EP0910651A1 - Expression genique induite par l'ozone chez les vegetaux - Google Patents

Expression genique induite par l'ozone chez les vegetaux

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Publication number
EP0910651A1
EP0910651A1 EP97928238A EP97928238A EP0910651A1 EP 0910651 A1 EP0910651 A1 EP 0910651A1 EP 97928238 A EP97928238 A EP 97928238A EP 97928238 A EP97928238 A EP 97928238A EP 0910651 A1 EP0910651 A1 EP 0910651A1
Authority
EP
European Patent Office
Prior art keywords
ozone
plants
dna sequence
gene
genes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97928238A
Other languages
German (de)
English (en)
Inventor
Roland Schubert
Heinrich Sandermann
Dietrich Ernst
Rüdiger Hain
Regina Fischer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gsf-Forschungszentrum fur Umwelt und Gesundheit G
Bayer CropScience AG
Original Assignee
Bayer AG
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE19654574A external-priority patent/DE19654574A1/de
Application filed by Bayer AG, Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH filed Critical Bayer AG
Publication of EP0910651A1 publication Critical patent/EP0910651A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance

Definitions

  • the present invention relates to new DNA sequences, a method for producing new plants which contain a new DNA sequence, the coding sequence of which is expressed after ozone induction, these new plants and the use of the DNA sequences for generating ozone. responsive gene expression in plants and plant cells.
  • the present invention relates to a new promoter, the specificity of which is increased by eliminating its ozone responsiveness.
  • ozone Although ozone penetrates the plant cell by diffusion through open stomata, the ozone concentration in the intercellular spaces of the leaf is almost zero, regardless of the ambient ozone concentration (Laisk e_t al. (1989) Plant Physiol. JK>, 1163-1167). It is currently believed that ozone reacts rapidly with components of the cell walls and the plasma lemma and into reactive oxygen species such as superoxide anions, hydroxyl radicals and hydrogen peroxide, which are used in ozone-treated plant material using electron is magnetic resonance spectroscopy were detected, converted (Mehlhorn et AJL. (1990) Physiology Plantarum 79th, 377-383).
  • ozone causes an increased expression of various disease resistance genes, namely some PR (pathogenesis-related) proteins (Ernst et al. (1992) Plant Mol. Biol. 2JD, 673 -682; Ernst et al ⁇ (1996) J. Plant Physiol. 148, 215-221; Eckey-Kaltenbach et al ⁇ . (1994) Plant Physiol. IM * 67-74).
  • PR pathogenesis-related
  • Stilbene synthesis enzymes catalyze the synthesis of stilbenes such as resveratrol and pininovin from one molecule of p-coumaroyl-CoA or cinnamoyl-CoA and three units of malonyl-CoA. Both resveratrol and pininovin have phytoalexin properties and antifungal activity and, as phytoalexins, together with other stilbenes derived from phenylpropane metabolism, have an important function in the defense against pathogens (Hart (1981) Annu. Rev. Phytopathol. 19, 437-458).
  • STS genes occur in some unrelated plant species, such as Peanut (Schröder et al. (1988) Eur. J. Biochem. 112, 161-169), Weinrebe (Hain et ah (1993) Nature 361, 153-156) and Kiefer (Fliegmann et al ⁇ . (1992) Plant Mol Biol. 1J3, 489-503), and are organized in larger gene families consisting of six or more genes (Lanz et al. (1990) Planta 181, 169-175; Wiese et al _ (1994) Plant Mol. Biol. 2_6, 667-677).
  • DNA sequences encoding stilbene synthase are known, for example, from European patent EP 0 309 862, German patent application DE-A-41 07 396, European patent application 0 464 461 and US patent 5,500,367. These documents describe the isolation of stilbene synthase genes and their use in the generation of transgenic plants. The resulting transgenic plants have an increased resistance to various plant pests, such as fungi, bacteria, insects, viruses and nematodes. Plamides containing STS genes were deposited with the German Collection of Microorganisms (DSM), mecaniccheroder Weg 1B, D-38124 Braunschweig, e.g. also the V ⁇ tl gene from grapevine in the plasmid pVstl under the accession number DSM 6002 (DE-A-41 07 396, EP-A-0 464 461, US patent 5,500,367).
  • DSM 6002 German Collection of Microorganisms
  • the STS expression it is desirable for the STS expression to be planted in transgenic (culture) plants which, owing to the STS genes introduced, have an increased resistance to disease, solely (and only) through the infestation of pathogens and not additionally (or previously) can be activated and controlled by undesirable environmental stress factors, such as ozone.
