OsUreD基因突变对水稻生长及其氮素营养生理的影响
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摘要
植物细胞中,脲酶活性的存在使得尿素经水解后能够成为可被直接利用的铵态氮。脲酶的活性需要金属镍离子的活化,同时也需要三种脲酶辅助蛋白UreD、UreG及UreF的协同作用。本文对T-DNA插入的水稻脲酶辅助蛋白基因Osured突变体的农艺性状、氮素营养生理以及相关的酶活性变化进行了较详细的研究,结果表明,种子萌发1~7d,Osured突变体中的脲酶活性未发生明显变化,但显著低于野生型约60%~80%;精氨酸酶活性显著增加,高于野生型约30%~40%。在水稻幼苗期和分蘖期,突变体则表现出生长受到抑制而植株矮小(其株高、根长显著低于野生型)、分蘖减少,脲酶活性也明显低于野生型约60%~90%。由此表明,OsUreD的功能对维持水稻正常脲酶活性的必要性;该基因的突变导致植物中脲酶及精氨酸酶活性改变等氮代谢途径的紊乱,可能是突变体水稻生长受阻以及分蘖数减少的重要分子生理学因子。
The existence of urease activity in plant cells makes hydrolize urea into ammonium that can be directly used by the plant.It has been reported that the activity of rice urease needs both the activation by nikel ion and co-ordination of three anncessory proteins i.e.Ure D,Ure G and Ure F.In this work,the agronomic traits,nitrogen nutritional physiology and related enzymes' activity were investigated in a rice Osured mutant,where Osured was deleted by a T-DNA insertion.This research revaled that during the seed germination of 1 ~ 7 d,the urease activity inthe mutant remained relatively stable and 60% ~ 80% lower than that of in wildtype(WT),but the activity of arginase increased by 30% ~ 40% as compared to the WT.At the seeding-and tillering-stage,the mutant line exhibited an obvious growth inhibition with being small in size,e.g.lower plant-height and root length,reduction in tiller numbers,and reduced urease activity by 60% ~ 90%.These results suggested that Os Ure D encoding protein should be critical for rice urease activation.Nitrogen metabolic disorder associeted with alteration of the activity of urease and arginase in the Osured mutation might be an important molecular physiological factor responsible for the supression of rice growth and tillering.
The existence of urease activity in plant cells makes hydrolize urea into ammonium that can be directly used by the plant.It has been reported that the activity of rice urease needs both the activation by nikel ion and co-ordination of three anncessory proteins i.e.Ure D,Ure G and Ure F.In this work,the agronomic traits,nitrogen nutritional physiology and related enzymes' activity were investigated in a rice Osured mutant,where Osured was deleted by a T-DNA insertion.This research revaled that during the seed germination of 1 ~ 7 d,the urease activity inthe mutant remained relatively stable and 60% ~ 80% lower than that of in wildtype(WT),but the activity of arginase increased by 30% ~ 40% as compared to the WT.At the seeding-and tillering-stage,the mutant line exhibited an obvious growth inhibition with being small in size,e.g.lower plant-height and root length,reduction in tiller numbers,and reduced urease activity by 60% ~ 90%.These results suggested that Os Ure D encoding protein should be critical for rice urease activation.Nitrogen metabolic disorder associeted with alteration of the activity of urease and arginase in the Osured mutation might be an important molecular physiological factor responsible for the supression of rice growth and tillering.
引文
[1]曹凤秋,刘国伟,王伟红,等.高等植物尿素代谢及转运的分子机理[J].植物学报,2009,44(3):273-282.Cao F Q,Liu G W,Wang W H,et al..Molecu Iar processes of urea metabolism and transport in higher plants[J].Chin.Bull.Bot.,2009,44(3):273-282.
[2]Liu L H,Ludewing U,Frommer W B,et al..At DUR3 encodes a new type of high-affinity Urea/H~+symporter in Arabidopsis[J].Plant Cell,2003,15(3):790-800.
[3]Liu L H,Ludewing U,Brigitte G,et al..Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis[J].Plant Physiol.,2003,133(3):1220-1228.
[4]Polacco J C,Holland M A.Roles of urease in plant cells[J].Int.Rev.Cytol.,1993,145:65-103.
