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WO1999022011A1 - Reduction de la teneur en chlorophylle dans des graines de plantes oleagineuses - Google Patents

Reduction de la teneur en chlorophylle dans des graines de plantes oleagineuses Download PDF

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Publication number
WO1999022011A1
WO1999022011A1 PCT/EP1998/006852 EP9806852W WO9922011A1 WO 1999022011 A1 WO1999022011 A1 WO 1999022011A1 EP 9806852 W EP9806852 W EP 9806852W WO 9922011 A1 WO9922011 A1 WO 9922011A1
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Prior art keywords
promoter
plants
seeds
plant
chlorophyll
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PCT/EP1998/006852
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German (de)
English (en)
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Bernhard Grimm
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Institut für Pflanzengenetik und Kulturpflanzenforschung
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Priority claimed from DE19752647A external-priority patent/DE19752647C1/de
Application filed by Institut für Pflanzengenetik und Kulturpflanzenforschung filed Critical Institut für Pflanzengenetik und Kulturpflanzenforschung
Priority to AU11563/99A priority Critical patent/AU1156399A/en
Priority to EP98954461A priority patent/EP1025248A1/fr
Priority to CA002306205A priority patent/CA2306205A1/fr
Publication of WO1999022011A1 publication Critical patent/WO1999022011A1/fr

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    • 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
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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • the present invention relates to methods for reducing the chlorophyll content in seeds of oil plants, in particular rapeseed, based on the expression of antisense genes in chlorophyll synthesis.
  • the invention further relates to seeds of oil plants which have a reduced chlorophyll content compared to wild type plants, and to the use of these seeds for the production of vegetable oils.
  • the cruciferous oilseed rape (Brassica napus) and turnip rape (Brassica rapa) are among the most important oil plants alongside soybeans and cottonseed.
  • the seeds of rapeseed contain around 40% fatty oil, the so-called rapeseed or turnip oil, which is usually obtained from the crushed seeds by pressing or extraction in a yield of approx. 40% and then subjected to refining.
  • the rapeseed oil obtained can then be used as cooking oil, mineral lubricant oil additive, after fat hardening for margarine production and as a raw material in the production of tree wax, plasters, leather greasing agents, etc.
  • Rapeseed oil is also known as a good source of C 20 and C 22 fatty acids, which are important as plastic processing and washing aids.
  • vegetable oils, and in particular rapeseed oil are also becoming increasingly important as biodiesel fuels.
  • biodiesel fuels In general, the areas of application of vegetable oils have been significantly expanded in recent years. With increasing environmental awareness, increasingly more environmentally friendly industrial products, such as lubricants and hydraulic oils, were developed.
  • the pigments contained in the seeds, especially the chlorophylls, and their photosensitive precursors have to be extracted in a complex manner. Apart from the fact that these extractions are time and cost intensive, they always mean a loss of the rapeseed oil yield. Although turnips have somewhat lower requirements than rapeseed in terms of vegetation duration and location, similar problems arise for these Brassicaceae in terms of seed ripening and excess chlorophyll content. The extraction and use of rapeseed oil largely corresponds to that for rapeseed oil.
  • ALA 5-aminolevulinic acid
  • glutamate via three enzyme activities (glutamyl tRNA synthetase, glutamyl tRNA reductase and glutamate 1-semialdehyde aminotransferase).
  • the protopophyrin IX is formed from hydroxymethylbilane by oxidation and side chain modifications via uroporphyrinogen III, coproporphyrinogen and protopophyrinogen IX.
  • the incorporation of a divalent metal cation produces magnesium protopophyrin IX, which is converted into chlorophyll a by further modifications, which include the incorporation of an additional isocyclic ring on ring B and the particularly important esterification of a propionate with a phytol chain.
  • European patent application 0 779 364 A2 describes an approach to reducing the chlorophyll content in transgenic plants, in which the transcript and protein content of the chlorophyll-binding proteins (chlorophyll a / b binding (CAB) proteins) of the antenna complex of Photosystem II (light harvesting complex associated with photosystem II, LHCII) is reduced.
