EP1102855A1 - Method for producing plants with increased flavonoid and phenolic compound content - Google Patents

Method for producing plants with increased flavonoid and phenolic compound content

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Publication number
EP1102855A1
EP1102855A1 EP00945714A EP00945714A EP1102855A1 EP 1102855 A1 EP1102855 A1 EP 1102855A1 EP 00945714 A EP00945714 A EP 00945714A EP 00945714 A EP00945714 A EP 00945714A EP 1102855 A1 EP1102855 A1 EP 1102855A1
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European Patent Office
Prior art keywords
plants
plant
flavonoids
activity
hydroxylase
Prior art date
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Application number
EP00945714A
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German (de)
French (fr)
Inventor
Wilhelm Rademacher
Klaus Krämer
Jürgen Schweden
Karin Herbers
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BASF SE
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BASF SE
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Publication of EP1102855A1 publication Critical patent/EP1102855A1/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9771—Ginkgophyta, e.g. Ginkgoaceae [Ginkgo family]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • 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
    • 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
    • C12N15/825—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 pigment biosynthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering

Definitions

  • the present invention relates to a process for increasing the content of flavonoids and phenolic constituents in plants, characterized in that a plant is produced using molecular genetic methods in which the activity of the enzyme flavanon-3-hydroxylase is reduced.
  • the method according to the invention is characterized in that the enzyme flavanone-3-hydroxylase by molecular biological methods (eg anti-sense construct, co-suppression, the expression of specific antibodies or the expression of specific inhibitors) in whole or in part, continuously or temporarily activity in the entire plant or in parts of the plant is reduced.
  • molecular biological methods eg anti-sense construct, co-suppression, the expression of specific antibodies or the expression of specific inhibitors
  • the invention furthermore relates to plants with an increased content of flavonoids and phenolic ingredients, characterized in that their enzymatic activity of the enzyme flavonon-3-hydoxylase is reduced.
  • the invention relates to the use of plants - produced by the process according to the invention - or parts of these plants as foods, food supplements or for the preparation of curative, health-promoting or strengthening agents (juices, teas, extracts, fermentation products) for humans and animals and for the manufacture of cosmetics.
  • curative, health-promoting or strengthening agents juices, teas, extracts, fermentation products
  • phenolic substances are found in plants, eg caffeic acid, ferulic acid, chlorogenic acid, gallic acid, eugenol, lignans, coumarins, lignin, stilbenes (polydatin, resveratrol), flavonoids (flavones, catechins, flavanones, anthocyanidins, isoflavones), polymethoxylated flavones. Accordingly, phenols are also a general component of many plant-based foods and stimulants. Certain phenolic substances are of particular importance because they can exert an antioxidative effect in human or animal metabolism after ingestion with food (Baum, B. 0 .; Perun, AL Antioxidant efficiency versus structure.
  • polyphenols have various effects on cell metabolism.
  • signal transduction enzymes such as protein kinase C, tyrosine protein kinase and phosphatidylinositol-3-kinase are modulated
  • a number of healing, health-promoting or strengthening agents are therefore already obtained from suitable plants, the effect of which is based on their content of phenolic substances (Gerritsen, ME; Carley, WW; Ranges, GE; Shen, CP; Phan, SA; Ligon, GF ; Perry, CA Flavonoids inhibit cyto-kine-induced endothelial cell adhesion protein gene expression.
  • the resveratrol (along with other components) contained in white wine, but especially in red wine, is effective, for example, against heart attacks, cardiovascular diseases and cancer (Gehm, BD; McAndrews, JM; Chien, P.-Y .; Jameson, JL Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for estrogen reeeptor.
  • polymethoxylated flavones from citrus fruits also have a potential antitumor effect (Chem, J.; Montanari, AM; Widmer, WW Two new polymethoxylated flavones, a class of compounds with potential anticancer activity, isolated from cold pressed dancy tangerine peel oil solids, J Agric Food Chem 45: 364-368, (1997)).
  • the object of the present invention was to find a simple and inexpensive method for increasing the content of flavonoids and phenolic constituents in crop plants.
  • Acylcyclohexadiones such as Prohexadione-Ca and Trinexapac-ethyl (older name: Cimectacarb) are used as bioregulators to inhibit plant growth. Their bioregulatory effect is due to the fact that they block the bio-synthesis of gibberellins that promote growth. Because of their structural relationship to 2-oxoglutaric acid, they inhibit certain dioxygenases that require 2-0xoglutaric acid as a co-substrate (Rademacher, W, Biochemical effects of plant growth retardants, in: Plant Biochemical Regulators, Gausman, HW (ed.) , Marcel Dekker, Inc., New York, pp. 169-200
  • Luteoliflavan is not normally found in apple tissue and eriodyctiol occurs only in small quantities as an intermediate in flavonoid metabolism.
  • the expected flavonoids catechin and cyanidine were not detectable in the treated tissue or only occurred in significant amounts.
  • PAL phenylalanine ammonium lyase
  • the flavonoids eriodictyol, proanthocyanidins which are substituted on the C atom 3 with hydrogen, eg luteoforol, luteoliflavan, apigeniflavan and tricetiflavan, as well as homogeneous and heterogeneous oligomers and polymers increasingly formed from the mentioned and structurally related substances.
  • the concentration of the glycosides of the flovonoids, the phenolic compounds, the chalcones and the stilbene is also increased.
  • genetically modified crop plants were generated in which the activity of F3H was reduced in whole or in part, permanently or temporarily, by anti-sense constructs, in the entire plant or in individual plant organs or tissues , so that the content of healing, health-promoting or strengthening ingredients was quantitatively and qualitatively improved.
  • the process according to the invention for increasing the content of flavonoids and phenolic compounds by expression of the flavonon-3-hydroxylase in an antisense orientation can be carried out successfully in the following crop plants, the process not being limited to the plants mentioned: grapevine, cherry , Tomato, plum, sloe, blueberry, strawberry, citrus (such as orange, grapefruit), papaya, red cabbage, broccoli, Brussels sprouts, cocoa, kale, carrot, parsley, celery, onions, garlic, tea, coffee, hops, soy , Rapeseed, oats, wheat, rye, Aronia melanocarpa, Ginko biloba.
  • the invention furthermore relates to plants with an increased content of flavonoids and phenolic constituents produced by the process according to the invention, characterized in that their enzymatic activity of the enzyme flavanon-3-hydroxylase is reduced.
  • flavonoids and phenolic constituents produced by the process according to the invention, characterized in that their enzymatic activity of the enzyme flavanon-3-hydroxylase is reduced.
  • other molecular genetic methods known from the literature such as co-suppression or the expression of specific antibodies, can also be used to achieve this effect .
  • the invention relates to the use of plants - produced by the process according to the invention - or parts of these plants as food, food supplements or for the production of curative, health-promoting or strengthening agents (juices, teas, extracts, fermentation products) for humans and animals and for Manufacture of cosmetics.
  • NF-kB redox-sensitive transcription factors
  • Ripe tomato fruits from Lycopersicon esculentum Mill.cv. Moneymakers were washed, dried and the pericarp was freed from seeds, middle columnella and wooden parts using a sterile blade.
  • the pericarp (approx. 50 g) was frozen in liquid nitrogen. The material was then crushed in a mixer. The comminuted material was mixed with 100 ml homogenizing medium in a pre-cooled mortar. The suspension was then transferred to centrifuge cups by pressing through sterile gauze cloths. Then 1/10 vol 10% SDS was added and mixed well. After 10 minutes on ice, 1 volume of phenol / chloroform was added, the centrifuge cup closed and mixed well.
  • the supernatant was transferred to a new reaction vessel. This was followed by three further phenol / chloroform extractions and one chloroform extraction. In the following, 1 vol 3 M NaAC and 2.5 vol ethanol were added. The nucleic acids were precipitated overnight at -20 ° C. The next morning, the nucleic acids were pelleted for 15 minutes at 10,000 rpm in the refrigerated centrifuge (4 ° C). The supernatant was discarded and the pellet resuspended in 5 ml of cold 3 M NaAc. This washing step was repeated twice. The pellet was washed with 80% ethanol. The completely dried pellet was taken up in about 0.5 ml of sterile DEPC water and the RNA concentration was determined photometrically.
  • RNA 20 ⁇ g of total RNA were first mixed with 3.3 ⁇ l of 3M sodium acetate solution, 2 ⁇ l of IM magnesium sulfate solution and made up to 100 ⁇ l of final volume with DEPC water.
  • a microliter RNase-free DNase (Boehringer Mannheim) was added and incubated at 37 ° for 45 min. After removing the enzyme by shaking with phenol / chloroform / isoamyl alcohol, the RNA was precipitated with ethanol and the pellet was taken up in 100 ⁇ l DEPC water. 2.5 ⁇ g RNA from this solution were transcribed into cDNA using a cDNA kit (Gibco BRL).
  • sequence of the oligonucleotide derived using the peptide sequence DHQAW was as follows: 5'-CTT CAC ACA (C / G / T) GC (C / T) TG (A / G) TG (A / G) TC-3.
  • the PCR reaction was carried out using the Perkin-Elmer tTth polymerase according to the manufacturer's instructions. 1/8 of the cDNA was used as template (corresponds to 0.3 ⁇ g RNA).
  • the PCR program was:
  • the fragment was cloned into Promega's vector pGEM-T according to the manufacturer's instructions.
  • the correctness of the fragment was checked by sequencing.
  • the PCR fragment was isolated using the restriction sites Ncol and Pstl present in the polylinker of the vector pGEM-T and the protruding ends were blunt-ended using the T4 polymerase. This fragment
  • Fragment A (529 bp) contains the 35S promoter of the CaMV (nucleotides 6909 to 7437 of the cauliflower mosaic virus).
  • Fragment B the fragment of the F3H gene in antisense orientation.
  • Fragment C (192 bp) contains the termination signal of the octopine synthase gene. Cloning of a larger cDNA fragment of Flavanone-3-hydroxylase from Lycopersicon esculentum Mill.cv. Moneymaker using the 5'RACE system.
  • a second antisense construct should be generated using a larger F3H fragment.
  • the 5'RACE method (System for Rapid amplification of cDNA ends) was used to clone a larger fragment of the F3H.
  • the cDNA first strand synthesis was carried out according to the manufacturer's instructions using the GSP-1 (gene-specific primer) 5'-TTCAC-CACTGCCTGGTGGTCC-3 '. Following an RNase digest, the cDNA was purified using the GlassMAX spin system from Life Tecgnologies TM in accordance with the manufacturer's instructions.
  • a cytosine homopolymer was added to the 3 'end of the purified single-stranded F3H cDNA using the terminal deoxynucleotydil transferase according to the manufacturer's instructions.
