The Responses Of Conchocel Of Porphyra Yezoensis To Heavy Metal Stress And Cloning And Expression Of The Resistant Gene GST

Porphyra yezoensis is one of the most economically important marine crops and is also considered as the representative model algae of the intertidal seaweeds. P. yezoensis has a strong capacity of stress resistance, which makes it an ideal species for the molecular mechanism research of alage stress resistance.Heavy metal pollutants are harmful to marine crops. The ability of algae to tolerate heavy metal is determined by multiple biochemical pathways. In this study, the conchocelis of P. yezoensis is taken as experiment material to investigate the effects of heavy metal (Cu2+ and Cd2+) on the contents of chlorophyll a, peroxidase (POD) activity and the soluble protein contents. The results showed that, P. yezoensis has a higher tolerance to Cd2+. With the increase of Cu2+ and Cd2+ concentration, the content of chlorophyll a, POD activity and the soluble protein content of the conchocelis appeared a tendency to increased first and then decreased except the groups exposed to 1mg/L Cu2+, and the higher the concentrations of the same heavy metal, the earlier the decrease occured.A resistant gene——glutathione S-transferases (GST) gene was studied further to search the molecular mechanism of the P. yezoensis stress resistance. GSTs is a kind of multifunctional detoxification enzyme which catalyses the conjugation of GSH to a range of electrophilic substrates. The GST gene of P. yezoensis was cloned by RT-PCR technique, the results of BLAST suggested that: this gene belongs to the gene family of microsomal glutathione S-transferaseⅢand multiple alignment indicated that the deduced amino acid sequence had 53.63% identity with those of the other species. The cloned cDNA contains an 417bp open reading frame, which encodes for a polypeptide of 138 amino acid residues with a molecular mass of 15.35kDa and a pI of 9.795. The predictions of signal peptide sequence and transmembrane helices in proteins indicated that the GST of P. yezoensis is a transmembrane protein with a signal peptide. The phylogenetic tree showed that it is homology with the Chlamydomonas reinhardtii (EDO96603.1) and Pfiesteria piscicida (ABI14355.1).Using gene recombination technology, the prokaryotic expression vector pGEX-4T-1/GST was successfully constructed and was transformed into E. coli DH5a. The differences between growth curves of the E. coli expressing GST and the control showed that the E. coli expressing GST protein had a higher heavy metal-ion (Cu2+ and Cd2+) tolerance. However, the growth of E. coli expressing GST was in accord with that of the control group basically when the heavy metal-ion is absent. These results indicate that GST may be involved in heavy metal detoxification.The results provide some references for researching the effects of heavy metal to alage. The work will contribute to a better understanding of alage GST and help to further analyze the functions of the gene and will make a different for studying resistant mechanism of GST gene in alage.