Studies Of Toll-Like Receptors And Their Expressions In Blunt Snout Bream Megalobrama Amblycephala

The blunt snout bream, Megalobrama amblycephala, commonly known as Wuchang fish, is one of the most economically important freshwater fish species endemic to China. M. amblycephala has been widely favored for its delicacy and recognized as a main aquaculture species in the freshwater polyculture system. Nevertheless, M. amblycephala is extremely vulnerable to varous pathogens, especially Aeromonas hydrophila, which results in huge economic losses in recent years. The innate immune system is the primary defense line against pathogen invasion in lower vertebrates. It can specially recognize the conserved pathogen-associated molecular patterns(PAMPs) on the invading pathogens through pattern recognition receptors(PRRs) on the surface of the cells, and effectively activate signaling transduction pathways, and finally produce the relevant immune responses to the infection. Toll- like receptors(TLRs) were the earliest characterized and the most widespreadly studied pattern recognition receptors(PRRs), which play an important role on linking innate and acquired immunity. Hence, the study of TLRs in M. amblycephala is essential for elucidation of evolution and regulation mechanism. The results are as followed:(1) 14 TLR genes of M. amblycephala were identited through the RNA-seq database and genome database:TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 a, TLR8 b, TLR9, TLR18, TLR19, TLR20, TLR21 and TLR22. Protein domain structural architectures show that Ma TLRs possess 3 canonical structural motifs of the TLR family, including an extracellular LRR domain, a TM region and a cytoplasmic TIR domain. Multiple sequence alignment of Ma TLRs show a highly conserved cysteine positions(CxCx24-29Cx17-20C) within the LRRCT domain, and 3 conserved boxes: Box1(YDAF?X(Y/F)), Box 2(LCXXXRD?X?G) and Box3(FWXXLR) within TIR domain. 3D models of MaTLR-TIRs show typically structural feature: α helix and β sheet. Molecular phylogenetic relationship showed that Ma TLR are composed of six major families: TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11 family.(2) Ma TLRs are constitutively expressed in 9 examined tissues but varies with tissue’s preferences. Mostly Ma TLR genes are relatively high expression in spleen and kidney tissues. After A. hydrophila infection, clearly pathological features(haemorrhages, vacuolization and lymphocyte hyperplasia) in head kidney, intestine, gill, liver and spleen tissues of M. amblycephala. In liver, spleen and head kidney, the expressions of MaTLR1/2/5/9/21/22 were induced and Ma TLR3/7/8a/8b/19 were down-regulated. But the expression profile of Ma TLR4/18/20 in various tissues were different.(3) The c DNA full- length of M. amblycephala TLR4 is 2862 bp, with an open reading frame of 2364 bp encoding 787 amino acids. Ma TLR4 is a typical TLR protein, including LRR domain, a transmembrane region and a TIR domain. An mRNA instability signal(ATTTA) and a typical polyadenylatio n signal(ATTAAA) are found in the 3?-UTR. MaTLR4 has the highest homology and the closest evolution relationship with the grass carp Tlr4.2. Ma TLR4 mRN A was ubiquitously expressed in all examined tissues and during all sampled developmental stages. After A. hydrophila infection, the expression of MaTLR4 and MaMyD88 are up-regulated on the transcription and protein level in head kidney and intestine tissues at 12 h. These results indicate that Ma TLR4 involved in immune response to pathogen invasion.