  • the 5 'extended F3H cDNA was amplified using a second gene-specific primer (GSP-2) which binds in the region 3' before the GSP-1 recognition sequence and thus enables a "nested” PCR.
  • GSP-2 second gene-specific primer
  • the 5 'primer was that of Manufacturer supplied "5 'RACE abrided anchor primer", which is complementary to the homopolymeric dC tail of the cDNA.
  • the cDNA fragment amplified in this way and designated as FSH extended was cloned into the vector pGEM-T from Promega according to the manufacturer's instructions.
  • the identity of the cDNA was confirmed by sequencing.
  • the FSHextended cDNA fragment was isolated using the restriction sites Ncol and Pstl present in the polylinker of the vector pGEM-T and the protruding ends under Conversion using T4 polymerase and blunt ends.
  • This fragment was cloned into a Smal (blunt) cut vector pBinAR (Höfgen and Willmitzer, 1990) (see Figure 3).
  • This vector mediates resistance to the antibiotic kanamycin in plants.
  • the DNA constructs obtained contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants.
  • Fragment A (529 bp) contains the 35S promoter of the CaMV (nucleotides 6909 to 7437 of the cauliflower mosaic virus).
  • Fragment B the fragment of the F3H gene in antisense orientation.
  • Fragment C (192 bp) contains the termination signal of the octopine synthesis gene.
  • Tomato seeds (Lycopersicon esculentum Mill. Cv. Moneymaker) were incubated by 10 minutes incubation in 4% sodium hypochlorite solution, then washed 3-4 times with sterile distilled water and placed on MS medium with 3% sucrose, pH 6, 1 for germination . After a germination period of 7-10 d, the cotyledons could be used for the transformation.
  • Day 2 Sterile filter paper was placed on the plates coated with the tobacco suspension culture without air bubbles. The cross-cut cotyledons were placed on top with the top down. The petri dishes were incubated for 3 days in the culture room.
  • Day 5 The agrobacterial culture (LBA4404) was sedimented by centrifugation at approx. 3000 g for 10 min and resuspended in MS medium so that the OD was 0.3. The cotyledon fragments were added to this suspension, which were incubated with gentle shaking for 30 minutes at room temperature. The cotyledon fragments were then dried off somewhat on sterile filter paper and placed back on their starting plates for the continued cultivation for 3 days in the culture room.
  • Example 2 in addition a partial fragment of flavanone-3-hydroxylase in antisense orientation, an amount between 10 and 20 mg was weighed exactly and so much DMSO was added that a stock solution of 10 mM total flavonoids was formed. Dilutions of these stock solutions were made in the culture medium immediately before the start of the test. The dilutions were made in 10 steps between 10 -4 and 10 " 8 M.
  • the hepatocyte cultures were held for a total of 22 h to demonstrate that cholesterol biosynthesis was influenced by test substances A and B. It was then incubated with serum-free Williams Medium E with the addition of 14 C acetate (only tracer amounts) with the test substances in the stated concentrations for 2 h. A control was carried out for each test series. The methodology is described in detail in Gebhardt (1991) and Gebhardt, Lipids 28: 613 -619 (1993). The tracer amounts of 14 C-acetate quickly exchange with the intracellular acetyl-CoA pool and therefore enable a trouble-free determination of the incorporation of 1 C-acetate in the sterol fraction, which consists of> 90% cholesterol (Gebhardt, 1993).
  • Samples A) from the non-genetically modified tomatoes showed no effects on cholesterol biosynthesis.
  • cholesterol synthesis was significantly inhibited by samples B from the tomatoes containing a partial fragment of flavanone 3-hydroxylase in the antisense orientation.

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Abstract

The invention relates to a method for increasing flavonoid content in plants. Said method is characterized that a plant is produced using molecular genetic methods in which the activity of enzyme flavanon-3-hydroxylase is reduced.

Description

Verfahren zur Herstellung von Pflanzen mit erhöhtem Gehalt an Flavonoiden und phenolischen VerbindungenProcess for the production of plants with an increased content of flavonoids and phenolic compounds
Beschreibungdescription
Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Erhöhung des Gehalts an Flavonoiden und phenolischen Inhaltsstoffen in Pflanzen dadurch gekennzeichnet, daß mit molekulargenetischen Methoden eine Pflanze hergestellt wird in der die Aktivität des Enzyms Flavanon-3-hydroxylase reduziert ist.The present invention relates to a process for increasing the content of flavonoids and phenolic constituents in plants, characterized in that a plant is produced using molecular genetic methods in which the activity of the enzyme flavanon-3-hydroxylase is reduced.
Desweiteren ist das erfindungsgemäße Verfahren dadurch gekennzeichnet, daß das Enzym Flavanon-3-hydroxylase durch molekular- biologische Verfahren (z.B. Anti-Sense-Konstrukt, Co-Suppression, der Expression spezifischer Antikörper oder der Expression spezifischer Inhibitoren) ganz oder teilweise, andauernd oder vorübergehend, in der gesamten Pflanze oder in Teilen der Pflanze in seiner Aktivität reduziert ist.Furthermore, the method according to the invention is characterized in that the enzyme flavanone-3-hydroxylase by molecular biological methods (eg anti-sense construct, co-suppression, the expression of specific antibodies or the expression of specific inhibitors) in whole or in part, continuously or temporarily activity in the entire plant or in parts of the plant is reduced.
Weiterer Gegenstand der Erfindung sind Pflanzen mit erhöhtem Gehalt an Flavonoiden und phenolischen Inhaltsstoffen dadurch gekennzeichnet, daß deren enzymatische Aktivität des Enzyms Flava- non-3-hydoxylase reduziert ist.The invention furthermore relates to plants with an increased content of flavonoids and phenolic ingredients, characterized in that their enzymatic activity of the enzyme flavonon-3-hydoxylase is reduced.
Darüberhinaus ist Gegenstand der Erfindung die Verwendung von Pflanzen - hergestellt nach dem erfindungsgemäßen Verfahren - oder von Teilen dieser Pflanzen als Nahrungsmittel, Nahrungser- gänzungsmittel oder zur Herstellung von heilenden, gesundheits- fördernden oder stärkenden Mitteln (Säfte, Tees, Extrakte, Fermentationsprodukte) für Mensch und Tier sowie zur Herstellung von Kosmetika.In addition, the invention relates to the use of plants - produced by the process according to the invention - or parts of these plants as foods, food supplements or for the preparation of curative, health-promoting or strengthening agents (juices, teas, extracts, fermentation products) for humans and animals and for the manufacture of cosmetics.
Verschiedene phenolische Substanzen kommen in Pflanzen vor, z.B. Kaffeesäure, Ferulasäure, Chlorogensäure, Gallussäure, Eugenol , Lignane, Cumarine, Lignin, Stilbene (Polydatin, Resveratrol) , Flavonoide (Flavone, Catechine, Flavanone, Anthocyanidine, Iso- flavone) , polymethoxylierte Flavone. Demgemäß sind Phenole auch genereller Bestandteil vieler pflanzlicher Nahrungs- und Genuß - mittel. Bestimmte phenolische Substanzen sind von besonderer Bedeutung, da sie nach Aufnahme mit der Nahrung im menschlichen oder tierischen Stoffwechsel eine antioxidative Wirkung ausüben können (Baum, B. 0.; Perun, A. L. Antioxidant efficiency versus structure. Soc. Plast . Engrs Trans 2: 250-257, (1962); Gardner, P.T.; McPhail, D.B. ; Duthie, G.G. Electron spin resonance spectroscopic assessment of the antioxidant potential of teas in aqueous and organic media . J. Sei . Food Agric. 76: 257-262, (1997); Rice-Evans, C. A. ; Miller, N. J. ; Pananga, G. Structure- antioxidant activity relationship of flavonoids and phenolic acids. Free Radio . Biol . Med. 20: 933-956, (1996); Salah, N. ; Miller, N. J.; Paganga, G.; Tijburg, L. ; Bolwell, G. P. ; Rice- Evans, C. Polyphenolic flavonoids as scavenger of aqueous phase radicals and as chain-breaking antioxidants . Arch Biochem Biophys 322: 339-346, (1995); Stryer, L. Biochemis try S . Francisco: Free- man, (1975); Vieira, 0.; Escargueil-Blanc, I.; Meilhac, 0.; Ba- sile, J. P.; Laranjinha, J.; Almeida, L.; Salvayre, R. ; Negre- Salvayre, A. Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL. Br J Pharmacol 123: 565-573, (1998)). Darüber hinaus haben Polyphenole vielfältige Wirkungen auf den Zellstoffwechsel. Unter anderem werden Enzyme der Signaltransduction wie Proteinkinase C, Tyro- sin-Proteinkinase und Phosphatidylinositol-3-kinase moduliertVarious phenolic substances are found in plants, eg caffeic acid, ferulic acid, chlorogenic acid, gallic acid, eugenol, lignans, coumarins, lignin, stilbenes (polydatin, resveratrol), flavonoids (flavones, catechins, flavanones, anthocyanidins, isoflavones), polymethoxylated flavones. Accordingly, phenols are also a general component of many plant-based foods and stimulants. Certain phenolic substances are of particular importance because they can exert an antioxidative effect in human or animal metabolism after ingestion with food (Baum, B. 0 .; Perun, AL Antioxidant efficiency versus structure. Soc. Plast. Engrs Trans 2: 250 -257, (1962); Gardner, PT; McPhail, DB; Duthie, GG Electron spin resonance spectroscopic assessment of the antioxidant potential of teas in aqueous and organic media. J. Sei. Food Agric. 76: 257-262, (1997); Rice-Evans, CA; Miller, NJ; Pananga, G. Structure- antioxidant activity relationship of flavonoids and phenolic acids. Free radio. Biol. Med. 20: 933-956, (1996); Salah, N.; Miller, NJ; Paganga, G .; Tijburg, L.; Bolwell, GP; Rice-Evans, C. Polyphenolic flavonoids as scavenger of aqueous phase radicals and as chain-breaking antioxidants. Arch Biochem Biophys 322: 339-346, (1995); Stryer, L. Biochemis try S. Francisco: Free-man, (1975); Vieira, 0 .; Escargueil-Blanc, I .; Meilhac, 0 .; Basile, JP; Laranjinha, J .; Almeida, L .; Salvayre, R.; Negre-Salvayre, A. Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL. Br J Pharmacol 123: 565-573, (1998)). In addition, polyphenols have various effects on cell metabolism. Among other things, signal transduction enzymes such as protein kinase C, tyrosine protein kinase and phosphatidylinositol-3-kinase are modulated
