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
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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。
【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.
【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.
引文
[1]Springer N M,Schmitz R J.Exploiting induced and natural epigenetic variation for crop improvement.Nat Rev Genet,2017,18(9):563.
[2]Shi J,Dong A,Shen W H.Epigenetic regulation of rice flowering and reproduction.Front Plant Sci,2015,5:803.
[3]洪苓苓,马旭东.组蛋白甲基化修饰的研究进展.临床血液学杂志,2010,23(1):54-56.Hong L L,Ma X D.Advances in histone methylation modification.J Clin Hematol,2010,23(1):54-56.(in Chinese)
[4]关录飞,吴笑女,徐启江.DNA甲基化及其对植物发育的调控.生物技术通讯,2008,19(4):632-634.Guan L F,Wu X N,Xu Q J.DNA methylation and its regulation effect during plant development.Lett Biotechnol,2008,19(4):632-634.(in Chinese with English abstract)
[5]郭广平,袁金玲,吴晓丽,顾小平.DNA甲基化在植物研究中的应用现状与前景.植物遗传资源学报,2011,12(3):425-430.Guo G P,Yuan J L,Wu X L,Gu X P.DNA methylation and its application in plant research.J Plant Genet Resour,2011,12(3):425-430.(in Chinese with English abstract)
[6]Finnegan E J,Kovac K A.Plant DNA methyltransferases.Plant Mol Biol,2000,43(2/3):189-201.
[7]Dowen R H,Pelizzola M,Schmitz R J,Lister R,Dowen JM,Nery J R,Dixon J E,Ecker J R.Widespread dynamic DNA methylation in response to biotic stress.Proc Natl Acad Sci,2012,109(32):E2183-91.
[8]Wang W S,Zhao X Q,Pan Y J,Zhu L H,Fu B Y,Li Z K.DNA methylation changes detected by methylationsensitive amplified polymorphism in two contrasting rice genotypes under salt stress.J Genet Genom,2011,38(9):419-424.
[9]赵云雷,叶武威,王俊娟,樊保香,宋丽艳.DNA甲基化与植物抗逆性研究进展.西北植物学报,2009,29(7):1479-1489.Zhao Y L,Ye W W,Wang J J,Fan B X,Song L Y.Review of DNA methylation and plant stress tolerance.Acta Bot Bor-Occ Sin,2009,29(7):1479-1489.(in Chinese with English abstract)
[10]Ou X F,Zhang Y H,Xu C M,Lin X Y,Zang Q,Zhuang TT,Jiang L L,Wettstein D V,Liu B.Transgenerational inheritance of modified DNA methylation patterns and enhanced tolerance induced by heavy metal stress in rice(Oryza sativa L.).Plo S One,2012,7(9):e41143.
[11]Goll M G,Bestor T H.Eukaryotic cytosine methyltransferases.Annu Rev Biochem,2005,74:481-514.
[12]Kakutani T,Jeddeloh J A,Flowers S K,Munakata K,Richards E J.Developmental abnormalities and epimutations associated with DNA hypomethylation mutations.Proc Natl Acad Sci USA,1996,93(22):12406-12411.
[13]Asao H,Eun S H,Okano Y,Moritoh S,One A,Okano Y,Yamaguchi K,Shimatani Z,Koizumi A,Terada R.Targeted disruption of an orthologue of DOMAINSREARRANGED METHYLASE 2,OsDRM2,impairs the growth of rice plants by abnormal DNA methylation.Plant J&Cell Mol Biol,2012,71(1):85.
[14]Baubec T,Pecinka A,Rozhon W,Scheid O M.Effective,homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine.Plant JCell&Mol Biol,2010,57(3):542-554.
[15]袁建民,木万福,麻继仙,杨龙,李易蓉,但忠,苏银玲.DNA甲基化抑制剂5-azaC对花椰菜生长发育的影响.江西农业学报,2018,30(2):42-45.Yuan J M,Mu W F,Ma J X,Yang L,Li Y R,Dan Z,Su YL.The effects of DNA methylation inhibitor 5-azaC on growth and development of cauliflower.Acta Agric Jiangxi,2018,30(2):42-45.(in Chinese with English abstract)
[16]汪媛媛.两种DNA甲基化抑制剂对菊花表型和DNA甲基化的影响.开封:河南大学,2012.Wang Y Y.The effect of two different DNA methylation inhibitors on curcumin phenotype and DNA methylation.Kaifeng:Henan University,2012.(in Chinese with English abstract)
[17]刘秋香.DNA甲基化抑制剂5-Aza-2′-deoxycytidine处理后水稻的DNA甲基化研究.杭州:浙江大学,2014.Liu Q X.The research on DNA methylation of rice treated with DNA methylation inhibitor 5-Aza-2′-deoxycytidine.Hangzhou:Zhejiang University,2014.(in Chinese with English abstract)
[18]Sano H,Kamada I,Youssefian S,Katsumi M,Wabiko H.A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA.Mol Gene Genet,1990,220(3):441-447.
