大花蕙兰子房授粉前后基因组DNA 胞嘧啶甲基化状态的MSAP 分析

应用甲基化敏感扩增多态性(Methylation sensitive amplified polymorphism, MSAP) 技术分析了大花蕙兰( Cymbidium hybridium) 授粉前后子房DNA 甲基化状态的变化(甲基化水平和甲基化差异模式) 。采用72 对引物进行选择性扩增, 共得到5892 条带, 其中748 条带为甲基化多态性带。结果显示DNA 甲基化在大花蕙兰子房发育过程中发生频繁, 从授粉前后子房的总扩增位点甲基化水平(14%和11. 4%) 和全甲基化率(9.5%和7.8% ) 来看, 授粉后都略低于未授粉子房, 表明子房在授粉后的发育过程中在某些位点发生了去甲基化。除甲基化水平有变化外, 大花蕙兰子房授粉前后的DNA 甲基化模式也存在较大差异, 共检测到14 种带型, 分为两大类( Ⅰ 和Ⅱ 型)。其中, 授粉前后DNA 甲基化状态保持不变的位点少, 只占25.6% , 归为Ⅰ型; 大部分检测位点( 占74.4% , 归为Ⅱ型) 的DNA 甲基化模式在授粉前后存在显著差异。上述结果表明, 大花蕙兰子房发育过程中以DNA 甲基化为代表的表观遗传调控起重要作用。本研究的开展将促进对与大花蕙兰子房发育相关的甲基化差异片段及受DNA 甲基化调控的关键基因的克隆, 进而为从表观遗传学这一新角度揭示大花蕙兰子房发育的分子机制奠定基础。

Analysis of DNA Cytosine Methylation between the Non-pollinated and Pollinated Ovaries of Cymbidium hybridium (Orchidaceae) Based on MSAP

The best known and most thoroughly studied epigenetic phenomena is DNA methylation, which plays an important role in regulating gene expression during plant regeneration and development. In this study, MSAP (methylation sensitive amplified polymorphism) technique was carried out to analyze differences of the methylation status between before and after pollination in ovaries of Cymbidium hybridium. 72 selective primer combinations were used to check the status of cytosine methylation DNA samples and a total of 5892 fragments were obtained . Thereinto, 748 fragments, each representing a recognition site cleaved by either or both of the isoschizomers ( Hpa Ⅱ and Msp Ⅰ ), were amplified , which were significant differences between the non-pollinated and the pollinated ovaries. The results demonstrated DNA methylation events occured in ovaries from Cymbidium hybridium . Both total and full methylation levels in the pollinated ovaries ( 11.4% ,7.8% ) were lower than those in the non-pollinated ovaries (14%, 9.5% ), which suggested some demethylations occurred. Furthermore, methylation patterns varied between the two ovaries. 14 types of MSAP patterns detected in the study belonged to two classes, type Ⅰ and Ⅱ. 25. 6% of detected fragments belonged to Type Ⅰ and 74.4% were type Ⅱ. Distinct patterns of DNA methylation arising through demethylation or de novo methylation might have specialized functions. This suggested the significance of epigenetic function in the development of orchid ovaries. The hypothesis that DNA methylation played a role in the C. hybridium ovary development would help to lucubrate the methylation status of special genes, clone the fragments with different methylation patterns, and shed novel insights into the molecular mechanisms of the ovary and floral development of orchids from the point of view of epigenetics.