  • This approach which is based on the expression of antisense genes for LHCII, therefore relates to proteins which bind already synthesized chlorophyll in Photosystem II, but not to enzymes which are directly involved in chlorophyll synthesis.
  • transgenic plant seeds with a reduced chlorophyll content compared to seeds of wild-type plants.
  • the present invention thus relates to the use of DNA sequences which code for enzymes in chlorophyll synthesis and whose targeted transfer to and expression in transgenic plant cells results in a reduction in chlorophyll synthesis. More specifically, the invention relates to the transfer of suitable antisense gene constructs to plants and their expression in the seed tissue.
  • the invention is based on experiments in which the amount of chlorophyll in plant seeds could be significantly reduced by the seed-specific expression of antisense genes against certain enzyme steps in tetrapyrrole synthesis and the resultant seed-specific inhibition of the activity of defined enzymes in chlorophyll synthesis.
  • Antisense genes which code complementary to the endogenous RNA for enzymes of the tetrapyrrole metabolism, reduce the endogenous RNA contents or the number of RNA molecules available for the subsequent protein biosynthesis.
  • a reduced content of endogenous RNA inevitably leads to a reduced translation, that is to say a reduced amount of protein, which in turn manifests itself in a reduced activity of the target enzyme.
  • the invention relates to all genes whose gene products catalyze steps of tetrapyrrole synthesis. Since the expression and activity of any enzyme involved in tetrapyrrole synthesis can be reduced or inhibited by targeted antisense RNA synthesis, the method according to the invention for reducing the chlorophyll content in sperm cells can use all genes of this metabolic pathway, ie also individually or in combination , be performed.
  • genes are primarily genes which are responsible for glutamyl tRNA synthetase, glutamyl tRNA reductase, glutamate 1 semialdehyde aminotransferase, magnesium chelatase or their subunits CHL I, Code CHL D, CHL H, chlorophyll synthetase and Mg protopophyrin monomethyl ester transferase.
  • the invention also relates to fragments of such genes involved in chlorophyll synthesis, the use of which within an antisense construct results in reduced activity of the target enzyme.
  • Such fragments could be referred to in the context of this invention as "antisense active" fragments, i.e. their transfer in the form of a suitable construct reduces the corresponding endogenous enzyme activity.
  • the invention relates to the use of alleles and derivatives of the genes according to the invention for reducing the chlorophyll content in plant seeds, thus also the use of nucleic acid molecules whose sequences differ from the genes according to the invention due to the degeneration of the genetic code and their transmission to plant cells in one go the
  • nucleic acid molecules which contain the antisense genes according to the invention or which have arisen from, or have been derived from, naturally occurring or by genetic engineering or chemical processes and synthesis processes.
  • This can be, for example, DNA or RNA molecules, cDNA, genomic DNA, mRNA etc.
  • GSA-AT glutamate 1 semialdehyde aminotransferase
  • GSA glutamate 1-semialdehyde
  • ALA aminolevulinic acid
  • GSA-AT The expression of a tobacco antisense RNA for GSA-AT has so far only been investigated in leaves of tobacco plants (Höfgen et al. (1994) Proc. Natl. Acad. Sei. USA 91, 1726-1730).
  • DNA sequences from two tobacco full-length cDNA clones are in the GenBank, Accession Nos. X65973 and X65974 are available.
  • a gene of chlorophyll synthesis or a fragment thereof preferably the coding region of such a gene or fragment, is in the antisense orientation, ie 3 '->5' orientation, with the 3 'end a promoter, i.e. a regulatory element that ensures the transcription of the linked gene in plant cells.
  • genes according to the invention can be expressed in plant cells under the control of constitutive, but also inducible or tissue or development-specific promoters. These are preferably seed-specific promoters, the use of which enables the targeted inhibition of chlorophyll synthesis in sperm cells.
  • seed-specific promoters that can be used in connection with the invention, mention should be made of the USP promoter (described, inter alia, in: Bäumlein et al. (1991) Mol. Gen. Genet. 459-467; Fiedler et al. ( 1993) Plant Mol. Biol. 22, 669-679; DE-C2-39 20 034), the napin promoter (Ericson et al. (1991) Eur. J. Biochem. 197, 741-746; Accession No. X 58142), the 2S albumin promoter (Krebbers et al. (1988) Plant Physiol. 87, 859-866; Accession No.