[19]段光明.叶绿素含量测定中Arnon公式的推导.植物生理学报,1992(3):221.Duan G M.Derivation of Arnon formula in determination of chlorophyll content.Plant Physiol Commun,1992(3):221.(in Chinese)
[20]Cheng C,Daigen M,Hirochika H.Epigenetic regulation of the rice retrotransposon Tos17.Mol Genet Genom,2006,276(4):378.
[21]黄菲,李雪梅,王文生,傅彬英.DNA甲基化在植物抗逆反应中的研究进展及其育种应用.中国农业科技导报,2013,15(6):83-91.Huang F,Li X M,Wang W S,Fu B Y.Research progress of DNA methylation in stress response and breeding in plant.J Agric Sci Technol,2013,15(6):83-91.(in Chinese with English abstract)
[22]Boyko A,Kovalchuk I.Epigenetic control of plant stress response.Environ Mol Mutag,2008,49(1):61-72.
[23]彭海,席婷,张静,吴先军.胁迫条件下植物DNA甲基化的稳定性.中国农业科学,2011,44(12):2431-2438.Peng H,Xi T,Zhang J,Wu X J.Stability of stress-induced DNA methylation in plant.Sci Agric Sin,2011,44(12):2431-2438.(in Chinese with English abstract)
[24]Hu L J,Li N,Xu C M,Zhong S L,Lin X Y,Yang J J,Zhou T Q,Yuliang A,Wu Y,Chen Y R,Cao X F,Zemach A,Rustgi S,Wettstein D V,Liu B.Mutation of a major CG methylase in rice causes genome-wide hypomethylation,dysregulated genome expression,and seedling lethality.Proc Natl Acad Sci USA,2014,111(29):10642-10647.
[25]Kankel M W,Ramsey D E,Stokes T L,Flowers S K,Haag J R,Jeddeloh J A,Riddle N C,Verbsky M L,Rechards E J.Arabidopsis MET1 cytosine methyltransferase mutants.Genetics,2003,163(3):1109.
[26]Wang Z,Gerstein M,Snyder M.RNA-Seq:Arevolutionary tool for transcriptomics.Nat Rev Genet,2010,10(1):57-63.
[27]仪治本,孙毅,牛天堂,梁小红,刘龙龙,赵威军,李炳林.高粱基因组DNA胞嘧啶甲基化在杂交种和亲本间差异研究.作物学报,2005,31(9):1138-1143.Yi Z B,Sun Y,Niu T T,Liang X H,Liu L L,Zhao W J,Li B L.Patterns of DNA cytosine methylation between hybrids and their parents in sorghum genome.Acta Agron Sin,2005,31(9):1138-1143.(in Chinese with English abstract)
[28]Song G S,Zhai H L,Peng Y G,Zhang L,Wei G,Chen XY,Xiao Y G,Wang L L,Chen Y J,Wu B,Chen B,Zhang Y,Chen H,Feng X J,Gong W K,Liu Y,Yin Z J,Wang F,Liu G Z,Xu H L,Wei X L,Zhao X L,Ouwerkerk P,Hankemeier T,Reijmers T,Heijden R,Lu C M,Wang M,Greef J,Zhu Z.Comparative transcriptional profiling and preliminary study on heterosis mechanism of superhybrid rice.Mol Plant,2010,3(6):1012-1025.
[29]Dowen R H,Pelizzola M,Schmitz R J,Lister R,Dowen JM,Nery J R,Dixon J E,Ecker J R.Widespread dynamic DNA methylation in response to biotic stress.Proc Natl Acad Sci USA,2012,109(32):E2183.
[30]Yu A,Lepere G,Jay F,Wang J Y,Bapaume L,Wang Y,Abraham A L,Penterman J,Fischer R L,Voinnet O,Navarro L.Dynamics and biological relevance of DNAdemethylation in Arabidopsis antibacterial defense.Proc Natl Acad Sci USA,2013,110(6):2389-2394.
[31]Akimoto K,Katakami H,Kim H J,Ogawa E,Sano C M,Wada Y,Sano H.Epigenetic inheritance in rice plants.Ann Bot,2007,100(2):205-217.