[5]Sirko A,Brodzik R.Plant ureases:Roles and regulation[J].Acta Biochim.Pol.,2000,47(4):1189-1195.
[6]Wiebke-Strohm B,Ligabue-Braun R,Rechenmacher C,et al..Structural and transcriptional characterization of a novel member of the soybean urease gene family[J].Plant Physiol.Biochem.,2016,101:96-104.
[7]Todd C D,Polacco J C.Update on ureide degradation in legumes[J].J.Exp.Bot.,2006,57(1):5-12.
[8]Witte C P,Davies H V.Leaf urea metabolism in potato.Urease activity profile and patterns of recovery and distribution of(15)N after foliar urea application in wild-type and urease-antisense transgenics[J].Plant Physiol.,2002,128(3):1129-1136.
[9]Polacco J C,Mazzafera P,Tezotto T.Opinion-Nickel and urease in plants:Still many knowledge gaps[J].Plant Sci.,2013,199-200(3):79-90.
[10]Hausinger R P.Biochemistry of Nickel[M].Holland:Plenum Press,1952.
[11]Lee M H,Mulrooney S B,Renner M J,et al..Klebsiella aerogenes urease gene cluster:Sequence of ure D and demonstration that four accessory genes(ure D,ure E,ure F,and ure G)are involved in nickel metallocenter biosynthesis[J].J.Bacteriol.,1992,174(13):4324-4330.
[12]Mulrooney S B,Hausinger R P.Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation[J].J.Bacteriol.,1990,172(10):5837-5843.
[13]Farrugia M A,Wang B,Feig M,et al..Mutational and computational evidence that a nickel-transfer tunnel in Ure D is used for activation of Klebsiella aerogenes urease[J].Biochemistry,2015,54(41):6392-6401.
[14]Freyermuth S K,Bacanamwo M,Polacco J C.The soybean Eu3 gene encodes an Ni-binding protein necessary for urease activity[J].Plant J.,2000,21(1):53-60.
[15]Witte C P,Romeis T.Identification of three urease accessory proteins that are required for urease activation in Arabidopsis[J].Plant Physiol.,2005,139(3):1155-1162.
[16]Carter E L,Flugga N,Boer J L,et al..Interplay of metal ions and urease[J].Metallomics,2009,1(3):207-221.
[17]Marco S,Barbara Z,Francesco M,et al..A model-based proposal for the role of Ure F as a GTPase-activating protein in the urease active site biosynthesis[J].Proteins,2007,68(3):749-761.
[18]Cao F Q,Werner A K,Dahncke K,et al..Identification and characterization of proteins involved in rice urea and arginine catabolism[J].Plant Physiol.,2010,154(1):98-108.
[19]Jeon J S,Lee S,Jung K H,et al..T-DNA insertional mutagenesis for functional genomics in rice[J].Plant J.,2000,22(6):561-570.
[20]Liu Y G,Chen Y.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J].Biotechniques,2007,43(43):649-656.
[21]Witte C P,Medina-Escobar N.In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction[J].Anal.Biochem.,2001,290(1):102-107.
[22]Wilson K,Long D,Swinburne J,et al..A dissociation insertion causes a semidominant mutation that increases expression of TINY,an Arabidopsis gene related to APETALA2[J].Plant Cell,1996,8(4):659-671.
[23]Wu L,Di D W,Zhang D,et al..Frequent problems and their resolutions by using thermal asymmetric interlaced PCR(TAILPCR)to clone genes in T-DNA tagged mutants[J].Biotechnol.Biotechnol.Equip.,2015,29(2):260-267.
[24]Wang W H,Khler B,Cao F Q,et al..Molecular and physiological aspects of urea transport in higher plants[J].Plant Sci.,2008,175(4):467-477.
[25]Gerendás J,Sattelmacher B.Influence of N and Ni supply on nitrogen metabolism and urease activity in rice(Oryza sativa L.)[J].J.Exp.Bot.,1998,49(326):1545-1554.
[2]Liu L H,Ludewing U,Frommer W B,et al..At DUR3 encodes a new type of high-affinity Urea/H~+symporter in Arabidopsis[J].Plant Cell,2003,15(3):790-800.