  • legumin promoter (Bäumlein et al. (1986) Nucl. Acids Res. 14, 2707-2720; Accession No. X 03677) and the Hordein promoter (Entwistle et al. (1991) Plant Mol. Biol. 17, 1217-1231; Accession No. X 60037).
  • the antisense constructs used according to the invention can additionally comprise enhancer sequences or other regulatory sequences. It is also an object of the invention to provide new plants, plant cells, parts or products which are distinguished by a reduced chlorophyll content compared to wild type plants.
  • the antisense nucleic acid molecules according to the invention are transferred to plants.
  • the transgenic oilseed rape plants are particularly preferably summer oilseed rape plants.
  • OO rapeseed plants are also suitable for the use of the processes according to the invention, i.e. Rapeseed plants that are free of erucic acid and low in glucosinolate.
  • the plants that are transformed with the nucleic acid molecules according to the invention and in which a smaller amount of chlorophylls are synthesized due to the integration of such a molecule into their genome can in principle be any plant. It is preferably an oil plant, such as oilseed rape and turnips, from which vegetable oil is obtained, in the production of which high chlorophyll contents are undesirable.
  • the invention relates in particular to propagation material from plants according to the invention, for example seeds, fruits.
  • Cuttings, tubers, rhizomes etc., this material may be above contains described transgenic plant cells, and parts of these plants such as protoplasts, plant cells and calli; seeds are particularly preferred.
  • the present invention is also based on the object of providing processes for producing plant cells and plants and parts thereof, in particular seeds, which are distinguished by a reduced chlorophyll content.
  • plant cells which have a reduced chlorophyll content due to the expression of an antisense gene construct according to the invention are produced by a process which comprises the following steps:
  • an expression cassette which comprises the following DNA sequences: a promoter which ensures transcription in plant cells; at least one nucleic acid sequence encoding an enzyme or a fragment thereof that is involved in chlorophyll synthesis, the nucleic acid sequence being coupled in antisense orientation to the 3 'end of the promoter; and optionally a termination signal for the termination of the transcription and the addition of a poly-A tail to the corresponding transcript which is coupled to the 5 'end of the nucleic acid sequence.
  • the invention relates to the use of the antisense constructions according to the invention for producing plants, in particular plant seeds, which have a reduced chlorophyll content.
  • the invention preferably relates to the use of the antisense constructions according to the invention for the production of seeds from oil plants, particularly preferably from rapeseed and turnips, which have a reduced chlorophyll content.
  • Another object of the invention is to record the possibilities of using the plants according to the invention or their cells, parts and products, in particular their seeds.
  • the invention relates in particular to the use of the plants according to the invention, in particular their seeds, for obtaining vegetable oils as raw materials for the chemical, cosmetic, pharmaceutical and food industries and as an energy source.
  • the plants according to the invention thus represent an important source for the production of vegetable oils, in particular rapeseed and turnip oils for a broad spectrum of commercial purposes.
  • Transformation selected plant functional promoter into consideration which fulfills the condition that the expression regulated by it leads to a reduced chlorophyll synthesis performance in plant cells.
  • promoters which ensure seed-specific expression appear particularly useful for this purpose. Examples of such promoters are the above-mentioned USP, napin, 2S-albumin, legumin and hordein promoters.
  • RNA + RNA is isolated from seed tissue and a cDNA library is created.
  • cDNA clones based on poly (A) * RNA molecules from a non-seed tissue are used to identify those clones from the first bank whose hybrid poly (A) + RNA Molecules are only expressed in the seed tissue. Promoters are then isolated with the help of these cDNAs identified in this way, which can then be used for the expression of the antisense.
  • cloning vectors which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells.
  • examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc.
