2022年5月5日，中国农业科学院蔬菜花卉研究所甘蓝青花菜团队在期刊International Journal of Molecular Sciences（2023IF=5.6）上发表了名为Global DNA Methylation and mRNA-miRNA Variations Activated by Heat Shock Boost Early Microspore Embryogenesis in Cabbage (Brassica oleracea)的研究论文，论文的第一作者为孔枞枞博士和苏贺楠博士，通讯作者为吕红豪研究员。
小孢子培养是单倍体育种的一部分，广泛应用于甘蓝等十字花科作物的育种。在培养过程中，热休克（Heat Shock，HS）处理是必不可少的。但是它在促进早期小孢子胚胎发生（microspore embryogenesis，ME）方面的分子作用尚不清楚。本研究结合甘蓝“01-88”小孢子在高温下（32℃，24h）和常温中（25℃，24h）的甲基化水平，miRNA和转录组图谱，研究了DNA甲基化和miRNA在ME早期的调控作用。
两种前处理的全基因组甲基化水平有显著差异，共发现508个差异甲基化区域，59.92%的差异甲基化区域与转录本水平有关，39.43%的miRNA基因组与甲基化水平有关。显著的关联分析表明，31个差异表达基因是甲基化和miRNA的靶标，它们主要参与活性氧反应和脱落酸信号转导，表明HS诱导DNA甲基化，miRNA可能通过影响ROS和ABA水平来影响ME。
Figure 1. Embryoids produced by isolated microspores under the 32 ◦C and 25 ◦C pre-treatments.
(A) Embryoids produced by isolated microspores cultured for 3 weeks after 24 h treatment at 32 ◦C;
(B) Embryoids produced by isolated microspores cultured for 3 weeks after 24 h treatment at 25 ◦C;
(C) The embryoid rates after the 32 ◦C and 25 ◦C treatments. Error bars represent standard error of
the mean. Significant differences has been marked with asterisks (** p < 0.01).
Figure 2. The cytosine methylation level (mC) percentage for each sequence context in HT32 and NT25.
Figure 3. Genomic methylation levels of HT32 and NT25. The first circle represents chromosome
name and scale. The second to fourth circles (purple, blue and green) represent the methylation
background display of CG, CHG and CHH in the corresponding chromosome intervals of HT32
groups, respectively; The fifth to seventh circles (purple, blue and green) represents the methylation
background display of CG, CHG and CHH in the corresponding chromosome intervals of NT25
group, respectively . The eighth circle represents the number of genes in the corresponding interval,
and the darker the color, the more genes there are in that region.
Figure 4. Comparative analysis of DNA methylation levels in different genomic regions between
HT32 and NT25. (A) CG methylation level. (B) CHG methylation level. (C) CHH methylation level.
Peak distribution in different genomic regions. Methylation density is the ratio of methylation peaks
to the corresponding sequence lengths.
Figure 5. GO and KEGG enrichment of DMR-associated genes. (A) GO enrichment of DMR-
associated genes. (B) KEGG enrichment of DMR-associated genes.
Figure 6. Correlation between differential methylation and level of transcript abundance.
(A) Differential expression levels of genes associated with hyper/hypo DMRs. (B) The number
of up/down-regulated genes associated with hyper/hypo DMRs.
Figure 7. The qRT-PCR validation and expression analysis of 9 genes. The gene expression was
normalized using actin and expressed relative to gene expression in HT32 sample (negative control).
Error bars represent standard error of the mean. Significant differences has been marked with
asterisks (** p < 0.01).
Figure 8. The interaction network of miRNA and target genes in plant hormone signal transduction
pathway (ko04075).
Figure 9. Correlation between differential methylation and level of miRNA abundance. (A) Fraction
of different mCs present in miRNAs. (B) Differential expression levels of miRNAs associated with
hyper/hypo DMRs
Figure 10. Genes with negative correlation with miRNA and methylome.
