MicroRNA168 (MIR168) is a key miRNA that targets the main RNA-induced silencing complex component Argonaute 1 (AGO1) to regulate plant growth and environmental stress responses. However, the regulatory functions of MIR168 need to be further elucidated in rice. In this paper, we generated clean OsMIR168a deletion mutants by CRISPR-Cas9 strategy. We then phenotypically and molecularly characterized these mutants. The rice OsMIR168a mutants grew rapidly at the seedling stage, produced more tillers and matured early. Compared to the wild-type plants, the mutants were shorter at maturity and produced smaller spikelets and seeds. Analysis of gene expression showed that the transcription levels of OsMIR168a’s target genes such as OsAGO1a, OsAGO1b and OsAGO1d were elevated significantly in the OsMIR168a mutants. Intriguingly, OsAGO18, a member of a new AGO clade that is conserved in monocots, was confirmed to be a target of OsMIR168a not only by informatic prediction but also by expression analysis and a cell-based cleavage assay in the OsMIR168a mutants. Many protein-coding genes and miRNAs showed differential expression in the OsMIR168a mutants, suggesting OsMIR168a exerts a major transcriptional regulatory role, likely through its potential target genes such as OsAGO1s and OsAGO18. KEGG enrichment analysis of these differentially expressed genes pointed to OsMIR168a’s involvement in important processes such as plant hormone signalling transduction and plant–pathogen interaction. These data collectively support that the complex regulation module of OsMIR168a-OsAGO1/OsAGO18-miRNAs-target genes contributes to agronomically important traits, which sheds light on miRNA-mediated crop breeding.

中文翻译：

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CRISPR-Cas9 介导的 OsMIR168a 敲除揭示了其在水稻中的多效性

MicroRNA168 (MIR168) 是一种关键的 miRNA，它靶向主要的 RNA 诱导的沉默复合物成分 Argonaute 1 (AGO1) 来调节植物生长和环境应激反应。然而，MIR168在水稻中的调控功能需要进一步阐明。在本文中，我们通过 CRISPR-Cas9 策略生成了干净的OsMIR168a缺失突变体。然后我们对这些突变体进行表型和分子表征。水稻OsMIR168a突变体苗期生长迅速，分蘖多，成熟早。与野生型植物相比，突变体在成熟时较短，产生较小的小穗和种子。基因表达分析表明，OsMIR168a的靶基因如OsAGO1a的转录水平，OsAGO1b和OsAGO1d在OsMIR168a突变体中显着升高。有趣的是，OsAGO18是在单子叶植物中保守的新 AGO 进化枝的成员，不仅通过信息预测，而且通过表达分析和OsMIR168a突变体中基于细胞的切割测定，证实它是OsMIR168a的靶标。许多蛋白质编码基因和miRNA 在OsMIR168a突变体中表现出差异表达，表明OsMIR168a可能通过其潜在的靶基因如OsAGO1s 和 OsAGO18发挥主要的转录调节作用. 对这些差异表达基因的 KEGG 富集分析表明OsMIR168a参与了植物激素信号转导和植物-病原体相互作用等重要过程。这些数据共同支持OsMIR168a - OsAGO1 / OsAGO18 - miRNA s-靶基因的复杂调控模块有助于农艺学上重要的性状，这为 miRNA 介导的作物育种提供了启示。