Thomson Bennett, Graciet Emmanuelle, Wellmer Frank
Smurfit Institute of Genetics, Trinity College Dublin, Dublin, Ireland.
Department of Biology, National University of Ireland Maynooth, Maynooth, Ireland.
Methods Mol Biol. 2017;1629:15-25. doi: 10.1007/978-1-4939-7125-1_2.
Assessing molecular changes that occur through altering a gene's activity is often hampered by difficulties that arise due to the typically static nature of the introduced perturbation. This is especially problematic when investigating molecular events at specific stages and/or in certain tissues or organs during Arabidopsis development. To circumvent these issues, we have employed chemically inducible artificial microRNAs (amiRNAs) for the specific knockdown of developmental regulators. For our own research, we have combined this gene perturbation approach with a floral induction system, which allows the simultaneous induction of a large number of flowers on the inflorescence of a single plant, and the ability to knock down a gene's activity at any given stage of development. To enable the plant community to avail of the full benefits of these systems, we describe, in this chapter, strategies for amiRNA-mediated gene perturbations and address some common problems that can be encountered when generating inducible amiRNA constructs, growing these plants, and collecting floral buds for analysis.
评估因改变基因活性而发生的分子变化，常常受到因引入的扰动通常具有静态性质而产生的困难的阻碍。在研究拟南芥发育过程中特定阶段和/或某些组织或器官中的分子事件时，这一问题尤为突出。为了规避这些问题，我们采用了化学诱导型人工微小RNA（amiRNA）来特异性敲低发育调节因子。在我们自己的研究中，我们将这种基因扰动方法与一种花期诱导系统相结合，该系统能够在单株植物的花序上同时诱导大量花朵开放，并能在发育的任何给定阶段敲低基因的活性。为了使植物学界能够充分利用这些系统的优势，在本章中，我们描述了amiRNA介导的基因扰动策略，并探讨了在构建可诱导amiRNA载体、种植这些植物以及收集花芽进行分析时可能遇到的一些常见问题。
