大豆GmGLDH基因的克隆、表达及生物信息学分析
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摘要
为了探究大豆L-半乳糖酸-1,4-内酯脱氢酶基因Gm GLDH-Glyma.02G166300 (以下简称GmGLDH)的功能,本研究采用同源克隆法获得大豆GmGLDH基因,开展了相关生物信息学分析,并对其非生物胁迫下表达模式进行调查。结果表明,GmGLDH基因包含一个长度为1 752 bp的ORF序列,编码584个氨基酸。GmGLDH蛋白为亲水性较强的蛋白;二级结构中以无规则卷曲和α螺旋结构为主;预测亚细胞定位GmGLDH主要分布在线粒体中,分析结果并未发现跨膜结构域和信号肽结构;该蛋白序列中存在磷酸化位点56个。进化分析表明,克隆的GmGLDH氨基酸序列与菜豆、苜蓿的同源关系较近。基因上游顺式调控元件分析表明该基因可能参与光应答、激素响应、高温响应等,但光处理下大豆子叶中Gm GLDH基因的表达变化不大,且和子叶中抗坏血酸含量无相关性;4种非生物胁迫处理下大豆叶片中Gm GLDH基因是先下调后上调表达,和抗坏血酸含量变化呈反相关。本研究为进一步研究GmGLDH基因在大豆抗逆中的作用提供了重要的帮助。
In o rder to explore the function of L-galactono-1, 4-lactone dehydrogenase gene Gm GLDH-Glyma.02 G166300(Hereinafter referred to as GmGLDH) in soybean, GmGLDH was cloned by homology cloning strategy.The bioinformatics analysis was carried out and the expression patterns under abiotic stress were investigated. The result showed that Gm GLDH contained an ORF region with 1 752 bp length, encoding 584 amino acids. The GmGLDH protein was a strong hydrophilic protein, with random coil and alpha helix as main components in secondary structure. The predicted subcellular localization of GmGLDH was mainly in the Mitochondria but the transmembrane domain and signal peptide structure were not found in the analysis. The sequence protein included 56 phosphorylation sites. Evolutionary analysis displayed that the protein had the closest genetic relationship with those GLDH of Phaseolus vulgaris L. and Medicago truncatula. The upstream cis-regulatory element analysis showed that the gene might be involved in light response, hormone response and high temperature response, but the expression of GmGLDH gene in soybean cotyledon had little change under light treatment, and had no correlation with ascorbic acid content in cotyledons. Under four abiotic stress, the expression of GmGLDH gene in soybean leaves was first down-regulated, and then up-regulated, which was inversely correlated with the change of ascorbic acid content. The study might provide important help for the further study on the role of GmGLDH in soybean stress resistance.
In o rder to explore the function of L-galactono-1, 4-lactone dehydrogenase gene Gm GLDH-Glyma.02 G166300(Hereinafter referred to as GmGLDH) in soybean, GmGLDH was cloned by homology cloning strategy.The bioinformatics analysis was carried out and the expression patterns under abiotic stress were investigated. The result showed that Gm GLDH contained an ORF region with 1 752 bp length, encoding 584 amino acids. The GmGLDH protein was a strong hydrophilic protein, with random coil and alpha helix as main components in secondary structure. The predicted subcellular localization of GmGLDH was mainly in the Mitochondria but the transmembrane domain and signal peptide structure were not found in the analysis. The sequence protein included 56 phosphorylation sites. Evolutionary analysis displayed that the protein had the closest genetic relationship with those GLDH of Phaseolus vulgaris L. and Medicago truncatula. The upstream cis-regulatory element analysis showed that the gene might be involved in light response, hormone response and high temperature response, but the expression of GmGLDH gene in soybean cotyledon had little change under light treatment, and had no correlation with ascorbic acid content in cotyledons. Under four abiotic stress, the expression of GmGLDH gene in soybean leaves was first down-regulated, and then up-regulated, which was inversely correlated with the change of ascorbic acid content. The study might provide important help for the further study on the role of GmGLDH in soybean stress resistance.
