SPCP2是由甘藷老化葉片分離出來的全長cDNA，藉由氨基酸序列比對與papain-like 半胱胺酸蛋白質分解酵素(papain-like cysteine protease) 類似。SPCP2 ORF(open reading frame)含有1101個核苷酸(366個氨基酸)，與奇異果(Actinidia deliciosa)、阿拉伯芥(Arabidopsis thaliana)、花椰菜(Brassica oleracea)、菜豆(Phaseolus vulgaris)、 野豌豆(Pisum sativa)、蠶豆(Vicia faba)、巢菜(Vicia sativa) 及 豇豆(Vigna mungo)等植物的半胱胺酸蛋白質分解酵素具有高度相似性(68%~83%)。SPCP2基因表現顯著增加於甘藷自然老化葉片及已萌芽的貯存塊根。利用轉殖系統將SPCP2外源表現於阿拉伯芥植株進行分析，發現外源表現SPCP2基因可促進轉殖阿拉伯芥植株提早開花時間、減少開花時rosette leaves葉片數目、增加轉殖植株種子發芽時之耐鹽性、乾旱處理時轉殖植株具有較高的光合作用效率 (Fv/Fm value) 、較高的相對水份含量及增加轉殖植株之耐旱性。根據上述結果結論甘藷papain-like半胱胺酸蛋白質分解酵素SPCP2是一個具有功能性的基因，外源表現甘藷SPCP2會改變轉殖阿拉伯芥植株生長發育特徵及耐乾旱與耐鹽分的反應。

Sweet potato SPCP2 is a full-length cDNA isolated from senescent leaves and encodes a putative papain-like cysteine protease. The SPCP2 contained 1101 nucleotides (366 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 68% to 83%) with plant cysteine proteases, including Actinidia deliciosa, Arabidopsis thaliana, Brassica oleracea, Phaseolus vulgaris, Pisum sativa, Vicia faba, Vicia sativa and Vigna mungo. SPCP2 gene expression was enhanced significantly in natural senescent leaves and in sprouting storage root. Transgenic Arabidopsis plants with ectopic constitutive SPCP2 expression showed earlier floral transition from vegetative to reproductive growth, reduced rosette leaves when flowering, enhanced germination percentage of transgenic progeny seeds in salt-containing MS medium, higher Fv/Fm value, higher relative water content and enhanced tolerance during drought treatment. Based on these results, we conclude that sweet potato papain-like cysteine protease, SPCP2, is a functional gene, and its expression causes altered developmental characteristics and enhances drought and salt stress responses in transgenic Arabidopsis plants.

第一章、前言…………………………………………………1
第一節 甘藷……………………………………………1
第二節 葉片老化的生理………………………………2
第三節 半胱胺酸蛋白質分解酵素的研究……………5
第四節 研究方向………………………………………8
第二章、材料與方法…………………………………………9
第一節 實驗材料………………………………………9
一、甘藷（Ipomoea batatas）………………………9
二、SPCP2 cDNA……………………………………9
三、SPCP2轉殖阿拉伯芥種子………………………10
四、轉殖阿拉伯芥植株的種植………………………10
第二節 實驗方法……………………………………11
一、甘藷塊根SPCP2基因表現的檢測……………11
二、對外源表現 SPCP2 轉殖植株生長發育性狀影響分析…………………………………………14
三、外源表現SPCP2對轉殖植株種子發芽於鹽分逆境之影響分析……………………………………14
四、外源表現 SPCP2 對轉殖植株乾旱逆境之影響分析………………………………………………15
五、分析SPCP2之Putative catalytic domain area 及Phylogenetic tree……………………………16
第三章、結果…………………………………………………18
第一節 甘藷自然老化葉片及已萌芽的貯存塊根顯著增加SPCP2基因表現……………………………18
第二節 外源表現SPCP2基因可促進轉殖阿拉伯芥植株提早開花………………………………………18
第三節 外源表現SPCP2基因可增加轉殖阿拉伯芥植株種子發芽之耐鹽性……………………………19
第四節 外源表現SPCP2基因可增加轉殖阿拉伯芥植株之耐旱性………………………………………20
第五節 甘藷SPCP2基因之Putative catalytic domain area 及 Phylogenetic tree analysis.……………………………………21
第四章、討論…………………………………………………23
第五章、參考文獻……………………………………………29
圖…………………………………………………………42

蘇正庭.甘藷葉片老化相關基因半胱胺酸蛋白分解酵素SPCP2於轉殖阿拉伯芥之研究.中國文化大學生物科技研究所碩士論文.2006
Azumi Y, Watanabe A. Evidence for a Senescence-Associated Gene Induced by Darkness. Plant Physiol. 1991;95(2):577-583.
