CDNA Cloning And Expression Analysis Of Platelet-derived Growth Factor Receptor A Gene(PDGFR?) In Porcine(Sus Scrofa)

Platelet-derived growth factor receptor(PDGFR?)is the member of tyrosine kinase receptor family and has a potential role of regulating lipid metabolism.The aim of this study was to clone cDNA of PDGFRa and analyze its mRNA expression level in different tissues and different periods of pig(Sus scrofa).The specific primers were designed based on the published nucleotide sequence of human(Homo sapiens),bovine(Bos taurus)and mouse(Mus musculus)in GenBank,and the whole cDNA of porcine PDGFRa gene was cloned using RT-PCR and RACE.Using bioinformatics software aligned the homology of nucleotide and amino acid sequence between different species,and predicted the structure and function of porcine PDGFRa protein.Analyzning the PDGFRa gene polymorphism existed in three different varieties(Jinhua,Duroc and Yorkshire).Using Real-time quantitative PCR(qRT-PCR)detected PDGFRa gene expression level in different organizations,different parts of muscles,different growth stages,different breeds.The main results were as follows:1?Porcine PDGFRa gene cloning and sequencing The cloned full-length cDNA of PDGFRa gene was 6360 bp(GenBank accession:KP329568)containing an open reading frame(ORF)of 3270 bp,2964bp 3' untranslated regions(UTR),126 bp 5' UTR,encoding a protein of 1089 amino acid.This gene spaned approximately 148 Mb on the genome,and contained 23 exons and 22 introns.2?The bioinformatics analysis of PDGFRa proteinThe homology analysis of porcine PDGFRa gene indicated that the nucleotides and amino acids had high similarity with human,bovine,sheep(Ovis aries)and mouse.Ten N-glycosylation sites and 75 phosphorylation sites were predicted in the translated PDGFRa protein,as well as one signal peptide.The protein structure analysis showed that PDGFRa protein had 2 transmembrane helices.The N-terminal of the PDGFRa protein was high hydrophobic,but the C-terminal showed high hydrophilism.The PDGFRa protein charged negatively,hydrophilic,belonged to membrane binding protein.3?The polymorphism analysis of PDGFRa geneAnalyzning the PDGFRa gene polymorphism existed in three different varieties(Jinhua,Duroc and Yorkshire).The sequencing results found 13 SNPs,including 10 SNPs causing amino acid change.4?The gene expression analysis of porcine PDGFRaReal-time quantitative PCR(qRT-PCR)results suggested that PDGFRa mRNA expressed in heart,liver,spleen,lung,kidney,small intestine and skeletal muscle.PDGFRa.mRNA expressed abundantly in spleen,followed by kidney and lung.Six kinds of muscle,including soleus muscle,supraspinatus muscle,biceps femoris muscle,psoas minor muscle,gastrocnemius muscle and loin muscle,were selected.The qRT-PCR results showed that the expression of slow muscles represented with soleus muscle was extremely higher than that in fast muscles represented with loin muscle(P<0.01).Different growth stages expression analysis showed that expression of porcine PDGFRa gene was extremely higher in the primary stage than that in weaning and adult stage(P<0.01).Comparing PDGFRa gene expression between different breeds,the qRT-PCR results showed that that expression of different muscles(loinmuscle,soleus muscle,supraspinatus muscle,biceps femoris muscle,psoas minor muscle)in Jinhua pigs was extremely higher than Duroc and Yorkshire(P<0.01).The expression of gastrocnemius muscle in Jinhua pigs was significantly higher than in Yorkshire(P<0.05).In conclusion,the research obtains the full-length cDNA of porcine PDGFRa gene,the polymorphism in different varieties,the rule of tissue expression and relative mRNA expression of PDGFRa gene in different growth stages and different breeds.The results provide a basic data for further understanding of the function of PDGFRa gene.