柑橘Vc合成相关酶和ERF基因的克隆与表达分析
摘要
维生素C (Vitamin C, Vc)是生物体中重要的抗氧化剂及辅酶因子,是人体健康和植物的生长发育中必不可少的物质；ERF(Ethylene-responsive element binding factor)是植物特有的一类转录因子,在参与植物的生长发育及调控抗逆境胁迫应答中具有重要作用。
本研究以‘鄂柑二号’(C. unshiu Marc. cv. Egan No.2)果肉为材料,克隆了柑橘Vc合成的五个相关基因及ERF基因；并分别测定了果实生长发育过程及环剥处理后果肉中Vc的含量及合成相关的基因表达变化,以及环剥后果肉中ERF基因的表达变化。主要研究结果如下：
1.从柑橘果肉中克隆得到了五个Vc合成相关的基因及ERF基因的cDNA全长：GDP-甘露糖焦磷酸化酶基因(CitGMP),该基因包含1086 bp的ORF,编码361个氨基酸,登录号为HQ224946；GDP-甘露糖-3’,5’-差向异构酶基因(CitGME),包含1134bp的ORF,编码377个氨基酸,登录号为HQ224947；磷酸二酯酶基因(CitGGP),登录号为HQ224948,包含1368bp的ORF,编码455个氨基酸；.L-半乳糖-1-磷酸磷酸酶基因(CitGPP),,包含813bp的ORF,编码270个氨基酸,登录号为HQ224949；L-半乳糖脱氢酶基因(CitGDH),登录号为HQ224950,包含954 bp的ORF,编码317个氨基酸；柑橘乙烯反应元件结合因子(CitERF) cDNA全长874bp, ORF编码207个氨基酸,登录号为HM064500。这些基因的氨基酸序列与其他植物的同一基因同源性较高,且具有各自相对保守的功能结构域。
2.随着果实的生长发育,纽荷尔果肉中的Vc含量逐渐上升,且基因表达结果显示纽荷尔中CitGME、CitGGP、CitGDH及CitGLDH表达总体呈现上升趋势,与鄂柑二号相比具有较高的转录活性,这与两个品种间Vc的含量及变化趋势一致,橙类果肉具有较高的Vc水平可能是由于橙类具有更强的Vc合成能力。
3.对成熟期的温州蜜柑进行了不同形式的环剥处理,结果表明,大枝环剥(BG)可提高果肉中Vc的含量,CitGME、CitGGP、CitGDH及CitGLDH的转录水平也显著上升；而结果枝环剥(FGD)后Vc含量降低,CitGDH和CitGLDH基因表达也明显下调,说明这些基因可能是柑橘Vc合成中的关键基因。
4.BG处理后柑橘果肉中CitERF的表达呈持续上升趋势,FDG处理后其表达先下降后上升,与环剥后TSS的变化趋势一致,说明CitERF的表达可能受果实成熟过程中糖分积累的诱导。
Vitamin C (Vc), as a well-known abundant antioxidant and enzymatic cofactors, plays crucial roles in maintaining human health, plant development and stress tolerance. Ethylene-responsive element binding factors (ERFs), are a member of the AP2/EREBP gene family of plant transcription factors. They have been found to be mainly involved in response to biotic and abiotic stresses and play a important role in plant development progresses.
In the present study, five genes involved in vitamin C biosynthesis and ERF genewere isolated from the Citrus unshiu; ascorbate accumulation and expression profiles of these genes were investigated during fruit developing in fruit pulp of'Egan No.2' Satsuma mandarin and'Newhall'orange. In addition, we also investigated the effects of girdling on citrus ascorbate accumulation and genes'expression during fruit ripening in pulp of Satsuma mandarin. The main results were as follows:
1. Five genes involved in L-galactose pathway and ERF gene were isolated from the citrus unshiu and the primary information of these genes was listed in the following: GDP-D-mannose pyrophosphorylase (CitGMP) has an open reading frame of 1,086bp, encoding 361amino acids and its GenBank accession number is HQ224946; GDP-Man-3', 5'-epimerase(CitGME), GenBank accession number is HQ224947, has anORF of 1, 134bp, encoding 377 amino acids; GDP-L-galactose phosphorylase (CitGGP), has an ORF of 1,368bp, encoding 455 amino acids and its GenBank accession number is HQ224948; L-galactose-1-P phosphatase(CitGPP) GenBank accession number is HQ224949, has an ORF of 813bp, encoding 270 amino acids and its; L-galactose dehydrogenase(CitGDH), has an ORF of 954bp, encoding 317 amino acids and its GenBank accession number is HQ224950. Citrus Ethylene-responsive element binding factor(CitERF) has an ORF of 874bp, encoding 207 amino acids and its GenBank accession number is HM064500. Further analysis indicated that predicted amino acid sequences of these genes showed high homology with their respective gene in other plant species.
2. Ascorbate accumulation in pulp of'Newhall'were higher than those in'Egan No.2' fruits; Gene expression by real-time PCR revealed that CitGME, CitGGP, CitGDH and CitGLDH transcripts in 'Newhall'increased roughly during the whole experimental period which were higher than those in'Egan No.2'.
3.'Guoqing No.1'Satsuma mandarins(C.unshiu cv. Guoqing No.1) was chose for girdling treatment at fruit green mature stage. As compared to control, it was found thatVc contents in fruit pulp were increased by BG (branch girdling), but those contents were decreased after FGD (fruit-bear-shoot girdling plus defoliation). Gene expression analysis indicated that transcripts of CitGME, CitGGP, CitGDH and CitGLDH were upregulated by BG treatment,which was positive correlated with Vc accumulation in this treatment;
4. CitERF mRNA level in fruit pulp increased obviously by BG, but its expression be reduced significantly by FGD during fruit ripening, which was correlated with total soluble solid(TSS) accumulation in this treatment.
