Effect Of Cd²⁺ On Photosynthesis Of Porphyridium Purpureum And Cloning And Expression Studies Of Phytochelatin Synthase Gene From The Alga

Phytochelatin is a heavy metal binding polypeptide which is widely exists in many organisms.As a key enzyme in the synthesis pathway of phytochelatin,phytochelatin synthase?PCS?can catalyze the synthesis of phytochelatin using GSH as substrate,and then phytochelatin effectively relieves the toxicity of heavy metals in the organism.P.purpureum,as primary producer in the water,would be decreased significantly on its photosynthesis if it lived in the water polluted by heavy metals.The aim of this experiment is to investigate the impact of Cd²⁺ on the photosynthesis of P.purpureum and the effect of PCS from P.purpureum?designated as PpPCS?in resistance to heavy metal stress.The designed experiments and results were as follows:1.P.purpureum was treated by different concentrations of Cd²⁺ for 24 h,48h,72 h,96h and 120 h and then the chlorophyll fluorescence parameters were measured by Multi color PAM.The results show that the parameters Fv/Fm,yield,ETR and qP decreased significantly with Cd²⁺ concentration increasing,and NPQ increased first and then decreased.The cell density,phycobiliprotein and ATP content decreased evidently.The results indicated that the photosynthesis of the alga was significantly inhibited by Cd²⁺ stress.2.Based on NCBI and P.purpureum genome database,the ORF of PpPCS was cloned successfully by reverse transcription PCR.The length of the gene is 1476 bp,and it encodes491 amino acids.3.Bioinformatics of PpPCS was analyzed by online software.The results showed that the gene belongs to phytochelatin synthase super family and the molecular weight of deduced protein was about 53.99 KDa,and the protein is acidic,instable and weakly hydrophilic.There was no signal peptide in the PpPCS sequence so that the gene might function in the cytoplasm of cell.Phosphorylation site analysis showed that the protein had serine phosphorylation sites,but no tyrosine phosphorylation sites.Secondary structure prediction showed that the protein has 16 alpha helixs and 8 beta sheets,inked by?-turns.4.P.purpureum was stressed by 100?mol/L Cd²⁺ for 0h,1h,2h,4h and 8h,then relative expression level of PpPCS gene was detected by real-time PCR method.The results showed that the relative expression level increased and then went down gradually,indicating that the PpPCS gene could be induced by Cd²⁺ and the ability of resistance to heavy metal stress was raised by means of regulating expression of the gene.5.The expression vector was constructed by fused PpPCS gene with pET-28 a prokaryotic vector,then the vector was transformed into E.coli BL21 expression strain for prokaryotic expression.The results of SDS-PAGE showed that target protein was successfully induced and expressed.However the protein was an inclusion body and insoluble.6.A eukaryotic expression vector was constructed by inserting PpPCS gene into pYES2 vector.The expression vector then was transformed into INVSc1 yeast expression strain for eukaryotic expression.The data showed that the transformed yeast strains were much more resist to the Cd²⁺ stress than the control.It was also found that with the increase of Cd²⁺concentration,the capacity of the transformed yeast to tolerate Cd²⁺ gradually raised.The results implied that the PpPCS gene functioned to confer the resistance to heavy metal stress in organisms.The results confirmed that the photosynthesis of the alga was significantly depressed by Cd²⁺ stress.It was also found that the yeast overexpressed PpPCS was more resistant to Cd²⁺stress by studies of cloning,bioinformatics analysis and expression of PpPCS gene.It lays an important foundation for the study of the resistance mechanism of algae.