MEKK Family Related Genes Involved In The Immune Response Of Two Pathogen Infections In Hybrid Snakehead

In eukaryotes,MAPKKK-MAPKK-MAPK three-stage cascade reaction(MAPK inasecasade)is used to gradually transmit the signals received on the cell surface to the cell interior,so as to regulate various physiological reactions in the cell.MEKK,as the upstream kinase of MAPK signal cascade system,can activate the downstream MAPKK under the action of H-Ras,TRAF2 and Caspase3,and then activate MAPK to regulate cell differentiation,proliferation,migration and apoptosis in the immune and inflammatory response play an important role.Hybrid snakehead(Channa maculata ?× Channa argus ?)is an important freshwater breed with fast growth,high yield and strong disease resistance.With the increase of culture density and water pollution,it is faced with many disease threats.The "sarcoidosis" and "sarcoidosis like" caused by the infection of Nocardia seriolae and Aeromonas schubert in hybrid snakehead have become a great threat to the breeding industry of hybrid snakehead,but the immune response mechanism of hybrid snakehead to these two pathogens is not clear.In this paper,we first cloned six genes of MEKK3,MEKK4,MLK3 and their related genes Caspase3,H-Ras and TRAF2.The ORF of Cma MEKK3 is 1851 bp,and the predicted code is 616 aa;the ORF of Cma MEKK4 is 4617 bp,and the predicted code is 1538 aa.The ORF of Cma MLK3 is 3144 bp and the predicted code is 1047 aa.The ORF of Cma Caspase3 is 854 bp with a predicted code of 283 aa;the ORF of Cma H-Ras is 564 bp with a predicted code of 187 aa;the ORF of Cma TRAF2 is 1566 bp with a predicted code of 521 aa.Secondly,we analyzed the expression distribution of the above 6 genes in the tissues of hybrid snakehead.The results showed that the expression of these 6 genes was high in the immune related tissues,among them,the expression of Cma MEKK3 was the highest in the headkidney,the expression of Cma MEKK4 and Cma MLK3 was the highest in the blood,the expression of Cma Caspase3 and Cma TRAF2 is the highest in spleen,and Cma H-Ras is the highest in hindgut.It is speculated that 6 genes of hybrid snakehead play an important role in immune response.In addition,the expression patterns of 6 gene in liver,spleen and head kidney were analyzed after infection of Nocardia seriolae and Aeromonas schubert.The results showed that they were up-regulated or down regulated in different degrees after infection of two pathogens,and their response to infection of two pathogens was different.It was speculated that they participated in the immune defense reaction of hybrid snakehead pathogenic bacteria infection,but their immune response patterns to different bacterial infection were different.Thirdly,the headkidney leukocytes of healthy hybrid snakehead were isolated and stimulated by poly(I: C),LPS and LTA to detect the expression of the target gene at different 6 time points.The results suggested that the response of genes to pathogen infection was not the same in cell and organism,and it was speculated that the response mechanism of every gene to exogenous stimulation was not the same in cell and organism.Finally,three genes,Cma MEKK3,Cma Caspase3 and Cma H-Ras,were subcellular mapped and double luciferase reporter gene detected.The results showed that Cma MEKK3 was expressed in the cytoplasm,Cmacaspase3 and Cma H-Ras were both expressed in the cytoplasm and nucleus.Cma MEKK3 and Cma Caspase3 had significant activation on NF-?B,Cma HRas had activation on transcription factors AP-1 and NF-?B.