WO1998006831A1 - Cellules vegetales et plantes transgeniques a formation acetyle-coa modifiee - Google Patents

Cellules vegetales et plantes transgeniques a formation acetyle-coa modifiee Download PDF

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WO1998006831A1
WO1998006831A1 PCT/EP1997/004311 EP9704311W WO9806831A1 WO 1998006831 A1 WO1998006831 A1 WO 1998006831A1 EP 9704311 W EP9704311 W EP 9704311W WO 9806831 A1 WO9806831 A1 WO 9806831A1
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acetyl
coa
transgenic plant
activity
coa hydrolase
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PCT/EP1997/004311
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German (de)
English (en)
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Lothar Willmitzer
Bernd Müller-Röber
Ursula La Cognata
Michael BÄUERLEIN
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority claimed from DE19632121A external-priority patent/DE19632121C2/de
Application filed by MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. filed Critical MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority to AU42039/97A priority Critical patent/AU739905B2/en
Priority to CA002263186A priority patent/CA2263186A1/fr
Priority to EP97940060A priority patent/EP0918849A1/fr
Publication of WO1998006831A1 publication Critical patent/WO1998006831A1/fr

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Definitions

  • the present invention relates to transgenic plant cells and plants with a modified acetyl-CoA metabolism compared to non-transformed plants and with a changed ability to form and utilize acetyl-CoA (acetyl-coenzyme A).
  • the change in the ability to produce and use acetyl-CoA is accomplished by the introduction and expression in plant cells of a DNA sequence encoding an acetyl-CoA hydrolase, preferably a deregulated or unregulated acetyl-CoA hydrolase.
  • the invention also relates to the use of DNA sequences which encode an acetyl-CoA hydrolase to increase the acetyl-CoA hydrolase activity in plant cells, in particular for the production of transgenic plant cells and plants, with modified ability to form and utilize acetyl -CoA.
  • the goals are, for example, the primary processes of photosynthesis, which lead to CO2 fixation, the transport processes, which are involved in the distribution of the photosassiates within the plant, as well as metabolic pathways, which are used to synthesize storage substances, e.g. starch, proteins, Fats, oils, rubber substances, or of secondary metabolites such as flavonoids, steroids, isoprenoids (eg flavorings), pigments or polyketides (antibiotics), or of plant pathogen defense agents.
  • the primary processes of photosynthesis which lead to CO2 fixation
  • the transport processes which are involved in the distribution of the photosassiates within the plant
  • metabolic pathways which are used to synthesize storage substances, e.g. starch, proteins, Fats, oils, rubber substances, or of secondary metabolites such as flavonoids, steroids, isoprenoids (eg flavorings), pigments or polyketides (antibiotics), or of plant pathogen defense agents.
  • a change in the acetyl-CoA formation rate is particularly important for the formation of starch, proteins, fats, oils, gums, or of secondary metabolites such as flavonoids, steroids, isoprenoids (flavorings, pigments), or polyketides (antibiotics). Since plants would be suitable for the production of various of the above-mentioned substances on a large scale due to different properties, there is a need for plants in which the formation and distribution of acetyl-CoA in the cells is changed in such a way that the formation of the above-described substances is influenced becomes.
  • the present invention is therefore based on the object of making available plant cells and plants with a modified ability to form and utilize acetyl-CoA, and also processes for their production.
  • the present invention relates to transgenic plant cells with an altered acetyl-CoA metabolism which, due to the expression of a foreign DNA sequence which encodes a protein with acetyl-CoA hydrolase activity, one compared to wild-type, ie non-transformed, cells have increased acetyl-CoA hydrolase activity.
  • the expression of such a DNA sequence leads to an increase in the intracellular acetyl-CoA hydrolase activity in the transgenic plant cells.
  • Acteyl-CoA hydrolases are enzymes that catalyze the following reaction:
  • the present invention is based on the fact that an increase in acetyl-CoA hydrolase activity in plant cells is indeed possible and leads to advantageous properties of the plant cells.
  • the increase in acetyl-CoA hydrolase activity in the mitochondria in the leaves of transgenic plants leads to an increase in the content of soluble sugars, such as, for example, glucose, fructose and sucrose, and in starch, and to a simultaneous reduction in the content of fatty acids.
  • soluble sugars such as, for example, glucose, fructose and sucrose
  • starch a simultaneous reduction in the content of fatty acids.
  • mitochondrial acetyl-CoA hydrolase activity enables a change in the partitioning of photoassimilates in the cells.
  • the acetyl-CoA hydrolase activity in the cells according to the invention is preferably increased by at least 50% and particularly preferably by 100% compared to non-transformed cells.
  • Special Another advantage is an increase in enzyme activity by more than 150% compared to non-transformed cells.
  • the increase in acetyl-CoA hydrolase activity leads to an increased concentration of acetate.
  • acetate can penetrate unregulated cellular membranes. It is therefore available in other cellular compartments in higher concentration as a substrate for the acetyl-CoA synthesis catalyzed by the acetyl-CoA synthetase. This means that it is possible, by increasing the acetyl-CoA hydrolase activity in one compartment, to change the intracellular distribution of acetyl-CoA and thus to influence the flow of metabolites in different biosynthetic pathways.
  • the present invention relates to transgenic plant cells in which the acetyl-CoA hydrolase activity is increased in the mitochondria.
  • acetyl-CoA is biosynthesized in the mitochondria through the pyrurate-dehydrogenase multienzy complex-catalyzed conversion of pyrurate.
  • the increase in acetyl-CoA hydrolase activity in the mitochondria leads to an increased concentration of acetate, which through cellular membranes in other compartments, e.g. can diffuse into the cytosol.
  • it can be used for the synthesis of acetyl-CoA, e.g. can be used by the acetyl-CoA synthetase.
  • the transgenic plant cells according to the invention therefore show an increased activity of the acetyl-CoA synthetase in the cytosol. This enzyme catalyzes the following reaction
  • the acetyl-CoA that is increasingly formed in the cytosol can be used, for example, for an increased synthesis of isoprenoids by means of valonic acid and isopentenyl pyrophoshate can be used (Bach, Lipids 30 (1995), 191-202).
  • the acetyl-CoA hydrolase activity is increased in the cytosol of the transgenic plant cells. This in turn leads to an increased acetate concentration. For example, this can lead to more acetate being available in the plastids and being converted into acetyl-CoA in these. This would increase acetyl-CoA as a substrate e.g. for fatty acid biosynthesis or isoprenoid synthesis.
