概要: Fertilization constitutes a critical step in the plant life cycle during which the gamete genomes undergo reprogramming in preparation for embryogenesis. However, it is unclear whether and how DNA methylation that epigenetically regulates gene expression is reprogramed during fertilization. Here, we characterized DNA methylation patterns and investigated the function of DNA glycosylases in rice eggs, sperms and unicellular zygotes. We found that DNA methylation is extensively reprogramed at non-CG sites in euchromatin regions upon fertilization, which intensifies during early embryogenesis. Genetic and genomic analysis revealed that rice DNA glycosylase genes DNG702, DNG701 and DNG704 demethylate at distinct and complementary loci in egg, sperm, and zygote genomes and are required for zygotic gene expression and development. The results indicate that active demethylation takes place in the gametes and the zygote to reprogram DNA methylation and zygotic genome activation in plant.
项目整体设计: BS-seq and RNA-seq of gametes and unicellular zygote of wild type, dng702, dng701/4 knock out mutant
Rice plants were grown in either greenhouse or paddy field
处理方案:
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提取方案:
Rice gametes and unicellular zygotes were isolated from mature anthers and ovaries as reported. Briefly, mature anthers were soaked in 45% (w/v) sucrose and then transferred into 15% (w/v) sucrose to release sperm pairs. For isolation of eggs and unicellular zygotes, ovaries of non-pollinated and pollinated florets (about 6.5 h after pollination) were manually dissected under a dissection microscope. The dissociated ovules were then transferred into fresh 0.53 M mannitol and broken to release eggs or zygotes. All isolated cells were stained by FDA (Fluoresceinc diacetate, Sangon, 596-09-8) and collected by a micromanipulator system (Eppendorf, TransferMan® 4r).
建库方案:
For RNA-seq library construction, mRNA isolated from egg, sperm, and unicellular zygote were reverse transcribed and amplified by utilizing SMART-Seq® v4 Ultra® Low Input RNA Kit (TAKARA, Cat. No.634889), cDNAs were sheared into 200-400 bp DNA fragments followed by purification using Agencourt AMPure Beads (Beckman Coulter, USA A63881). The rest steps were performed using the Truseq ChIP DNA library preparation kit (Illumina .IP-202-1012). BS-seq libraries were constructed using reported protocol (Clark et al., 2017).
