Cloning And Primary Functional Analysis Of TLR7 Subfamily And Signaling Pathway Related Genes In Nile Tilapia

Toll-like receptors(TLR)is an important pathogen recognized receptor(PRR)in innate immunity.The TLR7 subfamily is one of the important members of TLRs.including TLR7,TLR8 and TLR9,localized in endosome.It can recognize bacterial or viral nucleic acids,and play an important role in the immune response against disease through myeloid differentiation factor 88(My D88)dependent or TIR domain-containing adaptor-inducing interferon-β(TRIF)dependent signaling pathway.Tilapia(Oreochromis niloticus)is an excellent breed of freshwater aquaculture in China,and its annual aquaculture output accounts for about half of the world’s total output.However,the diseases in recent years have caused great economic losses to tilapia culture in China.In order to understand the immune system of tilapia and explore the mechanism of disease resistance and immunity,the c DNA sequence of TLR7 subfamily genes and their signaling pathway related genes TRIF and TIR domain-containing adaptor protein(TIRAP)of Nile tilapia(Oreochromis niloticus)were obtained by reverse transcription PCR(RT-PCR),and bioinformatic analysis was also performed.Quantitative real time PCR(q RT-PCR)was used to detect the tissue distribution of these genes in healthy in dividuals and individuals intraperitoneal injection with Streptococcus agalactiae,lipopolysaccharides(LPS)and polyinosinic polycytidylic acid(Poly I:C).The recombinant eukaryotic expression vector was constructed to analyze the subcellular localization of these genes in 293 T human(Homo sapiens)embryonic kidney cells and the activation of nuclear factor κB(NF-κB).The experimental results of this paper are as follows:1.Cloning and expression analysis of TLR7 subfamily genes in Nile tilapiaThe c DNA sequences of TLR7(Gen Bank accession no.MT338568),TLR8(Gen Bank accession no.MT338569)and TLR9(Gen Bank accession no.MT338570)were 5172 bp,3704 bp,3343 bp respectively.The the open reading frame(ORF)of the three genes were3162 bp,3111 bp,3237 bp respectively;encoding 1053,1036,1078 amino acids.Smart software analysis shows that TLR7,TLR8 and TLR9 encode proteins with typical structural characteristics of TLRs.The homology of amino acid sequence showed that TLR7,TLR8 and TLR9 of Nile tilapia were the most similar to those of Astatotilapia calliptera(98.09%,89.67% and 95.45%,respectively).The expression level of TLR7,TLR8 and TLR9 m RNAswere widespread in all the tested tissues and organs(liver,skin,heart,kindey,stomach,gill,brain,spleen,intestine,blood and muscle).The expression level of TLR7 was the highest in the brain,and that of TLR8 and TLR9 was the highest in the spleen.After artificial infection with S.agalactiae,TLR7 m RNA expression level increased significantly in intestine,gill,spleen,kidney and blood,and reached it’s peak value at 8 h of infection;m RNA expression level TLR8 increased significantly in intestine,gill,spleen and kidney,and decreased in blood;TLR9 m RNA expression level increased significantly in intestine,gill and kidney,and reached it’s peak at the earliest(8 h).After LPS stimulation,TLR7 and TLR9 m RNA expression level were down-regulated in intestine,up-regulated in liver,spleen and kidney;TLR8 m RNA expression level was up-regulated in kidney only after LPS stimulation for 5 days.After Poly I:C stimulation,TLR7 and TLR9 m RNA expression level were up-regulated in intestine,liver,spleen and kidney,and the highest expression was in liver;TLR8 m RNA expression level was up-regulated in intestine,liver and kidney,and down regulated in spleen.The result indicated that TLR7 subfamily genes play a role in the immune response and participate in the immune response of Nile tilapia.2.Cloning and expression analysis of TLR7 subfamily signaling pathway related genes in Nile tilapiaThe c DNA sequences of TRIF(Gen Bank accession no.MT199561)and TIRAP(Gen Bank accession no.MT338571)were 3135 bp and 3913 bp.The open reading frames(ORF)of the two genes were 1653 bp and 876 bp;encoding 550 and 291 amino acids,respectively.The analysis of amino acid sequence alignment with other species showed that the TRIF and TIRAP had the typical structure of three Signaling boxes in the TIR domain.The amino acid sequence homology showed that Nile tilapia TRIF and Neolamprologus brichardi TRIF had the highest similarity(94.00%);Nile tilapia TIRAP had the highest similarity to the Astatotilapia calliptera TIRAP(92.78%).The m RNA expression of TRIF and TIRAP genes were widespread in all the tested tissues and organs,and both were highest expressed in muscle.The expression of TRIF and TIRAP m RNAs were up-regulated in intestine,gill,spleen and kidney,but down regulated in blood.After LPS induction,TRIF and TIRAP m RNA expression levels were up-regulated in the liver and spleen,down regulated in the intestine,and the highest expression level of TIRAP m RNA was found in the kidney after 8 h stimulation.After Poly I:C stimulation,the expression of TRIF m RNA expression level peaked in the liver and spleen at 8 h and decreased significantly in the kidney;TIRAP m RNA expression level decreased in the intestine and increased in the liver,spleen and kidney.The result indicated that TRIF and TIRAP genes play important roles in the immune mechanism of Nile tilapia against pathogens.3.Functional analysis of TLR7 subfamily and signaling pathway related genes in Nile tilapiaIn order to explore the functions of TLR7、TLR8、TLR9、TRIF and TIRAP genes and their roles in signaling pathways,eukaryotic expression vectors of the above genes were constructed and transfected into 293 T cells for subcellular localization and dual-luciferase reporter assay.Subcellular localization showed that Nile tilapia TLR9 was distributed in the cytoplasm and nucleus.TLR8,TRIF and TIRAP distributed mainly in the cytoplasm.TLR7 did not show a significant fluorescent signal in 293 T cells.Dual-luciferase reporter assay showed that overexpression of TLR7,TLR8 and TLR9 in293 T cells had no significant effect on activity of NF-κB,but they could significantly enhance My D88-mediated NF-κB activity after co-transfection with My D88.Overexpression of TRIF in 293 T cells can significantly increase the activity of NF-κB,overexpression of TIRAP has no significant effect on the activity of NF-κB.The above results provide basic information for the study of the function of TLR7 Subfamily genes and their signaling pathway related genes in Nile tilapia.