Cloning And Functional Verification Of Glutathione S Transferase Gene From Macrocystis Pyrifera

With the rapid development of modern industry and agriculture,and the exploitation of mineral resources,pollution of ecological environments,especially by heavy metals in water,is becoming ever more serious.The toxicity of heavy metals to algae has become one of the key issues in pollution ecology.Among metals,copper（Cu）and cadmium（Cd）are common and easily accumulate in many Marine organisms,making them the priority pollutants of concern.Brown seaweeds are economically importan,Macrocystis pyrifera,one of the more common species,is widely distributed and they are exposed to a variety of environmental conditions and are capable of acclimating to a wide range of abiotic and biotic factors.Studies have shown that the increased GST expression and enzyme activity are basically consistent with the increase of heavy metal tolerance in plants,indicating that GSTs play a role in improving the metal tolerance of plants.So we took the GST gene from M.pyrifera,cloned the GST genes from M.pyrifera and construct them into the expression vector of Synechococcus elongatus PCC 7942 and expressed it in S.elongatus PCC 7942 to investigate the role of the GST genes from M.pyrifera in tolerance to heavy metal ions.Glutathione S-transferase（GST）comprise a large gene family which plays an important regulatory role in organisms’growth and development,and responses to environmental change.In this study,six complete GST genes（mpgst1,mpgst2,mpgst3,mpgst4,mpgst5,and mpgst6）were cloned from the gametophytes of M.pyrifera.Subsequent bioinformatics analysis showed that these six genes encoded for 202,216,288,201,205,and 201 amino acids,respectively.Phylogenetic analysis and domain analysis showed that mpgst3 differed from the other GST genes cloned,and MPGST3belongs to the Ure2p type GST.However,the other GSTs from M.pyrifera belong to the soluble GST family and form an independent branch with the other GST genes found in macroalgae,suggesting that new GST types were formed during macroalgal evolution.In order to investigate the role of GST gene in heavy metal tolerance in M.pyrifera,we used real-time quantitative PCR to study the expression of 6 mpgsts.GST genes were upregulated in M.pyrifera when 2.5 mg/L Cu²⁺were added to the medium.The expression of mpgst3 was the highest,and both showed a short-term expression phenomenon.We speculated that this might be the adaptive mechanism of M.pyrifera to buffer the acute copper excess.When different concentrations of Cd²⁺were applied to M.pyrifera,the GST gene expression was down-regulated.Six GST genes were transformed into S.elongatus PCC 7942,and genomic DNA of the transformed strain was extracted as template for PCR verification and GST enzyme activity of the transformed strain was determined to verify the heterologous expression.The results showed that all six mpgsts were successfully integrated into the genome of the transformed strain.Porous plate results showed that all the transformed strains（MG1-MG6）were copper resistant,but only the transformed strains containing mpgst1,mpgst4and mpgst6 genes（MG1,MG4 and MG6）were cadmium resistant.These results indicated that the cadmium tolerance of some mpgsts could not be reflected in the M.pyrifera,but in the S.elongatus PCC 7942.All transformed strains were subjected to 0.3mg/L copper ion stress,and the transformed strains MG1,MG4 and MG6 were subjected to 0.2 mg/L cadmium ion stress.The results showed much higher levels of growth,photosynthetic pigment content and Fv/Fm value of chlorophyll fluorescence parameters in the transformed strain than in the wild strain.These results indicated that the GST gene of M.pyrifera enhanced the tolerance to heavy metals of S.elongatus PCC 7942,and different mpgsts showed different resistance in various physiological indexes due to their types and functions.The results of this study lay a theoretical foundation for further research into the heavy metal ion stress resistance function of GST genes in M.pyrifera.