Function Of Epinephelus Coioides MKK4/6 And MiR-122 In Apoptosis

Orange-spot grouper has become an important economic marine fish in the southern coast of China and Southeast Asia.Singapore grouper iridovirus(SGIV)and Vibrio alginolyticus are highly pathogenics of marine fish,which can cause massive of grouper.In this study,orange-spot groupers were uesd as the research objects to study the expression characteristics of grouper MKK4/6 gene and miR-122 after SGIV and V.alginolyticus stimulation,the activation of AP-1/NF-κB,and the effect on virus-induced apoptosis.The results are listed as follows:1.Ec MKK4 and Ec MKK6 genes were identified from Epinephelus coioides,termed Ec MKK4-1,Ec MKK4-2,Ec MKK4-3 and Ec MKK6.Through conservative domains prediction,there were found that Ec MKK4 contain a S＿TKc domain and two ATP-binding motifs;Ec MKK6 contains a S＿TKc domain,a tyrosine kinase domain and a DVD domain.Based on the homology comparison and the phylogenetic trees that Ec MKK4 were closely related to Larimichthys crocea,as 97.15%,98.12% and 97.53%,Ec MKK6 was closely related to Laters calcarifer,as 98%.2.Ec MKK4/6 were distributed in eleven grouper tissues.Ec MKK4 had highly expressed in the intestine.Ec MKK6 had highly expressed in kidney.The expressions of Ec MKK4/6 were detected in the head kidney and spleen infected by SGIV virus and V.alginolyticus.The expressions of Ec MKK4 and Ec MKK6 were differentially.The results of the dual luciferase reporter showed that Ec MKK4/6 were significantly inhibited AP-1activity.Finally,Ec MKK4/6 inhibited cell apoptosis induced by SGIV.The above results indicated that Ec MKK4/6 were involved in the immune response to virus and bacterial stimulation,played important roles in pathogen immune.3.E.coioides miR-122 shared a high similarity to miR-122 s from mammals and other fish.Up-regulation of miR-122 was found during the SGIV infection.Up-regulation of miR-122 can inhibit the activity of AP-1,NF-κB and the pro-apoptotic factors,reduce caspase-3 protease activity,and ultimately repress cell apoptosis induced by SGIV.In addition,miR-122 promoted viral replication and proliferation shown that miR-122 significantly enhanced the severity of CPE,and progressed the transcription expression levels of viral genes and the protein expression level of MCP,and heightened new progeny virions.In conclusion: three Ec MKK4 and a Ec MKK6 were found in grouper;the evolution of Ec MKK4/6 was highly conserved;Ec MKK4/6 showed differential expression stimulated by SGIV and V.alginolyticus and inhibited SGIV-induced apoptosis which speculated that Ec MKK4/6 were involved in the innate immune response of grouper.In the process of SGIV infection,miR-122 regulated the induced apoptosis by down-regulating the expression of immune signals and inflammatory factors,thereby promoting viral replication.