超表达番茄GDP-D-甘露糖焦磷酸化酶基因马铃薯对温度胁迫的响应

doi:10.3864/j.issn.0578-1752.2011.23.024

摘要/Abstract

摘要： 【目的】研究番茄GDP-甘露糖焦磷酸化酶基因（GMPase）在马铃薯中的超表达对其抵御温度胁迫能力的影响。【方法】利用已经获得的超表达番茄GMPase的两个转基因马铃薯株系，用野生型马铃薯作对照，比较其在常温（25/20℃）、温度胁迫（10/5℃低温处理3 d，35/30℃高温处理3 d）及25/20℃恢复3 d后GMPase的相对表达量、抗坏血酸（AsA）含量及其相关生理指标的变化。【结果】在常温、温度胁迫与恢复阶段，与野生型植株相比，转基因马铃薯植株具有较高的GMPase相对表达量、GMPase活性、AsA和脱氢抗坏血酸（DHA）含量；高温和低温胁迫后，转基因马铃薯的脱氢抗坏血酸还原酶（DHAR）、单脱氢抗坏血酸还原酶（MDAHR）和抗坏血酸过氧化物酶（APX）活性均明显提高，丙二醛（MDA）和H2O2含量及细胞质膜透性均明显降低。【结论】番茄GMPase在马铃薯中的超表达可能有提高马铃薯植株抵御温度胁迫的能力。

关键词: 马铃薯, 抗坏血酸, GDP-甘露糖焦磷酸化酶, 转基因, 温度胁迫

Abstract: 【Objective】 The aim of the study was to clarify the function of the GDP-D-mannose pyrophosphorylase (GMPase) in the L-galactose pathway of ascorbic acid (AsA) synthesis in plants, and to make sure the relation of GMPase overexpression with ascorbic acid content and temperature-stress tolerance ability. 【Method】 Two transgenic potato strains of overexpressing tomato GMPase were obtained by agrobacterium-mediated transformation. The transgenic plants and wild-type plants were treated in temperature-stress conditions (10/5℃ as low-temperature stress, 35/30℃ as high-temperature stress) for 3 days then in the recover-condition (25/20℃) for 3 days. Real time RT-PCR was used to analyze the expression of GMPase gene. And ascorbic acid content and relative index to temperature stress were also determined. 【Result】Compared with wild-type plants, with the overexpression of GMPase gene in transgenic plants, the GMPase activity and contents of AsA and dehydroascorbate (DHA) in normal and temperature-stress conditions were increased as well as the activities of dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and ascorbate peroxidase (APX). Meanwhile, the malondialdehyde (MDA) and H2O2 contents and permeability were decreased in the transgenic plants. 【Conclusion】 The overexpression of GMPase may increase potato tolerance to temperature stress by increasing AsA level and lowering reactive oxygen species (ROS).

Key words: potato, ascorbic acid, GDP-D-mannose pyrophosphorylase, transgenic, temperature stress

李超汉, 李青竹, 史庆华, 白龙强, 郭晓青, 李霞, 于贤昌. 超表达番茄GDP-D-甘露糖焦磷酸化酶基因马铃薯对温度胁迫的响应[J]. 中国农业科学, 2011, 44(23): 4952-4961.

LI Chao-Han, LI Qing-Zhu, SHI Qing-Hua, BAI Long-Qiang, GUO Xiao-Qing, LI Xia, YU Xian-Chang. Effect of Tomato GMPase Overexpression on Tolerance of Potato Plants to Temperature Stress[J]. Scientia Agricultura Sinica, 2011, 44(23): 4952-4961.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2011.23.024

https://www.chinaagrisci.com/CN/Y2011/V44/I23/4952

参考文献

[1]Scandalios J G. Oxygen stress and superoxide dismutases. Plant Physiology, 1993, 101: 712-720.

[2]Asada K. Ascorbate peroidase: a hydrogen perioxide-scavenging enzyme in plants. Physiologia Plantarum, 1992, 85: 235-241.

[3]Elstner E F, Osswald W. Mechanisms of oxygen activation during plant stress. Proceedings of the Royal Society of Edinburgh, 1994, 102: 131-154.

