根據先前研究結果顯示ethephon誘導葉片老化及老化相關基因SPCP1的表現受reduced glutathione, EGTA及cycloheximide抑制(Chen et al., 2009)，此結果顯示calcium influx、reactive oxygen species (ROS)及新合成(de novo)蛋白質會影響ethephon的作用。因此根據此研究結果本研究利用扣減雜交和RACE-PCR的方法從ethephon處理6及24小時的混合樣品中共選殖了五個全長的cDNAs具有可能的功能為calmodulin (SPCAM)、catalase (SPCATA)、anionic peroxidase (SPPA)、ACC oxidase (SPACO) 和DSS1-like protein (SPDSS1)。SPCAM其ORF包含450個核甘酸，產生149個胺基酸，蛋白質結構具有四個明顯EF-hand motifs，與阿拉伯芥分離的calmodulins比較其胺基酸相似度從48%至100%，其中與CaM7相似度最高。SPCATA其ORF包含1479個核甘酸，產生492個胺基酸；與不同物種的catalases胺基酸相似度從71.2%至80.9%，其中與水筆仔的catalase相似度最高。SPPA其ORF包含1068個核甘酸，產生355個胺基酸；與甘藷其他已發表的同功酵素比較其胺基酸相似度從28.7%至97.5%，其中與甘藷的anionic peroxidase SWPA4相似度最高。SPACO其ORF包含930個核甘酸，產生309個胺基酸；與不同物種ACC oxidase比較其胺基酸相似度從62.3%至81.5%，其中與菸草的ACC oxidase相似度最高。SPDSS1其ORF包含228個核甘酸，產生75個胺基酸；與不同物種DSS1比較其胺基酸相似度從25.2%至62.3%，其中與玉米的DSS1相似度最高。在自然老化過程葉片的葉綠素含量及光合作用效率會顯著減少，然而選殖基因的表現量則有顯著增加；DAB染色結果顯示H2O2的量於全部黃化的葉片(S3)比其它葉片發育階段有顯著增加；Evan blue染色結果亦顯示全部黃化的葉片(S3)細胞死亡較年青的葉片(S0)為多。另外利用ethephon誘導葉片老化過程也有相類似的結果。ethephon處理的葉片其葉綠素含量及光合作用效率也比對照組顯著減少，然而選殖基因的表現量則比對照組顯著增加；DAB染色結果顯示H2O2的量於ethephon處理三天的葉片比其對照組有顯著增加；Evan blue染色結果亦顯示ethephon處理三天的葉片細胞死亡較其對照組亦有顯著增加。根據這些實驗的結果結論SPCAM、SPCATA、SPPA、SPACO和SPDSS1在自然葉片老化及ethephon誘導葉片老化過程中其基因表現量會顯著增加；其功能是否與ethephon誘導葉片老化過程calcium influx、ROS增加或清除、及後續的訊息傳導等有關將會討論。

According to our previous results, ethephon-induced sweet potato leaf senescence and senescence-associated gene SPCP1 expression was affected by reduced glutathione, EGTA, and cycloheximide (Chen et al., 2009). These data suggest that calcium influx, reactive oxygen species (ROS) and de novo synthesized proteins can affect ethephon-mediated effects. Therefore, PCR-selective substractive hybridization and RACE-PCR methods were used to clone 5 full-length cDNAs encoded putative calmodulin (SPCAM), catalase (SPCATA), anionic peroxidase (SPPA), ACC oxidase (SPACO), and DSS1-like protein (SPDSS1) from mixed samples of ethephon-treated leaves for 6 and 24 hours. The ORF of SPCAM contains 450 nucleotides and encodes 149 amino acids. There are 4 putative EF-motifs in the deduced protein structure. SPCAM exhibited amino acid sequence identity with isolated Arabidopsis calmodulins from 48% to 100%, and was completely the same as CaM7 calmodulin. The ORF of SPCATA contains 1479 nucleotides and encodes 492 amino acids. SPCAM exhibited high amino acid sequence identity with other plant catalases from 71.2% to 80.9%, and had the highest identity with mangrove catalase. The ORF of SPPA contains 1068 nucleotides and encodes 355 amino acids. SPPA exhibited amino acid sequence identity with other published sweet potato peroxidase isoforms from 28.7% to 97.5%, and had the highest identity with anionic peroxidase SWPA4. The ORF of SPACO contains 930 nucleotides and encodes 309 amino acids. SPACO exhibited high amino acid sequence