  • the desired sequence can be matched to a suitable one - 1:
  • Restriction interface to be introduced into the vector The plasmid obtained is used for the transformation of E. co // cells. Transformed E. coli cells are grown in a suitable medium and then harvested and lysed. The plasmid is recovered. Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences. Each plasmid DNA sequence can be cloned into the same or different plasmids.
  • a large number of known techniques are available for introducing DNA into a plant host cell, and the person skilled in the art can determine the appropriate method in each case without difficulty. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, the fusion of protoplasts, the direct gene transfer of isolated DNA to protoplasts, the electroporation of DNA, the introduction of DNA using the biolistic method as well as other options.
  • plasmids When injecting and electroporation of DNA into plant cells, there are no special requirements per se for the plasmids used. The same applies to direct gene transfer. Simple plasmids such as pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary. The usual selection markers are known to the person skilled in the art and it is no problem for him to select a suitable marker. Depending on the method of introducing desired genes into the plant cell, additional DNA sequences may be required.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but often the right and left boundary of the T-DNA contained in the Ti and Ri plasmid, must be connected as a flank region to the genes to be introduced .
  • the DNA to be introduced must be cloned into special plasmids, either in an intermediate or in a binary vector.
  • the intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA.
  • Intermediate vectors cannot replicate in agrobacteria.
  • the intermediate vector can be transferred to Agrobacterium tumefaciens using a helper phasmid (conjugation).
  • Binary vectors can replicate in E. coli as well as in Agrobacteria.
  • T-DNA border region They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al. (1978) Molecular and General Genetics 163, 181-187). Serving as host cell should contain a plasmid of Agrobacterium carrying a vz 'R region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells.
  • T-DNA for the transformation of plant cells has been intensively investigated and is sufficient in EP 120 515; Hoekema in: The Binary Plant Vector System, Offsetdrokkerij Kanters BV, Alblasserdam (1985) Chapter V; Fraley et al. (1993) Crit. Rev. Plant. Sci., 4, 1-46 and An et al. (1985) EMBO J. 4, 277-287.
  • plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Whole plants can then be regenerated from the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which may contain antibiotics or biocides for the selection of transformed cells.
  • the plants are regenerated using conventional regeneration methods using known nutrient media.
  • the plants thus obtained can then be examined for the presence of the introduced DNA.
  • Other ways of introducing foreign DNA using the biolistic method or by protoplast transformation are known (cf., for example, Willmitzer L.
  • the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It normally contains a selection marker which gives the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin and others.
  • the individually selected marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA.
  • the transformed cells grow within the plant in the usual way (see also McCormick et al. (1986) Plant Cell Reports 5, 81-84).
  • the resulting plants can be grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup.
  • the resulting hybrid individuals have the corresponding phenotypic properties. Seeds can be obtained from the plants.
  • Two or more generations should be grown to ensure that the phenotypic trait is stably maintained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved.
  • transgenic lines can be determined using conventional methods, which are homozygous for the new nucleic acid molecules and which investigate their phenotypic behavior with regard to a changed chlorophyll content and compare them with that of hemizygotic lines.
  • the transmission and expression of the antisense gene constructs according to the invention can be carried out with the aid of conventional molecular biological and biochemical methods. These techniques are known to the person skilled in the art and he is easily able to choose a suitable detection method, for example a Northern blot analysis for the qualitative and quantitative detection of RNA which is specific for the coding region of the respective antisense gene, or a Southern -Blot analysis to identify the transferred DNA sequences.
  • a suitable detection method for example a Northern blot analysis for the qualitative and quantitative detection of RNA which is specific for the coding region of the respective antisense gene, or a Southern -Blot analysis to identify the transferred DNA sequences.
  • transgenic plant cells or plants as well as parts and products thereof can then be examined for their chlorophyll content.
  • analysis methods are available:
  • a reduced chlorophyll content of the transgenic plants or their parts and products compared to wild-type plants can often also be seen with the naked eye or with the aid of optical aids.
  • Example 1 Preparation of an antisense construct based on DNA sequences which code for a glutamate 1 semialdehyde aminotransferase from tobacco
  • the vector pP30T which contains the USP promoter from Viceafaba, was a pUC18 derivative (see also Bäumlein et al. (1991) supra), cut with PstI and Bglll.
