Plant Isoprenoids pp 245-255

Part of the Methods in Molecular Biology book series (MIMB, volume 1153) | Cite as

High-Throughput Testing of Terpenoid Biosynthesis Candidate Genes Using Transient Expression in Nicotiana benthamiana

  • Søren Spanner Bach
  • Jean-Étienne Bassard
  • Johan Andersen-Ranberg
  • Morten Emil Møldrup
  • Henrik Toft Simonsen
  • Björn Hamberger
Protocol

Abstract

To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps optimized for high throughput, this in planta expression platform is particularly suited for testing large panels of candidate genes in all possible permutations.

Key words

Nicotiana benthamiana In planta Transient expression Agrobacterium High throughput Enzyme characterization Terpenoid pathway discovery Metabolic engineering 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Søren Spanner Bach
    • 1
  • Jean-Étienne Bassard
    • 1
    • 2
  • Johan Andersen-Ranberg
    • 1
    • 2
  • Morten Emil Møldrup
    • 3
  • Henrik Toft Simonsen
    • 1
  • Björn Hamberger
    • 1
    • 2
  1. 1.Plant Biochemistry Laboratory, Department of Plant and Environmental SciencesCopenhagen Plant Science Centre, University of CopenhagenCopenhagenDenmark
  2. 2.Center for Synthetic BiologyUniversity of CopenhagenCopenhagenDenmark
  3. 3.Section for Molecular Plant BiologyDynaMo Center of Excellence, University of CopenhagenCopenhagenDenmark

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