| The quality and yield of beef are closely related to its economic value,and intramuscular fat and skeletal muscle are closely related to the meat-producing ability and meat quality of beef.There are many factors that can regulate meat quality.At present,it is the main research direction to explore the key factors affecting the production of muscle and fat,explore its transcriptional regulation mechanism and clarify the molecular mechanism of meat quality traits.In the early stage of this study,through the correlation analysis of growth traits of Qinchuan cattle,we found that Forkhead Box 1 was closely related to the weight and height of Qinchuan cattle.According to the existing research,the FoxO1 gene plays an important role in animal skeletal muscle and fat production.As a key transcription promoter element,the combination and level of transcription factors play a decisive role in regulating gene expression.Therefore,this study took Qinchuan cattle as the research object,aiming at exploring the structure and expression profile analysis of bovine FoxO1 gene,finding out the core transcription factors of the key promoter of bovine FoxO1 gene,and clarifying the transcriptional regulation function of its promoter.The main findings are as follows:(1)The bovine FoxO1 gene was located on chromosome 12 with a length of 9 3695 bp;According to the prediction of bioinformatics software,FoxO1 gene encodes 665 amino acids with a molecular weight of 69958.93 kDa,and is an unstable protein.The coding product of FoxO1 gene has no transmembrane helix structure and is a water-soluble protein without signal peptide,which is a non-secretory protein.There were 3 CpG islands in the promoter region and 4 CpG islands in the CDS region.The+1 position of guanine(G)in the transcription initiation site of FoxO1 was determined,and the upstream 2 000 bp sequence of FoxO1 was analyzed as promoter sequence.Furthermore,qRT-PCR was used to explore the tissue expression profile of FoxO1 gene.The results showed that FoxO1 gene was highly expressed in liver,lung,subcutaneous fat,heart and kidney,and also in spleen,large intestine,reticular tissue,longissimus dorsi muscle,small intestine,rumen,abomasum and testis.Therefore,it can be preliminarily inferred that the gene plays a role in skeletal muscle and fat.(2)The 1 920 bp sequence and 7 deleted promoter fragments of bovine FoxO1 promoter were obtained by PCR amplification.Further,the double-luciferase reporter vector was constructed and transfected into mouse C2C12 and 3T3-L1 cell lines,respectively.By detecting the activity of luciferase reporter vector with different deletion fragments,it was found that the core region of the promoter was located in the-285/-27 region.Bioinformatics software predicted that there are binding sites for transcription factors MEF2A,SREBF1,HOXA5,KLF4 and KLF5 related to the growth and development of muscle and fat in the core region of the promoter.(3)Site-directed mutation of potential transcription factor binding sites showed that the enzyme activity decreased by 49.48%(P<0.01)compared with that of wild type.After mutation of SREBF1 site,the enzyme activity increased by 1.73%compared with that of wild type(P>0.05).After mutation of KLF4,the enzyme activity increased by 35.15%compared with that of wild type(P<0.01).Compared with the wild type,the enzyme activity decreased by 23.73%(P<0.01)and 33.75%(P<0.05)after mutation of HOXA5 and KLF5.The important roles of MEF2A,KLF4,HOXA5 and KLF5 in maintaining the promoter activity of FoxO1 gene were preliminarily determined.(4)Further,RNA interfering(RNAi)test was used to determine whether MEF2A,KLF4,HOXA5 and KLF5 on the promoter of bovine FoxO1 gene are cis-acting cis-acting or trans-acting trans-acting transcription elements.Co-transfected plasmids pGL3-258/+198 and siMEF2A.siHOXA5,siKLF4,siKLF5 and control Negative Control siRNA into C2C12 cell line,respectively.The results showed that the activity of pGL3-258/+198 promoter decreased by 39.64%by interfering with MEF2A(P<0.05).The activity of pGL3-285/+198 promoter was decreased by 40.43%(p<0.05)by interference with HOXA5.The activity of pGL3-285/+198 promoter decreased by 3.28%after KLF4 interference(P>0.05).The activity of pGL3-258/+198 promoter increased by 0.57%after KLF5 interference(P>0.05),which further confirmed that MEF2 A and HOXA5 played an important role in transcriptional regulation of FoxO1 gene.(5)To verify whether MEF2A and HOXA5 can bind to the core promoter region of FoxO1 gene,chromatin immunoprecipitation assay(ChIP)was used.PCR amplification was carried out on the immunoprecipitate after quantitative detection of the enriched product IP,and the corresponding target band was detected by agarose electrophoresis;The results of qRT-PCR showed that compared with NC,MEF2A and HOXA5 enriched groups were significantly higher in input(P<0.0001).The above results proved that MEF2A and HOXA5 could bind to the core region of bovine FoxO1 gene promoter.To sum up,this study found that MEF2A and HOXA5 can bind to the core region of bovine FoxO1 gene promoter,and they play an important role in transcription regulation.This study laid a theoretical foundation for the transcription regulation mechanism of FoxO1 gene in the development of beef muscle and fat. |
