WO1997003205A2 - Nouvelles sequences d'adn et leur utilisation dans l'identification d'agents pouvant induire des mecanismes de defense pour les plantes - Google Patents

Nouvelles sequences d'adn et leur utilisation dans l'identification d'agents pouvant induire des mecanismes de defense pour les plantes Download PDF

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WO1997003205A2
WO1997003205A2 PCT/EP1996/002744 EP9602744W WO9703205A2 WO 1997003205 A2 WO1997003205 A2 WO 1997003205A2 EP 9602744 W EP9602744 W EP 9602744W WO 9703205 A2 WO9703205 A2 WO 9703205A2
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Prior art keywords
dna sequence
plants
plant
dna
pat
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PCT/EP1996/002744
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German (de)
English (en)
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WO1997003205A3 (fr
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Rüdiger Hain
Regina Fischer
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Bayer Aktiengesellschaft
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Priority to JP9505454A priority Critical patent/JP2000500002A/ja
Priority to EP96922025A priority patent/EP0837945A2/fr
Priority to AU63048/96A priority patent/AU6304896A/en
Priority to KR19977000400A priority patent/KR970704354A/ko
Publication of WO1997003205A2 publication Critical patent/WO1997003205A2/fr
Publication of WO1997003205A3 publication Critical patent/WO1997003205A3/fr

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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)
    • C12N15/8216—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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers

Definitions

  • the invention relates to new deoxyribonucleic acid sequences (DNA sequences) and their use or the use of certain plants that use them
  • Stimuli such as wounding, warmth or exposure, light / dark cycles, Heavy metals react. These test systems are therefore relatively susceptible to faults and can to a considerable extent lead to misinterpretations and thus to incorrect results.
  • DNA sequence I The new DNA sequence (hereinafter referred to as "DNA sequence I") has now been found, which consists of the following components, which are in direct 5'-3'-
  • Agents that can induce plant defense mechanisms are chemical substances or microorganisms, their metabolic products and
  • Extracts from microorganisms preferably chemical substances.
  • chemical substances are to be understood as meaning synthetic or naturally occurring organic compounds, preferably low molecular weight organic substances, very particularly preferably those which do not naturally occur in plants as elicitors (ie as repellants or Defense mechanisms inducing substances) occur and especially highlighted those that do not naturally occur in plants. Synthetic organic low-molecular substances that do not occur in nature are particularly preferred.
  • Promoters of plant origin are preferably used as promoters which can be activated in plants and which can systemically induce plant defense mechanisms.
  • Such promoters of plant origin are preferably used which are suitable for leading to induced systemic resistances and at the same time can be activated by chemical resistance inducers (for example sodium salicylate, dichlorisonicotinic acid or probenazole).
  • chemical resistance inducers for example sodium salicylate, dichlorisonicotinic acid or probenazole.
  • a large number of promoters which can lead to induced systemic resistance are already known from the literature (cf., for example, EP-A 0 648 839, EP-A 0 516 958, EP-A 0 533 010, EP-A 0 309 862, EP-A 0 480 236, EP-A 0 464 461 and EP-A 0 412 381)
  • promoters for example Na salicylate, dichloroisonicotinic acid or probenazole
  • resistance inducers for example Na salicylate, dichloroisonicotinic acid or probenazole
  • Such promoters of plant origin are preferably used which can systemically induce plant defense mechanisms, but cannot be activated by other abiotic stimuli (such as, for example, by wounding, warmth, exposure or light / dark cycles),
  • the promoters are preferably used as constituent 1 of DNA sequence I, which are described in the European published publications listed above. Furthermore, the prpl-1 promoter from potato, which is also known from the literature, is preferably used. The promoters can also be used to increase the activity a section of the CaMV 35S RNA promoter (preferably position -46 to +8) is connected downstream
  • DNA sequence I uses a region of the Vstl promoter from Vitis vinifera (position +72 to -552) or a region of the prpl promoter from potato from position -402 to -130, the prpl promoter being used in a particularly preferred embodiment -Region the region with the position -46 to +8 of the CaMV 35S RNA promoter is connected
  • Vstl promoter originating from Vitis vinifera or genes containing this promoter are known from EP-A 0 464 461 and are contained on the plasmids pVStl, pVSt2 and pVStl2t3
  • the Esche ⁇ cha coli strain E coli Fier 1 pVStl contains the plasmid pVStl.
  • the Escherichia coli strain E coli Fier 2 pVSt2 contains the plasmid pVSt2 Der
  • Escherichia coli strain E coli Fier pVSt! 2t3 contains the plasmid pVSt! 2t3 this Strains were deposited in connection with the aforementioned EP application with the German Collection of Microorganisms (DSM), Mascheroder Weg lb, D-38124 Braunschweig, Federal Republic of Germany in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (Filing date: E. coli Fier 1 pVStl and Fier 2 pVSt2 June 18, 1990 and E. coli Fier pVStl2t3: February 11, 1991).
  • DSM German Collection of Microorganisms
  • the E. coli Fier 1 pVStl strain received the accession number DSM 6002
  • the E. coli Fier 2 pVSt2 strain received the accession number DSM 6003
  • the E coli Fier pVStl2t3 strain received the accession number 6346.
  • the promoter can be isolated from the microorganisms and the plasmids contained therein by the person skilled in the art using simple routine measures
  • Vstl gene was isolated from Vitis vinifera (R Hain et al (1993) Nature 361: 153-156; W. Wiese et al (1994) Plant Mol Biol 26 667-677) and its inducible expression in the homologous and heterologous system examined.
  • Vstl promoter region used in DNA sequence I was amplified using the PCR technique and then fused to the region coding for PAT as described below
  • the prp-1 promoter is from the publications by J Taylor et al (Mol Plant-Microbe Interactions (1990) 3 72-77) and N Martini et al (Mol Gen Genet
  • CaMV Cauliflower Mosaic Virus