引文
Alhagdow M.,Mounet F.,Gilbert L.,Nunes-Nesi A.,Garcia V.,Just D.,Petit J.,Beauvoit B.,Fernie A.R.,Rothan C.,and Baldet P.,2007,Silencing of the mitochondrial ascorbate synthesizing enzyme L-galactono-1,4-lactone dehydrogenase affects plant and fruit de velopment in tomato,Plant Physiol.,145(4):1408-1422
Arrigoni O.,1994,As corbate system in plant development,J.Bioenerg.Biomembr.,26(4):407-419
Asard H.,May J.M.,and Smirnoff N.,eds.,2005,Vitamin C:functions and biochemistry in animals and plants,second published,Taylor&Francis e-Library,London and New York,pp.17-21
Barth C.,Gouzd Z.A.,Steele H.P.,and Imperio R.M.,2010,Amutation in GDP-mannose pyrophosphorylase causes conditional hypersensitivity to ammonium,resulting in arabidopsis root growth inhibition,altered ammonium metabolism,and hormone homeostasis,J.Exp.Bot.,61(2):379-394
Conklin P.L.,2001,Recent advances in the role and biosynthesis of ascorbic acid in plants,Plant Cell&Environment,24(4):383-394
Conklin P.L.,Pallanca J.E.,Last R.L.,and Smirnoff N.,1997,L-ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1,Plant Physiol.,115(3):1277-1285
Foyer C.,Rowell J.,and Walker D.,1983,Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination,Planta,157(3):239-244
Fukunaga K.,Fujikawa Y.,and Esaka M.,2010,Light regulation of ascorbic acid biosynthesis in rice via light responsive cis-elements in genes encoding ascorbic acid biosynthetic enzymes,Bioscience Biosci.Biotechnol.Biochem.,74(4):888-891
Imai T.,Niwa M.,Ban Y.,Hirai M.,魺ba K.,and Moriguchi T.,2009,Importance of the L-galactonolactone pool for enhancing the ascorbate content revealed by L-galactonolactone dehydrogenase-overexpressing tobacco plants,Plant Cell Tissue&Organ Culture,96(1):105-112
Isherwood F.A.,Chen Y.T.,and Mapson L.W.,1953,Synthesis of L-ascorbic acid in plants and animals,Biochem.J.,171(4347):348-349
Li M.,Ma F.,Shang P.,Zhang M.,Hou C.,and Liang D.,2009,Influence of light on ascorbate formation and metabolism in apple fruits,Planta,230(1):39-51
Li M.J.,Gao J.,Ma F.W.,Liang D.,and Hou C.M.,2010,Relationship between expressions of GalDH and GalLDH and ascorbate content in apple fruits,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),43(2):351-357(李明军,高静,马锋旺,梁东,侯长明,2010,苹果果实GalDH和GalLDH基因的表达与As A的关系,中国农业科学,43(2):351-357)
Li S.N.,Su A.Y.,Yu Y.C.,Zhang C.,Ma Y.L.,Wang L.S.,Jin Y.M.,Li W.B.,and Wu X.X.,2015,Bioinformatics and expression analysis of regeneration related gene GmARF in soybean,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(10):2187-2193(李思楠,苏安玉,于以成,张超,马彦龙,王玲爽,金杨媚,李文滨,武小霞,大豆再生相关基因GmARF的生物信息学及表达分析,基因组学与应用生物学,34(10):2187-2193)
Liu Y.H.,Yu L.,and Wang R.Z.,2011,Level of ascorbic acid in transgenic rice for l-galactono-1,4-lactone dehydrogenase overexpressing or suppressed is associated with plant growth and seed set,Acta Physiologiae Plantarum,33(4):1353-1363
Livak K.J.,amd Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod,Method,25(4):402-408
Loewus M.W.,Bedgar D.L.,Saito K.,and Loewus F.A.,1990,Conversion of L-sorbosone to L-ascorbic acid by a NADP-dependent dehydrogenase in bean and spinach leaf,Plant Physiol.,94(3):1492-1495
