Efficient and specific inhibition of plant microRNA function by anti-microRNA oligonucleotides (AMOs) in vitro and in vivo
Efficient and specific inhibition of plant microRNA function by anti-microRNA oligonucleotides (AMOs) in vitro and in vivo
复制标题
通过抗 microRNA 寡核苷酸 (AMO) 在体外和体内有效且特异性地抑制植物 microRNA 功能
DOI：
10.1007/s00299-016-1933-y
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发表时间：
2016-04-01
影响因子：
6.2
通讯作者：
Tang, Tian
中科院分区：
文献类型：
--
作者：
He, Lian;Xie, Munan;Tang, Tian
抗microRNA寡核苷酸（Anti-microRNA oligonucleotides，AMOs）是植物体内和体外miRNA功能的有效抑制剂。尽管通过化学修饰的反义寡核苷酸敲除miRNA在动物和治疗研究中普遍存在，但在植物中从未报道过这样的应用。在这里，我们表明，蔗糖介导的2′-O-甲基（2′-O-Me）抗miRNA寡核苷酸（AMOs）的传递是一种有效的和序列特异性的抑制植物miRNA活性的方法，在体外和体内。对水稻原生质体和完整叶片施用AMO导致对miRNA的有效抑制，同时解除其靶基因的阻遏。AMOs引起来自同一家族的miRNA的同时抑制，但对来自不同家族的miRNA产生可忽略的影响。在水稻幼苗中，单剂量AMO处理赋予了至少7天的持久miRNA抑制。尽管AMO-miRNA-mimic混合物引起的多个miRNA的同时失调导致严重的根缺陷，但单个AMOs和miRNA mimics的表型效应可以忽略不计，这表明这些miRNA在调节网络中一起发挥作用以确保稳态。我们的研究结果验证了AMOs作为植物miRNA体内功能丧失研究的有效工具的实用性，并且这种方法可能被证明是一种非常有前途的通用方法，用于解开miRNA介导的基因调控网络。
Key messageAnti-microRNA oligonucleotides (AMOs) are efficient and sequence-specific inhibitors of plant miRNA function both in vitro and in vivo.AbstractMicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in developmental and physiological processes in plants and animals. Although miRNA knockdown by chemically modified antisense oligonucleotides prevails in animal and therapeutic studies, no such application has ever been reported in plants. Here, we show that sucrose-mediated delivery of 2′-O-methyl (2′-O-Me) anti-miRNA oligonucleotides (AMOs) is an efficient and sequence-specific way of inhibiting plant miRNA activity both in vitro and in vivo. Administration of AMOs to rice protoplasts and intact leaves resulted in efficient inhibition of miRNAs with concurrent de-repression of their target genes. AMOs caused simultaneous inhibition of miRNAs from the same family but exerted negligible effects on miRNAs from different families. In rice seedlings, a single-dose AMO treatment conferred long-lasting miRNA inhibition for at least 7 days. Although simultaneous dysregulation of multiple miRNAs by an AMO-and-miRNA-mimic mixture resulted in severe root defects, the phenotypic effects of individual AMOs and miRNA mimics were negligible, suggesting that those miRNAs function together in regulatory networks to ensure homeostasis. Our results validate the utility of AMOs as an efficient tool for plant miRNA loss-of-function studies in vivo, and this approach may prove to be a highly promising general method for unraveling miRNA-mediated gene-regulatory networks.