番茄抗坏血酸代谢相关酶基因DHAR1和MDHAR1在抗逆方面的研究
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摘要
非生物逆境(干旱、冷害、盐碱等)是农作物产量和品质的主要限制因素之一。在各种非生物逆境胁迫下,植物通常都会出现活性氧(ROS)胁迫。植物在进化过程中形成了一套有效的抗氧化体系来减轻ROS胁迫以维持植物的正常生理功能。抗坏血酸(AsA)是植物组织中主要的抗氧化物质。脱氢抗坏血酸还原酶(DHAR)和单脱氢抗坏血酸还原酶(MDHAR)是抗坏血酸代谢中再生的关键酶,对于维持抗坏血酸氧化还原状态起着重要作用。逆境诱导型启动子(如受干旱、高盐或冷胁迫诱导的拟南芥RD29A启动子)在避免组成型超表达抗逆有关基因的负面影响方面具有积极的作用。本研究以番茄这一重要蔬菜作物为材料,通过调控番茄抗坏血酸代谢过程中的DHAR1和MDHAR1的表达,利用超表达、诱导表达和RNAi技术分析转基因番茄的抗非生物逆境的特性,探讨这两个基因在抗逆中的具体功能,并期望能获得具有应用价值的抗多种逆境的番茄材料。本研究获得的主要结果如下:
1.从拟南芥中克隆了RD29A基因(AT5G52310)上游823bp的启动子序列IpeD29A。构建了一个含pRD29A的通用诱导表达载体pK2GW7I;
2.通过Gateway重组技术构建了番茄DHAR1、MDHAR1的超表达载体pKOED、pKOEMD和诱导表达载体pKDI、pKMDI;
3.通过农杆菌介导的基因转化技术,将有关载体导入番茄,获得转基因番茄苗44株。通过PCR验证T-DNA已经整合到番茄基因组;
4.通过RT-PCR对抗坏血酸代谢相关基因在番茄不同组织中的表达谱进行了分析,发现DHAR1和MDHAR1均为组成型表达,但在各组织中的表达量有差异;
5.对DHAR1和MDHAR1有关载体的转基因T_0代植株进行了初步抗逆性分析。并根据结果从每个载体转化的转基因后代中挑选出5个系,在T_1代中作了进一步的分析。初步发现超表达DHAR1和MDHAR1以及诱导表达DHAR1的转基因植株对提高番茄耐盐、耐旱、耐寒均有作用。初步结果表明盐胁迫下RD29A诱导型启动子在番茄中可以有效驱动下游基因的表达,对提高番茄的抗逆性具有重要意义。本研究获得了部分耐盐、耐寒、耐旱的转基因株系,为进一步的理论和育种研究提供了材料。
Abiotic stress including drought, chilling and salinity etc. is one of the major threats to crop production and quality. Reactive oxygen stress generally accompanies with various abotic stresses. Plants have developed their own antioxidant defense systems to alleviate ROS stress and maintain normal physiological process. Ascorbate acid (AsA) is a major antioxidant in plants. Dehydroascorbate reductase (DHAR) and Monodehydroascorbate reductase (MDHAR) are key enzymes in AsA regeneration and essential for maintaining the redox state of AsA. The stress-inducible promoter, such as RD29A promoter which is induced upon drought, salt or cold stress, may be useful to minimize side effects caused by constitutively expressing of stress tolerance-related genes in plant. This study aims to decipher the functions of DHAR1 and MDHAR1 by analyzing of their roles in abiotic stress using strategies of over expression, inducible expression and RNA inference, which may also lead to creation of tomato breeding materials with multiple stress tolerance. The main results were as follows:
1. The 823 bp upstream regulatory region( pRD29A) of RD29A gene(AT5G52310) from Arabidopsis thaliana genome was amplified using PCR technique. pK2GW7I, a binary vector with pRD29A was constructed.
2. The tomato inducible expression vectors- pKDI、pKMDI and over expression vectors- pKOED, pKOEMD were constructed for tomato DHAR1 and MDHAR1, respectively.
3. A total of 44 putative transgenic tomato plants were obtained. PCR analysis results indicated that T-DNA was integrated into the tomato genome.
4. RT-PCR analysis showed that DHAR1, MDHAR1 were constitutively expressed in different tissue of tomato, whereas the expression levels were different.
5. Tolerance to oxidative stress for transgenic plants was investigated in T_0 generation. Five lines for each construct from T_1 transgenic plants were further chosen for function analysis of the DHAR1 and MDHAR1. It was found that that overexpression of DHAR1, MDHAR1 or inducible expression of DHAR1 could improve salt, drought and chilling stress tolerance on tomato. The preliminary results indicated pRD29A functions in tomato. Several transgenic tomato lines with tolerance to salt, drought and chilling stress were obtained and could serve as materials for future research on abiotic stress tolerance.
1.从拟南芥中克隆了RD29A基因(AT5G52310)上游823bp的启动子序列IpeD29A。构建了一个含pRD29A的通用诱导表达载体pK2GW7I;
2.通过Gateway重组技术构建了番茄DHAR1、MDHAR1的超表达载体pKOED、pKOEMD和诱导表达载体pKDI、pKMDI;
3.通过农杆菌介导的基因转化技术,将有关载体导入番茄,获得转基因番茄苗44株。通过PCR验证T-DNA已经整合到番茄基因组;
4.通过RT-PCR对抗坏血酸代谢相关基因在番茄不同组织中的表达谱进行了分析,发现DHAR1和MDHAR1均为组成型表达,但在各组织中的表达量有差异;
5.对DHAR1和MDHAR1有关载体的转基因T_0代植株进行了初步抗逆性分析。并根据结果从每个载体转化的转基因后代中挑选出5个系,在T_1代中作了进一步的分析。初步发现超表达DHAR1和MDHAR1以及诱导表达DHAR1的转基因植株对提高番茄耐盐、耐旱、耐寒均有作用。初步结果表明盐胁迫下RD29A诱导型启动子在番茄中可以有效驱动下游基因的表达,对提高番茄的抗逆性具有重要意义。本研究获得了部分耐盐、耐寒、耐旱的转基因株系,为进一步的理论和育种研究提供了材料。
Abiotic stress including drought, chilling and salinity etc. is one of the major threats to crop production and quality. Reactive oxygen stress generally accompanies with various abotic stresses. Plants have developed their own antioxidant defense systems to alleviate ROS stress and maintain normal physiological process. Ascorbate acid (AsA) is a major antioxidant in plants. Dehydroascorbate reductase (DHAR) and Monodehydroascorbate reductase (MDHAR) are key enzymes in AsA regeneration and essential for maintaining the redox state of AsA. The stress-inducible promoter, such as RD29A promoter which is induced upon drought, salt or cold stress, may be useful to minimize side effects caused by constitutively expressing of stress tolerance-related genes in plant. This study aims to decipher the functions of DHAR1 and MDHAR1 by analyzing of their roles in abiotic stress using strategies of over expression, inducible expression and RNA inference, which may also lead to creation of tomato breeding materials with multiple stress tolerance. The main results were as follows:
1. The 823 bp upstream regulatory region( pRD29A) of RD29A gene(AT5G52310) from Arabidopsis thaliana genome was amplified using PCR technique. pK2GW7I, a binary vector with pRD29A was constructed.
2. The tomato inducible expression vectors- pKDI、pKMDI and over expression vectors- pKOED, pKOEMD were constructed for tomato DHAR1 and MDHAR1, respectively.
3. A total of 44 putative transgenic tomato plants were obtained. PCR analysis results indicated that T-DNA was integrated into the tomato genome.
4. RT-PCR analysis showed that DHAR1, MDHAR1 were constitutively expressed in different tissue of tomato, whereas the expression levels were different.
5. Tolerance to oxidative stress for transgenic plants was investigated in T_0 generation. Five lines for each construct from T_1 transgenic plants were further chosen for function analysis of the DHAR1 and MDHAR1. It was found that that overexpression of DHAR1, MDHAR1 or inducible expression of DHAR1 could improve salt, drought and chilling stress tolerance on tomato. The preliminary results indicated pRD29A functions in tomato. Several transgenic tomato lines with tolerance to salt, drought and chilling stress were obtained and could serve as materials for future research on abiotic stress tolerance.
引文
1.李合生.现代植物生理学.北京:高等教育出版社,2002:390-400
2.李合生.植物生理生化实验原理和技术.北京:高等教育出版社,2000:199-200;211-212:246-248
3.欧阳波,几种病程相关蛋白基因转化番茄的研究.[博士学位论文].武汉:华中农业大学图书馆,2002
4.杨婷,番茄盐胁迫下的生理生化特性及耐盐相关基因的分离.[硕士学位论文].武汉:华中农业大学图书馆,2006
5.邹礼平,番茄抗坏血酸生物合成与代谢途径中相关酶基因的克隆与调控.[博士学位论文].武汉:华中农业大学图书馆,2006
6.Agarwal P,Agarwal P K,Nair S,Sopory S K,Reddy M K.Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity.Mol Genet Genomics,2007,277:189-198
7.Amin E E,Naoyoshi K,Ghazi H B,Hironori K,Takeshi S,Toshiyuki S,Shinobu I,Kiyoshi T.verexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone,salt and polyethylene glycol stresses.Planta,2007,225:1255-1264
8.Asada K.Ascorbate peroxidase:a hydrogen peroxide-scavenging enzyme in plants.Physiol Plant,1992,85:235-241
9.Behnam B,Kikuchi A,Celebi-Toprak F,Kasuga M,Yamaguchi-Shinozaki K,Watanabe KN.Arabidopsis rd29A::DREB1A enhances freezing tolerance in transgenic potato.Plant Cell Rep,2007,26(8):1275-1282
10.Bhatnagar-Mathur P,Devi M J,Reddy D S,Lavanya M,Vadez V,Serraj R,Yamaguchi-Shinozaki K,Sharma K K.Stress-inducible expression of AtDREB1A in transgenic peanut(Arachis hypogaea L.) increases transpiration efficiency under water-limiting conditions.Plant Cell Rep,2007,26(12):2071-2082
11.Bhattachrjee S.Reactive oxygen species and oxidative burst:roles in stress,senescence and signal transduction in plant.Curr Sci,2005,89:1113-1121
12.Bozs'o Z,Ott P G,Szama'ri A',Zelleng A'C,Varga G,Besenyei E.Early detection of bacterium-induced basal resistance in tobacco leaves with diaminobenzidine and dichlorofluorescein diacetate. J Phytopathol. 2005, 153: 596-607
