Superoxide Dismutase Multigene Family In Bay Scallop Argopecten Irradians: Gene Structures, Expression And Polymorphism

The bay scallop Argopecten irradians, which was first introduced into China from the USA in 1982, has become one of the most important bivalves cultured in China. It now dominates scallop farming in China and accounts for about 70% of the total production. However, bay scallop cultivation has suffered seriously from high mortality. Understanding the immunity of bay scallops is crucial for managing diseases and developing sustainable scallop culture.It has been proved that one important immune defense reaction of mollusca hemocytes is phagocytosis when the organism is attacked by microorganisms or viruses. During the course of phagocytosis, the host glycolytic reactions get activated which in turns increase the consumption of oxygen and induce the production of a mass of reactive oxygen species (ROS). At the same time, the host also starts other immune response to defense the infection of pathogenys. All these immune response will need more ATP to support the energy which will also result in more ROS production from the electron transport chain. Though ROS can kill foreign invaders, the mass accumulation of these reactive molecules in organisms will damage many cell components such as lipids, proteins, and nucleic acids. So the rapid elimination of these excessive ROS is essential for the proper functioning of cells and the survival of mollusca. Superoxide dismutases (SOD) family are thought to be one of the first lines of antioxidant defense and are highly efficient in protecting cells and tissues against oxidative stress by catalyzing the dismutation of superoxide radicals to form hydrogen peroxide and molecular oxygen.To begin with, three unique and highly compartmentalized bay scallop superoxide dismutases genes (CuZnSOD, MnSOD, and ECSOD) have been cloned and charactereized. The full-length CuZnSOD cDNA of bay scallop (AiCuZnSOD) was comprised of 1047bp, containing a 459bp open reading frame (ORF) which encoded 152 amino acids. Sequence comparison showed that the AiCuZnSOD shares 77.5% and 75% similarity with CuZnSOD of Chlamys farreri and Crassostrea gigas respectively. 74.5% similarity with that of human. The full-length cDNA of MnSOD (AiMnSOD) consisted of 1207 bp with a 681 bp ORF encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the AiMnSOD shares 85% and 78.4% similarity with MnSOD of Mizuhopecten yessoensis and Haliotis discus discus, respectively. The full-length cDNA of ECSOD (AiECSOD) was 893 bp with a 657 bp ORF encoding 218 amino acids. The deduced amino acid sequence contained a putative signal peptide of 20 amino acids. Sequence comparison showed that AiECSOD had low degree of homology to ECSODs of other organisms, which shares 27.9% similarity with that of Brugia pahangi, 31.4% with similarity with Danio rerio, 28.6% with human, and only 28.1% with mollusca C. gigas.Then, the genomic length of the AiCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region contained many putative transcription factor binding sites such as ICSBP, Oct-1, Sp1, TBP, C/EBPbeta, C/EBPalp, NF-1, NFκB, GATA-1, AP-1, YY1 and SRF binding sits. The genomic length of the AiMnSOD gene was about 10692 bp containing 4 exons and 3 introns. The promoter region contained many putative transcription factor binding sites such as ICSBP, Oct-1, Sp1, TBP, C/EBPbeta, C/EBPalp, NF-muE1, GATA-1, Pit-1a, HNF-1, GLO, HOXA4 and Antp binding sits. The genomic length of the AiECSOD gene was about 5276 bp containing 5 exons and 4 introns. The promoter region contained many putative transcription factor binding sites such as c-Myb, Oct-1, Sp1, Kruppel-like, c-ETS, NFκB, GATA-1, AP-1, and Ubx binding sites.Moreover, a quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of SODs in different tissues and the temporal expression of SODs in scallop challenged with bacteria Vibrio angullarum. Higher-level mRNA expression of AiCuZnSOD was detected in the tissues of gill filaments, haemocytes and gonad. The expression of AiCuZnSOD was no significant change from 0h to 48h after the bay scallop injected with V. angullarum. Higher-level mRNA expression of MnSOD was detected in gill and mantle. The expression of AiMnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. High levels of expression were detected in haemocytes, but not in gonad and mantle. The expression of AiECSOD reached the highest level at 12 h post-injection with V. anguillarum and then returned to normal between 24 h to 48 h post-injection. The results indicated that bay scallop SODs was a constitutive and inducible protein. It could play an important role in the immune responses against V. anguillarum infection in different way.In addition, expression, purification of recombinant and the enzyme activity assay confirm AiCuZnSOD and can express in E. coli with high enzyme activity and the enzyme is stable in vitro. AiECSOD can also expresss in E.coli but with low enzyme activity.Finally, three SOD isoforms polymorphism and the possible association of SNPs with resistance/susceptibility to bacteria Vibrio angullarum infection were also studied. In promoters, exons and partially introns of three SODs, 59 SNPs were discovered. Compare to AiCuZnSOD and AiMnSOD, AiECSOD has the most SNPs, especially in promoter. Among them, -1739 T-C SNP in AiCuZnSOD promter, -498 A-T and -267 G-A SNPs in AiECSOD promoter were significant associations with resistance/susceptibility to bacteria V. angullarum infection (P<0.05). The polymorphism of 38 Thr-Lys in the extracellular targeting sequence in AiECSOD is also associated.