(Agullo, G. ; Gamet-payrastre, L.,- Manenti, S.; Viala, C; Remesy, C.; Chap, H.; Payrastre, B. Relationship between flavonoid struc- ture and inhibition of phosphatidylinositol 3-kinase: a compari- son with tyrosine kinase and protein kinase C inhibition. Biochem Pharmacol 53 :1649-1657, (1997); Ferriola, P. C; Cody, V.; Mid- dleton, E. Protein kinase C inhibition by plant flavonoids. Kine- tic mechanisms and structure activity relationship. Biochem Pharmacol 38: 1617-1624, (1989); Cushman, M. ; Nagarathman, D.; Burg, D. L.; Geahlen, R. L. Synthesis and protein-tyrosine kinase inhibitory activity of flavonoids analogues. J Λfeed Chem 34:(Agullo, G.; Gamet-payrastre, L., - Manenti, S .; Viala, C; Remesy, C .; Chap, H .; Payrastre, B. Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase : a comparison with tyrosine kinase and protein kinase C inhibition. Biochem Pharmacol 53: 1649-1657, (1997); Ferriola, P. C; Cody, V .; Middleton, E. Protein kinase C inhibition by plant flavonoids Kinetic mechanisms and structure activity relationship. Biochem Pharmacol 38: 1617-1624, (1989); Cushman, M.; Nagarathman, D .; Burg, DL; Geahlen, RL Synthesis and protein-tyrosine kinase inhibitory activity of flavonoids analogues J Λfeed Chem 34:
798-806, (1991); Hagiwara, M. ; Inoue, S.; Tanaka, T.; Nunoki, K. ; Ito, M. ; Hidaka, H. Differential effects of flavonoids as inhibitors of tyrosine protein kinases and serine/threonin protein kinases. Biochem Pharmacol 37: 2987-2992, (1988)), die in- duzierbare NO-Synthase downreguliert (Kobuchi, H.; Droy- efaix, M. T. ,- Christen, Y. ; Packer, L. Ginkgo biloba extract (EGb761) : inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7. Biochem Pharmacol 53: 897-903, (1997)) und die Genexpression von z. B. Interleukinen und Adhäsionsmolekülen (ICAM-1, VCAM-1) reguliert (Kobuchi, H. ; Droy-Lefaix, M. T.;798-806, (1991); Hagiwara, M.; Inoue, S .; Tanaka, T .; Nunoki, K.; Ito, M.; Hidaka, H. Differential effects of flavonoids as inhibitors of tyrosine protein kinases and serine / threonin protein kinases. Biochem Pharmacol 37: 2987-2992, (1988)), which downregulates inducible NO synthase (Kobuchi, H .; Droefaix, MT, - Christen, Y.; Packer, L. Ginkgo biloba extract (EGb761): inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7. Biochem Pharmacol 53: 897-903, (1997)) and the gene expression of e.g. B. Interleukins and adhesion molecules (ICAM-1, VCAM-1) regulated (Kobuchi, H.; Droy-Lefaix, M. T .;
Christen, Y.,- Packer, L. Ginkgo biloba extract (EGb761) : inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7. Biochem Pharmacol 53:897-903, (1997); Wolle, J.; Hill, R. R.; Ferguson, E. ; Devall, L. J.; Trivedi, B. K. ; Newton, R. S. ; Saxena, U. Selective inhibition of Tumor necrosis Factor- induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid. Lack of effect on trascriptional factor NF-kB. Atherioscler Thromb Vase Biol 16: 1501-1508, (1996)). Es gilt als gesichert, daß diese Wirkungen positiv sind zur Vorbeugung und Prävention von Infarkten, Herz-Kreislauferkrankungen, Diabetes, bestimmter verschiedener Krebsarten, Tumorarten und weiterer chronischer Krankheiten (Bertuglia, S.; Malandrino, S.; Colan- tuoni, A. Effects of the natural flavonoid delphinidin on diabe- tic microangiopathy. Arznei -Forsch/Drug Res 45 : 481 -485, (1995); Griffiths, K. ; Adlercreutz, H.; Boyle, P.; Denis, L.; Nicholson, R.I.; Morton, M.S. Nutri tion and Cancer Oxford: Isis Medical Media, (1996); Hertog, M. G. L.; Fesrens, E. J. M.; Hollman, P. C. K. ; Katan, M. B.; Kromhout, D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen eiderly study. The Lance t 342: 1007-1011, (1993); Kapiotis, S.; Hermann, M. ; Held, I.; Seelos, C. ; Ehringer, H. ,- Gmeiner, B. M. Genistein, the dietary-derived angiogenesis inhibitor, prevents LDL oxidation and protects endothelial cells from damage by atherogenic LDL. Arterioscler Thromb Vase Biol 17: 2868-74, (1997); Stampfer, M. J.; Hennekens, C. H. ; Manson, J. E.; Colditz, G. A. ; Rosner, B.; Willet, W. C. Vitamin E consumption and the risk of coronary di- sease in women. New Engl J Med 328 :1444-1449, (1993); Tijburg, L. B. M.; Mattern, T.; Folts, J. D. ; Weisgerber, U. M.; Katan, M. B. Tea flavonoids and cardiovascular diseases: a review. Crit Rev Food Sei Nutr 37: 771-785, (1997); Kirk, E. A.; Sutherland, P.; Wang, S. A.; Chait, A. ; LeBoeuf, R. C. Dietary isoflavones reduce plasma cholesterol and atherosclerosis in C57BL/6 mice but not LDL reeeptor-deficient mice. J Nutr 128: 954-9, (1998) - Zitate - ) . Aus geeigneten Pflanzen wird daher bereits eine Reihe von heilenden, gesundheitsfördernden oder stärkenden Mitteln gewonnen, deren Wirkung auf ihrem Gehalt an phenolischen Substanzen beruht (Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A. ; Ligon, G. F.; Perry, C. A. Flavonoids inhibit cyto- kine-induced endothelial cell adhesion protein gene expression. Am J Pathol 147: 278-292, (1995); Lin, J. K. ; Chen, Y. C. ; Huang, Y. T. ; Lin-Shiau, S. Y. Suppression of protein kinase C and nu- clear oncogene expression as possible molecular mechanisms of cancer chemoprevention by apigenin and curcumin. J Cell Biochem Suppl 28-29:39-48, 1997; Zi, X.; Mukhtar, H. ; Agarval, R. Novel cancer chemoprevenetive effects of a flavonoid antioxidant silymarin: inhibition of mRNA expression of an endogenous tumor promoter TNF alpha. Bioche-τι Biophys Res Comm 239:334-339, 1997. Bekannt ist weiterhin, daß bestimmte pflanzliche Nahrungsmittel oder aus ihnen hergestellte Genußmittel eine positive Wirkung gegen verschiedene Krankheiten ausüben. Das in Weißwein, besonders aber in Rotwein, enthaltene Resveratrol (nebst weiterer Komponen- ten) wirkt beispielsweise gegen Infarkte kardiovasculäre Erkrankungen und Krebs (Gehm, B.D.; McAndrews, J.M.; Chien, P.-Y.; Ja- meson, J.L. Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for estrogen reeeptor. Proc Natl Acad Sei USA 94: 14138-14143, (1997),- Jang, M.; Cai , L.; Udeani, G.O.; Slowing, K.V. ; Thomas, C.F.; Beecher, C.W.W. ; Fong, H.H.S; Farns - worth, N.R.; Kinghorn, A.D.; Mehtha, R.G.; Moon, R.C., Pezzuto, J.M. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Sci ence 275: 218-220, (1997). Eine ähnliche Wirkung weisen auch Substanzen wie Catechin, Epicatec- hin-3-gallat, Epigallocatechin und Epigallocatechin-3-gallat auf, die in Blättern von Tee ( Camellia sinensis) vorkommen. Ins- besondere aus nicht-fermentierten Teeblättern (Grüner Tee) hergestellte Getränke sind von positiver gesundheitlicher Relevanz (Hu, G. ; Han, C. ; Chen, J. Inhibition of oncogene expression by green tea and (-) -epigallocatechin gallate in mice. JVutr Cancer 24: 203-209; (1995); Scholz, E; Bertram, B. Camellia sinensis (L.) 0. Kuntze. Der Teestrauch. Z. Phytotherapie 17: 235-250 ,Christen, Y., - Packer, L. Ginkgo biloba extract (EGb761): inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7. Biochem Pharmacol 53: 897-903, (1997); Wolle, J .; Hill, RR; Ferguson, E.; Devall, LJ; Trivedi, BK; Newton, RS; Saxena, U. Selective inhibition of tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid. Lack of effect on trascriptional factor NF-kB. Atherioscler Thromb Vase Biol 16: 1501-1508, (1996)). It is certain that these effects are positive for the prevention and prevention of infarcts, cardiovascular diseases, diabetes, certain different types of cancer, tumors and other chronic diseases (Bertuglia, S .; Malandrino, S .; Colan- Tuoni, A. Effects of the natural flavonoid delphinidine on diabetic microangiopathy. Arzneimittel -Forsch / Drug Res 45: 481-485, (1995); Griffiths, K.; Adlercreutz, H .; Boyle, P .; Denis, L .; Nicholson, RI; Morton, MS Nutrition and Cancer Oxford: Isis Medical Media, (1996); Hertog, MGL; Fesrens, EJM; Hollman, PCK; Katan, MB; Kromhout, D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen eiderly study. The Lance t 342: 1007-1011, (1993); Kapiotis, S .; Hermann, M.; Held, I .; Seelos, C.; Ehringer, H., - Gmeiner, BM Genistein, the dietary-derived angiogenesis inhibitor, prevents LDL oxidation and protects endothelial cells from damage by atherogenic LDL. Arterioscler Thromb Vase Biol 17: 2868-74, (1997); Stampfer, MJ; Hennekens, CH; Manson, JE; Colditz, GA; Rosner, B .; Willet, WC Vitamin E consumption and the risk of coronary disease in women. New Engl J Med 328: 1444-1449, (1993); Tijburg, LBM; Mattern, T .; Folts, JD; Weisgerber, UM; Katan, MB Tea flavonoids and cardiovascular diseases: a review. Crit Rev Food Sei Nutr 37: 771-785, (1997); Kirk, EA; Sutherland, P .; Wang, SA; Chait, A.; LeBoeuf, RC Dietary isoflavones reduce plasma cholesterol and atherosclerosis in C57BL / 6 mice but not LDL reeeptor-deficient mice. J Nutr 128: 954-9, (1998) - quotes -). A number of healing, health-promoting or strengthening agents are therefore already obtained from suitable plants, the effect of which is based on their content of phenolic substances (Gerritsen, ME; Carley, WW; Ranges, GE; Shen, CP; Phan, SA; Ligon, GF ; Perry, CA Flavonoids inhibit cyto-kine-induced endothelial cell adhesion protein gene expression. Am J Pathol 147: 278-292, (1995); Lin, JK; Chen, YC; Huang, YT; Lin-Shiau, SY Suppression of protein kinase C and nuclear oncogene expression as possible molecular mechanisms of cancer chemoprevention by apigenin and curcumin. J Cell Biochem Suppl 28-29: 39-48, 1997; Zi, X .; Mukhtar, H.; Agarval, R. Novel cancer chemoprevenetive effects of a flavonoid antioxidant silymarin: inhibition of mRNA expression of an endogenous tumor promoter TNF alpha. Bioche-τι Biophys Res Comm 239: 334-339, 1997. It is also known that certain plant foods or stimulants made from them are positive Effect against exercise various diseases. The resveratrol (along with other components) contained in white wine, but especially in red wine, is effective, for example, against heart attacks, cardiovascular diseases and cancer (Gehm, BD; McAndrews, JM; Chien, P.