  • the isolated USP promoter fragment was then cloned into a BamHI / Pstl-cut pBluescript vector (-> pUSPblue). This vector construct pUSPblue was then cut with EcoRI and Xbal to obtain an EcoRI / Xbal promoter fragment.
  • the 35S CaMV promoter was removed from the binary vector BinAR by restriction digestion with EcoRI and Xbal and replaced by this seed-specific promoter by ligation of the EcoRI / Xbal vector fragment with the USP promoter fragment (-> pUSPbin).
  • the complete cDNA sequence (Xbal - 3 '- GSA-AT-cDNA - 5' -Sall) of the tobacco-GSA aminotransferase cut from the pBluescript with Xbal and Sall was used in the Xbal-Sall-cut pUSPbin.
  • the resulting binary vector was named pUSPASGSAT (see also Figure 2).
  • any vector suitable for plant transformation can be used to produce an antisense gene consisting of a fusion of a promoter, preferably a seed-specific promoter, which ensures transcription and translation in plant cells, and DNA sequences which code for enzymes involved in chlorophyll synthesis are used.
  • a promoter preferably a seed-specific promoter, which ensures transcription and translation in plant cells, and DNA sequences which code for enzymes involved in chlorophyll synthesis are used.
  • a recombinant culture of Agrobacterium tumefaciens was washed and in medium 1 (MS medium (Murashige and Skoog (1962) Physiol. Plant 15, 473), 2.5 mM MES pH 5.5, 1 mg / 1 benzylaminopurine (BAP), 0.1 mg / 1 naphthylacetic acid (NAA), 0.01 mg / 1 gibberelic acid (GA 3 ), 200 ⁇ M acetosyringone).
  • hypocotyl of 14-day-old Brassica " ⁇ pw ⁇ seedlings was cut into 0.5 to 1 cm long segments, with the bacterial suspension for 30 min. incubated and then stored for 5 days in the dark at 21 ° C. on medium 1 containing 0.7% agar.
  • hypocotyl was at 25 ° C on medium 2 (MS medium, 2.5 mM MES pH 5.7, 30 g / 1 sucrose, 1 mg / 1 kinetin, 1 mg / 1 2,4-dichloropheneoxyacetic acid (2nd , 4-D), 0.01 mg / 1 GA 3 , 500 mg / 1 polyvinylpyrrolidone (PVP), 5 mg / 1 AgNO 3 , 5 g / 1 agarose, 250 mg / 1 carbenicillin, 100 mg / 1 kanamycin; at 150 ⁇ M photon / ⁇ r / sec).
  • MS medium MS medium, 2.5 mM MES pH 5.7, 30 g / 1 sucrose, 1 mg / 1 kinetin, 1 mg / 1 2,4-dichloropheneoxyacetic acid (2nd , 4-D), 0.01 mg / 1 GA 3 , 500 mg / 1 polyvinylpyrrolidone (PVP), 5 mg / 1 AgNO 3 , 5
  • shoot induction was carried out on medium 3 (MS medium, 2.5 mM MES pH 5.5, 20 g / 1 sucrose, 40 mg / 1 adenine, 1 mg / 1 BAP, 0.1 mg / 1 NAA , 0.01 mg / 1 GA 3 , 500 mg / 1 PVP, 5 mg / 1 AgNO 3 , 100 mg / 1 kanamycin, 5 g / 1 agarose, 250 mg / 1 carbenicillin).
  • MS medium 2.5 mM MES pH 5.5, 20 g / 1 sucrose, 40 mg / 1 adenine, 1 mg / 1 BAP, 0.1 mg / 1 NAA , 0.01 mg / 1 GA 3 , 500 mg / 1 PVP, 5 mg / 1 AgNO 3 , 100 mg / 1 kanamycin, 5 g / 1 agarose, 250 mg / 1 carbenicillin).