  • the Escherichia coli strain E coli GSpETVgus27-l contains the plasmid pETVgus27-l, which contains the prp-1 promoter from potatoes (N Martini et al (1993) Mol Gen Genet 236 179-186 or WO 93/19 188) of this E. coli strain was obtained from the German Collection for Microorganisms (DSM), Mascheroder Weg lb, D-38124 Braunschweig, Federal Republic of Germany in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the
  • the prpl -1- Promoter region from position -402 to -130 (272 bp) together with the TATA box region of the CaMV 35S RNA promoter (position -46 to +8) can be isolated from the plasmid pEVgus27-l as an EcoRI / Hindlll restriction fragment.
  • PAT phosphinothricin-N-acetyl transferase
  • pat gene The sequences coding for PAT from Streptomyces viridochromogenes ("pat gene”) or from Streptomyces hygroscopicus ("bar gene”) are preferably used, particularly preferably the coding sequence of the pat gene.
  • the bar gene from S. hygroscopicus can also be used.
  • the bar gene is also known from the literature (M. DeBlock et al. (1987) EMBO J. 6: 2513-2518; C Thompson et al. (1987) EMBO J. 6: 2519-2523)
  • the bar gene from S. hygroscopicus is also described in EP-A 0 242 246.
  • the complete coding region of the bar gene from S hygroscopicus is present in the vector pBG39 as a 1.65 kB PstI / BamHI fragment in pUC 19.
  • pBG39 was published in accordance with EP-A 042 246 on December 12, 1985 at the German Collection of Mi k r o org a n i sm en (D S M) i n G otti n g e n (D eu t s ch l an d) m i t d e r
  • the coding region of the bar gene consists of 184 amino acids, which can be amplified with the aid of the PCR technique according to generally known methods and fused with corresponding promoter and termination sequences
  • bacterial gene is the start codon for translation
  • GTG has, instead of the ATG start codon used in eukaryotes.
  • the start code for expression in plants must therefore be changed accordingly.
  • the oligonucleotides (primers) used for the PCR amplification are selected accordingly (see example Bc, below).
  • the expression of both genes mediates resistance to phosphinothricin in plants
  • the pat gene from S. viridochromogenes, the message also in the German Patentan ⁇ DE-A-36 28 747 is described, for example, from total DNA of S.
  • DSM 40736 General collection of the German Collection for Microorganisms (DSM) Mascheroder Weg 1 b, D-38124 Braunschweig, Federal Republic of Germany or DMS 4112 (deposit under Budapest contract with the German Collection for Microorganisms (DSM) Masche ⁇ roder Weg 1 b, D-38124 Braunschweig, Federal Republic of Germany available (see. DE-A-36 42 829 AI and EP-A 0 275 957).
  • DSM 40736 general collection of the German Collection for Microorganisms (DSM) Mascheroder Weg 1 b, D-38124 Braunschweig, Federal Republic of Germany or DMS 4112 (deposit under Budapest contract with the German Collection for Microorganisms (DSM) Masche ⁇ roder Weg 1 b, D-38124 Braunschweig, Federal Republic of Germany available (see. DE-A-36 42 829 AI and EP-A 0 275 957).
  • the termination sequence used as component 3 of the DNA according to the invention includes all termination sequences which are functional in plants
  • the termination sequence of the nopaline synthase gene (“nos gene”) from Agrabacterium tumefaciens is used with particular preference.
  • the termination sequence of the nopaline synthase gene ("nos gene") from Agrobacterium tumefaciens to be used with particular preference as component 3 of the DNA sequence is known or can be obtained by generally known methods (cf. A. Depicker (1982) J. Mol. Appl. Genet. 1: 561-574).
  • DNA sequence II which consists of the following constituents, which follow one another in direct 5'-3 'orientation, and which is referred to below as "DNA sequence II":
  • DNA sequence III Agrobacterium tumefaciens gene
  • the DNA sequence I according to the invention and the DNA sequences II and III can be used in a particularly advantageous way to find agents which can induce plant defense mechanisms.
  • the corresponding coding sequences have always been constitutively expressed, which, as expected, leads to high resistance.
  • the constructs according to the invention, using inducible promoters in plants would produce sufficient resistance to phosphinothricin for the screening method according to the invention.
  • DNA sequence I and DNA sequences II and III are understood to mean the DNA sequences composed of the above-mentioned components 1 to 3. However, this term also includes the DNA
  • Sequences I, II and III derived DNA sequences a Derived DNA sequences are those DNA sequences which essentially correspond to the DNA sequences I, II and III and can be used according to the invention. These are, for example, DNA sections which contain the DNA sequences I, II or III in such a way that their function according to the invention is not or not significantly impaired. Such DNA segments can, for example arise when the DNA sequences I, II and III are cut out of a larger piece of DNA with the aid of restriction enzymes.