-Y .; Jameson, JL Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for estrogen reeeptor. Proc Natl Acad Sei USA 94: 14138-14143, (1997), - Jang, M .; Cai, L .; Udeani, GO; Slowing, KV; Thomas, CF; Beecher, CWW; Fong, HHS; Farns-worth, NR; Kinghorn, AD; Mehtha, RG; Moon, RC, Pezzuto, JM Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Sci ence 275: 218-220, (1997). Substances such as catechin, epicatec-hin-3-gallate, epigallocatechin and epigallocatechin-3-gallate, which occur in tea leaves (Camellia sinensis), have a similar effect. In particular, beverages made from non-fermented tea leaves (green tea) are of positive health relevance (Hu, G.; Han, C.; Chen, J. Inhibition of oncogene expression by green tea and (-) -epigallocatechin gallate in mice JVutr Cancer 24: 203-209; (1995); Scholz, E; Bertram, B. Camellia sinensis (L.) 0. Kuntze. The tea bush. Z. Phytotherapy 17: 235-250,
(1995); Yu, R. ; Jiao, J. J. ; Duh, J. L.; Gudehithlu, K.; Tan, T. H. ; Kong, A. N. Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the re- gulation of antioxidant responsive elements-mediated phase II en- zyme gene expression. Carcinigenesis 18: 451-456, (1997); Jankun, J. ; Selman, S.H.; Swiercz, R. Why drinking green tea could pre- vent cancer. iVature 387: 561, (1997). Darüber hinaus weisen auch polymethoxylierte Flavone aus Zitrusfrüchten eine potentielle an- titumorale Wirkung auf (Chem, J. ; Montanari, A.M. ; Widmer, W.W. Two new polymethoxylierte flavone, a class of compounds with potential anticancer activity, isolated from cold pressed dancy tangerine peel oil solids. J Agric Food Chem 45: 364-368, (1997) ) .(1995); Yu, R.; Jiao, J.J .; Duh, J. L .; Gudehithlu, K .; Tan, T.H .; Kong, A.N. Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant responsive elements-mediated phase II enzyme gene expression. Carcinigenesis 18: 451-456, (1997); Jankun, J.; Selman, S.H .; Swiercz, R. Why drinking green tea could prevent cancer. iVature 387: 561, (1997). In addition, polymethoxylated flavones from citrus fruits also have a potential antitumor effect (Chem, J.; Montanari, AM; Widmer, WW Two new polymethoxylated flavones, a class of compounds with potential anticancer activity, isolated from cold pressed dancy tangerine peel oil solids, J Agric Food Chem 45: 364-368, (1997)).
Aufgabe der vorliegenden Erfindung war es, ein einfaches und kostengünstiges Verfahren zur Erhöhung des Gehaltes an Flavonoiden und phenolischen Inhaltsstoffen in Kulturpflanzen zu finden.The object of the present invention was to find a simple and inexpensive method for increasing the content of flavonoids and phenolic constituents in crop plants.
Ausgehend von physiologischen Untersuchungen mit Wachstums - regulatoren aus der Gruppe der Acylcyclohexadione wurden nun überraschend gentechnologische Verfahren verfügbar, mit deren Hilfe sich Pflanzen erzeugen lassen, die sich durch einen erhöhten Gehalt an heilenden, gesundheitsfördernden oder stärkenden Inhaltsstoffen ausweisen.On the basis of physiological studies with growth regulators from the group of acylcyclohexadiones, genetic engineering methods have now surprisingly become available, with the help of which plants can be produced which are characterized by an increased content of curative, health-promoting or strengthening ingredients.
Acylcyclohexadione wie Prohexadion-Ca und Trinexapac-ethyl (ältere Bezeichnung: Cimectacarb) werden als Bioregulatoren zur Hemmung des pflanzlichen Längenwachstums eingesetzt. Ihre bioregu- latorische Wirkung kommt dadurch zustande, daß sie die Bio- Synthese von längenwachstumsfördernden Gibberellinen blockieren. Dabei hemmen sie aufgrund ihrer strukturellen Verwandtschaft zu 2-Oxoglutarsäure bestimmte Dioxygenasen, die 2-0xoglutarsäure als Co-Substrat benötigen (Rademacher, W, Biochemical effects of plant growth retardants, in: Plant Biochemical Regulators, Gaus- man, HW (ed.), Marcel Dekker, Inc., New York, pp. 169-200Acylcyclohexadiones such as Prohexadione-Ca and Trinexapac-ethyl (older name: Cimectacarb) are used as bioregulators to inhibit plant growth. Their bioregulatory effect is due to the fact that they block the bio-synthesis of gibberellins that promote growth. Because of their structural relationship to 2-oxoglutaric acid, they inhibit certain dioxygenases that require 2-0xoglutaric acid as a co-substrate (Rademacher, W, Biochemical effects of plant growth retardants, in: Plant Biochemical Regulators, Gausman, HW (ed.) , Marcel Dekker, Inc., New York, pp. 169-200
(1991) ) . Es ist bekannt, daß derartige Verbindungen auch in den Stoffwechsel von Phenolen eingreifen und so bei mehreren Pflan- zenarten eine Hemmung der Anthocyanbildung bewirken können (Rade- macher, W et al . , The mode of action of acylcyclohexanediones - a new type of growth retardant, in: Progress in Plant Growth Regulation, Karssen, CM, van Loon, LC, Vreugdenhil, D (eds.), Kluwer 5 Academic Publishers, Dordrecht (1992)). Derartige Effekte auf den Haushalt phenolischer Inhaltsstoffe werden als ursächlich für die Nebenwirkung von Prohexadion-Ca gegen Feuerbrand angegeben (Rade- macher, W et al., Prohexadione-Ca - a new plant growth regulator for apple with interesting biochemical features, Poster auf dem(1991)). It is known that such compounds also intervene in the metabolism of phenols and thus in several plants species can inhibit anthocyanin formation (Rademacher, W et al., The mode of action of acylcyclohexanediones - a new type of growth retardant, in: Progress in Plant Growth Regulation, Karssen, CM, van Loon, LC, Vreugdenhil, D (eds.), Kluwer 5 Academic Publishers, Dordrecht (1992)). Such effects on the household of phenolic ingredients are said to be the cause of the side effect of prohexadione-Ca against fire blight (Rademacher, W et al., Prohexadione-Ca - a new plant growth regulator for apple with interesting biochemical features, poster on the
10 25th Annual Meeting of the Plant Growth Regulation Society of America, 7.-10. Juli 1998, Chicago). A. Lux-Endrich (Dissertation Technische Universität München in Weihenstephan, 1998) findet im Verlauf ihrer Untersuchungen zum Wirkmechanismus von Prohexadion- Ca gegen Feuerbrand, daß es in Zellkulturen von Apfel durch Pro-10 25 th Annual Meeting of the Plant Growth Regulation Society of America, 7-10. July 1998, Chicago). A. Lux-Endrich (dissertation Technical University of Munich in Weihenstephan, 1998) finds in the course of her studies on the mechanism of action of prohexadione-Ca against fire blight that it can be found in cell cultures of apple by pro-
15 hexadion-Ca zu einer mehrfachen Erhöhung des Gehaltes an phenolischen Substanzen kommt und daß dabei eine Reihe von sonst nicht .vorhandenen Phenolen auftritt. Im Rahmen dieser Untersuchungen wurde weiterhin gefunden, daß unter dem Einfluß von Prohexadion-Ca relativ hohe Mengen von Luteoliflavan und Eriodyctiol15 hexadione-Ca leads to a multiple increase in the content of phenolic substances and that a number of phenols not otherwise present occur. In the course of these investigations it was also found that under the influence of prohexadione-Ca, relatively high amounts of luteoliflavan and eriodyctiol
20 in Sproßgewebe von Apfel auftreten. Luteoliflavan kommt in Apfel - gewebe normalerweise nicht vor und Eriodyctiol tritt als Inter- mediat des Flavonoidstoffwechsels nur in geringen Mengen auf. Die zu erwartenden Flavonoide Catechin und Cyanidin waren im behandelten Gewebe jedoch nicht nachweisbar oder traten nur in deut-20 occur in apple shoots. Luteoliflavan is not normally found in apple tissue and eriodyctiol occurs only in small quantities as an intermediate in flavonoid metabolism. However, the expected flavonoids catechin and cyanidine were not detectable in the treated tissue or only occurred in significant amounts.
25 lieh reduzierten Mengen auf (S. Römmelt et al, Vortrag 8th International Work- Shop on Fire Blight, Kusadasi, Türkei, 12.-15. Oktober 1998) .25 borrowed reduced quantities (S. Römmelt et al, lecture 8 th International WorkShop on Fire Blight, Kusadasi, Turkey, October 12-15, 1998).