  • An important aspect here is the switching off of undesired non-specific environmental stimuli, e.g. the induction of certain defense genes by ozone, UV light, heavy metals, extreme temperatures and other abiotic stimuli.
  • Another object of the invention is to provide options for eliminating ozone induction, ie for switching off desired stimulation of gene expression by ozone.
  • a further object of the invention is therefore to provide DNA sequences which can be used to generate promoters which can be induced in a targeted manner.
  • so-called reporter genes the expression of which can be detected by simple enzymatic tests and which are well known in biotechnological research, can be used as bio-monitors in a very simple and reliable manner.
  • a further object of the invention is to provide a system with the aid of which certain genes, the gene products of which are able to detoxify reactive oxygen species in cells, are “switched on” when necessary, that is to say when there is a high ozone load can.
  • the provision of DNA sequences which are responsible for ozone-responsive gene regulation is intended to enable ozone-inducible expression of such
  • Vstl promoter that only has the base pairs up to and including -280 (and which therefore lacks the upstream Vstl promoter sequences) is no longer ozone-inducible. As mentioned above, this truncated promoter is still able to mediate pathogen-induced gene expression of the coding sequence controlled by it (see Fischer (1994) supra).
  • the ozone-responsive DNA sequence region described here for the first time thus comprises the base pairs -270 to -430 of the Vstl promoter from grapevine.
  • the present invention thus relates to the DNA sequence defined in claim 1 (SEQ ID NO: 1):
  • the DNA sequence according to the invention is a DNA sequence that comes from grapevine, and particularly preferably from the stilbene synthase gene Vstl from grapevine (base pairs -270 to -430).
  • the invention further relates to a promoter region of the V ⁇ tl gene which lacks at least the DNA sequence which comprises the base pairs -270 to -430 of the Vstl gene.
  • it is a promoter region of the Vstl gene which only comprises the sequence region from the translation start to base pair -270 of the Vstl gene.
  • the promoter region, which lacks the sequence range from -270 to -430 of the Vstl gene, is particularly preferably able to mediate pathogen-inducible gene expression in plants.
  • the invention further relates to chimeric nucleic acid molecules into which the DNA sequence of the base pairs -270 to -430 of the Vstl gene or at least one fragment of this sequence region has been introduced.
  • the chimeric nucleic acid molecules of the invention particularly preferably enable the ozone-inducible expression of the coding regions contained in them in plants due to the presence of the DNA sequence of the base pairs -270 to -430 of the Vstl gene or at least a partial region thereof .
  • the nucleic acid molecules according to the invention can be any nucleic acid molecules, in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. They can be naturally occurring molecules or those produced by genetic engineering or chemical synthesis methods.
  • Ozone induction of naturally ozone-inducible genes in plants and plant cells by deletion of the DNA sequence according to claim 1 or at least one fragment thereof in the genes which naturally contain this DNA sequence or a derivative thereof or a DNA sequence homologous to it , can be switched off.
  • a further advantage of the invention consists in the fact that genes which are not or not substantially inducible by ozone can be expressed in plants and plant cells using the nucleic acid sequences according to the invention in an ozone-inducible manner.
  • the nucleic acid sequence which is responsible for the ozone-inducible expression, or at least a fragment thereof controls the expression of genes whose gene products are capable of reactive oxygen species which arise, inter alia, as a result of ozone in plant cells can detoxify.
  • this controls Nucleic acid sequence the expression of catalase and / or superoxide dismutase genes.
  • the DNA sequence responsible for the ozone-inducible gene expression controls the expression of reporter genes, which is measured for the quantitative and / or qualitative determination of ozone concentrations and for the evaluation of ozone effects.
  • reporter genes can e.g. the uidA gene from E ⁇ . coli which codes for the enzyme ⁇ -glucuronidase (GUS), luciferase genes or other genes customary in plant biotechnology. Suitable reporter genes are known to any person skilled in the field of biotechnology, biochemistry or molecular biology.
  • the present invention further relates to vectors which contain the abovementioned DNA sequences or promoter regions or parts thereof.
  • the present invention thus also relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors which are common in genetic engineering and which contain the nucleic acid molecules according to the invention described above and which can optionally be used for the transfer of the nucleic acid molecules according to the invention to plants or plant cells.
  • the invention also relates to transformed microorganisms, such as bacteria, viruses, felts, which contain the nucleic acid sequences according to the invention.
