草地早熟禾NADH-GOGAT基因的克隆及表达分析
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摘要
氮素是制约草坪草生长发育的重要因素之一,GS/GOGAT循环是植物氮同化的主要途径,谷氨酸合酶(glutamine:2-oxoglutarate amidotransferase,glutamate synthase,GOGAT)在植物氮素转化过程中发挥着重要的催化作用。本研究以草地早熟禾(Poa pratensis)为试材,基于二代测序RNA-seq相关数据,克隆得到草地早熟禾NADHGOGAT基因,并进行生物信息学分析。结果表明:草地早熟禾NADH-GOGAT基因序列为3 236bp,完整的开放阅读框(Open Reading Frame Finder,ORF)为1 338bp,编码445个氨基酸残基组成的蛋白;预测NADH-GOGAT蛋白的分子量为49.89KD,等电点(isoelectric point,pI)为6.11,无信号肽,含有1个Glu-syn-central结构域,属于Glu-syn-central超级家族;二级结构以无规则卷曲、α-螺旋、延伸链和β-转角为主;同源进化分析结果表明草地早熟禾NADH-GOGAT与硬粒黑小麦氨基酸序列相似度为97%。草地早熟禾NADH-GOGAT基因在叶部的相对表达量高于根与茎,低氮浓度更有利于该基因的表达。NADH-GOGAT在干旱胁迫和水氮互作中表达差异显著(P<0.05)。草地早熟禾NADH-GOGAT基因的克隆,及相关序列分析和基因表达,对进一步研究该基因的功能和草地早熟禾GS/GOGAT循环的调控过程具有一定意义。
Nitrogen nutrition is one of the most important factors restricting the growth and development of turfgrass.GS/GOGAT cycle is the main pathway of plant nitrogen assimilation,and GOGAT(glutamate synthase)plays an important catalytic role in plant nitrogen transformation.Based on transcriptome sequencing data,NADH-GOGAT gene of Kentucky bluegrass was cloned and analyzed by bioinformatics.The results showed that the NADH-GOGAT sequence of Kentucky bluegrass was 3 236 bp,and the complete ORF region was 1 338 bp,encoding aprotein composed of 445 amino acid residues.It is predicted that the protein has a molecular weight of 49.89 KD,an isoelectric point of 6.11,a signal-free peptide,and a Glu-syn-central domain,belonging to the Glu_syn_central superfamily.The secondary structure is mainly composed of irregular curl,alpha helix,extended chain and beta corner.Homology analysis showed that the highest similarity was 97% with the Secale cereale ×Triticum turgidum subsp.durumof NADH-GOGAT amino acid sequence.The relative expression of NADH-GOGAT gene in leaves of Kentucky bluegrass was higher than that of roots and stems.Low nitrogen concentration was more conducive to the expression of the gene.Cloning sequence characteristics and expression analysis of the NADH-GOGAT gene in Kentucky bluegrass have a certain significance for further study of the function,of this gene and the regulation of GS/GOGAT cycle.
Nitrogen nutrition is one of the most important factors restricting the growth and development of turfgrass.GS/GOGAT cycle is the main pathway of plant nitrogen assimilation,and GOGAT(glutamate synthase)plays an important catalytic role in plant nitrogen transformation.Based on transcriptome sequencing data,NADH-GOGAT gene of Kentucky bluegrass was cloned and analyzed by bioinformatics.The results showed that the NADH-GOGAT sequence of Kentucky bluegrass was 3 236 bp,and the complete ORF region was 1 338 bp,encoding aprotein composed of 445 amino acid residues.It is predicted that the protein has a molecular weight of 49.89 KD,an isoelectric point of 6.11,a signal-free peptide,and a Glu-syn-central domain,belonging to the Glu_syn_central superfamily.The secondary structure is mainly composed of irregular curl,alpha helix,extended chain and beta corner.Homology analysis showed that the highest similarity was 97% with the Secale cereale ×Triticum turgidum subsp.durumof NADH-GOGAT amino acid sequence.The relative expression of NADH-GOGAT gene in leaves of Kentucky bluegrass was higher than that of roots and stems.Low nitrogen concentration was more conducive to the expression of the gene.Cloning sequence characteristics and expression analysis of the NADH-GOGAT gene in Kentucky bluegrass have a certain significance for further study of the function,of this gene and the regulation of GS/GOGAT cycle.