Es kann als gesichert gelten, daß Prohexadion-Ca, Trinexapac-It can be considered certain that Prohexadione-Ca, Trinexapac-
30 ethyl und andere Acylcyclohexadione 2-Oxoglutarsäure-abhängige Hydroxylasen inhibieren, die im Stoffwechsel phenolischer Substanzen von Bedeutung sind. Dabei handelt es sich primär um Chal - consynthetase (CHS) und um Flavanon-3-hydroxylase (F3H) (W. Heller und G. Forkmann, Biosynthesis, in: The Flavonoids, Har-30 ethyl and other acylcyclohexadione inhibit 2-oxoglutaric acid-dependent hydroxylases, which are important in the metabolism of phenolic substances. These are primarily Chal-consynthetase (CHS) and Flavanon-3-hydroxylase (F3H) (W. Heller and G. Forkmann, Biosynthesis, in: The Flavonoids, Har-
35 borne, JB (ed.), Chapman and Hall, New York, 1988). Es kann jedoch nicht ausgeschlossen werden, daß Acylcyclohexadione auch weitere, bislang unbekannte, 2-Oxoglutarsäure-abhängige Hydroxylasen hemmen. Es dürfte ferner naheliegend sein, daß ein Mangel an Catechin, Cyanidin oder anderen Endprodukten der Flavonoid- 0 synthese von der Pflanze registriert wird und daß über einen35 borne, JB (ed.), Chapman and Hall, New York, 1988). However, it cannot be excluded that acylcyclohexadiones also inhibit other, previously unknown, 2-oxoglutaric acid-dependent hydroxylases. It should also be obvious that a lack of catechin, cyanidine or other end products of flavonoid synthesis is registered by the plant, and that a
Feedback-Mechanismus die Aktivität des Schlüsselenzyms Phenylala- ninammoniumlyase (PAL) erhöht wird. Durch die weiterhin existierende Hemmung von CHS und F3H können diese Flavonoid-Endprodukte jedoch nicht gebildet werden, und es kommt zu einer vermehrten 5 Bildung von Luteoliflavan, Eriodyctiol und anderer Phenole (Abb .1 ) . Durch die Reduktion der Enzymaktivität des Enzyms Flavanon-3-hy- droxylase (F3H) werden die Flavonoide Eriodictyol, Proanthocyani - dine, die am C-Atom 3 mit Wasserstoff substituiert sind, z.B. Lu- teoforol, Luteoliflavan, Apigeniflavan und Tricetiflavan, sowie homogene und heterogene Oligomere und Polymere aus den genannten und strukturell verwandten Substanzen vermehrt gebildet.Feedback mechanism the activity of the key enzyme phenylalanine ammonium lyase (PAL) is increased. However, the continued inhibition of CHS and F3H means that these flavonoid end products cannot be formed, and there is an increased formation of 5 luteoliflavan, eriodyctiol and other phenols (Fig. 1). By reducing the enzyme activity of the enzyme flavanon-3-hydroxylase (F3H), the flavonoids eriodictyol, proanthocyanidins, which are substituted on the C atom 3 with hydrogen, eg luteoforol, luteoliflavan, apigeniflavan and tricetiflavan, as well as homogeneous and heterogeneous oligomers and polymers increasingly formed from the mentioned and structurally related substances.
Erhöhte Konzentratonen der Phenole Hydroxyzimtsäure (p-Cumar- säure, Ferulasäure, Sinapinsäure) , Salicylsäure oder Umbellife- ron, einschließlich der aus ihnen gebildeten homogenen und heterogenen Oligomere und Polymere werden nach Reduktion der Enzymaktivität des Enzyms Flavanon-3-hydroxylase (F3H) in Pflanzen festgestellt. Ebenso erhöht sich die Konzentration der Chalcone, wie z.B. Phloretin, und der Stilbene, wie z.B. Resveratrol.Increased concentrations of the phenols hydroxycinnamic acid (p-coumaric acid, ferulic acid, sinapic acid), salicylic acid or umbelliferone, including the homogeneous and heterogeneous oligomers and polymers formed from them, are reduced after reducing the enzyme activity of the enzyme flavanone-3-hydroxylase (F3H) Plants found. The concentration of chalcones also increases, e.g. Phloretin, and the stilbene, e.g. Resveratrol.
Durch Reduktion der Enzymaktivität des Enzyms Flavanon-3 -hydroxylase wird auch die Konzentration der Glykoside der Flovonoide, der phenolischen Verbindungen, der Chalcone und der Stilbene erhöht.By reducing the enzyme activity of the enzyme flavanone-3-hydroxylase, the concentration of the glycosides of the flovonoids, the phenolic compounds, the chalcones and the stilbene is also increased.
Ausgehend von diesen Befunden und den daraus abgeleiteten Hypothesen wurden gentechnisch veränderte Kulturpflanzen erzeugt, in denen F3H durch Anti-Sense-Konstrukte ganz oder teilweise, dauerhaft oder vorübergehend, in der gesamten Pflanze oder in einzel- nen Pflanzenorganen oder -geweben in ihren Aktivitäten reduziert war, so daß hier der Gehalt an heilenden, gesundheitsfördernden oder stärkenden Inhaltsstoffen quantitativ und qualitativ verbessert war.Based on these findings and the hypotheses derived from them, genetically modified crop plants were generated in which the activity of F3H was reduced in whole or in part, permanently or temporarily, by anti-sense constructs, in the entire plant or in individual plant organs or tissues , so that the content of healing, health-promoting or strengthening ingredients was quantitatively and qualitatively improved.
Das erfindungsgemäße Verfahren zur Erhöhung des Gehalts an Flavonoiden und phenolischen Verbindungen durch Expression der Flava- non-3-hydroxylase in Antisens -Orientierung kann erfolgreich bei folgenden Kulturpflanzen ausgeübt werden, wobei das Verfahren nicht auf die genannten Pflanzen beschränkt ist: Weinrebe, Kir- sehe, Tomate, Pflaume, Schlehe, Blaubeere, Erdbeere, Zitrus- früchte (wie Orange, Grapefruit), Papaya, Rotkohl, Broccoli, Rosenkohl, Kakao, Grünkohl, Karotte, Petersilie, Sellerie, Zwiebeln, Knoblauch, Tee, Kaffee, Hopfen, Soja, Raps, Hafer, Weizen, Roggen, Aronia melanocarpa, Ginko biloba.The process according to the invention for increasing the content of flavonoids and phenolic compounds by expression of the flavonon-3-hydroxylase in an antisense orientation can be carried out successfully in the following crop plants, the process not being limited to the plants mentioned: grapevine, cherry , Tomato, plum, sloe, blueberry, strawberry, citrus (such as orange, grapefruit), papaya, red cabbage, broccoli, Brussels sprouts, cocoa, kale, carrot, parsley, celery, onions, garlic, tea, coffee, hops, soy , Rapeseed, oats, wheat, rye, Aronia melanocarpa, Ginko biloba.
Gegenstand der Erfindung sind darüberhinaus Pflanzen mit erhöhtem Gehalt an Flavonoiden und phenolischen Inhaltsstoffen hergestellt nach dem erfindungsgemäßen Verfahren dadurch gekennzeichnet, daß deren enzymatische Aktivität des Enzyms Flavanon-3 -hydroxylase reduziert ist. Alternativ zur Herstellung von Pflanzen, die mit Hilfe der Anti- sense- Technologie in ihrer Flavanon-3 -hydroxylase Aktivität reduziert sind, lassen sich auch andere literaturbekannte molekulargenetische Methoden wie Co-Suppression oder die Expression von spezifischen Antikörpern verwenden, um diesen Effekt zu erreichen.The invention furthermore relates to plants with an increased content of flavonoids and phenolic constituents produced by the process according to the invention, characterized in that their enzymatic activity of the enzyme flavanon-3-hydroxylase is reduced. As an alternative to the production of plants which are reduced in their flavanone-3-hydroxylase activity with the aid of antisense technology, other molecular genetic methods known from the literature, such as co-suppression or the expression of specific antibodies, can also be used to achieve this effect .
Darüberhinaus ist Gegenstand der Erfindung die Verwendung von Pflanzen - hergestellt nach dem erfindungsgemäßen Verfahren - oder von Teilen dieser Pflanzen als Nahrungsmittel, Nahrungsmittelergänzungsmittel oder zur Herstellung von heilenden, gesundheitsfördernden oder stärkenden Mitteln (Säfte, Tees, Extrakte, Fermentationsprodukte) für Mensch und Tier sowie zur Herstellung von Kosmetika.In addition, the invention relates to the use of plants - produced by the process according to the invention - or parts of these plants as food, food supplements or for the production of curative, health-promoting or strengthening agents (juices, teas, extracts, fermentation products) for humans and animals and for Manufacture of cosmetics.
Es wurde nun überraschend gefunden, daß unter dem Einfluß von erfindungsgemäß produzierten Pflanzen oder von Teilen dieser Pflanzen oder aus ihnen hergestellter Produkte (Tees, Extrakte, Fermentationsprodukte, Säfte etc.)It has now surprisingly been found that under the influence of plants produced according to the invention or of parts of these plants or products made from them (teas, extracts, fermentation products, juices etc.)
(1) die Antioxidative Kapazität in vi tro (Electron Spin Resonance (ESR) , LDL-Oxidation, Total Antioxidant Capacity, NO-Scaven- ging) verbessert wird;(1) the antioxidant capacity in vitro (electron spin resonance (ESR), LDL oxidation, total antioxidant capacity, NO scavenging) is improved;
(2) eine modulierende Wirkung auf Enzyme, vor allem Enzyme der Signal transduktion (Proteinkinase C, Tyrosin-Proteinkinase, Phosphatidylinositoi-3-Kinase) auftritt;(2) a modulating effect on enzymes, especially enzymes of signal transduction (protein kinase C, tyrosine protein kinase, phosphatidylinositoi-3-kinase) occurs;
(3) eine Modulation redoxsensitiver Transkriptionsfaktoren (NF- kB, AP-1) in Endothelzellen, Lymphocyten und glatten Muskel - zellen induziert wird;(3) modulation of redox-sensitive transcription factors (NF-kB, AP-1) is induced in endothelial cells, lymphocytes and smooth muscle cells;
(4) die Regulation der Genexpression von Targetgenen involviert in die Pathogenese inflammatorischer Erkrankungen (Cytokine IL-1 und IL-8, macrophage chemoattractant protein 1 (MCP-1) , Adhesionsfaktoren ICAM-1 und VCAM-1) moduliert wird;(4) the regulation of gene expression of target genes involved in the pathogenesis of inflammatory diseases (cytokines IL-1 and IL-8, macrophage chemoattractant protein 1 (MCP-1), adhesion factors ICAM-1 and VCAM-1) is modulated;
(5) eine antiaggregatorische Wirkung induziert wird;(5) an anti-aggregatory effect is induced;
(6) die Cholesterinsynthese in Hepatocyten gehemmt wird;(6) cholesterol synthesis in hepatocytes is inhibited;
(7) antiproliferative/antineoplastische Effekte bestehen. Beispi el 1(7) there are antiproliferative / antineoplastic effects. Example 1
Klonierung des Gens einer Flavanone-3-Hydroxylase aus Lycopersicon esculentum Mill.cv. Moneymaker.Cloning of a Flavanone-3-hydroxylase gene from Lycopersicon esculentum Mill.cv. Moneymaker.
