不同感温性甘蓝型冬油菜DNA甲基化差异及At4g02000-like结构预测
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摘要
为研究甘蓝型冬油菜(Brassica napus L.)在低温胁迫后DNA甲基化水平及模式的变化情况,以强抗寒的15TS306和14NS52-3等13个甘蓝型冬油菜为材料,利用甲基化敏感扩增多态性技术,选用12对引物组合对低温(4℃)胁迫后的冬油菜DNA甲基化水平及模式的变化情况进行检测,并对差异片段进行序列比对及克隆。结果发现:低温胁迫后,弱抗寒的14美切实7、14美切实16、14美切实20、14美切实3和美切实38等品系DNA甲基化水平有所升高,且具有较高的甲基化程度;而强抗寒的15TS306、14NS52-3、15TS309、14NS54-7和15TS312等品系去甲基化程度较高。经过对22条DNA甲基化特异片段的序列分析,有16条片段序列与已知和假定功能的酶及蛋白具有同源性,其中以At4g02000-like蛋白变化最为明显。克隆及生物信息学分析显示,At4g02000-like蛋白等电点9. 20,相对分子质量38. 89kD。甘蓝型冬油菜受低温胁迫后,抗寒性强的品种DNA甲基化水平降低,以去甲基化为主;抗寒性弱的材料DNA甲基化水平升高;而且油菜对低温环境的适应性与某些特定基因不同的甲基化模式密切相关。
To better understand the cold-resistance of winter rapeseed(Brassica napus L.),DNA methylation were investigated using MSAP(methylation sensitive amplified polymorphism) technology on 13 materials with different cold resistance under low temperature(4℃) stress.MSAP results showed that,after 4℃ treatment,the weak cold-resistance lines(14 Meiqieshi 7,14 Meiqieshi 16,14 Meiqieshi 20,14 Meiqieshi 3 and Meiqieshi 38 had increased DNA methylation level and had higher methylation degree.The strong cold-resistant lines(15 TS306,14 NS52-3,15 TS309,14 NS54-7 and 15 TS312) had higher degree of demethylation.Sequence analysis of the differentially methylated fragments showed that 16 of 22 fragments were similar to the known and putative functional enzymes.Among them,At4 g02000-like protein had the greatest variation.Cloning and bioinformatics results showed that its isoelectric point was 9.20 and the relative molecular weight was 39.89 kD.In summary,after low temperature stress,the strong cold-resistant lines had decreased DNA methylation level with dominant demethylation,while the weak cold-resistance lines had increased DNA methylation level with dominant methylation.In these winter rapeseeds,cold adaptability showed close correlation between differentially methylation patterns of some specific genes.
To better understand the cold-resistance of winter rapeseed(Brassica napus L.),DNA methylation were investigated using MSAP(methylation sensitive amplified polymorphism) technology on 13 materials with different cold resistance under low temperature(4℃) stress.MSAP results showed that,after 4℃ treatment,the weak cold-resistance lines(14 Meiqieshi 7,14 Meiqieshi 16,14 Meiqieshi 20,14 Meiqieshi 3 and Meiqieshi 38 had increased DNA methylation level and had higher methylation degree.The strong cold-resistant lines(15 TS306,14 NS52-3,15 TS309,14 NS54-7 and 15 TS312) had higher degree of demethylation.Sequence analysis of the differentially methylated fragments showed that 16 of 22 fragments were similar to the known and putative functional enzymes.Among them,At4 g02000-like protein had the greatest variation.Cloning and bioinformatics results showed that its isoelectric point was 9.20 and the relative molecular weight was 39.89 kD.In summary,after low temperature stress,the strong cold-resistant lines had decreased DNA methylation level with dominant demethylation,while the weak cold-resistance lines had increased DNA methylation level with dominant methylation.In these winter rapeseeds,cold adaptability showed close correlation between differentially methylation patterns of some specific genes.
引文
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[26]华扬,陈学峰,熊建华,等.水稻冷胁迫诱导的甲基化差异片段CIDM7的分离和分析[J].遗传,2005,27(4):595-600.
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[30] Meyer P. Transcriptional transgene silencing and chromatin components[J]. Plant Mol Biol,2000,43(2-3):221-234.
[31] Shan X H,Wang X Y,Yang G,et al. Analysis of the DNA methylation of maize(Zea mays L.)in response to cold stress based on methylation-sensitive amplified polymorphisms[J]. J Plant Biol,2013,56(1):32-38.
[32]孟德义.低温胁迫下冬小麦糖酵解代谢对外源SA的响应[D].哈尔滨:东北农业大学,2017.
[33] Singh B,Usha K. Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress[J]. Plant Growth Regulation,2003,39(2):137-141.
[34]陈秀华,王臻昱,李先平,等.谷胱甘肽S-转移酶的研究进展[J].东北农业大学学报,2013,44(1):149-153.
[2]侯献飞,孙万仓,方彦,等.甘蓝型冬油菜在西北寒旱区适应性分析[J].干旱地区农业研究,2016,34(6):63-68.
[3]王月,孙万仓,刘自刚,等.甘蓝型冬油菜在西北不同生态区适应性及生理生化反应[J].干旱地区农业研究,2015,33(4):197-205.
[4]李亚娇,郭九峰,王淑妍,等. DNA甲基化与植物生长发育的表观遗传调控研究进展[J].生态科学,2016,35(4):192-198.
[5]孟德斌,郭九峰,马梦宇,等.植物DNA甲基化与低温胁迫研究进展及展望[J].黑龙江农业科学,2017(8):119-125.
[6]邢潇晨.低温胁迫下黄瓜幼苗DNA甲基化与抗性的关系[D].石河子:石河子大学,2014.
[7] Steward N. Expression of Zm MET1,a gene encoding a DNA methyltransferase from maize,is associated not only with DNA replication in actively proliferating cells,but also with altered DNA methylation status in coldstressed quiescent cells[J]. Nucleic Acids Res,2000,28(17):3250-3259.
[8] Steward N,Ito M,Yamaguchi Y,et al. Periodic DNA methylation in maize nucleosomes and demethylation by environmental stress[J]. J Biol Chem,2002,277(40):37741-37746.
[9] Zhang L,Wang Y,Zhang X Z,et al. Dynamics of phytohormone and DNA methylation patterns changes during dormancy induction in strawberry(Fragaria×ananassa Duch.)[J]. Plant Cell Rep,2012,31(1):155-165.
[10] Finnegan E J,Genger R K,Peacock W J,et al. Dna methylation in plants[J]. Annu Rev Plant Physiol Plant Mol Biol,1998,49(1):223-247.
[11] Xiong L Z,Xu C G,Maroof M A S,et al. Patterns of cytosine methylation in an elite rice hybrid and its parental lines,detected by a methylation-sensitive amplification polymorphism technique[J]. Mol Gen Genet Mgg,1999,261(3):439-446.
[12] Xiong L Z,Liu K D,Dai X K,et al. Identification of genetic factors controlling domestication-related traits of rice using an F2population of a cross between Oryza sativa and O. rufipogon[J]. Theor Appl Genet,1999,98(2):243-251.
[13]李卫国,常天俊,龚红梅. MSAP技术及其在植物遗传学研究中的应用[J].生物技术,2008,18(1):83-87.
[14]谢涛,戎浩,蒋金金,等.人工合成甘蓝型油菜及其亲本的甲基化变异模式分析[J].作物学报,2016,42(4):513-524.
[15]刘丽君.冷胁迫下高山离子芥表观遗传修饰变化和ADH1功能研究[D].兰州:兰州大学,2017.
[16]何平,疏冕,蔡晓丹,等.栽培稻×药用野生稻种间杂种基因组DNA甲基化的遗传与变异研究[J].华北农学报,2017,32(4):19-31.
[17]高金燕.全基因组DNA甲基化对甘蓝型油菜春化作用的影响[D].郑州:郑州大学,2012.
[18]方婷婷.人工合成甘蓝型油菜与其二倍体亲本抗旱性及DNA甲基化差异研究[D].扬州:扬州大学,2016.
[19]杨光.玉米苗期冷响应分子机理的研究[D].长春:吉林大学,2010.
[20] Lindroth A M,Shultis D,Jasencakova Z,et al. Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3[J].Embo J,2004,23(21):4 146-4 155.
[21] Goll M G,Bestor T H. Eukaryotic cytosine methyltransferases[J]. Annu Rev Biochem,2005,74(1):481-514.
[22] Matzke M,Kanno T,Huettel B,et al. Targets of RNA-directed DNA methylation[J]. Curr Opin Plant Biol,2007,10(5):512-519.
[23] Kakutani T. Epi-alleles in plants:inheritance of epigenetic information over generations[J]. Plant Cell Physiol,2002,43(10):1 106-1 111.
[24] Choi C S,Sano H. Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants[J]. Mol Genet Genom,2007,277(5):589-600.
[25]杨美娜,杨瑰丽,郭涛,等.逆境胁迫下植物DNA甲基化及其在抗旱育种中的研究进展[J].中国农学通报,2013,29(6):6-11.
[26]华扬,陈学峰,熊建华,等.水稻冷胁迫诱导的甲基化差异片段CIDM7的分离和分析[J].遗传,2005,27(4):595-600.
[27] Wassenegger M. RNA-directed DNA methylation[M]//Plant Gene Silencing. Dordrecht:Springer Netherlands,2000:83-100.
[28]杨岐生.分子生物学基础[M].杭州:浙江大学出版社,1994.
[29] Kass S U,Pruss D,Wolffe A P. How does DNA methylation repress transcription?[J]. Trends Genet,1997,13(11):444-449.
[30] Meyer P. Transcriptional transgene silencing and chromatin components[J]. Plant Mol Biol,2000,43(2-3):221-234.
[31] Shan X H,Wang X Y,Yang G,et al. Analysis of the DNA methylation of maize(Zea mays L.)in response to cold stress based on methylation-sensitive amplified polymorphisms[J]. J Plant Biol,2013,56(1):32-38.
[32]孟德义.低温胁迫下冬小麦糖酵解代谢对外源SA的响应[D].哈尔滨:东北农业大学,2017.
[33] Singh B,Usha K. Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress[J]. Plant Growth Regulation,2003,39(2):137-141.
[34]陈秀华,王臻昱,李先平,等.谷胱甘肽S-转移酶的研究进展[J].东北农业大学学报,2013,44(1):149-153.