Molecular Cloning And Characterization Analysis Of NLR-C Gene In Common Carp (Cyprinus Carpio L.)

Innate immunity as a defense against microbial or other foreign body penetrates the firstline of body defense, plays an important role in the immune defense. Therefore it has beenplayed more and more attention by immune research workers, becomes a hotspot in theresearch of immunology. Nucleotide binding domain and leucine-rich repeat containingfamily (NLR) is an important intracellular pattern recognition receptor, which could be ableto identify a variety of pathogenic microbial ingredients, eliciting an immune response, toplay an important role in the innate immune functions. NLR-C is a specific gene family andonly exists in teleost fish, whether NLR-C play the same role in the fish innate immunity?Design primers based on the NLR-C gene cDNA of other fish, we cloned carp (Cyprinuscarpio L.) NLR-C by reverse transcription PCR and RACE, Several primer sets weredesigned based on this partial DNA sequence for both5’and3’RACE. This transcriptconsisted of3665bp with a predicted open reading frame (ORF) of3078bp, a5’untranslatedregion (UTR) of100bp and a3’UTR of487bp. The ORF encoded for1175amino acid.Conserved domain analysis of NLR-C using SMART program indicated that the polypeptidewas composed of a middle NACHT, three leucine-rich repeat (LRR) regions andB30.2(PRY/SPRY) motif which resembled that of NLR-C, which has the typicalcharacteristic structure of teleost fish NLR-C.Several NLR-C amino acid sequence comparison of the carp, zebrafish, paralichthysolivaceus, and other teleost fish demonstrated that these domains are highly conserved inteleost fish.The phylogenetic tree of the full-length, NACHT domain of NLR-C wereconstructed using the amino acids sequences from mammalian and fish NLRs. Thephylogenetic analysis of the full-length NLRs demonstrated that the carp NLR-C sequenceshowed high homology to the NLRs family, and fish NLR-C were clustered together but notwith other known NLRs. Moreover, it has been revealed that the fish NLR subfamily C is restricted only to teleost fish. NLR-C in the NACHT structural domain of evolutionarykinship with NLRC3recently, This observation supported the independent and parallelevolution among members of the NLR-family.The expression of carp NLR-C was determined by QRT-PCR in different tissues, theresults showed that the highest expression in the NLR-C genes in carp gills, skin, brain,spleen and also have a higher expression in the oral, foregut, hindgut, but lower level ingonad.After immersion stimulation with V.anguillarum, NLR-C gene expression varied fromdifferent time points and tissues, NLR-C gene expression in the hindgut and skin appeared tobe down-regulated more than ten times. And it showed no significant change in the liver,spleen and head-kidney, although NLR-C gene showed increase in expression afterup-regulation at3h in the liver, In the foregut and gill, NLR-C gene expression had a varyingdegree of increase, in the foregut of carp, the level of NLR-C gene expression reached thehighest value at3h and reached the highest value at6h in the gill.NLR-C mRNA in the liver, spleen, head kidney, hindgut, gills, skin and foregut appearedin varying degree of significant up-regulation after injection infection with V.anguillarum,which has increased significantly, and subsequently showed a recovery trend. It was notablethat NLR-C gene expression in the head kidney and spleen showed significant increase.NLR-C gene expression rapidly increased to the highest level in the head kidney at3h, andin the liver and spleen it was abruptly increased at6h post-infection. in foregut,themaximum value reached to the highest level at48h, and in the gills, skin and hindgut, themaximum value reached to the highest level at24h compared to PBS control group. Andafter immersion and injection stimulition with V.anguillarum，IL-1β mRNA increased tovarying degrees in the head kidney and hindgut of carp.The results show that NLR-C gene isinvolved in innate immune defense of carp, resisting the invasion of pathogenicmicroorganisms, which indicated that NLR-C gene might play a significant role in the innateimmune response against V.anguillarum in carp.