EP2109676A1 - Expression de ppase modifiee dans la betterave a sucre - Google Patents

Expression de ppase modifiee dans la betterave a sucre

Info

Publication number
EP2109676A1
EP2109676A1 EP04711306A EP04711306A EP2109676A1 EP 2109676 A1 EP2109676 A1 EP 2109676A1 EP 04711306 A EP04711306 A EP 04711306A EP 04711306 A EP04711306 A EP 04711306A EP 2109676 A1 EP2109676 A1 EP 2109676A1
Authority
EP
European Patent Office
Prior art keywords
sequence
promoter
seq
nucleic acid
ppase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04711306A
Other languages
German (de)
English (en)
Inventor
Steffen Greiner
Karsten Harms
Markwart Kunz
Mohammad Munir
Thomas Rausch
Markus Schirmer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suedzucker AG
Original Assignee
Suedzucker AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suedzucker AG filed Critical Suedzucker AG
Publication of EP2109676A1 publication Critical patent/EP2109676A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/8245—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 carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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
    • C12N9/14—Hydrolases (3)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to methods and means for producing an improved sugar beet, in particular a sugar beet, which has an increased sucrose content in its storage organ, a reduced sucrose breakdown during storage and an increased growth of the beet.
  • the invention relates to the use of at least two gene constructs for generating such a plant and the nucleotide sequences used.
  • sucrose degradation is primarily mediated by enzymatic hydrolysis through a wound-induced invertase, which is primarily located in the vacuoles of the beet cells.
  • Vacuum and / or cell wall-bound invertase isoforms are also induced in de novo wounds of beet tissue (Rosenkranz, H. et al., " . Exp. Bod. 52 (2001), 2381-2385).
  • This process can be achieved by expression an invertase inhibitor (WO 98/04722) or by expressing an antisense or a dsRNA construct for the vacuolar invertase (WO 02/50109).
  • the second and PPi-dependent degradation pathway is even preferentially followed in the plant cell under anaerobic conditions which occur during storage of the beet bodies, since ATP reserves which would be used up in the first degradation pathway of sucrose are thereby obtained.
  • previously known measures for reducing sucrose loss mainly concern the inhibition of the first degradation route (for example invertase inhibition), which - Except in wounded areas -
  • invertase inhibition for example invertase inhibition
  • Other known measures consist in the general reduction of enzymatic activity by storage at low temperatures, for example below 12 ° C., with high air humidity at the same time.
  • This object is achieved according to the invention by providing a process for the production of a transgenic plant, in particular a beet plant, preferably sugar beet (Beta vulgaris), with an increased sucrose content and preferably reduced sucrose degradation during storage.
  • a transgenic plant which can be obtained by this process and has an increased sucrose content and in particular a reduced sucrose breakdown during storage.
  • the object is also achieved according to the invention by providing at least one nucleic acid molecule coding for a protein with the biological activity of a soluble pyrophosphatase from Beta vulgaris, in particular a cytosolic pyrophosphatase (C-PPase), preferably the same pyrophosphatase, which is obtained by Inserting at least one nuclear localization sequence (NLS) is changed in its compartmentalization, and by providing at least one nucleic acid olecule which encodes a promoter of a vacuolar pyrophosphatase (V-PPase) from Beta vulgaris.
  • the method according to the invention for producing a transgenic beet plant with an increased sucrose content comprises
  • V-PPase vacuolar pyrophosphatase
  • C-PPase cytosolic or nuclear-localized soluble pyrophosphatase
  • a transgenic regenerated beet plant with an increased sucrose content in the beet is obtained, which has an increased sucrose content in the beet, preferably a reduced sucrose breakdown during storage, and / or preferably a beet body which is increased by increased meristem activity.
  • vacuolar sucrose in the cytosol is primarily due to the increased activity of the ⁇ pH-dependent sucrose transport from sucrose via the tonoplast membrane to the vacuole.
  • the pH gradient required for sucrose transport is largely due to the activity of the membrane-bound V PPase dependent. This shows high activity (K M ⁇ 10 ⁇ mol / 1) even at a low concentration of the substrate pyrophosphate, while the affinity of soluble PPases is significantly lower (K M > 100 ⁇ mol / 1).
  • Plant cell especially a transgenic plant, can be obtained with increased sucrose accumulation.
  • the expression mediated according to the invention reduces the pyrophosphate content in the plant cell.
  • Particularly preferred according to the invention is the measured expression, in particular overexpression, of transgenic cytosolic or nuclear localized together, preferably simultaneously, with the expression mediated according to the invention, in particular overexpression, of transgenic vacuolar pyrophosphatase.
  • the increased pyrophosphate breakdown in the cytosol and cell nucleus also promotes various synthetic activities in the meristems of the plant, which in turn has a growth-increasing effect, so that enlarged beet bodies. be preserved.
  • the sucrose content in the vacuole is advantageously increased by the increased activity of the V-PPase, the sucrose breakdown in the cytosol is significantly reduced and the activity of the meristems, in particular localized on the periphery of the growing beet body, is increased.
  • a transgenic plant obtainable in this way has an increased growth and, in particular, an increased sucrose content, in particular already at the time of harvest.
  • the storage-related breakdown of sucrose in the plant is reduced and the transgenic plant thus obtainable is more stable in storage.
