 14: 94-98. Misaka T., Kuroda M., Iwabuchi K., Abe K., Arai S. (1996) Soyacystatin, a novel cysteine proteinase inhibitor in soybean, is distinct in protein structure and gene organization from other cystatins of animal and plant origin. Eur. J. Biochem 240: 609-614. Mitsuhashi W., Oaks A. (1994) Development of endopeptidase activities in maize (Zea mays L.) endosperm. Plant Physiol. 104: 401-407. Murzin A. G. 1993. Sweet-testing protein monellin is related to the cystatin family of thiol proteinase inhibitors. J. Mol. Biol. 230: 689-694. Nagata K., Kudo N., Abe K., Arai S., Tanokura M. (2000) Three-dimensional solution structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. Japonica. Biochemistry 39: 14753-14760. Neurath H. 1984. Evolution of proteolytic enzymes. Science. 224: 350-357. Ojima A., Shiota H., Higashi K., Kamada H., Shimma T. I., Wada M. S. (1997) An extracellular insoluble inhibitor of cysteine proteinases in cell cultures and seeds of carrot. Plant Mol. Biol. 34: 99-109. Pernas M., López-Solanilla E., Sánchez-Monge R., Salcedo G., Rodríguez-Palenzuela P. (1999) Antifungal activity of a plant cystatin. MPMI. 12: 624-627. Pernas M., Sánchez-Monge R., Gómez L., Salcedo G. (1998) A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests. Plant Mol. Biol. 38:1235-1242. Plucknett D. L. (1983) Taxonomy of the genus Colocasia. In: J. K. Wang (Ed.). A review of Colocasia esculenta and its potentials. pp. 14-19. Univ Hawaii Press, Honolulu. Potgieter M. (1940) Taro ( Colocasia esculenta ) as a food. J. Amer. Diet. Assoc. 16: 536-540. Purcell J. P., Greenplate J. T., Sammons R. D. (1992) Examination of midgut luminal protenase activities in sex economically important insect. Insect Biochem. Mol. Biol. 22: 41-47. Purseglove J. W. (1972) Tropical crops monocotyledons, John Wiley and Sons. New York. Rawlings N. D., Barrett A. J. (1993) Evolutionary families of peptidases. Biochem. J. 290: 205-18. Richardson M. (1977) The proteinase inhibitors of plants and micro-organisms. Phytochemistry 16: 159-169. Ryan C. A. (1981) Protease inhibitor. In the biochemistry of plants. Vl 6. pp 351-370. New York: Acasemic press. Inc. Ryan C. A. (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu. Rev. Phytopathol. 28: 425-449. Ryan S. N., Laing W. A., McManus M. T. (1998) A cysteine proteinase inhibitor purified from apple fruit. Phytochemistry (Elsevier). 49: 957-963. Saili R. K., Bugawan T. L., Horn G. T., Mullis K. B., Erlich H. A. (1986) Analysis of enzymatically amplified β-globin and HLA-DQ α DNA with allele-specific oligonucleotide probes. Nature 324: 163-166. Salmia M. (1980) Inhibitors of endogenous proteinases in scots pine seed: fractionation and activity changes during germination. Physiol. Plant. 48: 266-270. Sambrook J., Fritsch E., Maniatis T. 1989. Molecular cloning: A laboratory manual. 