EP1252318A2 - Gene d'allene oxyde cyclase et son utilisation pour la preparation d'acide jasmonique - Google Patents

Gene d'allene oxyde cyclase et son utilisation pour la preparation d'acide jasmonique

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Publication number
EP1252318A2
EP1252318A2 EP01902398A EP01902398A EP1252318A2 EP 1252318 A2 EP1252318 A2 EP 1252318A2 EP 01902398 A EP01902398 A EP 01902398A EP 01902398 A EP01902398 A EP 01902398A EP 1252318 A2 EP1252318 A2 EP 1252318A2
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Prior art keywords
nucleic acid
plant
acid according
protein
expression
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German (de)
English (en)
Inventor
Jörg Ziegler
Irene Stenzel
Bettina Hause
Claus Wasternack
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Institut fur Pflanzenbiochemie
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Institut fur Pflanzenbiochemie
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P31/00—Preparation of compounds containing a five-membered ring having two side-chains in ortho position to each other, and having at least one oxygen atom directly bound to the ring in ortho position to one of the side-chains, one side-chain containing, not directly bound to the ring, a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, and the other side-chain having at least one oxygen atom bound in gamma-position to the ring, e.g. prostaglandins
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    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a nucleic acid which codes for a vegetable allenoxide cyclase and the use of the nucleic acid for the production of jasmonic acid in a biotechnological way.
  • jasmonates were viewed as the signals of altered gene expression in various plants in response to biotic and abiotic stress as well as the signal of certain developmental processes during plant development (Creelman, RA and Mullet, JE (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 355-381; Wasternack, C. and Parthier, B. (1997) Trends in Plant Science 2, 302-307).
  • Farmer and Ryan Farmer, EE and Ryan, CA (1990) Proc. Natl. Acad. Sci. USA 87, 7713-7716
  • JA was an essential intermediate in the wound-induced signaling cascade as a result of attack by herbivores Is tomato leaves.
  • Jasmonate has been identified as a trigger for numerous defense mechanisms in plants against pathogen infection or abiotic stress.
  • defense genes such as proteinase inhibitors
  • the synthesis of phytoalexins, alkaloids and fragrances are the most important answers to YES, which in most cases takes place through the up-regulation of specific enzymes (Ellard-Ivey, M. and Douglas CJ (1996) Plant Physiol. 112, 183-192; Feussner, I. et al. (1995) Plant J. 7, 949-957).
  • JA is biosynthesized via the oxylipine biosynthetic pathway, which begins with the incorporation of molecular oxygen into position 13 of linolenic acid catalyzed by a lipoxygenase.
  • the resulting fatty acid hydroperoxide 13 (S) -hydroperoxy-9 (Z), 11 (E), 15 (Z) -octadecatrienoic acid, 13-HPOT
  • AOS allene oxide synthase
  • This allene oxide is then cyclized by the allene oxide cyclase (AOC) to 9 (S), 13 (S) -OPDA.
  • AOC allene oxide cyclase
  • the authors Vick and Zimmermann proposed a similar biosynthetic pathway as early as 1983, but it was assumed that the formation of OPDA from 13-HPOT is catalyzed by a single enzyme, a so-called hydroperoxide cyclase (Vick, BA and Zimmermann, DC (1983) Biochem Biophys Res. Commun.
  • the products formed by lipoxygenases can be converted by a divinyl ether synthase, a reductase, a peroxygenase and one Hydroperoxide lyase (Blee, E. (1998) Prag. Lipid Res. 37, 33-72). Because of these reaction options and the nonspecific ketol binding by the AOS, the AOC can be regarded as the first enzyme that makes the jasmonate synthesis highly specific.
  • the object of the present invention was to provide nucleic acids and fragments thereof which enable a method for the highly specific production of the above-mentioned stereoisomer c / ' s - (+) - OPDA from the JA biosynthetic pathway.