  • an “increased sucrose content” is understood to mean a content of sucrose mainly in the storage tissue of plants, in particular beets, which is normally at least 5%, in particular min. at least 10%, preferably at least 20%, particularly preferably at least 30% above the average sucrose content in corresponding tissues of comparable known beets.
  • the average Sac charosegehalt in the storage tissue of the sugar beet obtainable according to more than 18% by weight, in particular more than 21% by weight.
  • an “increased” or “increased meristem activity” or “improved meristem growth” usually means an increase in the beet growth (based on the fresh weight) by at least 5%, preferably at least 10%, particularly preferably understood to be at least 19% compared to the average growth of comparable known beets.
  • a “transgene” is understood to mean a gene which can be transfected, ie transformed, in the form of DNA or RNA, preferably cDNA, into a eukaryotic cell, as a result of which foreign genetic information in particular is introduced into the transfected eukaryotic cell At least one is under the operational control of at least one regulatory element under a "gene”. Understanding, in particular, protein-coding nucleotide sequence, that is to say one or more information-carrying sections of DNA molecules.
  • transgenes are transiently present as nucleic acid molecule (s) or are integrated into the genome of the transfected cell, which naturally do not occur in this cell, or they are integrated at a location in the genome of this cell where they do not occur naturally, that is to say transgenes are located in a different genomic environment or are present in a copy number other than the natural number or are under the control of another promoter.
  • s nucleic acid molecule
  • the first transgene which codes for a V-PPase, in particular from Beta vulgaris, preferably comprises at least one nucleic acid molecule, the sequence of this nucleic acid molecule being selected from the group consisting of
  • a modified nucleotide sequence a modified nucleic acid molecule of the modified nucleotide sequence having the nuclein acid molecule hybridized with the nucleotide sequence according to a) or b) and thereby has a sequence identity of more than 80%, preferably more than 90%, 95% or 99%.
  • the second transgene which codes for a C-PPase, in particular from Beta vulgaris, preferably comprises at least one nucleic acid molecule, the sequence of this nucleic acid molecule being selected from the group consisting of
  • acid molecule hybridized with the nucleotide sequence according to a) or b) and thereby has a sequence identity of more than 80%, preferably more than 90%, 95% or 99%.
  • the nucleotide sequence of the aforementioned C-PPase nucleic acid molecule which is preferred according to the invention also comprises at least one nucleus localization sequence.
  • the at least one first transgene is arranged on a vector.
  • the at least one second transgene can preferably also be arranged on a vector. Both first and second transgenes are particularly preferably arranged on a vector, in particular the same vector.
  • the vector is present in isolated and purified form.
  • the second transgene is arranged on the vector in the 5 'to 3' direction before the first transgene.
  • at least one first transgene is arranged on at least one first vector and at least one second transgene is arranged on at least one second vector different from the first vector.
  • the first and second transgenes are transfected simultaneously into at least one plant cell, in particular beet cell, that is to say transformed.
  • the transformation is preferred through ballistic in- jection, that is, by means of biolistic transformation, carried out in a manner known per se.
  • the transformation takes place by electrotransformation, preferably by means of electroporation, in a manner known per se.
  • the transformation is carried out in a known manner by means of Agrobacteria, preferably using in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes, as transformation agents.
  • the transformation is carried out using viruses in a manner known per se.
  • vectors are understood to mean, in particular, liposomes, cosmids, viruses, baeteriophages, shuttle vectors and other vectors customary in genetic engineering. These are preferably understood to mean plasmids. In a particularly preferred variant, this is pBinAR -Vector (Höfgen and Willmitzer, 1990) These vectors preferably have at least one further functional unit which in particular effects or contributes to stabilization and / or replication of the vector in the host organism.
  • vectors are used in which at least one nucleic acid molecule according to the invention is under the functional control of at least one regulatory element.
  • regulatory element means such elements those that ensure the transcription and / or translation of nucleic acid molecules in prokaryotic and / or eukaryotic host cells, so that a polypeptide or protein is expressed.
  • Regulatory elements can be promoters, enhancers, silencers and / or transcription termination signals.
  • Regulatory elements which are functionally linked to a nucleotide sequence according to the invention can be nucleotide sequences which come from organisms or genes other than the protein-coding nucleotide sequence itself.
  • the inventor has according to the invention, the vector used with preference at least one further regulatory element, in particular at least one intrans enhancer.
  • the vectors used are preferably equipped to overexpress the first or second transgene or both transgenes. This is achieved in particular in that the at least one first and / or the at least one second transgene on the vector is operably linked to at least one promoter.
  • the promoter is also preferably a storage-specific promoter.
  • the pro motor on the aforementioned vector a combination of the properties of the aforementioned promoters.
  • the at least one promoter is a promoter from a beet plant, in particular from Beta vulgaris. This is preferably a promoter of the vacuolar pyrophosphatase (V-PPase promoter).