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Schwede T., Kopp J., Guex N., Peitsch M. C. (2003) Swiss-model: an automated protein homology-modeling server. Nucleic Acids Res. 31: 3381-3385. Siqueira-Junior C. L., Fernandes K. V. S., Machado O. L. T., Cunha M., Gomes V. M., Moura D. T. J. (2002) 87 kDa tomato cystatin exhibits properties of a defense protein and forms crystals of prosystemin overexpressing transgentic plants. Plant Physiol. Biochem. 40: 247-254. Soares-Costa A., Beltramini L. M., Thiemann O. H., Henrique-Silva F. (2002) A sugarcane cystatin: recombinant expression, purification, and antifungal activity. Biochem. Biophys. Res. Commun. 296: 1194-1199. Solomon M., Belenghi B., Delledonne M., Menachem E., Levine A. (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation fo programmed cell death in plants. Plant Cell 11: 431-443. Song I., Taylor M., Baker K., Batemam R. C. (1995) In inhibition of cysteine proteinases by Carica papaya cystatin produced in Escherichia coli. Gene 1995. 162: 221-224. Stubbs M. T., Laber B., Bode W., Huber R., Jerala R., Lenarcie B., Turk V. (1990) The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J. 9:1939-1947. Tate S., Ushioda T., Utsunomiya-Tate N., Shibuya K., Ohyama Y., Nakano Y., Kaji H., Inagaki F., Samejima T., Kainosho M. (1995) Solution structure of a human cystatin A variant, cystatin A2-98 M65L, by NMR spectroscopy. A possible role of the interaction between N- and C-termini to maintain the inhibitory active form of cystatin A. Biochemistry 34: 14637-14648. Thiele U., Assfalg-Machleidt I., Machleidt W., Auerewald E. A. (1990) N-terminal variants of recombinant stefin B: effect on affinity for papain and cathepsin B. Biol. Chem. Hoppe Seyler. 371 (Suppl.) 125-136. Travis J., Salvesen G. S. (1983) Human plasma proteinase inhibitors. Annu. Rev. Biochem. 52: 655-709. Turk V., and Bode W. (1991) The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett. 285: 213-219. Urwin P. E., Alkinson H. J., Waller D. A., McPherson M. J. (1995) Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida. Plant J. 8: 121-131. Urwin P. E., Lilley C. J., McPherson M. J., Alkinson H. J. (1997) Resistance to both cyst and root-knot nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin. Plant J. 12(2): 455-461. Waldron C., Wegrich L. M., Owens-Merlo P. A., Walsh T. A. (1993) Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cystatin proteinase inhibitor. Plant Mol. Biol. 23: 801-812. Walsh T. A., Strickland J. A. (1993) Proteolysis of the 85 kilodalton crystalline cysteine proteinase inhibitor from potato releases functional systatin domain. Plant Physiol. 103: 1227-1234. Watanabe H., Abe K., Emori Y., Hosoyama H., Arai S. (1991) Molucular cloning and gibberellin-induced expression of mulitiple cysteine proteinase of rice seeds (Oryzains). J. Biol. Chem.. 266: 16897-16902. Wu J. W., Haard N. F. (1998) Use of cysteine proteinase inhibitors from injured tomato leaves in whiting surimi. J. Food Biochem. 22: 383-398. Wu J. W., Haard N. F. (2000) Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants. Compara. Biochem. Physiol. Part C. 127: 209-220. Yamada T., Ohta H., Sinohara A., Iwamatsu A., Shimada H., Tsuchiya T., Masuda T., Takamiya K. I. (2000) A cysteine protease from maize isolated in a complex with cystatin. Plant Cell Physiol. 