Studies On The Mechanism Of DAZAP2 Regulates The Expression Of AMPs Through Hippo Signaling Pathway In Chinese Mitten Crab Eriocheir Sinensis

China is a big aquaculture country.In recent years,with the continuous expansion of breeding scale,breeding diseases have become increasingly prominent.How to effectively prevent the occurrence of diseases and improve the disease resistance of cultured species is an important issue in the current aquaculture industry.The immune system is an effective line of defense against the invasion of foreign pathogens.The study on the immune mechanism of cultured species not only helps to better understand the immune response mechanism of the cultured species,but also provides a scientific basis for the prevention and control of bacterial diseases.In this paper,an open reading frame(ORF)sequence of EsDAZAP2 was obtained based on the whole genome sequencing database and a large number of transcriptome libraries of Eriocheir sinensis.The full-length cDNA of EsDAZAP2 gene was amplified by RACE.Bioinformatics analysis found that DAZAP2 is very conserved among different species and has characteristic domains and motifs.Further studies showed that EsDAZAP2 was expressed in different tissues of Eriocheir sinensis.Both Staphylococcus aureus and Vibrio parahemolyticus could induce significant up-regulation of EsDAZAP2 in hemocytes,suggesting that this gene may be involved in immune response.To clarify whether EsDAZAP2 is involved in the innate immune response of Eriocheir sinensis,we knocked down the EsDAZAP2 gene by RNAi technology and stimulated crabs with bacteria.It was found that crabs with knockdown of EsDAZAP2 showed higher sensitivity to both Gram-positive bacteria and Gram-negtive bacteria.It had the higher bacterial load upon bacteria infection in EsDAZAP2 knockdown crabs.The results indicated that EsDAZAP2 plays an important role in the antibacterial immune response of Eriocheir sinensis.As an intracellular protein,EsDAZAP2 has the potential to regulate the antibacterial response through immune-related signaling pathways.To solve this problem,we first investigated whether EsDAZAP2 may regulate Toll,IMD,JAK/STAT signaling pathways,and select Dorsal,Relish,and Stat92 E as indicator proteins for the above signaling pathways.The results showed that both S.aureus and V.parahemolyticus could induce the above proteins to enter the nucleus from the cytoplasm,but EsDAZAP2 only regulates the nuclear transport of Dorsal,suggesting that EsDAZAP2 may be involved in the Toll signaling pathway.In order to further explore the relationship between EsDAZAP2 and Toll signaling pathway,the protein EsSav interacting with EsDAZAP2 was obtained by yeast two-hybrid library screening,and its protein interaction was verified by Co-IP assay.Sav is a key signaling molecule in the Hippo pathway,and studies have shown that it has the function of binding and promoting Hpo phosphorylation,thereby activating the Hippo signaling pathway.In order to clarify the binding mechnism of DAZAP2,Hpo and Sav,this study constructed a wild-type and functional domain deletion-type recombinant plasmid.By transfecting S2 cells and Co-IP experiments,EsDAZAP2 binds to the WW domain of EsSav,while EsHpo binds to SARAH domain.In order to clarify whether the Hippo signaling pathway is involved in the antibacterial response in Eriocheir sinensis,the crabs were stimulated with S.aureus and V.parahemolyticus,and the phosphorylation of Hpo and the downstream molecular Yki were detected by Western Blot and immunofluorescence experiments.After bacterial stimulation,the phosphorylation level of Hpo increased and Yki transferred from the nucleus to the cytoplasm.The results indicated that bacterial stimulation can activate the Hippo signaling pathway in the Chinese mitten crab.Through RNA interference experiments,it was found that Yki’s nuclear transfer was inhibited after interference with Hpo,and the transcription level of antimicrobial peptides in hemocytes was significantly down-regulated after bacterial stimulation.It was reported that Yki can regulate the expression of Cactus,an important component of the Toll signaling pathway in Drosophila.The transcriptional level of EsCact was significantly down-regulated with EsYki knockdown in the hemocytes of crabs.To further verify the relationship between the Hippo pathway and the Toll pathway,we detected the translocation of Dorsal with knockdown of Hpo,which inhibited Dorsal’s nuclear translocation.These results indicate that the Hippo signaling pathway is involved in the innate immune response of Eriocheir sinensis by regulating the expression of Cactus.To explore the relationship between EsDAZAP2 and Hippo signaling pathway,we knocked down EsDAZAP2 by RNA interference,and then stimulated the hemocytes of crabs with different bacteria to detect the phosphorylation level of Hpo and the translocation of Yki in hemocytes.The experimental results showed that knockdown of EsDAZAP2 would decrease phosphorylation levels of Hpo,as well as inhibition of Yki transfer from the nucleus to the cytoplasm.These results indicate that EsDAZAP2 is able to regulate the Hippo signaling pathway.In summary,this study clarified the important role of Hippo signaling pathway in the antibacterial immune response of Eriocheir sinensis,and found that DAZAP2 regulates the activation of Hippo signaling pathway by binding to the WW domain of Sav protein,which in turn affects the functional pathway of antimicrobial peptide expression.This study provides a new idea for the study of the regulation mechanism of antibacterial cover expression in invertebrates,and provides a scientific basis for the prevention and control of bacterial diseases of Eriocheir sinensis.