  • the transgenic plant cells according to the invention with an increased acetyl-CoA hydrolase activity in the mitochondria or the cytosol additionally show an increased activity of the acetyl-CoA synthetase in the plastids. This can increase the shift of acetate into the plastids and its conversion into acetyl-CoA. This is then available, for example, to an increased extent for fatty acid biosynthesis.
  • the plant cells described above have a reduced activity of citrate synthase in the mitochondria. This enzyme catalyzes the following reaction:
  • Another preferred embodiment of the present invention provides that the activity of citrate synthase is increased in the plant cells according to the invention described above in the mitochondria or the cytosol.
  • Such an increase in the activity of citrate synthase can lead to a change in the flow of metabolites to acetyl-CoA in a specific subcellular compartment, and can in particular cause an increase in the biosynthesis of fatty acids or lipids.
  • the plant cells according to the invention described above also have a reduced activity of the ATP citrate lyase in the cytosol. This enzyme catalyzes the following reaction:
  • Another embodiment of the present invention provides that the plant cells have an increased ATP citrate lyase activity in the cytosol.
  • Such an increase can result in an increased formation of cytosolic acetyl-CoAs, which can lead to an increased synthesis of isopentenyl pyrophosphate (IPP) and thus to an increased formation of terpenoids.
  • IPP isopentenyl pyrophosphate
  • transgenic plant cells described above have, due to the modified enzyme activities described, a modified ability to form and utilize acetyl-CoA compared to wild-type cells. This can be done, for example, through the Determination of the changed amounts or ratios of metabolic end products and metabolic intermediates, as described in the examples.
  • plant cells according to the invention can be produced which have altered amounts of isoprenoids, steroids, pigments, isoprenoids, flavonoids, hormones, fats, oils, proteins, rubber substances, polyketides or substances which are involved in the defense against plants, or their soluble content Sugars, such as glucose, fructose and sucrose, as well as changes in starch. Such cells can in turn be advantageous starting materials for further uses.
  • these cells can be used for the heterologous expression of further genes with the aim of intensifying the synthesis of economically relevant substances.
  • DNA sequences can be introduced which encode the enzymes for the synthesis of polyhydroxyalkanoic acids (eg PHB and PHA).
  • PHB and PHA polyhydroxyalkanoic acids
  • acetyl-CoA can be used in plants for the synthesis of such acids, which are of great economic importance.
  • Other examples of economically interesting substances are polyketides, flavorings, rubber, alkaloids, isoprenoids etc.
  • an acetyl-CoA hydrolase in oil-storing tissues of a plant, such as the endosperm or the cotyledons of seeds or in other oil-storing organs, the flow of the photoassimilates delivered in the seeds or organs in the direction of Formation of sugars, starches, fats, pigments, isoprenoids, polyketides, steroids, flavonoids, gums, substances that are involved in plant pathogen defense, proteins, and polymers such as polyhydroxyalkanoic acids (see e.g. Poirier et al., Bio / Technology 13 ( 1995), 142-150).
  • General advantages of the cells according to the invention are the possibility of influencing the partitioning of metabolic metabolites, in particular acetyl-CoA, on the content of metabolic end products such as starch and fats, the content and the composition. Settlement of secondary metabolites, the energy balance and on the content and composition of amino acids in the cells.
  • metabolic metabolites in particular acetyl-CoA
  • the transgenic plant cells are cells of oil-storing tissue, e.g. the endosperm or cotyledons of seeds or other oil-storing organs.
  • Such cells preferably have a fat content which is at least 3%, preferably at least 5% and particularly preferably at least 7% higher than that of corresponding cells from non-transformed plants.
  • the acetyl-CoA hydrolase activity in the cells according to the invention is preferably increased by introducing and expressing DNA sequences which code for an acetyl-CoA hydrolase.
  • DNA sequences which encode a protein with the enzymatic activity of an acetyl-CoA hydrolase, can be both prokaryotic, in particular bacterial, and eukaryotic DNA sequences, i.e. DNA sequences from plants, algae, fungi or animal organisms or sequences which encode acetyl-CoA hydrolases from such organisms.
  • the DNA sequences which encode an acetyl-CoA hydrolase are sequences which encode enzymes which are deregulated or unregulated in comparison to acetyl-CoA hydrolases normally found in plant cells.
  • Deregulated means that these enzymes are not regulated in the same way as the acetyl-CoA hydrolase enzymes normally formed in unmodified plant cells.
  • these enzymes are subject to other regulatory mechanisms, ie they are not inhibited to the same extent by the inhibitors present in the plant cells or are allosterically regulated by metabolites.
  • Deregulated preferably means that the enzymes have a higher activity than endogenously expressed acetyl-CoA hydrolases in plant cells.
  • unregulated means that the enzymes in plant cells are not subject to any regulation.
  • These enzymes encoded by the sequences can be both known enzymes occurring in nature which have different regulation by various substances and also enzymes which, by mutagenesis of DNA sequences, the known enzymes from bacteria, algae, fungi , Encode animals or plants.
  • the DNA sequences used encode proteins with the enzymatic activity of an acetyl-CoA hydrolase from fungi, in particular from fungi of the genus Saccharomyces.
  • DNA sequences which encode an acetyl-CoA hydrolase from Saccharomyces cerevisiae are preferably used. Such sequences are known and described (see Lee et al., Journal of Biological Chemistry 265 (1990), 7413-7418 (accession number M31036)).
  • DNA sequences which code for mitochondrial targeting sequences must be fused with the coding region of the acetyl-CoA hydrolase. Such sequences are known, for example from Braun et al. (EMBO J. 11 (1992), 3219-3227).
  • DNA sequences which code for proteins with the enzymatic activity of an acetyl-CoA hydrolase, for example from Neurospora crassa (see EMBL accession number M31521; Marathe et al., Molecular and Cellular Biology 10 (1990), 2638-2644) and, because of their properties, can also be used to produce the plant cells according to the invention. Care must be taken to ensure that the protein is formed in mitochondria or in the cytosol of the plant cell. Techniques for modifying such DNA sequences to localize them to ensure the synthesized enzymes in mitochondria and in the cytosol of plant cells are known to the person skilled in the art.