本研究以‘鄂柑二号’(C. unshiu Marc. cv. Egan No.2)果肉为材料,克隆了柑橘Vc合成的五个相关基因及ERF基因；并分别测定了果实生长发育过程及环剥处理后果肉中Vc的含量及合成相关的基因表达变化,以及环剥后果肉中ERF基因的表达变化。主要研究结果如下：
1.从柑橘果肉中克隆得到了五个Vc合成相关的基因及ERF基因的cDNA全长：GDP-甘露糖焦磷酸化酶基因(CitGMP),该基因包含1086 bp的ORF,编码361个氨基酸,登录号为HQ224946；GDP-甘露糖-3’,5’-差向异构酶基因(CitGME),包含1134bp的ORF,编码377个氨基酸,登录号为HQ224947；磷酸二酯酶基因(CitGGP),登录号为HQ224948,包含1368bp的ORF,编码455个氨基酸；.L-半乳糖-1-磷酸磷酸酶基因(CitGPP),,包含813bp的ORF,编码270个氨基酸,登录号为HQ224949；L-半乳糖脱氢酶基因(CitGDH),登录号为HQ224950,包含954 bp的ORF,编码317个氨基酸；柑橘乙烯反应元件结合因子(CitERF) cDNA全长874bp, ORF编码207个氨基酸,登录号为HM064500。这些基因的氨基酸序列与其他植物的同一基因同源性较高,且具有各自相对保守的功能结构域。
2.随着果实的生长发育,纽荷尔果肉中的Vc含量逐渐上升,且基因表达结果显示纽荷尔中CitGME、CitGGP、CitGDH及CitGLDH表达总体呈现上升趋势,与鄂柑二号相比具有较高的转录活性,这与两个品种间Vc的含量及变化趋势一致,橙类果肉具有较高的Vc水平可能是由于橙类具有更强的Vc合成能力。
3.对成熟期的温州蜜柑进行了不同形式的环剥处理,结果表明,大枝环剥(BG)可提高果肉中Vc的含量,CitGME、CitGGP、CitGDH及CitGLDH的转录水平也显著上升；而结果枝环剥(FGD)后Vc含量降低,CitGDH和CitGLDH基因表达也明显下调,说明这些基因可能是柑橘Vc合成中的关键基因。
4.BG处理后柑橘果肉中CitERF的表达呈持续上升趋势,FDG处理后其表达先下降后上升,与环剥后TSS的变化趋势一致,说明CitERF的表达可能受果实成熟过程中糖分积累的诱导。
Vitamin C (Vc), as a well-known abundant antioxidant and enzymatic cofactors, plays crucial roles in maintaining human health, plant development and stress tolerance. Ethylene-responsive element binding factors (ERFs), are a member of the AP2/EREBP gene family of plant transcription factors. They have been found to be mainly involved in response to biotic and abiotic stresses and play a important role in plant development progresses.
In the present study, five genes involved in vitamin C biosynthesis and ERF genewere isolated from the Citrus unshiu; ascorbate accumulation and expression profiles of these genes were investigated during fruit developing in fruit pulp of'Egan No.2' Satsuma mandarin and'Newhall'orange. In addition, we also investigated the effects of girdling on citrus ascorbate accumulation and genes'expression during fruit ripening in pulp of Satsuma mandarin. The main results were as follows:
1. Five genes involved in L-galactose pathway and ERF gene were isolated from the citrus unshiu and the primary information of these genes was listed in the following: GDP-D-mannose pyrophosphorylase (CitGMP) has an open reading frame of 1,086bp, encoding 361amino acids and its GenBank accession number is HQ224946; GDP-Man-3', 5'-epimerase(CitGME), GenBank accession number is HQ224947, has anORF of 1, 134bp, encoding 377 amino acids; GDP-L-galactose phosphorylase (CitGGP), has an ORF of 1,368bp, encoding 455 amino acids and its GenBank accession number is HQ224948; L-galactose-1-P phosphatase(CitGPP) GenBank accession number is HQ224949, has an ORF of 813bp, encoding 270 amino acids and its; L-galactose dehydrogenase(CitGDH), has an ORF of 954bp, encoding 317 amino acids and its GenBank accession number is HQ224950. Citrus Ethylene-responsive element binding factor(CitERF) has an ORF of 874bp, encoding 207 amino acids and its GenBank accession number is HM064500. Further analysis indicated that predicted amino acid sequences of these genes showed high homology with their respective gene in other plant species.
2. Ascorbate accumulation in pulp of'Newhall'were higher than those in'Egan No.2' fruits; Gene expression by real-time PCR revealed that CitGME, CitGGP, CitGDH and CitGLDH transcripts in 'Newhall'increased roughly during the whole experimental period which were higher than those in'Egan No.2'.
3.'Guoqing No.1'Satsuma mandarins(C.unshiu cv. Guoqing No.1) was chose for girdling treatment at fruit green mature stage. As compared to control, it was found thatVc contents in fruit pulp were increased by BG (branch girdling), but those contents were decreased after FGD (fruit-bear-shoot girdling plus defoliation). Gene expression analysis indicated that transcripts of CitGME, CitGGP, CitGDH and CitGLDH were upregulated by BG treatment,which was positive correlated with Vc accumulation in this treatment;
4. CitERF mRNA level in fruit pulp increased obviously by BG, but its expression be reduced significantly by FGD during fruit ripening, which was correlated with total soluble solid(TSS) accumulation in this treatment.
引文
1.安华明,陈力耕,樊卫国.高等植物中维生素C的功能、合成及代谢研究进展.植物学通报,2004,21(5)：608-617
2.安华明.刺梨高含量AsA的积累机制及其关键酶基因的克隆与表达.[博士学位论文].浙江：浙江大学图书馆,2004
3.杜盈,姚远,刘进元.两个水稻GDP-甘露糖-3',5'异构酶基因特征及表达模式的研究.生物化学与生物物理进展,2006,4：73-81
4.冯超,雷莹,刘永忠.柑橘L-半乳糖-1,4-内酯脱氢酶基因的克隆.华中农业大学学报,2009,28(6)：731-735
5.李明军.苹果和猕猴桃抗坏血酸形成与积累的生理和分子机理研究.[博士学位论文].陕西：西北农林科技大学大学图书馆,2009
6.刘永立,胡海涛,兰大伟.维生素C的生物合成及其基因调控研究进展.果树学报,2006,23(3)：431-436
7.祁春节,宋金田.2009年中国柑橘产销形势分析http://www.cncitrus.com/
8.王冬冬,朱延明,李勇等.电子克隆技术及其在植物基因工程中的应用.东北农业大学学报,2006,37(3)：403-408
9.张敏.苹果果实抗坏血酸合成酶基因的克隆及定量表达分析.[硕士学位论文].陕西：西北农林科技大学大学图书馆,2009
10.张伟玉,杨静慧,Wang-Pruski G等.不同转基因番茄GalUR的表达与AsA含量.西南大学学报(自然科学版),2008,30：43-47
11. Agarwal P, Agarwal P, Reddy M., Sopory S. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep,2006,25 (12):1263-1274
12. Agius F, Amaya I, Botella MA, Valpuesta V. Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression. J Exp Bot, 2005,56:37-46
13. Agius F, Gonzalez-Lamothe R, Caballero JL, et al. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid. Nature Biotech,2003,21: 177-181
14. Agius F, Lamothe RG, Caballero JL, et al. Engineering in creased vitaminC levels in Plants by over-expression of a D-galacturo- nic acid reduetase. Nature Biotechnology, 2003,21:177-181
15. Alhagdow M, Mounet F, Gilbert L, et al. Silencing of the mitochondrial ascorbate synthesizing enzyme L-Galactono-1,4-Lactone dehydrogenase affects plant and fruit development in Tomato. Plant Physiol,2007,145:1408-1422
16. Badejo AA, Fujikawa Y, Esak M. Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola(Malpighia glabra). J Plant physiol,2009,166:652-660
17. Badejo AA, Jeong ST, Goto-Yamamoto N. et al. Cloning and expression of GDP-D-mannose pyrophosphorylase gene and ascorbic acid content of acerola (Malpighia glabra L.)fruits at ripening stages. Plant Physiol Biochem,2007,45: 665-672
18. Badejo AA, Tanaka N, Esaka M, Analysis of GDP-D-mannose pyrophosphory-lase gene promoter from acerola(Malpighia glabra) and increase in ascorbate content of transgenic tobacco expressing acerola gene, Plant Cell Physiol,2008,49:126-132
19. Baig MM, Kelly S, Loewus F. Ascorbic Acid Biosynthesis in Higher Plants from 1-Gulono-1,4-lactoneand 1-Galactono-1,4-lactone. Plan Physiol,1970,46(2):277-280
20. Baldwin E A, Seymour GB, Taylor, JE, et al. Biochemistry of Fruit Ripening. In Citrus. Chapman & Hall,1993,107-137
21.Banno H, Ikeda Y, Niu QW, et al. Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration. Plant Cell,2001,13 (12):2609-2618.
