JP2003503032A - Method for producing plant with increased content of flavonoids and phenolic compounds - Google Patents

Abstract

  (57) [Summary] A method for increasing a flavonoid content in a plant, comprising preparing a plant having a reduced activity of the enzyme flavanone 3-hydroxylase by a technique of molecular genetics.

Description

Detailed Description of the Invention

The present invention relates to a method for increasing the content of flavonoid and phenol components in a plant, characterized by producing a plant in which the activity of the enzyme flavanone 3-hydroxylase is reduced by a method of molecular genetics. .

Further, in the above-mentioned method according to the present invention, the activity of the enzyme flavanone 3-hydroxylase in the whole or a part of the plant body is measured by a molecular biology method (for example
Antisense constructs, co-suppression, expression of specific antibodies, or expression of specific inhibitors), either fully or partially, permanently or temporarily.

The invention further relates to plants having an increased content of flavonoids and phenolic components, the enzymatic activity of the enzyme flavanone 3-hydroxylase being reduced.

Furthermore, the present invention provides a food composition for humans and animals, as a dietary supplement, or as a therapeutic composition, health promoting composition or tonic (squeezed juice, exudate, extract, fermentation product). A), as well as the production of cosmetics, of a plant or parts of these plants produced by the method according to the invention.

Various phenolic substances have been found in plants, for example, caffeic acid, ferulic acid, chlorogenic acid, gallic acid, eugenol, lignan, coumarin, lignin, stilbene (polydatin, resveratrol), flavonoid (flavone). , Catechins, flavanones, anthocyanidins, isoflavones), and polymethoxylated flavones. Therefore, phenol is also generally a component of many plant-derived foods and treats. Certain phenolic substances are of particular importance. Because they can show antioxidant effects on human or animal metabolism after being taken up with food [Baum, BO; Perun, AL Antiox.
idant efficiency versus structure. Soc. Plast. Engrs Trans 2: 250-257 (19
62); Gardner, PT; McPhail, DB; Duthie, GG Electron spin resonance
spectroscopic assessment of the antioxidant potential of teas in aqueous
and organic media. J. Sci. Food Agric. 76: 257-262, (1997) ； Rice-Evans,
CA; Miller, NJ; Pananga, G. Structure-antioxidant activity relations
hip of flavonoids and phenolic acids. Free Radic. 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: 33
9-346, (1995); Stryer, L. Biochemistry S. Francisco: Freeman, (1975); Vieira, O .; Escargueil-Blanc, I .; Meilhac, O .; Basile,
JP; Laranjinha, J .; Almeida, L; Salvayre, R .; Negre-Salvayre, A. Effe
ct of dietary phenolic compounds on apoptosis of human cultured endothel
ial cells induced by oxidized LDL. Br J Pharmacol 123: 565-573, (1998)]
. Moreover, polyphenols also have many effects on cellular metabolism. In particular, they modulate signaling enzymes such as protein kinase C, tyrosine protein kinases and phosphatidylinositol 3-kinase [
Agullo, G .; Gamet-payrastre, L .; Manenti, S .; Viala, C .; Remesy, C .; Cha
p, H .; Payrastre, B. Relationship between flavonoid structure and inhibi
tion of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase
and protein kinase C inhibition. Biochem Pharmacol 53: 1649-1657, (1997)
Ferriola, PC; Cody, V .; Middleton, E. Protein kinase C inhibition by
plant flavonoids. Kinetic mechanisms and structure activity relationshi
p. Biochem Pharmacol 38: 1617-1624, (1989); Cushman, M .; Nagarathman, D .;
Burg, DL; Geahlen, RL Synthesis and protein-tyrosine kinase inhibit
ory activity of flavonoids analogues. J. Meed Chem 34: 798-806, (1991) ； H
agiwara, M .; Inoue, S .; Tanaka, T .; Nunoki, K .; Ito, M .; Hidaka, H. Diff
erential effects of flavonoids as inhibitors of tyrosine protein kinases
and serine / threonin protein kinases. Biochem Pharmacol 37: 2987-2992, (1
988)], thereby down-regulating inducible NO synthesis [Kobuchi, H .;
Droy-Lefaix, MT; Christen, Y .; Packer, L. Ginkgo biloba extract (EGb7
61): inhibitory effect on nitric oxide production in the macrophage cell
line RAW 264.7. Biochem Pharmacol 53: 897-903, (1997)], and further regulates gene expression of eg interleukins and adhesion molecules (ICAM-1, VCAM-1) [Kobuchi, H .; Droy-Lefaix, MT; Christen, Y .; Packer, L. Ginkgo biloba
