Cloning And Primary Immune Functional Analysis Of MAPK Signaling Pathway Related Genes And Tbk1 In Nile Tilapia

Nile tilapia(Oreochromis niloticus)is an important freshwater cultured economic fish in China,and it is also one of the main economic fish in the world.However,the high mortality rate of Streptococcus agalactiae disease has had a huge impact on tilapia farming industry.Therefore,it is necessary to study the immune mechanism of tilapia against S.agalactiae.As the hub of innate immune signal pathway,joint molecules can help the body to resist and eliminate pathogens,which is very important in the signal transduction pathway.Therefore,in this study,the c DNA sequences of tbk1,p38,tak1 gene and its binding protein Tab1,Tab2 gene were cloned,and the tissue distribution of these genes in healthy fish and the expression changes of these genes in fish infected with S.agalactiae and treated with LPS and Poly I:C were detected at the level of m RNA.In addition,the eukaryotic expression vectors of the above five genes were constracted in He La cells to study their subcellular localization and signal pathway.The results are as follows:1 Cloning and expression analysis of tak1,tab1,tab2 and p38 genes in Nile tilapiaIn this study,The c DNA sequence of tak1 gene is 3492 bp,its ORF is 1809 bp,encoding 602 amino acids.The c DNA sequence of tab1 gene is 4001 bp,its ORF is1491 bp,which encodes 497 amino acids.The c DNA sequence of tab2 gene is 4792 bp,and its ORF is 2217 bp,encoding 738 amino acids.The c DNA sequence of p38 gene is1493 bp,and its ORF is 1083 bp,which encodes 360 amino acids.Tak1 has a S＿TKc domain and a coiled coil structure;Tab1 protein structure contains a PP2C＿SIG domain and a conservative PYVDXA/TXF sequence model;Tab2 has a CUE domain,coiled coil domain and Znf＿RBZ domain;P38 contains a highly conserved S＿TKc domain.Homology analysis showed that Tak1,Tab1 and P38 had the highest homology with Neolamprologus brichardi,and Tab2 had the highest homology with Simochromis diagramma(98.28%).Multiple sequence alignment showed that Tak1,Tab1,Tab2 and P38 were highly conserved.The phylogenetic tree results showed that the tak1,tab1,tab2 and p38 genes of Nile tilapia formed a branch with other bony fishes,and then integrated with mammals to form a large branch.The tissue expression anaylsis showed that the expression of tak1,tab1 and tab2 was the highest in the muscle,and the expression of p38 was the highest in the heart.The expression of tak1,tab1,tab2 and p38 was significantly induced in most of the tested tissues after stimulation with LPS,Poly I: C and S.agalactiae.These results suggest that tak1,tab1,tab2 and p38 play an important role in the disease resistance and immunity mechanism of Nile tilapia.2.Preliminary functional analysis of tak1,tab1,tab2 and p38 genes in Nile tilapiaIn order to explore the functions of tak1,tab1,tab2 and p38 in the signal pathway,subcellular localization and Co-IP protein interaction experiments were carried out.The results of subcellular localization showed that Tak1 was located in the cytoplasm,and Tab1,Tab2 and P38 had certain distribution in the cytoplasm and nucleus.Co-IP results showed that Tak1 did not interact with Traf6 protein,but it interacted with Tab1,and Tab1 had no interaction with P38.3.Cloning and functional analysis of tbk1 gene in Nile tilapiaThe full-length c DNA sequence of tbk1 gene is 3378 bp,and its ORF is 2172 bp,which encodes 723 amino acids.SMART software analysis showed that Tbk1 contained a highly conserved S＿TKc domain,a coiledcoil domain and a ubiquitin-like domain(ULD).Homology analysis showed that Tbk1 had the highest homology with Maylandia zebra and Astatotilapia calliptera,both of which were 97.59%.The phylogenetic tree results showed that Tbk1 was located in one branch with other bony fishes and mammals in another branch.The tissue expression analysis showed that the expression of tbk1 was the highest in the brain and the lowest in the liver.After stimulation with LPS,Poly I:C,and S.agalactiae,the expression of tbk1 in the tested tissues had significant changes.Subcellular localization showed that Tbk1-GFP was mainly present in the cytoplasm,so it was concluded that Tbk1 was a kind of cytoplasmic protein.Pull-down analysis showed that Tbk1 proteins interact with the Traf3 protein,and there was also interaction between Sting protein and Tbk1 protein.The above results lay a foundation for further exploring the mechanism of tbk1 participating in the signal pathway.