  • the DNA sequences I, II and III can also carry at their ends those DNA sequences which are adapted to their handling (eg so-called "linkers") Furthermore, one or more DNAs or DNA sections in the DNA sequences I, II or III can be replaced by other DNAs or DNA sections having essentially the same effect, as can be seen, for example the variation of DNA due to the degeneracy of the genetic code
  • the DNA sequence I or the DNA sequence II are for the use according to the invention.
  • Roots bleeding and seeds or their parts or cells (including
  • Protoplasts Protoplasts
  • calli etc. are brought into contact with the chemical substances to be investigated for their inducing effectiveness.
  • the enzyme phosphinothricin-N-acetyltransferase (PAT) is formed by the sequence of the pat or bar gene, which or its activity based on the phosphinoth ⁇ cin resistance of the test plants or in
  • Enzyme test can be determined qualitatively or quantitatively In this way it is possible to set up test systems that are easy to carry out, with the help of which larger numbers of test substances can be tested quickly and reliably for their inducing properties. A particular advantage of this
  • Test systems also maintains that substances with a positive result are not just that
  • Activate VStl or prpl-1 promoter but are generally suitable for inducing other promoters of plant pest defense genes, in particular promoters of phytoalexin genes and PR promoters.
  • promoters of plant pest defense genes in particular promoters of phytoalexin genes and PR promoters.
  • Suitable compounds are quickly and reliably determined, which can then optionally be used in a targeted manner for further investigations.
  • the method according to the invention enables simple optical assessment of the gene-activating potency of a test substance by observing the herbicidal agents
  • the present invention thus relates to a test method according to which it is simple to use
  • Routine procedures from a variety of organic compounds such can be selected which can induce plant pest defense genes and which are therefore suitable as plant protection agents for pest control.
  • the plant defense genes to be induced are preferably genes which are responsible for protecting the plants against attack by phytopathogenic microbial pests, preferably by fungi, bacteria or viruses, particularly preferably by phytopathogenic fungi.
  • the present invention thus also relates to the use of DNA sequence I (or DNA sequence II and II) and of transgenic plants or parts of plants which DNA sequence I (or DNA sequence II or III) or a DNA sequence derived therefrom contained in their genome to find chemical substances which may be suitable for inducing plant defense mechanisms against plant pests. Furthermore, the present invention also relates to the new use of the DNA sequence coding for phosphinothricin-N-acetyltransferase as a reporter gene in connection with inducible plant promoters in plants for the detection of agents which can indicate plant defense mechanisms.
  • the present invention thus also relates to a method for finding chemical substances which may be suitable for inducing plant defense mechanisms against plant pests, which is characterized in that transgenic plants or parts of plants which are found in their
  • Genome contain the DNA sequence I (or the DNA sequences II or III) or a DNA sequence derived therefrom, in contact with the chemical substances to be investigated for their inducing effectiveness, and the substances which have an increased expression of phosphinothricin -N-acetyltransferase and thus cause increased resistance to phosphinothricin.
  • test plants which can be used according to the invention and which contain DNA sequence I in their genome. It is preferable to use plants in which the resistance inductors found are to be used for pest control. These include, above all, plants which are of interest for agriculture and forestry, for growing ornamental plants, for growing medicinal plants and for growing plants. However, plants which are easy to handle in a test laboratory and are also quick and easy to use are particularly preferred as test plants. let multiply. Arabidopsis thaliana, tomato and tobacco, in particular tobacco, may be mentioned as particularly preferred transgenic plants.
  • the present invention also includes new plants (including plant parts and propagation material, such as seeds, offshoots, tubers, cells, protoplasts, calli, etc.), which contain the DNA sequence I (or the DNA sequence) in their genome.
  • Preferred new plants according to the invention are Arabidopsis thaliana, tomato, potato and tobacco, very preferably tobacco.
  • the present invention also relates to a method for producing the new transgenic plants according to the invention (including the plant parts and the propagation material), which is characterized in that
  • DNA sequence I (or the DNA sequences II or III) or a DNA sequence derived therefrom is inserted into the genome of plant cells (including protoplasts) and, if appropriate,
  • the present invention also includes the transgenic plants obtainable by the processes described above.