  • acetyl-CoA hydrolases contain sequences which are necessary for secretion or for a specific subcellular localization, for example for localization in the extracellular space or the vacuole, the corresponding DNA sequences must be deleted.
  • DNA sequences which code for an acetyl-CoA hydrolase can be isolated from any organism with the aid of the already known DNA sequences. Methods for the isolation and identification of such DNA sequences are known to the person skilled in the art, for example hybridization with known sequences or by polymerase chain reaction using primers which are derived from known sequences.
  • the enzymes encoded by the identified DNA sequences are then examined for their enzyme activity and regulation.
  • the regulatory properties of the proteins encoded by the DNA sequences can be changed further in order to obtain de-regulated or unregulated enzymes compared to acetyl-CoA hydrolases naturally occurring in plants.
  • the increase in acetyl-CoA synthase, citrate synthase or ATP citrate lyase activity in the plant cells according to the invention is preferably achieved by the introduction and expression of DNA sequences which code for such enzymes.
  • sequences can be sequences which produce such enzymes from prokaryotic, in particular bacterial, or from eukaryotic organisms, e.g. Encode plants, algae, fungi or animals.
  • such enzymes are deregulated or unregulated enzymes, as explained above in connection with the acetyl-CoA hydrolase.
  • These enzymes encoded by the sequences can be both known enzymes occurring in nature, which have a different regulation by different substances, as well as enzymes, which by mutagenesis of DNA sequences, the known enzymes from bacteria, algae, Encode mushrooms, animals or plants.
  • the DNA sequences encode an acetyl-CoA synthetase with the biological properties of an acetyl-CoA synthase from fungi, in particular from those of the genus Saccharomyces, and particularly preferably from Saccharomyces cerevisiae.
  • Such sequences are accessible, for example, under the GenEMBL database access numbers Z47725, M94729, L09598, X56211 for Saccharomyces cerivisiae, in particular under X76891.
  • DNA sequences which encode bacterial acetyl-CoA synthetases Such are e.g. accessible under the access numbers M97217, M87509 or M63968.
  • DNA sequences encoding a citrate synthase are known from various organisms. Sequences which encode vegetable citrate synthases are known, for example, for Arabidopsis thaliana (GenEMBL database access number X17528; Unger et al., Plant Mol. Biol. 13 (1989), 411-418), and for tobacco, potato and sugar beet (see WO 95/24487). Furthermore, sequences are known which code animal citrate synthases, for example from pigs (accession number M21197, Evans et al., Biochemistry 27 (1988), 4680-4686). Sequences are preferably used which contain a citrate synthase with the biological Properties of a citrate synthase from bacteria, especially E.
  • coli or fungi, in particular Saccharomyces cerevisiae, code.
  • Various sequences encoding bacterial citrate synthases are available, for example, under the GenEMBL database accession numbers: M33037, Z70021, M74818, Z70017, Z70009, Z70016, L38987, Z70014, Z70022, Z70019, Z70018, Z70020, Z70012, Z70010, Z70010 Z70013, Z70015, M36338, L33409, X66112, X60513, Z73101, M29728, M17149, L41815, Z34516, M73535, L14780, X55282, L75931 and D90117.
  • a preferred sequence used is that in Ner et al. (Biochemistry 22 (1983), 5243-5249), which published a citrate synthase from E. coli coded. Sequences that citrate synthases from E. encoding coli are available, for example, under accession numbers M28987 and M28988 (see also Wilde et al., J. Gen. Microbiol. 132 (1986), 3239-3251). Sequences encoding citrate synthases from fungi are available under accession numbers D63376 and D69731, those from S. cerevisiae in particular under the access numbers Z11113, Z48951, Z71255, M54982, X88846 and X00782. The latter is preferred.
  • DNA sequences encoding an ATP citrate lyase are known, for example, from Rat (Elshourbagy et al., J. Biol. Chem. 265 (1990), 1430-1435), human (Elshourbagy et al., Eur. J. Biochem 204 (1992), 491-499), C. elegans (Wilson et al., Nature 368 (1994), 32-38) and Arabidopsis thaliana (EMBL accession numbers T13771, Z18045, Z25661 and Z26232).
  • citrate synthase or ATP-citrate lyase in the cells according to the invention can be reduced by methods known to the person skilled in the art, for example by express sion of an antisense RNA, a specific ribozyme or by means of a cosuppression effect.
  • the DNA sequences which code for the enzymes described above in plant cells can in principle be placed under the control of any promoter which is functional in plant cells.
  • the expression of the said DNA sequences can generally take place in any tissue of a plant regenerated from a transformed plant cell according to the invention and at any time, but preferably takes place in those tissues in which an altered ability to form and utilize acetyl-CoA is advantageous either for the growth of the plant or for the formation of ingredients within the plant. Promoters which ensure specific expression in a specific tissue, at a specific time of development of the plant or in a specific organ of the plant therefore appear to be particularly suitable.
  • the DNA sequences are preferably under the control of promoters which ensure seed-specific expression. In the case of starch-storing plants, e.g. In maize, wheat, barley or other cereals, this changes the ability of acetyl-CoA to form and utilize in the seeds, and there is a change in the synthesis of seeds.
  • promoters are used for increasing the fatty acid biosynthesis as a result of an increased acetyl-CoA content in seeds of oil-forming plants such as oilseed rape, soybean, sunflower and oil palms, which are specifically active in the endosper or in the cotyledons of seeds which form, such as the Phaseolin promoter from Phaseolus is vulgar, the USP promoter from Vicia faba or the HMG promoter from wheat.
  • promoters for expressing the DNA sequences which are stored in Organs such as tubers or roots are active, for example in the storage root of the sugar beet or in the tuber of the potato.
  • the expression of the DNA sequences which encode an acetyl-CoA hydrolase leads to a redirection of biosynthetic pathways in the sense of the formation of more sugar or starch and a changed formation and utilization of acetyl-CoA in the direction of fatty acid biosynthesis.
  • the expression of the DNA sequences can take place under the control of promoters which are activated specifically at the time of flowering induction, or during flowering, or which are active in tissues which are necessary for flowering induction. Promoters can also be used which are activated at a time controlled only by external influences, e.g. by light, temperature, chemical substances ( ⁇ . for example WO 93/07279). For increasing the export rate of photoassimilates from the sheet, e.g. Promoters of interest that have a cell-specific expression. Such promoters are known (e.g. the promoter of the rolC gene from Agrobacterium rhizogenes).