13. Bray E A, Bailey-Serres J, and Weretilnyk E. Responses to abiotic stresses. Biochemistry and molecular biology of plants. American Society of Plant Physiologists, 2000: 1158-124
14. Chen M, Xu Z, Xia L, Li L, Cheng X, Dong J, Wang Q, Ma Y. Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene, GmDREB3, in soybean (Glycine max L.). J Exp Bot, 2009, 60(l):121-135
15. Chen Z and Gallie DR. Increasing tolerance to ozone by elevating foliar ascorbic acid confers greater protection against ozone than increasing avoidance. Plant Physiol, 2005,138:1673-1689
16. Chen Z and Gallie DR. The ascorbic acid redox state controls guard cell signaling and stomatal movement. Plant Cell, 2004,16:1143-1162
17. Chen Z, Young T E, Ling J, Chang S C, Gallie D R. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc Natl Acad Sci USA, 2003,100: 3525-3530
18. Chen Z and Gallie D R. Dehydroascorbate reductase affects non-photochemical quenching and photosynthetic performance. J Biol Chem, 2008, 283(31):21347-61
19. Chew O, Whelan J, and Millar A H. Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J Biol Chem, 2003, 278: 46869-46877
20. Christina L, Ute B, Neil J S, Damian P D and Geoffrey, B. F. Gene structure and expression pattern analysis of three monodehydroascorbate reductase (MDHAR) genes in Physcomitrella patens: Implications for the evolution of the MDHAR family in plants. Plant Molecular Biology, 2006, 60: 259-275
21. Dalai M, Tayal D, Chinnusamy V, Bansal K C. Abitotic stress and ABA-inducible Group 4LEA from Brassica napus plays a key role in salt and drought tolerance. Journal of Biotechnology, 2009, 139:137-145
22. De G L, Paciolla C, Tommasi F, Liso R, Arrigoni O. In vivo inhibition of L-galactono-1,4-lactone conversion to ascorbate by lycorine. J Plant Physiol, 1994, 144: 649-653
23. Desikan R S, Mackerness A H, Hancock J T, Neill S J. Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiol, 2001, 127:159-172
24. Doke N. Generation of superoxide anion by potato tuber protoplasts upon the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressor of hypersensitivity. Physiol Plant Pathol, 1983,27:311-322
25. Eskling M, Arvidsson P O, Akerlund H E. The xanthophylls cycle, its regulation and components. Physiol Plant, 1997, 100: 806-816
26. Grace S, Pace R, and Wydrzynski T. Formation and decay of monodehydro-ascorbate radicals in illuminated thylakoids as determined by EPR spectroscopy. Biochim. BiohysActa, 1995, 1229: 1555-165
27. Gossett D R, Banks S W, Millhollon E P, Lucas M C. Antioxidant response to nacl stress in a control and an NaCl-tolerant cotton cell line grown in the presence of paraquat, buthionine sulfoximine, and exogenous glutathione. Plant Physiol, 1996, 112:803-809
28. Hua Z M, Yang X, Fromm M E. Activation of the NaCl- and drought-induced RD29A and RD29B promoters by constitutively active Arabidopsis MAPKK or MAPK proteins. Plant Cell Environ, 2006, 29(9): 1761-1770
29. Jaglo KR, Kleff S, Amundsen KL, Zhang X, Haake V, Zhang JZ, Deits T, Thomashow MF. Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol, 2001, 127(3):910-917
30. Jimenez A, Hernandez J A, Del R L, Sevilla F. Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiol, 1997, 114: 275-284
31. Jyothilakshmi V, Swati T, Ram P, Ajit V, Ralf O. Monodehydroascorbate reductase 2 and dehydroascorbate reductase 5 are crucial for a mutualistic interaction between Piriformospora indica and Arabidopsis. J Plant Physiol, 2009 (In press)
32. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol, 1999, 17:229-230
33. Kato N and Esaka M. Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells. Physiologia Plantarum,1999,105: 321-329
34. Kozi A. The water-water cycle as alternative photon and electron sinks. Philos Trans R Soc Lond Biol Sci, 2000, 355, 1419-1431
35. Kwon S Y, Choi S M, Ahn Y O, Lee H S, Lee H B, Park Y M, Kwak S S. Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. Journal of Plant Physiology, 2003, 160: 347-353
36. Lee Y P, Kim S H, Bang J W, Lee H S, Kwak S S, Kwon S Y. Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts. Plant Cell Rep, 2007, 26: 591-598
37. Levitt J. Response of plants to environmental stress. New York: Academic Press, 1980,2(2): 102-106
38. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaquchi-Shinozaki K, Shinozaki K. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 1998,10: 1391-1406
39. Luwe M. Antioxidants in the apoplast and symplast of beech (Fagus sylvatica L.) leaves: seasonal variations and responses to changing ozone concentration in air. Plant Cell Env, 1996, 19: 321-328