22. Barber GA, The synthesis of guanosine 3,5-diphosphate-l-galactose by extracts of Chlorella pyrenoidosa. Arch Biochem,1975,167:718-722
23. Bartoli CG, Pastori GM, Foyer CH. Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiol,2000,123: 335-343
24. Bulley SM, Rassam M, Hoser D, et al. Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyl-transferase is a major control point of vitamin C biosynthesis. J Exp Bot,2009,60(3):765-778
25. Carreno J, Faraj S, Martinez A. Effects of girdling and covering mesh on ripening, colour and fruit characteristics of Italia grapes. J Hortic Sci Biotech,1998,73, 103-106
26. Conklin P L, Norris SR, Wheeler GL, et al. Last Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis, Proc Natl Acad Sci, 1999,4198-4203
27. Conklin PL, Gatzek S, Wheeler GL, et al. Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme. J Biol Chem, 2006,281:15662-15670
28. Conklin PL, Saracco SA, Norris SR, et al. Identification of ascorbic acid deficient Arabidopsis thaliana mutants. Genetics,2000,154:847-856
29. Cruz-Rus E, Botella MA, Valpuesta V, et al. Analysis of genes involved in 1-ascorbic acid biosynthesis during growth and ripening of grape berries. J Plant Physiol,2010, 167:739-748
30. Davey MW, Franck C, Keulemans J. Distribution, developmental and stress responses of antioxidant metabolism in malus. Plant Cell Environ,2004,27:1309-1320
31. Davey MW, Gilot C, Persiau G, et al. Ascorbate biosynthesis in Arabidopsis cell suspension cultures. Plant Physiol,1999,121:535-543
32. Davey MW, Sanmartin M, Kanellis A. Plant L-ascorbic acid:Chemistry, function, metabolism, bioavailability and effects of processing. Sci Food Agric,2000,80 825-860
33. Di Matteo A, Hancock RD, Ross HA, et al. Characterisation of Chlorella pyrenoi-dosa L-ascorbic acid accumulating mutants:Identification of an enhanced biosynthetic enzyme activity and cloning of the putative gene from Arabidopsis thaliana. Comp Biochem Physiol,2003,134:155
34. Dowdle J, Ishikawa T, Gatzek S, et al. Two genes in Arabidopsis thaliana encodin GDP-1-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J,2007,52:673-689
35. El-Sharkawy I, Sherif S, Mila I, et al. Molecular characterization of seven genes encoding ethylene-responsive transcriptional factors during plum fruit development and ripening. J Exp Bot,2009,60:907-922
36. Endres S, Tenhaken R. Myoinositol oxygenase controls the level of myoinositol in Arabidopsis, but does not increase ascorbic acid. Plant Physiol,2009,149:1042-1049
37. Franceschi VR, Tarlyn NM. L-Ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants. Plant Physiol,2002,130,649-656
38. Frei B, Lawson S. Vitamin C and cancer revisited. Proc Natl Acad Sci,2008,105, 11037-11038
39. Frossard E, Bucher M, Machler F, et al. Potential for increasing the content and bioavailability of Fe, Zn, and Ca in plants for human nutrition. Sci Food Agric,2000, 80:861-79
40. Gatzek S, Wheeler GL, Smirnoff N. Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis. Plant J,2002,30: 541-553
41. Gautier H, Massot C, Stevens R, et al. Regulation of tomato fruit ascorbate content is more highly dependent on fruit irradiance than leaf irradiance. Annals of Botany,2009, 103:495-504
42. Gilbert L, Alhagdow M, Nunes-Nesi A, et al. GDP-d-mannose-3,5-epimerase (GME) plays a key role at the intersection of ascorbate and non-cellulosic cell-wall biosynth esis in tomato. Plant J,2009,60:499-508
43. Giovannoni J. Molecular biology of fruit maturation and ripening. Annu. Rev.Plant Physiol. Plant Mol. Biol.2001,52,725-749
44. Gomez MLPA, Lajolo FM. Ascorbic acid metabolism in fruits:activity of enzymes involved in synthesis and degradation during ripening in mango and guava. J Sci Food Agr,2008,88:756-762
45. Goren R, Huberman M, Goldschmidt EE. Girdling:physiological and horticultural aspects. Horticultural Reviews,2003,30:1-36
46. Gournas C, Papageorgiou I, Diallinas G. the nucleobase-ascorbate transporter (NAT) family:genomics, evolution, structure-function relationships and physiological role. Mol BioSyst,2008,4:404-416
47. Grace SC, Logan BA. Acclimation of foliar antioxidant systems to growth irradiance in three broad-leaved evergreen species. Plant Physiol,1996,112:1631-1640
48. Gu YQ, Yang C, Thara VK, et al. Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the pto kinase. Plant Cell.2000,12 (5):771-786
49. Hancock RD, McRae D, Haupt S, et al. Synthesis of L-ascorbic acid in the phloem. BMC Plant Biology,2003,3:7
50. Hao D, Ohme-Takagi M, Sarai A. Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor (ERF Domain) in plant. J Biol Chem,1998,273 (41):26857-26861
51. Hebda PA, Barber GA, GDP-D-mannose:GDP-1-galactose epimerase from Chlor-ella pyrenoidosa. Meth Enzymol,1982,83:522-525
52. Imai T, Niwa M, Ban Y, et al. Importance of the L-galactonolactone pool for enhancing the ascorbate content revealed by L-galactonolactone dehydrogenase-overexpressing tobacco plants. Plant Cell, Tissue and Organ Culture,2009,96: 105-112
53. Ioannidi E, Kalamaki MS, Engineer C, et al. Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. J Exp Bot,2009,60(2):663-678
54. Isherwood FA, Chen YT, Mapson LW. Synthesis of 1-ascorbic acid in plants and animals. J Biochem,1954,56:1-21
55. Ishikawa T, Masumoto I, Iwasa N,et al. Functional characterization of D-galac-turonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis. Biosci Biotech Biochem.,2006,70:2720-2726
56. Ishikawa T, Nishikawa H, Gao Y, et al. The pathway via D-Galacturonate/L-Ga-lactonate is significant for ascorbate biosynthesis in Euglena gracilis:identifi cationand functional characterization of aldonolactonase. J Biol Chem,2008,283: 31133-31141
57. Jain AK, Nessler CL. Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol Breeding,2000,6:73-78
58. Jubany-Mari T, Munne-Bosch S, Alegre L. Redox regulation of water stress responses in field-grown plants. Role of hydrogen peroxide and ascorbate. Plant Physiol Bioch, 2010,48,351-358
