Molecular Characteristics,Expression And Methylation Analy-sis Of IGF2 And ZBED6 Genes In Liaoning Cashmere Goats

Liaoning cashmere goat is a unique cashmere goat,dual-purpose(cashmere and meat)type in China.In this study,the IGF2 gene and ZBED6 gene of Liaoning cashmere goat were cloned and sequenced by molecular biology techniques,the 5 ’regulatory region and the coding region of the gene are analyzed.The IGF2 gene and ZBED6 gene sequences of Liaoning cashmere goat was compared with other species sequences by using bioinformatics analysis software,and their CpG islands were predicted.This study investigated the effect of promoter methylation levels on gene expression.The secondary structure of protein and tertiary structure were pre-dicted,so did physical and chemical properties and expression profiles in different tissues.It lays a theoretical foundation for molecular breeding research of Liaoning Cashmere Goat.The main research works are as follows:1.Molecular cloning and molecular characterization of IGF2 and ZBED6The 5 ’regulatory region and coding region of IGF2 and ZBED6 genes in Liaoning cash-mere goats were amplified specifically,cloning and sequencing were also performed,It was found that the total length of the coding region in the IGF2 gene was 2090bp,encoded 179 amino acids.There was an A/T mutation occurred at 263bp in the 5 ’regulatory region,and mutation of A base deletion occurred at 974bp.There was a signal peptide sequence in this protein,which is a secretory protein with strong hydrophilicity though unstable.The total length of the coding region of ZBED6 gene was 4851 bp,encoded 980 amino acids,The A/G mutation occurred at 838bp in the coding region at 944bp;C/T mutation occurred at 1799bp;A/G muta-tion at 2276bp;C/T mutation at 2323bp;G/T mutation at 2333bp;A/G mutation at 2397bp;A/G mutation at 2480bp,signal peptide sequences were found in the protein,strong hydrophilicity though unstable.2.Expression levels of IGF2 and ZBED6The relative expression of muscle,heart,liver,spleen,lung,kidney and testis of Liaoning cashmere goat of IGF2 and ZBED6 was studied.Results showed that the expression of ZBED6 in muscle tissue is the highest,the expression of IGF2 was relatively low;IGF2 gene expression results:liver>Heart>kidney>muscle>lung>spleen>testis,ZBED6 gene expression results:muscle>heart>liver>kidney>testis>lung>spleen.3.Study on promoter methylation level of IGF2 and ZBED6The promoter region of muscle tissue of Liaoning cashmere goat was predicted.There were 34 CpG islands in the promoter region of IGF2 gene,and there were 28 CpG islands in the promoter region of ZBED6 gene.By bisulfite sequencing PCR(BSP),the level of IGF2 methylation in muscle tissues was significantly higher than that of ZBED6 methylation levels.The expression of IGF2 and ZBED6 were analyzed:in muscle tissue,the expression of IGF2 is regulated not only by the inhibitory factor ZBED6,but also by the methylation level of the promoter region:the higher its methylation level was,the lower its relative expression level was.These results show that IGF2 and ZBED6 genes played an important role in the regulation of muscle growth and development in Liaoning cashmere goats.IGF2 encoded 179 amino acids and ZBED6 encodes 980 amino acids;IGF2 and ZBED6 have differential expression and DNA methylation modification in different tissues.These differential genes are important for the growth,differentiation and metabolism of muscle cells.In this study,we studied the muscle growth and development of Liaoning cashmere goats at the molecular level,which provided a theoretical basis for the breeding of Liaoning cashmere goats.