| Infection by pathogenic microorganisms often caused the significant losses inaquaculture. Breeding aquaculture species with highly antibacterial infection is acost-effective way to prevent and control diseases.Natural resistance associated macrophageprotein (Nramp) gene as a candidate gene is closely related to disease resistance in fish. Itssequence information and expression pattern analysis have an important value on the Niletilapia antibacterial research.The natural resistance associated macrophage protein (Nramp) family are class of naturalimmune-related proteins which inhibited pathogenic bacteria infection in intracellular. Thefamily in the higher vertebrates have two members that were Nramp1and Nramp2withdifferent functions. Fish has the only one member that is Vertebrates Nramp2homolog. Inthis study, Nile tilapia was the experimental material. By the RACE, nested PCR, touchdownPCR and real-time quantitative reverse transcription PCR (qRT-PCR) detection methods, theNramp gene was cloned. Aeromonas hydrophila was artificial infected and Nramp geneexpression change research in vivo tissues after infection. Nile tilapia ovarian cells werecultured in vitro and the Nile tilapia ovarian cell line has been established. It laid a foundationfor the subsequent Nramp gene eukaryotic expression and other related research in Niletilapia, such as analysis of Nramp gene for the different expression model in vivo and in vitro,and to verify the function of Nramp gene. The main findings are as follows:(1) Obtained a840bp Nile tilapia Nramp gene cDNA sequence and submitted toGenbank (ONLTM, accession Number: KF006313). the sequence encoding280amino acids.The secondary structure of Nile tilapia Nramp gene containing six transmembrane segments,which are located in64-86,115-134,144-166,178-197,217-239and260-278. There is alow-complexity region in81-96. (2) The amino acid encoded by Nile tilapia Nramp sequence has a wealth ofphosphorylation sites: a protein kinase C phosphorylation sites (S/T-X-R/K) in the34th aminoacid site; in site17and157there are found two casein kinase II phosphorylation sites(S/T-X-X-D/E); in site255it is a tyrosine kinase phosphorylation site(R/K-X-X-D/E-X-X-X-Y); six N-cardamom acylation sites are in the site56,65,94,135,153and226which have the same type (G-E/D/R/K/H/P/F/Y/W-X-X-S/T/A/G/C/N-P).(3) The predicted Nile tilapia Nramp molecule spatial structure shows very similar in thespace structure with the Nramp molecule in model animal zebrafish, Nramp molecule inrainbow trout and Nramp1molecule in human. The result reflects highly conservedcharacteristics in the Nramp gene. Predicted Nile tilapia Nramp molecule has the typicalspatial transmembrane structure of membrane proteins.(4) Nile tilapia Nramp amino acid sequence similarity with the other16species between69%-93%, which with the highest homology in striped bass and turbot both are93%, whilethe lowest similarity are in Nramp1of human and mouse, that are70%and69%, respectively.The Phylogenetic tree of Nramp showed that Nile tilapia Nramp has a higher homology withthe Nramp2in mammals than that in Nramp1, reflects the level of the Nramp genehomology associated with its evolutionary history. Conserved105amino acids are among the280amino acid residues, its conservative rate is37.86%. It has three conserved cysteineresidues.(5) The qRT-PCR analysis in ck,5h,24h,48h,72h and96h showed that, Nramp gene inkidney, gill, spleen, muscle, fins, liver, blood, gonads, heart, intestine, brain and stomachwhich12tissues of Nile tilapia were all expressed and shown a constitutive expression pattern.The differential expression in Nramp gene reflects its correlation with the immune response.The Nramp has highest expression in the spleen, kidney, intestine, gill, liver, stomach andblood tissues, moderate expression in the heart, brain and gonadal tissues,while theexpression in the fin and muscle tissues is lowest. Expression changes in the liver at24h andbrain at72h shows the highly significant difference (p <0.01). Blood at5h, stomach at48hand liver at96h shows significant difference (p <0.05). (6) During in the96h after artificially infected with Aeromonas hydrophila, the trend ofNile tilapia Nramp gene expression has the biggest change in the liver, blood, intestines, gills,brain, stomach and heart, while the minimum magnitude in the gonads and fins which isrelatively stable. After5h infected with Aeromonas hydrophila by artificially, the Nramp geneincreased expression in gills, fins, heart and stomach, the blood is significantly upregulatedexpression (p <0.05). After24hours of infection, the increased expression in the kidney, fins,heart, intestine and brain, the expression in liver is highly significantly increased (p <0.01).After48hours of infection, the increased expression in the liver, blood and heart, theexpression of the stomach is significantly upregulated (p <0.05). After72hours infection, theexpression of the brain is highly significantly increased (p <0.01), the expression in all of theremained tissues increased except the fins, stomach and liver. At96hours infection, theexpression of the liver is highly significantly increased (p <0.01) and the expression allincreased except in the stomach, blood, muscles and fins.(7) The Nile tilapia ovary cells which isolated and cultured in vitro has been successfullysubcultured21times and has established its cell lines.The results of this study laid a foundation for further revealed that anti-infectivemechanism in Nile tilapia. It provides important reference for the subsequent study in Niletilapia Nramp gene difference expression between in vivo and in vitro, and further cultivationwith Nramp gene highly expressed transgenic Nile tilapia. |
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