Expression Analysis And Chromosomal Location Of Several Immune-related Genes Of NLR Family In Channel Catfish (Ictalurus Punctatus)

NLRs (nucleotide-binding domain, leucine-rich repeat containing receptors) is alarge family of intracellular pathogen recognition receptors (PRRs) that recognizepathogen associated molecular patterns (PAMPs)，it plays a significant role in innateimmunity. In the previous study, the identification and gene expression response toEdwardsiella ictaluri of NLRs subfamily A (NODs), including NOD1, NOD2,NLRC3, NLRC5and NLRX1were carried out in channel catfish （IctalurusPunctatus）. However, there are several pathogenes cause systemic infections andvarious diseases in channel catfish farming yield other than Edwardsiella ictaluri.Here we choose four representative pathogens: Edwardsiella tarda, Aeromonashydrophila, Streptococcus iniae and Channel Catfish Hemorrhage Reovirus (CCRV)to infect experimental channel catfish respectively, then genes expression of fiveNODs was determined by quantitative real-time PCR method. At the same time, inthis paper, we researched the chromosomal location of NOD1and NOD2, which veryimportant genes of NODs, with the fluorescence in situ hybridization technology, sothat further understand these genes more intuitively in the level of chromosomes,laying foundations for further research of channel catfish NODs. In addition, a familymember of inhibitor of apoptosis protein (IAP) termed baculoviral IAPrepeat-containing7(BIRC7) from channel catfish was identified by RACE.Quantitative real-time PCR was conducted to examine the expression profiles ofCcBIRC7in healthy tissues and responding to different pathogens (E.tarda, S. iniaeand CCRV). The main results are as follows:1. Expression profile of NODs after challenged with four different pathogensqRT-PCR results showed that in healthy catfish tissues, all the tested NODsgenes were found to be ubiquitously expressed in all12tested catfish tissues. Significant up-regulation of NOD1, NOD2, NLRC3, NLRC5mRNAs were observedin the intestine, liver and head kidney after infection with four different pathogens,whereas down-regulation was observed in the spleen after infection with A.hydrophila and CCRV. Expression of NLRX1gene was up-regulated in the intestine,liver and head kidney, but obviously decreased in the spleen after infection with fourpathogens. Among four different pathogens, S. iniae largely up-regulated NODsmRNAs, while CCRV only slightly enhanced NODs gene expression. Among fourimmune-related tissues, the order for NODs up-regulation was liver, head kidney,intestine, and spleen post various pathogens challenge. For the five NODs genes, thechange of NOD2is most significant while NLRX1gene expression is the smallestafter different pathogens challenge. All data suggest their involvement in the immuneresponses of catfish against the intracellular bacterial and virus pathogen in atissue-specific and pathogen-specific manner. The up-regulation and down-regulationof the tested genes showed a similarity of expression profiles after infection,suggesting the co-regulation of these genes, providing a basis for exploring theprecise immune-related molecular mechanism of NODs in catfish.2. Clone and identification of BIRC7and gene expression response to pathogensThe full length cDNA sequence of channel catfish BIRC7(CcBIRC7) was1686bp, containing a5’ UTR of93bp, a3’ UTR of399bp with a poly (A) tail and an ORFof1194bp encoding a putative protein of398amino acids. The putative CcBIRC7protein contains two BIR super-family conservative domains and a C-terminal RINGfinger motif. Phylogenetic analysis showed that catfish CcBIRC7was moderatelyconserved with other BIRC7. CcBIRC7was widely expressed in healthy tissues ofchannel catfish and with the highest expression in blood. E.tarda and S. iniae couldinduce CcBIRC7gene expression drastically in head kidney, liver and spleen whileCCRV virus could slightly induce CcBIRC7expression in head kidney and liver butreduce it in spleen. The result suggested CcBIRC7may play a potential role inchannel catfish innate immune system against bacterial and virus infections,especially as the anti-bacteria immune gene. This is the first report of BIRC7geneidentification and its expression in fish. 3. Chromosomal location of NOD1and NOD2NOD1and NOD2were located in channel catfish chromosomes respectivelywith BAC-FISH technology. The result showed that NOD1and NOD2located a pairof homologous chromosomes respectively, NOD1located at the end of the long arm,while NOD2is closed to the middle of chromosomes long arm, which confirm theprevious report that NOD1and NOD2are both single copy genes with southern blot.