title: A NLRC3-like gene from blunt snout bream (Megalobrama amblycephala): Molecular characterization, expression and association with resistance to Aeromonas hydrophila infection.
abstract: NLRC (the nucleotide-oligomerization domain (NOD)-like receptor subfamily C) consists of teleost-specific NLRs (NOD-like receptors) and plays pivotal roles in microbial recognition and regulation of innate immune response. In this study, we cloned and characterized a NLRC3-like gene (MamNLRC3-like) from blunt snout bream (Megalobrama amblycephala) by using the quantitative real-time PCR method, and analyzed the correlation between its polymorphisms and resistance to Aeromonas hydrophila infection. The full length cDNA of MamNLRC3-like was 2863 bp, with a 5'-UTR of 169 bp, ORF of 2301 bp and 3'-UTR of 393 bp, encoding 766 amino acid residues. MamNLRC3-like consisted of a conserved NACHT domain, a fish-specific NACHT associated domain (FISNA) and a PYRIN effective domain. During early developmental stages, MamNLRC3-like was most highly expressed at 39.4 hpf (hours post fertilization, hatching stage) and constitutively detected in various healthy tissues. The expression of MamNLRC3-like was strongly induced by A. hydrophila infection and peaked in the head kidney, liver, intestine, and trunk kidney at 12 h post infection. Six SNPs (single nucleotide polymorphisms) from MamNLRC3-like, with 2 in introns and 4 in exons, were identified by direct sequencing, in which 6515C/T was a novel non-synonymous mutation. HRM (high resolution melting) genotyping of the 6 loci showed that locus 6515C/T SNP was associated with the resistance/susceptibility of blunt snout bream to A. hydrophila infection (P < 0.01). The CC genotype fish were more resistant than CT carriers (P < 0.01). This study suggests that the MamNLRC3-like gene might play an important role in the innate immunity of blunt snout bream and could be used as a candidate marker for genetic selection of A. hydrophila-resistant blunt snout bream.
authors.affiliation: College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR China.
authors.forename: Fengjuan
authors.initials: F
authors.lastname: Zhou
authors.affiliation: College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR China.
authors.forename: Qifeng
authors.initials: Q
authors.lastname: Zhan
authors.affiliation: College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR China.
authors.forename: Zhujin
authors.initials: Z
authors.lastname: Ding
authors.affiliation: College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR China.
authors.forename: Lina
authors.initials: L
authors.lastname: Su
authors.affiliation: College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Fan
authors.affiliation: College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Cui
authors.affiliation: College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR China.
authors.forename: Nan
authors.initials: N
authors.lastname: Chen
authors.affiliation: College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China.
authors.forename: Weimin
authors.initials: W
authors.lastname: Wang
authors.affiliation: College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, PR China; Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province, Hunan University of Arts and Science, Changde 415000, PR China. Electronic address: liuhong59@mail.hzau.edu.cn.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
mesh_terms: D016980:Aeromonas hydrophila / Q000502:physiology; D000818:Animals; D003001:Cloning, Molecular; D003530:Cyprinidae / Q000196:embryology / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D060467:Disease Resistance; D005393:Fish Diseases / Q000235:genetics* / Q000276:immunology / Q000382:microbiology; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016905:Gram-Negative Bacterial Infections / Q000235:genetics / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000662:veterinary
chemical_list: D018076:DNA, Complementary; D029941:Fish Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger
keywords: Association analysis; High resolution melting; MamNLRC3-like; NLRC3-like; SNPs; Susceptibility/resistance