Molecular Characterization And Expression Analyses Of Five Genes In The Toll-like Receptor Signaling Pathway From Bostrychus Sinensis

Chinese black sleeper(Bostrychus sinensis)is an important economic marine fish in Fujian,Guangdong and Guangxi province in China.With the increasing density of intensive farming,the disease of the Chinese black sleeper has frequently occurred in recent years,which made huge economic losses.The innate immune system is the first effective defense against microbial pathogens.It recognizes the pathogen-associated molecular patterns(PAMPs)through a series of pattern recognition receptors(PRRs).Subsequently,the release of inflammatory cytokines and type I interferons is induced to respond to microbial pathogens.Toll-like receptors(TLRs)are a crucial family in PRRs that are type I transmembrane receptors.In order to understand the immune system of the Chinese black sleeper and the mechanism of TLR signaling pathway,we cloned five important genes in the TLR signaling pathway: TLR1(BsTLR1),TLR2(BsTLR2),TLR5(BsTLR5),MyD88(BsMyD88),and TIRAP(BsTIRAP).Subsequently,these genes were analyzed by bioinformatic.To further understand the role of these genes in innate immunity,their expression patterns in healthy fish tissues were analyzed by quantitative real-time PCR technique,as well as expression patterns after V.parahaemolyticus and Poly I:C stimulation.The specific research results are as follows:(1)The open reading frame of BsTLR1,BsTLR2,BsTLR5,BsMyD88 and BsTIRAP genes were 2385,2448,2646,867 and 648 bp,and encoding 794,815,881,288 and 215 amino acids(aa),respectively.All three TLR genes have a typical TLR protein domain: comprising LRR(Leucine-rich repeat),LRRCT(Leucine-rich repeat Cterminal),transmembrane region and TIR(Toll-IL 1-resistance)domain.BsMyD88 contains a death domain and a TIR domain.BsTIRAP contains a TIR domain.Multiple sequence alignment showed that BsTLR1,BsTLR2,BsTLR5,BsMyD88 and BsTIRAP were more similar to other fish species.The phylogenetic tree also showed that the five genes were closer to fish in evolutionary relationships,and have a certain genetic distance from mammals and birds.(2)Tissue distribution expression showed that BsTLR1,BsTLR2,BsTLR5,BsMyD88 and BsTIRAP were expressed in all detected tissues and had high relative expression levels in the immune tissues.BsTLR1 was highly express in head kidney and spleen and had the lowest relative expression level in the intestine;BsTLR2 was highly express in spleen and head kidney and had the lowest relative expression level in muscle;BsTLR5 was highly express in liver and head kidneys and had the lowest relative expression level in skin.BsMyD88 was highly express in liver and head kidneys and had the lowest relative expression level in intestine;BsTIRAP was highly expressed in peripheral blood and spleen and had the lowest relative expression level in muscle.(3)After using V.parahaemolyticus and Poly I:C to stimulate Chinese black sleeper,the expressions of BsTLR1,BsTLR2,BsTLR5,BsMyD88 and BsTIRAP were significantly up-regulated in peripheral blood,head kidney,liver and spleen,but their expression patterns were different,indicating that BsTLR1,BsTLR2,BsTLR5,BsMyD88 and BsTIRAP are involved in the immune response of V.parahaemolyticus and Poly I: C.