发菜固氮基因nifD和nifK克隆及其失水条件下的差异表达
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摘要
nifD和nifK编码钼铁固氮酶中的钼铁蛋白。为了解发菜nifD和nifK分子信息及对水分胁迫的响应机制,该研究设计了简并性引物克隆发菜nifD和nifK全长,进行原核表达和生物信息学分析,并对不同失水状态下发菜nifD和nifK在转录水平的差异表达和固氮酶活性的变化进行分析。结果表明,发菜nifD和nifK全长分别为1 443 bp和1 536 bp (登陆号为分别为KU886164和KU886165);将nifD和nifK在大肠杆菌中表达,分别获得一个约57 kD和58 kD的外源蛋白;生物信息学分析表明,nifD和nifK核苷酸序列和推译的氨基酸序列均与点形念珠藻(Nostoc punctiforme PCC 73102)高度一致性;nifD和nifK的二级结构主要有α-螺旋、β-折叠、β-转角和随机卷曲。此外,随着藻体含水量的逐渐降低,发菜nifD和nifK在转录水平上的表达量逐渐增加,但固氮酶活性呈现先增加后下降的趋势。研究结果为深入全面研究发菜固氮酶基因结构及其响应水分胁迫的固氮机理及氮代谢途径提供了基础。
Molybdenum-iron protein is coded by nif D and nif K in Mo-Fe nitrogenase. In order to understand molecular information of nifD and nifK and the mechanism in response to dehydration, the full length of nifD and nifK of Nostoc flagelliforme were cloned according to the degenerated primers, as well as prokaryotic expression was carried out and bioinformatics was analyzed. The study also discussed the differential expression at transcription level and changes of nitrogenase activities on dehydration of nifD and nifK of Nostoc flagelliforme under different dehydration situations. The results indicated that, the full length of nifD and nifK were 1 443 bp and 1 536 bp, respectively(GenBank access number is KU886164 and KU886165, respectively). Gene nifD and nifK were expressed in E. coli, and a 57 k D heterologous protein and a 58 kD heterologous protein were obtained, respectively. Bioinformatics analysis suggested that the nucleotide sequence and deduced amino acid sequence of nifD and nifK from Nostoc flagelliforme were similar to that of N. punctiforme PCC 73102 with high consistency. The secondary structure of nifD and nifK were made up of α helix, β-fold, random coil and β-turn. In addition, expression level of nifD and nifK gradually increased at transcription level with the decrease of water content of N. flagelliforme colonies, but nitrogenase activities increased firstly and then decreased. The results laid a foundation for further and comprehensive research on structure of nitrogenase gene and the molecular mechanism of nitrogen fixation as well as nitrogen metabolic pathway for N. flagelliforme in response to dehydration.
Molybdenum-iron protein is coded by nif D and nif K in Mo-Fe nitrogenase. In order to understand molecular information of nifD and nifK and the mechanism in response to dehydration, the full length of nifD and nifK of Nostoc flagelliforme were cloned according to the degenerated primers, as well as prokaryotic expression was carried out and bioinformatics was analyzed. The study also discussed the differential expression at transcription level and changes of nitrogenase activities on dehydration of nifD and nifK of Nostoc flagelliforme under different dehydration situations. The results indicated that, the full length of nifD and nifK were 1 443 bp and 1 536 bp, respectively(GenBank access number is KU886164 and KU886165, respectively). Gene nifD and nifK were expressed in E. coli, and a 57 k D heterologous protein and a 58 kD heterologous protein were obtained, respectively. Bioinformatics analysis suggested that the nucleotide sequence and deduced amino acid sequence of nifD and nifK from Nostoc flagelliforme were similar to that of N. punctiforme PCC 73102 with high consistency. The secondary structure of nifD and nifK were made up of α helix, β-fold, random coil and β-turn. In addition, expression level of nifD and nifK gradually increased at transcription level with the decrease of water content of N. flagelliforme colonies, but nitrogenase activities increased firstly and then decreased. The results laid a foundation for further and comprehensive research on structure of nitrogenase gene and the molecular mechanism of nitrogen fixation as well as nitrogen metabolic pathway for N. flagelliforme in response to dehydration.
