Cloning And Polymorphism Analysis Of OASL Gene In Gaoyou Duck And Study On The Effect Of DHAV-3 On OASL Expression

The OASL gene is one of the interferon-stimulating genes.Its protein family includes OAS1,OAS2,OAS3 and OASL.Up to now,only the OASL has been found in poultry.This article takes the OASL gene as the research object,clones the full sequence of Gaoyou duck OASL gene and conducts bioinformatics analysis.Then,PCR amplification was performed on the partial sequences of the OASL gene of four different populations of Beijing duck,Gaoyou duck,Putian duck and Shaoxin duck,and the polymorphism of the OASL gene between different populations of ducks was analyzed.In the end,The duckling hepatitis model was constructed to compare the distribution and expression of OASL gene in healthy ducklings and infected ducklings,in order to providing a relevant basis for the study of OASL molecular evolution and antiviral immune mechanism1 Cloning and bioinformatics analysis of Gaoyou duck OASL geneIn this experiment,Gaoyou duck was used as the research object,and the OASL gene’conserved sequences of Cherry Valley duck,Crested duck,chicken,and goose were used as templates to design 5 pairs of primers for different regions.A total of 4321bp base sequence was obtained by PCR amplification and sequence splicing,and the sequence encodes 443 amino acids.It is predicted by biological software that its secondary structure is composed ofα-helix,extended chain and random coil.The typical domain is N-terminal oligoadenylate synthase-like domain and C-terminal two ubiquitin Domain.This protein is a hydrophilic protein with no signal peptide site and transmembrane structure.The prediction of glycosylation sites revealed 3 potential N-glycosylation sites and 13 O-glycosylation sites Phylogenetic tree show that ducks and chickens are in the same branch,and the homology is as high as 100%.All mammals except Mus musculus belong to the same branch,and the relationship is close.This study provides a basis for an in-depth understanding of the genetic variation of OASL molecules and the molecular mechanism of exerting antiviral functions,so as to apply the research results to practice of antiviral breeding in poultry.2 Comparison of OASL gene polymorphismIn this experiment,the Beijing duck,Gaoyou duck,Putian duck and Shaoxin duck were used as the experimental objects.The sequence of the 5 ’regulatory region of the OASL gene was amplified and analyzed for polymorphism.A total of 5 single nucleotide polymorphism(SNP)sites(g.73C>T,g.95G>A,g.196T>C,g.280C>G,g.619C>G)were found.Calculate the polymorphic information value of each allele by Popgene32 software,The results showed that:Among the five polymorphic sites,except for Beijing ducks,the heterozygosity at g.73 and g.95 was higher.The heterozygosity of Shaoxin duck and Gaoyou duck at g.196 and g.619 was 0.The results of polymorphic information content showed that all five locis of Putian Duck were moderately polymorphic(0.25<PIC<0.5).The Gaoyou duck and Shaoxin duck were all moderate except g.196 and g.619.all five locis of Beijing Duck were low polymorphism(PIC<0.25).The results of linkage disequilibrium analysis showed that in g.73 and g.95,g.73 and g.280,g.95 and g.196,g.95 and g.280,g.95 and g.619,There was a strong linkage disequilibrium state(0.33<r2<1),and has a complete linkage disequilibrium state(r2=1)between g.196 and g.6193 Distribution and expression differences of OASL gene in healthy duck and challenged duck tissues,In this experiment,healthy ducklings were used as experimental subjects.Two-day-old ducklings were infected with DHAV-3 strain under natural conditions.Heart,liver,lung,small intestine,and pancreas were collected and total RNA was extracted from each tissue.RT-PCR and real-time fluorescence quantitative PCR was used to detect the expression level of OASL gene in various tissues,The results show that:in the control ducklings,The gene expression of the small intestine is the highest,followed by the pancreas,liver and lungs,and the relative expression of the heart is the lowest.In the ducklings affected with DHAV-3,The gene expression of the liver is the highest,followed by the small intestine,heart and lungs,and the relative expression of the pancreas is the lowest.The expression of OASL gene in various tissues increased significantly after infecting.The heart has the highest upregulation in all tissue.It suggests that the heart may be an important expression tissue for antiviral gene.Conclusion:(1)Gaoyou duck OASL gene consists of 4321 bp sequence,encoding 443 amino acids,It is predicted that there is no signal peptide and transmembrane structure,and it is a hydrophilic proteins,which has 3 potential N-glycosylation sites and 13 O-glycosylation sites.(2)Comparing the OASL gene polymorphism,on the whole,There is a strong linkage disequilibrium between g.73 and g.95,g.73 and g.280,g.95 and g.196,g.95 and g.280,g.95 and g.619,and a complete linkage disequilibrium between g.196 and g.619.Putian Duck has a higher degree of heterozygosity than Beijing Duck,Gaoyou Duck and Shaoxin Duck.(3)The expression level of OASL gene in liver,heart,lung,small intestine,and pancreas of DHAV-3 ducks was significantly up-regulated,and the heart has the most upregulation.