59. Katz E, Lagunes PM, Riov J, et al. Molecular and physiological evidence suggests the existence of a system Ⅱ-like path way of ethylene production in non-climacteric citrus fruit. Planta,2004,219:243-252
60. Keller R, Springer F, Renz A, et al. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. Plant J,1999,19:31-141
61. Kizis D, Lumbreras V, Pages M. Role of AP2/EREBP transcription factors in gene regulation during abiotic stress. FEBS Lett,2001,498 (2-3):187-189
62. Koch KE, AvigneWT. Postphloem, nonvascular transfer in citrus:kinetics, etabolism, and sugar gradients. Plant Physiol,1990, (4):1405-1416
63. Kojo S. Vitamin C:basic metabolism and its function as an index of oxidative stress. Curr. Med Chem,2004,11:1041-1064
64. Kotchoni SO, Larrimore KE, Mukherjee M, Kempinski CF, Barth C. Alterations in the Endogenous Ascorbic Acid Content Affect Flowering Time in Arabidopsis. Plant Physiol,2009,149:803-815
65. Laing W A, Frearson N, Bulley S, et al. Kiwifruit 1-galactose dehydrogenase: molecular,biochemical and physiological aspects of the enzyme. Funct Plant Biol, 2004,31:1015-1025
66. Laing WA, Bulley S, Wright M, et al. A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis. Proc Natl Acad Sci USA,2004,10: 16976-6981
67. Laing WA, Wright MA, Cooney J, et al. The missing step of the L-galactose path-way of ascorbate biosynthesis in plants, an L-galactose guanyltransferase, increa-ses leaf ascorbate content. Proc Natl Acad Sci USA,2007,104:9534-9539
68. Lee SK, Kader AA. Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Postharvest Biol Tec,2000,20,207-220
69. Li MJ, Ma F, Shang P, et al. Influence of light on ascorbate formation and metabolism in apple fruits. Planta,2009,230:39-51
70. Li MJ, Ma FW, Guo CM, et al. Ascorbic acid formation and expression profiling of the genes involved in its synthesis and recycling in apple leaves with different age. Plant Physiol Bioch,2010,48:216-224
71. Li MJ, Ma FW, Zhang M, et al. Distribution and metabolism of ascorbic acid in apple fruits(Malus domestica Borkh cv. Gala). Plant Sci,2008,174:606-612
72. Linster CL, Adler LT, Webb K, et al. A second GDP-L-galactose phosphorylase in Arabidopsis enroute to vitamin C:covalent intermediate and substrate require-ments for the conserved reaction. J Biol Chem,2008,283:18483-18492
73. Linster CL, Clarke SG. L-Ascorbate biosynthesis in higher plants:the role of VTC 2. Trends Plant Sci,2008,13:567-573
74. Liu YZ, Liu Q, Tao NG, et al. Efficient isolation of RNA from fruit peel and pulp of ripening navel orange(Citrus sinensis). Journal of Huazhong Agricultural University, 2006,25:300-304
75. Loewus FA, Kelly S. The metabolism of D-galacturonic acid and its methylester in the detached ripening strawberry. Arch Biochem.Biophys,1961,95:483-493
76. Lorence A, Chevone BI, Mendes P, et al. Myo-Inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol,2004,134:1200-1205
77. Loscos J, Matamoros MA, Becana M. Ascorbate and homoglutathione metabolism in common bean nodules under stress conditions and during natural senescence. Plant Physiol,2008,146:1282-1292
78. Major LL, Wolucka BA, Naismith JH. Structure and function of GDP-mannose-3',5'-epimerase:an enzyme which performs three chemical reactions at the same active site. J Am Chem Soc,2005,127:18309-18320
79. Mieda T, Yabuta Y, Rapolu M, et al. Feedback inhibition of spinach 1-galactose dehydrogenase by 1-ascorbate. Plant Cell Physiol,2004,45:1271-1279
80. Miiller-Moule Patricia. An expression analysis of the ascorbate biosynthesis enzyme VTC2. Plant Mol Bol,2008,68:31-41
81. Nagy, S. Vitamin C contents of citrus fruit and their products:a review. Agric. Food Chem,1980,28:8-18
82. Naidu KA. Vitamin C in human health and disease is still a mystery. An overview. Nutrition Journal,2003,2:7
83. Nakano T, Suzuki K, Fujimura T, et al. Genome-wide analysis of the ERF gene family in arabidopsis and rice. Plant Physiol,2006,140 (2):411-432
84. Oba K, Ishikawa S, Nishikawa M, et al. Purification and properties of L-galactono-gamma-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots. J Exp Bot,1995,117:120-124
85. Ohme-Takagi M, Shinshi H. Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell,19957 (2):173-182
86. Olmos E, Kiddle G, Pellny TK, et al. Modulation of plant morphology, root architecture, and cell structure by low vitamin C in Arabidopsis thaliana. J Exp Bot, 2006,57:1645-1655
87. Oxley D, Bacic A.Structure of the glycosyl-phosphatidylinositol anchor of an arabinogalacta protein from Pyrus communis suspension-cultured cells.Proc Natl Acad Sci USA,1999,96:14246-14251
88. Pateraki I, Sanmartin M, Kalamaki MS, et al. Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4-lactone dehydrogenase. J Exp Bot,2004,55:1623-1633
89. Pineau B, Layoune O, Danon A, et al. L-Galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I. J Biol Chem,2008,283: 32500-32505
90. Pirrello J, Jaimes-Miranda F, Sanchez-Ballesta MT, et al. S1-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination. Plant Cell Physiol,2006,47 (9):1195-1205
91. Radzio JA, Lorence A, Chevone BI, et al. L-Gulono-1,4-lactone oxidase expression rescues vitamin C-deficient Arabidopsis(vtc1) mutants. Plant Mol Biol,2003,53: 837-844
92. Reiter WD, Vanzin GF. Molecular genetics of nucleotide sugar interconversion pathways in plants. Plant Mol Biol,2001,47:95-113
93. Riechmann JL, Meyerowitz EM. The AP2/EREBP family of plant transcription factors. Biol. Chem,1998,379 (6):633-646
94. Rivas F, Gravina A, Agusti M. Girdling effects on fruit set and quantum yield efficiency of PSⅡ in two Citrus cultivars. Tree Physiol,2007,27527-535
95. Running JA, Burlingame RP, Berry A. The pathway of L-ascorbic acid biosyn thesis in the colourless microalga prototheca moriformis. J Exp Bot,2003,54:1841-1849
96. Sarkar N, Srivastava PK, Dubey VK. Understanding the Language of Vitamin C. Current Nutrition & Food Science,2009,5 (3):53-55