  • the calli which showed shoots, were placed on shoot extension medium (medium 4) in glass vessels (MS medium, 2.5 mM MES pH 5.7, 10 g / 1 sucrose, 0.0025 mg / 1 BAP, 100 mg / 1 kanamycin, 7 g / 1 agarose, 250 mg / 1 carbenicillin). After about 2-3 weeks, the shoots were transferred to medium 5 (MS medium, 2.5 mM MES pH 5.5, 7 g / 1 agarose) to form roots.
  • medium 4 MS medium, 2.5 mM MES pH 5.7, 10 g / 1 sucrose, 0.0025 mg / 1 BAP, 100 mg / 1 kanamycin, 7 g / 1 agarose, 250 mg / 1 carbenicillin.
  • transgenic oilseed rape plants were produced according to the following protocol: A recombinant culture of Agrobacterium tumefaciens (strain GV 3101) was washed and resuspended in MS medium with 2.5 mM MES pH 5.5. The hypocotyl of rape seedlings 5-7 days old was cut into explants approximately 7 mm long and precultivated in liquid CIM medium for 24 hours. The explants were then co-cultivated in 10 ml of the CIM medium with 50 ⁇ l of an overnight culture of the recombinant Agrobacterium strain for 2-3 days in the dark.
  • the CIM medium consists (per liter) of MS medium, 30 g sucrose, 500 mg MES pH 5.8, 1 mg 2.4 D, 1 mg kinetin.
  • the hypocotyl pieces were then washed and cultured on CIM medium with 5 g / 1 agarose, 20 mg kanamycin, 250 mg betabactyl, 250 mg carbenicillin for 7-10 days for callus induction.
  • the slightly swollen explants were then placed on SIM medium for shoot induction. The medium was renewed every 10-14 days.
  • the SIM medium consists (per liter) of MS medium, vitamins, 20 g sucrose, 500 mg MES pH 5.6-5.8, 2 mg zeatin, 2 mg BAP, 100 mg myo-inositol, 5 g agarose, 20 mg kanamycin, 500 mg betabactyl or carbenicillin. After the shoots had formed, the calli, which showed shoots, were placed in glass vessels with MS medium, 500 mg / 1 MES pH 5.7, 20 g / 1 sucrose, 20 mg / 1 kanamycin, 5 g / 1 agarose, 500 mg / 1 Carbenicillin transferred.
  • rapeseed plants can also be transformed using other techniques.
  • Moloney et al. (1989, Plant Cell Rep. 8. 238-242), in which an agrobacterial-mediated DNA transfer to cotyledons from 7 day old seedlings via the cut at the petiole.
  • the transformation of protoplasts using cells of different tissues is suitable (e.g. described in Thomzik (1993) In: Biotechnology in agriculture and forestry, Vol. 23, Plant protoplasts and genetic engineering IV (Bajaj, ed.) Springer-Verlag, Berlin, 170-182).
  • Plants that were transformed with the vector construct pUSPASGSAT were then examined for the expression of antisense RNA against GSA-AT.
  • chlorophyll contents and synthesis rates in the transgenic plants were determined.
  • chlorophylls were extracted from 100 mg of seed material ground in liquid nitrogen with buffered, ice-cold 80% acetone until the pellet had become colorless. The samples were diluted accordingly and the absorbance at 663, 646 and 750 nm was measured on the spectrophotometer. The formulas by Porra et al. (1989, supra).
  • Chlorophyll extraction carried out and separated by HPLC.
  • the extraction and separation was carried out according to the method of Gilmore and Yamamoto (1991, J. Chromatography 543, 137-145), modified by Kruse et al. (1995, EMBO J. 14, 3712-3720) as follows: 100 mg of seed material ground in liquid nitrogen were weighed and extracted with 100% acetone and 10 ⁇ M KOH until the pellet had become colorless (1 ⁇ 400 ⁇ l, 3 ⁇ 200 ⁇ l). The extracts were diluted 4: 1 with H 2 O for the HPLC runs in order to achieve sharper separations.
  • the chlorophylls were measured using a LiChrospher 100 HPLC RP 18 column (5 ⁇ m, Merck) at a flow of 1 ml / min. eluted with the following gradient: 100% mobile solvent A (780 ml acetonitrile; 80 ml MeOH; 30 ml Tris / HCl 0.1 M pH
  • Retention times that are known for the HPLC system can be assigned.