  • the DNA sequences which code for the enzymes described above are preferably linked, in addition to a promoter, to DNA sequences which ensure a further increase in transcription, for example so-called enhancer elements, or to DNA sequences which are in the transcribed region and which ensure a more efficient translation of the synthesized RNA into the corresponding protein.
  • Such regions can be obtained from viral genes or suitable plant genes or can be produced synthetically. They can be homologous or heterologous to the promoter used.
  • the coding DNA sequences are also linked to 3 'untranslated DNA sequences which ensure the termination of the transcription and the polyadenylation of the transcript.
  • sequences are known and described, for example that of the octopine synthase gene from Agrobacterium tumefaciens. These sequences are interchangeable.
  • the DNA sequences which are introduced and expressed in plant cells according to the invention are preferably stably integrated into the genome in the plant cells according to the invention.
  • the transgenic plant cells according to the invention can also be distinguished from non-transformed plant cells in that they have a foreign DNA stably integrated in the genome, the expression of which changes the acetyl-CoA hydrolase activity and, if appropriate, a further one of the above Enym activities described causes.
  • foreign DNA means that the DNA is either heterologous with respect to the transformed plant species, or the DNA, if it is homologous to it, is located at a location in the genome where it is not in non-transformed cells occurs. This means that the DNA is in a genomic environment in which it does not occur naturally.
  • the foreign DNA usually has the characteristic that it is recombinant, i.e. consists of several components that do not occur in nature in this combination.
  • the transgenic plant cells according to the invention can in principle be cells of any plant species. Of interest are both cells of monocotyledonous as well as dicotyledonous plant species, in particular cells that store starch or agricultural crops, such as e.g. Rye, oats, barley, wheat, potatoes, corn, rice, peas, sugar beets, tobacco, cotton, wine, tomatoes etc. or cells of ornamental plants.
  • starch or agricultural crops such as e.g. Rye, oats, barley, wheat, potatoes, corn, rice, peas, sugar beets, tobacco, cotton, wine, tomatoes etc. or cells of ornamental plants.
  • these are plant cells of oil-storing useful plants, such as, for example, rapeseed, sunflower, oil palm or soybean. Rapeseed is particularly preferred.
  • the present invention furthermore relates to transgenic plants which contain transgenic plant cells according to the invention. Such plants can be produced, for example, by regeneration from plant cells according to the invention by methods known to the person skilled in the art.
  • Plants containing cells according to the invention preferably have at least one of the following features:
  • the invention furthermore relates to propagation material of plants according to the invention which contains cells according to the invention. These include, for example, cuttings, fruit seeds, rhizomes, tubers, seedlings etc.
  • the present invention also relates to recombinant DNA molecules which contain the following elements:
  • the transfer of the DNA molecules which contain DNA sequences which encode one of the enzymes described above takes place according to methods known to the person skilled in the art, preferably using plasmids, in particular such plasmids which ensure stable integration of the DNA molecule into the genome of transformed plant cells, for example binary plasmids or Ti plasmids from the Agrojacteriujn tume facien ⁇ system.
  • plasmids in particular such plasmids which ensure stable integration of the DNA molecule into the genome of transformed plant cells
  • binary plasmids or Ti plasmids from the Agrojacteriujn tume facien ⁇ system for example binary plasmids or Ti plasmids from the Agrojacteriujn tume facien ⁇ system.
  • other systems for introducing DNA molecules into plant cells are also possible, such as the so-called biolistic method or the transformation of protoplasts (cf. Willmitzer L. (1993), Transgenic Plants, Bio
  • the present invention relates to the use of DNA sequences which encode a protein with the enzymatic activity of an acetyl-CoA hydrolase for expression in plant cells in order to increase the acetyl-CoA hydrolase activity in plant cells.
  • the invention relates to the use of such DNA sequences for the production of transgenic plant cells which, compared to non-transformed plant cells, have an altered ability to form sugars, starches, fats, pigments, isoprenoids, polyketides, steroids, flavonoids, rubber substances, substances which are involved in plant pathogen defense, have proteins and / or polymers such as polyhydroxyalkanoic acids.
  • the acetyl-CoA hydrolase activity is preferably increased in the mitochondria or in the cytosol of the plant cells.
  • FIG. 1 shows a schematic illustration of the 14.39 kb plasmid Bin-mHy-Int.
  • the plasmid contains the following fragments:
  • fragment A contains the EcoRI-A ⁇ p718 fragment of the promoter region of the 35S promoter of the
  • B fragment b (109 bp) comprises a DNA fragment with the coding region of the mitochondrial target sequence of the Proteins of the potato matrix processing peptidase (MPP) (Braun et al., EMBO J. 11 (1992), 3219-3227 (accession number X66284)).
  • MPP potato matrix processing peptidase
  • fragment C comprises a DNA fragment of the intron PIV2 from the plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet. 220 (1990), 245-250).
  • D '' Fragment D '' (1420 bp) comprises a DNA fragment with the coding region of the acetyl-CoA hydrolase gene (Lee et al., Journal of Biological Chemistry 265 (1990), 7413-7418), nucleotides 785 to 2194 (accession number M31036).
  • Fragment E (192 bp) comprises the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi-ACH5, nucleotides 11749-11939 (Gielen et al., EMBO J. 11 (1984), 3219-3227).
  • the plasmid contains the following fragments:
  • fragment A contains the EcoRI-Asp718 fragment of the promoter region of the 35S promoter of the "cauliflower osaic virus" (nucleotides 6909 to 7437) (Frank et al., Cell 21 (1980), 285-294).
  • fragment C comprises a DNA fragment of the intron PIV2 from the plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet. 220 (1990), 245-250).
  • D- * fragment D '(170 bp) comprises a DNA fragment with the 5' region of the coding region of the acetyl-CoA hydrolase gene (Lee et al., Journal of Biological Chemistry (1990) 265, 7413-7418), nucleotides 614 to 784 (accession number M31036).
  • fragment D (1420 bp) comprises a DNA fragment with the coding region of the acetyl-CoA hydrolase gene (Lee et al., Journal of Biological Chemistry (1990) 265, 7413-7418), nucleotides 785 to 2194 (accession number M31036).
  • fragment E comprises the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi-ACH5, nucleotides 11749-11939 (Gielen et al., EMBO J. 11 (1984), 3219-3227) .