97. Shi JX, Porat R, Goldschmidt EE, et al. Ethylene and volatile accumulation in citrus fruit. In:Ramina A., Chang C, Giovannoni J, et al. (Eds.), Advances in Plant Ethylene Research. Springer, Netherlands,2007, pp181-187
98. Siendones E,Gonzalez-Reyes J A, Santos-Ocana C, et al. Biosynthesis of ascorbic acid in kidney bean. L-Galactono-y-lactone dehy drogenase is an intrinsic protein located at the mitochondrial inner membrane. Plant Physiol,1999,120:907-912
99. Tabata K,Oba K, Suzuki K, et al. Generation and properties of ascorbic acid deficient trans genic tobacco cells expressing antisense RNA for L-galactono-1,4-lactonedehydrogenase. Plant J,2001,27:139-148
100. Tabata K, Takaoka T, Esaka M.Gene expression of ascorbic acid-related enzymes in tobacco. Phytochemistry,2002,61:631-635
101. Takeda S, Matsuokka M. Genetic approaches to crop improvement:responding to environmental and population changes. Nat Rev Genet,2008,9:444-457
102. Tamaoki M, Mukai F, Asai N, et al. Light-controlled expression of a gene encoding L-galactono-gamma-lactone dehydrogenase which affects ascorbate pool size in Arabidopsis thaliana. Plant Sci,2003,164:1111-1117
103. Taylor CA, Hampl JS, Johnston CS. Low intakes of vegetables and fruits, especially citrus fruits, lead to inadequate vitamin C intakes among adults. European Journal of Clinical Nutrition,2000,54:573-578
104. Tedone L, Hancock RD, Alberino S, et al. Long-distance transport of L-ascorbic acid in potato. BMC Plant Biol,2004,4:16
105. Tokunaga T, Miyahara K, Tabata K, et al. Generation and properties of ascorbic acid-overprod ucing transgenic tobacco cells expressin gsense RNA for L-galactono-1,4-lactonedehydroge nase. Planta,2005,220:854
106. Trainotti L, Pavanello A, Casadoro G. Different ethylene receptors show an increased expression during the ripening of strawberries:does such an increment imply a role for ethylene in the ripening of these non-climacteric fruits. J Exp Bot, 2005,56 (418):2037-2046
107. Trujillo LE, Sotolongo M, Menendez C, et al. SodERF3, a novel sugarcane ethylene responsive factor (ERF), enhances salt and drought tolerance when overexpressed in tobacco plants. Plant Cell Physiol,2008,49 (4):512-525
108. Wallerstein I, Goren R, Monselise SP. The effect of girdling on starch accumulation in sour orange seedlings. Can J Bot,1974,52:935-937
109. Wang A, Tan D, Takahashi A, et al. MdERFs, two ethylene-response factors involved in apple fruit ripening. J Exp Bot,2007,58 (13):3743-3748
110. Wang H, Huang Z, Chen Q, et al. Ectopic overexpression of tomato JERF3 in tobacco activates downstream gene expression and enhances salt tolerance. Plant Mol Biol,2004,55 (2):183-192
111. Watanabe K, Suzuki K, Kitamura S. Characterization of a GDP-D-mannose 3',5'-epimerase fromrice. Phytochemistry,2006,67:338-346
112. Wheeler G L, Jones MA, Smirnoff N. The biosynthetic pathway of vitamin C in higher plants. Nature,1998,393:365-369
113. Williams, LE, Retzlaff, WA, Yang, W, et al. Effect of girdling on leaf gas exchange, water status, and non-structural carbohydrates of field grown Vitis vinifera L. (cv. Flame Seedless). Am J Enol Vitic,2000,51:49-54
114. Wilson IBH. Glycosylation of proteins in plants and invertebrates. Curr Opin Struct Biol,2002,12:569-577
115. Wolucka BA, Goossens A, InzeD, et al. Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions. J Exp Bot,2005,56:2527-2538
116. Wolucka BA, Persian G, Van Doorsselaere J, et al. Partial purification and identification of GDP-mannose 3',5'-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proc Natl Acad Sci USA,2001,98: 14843-14848
117. Wolucka BA, Van Montagu M. GDP-Mannose 3',5'-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plant J Biol Chem,2003,278:47483-47490
118. Wolucka BA, Van Montagu M. The VTC2 cycle and the de novo biosynthesis pa-thways for vitamin C in plants:an opinion. Phytochemistry,2007,68:2602-2613
119. Yabuta Y, Maruta T, Nakamura A, et al. Conversion of L-galactono-1,4-lactone to L-ascorbate is regulated by the photosynthetic electron transport chain in Arabidopsis. Biosc Biotech Bioch,2008,72:2598-2607
120. Yabuta Y, Mieda T, Rapolu M, et al. Light regulation of ascorbate biosynthesis is dependent on the photosynthetic electron transport chain but independent of sugars in Arabidopsis. J Exp Bot,2007,58:2661-2671
121. Yabuta Y, oshimura K, Takeda T, et al. Molecular characterization of tobacco itochondrial-galactono-γ-lactone dehydro genase and its expressionin E.Coli. Plant Cell Physiol,2000,41:666-675
122. Zhang G, Chen M, Li L, et al. Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. J Exp Bot,2009,60:3781-3796
123. Zhang G., Chen M, Chen X, et al. Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.). J Exp Bot,2008,59 (15):4095-4107
2.安华明.刺梨高含量AsA的积累机制及其关键酶基因的克隆与表达.[博士学位论文].浙江：浙江大学图书馆,2004
3.杜盈,姚远,刘进元.两个水稻GDP-甘露糖-3',5'异构酶基因特征及表达模式的研究.生物化学与生物物理进展,2006,4：73-81
4.冯超,雷莹,刘永忠.柑橘L-半乳糖-1,4-内酯脱氢酶基因的克隆.华中农业大学学报,2009,28(6)：731-735
5.李明军.苹果和猕猴桃抗坏血酸形成与积累的生理和分子机理研究.[博士学位论文].陕西：西北农林科技大学大学图书馆,2009
6.刘永立,胡海涛,兰大伟.维生素C的生物合成及其基因调控研究进展.果树学报,2006,23(3)：431-436
7.祁春节,宋金田.2009年中国柑橘产销形势分析http://www.cncitrus.com/
8.王冬冬,朱延明,李勇等.电子克隆技术及其在植物基因工程中的应用.东北农业大学学报,2006,37(3)：403-408
9.张敏.苹果果实抗坏血酸合成酶基因的克隆及定量表达分析.[硕士学位论文].陕西：西北农林科技大学大学图书馆,2009
10.张伟玉,杨静慧,Wang-Pruski G等.不同转基因番茄GalUR的表达与AsA含量.西南大学学报(自然科学版),2008,30：43-47
11. Agarwal P, Agarwal P, Reddy M., Sopory S. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep,2006,25 (12):1263-1274
12. Agius F, Amaya I, Botella MA, Valpuesta V. Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression. J Exp Bot, 2005,56:37-46
13. Agius F, Gonzalez-Lamothe R, Caballero JL, et al. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid. Nature Biotech,2003,21: 177-181
14. Agius F, Lamothe RG, Caballero JL, et al. Engineering in creased vitaminC levels in Plants by over-expression of a D-galacturo- nic acid reduetase. Nature Biotechnology, 2003,21:177-181
