Polymorphism Of The 3’-UTR Of The Porcine BMP7 And BMP15 And Their Association With Reproductive Performance

Bone morphogenetic proteins(BMPs)are essential for female reproduction,and their dysregulation or dysfunction can cause follicular developmental block and ovulation disorders,and eventually lead to reduced fertility and even infertility.Whole-genome resequencing found that porcine BMP7 and BMP 15 genes are located in regions of high or low fertility breeds or groups with different genomic differences.They are potential candidate gene for pig reproductive traits,but the relationship between the mutation sites of porcine BMP7 and BMP 15 gene 3’-untranslated(3’-UTR)and their reproductive traits has not yet been clarified.This article intends to take the BMP 7 and BMP15 genes as research objects,identify the 3’-UTR of porcine BMP7 and BMP15 genes using RACE and sequencing technology,and screen for mutation sites in the 3’-UTR of BMP7 and BMP 15 genes using pool DNA sequencing technology.The mixed model was used to analyze the relationship between mutation site polymorphisms and reproductive traits in Large White pigs.The dual luciferase reporter system was used to analyze the regulation of mutations and miRNAs on the 3’-UTR activity of porcine BMP7 gene.It is hoped to provide a reference for analyzing the regulatory mechanism of the 3’-UTR of porcine BMP7 and BMP 15 genes,and to explore molecular markers for marker-assisted selection and genome selection in pigs.The results of this study included the following three parts.1.Identification and characterization of the 3’-UTR of the porcine BMP7 geneThe 3’-UTR of porcine BMP7 gene was isolated and identified by RACE technology.The sequence was 1538 bp in length and contained a typical poly A tail(26 bp in length)and polyA signal(AAUAAA,located 1095 bp before polyA tail).In addition,several classic RNA regulatory elements were found in the 3’-UTR sequence of porcine BMP7 gene,including an AU enrichment element(AREs,AAUUA),an GU enrichment element(GREs,UUGUU)and 9 potential miRNA response elements(MREs),etc.2.Polymorphism analysis of the 3’-UTR of BMP7 gene in PigsUsing pooled-DNA sequencing technology,two mutations were detected in the 3’-UTR of BMP7 gene in Large White pigs,including one point mutation c.1569A>G and one insertion/deletion mutation c.2251del_CTGGGGT.Polymorphism analysis showed that the dominant genotype of mutation c.1569A>G was AG(the genotype frequency was 0.450).and the dominant genotype of mutation c.2251 del_CTGGGGT was Ⅱ(the genotype frequency was 0.638).Association analysis of polymorphisms with four reproductive traits such as the total number of piglets born(TNB),the total number of piglets born alive(NBA),number of stillborns(NSB),and litter weight(LW)in large white pigs.Polymorphism of mutation c.1569A>G was significantly associated with LW in large white pigs,and LW of AG-genotype sows was significantly higher than that of AA-genotype sows.Polymorphism of mutation c.2251del_CTGGGGT was not significantly associated with four reproductive traits in large white pigs.In addition,the combined genotypes of two mutations were significantly associated with TNB,NSB,and LW in a Large White pig population.TNB of the AG/ID-genotype sows was significantly higher than that of the GG/ID-genotype sows(P<0.05),and NSB of the AG/ID-genotype sows was significantly higher than that of the AG/II-genotype sows(P<0.05).LW of AG/ID-genotype and AG/II-genotype sows is significantly higher than that of AA/II-genotype sows(P<0.05).In addition,LW of AG/ID-genotype was 0.57 higher than GG/ID-genotype sows,but the difference was not significant(P=0.0891).These results indicate that BMP7 is a candidate gene for reproductive traits in pigs.3.Regulation of the 3’-UTR activity of the porcine BMP7 geneTo investigate the effect of mutation c.1569A>G on the 3’-UTR activity of the porcine BMP7 gene,two reporter vectors of the porcine BMP7 3’-UTR contains allele A and G,pGL3-A and pGL3-G were constructed.Luciferase assay revealed that the luciferase activity of vector pGL3-A was higher than that of vector pGL3-G,but the difference was not significant.Using three online tools miRTarBase,miRWalk and miRcode,we predicted that miR-22-3p can bind to the 3’-UTR of the porcine BMP7 gene,and luciferase assay revealed that miR-22-3p can inhibit luciferase activity of reporter vector of the porcine BMP7 3’-UTR,but had no significant effect on the luciferase activity of reporter vector of the 3’-UTR with mutation-type miR-22-3p binding site,indicating that miR-22-3p can inhibits the 3’-UTR activity of the porcine BMP7 gene via miR-22-3p binding site within the 3’-UTR.4.Identification and polymorphism analysis of the 3’-UTR of the porcine BMP 15 geneWe next isolated a 1214-bp sequence of the 3’-UTR of the porcine BMP15 gene by PCR amplification and sequencing.The 3’-UTR of the porcine BMP 15 gene contained a ployA signal(AAUAAA)and several classic RNA regulatory elements,including 3 GU enrichment elements(GREs,UUGUU)and 10 potential miRNA response elements(MREs).One point mutation c.2366G>A was found in the 3’-UTR of BMP 15 gene using pooled-DNA sequencing technology.In the Large White pig population,GG is the dominant genotype(the genotype frequency was 0.732).The association analysis found that polymorphism of mutation c.2366G>A was not significantly associated with reproductive traits in a Large White pigs.Combined genotype analysis found that the combined genotypes of BMP15 c.2366G>A and BMP7 c.1569A>G or with BMP7 C.1569A G and c.2251de1_CTGGGGT are significantly associated with the reproductive traits of Large White pigs.LW of sows with genotypes AA/GA and AA/GG were significantly higher than that of sows with genotypeAG/GG TNB and NSB of sows with genotype AG/ID/GG were significantly higher than that of sows with genotype A G/II/GG.LW of sows carried genotypes AG/ID/GG and AG/II/GG were significantly higher than that of sows carried genotypes AA/II/GA and AA/II/GG.In conclusion,we identify the 3’-UTR of the porcine BMP7 gene and clone the 3’-UTR of the porcine BMP 15 gene.We confirm that BMP7 is a candidate gene for sow reproductive traits.,Mutations in the 3’-UTR were potential genetic markers in breeding of sow reproductive traits.