| Bone morphogenetic proteins-2(BMP2)and bone morphogenetic proteins-4(BMP4)are important members of the transforming growth factor superfamily(TGF-?)and are essential factors in the growth and development of animals.Its biological effects involve almost all systems in the whole body,especially in regulating the formation and growth of animal bone tissues.In view of the important functions of the BMP2 and BMP4 genes,this study uses the BMP2 and BMP4 genes as candidate genes affecting the growth traits of Qianbei Brown goats.From the molecular genetic point of view,the direct sequencing technology was used to detect the BMP2 and BMP4 genes of Qianbei Brown goats.Polymorphism,and screened out the loci that are significantly related to the growth traits of Qianbei Brown goats.From the perspective of epigenetics,the bisulfite treatment sequencing(BSP)technology was used to explore the methylation status of Cp G islands in the promoter region of BMP2 and BMP4 genes,and correlated with the m RNA expression detected by fluorescent quantitative PCR.To clarify the relationship between the degree of methylation and gene expression levels.From the perspective of cellular and molecular biology,construct eukaryotic expression vectors of BMP2 and BMP4 genes and transfect them into Qianbei Brown goat myoblasts.MTT method was used to detect the proliferation of cells after overexpression.Provide a theoretical reference for the biological role and mechanism of the main findings are as follows:1.BMP2 and BMP4 gene polymorphisms and their effects on the growth traits of Qianbei Brown goatsThe SNPs of BMP2 and BMP4 genes in 200 6-month-old Qianbei Brown goats(100 males and ewes)were detected by direct sequencing.Among them,3 SNPs were screened out for BMP2 gene,g.48698728 T >C,g.48700188 G>A and g.48700206G>A(located in intron 2);BMP4 gene screened a total of 5 SNPs sites,respectivelyg.33676037 T>C and g.36676044 G>A(Located in the promoter region),g.36679942G>A and g.36680005 G>T(located in intron 1),g.36681702 T>C(located in intron 2).According to the sequencing results,there are three genotypes in each mutation site,and the genetic characteristics were analyzed.Each mutation site belongs to moderate polymorphism(0.25<P<0.50).Chi-square(?2)test results showed that the genotype distribution of g.48698728T>C and g.48700206G>A mutation sites did not deviate from Hardy-Weinberg equilibrium(P>0.05),among the 3 SNPs of BMP2 gene..48700188G>A mutation site genotype distribution significantly deviated from Hardy-Weinberg equilibrium(P<0.01);among the 5 SNPs sites of BMP4 gene,each SNPs site did not deviate from Hardy-Weinberg equilibrium(P>0.05).Haplotype and linkage disequilibrium analysis showed that there were 5 haplotypes in the 3 SNPs loci of BMP2 gene in the population,3 of which had the highest frequency.Linkage disequilibrium effect;there are 10 haplotypes in the 5 SNPs of BMP4 gene in the population,4 of which have the highest frequency,g.33676037T>C and g.36681702T>C,g.36676044G>A and g.36679942G>A,g.36676044G>A and g.36680005G>T,g.36676044G>A and g.36681702T>C,g.36679942G>A and g.36680005G>T,g.36679942G>A and g.36681702T>C There is a strong linkage disequilibrium effect between C sites.The results of the correlation analysis showed that there were significant differences between BMP2 and BMP4 gene polymorphisms and multiple growth traits(P<0.05).Therefore,we can try to further study these SNPs as candidate genetic markers.2.Analysis of tissue expression profiles of BMP2 and BMP4 genes in Qianbei Brown goatsReal-time fluorescence quantitative PCR results showed that BMP2 and BMP4 genes were widely expressed in various tissues of Qianbei Brown goats,and the expression levels in lung tissues were significantly different(P<0.01).Among them,the order of BMP2 gene expression is: lung> hypothalamus> fat> muscle> kidney>small intestine> thymus> liver> stomach> heart> spleen;the order of BMP4 gene expression is: lung> muscle> kidney> Hypothalamus> Fat> Hypothalamus> SmallIntestine> Heart> Stomach> Spleen> Thymus> Liver.The expression levels of BMP2 and BMP4 genes in tissues of 6-month-old Qianbei Brown goats were significantly higher than those of 1.5-week-old Qianbei Brown goat tissues(P<0.05).3.The relationship between Cp G island methylation level and m RNA expression in BMP2 and BMP4 gene promoter regionsThe bisulfite treatment sequencing(BSP)technique was used to investigate the methylation status of Cp G islands in the promoter region of BMP2 and BMP4 genes in the lungs,muscles,and adipose tissues of Qianbei Brown goats at different growth and development stages.The results showed that the methylation levels of BMP2 and BMP4 genes in lung,muscle and adipose tissue were relatively low,and the methylation levels of the three tissues were different.At different growth and development stages,the methylation level in the lungs and muscles of 6-month-old Qianbei Brown goats was significantly lower than that in the lungs and muscles of1.5-year-old Qianbei Brown goats(P<0.05).Correlation analysis between BMP2 and BMP4 gene methylation degree and m RNA expression.Effect of overexpression of BMP2 and BMP4 genes on proliferation and differentiation of myoblasts4.Effect of overexpression of BMP2 and BMP4 genes on proliferation and differentiation of myoblastsUsing homologous recombination method to construct BMP2,BMP4 gene overexpression vectors p EGFP-N3-BMP2 and p EGFP-N3-BMP4,successfully transfected the identified Qianbei Brown goats myoblasts,q RT-PCR detection of BMP2,BMP4 genes And the differentiation marker genes Myo D,Myf5,Myf6 gene m RNA expression,the results showed that compared with the control group(p EGFP-N3 empty vector),the experimental group(overexpression vector)BMP2and BMP4 gene expression levels increased significantly(P<0.01),the expression levels of differentiation marker genes Myo D,Myf5,Myf6 were significantly increased(P<0.05).MTT method was used to detect the proliferation of cells that overexpressed or not overexpressed BMP2 and BMP4 genes,and found that overexpression of BMP2 and BMP4 genes had a certain role in promoting cell proliferation. |
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