玉米新选细胞质雄性不育系小孢子发育的细胞学观察及DNA甲基化分析

摘要

细胞质雄性不育在高等植物中普遍存在，是杂种优势利用的重要工具，为推动植物杂种优势的利用发挥了重要作用。文章以本课题组前期新选的玉米细胞质雄性不育系A1、A2及保持系18(红)为材料，利用石蜡切片技术对不育材料小孢子发育过程进行细胞学观察，采用高效液相色谱法(HPLC)对不同发育时期的叶片及不同发育时期的雄穗DNA进行甲基化分析，从细胞学和表观遗传学角度了解不育系A1、A2的败育机制。结果表明：不育材料A1、A2小孢子发生败育的主要时期为四分体时期至单核小孢子中期。在不育系A2中还存在另一种败育方式，即在花粉母细胞时期表现出败育特征。甲基化分析结果表明，保持系18(红)的叶片DNA甲基化水平从苗期到拔节期迅速上升，而不育系A1、A2叶片DNA甲基化水平基本保持不变；保持系雄穗DNA甲基化水平表现为从花粉母细胞时期到双核期逐渐升高，而不育材料A1、A2从花粉母细胞时期到双核期的雄穗DNA甲基化水平表现为先上升后下降的趋势，达到最高峰的时期均出现在小孢子发育的四分体时期。从小孢子发育的细胞学观察结果可以发现，小孢子败育的主要时期往往具有较高的甲基化水平，推测DNA甲基化水平变化可能与不育材料A1、A2的花粉败育有关。%Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants and has been applied in the commercial production of hybrid seeds. Two CMS lines A1 and A2 of maize were obtained previously by a trans-genic experiment. In this study, we conducted cytological observation of developmental microspores with CMS line A1, A2 and their maintainer line (18 red) using paraffin section technology. We also analyzed DNA methylation levels at different developmental stages using high performance liquid chromatography (HPLC). Our results showed that the pollen abortion of A1 and A2 mainly happened from the tetrad stage to the middle of mononuclear stage. Another abortive phenomenon found in CMS line A2 occurred at the pollen mother cell stage. The DNA methylation level of leaf increased rapidly from the seedling stage to the shooting stage in 18 red, while it remained constant in A1 and A2. For the tassel, the DNA methylation levels in 18 red increased gradually during the anther development, while a peak of DNA methylation level occurred in A1 and A2 at the tetrad stage, corresponding to the abortion period of micro-spore. This result suggested that the level of DNA methylation in the tassels is associated with the pollen abor-tion characteristics in CMS lines. In summary, our results implied a connection between pollen abortion and epige-netic regulation in maize CMS.