  • This task is accomplished by the provision of the DNA sequence responsible for ozone induction and the provision of promoters which lack this sequence and which for this reason are no longer capable of gene expression which can be induced by ozone through controlled genes in plants and plant cells.
  • these are transformed plants and plant cells in which genes are inducibly expressed, whose gene products are able to detoxify reactive oxygen species in plant cells. These are particularly preferably catalase and / or superoxide dimethyl genes.
  • plants and plant cells can be generated, which express so-called reporter genes after induction by ozone and which can optionally be used as bio-monitors.
  • the invention thus relates to transgenic plants which contain the recombinant nucleic acid molecules described above, integrated in the plant genome.
  • these plants can be any plant. It is preferably a monocot or dicot crop.
  • monocotyledonous plants are the plants belonging to the genera Avena (oat), Triticum (wheat), Seeale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, sorghum (millet), Zea ( Corn).
  • the dicotyledonous crops include cotton, legumes such as legumes and in particular alfalfa, soybean, rapeseed, tomato, sugar beet, potatoes, ornamental plants, trees.
  • Other useful plants can be fruit (in particular apples, pears, cherries, grapes, citrus, pineapple and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal and, for medicinal plants, rauwolfia and digitalis.
  • the invention also relates to propagation material from plants according to the invention, for example seeds, fruits, cuttings, tubers, rhizomes, etc., and parts of these plants, such as plant cells, protoplasts and calli.
  • the plant cells include differentiated and undifferentiated plant cells (including protoplasts), as well
  • Plant cells including protoplasts in which the nucleic acid molecules according to the invention are integrated into the plant genome or as autonomous molecules (including transient transformation).
  • the invention relates to host cells, in particular prokaryotic and eukaryotic cells, which have been transformed or infected with a recombinant nucleic acid molecule or a vector described above, and cells which are derived from such host cells and contain the described nucleic acid molecules or vectors .
  • the host cells can be bacteria or fungal cells as well as plant or animal cells.
  • the present invention is also based on the object
  • This object is achieved by methods with the aid of which it is possible to produce new plants and plant cells which do not have this naturally occurring ozone-induced gene expression.
  • the present invention is also based on the object of processes for the production of plants and plant cells which contain such genes and their expression naturally not or not significantly activated by ozone, after ozone stimulation, to provide.
  • This object is achieved by methods by means of which plants and plant cells can be produced which, after the introduction of the DNA sequences according to the invention or at least a partial region thereof into genes which are naturally not or only weakly ozone-inducible, express these genes after ozone stimulation.
  • plants or plant cells can be modified using conventional genetic engineering transformation methods in such a way that the new nucleic acid molecules are integrated into the plant genome, i.e. that stable transformants are generated.
  • plants or plant cells which, owing to the lack of the nucleic acid sequence according to the invention or at least one fragment thereof, no longer exhibit ozone induction of the gene (s) naturally containing this sequence, are produced by a process which comprises the following steps: a) deletion of the im Defined DNA sequence or a sequence which can be derived from this sequence or which is homologous to this sequence, or at least one fragment of this sequence from a gene which regulatory elements after the deletion of the DNA sequence according to the invention for the, if necessary regulated, transcription and translation in plant cells are indispensable, comprises at least one coding sequence, and optionally a termination signal for the termination of the transcription and the addition of a poly-A tail to the corresponding transcript; b) transformation of plant cells with the gene or nucleic acid molecule produced in step a), and c) optionally the regeneration of transgenic plants and, if appropriate, the multiplication of the plants.
  • step a) instead of deleting the sequence responsible for the ozone induction, this sequence or at least a part thereof, for example by mutagenesis, can be inactivated or blocked and remain in the gene in inactivated form.
  • all manipulation measures can be carried out with the aid of generally customary methods and aids of recombinant genetic engineering (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • the nucleic acid molecule which is transferred to plants or plant cells in step b) contains regulation elements which e.g. Enable pathogen-induced gene expression of the coding sequence.
  • plants or plant cells which, owing to the presence of the nucleic acid sequence according to the invention, which is essential or jointly responsible for ozone-induced expression of the genes containing them, or at least one fragment of this sequence, are produced by a process which comprises the following steps a) insertion of at least one DNA sequence according to the invention which can bring about an ozone-induced gene expression in plants, or a sequence which can be derived from this sequence or which is homologous to this sequence, or at least one fragment of this sequence in a gene , which is naturally not or not significantly expressed in an ozone-inducible manner b) transformation of plant cells with the gene or nucleic acid molecule produced in step a) which has all the elements which are naturally required for expression in plant cells, and c) if necessary the regeneration of transgenic cells Plants and gg f. the propagation of plants.
