Elevation of seed alpha-tocopherol levels using plant-based transcription factors targeted to an endogenous locus.
Synthetic zinc finger transcription factors (ZFP-TFs) were designed to upregulate the expression of the endogenous Arabidopsis gamma-tocopherol methyltransferase (GMT) gene. This gene encodes the enzyme responsible for the conversion of gamma-tocopherol to alpha-tocopherol, the tocopherol species with the highest vitamin E activity. Five three-finger zinc finger protein (ZFP) DNA binding domains were constructed and proven to bind tightly to 9 bp DNA sequences located in either the promoter or coding region of the GMT gene. When these ZFPs were fused to a nuclear localization signal and the maize C1 activation domain, four of the five resulting ZFP-TFs were able to upregulate the expression of the GMT gene in leaf protoplast transient assays. Seed-specific expression of these ZFP-TFs in transgenic Arabidopsis produced several lines with a heritable elevation in seed alpha-tocopherol. These results demonstrate that engineered ZFP-TFs comprised of plant-derived elements are capable of modulating the expression of endogenous genes in plants.
Van Eenennaam
Alison L
AL
Monsanto Company, 1920 5th Street, Davis, CA 95616, USA.
Li
Guofu
G
Venkatramesh
Mylavarapu
M
Levering
Charlene
C
Gong
Xiaosong
X
Jamieson
Andrew C
AC
Rebar
Edward J
EJ
Shewmaker
Christine K
CK
Case
Casey C
CC
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D018390:Gene Targeting / Q000379:methods; D024861:Genetic Enhancement / Q000379:methods; D005819:Genetic Markers; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D012639:Seeds / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology; D016335:Zinc Fingers / Q000235:genetics; D024502:alpha-Tocopherol / Q000378:metabolism*
D005819:Genetic Markers; D011994:Recombinant Proteins; D014157:Transcription Factors; D008780:Methyltransferases; C047259:gamma-tocopherol methyltransferase; D024502:alpha-Tocopherol