  • Process steps (a), (b) and (c) can be carried out in the usual manner by 'saw 1 using known processes and methods.
  • the organic chemical substances to be tested are preferably dissolved in water.
  • a solution is preferably first prepared in a physiologically acceptable organic solvent, such as acetone, dimethylformamide, dimethyl sulfoxide, methanol or ethanol. This solution is then diluted with water.
  • the test solution should preferably contain less than 5% (percent by weight), particularly preferably less than 1%, organic solvent.
  • concentration of the test substances in the test solution can fluctuate over a wide range and depends on the solubility of the test substances and the particular details of the test procedure, e.g.
  • test substances are preferred in a concentration of 5 ⁇ M to 20,000 ⁇ M, particularly preferably of
  • wetting agents For better application to the surface, one of the usual wetting agents can also be added to ensure better wetting of the plant material. Care must be taken to ensure that the wetting agent used itself has no inducing properties
  • the concentration of phosphinoth ⁇ cin can be varied over a wide range. Depending on the plant, plant age, part of the plant, genotypic and phenotypic factors etc., phosphinoth ⁇ cin concentrations can be used which correspond to the usual application rates between 0.1 kg / ha and 10 kg / ha phosphinoth ⁇ cin The optoimal application rate of phosphinoth ⁇ cin can easily be determined by simple series experiments
  • test plants can be used for the test method according to the invention in the form of the complete plants or else in the form of parts of plants, preferably in the form of leaves, leaf parts or stems or parts of stems.
  • the test plants which as such or in In the form of parts of plants or parts obtained therefrom (e.g. small pieces of leaf) should at least have reached the 4-leaf stage, i.e. have at least the first fully developed leaves growing after the cotyledons. In tobacco, this stage, starting from seeds, depending on the prevailing conditions after about 14 to 21 days in soil at 20 to 22 ° C and approx. 10,000 lux lighting reached. to protect the young plants from excessive evaporation of water, for example by covering, in the growing season.
  • the size and age of the test plants can largely be varied. Small and young plants which have reached the 4-leaf stage or the subsequent growth stage are expediently and preferably used.
  • the test plants are particularly preferably used in the 4-leaf stage.
  • the dissolved test substances can be brought into contact with the plants or the plant material in any way, e.g. by spraying or brushing.
  • the plant material can also be immersed or placed in the solutions of the test substances.
  • the roots of the intact plants, plant stems or petioles are immersed in the test solutions for a sufficiently long time, which allows the test substances to be taken up. Application is preferably via the roots.
  • the exposure time is preferably between 1 and 96, particularly preferably between 2 and 72 and very particularly preferably between 24 and 48 hours.
  • the incubation is preferably carried out under normal exposure or exposure in a conventional light cabinet, the light-dark rhythm customary for the plants (for example 16 hours light, 8 hours dark) expediently being observed.
  • the incubation is preferably carried out at temperatures between 15 and 30 ° C., particularly preferably between 18 and 28 ° C.
  • the application of the herbicide phosphinothricin can take place simultaneously with the treatment of the chemical substance to be tested or after various large time intervals after the treatment of the chemical substance to be tested.
  • test plants treated with the test substance or with phosphinothricin are incubated under greenhouse conditions (20-25 ° C., approx. 10,000 lux and 60% relative atmospheric humidity) and irrigated exclusively from below. Evaluation takes place 3-21 days after treatment with phosphinothricin.
  • the resistance-inducing influence of the test substance is assessed on the basis of the phosphinothricin resistance of the test plants in comparison to control plants which were only treated with phosphinothricin. Criteria such as leaf size, growth speed, leaf damage etc. can be used here.
  • the genetically Fourth potency of a test substance can be analyzed in more detail by determining the in vitro phosphinothincin-N-aceytltranferase activity
  • test substances are applied via the roots of young test plants.
  • the substances are absorbed by the roots and distributed over the lead tissue in the plant.
  • the test is then carried out and evaluated as described
  • phosphinothricin ie, for example, increased plant growth or a reduction in plant damage caused by phosphinothricin
  • a test substance is present which can be assumed to have the desired inducing properties.
  • the control plants are traded with water as a test solution, which may contain the organic solvent and / or surface-active agent used in the appropriate concentration.
