Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden.
Curr Protoc. 2023 Feb;3(2):e673. doi: 10.1002/cpz1.673.
Plastids are found in all plant cell types. However, most extraction methods to study these organelles are performed at the organ level (e.g., leaf, root, fruit) and do not allow for tissue-specific resolution, which hinders our understanding of their physiology. Therefore, IPTACT (Isolation of Plastids TAgged in specific Cell Types) was developed to isolate plastids in a tissue-specific manner in Arabidopsis thaliana (Arabidopsis). Plastids are biotinylated using one-shot transgenic lines, and tissue specificity is achieved with a suitable promoter as long as such a promoter exists. Cell-specific biotinylated plastids are then isolated with 2.8-µm streptavidin beads. Plastids extracted by IPTACT are suitable for RNA or protein isolation and subsequent tissue-specific OMICs analyses. This method provides the user with a powerful tool to investigate plastidial functions at cell-type resolution. Furthermore, it can easily be combined with studies using diverse genetic backgrounds and/or different developmental or stress conditions. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Promoter cloning and plant selection Basic Protocol 2: Isolation of biotinylated plastids Basic Protocol 3: Quality control of isolated plastids.
质体存在于所有植物细胞类型中。然而，大多数用于研究这些细胞器的提取方法都是在器官水平上进行的（例如，叶、根、果实），无法实现组织特异性分辨率，这阻碍了我们对其生理学的理解。因此，开发了 IPTACT（在特定细胞类型中标记质体的分离），以在拟南芥中以组织特异性的方式分离质体。使用单次转化系对质体进行生物素化，只要存在合适的启动子，就可以通过合适的启动子实现组织特异性。然后用 2.8-µm 链霉亲和素珠分离细胞特异性生物素化质体。通过 IPTACT 提取的质体适用于 RNA 或蛋白质分离以及随后的组织特异性 OMICs 分析。该方法为用户提供了一种强大的工具，可在细胞类型分辨率下研究质体功能。此外，它可以轻松与使用不同遗传背景和/或不同发育或胁迫条件的研究相结合。© 2022 作者。Wiley Periodicals LLC 出版的当前方案。基本方案 1：启动子克隆和植物选择基本方案 2：生物素化质体的分离基本方案 3：分离质体的质量控制。