  • FIG. 3 shows a Western blot for the detection of the expression of the acetyl-CoA hydrolase from Saccharomyces cerevisiae in transgenic tobacco leaves.
  • Fig. 4 shows three transgenic MB-Hyl lines compared to a control plant (left).
  • Fig. 6 shows leaves of a control plant
  • Fig. 7 shows a plant of a transgenic MB-Hyl line with flowers
  • FIG. 9 shows a schematic illustration of the 14, 25 kb Plas ids pTCSAS
  • A fragment A (528 bp) contains the EcoRI-Asp718 fragment of the promoter region of the 35S promoter of the "Cauliflower Mosaic Virus" (nucleotides 6909 to 7437) (Frank et al., Cell 21 (1980), 285-294).
  • B fragment B (1747 bp) comprises a DNA fragment with the coding region of the citrate synthase gene from tobacco in reverse orientation (nucleotides 1 to 1747) (accession number X84226).
  • fragment C comprises the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi-ACH5, nucleotides 11749-11939 (Gielen et al., EMBO J. 11 (1984), 3219-3227) .
  • the E. coli strain DH5 ⁇ (Bethesda Research Laboratories, Gaithersburgh, USA) was used.
  • the DNA was transferred by direct transformation using the method of Höfgen and Willmitzer (Nucleic Acids Res. 16 (1988), 9877).
  • the plasmid DNA of transformed agrobacteria was determined by the method of Birnboim and Doly (Nucleic Acids Res. 7 (1979), 1513-1523) isolated and analyzed by electrophoresis after suitable restriction cleavage.
  • the tobacco was transformed according to the method described in Rosahl et al. (EMBO J. 6 (1987), 1155-1159).
  • rapeseed Brassica napus
  • the leaves were then used for callus induction on MS medium with 1.6% glucose, 5 mg / 1 naphthylacetic acid, 0.2 mg / 1 benzylaminopurine, 250 mg / 1 claforan, 3 mg / 1 hygromycin and 0.80 % Bacto agar laid. After incubation at 25 ° C. and 3000 lux for one week, the leaves were inducible to shoot on MS medium with 1.6% glucose, 1.4 mg / 1 zeatin ribose, 20 mg / 1 naphthylacetic acid, 20 mg / 1 giberellic acid, 250 mg / 1 Claforan, 3 mg / 1 hygromycin and 0.80% Bacto Agar. 6. Plant husbandry
  • leaf samples were extracted in extraction buffer (50 mM Hepes-KOH pH 7.5; 5 mM MgCl 2 ; 1 mM EDTA; 1 mM EGTA; 10 mM DTT; 10% ( Vol. / Vol.) Glycerin; 0.1% (Vol. / Vol.) Triton X-100) homogenized. After centrifugation, the cell-free extracts were used for the enzyme activity measurement.
  • Reaction buffer 100 mM Na phosphate, pH 7.2
  • leaf samples were extracted in extraction buffer (50 M Hepes-KOH pH 7.5; 5 mM MgCl 2 ; 1 mM EDTA; 1 mM EGTA; 10 mM DTT; 10% (vol. / Vol.) Glycerin; 0.1% (Vol. / Vol.) Triton X-100) homogenized. After centrifugation, aliquots (10 ⁇ g protein) of the cell-free extracts were used for a Western blot.
  • extraction buffer 50 M Hepes-KOH pH 7.5; 5 mM MgCl 2 ; 1 mM EDTA; 1 mM EGTA; 10 mM DTT; 10% (vol. / Vol.) Glycerin; 0.1% (Vol. / Vol.) Triton X-100
  • Sucrose, glucose, fructose and starch were determined spectrophotometrically using coupled enzymatic reactions according to Stitt et al. (Methods in Enzymology, 174, 518-552).
  • the reaction buffer contained: 100 mM I idazol pH 6.9; 5 mM MgCl 2 ; 2mM NADP
  • the measurement is carried out at 30 ° C with 50 ⁇ l extract.
  • G6P glucose-6-phosphate
  • F1P fructose-1-phosphate
  • G1P glucose-1-phosphate
  • the pellet was disrupted with 400 ⁇ l of 0.2 N NaOH at 95 ° C. for one hour.
  • E ⁇ was neutralized with 70 ⁇ l of 1 N acetic acid at room temperature and the solid phase was separated from the liquid phase by centrifugation.
  • the starch was hydrolyzed using a range for starch determination (Boehringer, Mannheim) according to the manufacturer's instructions with the aid of amyloglucosidase and the released glucose was determined enzymatically.
  • Leaf disks each 1.1 cm in diameter or one tobacco seed in 1 ml of 1N HC1 in methanol at 80 ° C under a nitrogen atmosphere after adding 5 ⁇ g of meristylic acid as an internal standard for 15 Minutes in a sealed glass jar. After cooling to room temperature, 1 ml of 0.9% aqueous NaCl solution and 1 ml of n-hexane (p. A.) were added. After extraction of the aqueous phase, the organic phase was removed and concentrated with gaseous nitrogen.
  • the fatty acid methyl esters were separated and quantified by gas chromatography according to Browse et al. (Analytical Biochemistry 152: 141-145 (1986)).
  • Leaf disks (each 1.1 cm in diameter) were frozen in liquid nitrogen immediately after sampling. The following steps were carried out with the room light darkened.
  • the samples were homogenized with 250 ⁇ l of ice-cold 85% acetone in water, the suspension was then flushed with nitrogen gas for about 30 seconds and then stored on ice for 15 minutes. After centrifugation for 15 minutes at 4 ° C and 10,000 g, the supernatant was filtered through a Millipore-Millex-GV4 sterile filter attachment and then flushed with nitrogen gas at 4 ° C for 30 seconds. The samples were then stored at -70 ° C until measurement. The pigments were separated and quantified by high pressure liquid chromatography (HPLC) as described by Zhayer and Björkman (J. Chromatogr. 543 (1990), 137-145).
  • HPLC high pressure liquid chromatography
  • a ZORBAX ODS 5 ⁇ m non-endcapped 250 * 4.5 mm reversed phase column was used as the separation column.
