碱茅铵转运蛋白（PutAMT1；1）基因的克隆及功能研究
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摘要
铵转运蛋白(ammonium transporters,简称AMT)基因在众多生物中被克隆与鉴定,它是一种广泛存在于微生物、植物细胞及动物的细胞膜上主动转运铵离子的载体,分子量约为48kDa,含有10-11个跨膜域.不同氮素条件下,铵转运蛋白基因通过转录调控表现了对铵离子吸收转运的不同特点,使植物根系在较宽的浓度范围中吸收铵离子,对作物能合理有效的吸收氮素,为农业生产粮食增收提供了有利保障。本研究从碱茅的cDNA文库中克隆得到了铵转运蛋白基因(PutAMT1;1)。对基因的表达特性、酵母亚细胞定位、酵母突变体中铵转运蛋白的功能进行了解析,结果如下:
1.测序结果表明,PutAMT1;1基因cDNA全长为2129 bp,ORF区域为1500 bp,编码499个氨基酸残基,通过软件预测分析表明,PutAMT1;1基因分子量为55.47kDa,等电点为5.28,属于酸性膜蛋白,具有11个跨膜域。氨基酸序列同源比较的结果,PutAMT1;1与TaAMT1;1具有92%的同源性,系统进化树分析的结果表明PutAMT1;1归属于铵转运蛋白AMT1亚家族。
2.PUtAMT1;1基因的表达特性分析的结果表明,PutAMT1;1基因在碱茅叶、根、茎、穗、穗茎、叶鞘、花药和花八个器官中都有不同程度的表达,其中,在叶和花中的表达微弱,在花药中表达最强。在不同NH_4~+浓度和不同时间处理下,PutAMT1;1基因在碱茅叶和根中的表达与处理条件均有一定的应答关系。
3.将重组质粒pYES2-GFP-PutAMT1;1转入酵母细胞,PutAMT1;1与绿色荧光蛋白融合,同时以GFP为对照,用2%半乳糖诱导该基因表达,通过激光共聚焦显微镜观察的结果表明,在酵母细胞中GFP-PutAMT1融合蛋白明显存在于细胞膜上。
4.将PutAMT1;1基因构建到酵母表达载体pYES2上,获得重组质粒pYES2-PutAMT1;1,将pYES2(对照)和pYES2-PutAMT1;1转入酵母突变体31019b中,通过酵母突变体吸收NH_4~+功能互补的实验表明,pYES2(对照)酵母与pYES2-PutAMT1;1酵母在含不同浓度NH_4~+培养基上生长趋势没有明显的差别,这个结果暗示了全长PutAMT1;1基因在酵母中表达也许没有发挥吸收转运铵离子的作用。
Ammonium transporters are active transport carriers of ammonium ion which widely present in cell membrane of microorganisms,plant and animal cells,with molecular weight of which are about 48kDa,containing 10-11 transmembrane domains.Ammonium transporter genes(AMTs) have been cloned and identified in many organisms.In plant roots,AMTs show different characters in transportation and absorption of ammonium ion through transcriptional regulation,through which plant roots can absorb ammonium ions at a wide concentration range. In crops,AMTs can contribute to absorb nitrogen more effectively,which provide a favorable protection for improving of agricultural production.In this study,the PutAMT1 gene was cloned from a Puccinellia tenuiflora cDNA library,the gene expression characteristic and the function of PutAMT1in yeast mutant as well as the subcellular location of PutAMT1in yeast was analyses.The results were as follows,
1.Sequencing analysis revealed that the full length eDNA of PutAMT1;1 was 2129 bp with a 1500 bp open reading frame(ORF) which encodes a protein with 499 amino acids.The putative structure of the PutAMT1;1 protein showed that the PutAMT1;1 was a 55.47-kDa protein with 11 hypothetical transmembrane domains.And the isoelectric points(IEP) of this membrane protein was 5.28.Homology comparison showed that the homology of the amino acid sequences between PutAMT1;1 and TaAMT1;1 was 92%.From the evolutionary trees,we found that PutAMT1;1 belongs to Ammonium transporter(AMT) proteins of the AMT1 subfamily.