  • the 5-aminolevulinic acid synthesis capacity in the transgenic plants was determined. Since enzyme activities in the C5 pathway cannot be determined without purifying the enzymes, indirect methods were chosen to measure the ability of ALA formation from glutamate. On the one hand, the accumulation of ALA after incubation of LA was determined according to the following protocol: 100-300 mg of seed tissue were mixed with 40 mM levulinic acid, a potent inhibitor (substrate analog) of ALA dehydratase (ALAD), in 20 mM K 2 per batch HPO 4 / KH 2 PO 4 (pH 7.1) incubated in the light for 2-4 h.
  • ALAD potent inhibitor
  • the plant material was frozen in liquid nitrogen, homogenized and mixed well after adding 1 ml of 20 mM K 2 HPO 4 / KH 2 PO 4 (pH 7.1).
  • the ALA determination was carried out according to Mauzerall and Granick (1956, J. Biol. Chem. 219, 435-446). After centrifugation for 20 minutes at 15,000 g at 4 ° C., the same volume of 20 mM K 2 HPO 4 / KH 2 PO 4 (pH 7.1) and 100 ⁇ l of ethyl acetoacetate were pipetted into 250 ⁇ l of the supernatant. Samples that had been extracted without levulinic acid incubation at time t 0 served as a control. All samples were for exactly 10 min.
  • Fig. 1 shows the metabolic pathway of tetrapyrrole biosynthesis
  • Fig. 2 shows a restriction map of the binary described in Example 1
  • Vector pUSP-ASGSAT which contains a fusion of the USP promoter and the region coding for GSA aminotransferase in antisense orientation.
  • the vector pUSP-ASGSAT carries a kanamycin resistance gene as a plant selection marker.

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Abstract

L'invention concerne un procédé de réduction de la teneur en chlorophylle dans des graines de plantes oléagineuses, en particulier des graines de colza, procédé basé sur l'expression de gènes anti-sens de la synthèse chlorophyllienne. L'invention concerne également des graines de plantes oléagineuses présentant une teneur en chlorophylle réduite par rapport à des plantes d'espèces sauvages, ainsi que l'utilisation de ces graines pour l'obtention d'huiles végétales.
PCT/EP1998/006852 1997-10-29 1998-10-29 Reduction de la teneur en chlorophylle dans des graines de plantes oleagineuses WO1999022011A1 (fr)

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AU11563/99A AU1156399A (en) 1997-10-29 1998-10-29 Reduction of chlorophyll content in oil plant seeds
EP98954461A EP1025248A1 (fr) 1997-10-29 1998-10-29 Reduction de la teneur en chlorophylle dans des graines de plantes oleagineuses
CA002306205A CA2306205A1 (fr) 1997-10-29 1998-10-29 Reduction de la teneur en chlorophylle dans des graines de plantes oleagineuses

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DE19747739 1997-10-29
DE19747739.9 1997-10-29
DE19752647A DE19752647C1 (de) 1997-10-29 1997-11-27 Reduktiion des Chlorophyllgehaltes in Ölpflanzensamen
DE19752647.0 1997-11-27

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000075340A2 (fr) * 1999-06-04 2000-12-14 E.I. Du Pont De Nemours And Company Chelatase de magnesium
WO2001009355A2 (fr) * 1999-08-03 2001-02-08 Basf Aktiengesellschaft S-ADENOSYLMETHIONIN:Mg-PROTOPORPHYRIN-IX-O-METHYLTRANSFERASE VEGETALE, VEGETAUX A TENEUR EN CHLOROPHYLLE VARIABLE ET/OU A TOLERANCE AUX HERBICIDES VARIABLE, ET PROCEDE DE PRODUCTION