  • the gene is a catalase, superoxide dismutase or a common reporter gene.
  • Another object of the invention is to show advantageous uses of the nucleic acid sequences according to the invention.
  • the invention therefore encompasses uses of the new DNA molecules for the production of the plants and plant cells according to the invention described above, which are characterized either by the lack of an expression of a certain phenotypic characteristic which is normally influenced by ozone, or by the presence of the DNA sequence according to the invention Distinguish ozone-induced traits from non-transgenic plants and plant cells.
  • the invention encompasses the use of the nucleic acid molecules according to the invention for the production of plants which are characterized by an increased pathogen-induced but non-ozone-induced disease resistance.
  • the invention relates to the use of the nucleic acid sequences according to the invention or fragments thereof for the detection and identification of ozone-responsive nucleic acid elements.
  • ozone-responsive nucleic acid elements with the aid of common molecular biological methods, for example hybridization experiments or DNA-protein binding studies.
  • a first step for example, the poly (A) * RNA is isolated from a tissue that has been treated with ozone and a cDNA library is created.
  • a second step with the help of cDNA clones which are based on poly (A) * RNA molecules from an untreated tissue, those clones are identified from the acquired bank by means of hybridization whose corresponding poly (A) * RNA -Molecules are only induced during ozone treatment.
  • these identified cDNA ⁇ promoters are isolated, which via Ozone-reflective elements. Nucleic acid sequences and molecules can be useful tools in the study and characterization of these isolated promoters.
  • hybridization means hybridization under conventional hybridization conditions, preferably under stringent conditions, such as, for example, in Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • Nucleic acid molecules which hybridize with the molecules according to the invention can e.g. from genomic or from cDNA libraries.
  • nucleic acid molecules can be identified and isolated using the nucleic acid molecules according to the invention or parts of these molecules or the reverse complement of these molecules, e.g. by means of hybridization according to standard methods (see e.g. Sambrook et al, supra).
  • the invention thus also relates to the use of a DNA sequence according to the invention or of parts thereof for the identification and isolation of homologous sequences from plants or other organisms.
  • nucleic acid molecules which have the nucleotide sequences according to the invention or parts of these sequences exactly or essentially can be used as the hybridization probe.
  • the fragments used as the hybridization probe can also be synthetic fragments which were produced with the aid of the conventional synthetic techniques and whose sequence essentially corresponds to that of an inventive method Nucleic acid molecules. If genes which hybridize with the nucleic acid sequences according to the invention have been identified and isolated, a determination of the sequence and an analysis of the properties are necessary. For this purpose, a number of standard molecular, biochemical and biotechnological methods are available to the person skilled in the art.
  • the molecules hybridizing with the nucleic acid molecules according to the invention also include fragments, derivatives and allelic variants of the DNA molecules described above, which contain an ozone-responsive sequence, in active or inactivated form, or which are distinguished by the fact that they do not have this sequence have more.
  • derivative means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above at one or more positions and have a high degree of homology to these sequences.
  • Homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably over 80% and particularly preferably over 90%.
  • the deviations from the nucleic acid molecules described above can be caused by deletion, addition, substitution, insertion or recombination.
  • nucleic acid molecules which are homologous to the molecules described above and which are derivatives of these molecules are generally variations of these molecules which represent modifications which have the same biological function. These can be both naturally occurring variations, for example sequences from other organisms, or mutations, whereby these modifications can have occurred naturally or have been introduced by targeted mutagenesis. Furthermore, the variations can be synthetically produced sequences.
  • allelich variants can be both naturally occurring and synthetically produced variants or those produced by recombinant DNA techniques. To prepare the introduction of foreign genes into higher plants or their cells, a large number of cloning vectors are available which generate a replication signal for JL. coli and a marker gene for the selection of transformed Bakt ⁇ rien cells contain.
  • Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 and others.
  • the desired sequence can be inserted into the vector at a suitable restriction interface.
  • the plasmid obtained is used for the transformation of Ej_ coli. Cells. Transformed E_ j . coli cells are grown in a suitable medium and then harvested and lysed. The plasmid is recovered. Restriction analysis, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences. Each plasmid DNA sequence can be cloned into the same or different plasmids.