Barth C, De Tullio M, Conklin PL. The role of ascorbic acid in the control of flowering time and the onset of senescence. J Exp Bot .2006; 57:1657-1665.
Beers EP,Woffenden BJ,Zhao CS. Plant proteolytic enzymes: possible roles during proprammed cell death. Plant Molecular Biology.2000;44:399-415.
Bernoux M, Timmers T, Jauneau A, Brie C, de Wit PJGM, Marco Y, Deslandesa L. RD19, an Arabidopsis cysteine protease required for RRS1-R–mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector. Plant Cell .2008; 20: 2252–64.
Buchanan-Wollaston V, Page T, Harrison E, Breeze E, Lim PO. Comparative transcriptome analysis reveals significant differences in gene expression and signaling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. Plant J .2005; 42: 567-85.
Buchanan-Wollaston V. The molecular biology of leaf senescence. J Exp Bot 1997;48: 181-99.
Chen HJ, Hou WC, Yang CY, Huang DJ, Liu JS, Lin YH. Molecular cloning of two metallothionein-like protein genes with differential expression patterns from sweet potato (Ipomoea batatas (L.) Lam.) leaves. J Plant Physiol .2003; 160: 547-55.
Chen HJ, Su CT, Lin CH, Huang GJ, Lin YH. Expression of sweet potato cysteine protease SPCP2 altered developmental characteristics and stress responses in transgenic Arabidopsis plants. J Plant Physiol. 2010 Jul 1;167(10):838-47.
Chen HJ, Wen IC, Huang GJ, Hou WC, Lin YH. Expression of sweet potato asparaginyl endopeptidase caused altered phenotypic characteristics in transgenic Arabidopsis. Bot Stud. 2008; 49: 109-17
Chen HJ, Hou WC, Liu JS, Yang CY, Huang DJ, Lin YH. Molecular cloning and characterization of a cDNA encoding asparaginyl endopeptidase from sweet potato (Ipomoea batatas (L.) Lam) senescent leaves. J Exp Bot 2004; 55: 825-35.
Clouse SD. Plant hormones: brassinosteroids in the spotlight. Curr Biol. 1996;6(6):658-61.
Corbesier L, Bernier G, and Périlleux C. C : N ratio increases in the phloem sap during floral transition of the long-day plants Sinapis alba and Arabidopsis thaliana. Plant & cell physiology . 2002; 43(6):684
Coupe SA, Sinclair BK, Watson LM, Heyes JA, Eason JR. Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets. J Exp Bot .2003; 54: 1045 – 56.
Fischer J.Charakterisierung einer Cysteinproteinase undihrer cDNA aus Keimlingen der Saatwicke(Vicia sativa L.).Dissertation, Mathematisch-Naturwissenschaftliche Fakultat,Universitat Halle,Germany.1996.
Fischer J,Becker C,Hillmer S,Horstmann B,Neubohn B,Schlereth A,Senyuk V,Shutov A,Muntz K. The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns, intracellular localization and functions in globulin proteolysis. Plant Molecular Biology.2000;43:83-101
Flexas J ,Escalona JM, Medrano H. Water stress induces different levels of photosynthesis and electron transport rate regulation in grapevines.Plant,Cell and Env.1999;22:39-28.
Fujiki Y, Yoshikawa Y, Sato T, Inada N, Ito M, Nishida I, Watanabe A. Dark-inducible genes from Arabidopsis thaliana are associated with leaf senescence and repressed by sugars. Physiologia Plantarum. 2001;111: 345-352(8)
Gan S, Amasino RM . Inhibition of leaf senescence by autoregulated production of cytokinin. Science.1995;270: 1986-1988
Graham IA, Denby KJ, Leaver CJ. Carbon catabolite repression regulates glyoxylate cycle gene expression in cucumber. The Plant Cell.1992;4:349-357.
Gruis D, Selinger D.A,Curran J.M,Jung R.Redundant proteolytic mechanisms process seed storage proteins in the absence of seed-type members of the vacuolar processing enzyme family of cycteine proteases.2002.The Plant Cell.14:2863-2882
Hardwick JM, Shaw KE, Wills JW, Hunter E. Amino-terminal deletion mutants of the Rous sarcoma virus glycoprotein do not block signal peptide cleavage but can block intracellular transport.J Cell Biol. 1986;103(3):829-38.
