State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Ministry of China Key Laboratory of Biotechnology in Plant Protection, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China.
Biotechnol J. 2014 May;9(5):702-8. doi: 10.1002/biot.201300523. Epub 2014 Apr 10.
MicroRNAs (miRNAs) play key roles in plant development and defense against pathogens. To establish the function of individual miRNAs, gain-of-function analysis is usually done by overexpressing a specific miRNA in transgenic plants and has proved very effective. Loss-of-function analysis by the target mimic method is now also increasingly being used. The mimics expressed in the transgenic plants sequester a specific miRNA and lead to changed phenotypes that elucidate miRNA function. However, it takes time to obtain the transgenic plants. To avoid using transgenic plants, we have developed a virus-based miRNA suppression system (VbMS) based on a Tobacco rattle virus vector. The target mimic sequences of miR156, miR319, or miR164 were introduced into the viral genomic RNA, which was then inoculated to Arabidopsis thaliana plants. The resulting phenotypes were consistent with previous reports from transgenic plants, and the expression of targets of the miRNAs was also increased showing that the activity of the miRNAs had been inhibited. VbMS developed here is validated for loss-of-function analysis of miRNA in plants. Moreover, since only simple agroinfiltration rather than transformation is needed, VbMS is suitable for large-scale approaches to miRNA function analysis in plants.
MicroRNAs (miRNAs) 在植物发育和抵御病原体方面发挥着关键作用。为了确定单个 miRNAs 的功能，通常通过在转基因植物中过表达特定的 miRNA 来进行功能获得分析，这已被证明非常有效。现在，通过靶标模拟方法进行功能丧失分析也越来越被广泛使用。在转基因植物中表达的模拟物可以隔离特定的 miRNA，并导致阐明 miRNA 功能的表型变化。然而，获得转基因植物需要时间。为了避免使用转基因植物，我们基于烟草脆裂病毒载体开发了一种基于病毒的 miRNA 抑制系统（VbMS）。miR156、miR319 或 miR164 的靶标模拟序列被引入病毒基因组 RNA，然后将其接种到拟南芥植物中。由此产生的表型与来自转基因植物的先前报告一致，并且 miRNA 的靶标表达也增加，表明 miRNA 的活性已被抑制。这里开发的 VbMS 已被验证可用于植物中 miRNA 的功能丧失分析。此外，由于只需要简单的农杆菌浸润而不是转化，因此 VbMS 适合于大规模分析植物中 miRNA 功能的方法。