  • nucleic acid which codes for a protein with the activity of allene oxide cyclase and comprises a sequence which is selected from the following sequences:
  • nucleic acid which is obtainable by screening a plant gene bank with a probe and selection for allene oxide cyclase-positive clones;
  • nucleic acid which codes for a protein with the sequence selected from SEQ ID Nos. 2, 4, 6, 8,10, 12, 14, 16;
  • nucleic acid which hybridizes with a nucleic acid according to b);
  • nucleic acid which would hybridize with a nucleic acid according to b) taking into account the degeneration of the genetic code; e) derivatives of a nucleic acid according to a) to d) obtained by substitution, addition, inversion and / or deletion of one or more bases;
  • nucleic acid with at least 53%, preferably at least 70% identity to the coding region of the nucleic acid according to SEQ ID No. 1;
  • Allen oxide cyclase means an enzyme that catalyzes the cs (+) - OPDA synthesis from the substrate 12,13-EOT as shown in FIG. 1.
  • Probe means a nucleic acid that is suitable for searching gene banks. However, the term also means agents, preferably proteins, which can indicate the presence of the expression product Allen oxide cyclase. These include anti-Allen oxide cyclase antibodies.
  • Hybridization with one of the sequences which are preferred for a protein with the sequence selected from SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, preferably the sequence according to SEQ ID No. 1 below, is preferred Conditions: Hybridization with the sequence according to SEQ ID No. 1 at 2xSSC, 0.1% SDS at 50 ° C., preferably for at least 30 min, is preferred. A slightly more stringent, also preferred Hybridization takes place at 1xSSC, 0.1% SDS at 50 ° C.
  • hybridization in which the Express Hyb TM hybridization solution from Clontech (USA) is used is particularly preferred, hybridization being carried out at 60 ° C. for 18 h and then at 30 min. with 2xSSC, 0.1% SDS at 50 ° C followed by 30 min. 1xSSC, 0.1% SDS at 50 ° C.
  • % identical refers to identity at the nucleic acid level, which according to known methods, e.g. of computer-aided sequence comparisons (Basic local alignment search tool, S.F. Altschul et al., J. Mol. Biol. 215 (1990), 403-410).
  • % identity familiar to the person skilled in the art denotes the degree of relationship between two or more nucleic acid molecules, which is determined by the agreement between the sequences.
  • the percentage of “identity” results from the percentage of identical regions in two or more sequences, taking into account gaps and other sequence peculiarities.
  • the identity of related nucleic acid molecules can be determined using known methods. As a rule, special computer programs with algorithms that take account of the special requirements are used. Preferred methods for determining identity initially produce the greatest agreement between the sequences examined. Computer programs for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12 (12): 387 (1984); Genetics Computer Group Univer - City of Wisconsin, Madison, (WI)); BLASTP, BLASTN and FASTA (Altschul, S. et al., J. Molec Biol 215: 403/410 (1990)).
  • the BLAST X program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbuch, Altschul S., et al., NCB NLM NIH Bethesda MD 20894; Altschul, S., et al., J. Mol. 215: 403/410 (1990)).
  • NCBI National Center for Biotechnology Information
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • Preferred parameters for nucleic acid sequence comparison include the following:
  • Disagreement (mismatch) 0 gap value (gap penalty): 50 gap length value: (gap length penalty): 3
  • the GAP program is also suitable for use with the above parameters.
  • the above parameters are the error parameters (default parameters) for nucleic acid sequence comparisons.
  • gap opening penalties can be used. The selection will depend on the comparison to be performed and also on whether the comparison is carried out between sequence pairs, GAP or Best Fit being preferred, or between a sequence and an extensive sequence database, with FASTA or BLAST being preferred.
  • the nucleic acid according to the invention encodes a protein with the sequence as given in one of the sequences with SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, preferably in SEQ ID No. 2.
  • the specific sequence can be separated according to conventional methods by changes in the nucleic acid sequence (such as by substitution, addition, inversion or deletion). ner or more bases) produce protein variants. Whether a new protein variant still has the desired allene oxide cyclase activity can easily be demonstrated by means of appropriate enzyme tests, as detailed below.
  • the nucleic acid is a cDNA, but the genomic DNA is also suitable as the nucleic acid coding for all-oxide cyclase.
  • the following table shows a list of clones which have been isolated with the DNA according to the invention according to SEQ ID No. 1 of the method according to the invention.
  • fragments of the claimed nucleic acids according to the invention are distinguished by the fact that they can hybridize specifically with the abovementioned nucleic acid.
