Abstract
Nuclear proteins are involved in many critical biological processes within plant cells and, therefore, are in the focus of studies that usually begin with demonstrating that the protein of interest indeed exhibits nuclear localization. Thus, studies of plant nuclear proteins would be facilitated by a convenient experimental system for identification of proteins that are actively imported into the cell nucleus and visualization of their nuclear accumulation in vivo. To this end, we developed a system of vectors that allows screening for cDNAs coding for nuclear proteins in a simple genetic assay in yeast cells, and verification of nuclear accumulation in planta following one-step transfer and autofluorescent tagging of the identified clones into a multiple cloning site-compatible and reading frame-compatible plant expression vector. In a recommended third experimental step, the plant expression cassette containing the identified clone can be transferred, also by a one-step cloning, into a binary multigene expression vector for transient or stable coexpression with any other proteins.
Highlights
Nuclear proteins are involved in many critical biological processes within plant cells and, are in the focus of studies that usually begin with demonstrating that the protein of interest exhibits nuclear localization
We describe a system of vectors that allows (1) identification of nuclear proteins from products encoded by large numbers of cDNA clones, or even entire libraries, via a simple genetic assay in yeast cells, and (2) verification of nuclear accumulation in planta following one-step transfer and autofluorescent tagging of the identified clones into a multiple cloning site (MCS)-compatible and open reading frame (ORF)compatible plant expression vector
The cDNA can be either inserted into one of the pNIA-C vectors, or it can be cloned into the mixture of all three vectors, i.e. pNIA-Ca, pNIA-Cb, and pNIA-Cc (Fig. 2), which allows better representation of all three reading frames in the library
Summary
Nuclear proteins are involved in many critical biological processes within plant cells and, are in the focus of studies that usually begin with demonstrating that the protein of interest exhibits nuclear localization. Studies of plant nuclear proteins would be facilitated by a convenient experimental system for identification of proteins that are actively imported into the cell nucleus and visualization of their nuclear accumulation in vivo To this end, we developed a system of vectors that allows screening for cDNAs coding for nuclear proteins in a simple genetic assay in yeast cells, and verification of nuclear accumulation in planta following one-step transfer and autofluorescent tagging of the identified clones into a multiple cloning site-compatible and reading frame-compatible plant expression vector. In a recommended third experimental step, the plant expression cassette containing the identified clone can be transferred, by a one-step cloning, into a binary multigene expression vector for transient or stable coexpression— following biolistic delivery or Agrobacterium-mediated genetic transformation, respectively—with any other
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