Cloning And Primary Function Analysis Of TRIM35,TRIM39 And DDX43 Genes In Nile Tilapia

Tilapia is one of the most important farmed fish species in China.However,the outbreak of streptococcal diseases in recent years has seriously caused huge economic losses.DEx D/H box proteins have been found to involved in recognizing viral nucleic acids and activating downstream signaling pathways.Tripartite motif(TRIM)family proteins are important regulators and can be used as E3 ubiquitin ligases to participate in the regulation of various signaling pathways.In this study,the cloning,expression and function analysis of OnTRIM35,OnTRIM39 and OnDDX43 were performed,which provided basic data for the disease resistance mechanisms of Nile tilapia.The main results were as follows:1.Cloning and expression analysis of OnTRIM35 and OnTRIM39The cDNA sequence of Oreochromis niloticus TRIM35(OnTRIM35)was 3646 bp in length with a 1512 bp open reading frame(ORF),encoding a total of 503 amino acids polypeptide.The cDNA sequence of OnTRIM39 was 5025 bp long with a 1650 bp ORF,encoding 549 amino acids polypeptide.The SMART analysis showed that TRIM35 and TRIM39 are highly conserved in structure.They are all have a typical RING domain,a B-Box zinc finger domain,a coiled-coil domain and a PRY-SPRY domain.Phylogenetic and syntenic analysis respectively showed that OnTRIM35 had the highest identity with Maylandia zebra(96%)and OnTRIM39 had the highest identity with Astatotilapia calliptera(83.5%).Tissue expression analysis showed that OnTRIM35 and OnTRIM39 transcripts were broadly expressed in 10 tested tissues/organs.The highest expression of OnTRIM35 was detected in the heart and spleen,and OnTRIM39 was in the spleen and kidney.After challenged with Streptococcus agalactiae,OnTRIM35 and OnTRIM39 showed significant up-regulation changes at most time points in all tested tissues.After LPS and Poly I:C stimulated,the expression levels of OnTRIM35 and OnTRIM39 were significantly induced in tested tissues.These results indicated that OnTRIM35 and OnTRIM39 could be induced by Streptococcus agalactiae,LPS and Poly I:C,and participate in the host’s defense against pathogen infection.2.Preliminary function analysis of OnTRIM35 and OnTRIM39In order to investigate the functions of TRIM35 and TRIM39 genes and their roles in signaling pathways,the dual-luciferase assay,subcellular localization and pull-down assay was performed.The results showed that the overexpression of OnTRIM35 and OnTRIM39 could significantly enhance NF-κB activity.After co-transfection with OnTRAF3,the OnTRIM35-dependent NF-κB activity was significantly increased.The results of subcellular localization showed that OnTRIM35 and OnTRIM39 were mainly distributed in the cytoplasm.Pull-down results showed that there was interaction between OnTRIM35 and TRAF3.250 potential combine proteins of TRIM39 were obtained by pull-down and mass spectrometry,and OnTRIM39 interacted with I3K4I3 and I3KL45.3.Cloning and expression analysis of OnDDX43The cDNA sequence of OnDDX43 is 2338 bp in length,containing an ORF of 2064 bp,and encoding a polypeptide of 687 amino acids.The SMART analysis showed that OnDDX43 has a KH domain,a typical DEXD domain and a C-terminal HELICc domain.Phylogenetic and syntenic analysis showed that OnDDX43 has the highest homology similarity with Maylandia zebra,reaching 98%.Tissue expression analysis showed that OnDDX43 was ubiquitously expressed in all tissues/organs tested from healthy individuals,and the highest expression level was in muscle,following in the brain and spleen.The expression level of OnDDX43 could be induced by Streptococcus agalactiae,LPS and Poly I:C.4.Preliminary function analysis of OnDDX43Overexpression of OnDDX43 in 293 T cells can significantly enhance the activity of IFN-β.Subcellular localization analysis showed that OnDDX43 was localized in the nucleus and cytoplasm.GST-Pull-down showed that OnDDX43 interacted with TRIF or IPS-1.This result indicates that OnDDX43 combines with TRIF or IPS-1 to regulate the immune response in Nile tilapia.These studies provide a molecular basic information for elucidating the immune regulation mechanism of Nile tilapia.