  • the level of phosphinothricin resistance correlates usually with the strength of the induction power of the respective test substance
  • Test plants that can be used are available according to the generally customary methods
  • DNA sequence I or the DNA sequences II or III
  • the DNA transfer can be carried out according to the generally known methods the person skilled in the art can determine and carry out the appropriate method without difficulty
  • Phosphinothricin (PPT) resistant tobacco plants are grown under greenhouse conditions (22 ° C, 60% relative humidity, approx. 15,000 lux)
  • Blood is cultivated and self-harvested to obtain the F ⁇ l ⁇ al (Fl) generation.
  • Seeds of the Fl generation are preselected in sterile culture on LS medium with 75 ⁇ g / ml kanamycin (Sigma) and the resistant Fl seedlings are transferred to soil after 2 weeks For 6-14 days, the plants are sprayed with aqueous test solutions of various chemicals / active substances to be tested (5-20,000 ⁇ M) in order to be treated with PPT (0.1-10 kg / ha) at different intervals as a negative Control is carried out by wild-type SRI seedlings or tobacco seedlings which only contain the transferring plasmid with the kanamycin resistance gene (nptII), but without a promoter / pat or promoter / bar fusion, in parallel to
  • the growth of the plants is assessed after 5-20 days by measuring the largest leaf.
  • the leaves are examined for any leaf damage after treatment with the inducing chemicals Evalution in Different large intervals after the PPT treatment turned out to be extremely useful, since the time course of gene activation or PPT resistance induction can vary greatly due to the different chemical substances. If a substance shows a resistance-inducing effect, the time course and the level of gene activation can be analyzed and quantified with the aid of a simple enzyme test (M. DeBlock et al. (1987) EMBO J. 6: 2513-2518 and M. DeBlock et. al. (1989) Plant Physiol. 91: 694-701).
  • Tobacco plants which contain the DNA sequence III with the pat gene from Streptomyces viridochromogenes were subjected to the PPT resistance test: Homozygous seedlings of the F2 generation were treated with probenazole (1 mM), water 16 days after sowing Control test solution, and 5-chlorosalicylic acid (1 mM) treated. 24 hours later the plants were with
  • Tobacco plants that contain the DNA sequence III with the pat gene from Streptomyces viridochromogenes were subjected to the PPT resistance test: Homozygous F2 seedlings were treated with 10 mM sodium salicylate or water 20 days after sowing, 24 h later 4 kg / ha PPT were applied. Wild-type plants and plants which have the PPT resistance gene pat under the constitutive 35S RNA
  • Leaf size of the plants treated with Na salicylate corresponded to that of the control plants which contained the constitutive CaMV 35S RNA promoter. Wild type plants were so badly damaged by 4 kg / ha PPT that they stopped growing and died away a few days after the treatment.
  • the amplified fragment which bears the sequence of the Vstl promoter from Vitis vinifera from position +72 to -552, was cut with the restriction enzymes BamHI and Hindlll.
  • the BamHI / Hindlll-Vstl promoter fragment formed was cut together with a
  • pVstlPAT the coding region of the pat gene is under the control of the Vstl promoter (up to -552).
  • pVstlPAT corresponds to DNA sequence II.
  • the bacterial pat gene is already modified in the vector pWD26.41 in such a way that efficient expression in plants is possible.
  • the 900 bp fragment was fused with a Hindlll / BamHI oligonucleotide linker (see below), the linker being oriented such that its Hindlll restriction site was only generated by a subsequent Hindlll restriction digest.
  • the HindIII / BamHI oligonucleotide linker used consisted of the oligonucleotide according to SEQ ID No. 3 (see Fig. 3).
  • the bar gene is primed with the sequences according to SEQ ID No. 4 and SEQ ID No. 5 (cf. also FIGS. 5 and 6) were amplified in a PCR reaction and then trimmed with the restriction enzymes BamHI and EcoRI.
  • the resulting BamHI / HindIII (5 ' ->3') fragment consisting of the bar gene and a polyA sequence, is purified using generally customary methods and, in the next cloning step, together with the Vstl promoter, which is used as H ⁇ ndIII / BamHI (5 ' -> ' 3 ') fragment is present (see cloning strategy for the production of DNA sequence II with pat gene from S viridochromogenes), into the HindIII interface of the binary vector pPCV002 (C Koncz and J Schell ( 1986) Mol Gen Genet 204 383-396) ligated
  • the coding region of the bar gene is under the control of the Vstl promoter (up to -552, cf.
  • the bar gene is amplified in a PCR reaction with the polymer of the sequences according to SEQ ID No 6 and SEQ ID No 7 (cf. also FIGS. 7 and 8) and then cut with the restriction enzymes HindIII and BamHI
