The Role Of Suaeda Salsa SsNRT1.7 In The Nitrate Remobilization

Suaeda salsa L.is an annual euhalophyte with high salt tolerance.The NO₃^-content in the soils where S.salsa from intertidal zone and inland saline soils occurs is low,especially for that in intertidal zone.However,it is not clear how the two populations adapt to fluctuating low nitrogen environments.This study revealed the physiological and molecular mechanisms for the adaptation to low nitrogen environments in two S.salsa populations from different saline environments.The results are as follows:1.Effect of N starvation on NO₃^-remobilizationSeedlings of two S.salsa populations were precultured with 1 mM NO₃^--N under200 mM NaCl condition.After the seedlings were cultured with 1 mM NO₃^--N and N-free solution?N starvation?for additional 14 days,the expression of the gene related to NO₃^-remobilization SsNRT1.7,NO₃^-and chlorophyll content,and net photosynthetic rate were determined.The expression of SsNRT1.7 in the old and mature leaves was upregulated markedly during N starvation in the intertidal population compared to the inland population,but it was not the case in the young leaves.After N starvation,the decrease of NO₃^-and chlorophyll content,net photosynthetic rate in the young leaves and shoot dry weight in the intertidal population were lower than those in the inland population.SsNRT1.7 may play more important role in NO₃^-remobilization in the old or mature leaves in the intertidal population compared to the inland population during N starvation.As a result,more NO₃^-in the young leaves can better maintain photosynthesis in the intertidal population than the inland population in fluctuating low nitrogen environments.2.The clone of SsNRT1.7Based on the gene NRT1.7 obtained from the sequence in the transcriptome of S.salsa,the intermediate sequence was cloned?temporarily named SsNRT1.7?.The sequence was analyzed,and the results showed that the sequence was 81%similar to the NRT1/PTR FAMILY2.12 protein in Beta vulgaris L.Based on this sequence,SsNRT1.7 was amplified by 3'RACE and 5'RACE techniques.The full length of the gene is 2375 bp and the CDS region is 1851 bp.The coding region shares 55.83%homology with AtNRT1.7 in Arabidopsis.3.Bioinformatics analysis and subcellular localization of SsNRT1.7The gene SsNRT1.7 encodes 617 amino acids.The physicochemical properties of the protein were analyzed.The protein is a hydrophobic stable protein.The amino acid sequence was analyzed by SMART and the PTR2 conserved domain was presented.The transmembrane structure of the protein was analyzed by TMHMM software,and the protein has 12 transmembrane domains.The phylogenetic analysis of the protein sequence of this gene was carried out using MEGA software,and it was found that the protein was closely related to Beta vulgaris L.,Spinacia oleracea L.and Chenopodium quinoa.SsNRT1.7 gene was ligated into the expression vector pCAMBIA1300-35S-sGFP.The tobacco leaves was transiently transformed by agrobacterium,and the fusion of SsNRT1.7 and GFP in tobacco epidermal cells was observed by two-photon laser scanning confocal microscopy.The expression showed that the cells of the empty vector pCAMBIA1300-35S-sGFP were covered with green fluorescence,while the tobacco epidermal cells transfected with SsNRT1.7 showed only green fluorescence in the cell membrane,which indicates that SsNRT1.7 was localized in the cell membrane.4.Screening the Arabidopsis AtNRT1.7 mutant and SsNRT1.7 transgenic homozygous linesSALK₀22429 Arabidopsis mutant strains were selected and the double primer method was used for identification,then a homozygous SALK₀22429 plant was obtained.The SsNRT1.7 gene was ligated into the expression vector pCAMBIA1300-35S-sGFP.The agrobacterium-transformated homozygous mutant plants and WT plants were infested,and over-expressed and replenished plants were obtained.The positive plants were screened by hygromycin resistance,and in the DNA level was verified.Finally,homozygous overexpression and replenishment lines were obtained,and these plants with higher expression were screened according to the results of real-time PCR.