过表达ApGSMT2和ApDMT2基因的拟南芥和玉米耐盐性分析
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摘要
玉米是对土壤盐渍化中度敏感的作物,易受盐碱危害。甘氨酸甜菜碱作为一种主要的渗透保护物质,能够提高植物对多种非生物胁迫(如盐碱、干旱、低温等)的抗性。本工作前期从嗜盐隐杆藻中克隆得到两个参与甘氨酸甜菜碱合成的甲基转移酶基因ApGSMT2和ApDMT2,利用农杆菌介导法,将两个基因分别在拟南芥和玉米中共同过表达,获得转基因阳性株,收获T_1代转基因种子,经自交后得到T_2代种子。以拟南芥T_2代种子为试材,设置0、50、100、150、200 mmol/L NaCl处理,进行种子萌发试验,结果显示,不同盐浓度处理下,转基因拟南芥种子的萌发率显著高于未转基因对照植株,说明过表达ApGSMT2和ApDMT2基因对于提高拟南芥的耐盐性具有显著效果。进一步对T_2代转基因玉米株系幼苗的耐盐性进行试验,结果表明,180 mmol/L NaCl处理后,未转基因对照植株萎蔫,而转基因株系长势良好,其株高、根长、叶片相对含水量和鲜重显著高于对照,说明过表达ApGSMT2和ApDMT2基因显著提高了玉米对盐胁迫的耐受性,为利用基因工程技术创制玉米耐盐种质提供了理论依据。
Maize is a moderately sensitive crop to soil salinization and is vulnerable to saline-alkali damage. Glycine betaine(GB), as a major osmotic protective solute, has shown the ability to improve plant resistance to a variety of abiotic stresses, such as salinity, drought and low temperature. Two methyltransferase genes, ApGSMT2 and ApDMT2, which are involved in the synthesis of GB, were cloned from Aphanothece halophytica in our previous studies. The two genes were overexpressed in Arabidopsis and maize through Agrobacterium-mediated method, and the transgenic positive strains were obtained. The T_2 generation was obtained through selfing-cross from T_1 generation. With the T_2 seeds of Arabidopsis as materials, the germination test was conducted by setting the treatments of 0, 50, 100, 150 and 200 mmol/L NaCl. The germination rate of transgenic Arabidopsis seeds was significantly higher than that of wild-type plants under various concentrations of salt treatment. It indicated that overexpressing ApGSMT2 and ApDMT2 could significantly enhance the salt tolerance of Arabidopsis. The test was further conducted on the salt tolerance of T_2 maize seedlings. Under the treatment of 180 mmol/L NaCl, the transgenic maize seedlings developed better, while the control plants wilting. The plant height, root length, leaf relative water content and fresh weight of transgenic lines were significantly higher than those of untransformed control plants. These results demonstrated that ApGSMT2 and ApDMT2 overexpression significantly increased the tolerance of Arabidopsis and maize to salt stress.
Maize is a moderately sensitive crop to soil salinization and is vulnerable to saline-alkali damage. Glycine betaine(GB), as a major osmotic protective solute, has shown the ability to improve plant resistance to a variety of abiotic stresses, such as salinity, drought and low temperature. Two methyltransferase genes, ApGSMT2 and ApDMT2, which are involved in the synthesis of GB, were cloned from Aphanothece halophytica in our previous studies. The two genes were overexpressed in Arabidopsis and maize through Agrobacterium-mediated method, and the transgenic positive strains were obtained. The T_2 generation was obtained through selfing-cross from T_1 generation. With the T_2 seeds of Arabidopsis as materials, the germination test was conducted by setting the treatments of 0, 50, 100, 150 and 200 mmol/L NaCl. The germination rate of transgenic Arabidopsis seeds was significantly higher than that of wild-type plants under various concentrations of salt treatment. It indicated that overexpressing ApGSMT2 and ApDMT2 could significantly enhance the salt tolerance of Arabidopsis. The test was further conducted on the salt tolerance of T_2 maize seedlings. Under the treatment of 180 mmol/L NaCl, the transgenic maize seedlings developed better, while the control plants wilting. The plant height, root length, leaf relative water content and fresh weight of transgenic lines were significantly higher than those of untransformed control plants. These results demonstrated that ApGSMT2 and ApDMT2 overexpression significantly increased the tolerance of Arabidopsis and maize to salt stress.
引文
[1] Deinlein U,Stephan A B,Horie T,et al.Plant salt-tolerance mechanisms [J].Trends in Plant Science,2014,19(6):371-379.
