| Somatic cell cloning technology has been widely used in medicine,agriculture and animal husbandry,but the development rate of somatic cloned embryo is low,especially in mammals.Clonal cows may have abnormal pregnancy,increased allantoic effusion,and abnormal abdominal circumference during pregnancy.The placenta is an organ for exchanging substances between the mother and the fetus.The transfer of nutrients,the synthesis of essential nutrients and hormones,and the metabolic capacity directly affect the growth and development of the fetus.The study found that placental abnormalities occurred in almost all cloned mammals,which may be one of the main reasons for inhibiting the growth and development of cloned embryos.miRNAs are endogenous non-coding single-stranded RNAs that regulate gene expression in a sequence-specific manner.It is an important regulator of gene expression by combining with 3 'UTR of target mRNA to cut mRNA or inhibit its translation and regulate the expression of target gene in the post transcriptional.miRNA sequencing is an important method to find important related genes,establish regulatory networks,and clarify the mechanism of action.In this study,the Illumina high-throughput sequencing platform was used to compare the expression differences of miRNA and mRNA between the somatic cell cloned cows and the normalcow placenta,and the miRNAs and mRNAs with significant differences were screened and verified,and the differential expressions were analyzed using the biological information analysis method.The miRNAs and mRNAs were used for the significant enrichment analysis of GO functionand the KEGG Pathway significant enrichment analysis were used to find important genes and miRNAs related to the regulation of pregnancy maintenance,and a regulatory network of genes involved in pregnancy maintenance during the transgenic somatic cell nuclear transfer was established to study the regulation mechanism.In this study,two 180-day pregnant somatic cell cloned bovine placenta tissues were collected as the experimental group,and three 180-day naturally-inoculated bovine placenta tissues were used as the control group.After intensive sequencing of the collected bovine placental miRNAs by Illumina high-throughput sequencing,990 mature miRNAs were screened.There were 10 significantly differently expressed mi RNAs,of which 7were significantly up-regulated and 3 were significantly down-regulated.Fluorescentquantitative PCR was used to validate the 10 miRNAs that were significantly differently expressed.The quantitative results were consistent with the sequencing results.The target genes of 10 miRNAs with significant difference were analyzed by software and 1241 target genes were found.There were 9 biological processes,6 cell components and 5 molecular functions of GO that were significantly enriched in these target genes.The significantly enriched KEGG pathway had 7 articles.A total of 362 differentially expressed mRNAs were screened by transcriptome sequencing,of which 154 were up-regulated and 208 were down-regulated in the experimental group,which is consistent with 15 differentially expressed miRNA-targeted mRNAs.The target gene SLC14A2 of bta-miR-205 is related to urea transmembrance transport and the target gene BMP7 of bta-miR-2411-3p is related to ureteric bud development,and the target gene SLC16A4 of bta-miR-1298 is related to ion transmembrane transport.These are related to the increase of allantoic effusion and the abnormal increase of abdominal circumference.The above results can find genes and pathways related to pregnancy maintenance through miRNA and mRNA sequencing,and provide new solutions to improve the late stage of somatic cell cloning in bovine pregnancies,laying the foundation for revealing the molecular mechanism of pregnancy abnormalities. |
PDF Full Text Request