真空渗透转化拟南芥幼苗的瞬时转化。

Transient Transformation of A. thaliana Seedlings by Vacuum Infiltration.

机构信息

Departamento de Bioquímica y Biología Molecular, Estructura de Recerca Interdisciplinar en Biotecnología i Biomedicina (ERI BIOTECMED), Universitat de València, Burjassot (Valencia), Spain.

Faculty of Health and Life Sciences, Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK.

出版信息

Methods Mol Biol. 2021;2200:147-155. doi: 10.1007/978-1-0716-0880-7_6.

DOI:10.1007/978-1-0716-0880-7_6

PMID:33175376

Abstract

Transient expression in Arabidopsis thaliana seedlings allows fast expression of fluorescent markers for different subcellular compartments. This protocol describes a transient transformation assay with five-day-old seedlings using Agrobacterium tumefaciens-mediated vacuum infiltration. Three days after infiltration of the Agrobacterium containing an expression vector for a fluorescent marker of interest, cotyledon cells expressing the fluorescent protein can be imaged in a confocal microscope. This assay allows high-throughput screening of new constructs and the study of the localization of a large number of subcellular markers in Arabidopsis seedlings including wild-type, stable over-expressing and mutant lines.

摘要

拟南芥幼苗的瞬时表达可快速表达用于不同亚细胞区室的荧光标记物。本方案描述了一种使用农杆菌介导的真空渗透法对 5 天大的幼苗进行瞬时转化测定。在渗透含有感兴趣的荧光标记物表达载体的农杆菌 3 天后，可以在共聚焦显微镜下观察到表达荧光蛋白的子叶细胞。该测定法可用于新构建体的高通量筛选以及研究拟南芥幼苗中大量亚细胞标记物的定位，包括野生型、稳定过表达和突变系。