臺灣博碩士論文加值系統

抗壞血酸過氧化酶 (Ascorbate peroxidase, APX；EC 1.11.1.11)是一植物體內代謝過氧化氫且含有血基質 (heme) 之重要酵素，而其發現於高等植物、藻類、及藍綠藻中，並藉由抗壞血酸 (Ascorbic acid, AsA) 的氧化反應清除過氧化氫的危害。前人研究證實耐淹水之茄子栽培品種（Solanum melongena L.,var.Ping-tong Long Eggplant），其APX於淹水環境中扮演著調控因子的重要角色，且可有效的抵禦過氧化氫對於植物體的傷害。
本研究利用聚合酶鏈鎖反應（polymerase chain reaction, PCR）於前後兩端引子上加入限制酶切位，大量複製後，將耐淹水之茄子的抗壞血酸基因構築於載體 pGEM® T easy-vector 上，接著進行次選殖至表現載體pQE30上，並轉殖至大腸桿菌DH5α中大量表現。在大腸桿菌表現的APX與載體pQE30上的序列融合形成具有六個histidine殘基的標誌 (Tag)，可利用鎳親和性管柱進行快速的單一步驟快速純化，接著以膠體過濾層析進一步純化即可獲得高純度的抗壞血酸過氧化酶，用以進行APX的酵素活性及特性分析。SDS-PAGE及膠體過濾分析可觀察到變性 (denature)及原態的(native)酵素分子量分別為29 kDa及41kDa，而結果顯示抗壞血酸過氧化酶是以二聚體(dimer)的形式存在。APX的氧化作用將過氧化氫(H2O2)轉換成水(H2O)和氧(O2)，而AsA於波長290 nm具有吸光特性，因此藉由偵測波長290 nm的吸收值的減少可以換算出AsA的消耗量，並計算出酵素的活性。於不同pH值下分析APX的活性，顯示酵素的最適催化活性位於pH 6.0；於不同的溫度下分析酵素活性，顯示其最適催化溫度約為35℃。

Ascorbate peroxidase (APX, EC 1.11.1.11) is an important heme-containing enzyme that has been found in higher plants, algae, and some cyanobacteria, which scavenges hydrogen peroxide by preferentially oxidizing ascorbate. APX of flood-tolerant eggplant cultivar (Solanum melongena L., var. Ping-tong Long Eggplant) has been demonstrated to play a key role under flood and regulated the antioxidant availability to resist H2O2.In this study, eggplant APX was cloned into pGEM® T easy-vector, and then subcloned into pQE30 for over-expressing APX. The recombinant eggplant APX was contructed by fusing its N-terminus with a 6× Histidine tag; therefore, the recombinant APX could be quickly purified by Ni-chelatin chromatography in one step, followed bypurifing by gelfiltration to obtain a pure APX with high homogeneity for activity analysis and enzyme characterization. Analyses of denature and native APX by SDS-PAGE and gel filtration showed a molecular mass of 29 kDa and 41 kDa, respectively. It suggested the eggplant APX had a dimeric structure in nature. APX catalyzes the oxidation reaction of H2O2coupled to reduction reaction of Ascorbic acid (AsA). AsA possesses absorbance at 290 nm, so measurement of the decrease at 290 nm could be used to calculate the enzyme activity. Analysis of enzyme activity in different pHs and in different temperatures showed that the eggplant APX had a pH optima at pH 6.0, and the best catalyzing temperature at 35℃.