  • the at least one promoter is a promoter from Arabidopsis thaliana or a promoter from the cauliflower mosaic virus (CaMV), in particular the CaMV35S promoter.
  • the at least one promoter is a sucrose synthase promoter.
  • vacuolar pyrophosphatase preferred according to the invention preferably under the control of at least one CaMV35S promoter, leads to a significantly improved energization of the vacuole, that is to say to an increased pH gradient, which mainly leads to the increased accumulation of storage materials, in particular sucrose in the vacuole leads;
  • a significantly improved energization of the vacuole that is to say to an increased pH gradient, which mainly leads to the increased accumulation of storage materials, in particular sucrose in the vacuole leads;
  • the acidification of the vacuole caused by the preferred overexpression according to the invention increases the active saccharose transport into the lumen of the vacuole.
  • C-PPase preferred according to the invention ensures that the breakdown of cytosolic or nuclear pyrophosphate (PP ⁇ ) is significantly increased compared to a non-transformed beet cell.
  • -dependent sucrose breakdown or by activating various synthetic activities (see above) to an increased meristema activity together with the increased accumulation of storage substances, in particular sucrose, in the vacuole due to the overexpression of the V-PPase, an increase in the transgenic beet obtainable according to the invention preferably occurs before harvesting, ie when the plant grows sucrose content.
  • Another object of the present invention is a nucleic acid molecule which encodes a protein with the biological activity of a soluble pyrophosphatase, in particular from Beta vulgaris, in particular a cytosolic pyrophosphatase (C- _PPase) - preferably according to the universal genetic standard code known per se, where the sequence of this nucleic acid molecule is selected from the group consisting of
  • a modified nucleotide sequence wherein a modified nucleic acid molecule of the modified nucleotide sequence hybridizes with the nucleic acid molecule with the nucleotide sequence according to a) or b) and thereby a sequence identity of more than 80%, preferably more than 90%, 95% or 99%, having.
  • Another object of the present invention is a nucleic acid molecule which encodes a protein with the biological activity of a vacuolar pyrophosphatase, in particular from Beta vulgaris - preferably according to the universal genetic standard code known per se - the sequence of this nucleic acid molecule being selected from the group consisting of
  • nucleotide sequence which encodes the amino acid sequence which is shown in sequence ID No. 5, and its complementary nucleotide sequence
  • a modified nucleotide sequence a modified nucleic acid molecule of the modified nucleotide sequence with the nucleic acid molecule with the nucleotide sequence according to a) or b) hybridizes and has a sequence identity of more than 80%, preferably more than 90%, 95% or 99%.
  • Another object of the present invention is a nucleic acid molecule which codes for a promoter of the vacuolar pyrophosphatase (V-PPase) in particular from Beta vulgaris - preferably according to the universal genetic standard code known per se - the sequence of the nucleic acid molecule being selected from the group consisting of out
  • the Nuelein Acidmolekül is preferably a DNA molecule, 'for example, cDNA or genomic DNA, or an RNA molecule, for example mRNA.
  • the nucleic acid molecule preferably comes from the sugar beet Beta vulgaris.
  • the nuclein is preferably acid molecule in isolated and purified form.
  • the invention thus also includes modified nucleic acid molecules with a modified nucleotide sequence, which are obtainable, for example, by substitution, addition, inversion and / or deletion of one or some bases of a nucleic acid molecule according to the invention, in particular within the coding sequence of a nucleic acid is called nucleic acid molecules, which can be referred to as mutants, derivatives or functional equivalents of a nucleic acid molecule according to the invention.
  • Such manipulations of the sequences are carried out, for example, in order to specifically change the amino acid sequence encoded by a nucleic acid.
  • modified nucleic acids preferred according to the invention encode modified enzymes, in particular modified vacuolar and / or cytosolic pyrophosphates and / or in particular with modified enzymatic activity, and are used in particular for the transformation of plants used for agriculture, mainly to produce transgenic plants.
  • modified enzymes in particular modified vacuolar and / or cytosolic pyrophosphates and / or in particular with modified enzymatic activity
  • nucleic acid molecules according to the invention are inserted into plasmids and using standard methods of microbiology or Molecular biology is subjected to mutagenesis or a sequence change by recombination in a manner known per se.
  • sequence changes can also be achieved by adding natural or synthetic nucleic acid sequences.
  • synthetic nucleic acid sequences are adapters or linkers, which i.a. also be used to link nucleic acid fragments to these fragments.
  • the present invention also relates to naturally occurring sequence variants of the nucleic acid molecules according to the invention or used according to the invention.
  • formulations used in connection with the present invention analogous to the formulation “modified nucleic acid molecule which hybridizes with a nucleic acid molecule” mean that a nucleic acid molecule hybridizes with another, different nucleic acid molecule in a manner known per se under moderately stringent conditions.
  • hybridization with a radioactive gene probe in a hybridization solution for example: 25% formamide, 5 x SSPE, 0.1% SDS, 5 x Denhardt's solution, 50 mg / ml herring sperm DNA, based on the composition of the individual components