  • “Specific hybridization” means that the person skilled in the art can choose the hybridization conditions such that the fragment after hybridization only gives a signal with the above-mentioned nucleic acid and does not give a signal with other nucleic acids also present in the sample.
  • Such fragments can also be used for a specific amplification of the nucleic acid according to the invention by means of PCR.
  • the expression of the allene oxide cyclase can be inhibited in a host cell with the aid of the fragment, this being made possible, for example, by the fragment in an antisense orientation to a promoter.
  • the "construct" containing a nucleic acid according to the invention is a combination of the above-mentioned nucleic acid according to the invention and a further nucleic acid with which the nucleic acid according to the invention is naturally not linked.
  • the construct can encode AOC coding sequences together with e.g. regulatory sequences, vector sequences or sequences of fusion partners.
  • the sequence according to the invention is in a plasmid.
  • the host cells according to the invention containing the nucleic acid according to the invention are distinguished by the fact that they contain the nucleic acid in an amount / copy number which does not occur in the host type.
  • the host cells can also be cells whose wild type does not contain the nucleic acid according to the invention but which they have after the nucleic acid has been introduced.
  • the nucleic acids according to the invention can be introduced into any desired cells, including bacteria, as well as plant cells, animal cells, including insect cells, and human cells using conventional transformation techniques.
  • nucleic acid according to the invention into plant cells or e.g. Plant tissue or plant organs.
  • the host cell or the cell assembly contains the nucleic acid integrated into the genome.
  • the site of integration will not be the site where the wild type contains the allenic oxide cyclase gene, if present.
  • the protein is a fusion protein comprising the allene oxide cyclase or a fragment thereof and a non-alumina cyclase protein sequence.
  • Preferred fusion partners are sequences from the allen oxide synthase and reductase, as they occur in the Jamonat biosynthetic pathway.
  • the complete allene oxide cyclase is particularly preferred in connection with the complete allene oxide synthase or the OPDA reductase.
  • the skilled worker is familiar with the production of antibodies which are directed against the protein according to the invention. For this purpose, for example, the generation of antisera and the generation of polyclonal antibodies after immunization of a host organism with the protein according to the invention can be considered.
  • hybridoma technology can also be used to produce monoclonal antibodies.
  • new transgenic plants and parts of plants can now be produced for the first time by introducing the nucleic acid according to the invention into a starting plant cell and regenerating the more complex plant tissue down to the whole plant from the initially transformed plant cell. The techniques required for this are available to the person skilled in the art.
  • the nucleic acid according to the invention can also be used to influence the allene oxide cyclase activity in a plant or parts thereof.
  • the nucleic acid for example under a strong promoter, the allene oxide cyclase activity in the plant or in the plant cells can be increased. This effect can also be achieved by increasing the copy number of the sequences encoding allene oxide cyclase.
  • the nucleic acid according to the invention for example in an antisense orientation to a promoter, the endogenous expression of the alloxide cylase gene can be reduced or even completely blocked. This then leads to a reduction in the activity of all-oxide cyclase in the corresponding part of the plant.
  • the method according to the invention enables for the first time a highly selective production of the metabolite 9,13-cis - (+) - 12-oxophytodienic acid in large quantities.
  • This metabolite is the essential and crucial starting point for the production of jasmonic acid in its naturally occurring enantiomeric form in large quantities.
  • the metabolite mentioned was previously practically inaccessible because there was no possibility of selectively producing the cis - (+) - OPDA from the precursor molecule, 12,13-EOT. Only mixtures of different metabolites were accessible. These metabolite mixtures in turn prevented the synthesis of jasmonate in large quantities and high purity.
  • the nucleic acid according to the invention also enables the production of new transgenic crop plants with modified properties.
  • the new properties include increasing the pathogenic defense or pathogenic resistance of plants.
  • the increased expression of the AOC after introducing the nucleic acid according to the invention into the planting material leads to an increase in metabolites which are involved in the defense against pathogens. In the same way, an increased readiness to defend against Herbivore can be brought about. This effect is based on an increased induction of the wound response after introduction and expression of the nucleic acid according to the invention in the plant material.
  • the nucleic acids according to the invention allow the plant-beneficial insect-pest interaction to be optimized.
