Expression And Polymorphism Analysis Of MED4 Gene In Chinese Red Steppe Cattle And Its Effect On Lipid Metabolism

With the increasing demand of consumers for beef quality,the exploitation and utilization of beef cattle germplasm characteristics have attracted more and more attention from breeders.Grassland red cattle in China is the first meat and milk dual-purpose variety bred in China,which has excellent characteristics of roughage tolerance,strong stress resistance,tender meat quality and unique flavor.A number of genes affecting the meat quality characteristics of grassland red cattle,especially intramuscular fat deposition,have been found,but the specific functions of the genes need to be verified.MED4(mediator complex subunit 4,MED4)is one of the subunits of mediator complex,located on chromosome 12,and is also a vitamin D receptor protein.Vitamin D metabolism can be regulated by binding to vitamin D receptor,which further affects mineral ion homeostasis and bone integrity;studies have shown that MED4 gene plays an important role in animal growth,carcass traits and triglyceride deposition.Little is known about the effect of MED4 gene on bovine preadipocyte differentiation and lipid metabolism.In this study,a CDS sequence of MED4 gene was cloned from 118 20-month-old prairie red cattle,and its physicochemical properties and protein structure were analyzed by bioinformatic software.The expression differences of MED4 gene in different tissues of grassland red cattle were analyzed by q RT-PCR.The specific primers for exons were designed,and the SNP loci of MED4 gene were screened by PCR sequencing.The association analysis was carried out with meat quality traits such as tenderness,meat color,p H value,intramuscular fat content,protein content and ocular muscle area.The si RNA interference and overexpression vector of MED4 gene in prairie red cattle were constructed and transfected into preadipocytes of prairie red cattle.q RT-PCR and protein immunoblot were used to detect the expression of MED4 m RNA and protein levels;oil red O staining and triglyceride kit were used to detect lipid droplets and triglyceride content in preadipocytes,and the effect of MED4 gene on preadipocyte differentiation and fat formation in prairie red cattle was studied.The main test results are as follows:(1)The coding region sequence of MED4 gene was obtained.The full length of the sequence was 813 bp,encoding 270 amino acids.The relative molecular mass of the protein was about 29707.34,and the theoretical isoelectric point(p I)was 5.06.It belonged to soluble protein,which mainly existed in cytoplasm and nucleus(73.9%).Its secondary structure is mainly composed of α-helix,and the protein structure is relatively stable.The expression levels of MED4 gene in different tissues of grassland red cattle were significantly different(P<0.05),from high to low as kidney,lung,spleen,liver,subcutaneous fat,duodenum,heart,longissimus dorsi muscle,testis and rumen.(2)Two SNP loci in exon 7 of MED4 gene were found.A / G mutation existed at Chr12.17897166,resulting in amino acid changing from glycine(Gly)to serine(Ser);there was A/G mutation in Chr12.17897014,but did not lead to amino acid changes.The two loci were not in HardyWeinberg equilibrium.The results of correlation analysis with meat traits showed that there was no significant difference in meat traits among individuals with different genotypes produced by Chr12:17897166A>G.Different genotypes of Chr12:17897014A>G had significant differences in muscle protein content,tenderness,p H value and L value(P<0.05).(3)The expression level of MED4 m RNA in preadipocyte differentiation of grassland red cattle showed a rising-decreasingrising trend.Overexpression and interference experiments showed that MED4 gene overexpression group increased most significantly on the 0th and 4th day,and the expression trend of adipogenic gene PPARγ was consistent with MED4.In the MED4 gene interference group,the decline was the most obvious on the 4th and 8th days,and the expression of PAARγ was the most obvious only on the 8th day.Oil red O staining and triglyceride content determination showed that overexpression of MED4 gene promoted lipid droplet formation and triglyceride accumulation.MED4 gene interference inhibited lipid droplet formation and triglyceride accumulation.In summary,the complete CDS region of MED4 gene in grassland red cattle was obtained in this study,and the physicochemical properties and protein structure of MED4 gene were analyzed by bio-signal analysis,which clarified the expression differences of MED4 gene in different tissues.It was found that two SNP loci in exon 7 of MED4 gene could affect the meat quality traits of grassland red cattle.The promoting effect of MED4 gene on preadipocyte differentiation and adipogenesis in grassland red cattle was clarified.The results can provide reference for exploring MED4 gene function and improving meat quality of grassland red cattle.