Gene Identification And Immune Stress Mechanism Of MARCO Gene In Yellow Drums (Nibea Albifiora)

Yellow drum(Nibea albiflora)is a relatively common economic fish in offshore China.It is widely distributed in the East China Sea,the Yellow Sea,the Bohai Sea and the South China Sea.However,due to overfishing and severe damage of the marine environment,the wild resources of Nibea albiflora are gradually declining.Its breeding industry has gradually developed,but due to germplasm factors and the deterioration of the breeding environment,it inevitably leads to the frequent occurrence of diseases of breeding yellow croaker.Pathogen infection and body immune response are two important elements in the occurrence of diseases,which requires us to study the genes closely related to immune defense in the body on the basis of the development of new antibacterial drugs and treatment methods against pathogens.To maintain the sustainable development of the yellow aquaculture industry,the interaction mechanism between pathogens and immune molecules need to figure out for exploring the methods of improving the body’s resistance to stress and cultivating new disease-resistant breeding species.The scavenger receptor(MARCO),which is rich in collagen on macrophages,is an important gene in the scavenger receptor A family(SRA),which plays an important role in the natural immune system of organisms.However,there are still few reports on MARCO in fish.Therefore,MARCO was identified from Nibea albiflora,and its phylogeny,expression characteristics and subcellular localization were explored.The main research results obtained are as follows:1.Based on the transcriptome data of yellow drum,the full-length cDNA of 1788 bp for MARCO gene in scavenger receptor A family(named NaMARCO,GenBank accession number:MN243703.1)was identified,and its open reading frame(ORF)was 1266 bp encoding 421 amino acids.Multiple sequence alignment and phylogenetic tree analysis showed that the amino acid sequence of NaMARCO had high homology with MARCO of other tochiocephalidae fish.2.The expression of’NaMARCO could be detected in the six tissues of liver,spleen,kidney,muscle,gills and stomach.Among them,the highest expression of NaMARCO was in the spleen,followed by the kidney,which was the lowest in the stomach.3.The expression of NaMARCO gene was upregulated under the stress of Pseudomonas plecoglossicida with the highest at 48 hours(9.86 times)or PolyI:C at 2 h(5.17 times than that of the control group at 0 hours).After Vibrio alginolyticus or Vibrio parahaemolyticus infection the expression of NaMARCO reached the maximum level at 12h(20.20 times than that of the control group at 0 hours)and 4h hours a(10.98 times that of the control group at 0 hours)respectively.The results suggested that NaMARCO might be involved in the regulation of both inflammation priming and resolution.4.After transfecting the recombinant plasmid NaMARCO with green fluorescent protein was expressed in EPC cells(epithelial cells of carp).The results showed NaMARCO was expressed mainly on the cell membrane,the endoplasmic reticulum and the organelle membrane surrounding the nucleus appeared weak signals,which suggested that as an important kind of transmembrane scavenger receptors,had a potential role in the immune defense system of N.albiflora against the pathogen.5.To test whether the pathogenic bacteria used in this stuy can induce an immune response of yellow drum,the expression of SOD,CAT,GST and HSP70 were detected after being stimulated V.alginolyticus and V.parahaemolyticus in yellow drum.The results showed that they appeared a time-dependent manner in the spleen.The highest expression level of appeared at 12h with 11.33 times than that of the control group at 0 h under the stress of V.alginolyticus and at 8 hours for V.parahaemolyticus(8.61 times that of the control group at 0 hours).After being challenged with V.alginolyticus,CAT expressed significantly and got the peak at 2 h(10.94 times compared with the control group),but the peak appeared at 4 h by being infected with V.parahaemolyticus(4.46 times compared with the control group).The highest point of GST appeared at 24 h(13.30 times)after being challenged with Valginolyticus and 24 h(12.47 times)for V.parahaemolyticus challenge.The expression of HSP70 peaked at 12 h with a 18.44-fold increase,after which time point a bluff down-regulation presented.In the V.parahaemolyticus challenge assay,HSP70 was gradually induced and reach a significant level at 48 h with a 8.15-fold increase.Through the analysis of the above data,it could be known that the four immune stress factors participated in the immunity of yellow drums.The above experimental results showed that NaMARCO played an important role in the immune function of yellow croaker just like four immune stress factors(SOD,CAT,GST and HSP70)for the bacterial infection.Therefore,the experimental results in this article provide important basied for future research on the molecular mechanism of innate immunity in fish.