  • the expression of the nucleic acid according to the invention in the plant leads to a changed fragrance emission spectrum of the plant. Insects that contribute to the elimination of insects harmful to plants are also attracted (tritrophic interaction). Furthermore, by introducing the nucleic acid according to the invention into plant material, the biomass formation by the modified plants can be increased. This is based on an optimization of the mycorrhizability of roots in response to the expression of the nucleic acid according to the invention.
  • the carbohydrate balance of the plants can be changed by means of the nucleic acid according to the invention.
  • This is due to the fact that enzymes that are involved in the distribution of assimalate (eg invertases) are regulated by jasmonate.
  • a changed jasmonate level thus changes the activity of the enzymes mentioned. me.
  • an assimilate shift can be achieved, with an increased accumulation in the storage organs and during seed ripening being possible.
  • the nitrogen balance can be changed by means of the nucleic acid according to the invention, in particular if the nucleic acid according to the invention is expressed in the vegetative storage organs.
  • Vegetative storage proteins such as those found in soybeans, represent a form of N and protein storage. The expression of these proteins can be induced by wounds and jasmonates. As a result, increased AOC expression with increased jasmonate formation results in an increase in the protein content in vegetative storage organs.
  • UV protection of plants can be optimized with the aid of the nucleic acid according to the invention.
  • Anthocyanin formation is also inducible.
  • a targeted increase in the endogenous jasmonate level, for example in epidermal cells as a result of the increased AOC expression, can lead to an increase in the amount of anthocyanin in these cells.
  • Anthocyanins are known as UV protection agents.
  • plants with an increased anthocyanin content in epidermal tissue are less sensitive to UV stress.
  • nucleic acid according to the invention it is also possible to produce male sterility by introducing the nucleic acid according to the invention into pollen-forming tissue of the flower.
  • the nucleic acid is preferably used in an antisense orientation under a regulatory element, as a result of which the internal expression of the AOC is reduced.
  • Tissue-specific expression of the introduced nucleic acid can also be achieved by means of tissue-specific promoters.
  • ⁇ -Linolenic acid plays an important role in flower development.
  • ⁇ -Linolenic acid is the precursor of jasmonic acid in the tapetum, the pollen-forming tissue of the flower, and is the only unsaturated fatty acid there.
  • Mutants with disorders of the ⁇ - Linolenic acid formation is deficient in jasmonate.
  • These mutants are also male sterile, as are the jasmonate-insensitive mutants. It follows that male sterile lines can be produced by inhibiting jasmonate formation by lowering AOC expression. Since flowers, such as tomato flowers, have a high expression of specific invertases as well as high jasmonate contents, the properties of the flowers can be influenced by changing the jasmonate content.
  • Fig. 2 shows the nucleotide sequence of the allen oxide cyclase cDNA clone from tomato and the amino acid sequence derived therefrom.
  • Bold arrows indicate the sequences used for the RT-PCR.
  • Broken arrows indicate the sequences that were used to amplify a fragment encoding a truncated version of the allene oxide cyclase used in an overexpression assay.
  • Putative restriction interfaces for chloroplastic signal peptides which could be predicted using the computer program ChloroP V1.1, are underlined twice.
  • FIG. 3 shows the overexpression of a truncated tomato AOC protein in bacteria, the translation of which begins at the amino acid residue in position 64.
  • the corresponding partial cDNA was subcloned into the vector pJC20 and, for the purification of the protein, cloned into a vector pJC40 provided with a histidine tail.
  • the £. Was transfected. coli strain BL21 DE3 (pLysS) used. Extracts of bacteria grown with the vector pJC40 alone as well as extracts of bacteria transformed with the partial AOC cDNA (in vector pJC40) were grown either in the absence (-) or in the presence of IPTG (+) and then separated by gel electrophoresis on SDS-PAGE. The gel was stained with Coomassie Blue (A) or a corresponding blot was used an anti-AOC antibody (B). An arrow points to the AOC protein.
  • Figure 5 shows Southern blot analysis of tomato genomic DNA (A) and RFLP analysis of the AOC locus (B). The cleavage with the different restriction enzymes showed that the AOC gene is available as a single copy.
