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.
Highlights
Transient expression system is an important research approach for conducting cell-based assays in plant and animal cells
Our results demonstrated that PCRfragment based transient expression system (PCR-TES) is as vital as the plasmid transformation system, which was tested in polyethylene glycol (PEG)-mediated protoplasts transformation as well as in biolistic bombardment transformation with leave tissues
Results showed that GFP fluorescent signal was detected in the transformation with 35SGFP-NOS or p35S-GFP, no GFP signal was detectable in the negative control (Figure S1). These results indicated that PCRfragment based transient expression system (PCR-TES) could be used as an alternative way to assess gene expression in plant cells
Summary
Transient expression system is an important research approach for conducting cell-based assays in plant and animal cells. In comparison with stable transformation system a transient expression assay is of quick analyzing advantage, which may not interfere with the stability of host genome [1,2]; it is widely used for studying transient activities of genes in cells [3,4,5,6]. The polyethylene glycol (PEG) mediated transformation serves as an efficient system for analyzing gene regulation at the single cell level [6,11,12,13]. Using the mesophyll protoplasts transfection approach, the reconstitution of ABA receptor PYR/RCAR dependent signaling pathway is determined [11]. The importance of CDPKs’ activities in the innate immune signaling pathways has been concluded with transient expression assay in protoplasts of Arabidopsis [12]
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