目錄
中文摘要 Ⅰ
英文摘要 III
壹、 背景介紹 1
一、 活性氧族(ROS)與抗氧化防禦 1
二、 植物的淹水逆境生理 3
三、 植物在逆境下的保護機制 4
四、 抗壞血酸過氧化酶 7
五、 抗壞血酸過氧化酶目前的研究 8
六、 茄子 10
貳、 研究源起及目的 11
參、 材料與方法 13
一、 實驗材料 13
1. 茄子(Solanum melongena L.)抗壞血酸過氧化酶cDNA GENE 13
2. 菌株及載體 ...…………….…13
3. 試藥及實驗相關酵素 13
二、 儀器設備 14
三、 實驗方法 14
3.1. 基因選殖 14
小量質體製備 14
限制酶切割之分析 15
DNA 洋菜膠體電泳分析 15
DNA片段回收及純化 16
接合反應 17
製備勝認細胞 17
轉型作用 17
藍白篩選 18
3.2. 聚合酶鏈鎖反應(PCR) 19
3.3. 蛋白質純化 20
大量表現 20
超音波振盪法破菌 20
鎳親和性管柱層析法 20
離子交換層析法 22
膠體過濾層析法 22
蛋白質定量- Bradford法 23
蛋白質聚丙醯胺膠體電泳之分析 23
3.4. 酵素活性與特性分析 25
酵素活性測定 25
酵素動力學測定 26
蛋白質活性電泳 26
肆、 結果與討論 27
一、 重組茄子抗壞血酸過氧化酶之基因建構 27
二、 重組茄子抗壞血酸過氧化酶之大量表現與純化 28
2.1. 重組茄子抗壞血酸過氧化酶表現條件的探討 28
2.2. 重組茄子抗壞血酸過氧化酶的純化 29
2.3. 重組茄子抗壞血酸過氧化酶結構的分析 31
三、 重組茄子抗壞血酸過氧化酶最適活性條件分析 32
3.1. 重組茄子抗壞血酸過氧化酶的最適pH值分析 32
3.2. 重組茄子抗壞血酸過氧化酶的最適溫度分析 32
3.3. 重組茄子抗壞血酸過氧化酶的動力學參數的測定 33
伍、 未來展望 34
陸、 參考文獻 35
柒、 圖表 43

Alscher, R.G., J.L. Donahue, and C.L. Cramer. 1997. Reactive oxygen species and antioxidants: Relationships in green cells. Physiologia Plantarum. 100:224-233.
Alscher, R.G., N. Erturk, and L.S. Heath. 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of experimental botany. 53:1331-1341.
Battistuzzi, G., M. D'Onofrio, L. Loschi, and M. Sola. 2001. Isolation and Characterization of Two Peroxidases from Cucumis sativus. Archives of Biochemistry and Biophysics. 388:100-112.
Blokhina, O., E. Virolainen, and K.V. Fagerstedt. 2003. Antioxidants, Oxidative Damage and Oxygen Deprivation Stress: a Review. Annals of Botany. 91:179-194.
Bowler, C., M.V. Montagu, and D. Inze. 1992. Superoxide Dismutase and Stress Tolerance. Annual Review of Plant Physiology and Plant Molecular Biology. 43:83-116.
Bunkelmann, J.R., and R.N. Trelease. 1996. Ascorbate Peroxidase (A Prominent Membrane Protein in Oilseed Glyoxysomes). Plant Physiology. 110:589-598.
Carvalho, L.s.C., and S. Amâncio. 2002. Antioxidant defence system in plantlets transferred from in vitro to ex vitro: effects of increasing light intensity and CO2 concentration. Plant Science. 162:33-40.
Chatfield, M., and D.A. Dalton. 1993. Ascorbate peroxidase from soybean root nodules. Plant Physiol. 103:661-662.
Chen, G.-X., and K. Asada. 1989. Ascorbate Peroxidase in Tea Leaves: Occurrence of Two Isozymes and the Differences in Their Enzymatic and Molecular Properties. Plant and Cell Physiology. 30:987-998.
Chen, Y., H. Wang, X. Wang, A. Cao, and P. Chen. 2006. Cloning and expression of peroxisomal ascorbate peroxidase gene from wheat. Mol Biol Rep. 33:207-213.
Cheng, H., Q. He, Y. Huo, L. Hou, and J. Lv. 2009. Molecular cloning, characterization and expression analysis of CmAPX. Mol Biol Rep. 36:1531-1537.
Dai, Y.H., C.Y. Huang, L. Wen, D.C. Sheu, and C.T. Lin. 2012. Lemon Ascorbate Peroxidase: cDNA Cloning and Biochemical Characterization. Botanical Studies. 53:11-19.
