Identifying SNPs In The Porcine MiRNA Target Gene Seed Sequences

The swine litter size is the important factors affecting porcine reproductive traits and economic traits,Embryo implantation plays an important role in determining swine litter size.So it is very necessary to investigate the genes which have influence on the embryo implantation.The miRNAs can combine with target gene 3’UTR seed sequence,thus change the expression of genes in the transcription level.SNP at miRNA binding site in target genes can affect the efficiency of miRNA and target gene,ultimately affect the expression of genes.This research uses the whole genome sequencing data and the miRNA sequence to predict SNP which at miRNA binding site in target genes.We carried out in vitro luciferase reporter assays and found target SNP that is located in WNT5A affected significantly the luciferase activity by the predicted interacting miR-628-5p.The reports show that WNT5A plays an important role in the embryo implantation of mouse,The immunohistochemistry and qPCR technology were used to investigate the role of WNT5A.The main results are as follows:(1)We got 404 SNP which at miRNA binding site in target genes by TargetScan software combining 64 individuals genome sequencing and miRNA sequences of pig.These SNPs located in the sequence of seeds distribute more uniforme.(2)We got some SNPs which were consistent with the prediction by PCR and sequencing in the Duroc×LandracexYorkshire pigs.We carried out in vitro luciferase reporter assays and found target SNP that is located in WNT5A affected significantly the luciferase activity by the predicted interacting miR-628-5p,and SNP located in VAV3 affected significantly the luciferase activity by the predicted interacting miR-122.(3)We detected the specific expression and localization of WNT5A protein in porcine placenta of Yorkshire pigs on different stages of gestation by immunohistochemistry.The results showed that the WNT5A protein is expressed on endometrial epithelial cells and uterine glands epithelial cells on day 9 of gestation.On day 15 and 18 of gestation,the WNT5A protein is expressed on Trophoblast epithelial cells specific and the staning of WNT5A protein is very strong.On day of 26 gestation,the WNT5A protein is expressed on Trophoblast epithelial cells and uterine glands epithelial cells.On day of 50 gestation,the WNT5A protein is expressed on endometrial epithelial cells,Trophoblast epithelial cells and uterine glands epithelial cells,but the staning of WNT5A protein is weak.On day of 95 gestation,the WNT5A protein is expressed on Trophoblast epithelial cells and uterine glands epithelial cells,uterine glands epithelial cells staning of WNT5A protein is stronger than Trophoblast epithelial cells.(4)We confirmed the expression of WNT5A and miR-628-5p by RT-PCR.The results showed that WNT5A is expressed in the heart,liver,spleen,lung,kidney,muscle and chorionic tissues.miR-628-5p is expressed in the all tissues,but the expression is very low.(5)We detected the expression level of WNT5A in AA,AG and GG genotype ofchorion of Yorkshire pigs on day of 50 gestation by the real-time RT-PCR.Statistical analysis showed that WNT5A gene is the highest expressed in GG genotype,and the lowest in AG genotype.GG genotype is expressed higher than AA genotype,but not differentially.AA genotype is expressed significantly higher than AG genotype.(6)On 3’UTR of WNT5A SNP was assessed by BcgI-PCR-RFLP in the experimental Yorkshire pigs population,the results showed that WNT5A gene was not associated with birth weight but associated with total litter size.