  • a hybridization solution for example: 25% formamide, 5 x SSPE, 0.1% SDS, 5 x Denhardt's solution, 50 mg / ml herring sperm DNA, based on the composition of the individual components
  • the unspecifically bound probe is then removed, for example by washing the filter several times in 2 x SSC / 0.1% SDS at 42 ° C.
  • these hybridizing nucleic acid molecules preferred according to the invention have at least 80%, preferably at least 85%, 90%, 95%, 98% and particularly • preferably at least 99% homology, that is to say sequential identity at the nucleic acid level.
  • nucleotide sequences of the nucleic acid molecules to be compared are preferably compared over their entire sequence length.
  • the present invention also relates to a vector which is preferably used in the method according to the invention and which contains at least one of the sequences of the aforementioned nucleic acid molecules according to the invention.
  • this vector is preferably a viral vector.
  • this vector is preferably a plasmid, in a particularly preferred variant the pBinAR vector.
  • the vectors are preferably also recorded in which the at least one nucleic acid molecule according to the invention contained therein is operatively linked to at least one regulatory element which inhibits the transcription and synthesis of translatable nucleic acid molecules Pro and / or eukaryotic cells guaranteed.
  • regulatory elements are preferably promoters, enhancers, operators and / or transcription termination signals.
  • the aforementioned vectors according to the invention preferably additionally contain antibiotic resistance genes, herbicide resistance genes and / or other customary selection markers.
  • Another object of the present invention is a host cell which is transformed with at least one of the aforementioned vectors according to the invention, this host cell preferably a bacterial cell, a plant cell or an animal cell.
  • the present invention therefore also relates to a transgenic and preferably fertile plant which is obtained by the process according to the invention, where at least one of the cells of this plant is transformed and this plant is preferably increased by an increased sucrose content and / or an increased growth as a result Meristema activity are marked.
  • the invention also includes the progeny and further breeding obtained from the transformed plants according to the invention.
  • the present invention also relates to transgenic plant cells which have been transformed, ie transfected, with one or more nucleic acid molecule (s) according to the invention or used according to the invention, and transgenic plant cells len derived from such transformed cells.
  • Such cells contain one or more nucleic acid molecule (s) used or according to the invention, this (s) preferably being linked to regulatory DNA elements which ensure transcription in plant cells.
  • Such cells can be distinguished from naturally occurring plant cells in particular in that they contain at least one nucleic acid molecule according to the invention or used according to the invention which does not naturally occur in these cells and / or in that such a molecule integrates at one location in the genome of the cell is present on which it does not occur naturally, that is to say in a different genomic environment or in a copy number other than the natural number and / or is under the control of at least one other promoter.
  • the transgenic plant cells can be regenerated into whole plants using techniques known to those skilled in the art.
  • the plants obtainable by regeneration of the transgenic plant cells according to the invention are also the subject of the present invention.
  • the invention also relates to plants which contain at least one, but preferably a multiplicity, of cells which contain the vector systems according to the invention or the vector systems used according to the invention, but also derivatives or parts thereof, and which, due to the inclusion of these vector systems, derivatives or parts thereof, form a synthesis are capable of polypeptides (proteins) which cause a modified pyrophosphatase activity.
  • the invention thus makes it possible to provide plants of the most diverse types, genera, families, orders and classes, which in particular have the abovementioned characteristics.
  • the transgenic plants according to the invention are in principle monocotyledon or dicotyledonous plants such as Graminae, Pinidae, Magnoliidae, Ranunculidae, Caryophyllidae, Rosidae, Asteridae, Aridaee, Liliidae, Arecidae, Commelinidae and Gymnospermae but also algae, mosses , Ferns or calli, plant cell cultures etc., as well as parts, organs, tissues, harvesting or propagation materials' thereof.
  • they are preferably useful plants, in particular sucrose-synthesizing and / or storing plants such as the sugar beet.
  • Another object of the present invention is harvested material and propagation material of the aforementioned transgenic plants according to the invention, for example flowers, fruits, seeds, bulbs, roots, leaves, rhizomes, seedlings, cuttings, etc.
  • the present invention also relates to the use of at least one of the aforementioned nucleic acid molecules according to the invention for producing such a aforementioned transgenic plant with at least one transformed cell, in particular especially in connection with at least one of the aforementioned vectors.
  • sequence listing is part of this description and illustrates the present invention; it contains the sequences with SEQ ID No. 1 to 7:
  • SEQ ID No. 1 shows the 1041 nucleotides
  • SEQ ID No. 2 shows the 222 amino acid polypeptide sequence of the soluble beta-pyrophosphatase from Beta vulgaris (BSP1); SEQ ID No. 3 shows the 245 amino acids
  • DNA sequence of the nucleic acid molecule encoding the vacuolar beta pyrophosphatase from beta vulgaris of isoform I (bvpl); SEQ ID No. 5 ' shows the 764 amino acids
  • Polypeptide sequence of the vacuolar beta-pyrophosphatase from beta vulgaris of isoform I (BVP1); SEQ ID No. 6 shows the 1733 nucleotide DNA sequence of the bvpl promoter for the vacuolar beta-pyrophosphatase from Beta vulgaris of the isoform. I; SEQ ID No. 7 shows the 962 nucleotides
