Screening And Functional Study Of Highly Expressed LncRNA During Embryonic Genome Activation In Goat

During the early embryonic development,a defining transition of embryonic gene expression regulation gradually changing from maternal regulation to embryonic regulation is called maternal-zygotic transition.This transition requires embryo-wide embryonic genome activation(EGA).During the EGA process,the maternal m RNA is rapidly degraded,while the zygote gene is activated.During the development of somatic cloned embryos,the incomplete activation of the embryonic genome is an important factor affecting the developmental potential of cloned embryos.The latest research shows that long non-coding RNA(lncRNA)plays an important role in epigenetic modification and gene expression regulation.And it has been proved that long non-coding RNA in mice is a key regulator of the EGA process.Screening and studying the role of these lncRNAs in goat early embryos and somatic cloning embryos can play an important role in addressing the issue of the regulatory mechanism of EGA during goat early embryo development and improving the efficiency of cloned embryo development.Therefore,in this study,we collected 15 embryos from in vitro fertilized embryos at the two-cell(IVF-2c),8-cell stages(IVF-8c)and nuclear transfer embryos at 8-cell(SCNT-8c)stages for embryo transcriptome sequencing.,and differential expression analysis was performed.After that,the differentially expressed lncRNA was screened and the function of the predicted target gene was searched.Then,their expression patterns in early embryos were verified by quantitative PCR,and finally stage-specific lncRNA3720 that was significantly high expressed in IVF 8-cell compared with SCNT 8-cell was obtained.In the follow-up phase of the study,the effect of lncRNA3720 on embryonic development and its regulation on the expression of nearby target genes and key genes in embryonic development were verified by gene knockout and overexpression experiments.Then,the recombinant vector and overexpressing cell models was constructed,and the differentially expressed genes after overexpression of lncRNA3720 were analyzed and the changes of histone methylation level were detected at the cellular level.Finally,the embryo experiment verifies the effect of lncRNA3720 on the development rate of nuclear transfer embryos.The main results include the following four parts:(1)RNA sequencing analysis showed that lncRNA was abundantly expressed in goat early embryos,and lncRNA3720 is highly expressed in IVF 8-cell stages,while the expression level was low in IVF 2-cell stages and nuclear transfer embryo 8-cell stages,which proved that a large number of transcripts were detected at the 8-cell stage activation,the typical zygotic gene Zscan4 is also among them.GO enrichment shows that the differentially expressed genes of nuclear transfer embryos and in vitro fertilized embryos are mainly enriched in nucleic acid metabolism pathways.IVF-8c was compared with IVF-2c and SCNT-8c respectively,finally,12 common differentially expressed lncRNAs were obtained.(2)Transcriptome sequencing and qPCR quantitative analysis showed that lncRNA3720 was highly expressed in the IVF-8 cell stage,which was consistent with the pattern of embryonic genome activation genes;Knockdown of lncRNA3720 significantly decreased the blastocyst rate of the early embryo(P<0.05),but did not affect the the downstream genes indicates that it can indeed promote early embryo development but it does not affect the development of early embryos in cis.(3)The full length of lncRNA3720 was successfully amplified by RACE,then the interference and overexpression of lncRNA3720 can regulate the expression of zygote genes Zscan4 and EIF1 AX,indicating that lncRNA3720 perhaps was involved in the activation of little goat zygote genes.(4)The pcDNA3.1(+)-lncRNA3720 overexpression vector was successfully constructed and successfully transfected into goat fetal fibroblasts.the specific regulatory mechanism was verified at the cell level and 1071 differentially expressed genes was obtained by transcriptome sequencing,including 362 up-regulated genes and 709 down-regulated genes.Sequencing data analysis found that lncRNA3720 would reduce the level of OGT,and OGT is essential for the generation of H3K27me3.Western blot and immunofluorescence staining experiments have verified that the level of H3K27me3 is indeed reduced after lncRNA3720 overexpression.In addition,Compensation experiments proved that lncRNA3720 can improve the development rate of nuclear transfer embryos.In summary,the research shows that lncRNA is abundantly expressed in goat early embryos and plays an important role in maintaining early embryo development.The regulation of lncRNA on early embryo development can regulate the activation of related embryo genomes in early embryos by regulating the level of histone methylation Realization of genes and analysis of the role of specific lncRNA in epigenetic regulation have important guiding significance for the development of nuclear transfer embryos.