40. Maria C. Rubio, Pilar Bustos Sanmamed, Maria R. Clemente and Manuel Becana. Effects of salt stress on the expression of antioxidant genes and proteins in the model legume Lotus japonicus. New Phytologis, 2009, (181): 851-859
41. Marina L, Francisco J C, Juan B B, Luisa M S, and Luis A R. Peroxisomal monodehydroascorbate reductase. Genomic clone characterization and functional analysis under environmental stress conditions. Plant Physiology, 2005, 138: 2111-2123
42. Miramar M D, Costantini P, Ravagnan L, Saraiva L M, Haouzi D, Brothers G, Penninger J M., Peleato M L, Kroemer G and Susin S A,. NADH oxidase activity of mitochondrial apoptosis-inducing factor. J Biol Chem, 2001, 276: 16391-16398
43. Miranda J A, Avonce N, Suarez R, Thevelein J M, Van D P, Iturriaga G. A bifunctional TPS-TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis. Planta, 2007, 226(6): 1411-1421
44. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F. Reactive oxygen gene network of plants. Trends in Plant Science, 2004, 9: 490-498
45. Miyake C and Asada K. Ferredoxin-dependent photoreduction of the monodehydroascorbate radical in spinach thylakoids. 1994, Plant Cell Physiol, 35: 539-549
46. Neill S J, Desikan R, Clarke A, Hancock J T. Nitric oxide is a novel component of abscisic acid signaling in stomatal guard cells. Plant Physiol, 2002, 128: 13-16
47. Noctor G, Foyer C H. Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol, 1998, 49: 249-279
48. Padh H. Cellular functions of ascorbic acid. Biochem Cell Biol, 1990, 68: 1166-1173
49. Pasqualini S, Batini P, Ederli, Porceddu A, Piccioni C, De M F and Antonielli M. Effects of short-term ozone fumigation on tobacco plants: response of the scavenging system and expression of the glutathione reductase. Plant Cell Environ, 2001, 24: 245-252
50. Pellinen R, Palva T, Kangasjarvi J. Subcellular localization of ozone-induced hydrogen peroxide production in birch (Betula pendula) leaf cells. Plant J, 1999, 20: 349-356
51. Peter J E. Monodehyroascorbate reductase 4 is required for seed storage oil hydrolysis and postgerminative growth in Arabidopsis. The Plant Cell, 2007, 19: 1376-1387
52. Pignocchi C, Foyer C H. Apoplastic ascorbate metabolism and its role in the regulation of cell signaling. Curr Opin Plant Biol, 2003, 6: 379-389
53. Pino M T, Skinner J S, Park E J, Jeknic Z, Hayes P M, Thomashow M F and Chen T H. Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield. Plant Biotechnol J, 2007, 5: 591-604
54. Quan L J, Zhang Bo, Shi W W and Li Hong Yu. Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species network. Journal of Integrative Plant Biology, 2008, 50: 2-18
55. Rasmusson A G, Heiser V, Zabaleta E, Brennicke A, Grohmann L. Physiological, biochemical and molecular aspects of mitochondria complex in plants. Biochim Biophys Acta, 1998,1364: 101-111
56. Sakihama Y, Mano J, Sano S, Asada K, and Yamasaki H. Reduction of phenoxyl radicals mediated by monodehydroascorbate reductase. Biochem Biophys Res Commun, 2000,279: 949-954
57. Sano S and Asada K. cDNA cloning of monodehydroascorbate radical reductase from cucumber: a high degree of homology in terms of amino acid sequence between this enzyme and bacterial flavoenzymes. Plant Cell Physiol, 1994, 35: 425-437
58. Satoshi S, Satoru T, Yuko E, Tomomi I, M. A H, Chikahiro M L, Francisco J C, Juan B B, Luisa M S, and Luis A R. Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach. Biosci Biotechnol Biochem, 2005, 69: 762-772
59. Shao H B, Li Y Ch, Zhao H L, Cong M K. Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells. International Journal of Biological Sciences, 2008, 4: 8-14
60. Seiji Y, Masanori T, Takeshi S, Nobuyoshi N, Daisuke O, Motohide Ii, Mitsuko A, Akihiro K, Hiroshi K, Yasunori I and Hikaru S. Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana. Plant Cell Physiol, 2006, 47): 304-308
61. Shin S Y, Kim I S, Kim Y H, Park H M, Lee J Y, Kang H G, Yoon H S. Scavenging reactive oxygen species by rice dehydroascorbate reductase alleviates oxidative stresses in Escherichia coli. Mol Cells, 2008, 26: 616-620
62. Smirnoff N. The function and metabolism of ascorbic acid in plants. Ann Bot, 1996, 78: 661-669
63. Stevens R, Page D, Gouble B, Garchery C, Zamir D and Causse M. Tomato fruit ascorbic acid content is linked with monodehydroascorbate reductase activity and tolerance to chilling stress. Plant, Cell and Environment, 2008, 31: 1086-1096