引文
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[20]林郑和,钟秋生,陈常颂,等.不同氮浓度下茶树氮合成关键酶基因的表达分析[J].核农学报,2014,28(6):985-989
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[22]杨夕,郁松林,赵丰云,等.水分胁迫下硝酸钠对葡萄幼苗氮代谢酶活性的影响[J].北方园艺,2018(12):27-35
[23]李静静,陈雅君,张璐,等.水氮交互作用对草地早熟禾生理生化与坪用质量的影响[J].中国草地学报,2016,38(4):42-48
[24]孙佳林,张炜成,郑海霞,等.水氮交互作用下草地早熟禾生长特性的变化[J].草地学报,2015,23(6):1226-1232
[2]Tajul M I,Alam M M,Hossain S M,et al.Influence of plant population and nitrogen-fertilizer at various levels on growth and growth efficiency of maize[J].The Scientific World Journal,2013:1-9
[3]余佳玲,朱兆坤,张振华,等.不同供氮条件下谷氨酰胺合成酶与谷氨酸合成酶对油菜氮素再利用的影响[J].作物杂志,2014(6):81-85
[4]李彩凤,徐影,郭剑,等.甜菜(Beta vulgaris L.)叶片GOGAT与GS协同变化分析[J].东北农业大学学报,2015(4):17-22
[5]Ishiyama K,Inoue E,Watanabe-Takahashi A,et al.Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis[J].Journal of Biological Chemistry,2004,279(16):16598-605
[6]Cai H,Zhou Y,Xiao J,et al.Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice[J].Plant Cell Reports,2009,28(3):527-537
[7]Goodall A J,Kumar P,Tobin A K.Identification and expression analyses of cytosolic glutamine synthetase genes in barley(Hordeum vulgare L.)[J].Plant Cell Physiology,2013,54(4):492-505
[8]Martin A,Lee J,Kichey T,et al.Two Cytosolic Glutamine Synthetase Isoforms of Maize Are Specifically Involved in the Control of Grain Production[J].Plant Cell,2006,18(11):3252-3274
[9]Tabuchi M,Abiko T,Yamaya T.Assimilation of ammonium ions and reutilization of nitrogen in rice(Oryza sativa L.)[J].Journal of Experimental Botany,2007,58(9):2319-2327
[10]Cordoba E,Shishkova S,Vance C P,et al.Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa(Medicago sativa L.)[J].The Plant Journal,2010,33(6):1037-1049
[11]Lancien M,Martin M,Ming-Hsiun Hsieh,et al.Arabidopsis glt1-T mutant defines a role for NADH-GOGAT in the nonphotorespiratory ammonium assimilatory pathway[J].The Plant Journal,2002,29(3):347-358
[12]袁秀云,许申平,王默霏,等.蝴蝶兰PhTSJT1基因的克隆及在低温胁迫下的表达分析[J].基因组学与应用生物学,2019,38(2):707-713
[13]Lea P J,Miflin B J.Glutamate synthase and the synthesis of glutamate in plants[J].Plant Physiology Biochemistry,2003,41(6):555-564
[14]Nigro D,Blanco A,Anderson O D,et al.Characterization of Ferredoxin-Dependent Glutamine-Oxoglutarate Amidotransferase(FdGOGAT)Genes and Their Relationship with Grain Protein Content QTL in Wheat[J].Plos One,2014,9(8):e103869
[15]Vance C P,Miller S S,Gregerson R G,et al.Alfalfa NADH-dependent glutamate synthase:structure of the gene and importance in symbiotic N2fixation[J].Plant Journal,2010,8(3):345-358
[16]Yamaya T,Obara M,Nakajima H,et al.Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice[J].Journal of Experimental Botany,2002,53(370):917
[17]Forde B G,Lea P J.Glutamate in plants:metabolism,regulation,and signalling[J].Journal of Experimental Botany,2007,58(9):2339-2358
[18]Obara,M.Mapping of QTLs associated with cytosolic glutamine synthetase and NADH-glutamate synthase in rice(Oryza sativa L.)[J].Journal of Experimental Botany,2001,52(359):1209-1217
[19]Claros M G,Aguilar M L,Cánovas F M.Evidence for an operative glutamine translocator in chloroplasts from maritime pine(Pinus pinaster Ait.)cotyledons.[J].Plant Biology,2010,12(5):717-723
[20]林郑和,钟秋生,陈常颂,等.不同氮浓度下茶树氮合成关键酶基因的表达分析[J].核农学报,2014,28(6):985-989
[21]曹瑞霞,周心渝,李姣姣,等.外源NO对半夏组培苗次生代谢的调控[J].西南大学学报(自然科学版),2013,35(4):14-19
[22]杨夕,郁松林,赵丰云,等.水分胁迫下硝酸钠对葡萄幼苗氮代谢酶活性的影响[J].北方园艺,2018(12):27-35
[23]李静静,陈雅君,张璐,等.水氮交互作用对草地早熟禾生理生化与坪用质量的影响[J].中国草地学报,2016,38(4):42-48
[24]孙佳林,张炜成,郑海霞,等.水氮交互作用下草地早熟禾生长特性的变化[J].草地学报,2015,23(6):1226-1232