The Cellular Glutathione Peroxidase And Phospholipid Hydroperoxide Glutathione Peroxidase From Qihe Crucian Carp Carassius Auratus:Molecular Cloning,Characterization And Response To Aeromonas Hydrophila Infection

Glutathione peroxidase(GPx),as an important enzyme of antioxidant system,protects the integrity of biological macromolecular and cell membrane stability by removing the superabundant reactive oxygen species.Aquatic animals depended on the natural immunity because of the lack of specific immunity.In recent years,a large number of studies have confirmed that GPx participates in aquatic antioxidant process.Qihe crucian carp Carassius auratus is a precious fish in Henan Province,with the high nutritional and economic value.As a conerquence,it is an important to study the gene features and the expression response to pathogen for understanding the biological function of GPx.In this study,the completed cDNA sequence of GPx1 and GPx4 in Qihe crucian carp Carassius auratus was cloned by reverse transcription PCR(RT-PCR).The relative expression of GPx were detected by quantitative PCR(qPCR).We studied the express response in liver,gills,kidney,spleen and head kidney of Carassius auratusafter treated with A.hydrophila.These studies are very important for understanding the antioxidant defense mechanisms in fish,and provide a theoretical basis for disease control and prevention in aquaculture,especially in Qihe crucian carp Carassius auratus.The GPx1 and GPx4 full-length cDNA sequences were cloned with 889 bp and 875 bp,respectively.The open reading frame(ORF)of GPx1 with 576 bp length encodes 191 amino acids.The predicted molecular weight and the theoretical isoelectric point of the GPx1 protein were 21.61 KDa and 6.84,respectively.In the active centers,the binding sites for Se was the cysteine,respectively.And the two family signature sequences of the GPx1 were 65LGAPCNQF72 and 153WNFEKF158.The CuZnSOD is located in cytoplasm without transmembrane region and signal peptide.The full-length GPx4 had an ORF of 513 bp encoding 170 amino acids.The predicted molecular weight was 19.31 KDa,and the theoretical isoelectric point is 7.64.In the active centers,the binding sites for Sewas the cysteine.The two signature sequences were 71LGFPCNQF78 and136WNFTKF141.BLAST results suggest that the amino acid sequences of the two genes are conserved protein in different species.In the present study,the mRNA expression of GPx1 in 9 organs and tissues(liver,heart,muscle,spleen,intestines,head kidney,ovary,kidney and gill)from Qihe crucian carp Carassius auratus were detected by qPCR.The results showed that GPx1 genes was expressed in all organs and tissues,and there were significant differences in m RNA expression.The highest GPx1 mRNA expression level was detected in the liver.At the same time,the mRNA expression of GPx1 in each phase of embryonic development from Qihe crucian carp Carassius auratus were detected by qPCR.The results indicated that the mRNA expression of GPx1 increased gradually from the fertilized egg to embryonic development finally to incubate membrane.The Qihe crucian carp Carassius auratus injected with A.hydrophila,the changes of the GPx1 mRNA expression levels were detected in liver,head kidney,kidney,spleen and gills,at six time points(0 h,3 h,6 h,12 h,24 h and 48 h)by qPCR.The GPx1 gene in various organs and tissues showed a significant increase aftertreated by A.hydrophila(p< 0.01**).In kidney and spleen,mRNA levels significantly declined at the beginning,then went upward.But in headkidney and liver,the expression level increased at the beginning,then fell.The change of mRNA in gills are not obvious,indicating that the GPx1 in Qihe crucian carp Carassius auratus may be induced to play an important role in the immune response against the pathogen infection.Conclusion:1.The GPx1 and GPx4 full-length cDNA sequences were cloned from Qihe crucian carp Carassius auratus in this study.The full-length c DNA of GPx1 consisted of 889 nucleotides encoding 191 amino acids.And the full-length cDNA of GPx4 consisted of 875 nucleotides encoding 170 amino acids.2.The mRNA expression levels of GPx1 in Carassius.auratus in Qihe River indicated the obviously responses to the injection of A.hydrophila.It was suggested that GPx1 may play an important role in the immune response against the pathogen infection.3.The mRNA expression levels of GPx1 in Carassius.auratus in Qihe River between the pirod of embryonic development rises gradually,indicating that GPx1 may play an important part between the pirod of embryonic development and the first life phases.