identity with other plant ACC oxidases from 62.3% to 81.5%, and had the highest identity with tobacco ACC oxidase. The ORF of SPDSS1 contains 228 nucleotides and encodes 75 amino acids. SPDSS1 exhibited amino acid sequence identity with other DSS1 from 25.2% to 62.3%, and had the highest identity with maize DSS1. The chlorophyll contents and Fv/Fm values were significantly reduced, however, the isolated gene expression was remarkably enhanced in natural senescent leaves. DAB staining showed that H2O2 amount was remarkably elevated at S3 senescent leaves compared to leaves of the other developmental stages. Evan blue staining also demonstrated that S3 senescent leaf had more cell death compared to S0 young leaves. In addition ethephon-induced leaf senescence exhibited similar results. The chlorophyll contents and Fv/Fm values were significantly reduced, however, the isolated gene expression was remarkably enhanced in ethephon-treated leaves compared to dark control. DAB staining showed that H2O2 amount was remarkably elevated at 72 hours in ethephon-treated leaves compared to dark control. Evan blue staining also demonstrated that ethephon-treated leaf for 72 hours had more cell death compared to dark control. Based on these data we conclude that SPCAM, SPCATA, SPPA, SPACO and SPDSS1 gene expression were significantly increased in natural and ethephon-induced senescent leaves. The possible functions of these isolated genes in association with events in ethephon-induced leaf senescence, including calcium influx, ROS elevation or scavenge, and following signaling will be discussed.

目錄
頁次
目錄…………………………… …………… ………… I
內容目次…………………………………… ………… II
圖目錄………………………………………………… IV
縮寫表………………………………………………… VI
中文摘要……………………………………………… VII
英文摘要……………………………………………… IX

壹、 緒論…………… …………………………………………1
I. 老化的定義與過程……………………………………… 1
II. 影響葉片老化的因子…………………………………… 2
III. Ethylene 的合成及信息傳導途徑……………………… 6
IV. 與Ca2+-binding，H2O2 清除，乙烯合成及其他相關基因的
研究………………………………… …………………… 8
貳、材料與方法…………………… ………… ………………13
I. 實驗材料……………… ……………………………………13
II. 實驗方法……………………………………………………13
A. 葉片處理…………………………………………………13
B. RNA extraction………………………… ……… …… …15
C. PCR select cDNA subtraction … … … … ………………19
D. 差異性cDNA 選殖以及親緣樹分析……………………29
E. RACE PCR………………… ………………… …… ……33
F. RT-PCR……………………………………………………38
G. 色素測定…………………………………………………42
H. 光合作用測定……………………………… ……………43
I. DAB 染色及Evan blue ……………………………………43
参、結果…………………………………………………………45
I. 差異性表現之cDNA 片段選殖………… …… … ………45
II. 選殖全長cDNA 之核甘酸序列、胺基酸序列及親緣樹之分
析…………………………… …………… …………… …45
III. 自然老化的甘藷葉片會增加選殖基因的表現… … ……50
IV. Ethephon 誘導甘藷葉片老化及增加選殖基因的表現 …52
肆、討論…………………………………………………… ……55
參考文獻……………………………………………………… …60

參考文獻
Ageez A, Kazama Y, Sugiyama R, Kawano S (2005) Male-fertility genes expressed in male flower buds of Silene latifolia include homologs of anther-specific genes. Genes Genet Syst 80:403-413