  • SEQ ID No. 8 shows the 18-nucleotide DNA sequence of the sense primer according to
  • SEQ ID No. 9 shows the 22 nucleotide DNA sequence of the antisense primer according to Example 1.
  • SEQ ID No. 10 shows the 38 nucleotide
  • SEQ ID No. 11 shows the 38 nucleotide DNA sequence of the antisense primer according to Example 2.
  • SEQ ID No. 12 shows the 31-nucleotide DNA sequence of the sense primer according to Example 4.
  • SEQ ID No. 13 shows the 31-nucleotide DNA sequence of the antisense primer according to Example 4.
  • SEQ ID No. 14 shows the 30-nucleotide DNA sequence of the sense primer according to Example 5.
  • SEQ ID No. 15 shows the 31-nucleotide DNA sequence of the antisense primer according to Example 5.
  • SEQ ID No. 16 shows the 34-nucleotide DNA sequence of the sense primer according to
  • SEQ ID No. 17 shows the 35 nucleotide DNA sequence of the antisense primer according to Example 6.
  • SEQ ID No. 18 shows the 20 nucleotide
  • SEQ ID No. 19 shows the 21 nucleotides • DNA sequence of an antisense primer according to Example 7.
  • SEQ ID No. 20 shows the 24-nucleotide DNA sequence of a sense primer according to Example 7.
  • SEQ ID No. 21 shows the 20 nucleotide DNA sequence of an antisense primer according to Example 7.
  • Figure 1 shows fluorescence micrographs of transformed beet cells:
  • Figure la shows a transformed Beta vulgaris
  • FIG. 1b shows the subcellular localization of the RFP Control plasmids in the plastids and FIG. 1c shows the subcellular localization of soluble pyrophosphatase (BSPl) fused with GFP in the cytoplasmic and near-core regions of the protoplast;
  • BSPl soluble pyrophosphatase
  • FIG. 2 shows biochemical properties of soluble beta-pyrophosphatase (BSPL): FIG. 2a shows the pH dependence and FIG. 2b shows the temperature dependence of the enzyme activity, FIG. 2c shows the determination of the K m value for pyrophosphate (Eadie-Hofstee diagram) ;
  • FIG. 3 shows the proton pumping activity in beets stored for three months: FIG. 3a shows the V-PPase activity, FIG. 3b shows the V-ATPase activity;
  • FIG. 4 shows Western blot analysis for BSPl in leaf and beet
  • FIG. 5 shows a Western blot analysis of the V-PPase in the sugar beet (Beta vulgaris);
  • FIG. 6 shows the Northern blot analysis of V-PPase and V-ATPase in seedlings of the sugar beet
  • FIG. 7 shows the Northern blot analysis of the V-PPase in the stress treatment of suspension culture cells of the sugar beet
  • Figure 8 shows Northern blot analysis of expression patterns after sugar beet wounding
  • FIG. 9 shows the Northern blot analysis of the development-dependent expression of the polypeptide of the V-PPase from Beta vulgaris of Isoform II (BVP2);
  • FIG. 10 shows the schematic structure of recombinant vectors: FIG. 10a shows the vector obtained according to Example 4, FIG. 10
  • FIG. 10b the vector obtained according to Example 5
  • FIG. 10c the vector obtained according to Example 6.
  • Example 1 Isolation of the cDNA sequence of a soluble pyrophosphatase from Beta vulgaris L. (BSPl)
  • RNA The total RNA according to Logemann et al. was extracted from suspension culture cells from Beta vulgaris L. (Analyt. Biochem., 163 (1987), 16-20) isolated and transcribed into cDNA by means of reverse transcriptase. On the basis of sequence comparisons, degenerate primers were produced with the aid of which a 435 bp partial cDNA sequence from the coding region of the soluble pyrophosphatase from sugar beet (bspl) was amplified by PCR:.
  • Sense primer a 435 bp partial cDNA sequence from the coding region of the soluble pyrophosphatase from sugar beet
  • the sequence of the bspl full-length cDNA (1041 bp) was then determined by RLM-RACE technology (GeneRacer TM Kit, Vitrogen, Groningen, the Netherlands), which was accordingly derived from a 666 bp long ORF which is flanked by a 118 bp long 5'-UTR and a 257 bp long 3 'UTR.
  • the amino acid sequence encoded by the ORF of the bspl cDNA is shown in SEQ ID No. 2 and has 222 amino acids.
  • Tables 1 and 2 show the biochemical properties of BSPl and the influence of divalent cations on the activity of the BSPl:
  • FIG. 2a shows the results of the pH value determination (pH 8.5)
  • FIG. 2b the results of the temperature optimum determination (53 ° C.)
  • FIG. 2c the " results of the K M value determination (160 ⁇ mol / 1 PPi ).
  • the coding region was cloned into a modified pFF i9 G vector (Timmermanns et al., J. Biotech. 14 (1990), 333-344), which instead of the ⁇ - Glucoronidase structural gene carries the sequence of the "green fluorescent protein" (GFP) (Sheen, et al., Plant J. 8 (5) (1995), 777-784).
  • GFP green fluorescent protein
  • the sense primer used for this contains a “Kozak” sequence immediately before the start ATG in order to ensure optimal translation.
  • the antisense primer contains both a PstI and a Sall interface (underlined):
  • the cell walls were digested using lytic enzymes, and after a further 24 hours the transient expression of the two fusion proteins in the protoplasts was examined by fluorescence microscopy using an inverted microscope.
  • the GFP fusion protein was analyzed using a FITC filter (excitation: 450-490 nm, emission: 515 nm long pass), in the case of the dsRED fusion protein an XF137-2 filter (excitation: 540 + 30 nm, emission : 585 nm long pass) is used.
  • Figure 1 shows the subcellular localization of BSPl determined by fluorescence microscopic GFP analysis of transformed beet cells: It can be seen from Figure la that a transformed Beta vulgaris cell is indistinguishable from an untransformed one.
  • Figure lb relates to the RFP control plasmid. It can be seen that the plastids light up red (bright) due to the plastid signal peptide of the plastid ⁇ -ECS.
  • FIG. 1c the excitation of the GFP shows that the soluble pyrophosphatase fused to the GFP has no plastid signal peptide.
  • BSPl is obviously a cytosolic or core-localized soluble pyrophosphatase. This is also called C-PPase.
  • the coding sequence for the beta vulgaris C-PPase was amplified by PCR.
