Molecular Characteristics And Expression Analysis Of TLR1/TLR2/TLR4 In Qihe Crucian Carp (Carassius Auratus)

Qihe crucian carp,Carassius auratus,as a triploid freshwater fish,are widely cultivated in northern area of Henan Province in China.Recently,with the increasing of cultivated density,immune defense ability decreases in Qihe crucian carp,which are susceptible to be attacked by infectious pathogens,and the outbreak of fish disease has brought about extensive economic losses in aquaculture.Therefore,in order to understand the immune mechanism of fish against pathogen infection,it is necessary to study the natural immune response to pathogen.Toll like receptors are a class of important pattern recognition receptors encoded by germ-line gene and expressed in immune cells,which could recognize the conserved molecular structures of pathogens,known as pathogen-associated molecular patterns,and trigger the signaling pathway that activate immune cells in response to pathogen infection.In the present study,the full-length cDNA sequences of CaTLR1,CaTLR2 and CaTLR4 in Qihe crucian carp were cloned and characterized.The relative expression levels of CaTLR1,CaTLR2 and CaTLR4 at different stages of embryonic development,in various tissues of fish,and after fish were respectively challenged by LPS,PGN,PolyI:C,and A.hydrophila,were detected by real-time fluorescent quantitative PCR.In addition,the effects on TLRs after the adding APS or CGA into feed were studied to explore immune response to active components of Chinese Herbs.The main research results obtained are as follows:(1)The cDNA sequences of CaTLR1,CaTLR2 and CaTLR4 were cloned and characterized.The full-length cDNA sequence of CaTLR1 was 2754 bp,which included a 5'-UTR of 60 bp,an ORF of 2394 bp encoding a protein sequence of 797 aa,and a 3'-UTR of 300 bp.The theoretical pI was 6.57,and the predicted molecular weight of protein was 91.5 kDa.The full-length cDNA of CaTLR2 was 2605 bp,which included a 5'-UTR of 36 bp,an ORF of 2367 bp encoding a predicted polypeptide of 788 amino acid residues,and a 3'-UTR of 202 bp.The predicted molecular weight of protein was 90.3 kDa,and theoretical pI was 7.57.The full-length cDNA sequence of CaTLR4 was 2859 bp,which included a 5'-untranslated region(UTR)of 183 bp,an open reading frame(ORF)of 2463 bp which encodes a predicted polypeptide of 820 amino acid residues,and a 3'-UTR of 213 bp.The theoretical pI was 8.28 and the predicted molecular weight of protein was 94.2 kDa.The domain and 3D models predicted based on the amino acid sequences of CaTLR1,CaTLR2 and CaTLR4 showed that they all have typical characteristics of TLR,including LRR domain,transmembrane domain and TIR domain.Based on CaTLR1,CaTLR2 and CaTLR4 respectively,homology alignment and phylogenetic analysis indicated that Qihe crucian carp is the closest relationship to the other cyprinids.(2)CaTLR1,CaTLR2 and CaTLR4 were widely expressed at different stages of embryonic development and larval stage.High mRNA expression levels were detected in fertilized egg and at the cleavage stage,followed by a decrease,and the minimum level was found at the hatching stage.After hatching,the expression levels gradually increased and remained at relative high level from 5 to 25 dph,and continued to increase to the highest levels at 30 dph.Generally,CaTLR1,CaTLR2 and CaTLR4 appeared to be developmentally regulated and had obvious dynamic changes during embryonic development.The lowest level was found at the hatching stage indicating the poor immune defense ability in this period,and it is necessary to pay more attention to care for embryos to prevent embryos from pathogen infection.(3)CaTLR1,CaTLR2 and CaTLR4 were constitutively expressed in eleven tissues examined,but the relative expression levels in different tissues varied significantly.CaTLR1 expression was the highest in liver and muscle,moderate in skin and head kidney,and lower in gill and brain.CaTLR2 was highly expressed in heart and muscle,while the low levels were found in gill and brain.CaTLR4 was highly expressed in muscle and skin,followed by gill and intestine,while lower in head kidney and kidney.The lowest expression of CaTLR1,CaTLR2 and CaTLR4 were detected in ovary.(4)The mRNA expressions of CaTLR1,CaTLR2 and CaTLR4 in response to the challenges of LPS,PGN,PolyI:C and A.hydrophila respectively showed as follows: expression level of CaTLR1 mRNA was significantly up-regulated in spleen and in response to LPS and A.hydrophila in head kidney;CaTLR2 and CaTLR4 could be significantly induced by LPS,PGN,PolyI:C and A.hydrophila,although the expression patterns were varied among the different TLR genes after different stimulus.The expression differences indicate that these genes may play different roles in responding to different pathogen infections.(5)Either APS or CGA was added to feed Qihe crucian carp,and could up-regulate the basal expression of CaTLR1,CaTLR2 and CaTLR4,suggesting that it can enhance the non-specific immunity of fish.In addition,both APS and CGA can significantly up-regulate the expression of CaTLR2 and CaTLR4 after challenging with A.hydrophila and PolyI:C,and it was suggested that both APS and CGA can enhance immune responses of Qihe crucian carp to A.hydrophila and PolyI:C through the increasing TLR mRNA expression levels.