Expression Characteristics And Disease Resistance Of Natural Resistance Associated Macrophage Protein(Nramp) Gene From Grass Carp(Ctenopharyngodon Idellus)

In recent years, with the continuous expansion of aquaculture industry production scale as well as the deterioration of the culture environment, in the breeding process, grass carp outbreaks many disease, this not only harmed the production of grass carp, but also seriously hampered the development of cultivating grass carp. Breeding disease-resistant lines is an effective way to prevent the occurrence of disease and is a general trend for the healthy development of aquaculture industry. As a disease-resistant candidate gene, Natural resistance associated macrophage protein(Nramp) is closely related to disease resistance in fish and has an important value on the grass carp antibacterial research.The natural resistance associated macrophage protein(Nramp) family are class of natural immune-related proteins which inhibited pathogenic bacteria infection in intracellular. So far, The family in the mammals, such as humans and mice have two members that were Nramp1 and Nramp2 with different functions. In addition to rainbow trout(Nrampα and Nrampβ), the other fish have only one Nramp protein, the amino acid sequence alignment analysis showed that, the homology of the fish Nramp and mammals Nramp2 is higher than Nramp1, but fish Nramp has similar with Nramp1.In this study, grass carp was the experimental material. By Quantitative Real-time PCR, the expression of the gene was analyzed in different developmental stages, different tissues and four tissues at different time after challenge of grass carp with the Aeromonas hydrophila; the Nramp ORF area was obtained by RT-PCR technique, then expression and purification of grass carp Nramp protein. It laid a foundation for further study the function of Nramp, biological activity and regulatory mechanisms, as well as revealing the mechanisms of grass carp immune system against infection. The main findings are as follows:The level of Nramp mRNA in embryos gradually increases during embryogenesis from cleavage stage to fry stage after fertilization, with the highest level in fry stage. In adult fish, Nramp gene has different levels of expression in all thirteen tissues, Nramp transcripts were found to be highly abundant in liver, abundant in head kidney, spleen and intestine, least in eye and bladder.After challenged with Aeromonas hydrophil, uninjected groups as control, the level of Nramp mRNA in head kidney, liver, spleen and intestine increased significantly(P<0.05). Significantly up-regulated in head kidney at 12 h post-treatment, with a maximum level at 48 h post-infection, significantly down-regulated at 72 h and 96 h, the expression regressed to the initial level at 7d. The expression of Nramp in liver was significantly increased at 12 h post-treatment followed by a decrease at 24 h, raised again at 48 h, with a maximum level at 72 h post-infection and was still in the high expression level at 96 h and 7d. Significantly up-regulated in intestine at 12 h post-treatment, with a maximum level at 48 h post-infection, significantly down-regulated at 72 and 96h, the expression regressed to the initial level at 7d. In spleen, the level of Nramp mRNA increased at 4h post-infection, with a maximum level at 12 h, significantly down-regulated at 24 h and 48 h, the expression regressed to the initial level at 7d. The bacterial infection can improve the Nramp gene expression in immune-related organs, may suggest that Nramp play an important role in the innate immune response of grass carp.RNA was extracted from grass carp tissue and then was reverse transcribed to cDNA. The 642 bp ORF region of Nramp was amplified and ligated into pET-32a(+) expression vector to construct the prokaryotic expression vector of pET-32a-Nramp. Verified by enzyme digestion and DNA sequencing. pET-32a-Nramp was transformed into BL21, the results displayed that pET-32a-Nramp recombinant protein abundantly expressed in 0.5 mM/L IPTG induction after 6h and form inclusion bodies. By HisTrap HP, expected fragment size expressed proteins was purified, the purified protein was determined by western blotting using His-tag antibody. This laid a foundation for preparation of Nramp monoclonal antibodies, using immunohistochemistry and immunoblotting technology detecting the mRNA changes of Nramp gene in protein levels.Using PCR cloning sequencing screened grass carp Nramp gene polymorphism loci, comparative analysis found C2419 T mutations in Nramp gene 3 ’UTR region, occurred two genotypes AA and AB genotype. To study the correlation between polymorphism loci and grass carp disease resistance, 40 AA genotype and 40 AB genotype fish challenged with Aeromonas hydrophil, blooding sampling of the two genotypes in different time, detecting the Lysozyme activity, Superoxide dismutase activity, Alkaline phosphatase activity, Acid phosphatase activity. The enzyme activity of AA and AB genotype increased significantly(P>0.05), Alkaline phosphatase activity between AA genotype and AB genotype had no significant difference(P>0.05), the Lysozyme activity, Superoxide dismutase activity and Acid phosphatase activity of AA genotype and AB genotype were significant differences(P<0.05) at certain times. Preliminary research showed that the correlation between Nramp gene polymorphism loci and the disease resistance of grass carp need further study.