  • the primers used for this were the same as in the amplification described above for the pFF ⁇ 9 :: GFP construct (Example 2).
  • the cloning into the expression vector pQE30 -. (Qia-. Gen ® , Hilden) was carried out via BamRI / Sal.
  • the construct was transformed together with a pUBS520 plasmid (Brinkmann et al., Gene 85 (1) (1989), 109-114) into E. co! I-DH5 ⁇ cells.
  • the purification of BSPl was carried out under native conditions. The cells were disrupted using a French press.
  • the lysis buffer used contained 50 mmol / 1 MOPS (pH 8), 300 mmol / 1 NaCl, 10 mmol / 1 imidazole and 5 mmol / 1 MgCl 2 .
  • N-terminal Histidine-mediated binding to a nickel-NTA matrix was carried out in several washing steps with increasing imidazole concentration (20-75 mmol / 1) under otherwise the same buffer conditions.
  • the elution was carried out analogously using 100-250 mmol / 1 imidazole.
  • reaction buffer standard: 50 mmol / 1 Tris (pH 8.5), 1 mmol / 1 pyrophosphate, 2.5 mmol / 1 MgCl 2
  • staining solution 3.4 mmol / 1 ammonium molybdate in 0.5 mol / 1 sulfuric acid, 0.5 mol / 1 SDS, 0.6 mol / 1 ascorbic acid: 6: 2: 1, v / v / v
  • the absorption was measured at 820 nm (Rojas-Belträn et al. 39 (1999), 449-461).
  • the 1041 bp full length cDNA (SEQ ID NO. 1) of the soluble pyrophosphatase (bspl) by PCR was amplified.
  • the ends of the primers were with KpnI (sense Primer) or Xbal- (antisense primer)
  • Antisense primer CTA GTC TAG AAG CCT CCT AAA CCA AAC ATG A (SEQ ID No. 13)
  • the vector obtained is shown in FIG. 10a.
  • Example 5 Cloning of the vacuolar pyrophosphatase (V-PPase) into the transformation vector pBinAR
  • Amplificate (SEQ ID No. 4) then into the vector pCR ⁇ 2.1-T0PO ⁇ (Invitrogen, Groningen, Nieder- land) intermediate cloned.
  • the amplificate obtained contains the region coding for the beta-V-PPase (BVP1) (SEQ ID No. 5).
  • the restriction sites Kpnl and Xbal of the TOPO vector located to the left and right of the insertion site were used to cut out the sequence of the V-PPase and then to ligate into the MCS of the plant transformation vector pBinAR, which was also cut by Kpnl / Xbal.
  • the vector thus obtained is shown in FIG. 10b.
  • Example 6 Preparation of the double construct by cloning the sequences of V-PPase and C-PPase in pBinAR
  • the entire expression cassette of the C-PPase was amplified from the corresponding pBinAR construct by PCR.
  • it contains the CaMV35S promoter (540 bp) and the OCS terminator (196 bp).
  • the sense primer used for the amplification binds at the 5 'end of the CaMV35S promoter and has an Apal interface
  • the antisense primer engages at the 3' end of the OCS terminator and has a Clal interface (underlined):
  • Sense primer AAG TCG GGG CCC GAA TTC CCA TGG AGT CAA AGA T (SEQ ID No. 16)
  • Antisense primer AAG TCG GGG CCC GAA TTC CCA TGG AGT CAA AGA T (SEQ ID No. 16)
  • Antisense primer AAG TCG GGG CCC GAA TTC CCA TGG AGT CAA AGA T (SEQ ID No. 16)
  • the amplificate obtained using these primers was digested with Apal and Clal and then ligated into the V-PPase / pBinAR construct, which was also digested with Apal and Clal. These two interfaces are located here between the OCS terminator and the right border region of the T-DNA. Due to the position of the Apal and Clal interfaces, the two expression cassettes are in the opposite orientation in the pBinAR double construct. The double vector is shown in FIG. 10c.
  • the promoter sequence (SEQ ID No. 6) of the V-PPase isoform I (BSPl) was isolated using a genomic DNA bank, which was generated using the Lambda-ZAP-Xhol partial fill-in vector kit (Stratagene, Ams- terdam, The Netherlands) (Lehr et al., Plant Mol. Biol., 39 (1999), 463-475).
  • the promoter sequence (SEQ ID No. 7) of isoform II (BSP2) was determined by means of "inverse ⁇ -PCR. Genomic DNA was isolated from sugar beet leaves by the method of Murray and Thompson (Nucl. Acids Res. 8 (1980), 4321-4325). After digestion with the Tagl restriction enzyme, the ends of the cleavage products were ligated so that circular DNA molecules were formed. These served in a "PCR” as a template, the • sense primer originating from the region near the 5 'of the coding region, the antisense primer originating from the 5' UTR: sense primer:
  • the homogenate was filtered through 200 ⁇ m gauze and then centrifuged for 5 min at 4200 xg. The supernatant was centrifuged for 30 min at 300,000 xg in a Beckman 50.2 Ti rotor to obtain the microsomal fraction.
  • the pellets obtained were resuspended in 50 ml of homogenization medium. 25 ml each were underlaid with 8 ml of gradient medium (5 mmol / 1 HEPES (pH 7.5), 2 mmol / 1 DTT and 25% (w / w) sucrose) and centrifuged at 100,000 xg for 90 min.
  • 1 ml of interphase which represents the tonoplast fraction, was removed from both gradients with a pateur pipette and with dilution medium
  • V-PPase proton pump activity was determined according to Palmgren (Plant Physiol., 94 (1990), 882-886). 50 ⁇ g of tonoplast protein was used. Results :
  • FIGS 3a and 3b show the H + pumping activity in beets stored for three months:
  • the specific activity of the V-ATPase is about twice as high as that of the V-PPase.
  • Vigna radiata a polyclonal antiserum from rabbit directed against the V-PPase of the mung bean (Vigna radiata) was used (Maeshima and Yoshida, J. Biol. Chem., 264 (1989), 20068- 20073).
  • V-ATPase vacuolar adenosine triphosphatase
  • each 5 ug protein enriched tonoplast fraction were in a native '12% polyacrylamide gel separated e- lektrophoretisch.
  • C-PPase 0.5 g leaf and beet material was ground in liquid nitrogen and the homogenate was taken up directly in 1 ml reducing 2x application buffer (RotinLoadl, Roth, Düsseldorf). 5 ⁇ l crude extract (corresponding to 2.5 mg fresh weight equivalents) was separated in a 15% polyacrylamide gel.
  • FIG. 4 shows the results of a Western blot analysis for BSPL:
  • BSPl is present in both the beet and the leaf.
  • FIG. 5 shows the results of a Western blot analysis for the V-PPase: • The V-PPase can be detected in the beet of Beta vulgaris.
  • Example 10 RNA extraction and Northern blot analysis
