A Genome-wide Survey And Expression Analysis Of NLR Receptors In Miiuy Croaker

Innate immunity plays an important role in resistance against major bacterial disease in vertebrates. NLR receptor as the intracellular pattern recognition receptors is the bridge of the innate immune and specific immune. Several subfamilies of NLRs have been characterized in mammals, but studies of NLRs in teleost species have been lacking. NLRs include three distinct subfamilies: NLR-A, NLR-B and NLR-C subfamilies. In this study, we conducted the analysis about the three subfamilies in miiuy croaker comprehensively.NLR-A subfamily members includes five members: NOD1, NOD2, NLRC3(NOD3), NLRC5(NOD4) and NLRX1(NOD5). We obtained these genes from the miiuy croaker genome database and miiuy croaker transcriptome. Structural analysis showed that miiuy croaker NLR-A subfamily members own the feature of 5'UTR intron which may influence their role in enhancing translation level. Through the synteny analysis and the repertoire of NOD1 and NOD2 genes in vertebrates, we proposed the evolutionary pattern of NOD1 and NOD2 genes. NOD1 underwent the duplication and NOD2 underwent the absence. Meanwhile, comparative analysis revealed NLRX1 duplicated into NLRX1 a and NLRX1 b, then NLRX1 a was lost in actinopterygii and NLRX1 b formed NLRX1 that now we called. Both the current lineages of NOD1 and NOD2 genes in bonyfish underwent purifying selection indicating that NOD1 gene in the ancestor of bonyfish experienced positive selection. Simultaneously, molecular evolutionary analysis indicated that the ancestral lineages of NLRX1 in tetrapod and actinopterygii under positive selection pressure. The positively sites in actinopterygii are mainly located in NACHT domain which was the critical region for signal transduction, suggesting that the evolution of NLRX1 gene in the ancestor of actinopterygii is beneficial in immune response. Induction experiments with Vibrio anguillarum indicated the different expression levels of NOD1 and NOD2 in liver, spleen and kidney of miiuy croaker. The expressions of NOD1 and NOD2 increased more significantly after poly(I:C) stimulation, meanwhile, we carried out the expression analysis at the transcriptome level and the regulation of micro RNAs. Pathogens challenge demonstrated that the expressions of NLRC3 and NLRC5 in miiuy croaker were induced not only by Vibrio anguillarum but also by poly(I:C), whereas NLRX1 exhibited more sensitive response to bacteria than virus.Otherwise, a total of 50 predicted NLR-like sequences were identified in the miiuy croaker. Meanwhile, we identified the members of NLR-B and NLR-C subfamilies containing 2 and 48 genes respectively in miiuy croaker according to the phylogenetic tree. Compared with other teleosts except for zebrafish, NLR-C subfamily genes occurred expansion in miiuy croaker. The gene expansions of NLR-C subfamily may illustrate adaptive genome evolution in response to specific aquatic environments. Structural analysis showed that the N-terminus of NLR-C subfamily receptors has different characteristics of the domains including RING domain, FISNA domain or PYRIN domain. Interestingly, the C-terminus of 18 NLR-C subfamily members contains an extra B30.2 domain(named NLR-B30.2 genes) which plays an important role in antiviral immune recognition. Simultaneously, molecular evolutionary analysis indicated that the positively sites in miiuy croaker are mainly located in NACHT domain which was the vital region for signal transduction in immune response. Significantly, pathogens challenge in spleen and macrophages demonstrated that NLR-B30.2 genes exhibited more sensitive response to virus than bacteria, suggesting these genes play enhanced roles in innate antiviral immunity, which may represent a new family used for antiviral infection.In conclusion, this is the first time to identify the NLR-A,NLR-B and NLR-C subfamilies in miiuy croaker. The expansion of NLR receptors play an important role in the innate immune response and the positively selected sites illustrate the evolution in the process of constantly adapting to the environment. Expression analysis showed provides a large resource in the response to pathogens and diseases, meanwhile, it will pave the way for the aquaculture.