Polymorphism Analysis Of SFRP4 Gene And Its Effect On Differentiation Of Preadipocytes In Qinchuan Cattle

SFRP4(Secreted frizzled related protein 4)gene is a differentially expressed gene screened by transcriptome sequencing technology in our previous study(co-culture of Qinchuan cattle myoblasts and preadipocytes).In previous studies,SFRP4 has been identified as an adipokine,which plays a role in adipocyte differentiation and lipid metabolism.However,at present,there are few studies on the role of this gene in the differentiation of bovine preadipocytes and lipid metabolism,and its function in the differentiation of bovine preadipocytes is not clear.Recent studies have shown that the gene has differentially expressed in different regions fat of bovine body and is related to the differentiation of preadipocytes in mice.It is speculated that SFRP4 gene may affect the marbling formation of beef by affecting the differentiation of preadipocytes and the deposition of fat,thus affecting the quantity and quality of beef.Therefore,in this study,the bovine SFRP4 gene was used as the target gene,the SNP loci of SFRP4 were screened,and the association between SFRP4 gene and growth traits and meat quality traits of Qinchuan cattle was analyzed to explore the relationship between SFRP4 gene and growth and meat quality traits of Qinchuan cattle.In this study,si RNA mediated interference technology and adenovirus mediated overexpression technology were used to explore the effect of SFRP4 gene on adipogenic differentiation of preadipocytes in Qinchuan cattle,so as to provide a theoretical basis for further revealing the mechanism of SFRP4 gene regulating the growth and development of adipose tissue in Qinchuan cattle.The results of this study are shown as follows:1.In this study,A single nucleotide polymorphism(SNP)in the third intron of SFRP4gene(g.50017285 g >A)was genotyped by first generation sequencing technology,and its association with 11 body size traits and meat quality traits of 230 Qinchuan cattle was analyzed.The results showed that the polymorphism of this locus was significantly correlated with chest circumference,ischial end width,waist Angle width and eye muscle depth(P<0.05).G>A)was sequenced and genotyped,and its association with 11 body size traits and meat quality traits of 230 Qinchuan cattle was analyzed.The results showed that the polymorphism of this locus was significantly correlated with chest circumference,ischial end width,waist Angle width and eye muscle depth of Qinchuan cattle(P<0.05).2.Bioinformatics analysis showed that the amino acid sequence of bovine SFRP4 was100% identical to that of Zebu cattle,99% identical to that of sheep and goat,and 94% distant to that of mouse.The results of tissue expression profiling showed that the highest expression level of SFRP4 gene was found in subcutaneous fat and kidney of newborn cattle in Qinchuan,and the expression level was relatively low in colon and liver.The expression of SFRP4 gene was the highest in subcutaneous fat and muscle of adult Qinchuan cattle,which were significantly higher than those in other tissues(P < 0.01),and the expression level in liver was low.The overall expression of SFRP4 gene increased first and then decreased from day 0 to day 8 of preadipocyte differentiation.3.Knockdown of SFRP4 gene in preadipocyte significantly down-regulated the adipogenic differentiation-related factors,such as PPARγ,C/EBPβ,FABP4,PLIN2 and SCD1 at m RNA level,and significantly down-regulated PPARγ and C/EBPβ at protein level.Oil red staining results showed that the amount of lipid droplets in si-SFRP4 group was significantly lower than that in si-NC group on the 6th day of cell differentiation.Therefore,SFRP4 gene may affect the differentiation of preadipocytes in Qinchuan cattle by affecting the expression of adipogenic differentiation related genes.4.Overexpression of SFRP4 gene significantly up-regulated the m RNA level of adipogenic differentiation-related factors(PPARγ,C/EBPβ,FABP4,SCD1,PLIN2)and the protein level(PPARγ,C/EBPβ,FABP4,SCD1).Oil red O staining results showed that the number of lipid droplets in the Ad-SFRP4 group was significantly higher than that in the AdNC group on the 6th day of cell differentiation.In summary,this study found that SFRP4 gene polymorphism was significantly associated with some body size and some meat quality traits in Qinchuan cattle,and SFRP4 gene could promote adipogenic differentiation of preadipocytes.Therefore,SFRP4 gene may be an important candidate gene affecting lipogenesis in beef cattle.In this study,we investigated the effect of SFRP4 gene on the differentiation of bovine preadipocytes and revealed the regulatory relationship between SFRP4 gene and the expression of genes related to adipose differentiation and fat deposition.It provides some scientific basis for the improvement breeding of Qinchuan cattle.