Effect Of Salinity On Nitrate Uptake Of Suaeda Salsa

For the purpose of understanding the molecular and physiological mechanisms related to NO₃^--N uptake and transport in the euhalophyte Suaeda salsa which has high salinity tolerance and important economic value,transcriptom analysis and Non-invasive Micro-test Technology were performed at high salinity and low NO₃^-concentration in this study.The results are as follows: 1.Analysis and validation of transcriptome sequencing of S.salsa seedlings under high salinity and low nitrogen conditionsAfter cultured for one month,seedlings of S.salsa was subjected to high salinity?100 m M increasing to 300 m M NaCl?or control condition?0 m M NaCl?and then to perform transcriptom analysis.The results are as follows: A total of 41.63 gigabases?GB?clean base number were generated from the sample c DNA libraries with 85% at the Q30 level?an error probability of 0.1 %?.After de novo assembly,a total of 91,957 unigenes were obtained,and there were 25,264 unigenes with length > 1000 bp.We gained 10,915 SSR markers based on the gene structure analysis of unigene database,including ORF prediction,SSR analysis,and SNP analysis of samples.According to the bioinformatics annotations of GO,COG,Swiss-Prot,NR and KEGG database comparison,53,186 unigenes were obtained,and 8,840 differentially expressed genes?DEGs?were tested out in the roots of S.salsa by RNA sequencing?RNA-seq?when plants were treated with 300 m M NaCl?vs.0 m M?at low NO₃^-levels.Among these DEGs,6,686 genes were up-regulated and 2,154 genes were down-regulated.Interestingly,some key genes related to NO₃^-uptake?NRT2.1,NRT3.1 and several genes related to plasma membrane H+-ATPase?and transport?NRT1.5?were up-regulated by salinity.Therefore,we speculate that the accumulation of NO₃^-in the leaves of S.salsa under high salt and low nitrogen conditions may be due to the up-regulate of these key genes.Simultaneously,sixteen DEGs were selected and quantitative real-time PCR were performed and then the reliability of the RNA-seq data were confirmed,indicating that all these RNA-seq data can be used for further analysis.2.The effect of NaCl on nitrate uptake of S.salsaKey genes related to NO₃^-uptake?NRT2.1,NRT3.1 and gene related to plasma membrane H+-ATPase?and transport?NRT1.5?were up-regulated by salinity under low NO₃^-levels?0.5 m M NO₃^--N?,and the up-regulation was particularly significant at 200 m M NaCl.Meanwhile,dry weight of aboveground and underground of S.salsa were increased significantly at 200 m M NaCl.In the present study,200 m M NaCl increased while 400 m M NaCl did not decrease the concentration of NO₃^-in S.salsa roots and xylem sap.In addition,the NMT analysis showed that NO₃^-fluxes were generally similar in the roots treated with different concentrations of NaCl?0 and 200 m M?.Compared with 0 m M and 200 m M NaCl,400 m M NaCl tended to induce NO₃^-efflux more significantly from S.salsa roots.This indicates that 200 m M NaCl may promote the transport of NO₃^-from S.salsa roots to shoots and not significantly inhibit NO₃^-uptake.This can confirmed our hypothesis that S.salsa can efficiently absorb and transport NO₃^-under high soil salinity and low NO₃^-conditions.This trait may partly explain why S.salsa and other euhalophytes not only tolerate high salinity but also obtain their highest biomass when treated with 200-300 m M NaCl,i.e.,when treated with salt concentrations that would kill normal crops.3.Preliminary clone of the gene Ss NRT2.1 related to high-affinity NO₃^-transporter protein of S.salsaPrimers were designed based on the sequence of NRT2.1?2346 bp?which was related to high-affinity NO₃^-transporter protein and produced by the transcriptome sequence of S.salsa,and to amplify specific DNA fragment using c DNA prepared from S.salsa roots treated with 200 m M NaCl by PCR.Three middle fragments have been obtained,and the blast results on the NCBI are as follows: The first middle fragment was similar to the homologous genes of Chrysanthemum,Amborella trichopoda,Nicotiana tabacum and Populus euphratica and the homology is as high as 94%,indicating that the fragment is likely to be the middle fragment of S.salsa NRT2.1?temporarily named as Ss NRT2.1?.The second middle fragment was similar to the homologous genes of Brachypodium distachyon,Setaria italica,Cicer arietinum and Gossypium arboretum and the homology is as high as 96%,but most function of them was not sure.However,the third middle fragment was predominantly similar to the NRT2-related genes,such as Ricinus communis,Physcomitrella patens,Jatropha curcas,Sesamum indicum,Populus euphratica,Fragaria vesca and Nicotiana attenuata,and this fragment need to be further studied.At present,RACE is widely used to clone the full-length of gene,and its operation is simple.Therefore,the middle fragment of S.salsa NRT2.1 was used to obtain the full-length of Ss NRT2.1 by RACE,and then to determine the function of this gene.