Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan.
Plant Sci. 2011 Jun;180(6):789-95. doi: 10.1016/j.plantsci.2011.02.003. Epub 2011 Feb 13.
Dynamin family proteins in eukaryotic cells assemble into rings or spirals on the surface of membranes and pinch the membranes. We found 21 dynamin-related protein (DRP) genes in the Physcomitrella patens genome. Phylogenetic analysis indicated that three of them (PpDRP5B-1, PpDRP5B-2, and PpDRP5B-3) showed robust monophyly with Arabidopsis thaliana DRP5B and Cyanidioschyzon merolae CmDnm2, both of which are related to plastid division. Quantitative RT-PCR analysis showed that the amounts of DRP5B-3 transcripts were 14-fold and 8-fold higher than those of DRP5B-1 and DRP5B-2, respectively. We generated PpDRP5B knockout transformants for each of these genes. Subapical protonemata cells in wild-type plants had an average of 47 chloroplasts. The cells in the PpDRP5B-3 knockout transformant had slightly enlarged chloroplasts, with an average chloroplast number of 28, whereas the PpDRP5B-1 and 5B-2 knockout lines had no effect on chloroplast number in P. patens. To analyze function of each PpDRP5B gene, we generated double- and triple-knockout lines. Whereas there were 32 chloroplasts in a cell of the PpDRP5B-1/5B-2 double-knockout lines, the triple-knockout line had only a few macrochloroplasts. A transient expression assay with the triple-knockout line demonstrated that the PpDRP5B-3 gene could recover the normal chloroplast phenotype.
真核细胞中的动力蛋白家族蛋白在膜表面组装成环或螺旋，并将膜缢缩。我们在石莼基因组中发现了 21 个动力相关蛋白 (DRP) 基因。系统发育分析表明，其中 3 个（PpDRP5B-1、PpDRP5B-2 和 PpDRP5B-3）与拟南芥 DRP5B 和蓝藻 Cyanidioschyzon merolae CmDnm2 表现出强烈的单系性，它们都与质体分裂有关。定量 RT-PCR 分析表明，DRP5B-3 转录本的含量分别比 DRP5B-1 和 DRP5B-2 高 14 倍和 8 倍。我们生成了这 3 个基因的 PpDRP5B 敲除转化体。野生型植物亚顶生原丝体细胞平均有 47 个叶绿体。PpDRP5B-3 敲除转化体的细胞叶绿体略有增大，平均叶绿体数量为 28，而 PpDRP5B-1 和 5B-2 敲除系对石莼的叶绿体数量没有影响。为了分析每个 PpDRP5B 基因的功能，我们生成了双敲除和三敲除系。PpDRP5B-1/5B-2 双敲除系的一个细胞中有 32 个叶绿体，而三敲除系只有少数大叶绿体。对三敲除系的瞬时表达分析表明，PpDRP5B-3 基因可以恢复正常的叶绿体表型。