  • Beta vulgaris suspension culture cells were grown in "Ga borg B 5 " medium with 2% sucrose, with the addition of the following phytohormones: 0.2 mg / 1 kinetin, 0.5 mg / 1 naphthylacetic acid (NAA), 0.5 mg / 1 Indole-3-acetic acid (IAA) and 2 mg / 1 2,4-dichlorophenoxyacetic acid (2,4-D).
  • 6-day-old cells were first transferred to fresh medium and, after two more days, transferred to 0.9% agar plates, where they were left for 3 days.
  • the plates contained Gamborg B 5 medium with 2% sucrose, but additionally 125 mmol / 1 mannitol and 125 mmol / 1 sorbitol.
  • the cells were grown in places without mannitol and sorbitol, without phytohormones, without sucrose, without phosphate or with 100 mmol / 1 KCl or NaCl.
  • beta vulgaris seeds diploid hybrids, KWS, Einbeck
  • the dishes were covered with a plastic hood and then kept in the dark at 23 ° C (control plants germinated under light with a light / dark rhythm of 12/12 h).
  • the plants germinated in the dark were exposed to the light and their hypocotyl divided into tips (upper 0.5 cm) and base and their cotyledons at the times 0, 3, 6, 9 and 12 h after the start of the exposure harvested.
  • some of the plants were left in the dark for a further 24 h before appropriate control samples were taken.
  • V-PPase V-PPase
  • RNA was determined using the method of Logemann et al. (Analyt. Biochem., 163 (1987), 16-20). Each with 15 ug of RNA per lane was separated in a ica l, 4% Aga rosegel e- lektrophoret with a formaldehyde content of 2% '. The RNA was then transferred by capillary transfer to a nylon membrane (Duralon, Stratagene, Amsterdam) and fixed thereon by UV (Crosslinker, Stratagene, Amsterdam). The detection was carried out with Biotin-labeled probes according to Löw and Rausch
  • FIG. 6 shows a Northern blot analysis of V-PPase and V-ATPase transcripts in different tissues of 6-day-old, etiolated sugar beet seedlings which, following dark growth, had a different exposure time (0, 3, 6, 9 and 12 h, respectively) ) had been suspended.
  • some dark germs were left in the dark for a further 24 h, i.e. a total of 7 days, in order to compare their transcript quantities (lane 9 or 15) with those of the 6-day-old, seeded seedlings without light contact (lane 4 or 10) can.
  • a further control was 6-day-old light germs, which had grown under a 12/12 h light / dark rhythm at 160 ⁇ mol photons per m 2 / s (lanes 3 and 16). 15 ⁇ g of RNA were applied in each case.
  • FIG. 6 shows the results of a Northern blot analysis for the expression of V-PPase and V-ATPase in beta seedlings.
  • V-PPase is in tissues with a high division rate (hypocotyl tip) or synthesis performance (cotyledons) strongly expressed, while the expression in differentiated tissues (hypocotyl base) is low.
  • V-ATPase The subunits of V-ATPase are ' expressed less strongly in the cotyledons than in the hypocotyl base, regardless of the degree of exposure. In the divisionally active area of the hypocotyl peaks, expression is high in etiolated seedlings, but increases already a few hours after exposure. from.
  • FIGS. 7a and 7b show the results of a Northern blot analysis of the effects of various stress treatments on the transcript levels of vacuolar pyrophosphatase (isoform I and II) in suspension culture cells from Beta vulgaris L.
  • FIG. 8 shows the results of a Northern blot analysis, from which a contradicting expression pattern of V-ATPase and V-PPase genes in beta beets emerges after being wounded.
  • Example 11 Expression of V-PPase and C-PPase in Arabidopsis thaliana
  • the vacuolar pyrophosphatase (V-PPase) from Beta vulgaris or the simultaneous overexpression of both pyrophosphatases on the growth, in particular the rosette growth, of Arabidopsis thaliana, j transgenic Arabidopsis plants are provided in each case with the abovementioned methods according to the invention.
  • the pBinAR vectors used for this purpose contained the CaMV35S promoter in addition to the full-length cDNA of the respective pyrophosphatase.
  • the overexpression of the pyrophosphatases in the transgenic Arabidopsis plants leads to a significant increase in the ' total shoot dry weight (rosette) of these plants compared to the wild Arajidopsis type.
  • the simultaneous overexpression of both cytosolic and vacuolar pyrophosphatase in Arabidopsis thaliana shows a particularly strong effect on the total shoot dry weight; an increase of about 26% was achieved.
  • the transgenic plant obtainable according to the invention has increased growth as a result of increased meristema activity.
  • transgenic beta vulgaris beets were provided in each case using the abovementioned methods according to the invention in addition to the full-length cDNA of the respective pyrophosphatase, also the CaMV35S promoter.
  • the overexpression of the respective pyrophosphatases took place under the control of this CaMV35S promoter.
  • the influence on the fresh beet weight of Beta vulgaris compared to Beta vulgaris wild type 6 B 2840 was examined (Table 4).
  • transgenic beet plants obtainable according to the invention have an increased sucrose content and an increased growth as a result of increased meristem activity.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Nutrition Science (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne un procédé et un système pour produire une betterave à sucre améliorée, en particulier une betterave à sucre qui a une teneur supérieure en saccharose, une vitesse de dégradation limitée du saccharose au cours du stockage, et une croissance améliorée. L'invention a également pour objet l'utilisation d'au moins deux constructs génétiques pour produire un végétal de ce type ainsi que des séquences nucléotidiques impliquées.
EP04711306A 2003-03-20 2004-02-14 Expression de ppase modifiee dans la betterave a sucre Withdrawn EP2109676A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10313795A DE10313795A1 (de) 2003-03-20 2003-03-20 Veränderte PPase-Expression in Zuckerrübe
PCT/EP2004/001405 WO2004083440A1 (fr) 2003-03-20 2004-02-14 Expression de ppase modifiee dans la betterave a sucre

