title: 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.
authors.affiliation: State Key Laboratory of Hybrid Rice, Department of Cell and Developmental Biology, College of Life Sciences, Wuhan University, Wuhan, PR China.
authors.forename: Yuming
authors.initials: Y
authors.lastname: Lu
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Wu
authors.forename: Yanping
authors.initials: Y
authors.lastname: Wang
authors.forename: Yuqing
authors.initials: Y
authors.lastname: He
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
mesh_terms: D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D018274:Electroporation; D015870:Gene Expression; D059828:Plant Cells / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D004247:DNA; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases