Influence of DNA Methylation Inhibitor 5-Aza-2′-deoxycytidine on DNA Methylation and Seedling Development of Rice

doi:10.16819/j.1001-7216.2019.9001

Abstract

Abstract:

【Objective】 DNA methylation is an epigenetic modification broadly existed in higher plants. DNA methylation plays an important role in regulating gene expression, maintaining the genomic stability and modulating plant development. Here, we used a DNA methylation inhibitor, 5-Aza-2′-deoxycytidine (AZA), to study how the DNA methylation status influences rice development. 【Method】We applied AZA to the rice seedlings to explore its influence on seedling development. 【Results】AZA-treatment resulted in overall hypomethylation of the rice genome. AZA-treated plants were strikingly shorter than the control, indicating AZA treatment disturbed normal development of the plants. However, the seed germination was not significantly affected. Expression of several key genes involved in DNA methylation could be repressed by AZA treatment. In addition, some genes involving defense and photosynthesis pathways were also down-regulated. 【Conclusion】Our findings confirm that AZA is a potent DNA methylation inhibitor and suggest that to maintain proper DNA methylation status is important for rice development.

Key words: DNA methylation, seedling growth, gene expression, rice

摘要：

【目的】DNA甲基化是高等植物中普遍存在的一种表观修饰形式,其在调节基因表达、维持基因组稳定以及调控植物生长发育等方面发挥重要作用。本研究拟对基因组甲基化如何影响水稻发育进行解析。【方法】利用DNA甲基化抑制剂5-氮脱氧胞苷(AZA, 5-Aza-2′-deoxycytidine)处理水稻幼苗,研究DNA甲基化抑制剂对水稻幼苗生长发育及相关基因表达的影响。【结果】AZA处理后水稻基因组甲基化水平下降、植株发育迟缓,但种子萌发并不受AZA处理的影响;DNA和组蛋白表观修饰相关基因的表达受到AZA处理抑制。此外,防卫反应和光合通路相关基因表达也受到AZA处理的影响,暗示DNA甲基化在这些基因的表达调控中可能发挥作用。【结论】这些结果表明AZA是一种有效的DNA甲基化抑制剂,AZA处理可以破坏水稻基因组甲基化水平的正常状态,从而影响水稻的正常发育。

关键词: DNA甲基化, 幼苗生长, 基因表达, 水稻

CLC Number:

Q755
S511.01

Hui DENG, Zhiguo E, Baixiao NIU, Lei WANG, Chen CHEN. Influence of DNA Methylation Inhibitor 5-Aza-2′-deoxycytidine on DNA Methylation and Seedling Development of Rice[J]. Chinese Journal OF Rice Science, 2019, 33(2): 108-117.

邓卉, 鄂志国, 牛百晓, 王磊, 陈忱. DNA甲基化抑制剂5-氮脱氧胞苷对水稻基因组甲基化及幼苗生长发育的影响[J]. 中国水稻科学, 2019, 33(2): 108-117.

Figures/Tables 7

Table 1 Primers used in the study.

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
MET1-1-qrt-f	GCATGTGCTTCCATCCTGAG	Lsi6-qrt-f	TTCCCTCCTAACCTCCTCAA
MET1-1-qrt-r	ATCTGCCTGTGCTTGTTCTG	Lsi6-qrt-r	CCTGCGAAGATCGACGTAAT
MET1-2-qrt-f	GCGCCAGTAAACTCCTACTT	LOC_Os07g05360-qrt-f	GTTGATGGGTACAGCCCAATA
MET1-2-qrt-r	CCCAATCCAGCCTACCATAAA	LOC_Os07g05360-qrt-r	GAGTGATTGCCCAGAGAAGTAA
CMT3-qrt-f	ATCTGCCTGTGCTTGTTCTG	LOC_Os07g05365-qrt-f	GCCAGCAAGTATGGAGCTAAT
CMT3-qrt-r	ATCTGCCTGTGCTTGTTCTG	LOC_Os07g05365-qrt-r	AGAGCAACAACCCTGTCTTTC
CMT2-qrt-f	TCAGGTTGTGGTGGCTTTAC	LOC_Os08g40160-qrt-f	CATTCCACAAGACTGGCTGAT
CMT2-qrt-r	CTCTGTGTCTTTGGAGGTTGAG	LOC_Os08g40160-qrt-r	GTGATGGCTACGCTGACATT
DNMT2-qrt-f	GACACCTACATTCCTAACATTGG	LOC_Os10g21192-qrt-f	GATAGCCAAGGTCGCGTTATTA
DNMT2-qrt-r	TCAGCGACATTCAGACTTATTG	LOC_Os10g21192-qrt-r	AGAGGGAAGTTGTGAGCATTAC
DRM2-qrt-f	CGTGCGGCATCTTACTACTGA	LOC_Os10g39880-qrt-f	GAGAGAGAACGCGAAAGTACAA
DRM2-qrt-r	ATCTCGGTGATGGCGGTTG	LOC_Os10g39880-qrt-r	ACCGAACCATCCGATGTAAAG
JIOsPR10-qrt-f	CCTCAGCCATGCCATTCA	LOC_Os07g37030-qrt-f	GCTCGCAGTACAACAACCA
JIOsPR10-qrt-r	CTTGGTGATCTCGTCCTTCAC	LOC_Os07g37030-qrt-r	CCACGGCACGAAGAGAAC
RSOsPR10-qrt-f	GCACTGTCACCACCATGAA	LOC_Os01g64120-qrt-f	CCACGCCGATCACCAATTA
RSOsPR10-qrt-r	CGAGCTCATACTCCACGTTTATC	LOC_Os01g64120-qrt-r	CTGAACCTGCCGGAGAAC
OsPR10a-qrt-f	GCCGCAAGTCATGTCCTAAA	LOC_Os07g30670-qrt-f	CGACAACTACATCGACCTCTAC
OsPR10a-qrt-r	CGAGGTAGTCCTCGATCATCTT	LOC_Os07g30670-qrt-r	AGTTCACCGCCTTCAACTAC
Lsi2-qrt-f	CTGGAGATGTCGGAGAACATAAC	Actin-f	CCAGGTATCGCTGACCGTAT
Lsi2-qrt-r	CCACCTCATCACCCATAAGAAG	Actin-r	GGAAAGTGCTGAGTGAGGCT
Lsi3-qrt-f	CGTTCGGTGTGTTCTGGAT	Tos17methy-f	GTTGATTATAGGGGATGATTTGGAGTATATTGTTT
Lsi3-qrt-r	GCCGAGGAGGAAGGAAATAAA	Tos17methy-r	CATAAAACACAAAACAAATAACCATAATAAACTA

Fig. 1. Effects of AZA treatment on genomic methylation. A, Digestion of the genomic DNA by methylation-sensitive MspⅠ; B, Methylation level of transposons Tos17chr7; C, Methylation pattern of transposons Tos17chr7 detected by bisulfate sequencing.

Fig. 2. Relative expression level of DNA methylation related genes in AZA-untreated and -treated rice seedlings. Values are Mean±SD(n=3).

Fig. 3. Phenotypic analysis of the AZA-treated rice seedlings. A, Germination rate, values are mean±SD(n=50); B, Morphology of AZA treated and untreated plants at 2 days after germination(DAI) and 7DAI; C, Leaves of AZA treated and untreated plants; D, Chlorophyll contents, values are Mean±SD(n=3), **indicates significant difference at 0.01 level; E, Chloroplast structure, bar=0.5 μm.

Fig. 5. Leaf phenotype and relative expression level of silicon transporter. A, Semi-thin section of leaf of AZA-untreated plants, *indicate vascular bundle, bar=200 μm; B, Semi-thin section of leaf of AZA-treated plants, *indicate vascular bundle, bar=200 μm; C, Higher magnification view of leaf of AZA-untreated plant; D, Higher magnification view of leaf of AZA-treated plant; E, Scanning electron microscope (SEM) analysis of the leaf epidermal cells, Bar=25 μm; F, Cell length and width of the leaf epidermal cells, values are mean±SD(n=100); G, Relative expression level of silicon transporter, values are mean±SD(n=3).

Fig. 6. Relative expression level of pathogenesis-related genes and contents of resistance-related hormones. A, Relative expression level of pathogenesis-related genes JIOsPR10, RSOsPR10 and OsPR10a, values are mean±SD(n=3); B, Contents of abscisic acid (ABA), salicylic acid(SA), jasmonic acid(JA) in AZA-untreated and AZA-treated plants, values are mean±SD(n=3).

Fig. 4. Relative expression level of photosynthetic genes in AZA-untreated and AZA-treated plants. The error bars indicate standard deviations(n=3). **indicates significant difference at 0.01 level; ns indicates no statistical significance.

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