2.Expression characteristic analysis of PutAMT1;1 showed the gene expressed in leaf,root, stem,fringe,earstem,leaf sheath,anther and flower in Puccinellia tenuifolra at different level, it expressed at a low level in leaf and flower,and the highest in anther.When treated with dif ferent concentration of NH_4~+ or different time course,PutAMT1;1 transcriptional expression showed some responsive relation with treatment condition in Puccinellia tenuifolra leaf and root.
3.Construct PutAMT1;1 into yeast expression vector pYES2,we obtain the recombinant plasmid pYES2-PutAMT1;1.The recombinant plasmid,pYES2-GFP-PutAMT1;1 was transferrred into yeast cells,with pYES2-GFP as a control.Expression of the fusion gene was induced using 2%galactose.Observing from the laser scanning confocal microscope,we recognize the fusion protein GFP-PutAMT1 obviously exist in the yeast cell membrane.
4.The plasmid pYES2(control) and pYES2-PutAMT1;1 were transferred into yeast mutant 31019b respectively.Functional complementation analysis of yeast mutant absorbing NH4_4~+ indicated that pYES2-PutAMT1;1 and control show no significant difference of growth tendency in medium supplied with different concentration of NH_4~+,which suggest that the expression of the full length PutAMT1;1 gene in yeast may can't play an important role in the NH_4~+ transportation.
1.测序结果表明,PutAMT1;1基因cDNA全长为2129 bp,ORF区域为1500 bp,编码499个氨基酸残基,通过软件预测分析表明,PutAMT1;1基因分子量为55.47kDa,等电点为5.28,属于酸性膜蛋白,具有11个跨膜域。氨基酸序列同源比较的结果,PutAMT1;1与TaAMT1;1具有92%的同源性,系统进化树分析的结果表明PutAMT1;1归属于铵转运蛋白AMT1亚家族。
2.PUtAMT1;1基因的表达特性分析的结果表明,PutAMT1;1基因在碱茅叶、根、茎、穗、穗茎、叶鞘、花药和花八个器官中都有不同程度的表达,其中,在叶和花中的表达微弱,在花药中表达最强。在不同NH_4~+浓度和不同时间处理下,PutAMT1;1基因在碱茅叶和根中的表达与处理条件均有一定的应答关系。
3.将重组质粒pYES2-GFP-PutAMT1;1转入酵母细胞,PutAMT1;1与绿色荧光蛋白融合,同时以GFP为对照,用2%半乳糖诱导该基因表达,通过激光共聚焦显微镜观察的结果表明,在酵母细胞中GFP-PutAMT1融合蛋白明显存在于细胞膜上。
4.将PutAMT1;1基因构建到酵母表达载体pYES2上,获得重组质粒pYES2-PutAMT1;1,将pYES2(对照)和pYES2-PutAMT1;1转入酵母突变体31019b中,通过酵母突变体吸收NH_4~+功能互补的实验表明,pYES2(对照)酵母与pYES2-PutAMT1;1酵母在含不同浓度NH_4~+培养基上生长趋势没有明显的差别,这个结果暗示了全长PutAMT1;1基因在酵母中表达也许没有发挥吸收转运铵离子的作用。
Ammonium transporters are active transport carriers of ammonium ion which widely present in cell membrane of microorganisms,plant and animal cells,with molecular weight of which are about 48kDa,containing 10-11 transmembrane domains.Ammonium transporter genes(AMTs) have been cloned and identified in many organisms.In plant roots,AMTs show different characters in transportation and absorption of ammonium ion through transcriptional regulation,through which plant roots can absorb ammonium ions at a wide concentration range. In crops,AMTs can contribute to absorb nitrogen more effectively,which provide a favorable protection for improving of agricultural production.In this study,the PutAMT1 gene was cloned from a Puccinellia tenuiflora cDNA library,the gene expression characteristic and the function of PutAMT1in yeast mutant as well as the subcellular location of PutAMT1in yeast was analyses.The results were as follows,
1.Sequencing analysis revealed that the full length eDNA of PutAMT1;1 was 2129 bp with a 1500 bp open reading frame(ORF) which encodes a protein with 499 amino acids.The putative structure of the PutAMT1;1 protein showed that the PutAMT1;1 was a 55.47-kDa protein with 11 hypothetical transmembrane domains.And the isoelectric points(IEP) of this membrane protein was 5.28.Homology comparison showed that the homology of the amino acid sequences between PutAMT1;1 and TaAMT1;1 was 92%.From the evolutionary trees,we found that PutAMT1;1 belongs to Ammonium transporter(AMT) proteins of the AMT1 subfamily.
