Improved T-DNA vector for tagging plant promoters via high-throughput luciferase screening.
Transferred DNA (T-DNA) tagging is a powerful tool for tagging and in planta characterization of plant genes on a genome-wide scale. An improved promoter tagging vector is described here, which contains the codon-optimized luciferase (luc+) reporter gene 31 bp from the right border of the T-DNA. Compared to the wild-type luciferase gene, this construct provides significantly increased reporter gene expression and a 40 times higher tagging frequency. The utility of the construct is demonstrated in banana, a tropical monocot species, by screening embryogenic cell colonies and regenerated plants with an ultrasensitive charged-coupled device (CCD) camera. The improved vector resulted in a luciferase activation frequency of 2.5% in 19,000 cell colonies screened. Detailed molecular analysis of flanking DNA sequences in a tagged line revealed insertion of the luciferase tag in a novel gene with near-constitutive expression.
Remy
Serge
S
Laboratory of Tropical Crop Improvement, Katholieke Universiteit Leuven, Leuven, Belgium. serge.remy@biw.kuleuven.be
Thiry
Els
E
Coemans
Bert
B
Windelinckx
Saskia
S
Swennen
Rony
R
Sági
László
L
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049409:Luciferases, Firefly / Q000235:genetics* / Q000378:metabolism; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D028521:Musa / Q000201:enzymology / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016324:Sequence Tagged Sites
D004269:DNA, Bacterial; D011994:Recombinant Proteins; C036483:T-DNA; D049409:Luciferases, Firefly