  • the pigments were detected by measuring the absorption at 450 nm in a range of 0.04 absorption units (AUFS). 10. Determination of citrate synthase activity in leaves of tobacco plants
  • leaf samples were extracted in extraction buffer (50 mM Hepes-KOH pH 7.5; 5 mM MgCl 2 ; 1 mM EDTA; 1 mM EGTA; 10% (v / v) glycerin; 0 , 1% (vol. / Vol.) Triton X-100; 4 mU / ml ⁇ .2-macroglobulin) homogenized. After centrifugation, the cell-free extracts were used for the enzyme activity measurement.
  • extraction buffer 50 mM Hepes-KOH pH 7.5; 5 mM MgCl 2 ; 1 mM EDTA; 1 mM EGTA; 10% (v / v) glycerin; 0 , 1% (vol. / Vol.) Triton X-100; 4 mU / ml ⁇ .2-macroglobulin
  • Reaction temperature 30 ° C protein used: ⁇ 60 ⁇ g
  • the absorption was measured at 412 nm.
  • the coding region of the acetyl-CoA hydrolase gene from Saccharomyces cerevisiae was determined using the polymerase chain reaction (PCR) starting from genomic Saccharomyces cerevisiae DNA using the primers AcCoHyl (5 '-GTCAGGATCCATGACAATTTCTAATTTGT IDAAGCAGAGA. 3A)
  • AcC ⁇ Hy2 (5 » -GTCAGGATCCCTAGTCAACTGGTTCCCAGCTGTCGACCTT-3 ') (Seq ID No. 2) a plified.
  • the sequence of the acetyl-CoA hydrolase from Saccharomyces cerevisiae is entered in the GenEmbl database with the accession number M31036.
  • the cloning of the acetyl-CoA hydrolase gene is described in Lee et al. (Journal of Biological Chemistry 265 (1990), 7413-7418).
  • the amplified fragment corresponds to the region from nucleotides 614 to 2194 of this sequence (accession number M31036).
  • a BamHI interface was inserted at the 5 'end and at the 3' end.
  • the 1590 bp Ba HI cut PCR fragment was cloned into the BamHI site of the vector pUC9-2 via the additional cleavage sites.
  • the intron PIV2 (189 bp) from the plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet. 220 (1990), 245-250) via PCR using the primer GUS-1 (5 1 - gtatacgtaagtttctgcttctac-3 ' ) (Seq ID No. 3) and GUS-2 (5 * - gtacagctgcacatcaacaaattttgg-3 ') (Seq ID No.
  • the plasmid pUC-Hynt was cut with BamHI and the 1779 bp acetyl-CoA hydrolase fragment (with inserted intron) was cloned into the BamHI site of the plasmid pAM.
  • the plasmid obtained in this way was given the name pAM-Hylnt.
  • the plasmid pAM was prepared as described below.
  • the mitochondrial target sequence (111 bp) of the protein of the "matrix processing peptidase” (MPP) from potato was determined using the primers Mito-TPl (5'-GATCGGTACCATGTACAGATGCGCATCGTCT-3 ') (Seq ID No. 5) and Mito-TP2 (5 '-GTACGGATCCCTTGGTTGCAACAGCAGCTGA-3') (Seq ID No. 6) amplified by PCR.
  • the plasmid pMPP (Braun et al., EMBO J. 11 (1992), 3219-3227) served as the template for the PCR.
  • the amplified fragment corresponds to the region of the Nucleotides 299 to 397 of the MPP cDNA (Braun et al., See above; EMBL accession number: X66284).
  • An Asp718 interface was inserted at the 5 • end and a BamHI interface at the 3 'end.
  • the PCR fragment was cut with Asp718 and BamHI and the resulting 109 bp fragment was then cloned into the vector pA7 cut with Asp718 and BamHI (from Schaewen, A. (1989) dissertation, Freie (2015) Berlin).
  • the plasmid pAM-Hylnt was cut with Asp718 and Xbal and the 1887 bp fragment consisting of the coding region for the targeting peptide of the potato "Matrix processing peptidase" and the coding region for the acetyl-CoA hydrolase from Saccharomyces cerevisiae (with inserted intron ) isolated and the 5 'overhangs of this fragment filled to blunt ends using T4 DNA polymerase.
  • the fragment thus produced was cloned into the Smal interface of the binary plasmid pBinAR-Hyg.
  • the coding regions of the targeting peptide of the potato "matrix processing peptidase” were oriented towards the 35S RNA promoter of the cauliflower mosaic virus. This resulted in the plasmid Bin-mHy-Int (see FIG. 1), which was used for the transformation of tobacco (Nicotiana tabacu SNN) and potato (Solanum tuberosum L. cv. Desiree) as described above.
  • the BamHI fragment of the plasmid pUC-Hy-Int was cloned into the BamHI site of the plasmid pA7 (from Schaewen, A. (1989) dissertation, Free University Berlin).
  • the 5 'end of the coding region of the acetyl-CoA hydrolase was oriented towards the 35S RNA promoter.
  • the plasmid thus produced was given the name pA7-Hy-Int.
  • the plasmid pA7-Hy-Int was cut with Kpnl and Xbal, the 1778 bp fragment consisting of the coding region for the acetyl-CoA hydrolase from Saccharomyces cerevisiae (with inserted intron) and then cloned into the binary plasmid pBinAR-Hyg cut with Kpn 1 and Xbal (deposit number: DSM 9505; deposit date: 20.10.1994).
  • This resulted in the plasmid Bin-Hy-Int (see FIG. 2), which was used for the transformation of tobacco (Nicotiana tabacum SNN) and potato (Solanum tuberosu L. cv. Desiree).
  • MB-Hyl-81 3.25 ⁇ 0.79 The enzyme activities shown here are the mean of at least eight measurements based on at least three independent plants of the transgenic line mentioned.
  • FettKontrollMB-Hyl-39 MB-Hyl-78 MB-Hyl-81 acid plant [ ⁇ mol / g [ ⁇ mol / g [ ⁇ mol / g type [ ⁇ mol / g (dry (dry (dry (dry weight) weight)] weight)] weight) ]
  • the stated values represent mean values and the standard deviations from 2 independent measurements each.
  • the values given represent mean values and the standard deviations from at least 6 independent measurements.
  • the analysis of the weight of 200 seeds in each case showed that there was no significant difference between the seeds of the transgenic plant MB-Hyl-39 and the seeds of control plants (cf. Table IV ).