  • Figure 6 shows Northern blot analysis of AOC mRNA accumulation in wounded tomato leaves. 10 ⁇ g total RNA was loaded per lane. It originated from non-wounded (water) or wounded leaves that were harvested at different times after the start of the experiment.
  • Fig. 5A The results of the cleavages are shown in Fig. 5A. They correspond to the mapping data, according to which a locus was determined on chromosome 2 for AOC (FIG. 5B).
  • AOC is the enzyme that catalyzes the step in the biosynthesis of JA in which the "right", naturally occurring stereo
  • FIG. 6 the level of AOC mRNA in tomato leaves increases 30 min after the tomato leaves are wounded. The maximum induction could be observed after two hours and the control amount was reached again after 8 hours. This result correlates well with the amounts of JA that were measured after wounding and that also showed transient accumulation with an increase after 1 hour. This result demonstrates an important physiological function of AOC in regulating JA levels during wound response in tomato plants.
  • AOC Another very important function of the AOC seems to be the influence on the development of flower organs.
  • a high expression was found in pistil tissue, ripe flower petals, red fruits and, in smaller quantities, in stamens. In contrast, no expression was detected in young developing flower buds.
  • Mutants that are defective in the JA signal transduction chain, such as Arabidopsis coil or the fadZ-2 fad7-2 fadQ mutants, are male sterile. This shows the importance of JA in flower development. It has also been shown that AOS genes (the synthase genes) are strongly expressed in flower organs of Arabidopsis thaliana, which suggests that JA may be synthesized in flowers.
  • Tomato plants were grown for 8 weeks, the secondary leaf cut off, wounded with a standard toothed wheel and then incubated in distilled water under continuous white light (at an intensity of 120 ⁇ m • m "2 • s " 1) ). Analysis of endogenous JA and OPDA amounts
  • the AOS activity was measured in 50 mM potassium phosphate with pH 6.7 in a total volume of 1 ml. The reaction was started by the addition of the fatty acid hydroperoxide and the decrease in absorbance at 235 nm was measured. For the determination of the kinetic parameters of AOS from maize, the initial reaction rate at 14 concentrations in the range from 5 to 90 ⁇ m was determined for each substrate. The K m and v a ⁇ values were calculated directly using the Michaelis-Menten equation and plotted according to the Eadie-Hofstee diagram. ANALYSIS OF THE CYCLE PRODUCTS OF THE ALLENOXIDE
  • Fatty acid hydroperoxides (150 ⁇ m) were at 0 ° C for 10 min. with 0.5 ml of a suspension of corn seed membranes (0.5 mg protein; 28 nkat using 13 (S) -HPOT as substrate) in 0.1 m potassium phosphate buffer pH 6.7 and 2.5 ml of the AOC preparation in Ammonium sulfate / 20 mm tris-HCL buffer pH 7.5 (total assay volume: 5 ml) stirred.
  • the activities of the AOC from corn or potatoes that were added to this assay were equivalent to 178 nmol PDA or 156 nmol PDA, as measured by the method based on RP radio HPLC.
  • Controls in which the AOC solution was replaced by potassium phosphate buffer were carried out in parallel.
  • the incubations were terminated by adding 10 ml of ethanol and the reaction products were extracted with diethyl ether, methylated and subjected to TLC (using the solvent system: ethyl acetate / toluene 15:18 vol / vol.).
  • the bands were visualized under UV light after spraying with 2-6-dichlorofluorescein and the R f of the cyclopentenone band (the methyl ester of 12-oxo-PDA and its homologues and analogs) was recorded.
  • the bacterial suspension was induced by 1 mM IPTG for 4 hours, then centrifuged, subjected to two freezing and thawing cycles and then by Ultrasound lysed in 20 mM Tris-HCl pH 7.5, 0.5 M NaCl, 0.1% Tween 20 and 10% glycerol. After a further centrifugation, the supernatants were used further for measuring the AOC activity.
  • the enzyme activity was measured on the one hand using a radioactive assay according to the method as specified below, and by determining the enantiomeric composition of OPDA.
  • the hybridization conditions were as follows: It was hybridized using the Expression Hyb TM hybridization solution from Clontech (USA) at 60 ° C. for 18 h and then for 30 min.