extract (EGb761): inhibitory effect on nitric oxide production in the m
acrophage cell line RAW 264.7. Biochem Pharmacol 53: 897-903, (1997); Wol
le, J .; Hill, RR; Ferguson, E .; Devall, LJ; Trivedi, BK; Newton, R
.S .; Saxena, U. Selective inhibition of Tumor necrosis Factor-induced va
scular cell adhesion molecule-1 gene expression by a novel flavonoid. La
ck of effect on transcriptional factor NF-kB. Atherioscler Thromb Vasc B
iol 16: 1501-1508, (1996)]. These effects are now found to be beneficial in the prevention of infarction, cardiovascular disease, diabetes, various specific cancers, tumors and other chronic diseases [Bertuglia, S .; Malandrino, S. Colantuoni, A. Effects
of the natural flavonoid delphinidin on diabetic microangiopathy. Arznei
-Forsch / Drug Res. 45: 481-485, (1995); Griffiths, K .; Adlercreutz, H .; Bo
yle, P .; Denis, L .; Nicholson, RI; Morton, MS Nutrition and Cancer O
xford: Isis Medical Media, (1996); Hertog, MGL; Fesrens, EJM; Holl
man, PCK; Katan, MB; Kromhout, D. Dietary antioxidant flavonoids an
d risk of coronary heart disease: the Zutphen elderly study. The Lancet
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 da
mage by atherogenic LDL. Arterioscler Thromb Vasc 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; Matter
n, T .; Folts, JD; Weisgerber, UM; Katan, MB Tea flavonoids and car
diovascular diseases: Review. Crit Rev Food Sci Nutr 37: 771-785, (1997) ； Ki
rk, EA; Sutherland, P .; Wang, SA; Chait, A .; LeBoeuf, RC Dietary i
soflavones reduce plasma chloresterol and atherosclerosis in C57BL / 6 mic
e but not LDL receptor-deficient mice. J Nutr 128: 954-9, (1998) -Cited document-]. Therefore, a series of therapeutic compositions, health-enhancing compositions or tonics whose effects are based on their phenolic content have already been obtained from suitable plants [Gerritsen, ME; Carley, WW; Ranges, GE; Shen, CP;
Phan, SA; Ligon, GF; Perry, CA Flavonoids inhibit cytokine-induce
d endothelial cell adhesion protein gene expression. Am J Pathol 147: 278
-292, (1995); Lin, JK; Chen, YC; Huang, YT; Lin-Shiau, SY Suppre
ssion of protein kinase C and nuclear oncogene expression as possible mo
lecular 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 chemopreventive effects of a flavonoid antioxidant silymar
in: inhibition of mRNA expression of an endogenous tumor promoter TNF al
pha. Biochem Biophys Res Comm 239: 334-339, 1997]. Further, it is known that foods derived from a specific plant or luxury products prepared from them have a positive effect on various diseases. For example, resveratrol, which is found (in addition to other ingredients) in white wines, but especially in red wines, acts against infarction, cardiovascular disease 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 receptor.
Proc Natl Acad Sci USA 94: 14138-14143, (1997); Jang, M .; Cai, L .; Udean
i, GO; Slowing, KV; Thomas, CF; Beecher, CWW; Fong, HHS; Farn
sworth, NR; Kinghorn, AD; Mehtha, RG; Moon, RC; Pezzuto, JM Ca
ncer chemopreventive activity of resveratrol, a natural product derived
from grapes. Science 275: 218-220, (1997)]. Similar effects have been found in substances such as catechin, epicatechin-3-gallate, epigallocatechin and epigallocatechin-3-gallate, all of which are tea (Camellia sinensi).
s) found in leaves. Beverages made from unfermented tea leaves (green tea) are especially health-promoting [Hu, G .; Han, C .; Chen, J. Inhibition of oncogene express
ion by green tea and (-)-epigallocatechin gallate in mice. Nutr cancer 2
4: 203-209; (1995); Scholz, E .; Bertram, B. Camellia sinensis (L.) O. Ku
ntze. Der Teestrauch [the tea shrub]. Z. Phytotherapie 17: 235-250, (1995
); Yu, R .; Jiao, JJ; Duh, JL; Gudehithlu, K .; Tan, TH; Kong, AN
Activation of mitogen-activated protein kianses by green tea polyphenols
: potential signaling pathways in the regulation of antioxidant responsi
ve elements-mediated phase II enzyme gene expression. Carcinigenesis 18:
451-456, (1997); Jankun, J .; Selman, SH; Swiercz, R. Why drinking gree
n tea could prevent cancer. Nature 387: 561, (1997)]. In addition, polymethoxylated flavones from citrus fruits also show potential antitumor activity [Chem, J .; Montanari, AM; Widmer, WW Two new polymethoxylierte flav
one, a class of compounds with potential anticancer activity, isolated f
rom cold pressed dancy tangerine peel oil solids. J Agric Food Chem 45: 3
64-368, (1997)].