Balestrasse KB, Yannarelli GG, Noriega GO, Batlle A, Tomaro ML (2008) Heme oxygenase and catalase gene expression in nodules and roots of soybean plants subjected to cadmium stress. Biometals 21: 433-441
Brosché M, Fant C, Bergkvist S, W., Strid H, Svensk A, Olsson O, Strid Å (1999) Molecular markers for UV-B stress in plants: alteration of the expression of four classes of genes in Pisum sativum and the formation of high molecular mass RNA adducts. Biochim Biophys Acta 1447: 185-198
Buchanan-Wollaston V, Page T, Harrison E, Breeze E, Lim PO, Nam HG, Lin J, Wu S, Swidzinski J, Ishizaki K, Leaver C, J. (2005) Comparative transcriptome analysis reveals significant differences in gene expression and signaling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. Plant J. 42: 585-567
Chen H, Hou W, Yang C, Huang D, Liu J, Lin Y (2003) Molecular cloning of two metallothionein-like protein genes with differential expression patterns from sweet potato (Ipomoea batatas) leaves. J Plant Physiol 160: 547-555
Chen HJ, Huang DJ, Hou WC, Liu JS, Lin YH (2006) Molecular cloning and characterization of a granulin-containing cysteine protease SPCP3 from sweet potato (Ipomoea batatas) senescent leaves. J Plant Physiol. 163:863-76
Chen H, Tsai Y, Chen W, Huang G, Huang S, Lin Y (2010) Ethephon-mediated effects on leaf senescence are affected by reduced glutathione and EGTA in sweet potato detacehed leaves. BS (accepted)
Chen Y, Lin H, Jeng S (2008) Calcium influxes and mitogen- activated protein kinase kinase activation mediate ethylene inducing ipomoelin gene expression in sweet potato. Plant Cell Environ 31: 62-72
Collin GB, Marshall JD, Naggert JK, Nishina PM (1999) TGFA: exon-intron structure and evaluation as a candidate gene for Alström syndrome. Clin Genet 55:61-2
Coupe SA, Taylor JE, Roberts JA (1997) Temporal and spatial expression of mRNAs encoding pathogenesis-related proteins during ethylene-promoted leaflet abscission in Sambucus nigra. Plant Cell Environ 20: 1517-1524
Dray E, Siaud N, Dubois E, Doutriaux MP (2006) Interaction between Arabidopsis Brca2 and its partners Rad51, Dmc1, and Dss1. Plant Physiol 140:1059-69.
Espinoza C, Medina C, Somerville S, Arce-Johnson P (2007) Senescence-associated genes induced during compatible viral interactions with grapevine and Arabidopsis. J Exp Bot 58: 3197-3212
Gilroy S, Blowers DP, Trewavas AJ (1987). Calcium: a regulatory system emerges in plant cells. Development 100:181-184
Giri AP, Wünsche H, Mitra S, Zavala JA, Muck A, Svatos A, Baldwin IT (2006) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. VII. Changes in the plant''s proteome. Plant Physiol.142:1621-41
Grantz AA, Brummell DA, Bennett AB (1995) Ascorbate free radical reductase mRNA levels are induced by wounding. Plant Physiol 108:411-8