15. Alhagdow M, Mounet F, Gilbert L, et al. Silencing of the mitochondrial ascorbate synthesizing enzyme L-Galactono-1,4-Lactone dehydrogenase affects plant and fruit development in Tomato. Plant Physiol,2007,145:1408-1422
16. Badejo AA, Fujikawa Y, Esak M. Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola(Malpighia glabra). J Plant physiol,2009,166:652-660
17. Badejo AA, Jeong ST, Goto-Yamamoto N. et al. Cloning and expression of GDP-D-mannose pyrophosphorylase gene and ascorbic acid content of acerola (Malpighia glabra L.)fruits at ripening stages. Plant Physiol Biochem,2007,45: 665-672
18. Badejo AA, Tanaka N, Esaka M, Analysis of GDP-D-mannose pyrophosphory-lase gene promoter from acerola(Malpighia glabra) and increase in ascorbate content of transgenic tobacco expressing acerola gene, Plant Cell Physiol,2008,49:126-132
19. Baig MM, Kelly S, Loewus F. Ascorbic Acid Biosynthesis in Higher Plants from 1-Gulono-1,4-lactoneand 1-Galactono-1,4-lactone. Plan Physiol,1970,46(2):277-280
20. Baldwin E A, Seymour GB, Taylor, JE, et al. Biochemistry of Fruit Ripening. In Citrus. Chapman & Hall,1993,107-137
21.Banno H, Ikeda Y, Niu QW, et al. Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration. Plant Cell,2001,13 (12):2609-2618.
22. Barber GA, The synthesis of guanosine 3,5-diphosphate-l-galactose by extracts of Chlorella pyrenoidosa. Arch Biochem,1975,167:718-722
23. Bartoli CG, Pastori GM, Foyer CH. Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiol,2000,123: 335-343
24. Bulley SM, Rassam M, Hoser D, et al. Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyl-transferase is a major control point of vitamin C biosynthesis. J Exp Bot,2009,60(3):765-778
25. Carreno J, Faraj S, Martinez A. Effects of girdling and covering mesh on ripening, colour and fruit characteristics of Italia grapes. J Hortic Sci Biotech,1998,73, 103-106
26. Conklin P L, Norris SR, Wheeler GL, et al. Last Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis, Proc Natl Acad Sci, 1999,4198-4203
27. Conklin PL, Gatzek S, Wheeler GL, et al. Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme. J Biol Chem, 2006,281:15662-15670
28. Conklin PL, Saracco SA, Norris SR, et al. Identification of ascorbic acid deficient Arabidopsis thaliana mutants. Genetics,2000,154:847-856
29. Cruz-Rus E, Botella MA, Valpuesta V, et al. Analysis of genes involved in 1-ascorbic acid biosynthesis during growth and ripening of grape berries. J Plant Physiol,2010, 167:739-748
30. Davey MW, Franck C, Keulemans J. Distribution, developmental and stress responses of antioxidant metabolism in malus. Plant Cell Environ,2004,27:1309-1320
31. Davey MW, Gilot C, Persiau G, et al. Ascorbate biosynthesis in Arabidopsis cell suspension cultures. Plant Physiol,1999,121:535-543
32. Davey MW, Sanmartin M, Kanellis A. Plant L-ascorbic acid:Chemistry, function, metabolism, bioavailability and effects of processing. Sci Food Agric,2000,80 825-860
33. Di Matteo A, Hancock RD, Ross HA, et al. Characterisation of Chlorella pyrenoi-dosa L-ascorbic acid accumulating mutants:Identification of an enhanced biosynthetic enzyme activity and cloning of the putative gene from Arabidopsis thaliana. Comp Biochem Physiol,2003,134:155
34. Dowdle J, Ishikawa T, Gatzek S, et al. Two genes in Arabidopsis thaliana encodin GDP-1-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J,2007,52:673-689
35. El-Sharkawy I, Sherif S, Mila I, et al. Molecular characterization of seven genes encoding ethylene-responsive transcriptional factors during plum fruit development and ripening. J Exp Bot,2009,60:907-922
36. Endres S, Tenhaken R. Myoinositol oxygenase controls the level of myoinositol in Arabidopsis, but does not increase ascorbic acid. Plant Physiol,2009,149:1042-1049
37. Franceschi VR, Tarlyn NM. L-Ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants. Plant Physiol,2002,130,649-656
38. Frei B, Lawson S. Vitamin C and cancer revisited. Proc Natl Acad Sci,2008,105, 11037-11038
39. Frossard E, Bucher M, Machler F, et al. Potential for increasing the content and bioavailability of Fe, Zn, and Ca in plants for human nutrition. Sci Food Agric,2000, 80:861-79
40. Gatzek S, Wheeler GL, Smirnoff N. Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis. Plant J,2002,30: 541-553
41. Gautier H, Massot C, Stevens R, et al. Regulation of tomato fruit ascorbate content is more highly dependent on fruit irradiance than leaf irradiance. Annals of Botany,2009, 103:495-504
42. Gilbert L, Alhagdow M, Nunes-Nesi A, et al. GDP-d-mannose-3,5-epimerase (GME) plays a key role at the intersection of ascorbate and non-cellulosic cell-wall biosynth esis in tomato. Plant J,2009,60:499-508
43. Giovannoni J. Molecular biology of fruit maturation and ripening. Annu. Rev.Plant Physiol. Plant Mol. Biol.2001,52,725-749
44. Gomez MLPA, Lajolo FM. Ascorbic acid metabolism in fruits:activity of enzymes involved in synthesis and degradation during ripening in mango and guava. J Sci Food Agr,2008,88:756-762
45. Goren R, Huberman M, Goldschmidt EE. Girdling:physiological and horticultural aspects. Horticultural Reviews,2003,30:1-36
46. Gournas C, Papageorgiou I, Diallinas G. the nucleobase-ascorbate transporter (NAT) family:genomics, evolution, structure-function relationships and physiological role. Mol BioSyst,2008,4:404-416
47. Grace SC, Logan BA. Acclimation of foliar antioxidant systems to growth irradiance in three broad-leaved evergreen species. Plant Physiol,1996,112:1631-1640
48. Gu YQ, Yang C, Thara VK, et al. Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the pto kinase. Plant Cell.2000,12 (5):771-786
49. Hancock RD, McRae D, Haupt S, et al. Synthesis of L-ascorbic acid in the phloem. BMC Plant Biology,2003,3:7
50. Hao D, Ohme-Takagi M, Sarai A. Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor (ERF Domain) in plant. J Biol Chem,1998,273 (41):26857-26861
51. Hebda PA, Barber GA, GDP-D-mannose:GDP-1-galactose epimerase from Chlor-ella pyrenoidosa. Meth Enzymol,1982,83:522-525