  • a large number of known techniques are available for introducing DNA into a plant host cell, the person skilled in the art being able to determine the appropriate method in each case without difficulty. These techniques include the transformation of plant cells with T-DNA using Aqrobacterium tumefaciens or A ⁇ robacterium rhizoqene ⁇ as a transformation agent, the fusion of protoplasts, the direct gene transfer of isolated DNA into protoplasts, the electroporation of DNA, the introduction of DNA using the biolistic method as well as other options. Both stable and transient transformants can be generated.
  • Customary selection markers are those which impart to the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonyl-urineoff, gentamycin or phosphinotricin, among others.
  • an antibiotic such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonyl-urineoff, gentamycin or phosphinotricin, among others.
  • the Ti or Ri pliffmid is used for the transplantation of the plant cell, at least the right boundary, but often the right and left boundary of the T-DNA contained in the Ti and Ri pliffmid, must be linked as a flank region with the genes to be introduced .
  • the DNA to be introduced must be cloned into special plasmids, either in an intermediate or in a binary one
  • the intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Aqrobacterium tumefacien ⁇ (conjugation). Binary vectors can replicate in E_ ⁇ coli as well as in Agrobacteria. They contain a selection marker gene and a linker or
  • Polylinkers which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al (1978) Molecular and General Genetics 163, 181-187).
  • the agrobacterium serving as the host cell is said to contain a plasmid which carries a vir reqion. The pre-region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present.
  • the Agrobacterium transformed in this way is used for the transformation of plant cells.
  • T-DNA for the transformation of plant cells has been intensively investigated and is sufficiently described in EP 120 515; Hoekema in: The Binary Plant Vector System, Offsetdrokkerij Kanter ⁇ BV, Alblwie ⁇ erdam (1985) Chapter V; Fraley et al (1993) Crit. Rev. Plant. Sci., 4, 1-46 and An et al (1985) EMBO J. 4, 277-287.
  • plant explants can expediently be cultivated with Aqrobacterium turnefacien ⁇ or Aqrobacterium rhizoqene ⁇ .
  • Whole plants can then be regenerated again from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or suspension cells cultivated plant cells) in a suitable medium which can contain antibiotics or biocides for the selection of transformed cells .
  • the plants are regenerated using conventional regeneration methods using known nutrient media.
  • the plants or plant cells obtained can then be examined for the presence of the introduced DNA.
  • Other ways of introducing foreign DNA using the biological one are possible.
  • the transformed cells grow within the plant in the usual way (see also McCormick et al (1986) Plant Cell Reports 5, 81-84).
  • the resulting plants can be grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup.
  • the resulting hybrid individuals have the corresponding phenotypic properties.
  • Two or more generations should be attracted to ensure that the phenotypic characteristic is stably retained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved.
  • transgenic lines can be determined using conventional methods, which are homozygous for the new nucleic acid molecules and which investigate their phenotypic behavior with regard to an existing or non-existing ozone-responsiveness and compare it with that of hemizygotic lines.
  • plant cells which contain the nucleic acid molecules according to the invention can also be further cultivated as plant cells (including protoplasts, calli, suspension cultures, etc.).
  • Another object of the present invention is the use of the nucleic acid molecules according to the invention or parts of these molecules or the reverse complements of these molecules for the identification and isolation of homologous molecules, the ozone-responsive elements comprise from plants or other organisms.
  • the ozone-responsive elements comprise from plants or other organisms.
  • the 5 'non-coding sequence region of the Vstl gene from grapevine referred to below as the promoter consists of 1570 base pairs.
  • This promoter whose sequence i.a. Known from German DE 41 07 396 and Fischer (1994) supra, using a conventional polymer chain reaction using the following oligonucleotides as primer and the plasmid pV ⁇ tl containing the complete Vstl gene (DE-A-41 07 396; Fischer (1994) supra) amplified as template:
  • the PCR reaction was performed according to the Perkin Elmer protocol (Norwalk, USA) using the Perkin Elmer native Taq DNA polymerase.
  • the DNA fragment amplified under customary PCR conditions was then subjected to the restriction enzymes HindII (the restriction portion is contained at the 5 'end of primer 1) and BamHI (the restriction site is contained at the 5' end of primer 2).
  • HindII the restriction portion is contained at the 5 'end of primer 1
  • BamHI the restriction site is contained at the 5' end of primer 2
  • resected and together with the BamHI / EcoRI fragment from the vector pBI101.2 (Jeffer ⁇ on (1987) Plant Mol. Biol. Reporter 5_, 387-405), which contains the / 8-glucuronidase reporter gene (GUS, uidA) from E_j. coli together with the termination signal de ⁇ nos gene from A.