[4]陈坤明, 宫海军, 王锁民. 植物抗坏血酸的生物合成、转运及其生物学功能.西北植物学报, 2004, 24(2): 329-336.

Chen K M, Gong H J, Wang S M. Biosynthesis, transport and function of ascorbates in plants. Northwest Plant Journal, 2004, 24(2): 329-336. (in Chinese)

[5]Manisha G, Ann C, Jaco V, Herman C. Copper affects the enzymes of ascorbate-glutathione cycle and its related metabolites in the roots of Phaseolus vulgaris. Physiologia Plantarum, 1999, 106(3): 262-267.

[6]Drazkiewicz M, Sko′rzyn′Ska-Polite E, Krupa Z. Response of the ascorbate-glutathione cycle to excess copper in Arabidopsis thaliana. Plant Science, 2003, 164: 195-202.

[7]Pukacka S, Ratajczak E. Antioxidative response of ascorbate- glutathione pathway enzymes and metabolites to desiccation of recalcitrant Acer saccharinum seeds. Journal of Plant Physiology, 2006, 163: 1259-1266.

[8]Zou L P, Li H X, Ouyang B, Zhang J H, Ye Z B. Cloning, expression, and mapping of GDP-D-mannose pyrophosphorylase cDNA from tomato (Lycopersicon esculentum L.). Acta Genetica Sinica, 2006, 33 (8): 757-764.

[9]Conklin P L, Norris S R, Wheeler G L , Williams E H, Smirnoff N, Last R L. Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96: 4198-4203.

[10]Huang C, He W, Guo J, Su P, Zhang L. Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant. Journal of Experimental Botany, 2005, 56: 3041-3049.

[11]Badejo A A, Jeong S T, Goto-Yamamoto N, Esaka M. Cloning and expression of GDP-D-mannose pyrophosphorylase gene and ascorbic acid content of acerola (Malpighia glabra L.)fruits at ripening stages. Plant Physiology and Biochemistry, 2007, 45:665-672.

[12]Keller R, Springer F, Renz R, Kossmann J. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. The Plant Journal, 1999, 19:131-141.

[13]Lin L L, Shi Q H, Wang H S, Qin A G, Yu X C. Over-expression of tomato GDP-Mannose pyrophosphorylase (GMPase) in potato increases ascorbate content and delays plant senescence. Agricultural Sciences in China, 2011, 10(4): 101-105.

[14]Nicot N, Hausman J F, Hoffmann L, Evers D. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. Journal of Experimental Botany, 2005, 56: 2907-2914.

[15]Bubner B, Baldwin I T. Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell Reports, 2004, 23: 263-27.

[16]Takahama U. Regulation of peroxidase-dependent oxidation of phenolics by ascorbic acid: Different effects of ascorbic acid on the oxidation of coniferyl alcohol by the apoplastic soluble and cell wall-bound peroxidases from epicotyls of Vignaangularis. Plant and Cell Physiology, 1994, 34:809-817.

[17]Chen Z, Young T E, Ling J, Chang S C, Gallie D R. Increasing vitamin C content of plants through enhanced ascorbate recycling. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100: 3525-3530.

[18]Jiménez A, Hernández J A, Rio L A, Sevilla F. Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiology, 1997, 114: 275-284.

[19]Pignocchi C, Kiddle G, Hernández I, Foster S J, Asensi A, Tahar Taybi T, Barnes J, Foyer C H. Ascorbate oxidase-dependent changes in the redox state of the apoplast modulate gene transcript accumulation leading to modified hormone signaling and orchestration of defense processes in tobacco. Plant Physiology, 2006, 141: 423-435.

[20]赵世杰, 许长成, 邹琦. 植物组织中丙二醛测定方法的改进. 植物生理学通讯, 1994, 30(3): 207-210.

Zhao S J, Xu C C, Zou Q. Improvements of method for measurement of malondialdehyde in plant tissues. Plant Physiology Communications, 1994, 30(3): 207-210. (in Chinese)

[21]Petrie S E, Jaekson T L. Effects of nitrogen fertilization on manganese concentration and yield of barley and oats. Soil Science Society of America Journal, 1984, 48: 319-322.

