| Autor: |
Bade, Jacob, van Grinsven, Emiel, Custers, Jerome, Hoekstra, Sietske, Ponstein, Anne |
| Zdroj: |
Plant Molecular Biology; May 2003, Vol. 52 Issue: 1 p53-68, 16p |
| Abstrakt: |
A simple strategy to identify and isolate new promoters suitable for driving the expression of selectable marker genes is described. By employing a Brassica napushypocotyl 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 vitroleaf 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 napushypocotyl and Solanum tuberosumstem 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 ArabidopsisBAC clones, one ArabidopsiscDNA and one Brassica napuscDNA. The results presented in this paper illustrate the strength of combined methods for identification, isolation and testing of new plant promoters. |
| Databáze: |
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