52. Imai T, Niwa M, Ban Y, et al. Importance of the L-galactonolactone pool for enhancing the ascorbate content revealed by L-galactonolactone dehydrogenase-overexpressing tobacco plants. Plant Cell, Tissue and Organ Culture,2009,96: 105-112
53. Ioannidi E, Kalamaki MS, Engineer C, et al. Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. J Exp Bot,2009,60(2):663-678
54. Isherwood FA, Chen YT, Mapson LW. Synthesis of 1-ascorbic acid in plants and animals. J Biochem,1954,56:1-21
55. Ishikawa T, Masumoto I, Iwasa N,et al. Functional characterization of D-galac-turonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis. Biosci Biotech Biochem.,2006,70:2720-2726
56. Ishikawa T, Nishikawa H, Gao Y, et al. The pathway via D-Galacturonate/L-Ga-lactonate is significant for ascorbate biosynthesis in Euglena gracilis:identifi cationand functional characterization of aldonolactonase. J Biol Chem,2008,283: 31133-31141
57. Jain AK, Nessler CL. Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol Breeding,2000,6:73-78
58. Jubany-Mari T, Munne-Bosch S, Alegre L. Redox regulation of water stress responses in field-grown plants. Role of hydrogen peroxide and ascorbate. Plant Physiol Bioch, 2010,48,351-358
59. Katz E, Lagunes PM, Riov J, et al. Molecular and physiological evidence suggests the existence of a system Ⅱ-like path way of ethylene production in non-climacteric citrus fruit. Planta,2004,219:243-252
60. Keller R, Springer F, Renz A, et al. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. Plant J,1999,19:31-141
61. Kizis D, Lumbreras V, Pages M. Role of AP2/EREBP transcription factors in gene regulation during abiotic stress. FEBS Lett,2001,498 (2-3):187-189
62. Koch KE, AvigneWT. Postphloem, nonvascular transfer in citrus:kinetics, etabolism, and sugar gradients. Plant Physiol,1990, (4):1405-1416
63. Kojo S. Vitamin C:basic metabolism and its function as an index of oxidative stress. Curr. Med Chem,2004,11:1041-1064
64. Kotchoni SO, Larrimore KE, Mukherjee M, Kempinski CF, Barth C. Alterations in the Endogenous Ascorbic Acid Content Affect Flowering Time in Arabidopsis. Plant Physiol,2009,149:803-815
65. Laing W A, Frearson N, Bulley S, et al. Kiwifruit 1-galactose dehydrogenase: molecular,biochemical and physiological aspects of the enzyme. Funct Plant Biol, 2004,31:1015-1025
66. Laing WA, Bulley S, Wright M, et al. A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis. Proc Natl Acad Sci USA,2004,10: 16976-6981
67. Laing WA, Wright MA, Cooney J, et al. The missing step of the L-galactose path-way of ascorbate biosynthesis in plants, an L-galactose guanyltransferase, increa-ses leaf ascorbate content. Proc Natl Acad Sci USA,2007,104:9534-9539
68. Lee SK, Kader AA. Preharvest and postharvest factors influencing vitamin C content of horticultural crops. Postharvest Biol Tec,2000,20,207-220
69. Li MJ, Ma F, Shang P, et al. Influence of light on ascorbate formation and metabolism in apple fruits. Planta,2009,230:39-51
70. Li MJ, Ma FW, Guo CM, et al. Ascorbic acid formation and expression profiling of the genes involved in its synthesis and recycling in apple leaves with different age. Plant Physiol Bioch,2010,48:216-224
71. Li MJ, Ma FW, Zhang M, et al. Distribution and metabolism of ascorbic acid in apple fruits(Malus domestica Borkh cv. Gala). Plant Sci,2008,174:606-612
72. Linster CL, Adler LT, Webb K, et al. A second GDP-L-galactose phosphorylase in Arabidopsis enroute to vitamin C:covalent intermediate and substrate require-ments for the conserved reaction. J Biol Chem,2008,283:18483-18492
73. Linster CL, Clarke SG. L-Ascorbate biosynthesis in higher plants:the role of VTC 2. Trends Plant Sci,2008,13:567-573
74. Liu YZ, Liu Q, Tao NG, et al. Efficient isolation of RNA from fruit peel and pulp of ripening navel orange(Citrus sinensis). Journal of Huazhong Agricultural University, 2006,25:300-304
75. Loewus FA, Kelly S. The metabolism of D-galacturonic acid and its methylester in the detached ripening strawberry. Arch Biochem.Biophys,1961,95:483-493
76. Lorence A, Chevone BI, Mendes P, et al. Myo-Inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol,2004,134:1200-1205
77. Loscos J, Matamoros MA, Becana M. Ascorbate and homoglutathione metabolism in common bean nodules under stress conditions and during natural senescence. Plant Physiol,2008,146:1282-1292
78. Major LL, Wolucka BA, Naismith JH. Structure and function of GDP-mannose-3',5'-epimerase:an enzyme which performs three chemical reactions at the same active site. J Am Chem Soc,2005,127:18309-18320
79. Mieda T, Yabuta Y, Rapolu M, et al. Feedback inhibition of spinach 1-galactose dehydrogenase by 1-ascorbate. Plant Cell Physiol,2004,45:1271-1279
80. Miiller-Moule Patricia. An expression analysis of the ascorbate biosynthesis enzyme VTC2. Plant Mol Bol,2008,68:31-41
81. Nagy, S. Vitamin C contents of citrus fruit and their products:a review. Agric. Food Chem,1980,28:8-18
82. Naidu KA. Vitamin C in human health and disease is still a mystery. An overview. Nutrition Journal,2003,2:7
83. Nakano T, Suzuki K, Fujimura T, et al. Genome-wide analysis of the ERF gene family in arabidopsis and rice. Plant Physiol,2006,140 (2):411-432
84. Oba K, Ishikawa S, Nishikawa M, et al. Purification and properties of L-galactono-gamma-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots. J Exp Bot,1995,117:120-124
85. Ohme-Takagi M, Shinshi H. Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell,19957 (2):173-182
86. Olmos E, Kiddle G, Pellny TK, et al. Modulation of plant morphology, root architecture, and cell structure by low vitamin C in Arabidopsis thaliana. J Exp Bot, 2006,57:1645-1655
87. Oxley D, Bacic A.Structure of the glycosyl-phosphatidylinositol anchor of an arabinogalacta protein from Pyrus communis suspension-cultured cells.Proc Natl Acad Sci USA,1999,96:14246-14251
88. Pateraki I, Sanmartin M, Kalamaki MS, et al. Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4-lactone dehydrogenase. J Exp Bot,2004,55:1623-1633