Hadfield KA, Bennett AB (1997) Programmed senescence of plant organs. Cell Death Differ 4: 662-670
Hattori K, Hirayama M, Suzuki H, Hamamoto H, Sekimizu K, Tamura HO (2007) Cloning and expression of a novel sulfotransferase with unique substrate specificity from Bombyx mori. Biosci Biotechnol Biochem 71:1044-51
Hepler PK, and Wayne RO (1985) Calcium and plant development.Annu Rev Plant Physiol 36: 397-439
Hodges DM, Forney CF (2000) The effects of ethylene, depressed oxygen and elevated carbon dioxide on antioxidant profiles of senescing spinach leaves. J Exp Bot 51: 645-655
Huang YJ, To KY, Yap MN, Chiang WJ, Suen DF, Chen SC (2001) Cloning and characterization of leaf senescence up-regulated genes in sweet potato. Physiol Plant 113:384-391

Ihara-Ohori Y, Nagano M, Muto S, Uchimiya H, Kawai-Yamada M (2007) Cell Death Suppressor Arabidopsis Bax Inhibitor-1 Is Associated with Calmodulin Binding and Ion Homeostasis. Plant Physiol 143: 650-660
Jang I, Park S, Kim K, Kwon S, Kim J, Kwak S (2004) Differential expression of 10 sweetpotato peroxidase genes in response
to bacterial pathogen, Pectobacterium chrysanthemi. Plant Physiol Biochem 42: 451-455
Jing HC, Schippers JHM, Hille J, Dijkwel PP (2005) Ethylene-induced leaf senescence depends on age-related changes and OLD genes in Arabidopsis. J Exp Bot 56: 2915-2923
Kim KY, Huh GH, Lee HS, Kwon SY, Hur Y, Kwak SS (1999) Molecular characterization of cDNAs for two anionic peroxidases from suspension cultures of sweet potato. Mol Gen Genet 261:941-7
Kim MC, Chung WS, Yun DJ, Cho MJ (2009) Calcium and Calmodulin-Mediated Regulation of Gene Expression in Plants. Mol Plant 2: 13-21
Kim YH, Kim CY, Song WK, Park DS, Kwon SY, Lee HS, Bang JW, Kwak SS (2008) Overexpression of sweetpotato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco. Planta 227: 867-881
Kim YS, Choi D, Lee MM, Lee SH, Kim WT (1998) Biotic and abiotic stress-related expression of 1-aminocyclopropane-1-carboxylate oxidase gene family in Nicotiana glutinosa L. Plant Cell Physiol 39: 565-573
Kim YS, Kim HS, Lee YH, Kim MS, Oh HW, Hahn KW, Joung H, Jeon JH (2008) Elevated H2O2 production via overexpression of a chloroplastic Cu/ZnSOD gene of lily (Lilium oriental hybrid ‘Marco Polo’) triggers ethylene synthesis in transgenic potato. Plant Cell Reports 27: 973-983
Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant Biol 58: 36-115
Lin Z, Zhong S, Grierson D (2009) Recent advances in ethylene research. J Exp Bot 60: 3311-3336
Liu J, Lee-Tamon SH, Reid DM (1997) Differential and wound-inducible expression of 1-aminocylopropane-1-carboxylate oxidase genes in sunflower seedlings. Plant Mol Biol 34: 923-933
Mannen T, Andoh T, Tani T (2008) Dss1 associating with the proteasome functions in selective nuclear mRNA export in yeast. Biochem Biophys Res Commun 365:664-71.
