A Study On The Effect Of Nitrate Assimilation On The Salt Tolerance Of Zostera Marina

Zostera marina is a representative species of perennial seaweed and an important halophyte widely distributed in the northern hemisphere.It evolved from terrestrial photosynthetic organisms re-entering the ocean during evolution,and its unique evolutionary history resulted in a set of special salt tolerance mechanisms.Its salt tolerance is strong,and its evolutionary status is far higher than other halophytes in the ocean(such as algae),so it is a good material for studying the salt tolerance mechanism of higher plants.It was found that nitrate assimilation changes most significantly among the key genes and metabolic pathways that might be involved in Z.marina salt tolerance through the transcriptome analysis in the early stage of the research group.In this study,bioinformatics methods,RT-PCR,physiological and biochemical techniques were used to study the molecular response and physiological response of two key enzyme genes:nitrate reductase gene(NR)and nitrite reductase gene(NiR)to salt stress during the nitrate assimilation of Z.marina,,exploring the effect of nitrate assimilation on salt tolerance of Z marina.The research contents of this thesis are mainly divided into three aspects:1.Bioinformatics analysis of NR and NiR in nitrate assimilation processSequence analysis results showed that the coding sequence of NR and NiR was 2628 bp and 1773 bp,encoding 875 and 590 amino acids,with molecular weights of 98.988 kDa and 65.958 kDa,respectively.It was mainly composed of C,H,O and N elements,in addition to a small amount of S elements.The base composition of NR and NiR was similar,and the content of G+C was slightly lower than that of A+T,which indicated that NR and NiR had similar DNA density and thermal stability.The isoelectric points of NR and NiR were 6.35 and 6.46 respectively.The instability indices of NR and NiR were 39.30 and 43.49 respectively.The total average hydrophobicity of NR and NiR were-0.375 and-0.314,respectively,which were hydrophilic proteins.Both of them had similar secondary structures,which were consisted of three forms:alpha helix,straight chain extension and random curl and there was no beta folding.Transmembrane analysis showed that both NR and NiR were non-transporter proteins without transmembrane distribution.The prediction of subcellular localization showed that NR was mainly distributed on cytoplasm,chloroplast membrane,mitochondria and nuclear membrane,while TNiR was mainly distributed on chloroplast membrane,mitochondria and nuclear membrane.The sequence similarity of NR gene of Z.marina was less than 65%and that of NiR gene was less than 74%.It had low affinity with the studied species.This indicated that the NR and NiR of Z.marina had some evolutionary similarities,low homology with other species and high evolutionary specificity.2.Analysis of expression patterns of the key enzymes gene NR and NiR of nitrate assimilation under salt treatmentPrevious studies had shown that nitrate assimilation might affect the salt tolerance of Z.marina.In this study,the effects of different concentrations of sodium chloride on the activity and expression pattern of NR and NiR were investigated by RT-PCR.When the salinity was 10%,the expression levels of NR and NiR genes fluctuated with the increase of treatment time,and the change was not significant.When the salinity was 20‰,the expression of NR and NiR was induced.The highest expression of NR was about 2-2.5 times of the initial expression(t=0.5h),and that of NiR was 4-5 times of the initial expression(t=3h).When the salinity was 40‰,the expression of NR and NiR genes showed different degrees of up-regulation with the increase of treatment time,and the expression of NR increased as a whole,and the highest expression was about 5-7 times of the initial expression(t=6h),that of NiR can reach 13-16 times of the initial expression(t=6h).This indicated that the gene expression of NR and MiR,two key genes in nitrate assimilation,was insensitive to low salinity and did not respond positively.NR and NiR genes began to be induced to express with the increase of salinity.In the high salt environment,the gene expression of NR and NiR was very active,and the response to high salinity was very positive.Therefore,nitrate assimilation of Z.marina is closely related to its salt tolerance.3.Effect of different nitrogen forms on salt tolerance of Z.marinaIn this study,different forms of nitrogen and nitrate reductase inhibitor Na2WO4 were added to explore the effect of Z.marina nitrate assimilation on physiology and growth.The results showed that the growth rate of leaf length,leaf width,fresh weight,chlorophyll and proline content of mixed nitrogen source culture were higher than those of single ammonium nitrogen source culture in Z.marina,and the difference was reduced by adding Na2WO4,while the change of MDA content was opposite.This indicated that the addition of nitrate nitrogen source enhanced the salt tolerance of Z.marina to some extent,while the presence of nitrate reductase inhibitor Na2WO4 reduced the salt tolerance of Z marina,indicating that the nitrate assimilation process enhanced the salt tolerance of Z.marina.Compared with single nitrate nitrogen source culture,adding Na2WO4 decreased the growth rate of leaf length,leaf width,fresh weight,chlorophyll and proline content,and increased the content of malondialdehyde in Z marina.This indicated that the effect of nitrate nitrogen source on salt tolerance of Z.marina was not through nitrate signal,but possibly through the key enzymes in nitrate assimilation process to influence salt tolerance of Z marina,which provided some clues for further study of the molecular mechanism of salt tolerance of Z.marina.