Dalton, D.A., F.J. Hanus, S.A. Russell, and H.J. Evans. 1987. Purification, Properties, and Distribution of Ascorbate Peroxidase in Legume Root Nodules. Plant Physiology. 83:789-794.
del Rio, L.A., F.J. Corpas, L.M. Sandalio, J.M. Palma, M. Gomez, and J.B. Barroso. 2002. Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes. Journal of experimental botany. 53:1255-1272.
del Rio, L.A., L.M. Sandalio, D.A. Altomare, and B.A. Zilinskas. 2003. Mitochondrial and peroxisomal manganese superoxide dismutase: differential expression during leaf senescence. Journal of experimental botany. 54:923-933.
Desikan, R., J. Hancock, and S. Neill. 2004. Oxidative stress signalling. In Plant Responses to Abiotic Stress. Vol. 4. H. Hirt and K. Shinozaki, editors. Springer Berlin Heidelberg. 121-149.
Elia, M.R., G. Borraccino, and S. Dipierro. 1992. Soluble ascorbate peroxidase from potato tubers. Plant Science. 85:17-21.
Ferreira, R.R., R.F. Fornazier, A.P. Vitória, P.J. Lea, and R.A. Azevedo. 2002. changes in antioxidant enzyme activities in soybean under cadmium stress. Journal of Plant Nutrition. 25:327-342.
Gechev, T., I. Gadjev, F. Van Breusegem, D. Inzé, S. Dukiandjiev, V. Toneva, and I. Minkov. 2002. Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes. CMLS, Cell. Mol. Life Sci. 59:708-714.
Groden, D., and E. Beck. 1979. H2O2 destruction by ascorbate-dependent systems from chloroplasts. Biochimica et biophysica acta. 546:426-435.
Gupta, S.D. 2010. Reactive Oxygen Species and Antioxidants in Higher Plants.
Ishibashi, Y., T. Tawaratsumida, K. Kondo, S. Kasa, M. Sakamoto, N. Aoki, S.-H. Zheng, T. Yuasa, and M. Iwaya-Inoue. 2012. Reactive Oxygen Species Are Involved in Gibberellin/Abscisic Acid Signaling in Barley Aleurone Cells. Plant Physiology. 158:1705-1714.
Ishikawa, T., K. Sakai, K. Yoshimura, T. Takeda, and S. Shigeoka. 1996. cDNAs encoding spinach stromal and thylakoid-bound ascorbate peroxidase, differing in the presence or absence of their 3'-coding regions. FEBS Lett. 384:289-293.
Ishikawa, T., K. Yoshimura, K. Sakai, M. Tamoi, T. Takeda, and S. Shigeoka. 1998. Molecular Characterization and Physiological Role of a Glyoxysome-Bound Ascorbate Peroxidase from Spinach. Plant and Cell Physiology. 39:23-34.
Kanematsu, S., and K. Asada. 1990. Characteristic Amino Acid Sequences of Chloroplast and Cytosol Isozymes of CuZn-Superoxide Dismutase in Spinach, Rice and Horsetail. Plant and Cell Physiology. 31:99-112.
Kawakami, S., Y. Matsumoto, A. Matsunaga, S. Mayama, and M. Mizuno. 2002. Molecular cloning of ascorbate peroxidase in potato tubers and its response during storage at low temperature. Plant Science. 163:829-836.
Kelly, G.J., and E. Latzko. 1979. Soluble ascorbate peroxidase. Naturwissenschaften. 66:617-618.
Kieselbach, T., M. Bystedt, P. Hynds, C. Robinson, and W.P. Schroder. 2000. A peroxidase homologue and novel plastocyanin located by proteomics to the Arabidopsis chloroplast thylakoid lumen. FEBS Lett. 480:271-276.
Kim, I.-J., and W.-I. Chung. 1998. Molecular characterization of a cytosolic ascorbate peroxidase in strawberry fruit. Plant Science. 133:69-77.
Kubo, A., H. Saji, K. Tanaka, K. Tanaka, and N. Kondo. 1992. Cloning and sequencing of a cDNA encoding ascorbate peroxidase from Arabidopsis thaliana. Plant molecular biology. 18:691-701.
Laloi, C., K. Apel, and A. Danon. 2004. Reactive oxygen signalling: the latest news. Current Opinion in Plant Biology. 7:323-328.