Publications (1)

Publication Number Publication Date
EP2109676A1 true EP2109676A1 (fr) 2009-10-21

Family

ID=32946238

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04711306A Withdrawn EP2109676A1 (fr) 2003-03-20 2004-02-14 Expression de ppase modifiee dans la betterave a sucre

Country Status (4)

Country Link
US (1) US20070067873A1 (fr)
EP (1) EP2109676A1 (fr)
DE (1) DE10313795A1 (fr)
WO (1) WO2004083440A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101457234A (zh) * 2007-12-14 2009-06-17 中国科学院遗传与发育生物学研究所 一种提高植物产量的方法及其表达盒
DE102008064184A1 (de) * 2008-12-22 2010-08-12 Südzucker AG Mannheim/Ochsenfurt Verfahren zur Steigerung des Saccharoseertrages beim landwirtschaftlichen Anbau von Zuckerrüben und Zuckerrohr
US20100257639A1 (en) * 2009-02-26 2010-10-07 Robert Edward Bruccoleri Methods and compositions for altering sugar beet or root crop storage tissue
CA2764073A1 (fr) * 2009-06-30 2011-01-20 E. I. Du Pont De Nemours And Company Graines de plante avec niveaux de compose de stockage alteres, produits de construction apparentes et procedes mettant en jeu des genes codant pour la pyrophosphatase cytosolique

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4035756A1 (de) * 1990-11-08 1992-05-14 Inst Genbiologische Forschung Neue plasmide zur herstellung von im habitus und ertrag veraenderten transgenen pflanzen
DE4317596A1 (de) * 1993-05-24 1994-12-01 Schering Ag DNA-Sequenzen und Plasmide zur Herstellung von Zuckerrüben mit veränderter Saccharose-Konzentration
WO1994028146A2 (fr) * 1993-05-24 1994-12-08 Hoechst Schering Agrevo Gmbh Sequences d'adn et plasmides destines a la production d'une betterave a concentration de sucre modifiee

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004083440A1 *

Also Published As

Publication number Publication date
WO2004083440A8 (fr) 2005-02-17
WO2004083440A1 (fr) 2004-09-30
US20070067873A1 (en) 2007-03-22
DE10313795A1 (de) 2004-10-07

Similar Documents

Publication Publication Date Title
DE69433941T2 (de) Gen für die fettsäure-desaturase, besagtes gen enthaltender vektor, eine pflanze, in die besagtes gen eingebracht ist, und ein verfahren zur schaffung besagter pflanze
DE69634699T2 (de) Methode zur regulation der ligninzusammensetzung in pflanzen
DE4035756A1 (de) Neue plasmide zur herstellung von im habitus und ertrag veraenderten transgenen pflanzen
EP0462065A2 (fr) Nouvelles séquences de signal
DE19502053A1 (de) Verfahren und DNA-Moleküle zur Steigerung der Photosyntheserate in Pflanzen, sowie Pflanzenzellen und Pflanzen mit gesteigerter Photosyntheserate
EP2298917B1 (fr) Promoteurs spécifiques aux tissus
DE19752647C1 (de) Reduktiion des Chlorophyllgehaltes in Ölpflanzensamen
WO2004083440A1 (fr) Expression de ppase modifiee dans la betterave a sucre
DE19852757A1 (de) Promotoren zur Genexpression in Wurzeln von Pflanzen
DE19853778C1 (de) DNA-Sequenzen kodierend einen Glutamat/Malat-Translokator, Plasmide Bakterien, Hefen und Pflanzen enthaltend diesen Transporter
DE69837396T2 (de) Genetisches verfahren
DE69730984T2 (de) Promotor aus tabak
DE19940270C2 (de) Verfahren zur Herstellung von Pflanzen mit gesteigerter Photosyntheserate
DE60008962T2 (de) Klonierung einer N-Methyltransferase, die an der Koffeinbiosynthese beteiligt ist
DE10063879B4 (de) Nucleinsäuren, die vacuoläre Invertasen codieren, Pflanzenzellen und Pflanzen, die diese enthalten sowie ihre Verwendung
EP0927246A2 (fr) Adenylosuccinate-synthetase
WO1999022011A1 (fr) Reduction de la teneur en chlorophylle dans des graines de plantes oleagineuses
EP1307561B1 (fr) Acides nucleiques a l'aide desquels peuvent etre produits des vegetaux a teneur en metabolites modifiee
DE19600357C1 (de) DNA-Sequenz kodierend einen Phosphoenolpyruvat-Phosphat-Translokator, Plasmide, Bakterien, Hefen und Pflanzen enthaltend diesen Transporter
EP1156117A2 (fr) Procédé d'isolation des modulateurs d'enzymes de la biosynthèse des caroténoides
WO1999011805A1 (fr) Molecules d'adn recombinees et procede pour augmenter la teneur en huile dans des plantes
EP0979874A2 (fr) Séquence de protéine et d'ADN encodant la pinosylvine-3-O-méthyltransférase (PMT)
WO2003014364A1 (fr) Procede pour influencer l'absorption de substances minerales chez des plantes transgeniques
WO2002066661A1 (fr) Procede pour realiser et transformer des agglomerations de mitochondries
DE10050233A1 (de) Verfahren zur genetischen Modifizierung einer Pflanze

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20051020

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20090901