  • the recombinant protein expressed by the expression vector pJC40 was purified by affinity chromatography on a nitrilotriacetic acid agarose (NiNTA, Qiagen). The purity of the protein was checked by SDS-PAGE gel electrophoresis. The purified recombinant AOC enzyme was then used to produce a rabbit polyclonal antibody.
  • Northern blot analysis was carried out according to Maniatis et al. Molecular cloning: A Laboratory Manual, Cold Spring Harbor (1989) with 10 ⁇ g total RNA per lane. The blots obtained were hybridized at 65 ° C. for 16 hours with a radioactively labeled sample of the tomato AOC cDNA clone, consisting of the full-length cDNA sequence (see FIG. 2). Hybridization with the sequence according to SEQ ID No. 1 at 2xSSC, 0.1% SDS at 50 ° C., preferably for at least 30 min, is preferred. A somewhat more stringent, also preferred hybridization takes place at 1xSSC, 0.1% SDS at 50 ° C.
  • Hybridization is particularly preferred who uses the Express Hyb TM hybridization solution from Clontech (USA), hybridizing at 60 ° C. for 18 h and then at 30 min. with 2xSSC, 0.1% SDS at 50 ° C followed by 30 min. IxSSC, 0.1% SDS at 50 ° C. A uniform gel loading of the samples was checked via the ethidium bromide staining of the ribosomal RNA.
  • RNA isolation Tissues in which the endogenous JA concentration accumulates due to induction were used for RNA isolation. So were sorbitol-stressed leaves from barley (Lehmann et al., Planta 197 p. 156, 1995) and wounded tomato leaves (Pena-Cortes et al., Proc. Natl. Acad. Sei USA, p. 4106, 1995) as the starting material for further RNA isolation. While no specific PCR products could be obtained from barley, a weak band of approximately 900 bp fragment size was amplified after RT-PCR with RNA from wounded tomato leaves.
  • This PCR product was used as a probe for the screen of the tomato cDNA library, which led to the isolation of a 1 kb cDNA clone. This size roughly corresponded to the signal size that could be detected in the Northern blot analyzes. From this it could be concluded that the isolation was a full length clone.
  • the first start codon is located at position 47 and preceded by a stop codon at position 16.
  • the protein coding region comprises 732 bp which code for a protein of 244 amino acids with a calculated molecular weight mass of 26 kDa (FIG. 2).
  • the approximately 4 kDa deviation of the derived molecular weight from the tomato protein was determined by SDS-PAGE gel electrophoresis and could, at least in part, be the result of post-translational removal of amino acids at the N-terminus of the tomato protein.
  • the AOC activity of the recombinant protein was sensitive to proteinase K digestion and could be inhibited by 12,13-epoxyoctadecenoic acid, a specific AOC inhibitor, which further supports the specificity of the recombinant protein.
  • a specific property of the AOC reaction is the competing reaction between the chemical splitting of the unstable allen oxide substrate, resulting in a racemic mixture of OPDA, and the enzymatic conversion to enantiomeric OPDA.
  • a protein can therefore ultimately only be identified as AOC if the specific enantiomer 9 (S), 13 (S) -OPDA has been formed and detected.
  • AOS from flax (Song et al., Proc. Natl. Acad. Sei 90, S 8519, 1993) and Arabidopsis (Laudert et al., Plant. Mol. Biol., 31, p. 323, 1996) also showed possible ones chloroplastic signal peptides and, like the AOS, were purified from barley together with chloroplasts. Enzyme activities of LOX, hydroperoxide lyase and AOS could be demonstrated by biochemical data in the outer envelope membrane of chloroplasts (Blee and Joyard, Plant Physiol. 110, p. 445, 1996). In the case of AOS and LOX, their chloroplastic localization could also be confirmed immunocytochemically.
  • the amounts of OPDA and linolenic acid rose to 187 ng / g (90-fold) and 1813 ng / g (9-fold) within 30 and 180 min. after mechanical tissue wound.
  • Steric analysis of wound-induced OPDA showed exclusive formation (> 99%) to the 9 (S), 13 (S) stereoisomer. It could be concluded that the AOS and AOC are either localized in the chloroplast or are loosely associated in the chloroplast.
