Study On The Regulation Function Of DNA Methylation In Early Embryonic Development Of Bombyx Mori

As an important economic insect and model organism,the diapause phenomenon in the embryonic development of Bombyx mori has been widely concerned.Up to now,scientists have expounded the mechanism of diapause in Bombyx mori from the aspects of physiology,biochemistry,molecular biology and genetics.Studies on DNA methylation of Bombyx mori showed that about 0.11% of thymine contained methylation modification,with only DNA methyltransferase 1 and DNA methyltransferase 2 present.It is generally believed that DNA methylation is closely related to embryonic development.However,the regulatory role of DNA methylation in the development of Bombyx mori embryo,especially the embryo diapause,has been seldom reported.In this study,the expression patterns of DNA methylase in the embryonic development of Bombyx mori were analyzed with the bivoltine strain P50 and the polymorphic strain Nistari as research materials.At the same time,WGBS and RNA-seq technology were used to study the DNA methylation pattern and expression level before and after the removal of diapause in Bombyx mori embryos,and the pathways and genes related to embryo development and removal of diapause were further screened by association analysis.Finally,the G2E3 gene selected and related to periodic regulation were analyzed in depth.Main research results and conclusions are as follows:1.Analysis of the expression of DNA methyltransferaseBm DNMTs is highly expressed in the embryonic stage of silkworm,especially in the early embryonic development stage.Tissue expression results showed that Bm DNMTs were significantly up-regulated in silkworm pupa and adult stage.Meanwile,Bm DNMTs expression in P50 embryos after diapause removal was significantly up-regulated.By further analyzing the expression levels of Bm DNMTs at different developmental stages in P50 and Nistari show that DNA methylase showed a peak value at 24 h,and the expression pattern of DNA methylase was similar to Nistari after artificial diapause removal of P50.2.Differentially expressed genes and pathways in diapause eggs and artificially released diapause eggs were analyzed based on RNA-seq techniqueBivoltine strains P50 as material,using the comparison the transcriptome technology of diapause eggs and remove HCl diapause eggs to analyze differentially expressed genes,identified 1732 differentially expressed genes,in which 856 genes up-regulated,and 876 genes down-regulated.GO and KEGG analysis shows that these genes mainly involved in metabolism of matter and energy,cell skeleton and apoptosis related pathways.3.WGBS technique was used to analyze the modification characteristics and differential variation of DNA methylation during embryo development and removal of diapauseWGBS technology was used to analyze Methylation of diapause eggs and HCl diapause removal eggs for Bivoltine strains P50.The results showed that methylated cytosine in Bombyx mori embryo accounted for 0.20-0.24% of cytosine in the whole genome,and nearly 98.5% m C occurred in CG sequence unit.DNA methylation levels in diapause embryos stimulated by HCl were significantly up-regulated compared to those entering diapause embryos.Moreover,it was found that the high level of modification of CG methylation in the gene translation region may promote embryo development by enhancing gene transcription.Meanwhile,a total of 43,781 DMRs was identified by WGBS methylation sequencing,and the genes involved were mainly enriched in endoplasmic reticulum protein processing,cell cycle,RNA transport and other pathways.A total of 99 DMPs were identified,mainly enriched in hippo signaling pathway,Wnt signaling pathway and m TOR signaling pathway.4.Correlation analysis of RNA-seq and WGBS data to analyze DNA methylation regulation of early embryonic development of Bombyx moriAssociation analysis of WGBS and RNA-seq databases identified 364 associated genes.KEGG and GO analysis showed that these genes were mainly involved in material metabolism,energy metabolism,protein phosphorylation,cell cycle regulation and other pathways.23 MTGs were identified and correlated with metabolic synthesis,mainly enriched to n-glycan biosynthesis,glycosaminoglycan biosynthesis,glutathione metabolism,regulation of lipolysis in adipocytes,biosynthesis of amino acids and glyoxylate and dicarboxylate metabolism pathways.Morover,6 MTGs were correlated with the phosphatidylinositol signaling pathway,and 16 MTGs related to cell cycle regulation,mainly enrichment in G1/S transition of the mitotic cell cycle,the regulation of apoptotic the processes and signalling pathways regulating stem cell pluripotency pathways.5.Relationship between DNA methylation and cell cycle regulation in Bombyx moriAfter cell-level intervention of Bm DNMTs,the cells were retained in G1 and G2/M phases,while the content of S phase was significantly reduced.It was preliminarily determined that DNA methylation of silkworm was involved in the regulation of cell cycle.Through BS-seq and RT-q PCR,the methylation and expression of G2E3 in diapause removal eggs were significantly up-regulated compared with diapause eggs,suggesting that G2E3 in silkworm embryos may affect transcription level and regulate cell cycle changes through methylation modification to regulate diapause.In conclusion,this study based on the analysis of DNMTs in different organizations,different strain and different stages of embryonic development.Meanwhile,based on WGBS and RNA-Seq data,modification rules of DNA methylation of diapause eggs and artificial removal of diapause eggs in silkworm were analyzed,And the pathways involved are analyzed,the system analysis of DNA methylation in silkworm embryonic development and regulation mechanism in the process of remove embryo diapause.Finally,the related metabolic pathways of the associated genes was systematically analyzed and the regulation mechanism of DNA methylation in silkworm embryo development and embryo diapause removal was tentatively discussed.