Kumar M B Arun, Martin Ruth C, Nonogaki Hiro
Directorate of Seed Research, Kushmaur (Vill), Kaithauli (P.O.), Uttar Pradesh, India.
Methods Mol Biol. 2011;773:199-213. doi: 10.1007/978-1-61779-231-1_13.
MicroRNAs (miRNAs) play an important role in gene regulation in many plant tissues and organs during various developmental stages. Previous studies have suggested the importance of gene regulation by miRNA in seeds. Characterizing the expression of miRNAs and their target genes in dormant and germinating seeds helps to gain a better understanding of the regulatory role of miRNAs during seed dormancy and germination. This can be achieved by implementing a simple miRNA extraction method using fractionation with isopropanol and Northern blot analysis using nonradioactive miRNA probes. Functional analysis of miRNA target genes potentially associated with seed dormancy and germination can be examined using mutant seeds in which specific miRNAs are deregulated by introducing silent mutations in the miRNA target sites of these genes.
微小RNA（miRNA）在许多植物组织和器官的不同发育阶段的基因调控中发挥着重要作用。先前的研究表明，miRNA在种子中的基因调控具有重要意义。表征miRNA及其靶基因在休眠和萌发种子中的表达，有助于更好地理解miRNA在种子休眠和萌发过程中的调控作用。这可以通过采用一种简单的miRNA提取方法来实现，该方法利用异丙醇分级分离，并使用非放射性miRNA探针进行Northern印迹分析。对于可能与种子休眠和萌发相关的miRNA靶基因的功能分析，可以使用突变种子进行研究，在这些突变种子中，通过在这些基因的miRNA靶位点引入沉默突变来解除特定miRNA的调控。
