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

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.

• Publication year

  2005

• Venue

  Plant Methods

• Publication date

  2005-12-18

• Fields of study

  Biology, Medicine, Environmental Science

• Identifiers
  DOI 10.1186/1746-4811-1-13PMID 16359558PMCID 1334188
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• The Arabidopsis Transcription Factor MYB12 Is a Flavonol-Specific Regulator of Phenylpropanoid Biosynthesis1

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• Agrobacterium T-DNA integration: molecules and models.

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• Kiwifruit L-galactose dehydrogenase: molecular, biochemical and physiological aspects of the enzyme.

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• A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis.

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• Extraction of Proteins from Plant Tissues

  2004cited by this paper
• Exploring plant genomes by RNA-induced gene silencing

  2003cited by this paper
• An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.

  2003cited by this paper
• Functional genomics of P450s.

  2003cited by this paper
• Engineering of a Water-Soluble Plant Cytochrome P450, CYP73A1, and NMR-Based Orientation of Natural and Alternate Substrates in the Active Site1

  2003cited by this paper
• Requirements for intron-mediated enhancement of gene expression in Arabidopsis.

  2002cited by this paper
• Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium.

  2002cited by this paper
• Molecular Cloning: A Laboratory Manual

  2001cited by this paper
• The R2R3-MYB gene family in Arabidopsis thaliana.

  2001cited by this paper
• Construct design for efficient, effective and high-throughput gene silencing in plants.

  2001cited by this paper
• A genomic perspective on plant transcription factors.

  2000cited by this paper
• Phytochromes and light signal perception by plants—an emerging synthesis

  2000cited by this paper
• Technical Focus:a guide to Agrobacterium binary Ti vectors.

  2000cited by this paper
• Gene expression: Total silencing by intron-spliced hairpin RNAs

  2000influential reference
• pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation

  2000influential reference
• Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway.

  2000cited by this paper
• Activation Tagging Identifies a Conserved MYB Regulator of Phenylpropanoid Biosynthesis

  2000cited by this paper
• Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants.

  1999cited by this paper
• Overexpression of L-Phenylalanine Ammonia-Lyase in Transgenic Tobacco Plants Reveals Control Points for Flux into Phenylpropanoid Biosynthesis

  1996cited by this paper
• Cinnamate 4‐hydroxylase from Catharanthus roseus and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli

  1995cited by this paper
• Transfer of T-DNA from Agrobacterium to the Plant Cell

  1995cited by this paper
• Plant transformation and expression vectors

  1993cited by this paper
• A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome

  1992cited by this paper
• Basic local alignment search tool.

  1990cited by this paper
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