Application of Yeast-Two Hybrid Assay to Chemical Genomic Screens: A High-Throughput System to Identify Novel Molecules Modulating Plant Hormone Receptor Complexes
Application of Yeast-Two Hybrid Assay to Chemical Genomic Screens: A High-Throughput System to Identify Novel Molecules Modulating Plant Hormone Receptor Complexes
复制标题
DOI：
10.1007/978-1-62703-592-7_4
复制
发表时间：
2014-01-01
期刊：
影响因子：
--
通讯作者：
Chini, Andrea
中科院分区：
文献类型：
--
作者：
Chini, Andrea
植物激素是调节植物发育、适应环境和生存的内源性信号分子。在内部或外部刺激下，激素迅速积累和感知，进而激活特定的信号级联，调节适当的生理反应。近十年来，对植物激素感知机制的研究取得了很大进展。在不同的方法方法中，酵母双杂交（Y2H）试验在植物激素感知复合物的鉴定和分析中发挥了关键作用。Y2H测定是一种快速、直接的技术，可以很容易地用于鉴定高通量方式直接调节植物激素感知复合物的小分子。然而，Y2H化学筛选往往会分离假阳性分子，因此需要在植物中进行二次筛选，以确认推定阳性命中的真正生物活性。这种两步筛选方法可以大大节省时间和体力劳动。本章重点介绍了基于y2h的化学基因组高通量筛选在植物激素感知复合物中的应用前景。具体来说，本文描述了用于进行化学基因组筛选以鉴定植物激素茉莉酸缀合形式茉莉酸异亮氨酸（JA-Ile）的激动剂和拮抗剂分子的方法。还说明了一种简单的植物确认试验。然而，这种方法可以很容易地扩展到检测干扰其他植物激素受体复合物的新化合物。最后，本文描述的高通量方法也可以用于鉴定干扰植物复合体蛋白-蛋白相互作用的分子，而不是激素受体。
Phytohormones are endogenous signalling molecules that regulate plant development, adaptation to the environment, and survival. Upon internal or external stimuli, hormones are quickly accumulated and perceived, which in turn activates specific signalling cascades regulating the appropriate physiological responses. In the last decade, great advances in understanding plant hormone perception mechanisms have been achieved. Among different methodological approaches, yeast-two hybrid (Y2H) assays played a pivotal role in the identification and analysis of plant hormone perception complexes. The Y2H assay is a rapid and straightforward technique that can be easily employed to identify small molecules directly modulating plant hormone perception complexes in a high-throughput manner. However, an Y2H chemical screen tends to isolate false positive molecules, and therefore a secondary in planta screen is required to confirm the genuine bioactivity of putative positive hits. This two-step screening approach can substantially save time and manual labor. This chapter focuses on the prospects of Y2H-based chemical genomic high-throughput screens applied to plant hormone perception complexes. Specifically, the method employed to carry out a chemical genomic screen to identify agonist and antagonist molecules of the phytohormone jasmonic acid in its conjugated form jasmonic acid-isoleucine (JA-Ile) is described. An easy in planta confirmation assay is also illustrated. However, this methodology can be easily extended to detect novel chemical compounds perturbing additional plant hormone receptor complexes. Finally, the high-throughput approach described here can also be implemented for the identification of molecules interfering with protein-protein interaction of plant complexes other than hormone receptors.