Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

By R. Hellens, A. Allan, E. Friel, K. Bolitho, K. Grafton, M. Templeton, Sakuntala Karunairetnam, A. Gleave, W. Laing. Plant Methods, 2005

1,492 citations recorded by the source corpus.

BackgroundWe describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.ResultsWe give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs.ConclusionIn developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

Subfields: Biotechnology, Molecular Biology, Plant Science

View Paper: https://www.semanticscholar.org/paper/fdc823980ebcf3c4e001a1fe586a9b02045739a3

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Citations in the corpus

A Conserved Network of Transcriptional Activators and Repressors Regulates Anthocyanin Pigmentation in Eudicots[C][W][OPEN] (2014, methodology): “cloned into the XhoI-NcoI sites within the dual luciferase plasmid p0800LUC (Hellens et al., 2005).”
Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus (2011, neutral): “Full-length DkADH1 and DkPDC2 coding sequences were constructed into pGreenII 0029 62-SK (Hellens et al., 2005).”
An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae (2010, methodology): “The promoter of Arabidopsis DFR (TT3, AT5g42800) was isolated from genomic Arabidopsis DNA and cloned into pGreenII 0800-LUC vector [46].”
Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10 (2007, methodology): “…ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 414–427 Transient luminescent assays of apple transcription factor activity The dual luciferase system has been demonstrated to provide a rapid method of transient gene expression analysis (Hellens et al., 2005; Matsuo et al., 2001).”