T-DNA tagging in Brassica napus as an efficient tool for the isolation of new promoters for selectable marker genes.
A simple strategy to identify and isolate new promoters suitable for driving the expression of selectable marker genes is described. By employing a Brassica napus hypocotyl transformation protocol and a promoterless gus::nptII tagging construct, a series of 20 kanamycin-resistant tagged lines was produced. Most of the regenerated plants showed hardly any GUS activity in leaf, stem and root tissues. However, expression was readily restored in callus tissue induced on in vitro leaf segments. Genomic sequences upstream of the gus::nptII insertions were isolated via plasmid rescue. Three clones originating from single copy T-DNA lines were selected for further evaluation. The rescued plasmids were cloned as linear fragments in binary vectors and re-transformed to Brassica napus hypocotyl and Solanum tuberosum stem segments. The new sequences maintained their promoter activity, demonstrated by transient and stable GUS activity after transformation. Furthermore, the promoters provided sufficient expression of the nptII gene to yield transgenic plants when using kanamycin as selective agent. Database searching (BLASTN) revealed that the promoters have significant homology with three Arabidopsis BAC clones, one Arabidopsis cDNA and one Brassica napus cDNA. The results presented in this paper illustrate the strength of combined methods for identification, isolation and testing of new plant promoters.
Bade
Jacob
J
Syngenta Mogen B.V., Leiden, The Netherlands. jacob.bade@bfactory.nl
van Grinsven
Emiel
E
Custers
Jerome
J
Hoekstra
Sietske
S
Ponstein
Anne
A
D001483:Base Sequence; D029688:Brassica napus / Q000187:drug effects / Q000235:genetics*; D046508:Culture Techniques; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017343:Genes, Plant / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D007210:Indoleacetic Acids / Q000494:pharmacology; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D018744:DNA, Plant; D007210:Indoleacetic Acids; D011993:Recombinant Fusion Proteins; C036483:T-DNA; D005966:Glucuronidase