干旱胁迫下高羊茅基因组甲基化分析

摘要

DNA 甲基化是真核细胞基因组重要的一种表观遗传调控。DNA 甲基化影响基因表达，在植物逆境胁迫适应中起着重要作用。高羊茅是禾本科单子叶羊茅属植物，具有良好的耐寒、耐热性，大量用作运动场草坪和防护草坪，也可用做牧草。干旱是限制高羊茅分布和产量的主要因素之一。本研究利用甲基化敏感扩增多态性技术（MSAP），比较了高羊茅在干旱胁迫15 d 后基因组胞嘧啶甲基化的变化。在干旱胁迫下，植株生长受到严重抑制。MSAP 分析显示，10对选择性扩增引物，共扩增出475个 CCGG 位点，其中131个位点发生了甲基化或去甲基化变化，占27．58％。对照组和干旱处理组，总甲基化水平分别是43．16％和42．11％，显示干旱胁迫诱导高羊茅总的甲基化率下降了1．05％。对差异条带进行归类分析，并克隆、测序，获得13条不同变化类型的序列。序列同源分析显示，大多数序列是参与胁迫应答的基因片段。其中2个片段，Fa6和 Fa7为干旱诱导下发生甲基化的位点，分别与小麦和大麦的一个逆转录转座子具有较高的同源性，在干旱胁迫下它们的甲基化程度增加，表明干旱诱导二者进一步甲基化。甲基化的转座子在维持基因组稳定性具有重要作用。综上，干旱胁迫诱导的甲基化的变化，可能参与高羊茅对环境的适应性调节。%DNA methylation is one of the most important mechanisms in the epigenetic regulation in eukaryotic cells.DNA methylation affects gene expression and plays an important role in plants adapting to adverse envi-ronments.Tall fescue (Festuca arundinacea ),a gramineous monocot,has superior resistance to cold and heat. It is widely used for sports turf,erosion control,and as a forage grass.However,the distribution and yield of tall fescue is constrained by drought stress.In this paper,the growth was measured and the DNA methylation profile of tall fescue (cv.Barlexas)evaluated using methylation-sensitive amplification polymorphism (MSAP) after 15 d drought treatment.The growth of the plants was seriously restricted under water-deficit challenge. Genome-wide,a total of 475 CCGG sites were detected by MSAP using 10 selective primer pairs in tall fescue seedlings.Among these sites,131 sites (27.58%)showed drought-induced methylation and demethylation. The total methylation level was 43.16% in control and 42.11% in drought-treatment,which indicates a 1.05%decrease when exposed to drought.The sequences of 13 amplified fragments were successfully obtained,repre-senting the differential types of methylation changes.Homologous analysis showed that most of the fragments were relevant to stress.Two sequences (Fa6 and Fa7)with enhanced methylation induced by drought were ho-mologous to a retrotransposon of Triticum aestivum and Hordeum vulgare ,respectively,showing drought in-duced methylation of the retrotransposons.The methylation of transposons is vital in maintaining DNA stabili-ty.In summary,drought stress decreased the total DNA methylation level in the tall fescue genome,and the altered methylation in response to stress was probably involved in environmental stress acclimation.