It is an object of the present invention to find a simple and inexpensive way to increase the content of flavonoids and phenolic components in cultivated plants.

The inventors have surprisingly achieved this object by the genetic engineering methods currently available, based on physiological studies on growth regulators from the acylcyclohexanedione group, and It has been found that the method can be used to produce plants characterized by an increased content of therapeutic, health-promoting, or tonic ingredients.

Acylcyclohexanediones such as prohexadione-calcium and trinexapac-ethyl (formerly known as Simectacurb) are used as bioregulators to control the vertical growth of plants. They exhibit bioregulatory effects because they interfere with the biosynthesis of gibberellin, which promotes longitudinal growth. Due to their structural association with 2-oxoglutarate, they inhibit certain dioxygenases that require 2-oxoglutarate as a co-substrate [Rademacher
, W, Biochemical effects of plant growth retardants, Plant Biochemical R
Among egurators, Gausman, HW (ed.), Marcel Dekker, Inc., New York, pp. 169-200
(1991)]. It is known that such compounds are also involved in phenol metabolism and therefore can inhibit anthocyanin production in various plant species [Ra
demacher, W. et al., The mode of action of acylcyclohexanediones-a new type
of growth retardant, Progress in Plant Growth Regulation, Karssen, CM
, van Loon, LC, Vreugdenhil, D (ed.), Kluwer Academic Publishers, Dordrech
t (1992)]. Such effects on the balance of the phenolic component are thought to be responsible for the side effects of prohexadione-calcium on burns [Radema
cher, W. et al., prohexadione-calcium − a new plant growth regulator for app
le with interesting biochemical features, 25 ^th Annual Meeting of the Pla
nt Growth Regulation Society of America, 7-10 July 1998, poster at Chicago]. A. Lux-Endrich (Technical University of Munich, We
Ph.D. dissertation at ihenstephan, 1998) found that the content of phenolic substances increased several-fold in apple cell cultures during the study of the mechanism of action of prohexadione-calcium on burn injury caused by prohexadione-calcium. However, in the process, they found a series of phenols that are not normally present. It was also found in these studies that prohexadione-calcium produced relatively high amounts of luteoliflavan and eriodictyol in shoot tissues of apple. Luteoli flavan is not normally present in apple tissue, and eriodictyol is only present in small amounts only as an intermediate in flavonoid metabolism. However, in the treated tissue, the expected flavonoids and is catechin and or cyanidin was undetectable, or was only present at a significantly lower amount [S. Rommelt et ^{al, 8 th International Workshop on Fire Blight} ,
Kusadasi, Turkey, documents submitted 12-15 October 1998].

