Cloning And Expression Analysis Of Toll-like Receptors(TLRs) And Signaling Pathway Related Genes Form Soiny Mullet (Liza Haematocheila)

Toll-like receptors(TLR)are important pattern recognition receptors(PRRs)of organisms,which play an important immune role in resisting the invasion of pathogenic microorganisms.Soiny mullet(sm,Liza haematocheila)is an important economic aquaculture fish in our country's coastal areas.It is a representative species of Mulletidae in the order of Mugilformes.With the expansion of mullet farming,reports on mullet diseases have increased year by year.The use of immunological methods to prevent mullt disease is of great significance for green and healthy farming of mullt.However,the current research on the immune system of flathead fish including mullt is almost blank.Fully understanding the mullt immune system is of practical significance for vaccine evaluation and genetic breeding.In this paper,we first cloned the genes of sm TLR13,sm TLR18,sm TLR22,sm TAK1 and sm TRAF6,and analyzed the sequence characteristics of the above genes.Soiny mullet TLR13,TLR18,TLR22,TAK1 and TRAF6 c DNA sequence lengths are: 2908 bp,2753 bp,3125 bp,1914 bp and 3416 bp.Open Reading Frame nucleotide sequences are: 2739 bp,2622 bp,2871 bp,1800 bp and 1716 bp.The number of encoded amino acids is inferred to be: 913 aa(amino acids),873 aa,956 aa,599 aa and 572 aa.The theoretical molecular weights of the proteins are: 106.35 k Da,100.59 k Da,109.8 k Da,66.9 k Da and 64.76 k Da.Predicted isoelectric points(p I)are: 6.28,8.47,4.79,4.89,and 5.89.Aliphatic indexes are: 103.21,91.27,29.60,26.59,and 64.63.Grand average of hydropathicity(GRAVY)are:-0.064,-0.245,0.861,0.915,and-0.517.SMART predicts that sm TLR13,sm TLR18,sm TLR22,sm TAK1,and sm TRAF6 have typical,conserved family domains;similarity analysis results prove sm TLR13,sm TLR18,sm TLR22,sm TAK1 and sm TRAF6 have higher sequence similarity of other fish.It shows that the cloned gene is indeed the homologue of the above genesPhylogenetic tree analysis showed that sm TLR13,sm TLR18,sm TLR22,sm TAK1,and sm TRAF6 were clustered in one branch with other fish-related genes.The related genes of freshwater fish and marine fish were spontaneously formed with mullt,indicating that the above genes exist in freshwater fish and marine fish.Fluorescence quantitative PCR detected the expressions of sm TLR13,sm TLR18,sm TLR22,sm TAK1 and sm TRAF6 in different tissues of healthy mullt.The results showed that sm TLR13,sm TLR18,sm TLR22,sm TAK1 and sm TRAF6 were expressed in all detected tissues.Further,the induced expression of the above genes in Streptococcus dysgalactiae was analyzed.The results showed that after Streptococcus dysgalactiae infection,the expression levels of sm TLR13,sm TLR18,sm TLR22,sm TAK1 and sm TRAF6 were significantly up-regulated in different tissues.In this study,soiny mullt was used as the experimental object to clone and analyze the expression of TLR13,TLR18,TLR22,TAK1 and TRAF6 for the first time,and to clarify the expression change rules of these genes in mullt during bacterial infection.The research results will lay a solid foundation for an in-depth understanding of the immune function and mechanism of fish TLRs signaling pathway.