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Plant Molecular Biology

, Volume 57, Issue 3, pp 411–423 | Cite as

Gene regulation in planta by plant-derived engineered zinc finger protein transcription factors

  • Rachel Holmes-Davis
  • Guofu Li
  • Andrew C. Jamieson
  • Edward J. Rebar
  • Qiang Liu
  • Yanhong Kong
  • Casey C. Case
  • Philip D. GregoryEmail author
Article

Abstract

The ability to modify plant traits is of great commercial potential in agricultural biotechnology. To this end we have engineered plant-based zinc finger protein transcription factors (ZFP TFs) that minimize the use of non-plant DNA sequences. This novel architecture supports the use of tandem arrays of zinc-finger DNA recognition domains such that the ZFP TF binds a contiguous DNA target site – thus emulating the design of ZFP TFs described previously for mammalian gene regulation. We show that this plant-based ZFP TF architecture supports high affinity DNA binding while allowing the specificity of the DNA-protein interaction to be determined by the amino acid sequences of the recognition helices. This plant-based backbone thus supports the use of previously characterized DNA recognition helices originally identified in a mammalian ZFP context without using mammalian DNA sequences. Moreover, we show that plant-based ZFP TFs employing this new architecture can up-regulate endogenous ADH activity by > 20-fold in transgenic Arabidopsis. Thus plant-based ZFP TFs are shown to be potent regulators of gene expression in vivo.

Keywords

alcohol dehydrogenase Arabidopsis gene regulation protein engineering transcriptional regulation transgenic plants zinc finger proteins zinc motifs 

Abbreviations

ACT2

Actin 2

ADH

Alcohol dehydrogenase

bp

base pair

BSA

bovine serum albumin

C2H2

Cys2-His2

CaMV35S

cauliflower mosaic virus 35S promoter

DTT

dithiothreitol

EMSA

electrophoretic mobility shift assay

GMT

γ-tocopherol methyltransferase

Kd

dissociation constant

Kdns

nonspecific dissociation constant

NOS

nopaline synthase

PAGE

polyacrylamide gel electrophoresis

T0

original plant used in flora-dip transformation

T1

original hemizygous transformant obtained from selection

T3

third generation homozygous transformant

ZFP

zinc finger protein

ZFP TF

zinc finger protein transcription factor

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Copyright information

© Springer 2005

Authors and Affiliations

  • Rachel Holmes-Davis
    • 1
  • Guofu Li
    • 2
  • Andrew C. Jamieson
    • 1
  • Edward J. Rebar
    • 1
  • Qiang Liu
    • 3
  • Yanhong Kong
    • 1
  • Casey C. Case
    • 1
  • Philip D. Gregory
    • 4
    Email author
  1. 1.Plant Gene Expression CenterUSDAAlbanyUSA
  2. 2.Agronomic Traits, Trait and Technology DevelopmentPioneer Hi-Bed InternationalJohnstonUSA
  3. 3.Avidia Research InstituteMountain ViewUSA
  4. 4.Point Richmond Tech CenterSangamo BioSciences Inc.,RichmondUSA

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