[0009] Prohexadione-calcium, trinexapac-ethyl and other acylcyclohexanediones are now found to inhibit 2-oxoglutarate-dependent hydroxylases, which play an important role in the metabolism of phenolic substances. ing. These are primarily chalcone synthetase (CHS) and flavanone 3-hydroxylase (F3H) [W. Heller and G. Forkmann, Biosynthesis, The
In Flavonoids, Harborne, JB (eds.), Chapman and Hall, New York, 1998].
However, it cannot be ignored that acylcyclohexanedione also inhibits other as yet unknown 2-oxoglutarate-dependent hydroxylases. Furthermore, it appears that deficiencies in catechin, cyanidin, and other end products of flavonoid synthesis appear in plants, and that the activity of the key enzyme phenylalanine ammonium lyase (PAL) is increased by a feedback mechanism. . However, due to the continued inhibition of CHS and F3H, these flavonoid end products could not be produced, resulting in increased production of luteoliflavan, eriodictythiol and other phenols (FIG. 1).

Due to the reduced enzymatic activity of the enzyme flavanone 3-hydroxylase (F3H), the flavonoids eriodictyol, proanthocyanidins (substituted by hydrogen at the C ₃ atom), such as luteoferol, luteoliflavan , Apigeniflavan and tricetiflavan, as well as homogeneous and heterogeneous oligomers and polymers of the above structurally related substances.

[0011] In plants, after the enzymatic activity of the enzyme flavanone 3-hydroxylase (F3H) decreases, the phenol hydroxycinnamic acid (p-coumaric acid, ferulic acid, sinapinic acid), salicylic acid, or umbelliferone ( Increasing concentrations of homogenous or heterogeneous oligomers and polymers produced from them) are seen. Similarly, the concentrations of chalcones (eg phloretin) and stilbenes (eg resveratrol) are increased.

As the enzymatic activity of the enzyme flavanone 3-hydroxylase decreases, so does the concentration of flavonoid glycosides, phenolic compounds, chalcones and stilbenes.

Based on these findings and hypotheses derived from them, F3H activity in whole plants or individual plant organs or tissues can be reduced completely or partially by antisense constructs, permanently or temporarily. Genetically modified cultivated plants were produced in which the content of therapeutic, health-promoting or tonic components was improved in quantity and quality.

The method according to the invention for increasing the content of flavonoids and phenolic compounds by expressing flavanone 3-hydroxylase in the antisense orientation can be successfully applied to the following cultivated plants, which method: Are not limited to the plants mentioned: grape vines, cherries, tomatoes, plums, limbs, cowberry, strawberries, citrus fruits (eg oranges, grapefruits), popo, red cabbage, broccoli, mecha cabbage, cacao, kale, carrots. , Parsley, celeriac / celery, onion, garlic, tea, coffee, hops,
Soybean, canola, oats, wheat, rye, caydon (Aronia melanoc
arpa), Ginkgo biloba.

The invention further relates to plants produced according to the method of the invention and having an increased content of flavonoids and phenolic compounds, which have a reduced enzymatic activity of the enzyme flavanone 3-hydroxylase.

Alternative methods for producing plants whose flavanone 3-hydroxylase activity is reduced using antisense techniques are known from the literature such as co-suppression or expression of specific antibodies. It is also possible to achieve this effect using other methods of molecular genetics.

Furthermore, the present invention relates to foods for humans and animals, as dietary supplements or as therapeutic compositions, health promoting compositions or tonics (squeezed juices, exudates, extracts, fermentation products). The use of plants or parts of plants produced by the method according to the invention for the manufacture of, as well as for the manufacture of cosmetics.

Surprisingly, here the plants or parts of these plants produced according to the invention or the products produced therefrom (tea, extracts, fermentation products, juices etc.) have the following effects: It was discovered.

