Cloning And Fusctional Verification Of Key Enzyme Genes For Nitrate Assimilation In Zostera Marina L.

As a typical representative of seagrass,Zosteria marina L.is separated from terrestrial monocotyledons and then returned to the marine.In order to adapt to the completely immersed marine environment,its genome,morphological characteristics,physiological processes and reproduction modes all have huge changes during the evolution.Based on its unique evolutionary status and extremely high salt tolerance,and being the first marine angiosperm to be completely sequenced,Zosteria marina has become a representative species for studying the salt adaptation mechanisms of plants.The transcriptome analysis of Zosteria marina in our previous work found that the expression of key enzymes in the nitrate assimilation process were significantly upregulated after NaCl treatment.The results of qRT-PCR analysis of nitrate reductase(NR)and nitrite reductase(NiR),the key enzymes in the process of nitrate assimilation,also showed that the transcripts levels of these two genes increased significantly under high salinity.In subsequent physiological experiments,we further found that the growth of eelgrass was seriously affected after the process of nitrate assimilation was blocked.All these previous experimental results indicate that the process of nitrate assimilation is involved in high salt stress resistance,but still lack molecular evidence.In order to identify the role of the key enzymes in nitrate assimilation to affect salt tolerance,ZmNR and ZmNiR genes,were cloned from Zosteria marina,and investigated the biological functions of ZmNR and ZmNiR in response to NaCl stress by heterologous expression these genes in Bacillus coli(Escherichia coli)and tobacco(Nicotiana benthamiana)using molecular biology and transgenic technology.Finally,salt tolerance phenotype was identified with physiological experiments.The main research contents were as follows:1.The ORF of ZmNR and ZmNiR genes were cloned from the eelgrass leaves by RTPCR,which were inserted into the pEASY-BluntE1 expression vector and transformed in E.coli Rosetta(DE3)to obtain the recombinant strains of ZmNR and ZmNiR,respectively.After the recombinants were induced by IPTG,the expressed protein was analyzed by SDS-PAGE and the enzyme activity was detected.The results showed that the target genes were successfully expressed in E.coli.The growth rates of ZmNR and ZmNiR recombinant strains in normal and salt stress medium were higher than those in the control group,which indicated that the expression of ZmNR and ZmNiR genes in E.coli improved its salt tolerance.2.Using the tobacco as explant,ZmNR and ZmNiR genes were introduced into tobacco by Agrobacterium mediated-transformation method,and the homozygous transgenic lines of T2 generation were obtained by PCR and enzyme activity detection,respectively.The results of salt tolerance analysis of transgenic plants showed that ZmNR gene increased root length,plant height,chlorophyll content,proline content and decreased malondialdehyde(MDA)content of transgenic tobacco under normal nutrient and salt solution.While ZmNiR gene has only a weak promoting effect in the above indexes,which may be related to its lower enzyme activity,but it significantly increases the activity of antioxidant enzymes(SOD,CAT).This demonstrated that ZmNR and ZmNiR genes improve the tolerance of transgenic tobacco to salt stress in different mechanisms.3.To investigate whether NO is involved in ZmNR and ZmNiR genes-triggered plant tolerance against NaCl stress,we also determined endogenous NO levels in transgenic and wild type leaves.The results showed that there was no difference in the NO content among the three lines in normal nutrient solution,but the NO content of ZmNR transgenic lines was higher than that of wild-type tobacco under salt stress,and there was no significant difference between ZmNiR transgenic line and wild-type tobacco.It suggests that the mechanism of eelgrass ZmNR gene involved in the process of salt tolerance in tobacco may be related to the mediation of NO signaling,but it is not the only regulatory pathway.In summary,this study confirmed the conclusion that the nitrate assimilation process of eelgrass plays an important role in eelgrass responses to NaCl stress at molecular level.It not only provides a theoretical basis for further understanding the molecular mechanism of salt-tolerance in plants,but also provides potential molecular genetic targets for breeding plants or crops resistant to salt stress through genetic engineering.It also has important practical significance for the future development of marine industry and the cultivation of terrestrial salt-tolerant crops.