  • transgenic plants MB-Hyl-39, MB-Hyl-78, MB-Hyl-81 were grown in the greenhouse, it was also found that the transgenic plants had a different phenotype compared to control plants. In particular, in the case of the transgenic plants, reduced growth, the formation of several shoots and a mosaic-like change in the leaf color were found (see FIGS. 4 and 5). For a more precise analysis of the leaf color, the levels of chlorophyll a and b, as well as of the carotenoids Zeaxanthin, antheraxanthin and violoxanthin determined (see table VIII and IXa and b).
  • Violaxanthin Antheraxanthin Zeaxanthin ( ⁇ mo ⁇ / g ( ⁇ mol / g ( ⁇ mol / g (dry weight (dry weight (dry weight) weight) weight)
  • the stated values represent mean values and the standard deviations from 3 independent measurements each.
  • Plasmid pTCS obtained contains a 1747 bp cDNA fragment with the coding region of the citrate synthase gene from tobacco (accession number X84226) in the EcoRI interface of the pBluescript SK vector.
  • the BamHI fragment of the plasmid pTCS was cloned into the BamHI / SalI sites of the binary plasmid BinAR.
  • the 3 'end of the coding region of the citrate synthase was oriented towards the 35S RNA promoter.
  • the plasmid thus produced was given the name pTCSAS.
  • pTCSAS was used for the transformation of tobacco (Nicotiana tabacum SNN).
  • Regenerated tobacco plants which had been transformed with the plasmid pTCSAS were transferred into soil and selected by measuring the citrate synthase activity in leaves.
  • Several genotypes were identified that clearly showed a reduction in citrate synthase activity (see Table X).
  • Several of the selected transgenic lines were analyzed for citrate synthase activity in leaves. In some lines, a specific citrate synthase activity reduced by up to six times compared to the control plants was measured (e.g. TCSAS-14, TCSAS-17; TCSAS-26; TCSAS-43; TCSAS-48; see Table X).
  • the enzyme activities shown here are the average of at least 18 measurements from at least nine independent plants.
  • the above-mentioned genotypes TCSAS-17 and TCSAS-26 were amplified and 6 plants each were transferred to a greenhouse.
  • the values given represent mean values and the standard deviations from 10 independent measurements in each case.
  • the coding region of the acetyl-CoA-Hvdrolase gene from Saccharomyces cerevisiae was isolated by means of the polymerase chain reaction (PCR) as described in Example 1.
  • the plasmid pAM-Hylnt was cut with Asp718 and Hindlll and the 1931 kb fragment consisting of the coding region of the targeting peptide of the potato "Matrix processing peptidase" and the coding region for the acetyl-CoA hydrolase from Saccharomyces cerevisiae (with inserted intron) was isolated. The fragment isolated in this way was cloned into the Asp718 / HindIII cleavage sites of the binary plasmid pUSP-Bin19. This vector contains the USP promoter (Fiedler et al., Plant Mol. Biol.
  • the coding region of the acetyl-CoA proteinase gene (De Virgilio et al., Yea ⁇ t 8 (1992), 1043-1051) from Saccharomyces cerevisiae was determined using the polymerase chain reaction (PCR) starting from genomic S. cerevisiae DNA using the Primers ACS1 (5 » GAT CAA GCT TAT GTC GCC CTC TGC CGT ACA ATC -3 • ; Seq ID No. 7) and ACS2 (5'- GAT CAA GCT TTC ATC ATT ACA ACT TGA CCG ATC C-3 •, Seq ID No. 8) amplified.
  • PCR polymerase chain reaction
  • the sequence of the acetyl-CoA synthetase is entered in the GenEMBL database with the access number X66425.
  • the cloning of the acetyl-CoA synthetase gene is described in De Virgilio (loc. Cit.).
  • the amplified fragment corresponds to the region from nucleotides 162 to 2303 of this sequence.
  • a HindIII interface was inserted at the 5 'end and at the 3' end.
  • the 2151 bp HindIII fragment was cloned into the HindIII site of the vector pSK-TP via the additional cleavage sites.
  • This plasmid contains a DNA sequence which encodes the plastid transit peptide of ferredoxin: NADP + oxireductase from spinach.
  • the plasmid pSK-TP-ACS was cut with Asp718, the interfaces filled in with the help of T4-DNA polymerase to a smooth end and then cut Xbal.
  • the 2380 kb fragment thus isolated, consisting of the coding region of the targeting peptide of spinach ferrodoxin: NADP + oxireductase and the coding region for the acetyl-CoA synthetase from S.
  • the coding region of the ATP: citrate lyase gene from Rattus norvegicus was determined using the polymerase chain reaction (PCR) starting from cDNA from Rattus norvegicus using the primer ACLY1 (5'-ACT GAA GCC TAT GTC AGC CAA GGC AAT TTC AGA GCA -3 ', Seq ID No. 9) and ACLY2 (5'- ACT GAA GCC TTT ACA TGC TCA TGT GTT CCG GGA GAA C -3', Seq ID No. 10).
  • the sequence of the ATP: citrate lyase is in the GenEMBL database with the accession number J05210 registered.
  • the cloning of the ATP: citrate lyase gene from Rattus norvegicus is described in Elshourbagy et al. (J. Biol. Chem. 265 (1990), 1430-1435).
  • the amplified fragment corresponds to the region from nucleotides 73 to 3375 of this sequence.
  • a HindIII interface was inserted at the 5 'end and at the 3' end.
  • the 3312 bp HindIII fragment was cloned via the additional interfaces into the HindIII interface of the vector pSK-TP.
  • the plasmid pSK-TP-ACLY was cut with Asp718, filled in with blunt ends using T4 DNA polymerase and then cut with Xbal.
  • the 3494 kb fragment isolated in this way consisting of the coding region of the targeting peptide of spinach ferredoxin: NADP + oxireductase and the coding region for the ATP: citrate lyase from Rattus norvegicus, was inserted into the Smal / Xbal interfaces of the binary plasmid pBin-USP -Hyg cloned.
  • the resulting plasmid pBin-USP / Hyg-TP-ACLY was used for the transformation of oilseed rape as described above.
  • PCR polymerase chain reaction
  • the amplified fragment corresponds to the coding region from nucleotides 1 to 1284 of this sequence.
  • a BamHI interface was inserted at the 5 'end and at the 3' end.
  • the 1294-long BamHI fragment was inserted into the BamHI section via the additional cleavage sites. site of the vector pAM cloned.
