Association Analysis Of BMP2 And PDGF-D Polymorphisms With Tail Type In Sheep And Functional Verification At The Cellular Level

Genome-wide resequencing analysis of domestic and foreign sheep breeds were carried out in the early stage.Some genes related to sheep tail type were screened.BMP2(bone morphogenetic protein 2)and PDGF-D(platelet-derived growth factor D)were significantly selected.Therefore,in this study,BMP2 and PDGF-D were selected as candidate genes for molecular and cellular level verification,aiming to provide basic research data for the regulation of fat deposition in sheep tail and provide available genetic markers for molecular breeding.DNA samples from 30 individuals of Tibetan sheep and Hu sheep were selected respectively to constructed DNA pools(DNA pool of Hu sheep and DNA pool of Tibetan sheep).A replication experiment was performed within the BMP2 and PDGF-D gene exons,the 5’untranslated regions(within 1000bp)and the 3’ untranslated regions(within 1000bp).Then,the target fragment was sequenced and filtrated.The selected SNPs were genotyped by flight mass spectrometry.Haploview 4.1 software was used for linkage disequilibrium analysis of mutation sites.SAS 9.2 software was used to associate polymorphic loci with tail traits.At the same time,the preadipocytes were isolated from the adipose tissue of unborn lamb tail.Subsequently,preadipocytes were cultured and expanded for the next experiments.Then,the BMP2 and PDGF-D genes were stably overexpressed in the preadipocytes using the lentiviral vector.Insulin was used to induce differentiation.And the expression level of peroxisome proliferator-activated receptor γ(PPARγ)and lipoprotein lipase(LPL),which were the marker genes in the differentiation of adipocytes,was detected by q PCR and Western Blot.The mature adipocytes were conduct oil red O staining to observe lipid droplets.Results as follows:1)Three SNPs were found downstream of BMP2 gene: g.48401619 T>A,g.48401272 C>A,g.48401136 C>T;Two SNPs were detected in PDGF-D gene: g.4122606 C>G was at the intron region.g.3852134 C>T was at promoter region.2)g.48401619 T>A,g.48401272 C>A and rs1 g.48401136 C>T 8 of BMP2 gene were significantlyassociated with tail length(P<0.05);g.4122606 C>G of PDGF-D gene was significantly associated with tail length(P<0.05).g.3852134 C>T was significantly associated with tail width(P<0.05).3)Among the three sites of BMP2 gene(g.48401619 T>A,g.48401272 C>A and g.48401136 C>T),there is a strong linkage between g.48401272 C>A and g.48401136 C>T.It constitutes three haplotypes and six combined genotypes.Among them,CACT type and CCCT type individuals were superior in tail length,tail width and tail circumference,while the average value of tail traits in CCCC type individual was larger than other genotypes.4)The isolated primary ovine preadipocytes were irregular or fusiform,and they still had strongproliferative ability and differentiation ability after passage.5)In BMP2 overexpression group,the expression levels of PPARγ and LPL were up-regulated at them RNA level,and the expression of PPARγ in test groups was significantly higher than control groups on day 0 and the day 1(P<0.05),the expression of LPL in test groups was significantly higher thancontrol group on day 1 and day 7(P<0.01);but the difference in protein level was not obvious.BMP2 has certain promoting influence on differentiation of ovine preadipocyte.6)In PDGF-D overexpression group,the expression of PPARγ and LPL was significantly up-regulated(P<0.05).The expression of PPARγ in test group was significant higer than control group at all stages,except day 0.The expression level of LPL in test group was significant high than control group atday 0,1,5,7.The results of wetern blot showed that the expression level of PDGF-D and PPARγwere higher than control group.PDGF-D has a significant promoting effect on the adipogenesis ofpreadipocytes in vitro.