  • turnefacJens contains, via which HindIII and EcoRI restriction portions of the pUCl ⁇ polylinker in the pUCl ⁇ plasmid (Yani ⁇ ch-Perron et aJU (1985) Gene 33., 103-119; available B. subcloned by Boehringer Mannheim). All cloning steps were carried out using common molecular biological techniques and aids (for example Sambrook et al. (1989) supra); Restriction enzymes and other enzymes used for the cloning were obtained from Boehringer Mannheim.
  • the resulting pUC clone thus contains a translation fusion of the ⁇ V ⁇ tl promoter ⁇ with the GUS gene, in which the first five STS codons in frame are linked to the GUS gene via a BamHI cut.
  • the sequence region of the fusion transition and the sequence of the V ⁇ tl promoter were determined using the enzymatic chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467) using the T7 sequencing -Kit from Pharmacia (Freiburg) checked for correctness.
  • the plasmid designations result from the approximate length of the respective promoter fragment, calculated from the transcription type, which is 73 base pairs away from the start codon (Hain et al. (1993) supra; Fischer (1994) supra).
  • the restriction interfaces used for the gradual shortening of the V ⁇ tl promoter are also shown schematically in FIG. 1.
  • pl500GUS The Hindlll / EcoRI fragment, which contains the complete STS promoter together with the GUS gene, was isolated from the plasmid pUC-Vstl / GUS described above and inserted between the restriction sites Hindlll and EcoRI in the polylinker of pPCV002.
  • - pl060GUS An 1130 bp promoter fragment was olated via the restriction sites BamHI and Sspl from pUC-V ⁇ tl / GUS i ⁇ and cloned in BamHI / HincII-linearized pUCl ⁇ (-> pUC1130).
  • the GUS gene was then isolated as BamHI / EcoRI fragment from pUC-V ⁇ tl / GUS and the BamHI and EcoRI cleavage site of pUC1130 was cloned in between. Finally, the fusion was isolated via a HindIII / EcoRI double digest and inserted into the polylinker of pPCV002 via the same interfaces. - p930GTJS: The 1.0 kb promoter fragment was over the
  • Restriction enzymes BamHI and HincII isolated from pUC-Vstl / GUS and inserted into the polylinker of pUCl ⁇ via the same restriction sites (-> pUClOOO). The cloning was then carried out analogously to pl060GUS.
  • p740GUS The 1.6 kb H ⁇ dlll / BamHI promoter fragment was isolated from the plasmid pUC-V ⁇ tl / GUS and digested with the restriction enzyme Dral. The resulting 810 bp. BamHI / Dral fragment was then cloned into pUCl ⁇ via the restriction sites BamHI and HincII (-> pUC810).
  • the GUS gene was then isolated as BamHI / EcoRI fragment from pUC-V ⁇ tl / GUS and the BamHI and EcoRI cleavage sites of pUC810 were cloned in between. Finally, the fusion was isolated via a HindIII / EcoRI double digest and inserted into the polylinker of pPCV002 via the same interfaces.
  • pUC-V ⁇ tl / GUS was digested with the restriction enzyme AfIII and the overhanging 5 'ends were filled with Klenow enzyme (from Boehringer Mannheim; dNTPs also from Boehringer Mannheim) according to the manufacturer's information. Subsequently, the restriction enzyme BamHI was cut and the resulting 620 bp promoter fragment was ligated in BamHI / HincII-linearized pUCl ⁇ (-> pUC620). After that, the cloning was carried out as continued for pl060GUS.
  • p500GUS A 570 bp. long ⁇ promoter fragment was isolated from pUC-V ⁇ tl / GUS via a BamHI / HaelII double digest and in
  • p280GTJS pUC-Vstl / GUS was digested with the restriction enzyme Banll, the overhanging ends were filled in with Klenow enzyme and the linearized vector was digested again with the restriction enzyme BamHI. After the isolation of the 350 bp long blunt end / BamHI promoter fragments, the cloning was continued in analogy to pl060GUS.
  • pI40GUS The pUC subclone pUC620 described above was digested with the restriction enzymes Neil and P ⁇ tl and the linearized vector was then purified and religated. This resulted in the deletion of the promoter sequences from -550 to -140 (-> pUC210).
