The Study On Genomic Imprinting And Regulation Mechanism Of LINC24065 And GPR1-ZDBF2 Region In Bovine

Genomic imprinting is an epigenetic phenomenon which caused the parent-of-originspecific expression of imprinted genes by different epigenetic modifications of chromosomes.The monoallelic expression genes are named imprinted genes.In mammals,imprinted genes play an important role in the regulation of fetal growth and development,placental function and postnatal behavior.In recent years,with extensive and in-depth research,imprinted genes has been found to be very important for economic traits and breeding of animals.The expression disorder of imprinted gene can result in birth-death and abnormal development in somatic cell nuclear transfer(SCNT)animals.However,most of the imprinted genes are researched in humans and mice,but few in livestock.In this study,I analyzed the imprinting status of a non-coding RNA in DLK1-DIO3 region and three genes in GPR1-ZDBF2 region,and identified two different methylation regions(DMRs)in GPR1-ZDBF2,respectively in somatic cell nuclear transfer bovine,adult bovine and calves at brith.The paper results as follows:1.Using RACE and RT-PCR,I identified six variants(V1-V6)of LINC24065 which is a long intergenetic non-coding RNA,between MEG8 and MEG9 of DLK1-DIO3 region in bovine.LINC24065 was detected in seven analyzed tissues(brain,kidney,liver,spleen,lung,heart and muscle)and placentas of cattle.But in SCNT calves,LINC24065-V3 was not detected and the other five variants showed tissue-specific expression.Imprinting status analysis indicated that LINC24065 showed monoallelic expression in cattle and SCNT calves,however,in the brain of SCNT calves,parent-specific expression was different from that of other six tissues.2.GPR1 gene was obtained from chromosome 2 of bovine.Sequence analysis revealed that GPR1 had four variants(V1-V4)and expressed in eight cattle tissues tested.GPR1 was determined parternal specific expression in all cattle tissues tested,and allelic expression in placenta by RT-PCR based on SNP.3.We obtained the 3'UTR and the complete open reading frame(ORF)of ZDBF2 from bovine brain.ZDBF2 was paternal specific expression in both calves and adult bovine tissues and allelic expression in bovine placenta.ZDBF2 were tested allelic expression only in five tissues(liver,spleen,kidney,muscle and brain)of SCNT bovine.4.LIZ,a long intergenetic non-coding RNA,was identified upstream of ZDBF2 according to ESTs.Using RT-PCR,LIZ was not detected in all tissues of newborn and adult cattle,but detected in sperm and placenta.LIZ was confirmed parternal special expression in bovine placenta.5.According to the sequence traits of DMRs identified in GPR1-ZDBF2 region of human and mouse and the promoter location ZDBF2 predicted,we predicted two DMRs in GPR1-ZDBF2 region of bovine.One is G-DMR located in intron 3 of GPR1 gene,the other is Z-DMR located 17 kb upstream of ZDBF2 gene promoter.using bisulfite sequencing,the methylation status of G-DMR and Z-DMR was analyzed.Z-DMR is methylated in bovine sperm and parental-specific methylation in placenta and brain of bovine,but unmethylated in oocyte.G-DMR is completely methylated in oocyte and brain of bovine,hardly methylated in sperm,and paternal allele-specific methylation in bovine placenta.The methylation status of G-DMR was consistent with parternal special expression of LIZ,indicating that G-DMR regulated the expression of LIZ gene.In the two predicted DMRs,SCNT bovine showed significant differences only in G-DMR region compare to the newborn bovine.Parent-specific methylation was lost in brain,which corresponded to the abnormal expression of the imprinted genes in SCNT cattle,suggesting that G-DMR may be the imprinted regulatory region(ICR)of GPR1-ZDBF2 region in bovine.In addition,CpG island(CGI)was found to have low methylation in brain,sperm,oocyte and placenta of bovine indicating that the CGI region was not involved in the regulation of GPR1-ZDBF2 imprinting region.These results increased the number of imprinted genes in bovine,provide a theoretical basis for revealing the molecular mechanism of abnormal development and birth and death of somatic cell nuclear transfer animals.