Study On Molecular Characterization,Function And Regulation Of Miiuy Croaker TLR1

Vertebrates are constantly threatened by the invasion of microorganisms and have evolved systems of immune defense to eliminate infective pathogens in the body. The immune system is indispensable for vertebrates disease prevention. The vertebrate immune system is comprised of two branches: innate and acquired immunity. The innate immune system is the first line of host defense against pathogens. The innate immune system recognizes microorganisms via a limited number of germline-encoded pattern-recognition receptors(PRRs). Thereinto, Toll-like receptors are one important PRR in recognizing pathogen-associated molecular patterns(PAMPs), activating organism immune response. Fish, as low vertebrate, the acquired immune system is not well-developed, however, the relatively developed innate immune system plays an indispensable role in the host defense. In this study, we identified TLR1 gene in miiuy croaker, and we analyzed the sequence characteristics, function, evolution, signaling pathway and the miRNA regulation of miiuy croaker TLR1. The main research conclusions are as follows:(1) The full length miiuy croaker TLR1 cDNA is 3393 bp and contains 314 bp 5'-UTR sequence, 2406 BP open reading frame(ORF) encoding 801 amino acids, and 673 bp 3'-UTR. Miiuy croaker TLR1 gene sequence contains two introns and one intron of 4465 bp sequence. Miiuy croaker TLR1 gene has only one intron and the intron located in 5'-UTR which is very close to the initiating ATG codon. The theoretical molecular weight of mmiTLR1 is 90.7 kDa. Miiuy croaker TLR1 contains a signal peptide, N-terminal leucine-rich repeats(LRRs), a leucine rich repeat C-terminal domain(LRR-CT), a transmembrane region, and a Toll/IL-1 receptor domain(TIR).(2) Branch site model of molecular evolution analysis showed that 2 positively selected sites in ancestral lineages of teleosts were found, but none positively selected sites in the ancestral lineage of mammals, indicating TLR1 gene in the ancestor of teleost under positive selection pressure. But site models for molecular evolution analysis found that mammalian TLR1 has nine positively selected sites, but teleosts has no positively selected sites.(3) Phylogenetic tree analysis showed that the evolutionary relationship between TLR1 and TLR6 was closer than that of TLR1 and TLR10. Further comparative genomic analysis revealed that TLR1, TLR6, and TLR10 are adjacent to each other in the genome, and the evolutionary relationships of TLR1, TLR6 and TLR10 were inferred by comparative genomics methods. Then we suspected that the generation mammal TLR1 subfamily was the result of two rounds gene duplications and the first duplication occurred prior to the divergence of amphibian and the second prior to the divergence of eutherian. Moreover, the first duplication gives rise to TLR10 and TLR1/TLR6, and the second gives rise to TLR1 and TLR6.(4) qRT-PCR showed that miiuy croaker TLR1 was highly expressed in liver, and also high in immune related tissues(such as the spleen and kidney). Further Vibrio anguillarum injection showed the immune response of miiuy croaker TLR1.(5) Although the TLR signaling pathway is well studied in mammals, few studies have been involved in signaling pathways in fish. Miiuy croaker TLR1 can activate NF-?B reporter depend on TIRAP and MyD88 for signal transmission and immunocytochemistry showed the predicted interaction of MyD88 with miiuy croaker TLR1 TIR domain. Luciferase reporter assays showed that TLR1 makes response to LPS stimulation, and the shRNA for TLR1 silence further confirmed this result.(6) Bioinformatics analysis revealed that miiuy croaker TLR1 is one target of miR-200a-3p. Expression level of miR-200a-3p and miiuy croaker TLR1 after Vibrio anguillarum stimulation in miiuy croaker tissues and LPS stimulated miiuy croaker macrophages are in opposite trend. That showed that miiuy croaker TLR1 may be one of the targets of miRNA miR-200a-3p.