| Epigenetic modification which refers to external DNA modification that do not change the nucleotide sequence but affect the gene expression. Examples of epigenetic modification are DNA methylation and histone modification. DNA methylation is an important epigenetic modification involving the regulations of plant development, genomic imprinting, response to biotic and abiotic stresses, and etc. The jointing is a developmental stage of maize. During jointing, maize internode cells are quickly elongation accompanied by a sharp changes in plant morphology and cell physiology. The un-elongated internode (BE) and elongating internode (AE1) of maize inbred B73were selected as the studied materials by cytological observation. Furthermore, methylation immunoprecipitation (Me-DIP) was used to analysis the genome-wide methylation and the differentially methylated genes (DMGs) between the studied materials, and the DMGs were validated by the bisulfate sequencing. In addition, we assayed the genes expressions in BE and AE1cells by RNA-seq, RT-PCR and QPCR. Our objective is to elucidate the effect of DNA methylation in regulation of internode elongation of maize. The main results are as follows:1. The cytological observation revealed that the length of cells in the AE1(-120um) was significantly longer than in BE (-40um)(p=1.1e-18), indicating the cells in AE1tissue were expanding quickly, while cells in the BE were slow and constant development. Thus, the BE and AE1tissues were selected for assay of DNA methylation and gene expression.2. Me-DIP showed that the average methylation density was generally higher in gene-body than that in promoter, within the gene body, methylation level of intron was highest. Total2502differentially methylated genes (DMGs) were identified among the two tissues. Futhermore, we found that many DMGs were enriched in pathways related to development and metabolism. In particular, the methyl indole-3-acetate interconversion pathway-associated genes were demethylated obviously, in contrast, the ethylene biosynthesis-associated genes were up-methylated in promoter.3. The methylation pattern of36DMGs were analysed by bisulfate sequencing which validated that the methylation of11DMGs was significantly different between the un-elongated and elongating internode cells(p<0.05). The methylation contexts of those DMGs were50%for CHH,28.6%for CHG, and21.4%for CG.4. RNA-seq revealed1246differentially expressed genes (p<0.05), of which96were overlapped with DMGs. For DEGs, only3.7%(96/2596) gene expressions were significantly regulated by DNA methylation. Furthermore, RT-PCR and QPCR showed that all DEGs performed the different expression levels at the different stages of internode elongation. Most of DMGs showed the highest expression level at the AE1which grew rapidly, and then were down-regulated expression at AE2and AE3. Taken together, the results of Me-DIP and RNA-seq found that methylation at the promoter region may be a key regulation for gene expression.5. The result of bioinformatics revealed that a part of DMGs were involved in the formation and division of intracellular organelle directly, and majority of DMGs were found related with the signaling (phosphorylation signaling pathway, etc.) and metabolic process (oligosaccharide biosynthetic process). Compared with BE, more genes involved with the regulation of oxidation reduction metabolic process, less genes related with the cellular component and signaling in elongating cell (AE1). In summary, in elongating cells, genes belonging to cellular component categories were selectively modified by demethylation, and used the material and energy which were accumulated at BE to promote the internode elongation. |
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