拟南芥原生质体中荧光融合蛋白的瞬时表达。

Transient Expression of Fluorescent Fusion Proteins in Arabidopsis Protoplasts.

机构信息

Centre for Cell and Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong.

出版信息

Methods Mol Biol. 2021;2200:157-165. doi: 10.1007/978-1-0716-0880-7_7.

DOI:10.1007/978-1-0716-0880-7_7

PMID:33175377

Abstract

Transient expression using protoplasts isolated from Arabidopsis suspension culture cells is a fast and useful tool for analyzing protein subcellular localization and dynamics in plant cells. Recently, super-resolution imaging techniques such as N-SIM (Nikon, Structured Illumination Microscopy) are widely used in cell biology study, allowing cell biologists to obtain unattainable details and relationships of cell structures and functions by conventional confocal imaging. To facilitate the usage of protoplasts transient expression and super-resolution imaging for protein localization and dynamic analysis in plant cell biology research, here we describe updated protocols of protoplasts isolation from Arabidopsis suspension culture cells and transient expression assay for protein trafficking and localization study. Further, using GFP-tagged ERES (Endoplasmic Reticulum Exit Site) marker proteins and RFP-tagged Golgi marker as examples, we illustrate the major tools and methods for protein localization analysis using super-resolution imaging.

摘要

使用从拟南芥悬浮培养细胞中分离的原生质体进行瞬时表达是分析植物细胞中蛋白质亚细胞定位和动态的快速而有用的工具。最近，超分辨率成像技术，如 N-SIM（尼康，结构照明显微镜），在细胞生物学研究中得到了广泛应用，使细胞生物学家能够通过传统的共聚焦成像获得无法获得的细胞结构和功能的细节和关系。为了促进原生质体瞬时表达和超分辨率成像在植物细胞生物学研究中用于蛋白质定位和动态分析的应用，我们在这里描述了从拟南芥悬浮培养细胞中分离原生质体和进行瞬时表达测定以研究蛋白质运输和定位的更新方案。此外，我们还使用 GFP 标记的 ERES（内质网出口位点）标记蛋白和 RFP 标记的高尔基体标记蛋白作为示例，说明了使用超分辨率成像进行蛋白质定位分析的主要工具和方法。