Scientific Imaging Section, Okinawa Institute of Science and Technology Graduate University (OIST), Okinawa, Japan.
Science and Technology Group, OIST, Okinawa, Japan.
Methods Mol Biol. 2022;2509:93-104. doi: 10.1007/978-1-0716-2380-0_6.
Small RNAs specifically expressed in reproductive tissues are key regulators of germline development in eukaryotes. Rice microRNA2118 (miR2118), which is enriched during reproduction in grasses, is a trigger to produce phased small interfering RNAs (phasiRNAs). These phasiRNAs demonstrate the temporal regulation with premeiotic phasiRNAs and meiotic phasiRNAs in anther development. Furthermore, the site-specific regulation via miR2118 and phasiRNAs is of importance in soma and germ development in anthers. Accordingly, histological imaging methods are essential tools for understanding spatiotemporal regulation during reproduction and elucidating the reproductive roles of miRNAs and phasiRNAs. We successfully developed a method to visualize the three-dimensional (3D) structure of entire rice anthers, which can also be used for distinguishing the internal structure of the anthers in other plants. Here, we describe the detailed methods of in situ hybridization for miR2118 localization and the visualization of the 3D structure of entire anthers of rice.
生殖组织中特异性表达的小 RNA 是真核生物生殖系发育的关键调节剂。在禾本科植物生殖过程中富集的水稻 microRNA2118（miR2118）是产生相分离小干扰 RNA（phasiRNA）的触发物。这些 phasiRNA 在花药发育过程中表现出与减数分裂前 phasiRNA 和减数分裂 phasiRNA 的时间调节。此外，通过 miR2118 和 phasiRNA 的特异性调控对花药中的体细胞和生殖细胞发育具有重要意义。因此，组织学成像方法是理解生殖过程中时空调节以及阐明 miRNA 和 phasiRNA 生殖作用的重要工具。我们成功开发了一种可视化整个水稻花药三维（3D）结构的方法，该方法也可用于区分其他植物花药的内部结构。在这里，我们描述了 miR2118 定位的原位杂交和水稻整个花药 3D 结构可视化的详细方法。
