Polymorphism Analysis Of Bovine CDC10 Gene And Its Mechanism Of Regulating Proliferation And Differentiation Of Intramuscular Preadipocytes

Intramuscular fat content and marbling that affecting beef quality are important economic traits.Therefore,it is very important to identify key candidate genes and molecular markers of meat quality and to reveal the molecular mechanisms of proliferation and differentiation of intramuscular preadipocyte cells for improving beef quality in beef cattle.CDC10(cell division cycle 10,also known as SEPT7)gene involved in physiological processes such as intracellular material transport,cell division,cell cycle regulation,and cell apoptosis,is one of the septins family.Our previous study found that the expression levels of the CDC10 gene were significantly higher in high-marbling than that in low-marbling,which suggested that the CDC10 gene could be considered as an important candidate functional gene for beef quality.In order to analyze the associations of CDC10 gene with meat quality and carcass traits in Chinese cattle breeds,this study selected 5 samples of each of Chinese Simmental,Mongolia cattle,Luxi cattle and Qinchuan cattle breeds for direct sequencing to identify mutation.And then the mutations of 367 Qinchuan cattle were genotyped by Mass ARRAY technology.The genetic diversity of the mutations and the associations of mutations with meat quality and carcass traits were analyzed in the Qinchuan cattle population.Meanwhile,to reveal the molecular mechanism of the CDC10 gene-regulating proliferation and differentiation of intramuscular preadipocyte in beef cattle,si RNA was used to interfere the expression of the CDC10 gene,and a lentiviral overexpression vector was constructed to overexpress the expression of CDC10 gene.And the Ed U proliferation assay,flow cytometry analysis,oil red O staining,real-time q PCR and western blot were used to detect the effects of interference and overexpression of CDC10 gene on proliferation and differentiation of 3T3-L1 and intramuscular preadipocytes.The results are as follows(1)in this study,9 mutations of CDC10 were found.The g.61654918A>G,g.61654835A>G,g.61634492A>G,g.61623940A>G and g.61619298T>G mutations are located in intron 3,exon 4,intron 7,intron 11 and intron 12 of CDC10 gene,respectively.The g.61612194G>A,g.61611958A>G,g.61611679A>T and g.61611524C>T mutations are located in 3’UTR region of CDC10 gene,respectively.Association analysis showed that the g.61619298T>G SNP was significantly associated with ultrasound loin muscle depth(P < 0.05),the g.61611679A>T SNP was significantly associated with ultrasound loin muscle area(P < 0.05),the g.61710450G>C SNP was significantly associated with back fat thickness(P < 0.05)and ultrasound loin muscle depth(P < 0.01)in Qinchuan cattle.(2)Ed U proliferation assay,flow cytometry,real-time q PCR and western blot analyses showed that interference of CDC10 had no significant effect on the proliferation of 3T3-L1 and intramuscular preadipocyte,while overexpression of CDC10 increased proliferation of 3T3-L1 and intramuscular preadipocytes.(3)The results of oil red O staining,real-time q PCR and western blot showed that interference of CDC10 inhibited differentiation of 3T3-L1 and intramuscular preadipocytes,while overexpression of CDC10 increased differentiation of 3T3-L1 and intramuscular preadipocytes.The experimental results of this study showed that g.61619298T>G,g.61611679A>T and g.61710450G>C mutations were significantly associated with meat quality and carcass traits of Qinchuan cattle,those mutations may facilitate effective marker-assisted selection to improve the quality of Qinchuan cattle.In addition,this study peliminary explain the effects of CDC10 gene on the proliferation and differentiation of 3T3-L1 and intramuscular preadipocyte,which provided theoretical basis of science for the improvement of beef quality.