Reife Tomatenfrüchte von Lycopersicon esculentum Mill.cv. Moneymaker wurden gewaschen, getrocknet und mittels einer sterilen Klinge das Perikarp von Samen, mittlere Kolumnella und Holzteilen befreit. Das Perikarp (ca. 50 g) wurde in fluessigem Stickstoff eingefroren. Das Material wurde anschliessend in einem Mixer zerkleinert. Das zerkleinerte Material wurde in einem vorgekühlten Mörser mit 100 ml Homogenisierungs-Medium versetzt und gemischt. Die Suspension wurde dann in Zentrifugenbecher überführt, indem sie durch sterile Mulltücher gepreßt wurde. Anschließend wurde 1/10 Vol 10% SDS hinzugefügt und gut gemischt. Nach 10 Minuten auf Eis, wurde 1 Vol Phenol/Chloroform zugegeben, der Zentrifugenbecher verschlossen und gut gemischt. Nach 15 inütiger Zentrifugation bei 4000 rpm wurde der Überstand in ein neues Reaktionsgefäß überführt. Es schlössen sich drei weitere Phenol/ Chloroform Extraktionen und eine Chloroform Extraktion an. Im folgenden wurde 1 Vol 3 M NaAC und 2.5 Vol Ethanol zugegeben. Die Fällung der Nukleinsäuren erfolgte über Nacht bei -20°C. Am nächsten Morgen wurden die Nukleinsäuren für 15 Minuten bei 10000 rpm in der Kühlzentrifuge (4°C) pelletiert. Der Überstand wurde ver- worfen und das Pellet in 5-lo ml kaltem 3 M NaAc resuspendiert. Dieser Waschschritt wurde zweimal wiederholt. Das Pellet wurde mit 80%igem Ethanol gewaschen. Das vollständig getrocknet Pellet wurde in ca. 0,5 ml sterilem DEPC Wasser aufgenommen und die RNA- Konzentration photometrisch bestimmt.Ripe tomato fruits from Lycopersicon esculentum Mill.cv. Moneymakers were washed, dried and the pericarp was freed from seeds, middle columnella and wooden parts using a sterile blade. The pericarp (approx. 50 g) was frozen in liquid nitrogen. The material was then crushed in a mixer. The comminuted material was mixed with 100 ml homogenizing medium in a pre-cooled mortar. The suspension was then transferred to centrifuge cups by pressing through sterile gauze cloths. Then 1/10 vol 10% SDS was added and mixed well. After 10 minutes on ice, 1 volume of phenol / chloroform was added, the centrifuge cup closed and mixed well. After centrifugation at 4000 rpm for 15 minutes, the supernatant was transferred to a new reaction vessel. This was followed by three further phenol / chloroform extractions and one chloroform extraction. In the following, 1 vol 3 M NaAC and 2.5 vol ethanol were added. The nucleic acids were precipitated overnight at -20 ° C. The next morning, the nucleic acids were pelleted for 15 minutes at 10,000 rpm in the refrigerated centrifuge (4 ° C). The supernatant was discarded and the pellet resuspended in 5 ml of cold 3 M NaAc. This washing step was repeated twice. The pellet was washed with 80% ethanol. The completely dried pellet was taken up in about 0.5 ml of sterile DEPC water and the RNA concentration was determined photometrically.
20 μg gesamt RNA wurden zunächst mit 3,3 μl 3M Natriumacetat-Lö- sung, 2 μl IM Magnesiumsulfat-Lösung versetzt und auf 100 μl Endvolumen mit DEPC Wasser aufgefüllt. Dazu wurde ein Microliter Rnase freie Dnase (Boehringer Mannheim) gegeben und 45 min bei 37° Grad inkubiert. Nach Entfernen des Enzyms durch ausschütteln mit Phenol/Chloroform/Isoamylalkohol wurde die RNA mit Ethanol gefällt und das Pellet in 100 μl DEPC Wasser aufgenommen. 2 , 5 μg RNA aus dieser Lösung wurden mittels eines cDNA-Kits (Gibco BRL) in cDNA umgeschrieben.20 μg of total RNA were first mixed with 3.3 μl of 3M sodium acetate solution, 2 μl of IM magnesium sulfate solution and made up to 100 μl of final volume with DEPC water. A microliter RNase-free DNase (Boehringer Mannheim) was added and incubated at 37 ° for 45 min. After removing the enzyme by shaking with phenol / chloroform / isoamyl alcohol, the RNA was precipitated with ethanol and the pellet was taken up in 100 μl DEPC water. 2.5 μg RNA from this solution were transcribed into cDNA using a cDNA kit (Gibco BRL).
Unter Verwendung von Aminosäuresequenzen die aus für Flava- none-3-Hydroxylase kodierenden cDNA Klonen abgeleitet wurden, konnten konservierte Bereiche in der Primärsequenz identifiziert werden (Britsch et al., Eur. J. Biochem. 217, 745 -754 (1993), die als Grundlage für das Design von degenerierten PCR Oligo- nukleotiden dienten. Das 5' Oligonukleotid wurde unter Verwendung der Peptidsequenz SRWPDK (Aminosäure 147-152 in der Sequenz FL3H PETHY aus Petunia ybrida ermittelt und hatte f olgende Se quenz :Using amino acid sequences derived from cDNA clones coding for flavanone-3-hydroxylase, conserved regions in the primary sequence could be identified (Britsch et al., Eur. J. Biochem. 217, 745-754 (1993), served as the basis for the design of degenerate PCR oligonucleotides The 5 'oligonucleotide was generated using the peptide sequence SRWPDK (amino acid 147-152 in the sequence FL3H PETHY from Petunia ybrida and had the following sequence:
5 ' -TCI (A/C) G (A/G) TGG CC (A/C/G) GA (C/T) AA (A/G) CC-3 .5 '-TCI (A / C) G (A / G) TGG CC (A / C / G) GA (C / T) AA (A / G) CC-3.
Die Sequenz des unter Verwendung der Peptidsequenz DHQAW (Aminosäure 276281 in der Sequenz FL3H PETHY aus Petunia hybridaj abgeleiteten Oligonukleotides lautete wie folgt: 5'-CTT CAC ACA (C/ G/T) GC (C/T) TG (A/G) TG (A/G)TC-3.The sequence of the oligonucleotide derived using the peptide sequence DHQAW (amino acid 276281 in the sequence FL3H PETHY from Petunia hybridaj was as follows: 5'-CTT CAC ACA (C / G / T) GC (C / T) TG (A / G) TG (A / G) TC-3.
1010
Die PCR-Reaktion wurde unter Verwendung der tTth-Polymerase von Perkin-Elmer nach Herstellerangaben durchgeführt. Als Template wurden 1/8 der cDNA eingesetzt (entspricht 0,3 μg RNA). Das PCR- Programm lautete:The PCR reaction was carried out using the Perkin-Elmer tTth polymerase according to the manufacturer's instructions. 1/8 of the cDNA was used as template (corresponds to 0.3 μg RNA). The PCR program was:
1515
30 Zyklen30 cycles
94 Grad 4 sec94 degrees 4 sec
40 Grad 30 sec40 degrees 30 sec
72 Grad 2 min72 degrees 2 min
20 72 Grad 10 min20 72 degrees 10 min
Das Fragment wurde nach Herstellerangaben in den Vektor pGEM-T von Promega kloniert.The fragment was cloned into Promega's vector pGEM-T according to the manufacturer's instructions.
25 Die Richtigkeit des Fragmentes wurde durch Sequenzierung überprüft. Das PCR Fragment wurde unter Verwendung der im Polylinker des Vektors pGEM-T vorhandenen Restriktionsschnittstellen Ncol und Pstl isoliert und die überstehenden Enden unter Verwendung der T4-Polymerase in glatte Enden überführt. Dieses Fragment25 The correctness of the fragment was checked by sequencing. The PCR fragment was isolated using the restriction sites Ncol and Pstl present in the polylinker of the vector pGEM-T and the protruding ends were blunt-ended using the T4 polymerase. This fragment
30 wurde in einen Smal (blunt) geschnittenen Vektor pBinAR (Höfgen und Willmitzer, Plant Sei. 66: 221 -230 (1990)) kloniert (siehe Abbildung 2). Dieser enthält den 35S-Promotor des CaMV (Blumen- kohlmosaikvirus) (Franck et al . , Cell 21: 285 - 294 (1980)) und das Terminationssignal des Octopin-Synthase Gens (Gielen et al . ,30 was cloned into a Smal (blunt) cut vector pBinAR (Höfgen and Willmitzer, Plant Sei. 66: 221-230 (1990)) (see Figure 2). This contains the 35S promoter of the CaMV (cauliflower mosaic virus) (Franck et al., Cell 21: 285-294 (1980)) and the termination signal of the octopine synthase gene (Gielen et al.,
35 EMBO J. 3: 835 - 846 ( 1984)). Dieser Vector vermittelt in Pflanzen Resistenz gegen das Antibiotikum Kanamycin. Die erhaltenen DNA Konstrukte enthielten das PCR Fragment in Sense und Antisense Orientierung. Das Antisensekonstrukt wurde zur Erzeugung transgener Pflanzen eingesetzt. 035 EMBO J. 3: 835-846 (1984)). This vector mediates resistance to the antibiotic kanamycin in plants. The DNA constructs obtained contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants. 0
Abbildung 2: Fragment A (529 bp) beinhaltet den 35S-Promotor des CaMV (Nukleotide 6909 bis 7437 des Blumenkohlmosaikvirus) . Fragment B das Fragment des F3H Gens in Antisense-Orientierung. Fragment C (192 Bp) enthält das Terminationssignal des Octopin-Syn- 5 thase Gens. Klonierung eines größeren cDNA Fragmentes der Flavanone-3-Hydro- xylase aus Lycopersicon esculentum Mill.cv. Moneymaker unter Verwendung des 5'RACESystems.Figure 2: Fragment A (529 bp) contains the 35S promoter of the CaMV (nucleotides 6909 to 7437 of the cauliflower mosaic virus). Fragment B the fragment of the F3H gene in antisense orientation. Fragment C (192 bp) contains the termination signal of the octopine synthase gene. Cloning of a larger cDNA fragment of Flavanone-3-hydroxylase from Lycopersicon esculentum Mill.cv. Moneymaker using the 5'RACE system.
i Um auszuschließen, dass die Erzeugung von Pflanzen mit reduzierter mRNA Fließgleichgewichtsmenge der F3H aufgrund der geringen Größe des im Antisensekonstrukt verwendeten F3H PCR Fragmentes nicht erfolgreich ist, sollte ein zweites Antisense-Konstrukt unter Verwendung eines größeren F3H Fragmentes erzeugt werden.i In order to rule out that the generation of plants with a reduced mRNA flow equilibrium amount of the F3H is not successful due to the small size of the F3H PCR fragment used in the antisense construct, a second antisense construct should be generated using a larger F3H fragment.
Zum Zweck der Klonierung eines größeren Fragmentes der F3H wurde die 5'RACE Methode (System for Rapid amplification of cDNA ends) angewendet .The 5'RACE method (System for Rapid amplification of cDNA ends) was used to clone a larger fragment of the F3H.
