Preliminary Study On The Physiological Changes And Regulatory Mechanism Of Neopyropia Yezoensis Under Different Salinity Conditions

The most important marine resource crop Neopyropia yezoensis,habitats in the intertidal environment,the borderline of ocean and land.During day time it is exposed to the sea and light for a large amount of time due to tidal action of sea.The evaporation from water surface leads to rise in the salt concentration of associated water with N.yezoensis.In addition,due to heavy rain the water level becomes high which reduces the salt concentration by flushing away the surface water of the thallus.In order to overcome these environmental effects and further improvement in the economic value of N.yezoensis through genetic modification,experimentally it was evaluated to explore the effect of salinity stress on the normal growth and development of N.yezoensis to build a "blue granary".A salt-tolerant gene,SnRK,was extracted from N.yezoensis and analyzed by exploring the regulatory mechanism of oxidative damage by salt stress in N.yezoensis.By measuring the content of phycobilin and photosynthetic pigment in thallus,we found that under 0‰,15‰,70‰ and 110‰ salinity stresses showed an up-level as compared with the control group under 30‰ salt concentration without damage to the physiological system of N.yezoensis.In addition,to weight the N.yezoensis under different seawater salt concentrations and calculation of its relative growth,the experimental results showed that N.yezoensis could only maintain a high relative growth rate under the normal seawater concentration of 30‰,and vice versa in other salinity stress.Furthermore,the growth and regeneration of protoplasts of phyllode cells of Porphyrophyllacordiformis was observed under seawater salt concentration of 30‰,it was concluded that the protoplasts cultured in 30‰ salinity could survive and develop into phyllode seedlings.In this study,we used the existing nitrogen-enriched transcriptome data of N.yezoensis in our laboratory,combined with KEGG,GO,Siwss-Prot and other databases to successfully excavate the plant sucrose non-fermentation-related protein kinase(SnRK)gene related to stress and salt tolerance in N.yezoensis,and designed primers to clone its CDS full-length.The full-length CDS sequence of SnRK gene is 2745bp,which has been uploaded to the NCBI Genebank database under the accession number OP852655.The related bioinformatics analysis of the gene was carried out and the results showed that the plasmid empty yeast had no growth trend at 1.3M NaCl concentration,while the SnRK-transformed yeast could still grow at this concentration.This concluded that the SnRK gene in N.yezoensis does have a salt tolerance function.At the same time,real-time fluorescence quantitative PCR was performed on N.yezoensis under 0‰,15‰,30‰,70‰ and 110‰ seawater salt concentrations.The results showed that the expression of SnRK gene in N.yezoensis was significantly increased after 2 hours of salt stress compared with the control group(P<0.001),and the change trend of its expression was approximately increased first and then decreased.According to oxidative stress,in order to explore the content changes of related substances in N.yezoensis when it experiences salinity stress so as to preliminarily explore its regulatory mechanism,this experiment used HPLC-MS/MS to measure the change of ABA content in N.yezoensis under salt stress.The results showed that the content of ABA in N.yezoensis was basically unchanged under the salinity of the control group 30.However,in the 0 salinity group,the difference was extremely significant at 6h(P<0.0001),and the peak value of ABA content at 12h was 18.02 times that of the control group.The peak value of ABA content in salinity group at 12h was 6.93 times higher than that in control group.The peak content of the 70 salinity group at 12h was 11.54 times higher than that of the control group.The content of the 110 salinity group also showed a continuous upward accumulation trend,and the peak content at 12h was 16.57 times that of the control group.In addition,the expression level of SnRK gene,which has the function of salt tolerance,was significantly increased at the early stage of N.yezoensis stress after ABA treatment.At the same time,we used DCFH-DA probe method to analyze the change trend of ROS in N.yezoensis under salt stress.The results showed that the content of ROS in the 0 salinity experimental group was 14.21 times that in the 30 salinity control group after 24 hours,and the content of ROS in the 15 salinity experimental group was 11.44 times that in the control group after 24 hours.At the salinity of 70,the content was 14.93 times higher than that of the control group at 24h.At the salinity of 110,the content was 16.57 times higher than that of the control group at 24h.In conclusion,salinity stress led to the continuous accumulation of ROS in the leaf body,causing oxidative stress response in N.yezoensis.When ABA was added,the activities of antioxidants CAT,GSH,SOD and APX in N.yezoensis were significantly increased compared with those without ABA.For the first time,we cloned a salt-tolerant SnRK gene in N.yezoensis,demonstrated its function in salinity tolerance and found that the SnRK gene does respond to salinity stress.Although N.yezoensis has a certain tolerance to salinity stress in the short term,it is still subjected to oxidative stress damage.At this time,N.yezoensis will increase the content of ABA to enhance the activity of related antioxidants to resist oxidative damage.Moreover,it could up-regulate the expression of SnRK gene,which has the function of salinity tolerance,to further enhance the salinity tolerance of N.yezoensis.