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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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