Molecular Cloning And Expression Profiling Of MDA5 And LGP2 Genes From Grass Carp Ctenopharyngodon Idella

Grass carp Ctenopharyngodon idella is considered an economically important aquaculture species in China with the largest production among the"Great Four Cultured Fish". Despite its fast growth, delicate taste and low production cost, the toughest challenge is that grass carp is susceptible to a wide variety of diseases, and the annual loss reaches billion yuan. Grass carp hemorrhage caused by Grass Carp Reovirus (GCRV) is accused of a threat to grass carp health. However, the available vaccines are subject to be less effective because of the primitive specific immunity of grass carp. Thus, identifying critical molecules in the innate immunity networks will become the basis for disease-resistant breeding in the future.In the phylogenetic history, a number of homologs executing antiviral roles are revolutionarily conserved in metazoans. The remarkable examples include: TLR (Toll-like receptors) and RLR (RIG-I-like receptors) signaling pathways. The RLR pathways have recently been recognized as a critical antiviral mechanism in mamamals. There are three members referred to as RLRs: RIG-I (Retinoic acid-inducible gene-I), MDA5 (Melanoma differentiation-associated gene 5) and LGP2 (Laboratory of genetics and physiology 2), which play separate roles in recognition of cytosolic nuclear acids and activate a series of host immune response to viruses. It is thereby significant to uncover the roles of fish RLRs in combating aquatic viruses.In the present study, degenerate primers were designed based on sequence alignement to produce core amplicons, followed by RACE (Rapid amplification of cDNA end), semi-quantitative RT-PCR (sqRT-PCR), quantitative reat-time PCR as well as bioinformatic analysis in succession. The complete cDNA of CiMDA5 and CiLGP2 genes was cloned to explore their roles in anti-GCRV and the phylogeny of RLRs. This research provides new insights to the unknown fish RLR family. Results are as follows:(1) Molecular cloning and immune responsive expression of CiMDA5 geneThe cytoplasmic helicase protein MDA5 recognizes long molecules of viral double-stranded RNA (dsRNA) and single-stranded RNA with 5' triphosphate and mediates type I interferon secretion. In the present study, the first MDA5 gene in fish was cloned and characterized from grass carp. The full length of CiMDA5 cDNA comprises 3233 nucleotides in length and encodes a polypeptide of 961 amino acids. The deduced amino acid sequence contained six main structural domains: a CARD (Caspase activation and recruitment domain), a DEXDc (DEAD/DEAH box helicase domain), a ResIII (Conserved restriction domain of bacterial type III restriction enzyme), two HELICc (Helicase superfamily c-terminal domain) and a RD (Regulatory domain). The CiMDA5 mRNA was widespread expression in the tested tissues, was high level in spleen, skin and gill tissues, and was up-regulated by GCRV injection by sqRT-PCR assay. The CiMDA5 transcripts in spleen were significantly up-regulated at 12 h (1.80 folds, P<0.05), reached the crest at 24 h (7.48 folds, P<0.05), and then recovered to normal level at 48 h post-injection (P>0.05) of GCRV. In liver, the temporal expression of CiMDA5 mRNA was significantly increased at 48 h (5.00 folds, P<0.05) and returned to control level at 72 h (P>0.05) after GCRV challenge.(2) Identification and expression profiling analysis of CiLGP2 cDNALGP2 is homogenous to RIG-I and MDA5 without a CARD required for signal transduction, plays a pivotal role in modulating signaling by RIG-I and MDA5 for interferon synthesis. In this study, a novel LGP2 gene from grass carp designated as CiLGP2 was isolated and characterized. The full-length cDNA of CiLGP2 was of 2920 bp with five instability motifs (ATTTA). The open reading frame was of 2043 bp and encoded a polypeptide of 680 amino acids, including five main overlapping structural domains: two DEXDc, one ResIII, one HELICc and one RD. There was one moreα-helix in the RD, compared with that in human. The CiLGP2 mRNA was ubiquitous expression in the tested tissues, was high level in spleen, skin, heart and intestine tissues, and was up-regulated by GCRV injection by sqRT-PCR assay. The CiLGP2 expression in spleen was significantly up-regulated at 12 h (14.50 folds, P<0.05), reached the crest at 24 h (19.00 folds, P<0.05), and then dropped a little at 48 h (10.40 folds) post-injection of GCRV and kept this level till the following test period (P<0.05). In liver, the temporal expression of CiLGP2 mRNA was significantly increased at 24 h (3.80 times, P<0.05), reached peak at 48 h (10.70 times, P<0.05), and then decreased a little bit at 72 h (5.80 times, P<0.05) and kept this high level till the test finish (P<0.05).These data implied that the expression of CiMDA5 and CiLGP2 genes is associated with the antiviral innate immune defense to GCRV; meanwhile, the results lay a foundation for the further research of RLR signaling pathways in fish. These two cytoplasmic receptors are critical in virus recognition; in-depth understanding of them will be conducive to the development of effective measures against viral infection in aquaculture.