(1) The in vitro antioxidant ability [electron spin resonance (ESR), LDL oxidation, comprehensive antioxidant ability, NO removal] is improved; (2) enzyme, especially signal transduction enzyme (protein kinase) C, tyrosine protein kinase, phosphatidylinositol 3-kinase) have a modulating effect; (3) redox-sensitive transcription factor (NF-kB, AP-1) modulation in endothelial cells, lymphocytes and smooth muscle cells (4) Gene expression of target genes involved in the pathogenesis of inflammatory diseases (cytokines IL-1 and IL-8, macrophage chemoinducible protein 1 (MCP-1), adhesion factors ICAM-1 and VCAM-1) (5) Induction of anti-aggregation effect; (6) Inhibition of cholesterol synthesis in hepatocytes; (7) Anti-proliferative / anti-neoplastic effect Appear.

Example 1 Cloning of flavanone 3 -hydroxylase gene from tomato (Lycopersicon esculentum Mill; variety Moneymaker). Ripe tomato fruits of tomato (Lycopersicon esculentum Mill; variety Moneymaker) were washed, drained and sterilized with a knife. The pericarp was used to separate the seed, the central columella and the xylem. Pericarp (about 50 g) was frozen in liquid nitrogen. The material was then chopped with a blender. 100 m of the chopped material in a pre-cooled mortar
l of homogenizing solvent and mixed. The suspension was then transferred to a centrifuge flask by squeezing through sterile gauze. Then 1/10 volume of 10% SDS was added and the material was mixed well. After 10 minutes on ice, 1 volume of phenol / chloroform was added, the centrifuge flask was sealed and the contents were mixed well. 1 at 4000 rpm
After centrifugation for 5 minutes, the supernatant was transferred to a new reaction vessel. This was followed by another 3 phenol / chloroform extractions and 1 chloroform extraction.
Then 1 volume of 3M NaAc and 2.5 volumes of ethanol were added. Nucleic acid -20 ℃
It was allowed to settle overnight. The next morning, the nucleic acids were pelleted in a refrigerated centrifuge (4 ° C) for 15 minutes at 10,000 rpm. The supernatant was discarded and the pellet resuspended in 5-10 ml cold 3M NaAc. This washing step was repeated twice. The pellet was washed with 80% ethanol. Once completely dry, the pellet was taken up in about 0.5 ml of sterile DEPC water and RNA concentration was measured spectrophotometrically.

20 μg of total RNA was first treated with 3.3 μl of 3M sodium acetate solution, 2 μl of 1M magnesium sulfate and the mixture was brought to a final volume of 100 μl with DEPC water. 1
μl RNase-free DNase (Boehringer Mannheim) was added to this and the mixture was incubated at 37 ° C.
Incubated for 45 minutes. The enzyme was removed by adding phenol / chloroform / isoamyl alcohol and shaking to extract, then RNA was precipitated with ethanol and the pellet was taken up in 100 μl of DEPC water. 2.5 μg RNA from this solution
It was transcribed using a cDNA kit (Gibco BRL) to obtain cDNA.

Amino acid sequences derived from a cDNA clone encoding flavanone 3-hydroxylase were used to identify conserved regions within the primary sequence [Britsch et al., Eur. J. Bioch.
em. 217, 745-754 (1993)], which served as the basis for designing degenerate PCR oligonucleotides. Peptide sequence SRWPDK (Petunia hybrid (Pet
unia hybrida) The 5'oligonucleotide was determined using amino acids 147 to 152) in the sequence FL3H PETHY and had the following sequence.

[0023]   5'-TCI (A / C) G (A / G) TGG CC (A / C / G) GA (C / T) AA (A / G) CC-3

The sequence of the oligonucleotides derived by using the peptide sequence DHQAVV (amino acids 276-281 in the Petunia hybrida sequence FL3H PETHY) was as follows: 5'-CTT CAC ACA (C / G / T) GC (C / T) TG (A / G) TG (A /
G) TC-3.