  • the plasmid pAM-CS was cut with Asp718 and HindIII and filled in to blunt ends with the T4-DNA polymerase, and the 1393 kb fragment consisting of the coding region of the targeting peptide of the potato "matrix processing peptidase" and the coding region for the citrate synthase from E. coli isolated.
  • the fragment isolated in this way was cloned into the Smal sites of the binary plasmid pUSP-Bin19 (see Example 6).
  • the resulting plasmid pBin-USP-MTPCS was used on the one hand for the transformation of tobacco plants and on the other hand of rapeseed plants as described above.
  • Plants that co-express an acetyl-CoA hydrolase from yeast with mitochondrial targeting and an acetyl-CoA synthetase from yeast with plastid targeting were regenerated and grown in the greenhouse. The seeds of these plants were examined for their total fatty acid content as described above. An increase of approx. 5% with respect to the total fatty acid content (per seed) was found in comparison to non-transformed plants.

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Abstract

L'invention concerne des cellules végétales et des plantes transgéniques dont l'activité de l'hydrolase acétyle-coenzyme A est augmentée. Cette augmentation de l'activité de l'hydrolase acétyle-coenzyme A est obtenue par introduction et expression d'une séquence d'ADN codant une hydrolase acétyle-coenzyme A, de préférence une hydrolase acétyle-coenzyme A dérégulée ou non régulée, dans des cellules végétales. L'invention concerne en outre des procédés et des molécules d'ADN recombinées pour préparer des cellules végétales et des plantes dont l'activité de l'hydrolase acétyle-coenzyme A est augmentée.
PCT/EP1997/004311 1996-08-08 1997-08-07 Cellules vegetales et plantes transgeniques a formation acetyle-coa modifiee WO1998006831A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999045122A1 (fr) * 1998-03-06 1999-09-10 Metabolix, Inc. Modification du metabolisme des acides gras dans des plantes
WO2000000619A2 (fr) * 1998-06-26 2000-01-06 Iowa State University Research Foundation, Inc. MATERIAUX ET PROCEDES PERMETTANT D'ALTERER LES NIVEAUX D'ENZYMES ET D'ACETYLE CoA CHEZ LES PLANTES
WO2000011199A1 (fr) * 1998-08-20 2000-03-02 Pioneer Hi-Bred International, Inc. Compositions et procedes de modification d'un mecanisme d'action metabolique de l'acetyl-coa d'une plante
WO2002004648A2 (fr) * 2000-07-06 2002-01-17 Pioneer Hi-Bred International, Inc. Procedes de regulation de la beta-oxydation dans les vegetaux
WO2002008433A2 (fr) * 2000-07-21 2002-01-31 Washington State University Research Foundation Thioestérases d'acyl-coenzyme a
US6586658B1 (en) 1998-03-06 2003-07-01 Metabolix, Inc. Modification of fatty acid metabolism in plants
EP1411125A1 (fr) * 1998-06-26 2004-04-21 Iowa State University Research Foundation, Inc. Matières et méthodes pour la modification des taux d'enzymes et acétyl-CoA dans les plantes
WO2008043849A2 (fr) * 2006-10-13 2008-04-17 Basf Plant Science Gmbh Plantes présentant un rendement accru

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Publication number Priority date Publication date Assignee Title
WO1999045122A1 (fr) * 1998-03-06 1999-09-10 Metabolix, Inc. Modification du metabolisme des acides gras dans des plantes
US6586658B1 (en) 1998-03-06 2003-07-01 Metabolix, Inc. Modification of fatty acid metabolism in plants
EP1411125A1 (fr) * 1998-06-26 2004-04-21 Iowa State University Research Foundation, Inc. Matières et méthodes pour la modification des taux d'enzymes et acétyl-CoA dans les plantes
WO2000000619A2 (fr) * 1998-06-26 2000-01-06 Iowa State University Research Foundation, Inc. MATERIAUX ET PROCEDES PERMETTANT D'ALTERER LES NIVEAUX D'ENZYMES ET D'ACETYLE CoA CHEZ LES PLANTES
WO2000000619A3 (fr) * 1998-06-26 2000-06-15 Univ Iowa State Res Found Inc MATERIAUX ET PROCEDES PERMETTANT D'ALTERER LES NIVEAUX D'ENZYMES ET D'ACETYLE CoA CHEZ LES PLANTES
US7524678B2 (en) 1998-06-26 2009-04-28 Iowa State University Research Foundation, Inc. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants
US6942994B2 (en) 1998-06-26 2005-09-13 Iowa State University Research Foundation, Inc. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants
US6764851B2 (en) 1998-06-26 2004-07-20 Iowa State University Research Foundation, Inc. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants
WO2000011199A1 (fr) * 1998-08-20 2000-03-02 Pioneer Hi-Bred International, Inc. Compositions et procedes de modification d'un mecanisme d'action metabolique de l'acetyl-coa d'une plante
US6566584B1 (en) 1998-08-20 2003-05-20 Pioneer Hi-Bred International, Inc. Compositions and methods for altering an acetyl-CoA metabolic pathway of a plant
WO2002004648A2 (fr) * 2000-07-06 2002-01-17 Pioneer Hi-Bred International, Inc. Procedes de regulation de la beta-oxydation dans les vegetaux
US6914170B2 (en) 2000-07-06 2005-07-05 Pioneer Hi-Bred International, Inc. Methods for regulating beta-oxidation in plants
WO2002004648A3 (fr) * 2000-07-06 2002-08-29 Pioneer Hi Bred Int Procedes de regulation de la beta-oxydation dans les vegetaux
WO2002008433A3 (fr) * 2000-07-21 2003-08-28 Gregory B Tilton Thioestérases d'acyl-coenzyme a
US6878861B2 (en) 2000-07-21 2005-04-12 Washington State University Research Foundation Acyl coenzyme A thioesterases
WO2002008433A2 (fr) * 2000-07-21 2002-01-31 Washington State University Research Foundation Thioestérases d'acyl-coenzyme a
WO2008043849A2 (fr) * 2006-10-13 2008-04-17 Basf Plant Science Gmbh Plantes présentant un rendement accru
WO2008043849A3 (fr) * 2006-10-13 2008-09-04 Basf Plant Science Gmbh Plantes présentant un rendement accru
US8344205B2 (en) 2006-10-13 2013-01-01 Basf Plant Science Gmbh Plants with increased yield

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