  • the resulting III / BamHI (5 -> 3) fragment (558 base pairs) is fused with the polyadenylation signal of the 35S RNA from Cauhflower Mosaic Virus, which was previously described as a BamHI / III III (5 -> 3 ') fragment (approx. 250 bp) from the vector pRT104 (R Topfer et al (1987) Nucl Acids Res 15 5890)
  • the resulting HindIII fragment consisting of the bar gene and a polyA sequence, is purified using generally customary methods and in the next cloning step into the HindIII restriction site of the vector pPRPl-lPro (see Fig. 4) ligated.
  • the coding region of the bar gene is under the control of the prpl-1 promoter from S. tuberosum (cf. DNA sequence III).
  • Nicotiana tabacum (Petit Havana SRI) is propagated as a sterile shoot culture on a hormone-free LS medium (Linsmaier and Skoog 1965). At intervals of approx. 6 - 8 weeks, shoot sections are transferred to fresh LS medium. The shoot cultures are kept in a culture room at 24-26 ° C under 12 h light (1000-3000 lux)
  • leaf protoplasts For the isolation of leaf protoplasts, approx. 2 g of leaves (approx. 3-5 cm long) are cut into small pieces (0.5 cm x 1 cm) with a fresh razor blade.
  • the leaf material is in 20 ml enzyme solution consisting of K3
  • the pre-cleaned protoplasts are made up to 10 ml with fresh K3 medium (0.4 M sucrose as an osmotic agent) and floated again.
  • the washing medium is suctioned off and the protoplasts are used for culture or subsequent infection with agrobacteria (coculture) diluted to 1 - 2 x 10 5 / ml.
  • the protoplast concentration is determined in a number chamber
  • prpl-1-PAT or Vstl -PAT or another suitable Agrob strain which contains prpl-1-PAT or Vstl-PAT, in minimal A (Am) medium (density approx. IO 9 Agrobacteria / ml) to 3 ml Regenerating protoplasts given.
  • the coculture time is 3-4 days at 20 ° C. in the dark.
  • the tobacco cells are filled in 12 ml centrifuge tubes, diluted to 10 ml with sea water (600 mOsm / kg) and pelleted at 60 ⁇ g for 10 minutes. This washing process is repeated a further 1-2 times in order to remove most of the agrobacteria.
  • the cell suspension is at a density of 5 ⁇ 10 4 / ml in K3 medium (0.3 m sucrose) with 1 mg / 1 NAA (Naphthyl-1-acetic acid), 0.2 mg / 1 kinetin and 500 mg / 1 of the cephalosporin antibiotic cefotaxime are cultivated.
  • K3 medium 0.3 m sucrose
  • NAA Naphthyl-1-acetic acid
  • 0.2 mg / 1 kinetin 500 mg / 1 of the cephalosporin antibiotic cefotaxime are cultivated.
  • the cell suspension is diluted every week with fresh K3 medium and the osmotic value of the medium is gradually increased by 0.05 m Sucrose (approx)
  • kanamycin 100 mg / 1 kanamycin sulfate
  • Kanamycin-resistant colonies can be 3-4 weeks after the start the selection can be distinguished from the background of the remaining colonies
  • Agarose low melting agarose, marine colloids
  • agarose is autoclaved dry and briefly boiled in the microwave after adding K3 medium. After the agarose has solidified, the agarose slices ("beads") with the embedded tobacco microcalli are placed in 10 cm for further culture and selection
  • Petri dishes were transferred and 10 ml K3 medium (0.3 M sucrose, 1 mg / 1 NAA, 0.2 mg / 1 kinetin) and 100 mg / 1 kanamycin sulfate were added.
  • the liquid medium is changed every week. The osmotic value of the medium is gradually reduced.
  • the exchange medium (K3 + Km) increases by 0.05 M per week
  • kanamycin-resistant colonies have reached a diameter of approx. 0.5 cm
  • half is placed on regeneration medium (LS medium, 2% sucrose, 0.5 mg / 1 benzylaminopu ⁇ n BAP) and in 12 h light (3000 - 5000 lux) and 24 ° C in the culture room
  • the other half is propagated as a callus culture on LS medium with 1 mg / 1 NAA, 0.2 mg / 1 kinetin, 0.1 mg / 1 BAP and 100 mg / 1 kanamycin sulfate if the regenerated Sprouts are approx. 1 cm in size, they are cut off and placed on 1/2 LS medium (1% sucrose, 0.8% agar) without growth regulators for rooting.
  • the sprouts are placed on 1/2 MS medium with 100 mg / 1 Kanamycin sulfate rooted and later converted into soil
  • leaves of approx. 2-3 cm in length are punched out of sterile shoot cultures into discs of 1 cm in diameter and with a suspension of appropriate agrobacteria, GV3101 C58CIpMP90RK :: prpl-l-PAT or Vstl-PAT, (approx.
  • DNA sequence 1 (or II and III) was determined by Northern blot analysis, Southern blot analysis, phosphinothricin-N-acetyltransferase activity (M. DeBlock et al (1987) EMBO J 6 25 13-2518 and M DeBlock et al (1989) Plant Physiol 91: 694-701) and phosphinothricin tolerance on food medium containing phosphinothricin confirmed
  • Vitamins Ca-panthotenate 1 mg / 1 biotin 10 mg / I
  • Nicotiana tabacum occidental Havana SRI On the culture of Nicotiana tabacum occidental Havana SRI, Nicotiana tabacum Wiscon ⁇ sin 38, and Nicotiana plumbaginifolia protoplasts (Nagy and Maliga, 1976)
  • Linsemaier and Skoog (LS) Medium is Murashige and Skoog Medium (Murashige and Skoog, 1962) with the following modifications:
  • Thiamine is weighed in a higher concentration 0.4 mg / 1 instead of 0.1 mg / 1;
  • Glycine, pyridoxine and nicotinic acid are missing.

