转GsGST13/SCMRP基因双价苜蓿的耐盐性分析

doi:10.11686/cyxb20140131

摘要/Abstract

摘要： 本研究是将从野大豆盐碱胁迫基因表达谱中筛选得到的GsGST13基因和采用生物信息学方法改造的高甲硫氨酸蛋白基因SCMRP构建成双价植物表达载体，通过农杆菌介导法转化农菁1号苜蓿，获得超量表达的转基因苜蓿，并对其中2个转基因苜蓿株系进行耐盐性分析及氨基酸组分分析。结果显示，转基因苜蓿具有较强的耐盐性，表现为随着盐浓度的升高，野生型苜蓿盐害不断加重，生长发育受抑制，叶片逐渐变黄、卷曲、萎蔫，而转基因苜蓿只受到轻微影响，仍能正常生长。转基因株系的丙二醛含量和过氧化氢含量显著低于非转基因株系(P<0.05)，而株高，鲜重，GST活性和SOD活性显著高于非转基因对照(P<0.05，P<0.01);同时株系G₁₆和G₅₀的含硫氨基酸含量分别比野生型植株提高了0.57%和0.52%。说明超量表达GsGST13/SCMRP 基因增强了苜蓿的耐盐性，并提高了含硫氨基酸含量。

Abstract: The GsGST13 gene was selected based on the Glycine soja gene transcriptome of salt-alkaline stress. The high-sulfur-containing amino acids gene SCMRP was designed using the bioinformatics codes. The plant binary expression vector (pBEOGST13/SCMRP) was designed and transferred into alfalfa (Medicago sativa cv. Nongjing No.1) using the Agrobacterium-mediated method. Many over-expression transgenic alfalfa lines were obtained, and two of them were selected to analyse the salt resistance and sulfur amino acids contents. Transgenic alfalfa lines showed enhanced tolerance to NaCl stress compared with wild-type plants. Under NaCl stress, the growth of wild-type plants was inhibited, the blade gradually became yellow and crimp panama, but there was only a slight effect on the transgenic lines, which had normal growth under stress. The malondialdehyde and hydrogen peroxide contents of transgenic lines were significantly lower than those of non-transgenic lines (P<0.05). Moreover, plant height, fresh weight, GST activity and SOD activity of transgenic alfalfa were significantly (P<0.05, P<0.01 respectively) higher than those of non-transgenic lines under salt stress conditions. Further, the sulfur amino acids contents of the G₁₆ and G₅₀ transgenic alfalfa lines were higher than those of wild type plants, by 0.57% and 0.52%, respectively. Over-expression of the GST13 and SCMRP in alfalfa improved the tolerance to salinity stress and increased the content of sulfur amino acids.

中图分类号:

吴婧，才华，柏锡，纪巍，魏正巍，唐立郦，赵阳，朱延明. 转GsGST13/SCMRP基因双价苜蓿的耐盐性分析[J]. 草业学报, 2014, 23(1): 257-265.

WU Jing, CAI Hua, BAI Xi, JI Wei, WEI Zheng-wei, TANG Li-li,
ZHAO Yang, ZHU Yan-ming. An analysis of salt tolerance of transgenic alfalfa with the GsGST13/SCMRP gene[J]. Acta Prataculturae Sinica, 2014, 23(1): 257-265.

