Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland.
UMR 5168, Université Grenoble Alpes, F-38041, Grenoble, France.
Plant J. 2015 Jul;83(2):344-58. doi: 10.1111/tpj.12878. Epub 2015 Jun 8.
Understanding how flowers develop from undifferentiated stem cells has occupied developmental biologists for decades. Key to unraveling this process is a detailed knowledge of the global regulatory hierarchies that control developmental transitions, cell differentiation and organ growth. These hierarchies may be deduced from gene perturbation experiments, which determine the effects on gene expression after specific disruption of a regulatory gene. Here, we tested experimental strategies for gene perturbation experiments during Arabidopsis thaliana flower development. We used artificial miRNAs (amiRNAs) to disrupt the functions of key floral regulators, and expressed them under the control of various inducible promoter systems that are widely used in the plant research community. To be able to perform genome-wide experiments with stage-specific resolution using the various inducible promoter systems for gene perturbation experiments, we also generated a series of floral induction systems that allow collection of hundreds of synchronized floral buds from a single plant. Based on our results, we propose strategies for performing dynamic gene perturbation experiments in flowers, and outline how they may be combined with versions of the floral induction system to dissect the gene regulatory network underlying flower development.
几十年来，发育生物学家一直致力于研究花朵如何从未分化的干细胞中发育而来。揭示这一过程的关键是详细了解控制发育转变、细胞分化和器官生长的全局调控层次结构。这些层次结构可以从基因干扰实验中推断出来，这些实验确定了在特定调控基因被破坏后对基因表达的影响。在这里，我们测试了在拟南芥花发育过程中进行基因干扰实验的实验策略。我们使用人工 microRNA（amiRNA）来破坏关键花调控因子的功能，并在植物研究界广泛使用的各种诱导启动子系统的控制下表达它们。为了能够使用各种诱导启动子系统以特定阶段分辨率进行全基因组实验来进行基因干扰实验，我们还生成了一系列花诱导系统，这些系统允许从单个植物中收集数百个同步的花蕾。基于我们的结果，我们提出了在花朵中进行动态基因干扰实验的策略，并概述了如何将它们与花诱导系统的版本结合起来，以剖析花发育的基因调控网络。