Abstract

L'invention concerne de nouvelles séquences d'acide désoxyribonucléique (séquences d'ADN) ainsi que leur utilisation ou l'utilisation de certaines plantes contenant ces séquences d'ADN pour trouver des agents pouvant induire des mécanismes de défense dans les plantes.
PCT/EP1996/002744 1994-07-21 1996-06-24 Nouvelles sequences d'adn et leur utilisation dans l'identification d'agents pouvant induire des mecanismes de defense pour les plantes WO1997003205A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP9505454A JP2000500002A (ja) 1995-07-10 1996-06-24 新規なｄｎａ配列及び植物における防御誘導因子を同定するためのこれらの使用
EP96922025A EP0837945A2 (fr) 1995-07-10 1996-06-24 Nouvelles sequences d'adn et leur utilisation dans l'identification d'agents pouvant induire des mecanismes de defense pour les plantes
AU63048/96A AU6304896A (en) 1995-07-10 1996-06-24 Novel dna sequences and their use for identifying defence-inducing agents in plants
KR19977000400A KR970704354A (en) 1994-07-21 1997-01-21 Method of combating harmful fungi

Applications Claiming Priority (2)

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DE1995125034 DE19525034A1 (de) 1995-07-10 1995-07-10 DNA-Sequenzen und ihre Verwendung
DE19525034.6 1995-07-10

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WO1997003205A3 WO1997003205A3 (fr) 1997-03-13

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WO1999027114A1 (fr) * 1997-11-21 1999-06-03 Calgene Llc Promoteur de l'expression genique sensible au stress et aux pathogenes
WO2000029596A1 (fr) * 1998-11-17 2000-05-25 Monsanto Company Plantes metabolisant les phosphonates

Citations (4)

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EP0332104A2 (fr) * 1988-03-08 1989-09-13 Ciba-Geigy Ag Sèquences d'ADN et gènes chimiquement regulables, et leur emploi
WO1993005164A1 (fr) * 1991-09-02 1993-03-18 The University Of Leicester Promoteurs specifiques de certains cals vegetaux
WO1993019188A1 (fr) * 1992-03-20 1993-09-30 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Gene chimerique sensible aux champignons
WO1994024295A1 (fr) * 1993-04-12 1994-10-27 Ciba-Geigy Ag Regulation exogene de l'expression des genes chez des plantes

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EP0332104A2 (fr) * 1988-03-08 1989-09-13 Ciba-Geigy Ag Sèquences d'ADN et gènes chimiquement regulables, et leur emploi
WO1993005164A1 (fr) * 1991-09-02 1993-03-18 The University Of Leicester Promoteurs specifiques de certains cals vegetaux
WO1993019188A1 (fr) * 1992-03-20 1993-09-30 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Gene chimerique sensible aux champignons
WO1994024295A1 (fr) * 1993-04-12 1994-10-27 Ciba-Geigy Ag Regulation exogene de l'expression des genes chez des plantes

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PLANT MOLECULAR BIOLOGY, Bd. 26, 1994, Seiten 667-677, XP002019713 WIESE, W. ET AL.: "Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment" in der Anmeldung erw{hnt *
PLANT MOLECULAR BIOLOGY, Bd. 26, Nr. 3, November 1994, Seiten 1007-1012, XP002019712 TAKIMOTO, I. ET AL.: "Non-systemic expression of a stress-responsive maize polyubiquitin gene (Ubi-1) in transgenic rice plants" *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999027114A1 (fr) * 1997-11-21 1999-06-03 Calgene Llc Promoteur de l'expression genique sensible au stress et aux pathogenes
US6072103A (en) * 1997-11-21 2000-06-06 Calgene Llc Pathogen and stress-responsive promoter for gene expression
WO2000029596A1 (fr) * 1998-11-17 2000-05-25 Monsanto Company Plantes metabolisant les phosphonates
US6448476B1 (en) 1998-11-17 2002-09-10 Monsanto Technology Llc Plants and plant cells transformation to express an AMPA-N-acetyltransferase
EP2042603A1 (fr) * 1998-11-17 2009-04-01 Monsanto Technology, LLC Plantes métabolisant le phosphonate
US7554012B2 (en) 1998-11-17 2009-06-30 Monsanto Technology Llc Plants and plant cells exhibiting resistance to AMPA, and methods for making the same

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JP2000500002A (ja) 2000-01-11
WO1997003205A3 (fr) 1997-03-13
EP0837945A2 (fr) 1998-04-29
DE19525034A1 (de) 1997-01-16
AU6304896A (en) 1997-02-10

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