Preliminary Study On Physiological Response And MAPK Signaling Pathway Under Nitrogen Stress Of Ulva Prolifera

Ulva prolifera has a complex life cycle and a variety of breeding methods.Coupled with its hollow tubular structure,it can cope with the stress of nature and make it one of the dominant green tide species.At present,the effects of natural stress on the growth and reproduction of U.prolifera are mainly focused on temperature,illumination and salinity.However,less research has been done on nutrient stress.Nitrogen,as the key nutrient element limiting the growth of U.prolifera,plays a decisive role in the growth of U.prolifera.It has the great research significance.However,in the current research,on the one hand,the influence of nitrogen elements only stays in the original basis of seawater with nitrate nitrogen or ammonium nitrogen,that is,the control of nitrogen concentration in seawater is not accurate,on the other hand,the research is limited to the detection of physiological indicators such as growth,and it does not go deep into the molecular level.The purpose of this experiment is to supplement the effect of nitrogen stress on the physiological metabolism of U.prolifera and to study the molecular mechanism of U.prolifera's response to nitrogen stress from the molecular levelTaking floating U.prolifera as the research object,this paper firstly supplemented the amplification of key enzyme genes in nitrogen assimilation.The process of nitrogen assimilation refers to the process in which plants absorb nitrate and ammonium nitrogen from the environment and convert them into nitrogenous compounds such as amino acids and proteins.Nitrate assimilation involves four key enzymes:Nitrate Reductase(NR),Nitrite Reductase(NiR),Glutamine Synthetase(GS)and Glutamate Synthase(GOGAT),of which NR has obtained the full length This experiment is carried out in accordance with the laboratory transcriptome data,and,for the first time,the researcher found the cDNA sequence of GS1(1143 bp,GenBank accession number,MN496139);total cDNA amplified with Fd-NiR(1698 bp,GenBank accession number,MN496140);partial CDS area amplified with Fd-GOGAT(2627 bp,GenBank accession number,MN496141).And these sequences were analyzed by protein physicochemical properties and three-dimensional structure.This experiment complemented the vacancy of U.prolifer a in the gene cloning of nitrogen assimilation key enzyme.It is a preparation for further study on the expression regulation of key enzymes in nitrogen assimilation under nitrogen stress/enrichment conditions.Primers were designed based on the amplified key enzyme sequence of nitrogen assimilation,and qRT-PCR experiments were performed to detect the growth and chloroplast content changes of U.prolifera.The purpose was to study the growth status and expression of key enzymes for nitrogen assimilation of U.prolifera under nitrogen stress.The study found that under the condition of nitrogen deprivation,the relative growth rate and chlorophyll content of U.prolifera will decrease,but it will still grow steadily in the short term.The expression of key nitrogen assimilation enzymes also increased slightly after a certain steady expression.Combined with other studies,it is possible that U.prolifera uses stored pigments and proteins as internal nitrogen sources in a short period of time to meet basic physiological and metabolic needs during nitrogen deficiency in order to survive the period of nitrogen deficiency.In order to study the role of MAPK signaling pathway in the response of U.prolifera to nitrogen stress,SB239063 inhibitor was selected to inhibit p38MAPK ?/?,and its protoplasts were observed for mortality and cell wall regeneration.First of all,by adjusting the composition of protoplast culture medium,it was found that the survival rate and cell wall regeneration rate of U.prolifera were increased under the condition of nitrogen enrichment,while the environment of nitrogen deficiency was the opposite.Secondly,Western blotting was used to detect the phosphorylation level of MAPK under nitrogen stress.It was found that nitrogen deprivation could induce the phosphorylation of MAPK,and the phosphorylation level increased with the extension of treatment time.Subsequently,Western blotting was used to detect the inhibition of SB239063 inhibitor on MAPK phosphorylation.It was found that the inhibition concentration was significantly determined by the decrease of MAPK phosphorylation level under the treatment of 10?m inhibitor SB239063,and the inhibitory action concentration was determined accordingly.Finally,SB239063 was used to inhibit the MAPK signal pathway of protoplasts,and the results of their mortality and cell wall regeneration were counted.From the results,it can be inferred that under the condition of nitrogen deprivation,MAPK regulates downstream factors through phosphorylation response,which reduces the mortality and regeneration rate of protoplasts,slows down the physiological and biochemical processes of cells,and reduces consumption to survive the adverse environment.It explains why U.prolifera did not die and continue to proliferate in the sea when nitrogen is deficient.This experiment is an attempt of MAPK signal pathway in response to abiotic stress of U.prolifera.At the same time,it paves the way for further study on the mechanism of signal pathway response to adverse environment in U.prolifera.