Response Patterns Of Toll-like Receptor Family To LPS And Cloning Of TLR4a/TLR4b And Expression Analyses In Yellow River Carp(Cyprinus Carpio Haematopterus)

Yellow river carp(Cyprinus carpio haematopterus)is one of the most widely cultivated and commercially important freshwater fish species in China.However,the environmental factor stress and bacteria,especially in intensive fish farming condition,could result in the injury of liver and the descent of immune ability,and subsequently,the outbreak of the disease,causing the large economic losses.Toll-like receptors(TLRs),as the key pattern recognition receptors,play an important role in recognizing various pathogen-associated molecular patterns and initiating signal transduction.Bacterial lipopolysaccharide(LPS),a cell wall component characteristic of Gram-negative bacteria,is a representative PAMP that allows animal cells to recognize bacterial invasion and trigger innate immune responses.Higher animals are extremely sensitive to LPS even at low doses,but lower vertebrates like fish are often resistant to LPS shock.LPS has been found to be responsible for the pathogenicity of several bacterial diseases,especially of Gram-negative origin,in fish.However,there is no clear understanding with the of immune response to LPS challenge.To determine the immune response of the TLR family against LPS challenge is very important for understanding the roles of TLRs in fish.The expression levels of 18 TLRs were detected in liver after LPS challenge,the expressions of certain TLRs(TLR1,TLR4,TLR5 M,TLR20,and TLR22)were significantly up-regulated in liver.However,TLR3,TLR18,TLR19,and TLR25 were generally down-regulated after LPS challenge.With regard to the members of the TLR7 subfamily(TLR7,TLR8,and TLR9),TLR2 and TLR21,no significant change was observed at any concentrations,at most time points,compared with the control.This is the first report to investigate the expression patterns of a complete set of TLRs in freshwater fish during LPS stimulation.Complete cDNA sequences of TLR4 a and TLR4 b in Yellow River carp were cloned and characterized.(1)The full-length cDNA of TLR4 a was 2836 bp,with a 123 bp 5’-UTR,a 388 bp 3’-UTR,and a 2325 bp ORF encoding an 794 amino acid polypeptide with a Mw of 121.5 k D and a p I of 5.73.Protein structure analysis indicated that TLR4 a possesses 6 LRR motifs,a LRR-CT motif,a transmembrane region,and a TIR domain.(2)The full-length cDNA of TLR4 b was 2897 bp,with a 198 bp 5’-UTR,a 233 bp 3’-UTR,and a 2466 bp ORF encoding an 821 amino acid polypeptide with a Mw of 123.1 k D and a p I of 5.81.Protein structure analysis indicated that TLR4 b possesses 8 LRR motifs,a LRR-CT motif,a transmembrane region,and a TIR domain.(3)Homology alignment based on full-length amino acid sequences indicated that TLR4 proteins were highly conserved with other fish ortholog,and the close evolutionary relationship may possibly suggest their vital functions in fish.Evolution analysis showed the high homology with other cyprinid fish,ecpecially with TLR4 a and TLR4 b of Danio rerio.Tissue specific expression analysis showed that TLR4 a and TLR4 b were expressed in all 12 tested tissues(Spleen,brain,trunk kidney,head kidney,muscle,liver,gill,fin,intestine,heart,blood,skin).TLR4 a and TLR4 b were higher expressed in head kidney and spleen,but very lower expressed in blood and muscle.In response to lipopolysaccharide(LPS)challenge,the expressions of TLR4 a and TLR4 b were significantly up-regulated in head kidney,spleen and liver,but no significant change was observed in gill.The TLR4 a and TLR4 b expression level were the highest at 6 h at higher LPS doses(8 mg/m L)in head kidney and spleen.In liver,the TLR4 a and TLR4 b expression level were the highest at at 6 h at low concentration(2 mg/m L),with 28.64-fold and 25.39-fold increase,respectively.The result showed TLR4 a and TLR4 b play an important role in the immune defense to LPS,the m RNA expression level there is an obvious tissue specificity and related to the LPS injection concentration.