Lorence A.,Chevone B.I.,Mendes P.,and Nessler C.L.,2004,myoI nositol oxygenase offers a possible entry point into plant ascorbate biosynthesis,Plant Physiol.,134(3):1200-1205
Nascimento J.R.O.D.,Higuchi B.K.,Gómez M.L.P.A.,Oshiro R.A.,and Lajolo F.M.,2005,I-Ascorbate biosynthesis in strawberries:l-Galactono-1,4-lactone dehydrogenase expression during fruit development and ripening,Postharvest Biology&Technology,38(1):34-42
覫stergaard J.,Persiau G.,Davey M.W.,Bauw G.,and Montagu M.V.,1997,Isolation of a cDNA coding forl-galactono-γ-lactone dehydrogenase,an enzyme involved in the biosynthesis of ascorbic acid in plants,J.Biol.Chem.,272(48):30009-30016
Pateraki I.,Sanmartin M.,Kalamaki M.S.,Gerasopoulos D.,and Kanellis A.K.,2004,Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4-lactone dehydrogenase,J.Exp.Bot.,55(403):1623-1633
Qin C.,2008,Molecular mechanisms of the increased NH4+sensitivity of mutant hsnl in Arabidopsis,Dissertation for Ph.D.,Zhejiang University,Supervisor:Wu P.,pp.57-58(秦诚,2008,拟南芥NH+4超敏突变体hsn1分子机制的相关研究,博士学位论文,浙江大学,导师:吴平,pp.57-58)
Sasaki-Sekimoto Y.,Taki N.,Obayashi T.,Aono M.,Matsumoto F.,Sakurai N.,Suzuki H.,Hirai M.Y.,Noji M.,Saito K.,Masuda T.,Takamiya K.,Shibata D.,and Ohta H.,2005,Coordinated activation of metabolic pathw ays for antioxidants and defence compounds by jasmonates and their roles in stress tolerance in Arabidopsis,Plant J.,44(4):653-668
Schmittgen T.D.,and Livak K.J.,2008,Analyzing real-time PCRdata by the comparative CT method,Nature Protocols,3(6):1101-1108
Tabata K.,Takaoka T.,and Esaka M.,2002,Gene expression of ascorbic acid-related enzymes in tobacco,Phytochemistry,61(6):631-635
Tamaoki M.,Mukai F.,Asai N.,Nakajima N.,Kubo A.,Aono M.,and Saji H.,2003,Light-controlled expression of a gene encoding l-galactono-γ-lactone dehydrogenase which affects ascorbate pool size in Arabidopsis thaliana,Plant Science,164(6):1111-1117
Tokunaga T.,Miyahara K.,Tabata K.,and Esaka M.,2005,Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for l-galactono-1,4-lactone dehydrogenase,Planta,27(2):854-863
Wheeler G.L.,Jones M.A.,and Smirnoff N.,1998,The biosynthetic pathway of vitamin C in higher plants,Nature,393(6683):365-369
Wolucka B.A.,and Van M.M.,2003,GDP-mannose 3',5'-epimerase forms GDP-L-gulose,a putative intermediate for the de novo biosynthesis of vitamin C in plants,Journal Biological Chemistry,278(48):47483-47490
Wolucka B.A.,Goossens A.,and InzéD.,2005,Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions,J.Exp.Bot.,56(419):2527-2538
Yabuta Y.,Maruta T.,Nakamura A.,Mieda T.,Yoshimura K.,Ishikawa T.,and Shigeoka S.,2008,Conversion of L-galactono-1,4-lactone to L-ascorbate is regulated by the photosynthetic electron transport chain in Arabidopsis,Biosci.Biotechnol.Biochem.,72(10):2598-2607
Yabuta Y.,Yoshimura K.,Takeda T.,and Shigeoka S.,2000,Molecular characterization of tobacco mitochondrial l-galactono-γ-lactone dehydrogenase and its expression in Escherichia coli,Plant and Cell Physiol.,41(6):666-675