[3]Liu L H,Ludewing U,Brigitte G,et al..Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis[J].Plant Physiol.,2003,133(3):1220-1228.
[4]Polacco J C,Holland M A.Roles of urease in plant cells[J].Int.Rev.Cytol.,1993,145:65-103.
[5]Sirko A,Brodzik R.Plant ureases:Roles and regulation[J].Acta Biochim.Pol.,2000,47(4):1189-1195.
[6]Wiebke-Strohm B,Ligabue-Braun R,Rechenmacher C,et al..Structural and transcriptional characterization of a novel member of the soybean urease gene family[J].Plant Physiol.Biochem.,2016,101:96-104.
[7]Todd C D,Polacco J C.Update on ureide degradation in legumes[J].J.Exp.Bot.,2006,57(1):5-12.
[8]Witte C P,Davies H V.Leaf urea metabolism in potato.Urease activity profile and patterns of recovery and distribution of(15)N after foliar urea application in wild-type and urease-antisense transgenics[J].Plant Physiol.,2002,128(3):1129-1136.
[9]Polacco J C,Mazzafera P,Tezotto T.Opinion-Nickel and urease in plants:Still many knowledge gaps[J].Plant Sci.,2013,199-200(3):79-90.
[10]Hausinger R P.Biochemistry of Nickel[M].Holland:Plenum Press,1952.
[11]Lee M H,Mulrooney S B,Renner M J,et al..Klebsiella aerogenes urease gene cluster:Sequence of ure D and demonstration that four accessory genes(ure D,ure E,ure F,and ure G)are involved in nickel metallocenter biosynthesis[J].J.Bacteriol.,1992,174(13):4324-4330.
[12]Mulrooney S B,Hausinger R P.Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation[J].J.Bacteriol.,1990,172(10):5837-5843.
[13]Farrugia M A,Wang B,Feig M,et al..Mutational and computational evidence that a nickel-transfer tunnel in Ure D is used for activation of Klebsiella aerogenes urease[J].Biochemistry,2015,54(41):6392-6401.
[14]Freyermuth S K,Bacanamwo M,Polacco J C.The soybean Eu3 gene encodes an Ni-binding protein necessary for urease activity[J].Plant J.,2000,21(1):53-60.
[15]Witte C P,Romeis T.Identification of three urease accessory proteins that are required for urease activation in Arabidopsis[J].Plant Physiol.,2005,139(3):1155-1162.
[16]Carter E L,Flugga N,Boer J L,et al..Interplay of metal ions and urease[J].Metallomics,2009,1(3):207-221.
[17]Marco S,Barbara Z,Francesco M,et al..A model-based proposal for the role of Ure F as a GTPase-activating protein in the urease active site biosynthesis[J].Proteins,2007,68(3):749-761.
[18]Cao F Q,Werner A K,Dahncke K,et al..Identification and characterization of proteins involved in rice urea and arginine catabolism[J].Plant Physiol.,2010,154(1):98-108.
[19]Jeon J S,Lee S,Jung K H,et al..T-DNA insertional mutagenesis for functional genomics in rice[J].Plant J.,2000,22(6):561-570.
[20]Liu Y G,Chen Y.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J].Biotechniques,2007,43(43):649-656.
[21]Witte C P,Medina-Escobar N.In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction[J].Anal.Biochem.,2001,290(1):102-107.
[22]Wilson K,Long D,Swinburne J,et al..A dissociation insertion causes a semidominant mutation that increases expression of TINY,an Arabidopsis gene related to APETALA2[J].Plant Cell,1996,8(4):659-671.
[23]Wu L,Di D W,Zhang D,et al..Frequent problems and their resolutions by using thermal asymmetric interlaced PCR(TAILPCR)to clone genes in T-DNA tagged mutants[J].Biotechnol.Biotechnol.Equip.,2015,29(2):260-267.
[24]Wang W H,Khler B,Cao F Q,et al..Molecular and physiological aspects of urea transport in higher plants[J].Plant Sci.,2008,175(4):467-477.
[25]Gerendás J,Sattelmacher B.Influence of N and Ni supply on nitrogen metabolism and urease activity in rice(Oryza sativa L.)[J].J.Exp.Bot.,1998,49(326):1545-1554.