64. Taise S, Chikahiro M and Akiho Y. Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts. Eur J Biochem, 2003, 270: 921-928
65. Takahashi M, Shiraishi T, Asada K. Superoxide production in aprotic interior of chloroplast thylakoids. Arch Biochem Biophys, 1988, 267: 714-722
66. Vanacker H, Carver T L W, Foyer C H. Pathogeninduced changes in the atioxidant status of the apoplast in barley leaves. Plant Physiol, 1998, 117: 1103-1114
67. Veljovic-Jovanovic S D, Pignocchi C, Noctor G, Foyer C H. Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol, 2001, 127: 426-435
68. Umezawa.Y R, Maruvama K, Yamaquchi K, Shinozaki K. SRK2C, a SNF1-related protein kinase2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana. Proc Natl Acad Sci USA, 2004, 101: 17306-17311
69. Urano J, Nakagawa T, Maki Y, Masumura T, Tanaka K, Murata N, Ushimaru T. Molecular cloning and characterization of a rice dehydroascorbate reductase. FEBS Lett, 2000, 466: 107-111
70. Veljovic-Jovanovic S D, Pignocchi C, Noctor G, Foyer C H. Low ascorbic acid in the vtc-1 mutant of arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol, 2001, 127:426-435
71. Vethanayagam J G, Green E H, Rose R C and Bode A M. Glutathione-dependent ascorbate recycling activity of rat serum albumin. Free Rad Biol, 1999, 26: 1591-1598
72. Walters D R. Polyamines and plant disease. Phytochemistry, 2003, 64: 97-107
73. Wang W, Vinocur B, and Airman A. Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta, 2003, 218:1-14
74. Washburn M P. and Wells W W. Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase. Biochem Biophys Res Commun, 1999, 257: 567-571
75. Xu D P , Washburn M P , Sun G P and Wells W W. Purification and characterization of a glutathione dependent dehydroascorbate reductase from human erythrocytes. Biochem Biophys Res Commun, 1996,221: 117-121
76. Yabuta Y, Yoshimura K, Takeda T, Shigeoka S. Molecular characterization of tobacco mitochondrial L-galactono-γ-lactone dehydrogenase and its expression in E.Coli. Plant Cell Physiol, 2000, 41: 666-675
77. Yamaguchi-Shinozaki K and Shinozaki K. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature, or high-salt stress. The Plant Cell, 1994, 6: 251-264
78. Yamaguchi-Shinozaki K and Shinozaki K. Arabidopsis DNA encoding two desiccationresponsive rd29 genes.Plant Physiol, 1993,101: 1119-1120
79. Yoshihiro N, Kazuo N, Zabta K S, Yoh S, Takashi F, Hiroshi A, Mari N, Kazuo S and Kazuko Y S. Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses. The Plant Journal, 2003, 34: 137-148
80. Yang T, Poovaiah B W. Hydrogen peroxide homeostasis activation of plant catalase by calcium/calmodulin. Plant Biol, 2002, 99: 4097-4102
81.Yoon H S, Lee H, Lee I A, Kim K Y, Jo J. Molecular cloning of the monodehydroascorbate reductase gene from Brassica campestris and analysis of its mRNA level in response to oxidative stress. Biochim Biophys Acta, 2004, 1658: 181-186
82. Zhang GY, Kang J G , Zhang YG , Cheng Z H , Wang X W. Gene cloning and expression analysis of a male sterility related gene BoDHAR from broccoli, Sheng Wu Gong ChengXue Bao, 2006, 22: 751-756
83. Zhao J, Ren W, Zhi D, Wang L, Xia G. Arabidopsis DREB1A/CBF3 bestowed transgenic tall fescue increased tolerance to drought stress. Plant Cell Rep, 2007, 26: 1521-1528
84. Zhong C and Daniel R G. Dehydroascorbate reductase affects leaf growth, development, and function. Plant Physiology, 2006, 142: 775-787
85. Zhu J, Jeong J C, Zhu Y, Sokolchik I, Miyazaki S, Zhu J K, Hasegawa P M, Bohnert H J, Shi H, Yun D J, Bressan R A. Involvement of Arabidopsis HOS15 in histone deacetylation and cold tolerance. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105: 4945-4950
2.李合生.植物生理生化实验原理和技术.北京:高等教育出版社,2000:199-200;211-212:246-248
3.欧阳波,几种病程相关蛋白基因转化番茄的研究.[博士学位论文].武汉:华中农业大学图书馆,2002
4.杨婷,番茄盐胁迫下的生理生化特性及耐盐相关基因的分离.[硕士学位论文].武汉:华中农业大学图书馆,2006
5.邹礼平,番茄抗坏血酸生物合成与代谢途径中相关酶基因的克隆与调控.[博士学位论文].武汉:华中农业大学图书馆,2006
6.Agarwal P,Agarwal P K,Nair S,Sopory S K,Reddy M K.Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity.Mol Genet Genomics,2007,277:189-198
7.Amin E E,Naoyoshi K,Ghazi H B,Hironori K,Takeshi S,Toshiyuki S,Shinobu I,Kiyoshi T.verexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone,salt and polyethylene glycol stresses.Planta,2007,225:1255-1264
8.Asada K.Ascorbate peroxidase:a hydrogen peroxide-scavenging enzyme in plants.Physiol Plant,1992,85:235-241