Zou L.P,Li H.X.,Ouyang B.,Zhang J.H.,and Ye Z.B.,2006,Cloning,expression,and mapping of GDP-D-mannose pyrophosphorylase cDNA from tomato(Lycopersicon esculentum),Acta Genetica Sinica 33(8):757-764
Arrigoni O.,1994,As corbate system in plant development,J.Bioenerg.Biomembr.,26(4):407-419
Asard H.,May J.M.,and Smirnoff N.,eds.,2005,Vitamin C:functions and biochemistry in animals and plants,second published,Taylor&Francis e-Library,London and New York,pp.17-21
Barth C.,Gouzd Z.A.,Steele H.P.,and Imperio R.M.,2010,Amutation in GDP-mannose pyrophosphorylase causes conditional hypersensitivity to ammonium,resulting in arabidopsis root growth inhibition,altered ammonium metabolism,and hormone homeostasis,J.Exp.Bot.,61(2):379-394
Conklin P.L.,2001,Recent advances in the role and biosynthesis of ascorbic acid in plants,Plant Cell&Environment,24(4):383-394
Conklin P.L.,Pallanca J.E.,Last R.L.,and Smirnoff N.,1997,L-ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1,Plant Physiol.,115(3):1277-1285
Foyer C.,Rowell J.,and Walker D.,1983,Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination,Planta,157(3):239-244
Fukunaga K.,Fujikawa Y.,and Esaka M.,2010,Light regulation of ascorbic acid biosynthesis in rice via light responsive cis-elements in genes encoding ascorbic acid biosynthetic enzymes,Bioscience Biosci.Biotechnol.Biochem.,74(4):888-891
Imai T.,Niwa M.,Ban Y.,Hirai M.,魺ba K.,and Moriguchi T.,2009,Importance of the L-galactonolactone pool for enhancing the ascorbate content revealed by L-galactonolactone dehydrogenase-overexpressing tobacco plants,Plant Cell Tissue&Organ Culture,96(1):105-112
Isherwood F.A.,Chen Y.T.,and Mapson L.W.,1953,Synthesis of L-ascorbic acid in plants and animals,Biochem.J.,171(4347):348-349
Li M.,Ma F.,Shang P.,Zhang M.,Hou C.,and Liang D.,2009,Influence of light on ascorbate formation and metabolism in apple fruits,Planta,230(1):39-51
Li M.J.,Gao J.,Ma F.W.,Liang D.,and Hou C.M.,2010,Relationship between expressions of GalDH and GalLDH and ascorbate content in apple fruits,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),43(2):351-357(李明军,高静,马锋旺,梁东,侯长明,2010,苹果果实GalDH和GalLDH基因的表达与As A的关系,中国农业科学,43(2):351-357)
Li S.N.,Su A.Y.,Yu Y.C.,Zhang C.,Ma Y.L.,Wang L.S.,Jin Y.M.,Li W.B.,and Wu X.X.,2015,Bioinformatics and expression analysis of regeneration related gene GmARF in soybean,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(10):2187-2193(李思楠,苏安玉,于以成,张超,马彦龙,王玲爽,金杨媚,李文滨,武小霞,大豆再生相关基因GmARF的生物信息学及表达分析,基因组学与应用生物学,34(10):2187-2193)
Liu Y.H.,Yu L.,and Wang R.Z.,2011,Level of ascorbic acid in transgenic rice for l-galactono-1,4-lactone dehydrogenase overexpressing or suppressed is associated with plant growth and seed set,Acta Physiologiae Plantarum,33(4):1353-1363
Livak K.J.,amd Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod,Method,25(4):402-408
Loewus M.W.,Bedgar D.L.,Saito K.,and Loewus F.A.,1990,Conversion of L-sorbosone to L-ascorbic acid by a NADP-dependent dehydrogenase in bean and spinach leaf,Plant Physiol.,94(3):1492-1495
Lorence A.,Chevone B.I.,Mendes P.,and Nessler C.L.,2004,myoI nositol oxygenase offers a possible entry point into plant ascorbate biosynthesis,Plant Physiol.,134(3):1200-1205