Hattori T,Nakagawa T ,Maeshima M, Nakamura K , Asahi T. Molecular cloning and nucleotide sequence of cDNA for sporamin, the major soluble protein of sweet potato tuberous roots. Plant Mol Biol. 1985；5:313-20
Hou WC, Chen YC, Chen, HJ, Lin YH, Yang LL, Lee MH. Antioxidant activities of trypsin inhibitor, a 33 kDa root storage protein of sweet potato (Ipomoea batatas(L.) Lam. cv. Tainong 57). J Agri Food Chem .2001; 49: 2978-81.
Hou WC, Chen HJ, Lin YH. Antioxidant peptides with angiotensin converting enzyme inhibitory activities and applications for angiotensin converting enzyme purification. J Agri Food Chem .2003; 51: 1706-09.
Huang DJ, Hou WC, Chen HJ, Lin YH. Sweet potato (Ipomoea batatas [L.] Lam. „Tainong 57) storage root mucilage exhibited angiotensin converting enzyme inhibitory activity in vitro. Bot Stud .2006; 47: 397-402.
Huang GJ, Chen HJ, Chang YS, Shue MJ, Lin YH. Recombinant sporamin and its synthesized peptides with antioxidant activities in vitro. Bot Stud. 2007; 48: 133-40.
Iturbe-Ormatxe, Escuredo PR, Arrese-lgor C, Becana M. Oxidative damage in pea plant exposed to water deficit or paraquat.Plant Physiol. 1998;116:173-181.
Kautsky H., Hirsch A. Das Fluoreszenzverhalten gruner Pflanzen . Biochem Z. 1934; 274: 422–434.
Koizumi M, Yamaguchi-Shinozaki K, Shinozaki K. Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana. Gene. 1993; 129: 175-82.
Lee BH, Ko JH, Lee S, Lee Y, Pak JH, Kim JH. The Arabidopsis GRF-INTERACTING FACTOR gene family performs an overlapping function in determining organ size as well as multiple developmental properties .Plant Physiol. 2009 ;151 (2) 655-668
Li L.and Stden JV. Effects of plant growth regulators in the antioxidant system in callus of two maize cultivars subjected to water stress.Plant Growth Regul.1998;24:55-66.
Makino A, Osmond B. Effects of Nitrogen Nutrition on Nitrogen Partitioning between Chloroplasts and Mitochondria in Pea and Wheat. Plant Physiol. 1991;96(2):355-362.
Maria I, Gama1 CS , Rui P, Leite Jr, Antonio R. Cordeiro and Daniel JC. Transgenic sweet potato plants obtained byAgrobacterium tumefaciens-mediated transformation. Plant Cell, Tissue and Organ Culture .1996;46:237-244.
Martinez DE, Costa ML, Gomez FM, Otegui MS,Guiamet JJ. “Senescence-associated vacuoles” are involved in degradation of chloroplast proteins in tobacco leaves.Plant J.2008.
Matile P, Hortensteiner S, Thomas H. CHLOROPHYLL DEGRADATION.Annu Rev Plant Physiol Plant Mol Biol. 1999;50:67-95.
Meeks,Wanger DR. Gene Expression in the early floral meristem. The Plant Cell. 1999;5:1167-1174
Minamikawa T, Toyooka K, Okamoto T, Hara-Nishimura I, Nishimura M. Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: Immunocytochemical and ultrastructural observations. Protoplasma .2001;218: 144-53.
Mitsuhashi W, Koshiba T, Minamikawa T. Separation and characterization of two endopeptidases from cotyledons of germinating Vigna mungo seeds. Plant Physiol .1986; 80: 628-34.
Muller P, Li XP, Niyogi KK. Non-photochemical quenching. A response to excess light energy.Plant Physiol. 2001;125:1558-1566
Noh SA, Lee HS, Huh EJ, Huh GH, Peak KH, Shin JS, Bae JM. SRD1 is involved in the auxin-mediated initial thickening growth of storage root by enhancing proliferation of metaxylem and cambium cells in sweet potato (Ipomoea batatas).J of Experimental Botany.2010;61(5):1337-1349.
Okamoto T, Minamikawa T. Molecular cloning and characterization of Vigna mungo processing enzyme 1 (VmPE-1), an asparaginyl endopeptidase possibly involved in post-translational processing of a vacuolar cysteine endopeptidase (SH-EP). Plant Mol Biol. 1999; 39: 63-73.
Okamoto T, Toyooka K, Minamikawa T. Identification of a Membrane-associated Cysteine Protease with Possible Dual Roles in the Endoplasmic Reticulum and Protein Storage Vacuole. THE JOURNAL OF BIOLOGICAL CHEMISTRY .2001; 276: 742–751
Okamoto T, Minamikawa T. Purification of a processing enzyme (VmPE-1) that is involved in post-translational processing of a plant cysteine endopeptidase (SH¬-EP). Eur J Biochem. 1995; 231: 300-5.