[22]Wheeler G L, Jones M A, Smirnoff N. The biosynthetic pathway of vitamin C in higher plants. Nature, 1998, 393: 365-369.

[23]Smirnoff N, Wheeler G L. Ascorbic acid in plants: biosynthesis and function. Critical Reviews in Biochemistry and Plant Molecular Biology, 2000, 35:291-314.

[24]Noctor G, Foyer C H. Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology, 1998, 49:249-279.

[25]Badejo A A, Tanaka N, Esaka M. Analysis of GDP-D-mannose pyrophosphorylase gene promoter from acerola (Malpighia glabra L.) and increase in ascorbate content of transgenic tobacco expressing acerola gene. Plant and Cell Physiology, 2008, 49:126-132.

[26]秦爱国, 高俊杰, 于贤昌. 温度胁迫对马铃薯叶片抗坏血酸代谢系统的影响. 应用生态学报, 2009, 20(12): 2964-2970.

Qin A G, Gao J J, Yu X C. Effects of high and low-temperature stresses on ascorbic acid metabolism system in potato leaves. Chinese Journal of Applied Ecology, 2009, 20(12): 2964-2970. (in Chinese)

[27]Liebler D C, Kling D S, Reed D J. Antioxidants protection of phospholipid bilayer by α-tocopherol. Control of α-tocopheol status and lipid peroxidation by ascorbic acid and glutathione. Journal of Biological Chemistry, 1986, (26):12114-12119.

[28]Tokunaga T, Miyahara K, Tabata K, Esaka M. Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for L-galactono-1, 4-lactone dehydrogenase. Plantarum, 2005, 220: 854-863.

[29]Smirnoff N, Colville L. Antioxidant status, peroxidase activity, and PR protein transcript levels in ascorbate-deficient Arabidopsis thaliana vtc mutants. Journal of Experimental Botany, 2008, 59(14): 3857-3868.

[30]Arial D, Jounechani R S. Impact of exogenous ascorbic acid on antioxidant activity and some physiological traits of common bean subjected to salinity stress. Notulae Botanicae Horti Agrobotanici Cluj- Napoca, 2009, 37(2):165-172.

[31]谢居清, 李国学, 王效科, 郑启伟, 冯兆忠. 外源抗坏血酸对臭氧胁迫下水稻光合及生长参数的影响. 中国生态农业学报, 2009, 17(6):1176-1181.

Xie J Q, Li G X, Wang X K, Zheng Q W, Feng Z Z. Effect of exogenous ascorbic acid on photosynthesis and growth of rice under O3 stress. Chinese Journal of Eco-Agriculture, 2009, 17(6):1176-1181. (in Chinese)

[32]Shigeoka S, Ishikawa T, Tamoi M, Yabuta Y, Yoshimura K. Regulation and function of ascorbate peroxidase isoenzymes. Journal of Experimental Botany, 2002, 53: 1305-1319.

[33]Yoshimura K, Yabuta Y, Tamoi M. Alternatively spliced mRNA variants of chloroplast ascorbate peroxidase isoenzymes in spinach leaves. Biochemistry, 1999, 338: 41-48.

[34]Eltayeb A E, Kawano N, Badawi G H. Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol. Physiologia Plantarum, 2006, 127: 57-65.

[35]Yoon H S, Lee H, Lee I A, Kim K Y, Jo J. Molecular cloning of the monodehydroascorbate reductase gene from Brassica campestrsi and analysis of its mRNA level in response to oxidative stress. Biochimica and Biophysica Acta, 2004, 1658: 181-186.

[36]马春花, 马锋旺, 李明军, 束怀瑞. 外源抗坏血酸对离体苹果叶片衰老的影响. 园艺学报, 2006, 33(6):1179-1184.

Ma C H, Ma F W, Li M J, Shu H R. Effects of exogenous ascorbic acid on senescence of detached apple leaves. Acta Horticulturae Sinica, 2006, 33(6):1179-1184. (in Chinese)

[37]Ma Y H, Ma F W, Zhang J K, Li M J, Wang Y H, Dong L. Effects of high temperature on activities and gene expression of enzymes involved in ascorbate-glutathione cycle in apple leaves. Plant Science, 2008, 175:761-766.