Molecular Cloning And Expression Analysis Of Tlr3 Signaling Pathway Related Genes From Golden Pompano(trachinotus Ovatus)

Innate immune system is the first protective barrier against pathogen invasion by recognizing PAMPs through the pattern recognition receptors(PRRs).TLRs are an important family of PRRs and belong to type I transmembrane protein,which can recognize various PAMPs,subsequently leading to the activation of MyD88/TRIF-dependent signaling pathway.The TRIF-dependent signaling pathway starts with TLR3 recognizing PAMPs,then TLR3 recruits adaptor protein TRIF which in turn activates TBK1 and phosphorylates IRF3/7,finally induces IFN-β expression.Besides,TRIF can recruit and activates TRAF6,resulting in NF-κB activation,which in turn upregulate the expression levels of pro-inflammatory cytokines to exert immune function.Golden pompano(Trachinotus ovatus)is an commercially important marine cultured fish species in South China.With the expansion of intensive farming,infectious diseases outbreak frequently,which seriously affect the development of the golden pompano aquaculture industry.Due to important roles of TLR3 signaling pathway play in innate immunity,the study of TLR3 and its related genes(TroTRIF,TroTRAF6 and TroTBKl)in Golden pompano will provide theoretical support for disease control and selecting resistant varieties.In this study,the full-length cDNAs of TroTLR3 signaling pathway related genes were cloned.The sequence characteristics,functional sites and protein secondary structure of these genes were analyzed by using bioinfonnatics.Analysis of the genetic evolution of four genes.Detecting the expression patterns of four genes in different tissues of healthy fish.To investigate the expression patterns of four genes in immune-related or non-immune-related tissues under stimLulating conditions.Prokaryotic and eukaryotic expression recombinant plasmids of four genes were constructed.The main results are as follows:1.TroTLR3,TroTRIF,TroTRAF6 and TroTBKl genes were cloned by RT-PCR and RACE-PCR.The full-length cDNAs of genes mentioned above were 3415 bp,2297 bp,2280 bp and 2482 bp in length,with ORFs of 2772 bp,1770 bp,1716 bp,and 2172 bp,respectively.TroTLR3,TroTRIF,TroTRAF6 and TroTBKl encoding 923,589,573,and 723 amino acid residues,respectively.Protein structure prediction showed that TroTLR3 had 11 LRRs domain,3 LRR-TYP motifs,a LRR-CT motif,a TM and a TIR domain.TroTRIF contains a TIR domain similar to TLRs.TroTRAF6 contains 4 characteristic functional areas:RING,Zf-TRAF,CC and MATH.TroTBKl consists of three domains:KD,ULD and CC.Homology and evolutionary analysis indicated that TroLR3,TroTRIF,TroTRAF6 and TroTBK1 protein were highly homologous to that of other fish specieses,shared 72.2-84.8%,50.6-78.9%,84.6-91.4%and 97.4-99.4%identity,respectively.The NJ phylogenetic tree showed that TroTLR3,TroTRIF,TroTRAF6 and TroTBKl were clustered with their counterparts from other fish,respectively,indicating that their genetic relationships were close and relatively conserved among different fish species.2.The expression of TroTLR3,TroTRIF,TroTRAF6 and TroTBK1 in Brain,Gill,Stomach,Intestine,Head kidney,Liver,Heart,Spleen and Muscle was examined using qPCR.The results showed that the expression of four genes were detected in all the examined tissues.Among them,TroTLR3 had the highest expression level in the liver,followed by the head kidney,and the lowest expression level in muscle.TroTRIF and TroTRAF6 had the highest expression level in intestinal,but TroTRIF had the lowest expression level in heart,while TroTRAF6 was the lowest in muscle.And the highest expression level of TroTBKl was detected in spleen,whereas the lowest expression was detected in brain.These results indicate that the expression of four genes in immune-related tissues is higher than in non-immune-related tissues,suggesting that TroTLR3 signaling pathway related genes play an important role in the body’s innate immune response.3.After challenged with LPS,TroTLR3 expression levels showed different changes in various tissues including head-kidney,spleen,liver,gill and muscle.In the immune-related tissues(Head Kidney,Spleen,Liver and Gill),the mRNA expression level of TroTLR3 was significantly up-regulated compared with the control group(P<0.05).However,there was no significant change in the expression level of muscles.The expression level of the TroTRIF gene is similar to that of TroTLR3.In the five tissues examined,the expression of TroTRAF6 were up-regulated after infected with LPS.In the head kidney,the expression of TroTBK1 was significant up-regulated,while the expression level in other tissues was up-regulated but not significant.After polyI:C stimulation,the expression levels of TroTLR3 in the spleen,head kidney,gill and liver were significantly up-regulated(P<0.05).The expression level of TroTRIF in spleen and liver was significantly up-regulated,and the expression level was continuously high.Although the expression level in gill and muscle was up-regulated,it was not significant.The expression level of TroTRAF6 was significantly up-regulated in different tissues.The expression levels of TroTBK1 in the spleen,head kidney,liver and sputum were also significantly up-regulated(P<0.05).After stimulation by Vibrio alginolyticus,there was no significant change in the expression level of TroTLR3 in the head kidney and muscle,and the expression levels in thespleen and liver were significantly up-regulated.The expression level of TroTRIF was significantly up-regulated in the head kidney,spleen and gill,and there was no significant change in liver and muscle.TroTRAF6 was significantly up-regulated in all five tissues examined,and the expression level in the head kidney was significantly up-regulated by 23.45-fold at 12 h and continued to be highly expressed.The expression levels of TroTBK1 in the spleen,head kidney and muscle were up-regulated to varying degrees,up from 1.17 to 5.38 times.These results indicate that TroTLR3 and related molecules are involved in the 11immune response of the body against LPS,polyI:C and Vibrio alginolyticus.4.4.The ORFs of TroTLR3 and signaling pathway related genes were cloned into pET-32a and pEGFP-N1 expression vectors,respectively,to complete the construction of prokaryotic expression and eukaryotic expression recombinant plasmids.Initial expression of TroTRIF and TroTRAF6 proteins laid the foundation for functional studies of these genes.