9.Behnam B,Kikuchi A,Celebi-Toprak F,Kasuga M,Yamaguchi-Shinozaki K,Watanabe KN.Arabidopsis rd29A::DREB1A enhances freezing tolerance in transgenic potato.Plant Cell Rep,2007,26(8):1275-1282
10.Bhatnagar-Mathur P,Devi M J,Reddy D S,Lavanya M,Vadez V,Serraj R,Yamaguchi-Shinozaki K,Sharma K K.Stress-inducible expression of AtDREB1A in transgenic peanut(Arachis hypogaea L.) increases transpiration efficiency under water-limiting conditions.Plant Cell Rep,2007,26(12):2071-2082
11.Bhattachrjee S.Reactive oxygen species and oxidative burst:roles in stress,senescence and signal transduction in plant.Curr Sci,2005,89:1113-1121
12.Bozs'o Z,Ott P G,Szama'ri A',Zelleng A'C,Varga G,Besenyei E.Early detection of bacterium-induced basal resistance in tobacco leaves with diaminobenzidine and dichlorofluorescein diacetate. J Phytopathol. 2005, 153: 596-607
13. Bray E A, Bailey-Serres J, and Weretilnyk E. Responses to abiotic stresses. Biochemistry and molecular biology of plants. American Society of Plant Physiologists, 2000: 1158-124
14. Chen M, Xu Z, Xia L, Li L, Cheng X, Dong J, Wang Q, Ma Y. Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene, GmDREB3, in soybean (Glycine max L.). J Exp Bot, 2009, 60(l):121-135
15. Chen Z and Gallie DR. Increasing tolerance to ozone by elevating foliar ascorbic acid confers greater protection against ozone than increasing avoidance. Plant Physiol, 2005,138:1673-1689
16. Chen Z and Gallie DR. The ascorbic acid redox state controls guard cell signaling and stomatal movement. Plant Cell, 2004,16:1143-1162
17. Chen Z, Young T E, Ling J, Chang S C, Gallie D R. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc Natl Acad Sci USA, 2003,100: 3525-3530
18. Chen Z and Gallie D R. Dehydroascorbate reductase affects non-photochemical quenching and photosynthetic performance. J Biol Chem, 2008, 283(31):21347-61
19. Chew O, Whelan J, and Millar A H. Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J Biol Chem, 2003, 278: 46869-46877
20. Christina L, Ute B, Neil J S, Damian P D and Geoffrey, B. F. Gene structure and expression pattern analysis of three monodehydroascorbate reductase (MDHAR) genes in Physcomitrella patens: Implications for the evolution of the MDHAR family in plants. Plant Molecular Biology, 2006, 60: 259-275
21. Dalai M, Tayal D, Chinnusamy V, Bansal K C. Abitotic stress and ABA-inducible Group 4LEA from Brassica napus plays a key role in salt and drought tolerance. Journal of Biotechnology, 2009, 139:137-145
22. De G L, Paciolla C, Tommasi F, Liso R, Arrigoni O. In vivo inhibition of L-galactono-1,4-lactone conversion to ascorbate by lycorine. J Plant Physiol, 1994, 144: 649-653
23. Desikan R S, Mackerness A H, Hancock J T, Neill S J. Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiol, 2001, 127:159-172
24. Doke N. Generation of superoxide anion by potato tuber protoplasts upon the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressor of hypersensitivity. Physiol Plant Pathol, 1983,27:311-322
25. Eskling M, Arvidsson P O, Akerlund H E. The xanthophylls cycle, its regulation and components. Physiol Plant, 1997, 100: 806-816
26. Grace S, Pace R, and Wydrzynski T. Formation and decay of monodehydro-ascorbate radicals in illuminated thylakoids as determined by EPR spectroscopy. Biochim. BiohysActa, 1995, 1229: 1555-165
27. Gossett D R, Banks S W, Millhollon E P, Lucas M C. Antioxidant response to nacl stress in a control and an NaCl-tolerant cotton cell line grown in the presence of paraquat, buthionine sulfoximine, and exogenous glutathione. Plant Physiol, 1996, 112:803-809
28. Hua Z M, Yang X, Fromm M E. Activation of the NaCl- and drought-induced RD29A and RD29B promoters by constitutively active Arabidopsis MAPKK or MAPK proteins. Plant Cell Environ, 2006, 29(9): 1761-1770
29. Jaglo KR, Kleff S, Amundsen KL, Zhang X, Haake V, Zhang JZ, Deits T, Thomashow MF. Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol, 2001, 127(3):910-917
30. Jimenez A, Hernandez J A, Del R L, Sevilla F. Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiol, 1997, 114: 275-284
31. Jyothilakshmi V, Swati T, Ram P, Ajit V, Ralf O. Monodehydroascorbate reductase 2 and dehydroascorbate reductase 5 are crucial for a mutualistic interaction between Piriformospora indica and Arabidopsis. J Plant Physiol, 2009 (In press)
32. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol, 1999, 17:229-230
33. Kato N and Esaka M. Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells. Physiologia Plantarum,1999,105: 321-329