引文
Burgess B.K.,and David J.L.,1996,Mechanism of molybdenum nitrogenase,Chem.Rev.,96(7):983-1012
César P.C.,Emilio J.V.,Mónica N.R.,Carlos E.E.,and Luis M.R.,2014,Kinetics of nif gene expression in a nitrogen-fixing bacterium,Journal of Bacteriology,3(196):595-603
Chen Q.H.,Han Y.L.,Ma Y.,Yan Y.L.,Ping S.Z.,and Lu W.,2013,Research progress on structure and evolution of biological nitrogen-fixation gene cluster,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and Technology),15(4):129-138(陈清华,韩云蕾,马尧,燕永亮,平淑珍,陆伟,2013,生物固氮基因簇结构与进化研究进展,中国农业科技导报,15(4):129-138)
Dos S.P.,and Dean D.R.,2011,Coordination and finetuning of nitrogen fixation in Azotobacter vinelandii,Molecular Microbiology,79(5):1132-1135
Fani R.,Gallo R.,and LióP.,2000,Molecular evolution of nitrogen fixation:the evolutionary history of the nifD,nifK,nifE,and nifN gene,J.Mol.Evol.,51(1):1-11
Gao K.S.,1998,Chinese studies on the edible blue-green alga,Nostoc flagelliforme:a review,Journal of Applied Phycology,10(1):37-49
Gao X.,Liu K.,and Qiu B.S.,2011,An investigation on the genetic background of Nostoc flagelliforme by similarity analysis of its partial genomic DNA and phylogenetic comparison of deduced related species,Acta Physiologiae Plantarum,33(1):1301-1318
Hartmann L.S.,and Bamum S.R.,2010,Inferring the evolutionary history of Mo-dependent nitrogen fixation from phylogenetic studies of nifK and nifDK,Journal of Molecular Evolution,71(1):70-85
Hyoungseok L.,Si B.S.,Ho B.K.,and Chung S.A.,2001,Sequence analysis and expression patterns of two nifA genes from Frankia EuIK1,Australian Journal of Plant Physiology,28(8):939-949
Junichi I.,Kenshiro O.,Wataru S.,Mitsuo S.,Takao L.,Yuichi H.,Masahira H.,and Moriya O.,2015,Distribution and evolution of nitrogen fixation genes in the Phylum Bacteroidetes,Microbes and Environments,1(30):44-50
Kennedy C.,and Dean D.,1 992,The nifU,nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii,Molecular and General Genetics,231(3):494-498
Lammers P.J.,and Haselkorn R.,1983,Sequence of the nifD gene coding for the alpha subunit of dinitrogenase from the cyanobacterium Anabaena,Biochemistry,80(15):4723-4727
Liang W.Y.,Wang L.X.,Zhang Y.P.,Lei X.T.,You X.R.,Yang J.,Cheng C.,Zhou Y.W.,and Chen W.,2013,Comparative proteomic and physiological analysis of diurnal changes in Nostoc flagelliforme,Journal of Applied Phycology,25(6):1709-1721
Liang W.Y.,Yang J.,Wu S.J.,Jiao G.F.,Wang S.P.,and Xu T.T.,2015,Clone and expression of hypothetical protein NXL-01related to drought-tolerant in Nostoc flagelliforme,Xibei Zhiwu Xuebao(Acta Bot.Boreal.Occident.Sin.),35(1):44-49(梁文裕,杨佳,吴诗杰,焦广飞,王淑萍,徐婷婷,2015,发菜耐旱相关蛋白NXL-01的基因克隆与表达分析,西北植物学报,35(1):44-49)
Liang W.Y.,Zhou Y.W.,Wang L.X.,You X.R.,Zhang Y.P.,Cheng C.,and Chen W.,2012,Ultrastruc tural,physiological and proteomic analysis of nostoc flagelliforme in response to dehydration and rehydration,Journal of Proteomics,75(16):5604-5627
Livak K.J.,and Schmittgeen T.D.,2001,Analysis of relative gene expression data using real time quantitative PCR and the2-△△CT methods,Methods,25(4):402-408
Morandell S.,Stasyk T.,Grosstessner-Hain K.,Elisabeth R.Mechtler K.B.,and Huber L.A.,2006,Phosphoproteomics strategies for the functional analysis of signal transduction,Proteomics,6(14):4047-4056
Sathish K.,and Bhaskara R.,2012,Biological nitrogen fixation:a revie,International Journal of Advanced Life Sciences,1(1):1-9