参考文献

Reference:
[1]Wang J, Liu X N. Evaluation on salt tolerance to salt-tolerant variant materials of three alfalfa varieties[J]. Pratacultural Science, 2011, 28(1): 79-84. 
[2]Guo H Q, Ren W B, Xu Z, et al. Advances in genetic transformation in alfalfa[J]. Acta Agriculturae Nucleatae Sinica, 2010, 24(1): 55-61. 
[3]Liu Y, Zhang C M, Xie X R, et al. Effect of drought stress on polyamine metabolism in the leaves and roots of alfalfa[J]. Acta Prataculturae Sinica, 2012, 21(6): 102-107.
[4]Bao A K, Du B Q, Wang S M. Advances on physiological mechanisms of alfalfa resistant to salt and drought[J]. Pratacultural Science, 2011, 28(9): 1700-1705. 
[5]Zhang L Q, Zhang J F, Ha S A G L. Research progress on alfalfa salt tolerance[J]. Acta Prataculturae Sinica, 2012, 21(6): 296-305. 
[6]Wang Z Y, Cai H, Bai X, et al. Isolation of GsGST19 from Glycine soja and Analysis of Saline-Alkaline Tolerance for Transgenic Medicago sativa[J]. Acta Agronomica Sinica, 2012, 38(6): 971-979. 
[7]Dean D F, Goodwin P H, Hsiang T. Induction of glutathione Stransferase genes of Nicotiana benthamianafollowing infection by Colletotrichum destructivum and C. orbiculare and involvement of one in resistance[J]. Journal of Experimental Botany, 2005, 56: 1525-1533. 
[8]Ji W, Zhu Y M, Li Y, et al. Over expression of a glutathione Stransferase gene, GsGST, from wild soybean (Glycine soja) enhances drought and salt tolerance in transgenic tobacco[J]. Biotechnology Letters, 2010, 32: 1173-1179. 
[9]Lo Piero A R, Puglisi I, Rapisarda P, et al. Anthocyanin accumulation and related gene expression in red orange fruit induced by low temperature storage[J]. Journal of Agricultural and Food Chemistry, 2005, 53: 9083-9088.
[10]Shigeoka S, Ishikawa T, Tamoi M, et al. Regulation and function of ascorbate peroxidase isoenzymes[J]. Journal of Experimental Botany, 2002, 53: 1305-1319. 
[11]Diao G, Wang Y, Wang C, et al. Cloning and functional characterization of a novel glutathione Stransferase gene from Limonium bicolor[J]. Plant Molecular Biology Reporter, 2011, 29: 77-87. 
[12]Ben P D, David P D, Douglas J B, et al. Induction of glutathiones transferases in Arabidopsis by herbicide safeners[J]. Plant Physiology, 2002, 130: 1497-1505. 
[13]Wang G Y, Liu D Q, Ge F, et al. The role of GSTs in abiotic stress resistance in plants[J]. Plant Physiology Communications, 2010, 46(9): 890-894.
[14]Ge Y, Li Y, Lv D K, et al. Alkaline stress response in Glycine sojaleaf identifies specific transcription factors and ABA mediated signaling factors[J]. Functional Integrative Genomics, 2011, 11: 369-379. 
[15]Wang Z Y, Song F B, Cai H, et al. Over expressing GsGST 14 from Glycine soja enhances alkaline tolerance of transgenic Medicago sativa[J]. Biologia Plantarum, 2012, 56(3): 516-520. 
[16]Lü D Y, Fan Y L, Yu M M, et al. Regeneration of HNP transgenic alfalfa plants by agrobacterium mediated gene transfer[J]. Acta Genetica Sinica, 2000, 27(4): 331-337.
[17] Zhai H, Bai X, Zhu Y M, et al. Protokaryotic expression of SCMRP gene and preparation of polyclonal antibody[J]. Journal of Northeast Agricultural University, 2009, 40(7): 60-65.
[18]Zhang Y X, Tang F L, Zhang H Q, et al. Breeding of new variety nongjing N0.1 of Medicago sativa L.by magnetic field freespace[J]. Acta Agriculturae Nucleatae Sinica, 2006, 21(1): 34-37. 