34. Kozi A. The water-water cycle as alternative photon and electron sinks. Philos Trans R Soc Lond Biol Sci, 2000, 355, 1419-1431
35. Kwon S Y, Choi S M, Ahn Y O, Lee H S, Lee H B, Park Y M, Kwak S S. Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. Journal of Plant Physiology, 2003, 160: 347-353
36. Lee Y P, Kim S H, Bang J W, Lee H S, Kwak S S, Kwon S Y. Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts. Plant Cell Rep, 2007, 26: 591-598
37. Levitt J. Response of plants to environmental stress. New York: Academic Press, 1980,2(2): 102-106
38. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaquchi-Shinozaki K, Shinozaki K. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 1998,10: 1391-1406
39. Luwe M. Antioxidants in the apoplast and symplast of beech (Fagus sylvatica L.) leaves: seasonal variations and responses to changing ozone concentration in air. Plant Cell Env, 1996, 19: 321-328
40. Maria C. Rubio, Pilar Bustos Sanmamed, Maria R. Clemente and Manuel Becana. Effects of salt stress on the expression of antioxidant genes and proteins in the model legume Lotus japonicus. New Phytologis, 2009, (181): 851-859
41. Marina L, Francisco J C, Juan B B, Luisa M S, and Luis A R. Peroxisomal monodehydroascorbate reductase. Genomic clone characterization and functional analysis under environmental stress conditions. Plant Physiology, 2005, 138: 2111-2123
42. Miramar M D, Costantini P, Ravagnan L, Saraiva L M, Haouzi D, Brothers G, Penninger J M., Peleato M L, Kroemer G and Susin S A,. NADH oxidase activity of mitochondrial apoptosis-inducing factor. J Biol Chem, 2001, 276: 16391-16398
43. Miranda J A, Avonce N, Suarez R, Thevelein J M, Van D P, Iturriaga G. A bifunctional TPS-TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis. Planta, 2007, 226(6): 1411-1421
44. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F. Reactive oxygen gene network of plants. Trends in Plant Science, 2004, 9: 490-498
45. Miyake C and Asada K. Ferredoxin-dependent photoreduction of the monodehydroascorbate radical in spinach thylakoids. 1994, Plant Cell Physiol, 35: 539-549
46. Neill S J, Desikan R, Clarke A, Hancock J T. Nitric oxide is a novel component of abscisic acid signaling in stomatal guard cells. Plant Physiol, 2002, 128: 13-16
47. Noctor G, Foyer C H. Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol, 1998, 49: 249-279
48. Padh H. Cellular functions of ascorbic acid. Biochem Cell Biol, 1990, 68: 1166-1173
49. Pasqualini S, Batini P, Ederli, Porceddu A, Piccioni C, De M F and Antonielli M. Effects of short-term ozone fumigation on tobacco plants: response of the scavenging system and expression of the glutathione reductase. Plant Cell Environ, 2001, 24: 245-252
50. Pellinen R, Palva T, Kangasjarvi J. Subcellular localization of ozone-induced hydrogen peroxide production in birch (Betula pendula) leaf cells. Plant J, 1999, 20: 349-356
51. Peter J E. Monodehyroascorbate reductase 4 is required for seed storage oil hydrolysis and postgerminative growth in Arabidopsis. The Plant Cell, 2007, 19: 1376-1387
52. Pignocchi C, Foyer C H. Apoplastic ascorbate metabolism and its role in the regulation of cell signaling. Curr Opin Plant Biol, 2003, 6: 379-389
53. Pino M T, Skinner J S, Park E J, Jeknic Z, Hayes P M, Thomashow M F and Chen T H. Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield. Plant Biotechnol J, 2007, 5: 591-604
54. Quan L J, Zhang Bo, Shi W W and Li Hong Yu. Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species network. Journal of Integrative Plant Biology, 2008, 50: 2-18
55. Rasmusson A G, Heiser V, Zabaleta E, Brennicke A, Grohmann L. Physiological, biochemical and molecular aspects of mitochondria complex in plants. Biochim Biophys Acta, 1998,1364: 101-111
56. Sakihama Y, Mano J, Sano S, Asada K, and Yamasaki H. Reduction of phenoxyl radicals mediated by monodehydroascorbate reductase. Biochem Biophys Res Commun, 2000,279: 949-954
57. Sano S and Asada K. cDNA cloning of monodehydroascorbate radical reductase from cucumber: a high degree of homology in terms of amino acid sequence between this enzyme and bacterial flavoenzymes. Plant Cell Physiol, 1994, 35: 425-437
58. Satoshi S, Satoru T, Yuko E, Tomomi I, M. A H, Chikahiro M L, Francisco J C, Juan B B, Luisa M S, and Luis A R. Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach. Biosci Biotechnol Biochem, 2005, 69: 762-772
59. Shao H B, Li Y Ch, Zhao H L, Cong M K. Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells. International Journal of Biological Sciences, 2008, 4: 8-14
