Molecular cloning and functional characterization of peroxiredoxin 4 (prx 4) in freshwater crayfish, Procambarus clarkii

Peroxiredoxin (Prx), which is a newly discovered member of the antioxidant protein family, performs important biological functions in intracellular signal transduction. In the present study, a peroxiredoxin 4 gene was cloned from crayfish for the first time and named Pc-prx 4. According to the amino acid sequence signature, Pc-Prx 4 was identified as the typical 2-Cys Prx molecule, which possessed two conserved cysteines (Cys98 and Cys219). Time-course expression patterns post V. harveyi infection revealed that Pc-prx 4 was likely related to crayfish innate immune defense responses. In particular, the highest fold upregulation of the Pc-prx 4 mRNA transcript reached approximately 170 post V. harveyi infection in the crayfish hepatopancreas. The results of the mixed functional oxidase assay showed that rPc-Prx 4△ could resist the damaging effect of reactive oxygen species generated from the thiol/Fe3+/O2- reaction system to some extent. In addition, the results of the RNAi assay revealed that the crayfish survival rate was obviously increased post injection of V. harveyi when Pc-prx 4 was knocked down. Further study revealed that both hemolymph melanization and PO activity were strengthened to different degrees in the RNAi assay. Therefore, we speculated that the increase in the crayfish survival rate was likely due to the increase in hemolymph melanization. The obviously reinforced hemolymph melanization was directly caused by the upregulation of hemolymph PO activity, which was induced by the knockdown of Pc-prx 4. However, further studies are still indispensable for illuminating the molecular mechanism of Pc-prx 4 in the crayfish innate immune defense system.