Sequence Analysis And Functional Verification Of C/EBPα Gene Related To Fat Deposition In Black Angus Cattle

Black Angus cattle and Simmental cattle were used as experimental subjects in this study.Transcriptome technology and bio informatics methods were used to screen differentially expressed genes related to fat deposition between varieties.In order to further study the differential gene C/EBPα,qRT-PCR was used to detect the mRNA expression levels of C/EBPα gene between the two varieties of longissimus dorsi muscle tissues and different tissues of black Angus cattle.Bioinformatics was used to analyze and predict the coding protein,structure,physical and chemical properties and evolutionary tree of CDS region of C/EBPα gene in different variable shear bodies.The C/EBPα gene of black Angus cattle was used as the target gene and bovine myoblasts were used as experimental materials to analyze the mutual regulatory relationship between C/EBPα gene and related genes related to adipocyte differentiation.The main research results are as follows:(1)Transcriptomics technology was used to analyze the differential genes between the black Angus cattle and Simmental cattle,and the pathway of C/EBPα gene was identified to be related to fat deposition.The network interaction of differential expression gene C/EBPα protein was constructed,and it was found that genes related to fat deposition such as PPARG,CEBPE and CEBPG interacted.(2)The mRNA expression levels of C/EBPα gene in the longissimus dorsi tissues of the two breeds and different tissues of Angus cattle were detected.The expression levels of Angus cattle in the longissimus dorsi tissues were significantly higher than those of Simmental cattle(P<0.01).The expression levels of C/EBPα gene in the longissimus dorsi and spleen of Angus cattle were significantly higher than those in kidney,subcutaneous fat,liver,heart and rumen tissues(P<0.01).(3)The CDS region sequence of C/EBPα gene in Angus cattle was cloned,and three kinds of variable shear bodies were found.The cloning and sequencing showed that there were base deletions in all three base sequences,with the full length of 570 bp,564 bp and 780 bp,respectively.The number of deletions compared with the CDS region was 39 bp,45 bp and 45 bp,respectively,encoding 189,187 and 259 amino acids,respectively.The mutation base sequence of CEBPA3 due to the termination codon continued to encode amino acids backward,and all had strong hydrophilic regions.Both of them had no transmembrane structure and were not transmembrane proteins.They were non-secretory proteins with one CpG island.There were 17 phosphorylation sites in CEBPA1 and CEBPA2,and 20 phosphorylation sites in CEBPA3.(4)The C/EBPa gene of Black Angus cattle was transfected into myoblasts.The expression of CEBPB,LPL,FAS,PPARG,ACCα,SCD and CFD genes was positively regulated by C/EBPα gene in myoblasts by real-time fluorescence quantitative analysis.There was no obvious regulation on the expression of FABP4 and FATPl genes.The results will provide a theoretical basis for molecular breeding of Ningxia Angus cattle,and provide a theoretical support for the in-depth study of C/EBPα gene regulating fat deposition.