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DEVELOPMENT OF INDUCIBLE GENE EXPRESSION SYSTEMS FOR HIGHER PLANTS AND PLANT CELL CULTURES; 2. ISOLATION OF TRANSPOSABLE ELEMENTS FROM PETUNIA HYBRIDA

Objective

THE GOAL OF PROJECT 1 IS TO USE INDUCIBLE PLANT REGULATORY SEQUENCES FOR THE CONTROLLED EXPRESSION OF 2 USEFUL GENETIC PROPERTIES :

- THE CONSTRUCTION OF RESISTANCE GENES WHICH COULD BE SWITCHED ON AFTER FUNGAL INFECTIONS, THEREFORE REDUCING THE DEPENDENCE ON CHEMICAL FUNGICIDES.
- THE USE OF "STRESS BOX" TO SWITCH ON THE PRODUCTION PHASE OF CELL CULTURES ENGINEERED FOR THE SYNTHESIS OF A GERE PRODUCT IN MASSIVE AMOUNTS.

THE GOAL OF PROJECT 2 IS TO DEVELOP A GENERAL STRATEGY FOR THE CLONING OF TRANSPOSABLE ELEMENTS FROM DICOTYLETONOUS 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.

Research was carried out in order to develop a gene expression system based on the flavonoid specific chalcone synthase (chs) genes from petunia and soybean chs is involved in flower pigmentation (in petunia) and phytoalexin synthesis (in soybean). Primary approaches include: isolation of chs genes, analysis of chs genes, analysis of tissue specific expression, construction of chimaeric genes (gus, CAT, NPTII), promoter deletion analysis in transient expression systems and stable transformants. These findings will extend understanding of the factors that control gene expression in higher plants.

10 chs genes were cloned from petunia and sequenced. chsA and J contribute 90 and +/- 10% to the chs messenger ribonucleic acid (mRNA) level, respectively. Both are flower specific, gibberellic acid (GA) sensitive and in young seedlings and tissue cultures inducible by ultraviolet (UV) light. Unexpectedly, expression of chsA and J was also found in reproductive tissues and seeds. In chsJ-gus functions, the chs promoter remained GA-inducible in flowers and UV-inducible in plantlets. By deletion analysis it was shown that a promoter as short as 100 nucleotides was still UV-inductible and flower specific. 3 soybean chs genes were characterized. Only chs1 was found to be UV-inducible and elicitor inducible in soybean seedlings, whereas in cell culture all chs genes were elicitor inducible. Promoter deletion analysis of ch1-gus fusions in transient expression systems of soybean and parsley protoplasts showed that sequences up to -178 were needed to confer elicitor inducibility in both systems and UV inducibility in parsley.
THE AIM OF PROJECT 1 IS TO DEVELOP INDUCIBLE GENE EXPRESSION SYSTEMS WITH A WIDE HOST RANGE FOR PLANTS AND PLANT CELL CULTURES. THIS SYSTEM WILL BE DIRECTLY TESTED BY GENERATING PATHOGEN-RESISTANT SOYBEAN LINES (CONTRIBUTION KOLN) AND BY IMPROVING PRODUCTIVITY IN CELL SUSPENSION CULTURES (CONTRIBUTION AMSTERDAM). THE QUESTIONS TO ANSWER WILL BE : WHAT IS THE MOLECULAR MECHANISM OF THE INDUCTION OF GENE EXPRESSION BY STRESS (TEMPERATURE SHOCK, FUNGAL ELICITOR) IS IT POSSIBLE TO CONSTRUCT CHIMERIC GENES, THE EXPRESSION OF WHICH WILL BE STIMULATED BY STRESS WILL STRESS INDUCTION WORK IN A BROAD RANGE OF TRANSFORMED SPECIES

PROJECT 2 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 INACTIVITING 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 Scheme

CSC - Cost-sharing contracts

Coordinator

Vrije Universiteit
Address
De Boelelaan 1087
1081 HV Amsterdam
Netherlands

Participants (1)

MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V.
Germany
Address
Carl-von-linné-weg 10
50829 Koeln