[32]Deleris A,Halter T,Navarro L.DNA methylation and demethylation in plant immunity.Ann Rev Phytopathol,2016,54(1):579-603.
[2]Shi J,Dong A,Shen W H.Epigenetic regulation of rice flowering and reproduction.Front Plant Sci,2015,5:803.
[3]洪苓苓,马旭东.组蛋白甲基化修饰的研究进展.临床血液学杂志,2010,23(1):54-56.Hong L L,Ma X D.Advances in histone methylation modification.J Clin Hematol,2010,23(1):54-56.(in Chinese)
[4]关录飞,吴笑女,徐启江.DNA甲基化及其对植物发育的调控.生物技术通讯,2008,19(4):632-634.Guan L F,Wu X N,Xu Q J.DNA methylation and its regulation effect during plant development.Lett Biotechnol,2008,19(4):632-634.(in Chinese with English abstract)
[5]郭广平,袁金玲,吴晓丽,顾小平.DNA甲基化在植物研究中的应用现状与前景.植物遗传资源学报,2011,12(3):425-430.Guo G P,Yuan J L,Wu X L,Gu X P.DNA methylation and its application in plant research.J Plant Genet Resour,2011,12(3):425-430.(in Chinese with English abstract)
[6]Finnegan E J,Kovac K A.Plant DNA methyltransferases.Plant Mol Biol,2000,43(2/3):189-201.
[7]Dowen R H,Pelizzola M,Schmitz R J,Lister R,Dowen JM,Nery J R,Dixon J E,Ecker J R.Widespread dynamic DNA methylation in response to biotic stress.Proc Natl Acad Sci,2012,109(32):E2183-91.
[8]Wang W S,Zhao X Q,Pan Y J,Zhu L H,Fu B Y,Li Z K.DNA methylation changes detected by methylationsensitive amplified polymorphism in two contrasting rice genotypes under salt stress.J Genet Genom,2011,38(9):419-424.
[9]赵云雷,叶武威,王俊娟,樊保香,宋丽艳.DNA甲基化与植物抗逆性研究进展.西北植物学报,2009,29(7):1479-1489.Zhao Y L,Ye W W,Wang J J,Fan B X,Song L Y.Review of DNA methylation and plant stress tolerance.Acta Bot Bor-Occ Sin,2009,29(7):1479-1489.(in Chinese with English abstract)
[10]Ou X F,Zhang Y H,Xu C M,Lin X Y,Zang Q,Zhuang TT,Jiang L L,Wettstein D V,Liu B.Transgenerational inheritance of modified DNA methylation patterns and enhanced tolerance induced by heavy metal stress in rice(Oryza sativa L.).Plo S One,2012,7(9):e41143.
[11]Goll M G,Bestor T H.Eukaryotic cytosine methyltransferases.Annu Rev Biochem,2005,74:481-514.
[12]Kakutani T,Jeddeloh J A,Flowers S K,Munakata K,Richards E J.Developmental abnormalities and epimutations associated with DNA hypomethylation mutations.Proc Natl Acad Sci USA,1996,93(22):12406-12411.
[13]Asao H,Eun S H,Okano Y,Moritoh S,One A,Okano Y,Yamaguchi K,Shimatani Z,Koizumi A,Terada R.Targeted disruption of an orthologue of DOMAINSREARRANGED METHYLASE 2,OsDRM2,impairs the growth of rice plants by abnormal DNA methylation.Plant J&Cell Mol Biol,2012,71(1):85.
[14]Baubec T,Pecinka A,Rozhon W,Scheid O M.Effective,homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine.Plant JCell&Mol Biol,2010,57(3):542-554.
[15]袁建民,木万福,麻继仙,杨龙,李易蓉,但忠,苏银玲.DNA甲基化抑制剂5-azaC对花椰菜生长发育的影响.江西农业学报,2018,30(2):42-45.Yuan J M,Mu W F,Ma J X,Yang L,Li Y R,Dan Z,Su YL.The effects of DNA methylation inhibitor 5-azaC on growth and development of cauliflower.Acta Agric Jiangxi,2018,30(2):42-45.(in Chinese with English abstract)
[16]汪媛媛.两种DNA甲基化抑制剂对菊花表型和DNA甲基化的影响.开封:河南大学,2012.Wang Y Y.The effect of two different DNA methylation inhibitors on curcumin phenotype and DNA methylation.Kaifeng:Henan University,2012.(in Chinese with English abstract)
[17]刘秋香.DNA甲基化抑制剂5-Aza-2′-deoxycytidine处理后水稻的DNA甲基化研究.杭州:浙江大学,2014.Liu Q X.The research on DNA methylation of rice treated with DNA methylation inhibitor 5-Aza-2′-deoxycytidine.Hangzhou:Zhejiang University,2014.(in Chinese with English abstract)
[18]Sano H,Kamada I,Youssefian S,Katsumi M,Wabiko H.A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA.Mol Gene Genet,1990,220(3):441-447.
