Hayashida A, Takechi K, Sugiyama M, Kubo M, Itoh R D, Takio S, Fujita T, Hiwatashi Y, Hasebe M, Takano H
Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan.
Plant Biol (Stuttg). 2005 May;7(3):300-6. doi: 10.1055/s-2005-837691.
Eleven mutant lines exhibiting decreased numbers of chloroplasts per cell were isolated from 8 800 tagged mutant lines of Physcomitrella patens by microscopic observations. Chloronema subapical cells in wild-type plants had a mean of 48 chloroplasts, whereas chloroplast numbers in subapical cells in mutant lines 215 and 222 decreased to 75 % of that in the wild type. Seven mutant lines - 473, 122, 221, 129, 492, 207, and 138 - had about half as many chloroplasts as the wild type. Mutant line 11 had a few remarkably enlarged chloroplasts, and mutant line 347 had chloroplasts of various sizes. Whereas the cell volume was the same as in the wild type in mutant lines 222, 473, 221, 129, 492, and 207, the cell volume of the other mutants increased. The chloroplast number of leaf cells was the same as that of chloronema cells in each mutant line when gametophores could be formed. Treatment with ampicillin decreased the number of chloroplasts in all mutant lines. Southern hybridization using DNA in tags as probes showed that only one insertion occurred in mutant lines 473 and 221. To determine whether the tagged DNA inserted into the known genes for plastid division, we isolated the PpMinD1, PpMinD2, and PpMinE1 genes. Genomic polymerase chain reaction analysis showed that the PpFtsZ and PpMinD/E genes were not disrupted by the insertion of the tags in mutant lines 11 and 347, respectively.
通过显微镜观察，从8800个带有标签的小立碗藓突变株系中分离出11个每个细胞中叶绿体数量减少的突变株系。野生型植株的绿丝体亚顶端细胞平均有48个叶绿体，而突变株系215和222的亚顶端细胞中的叶绿体数量减少到野生型的75%。7个突变株系——473、122、221、129、492、207和138——的叶绿体数量约为野生型的一半。突变株系11有一些明显增大的叶绿体，突变株系347有各种大小的叶绿体。在突变株系222、473、221、129、492和207中，细胞体积与野生型相同，而其他突变体的细胞体积增大。当可以形成配子体时，每个突变株系中叶细胞的叶绿体数量与绿丝体细胞的相同。用氨苄青霉素处理会减少所有突变株系中的叶绿体数量。以标签中的DNA为探针进行Southern杂交表明，在突变株系473和221中仅发生了一次插入。为了确定插入的标签DNA是否插入到已知的质体分裂基因中，我们分离了PpMinD1、PpMinD2和PpMinE1基因。基因组聚合酶链反应分析表明，在突变株系11和347中，标签的插入分别没有破坏PpFtsZ和PpMinD/E基因。