Stewart W.D.,Fipzegrald G.P.,and Burris R.H.,1968,Acetylene reduction by nitrogen fixing blue-green algae,Archives Microbiology,62(1):336-348
Svetlana D.,Peter R.,and Werner L.,2004,Nif H and nif D phylogenies:an evolution basis for understanding nitrogen fixation capabilities of methanotrophic bacteria,Microbiology,150(23):1301-1313
Tine E.T.,Ole N.J.,and Martin R.L.,2009,Analytical strategies for phosphoproteomics,Proteomics,9(1):1451-1468
Wang L.Y.,Zhang L.H.,Liu Z.Z.,Zhao D.H.,Liu X.M.,Zhang B.,Xie J.B.,Hong Y.Y.,Li P.F.,Chen S.F.,Dixon R.,and Li J.L.,2013,A minimal nitrogen fixation gene cluster from Paenibacillus sp.WLY78 enables expression of active nitrogenase in Escherichia coli,PLoS Genetics,9(10):1-11
Wu G.,2011,Biosynthesis of poly-hydroxybutyrate(PHB)is controlled by CydR(Fnr)in the obligate aerobe Azotobacter vinelandii FEMS,Microbiol Lett.,109(12):215-220
Xie J.B.,Du Z.,Bai L.,Tian C.,Zhang Y.,Xie J.Y.,Wang T.,Liu X.,Chen X.,Cheng Q.,Chen S.,and Li J.,2014,Comparative genomic analysis of N2-fixing and non-N2-fixing Paenibacillus spp:organization,evolution and expression of the nitrogen fixation genes,PLoS Genetics,10(3):1-17
Zhang K.K.,Shao M.,Wu C.X.,Yang L.T.,Li Y.R.,and Xing Y.X.,2015,Cloning,prokaryotic expression and purification of nifD gene from sugarcane endogenous nitrogen-fixing bacteria Klebsiella variicola DX120E,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):512-520(张琨琨,邵敏,吴朝兴,杨丽涛,李杨瑞,邢永秀,2015,甘蔗内生固氮菌Klebsiella variicola DX120E nifD基因的克隆、原核表达及纯化,基因组学与应用生物学,34(3):512-520)
Zhong Z.P.,Shi D.J.,Wang F.Z.,and Cui Z.Y.,1992,Effect of temperature,water content and light intensity on nitrogenase activity of Nostoc flagelliforme,Zhiwu Xuebao(Acta Botanica Sinica),34(3):219-225(钟泽璞,施定基,王发珠,崔志有,1992,温度,含水量和光照对发菜固氮酶活性的影响,植物学报,34(3):219-225)
César P.C.,Emilio J.V.,Mónica N.R.,Carlos E.E.,and Luis M.R.,2014,Kinetics of nif gene expression in a nitrogen-fixing bacterium,Journal of Bacteriology,3(196):595-603
Chen Q.H.,Han Y.L.,Ma Y.,Yan Y.L.,Ping S.Z.,and Lu W.,2013,Research progress on structure and evolution of biological nitrogen-fixation gene cluster,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and Technology),15(4):129-138(陈清华,韩云蕾,马尧,燕永亮,平淑珍,陆伟,2013,生物固氮基因簇结构与进化研究进展,中国农业科技导报,15(4):129-138)
Dos S.P.,and Dean D.R.,2011,Coordination and finetuning of nitrogen fixation in Azotobacter vinelandii,Molecular Microbiology,79(5):1132-1135
Fani R.,Gallo R.,and LióP.,2000,Molecular evolution of nitrogen fixation:the evolutionary history of the nifD,nifK,nifE,and nifN gene,J.Mol.Evol.,51(1):1-11
Gao K.S.,1998,Chinese studies on the edible blue-green alga,Nostoc flagelliforme:a review,Journal of Applied Phycology,10(1):37-49
Gao X.,Liu K.,and Qiu B.S.,2011,An investigation on the genetic background of Nostoc flagelliforme by similarity analysis of its partial genomic DNA and phylogenetic comparison of deduced related species,Acta Physiologiae Plantarum,33(1):1301-1318
Hartmann L.S.,and Bamum S.R.,2010,Inferring the evolutionary history of Mo-dependent nitrogen fixation from phylogenetic studies of nifK and nifDK,Journal of Molecular Evolution,71(1):70-85
Hyoungseok L.,Si B.S.,Ho B.K.,and Chung S.A.,2001,Sequence analysis and expression patterns of two nifA genes from Frankia EuIK1,Australian Journal of Plant Physiology,28(8):939-949
Junichi I.,Kenshiro O.,Wataru S.,Mitsuo S.,Takao L.,Yuichi H.,Masahira H.,and Moriya O.,2015,Distribution and evolution of nitrogen fixation genes in the Phylum Bacteroidetes,Microbes and Environments,1(30):44-50