2.Expression characteristic analysis of PutAMT1;1 showed the gene expressed in leaf,root, stem,fringe,earstem,leaf sheath,anther and flower in Puccinellia tenuifolra at different level, it expressed at a low level in leaf and flower,and the highest in anther.When treated with dif ferent concentration of NH_4~+ or different time course,PutAMT1;1 transcriptional expression showed some responsive relation with treatment condition in Puccinellia tenuifolra leaf and root.
3.Construct PutAMT1;1 into yeast expression vector pYES2,we obtain the recombinant plasmid pYES2-PutAMT1;1.The recombinant plasmid,pYES2-GFP-PutAMT1;1 was transferrred into yeast cells,with pYES2-GFP as a control.Expression of the fusion gene was induced using 2%galactose.Observing from the laser scanning confocal microscope,we recognize the fusion protein GFP-PutAMT1 obviously exist in the yeast cell membrane.
4.The plasmid pYES2(control) and pYES2-PutAMT1;1 were transferred into yeast mutant 31019b respectively.Functional complementation analysis of yeast mutant absorbing NH4_4~+ indicated that pYES2-PutAMT1;1 and control show no significant difference of growth tendency in medium supplied with different concentration of NH_4~+,which suggest that the expression of the full length PutAMT1;1 gene in yeast may can't play an important role in the NH_4~+ transportation.
引文
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[2]Gazzarrini S.,Lejay L.,Gojon A.,Ninnemann O.,Frommer W.B.,and Von Wire'n N.,1999,Three functional transporters for constitutive,diurnally regulated,and starvation -induced uptake of ammonium into Arabidopsis roots,Plant Cell,11(5):937-947.
[3]Ninnemann O.,Jauniaux J.C.,and Frommer W.B.,1994,Identification of a high affinity NH_4~+ transporter from plants,EMBOJ.,13(15):3464-3471.
[4]Suenaga A.,Moriya K.,Sonoda Y.,Ikeda A.,Von Wire'n N.,Hayakawa T.,Yamaguchi J.,and Yamaya T.,2003,Constitutive expression of a novel,type ammonium transporter OsAMT2 in rice plants,Plant Cell Physoil.,44(2):206-211.
[5]Gansel X.,Munos S.,Tillard P.,and Gojon A.,2001,Differential regulation of the NO_3~-and NH_4~+ transporter genes AtNrt2.1 and AtAmt1.1 in Arabidopsis:relation with longdistance and local controls by N status of the plant,Plant J.,26(2):143-155.
[6]Sonoda Y.,Ikeda A.,Saiki S.,Von Wire'n N.,Yamaya T.,and Yamaguchi J.,2003a,Disti -nct expression and function of three ammonium transporter genes(OsAMT1;1-1;3) in rice,Plant Cell Physoil.,44(7):726-734.
[7]Kaiser B.N.,Rawat S.R.,Siddiqi M.Y.,Masle J.,and Glass A.D.,2002,Functional analysis of an Arabidopsis T-DNA"knockout"of the high-affinity NH_4~+ transporter AtAMT1;1,Plant Physiol.,130(3):1263-1275.
[8]Lauter F.R.,Ninnemann O.,Bucher M.,Riesmeier J.W.,and Frommer W.B.,1996,Prefe -rential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato,Proc.Natl.Acad.Sci.,USA,93(15):8139-8144.
[9]Von Wire'n N.,Gazzarrini S.,Gojon A.,and Frommer W.B.,2000a,The molecular physicology of ammonium uptake and retrieval,Curr.Opin.Plant Biol.,3(3):254-261.
[10]Salvemini F.,Marini A.,Riccio A.,Patriarca E.J.,and Chiurazzi M.,2001,Functional cha -racterization of an ammonium transporter gene from Lotus japonicus,Gene,270(1-2):237-243.
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