Analysis Of DNA Methylation And MRNA Expression Levels In Skin Tissues Of Bashibai Sheep With Different Wool Colors

In this study,black and white phenotypic one-year-old Bashibai sheep of Xinjiang have been selected as the research objects.Transcriptome sequencing has been used to screen the differentially expressed genes in phenotypic skin tissues of the two groups.Fluorescence quantitative PCR has been used to study the expression levels of differentially expressed genes in skin tissues of Bashibai sheep with different hair colors.At the same time,the influence of DNA methylation in promoter region of differentially expressed genes on their mRNA expression level have been investigated.This study provides a reference for further understanding the molecular mechanism of DNA methylation regulating gene expression affecting wool color production.(1)Transcriptome sequencing analysis of sheep skin tissue with black and white phenotypes revealed 183 differentially expressed genes.At the same time,the enrichment analysis of differentially genes showed that the up-regulated genes are significantly enriched in single biological process,cell,cell component,etc.And c GMP-PKG signaling pathway,as well as melanin metabolism and other signaling pathways.(2)Seven target genes,including LEF1,WNT2,YWHAZ,HPS6,SLC45A2,OCA2 and TYR have been screened based on differentially expressed genes.The mRNA expression levels of target genes in different wool color skin tissues of Bashibai sheep have been studied by fluorescence quantitative PCR.The results have showed that the expression levels of 6 genes in the skin tissue of Bashibai sheep with black wool color are significantly higher than those of Bashibai sheep with white coat(P < 0.01),and there is no significant difference in the expression levels of HPS6 in the skin tissue of the two phenotypes(P > 0.05).The expression levels of the seven genes are consistent with the results of transcriptome sequencing.The results of transcriptome sequencing are reliable and can be further tested.(3)To investigate the DNA methylation level in the LEF1 promoter region.By predicting the CpG island in part of the promoter region of LEF1 gene.Three CpG islands have been found and the target fragments have been screened out.Methylation tests have been performed using the bisulfite sequencing method.The results showed that 5 of the 14 potential DNA methylation sites of the target fragment were methylated.The methylation rates of 5 CpG sites in the black phenotype are 2%,2%,2%,6%,and 2%,respectively.The methylation probability of 5 CpG sites in the white phenotype was 0.(4)The relationship between the mRNA expression of LEF1 gene and the DNA methylation level in the promoter region of black and white wool color Bashibai sheep has been analyzed.Because the DNA methylation level in the promoter region of LEF1 gene of the two wool colors were very low and the difference was not significant(P > 0.05).The results showed that the methylation level of 5 CpG sites in the promoter region of LEF1 gene have no effect on its mRNA expression.In summary,based on transcriptome sequencing results,this study analyzed the expression of 7 genes,LEF1,WNT2,YWHAZ,HPS6,SLC45A2,OCA2 and TYR,in Bashiba sheep with different hair colors and proved that the DNA methylation level in the LEF1 promoter region had no effect on its expression level.