  • the GUS gene was then isolated as a BamHI / EcoRI fragment from pUC-V ⁇ tl / GUS and the BamHI and EcoRI sections of pUC1130 were cloned in between. Finally, the foot was over a Hindlll / EeoRI
  • p40GUS The 110 bp promoter fragment was isolated together with the GUS gene by means of a Nhel / EcoRI double digest from pUC-Vstl / GUS and the fragment was then cloned into Xbal / EcoRI-linearized pPCV002.
  • the construct pl500GUS was digested with the restriction enzyme BamHI and the purified vector was then religated.
  • the BamHI cut parts which the vector pPCV002 naturally contains in its multiple cloning site next to the Hindlll cut parts (Koncz and Schell (1986) ⁇ upra), have thus eliminated the entire promoter fragment.
  • NicotJana tabacum cv. Petit Havana SRI (Maliga et al. (1973) Nature New Biol. 244, 29-30) was grown as a sterile shoot culture on hormone-free 1/2 LS medium (Lin ⁇ maier and Skoog (1965) Physiol.
  • the agrobacterial cultures used for the transformation of the tobacco leaf pieces were prepared as follows. First, the pPCVO02 derivatives described above were used in the E ⁇ . coli mobilization strain S17-1 (Simon et al. (1983) Bio / Technology 1, 784-790). The manufacturing took place
  • the transgenic tobacco shoots each containing one of the Vstl promoter / GUS fusions described above, were propagated ⁇ terilly via shoot culture, partially transferred to soil and in the greenhouse under normal conditions (22 ° C, 60% relative humidity, approx. 15,000 lux) cultivated until flowering.
  • the flowers were protected from cross-pollination by parchment bags and ripe seed capsules with seeds of the Fl generation were harvested after 4-6 weeks.
  • the Fl generation seed was designed on LS medium with 75 ⁇ g / ml kanamycin. This was done under sterile conditions directly from the capsule or after surface sterilization (1. brief washing with sterile water; 2. 2 min. Incubation in 70% ethanol; 3. 10 min.
  • the resistant Fl seedlings were transferred to earth, hardened and further cultivated under suitable conditions.
  • transgenic, canine-resistant tobacco seedlings were first transferred from agar plates into flower pots with a 2: 1 mixture of standard substrate (type T / ceremoniesstorfer / Lauterbach) with perlite and for 3 weeks in a climatic chamber with a 12-hour day / Night cycle, 15,000 lux, 25 ° C day temperature, 20 ° C night temperature, 65-70% humidity and filtered air incubated.
  • the fluorometric analysis of the GUS enzyme activity was carried out strictly according to Jefferson (1987) supra or Jefferson et al. (1987) EMBO J. 6, 3901-3907 using 4-methyl-umbelliferyl-glucuronide (MUG, Sigma, Kunststoff).
  • the concentration of the product 4-methylumbelliferone (MU) was measured with a fluorescence photometer (Perkin Elmer LS-2 B filter fluorimeter) in a quartz flow cell.
  • the GUS activity in plant extracts was calculated in pmol MU x mg " 1 x min" 1 .
  • the protein concentration in the tobacco leaf extracts was analyzed according to Bradford (1976). Biochem. 12_, 248-254.
  • Fl tobacco plants 11 weeks old from the stably transformed tobacco line, which contains the Vstl promoter / GUS fusion construct with the complete promoter region (plSOOGUS), were used in ozone gassing experiments.
  • GUS enzyme activities were measured fluorometrically in crude extracts from leaves, which were at different times during a 10-hour ozone exposure with different ozone concentrations (0.1 ⁇ l / 1 ozone, 0.2 ⁇ l / l ozone and 0.4 ⁇ l / 1 ozone) and one 14-hour post-incubation phase in pollutant-free air were determined.
  • transgenic tobacco lines which contain the 5 'deletions of the V ⁇ tl promoter described above in fusion with the bacterial GUS reporter gene were identified for the identification of ice-regulatory sequences which are responsible for the observed strong ozone induction of the STS promoter. analyzed as independent FO plants in ozone fumigation experiments. The primary rain rate was cultivated in sterile culture for several months and propagated via shoot culture. Fl tobacco plants (11 weeks old) of these stable transformants were also examined for ozone-induced GUS expression.
  • the transparent tobacco plants were treated with 0.1 ⁇ l / 1 ozone for 10 h and then incubated for a further 2 h in air free of harmful substances.
  • the GUS activity was determined in crude extracts from middle-aged leaves and compared with the enzyme activity in untreated control plants.
  • the results of the fluorometric analysis of the GUS enzyme activities are shown in Table 1. While the GUS activity with increasing shortening of the promoter region from -1500 to -430 results both in ozone-treated test plants and in untreated control plants in a slight decrease in the ozone induction (induction ⁇ factor drops from approx.
