Research On Genetic Mechanism Of Developmental Abnormalities In Pig Cloned Early Embryos Using Transcriptome And DNA Methylation Sequencing

Somatic cell nuclear transfer(SCNT) is an important means for the efficient propagation of high-quality germplasm and the creation of new breeding materials.It has important application value in genetic improvement of agricultural animals.In pig breeding,SCNT technology can be used to replicate and expand elite individual boars to produce cloned individuals that are as good as donors.In addition,SCNT combined with gene editing technology can create new materials for pig breeding with superior traits.However,the current efficiency of pig SCNT remains low,mainly due to incomplete epigenetic reprogramming of somatic cells,which leads to abnormal expression of key genes during embryonic development.Therefore,this study aims to reveal the molecular mechanism of early developmental abnormalities in porcine cloned embryos,to find key genes that cause early developmental arrest of porcine cloned embryos,and to explore new methods for improving pig cloning efficiency.In this study,we used single-cell transcriptome sequencing technology to analyze the gene expression map of seven consecutive developmental stages(oocyte,1-cell,2-cell,4-cell,8-cell,morula,and blastocyst stage)of porcine cloned embryos and in vivo fertilized embryos.The results show that the porcine embryo genome activation mainly occurred in the 2-cell to 8-cell stage;a large number of possible candidate genes for abnormal development of pig cloned embryos were identified,of which TET1 and DRAP1 were key genes affecting the developmental potential of mature oocytes in vitro;KLF17,OLIG3,ZFP37,and ZFP42 are key genes that hinder the genomic activation of porcine 4-cell cloned embryos.By comparing the expression patterns of H3K4me3,H3K9me3,and H3K27me3 related histone methyltransferase and demethylase genes of pig cloned and in vivo fertilized embryos,we found that KDM4 A,KDM4D and KDM5 B genes were abnormally low expressed in 4-cell cloned embryos,and KMT2 A and SUV39H2 genes were abnormally highly expressed in 8-cell cloned embryos.Abnormal expression of these genes may be an important cause of abnormal histone modification in pig cloned embryos.We further utilized single-cell whole-genome methylation sequencing technique to construct a single-base resolution DNA methylation map of porcine cloned and in vivo fertilized embryos at 4-cell and 8-cell stage.The methylation pattern of cloned embryos and in vivo fertilized embryos showed 7738 differentially methylated regions(DMRs),of which 6403 were hypermethylated regions and 1335 were hypomethylated regions,indicating that there are not only a large number of abnormal hypermethylated regions in porcine 4-cell cloned embryos,but also many abnormally hypomethylated regions;24791 DMRs were identified between 8-cell cloned embryos and in vivo fertilized embryos,of which 24014 DMRs were hypermethylated regions and 777 DMRs are hypomethylated regions,suggesting that the abnormal DNA methylation of porcine 8-cell cloned embryos may be mainly caused by a large number of abnormally hypermethylated regions.The KEGG pathway analysis revealed that the differentially methylated genes of cloned and in vivo fertilized embryos were significantly enriched in NF-κB signaling pathway and oxidative phosphorylation signaling pathways,which play important roles in cell differentiation,proliferation and apoptosis.Finally,we tried to explore the effect of inhibition of histone methylation or ubiquitination on the developmental efficiency of porcine cloned embryos by overexpressing KDM4 A,KDM5B,KDM6 B,USP21 or USP29.It showed that simultaneous overexpression of KDM4 A and KDM5 B significantly increased blastocyst rate and total number of cells in blastocysts;overexpression of KDM4 A,KDM5B or USP29 respectively only significantly increased the total number of cells in blastocysts,but had no significant effect on blastocyst rate;overexpression of USP21 or KDM6 B had no significant effect on the blastocyst rate and the total number of cells in blastocysts.By using transcriptome sequencing analysis,we found that overexpression of KDM4 A,KDM5B or simultaneous overexpression of KDM4 A and KDM5 B partially corrected expression level of the abnormal expressed genes in the control cloned embryos,most of which were significantly enriched in histone lysine metabolism pathways.By using immunofluorescence staining,we found that overexpression of KDM4 A significantly reduced the H3K9me3 fluorescence signal of 2-cell,4-cell and 8-cell cloned embryos.Overexpression of KDM5 B significantly reduced the H3K4me3 fluorescence signal of 2-cell and 4-cell cloned embryos,while overexpressing KDM4 A and KDM5 B simultaneously decreased H3K9me3 and H3K4me3 fluorescence signals.By microinjecting the in vitro synthesis of KDM4 A and KDM5 B mRNA into 1-cell embryos,we increased the birth efficiency of cloned pigs by 1.96 times.In this study,we compared the transcriptome and DNA methylation profiles of porcine early cloned and in vivo fertilized embryos,and deeply analyzed the abnormal pattern of early development of pig cloned embryos.We also increased the efficiency of porcine SCNT by overexpressing histone methylation related genes in 1-cell cloned embryos.This study provides an important theoretical basis for further understanding the abnormal development of pig cloned embryos,and provides a new idea for improving the efficiency of porcine SCNT.