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

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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.

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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

K d :

dissociation constant

K ns d :

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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Correspondence to Philip D. Gregory.

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Holmes-Davis, R., Li, G., Jamieson, A.C. et al. Gene regulation in planta by plant-derived engineered zinc finger protein transcription factors. Plant Mol Biol 57, 411–423 (2005). https://doi.org/10.1007/s11103-004-7820-x

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  • DOI: https://doi.org/10.1007/s11103-004-7820-x

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