Kennedy C.,and Dean D.,1 992,The nifU,nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii,Molecular and General Genetics,231(3):494-498
Lammers P.J.,and Haselkorn R.,1983,Sequence of the nifD gene coding for the alpha subunit of dinitrogenase from the cyanobacterium Anabaena,Biochemistry,80(15):4723-4727
Liang W.Y.,Wang L.X.,Zhang Y.P.,Lei X.T.,You X.R.,Yang J.,Cheng C.,Zhou Y.W.,and Chen W.,2013,Comparative proteomic and physiological analysis of diurnal changes in Nostoc flagelliforme,Journal of Applied Phycology,25(6):1709-1721
Liang W.Y.,Yang J.,Wu S.J.,Jiao G.F.,Wang S.P.,and Xu T.T.,2015,Clone and expression of hypothetical protein NXL-01related to drought-tolerant in Nostoc flagelliforme,Xibei Zhiwu Xuebao(Acta Bot.Boreal.Occident.Sin.),35(1):44-49(梁文裕,杨佳,吴诗杰,焦广飞,王淑萍,徐婷婷,2015,发菜耐旱相关蛋白NXL-01的基因克隆与表达分析,西北植物学报,35(1):44-49)
Liang W.Y.,Zhou Y.W.,Wang L.X.,You X.R.,Zhang Y.P.,Cheng C.,and Chen W.,2012,Ultrastruc tural,physiological and proteomic analysis of nostoc flagelliforme in response to dehydration and rehydration,Journal of Proteomics,75(16):5604-5627
Livak K.J.,and Schmittgeen T.D.,2001,Analysis of relative gene expression data using real time quantitative PCR and the2-△△CT methods,Methods,25(4):402-408
Morandell S.,Stasyk T.,Grosstessner-Hain K.,Elisabeth R.Mechtler K.B.,and Huber L.A.,2006,Phosphoproteomics strategies for the functional analysis of signal transduction,Proteomics,6(14):4047-4056
Sathish K.,and Bhaskara R.,2012,Biological nitrogen fixation:a revie,International Journal of Advanced Life Sciences,1(1):1-9
Stewart W.D.,Fipzegrald G.P.,and Burris R.H.,1968,Acetylene reduction by nitrogen fixing blue-green algae,Archives Microbiology,62(1):336-348
Svetlana D.,Peter R.,and Werner L.,2004,Nif H and nif D phylogenies:an evolution basis for understanding nitrogen fixation capabilities of methanotrophic bacteria,Microbiology,150(23):1301-1313
Tine E.T.,Ole N.J.,and Martin R.L.,2009,Analytical strategies for phosphoproteomics,Proteomics,9(1):1451-1468
Wang L.Y.,Zhang L.H.,Liu Z.Z.,Zhao D.H.,Liu X.M.,Zhang B.,Xie J.B.,Hong Y.Y.,Li P.F.,Chen S.F.,Dixon R.,and Li J.L.,2013,A minimal nitrogen fixation gene cluster from Paenibacillus sp.WLY78 enables expression of active nitrogenase in Escherichia coli,PLoS Genetics,9(10):1-11
Wu G.,2011,Biosynthesis of poly-hydroxybutyrate(PHB)is controlled by CydR(Fnr)in the obligate aerobe Azotobacter vinelandii FEMS,Microbiol Lett.,109(12):215-220
Xie J.B.,Du Z.,Bai L.,Tian C.,Zhang Y.,Xie J.Y.,Wang T.,Liu X.,Chen X.,Cheng Q.,Chen S.,and Li J.,2014,Comparative genomic analysis of N2-fixing and non-N2-fixing Paenibacillus spp:organization,evolution and expression of the nitrogen fixation genes,PLoS Genetics,10(3):1-17
Zhang K.K.,Shao M.,Wu C.X.,Yang L.T.,Li Y.R.,and Xing Y.X.,2015,Cloning,prokaryotic expression and purification of nifD gene from sugarcane endogenous nitrogen-fixing bacteria Klebsiella variicola DX120E,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):512-520(张琨琨,邵敏,吴朝兴,杨丽涛,李杨瑞,邢永秀,2015,甘蔗内生固氮菌Klebsiella variicola DX120E nifD基因的克隆、原核表达及纯化,基因组学与应用生物学,34(3):512-520)
Zhong Z.P.,Shi D.J.,Wang F.Z.,and Cui Z.Y.,1992,Effect of temperature,water content and light intensity on nitrogenase activity of Nostoc flagelliforme,Zhiwu Xuebao(Acta Botanica Sinica),34(3):219-225(钟泽璞,施定基,王发珠,崔志有,1992,温度,含水量和光照对发菜固氮酶活性的影响,植物学报,34(3):219-225)