[19]Sheng H, Zhu Y M, Li J, et al. Genetic transformation of DREB2A gene into alfalfa[J]. Pratacultural Science, 2007, 24(3): 40-45.
[20]Wei Z W, Zhu Y M, Hua Y, et al. Transgenic alfalfa with GsPPCK1 and its alkaline tolerance analysis[J]. Acta Agronomica Sinica, 2013, 39(1): 68-75. 
[21]Zhang L Q, Ao D H, Shi W G, et al. Analysis of physiological and biochemical characteristics of T1 salt-tolerant transgenic alfalfa transformed with the Rhizophora apiculatatotal total DNA via the pollen-tube pathway[J]. Acta Prataculturae Sinica, 2012, 21(2): 149-155.
[22]Rao M V, Paliyath G, Ormrod D P, et al. Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2 metabolizing enzymes[J]. Plant Physiology, 1997, 115: 137-149. 
[23]Mauch F, Dudlar R. Differential induction of distinct glutathione Stransferases of wheat by xenobiotics and by pathogen attack[J]. Plant Physiology, 1993, 102: 1193-1201. 
[24]Health R L, Packer L. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation[J]. Archives of Biochemistry and Biophysics, 1968, 125: 189-198. 
[25]Talarczyk A, Krzymowska M, Borucki W, et al. Effect of yeast CAT1 gene expression on response of tobacco plants to tobacco mosaic virus infection[J]. Plant Physiology, 2002, 129: 1032-1044. 
[26]Liu Q G, Tian L P, Jiang H, et al. The content of amino acid in the leaf protein of alfalfa and its nutritive analysis[J]. Journal of Henan University of Technology, 2005, 26(2): 36-39. 
[27]Zhang Y F, Yin B. Influences of salt and alkali mixed stresses on antioxidative activity and MDA content of Medicago sativa at seedling stage[J]. Acta Prataculturae Sinica, 2009, 18(1): 46-50. 
[28]Li Y, Sun Y, Yang Q C, et al. Construction and transformation of an over-expression plasmid of the MsZIP gene from Medicago sativa[J]. Acta Prataculturae Sinica, 2012, 21(6): 182-189.
[29]Li W F, Wang D L, Jin T C, et al. The vacuolar Na+/H+ antiporter gene SsNHX1 from the halophvte Salsola sodaconfers salt tolerance in transgenic alfafla（Medicago sativa L.)[J]. Plant Molecular Biology Reporter, 2011, 29: 278-290. 
[30]Bao A K, Wang S M, Wu G Q, et al. Overexpression of the ArabidopsisH+ PPase enhanced resistance to salt and drought stress in transgenic aflafla（Medicago sativa L.)[J]. Plant Science, 2009, 176: 232-240. 
[31]Yang X, Su W, Liu J P, et al. Biochemical and physiological characterization of a tau class glutathione transferase from rice（Oryza sativa)[J]. Plant Physiology and Biochemistry, 2009, 47: 1061-1068. 
[32]Lo Piero A R, Puglisi I, Petrone G. Gene isolation, analysis of expression, and in vitro synthesis of glutathione Stransferase from orange fruit [Citrus sinensisL.(Osbeck)][J]. Journal of Agricultural and Food Chemistry, 2006, 54: 9227-9233. 
[33]Quan X Q, Gao W. Enzymatic mechanism of scavenging reactive oxygen species of euhalophytes[J]. Journal of Anhui Agricultural Sciences, 2003, 31(2): 320-322.
[34]Saalbach G, Jung E, Saalbach I, et al. Construction of storage protein genes with increased number of methionine codons and their use in transformation experiments[J]. Biochemie und Physiologie der Pflanzen, 1988, 183(2-3): 211-218. 
[35]Li L, Liu S M, Hu Y L, et al. Gliadin genes into maize 10ku improve potato tubers sulfur amino acid content[J]. Chinese Science Bulletin, 2000, 45(12): 1313-1317.