41: 185-191. Yamada T., Kondo A., Ohta H., Masuda T., Shimada H., Takamiya K. I. (2001) Isolation of the protease component of maize cysteine protease-cystatin complex: release of cystatins is not crucial for the activation of the cysteine protease. Plant Cell Physiol. 42: 710-176. Yeh K. W., Juang R. H., Su J. C. (1991) A rapid and efficient method for RNA isolation from plants with high carbohydrate content. Focus 13:102-103. Yeh K. W., Chen J.C., Lin M. I., Chen Y. M., Lin C. Y. (1997a) Functional activity of sporamin from sweet potato (Ipomoea batatas Lam.): a tuber storage protein with trypsin inhibitory activity. Plant Mol. Biol. 33(3): 565-70. Yeh K. W., Lin M. I., Tuan S. J., Chen Y. M., Lin C. J., Kao S. S. (1997b) Sweet potato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura. Plant Cell Rep. 16: 696-699. Zhao Y., Botella M. A., Subramanian L., Niu X., Nielsen S. S., Bressan R. A., Hasegawa P. M. (1996) Two wound-inducible soybean cysteine proteinase inhibitor have greater insect digestive proteinase inhibitor activities than a constitutive homology. Plant Physiol. 111: 1299-1306. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39146 | - |
dc.description.abstract | 芋頭為一傳統食用作物,主要食用部位為球莖及葉部;一般而言,球莖在發育過程中所含有之貯存性蛋白質除了扮演基因之表現與調控外,亦含有抗病及抗菌功能的蛋白酶抑制劑。本實驗在檢測不同芋頭品種後發現高雄1號品種含有一種硫醇型的蛋白質酶抑制劑, 利用5’-RACE及3’-RACE進行PCR反應,得到全長的tarocystain 基因,命名為CeCPI, CeCPI全長共有1,008 bp,其open reading frame (ORF)為618 bp,可轉譯成205 胺基酸,分子量約29 kDa,其序列特徵為: Gly5 (G)位在N-端序列之第5個胺基酸; 第一個binding loop(L1)區域起始於第49個胺基酸位置,含有一個五胜肽(pentapeptide)序列 Gln49-Val50-Val51-Ser52-Gly53 (Q-V-V-S-G); 第二個binding loop(L2) 則在Trp 80 (W80)之後; 在靠近C-端序列處尚有一個不含cysteine 的contact point -(W114); 此外, 在鄰近N-端第22個胺基酸起含有一個保守性的胺基酸序列ARFAVDEHNKK,因此,認為CeCPI屬於phytocystatin之一種。進行胺基酸演譯序列相似度之比較,就芋頭而言,芋頭為網狀脈葉但屬於單子葉植物,而其CeCPI基因序列之胺基酸演譯及分子量大小,與雙子葉植物之相似度高於單子葉植物。
重組CeCPI 蛋白質當濃度在80 mg/ml時,即可抑制白絹菌(Sclerotium rolfsii)菌核之生長,當重組CeCPI蛋白質濃度達150 ~ 200 mg/ml以上,白絹病菌菌核之菌絲的生長完全的受到抑制。以不同濃度重組CeCPI蛋白質檢測其他真菌性病害,發現重組CeCPI蛋白質對某些真菌性病害,如芸苔鏈格孢菌(Alternaria brassica),瓜果腐霉(Pythium aphanidermatum)及 立枯絲核菌(Rhizoctonia solani)具有明顯抑制其菌絲生長之毒性。 以水稻OC-I為模組,使用SWISS-Model程式分析CeCPI蛋白質結構模式,由3D構造分析,得到重組CeCPI蛋白質由6個α-helix (α1、α2、α3、α4、α5、α6)及7個antiparallelβ-sheet(β1、β2、β3、β4、β5、β6、β7)組成。檢測定點突變後的數種CeCPI蛋白質(site-directed mutant protein)對木瓜酶的抑制活性是否受影響,結果當保守區(L1) Q49位置一旦突變, 抑制木瓜酶的活性則完全喪失,而且對抑制白絹病菌菌核生長之抗性也消失,但在C-端保守區(W80、W114)的突變體雖然對木瓜酶抑制活性部份減弱,但對白絹病菌菌絲仍顯著抑制其生長; 顯然CeCPI 蛋白質對木瓜酶的抑制活性與抑菌活性是二個不同的分子機制。 | zh_TW |
dc.description.abstract | Taro (Colocasia esculenta L. Schott) is one of the oldest cultivated crops grown for its edible corms and leaves. Taro corm is a well-differentiated organ and contains approximately 1.4 ~ 3.0% storage proteins on a fresh weight basis. In order to investigate the proteinase inhibitors of storage protein in corm of four local taro cultivars, the affinity analysis of the cystatin/papain was performed by using the mild denaturing gelatin /PAGE. Results from substrate polyacrylamide gel electrophoresis revealed that the cultivar-Kaohsiung No. 1 contains cystatin in corm.
