A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny
A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny
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DOI：
10.1111/j.1365-313x.2012.05009.x
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发表时间：
2012-08-01
期刊：
影响因子：
7.2
通讯作者：
Iida, Shigeru
中科院分区：
文献类型：
--
作者：
Ono, Akemi;Yamaguchi, Katsushi;Iida, Shigeru
植物中促进DNA甲基化和去甲基化的基因主要在拟南芥中被表征。拟南芥DNA去甲基化是由双功能DNA酶介导的，双功能DNA酶具有糖基化酶活性和裂解酶活性，糖基化酶活性去除5-甲基胞嘧啶，裂解酶活性在脱碱基位点切割双链DNA。同源重组促进的敲入靶向的ROS 1a基因，最长的6个推定的DNA去甲基化酶基因在水稻基因组中，通过融合其内源启动子的GUS报告基因，导致可再现地破坏的ROS 1a在初级（T0）转基因植物在杂合条件下。这些T0植株在营养生长期没有表现出明显的形态学表型，GUS染色显示，在花粉、未受精胚珠和分生组织细胞中表达ROS 1a。有趣的是，无论是母亲还是父亲敲入无效等位基因，ros 1a-GUS 1，几乎检测到的后代，这样一个不可传递的无效突变是难以分离的常规诱变技术，通常用于识别和分离后代群体中的突变体。即使在野生型父本ROS 1a等位基因的存在下，母本ros 1a-GUS 1等位基因也会导致早期胚乳发育失败，导致胚胎发育不完全，胚胎发生产生不规则但有活力的胚胎，这些胚胎未能完成种子休眠，这意味着ROS 1a在胚乳发育中的母本和父本贡献不相等。父本的ros 1a-GUS 1等位基因没有传递给后代，可能是因为受精前的雄配子体缺陷。因此，ROS 1a在雄配子体和雌配子体中都不可或缺，DNA去甲基化在雄配子体和雌配子体中都起重要作用。
Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T0) transgenic plants in the heterozygous condition. These T0 plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.