EP2522722A3 (fr) * 2005-12-09 2012-12-12 BASF Plant Science GmbH Molécules d'acide nucléique codant des polypeptides impliquées dans la régulation du métabolisme du sucre et des lipides et procédés d'utilisation VIII
CN118406702A (zh) * 2024-05-16 2024-07-30 华中农业大学 一种甘蓝型油菜BnaCHL基因及其突变体的制备方法和应用

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
CN111066437B (zh) * 2019-12-11 2021-11-05 中国烟草总公司郑州烟草研究院 一种用于降低烟叶中氯离子含量的土壤调节液及其施用方法

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EP0779364A2 (fr) * 1995-12-12 1997-06-18 Her Majesty in Right of Canada, represented by The Minister of Agriculture and Agri-Food Canada ARN-antisens de transcrit CAB pour réduire la teneur en chlorophylle de plantes

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EP0779364A2 (fr) * 1995-12-12 1997-06-18 Her Majesty in Right of Canada, represented by The Minister of Agriculture and Agri-Food Canada ARN-antisens de transcrit CAB pour réduire la teneur en chlorophylle de plantes

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BOUGRI,O. AND GRIMM, B.: "members of a low-copy number gene family encoding glutamyl-tRNA reductase are differentially expressed in barley", THE PLANT JOURNAL, vol. 9, no. 6, 1996, pages 867 - 878, XP002093836 *
GRIMM B ET AL: "MOLECULAR BIOLOGY OF CHLOROPHYLL BIOSYNTHESIS AND ITS MANIPULATION BY EXPRESSION OF ANTISENSE RNA", JOURNAL OF CELLULAR BIOCHEMISTRY. SUPPLEMENT, 1994, pages 93, XP002058315 *
HÖFGEN,R., ET AL.: "a visible marker for antisense mRNA expression in plants: inhibition of chlorophyll synthesis with a glutamate-1-semialdehyde aminotransferase antisense gene", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 91, March 1994 (1994-03-01), pages 1726 - 1730, XP002093834 *
KRUSE ET AL: "isolation and characterisation of tobacco (Nicotiana tabacum) cDNA clones encoding proteins involved in Magnesium chelation into protoporphyrin IX", PLANT MOLECULAR BIOLOGY, no. 35, 1997, pages 1053 1053, XP002076483 *
MOCK. H.P. AND GRIMM, B.: "reduction of uroporphyrinogen dearboxylase by antisense RNA expression affects activities of other enzymes involved in tetrapyrrole biosynthesis and leads to light-dependent necrosis", PLANT PHYSIOLOGY, vol. 113, 1997, pages 1101 - 1112, XP002093835 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000075340A2 (fr) * 1999-06-04 2000-12-14 E.I. Du Pont De Nemours And Company Chelatase de magnesium
WO2000075340A3 (fr) * 1999-06-04 2001-07-05 Du Pont Chelatase de magnesium
US6570063B1 (en) 1999-06-04 2003-05-27 E.I. Dupont De Nemours And Company Magnesium chelatase
WO2001009355A2 (fr) * 1999-08-03 2001-02-08 Basf Aktiengesellschaft S-ADENOSYLMETHIONIN:Mg-PROTOPORPHYRIN-IX-O-METHYLTRANSFERASE VEGETALE, VEGETAUX A TENEUR EN CHLOROPHYLLE VARIABLE ET/OU A TOLERANCE AUX HERBICIDES VARIABLE, ET PROCEDE DE PRODUCTION
WO2001009355A3 (fr) * 1999-08-03 2001-09-07 Basf Ag S-ADENOSYLMETHIONIN:Mg-PROTOPORPHYRIN-IX-O-METHYLTRANSFERASE VEGETALE, VEGETAUX A TENEUR EN CHLOROPHYLLE VARIABLE ET/OU A TOLERANCE AUX HERBICIDES VARIABLE, ET PROCEDE DE PRODUCTION
EP2522722A3 (fr) * 2005-12-09 2012-12-12 BASF Plant Science GmbH Molécules d'acide nucléique codant des polypeptides impliquées dans la régulation du métabolisme du sucre et des lipides et procédés d'utilisation VIII
CN118406702A (zh) * 2024-05-16 2024-07-30 华中农业大学 一种甘蓝型油菜BnaCHL基因及其突变体的制备方法和应用

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