The Copper Zinc Superoxide Dismutase And Manganese Superoxide Dismutase From Qihecrucian Carp Carassius Auratus: Molecular Cloning, Characterization And Response To Aeromonas Hydrophila Infection

Superoxide Dismutase(SOD), as an important enzyme of antioxidant system, protects the integrity of biological macromolecular and cell membrane stability by removing the surplus reactive oxygen species. Aquatic animals mainly depended on the natural immunity because of the lack of specific immunity. In recent years, a large number of studies have confirmed that SOD participates in aquatic antioxidant defense. Qihe crucian carp Carassius auratus is a precious fish in Henan Province, with the high nutritional and economic value. Therefore, it is an important to study the gene features and the expression response to pathogen for understanding the biological function of SOD. In this study, the completed c DNA sequence of Cu Zn SOD and Mn SOD in Qihe crucian carp Carassius auratus was cloned by reverse transcription PCR(RT-PCR). The relative expression of SOD in 10 kinds of organs and tissues from Qihe crucian carp Carassius auratus were detected by quantitative PCR(q PCR). We studied the express response in liver, gills, kidney, spleen and head kidney of Carassius auratus after treated with A.hydrophila and LPS. At the same time, the corresponding enzyme activities were measured by hydroxylamine. 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 Cu Zn SOD and Mn SOD full-length c DNA sequences were cloned with 759 bp and 960 bp, respectively. The open reading frame(ORF) of Cu Zn SOD with 465 bp length encodes 154 amino acids. The predicted molecular weight and the theoretical isoelectric point of the Cu Zn SOD protein were 16.11 KDa and 5.95, respectively. In the active centers, the binding sites for Cu2+ and Zn2+ were: His-47,-49,-64,-121 and His-64、-72、-81、Asp-84, respectively. And the two family signature sequences of the Cu Zn SOD were 45GFHVHAFGDNT55 and 139GNAGGRLACGVI150. The two glycosylation sites were N16 and N69. In addition, the cysteine C58 and C147 formed a disulfide bond. The Cu Zn SOD is located in cytoplasm without transmembrane region and signal peptide. The full-length Mn SOD had an ORF of 675 bp encoding 224 amino acids. The predicted molecular weight was 24.87 KDa, and the theoretical isoelectric point is 8.28. In the active centers, the binding sites for Mn2+ were: His-52,-100, Asp-185, and His-189. The signature sequence was 185DVWEHAYY192. There was a 26 bp-long Mitochondrial Targeting Sequence(MTS) in the front of the sequence(1MLCRVGYVRRCAATLNPILGAVASKQ26). BLAST results suggest that the amino acid sequences of the two genes are conserved protein in different species. Phylogenetic tree showed that Cu Zn SOD in Qihe crucian carp Carassius auratus gathered in the branch of cytoplasm Cu Zn SOD, and Mn SOD gathered in the branch of mitochondrial Mn SOD.In the present study, the m RNA expression of Cu Zn SOD and Mn SOD in 10 organs and tissues(liver, heart, muscle, spleen, intestines, skin, head kidney, brain, kidney and gill) from Qihe crucian carp Carassius auratus were detected by q PCR. The results showed that both SOD genes were expressed in all organs and tissues, and there were significant differences in m RNA expression. The highest SOD m RNA expression level was detected in the liver. The Qihe crucian carp Carassius auratus injected with A.hydrophila and LPS,the changes of the two SOD m RNA 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 q PCR, and the SOD activities were also detected correspondingly. The two SOD genes in various organs and tissues indicated the obviously responses after treated by A.hydrophila and LPS in some special time points.The expression level increased at the beginning, then fell, then rose again in spleen and kidney. But in head kidney, m RNA levels significantly declined at the beginning, then went upward. The total SOD activities in various tissues showed the similar change at different time points with the m RNA levels, indicating that the two SODs 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 Cu Zn SOD and Mn SOD full-length c DNA sequences were cloned from Qihe crucian carp Carassius auratus in this study. The full-length c DNA of Cu Zn SOD consisted of 759 nucleotides encoding 154 amino acids. And the full-length c DNA of Mn SOD consisted of 960 nucleotides encoding 224 amino acids.2. The m RNA expression levels and enzyme activities of Cu Zn SOD and Mn SOD in Qihe crucian carp Carassius auratus indicated the obviously responses to the injection of A.hydrophila and LPS in some special time points. It was suggested that Cu Zn SOD and Mn SOD may play an important role in the immune response against the pathogen infection.