  • the additional deletion of the promoter region between -430 and -280 results in a drastic reduction in GUS expression in ozone-treated test plants. While plants in which the GUS gene is under the control of the -430-5 'deletion promoter show a 10-fold ozone induction compared to the control plants, in plants the expression of the GUS gene by the shorter -280- 5 'deletion promoter is controlled, only a maximum 2-fold induction of the GUS expression by ozone is observed. As a result, the promoter region contains the Vstl gene Grapevine comprising the base pairs -430 to -280 cis-active elements which are essential for a pronounced ozone-induced expression of the STS gene.
  • Figure 1 Restriction map of the Vstl promoter / GUS translation foot in the plasmid pUC-Vstl / GUS.
  • the plasmid contains a translation fusion of the Vstl promoter from the grapevine with the GUS gene from E ⁇ coli.
  • Table 1 GUS enzyme activities were determined fluorometrically in protein extracts from middle-aged leaves of ozone-treated (+) and untreated (-) independent tobacco teardrop formants.
  • the transgenic tobacco lines contain various 5 'deletions of the Vstl promoter in fusion with the bacterial GUS reporter gene.
  • FO transformants and FL plants (11 weeks old) were gassed with 100 nl / 1 ozone for 10 h and then incubated for 2 h in air free of harmful substances. Mean values ⁇ standard error; all analyzes were carried out as double experiments.
  • GUS enzyme activities were determined fluorometrically in protein extracts from middle-aged leaves of ozone-treated (+) and untreated (-) independent tobacco transformants.
  • the transgenic tobacco lines contain various 5 'deletions of the V ⁇ tl promoter in fusion with the bacterial GUS reporter gene.
  • F0 transformants and FL plants (11 weeks old) were gassed with 100 nl / 1 ozone for 10 h and then post-incubated in pollutant-free air for 2 h. Mean values ⁇ standard error; all analyzes were carried out as double tests.
  • GCAATCCCAC GGGAGGGAAG CTGCTACCAA CCTTCGTAAT GTT ⁇ ATGAAA TCAAAGTCAC 120

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Abstract

La présente invention concerne de nouvelles séquences d'ADN, une méthode pour produire de nouveaux végétaux contenant une nouvelle séquence d'ADN, leur séquence de codage étant exprimée après induction par l'ozone. L'invention concerne également ces nouveaux végétaux ainsi que l'utilisation des séquences d'ADN pour produire l'expression génique en réaction à l'ozone chez les végétaux et les cellules végétales. En outre, elle concerne un nouveau promoteur dont la spécificité est accrue par l'élimination de sa sensibilité à l'ozone.
EP97928238A 1996-06-25 1997-06-18 Expression genique induite par l'ozone chez les vegetaux Withdrawn EP0910651A1 (fr)

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DE19625347 1996-06-25
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DE19625347 1996-06-25
DE19625330 1996-06-25
DE19635569 1996-09-02
DE19635569 1996-09-02
DE19654574 1996-12-27
DE19654574A DE19654574A1 (de) 1996-06-25 1996-12-27 Ozon-Induzierte Genexpression in Pflanzen
PCT/EP1997/003187 WO1997049823A1 (fr) 1996-06-25 1997-06-18 Expression genique induite par l'ozone chez les vegetaux

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JP2002505109A (ja) 1998-03-06 2002-02-19 シンジェンタ モーヘン ビー． ブイ． 植物における病原体抵抗性の誘導のための方法
KR100893144B1 (ko) * 2007-06-20 2009-04-16 한국생명공학연구원 감자 유래의 스트레스에 의해 유도되어지는 ＳｔＧＲＸ２유전자
US20130055471A1 (en) 2009-12-15 2013-02-28 Edwin Henricus Antonius HOLMAN Transgenic Ozone-Resistant Plants
KR101451834B1 (ko) 2013-05-14 2014-10-16 한국원자력연구원 이온화에너지원 표지 유전자 OsUNP2 및 이를 이용한 형질전환 지표식물
CN116406590B (zh) * 2023-03-31 2023-12-22 东北农业大学 一种利用片段化番茄esDNA提高番茄植株抗性、防治灰霉病的方法及应用

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DE4234131C2 (de) * 1992-10-09 1995-08-24 Max Planck Gesellschaft Transgener pathogen-resistenter Organismus
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CA2257832A1 (fr) 1997-12-31

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