| The early embryo of ovine cleavage stage has experienced four stages: the formation of fertilized egg,the activation of zygote genome and the compaction of blastomeres,which involve the drastic changes of embryo transcription structure.It is very important to study the transcription group of 8-cell,16-cell and morula stage ovine IVF embryo,so as to understand the growth and development of early embryo.In order to obtain sheep IVF embryo samples for transcriptome sequencing and optimize the culture system of IVF,Single cell lysate and RNA Isolation Kit were used to obtaine RNA,and effective methods for obtaining single embryo RNA were screened,purifies m RNA,reverses m RNA into c DNA,and Smart-Seq2 amplifys c DNA to construct sheep IVF 8-cell,16-cell,morula stage single embryo transcriptome sequencing library,The transcriptome was sequenced by high-throughput sequencing technology of Illumina hiseq xten,+the differentially expressed genes in different cleavage embryos were screened,and go function analysis and KEGG analysis were carried out.The results are as follows:1.The maturation rate of ovine oocytes was 76.60%,cleavage rate was 63.24%,morula rate was 30.81%,which was significantly higher than that of 0 μg/m L,5 μg/m L,15 μg/m L,20 μg/m L LH(P < 0.05).2.Using single cell lysate to split single embryo of sheep,purify m RNA,reverse convert mRNA into c DNA,amplify c DNA by Smart-seq2,detect the concentration of PCR products are more than 60 ng,and the size of c DNA fragments is concentrated between 1 kbp and 2 kbp,successfully construct the transcription group sequencing Library of single embryo of sheep IVF 8-cell,16-cell and morula stage,and use picopure ? RNA Isolation Kit extracted single embryo RNA and detected RNA quality,Rin < 1.5 and 28 s / 18 S < 0.2,which did not meet the requirements of c DNA amplification and could not construct transcriptome sequencing library.3.Using DEGSeq software to analyze the differentially expressed genes,with FC > 2or < 0.5 and Q-value <0.05 as the screening criteria,170 DEGs were screened in 8-cell vs 16-cell of IVF sheep embryo,of which 126 were up-regulated and 44 were down regulated;417 DEGs were found in 16-cell vs morula,of which 47 were up-regulated and 370 down-regulated;126 were down regulated and 36 were up regulated in 8-cell vs morula.Go functional analysis of differentially expressed genes mainly involves the synthesis of nucleotide compounds,metabolic regulation,organic ring compounds biosynthesis,RNA metabolism,compound metabolism and other biological processes.KEGG analysis of the differentially expressed genes showed that8-cell vs 16-cell DEGs was involved in 126 pathways,and 11 pathways were involved in stem cell pluripotency and cell proliferation and differentiation;16-cell vs morula DEGs was involved in 206 pathways,and 2pathways were involved in spliceosome and stem cell pluripotency;8-cell vs morula DEGs was not. |
