Cloning,Expression Analysis Of IgT And PIgR Immunorelated Genes In Half-smooth Tongue Sole (Cynoglossus Semilaevis)

Half-smooth tongue sole(Cynoglossus semilaevis)is mainly distributed in the coastal areas of China,and is an important marine fish cultured in China.Because of its delicious tenderness,high nutritional value and rapid growth,it is popular and widely cultivated.With the increase in the number of cultured animals,the threat of diseases has become increasingly serious.In particular,gill,skin and intestinal tracts have been most severely festered by Vibrio harveyi,causing serious economic losses to the aquaculture industry.At the same time,the main living environment of fish is in the water,in which tissues such as skin,gills and intestines,which are covered by a large area of mucous membranes,are the first places that pathogens infect the fish.Therefore,the mucous membrane not only has a simple physical isolation,but its local mucosal immune defense is more important for the infection of pathogens.Due to the low evolutionary status of fish and its own characteristics,the acquired immunity mechanism of fish has not been perfected,making non-specific immunity play a more important role than the specific immunity in resisting the invasion of foreign pathogens.Therefore,it is extremely urgent to study the immune genes of the half-smooth tongue sole from the aspects of molecular immune response mechanisms.IgT is a novel immunoglobulin gene that is different from IgM and IgD of teleoste,belonging to the immunoglobulin superfamily.In this study,based on the predicted sequence of the immunoglobulin tua(IgT),the full-length cDNA sequence of IgT was obtained in half-smooth tongue sole(Cynoglossus semilaevis),which was 2033 bp including an open reading frame(1794 bp,encoding 597 animo acids),the 5'UTR region(99 bp)and the 3 'UTR region(140 bp).Conserved domain analysis revealed that the IgT amino acid of half-smooth tongue contains a signal peptide,four heavy chain domain(CH)and one transmembrane domain.According to the protein sequence homology alignment and phylogenetic tree analysis,we found that the IgT of half-smooth tongue sole has highly similarity with the Japanese flounder(Paralichthy solivaceus).Real-time PCR analysis showed that the IgT gene expressed in the healthy half-smooth tongue sole with the highest relative expression in gill,with the higher relative expression in mucus,intestine,spleen and kidney,and the lowest relative expression in muscle.After challenge with Vibrio harveyi,the IgT gene initially increased and then decreased in 6 immune tissues of the half-smooth tongue sole,with the peak time point in skin and liver is 12 h,and the peak time point in kidney is 24 h,the peak time point in gill and spleen is 72 h,the expression trend of the IgT gene in the intestine is continually upregulated.qRT-PCR results of different tissues of IgT genes in resistant family and susceptible family show the IgT gene relative expression levels in spleen,intestine,sputum,and mucus of resistant family were higher than susceptible family,whereas IgT expression levels in kidney and liver did not differ significantly between resistant and susceptible families.These experimental results fully demonstrate that the IgT gene plays an important immune role in the process of resisting pathogens in the half-smooth tongue sole.The candidate genes were provided for the evaluation of non-invasion and disease resistance of fish,the selection of disease-resistance-related gene markers,the cultivation of disease-resistant varieties,and the selection of disease-resistant families.Poly immunoglobulin receptor(pIgR)is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily and exists as a membrane integrin that mediates poly immunoglobulin(pIgs)in mucosal epithelial cells.Polar operation,playing an important role in both non-specific immunity and specific immunity.In this study,based on the predicted sequence of the polymeric immunoglobulin receptor(pIgR),the full-length cDNA sequence of pIgR was obtained in half-smooth tongue sole(Cynoglossus semilaevis),which was 1419 bp including an open reading frame(1020 bp,encoding 339 animo acids),the 5'UTR region(109 bp)and the 3 'UTR region(290 bp).Conserved domain analysis revealed that the pIgR amino acid of half-smooth tongue sole contains a signal peptide,two Ig-like domains(ILDs)and one transmembrane domain.According to the protein sequence homology alignment and phylogenetic tree analysis,we found that the pIgR of half-smooth tongue sole has the highest homology and similarity with the turbot(Scophthalmus maximus)and the Japanese flounder(Paralichthy solivaceus).qRT-PCR analysis showed that the pIgR gene expressed in 9 tissues of healthy halfsmooth tongue sole with the highest relative expression in gill,and the lowest relative expression in muscle.After challenge with Vibrio harveyi,the pIgR gene initially increased and then decreased in 5 tissues(liver,spleen,kidney,intestine and gill)of the half-smooth tongue sole,with the peak time point in spleen and gill is 48 h,and the peak time point in liver,kidney and intestine is 72 h.Unlike the internal organs,the expression trend of the pIgR gene in the skin is continually upregulated.These experimental results fully indicate that pIgR is involved in the immune response process of half-smooth tongue sole to the bacterial pathogen,and contribute to discovering the anti-bacterial gene maker.