Association Analysis Between Polymorphisms Of The CDC10 Gene And Growth Traits In Qinchuan Cattle

The cell divison cycle 10(CDC10,also known as SEPT7)gene is a member of the septins family.The functions of septins mainly include intracellular material transport,cell membrane reconstruction and chromosome separation.CDC10 gene is located in the genomic QTL regions for growth traits in several beef cattle breeds,and has a certain universality in regulating muscle growth and development and affecting growth traits of beef cattle.Thus,CDC10 gene could be considered as an important candidate functional gene for growth traits in Chinese local yellow cattle.In order to find the species-specific mutation of CDC10 gene associated with growth traits of Qinchuan cattle,this study firstly sequence the promoter region of CDC10 gene in Qinchuan,Luxi,Mongolia and Chinese Simmental cattle breeds.And then,we perform direct sequencing and MassARRAY to detect genotype of 384 Qinchuan cattle and analysis between polymorphisms and grotwh traits in Qinchuan cattle.Meanwhile,in order to further reveal the molecular mechanism of CDC10 gene affecting the proliferation and differentiation of bovine myoblasts,this study construct the lentivirus overexpression vector and synthesize the siRNA interference sequence of CDC10.Through overexpression and interference of CDC10 gene,we detect the influence of CDC10 expression on the proliferation of bovine myoblasts using EdU,and the influence on the differentiation of bovine myoblasts using MyHC immunofluorescence,as well as the expression changes of cell proliferation and differentiation related genes using real-time PCR.Furthermore,we perform bioinformatic analyzes to predict the transcription factor binding motifs at the g.61710450G>C SNP(single nucleotide polymorphism)site in the promoter of CDC10 gene,also perform EMSA to observe the transcription factors binding situation at the g.61710450G>C SNP site of CDC10 gene.The results are as follows: 1)There are 7 SNPs were identified in the promoter of CDC10.Among them,the g.61710450G>C SNP was significantly associated with body length,hip width and pin bone width(P<0.05),and was significantly associated with body weight,chest circumference and withers height of Qinchuan cattle(P<0.01).The g.61711075T>G SNP was significantly correlated with the body weight and chest circumference of Qinchuan cattle(P<0.05).There was no association between other SNPs and growth traits of Qinchuan cattle.2)Overexpression of CDC10 gene significantly promoted the proliferation of bovine myoblasts(P<0.05),and interference with CDC10 gene significantly inhibited the proliferation of bovine myoblasts(P<0.05).Meanwhile,the expression of CDC10 was positively correlated with the cell proliferation related genes MEK,ERK1 and ERK2(P<0.05).3)Overexpression of CDC10 gene significantly inhibited the differentiation of bovine myoblasts(P<0.05),while interference with CDC10 gene resulted in the opposite result(P<0.05).The expression level of CDC10 gene was negatively correlated with the cell differentiation related genes MyoD,MyoG and MyHC(P<0.05).4)Bioinformatics analysis showed that when the g.61710450G>C in the upstream of CDC10 promoter is G allele,the binding motif of GABPA,RORA-1,ELK4,FEV and ELF1 transcription factors could be formed.When is C allele,the binding motif of Myog,Myod1,Tcf12,NHLH1,Tcf3,Bhlhe40 and Arnt could be formed.5)EMSA results showed that the G allele probe of the g.61710450G>C SNP site had transcription factor binding bands in fetal cattle sample.The results of this study suggest that the g.61710450G>C and g.61711075T>G of the CDC10 gene may influence growth traits in Qinchuan cattle,which could be used as effective molecular markers for Qinchuan cattle breeding.The CDC10 gene could promote the proliferation of bovine myoblasts and inhibit the differentiation of bovine myoblasts.We also preliminarily verified that the G allele of the g.61710450G>C SNP site could bind transcription factors in the fetal cattle,which bring new insights into the regulatory mechanism of CDC10 gene affecting muscle growth and development in beef cattle.