Institute of Plant Science and Resources, Okayama University, Kurashiki, Japan.
PLoS One. 2012;7(12):e51315. doi: 10.1371/journal.pone.0051315. Epub 2012 Dec 7.
Due to the ease with which chromosomes can be observed, the Allium species, and onion in particular, have been familiar materials employed in cytogenetic experiments in biology. In this study, centromeric histone H3 (CENH3)-coding cDNAs were identified in four Allium species (onion, welsh onion, garlic and garlic chives) and cloned. Anti-CENH3 antibody was then raised against a deduced amino acid sequence of CENH3 of welsh onion. The antibody recognized all CENH3 orthologs of the Allium species tested. Immunostaining with the antibody enabled clear visualization of chromosome behavior during mitosis in the species. Furthermore, three-dimensional (3D) observation of mitotic cell division was achieved by subjecting root sections to immunohistochemical techniques. The 3D dynamics of the cells and position of cell-cycle marker proteins (CENH3 and α-tubulin) were clearly revealed by immunohistochemical staining with the antibodies. The immunohistochemical analysis made it possible to establish an overview of the location of dividing cells in the root tissues. This breakthrough in technique, in addition to the two centromeric DNA sequences isolated from welsh onion by chromatin immuno-precipitation using the antibody, should lead to a better understanding of plant cell division. A phylogenetic analysis of Allium CENH3s together with the previously reported plant CENH3s showed two separate clades for monocot species tested. One clade was made from CENH3s of the Allium species with those of Poaceae species, and the other from CENH3s of a holocentric species (Luzula nivea). These data may imply functional differences of CENH3s between holocentric and monocentric species. Centromeric localization of DNA sequences isolated from welsh onion by chromatin immuno-precipitation (ChIP) using the antibody was confirmed by fluorescence in situ hybridization and ChIP-quantitative PCR.
由于染色体易于观察，葱属植物，特别是洋葱，一直是生物学中细胞遗传学实验的常用材料。在这项研究中，从四个葱属物种（洋葱、葱、大蒜和韭菜）中鉴定出着丝粒组蛋白 H3（CENH3）编码 cDNA 并进行了克隆。然后针对葱的 CENH3 推导的氨基酸序列制备了抗 CENH3 抗体。该抗体识别测试的葱属所有 CENH3 同源物。用该抗体进行免疫染色可在物种的有丝分裂过程中清楚地观察染色体行为。此外，通过对根切片进行免疫组织化学技术，实现了有丝分裂细胞分裂的三维（3D）观察。用抗体进行免疫组织化学染色清楚地揭示了细胞的 3D 动力学和细胞周期标记蛋白（CENH3 和α-微管蛋白）的位置。免疫组织化学分析使建立根组织中分裂细胞的位置概览成为可能。这项技术的突破，除了用抗体通过染色质免疫沉淀从葱中分离出的两个着丝粒 DNA 序列外，应该有助于更好地理解植物细胞分裂。对葱属 CENH3 与先前报道的植物 CENH3 的系统发育分析表明，测试的单子叶植物有两个独立的分支。一个分支由葱属的 CENH3 和禾本科的 CENH3 组成，另一个分支由一个着丝粒的物种（Luzula nivea）的 CENH3 组成。这些数据可能暗示着着丝粒的功能差异 CENH3s 在全着丝粒和单核物种之间。用抗体通过染色质免疫沉淀（ChIP）从葱中分离的 DNA 序列的着丝粒定位通过荧光原位杂交和 ChIP 定量 PCR 得到了证实。
