Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay

Abstract

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

High-efficiency nuclear transformation of the oleaginous marine Nannochloropsis species using PCR product

“…In plant cells, transformation using PCR fragments gave rise to 10% low efficiencies compared with those using plasmids. 34) However, our results showed that PCR fragment-based transformation improved the transformation efficiency in eukaryotic microalgae Nannochloropsis species, indicating that replacing linearized vectors with PCR fragments might give rise to a higher transformation efficiency in other microalgae.…”

“…Although Kilian et al 16) indicated that transformation with these two linear DNA fragments was efficient in N. oceanica W2J3B, the detailed data were not provided. In mammalian 32,33) and plant cells, 34) transformation with PCR fragments is as effective as the plasmid, but this transformation protocol was only used for the gene transient expression assay. In order to establish a time-saving and highly efficient transformation protocol, PCR fragments containing only a selection marker cassette were used to transform all the marine Nannochloropsis strains.…”

Properties and functions of calcium‐dependent protein kinases and their relatives inArabidopsis thaliana

“…AtCPK32 was shown to interact with and phosphorylate ABF4 in vitro on S110, which was important for the ABA‐induced transactivation activity of ABF4 (Choi et al ., ). Similarly, AtCPK4 phosphorylated ABF2 in vitro and stimulated its activity in a protoplast transient assay (Lu et al ., ). Interestingly, the ABA signaling pathway could be reconstituted by additionally coexpressing the ABA receptor and the protein phosphatase 2C (PP2C) to demonstrate that AtCPK4 activated ABF2 upon ABA perception.…”

A transient gene expression system in Populus euphratica Oliv. protoplasts prepared from suspension cultured cells

“…Additionally, transfected protoplasts with specific fluorescence could be obtained with high purity using fluorescence-activated cell sorting (Birnbaum et al 2005;Birnbaum 2009, 2010). Because PCR-amplified dsDNA fragments (Lu et al 2013), microRNAs (Bai et al 2014), and double-stranded RNAs (Cao et al 2014) can also be introduced into protoplasts directly by the PEGmediated method, this protoplast transient gene expression system in P. euphratica could be used not only to investigate protein localization, protein function, and signal transduction, but also to study hierarchical gene regulators such as miRNAs involved in the salinity resistance of P. euphratica ).…”