| This paper takes Kazakh sheep as the research object,and the DNA methylation levels of MSTN gene in different tissues of 6 months old(two biceps femoris,triceps femoris,semitendinosus,semimembranous muscle,longissimus dorsi and tail fat)and muscle and adipose tissue of 1-5 months old were detected by bisulfite sequencing.Furthermore,the correlation analysis was conducted between the expression level of MSTN gene,the expression level of MSTN/Smad signaling pathway gene,and the growth index.Meanwhile,the DNA methylation level of MSTN gene was compared with Bashbay sheep in order to explore the regularity of DNA methylation of MSTN gene in different tissues of Kazakh sheep at different stages,and provide scientific basis for genetic breeding of Kazakh sheep.1.DNA methylation level of MSTN gene in muscle and adipose tissue of 1-5 month old Kazakh sheepMuscle tissue:The DNA methylation level of the MSTN gene from 1 to 5 months old is highly methylated.The DNA methylation probability of MSTN gene in muscle tissues from 1 to 5 months old was 74.2%,71.6%,80.5%,81.5%,and 77.4%.fat tissue:The DNA methylation probabilities of the MSTN gene in fat tissue from 1 to 5 months old were 78.4%,75.8%,76.3%,78.9%,and 83.2%.The expression of MSTN gene in muscle tissues of Kazakh sheep aged 1 and 2 months was medium negatively correlated with the probability of DNA methylation,and the correlation coefficients were r=0.46 and R=0.50(P>0.05).The expression of MSTN gene at 3,4,and 5 months of age was significantly negative correlated with the probability of DNA methylation,and the correlation coefficients were r=0.62,r=0.57 and R=0.60(P<0.05).The expression of MSTN gene in fat tissue aged 1,2,3,and 4 months was negatively correlated with the probability of DNA methylation,and the correlation coefficients were r=0.60(P>0.05),r=0.56(P<0.05),r=0.52 and r=0.55(P>0.05).There was a significantly negatively correlation with 5 month old(r=-0.8,P<0.05).2.DNA methylation level of MSTN gene in 6 month old tissues of Kazakh sheepThe methylation level of muscle tissue in 6-month-old Kazakh sheep weas low and the highest of adipose tissue was.The DNA methylation probabilities of two biceps femoris,triceps femoris,semitendinosus,semimembranous muscle,longissimus dorsi and tail fat were 34.7%,22.6%,23.3%,28%,42.6%,and 76.4%,respectively.The probability of DNA methylation in 6-month-old Kazakh sheep was negatively correlated with the total expression of MSTNgene(r=-0.58,P<0.05).The DNA methylation probability of MSTN gene in two biceps femoris,triceps femoris,semitendinosus,semimembranous muscle,longissimus dorsi and tail fat was negatively correlated with the expression of MSTN gene,and the correlation coefficients were r=-0.56,r=-0.48,r=-0.50,r=-0.58 and r=-0.50.The expression of MSTN gene in adipose tissue was negatively correlated with the probability of DNA methylation(r=-0.74,P<0.05).3.Correlation between DNA methylation probability of MSTN gene and expression of MSTN/Smad signaling pathway genes in 6-month-old tissuesCorrelation between the probability of DNA methylation of MSTN gene and the expression of MSTN/Smad signaling pathway in the age of 6-month-old Kazakh sheep.The overall expression of Smad3 signaling pathway gene and biceps femoris,triceps,The DNA methylation probabilities of MSTN gene in semimembranosus,semitendinosus,longissimus dorsi and adipose tissue were weakly negatively correlated.The gene expression of Smad4 signaling pathway was negatively correlated with the DNA methylation probability of MSTN gene in the biceps femoris and semimembranosus muscle(r=-0.31,P<0.05).The gene expression of Smad7 signaling pathway was negatively correlated with the DNA methylation probability of MSTN gene in semimembrane muscle(r=-0.31,P>0.05).The expression level of TGFBRⅡ gene was weakly negatively correlated with the DNA methylation probability of MSTN gene in each tissue.The expression of TGFBRⅡ gene was significantly different from that of the longissimus dorsi muscle,showing a negative correlation(r=-0.45,P<0.05).4.Correlation between DNA methylation probability of MSTN gene and growth index in 6 month old tissuesCorrelation between DNA methylation probability and growth index of 6-month-old Kazakh sheep MSTN,in general,Kazakh sheep biceps,triceps,semimembranosus,semitendinosus,longissimus dorsi,adipose tissue The DNA methylation probability of MSTN gene was positively correlated with body weight r=0.61(P<0.05),r=0.45,r=0.39,r=0.32,r=0.30(P>0.05),r=0.63(P<0.05).In addition to the semitendinosus,the DNA methylation probabilities of the biceps femoris,triceps,semimembranosus,longissimus dorsi,and adipose tissue MSTN genes were positively correlated r=0.31,r=0.3(P>0.05),r=0.62(P<0.05),r=0.5(P>0.05),r=0.61(P<0.05).There was a strong positive correlation between the DNA methylation of the MSTN gene and the semitendinosus muscle(r=0.65,P<0.05).The DNA methylation probability of body length and semi-membrane muscle and longissimus dorsi muscle MSTN gene was positively correlated with r=0.39(P>0.05)and r=0.41(P<0.05).There was a positive correlation between body height and DNA methylation of semi-membrane muscle and semitendinosus MSTN gene r=0.42(P>0.05),r=0.48(P<0.05).5.Comparison analysis of DNA methylation level of MSTN gene in different tissues of 6 month old Kazakh sheep and 5 month old bashbae sheepThe expression levels of MSTN gene in both biceps,triceps,semimembranosus and semitendinosus muscles were negatively correlated with DNA methylation probability(p=-0.5,r=-0.5,P<0.05).The expression of MSTN gene in the longissimus dorsi muscle of Kazakh sheep was negatively correlated with the probability of DNA methylation(r=-0.5,P>0.05),but there was a strong negative correlation between the longissimus dorsi of Bashibai sheep(r=-0.87,P<0.05).The expression of MSTN gene in Kazakh sheep adipose tissue was significantly negatively correlated with the probability of DNA methylation(r=-0.74,P<0.05).The expression of MSTN gene and the probability of DNA methylation in adipose tissue of Bashibai sheep were Strong negative correlation(r=-0.99,P<0.05),although both varieties were negatively correlated,the correlation coefficient of Bashbai sheep was higher.Compared with the body length,the DNA methylation probability of the MSTN gene in Kazakh sheep is stronger than that of Bashbai sheep,and the correlation with chest circumference is weaker than that of Bashbai sheep.6.SNP site of MSTN gene in Sac and Bashbai sheepThe SNP39,SNP40,and SNP41 mutation sites of the MSTN gene were verified by high-throughput sequencing.There were three genotypes at the SNP39 mutation site:AA,TG,GG,and the AA genotype frequencies of Kazakh sheep and Bashba sheep were 68.5%and 70.8%,respectively,and the TG genotype frequencies were 26.2%and 20.8%,respectively.The GG genotype frequencies were 5.4%and 8.5%,respectively.There were three genotypes at the SNP40 mutation site:AA,AT,TT,and the TT genotype frequencies of Kazakh sheep and Bashbai sheep were 72.8%and 79.0%,respectively.The frequencies of TA genotypes were 18.4%and 15.3%,respectively.The frequencies of AA genotypes were 8.8%and 5.65%,respectively.There are two genotypes of SNP41 mutation site:AAG,AA,which is no mutation in Bashba sheep.The frequency of AAG genotype of Kazakh sheep is 89.2%,and the frequency of AA genotype is 10.8%. |
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