  • the reaction mixture consisted of the 100,000 g pellet of the tissue homogenate investigated with an AOS activity of 7 nkat and the sample to be investigated, resuspended in 50 mM Tris-HCl (pH 7.5). The volume was made up to 625 ⁇ l with 50 mM Tris-HCl (pH 7.5) and the enzyme reaction was started by adding 2.6 ⁇ l 10 mM [1- 1 C] 13 (S) -HPOT (final concentration 41 ⁇ M; 0 , 83 kBq [0.022 ⁇ Ci]). The mixture was incubated on ice for 10 min.
  • the reaction was stopped by adding 750 ⁇ l of MeOH, the reaction mixture was acidified with 2N HCl and the products were extracted with 4 ml of diethyl ether. The organic phase was evaporated and the residue was taken up in 110 ⁇ l acetonitrile / H 2 O / HOAc (55/45 / 0.02 v / v / v).
  • the HPLC separation of the reaction products was carried out isocratically (acetonitrile / H 2 O / HOAc 55/45 / 0.02 vol./vol./vol.) On a Eurospher 100 C18 column (5 ⁇ m, 200 ⁇ 4.6 mm; Knauer Berlin) at a flow rate of 1 ml / min.
  • the products were quantified by measuring the radioactivity using a flow cell filled with a solid scintillator (yttrium silicate 0.4 ml, RSM 100 radioactivity monitor analyzer, Raytest Isotopenmeßtechnik GmbH, Strubenhardt).
  • the amount of OPDA was compared on the one hand with the peak area of the ⁇ -ketol and on the other hand with the sum of all peak areas appearing on the chromatogram and the AOC activity was calculated as follows:
  • the AOC enzyme test was carried out with non-radioactive labeled 13 (S) -HPOT.
  • S non-radioactive labeled 13
  • the methylated reaction products were separated by GC on an SPB-1 column (30 m, 0.25 mm, 0.25 ⁇ m view; Supleco, Deisenhofen) , The injection temperature was 100 ° C, which was increased to 175 ° C at 15 ° C / min. After 2 min at 175 ° C, the temperature was raised to 230 ° C at 2.5 ° C / min.
  • the extracted reaction products were first heated at 190 ° C. for 10 min and converted into the methyl ester by adding 1 ml of ethereal diazomethane.
  • the separation of the methylation products was carried out on an HP 6890 GC system (Hewlett Packard) on a Megadex-5 GC column (12 m, 0.25 mm, coating thickness 0.25 ⁇ m) with a helium flow of 2 ml / min.
  • the temperature during the injection was 150 ° C., which remained constant for the first 15 minutes and was subsequently increased to 200 ° C. at 1 ° C./min.
  • Table 1 AOC activity and composition of the OPDA as obtained using the recombinant AOC

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Abstract

La présente invention concerne un acide nucléique codant pour une allène oxyde cyclase (AOC) végétale. La préparation du nouveau clone d'ADNc permet aujourd'hui pour la première fois de préparer en quantités importantes de l'acide jasmonique de grande pureté, par la voie de la biotechnologie.
EP01902398A 2000-02-02 2001-02-02 Gene d'allene oxyde cyclase et son utilisation pour la preparation d'acide jasmonique Withdrawn EP1252318A2 (fr)

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DE10004468 2000-02-02
DE10004468A DE10004468A1 (de) 2000-02-02 2000-02-02 Allenoxidcyclasegen und dessen Verwendung zum Herstellen von Jasmonsäure
PCT/EP2001/001148 WO2001057224A2 (fr) 2000-02-02 2001-02-02 Gene d'allene oxyde cyclase et son utilisation pour la preparation d'acide jasmonique

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PT102979A (pt) * 2003-06-26 2004-12-31 Castania Sociedade Agroflorest Genes codificantes de oxido de aleno ciclase (aoc), cistatina, beta-1,3-glucanasee de proteina taumatin-like isolados de castanheiro europeu (castanea sativa mill.)
CN110305884B (zh) * 2019-08-05 2022-11-04 云南省烟草农业科学研究院 一种提高烟草叶片茉莉酸含量的基因NtAOS1及其克隆方法与应用
WO2023204106A1 (fr) * 2022-04-19 2023-10-26 Sumitomo Chemical Company, Limited Souche de levure pour la production d'acide jasmonique

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