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

Abstract

Background Genetically 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. Results Here 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. Conclusions The 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.

An Easy and Rapid Transformation Protocol for Transient Expression in Cotton Fiber

“…Having validated that proteins could be transiently expressed in cotton fibers, we further investigated whether the expressed proteins were correctly subcellular localized in fiber cells. A nuclear-localized protein, histone B2 (AtHTB2) ( Zhang et al, 2020 ), and a plasma membrane-localized aquaporin, AtPIP2A ( Qiu et al, 2020 ), were ligated with mCherry protein at the N-terminal, respectively, to form 35S:: AtHTB2 -mCherry and 35S:: AtPIP2A -mCherry constructs. Agrobacterium -mediated transient transformation with the 35S:: AtHTB2 -mCherry construct produced a strong fluorescence signal confined to nuclear ( Figure 6A ), while fiber cell expressing AtPIP2A-mCherry showed strong fluorescence at the plasma membrane ( Figure 6B ).…”

“…To test the application of subcellular localization in cotton fiber cells via the developed Agrobacterium -mediated transient transformation, a nuclear-localized protein histone B2, AtHTB2 ( Zhang et al, 2020 ), and a plasma membrane-localized aquaporin, AtPIP2A ( Qiu et al, 2020 ), were selected. Open reading frames of these two proteins without stop codon were fused with mCherry to generate 35S:: AtH2B -mCherry and 35S:: AtPIP2A -mCherry constructs, respectively.…”