| Nucleotide-binding oligomerization domain NOD1 and NOD2 are cytoplasmic pattern-recognition receptors recognizing bacteria. The researches on NOD1 and NOD2 has been mainly focused in mammals, whereas little in fish. In addition to the IFN-γgene, an IFN-γrelated gene neighbouring the authentic IFN-γgene in the genome was identified in teleost fish, and named as IFN-γrel. The literature on IFN-γrel is rather limited in fish. In the present study, the complete cDNA and genomic sequences of grass carp NOD1 (gcNOD1), NOD2 (gcNOD2) and IFN-γrel (gcIFN-γrel) were cloned using RACE and genome walking techniques, together with expression analysis following reovirus infection or injection of LPS, polyI:C and PGN. The studies for the further study of these genes laid the foundation in anti-microbial infection of the basis functions.The gcNOD1 cDNA contains 3215 bp. The predicted open reading frame (ORF) contains 2813 bp and encodes a putative 937-aa protein with a 350 bp 5'-untranslated region (UTR) and a 52 bp 3'-UTR. The cloned gcNOD2 cDNA consists of 3130 bp with an 81 bp 5'-UTR, a 2949 bp ORF encoding a 982 aa peptide and a 103 bp 3'-UTR.The gcNODs have very similar domains and architectures as the zebrafish and mammalian counterparts and mainly consists of three different domains:(1) LRR:the leucine-rich repeat lie on the C-terminal of NOD molecular, consisting of 2 LRR domains in gcNOD1 and 7 tandem LRR domains in gcNOD2; (2) NACHT:a central domain of NOD molecular; (3) Effector domain:an N-terminal effector domain that contains one CARD domain in gcNOD2, while For gcNOD2,2 N-terminal CARD domains. The gcNOD1 gene consists of 11 exons, with 10 intervening introns, spaced over approximately 9 kb of genomic sequence.The gcNOD2 gene has a length of approximately 5 kb, and is composed of ten exons and nine introns. gcNOD1 protein was predicted as a non-secretory protein without a signal peptide, whereas the gcNOD2 protein surprisingly possesses a putative signal sequence of 24 aa in length, that is predicted to be cleaved between Ala24 and Glu25.The alignment of gcNODs with zebrafish, human, pig and mouse NODs showed that NACHT domain is most conserved among three domains of vertebrate NODs, with the highest identity varying from among 58.0-92.5% for NOD1 and 56.7-90.1% for NOD2. The identities of full-length, CARDs, NACHT and LRRs between gcNOD1 and NOD1 from other species were higher than that of gcNOD2 and NOD2 from other species.Real-time quantitative PCR showed that in healthy grass carp, the expressions of gcNOD1 and gcNOD2 were observed in all the tissues examined such as brain, liver, head kidney, gill, intestine and blood. Expressions of gcNODl and gcNOD2 gene were examined by Real-time quantitative PCR in grass carp after i.p. injection with PBS, LPS, PGN or polyI:C. These stimulus can significantly induce the expression of gcNOD1 and gcNOD2 in all tested tissues. By using real-time quantitative RT-PCR analysis, the induced fold changes of gcNODl by reovirus (GCRV) was highest on day 1, then gradual decreased, while the highest induced fold changes of gcNOD2 in spleen were observed on day 2. The positive of gcNODl protein were detected in the tissues examined such as in intestine, liver, spleen, kidney, brain and blood by using immuno-histochemistry.The cloned gcIFN-γrel cDNA consists of 861 bp with a 39 bp 5'-UTR, a 504 bp open reading frame encoding a 167 aa peptide and a 316 bp 3'-UTR. The 3'-UTR of the gcIFN-γrel gene is extremely AT rich (75.2%) and contains 5 mRNA instability motifs (ATTTA). A potential polyadenylation signal (AATAAA) is located 13 bp upstream from the poly [A] tail. The gcIFN-γrel gene has a length of approximately 2 kb, composed of four exons and three introns.The gcIFN-yrel protein possesses a putative signal sequence of 24 aa in length. Fish IFN-yrel proteins (166-171 aa) are shorter than the IFN-γcounterparts (180-194 aa) in length and thus lack a classical nuclear localisation signal (NLS) seen at the C-terminal region. The gcIFN-γrel protein has 38-63 aa identities/63-78% aa similarities with other known fish IFN-γrel,18-22 aa identities/41-53% aa similarities with other known fish IFN-γ. In a phylogenetic tree based on amino acid sequences from fish IFN-γand IFN-γrel, the gcIFN-yrel clustered with fish IFN-yrel including channel catfish, common carp, and zebrafish, with 76% bootstrap value.Expression of gcIFN-γrel gene was examined by Real-time quantitative PCR in grass carp after i.p. injection with LPS, PGN or polyI:C. LPS, PGN and polyI:C can significantly induce gcIFN-γrel expression in all tested tissues. In spleen and trunk kidney, the gcIFN-γrel transcripts increased more by injection of LPS than polyI:C. Conversely, polyI:C was more potent than LPS in inducing gcIFN-γrel expression in blood and head kidney. Unlike LPS and polyI:C, PGN resulted in 39-fold increase in spleen, the highest among the four tissues. The expression level of the gcNOD1, gcNOD2 and gcIFN-γrel gene was examined by real time PCR in grass carp after i.p. injection with PGN. Similar to the expression pattern of gcIFN-γrel induced by PGN, the highest expression of gcNOD2 was seen in spleen, then blood, head kidney and trunk kidney. In contrast, the expression of gcNOD1 was significantly increased only in spleen. Real-time quantitative PCR showed after inoculating GCRV, the gcIFN-γrel gene was induced in spleen of grass carp over the 7 days period, with the highest transcript level observed at day 2.
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