Gateway-compatible vectors for functional analysis of proteins in cell type specific manner

Liu Zhang,Shuang Wu,Yang Zhao,Xugang Li,Haiyan Liang,Kimberly L Gallagher

doi:10.1186/s13007-020-00635-z

Abstract

BackgroundGenetically encoded fluorescent proteins are often used to label proteins and study protein function and localization in vivo. Traditional cloning methods mediated by restriction digestion and ligation are time-consuming and sometimes difficult due to the lack of suitable restriction sites. Invitrogen developed the Gateway cloning system based on the site-specific DNA recombination, which allows for digestion-free cloning. Most gateway destination vectors available for use in plants employ either the 35S or ubiquitin promoters, which confer high-level, ubiquitous expression. There are far fewer options for moderate, cell-type specific expression.ResultsHere we report on the construction of a Gateway-compatible cloning system (SWU vectors) to rapidly tag various proteins and express them in a cell-type specific manner in plants. We tested the SWU vectors using the HISTONE (H2B) coding sequence in stable transgenic plants.ConclusionsThe SWU vectors are a valuable tool for low cost, high efficiency functional analysis of proteins of interest in specific cell types in the Arabidopsis root.

Highlights

Encoded fluorescent proteins are often used to label proteins and study protein function and localization in vivo
We have developed a flexible series of Gateway enabled plant expression vectors for cell specific gene transcription in the root of Arabidopsis thaliana
Here we reported the construction of Gateway (Invitrogen) compatible cloning system for cell-specific expression in roots of Arabidopsis thaliana

Summary

Introduction

Encoded fluorescent proteins are often used to label proteins and study protein function and localization in vivo. Some techniques such as BIFC, which are performed in heterologous plant systems are benefitted by the broad host range that the 35S or ubiquitin promoters provide [6,7,8,9]. Despite their wide usage, constitutive promoters have their drawbacks. For the 35S and the UBQ10 promoters, we have found this to be true in the root meristem For these reasons, we often choose to use promoters that show cell or tissue specific patterns of gene whenever possible. We have developed a flexible series of Gateway enabled plant expression vectors for cell specific gene transcription in the root of Arabidopsis thaliana

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Conclusion