Analysis Of Imprinting And Methylation Status Of MKRN3、NDN And MAGEL2 Genes In Dolang Sheep

Genomic imprinting is an epigenetic phenomenon that causes alleles originating from both parents to express only one of the alleles,and this phenomenon is regulated mainly through DNA differential methylation.Imprinted genes can be involved in mediating cell signaling and endocrine processes that influence the onset of puberty in animals.Puberty is a marker for the onset of animal fertility,and the early onset of puberty is closely related to the reproductive capacity of animals.In this study,we first obtained the sequences of CDS region of MKRN3,NDN and MAGEL2 genes by clonal sequencing and performed bioinformatics analysis,and examined the m RNA expression of the three genes in hypothalamic-pituitarygonadal axis before and after puberty initiation using fluorescence quantitative PCR.Next,the allelic expression status of the three genes in sheep was analyzed by direct sequencing of the SNP based RT-PCR products.Finally,the methylation status of Cp G islands in the promoters was examined by cloning the sequences of the promoter regions of the three genes and using bisulfite sequencing(BSP).The main results of this study are as follows:The CDS sequences of MKRN3,NDN and MAGEL2 genes were obtained by cloning and uploaded to Gen Bank to obtain the accession numbers OQ632815,OP619999 and OP680992,respectively.MKRN3,NDN and MAGEL2 proteins are all unstable hydrophilic proteins without transmembrane structures and signal peptides.The fluorescence quantification results showed that MKRN3 m RNA was the most highly expressed in the prepubertal hypothalamus,and NDN and MAGEL2 m RNA were the most highly expressed in the pubertal hypothalamus.The expression of MKRN3 m RNA in hypothalamus,pituitary,ovary,uterus and oviduct significantly decreased from prepubertal to pubertal(p<0.05);The expression of NDN and MAGEL2 m RNA in hypothalamus,pituitary,ovary and oviduct significantly increased from prepubertal to pubertal and decreased significantly after pubertal(p<0.05).This experiment identified two SNP loci on the MKRN3 exon of Dolang sheep by PCR product sequencing,and the SNP loci determined that the MKRN3 gene was expressed as a single allele in Dolang sheep tissues(heart,liver,spleen,lung,kidney,hypothalamus,pituitary gland,ovary,uterus and oviduct);A single SNP locus was identified in each of the exons of NDN and MAGEL2 genes,and the SNP locus identified NDN and MAGEL2 genes as single alleles in all tissues of Dolang sheep.The genotypes of the umbilical cord tissues of newborn lambs and the corresponding parents determined that MKRN3,NDN and MAGEL2 genes were all paternally expressed maternally imprinted genes.In this study,the promoter sequences of MKRN3,NDN and MAGEL2 genes were obtained by cloning,respectively,and it was predicted that a Cp G island of 208 bp in length containing 17 Cp G sites existed in the MKRN3 promoter sequence,and a Cp G island of 363 bp in length containing 40 Cp G sites existed in the NDN promoter sequence.The MAGEL2 gene promoter region sequence does not have a Cp G island.The methylation status of MKRN3 and NDN promoter Cp G islands in hypothalamus,ovary and spleen of Dolang sheep was examined by bisulfite sequencing(BSP),and significant differentially methylated regions(DMR)were observed in all three tissues.In summary,the sequences of CDS region of MKRN3,NDN,and MAGEL2 genes were obtained by cloning in this study.QPCR results revealed that MKRN3,NDN,and MAGEL2 genes may be involved in puberty initiation in Dolang sheep.It was verified that MKRN3,NDN and MAGEL2 genes were paternally expressed maternally imprinted genes in Dolang sheep,and the imprinting of MKRN3 and NDN genes was regulated by DNA methylation.The results of this experiment not only enriched the imprinted genes on sheep,but also laid the foundation for further study of the functions of MKRN3,NDN,and MAGEL2 genes in puberty.