Molecular Characteristics,polymorphism And Expression Analysis Of MHC Ⅱ In Yellow Catfish(Pelteobagrus Fulvidraco)

Due to the increasing breeding density and the deterioration of the breeding environment,there has been an outbreak of columnar disease caused by Flavobacterium columnare,causing heavy losses to the yellow catfish(Pelteobagrus fulvidraco)breeding industry.Breeding of disease-resistant strains of yellow catfish is one of the effective measures to effectively prevent columnar disease,and the research on the molecular genetic basis of disease resistance is the prerequisite for disease-resistant breeding of the fish.Major histocompatibility complex(MHC)is an important immune-related gene family in vertebrates and plays an important role in resisting the invasion of pathogens.At present,the molecular biological characteristics and function of yellow catfish MHC Ⅱ in anti-bacterial immunity are still unclear.Therefore,based on the transcriptome data of yellow catfish,we cloned and characterized the c DNA sequences of MHC Ⅱα and MHC Ⅱβ.Quantitative Real-time PCR(qPCR)technology was used to detect the relative expression levels of MHC Ⅱα and MHC Ⅱβ genes in different tissues,embryos and early development stages of yellow catfish.We also used HE staining methods to evaluate the gill histopathological changes after infection of F.columnare.Additionally,expression level changes of yellow catfish MHC Ⅱα and MHC Ⅱβ were assessed in immune and digestive tissues responding to F.columnare challenge.The main research results obtained are as follows:1.Cloning,polymorphism and molecular characteristic analysis of MHC Ⅱα/β gene in yellow catfishThe MHC Ⅱα sequence of yellow catfish was 1502 bp,containing an 89 bp5’-untranslated region(UTR),a 705 bp 3’UTR and a 708 bp open reading frame(ORF)that encoded 235 amino acids.Its amino acid sequence includes signal peptide,α1 domain,α2 domain,connecting peptide,transmembrane region and intracellular region.The MHC Ⅱβ sequence of yellow catfish was 1841 bp,containing a 78 bp 5’-UTR,a 1016 bp 3’UTR and a 747 bp ORF that encoded 248 amino acids.Its amino acid sequence includes signal peptide,β1 domain,β2 domain,connecting peptide,transmembrane region and intracellular region.The secondary structure analysis revealed that the MHC Ⅱ molecule contains special conservative sequences,such as protein kinase C-phosphorylation site,casein kinase Ⅱ phosphorylation site,N-glycosylation sites and cysteine?residues,which is similar to that of other fishes.These special structures are closely related to the biological functions of MHC Ⅱ.Polymorphism analysis of the MHC Ⅱ c DNA sequences of six yellow catfish showed that MHC Ⅱα has 13 alleles,which encode 13 different amino acid sequences and named Pefu-DAA*0101-0801.MHC Ⅱβ has 11 alleles,which encode 11 different amino acid sequences,named Pefu-DAB*0101-0901.The similarity of the MHC Ⅱα amino acid sequence between yellow catfish and other vertebrates went from 30.0% to 71.5%.It shared the highest similarity with that of long-snout catfish(Leiocassis longirostris),which belongs to Siluriformes,up to71.5%.However,the amino acid sequence of MHC Ⅱα from yellow catfish shared lower similarity with that of amphibians,reptiles,birds and mammals,and the similarity was between 30% and 32.7%.The similarity of the MHC Ⅱβ amino acid sequence between yellow catfish and other vertebrates went from 33.2% to 83.5%.MHC Ⅱβ of yellow catfish has the highest similarity with that of long-snout catfish,up to 83.5%,and shared lower similarity with that of amphibians,reptiles,birds and mammals,between 33.2% and 36.3%.2.The mRNA expression levels of MHC Ⅱα/β in different tissues of yellow catfishThe results of qPCR showed that yellow catfish MHC Ⅱα is expressed in all tissues.The highest expression levels were detected in spleen and head kidney,followed by gills,body kidneys,intestines,and the lower expression in liver,stomach and other tissues.Similarly,MHC Ⅱβ was highly expressed in the spleen,head kidney,and gills,and moderately expressed in the body kidneys,intestines,and lowly expressed in the liver and stomach.3.The mRNA expression levels of MHC Ⅱα/β in different embryonic development stagesYellow catfish MHC Ⅱα/β has a higher expression levels from the fertilized egg to the 32-cell stage,and the expression level declined to a low level since the multi-cell stage until to 3d after hatching,and then the expression level began to increase at 4d after hatching.4.Histopathological changes in the gills after infected with F.cloumnareHE staining results showed that the gill morphology began to change significantly at 6 h after infection of F.cloumnare,and the length-width ratio of the secondary lamellae got smaller than that of the control group,especially at 24 h after infection,and simultaneously,symptoms of cell proliferation also appeared.5.Expression level changes of MHC Ⅱα/β in immune-related tissues of yellow catfish after infection with F.cloumnareYellow catfish MHC Ⅱα/β is significantly up-regulated in the midgut,hindgut,body kidney and spleen after infection of F.columnare,and the change trend of the expression level is firstly up-regulated,then stabilized,while the expression changes in the foregut and head kidney are not significant,implying that the MHC Ⅱ molecules on the antigen-presenting cells(APC)in the foregut and head kidney may not play a significant role in the resistance to F.cloumnare infection.In the liver and gills,the expression level of MHC Ⅱ remained high from 3h to 7d after infection,which indicates that the liver and gills have long-lasting immune response to the infection of exogenous pathogens.