[19]段光明.叶绿素含量测定中Arnon公式的推导.植物生理学报,1992(3):221.Duan G M.Derivation of Arnon formula in determination of chlorophyll content.Plant Physiol Commun,1992(3):221.(in Chinese)
[20]Cheng C,Daigen M,Hirochika H.Epigenetic regulation of the rice retrotransposon Tos17.Mol Genet Genom,2006,276(4):378.
[21]黄菲,李雪梅,王文生,傅彬英.DNA甲基化在植物抗逆反应中的研究进展及其育种应用.中国农业科技导报,2013,15(6):83-91.Huang F,Li X M,Wang W S,Fu B Y.Research progress of DNA methylation in stress response and breeding in plant.J Agric Sci Technol,2013,15(6):83-91.(in Chinese with English abstract)
[22]Boyko A,Kovalchuk I.Epigenetic control of plant stress response.Environ Mol Mutag,2008,49(1):61-72.
[23]彭海,席婷,张静,吴先军.胁迫条件下植物DNA甲基化的稳定性.中国农业科学,2011,44(12):2431-2438.Peng H,Xi T,Zhang J,Wu X J.Stability of stress-induced DNA methylation in plant.Sci Agric Sin,2011,44(12):2431-2438.(in Chinese with English abstract)
[24]Hu L J,Li N,Xu C M,Zhong S L,Lin X Y,Yang J J,Zhou T Q,Yuliang A,Wu Y,Chen Y R,Cao X F,Zemach A,Rustgi S,Wettstein D V,Liu B.Mutation of a major CG methylase in rice causes genome-wide hypomethylation,dysregulated genome expression,and seedling lethality.Proc Natl Acad Sci USA,2014,111(29):10642-10647.
[25]Kankel M W,Ramsey D E,Stokes T L,Flowers S K,Haag J R,Jeddeloh J A,Riddle N C,Verbsky M L,Rechards E J.Arabidopsis MET1 cytosine methyltransferase mutants.Genetics,2003,163(3):1109.
[26]Wang Z,Gerstein M,Snyder M.RNA-Seq:Arevolutionary tool for transcriptomics.Nat Rev Genet,2010,10(1):57-63.
[27]仪治本,孙毅,牛天堂,梁小红,刘龙龙,赵威军,李炳林.高粱基因组DNA胞嘧啶甲基化在杂交种和亲本间差异研究.作物学报,2005,31(9):1138-1143.Yi Z B,Sun Y,Niu T T,Liang X H,Liu L L,Zhao W J,Li B L.Patterns of DNA cytosine methylation between hybrids and their parents in sorghum genome.Acta Agron Sin,2005,31(9):1138-1143.(in Chinese with English abstract)
[28]Song G S,Zhai H L,Peng Y G,Zhang L,Wei G,Chen XY,Xiao Y G,Wang L L,Chen Y J,Wu B,Chen B,Zhang Y,Chen H,Feng X J,Gong W K,Liu Y,Yin Z J,Wang F,Liu G Z,Xu H L,Wei X L,Zhao X L,Ouwerkerk P,Hankemeier T,Reijmers T,Heijden R,Lu C M,Wang M,Greef J,Zhu Z.Comparative transcriptional profiling and preliminary study on heterosis mechanism of superhybrid rice.Mol Plant,2010,3(6):1012-1025.
[29]Dowen R H,Pelizzola M,Schmitz R J,Lister R,Dowen JM,Nery J R,Dixon J E,Ecker J R.Widespread dynamic DNA methylation in response to biotic stress.Proc Natl Acad Sci USA,2012,109(32):E2183.
[30]Yu A,Lepere G,Jay F,Wang J Y,Bapaume L,Wang Y,Abraham A L,Penterman J,Fischer R L,Voinnet O,Navarro L.Dynamics and biological relevance of DNAdemethylation in Arabidopsis antibacterial defense.Proc Natl Acad Sci USA,2013,110(6):2389-2394.
[31]Akimoto K,Katakami H,Kim H J,Ogawa E,Sano C M,Wada Y,Sano H.Epigenetic inheritance in rice plants.Ann Bot,2007,100(2):205-217.
[32]Deleris A,Halter T,Navarro L.DNA methylation and demethylation in plant immunity.Ann Rev Phytopathol,2016,54(1):579-603.