将mRNA翻译成功能性蛋白质是植物生长和发育的许多方面的基本过程。然而,翻译调节在不同组织类型的植物中的作用,包括种子,由于缺乏针对这些过程的方法,因此并不为人所知。研究种子翻译组可以揭示种子特异性调节机制,为育种努力增强种子性状提供有价值的见解。多聚体谱分析是用于研究被翻译的mRNA的广泛使用的技术。然而,传统的方法耗时长,多聚体回收率低,因此,它需要大量的起始材料。这对于种子数量有限的物种或突变体尤其具有挑战性。此外,由于干扰常规RNA提取方案的各种化合物的丰度,种子多体部分通常产生低质量的RNA。在这里,我们提出了一种强大的多聚体提取方法,该方法结合了大小排阻步骤,以简化多聚体浓度,并采用针对种子优化的快速RNA提取方法。该协议适用于多种植物物种,并提供更高的速度和鲁棒性,与常规方法相比,需要少于一半的种子组织和时间,同时确保下游实验的高质量RNA的高多聚体回收率和产量。这些特征使该协议成为研究种子翻译效率的理想工具,并在各种植物物种和组织中具有广泛的适用性。©2024Wiley期刊有限责任公司。基本方案1:种子的稳健多聚体提取基本方案2:快速级分总RNA提取。
Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed-specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time-consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size-exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high-quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Robust polysome extraction for seeds Basic Protocol 2: Rapid fraction total RNA extraction.