Lin, K.-H., H.-F. Lo, C.-H. Lin, and M.-T. Chan. 2007. Cloning and Expression Analysis of Ascorbate Peroxidase Gene from Eggplant under Flooding Stress. Botanical Studies. 48:25-34.
Lin, K.-H., C.-C. Weng, H.-F. Lo, and J.-T. Chen. 2004. Study of the root antioxidative system of tomatoes and eggplants under waterlogged conditions. Plant Science. 167:355-365.
Lin, L., X. Wang, and Y. Wang. 2006. cDNA Clone, fusion expression and purification of the novel gene related to ascorbate peroxidase from Chinese wild Vitis pseudoreticulata in E. coli. Mol Biol Rep. 33:197-206.
Liu, Y.J., Y. Yuan, Y.Y. Liu, Y. Liu, J.J. Fu, J. Zheng, and G.Y. Wang. 2012. Gene families of maize glutathione-ascorbate redox cycle respond differently to abiotic stresses. J Plant Physiol. 169:183-192.
Lu, H., R.L. Han, and X.N. Jiang. 2009. Heterologous expression and characterization of a proxidomal ascorbate peroxidase from Populus tomentosa. Mol Biol Rep. 36:21-27.
Makino, A., C. Miyake, and A. Yokota. 2002. Physiological Functions of the Water–Water Cycle (Mehler Reaction) and the Cyclic Electron Flow around PSI in Rice Leaves. Plant and Cell Physiology. 43:1017-1026.
May, M.J., T. Vernoux, C. Leaver, M.V. Montagu, and D. Inzé. 1998. Glutathione homeostasis in plants: implications for environmental sensing and plant development. Journal of experimental botany. 49:649-667.
Mittler, R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends in plant science. 7:405-410.
Mittler, R., S. Vanderauwera, M. Gollery, and F. Van Breusegem. 2004. Reactive oxygen gene network of plants. Trends in plant science. 9:490-498.
Mittler, R., and B.A. Zilinskas. 1991. Purification and Characterization of Pea Cytosolic Ascorbate Peroxidase. Plant Physiology. 97:962-968.
Mori, I.C., and J.I. Schroeder. 2004. Reactive Oxygen Species Activation of Plant Ca2+ Channels. A Signaling Mechanism in Polar Growth, Hormone Transduction, Stress Signaling, and Hypothetically Mechanotransduction. Plant Physiology. 135:702-708.
Neill, S., R. Desikan, and J. Hancock. 2002. Hydrogen peroxide signalling. Curr Opin Plant Biol. 5:388-395.
Nishikawa, F., M. Kato, R. Wang, H. Hyodo, Y. Ikoma, M. Sugiura, and M. Yano. 2003. Two ascorbate peroxidases from broccoli: identification, expression and characterization of their recombinant proteins. Postharvest Biology and Technology. 27:147-156.
Ohya, T., Y. Morimura, H. Saji, T. Mihara, and T. Ikawa. 1997. Purification and characterization of ascorbate peroxidase in roots of Japanese radish. Plant Science. 125:137-145.
Polle, A. 2001. Dissecting the superoxide dismutase-ascorbate-glutathione-pathway in chloroplasts by metabolic modeling. Computer simulations as a step towards flux analysis. Plant Physiol. 126:445-462.
Raven, E.L. 2003. Understanding functional diversity and substrate specificity in haem peroxidases: what can we learn from ascorbate peroxidase? Nat Prod Rep. 20:367-381.
Rudell, D.R., D.A. Buchanan, R.S. Leisso, B.D. Whitaker, J.P. Mattheis, Y. Zhu, and V. Varanasi. 2011. Ripening, storage temperature, ethylene action, and oxidative stress alter apple peel phytosterol metabolism. Phytochemistry. 72:1328-1340.
Schantz, M.-L., H. Schreiber, P. Guillemaut, and R. Schantz. 1995. Changes in ascorbate peroxidase activities during fruit ripening in Capsicum annuum. FEBS Letters. 358:149-152.