Okamoto T, Shimada T, Hara-Nishimura I, Nishimura M, Minamikawa T. C-terminal KDEL sequence of A KDEL-tailed cysteine proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle and in efficient vacuolar transport of sulfhydryl-endopeptidase1. Plant Physiol. 2003; 132: 1892–900.
Poueymiro M, Genin SP. Secreted proteins from Ralstonia solanacearum: a hundred tricks to kill a plant. Curr Opin Microbiol. 2009; 12: 44–52.
Powers JC, Asgian JL, Ekici OD, James KE. Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem Rev 2002; 102:4639-4750.
Quirino BF, Noh YS, Himelblau E, Amasino RM. Molecular aspects of leaf senescence. Trends Plant Sci .2000; 5: 278-82.
Ranwala AP, Miller WB. Preventive mechanisms of gibberellin4+7 and light on low temperature-induced leaf senescence in Lilium cv. Stargazer. Postharvest Biology and Technology. 2000;19:85-92
Raper CDJ, Thomas JF, Tolley-Henry L, Rideout JW. Assessment of an apparent relationship between availability of soluble carbohydrates and reduced nitrogen during floral initiation in tobacco. Bot Gaz .1988; 149: 289-94.
Rideout JW, Raper CD, Miner GS. Changes in ratio of soluble sugars and free amino nitrogen in the apical meristem during floral transition of tobacco. Int J Plant Sci. 1992; 153: 78-88.
Schlereth A, Becker C, Horstmann C, Tiedemann J, Muntz K. Comparison of globulin mobilization and cysteine proteinases in embryonic axes and cotyledons during germination and seedling growth of vetch (Vicia sativa L.). J Exp Bot. 2000; 51: 1423-33.
Schlereth A, Standhardt D, Mock HP, Muntz K. Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination. Planta .2001; 212: 718-27.
Senyuk V, Rotari V, Becker C, Zakharov A, Horstmann C, Muntz K, Vaintraub I. Does an asparaginyl-specific cysteine endopeptidase trigger phaseolin degradation in cotyledons of kidney bean seedlings? Eur J Biochem 1998; 258: 546-58.
Sihachakr D, Haïcour R, Cavalcante Alves JM, Umboh I, Nzoghé D, Servaes A, and Ducreux G. Plant regeneration in sweet potato (Ipomoea batatas L.,Convolvulaceae). Euphytica .1997;96: 143-152.
Smart CM. Gene expression during leaf senescence. New Phytol .1994;126:419-488
Solomon M,Belenghi B,Delledonne M,Menachem E,Levine A. The involvement of cysteine proteases and protease inhibitor genes in the regulation of proprammed cell death in plant. The Plant Cell .1999;11:431-443
Taiz and Zeiger . Plant Physiology. The Benjamin/Cummings Publishing Company, Inc. 1991
Taiz and Zeiger .Ethylene: The gaseous hormone. Plant Physiology .2002. 3rd ed. Sinauer Association, Inc., Massachusetts. 521pp.
Toyooka k,Okamoto T,Minamikawa T. Mass transport of proform of a KDEL-tailed cysteine proteinase (SH-EP) to protein storage vacuoles by endoplastic reticulum-derived vesicle is involved in protein mobilization in germinating seeds. J Cell Biol.2000;148:453-63.
Vierstra RD. Proteolysis in plants: mechanisms and functions. Plant Mol.Biol. 1996; 32: 275-302.
Watanabe, H., Abe, K., Emori, Y., Hosoyama, H., and Arai, S. Molecular cloning and gibberellin-induced expression of multiple cysteine proteinases of rice seeds (Oryzains). J. Biol. Chem. 1991;266: 16897–16902.
Xiong L, Lee H, Ishitani M, Zhu JK. Regulation of osmotic stress-responsive gene expression by the LOS6/ABA1 locus in Arabidopsis. J Biol Chem. 2002; 277: 8588-96.
Xu FX, Chye ML. Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal. Plant J. 1999; 17: 321–7.
Yamauchi D, Kobayashi M, NakaiT ,Kubota K .Cloning of cDNAs for DELLA proteins suppressing cysteine proteinase genes in germinated cotyledons of common bean seeds. Plant Biotechnology.2007;24:513-517.

Yoshida S. Molecular regulation of leaf senescence. Curr Opin Plant Biol 2003; 6: 79