[2] Farooq M,Hussain M,Wakeel A,et al.Salt stress in maize:effects,resistance mechanisms,and management.A review [J].Agronomy for Sustainable Development,2015,35(2):461-481.
[3] 赵韦.土壤盐碱化对玉米胁迫的研究进展[J].黑龙江农业科学,2019(1):140-142.
[4] Sakamoto A,Murata N.The role of glycinebetaine in the protection of plants from stress:clues from transgenic plants [J].Plant Cell and Environment,2002,25(2):163-171.
[5] 郭嘉,孙传波,杨向东,等.耐盐碱转基因玉米的获得及其抗性分析[J].玉米科学,2016,24(6):24-29.
[6] Hayashi H,Alia A,Mustardy L A,et al.Transformation of Arabidopsis thaliana with the codA gene for choline oxidase;accumulation of glycinebetaine and enhanced tolerance to salt and cold stress [J].The Plant Journal,1997,12(1):133-142.
[7] Chen T H H,Murata N.Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes [J].Current Opinion in Plant Biology,2002,5(3):250-257.
[8] Chen T H H,Murata N.Glycinebetaine:an effective protectant against abiotic stress in plants [J].Trends in Plant Science,2008,13(9):499-505.
[9] Yang X,Liang Z,Lu C.Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants [J].Plant Physiology,2005,138(4):2299-2309.
[10] Wani S H,Singh N B,Haribhushan A,et al.Compatible solute engineering in plants for abiotic stress tolerance — role of glycine betaine [J].Current Genomics,2013,14(3):157-165.
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[12] Rathinasabapathi B,Burnet M,Russell B L,et al.Choline monooxygenase,an unusual ironsulfur enzyme catalyzing the first step of glycine betaine synthesis in plants:prosthetic group characterization and cDNA cloning [J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(7):3454-3458.
[13] Nyyssola A,Kerovuo J,Kaukinen P,et al.Extreme halophiles synthesize betaine from glycine by methylation [J].The Journal of Biological Chemistry,2000,275(29):22196-22201.
[14] Waditee R,Tanaka Y,Aoki K,et al.Isolation and functional characterization of N-methyltransferases that catalyze betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica[J].The Journal of Biological Chemistry,2003,278(7):4932-4942.
[15] Waditee R,Bhuiyan M N,Rai V,et al.Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis [J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(5):1318-1323.
[16] Waditee-Sirisattha R,Singh M,Kageyama H,et al.Anabaena sp.PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt [J].Archives of Microbiology,2012,194(11):909-914.
[17] Lai S J,Lai M C,Lee R J,et al.Transgenic Arabidopsis expressing osmolyte glycine betaine synthesizing enzymes from halophilic methanogen promote tolerance to drought and salt stress [J].Plant Molecular Biology,2014,85(4/5):429-441.
[18] Niu X,Xiong F,Liu J,et al.Co-expression of ApGSMT and ApDMT promotes biosynthesis of glycine betaine in rice(Oryza sativa L.) and enhances salt and cold tolerance [J].Environmental and Experimental Botany,2014,104:16-25.
[19] He Y,He C M,Li L H,et al.Heterologous expression of ApGSMT2 and ApDMT2 genes from Aphanothece halophytica enhanced drought tolerance in transgenic tobacco [J].Molecular Biology Reports,2011,38(1):657-666.
[20] Song J,Zhang R,Yue D,et al.Co-expression of ApGSMT2g and ApDMT2g in cotton enhances salt tolerance and increases seed cotton yield in saline fields [J].Plant Science,2018,274:369-382.
[21] He C,He Y,Liu Q,et al.Co-expression of genes ApGSMT2 and ApDMT2 for glycinebetaine synthesis in maize enhances the drought tolerance of plants [J].Molecular Breeding,2013,31(1):559-573.
[22] 何春梅,王娟,董瑞,等.玉米ZmGS5基因的克隆及其对转基因拟南芥种子发育的影响[J].浙江农业学报,2019,31(4):513-518.
[23] Manoli A,Sturaro A,Trevisan S,et al.Evaluation of candidate reference genes for qPCR in maize [J].Journal of Plant Physiology,2012,169(8):807-815.
[24] Apse M P,Blumwald E.Engineering salt tolerance in plants [J].Current Opinion in Biotechnology,2002,13(2):146-150.
[25] Agarwal P K,Shukla P S,Gupta K,et al.Bioengineering for salinity tolerance in plants:state of the art [J].Molecular Biotechnology,2013,54(1):102-123.
[2] Farooq M,Hussain M,Wakeel A,et al.Salt stress in maize:effects,resistance mechanisms,and management.A review [J].Agronomy for Sustainable Development,2015,35(2):461-481.
