The Selenium-dependent Glutathione Peroxidase From Mytilus Coruscus:Cloning And Expression Upon Cu²⁺ And LPS Exposure

Marine organisms mostly live in the aquatic environment,which face more severe environmental stresses than the terrestrial environment.These environmental stresses include biological stresses?parasites,bacteria,fungi and viruses?and abiotic stresses?heavy metals,organic and inorganic pollution,etc.?.Screening local species as environmental indicator organisms and carrying out in-depth research on biomarkers are the current research hotspots in the field of environmental monitoring,which can provide a powerful supplement for conventional marine pollution monitoring.Some shellfish have been used as environmental indicators in many countries due to their characteristics of solid living,filter feeding and strong tolerance to environmental pollution.In this study,the full-length cDNA of glutathione peroxidase?GPx?was cloned from Mytilus coruscus,and its expression under copper and lipopolysaccharide stress was analyzed.The results may provide theoretical basis for revealing the stress mechanism of glutathione peroxidase in M.coruscus in response to external stress,and also provide support for McGPx possibility as a marine marker.In our study,the GPx complete cDNA sequence of M.coruscus was firstly obtained by homologous cloning and RACE and named McGPx.The full-length cDNA of McGPx is 970 bp containing an ORF of 594 bp,it encodes 197 amino acids.The M.coruscu GPx contains a ²⁹²TGA²⁹⁴ codon,corresponding to selenocysteine?Sec?residue at the 46th codon,indicating that it is a selenium dependent glutathione peroxidase.In the 3?-UTR,characteristic SECIS element?91bp?identified by SECISearch3 program.Sequence characterization revealed that McGPx contains a characteristic GPx signature motif(⁷⁰LGFPCNQF⁷⁷),an active site motif(³⁴GKVILVENVASLUGTT⁴⁹).The conservation of(H⁷⁹,N⁸²,S⁸⁴,E⁸⁷,N⁹⁰,H⁹⁴)are observed in the McGPx sequence,and those may be used to form similar loop structures.Two potential N-glycosylation site ²⁸NFSD³¹,⁸²NGSG⁸⁵ were identified.The McGPx amino acid sequence exhibited the highest level of identity?96%?with Mytilus galloprovincialis GPx,and shares 71%with Ruditapes philippinarum and Sinanodonta woodiana GPx.Phylogenetic analysis showed that McGPx share a high level of identities and closer relationship with other selected invertebrate GPx,Especially the M.galloprovincialis.Real-time RT-PCR was employed to study the tissue-specific expression of McGPx mRNA with?-actin as internal control.In untreated M.coruscus,McGPx mRNA were expressed in all six tissues,with the highest expression level in hemocyte,moderate levels in digestive gland and gill,the lowest gonad.Under the stress of Cu²⁺,McGPx could be rapidly respond and expressed in gill and digestive gland,and the peak value of McGPx were reached at 16 d and 23 d,respectively.The results suggested that McGPx might play an important role in protecting tissues from oxidative stress.After LPS challenge,the expression pattern of McGPx in the gill increased and then decreased,and reached the peak at 24 h.At 3 h,the change of McGPx in the digestive gland was not obvious,while the change of McGPx in the gill was significant?4.13-fold compare to control group?.The results suggested that McGPx was implicated in the immune response induced by LPS.These results suggest that McGPx perhaps play an important role in maintaining cellular redox homeostasis and protecting M.coruscus against copper and LPS toxicity.It can also be inferred that GPx has the potential to be used as molecular biomarkers for assessing marine environmental pollution.