Subsequently, a cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from corm of taro using both degenerated primers/RT-PCR amplification and 5’- / 3’- RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 205 amino acid residues, with deduced MW. 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to eudicots rather than to monocots, despite taro belongs to monocot. Recombinant GST-CeCPI fusion protein was overexpressed in E.coli and its inhibitory activity against papain was identified on gelatin/ SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. The investigation of its antifungal activity clearly revealed the toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at the concentration of 80 μg of recombinant CeCPI per ml. Moreover, the mycelium growth was completely inhibited and the sclerotia lysed at the concentration of 150 μg – 200 μg per ml. Further studies have demonstrated that the recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium. The predicted tertiary structure of CeCPI was modeled based on the known crystal structure of the rice OC-I, which shares 50.5% (48/95) sequence identity with the taro cystatin. The data indicate that tarocystatin consists of six | en |
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dc.description.tableofcontents | 中文摘要……………………………………………………….. 6
英文摘要……………………………………………………….. 8 縮寫對照表…………………………………………………….. 10 緒言…………………………………………………………….. 12 前人研究……………………………………………………….. 13 一、芋之起源與特性…………………………………………….. 13 二、硫醇型蛋白酶(thiol protease)之作用機……………….. 15 三、植物之防禦及蛋白酶抑制因子………………………….… 17 四、cystatin的種類與特性…………………………………….. 20 五、Phytocystatin 結構與分析………………………………… 24 材料與方法 …………………………………………………… 27 第一部份:試驗材料……………………………………………. 27 一、植物品種……………………………………………………. 27 二、試驗菌株……………………………………………………. 27 第二部份:試驗方法……………………………………………… 28 一、半胱胺酸蛋白酶抑制劑之純化及生化分析………………… 28 (一)芋頭總蛋白質萃取及分析…………………………………. 28 1. 芋頭總蛋白質萃取……………………………………………. 28 2. 蛋白質定量分析………………………………………………. 28 (二)半胱胺酸蛋白酶抑制劑之分析……………………………. 29 1. 半胱胺酸蛋白酶抑制劑之膠體活性染色分析………………. 29 2. 半胱胺酸蛋白酶抑制劑活性定量分析………………………. 30 3. SDS-聚丙烯醯氨膠體電泳…………………………………… 31 4. 西方墨點轉印分析……………………………………………. 33 (1)蛋白質轉印………………………………………………… 33 (2)免疫偵測…………………………………………………… 34 二、基因選殖及基因特性分析…………………………………. 35 (一)總RNA之製備…………………………………………….... 35 (二)分離poly(A)+ mRNA之製備……………………………..… 36 (三)CeCPI基因之選殖…………………………………………… 38 1. 第一股cDNA合成……………………………………………. 38 2. 第二股cDNA合成…………………………………………… 38 3. Adaptor ligation………………………………………….. 39 4. Rapid Amplification of cDNA ends (RACE)……………… 39 5. RNA / DNA 片段回收及純化………………………………… 40 (四)大腸桿菌轉形勝任細胞(competent cell)的製備………… 42 (五)大腸桿菌的接合(ligation) 與轉形作用(transformation)43 1. Ligated to pGEM-T Easy Vector …………………………. 43 2. Transformed to E.coli XL1-blue………………………… 44 (六)細菌質體DNA的小量製備………………………………….. 45 (七)南方墨點轉印分析…………………………………………. 46 1. 芋頭球莖genomic DNA 萃取……………………………….. 46 2. PCR方式之探針製備 (非放射性DIG標記探針製備)……… 47 3. 基因組DNA (genomic DNA)轉漬…………………………… 47 4. 雜合反應………………………………………………………. 48 5. 呈色反應………………………………………………………. 49 三、基因表現分析………………………………………………… 51 (一)北方墨點轉印分析…………………………………………… 51 1. PCR方式之探針製備 (非放射性DIG標記探針製備)…….. 