Nascimento J.R.O.D.,Higuchi B.K.,Gómez M.L.P.A.,Oshiro R.A.,and Lajolo F.M.,2005,I-Ascorbate biosynthesis in strawberries:l-Galactono-1,4-lactone dehydrogenase expression during fruit development and ripening,Postharvest Biology&Technology,38(1):34-42
覫stergaard J.,Persiau G.,Davey M.W.,Bauw G.,and Montagu M.V.,1997,Isolation of a cDNA coding forl-galactono-γ-lactone dehydrogenase,an enzyme involved in the biosynthesis of ascorbic acid in plants,J.Biol.Chem.,272(48):30009-30016
Pateraki I.,Sanmartin M.,Kalamaki M.S.,Gerasopoulos D.,and Kanellis A.K.,2004,Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4-lactone dehydrogenase,J.Exp.Bot.,55(403):1623-1633
Qin C.,2008,Molecular mechanisms of the increased NH4+sensitivity of mutant hsnl in Arabidopsis,Dissertation for Ph.D.,Zhejiang University,Supervisor:Wu P.,pp.57-58(秦诚,2008,拟南芥NH+4超敏突变体hsn1分子机制的相关研究,博士学位论文,浙江大学,导师:吴平,pp.57-58)
Sasaki-Sekimoto Y.,Taki N.,Obayashi T.,Aono M.,Matsumoto F.,Sakurai N.,Suzuki H.,Hirai M.Y.,Noji M.,Saito K.,Masuda T.,Takamiya K.,Shibata D.,and Ohta H.,2005,Coordinated activation of metabolic pathw ays for antioxidants and defence compounds by jasmonates and their roles in stress tolerance in Arabidopsis,Plant J.,44(4):653-668
Schmittgen T.D.,and Livak K.J.,2008,Analyzing real-time PCRdata by the comparative CT method,Nature Protocols,3(6):1101-1108
Tabata K.,Takaoka T.,and Esaka M.,2002,Gene expression of ascorbic acid-related enzymes in tobacco,Phytochemistry,61(6):631-635
Tamaoki M.,Mukai F.,Asai N.,Nakajima N.,Kubo A.,Aono M.,and Saji H.,2003,Light-controlled expression of a gene encoding l-galactono-γ-lactone dehydrogenase which affects ascorbate pool size in Arabidopsis thaliana,Plant Science,164(6):1111-1117
Tokunaga T.,Miyahara K.,Tabata K.,and Esaka M.,2005,Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for l-galactono-1,4-lactone dehydrogenase,Planta,27(2):854-863
Wheeler G.L.,Jones M.A.,and Smirnoff N.,1998,The biosynthetic pathway of vitamin C in higher plants,Nature,393(6683):365-369
Wolucka B.A.,and Van M.M.,2003,GDP-mannose 3',5'-epimerase forms GDP-L-gulose,a putative intermediate for the de novo biosynthesis of vitamin C in plants,Journal Biological Chemistry,278(48):47483-47490
Wolucka B.A.,Goossens A.,and InzéD.,2005,Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions,J.Exp.Bot.,56(419):2527-2538
Yabuta Y.,Maruta T.,Nakamura A.,Mieda T.,Yoshimura K.,Ishikawa T.,and Shigeoka S.,2008,Conversion of L-galactono-1,4-lactone to L-ascorbate is regulated by the photosynthetic electron transport chain in Arabidopsis,Biosci.Biotechnol.Biochem.,72(10):2598-2607
Yabuta Y.,Yoshimura K.,Takeda T.,and Shigeoka S.,2000,Molecular characterization of tobacco mitochondrial l-galactono-γ-lactone dehydrogenase and its expression in Escherichia coli,Plant and Cell Physiol.,41(6):666-675
Zou L.P,Li H.X.,Ouyang B.,Zhang J.H.,and Ye Z.B.,2006,Cloning,expression,and mapping of GDP-D-mannose pyrophosphorylase cDNA from tomato(Lycopersicon esculentum),Acta Genetica Sinica 33(8):757-764