THE GOAL OF THIS PROJECT IS TO DEVELOP A GENERAL STRATEGY FOR THE CLONING OF TRANSPOSABLE ELEMENTS FROM DICOTYLEDONOUS PLANTS. ISOLATED TRANSPOSABLE ELEMENTS WILL BE VERY POWERFUL TOOLS FOR THE ISOLATION OF PLANT GENES, PARTICULARLY THOSE WHERE THE CLONING VIA MRNA CANNOT BE APPLIED, INCLUDING MOST GENES CONTROLLING METABOLISM GROWTH, DEVELOPMENT AND OTHER PROPERTIES IMPORTANT FOR AGRICULTURE. IF ACTIVE IN OTHER PLANT SPECIES, TRANSPOSABLE ELEMENTS MIGHT ENABLE THE TAGGING AND SUBSEQUENT ISOLATION OF AGRONOMICALLY IMPORTANT GENES IN CROP SPECIES FROM WHICH NO TRANSPOSONS HAVE BEEN ISOLATED.
Research was carried out involving the isolation and characterization of transposable elements from Petunia hybrida. An attempt was made to select for an insertion event into a gene that has already been isolated and characertized such as alcohol dehydrogenase and a colour gene, a gene transformed in specific Petunia lines, T-DNA gene 2, beta-glucuronidase, nitrate reductase, regulatory genes. An attempt was also made to try to isolate a gene, which already harbours a transposable element. To this end, a Petunia flower colour gene that encodes the enzyme dihydroflavanol-4-reductase was isolated and characterized.
To accelerate the selection of transposon induced mutations, haploid transposon bearing lines were developed; every insertion event can be recognized directly in such a haploid line.
Many interesting genes were isolated from Petunia; alcohol dehydrogenase and dihydroflavonol-4-reductase genes and regulatory genes that are being characterized at a functional level. The dihydroflavonol-4-reductase gene harboured a transposable element (dTphl) which has been isolated and characterized to some extent. The experiment to trap transposable elements in the introduced T-deoxyribonucleic acid (T-DNA) gene 2 by selecting for inactivation of gene 2 were isolated. In 27 of these, no alteration in gene 2 structure could be detected; inactivation appeared to be due to methylation of the insert DNA.
The isolation of haploid petunia lines that bear an active transposable element will be very useful in the direct selection of inactivation events in, for example, alcohol dehydrogenase and nitrate reductase genes.
THE PROJECT WILL DEVELOP TWO STRATEGIES THAT BOTH INVOLVE THE SELECTION OF TRANSPOSON INSERTIONS INTO GENES FOR WHICH PROBES ARE AVAILABLE :
- IN AMSTERDAM, IT WILL BE INVESTIGATED WHETHER INSERTIONS OF TRANSPOSABLE ELEMENTS CAN BE OBTAINED IN THE PETUNIA POLLEN SPECIFIC ADH GENE;
- IN BRUSSELS, INSERTIONS INACTIVATING GENE 2 OF THE T-DNA OF AGROBACTERIUM TUMEFACIENS WILL BE LOOKED FOR, BY SCREENING FOR A RECOVERED RESISTANCE TO HIGH CONCENTRATIONS OF AUXIN.
Funding SchemeCSC - Cost-sharing contracts