Verlängerung des F3H PCR Fragmentes durch die 5 'RACE-Methode unter Verwendung des 5 'RACE System for rapid amplification of cDNA ends, Version 2-0 von Life Tecgnologies™.Extension of the F3H PCR fragment by the 5 'RACE method using the 5' RACE System for rapid amplification of cDNA ends, Version 2-0 from Life Tecgnologies ™.
Aus reifen Tomatenfrüchten von Lycopersicon esculentum Mill.cv. Moneymaker wurde gesamt RNA isoliert (siehe oben) .From ripe tomato fruits from Lycopersicon esculentum Mill.cv. Moneymaker total RNA was isolated (see above).
Die cDNA Erststrang-Synthese wurde nach Herstellerangaben unter Verwendung des GSP-1 (Gen spezifischer Primer) 5'-TTCAC- CACTGCCTGGTGGTCC-3 ' durchgeführt. Im Anschluß an einen Rnase Ver- dau, wurde die cDNA unter Anwendung des GlassMAX spin Systems von Life Tecgnologies™ gemäß den Herstellerangaben aufgereinigt.The cDNA first strand synthesis was carried out according to the manufacturer's instructions using the GSP-1 (gene-specific primer) 5'-TTCAC-CACTGCCTGGTGGTCC-3 '. Following an RNase digest, the cDNA was purified using the GlassMAX spin system from Life Tecgnologies ™ in accordance with the manufacturer's instructions.
An das 3 'Ende der gereinigten einzelsträngigen F3H cDNA wurde unter Verwendung der terminalen deoxynukleotydil-Transferase ge- maß den Herstellerangaben ein Cytosin Homopolymer addiert.A cytosine homopolymer was added to the 3 'end of the purified single-stranded F3H cDNA using the terminal deoxynucleotydil transferase according to the manufacturer's instructions.
Die Amplifikation der 5' verlängerten F3H cDNA erfolgte unter Verwendung eines zweiten Gen spezifischen Primers (GSP-2) der im Bereich 3' vor der GSP-1 Erkennungsequenz bindet und somit eine , ,nested" PCR ermöglichte. Als 5 'Primer wurde der vom Hersteller gelieferte "5' RACE abrided anchor primer" verwendet, der komplementär zum homopolymeren dC-Schwanz der cDNA ist.The 5 'extended F3H cDNA was amplified using a second gene-specific primer (GSP-2) which binds in the region 3' before the GSP-1 recognition sequence and thus enables a "nested" PCR. The 5 'primer was that of Manufacturer supplied "5 'RACE abrided anchor primer", which is complementary to the homopolymeric dC tail of the cDNA.
Das so amplifizierte und als FSHextended bezeichnete cDNA Fragment wurde nach Herstellerangaben in den Vektor pGEM-T von Promega kloniert.The cDNA fragment amplified in this way and designated as FSH extended was cloned into the vector pGEM-T from Promega according to the manufacturer's instructions.
Die Identität der cDNA wurde durch Sequenzierung bestätigt.The identity of the cDNA was confirmed by sequencing.
Das FSHextended cDNA Fragment wurde unter Verwendung der im Polylinker des Vektors pGEM-T vorhandenen Restriktionsschnittstellen Ncol und Pstl isoliert und die überstehenden Enden unter Verwendung der T4-Polymerase und glatter Enden überführt. Dieses Fragment wurde in einen Smal (blunt) geschnittenen Vektor pBinAR (Höfgen und Willmitzer, 1990) kloniert (siehe Abbildung 3). Dieser enthält den 35S-Promotor des CaMV (Blumenkohlmosaikvirus) (Franck et al . , 1980) und das Terminationssignal des Octopin-Syn- thase Gens (Gielen et al., 1984). Dieser Vektor vermittelt in Pflanzen Resistenz gegen das Antibiotikum Kanamycin. Die erhaltenen DNA Konstrukte enthielten das PCR Fragment in Sense und Antisense Orientierung. Das Antisensekonstrukt wurde zur Er- zeugung transgener Pflanzen eingesetzt.The FSHextended cDNA fragment was isolated using the restriction sites Ncol and Pstl present in the polylinker of the vector pGEM-T and the protruding ends under Conversion using T4 polymerase and blunt ends. This fragment was cloned into a Smal (blunt) cut vector pBinAR (Höfgen and Willmitzer, 1990) (see Figure 3). This contains the 35S promoter of the CaMV (cauliflower mosaic virus) (Franck et al., 1980) and the termination signal of the octopine synthase gene (Gielen et al., 1984). This vector mediates resistance to the antibiotic kanamycin in plants. The DNA constructs obtained contained the PCR fragment in sense and antisense orientation. The antisense construct was used to generate transgenic plants.
Abbildung 3: Fragment A (529 bp) beinhaltet den 35S-Promotor des CaMV (Nukleotide 6909 bis 7437 des Blumenkohlmosaikvirus) . Fragment B das Fragment des F3H Gens in Antisense-Orientierung. Frag- ment C (192 Bp) enthält das Terminationssignal des Octopin-Syn- thase Gens .Figure 3: Fragment A (529 bp) contains the 35S promoter of the CaMV (nucleotides 6909 to 7437 of the cauliflower mosaic virus). Fragment B the fragment of the F3H gene in antisense orientation. Fragment C (192 bp) contains the termination signal of the octopine synthesis gene.
Beispiel 2Example 2
Herstellung transgener Lycopersicon esculentum Mill.cv. Moneymaker die ein Teilfragment der Flavanone-3-Hydroxylase in Antisense Orientierung exprimieren.Production of transgenic Lycopersicon esculentum Mill.cv. Moneymakers that express a partial fragment of flavanone 3-hydroxylase in an antisense orientation.
Es wurde die Methode nach Ling et al.. Plant Cell Report 17, 843 - 847 ( 1998 ) genutzt. Die Kultivierung erfolgt bei ca. 22°C unter einem 16 h - Licht / 8 h - Dunkel - Regime.The method according to Ling et al. Plant Cell Report 17, 843-847 (1998) was used. The cultivation takes place at approx. 22 ° C under a 16 h light / 8 h dark regime.
Tomatensamen (Lycopersicon esculentum Mill. cv. Moneymaker) wurden durch 10 minütige Inkubation in 4%iger Natriumhypochlorit - lösung inkubiert, anschließend 3 - 4 mal mit sterilem destilliertem Wasser gewaschen und auf MS Medium mit 3 % Saccharose, pH 6 , 1 zur Keimung ausgelegt. Nach einer Keimdauer von 7 - 10 d konnten die Kotyledonen für die Transformation eingesetzt werden.Tomato seeds (Lycopersicon esculentum Mill. Cv. Moneymaker) were incubated by 10 minutes incubation in 4% sodium hypochlorite solution, then washed 3-4 times with sterile distilled water and placed on MS medium with 3% sucrose, pH 6, 1 for germination . After a germination period of 7-10 d, the cotyledons could be used for the transformation.
Tag 1: Petrischalen mit dem Medium "MSBN" wurden mit 1,5 ml einer ca. 10 d alten Tabaksuspensionskultur überschichtet. Die Platten wurden mit Folie abgedeckt und bis zum nächsten Tag bei Raumtemperatur inkubiert.Day 1: Petri dishes with the medium "MSBN" were overlaid with 1.5 ml of an approximately 10 day old tobacco suspension culture. The plates were covered with foil and incubated at room temperature until the next day.
Tag 2: Auf die mit der Tabaksuspensionskultur beschichteten Platten wurde steriles Filterpapier luftblasenfrei aufgelegt. Darauf wurden die quer geschnittenen Keimblätter mit der Oberseite nach unten aufgelegt. Die Petrischalen wurden für 3 Tage im Kulturenraum inkubiert. Tag 5: Die Agrobakterienkultur (LBA4404) wurde durch Zentri- fugation bei ca. 3000g für 10 min sedimentiert und in MS-Mediu resuspendiert, so daß die OD 0,3 beträgt. In diese Suspension wurden die Keimblattstückchen gegeben, die unter leichtem Schüt- teln für 30 Minuten bei Raumtemperatur inkubiert wurden. Anschließend wurden die Keimblattstückchen auf sterilem Filterpapier etwas abgetrocknet und wieder zurück auf ihre Ausgangsplatten für die Fortsetzung der Cocultivierung für 3 Tage im Kulturenraum gelegt.Day 2: Sterile filter paper was placed on the plates coated with the tobacco suspension culture without air bubbles. The cross-cut cotyledons were placed on top with the top down. The petri dishes were incubated for 3 days in the culture room. Day 5: The agrobacterial culture (LBA4404) was sedimented by centrifugation at approx. 3000 g for 10 min and resuspended in MS medium so that the OD was 0.3. The cotyledon fragments were added to this suspension, which were incubated with gentle shaking for 30 minutes at room temperature. The cotyledon fragments were then dried off somewhat on sterile filter paper and placed back on their starting plates for the continued cultivation for 3 days in the culture room.
Tag 8: Die cocultivierten Keimblattstückchen wurden auf MSZ2K50+ß gelegt und für die nächsten 4 Wochen im Kulturenraum inkubiert. Danach erfolgte die Subkultivierung.Day 8: The cocultivated pieces of cotyledon were placed on MSZ2K50 + ß and incubated for the next 4 weeks in the culture room. Subcultivation then took place.
Sich bildende Sprosse wurden auf Wurzelinduktionsmedium gebracht.Rungs that formed were placed on root induction medium.
Nach erfolgreicher Bewurzelung konnten die Pflanzen getestet und ins Gewächshaus überführt werden.After successful rooting, the plants could be tested and transferred to the greenhouse.
Beispiel 3Example 3
Hemmung der Cholesteri biosynthese in Kulturen primärer Rattenhe- patocytenInhibition of cholesterol biosynthesis in cultures of primary rat hepatocytes
Herstellung der StammlösungenProduction of stock solutions
Vom Lyophilisat reifer Tomaten der Sorte "Moneymaker" enthaltendContaining ripe tomatoes of the "Moneymaker" variety
A) allein das native Flavanon- 3 -hydroxylase Gen (Kontrolle) sowieA) only the native flavanone 3-hydroxylase gene (control) and
B) wie in Beispiel 2 beschrieben zusätzlich ein Teilfragment der Flavanon-3 -hydroxylase in Antisens Orientierung wurde eine Menge zwischen 10 und 20 mg exakt abgewogen und mit soviel DMSO versetzt, daß eine Stammlösung von 10 mM Gesamtflavonoide entstand. Von diesen Stammlösungen wurden unmittelbar vor Testbeginn Verdünnungen im Kulturmedium hergestellt. Die Verdünnungen er- folgten in 10er Schritten zwischen 10-4 und 10"8 M.B) as described in Example 2, in addition a partial fragment of flavanone-3-hydroxylase in antisense orientation, an amount between 10 and 20 mg was weighed exactly and so much DMSO was added that a stock solution of 10 mM total flavonoids was formed. Dilutions of these stock solutions were made in the culture medium immediately before the start of the test. The dilutions were made in 10 steps between 10 -4 and 10 " 8 M.
