Molecular Characteristics Of TLR9/TLR21/TLR25 And Its Expression Responses To Chinese Herbal Medicine Addition In Qihe Crucian Carp Carassius Auratus

During the evolution process,non-specific immunity is a stable and inherited defense to resist the invasion of microorganisms,which is the basic and main immune defense system in fish.Toll-like receptor(TLR)is an oldest and most conserved molecule in the immune system,which plays an important role in identifying pathogen-specific PAMPs and triggering immune responses,and then activating downstream nuclear factors or interferon regulators through My D88 dependent or independent pathway.TLR could stimulate cells to produce effectors,such as tumor necrosis factor,interleukin and interferon,and eventually kill foreign pathogens.Qihe crucian carp,as a natural triploid fish,is popularly cultivated for high nutritional value in north area of Henan Province.However,in recent years,with the improvement of aquaculture technology,the expanded scale of intensive aquaculture with high density,and the increased species,the water environment has been led to the deterioration and fish diseases continuously increased.Toll-like receptors play an important role in preventing pathogen invasion,however,the functions of some TLRs are still unclear in fish.Therefore,TLR9,TLR21 and TLR25 gene in Qihe crucian carp are studied to ascertain molecular characteristics of TLRs and immune responses after the addition of active ingredients of Chinese herbal medicine,and to explore its potential function in congenital immunity.In this study,Qihe crucian carp was used as an experimental fish.The full length of TLR9,TLR21 and TLR25 genes were cloned by RT-PCR and RACE,and sequence characteristics and distribution in healthy fish were analyzed.Active constituent of Chinese herbal medicine—APS and CGA,were added to feed Qihe crucian carp to investigate its effect on TLR gene expression;After challenges with Aeromonas hydrophila and poly(I:C),the effects on the immune response of three genes were respectively analyzed to study the expression differences among three TLR genes after feeding with Chinese herbal medicine,in order to explore the effect of Chinese herbal medicine on non-specific immunity.The results of this study are as follows:1.The full-length c DNA sequence of Qihe crucian carp Ca TLR9 is 3520 bp,including the Open Reading Frame(ORF)3195 bp,encoding 1064 amino acids,5? untranslated regions(5?-UTR)129 bp,and 3? untranslated regions(3?-UTR)236 bp.SMART analysis revealed that the protein structure consists of a signal peptide,15 LRR motifs,a transmembrane domain and a TIR domain.The high similarity of TLR9 is in cyprinids with 80% amino acid identity.phylogenetic tree constructed based on TLR9 also showed that cyprinids were clustered a clade.Ca TLR9 gene is widely expressed in tested tissues,with the highest expression in blood,followed by muscle,liver and skin,and the lowest expression in ovary After fish were stimulated by A.hydrophila and poly(I:C),respectively,gene expressions of Ca TLR9 were up-regulated at 6,12 and 24 h in liver,spleen and head kidney.It was demonstrated that the Ca TLR9 is highly conserved in fish and plays an important role in immune defenses against pathogens in Qihe crucian carp.2.The full-length c DNA sequence of Ca TLR21 is 3290 bp,including ORF 2946 bp,encoding 981 amino acids,5?-UTR 143 bp,and 3?-UTR 201 bp.SMART analysis revealed that the protein structure consists of a signal peptide,15 LRR motifs,a transmembrane domain,and a TIR domain.The homology analysis showed that Ca TLR21 was the highest homology with common carps,and phylogenetic tree constructed based on TLR21 showed that Qihe crucian carp and common carp were clustered into one branch.Gene Ca TLR21 were widely expressed in tested tissues,with the highest expression in liver and muscle,followed by heart,head kidney,skin and kidney,and the lowest expression in ovary.After A.hydrophila infection,gene expressions were significantly down-regulated in the spleen,and up-regulated in liver and head kidney;after poly(I:C)stimulation,gene expressions were upregulated in different degrees in liver,spleen,and head kidney.These results indicated that the Ca TLR21 was obvious in immune response to pathogens.3.The full-length c DNA sequence of Ca TLR25 is 2596 bp,including ORF 2456 bp,encoding 817 amino acids,5?-UTR 38 bp,and 3?-UTR 104 bp.SMART analysis revealed that the protein structure consists of a signal peptide,7 LRR motifs,a transmembrane domain,and a TIR.Based on TLR25 homology and phylogenetic relationship analysis,Qihe crucian carp was closest to the other fish of cyprinids.The gene Ca TLR25 is widely expressed in tested tissues,with the highest expressed in blood,followed by muscle,skin,and head kidney,and the lowest expressed in ovary.After infection with A.hydrophila and poly(I:C),the gene Ca TLR25 was highly expressed in liver,spleen and head kidney.It was indicated that Ca TLR25 was highly conserved in evolution,with an important biological function,which was involved in immune response to pathogens in Qihe crucian carp.4.After Qihe crucian carp were fed with the addition of APS(Astragalus polysaccharide)or CGA(Chlorogenic Acid)for 6 weeks,the results showed that APS and CGA could increase genes expression in immune organs,respectively.After infection with A.hydrophila and poly(I:C),gene expressions in the spleen,liver,and head kidney showed that Chinese herbal medicine could regulate the expressions of gene TLR9,TLR21 and TLR25.These results indicated that,in the early immune response,APS supplementation could promote fish immune reaction and was beneficial for fish to eliminate pathogens.With regard to Qihe crucian carp fed with CGA,after pathogen stimulation,the expressions of TLR9,TLR21 and TLR25 were inhibited and the peak of expression was delayed.The results suggested that APS and CGA could promote the expressions of TLRs,and showthe different effects on TLRs expressions in response to pathogen infection.