60. Seiji Y, Masanori T, Takeshi S, Nobuyoshi N, Daisuke O, Motohide Ii, Mitsuko A, Akihiro K, Hiroshi K, Yasunori I and Hikaru S. Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana. Plant Cell Physiol, 2006, 47): 304-308
61. Shin S Y, Kim I S, Kim Y H, Park H M, Lee J Y, Kang H G, Yoon H S. Scavenging reactive oxygen species by rice dehydroascorbate reductase alleviates oxidative stresses in Escherichia coli. Mol Cells, 2008, 26: 616-620
62. Smirnoff N. The function and metabolism of ascorbic acid in plants. Ann Bot, 1996, 78: 661-669
63. Stevens R, Page D, Gouble B, Garchery C, Zamir D and Causse M. Tomato fruit ascorbic acid content is linked with monodehydroascorbate reductase activity and tolerance to chilling stress. Plant, Cell and Environment, 2008, 31: 1086-1096
64. Taise S, Chikahiro M and Akiho Y. Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts. Eur J Biochem, 2003, 270: 921-928
65. Takahashi M, Shiraishi T, Asada K. Superoxide production in aprotic interior of chloroplast thylakoids. Arch Biochem Biophys, 1988, 267: 714-722
66. Vanacker H, Carver T L W, Foyer C H. Pathogeninduced changes in the atioxidant status of the apoplast in barley leaves. Plant Physiol, 1998, 117: 1103-1114
67. Veljovic-Jovanovic S D, Pignocchi C, Noctor G, Foyer C H. Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol, 2001, 127: 426-435
68. Umezawa.Y R, Maruvama K, Yamaquchi K, Shinozaki K. SRK2C, a SNF1-related protein kinase2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana. Proc Natl Acad Sci USA, 2004, 101: 17306-17311
69. Urano J, Nakagawa T, Maki Y, Masumura T, Tanaka K, Murata N, Ushimaru T. Molecular cloning and characterization of a rice dehydroascorbate reductase. FEBS Lett, 2000, 466: 107-111
70. Veljovic-Jovanovic S D, Pignocchi C, Noctor G, Foyer C H. Low ascorbic acid in the vtc-1 mutant of arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol, 2001, 127:426-435
71. Vethanayagam J G, Green E H, Rose R C and Bode A M. Glutathione-dependent ascorbate recycling activity of rat serum albumin. Free Rad Biol, 1999, 26: 1591-1598
72. Walters D R. Polyamines and plant disease. Phytochemistry, 2003, 64: 97-107
73. Wang W, Vinocur B, and Airman A. Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta, 2003, 218:1-14
74. Washburn M P. and Wells W W. Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase. Biochem Biophys Res Commun, 1999, 257: 567-571
75. Xu D P , Washburn M P , Sun G P and Wells W W. Purification and characterization of a glutathione dependent dehydroascorbate reductase from human erythrocytes. Biochem Biophys Res Commun, 1996,221: 117-121
76. Yabuta Y, Yoshimura K, Takeda T, Shigeoka S. Molecular characterization of tobacco mitochondrial L-galactono-γ-lactone dehydrogenase and its expression in E.Coli. Plant Cell Physiol, 2000, 41: 666-675
77. Yamaguchi-Shinozaki K and Shinozaki K. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature, or high-salt stress. The Plant Cell, 1994, 6: 251-264
78. Yamaguchi-Shinozaki K and Shinozaki K. Arabidopsis DNA encoding two desiccationresponsive rd29 genes.Plant Physiol, 1993,101: 1119-1120
79. Yoshihiro N, Kazuo N, Zabta K S, Yoh S, Takashi F, Hiroshi A, Mari N, Kazuo S and Kazuko Y S. Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses. The Plant Journal, 2003, 34: 137-148
80. Yang T, Poovaiah B W. Hydrogen peroxide homeostasis activation of plant catalase by calcium/calmodulin. Plant Biol, 2002, 99: 4097-4102
81.Yoon H S, Lee H, Lee I A, Kim K Y, Jo J. Molecular cloning of the monodehydroascorbate reductase gene from Brassica campestris and analysis of its mRNA level in response to oxidative stress. Biochim Biophys Acta, 2004, 1658: 181-186
82. Zhang GY, Kang J G , Zhang YG , Cheng Z H , Wang X W. Gene cloning and expression analysis of a male sterility related gene BoDHAR from broccoli, Sheng Wu Gong ChengXue Bao, 2006, 22: 751-756
83. Zhao J, Ren W, Zhi D, Wang L, Xia G. Arabidopsis DREB1A/CBF3 bestowed transgenic tall fescue increased tolerance to drought stress. Plant Cell Rep, 2007, 26: 1521-1528
84. Zhong C and Daniel R G. Dehydroascorbate reductase affects leaf growth, development, and function. Plant Physiology, 2006, 142: 775-787
85. Zhu J, Jeong J C, Zhu Y, Sokolchik I, Miyazaki S, Zhu J K, Hasegawa P M, Bohnert H J, Shi H, Yun D J, Bressan R A. Involvement of Arabidopsis HOS15 in histone deacetylation and cold tolerance. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105: 4945-4950