| There is an important transition during mammalian early embryonic development,which is the transition from maternal to zygotic.During the transition,with the consumption of maternal material,the zygote genome gradually begins to activate transcription,called zygote genome activation(ZGA).In this process,a series of physiological processes such as epigenetic modification and changes in chromatin state are involved.ZGA is a very important process for embryo development.With the development of high-throughput sequencing technology,more and more studies have found that there are many non-coding RNAs in the genome.More than 200 nucleotides in length is called long non-coding RNA(long non-coding RNA,lnc RNA).The traditional view holds that lnc RNAs are "junk" in gene sequences.However,there is growing evidence show that lnc RNAs are important for regulating gene expression.The differential expression of some lnc RNAs in early embryos plays an important role in regulating embryonic development,while their functions and regulatory mechanisms for embryonic development have not yet been recognized.In this study,we first screened long non-coding RNAs that were differentially expressed in the previous sequencing,and the predicted target genes were reviewed in the literature.We selected long non-coding RNA TCONS?00135926(hereinafter referred to as lnc5926)based on the target gene and embryo development;then,the expression pattern of lnc5926 in the embryo was verified by quantitative PCR,and the role of lnc5926 in embryonic development was verified by knockdown experiment;we transfected cells by overexpression vector of lnc5926,performed transcriptome sequencing at the cellular level to analyze the differentially expressed genes and effects of on embryo development;the levels of target genes,cell and embryo sequencing repeat differentially expressed genes and zygotic genome activation-related genes were detected after embryonic knockdown and overexpression of lnc5926;finally,to study how lnc RNA5926 affects embryo development,we detect the level of histone lysine methylation and acetylation at the cellular level.The study obtained the following results:(1)Lnc5926 dynamically change during the early embryonic development of goats,which is in line with the law of genes of the zygotic genome activation;decreasing the level of lnc5926 significantly reduced the blastocyst development rate;(2)The full length of lnc5926 gene was obtained through RACE experiments.Overexpression vectors pc DNA3.1(+)-lnc5926 were successfully constructed,GFF cells were transfected,and cell samples overexpressing lnc5926 gene were obtained.Cell transcriptome sequencing showed a total of 399 differentially expressed genes,which included 76 up-regulated genes,323 down-regulated genes.Compared with embryo sequencing data,2 genes were up-regulated in both the 8-cell stage and over-expressing cells,and 1 gene were down-regulated;(3)Through knockdown and overexpression experiments,it was proved that lnc5926 has important regulatory functions on zygote genome activation genes Zscan4 and Eif1ax;(4)The effects of lnc5926 on the level of H3K9 trimethylation(H3K9 me3)and H3K9 acetylation(H3K9 Ac)were analyzed by immunofluorescence staining and Western Blot.The results showed that overexpression of lnc5926 can reduce the level of fetal fibroblast genome H3K9 me3 and increase the level of the genome H3K9 Ac.Based on the above results,long non-coding RNA is an important component in the goat genome,which can affect the expression level of zygotic genome activation-related genes and embryo development rate,and may change the level of histone methylation and acetylation modification.Encoding RNA plays an important role in early embryo development.This research discovery provides new insights into the effects of long non-coding RNA on mammalian embryo development and its mechanism,and lays the foundation for further exploration of the regulatory mechanism of embryo development. |
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