Characterisation of novel target promoters for the dexamethasone-inducible/tetracycline-repressible regulator TGV using
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O R I GI N A L P A P E R
S. BoÈhner á C. Gatz
Characterisation of novel target promoters for the dexamethasoneinducible/tetracycline-repressible regulator TGV using luciferase and isopentenyl transferase as sensitive reporter genes Received: 4 July 2000 / Accepted: 25 August 2000 / Published online: 29 November 2000 Ó Springer-Verlag 2000
Abstract The chimeric transcriptional activator TGV mediates dexamethasone (dx)-inducible and tetracycline (tc)-repressible transgene expression in tobacco (dx-on/ tc-o system). The expression pro®les of four dierent synthetic target promoters, comprising multiple TGV binding sites upstream of a core promoter, were characterised using the sensitive luciferase assay. Induction factors of over 1000 were measured in roots and leaves of over 30% of the transgenic plants, irrespective of the promoter used. Promoters PTF and PTax, which carry the )48 to +1 region of the Cauli¯ower Mosaic Virus 35S promoter, showed higher expression levels in both the uninduced and induced states than PTop10 and PTFM, which harbour several point mutations in this region. Moreover, PTax expressed higher background activities than PTF, indicating that the sequence of the synthetic regulatory region can in¯uence background levels. The usefulness of the dx-on/tc-o system for experiments addressing gene function was demonstrated by using it to control the expression of isopentenyl transferase. This enzyme catalyses the rate-limiting step in cytokinin biosynthesis and causes phenotypic eects even at low expression levels. Only dx-induced transgenic plants displayed phenotypic alterations indicative for increased cytokinin synthesis (e.g. outgrowth of lateral buds). Simultaneous treatment of selected buds with the antiinducer tc suppressed bud growth. This result suggests that cytokinins cannot serve as mobile signals to elicit the release of apical dominance in tissues compromised for enhanced cytokinin synthesis.
Communicated by G. JuÈrgens S. BoÈhner á C. Gatz (&) Albrecht-von-Haller-Institut fuÈr P¯anzenwissenschaften, Georg-August-UniversitaÈt GoÈttingen, Untere KarspuÈle 2, 37073 GoÈttingen, Germany E-mail: [email protected] Tel.: +49-551-397843 Fax: +49-551-397820
Key words Dexamethasone á Tetracycline á Cytokinin á Synthetic promoters á Chemically inducible gene expression
Introduction The transfer of foreign genes into the genomes of higher plants has become a powerful tool for addressing important biological problems. Most transgenes are transcribed constitutively from a strong promoter, such as the 35S promoter of the cauli¯ower mosaic virus (PCaMV 35S). For some purposes, non-constitutive expression of the transgene is desired to generate inducible phenotypes (Gatz and Lenk 1998). The ability to control the expression of a gene via a highly speci®c mechanism is particularly required if the gene product of interest interferes with regeneration, growth and/or reproduction. A number of chemically inducible expression systems are available, namely tetracycline (tc)-regulated systems (Gatz et al. 19
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