51 2. RNA 的轉漬…………………………………………………… 51 3. 雜合反應…………………………………………………….… 52 4. 呈色反應…………………………………………………….… 52 (二)融合蛋白質之製備…………………………………………… 52 1. 以PCR方式合成融合蛋白質…………………………….… 52 2. PCR產物回收及純化………………………………………… 53 3. 表現載體pGEX-2TK Vector之準備………………….…….. 53 4. Ligation of pGEX-2TK vector and DNA fragment ……… 54 5. 轉殖(transformed)至E.coli XL1-blue………………….. 54 6. 大量純化GST融合蛋白質………………………………….. 55 7. 水解GST融合蛋白質……………………………………….. 56 8. 抗體之製備…………………………………………………… 56 (三)定點突變基因之表現………………………………………… 57 1. Site-specific mutagenesis by overlap extension …… 57 2. Allele-specific oligonucleotides: ASO………………. 59 (1)探針的製備: (非放射性DIG標記探針的製備)……………. 60 (2) 質體DNA的轉漬………………………………………….. 60 (3)雜合反應…………………………………………………….. 60 (4)呈色反應……………………………………………..……… 61 四、對外源病原菌抗性檢定……………………………………… 62 Tarocystatin抗菌性檢定…………………………………….…. 62 五、芋頭半胱胺酸蛋白酶抑制劑結構分析……………………… 63 芋頭cystatin 蛋白質3D結構模式分析…………………………. 63 結 果…………………………………………………………… 64 第一部份;半胱胺酸蛋白酶抑制劑之檢定……………………… 64 分析不同品種芋頭球莖中粗抽取物對半胱胺酸蛋白酶活性抑制之效果……………………………………………………………… 64 第二部份:Tarocystatin 基因選殖及特性之研究…………… 65 一、Tarocystatin 基因之選殖…………………………………. 65 (一)利用RT-PCR選殖部分片段之tarocystation基因………… 65 (二)利用RACE技術延長tarocystatin 之5’-端和3’-端序列. 66 (三)全長之tarocystatin cDNA之選殖…………………………. 67 二、Tarocystatin 基因之序列比較分析……………………… 67 三、南方轉印分析………………………………………………… 68 第三部份:Tarocystatin 基因表現之研究……………………. 70 一、芋頭各部位組織及成熟球莖貯藏過程之CeCPI基因表現.. 70 (一) CeCPI基因表現具有組織專一性………………………….. 70 (二) 球莖貯藏過程中CeCPI基因表現…………………….…… 70 二、抗體之製備及融合蛋白質之表現及特性…………………… 71 第四部份:Tarocystatin 對微生物之抗性研究………………. 74 一、重組CeCPI蛋白質對白絹病菌抗性之檢定……………..… 74 二、重組CeCPI蛋白質對真菌性及細菌性病害抗性之檢定….. 75 第五部份:Tarocystatin 蛋白質結構分析及對微生物之抗性研究76 一、Tarocystatin (CeCPI) 3D立體結構分析………………… 76 二、二級結構與抗菌毒性之探討………………………………. 77 討 論………………………………………………………….. 80 第一部份:半胱胺酸蛋白酶抑制劑之檢定……………………… 80 不同品種芋頭球莖粗抽取物對半胱胺酸蛋白酶活性抑制之效果 80 第二部份:Tarocystatin基因之選殖及特性之研究………… 81 一、Tarocystatin基因之選殖………………………………….. 81 二、Tarocystatin基因特性分析……………………………..… 82 第三部份:Tarocystatin 基因表現之研究…………….……… 83 第四部份:Tarocystatin 對微生物抗性之研究………….….. 85 第五部份:Tarocystatin 蛋白結構分析對微生物抗性之研究. 87 結 語…………………………………………………………. 89 參 考 文 獻………………………………………………...… 91 表……………………………………………………………….. 106 圖……………………………………………………………….. 108 附錄一……………………………………………………….… 133 附錄二…………………………………………………………. 134 附錄三……………………………………………………….… 135 | |
dc.language.iso | zh-TW | |
dc.title | 芋頭半胱胺酸蛋白酶抑制劑(Tarocystatin)之基因選殖、表現特性及對微生物抗性的分子機制研究 | zh_TW |
dc.title | Molecular Cloning, Recombinant Gene Expression and Antifungal Activity of Cystatin from Taro
(Colocasia esculenta L. Schott ) | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 蔡嘉寅(C. Y. Tsai),陳益明(Yih-Ming Chen),宋賢一(Hsien-Yi Sung),高景輝(Ching-Huei Kao),王仕賢(S. S. Wang),林棋財(Chi-Tsai Lin),陳志成(C. Will Chen) | |
dc.subject.keyword | 半胱胺酸蛋白酶,對微生物抗性,半胱胺酸蛋白酶,芋頭,抑制劑, | zh_TW |
dc.subject.keyword | taro,cysteine proteinase inhibitor(cystatin),cysteine proteinase,antifungal, | en |
dc.relation.page | 135 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-01-28 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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