[3] 赵韦.土壤盐碱化对玉米胁迫的研究进展[J].黑龙江农业科学,2019(1):140-142.
[4] Sakamoto A,Murata N.The role of glycinebetaine in the protection of plants from stress:clues from transgenic plants [J].Plant Cell and Environment,2002,25(2):163-171.
[5] 郭嘉,孙传波,杨向东,等.耐盐碱转基因玉米的获得及其抗性分析[J].玉米科学,2016,24(6):24-29.
[6] Hayashi H,Alia A,Mustardy L A,et al.Transformation of Arabidopsis thaliana with the codA gene for choline oxidase;accumulation of glycinebetaine and enhanced tolerance to salt and cold stress [J].The Plant Journal,1997,12(1):133-142.
[7] Chen T H H,Murata N.Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes [J].Current Opinion in Plant Biology,2002,5(3):250-257.
[8] Chen T H H,Murata N.Glycinebetaine:an effective protectant against abiotic stress in plants [J].Trends in Plant Science,2008,13(9):499-505.
[9] Yang X,Liang Z,Lu C.Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants [J].Plant Physiology,2005,138(4):2299-2309.
[10] Wani S H,Singh N B,Haribhushan A,et al.Compatible solute engineering in plants for abiotic stress tolerance — role of glycine betaine [J].Current Genomics,2013,14(3):157-165.
[11] Rhodes D,Hanson A D.Quaternary ammonium and tertiary sulfonium compounds in higher plants [J].Annual Review of Plant Physiology and Plant Molecular Biology,1993,44:357-384.
[12] Rathinasabapathi B,Burnet M,Russell B L,et al.Choline monooxygenase,an unusual ironsulfur enzyme catalyzing the first step of glycine betaine synthesis in plants:prosthetic group characterization and cDNA cloning [J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(7):3454-3458.
[13] Nyyssola A,Kerovuo J,Kaukinen P,et al.Extreme halophiles synthesize betaine from glycine by methylation [J].The Journal of Biological Chemistry,2000,275(29):22196-22201.
[14] Waditee R,Tanaka Y,Aoki K,et al.Isolation and functional characterization of N-methyltransferases that catalyze betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica[J].The Journal of Biological Chemistry,2003,278(7):4932-4942.
[15] Waditee R,Bhuiyan M N,Rai V,et al.Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis [J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(5):1318-1323.
[16] Waditee-Sirisattha R,Singh M,Kageyama H,et al.Anabaena sp.PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt [J].Archives of Microbiology,2012,194(11):909-914.
[17] Lai S J,Lai M C,Lee R J,et al.Transgenic Arabidopsis expressing osmolyte glycine betaine synthesizing enzymes from halophilic methanogen promote tolerance to drought and salt stress [J].Plant Molecular Biology,2014,85(4/5):429-441.
[18] Niu X,Xiong F,Liu J,et al.Co-expression of ApGSMT and ApDMT promotes biosynthesis of glycine betaine in rice(Oryza sativa L.) and enhances salt and cold tolerance [J].Environmental and Experimental Botany,2014,104:16-25.
[19] He Y,He C M,Li L H,et al.Heterologous expression of ApGSMT2 and ApDMT2 genes from Aphanothece halophytica enhanced drought tolerance in transgenic tobacco [J].Molecular Biology Reports,2011,38(1):657-666.
[20] Song J,Zhang R,Yue D,et al.Co-expression of ApGSMT2g and ApDMT2g in cotton enhances salt tolerance and increases seed cotton yield in saline fields [J].Plant Science,2018,274:369-382.
[21] He C,He Y,Liu Q,et al.Co-expression of genes ApGSMT2 and ApDMT2 for glycinebetaine synthesis in maize enhances the drought tolerance of plants [J].Molecular Breeding,2013,31(1):559-573.
[22] 何春梅,王娟,董瑞,等.玉米ZmGS5基因的克隆及其对转基因拟南芥种子发育的影响[J].浙江农业学报,2019,31(4):513-518.
[23] Manoli A,Sturaro A,Trevisan S,et al.Evaluation of candidate reference genes for qPCR in maize [J].Journal of Plant Physiology,2012,169(8):807-815.
[24] Apse M P,Blumwald E.Engineering salt tolerance in plants [J].Current Opinion in Biotechnology,2002,13(2):146-150.
[25] Agarwal P K,Shukla P S,Gupta K,et al.Bioengineering for salinity tolerance in plants:state of the art [J].Molecular Biotechnology,2013,54(1):102-123.