Molecular Cloning,Tissue Expression,Polymorphism And Association Analysis Of Reproduction Related Genes-FKBP4,NPM2 And FZD2 In Gaoligong Pigs

Sow fecundity plays an important role in pig production and breeding.FKBP4,NPM2 and FZD2 genes are important genes related to reproduction.In this study,the FKBP4,NPM2 and FZD2 genes of Gaoligong pigs were cloned by RACE-PCR.The DNA and protein sequence characteristics of Gaoligong pig FKBP4,NPM2 and FZD2 genes were analyze by bioinformatics tools.Subsequently,the tissue expression of these three genes were detected by real-time PCR.Finally,To explore whether these genes can be used as reproduction-related candidate genes of Gaoligongshan pig,through PCR+sequencing method,the polymorphism of the coding region sequences of these three gene were investigated in 200 Gaoligongshan pig which have litter size records.The experimental results are described as below:1.FKBP prolyl isomerase 4(FKBP4),also known as FK506 binding protein 52(FKBP52),is a widely expressed peptide prolyl isomerase.In this study,a 1577 bp c DNA sequence of the Gaoligongshan pig FKBP4 gene was cloned by RACE-PCR.Gene prediction through Gen Scan software showed that this 1577 bp c DNA sequence encodes a complete single gene of 456 amino acids.The isoelectric point is 4.90 and the molecular weight is 12.67 k Da.The protein encoded by this gene has 92.81%,92.59%,92.16%,92.16%and 91.94%high homology with that of Eurasian badger,North American river otter,red fox,California sea lion and spotted seal respectively.This protein has two FKBPs＿C superfamily conservative domain.Computer aided analysis showed that the genome sequence of the gene contained 10 exons and 9 introns,with a total length of 7441bp.Further analysis showed that there were 7 mutation sites in the coding region of pig FKBP4 gene.Through PCR amplification and sequencing of FKBP4 gene coding region in 200 Gaoligongshan pigs,no polymorphism were detected.Tissue expression analysis showed that porcine FKBP4 was highly expressed in spleen and fat,and moderately expressed in heart,muscle,spleen,lung,liver,ovary,and kidney.2.NPM2 protein(Nuclear Phosmin/Nuclear Plasmin 2),also known as nuclear fibrinolytic protein,is a gene expressed in nuclear chaperone protein,mainly expressed in oocytes and eggs.In this study,a 991-bp c DNA sequence of porcine NPM2 gene was obtained,and this sequence encodes a complete single gene of 219 amino acids,with a protein isoelectric point of 5.05 and a molecular weight of 7.90 k Da.The protein encoded by this gene has 83.33%,79.09%,79.09%,77.03%,76.82% high homology with that of small headed porpoise,wild bactrian camel,alpaca,longan camel,and oblique abdomen lizard,respectively.The protein has a conserved Nucleoplasmin like domain.Computer aided analysis showed that the genome sequence of the gene contained 8 exons and 7introns,with a total length of 13898-bp.Further analysis showed that there were 5 variants and 4 mutation sites in the coding region of pig NPM2 gene.Through PCR amplification and sequencing of the coding region of NPM2 gene in 200 Gaoligongshan pigs,no polymorphism were detected.Tissue expression analysis showed that porcine NPM2 was highly expressed in muscle,spleen,ovary,and kidney,and moderately expressed in heart,lung,liver,and fat.3.Frizzled2(FZD2),some studies have indicated that the FZD2 gene affects ovarian development.In this study,a 1941-bp FZD2 gene c DNA sequence was obtained by RACE-PCR from Gaoligongshan pig.Genetic prediction of the c DNA sequence was performed using Gen Scan software.It was found that the gene encodes a completely single gene with 558 amino acids,with a protein isoelectric point of 4.85,and a molecular weight of 16.32 k Da.The protein encoded by this gene has 97.85%,97.85%,97.67%,and 97.31%homology with that of cattle,lancenose bat,American red deer,large hoofed bat,and marmoset,respectively.This protein has a Cysteine-rich Wnt-binding domain and a CRD＿Domain cysteine-rich domain.Computer assisted analysis showed that the genomic sequence of the gene contained an exon with a total length of 1649 bp.Further analysis showed that there were 24 mutation sites in the coding region of porcine FZD2 gene.By PCR and sequencing the coding region of the FZD2 gene of 200 Gaoligongshan pigs,we found FZD2 gene C166 A mutations were associated with the litter size of Gaoligongshan pigs The total litter size(TNB)and live litter size(NBA)of the AA genotype sows were significantly higher than those of the CC genotype and the CA genotype(P<0.05).Tissue expression analysis showed that porcine FZD2 was highly expressed in the heart,spleen,and ovary,and moderately expressed in muscle,lung,liver,fat,and kidney.These results established a molecular foundation for further study of porcine FKBP4,NPM2 and FZD2 genes as Gaoligongshan pig reproduction related candidate genes.