Molecular Mechanism Of Bovine KLF6 Gene In The Regulation Of Adipogenesis In Qinchuan Beef Cattle

Marbling affects meat tenderness and is a major determinant of beef carcass quality.As an excellent local cattle breed in my country,Qinchuan beef cattle have the advantages of resistance to rough feeding and strong stress resistance.However,their meat quality traits are significantly different from those of foreign specialized beef cattle breeds such as Wagyu and Angus cattle.How to improve the carcass quality(such as back fat thickness,intramuscular fat content and eye muscle area)of local cattle is an urgent problem to be solved in beef cattle breeding.The proportion of marbling is closely related to the proliferation and differentiation process of adipocytes,and exploring the process of adipocyte proliferation and differentiation at the molecular level is of great significance for guiding the breeding of local yellow beef.Previous studies have found that the transcription factor KLF6 may be an important candidate functional gene affecting meat quality traits.However,its role in beef cattle lipogenesis remains unknown.In this study,marker genes related to the regulation of marbling-a very important carcass quality traits of beef cattle were analyzed,and it was found that SIRT4,SIRT6,SIRT7,CRTC3,ABHD5,KLF6,H-FABP and ELOVL6 may affect beef quality especially adipogenesis through metabolic regulation,fatty acid oxidation,fat deposition and fat synthesis regulation,which ultimately affect adipogenesis.Adipogenesis is a main regulator for one of the most important factor of meat quality traits known as intramuscular fat(IF)or marbling.On this basis,this study focused on exploring the role of KLF6 gene in Qinchuan beef cattle carcass quality trait such as IF which is a basic indicator of all carcass quality parameters.Therefore,the effect of KLF6 gene on bovine adipocyte adipogenic differentiation was analyzed and its potential molecular mechanism were explored.The main research results are as follows:(1)Genetic polymorphisms in the KLF6 coding region were significantly correlated with body size and meat quality traits of Qinchuan beef cattleBioinformatics analysis of KLF6 protein showed that KLF6 protein was highly conserved in different animals.The tissue expression profile of KLF6 gene was constructed by real-time quantitative PCR,and it was found that KLF6 gene was highly expressed in the kidney,lung and liver tissues of Qinchuan beef cattle.Further,based on PCR amplification and sequencing,three new SNPs were found in the second exon region of KLF6 gene,g.3521G>A,g.3332C>G and g.3413C>T.Genetic parameter analysis found that the three new SNPs were in line with the Hardy-Weinberg equilibrium(P < 0.05).Through correlation analysis with Qinchuan beef cattle body size and meat quality traits,it was found that SNP1,SNP2 and SNP3(with genotypes CC,CT,GG,respectively)were significantly correlated with carcass traits and body size traits(P<0.05).Association analysis results showed that the Hap1/4 combination haplotype(-CTA-GTA-)was significantly higher than other combination haplotypes in Qinchuan beef cattle in terms of body length and body height,and the Hap1/4 combination haplotype had intramuscular fat content and eye muscle area.The traits were significantly higher than other combined haplotypes.(2)KLF6 inhibits adipogenic differentiation of bovine adipocytesBovine adipocytes were transfected with si RNA-KLF6 in vitro,and adipocytes were harvested and RNA-sequenced 9 days after induction of differentiation.A total of 171 differentially expressed genes(DEGs)were found,including 109 up-regulated genes and 62down-regulated genes.KEGG pathway analysis found that DEGs are associated with lipid metabolism,carbohydrate metabolism,cell growth and death,cancer,as well as calcium,AMPK(Adenosine Monophosphate-Activated Protein Kinase),PI3K-Akt(Phosphatidylinositol 3-kinase),PPAR(Peroxisome proliferator-activated)receptors),MAPK(mitogen-activated protein kinase),c AMP(Cyclic adenosine monophosphate),and Wnt(Wingless-related integration site)signaling pathways.Similarly,KLF6 gene interference significantly up-regulated genes related to adipogenesis,adipocyte differentiation,and bovine adipocyte homeostasis,especially affected cell apoptosis,and affected the negative regulation of apoptotic pathways,programmed cell death,and growth.The above results indicated that KLF6 gene plays an important role in regulating lipid metabolism in bovine adipocytes,and can be used as a potential candidate marker gene for Qinchuan beef cattle molecular marker-assisted breeding of beef cattle breeds.These findings provide a basis for further research on the molecular mechanism of KLF6 gene regulation of adipogenesis.