Function Evolution Of Glutathione S-transferase Gene Family From Ulva Prolifera

Since 2007,the yellow sea green tide has been erupting for 12 consecutive years,causing great harm and economic loss to the Marine ecological environment and coastal residents.Ulva prolifera is the dominant species of the yellow sea green tide.Dominant species gain competitive advantage by showing obvious adaptability to a wide range of irradiance,temperature and salinity,or physiological adaptability to various environmental conditions.One of the characteristics of plant antioxidant system is to show the growth characteristics of broad temperature and broad salt by improving its tolerance to the environment.Ulva prolifera has the growth characteristics of wide temperature and wide salt,so we selected glutathione transferase(GST),an important member of the antioxidant system,from the aspect of plant antioxidant stress.The expression of glutathione transferase in Ulva prolifera was studied to determine whether glutathione transferase in Ulva prolifera was involved in antioxidant stress to improve Ulva prolifera's tolerance to adverse environment.Glutathione S-transferase is a kind of protein superfamily widely existing in organisms,which plays an important role in detoxification and stress tolerance.However,previous studies on GST mainly focus on animals and higher plants,and there are few studies on algae.There has been no report on Ulva prolifera.In this study,Ulva prolifera was taken as the research object.Through obtaining GST gene sequence,bioinformatics analysis of gene and protein sequence,protein expression purification and enzyme activity determination,molecular evolution,gene expression and protein function analysis of Ulva prolifera GST gene family were conducted,and the following results were obtained:1.GST gene cloning:we obtained 5 Ulva prolifera GST genes:UpGST1,UpGST2,UpGST3,UpGST4,UpGST5.The amino acid sizes were 220,222,242,156 and 244,respectively.2.GST bioinformatics analysis:the physical and chemical properties of amino acids,transmembrane domains,signal peptides,phosphorylation sites,and secondary and tertiary structures of proteins were predicted and analyzed using a variety of tools.The results showed that UpGSTl,UpGST2,UpGST3 and UpGST5 were all fat-soluble hydrophilic proteins without transmembrane domain and signal peptide,and had 19,20,24 and 15 phosphorylation sites,respectively.UpGST4 is a fat-soluble hydrophobic protein with 3 transmembrane domains,no signal peptide and 13 phosphorylation sites.Through the analysis of protein domains,it was found that UpGST1,UpGST2,UpGST3 and UpGST5 all have unique C-terminal and N-terminal domains of soluble glutathione S-transferase,which proved that they belong to soluble proteins.The UpGST4 domain is characterized by the microsomal glutathione S-transferase of the MAPEG family,which is a small protein with 156 amino acids.3.Prokaryotic expression analysis:expression vectors of pET28a-GST1,pET28a-GST2 and pET28a-GST3 were successfully constructed and expressed after IPTG induction,and the expression band size was consistent with the theoretical value.4.Determination of enzyme activity:after the induced expression bacteria was broken by ultrasound and purified by Ni column,the purified protein was obtained,and its size was consistent with the theoretical value after SDS-page electrophoresis detection.The three recombinant proteins showed activity to CDNB by enzyme activity assay.5.GST expression analysis:the 18S rDNA gene was used as reference,to analyze the 24 h expression of five GST genes under seven different stress conditions including high temperature,low temperature,high light,low light,high salt,low salt and high intensity ultraviolet radiation by qPCR.It was found that all 5 GST genes were expressed and the expression was induced by stress conditions.