Molecular cloning, characterization of copper/zinc superoxide dismutase and expression analysis of stress-responsive genes from Eisenia fetida against dietary zinc oxide.

The full length cDNA of copper/zinc superoxide dismutase (Cu/Zn-SOD) from Eisenia fetida (E. fetida) was cloned (GenBank accession no. JN579648). Sequence characterization revealed that the cDNA contained characteristic Cu/Zn-SOD family signatures ((45)GFHVHEFGDNT(55) and (138)GNAGGRLACGVI(149)), cysteines (Cys-58 and-146) predicted to form one disulphide bond, Cu-binding (His-47, -49, -64 and -120) and Zn-binding (His-64, -72, -81 and Asp-84). They were essential for the structure and function of Cu/Zn-SOD. Differential expression of stress-responsive genes like Cu/Zn-SOD, catalase (CAT), heat shock protein 70 (Hsp70) and metallothionein (MT) was applied as potential biomarkers to assess their efficacy for the ecotoxicological effects of dietary zinc oxide (ZnO) on E. fetida. The results showed that the expression of Cu/Zn-SOD and MT increased to reach the highest levels of 6.22 and 7.68 fold in a dose-dependent manner at day 10 respectively. The highest expression of 3.03 fold of CAT was registered at day 10. The transient expression of Hsp70 without consistent time- or/and dose-dependent was observed. It implied that the transcriptional patterns of Cu/Zn-SOD, CAT and MT could serve as early warning signals in ecotoxicological assessment of dietary ZnO on earthworms while the expression of Hsp70 was not well done, which is helpful to monitoring and regulation of ZnO in veterinary application.