Gene Clone And Function Analysis Of Puccinellia Tenuifolra Ammonium Transporters (PutAMT1;1)

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 NH₄⁺ 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 NH4₄⁺ indicated that pYES2-PutAMT1;1 and control show no significant difference of growth tendency in medium supplied with different concentration of NH₄⁺,which suggest that the expression of the full length PutAMT1;1 gene in yeast may can't play an important role in the NH₄⁺ transportation.