Expression,Localization And Function Analyses Of High-affinity Nitrate Transporter(CsNRT2.1) In Cucumber

Nitrogen(N)is a macronutrient that plays a crucial role in plant growth,development and productivity,it is also a component of nucleotides,amino acids,chlorophyll and phytohormone.Plants can acquire N through their roots from the soil under nitrate(N03--N),ammonium(NH4+-N)and peptides froms.In general,NO3--N has much higher value compared with NH4+ in aerobic agricultural soils.Therefore,N03-is the the major N source for higher plants.NO3-availability in the soil varies drastically depending on various factors including soil physical properties and microbial activity.To cope with the temporal and spatial fluctuation of the soil NO3-,higher plants have evolved high-and low-affinity NO3-transport systems(HATS and LATS,respectively)to ensure efficiency of the uptake of NO3-into roots.At the molecular level,NRT1/PTR(NPF)and NRT2 gene family encode the LATS and HATS,respectively.Cucumber(Cucumis sativus L.)is an important vegetable crop cultivated in China,and the greenhouse is the major horticultural facilities for cucumber growth.However,the relatively monotonous planting structure causes soil degradation and continuous cropping obstacles to limited plant growth.Unfortunately,farmers often apply excessive N fertilizers to obtain high yields.Consequently,redundant N can release to the environment through leaching,runoff and emission,which caused serious environmental problems,such as soil degradation,freshwater contamination,and natural resource consumption.The study of cucumber nitrogen was restricted to physiolocical level.Therefore,study of molecular mechanism of cucumber NO3-absorption has important theoretical and practical significance for guiding efficient fertilization and breed improvement.In this study,we isolated and elucidated the expression,location,and function of cucumber NRT2.1 gene(CsNRT2.1)through the combination of phylogenetic analyses,quantitative real-time polymerase chain reaction,greenhou fluorescent protein fusion protein,and 15N stable isotope tracer technique.The mainly results are as follows:1.A putative cDNA sequences encoding CsNRT2.1(GeneBank Accession No.MH213459)was isolated from cucumber roots by BLAST and RT-PCR.It contained a 1593 bp open reading frame(ORF)consists of three exons and two introns,encodes a protein composed of 530 amino acids.The protein sequence alignment predicted that CsNRT2.1 is expressed in the cytoplasmic membrane with 12 transmembrane domains.CsNRT2.1 had typical N037 NO2-transporter and MFS family structure.The expression analysis of CsNRT2.1 in different plant organs showed that CsNRT2.1 was mainly expressed in mature roots and apical meristem,with a small amount of expression in stem,male flower,female flower and young leaf.2.N treatment of cucumber seedlings with different concentrations,forms and sample times indicated that the expression of CsNRT2.1 had a dual regulation.Both N-depleted and N03--repleetd could induced the expression of CsNRT2.1,and the induction showed a same tendency of first increasing and then decreasing,while NH4+ as a N metabolite had a strong inhibition effect on the expression of CsNRT2.1.3.Immunolocalization analysis indicated that CsNRT2.1 was mainly localized in the root meristem and epidermal cells of mature regions.Subcellular localization of CsNRT2.1 in Arabidopsis mesophyll protoplasts and tobacco leaf epidermal cell indiacted that CsNRT2.1 was localized in the plasma membrane.4.NO3-influx of WT and the two RNAi lines was determined by using 15N stable isotope tracer technique.The results showed that decreasing the knock-down of CsNRT2.1 resulted in a signicicantly decrease of the NO3-uptake at low NO3-concentration(10-500 P M),but no difference at high NO3-conditions(1,5,10 and 20 mM).It suggested CsNRT2.1 is a typical high affinity N03-transporters.5.Compared with WT,the decrease gene expression of CsNRT2.1 in transgenic plants significantly affected the root morphology of cucumber seedlings at 0.5mm NO3-,which mainly exhibited the decrease of main root length,lateral root length and number of the secondary lateral root.