Effect Of Salinity On Nitrate Accumulation And Reduction In Suaeda Salsa

The mechanisms of NO₃^- accumulation and reduction in the euhalophyte Suaeda salsa were investigated under high salinity and low nitrate conditions by transcriptome sequencing and quantitative real-time PCR combined with the determination of certain physiological indexes.The aim of the current study was to provide a theoretical basis for the understanding of nitrogen nutrition in halophytes.The results are as follows:1.The effects of different concentrations of Na Cl on nitrate accumulation and reduction of S.salsaSix genes which may be related with NO₃^-accumulation or reduction were selected from the results of transcriptome sequencing,including genes of CLC and NR.The real-time PCR results showed that at 0.5 m M NO₃^-,the relative expression of CLC-b and CLC-c2 were higher at 200 m M Na Cl than 0 m M Na Cl,but 200 m M Na Cl had no significant effect on the expression of CLC-c1.The relative expression of CLC-b decreased at 400 m M Na Cl,but the expression of CLC-c1 and CLC-c2 were higher than 0 m M Na Cl.Compared to control,the relative expression of NR-1 and NR-2 were higher,but the value for NR-3 was lower at 200 m M Na Cl.The relative expression of NR-1 and NR-3 decreased,but the value of NR-2 increased at 400 m M Na Cl compared to 0 m M Na Cl.At the concentration of 0.5 m M NO₃^-,200 m M Na Cl increased the NO₃^-content and NR activity in the leaves of S.salsa,but 400 m M Na Cl decreased the value.The trends of NO₃^-content and NR activity was consistent with the results of real-time PCR in CLC-b and NR-1.In the present study,the gene of CLC-b which may be related to NO₃^-accumulation in S.salsa was investigated?temporarily named Ss CLC-1?.Compared to 0 m M Na Cl,200 m M Na Cl had no adverse effect on the content of chlorophyll,free amino acids and soluble protein,while 400 m M Na Cl decreased the chlorophyll content,but increased the contents of free amino acids and soluble protein in the leaves of S.salsa.The current results indicate that S.salsa has high capacity to storage NO₃^-in the leaves,and high ability in NO₃^-reutilization to maintain nitrogen metabolism under low nitrate and high saline conditions.2.Cloning of Ss CLC-1 from S.salsaAccording to the sequences of CLC-b from former transcriptome sequencing and the results of the blast,two primers were designed to amplify specific DNA fragment using c DNA prepared from S.salsa leaves by PCR.The 5' and 3' fragment of the c DNA was isolated by 5' and 3' RACE.Then the full length was cloned by two specific primers.The c DNA sequence of Ss CLC-1 was 2,427 bp which was deduced to encode a peptide of 808 amino acids whose predicted molecular mass was 8.9 k Da and isoelectric point was 8.38,including a total of 12,717 atoms.The molecular formula is C4056H6429N1053O1144S35.The primary structure of the protein showed that the gene encoded 73 acidic amino acids,78 basic amino acids,326 hydrophobe amino acids,and 254 polar amino acids.The protein had an unstable index of 50.67 and a fat index of 97.83.Therefore,the protein is classified as stable protein.The hydrophobicity and hydrophilicity scale indicate it is a hydrophilic protein.The protein secondary structure included 43.81% Random coil,33.42% Alpha helix,and 22.77% Extended strand.By sequence homologous comparison,the c DNA sequence of Ss CLC-1 showed the highest identity with CLC-1 from Suaeda altissima,i.e.,the homology was 95%.The homology of the sequence for CLC-b of Beta vulgaris,and CLC-a of Populus euphratica was 87% and 81%,respectively.The function of Ss CLC-1 should be further investigated.