参考文献：
[1]王珺, 柳小妮. 3个紫花苜蓿品种耐盐突变材料的耐盐性评价[J]. 草业科学, 2011, 28(1): 79-84. 
[2]郭慧琴, 任卫波, 徐柱, 等. 紫花苜蓿转基因研究进展[J]. 核农学报, 2010, 24(1): 55-61. 
[3]刘义, 张春梅, 谢晓蓉, 等. 干旱胁迫对紫花苜蓿叶片和根系多胺代谢的影响[J]. 草业学报, 2012, 21(6): 102-107. 
[4]包爱科, 杜宝强, 王锁民. 紫花苜蓿耐盐、抗旱生理机制研究进展[J]. 草业科学, 2011, 28(9): 1700-1705. 
[5]张立全, 张凤英, 哈斯阿古拉. 紫花苜蓿耐盐性研究进展[J]. 草业学报, 2012, 21(6): 296-305. 
[6]王臻昱, 才华, 柏锡, 等. 野生大豆GsGST19 基因的克隆及其转基因苜蓿的耐盐碱性分析[J]. 作物学报, 2012, 38(6): 971-979. 
[7]Dean D F, Goodwin P H, Hsiang T. Induction of glutathione S-transferase genes of Nicotiana benthamianafollowing infection by Colletotrichum destructivum and C. orbiculare and involvement of one in resistance[J]. Journal of Experimental Botany, 2005, 56: 1525-1533. 
[8]Ji W, Zhu Y M, Li Y,et al. Over expression of a glutathione S-transferase gene, GsGST, from wild soybean（Glycine soja) enhances drought and salt tolerance in transgenic tobacco[J]. Biotechnology Letters, 2010, 32: 1173-1179. 
[9]Lo Piero A R, Puglisi I, Rapisarda P,et al. Anthocyanin accumulation and related gene expression in red orange fruit induced by low temperature storage[J]. Journal of Agricultural and Food Chemistry, 2005, 53: 9083-9088.
[10]Shigeoka S, Ishikawa T, Tamoi M,et al. Regulation and function of ascorbate peroxidase isoenzymes[J]. Journal of Experimental Botany, 2002, 53: 1305-1319. 
[11]Diao G, Wang Y, Wang C,et al. Cloning and functional characterization of a novel glutathione S-transferase gene from Limonium bicolor[J]. Plant Molecular Biology Reporter, 2011, 29: 77-87. 
[12]Ben P D, David P D, Douglas J B,et al. Induction of glutathiones-transferases in Arabidopsis by herbicide safeners[J]. Plant Physiology, 2002, 130: 1497-1505. 
[13]王光勇, 刘迪秋, 葛锋, 等. GST在植物非生物逆境胁迫中的作用[J]. 植物生理学通讯, 2010, 46(9): 890-894. 
[14]Ge Y, Li Y, Lv D K,et al. Alkaline-stress response in Glycine soja leaf identifies specific transcription factors and ABA-mediated signaling factors[J]. Functional Integrative Genomics, 2011, 11: 369-379. 
[15]Wang Z Y, Song F B, Cai H,et al. Over-expressing GsGST14 from Glycine soja enhances alkaline tolerance of transgenic Medicago sativa[J]. Biologia Plantarum, 2012, 56(3): 516-520. 
[16]吕德扬, 范云六, 俞梅敏, 等. 苜蓿高含硫氨基酸蛋白转基因植株再生[J]. 遗传学报, 2000, 27(4): 331-337. 
[17]翟红, 柏锡, 朱延明, 等. SCMRP基因原核表达及多克隆抗体制备[J]. 东北农业大学学报, 2009, 40(7): 60-65. 
[18]张月学, 唐凤兰, 张弘强, 等. 零磁空间处理选育紫花苜蓿品种农菁1号[J]. 核农学报, 2006, 21(1): 34-37. 
[19]盛慧, 朱延明, 李杰, 等. DREB2A基因对苜蓿遗传转化的研究[J]. 草业科学, 2007, 24(3): 40-45. 
[20]魏正巍, 朱延明, 化烨, 等. 转GsPPCK1基因苜蓿植株的获得及其耐碱性分析[J]. 作物学报, 2013, 39(1): 68-75. 
[21]张立全, 敖登花, 师文贵, 等. 转红树总DNA紫花苜蓿T1代耐盐株系的生理生化特性分析[J]. 草业学报, 2012, 21(2): 149-155. 
[22]Rao M V, Paliyath G, Ormrod D P,et al. Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2 metabolizing enzymes[J]. Plant Physiology, 1997, 115: 137-149. 
[23]Mauch F, Dudlar R. Differential induction of distinct glutathione S-transferases of wheat by xenobiotics and by pathogen attack[J]. Plant Physiology, 1993, 102: 1193-1201. 
[24]Health R L, Packer L. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation[J]. Archives of Biochemistry and Biophysics, 1968, 125: 189-198. 
[25]Talarczyk A, Krzymowska M, Borucki W,et al. Effect of yeast CAT1 gene expression on response of tobacco plants to tobacco mosaic virus infection[J]. Plant Physiology, 2002, 129: 1032-1044. 
[26]刘青广, 田丽萍, 姜红, 等. 苜蓿叶蛋白中氨基酸的含量及营养分析[J]. 河南工业大学学报, 2005, 26(2): 36-39. 
[27]张永峰, 殷波. 混合盐碱胁迫对苗期紫花苜蓿抗氧化酶活性及丙二醛含量的影响[J]. 草业学报, 2009, 18(1): 46-50. 
[28]李燕, 孙彦, 杨青川, 等. 紫花苜蓿MsZIP基因超表达载体的构建及转基因苜蓿检测[J]. 草业学报, 2012, 21(6): 182-189. 
[29]Li W F, Wang D L, Jin T C,et al. The vacuolar Na+/H+ antiporter gene SsNHX1 from the halophvte Salsola soda confers salt tolerance in transgenic alfafla（Medicago sativa L.)[J]. Plant Molecular Biology Reporter, 2011, 29: 278-290. 
[30]Bao A K, Wang S M, Wu G Q,et al. Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic aflafla（Medicago sativa L.)[J]. Plant Science, 2009, 176: 232-240. 
[31]Yang X, Su W, Liu J P,et al. Biochemical and physiological characterization of a tau class glutathione transferase from rice（Oryza sativa)[J]. Plant Physiology and Biochemistry, 2009, 47: 1061-1068. 
[32]Lo Piero A R, Puglisi I, Petrone G. Gene isolation, analysis of expression, and in vitro synthesis of glutathione S-transferase from orange fruit [Citrus sinensis L. (Osbeck)][J]. Journal of Agricultural and Food Chemistry, 2006, 54: 9227-9233. 
[33]全先庆, 高文. 盐生植物活性氧的酶促清除机制[J]. 安徽农业科学, 2003, 31(2): 320-322. 
[34]Saalbach G, Jung E, Saalbach I,et al. Construction of storage protein genes with increased number of methionine codons and their use in transformation experiments[J]. Biochemie und Physiologie der Pflanzen, 1988, 183(2-3): 211-218. 
[35]李雷, 刘松梅, 胡鸯雷, 等. 导入玉米10ku醇溶蛋白基因提高马铃薯块茎中含硫氨基酸的含量[J]. 科学通报, 2000, 45(12): 1313-1317.