Herstellung der HepatocytenkulturenPreparation of the hepatocyte cultures
Primäre Hepatocyten wurden aus den Lebern von männlichen Spraque- Dawley Ratten (240-290 g) mittels Collagenase-Perfusion gewonnen (Gebhardt et al., Arzneimittel-Forschung/Drug Res. 41: 800 -804 (1991) 1990) . Die Kultivierung erfolgte in Collagen-beschichteten Petrischalen (6-well Plates, Greiner, Nürtingen) mit einer Zelldichte von 125.000 Zellen/cm2 in Williams Medium E mit 10 % Kälberserum. Nähere Angaben insbesondere zum Kulturmedium finden sich bei Gebhardt et al., Cell Biol. Toxicol. 6: 369 - 372 (1990) und Mewes et al . , Cancer Res. 53: 5135 - 5142 (1993). Die Kulturen wurden nach 2 h auf serum-freies Medium mit Zusatz von 0,1 μM Insulin gewechselt. Sie wurden nach weiteren 20 h für die Versuche eingesetzt. Die Testsubstanzen wurden in je drei unabhängigen Kulturen von 2-3 Ratten getestet.Primary hepatocytes were obtained from the liver of male Spraque-Dawley rats (240-290 g) by means of collagenase perfusion (Gebhardt et al., Arzneimittel-Forschung / Drug Res. 41: 800-804 (1991) 1990). The cultivation was carried out in collagen-coated petri dishes (6-well plates, Greiner, Nürtingen) with a cell density of 125,000 cells / cm2 in Williams Medium E with 10% calf serum. Further information, in particular on the culture medium, can be found in Gebhardt et al., Cell Biol. Toxicol. 6: 369-372 (1990) and Mewes et al. , Cancer Res. 53: 5135-5142 (1993). The Cultures were changed after 2 h to serum-free medium with the addition of 0.1 μM insulin. They were used for the experiments after a further 20 h. The test substances were tested in three independent cultures of 2-3 rats.
Inkubation der Leberzellkulturen mit den Testsubstanzen A und BIncubation of the liver cell cultures with the test substances A and B
Für den Nachweis einer Beeinflussung der Cholesterin-Biosynthese durch die Testsubstanzen A und B wurden die Hepatocytenkuturen insgesamt für 22 h gehalten. Anschließend wurde mit serum-freiem Williams Medium E unter Zusatz von 14C-Acetat (nur Tracermengen) für 2 h mit den Testsubstanzen in den angegebenen Konzentrationen inkubiert. Bei jeder Testserie wurde eine Kontrolle mitgeführt. Die Methodik ist bei Gebhardt (1991) und Gebhardt, Lipids 28: 613 -619 (1993) detailliert beschrieben. Die Tracermengen von 14C-Ace- tat tauschen schnell mit dem intrazellulären Acetyl-CoA Pool aus und ermöglichen deshalb eine störungsfreie Bestimmung des Einbaus von 1C-Acetat in die Sterolfraktion, die zu >90 % aus Cholesterin besteht (Gebhardt, 1993).The hepatocyte cultures were held for a total of 22 h to demonstrate that cholesterol biosynthesis was influenced by test substances A and B. It was then incubated with serum-free Williams Medium E with the addition of 14 C acetate (only tracer amounts) with the test substances in the stated concentrations for 2 h. A control was carried out for each test series. The methodology is described in detail in Gebhardt (1991) and Gebhardt, Lipids 28: 613 -619 (1993). The tracer amounts of 14 C-acetate quickly exchange with the intracellular acetyl-CoA pool and therefore enable a trouble-free determination of the incorporation of 1 C-acetate in the sterol fraction, which consists of> 90% cholesterol (Gebhardt, 1993).
Analytik zur Beeinflussung der Cholesterin-BiosyntheseAnalytics for influencing cholesterol biosynthesis
Der Einbau von 1C-Acetat in die Sterolfraktion (nicht-verseifbare Lipide) wurde nach Gebhardt (1991) gemessen. Bei der verwendeten Extraktion mittels Extrelut®-Säulen (Merck, Darmstadt) wird das 14C-Acetat (und daraus in geringer Menge entstehende andere niedermolekulare Metabolite) zu mehr als 95 % abgetrennt. Dieser Test kann vergleichende Angaben über die relative Syntheserate von Cholesterin und Vorlaufer-Sterolen unter dem Einfluss von Testsubstanzen machen (Gebhardt, 1993) .The incorporation of 1 C-acetate into the sterol fraction (non-saponifiable lipids) was measured according to Gebhardt (1991). When using extraction using Extrelut ® columns (Merck, Darmstadt), more than 95% of the 14 C-acetate (and other small-molecule metabolites resulting from it) is separated off. This test can provide comparative information on the relative synthesis rate of cholesterol and precursor sterols under the influence of test substances (Gebhardt, 1993).
Visuelle und mikrobielle Überprüfung der Qualität der Hepatocy- tenkul urenVisual and microbial inspection of the quality of the hepatocyte cultures
Alle verwendeten Kulturen wurden vor und nach der Testinkubation visuell am Mikroskop auf Kontamination mit Mikroorganismen und auf die Integrität der Zellmonolayer überprüft. Bei keiner der Proben wurde eine erkennbare Veränderung der Zellmorphologie (insbesondere bei den höheren Konzentrationen) beobachtet. Dies schließt eine Beeinflussung der Testergebnisse durch zytotoxische Wirkungen der Testsubstanzen weitgehend aus.All cultures used were checked visually on the microscope for contamination with microorganisms and for the integrity of the cell monolayer before and after the test incubation. No discernible change in cell morphology (especially at the higher concentrations) was observed in any of the samples. This largely precludes the test results from being influenced by the cytotoxic effects of the test substances.
Die bei allen Kulturen routinemäßig durchgeführten Sterilitätstests ergaben keinerlei Hinweise auf eine Kontamination mit Mi- kroorganismen. ErgebnisseThe sterility tests carried out routinely for all cultures showed no evidence of contamination with microorganisms. Results
Proben A) aus den nicht genetisch modifizierten Tomaten (Kontrolle) zeigte keinerlei Wirkungen auf die Cholesterinbio- synthese. Dagegen wurde die Cholesterinsynthese durch Proben B aus den ein Teilfragment der Flavanon-3 -hydroxylase in Antisens- Orientierung enthaltenden Tomaten signifikant inhibiert. Samples A) from the non-genetically modified tomatoes (control) showed no effects on cholesterol biosynthesis. In contrast, cholesterol synthesis was significantly inhibited by samples B from the tomatoes containing a partial fragment of flavanone 3-hydroxylase in the antisense orientation.

Claims

Patentansprüche claims
1. Verfahren zur Erhöhung des Gehalts an Flavonoiden und phenolischen Inhaltsstoffen in Pflanzen dadurch gekennzeichnet, daß mit molekulargenetischen Methoden eine Pflanze hergestellt wird in der die Aktivität des Enzyms Flavanon-3-hydroxylase reduziert ist.1. A method for increasing the content of flavonoids and phenolic ingredients in plants, characterized in that a plant is produced using molecular genetic methods in which the activity of the enzyme flavanone-3-hydroxylase is reduced.
2. Verfahren gemäß Anspruch 1 zur Erhöhung des Gehalts an Flavonoiden und phenolischen Inhaltsstoffen in Pflanzen dadurch gekennzeichnet, daß das Enzym Flavanon-3-hydroxylase durch molekularbiologische Verfahren (z.B. Anti-Sense-Konstrukt, Co-Suppression, der Expression spezifischer Antikörper oder der Expression spezifischer Inhibitoren) ganz oder teilweise, andauernd oder vorübergehend, in der gesamten Pflanze oder in Teilen der Pflanze in seiner Aktivität reduziert ist.2. The method according to claim 1 for increasing the content of flavonoids and phenolic ingredients in plants, characterized in that the enzyme flavanone-3-hydroxylase by molecular biological methods (eg anti-sense construct, co-suppression, the expression of specific antibodies or expression specific inhibitors) is reduced in its activity in whole or in part, continuously or temporarily, in the entire plant or in parts of the plant.
3. Verfahren gemäß den Ansprüchen 1 und 2, dadurch gekennzeich- net, daß es sich bei den Pflanzen um Weinrebe, Kirsche, Tomate, Pflaume, Schlehe, Blaubeere, Erdbeere, Zitrusfrüchte3. The method according to claims 1 and 2, characterized in that the plants are grapevine, cherry, tomato, plum, sloe, blueberry, strawberry, citrus
(wie Orange, Grapefruit), Papaya, Rotkohl, Broccoli, Rosenkohl, Kakao, Grünkohl, Karotte, Petersilie, Sellerie, Zwiebeln, Knoblauch, Tee, Kaffee, Hopfen, Soja, Raps, Hafer, Wei- zen, Roggen, Aronia melanocarpa, Ginkgo biloba handelt.(such as orange, grapefruit), papaya, red cabbage, broccoli, Brussels sprouts, cocoa, kale, carrot, parsley, celery, onions, garlic, tea, coffee, hops, soy, rapeseed, oats, wheat, rye, Aronia melanocarpa, Ginkgo biloba acts.
4. Pflanze mit erhöhtem Gehalt an Flavonoiden und phenolischen Inhaltsstoffen hergestellt nach einem Verfahren gemäß den Ansprüchen 1 - 3 dadurch gekennzeichnet, daß die enzymatische Aktivität des Enzyms Flavanon-3-hydoxylase reduziert ist.4. Plant with an increased content of flavonoids and phenolic ingredients produced by a process according to claims 1-3, characterized in that the enzymatic activity of the enzyme flavanone-3-hydoxylase is reduced.
5. Verwendung von Pflanzen oder von Teilen dieser Pflanzen hergestellt nach einem Verfahren gemäß den Ansprüchen 1 - 3 als Nahrungsmittel, Nahrungsergänzungsmittel oder zur Herstellung von heilenden, gesundheitsfördernden oder stärkenden Mitteln (Säfte, Tees, Extrakte, Fermentationsprodukte) für Mensch und Tier sowie zur Herstellung von Kosmetika. 5. Use of plants or parts of these plants produced by a process according to claims 1-3 as foods, food supplements or for the production of curative, health-promoting or strengthening agents (juices, teas, extracts, fermentation products) for humans and animals and for the production of cosmetics.
EP00945714A 1999-06-17 2000-06-07 Method for producing plants with increased flavonoid and phenolic compound content Withdrawn EP1102855A1 (en)

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US20140072692A1 (en) * 2012-06-07 2014-03-13 Ichiro Yamada Garlic egg yolk composition
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