Munné-Bosch S, Alegre L (2004) Die and let live: leaf senescence contributes to plant survival under drought stress. Functional Plant Biology 31:
203-216
Nakayama A, Park S, Zheng-Jun X, Nakajima M, Yamaguchi I (2002) Immunohistochemistry of Active Gibberellins and Gibberellin-Inducible -Amylase in Developing Seeds of Morning Glory. Plant Physiol 129: 1045-1053
Navabpour S, Morris K, Allen R, Harrison E, Mackerness SAH, Buchanan-Wollaston V (2003) Expression of senescence-enhanced genes in response to oxidative stress. Journal of Experimental Botany 54: 2285-2292
Niewiadomska E, Polzien L, Desel C, Rozpadek P, Miszalski Z, Krupinska K (2008) Spatial patterns of senescence and development-dependent distribution of reactive oxygen species in tobacco (Nicotiana tabacum) leaves . J Plant Physiol 166: 1057-1068
Ohtsubo N, Mitsuhara I, Koga M, Seo S, Ohashi Y (1999) Ethylene Promotes the Necrotic Lesion Formation and Basic PR Gene Expression in TMV-Infected Tobacco. Plant Cell Physiol 40: 808-817
Oshima Y, Kamigaki A, Nakamori C, Mano S, Hayashi M, Nishimura M, Esaka M (2008) Plant catalase is imported into peroxisomes by Pex5p but is distinct from typical PTS1 import. Plant Cell Physiol 49: 671-677
Park SY, Ryu SH, Kwon SY, Lee HS, Kim JG, Kwak SS (2003) Differential expression of six novel peroxidase cDNAs from cell cultures of sweetpotato in response to stress. Mol Gen Genomics 269: 542-552
Passardi F, Cosio C, Penel C, Dunand C (2005) Peroxidases have more functions than a Swiss army knife. Plant Cell Ref 24: 255-265
Pooviah BW, and Reddy ASN (1987) Calcium messenger systemsin plants. CRC Crit. Rev. Plant Sci 6: 47-103
Raz V and Fluhr R (1992) Calcium requirement for ethylene-dependent responses. Plant Cell 4: 1123-1130
Reilly K, Bernal D, Cortés DF, Gómez-Vásquez R, Tohme J, Beeching JR (2007) Towards identifying the full set of genes expressed during cassava post-harvest physiological deterioration. Plant Mol Biol 64:187-203.
Saika H, Okamoto M, Miyoshi K, Kushiro T, Shinoda S, Jikumaru Y, Fujimoto M, Arikawa T, Takahashi H, Ando M, Arimura S, Miyao A, Hirochika H, Kamiya Y, Tsutsumi N, Nambara E, Nakazono M (2007) Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8''-hydroxylase in rice. Plant Cell Physiol 48:287-298
Shao H, Chu L, Lu Z, Kang C (2008) Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells. Int J Biol Sci 4: 8-14
Shi YH, Zhu SW, Mao XZ, Feng JX, Qin YM, Zhang L, Cheng J, Wei LP, Wang ZY, Zhu YX (2006) Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation. Plant Cell 18:651-664
Spanu P, Reinhardt D, Boller T (1991) Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes. EMBO J. 1991 10:2007-13
Steffens B and Sauter M (2009) Epidermal Cell Death in Rice Is Confined to Cells with a Distinct Molecular Identity and Is Mediated by Ethylene and H2O2 through an Autoamplified Signal Pathway. Plant Cell 21: 184-196
Ulmasov T, Hagen G, Guilfoyle TJ (1999) Activation and repression of transcription by auxin-response factors. Proc Natl Acad Sci U S A. 96: 5844-9.
Van der Straeten D, Rodrigues-Pousada RA, Goodman HM, Van Montagu M (1991) Plant enolase: gene structure, expression, and evolution. Plant Cell 3: 719-735
Wei SJ, Williams JS, Dang H, Darden TA, Betz BL, Humble MM, Chang FM, Trempus CS, Johnson K, Cannon RE, Tennant RW (2008) Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation. J Mol Biol 383: 693-712
Willekens H, Camp VW, Montagu VM, Inzé D, Langebartels C, Sandermann JH (1994) Ozone, sulfur dioxide, and ultraviolet B have similar
effects on mRNA accumulation of antioxidant genes in Nicotiana plumbaginifolia L. Plant Physiol 106: 1007-1014
Yamakawa H, Mitsuhara I, Ito N, Seo S, Kamada H, Ohashi Y (2001) Transcriptionally and post-transcriptionally regulated response of 13 calmodulin genes to tobacco mosaic virus-induced cell death and wounding in tobacco plant. Eur J Biochem 268:3916-3929
Zhang F, Wang Y, Lou Z, Dong J (2007) Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza). Chemosphere 67: 44-50
Zimmenrmeann P, Heinlein C, Orendi G, Zentgraf U (2006) Senescence-specific regulation of catalases in Arabidopsis thaliana (L.) Heynh. Plant Cell Environ 29: 1049-1060