Effects Of FBXO32 Gene On Differentiation Of Preadipocytes In Chinese Grassland Red Cattle And Its Genetic Polymorphism

FBXO32（F-box protein 32,also known as Atrogin-1）protein is an E3 ubiquitin protein ligase,a member of the F-box protein family.The expression of this gene is regulated by FOXO transcription factor and affects skeletal muscle atrophy.The FBXO32 gene is also closely related to various diseases,and it is regarded as a tumor suppressor,which has an inhibitory effect on some cancers.At present,the research on FBXO32 gene at home and abroad mainly focuses on humans and mice,and there is very little research on bovine FBXO32 gene.As a dairy and meat breed,Chinese grassland red cattle has the advantages of strong adaptability,delicious meat and high fecundity,but compared with some excellent breeds at home and abroad,there are still some deficiencies.The differentiation of preadipocytes is critical for adipogenesis,and by intervening in the cells during the differentiation stage,adipose tissue formation may be affected.Intramuscular fat content is an important indicator for evaluating beef quality,so how to effectively improve intramuscular fat content and make beef have better marbling has become the current focus of research.In this study,the effect of FBXO32 gene on the differentiation of adipocytes in Chinese grassland red cattle was investigated,in order to provide support for the application of this gene in animal husbandry.In this study,the complete CDS sequence of Chinese grassland red cattle FBXO32gene was successfully cloned by molecular cloning technology,with a length of 1068bp,encoding a total of 355 amino acids,a relative molecular mass of 41595.14,and an instability coefficient of 45.55,indicating that the protein is unstable.The maximum value of protein hydrophilicity was 2.678,and the maximum value of hydrophobicity was 2.011,indicating that FBXO32 protein was a hydrophilic protein.There is no signal peptide cleavage site,indicating that the protein is not a secreted protein.FBXO32protein has 28 potential phosphorylation sites and 4 O-glycosylation sites.The predicted secondary structure of the protein contained 38.2%α-helix,40.73%β-turn,16.3%β-sheet and 4.77%irregular coil,and the results were consistent with the tertiary structure.The detection of FBXO32 gene expression in different tissues of 18-month-old bulls showed that:FBXO32 gene was expressed to different degrees in various tissues of Chinese grassland red cattle.The expression level was lower in liver,kidney,subcutaneous fat and testis tissue,and the highest expression level was in the longissimus dorsi muscle.In order to explore the effect of FBXO32 gene on the differentiation of Chinese grassland red cattle preadipocytes,and to regulate the expression level of FBXO32 in Chinese grassland red cattle preadipocytes,q RT-PCR and Western blot were used to detect the expression of m RNA and protein.The results showed that:The m RNA expression of FBXO32 gene increased in the process of adipocyte differentiation;overexpression of FBXO32 gene could significantly increase the m RNA level and protein expression of FBXO32 gene and the m RNA level of LPL gene（P<0.01）,and significantly increased the content of triglycerides and the size of lipid droplets（P<0.01）;the opposite result was obtained by interfering with FBXO32 gene.In order to study the correlation between FBXO32 gene polymorphisms and meat quality traits of Chinese grassland red cattle,120 Chinese grassland red cattle were selected as the research objects,and the polymorphism of FBXO32 intron 4 was detected by direct sequencing of PCR products.Using SPSS 21 software to compare association analysis of polymorphic loci with Centrifugal water loss rate,drip water loss rate,pressed water,muscle tenderness,intramuscular fat content,initial water content,protein content,marbling,eye muscle area,p H₂₄ value,cooked meat rate and other meat quality indicators was carried out.The results showed that a C\T mutation site was detected at the fourth intron g.16336603 of FBXO32 gene,CC was the dominant genotype,and allele C was the dominant gene.χ² test showed that the locus was in Hardy-Weinberg equilibrium in the tested population.The results of correlation analysis showed that the polymorphism of this locus was significantly correlated with meat quality indicators such as centrifugal dehydration rate,intramuscular fat content,marble pattern and p H₂₄ value（P<0.05）,but there was no significant correlation with other meat quality indicators（P>0.05）.In conclusion,this study successfully cloned the FBXO32 gene CDS of Chinese grassland red cattle,carried out bioinformatics analysis of the FBXO32 gene,explored the effect of this gene on the differentiation of Chinese grassland red cattle preadipocytes,and analyzed the population of Chinese grassland red cattle.The correlation between polymorphism loci of FBXO32 gene and meat quality traits,it provides a reference material for the subsequent physiological function of FBXO32gene and its use as a molecular marker related to meat quality.