Effect Of Cadmium On The Gene Transcription Regulation Function Of Nuclear Transcription Factor ShDorsal In The Freshwater Crab Sinopotamon Henanense

Dorsal is the nuclear transcription factor of the invertebrate Toll signaling pathway that mediates the production of antimicrobial peptides in response to pathogenic microbial insult,and plays an important role in invertebrate innate immunity.In the current study,the dorsal from the freshwater crab Sinopotamon henanense(Shdorsal)was identified for the first time and analyzed by bioinformatics.The total length of the Shdorsal was 2483 bp,of which the open reading frame was 1644 bp,encoding 547 amino acids.Structural prediction of Dorsal proteins revealed that ShDorsal contains conserved RHD and IPT domains and belonged to the class II NF-κB transcription factor.ShDorsal shared high homology with other crustaceans and clustered with Scylla paramamosain Dorsal(Sp Dorsal)and Portunus trituberculatus Dorsal(Pt Dorsal)as one clade.In addition,Shdorsal were detected by realtime PCR in all seven tissues,and had the highest expression in hemolymph.To verify the transcriptional regulation of downstream-related antimicrobial peptide genes by Shdorsal,real-time PCR method were used to detect the expression of five antimicrobial peptide genes after interfering with the Shdorsal gene in S.henanense.Afterwards,we probed the immune response of Shdorsal to the Aeromonas hydrophila challenge.Shdorsal expression levels in gill and hemolymph of crabs after bacterial challenge was significantly different from those of controls,indicating that Shdorsal were significantly responsive to A.hydrophila challenge.In addition,the effect of cadmium on the Shdorsal response to A.hydrophila challenge was explored at both the gene and protein levels.Shdorsal expression levels in gill and hemolymph of crabs were significantly reduced after challenged with A.hydrophila and different concentrations of cadmium,which suggested that cadmium had an inhibitory effect on Shdorsal function in crabs and further affected antibacterial immunity in crabs.Finally,the immunohistochemical experiment explored the immune response of ShDorsal to cadmium exposure and A.hydrophila challenge at the protein level.The expression of ShDorsal protein in the gill tissue of S.henanense increased,and the obvious nuclear translocation phenomenon could also be observed in the hemocytes,indicating that ShDorsal protein could participate in the immune response of S.henanense to gram-negative bacteria and cadmium stress.In summary,our findings investigated the antimicrobial characteristics and the mechanism of Shdorsal in response to cadmium,supplemented the regulation mechanism of Toll signaling pathway in response to cadmium stress,and provided scientific basis for in-depth understanding of the natural immune mechanism of crustaceans.