Seppanen, M.M., T. Cardi, M. Borg Hyokki, and E. Pehu. 2000. Characterization and expression of cold-induced glutathione S-transferase in freezing tolerant Solanum commersonii, sensitive S. tuberosum and their interspecific somatic hybrids. Plant science : an international journal of experimental plant biology. 153:125-133.
Sharp, K.H., Mewies, M., Moody, P.C., Raven, E.L. (2003) Crystal structure of theascorbate peroxydases-ascoebate complex. Nat Struct Biol 10: 303-307.
Shigeoka, S., T. Ishikawa, M. Tamoi, Y. Miyagawa, T. Takeda, Y. Yabuta, and K. Yoshimura. 2002. Regulation and function of ascorbate peroxidase isoenzymes. Journal of experimental botany. 53:1305-1319.
Shigeoka, S., Y. Nakano, and S. Kitaoka. 1980. Metabolism of hydrogen peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase. The Biochemical journal. 186:377-380.
Simova-Stoilova, L., K. Demirevska, A. Kingston-Smith, and U. Feller. 2012. Involvement of the leaf antioxidant system in the response to soil flooding in two Trifolium genotypes differing in their tolerance to waterlogging. Plant science : an international journal of experimental plant biology. 183:43-49.
Smith, M.W., and R.F. Doolittle. 1992. A comparison of evolutionary rates of the two major kinds of superoxide dismutase. Journal of molecular evolution. 34:175-184.
Sohn, S.-I., J.-C. Kim, K.-W. Lee, H.-I. Rhee, and M.-H. Wang. 2002. Molecular cloning and expression of a cDNA encoding cytosolic ascorbate peroxidase fromPimpinella brachycarpa. Journal of Plant Physiology. 159:1029-1035.
Szarka, A., B. Tomasskovics, and G. Banhegyi. 2012. The Ascorbate-glutathione-alpha-tocopherol Triad in Abiotic Stress Response. International journal of molecular sciences. 13:4458-4483.
Takahashi, M.A., and K. Asada. 1983. Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids. Arch Biochem Biophys. 226:558-566.
Terazawa, K., Y. Maruyama, and Y. Morikawa. 1992. Photosynthetic and stomatal responses ofLarix kaempferi seedlings to short-term waterlogging. Ecol. Res. 7:193-197.
Vranova, E., D. Inze, and F. Van Breusegem. 2002. Signal transduction during oxidative stress. Journal of experimental botany. 53:1227-1236.
Wada, K., T. Tada, Y. Nakamura, Y. Yabuta, K. Yoshimura, T. Takeda, S. Shigeoka, and K. Nishimura. 2002. Crystallization and preliminary X-ray diffraction analysis of chloroplastic ascorbate peroxidase of tobacco plants. Acta Crystallographica Section D. 58:559-561.
Willekens, H., D. Inzé, M. Montagu, and W. Camp. 1995. Catalases in plants. Mol Breeding. 1:207-228.
Wong, V., S. Armknecht, and A. Zhitkovich. 2012. Metabolism of Cr(VI) by ascorbate but not glutathione is a low oxidant-generating process. Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS). 26:192-196.
Yanagawa, Y., and S. Komatsu. 2012. Ubiquitin/proteasome-mediated proteolysis is involved in the response to flooding stress in soybean roots, independent of oxygen limitation. Plant Science. 185–186:250-258.
Zsigmond, L., B. Tomasskovics, V. Deak, G. Rigo, L. Szabados, G. Banhegyi, and A. Szarka. 2011. Enhanced activity of galactono-1,4-lactone dehydrogenase and ascorbate-glutathione cycle in mitochondria from complex III deficient Arabidopsis. Plant physiology and biochemistry : PPB / Societe francaise de physiologie vegetale. 49:809-815.
林俊宏. 2004. 選殖及分析淹水逆境下茄子中APX-like的基因. In 生物科技研究所. 碩士. 中國文化大學.
翁嘉成. 2003. 番茄與茄子淹水逆境下根部抗氧化系統之研究. In 生物科技研究所. 碩士. 中國文化大學.
曹幸之、羅筱鳳. 2002. 蔬菜(Ⅱ). 復文書局. P.186~190.