89. Pineau B, Layoune O, Danon A, et al. L-Galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I. J Biol Chem,2008,283: 32500-32505
90. Pirrello J, Jaimes-Miranda F, Sanchez-Ballesta MT, et al. S1-ERF2, a tomato ethylene response factor involved in ethylene response and seed germination. Plant Cell Physiol,2006,47 (9):1195-1205
91. Radzio JA, Lorence A, Chevone BI, et al. L-Gulono-1,4-lactone oxidase expression rescues vitamin C-deficient Arabidopsis(vtc1) mutants. Plant Mol Biol,2003,53: 837-844
92. Reiter WD, Vanzin GF. Molecular genetics of nucleotide sugar interconversion pathways in plants. Plant Mol Biol,2001,47:95-113
93. Riechmann JL, Meyerowitz EM. The AP2/EREBP family of plant transcription factors. Biol. Chem,1998,379 (6):633-646
94. Rivas F, Gravina A, Agusti M. Girdling effects on fruit set and quantum yield efficiency of PSⅡ in two Citrus cultivars. Tree Physiol,2007,27527-535
95. Running JA, Burlingame RP, Berry A. The pathway of L-ascorbic acid biosyn thesis in the colourless microalga prototheca moriformis. J Exp Bot,2003,54:1841-1849
96. Sarkar N, Srivastava PK, Dubey VK. Understanding the Language of Vitamin C. Current Nutrition & Food Science,2009,5 (3):53-55
97. Shi JX, Porat R, Goldschmidt EE, et al. Ethylene and volatile accumulation in citrus fruit. In:Ramina A., Chang C, Giovannoni J, et al. (Eds.), Advances in Plant Ethylene Research. Springer, Netherlands,2007, pp181-187
98. Siendones E,Gonzalez-Reyes J A, Santos-Ocana C, et al. Biosynthesis of ascorbic acid in kidney bean. L-Galactono-y-lactone dehy drogenase is an intrinsic protein located at the mitochondrial inner membrane. Plant Physiol,1999,120:907-912
99. Tabata K,Oba K, Suzuki K, et al. Generation and properties of ascorbic acid deficient trans genic tobacco cells expressing antisense RNA for L-galactono-1,4-lactonedehydrogenase. Plant J,2001,27:139-148
100. Tabata K, Takaoka T, Esaka M.Gene expression of ascorbic acid-related enzymes in tobacco. Phytochemistry,2002,61:631-635
101. Takeda S, Matsuokka M. Genetic approaches to crop improvement:responding to environmental and population changes. Nat Rev Genet,2008,9:444-457
102. Tamaoki M, Mukai F, Asai N, et al. Light-controlled expression of a gene encoding L-galactono-gamma-lactone dehydrogenase which affects ascorbate pool size in Arabidopsis thaliana. Plant Sci,2003,164:1111-1117
103. Taylor CA, Hampl JS, Johnston CS. Low intakes of vegetables and fruits, especially citrus fruits, lead to inadequate vitamin C intakes among adults. European Journal of Clinical Nutrition,2000,54:573-578
104. Tedone L, Hancock RD, Alberino S, et al. Long-distance transport of L-ascorbic acid in potato. BMC Plant Biol,2004,4:16
105. Tokunaga T, Miyahara K, Tabata K, et al. Generation and properties of ascorbic acid-overprod ucing transgenic tobacco cells expressin gsense RNA for L-galactono-1,4-lactonedehydroge nase. Planta,2005,220:854
106. Trainotti L, Pavanello A, Casadoro G. Different ethylene receptors show an increased expression during the ripening of strawberries:does such an increment imply a role for ethylene in the ripening of these non-climacteric fruits. J Exp Bot, 2005,56 (418):2037-2046
107. Trujillo LE, Sotolongo M, Menendez C, et al. SodERF3, a novel sugarcane ethylene responsive factor (ERF), enhances salt and drought tolerance when overexpressed in tobacco plants. Plant Cell Physiol,2008,49 (4):512-525
108. Wallerstein I, Goren R, Monselise SP. The effect of girdling on starch accumulation in sour orange seedlings. Can J Bot,1974,52:935-937
109. Wang A, Tan D, Takahashi A, et al. MdERFs, two ethylene-response factors involved in apple fruit ripening. J Exp Bot,2007,58 (13):3743-3748
110. Wang H, Huang Z, Chen Q, et al. Ectopic overexpression of tomato JERF3 in tobacco activates downstream gene expression and enhances salt tolerance. Plant Mol Biol,2004,55 (2):183-192
111. Watanabe K, Suzuki K, Kitamura S. Characterization of a GDP-D-mannose 3',5'-epimerase fromrice. Phytochemistry,2006,67:338-346
112. Wheeler G L, Jones MA, Smirnoff N. The biosynthetic pathway of vitamin C in higher plants. Nature,1998,393:365-369
113. Williams, LE, Retzlaff, WA, Yang, W, et al. Effect of girdling on leaf gas exchange, water status, and non-structural carbohydrates of field grown Vitis vinifera L. (cv. Flame Seedless). Am J Enol Vitic,2000,51:49-54
114. Wilson IBH. Glycosylation of proteins in plants and invertebrates. Curr Opin Struct Biol,2002,12:569-577
115. Wolucka BA, Goossens A, InzeD, et al. Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions. J Exp Bot,2005,56:2527-2538
116. Wolucka BA, Persian G, Van Doorsselaere J, et al. Partial purification and identification of GDP-mannose 3',5'-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proc Natl Acad Sci USA,2001,98: 14843-14848
117. Wolucka BA, Van Montagu M. GDP-Mannose 3',5'-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plant J Biol Chem,2003,278:47483-47490
118. Wolucka BA, Van Montagu M. The VTC2 cycle and the de novo biosynthesis pa-thways for vitamin C in plants:an opinion. Phytochemistry,2007,68:2602-2613
119. Yabuta Y, Maruta T, Nakamura A, et al. Conversion of L-galactono-1,4-lactone to L-ascorbate is regulated by the photosynthetic electron transport chain in Arabidopsis. Biosc Biotech Bioch,2008,72:2598-2607
120. Yabuta Y, Mieda T, Rapolu M, et al. Light regulation of ascorbate biosynthesis is dependent on the photosynthetic electron transport chain but independent of sugars in Arabidopsis. J Exp Bot,2007,58:2661-2671
121. Yabuta Y, oshimura K, Takeda T, et al. Molecular characterization of tobacco itochondrial-galactono-γ-lactone dehydro genase and its expressionin E.Coli. Plant Cell Physiol,2000,41:666-675
122. Zhang G, Chen M, Li L, et al. Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. J Exp Bot,2009,60:3781-3796
123. Zhang G., Chen M, Chen X, et al. Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.). J Exp Bot,2008,59 (15):4095-4107