PCR reactions were performed using Perkin Elmer tTth polymerase according to the manufacturer's instructions. The template used was 1/8 of cDNA (corresponding to 0.3 μg of RNA). The PCR program was as follows.

[0026]   30 cycles   94 ° C, 4 seconds   40 ° C, 30 seconds   72 ° C, 2 minutes   72 ° C, 10 minutes

This fragment was cloned into the vector pGEM-T from Promega according to the manufacturer's instructions.

The fragment was confirmed to be correct by sequencing. Restriction cleavage sites NcoI and PstI
(Present in the polylinker of vector pGEM-T) is used to isolate the PCR fragment,
The overhang was made blunt with T4-polymerase. This fragment was digested with SmaI (blunt) vector pBinAR [Hofgen and Willmitzer, Plant Sci. 66: 221-230 (1990)].
(See Figure 2). This vector contains a CaMV (cauliflower mosaic virus) 35S promoter [Franck et al., Cell 21: 285-294 (1980)] and a termination signal derived from the octopine synthase gene [Gielen et al., EMBO J. 3: 835-.
846 (1984)]. This vector mediates resistance to the antibiotic kanamycin in plants. The resulting DNA construct contained the PCR fragment in sense and antisense orientations. The antisense construct was used for the production of transgenic plants.

FIG. 2: Fragment A (529 bp) contains the CaMV 35S promoter (nucleotides 6909-7437 of the cauliflower mosaic virus). Fragment B contains a fragment of the F3H gene in the antisense orientation. Fragment C (192 bp) contains the termination signal for the octopine synthase gene.

Tomatoes (Lycopersicon esculentum Mill; variety Moneymaker) using 5'RACE system
Cloning of a Larger cDNA Fragment of Flavanone 3-Hydroxylase from Escherichia coli Avoids the Generation of Plants with Reduced F3H mRNA Flow Balance, Due to the Small Size of F3H PCR Fragments Used in Antisense Constructs In order to make a second antisense construct with a larger F3H fragment.

The 5'RACE method (a system for rapid amplification of cDNA ends) was used for cloning larger F3H fragments.

Extension of F3H PCR fragment by 5'RACE method using 5'RACE system ( Version 2 to 0 of Life Technologies ^™ ) for rapid amplification of cDNA ends Total tomato (Lycopersicon esculentum Mill; cultivar) Moneymaker) isolated from ripe tomato fruit (see above).

The synthesis of the first strand of the cDNA is performed by GSP-1 (gene specific primer) 5'-TTCACCACTGCCTG
Performed according to the manufacturer's instructions using GTG GTCC-3 '. After digestion with RNase, the cD
NA was purified using the GlassMAX spin system from Life Technologies ^™ according to the manufacturer's instructions.

Cytosine homopolymer was added to the 3'end of the purified single-stranded F3H cDNA using terminal deoxynucleotidyl transferase according to the manufacturer's instructions.

The 5′-extended F3H cDNA was used as a second gene-specific primer (GSP-2) (this was GSP-1).
It was attached to the region 3'upstream of the recognition sequence, thereby allowing a "nested" PCR to be performed). The 5'primer used was the "5'RACE Cleavage Anchor Primer", which was provided by the manufacturer and the cDNA
Is complementary to the homopolymeric dC tail of.

The cDNA thus amplified was named F3H _extended and cloned into the vector pGEM-T of Promega according to the manufacturer's instructions.

[0037]   The identity of the cDNAs was confirmed by sequencing.

The F3H _extended cDNA fragment was isolated using the restriction cleavage sites NcoI and PstI (present within the polylinker of the vector pGEM-T) and its overhangs made blunt-ended using T4-polymerase. This fragment was cloned into the SmaI (blunt) cut vector pBinAR (Hofgen and Willmitzer, 1990) (see Figure 3). This vector is Ca
It contains the MV (cauliflower mosaic virus) 35S promoter (Franck et al., 1980) and the termination signal from the octopine synthase gene (Gielen et al., 1984). This vector mediates resistance to the antibiotic kanamycin in plants. The resulting DNA construct contained the PCR fragment in sense and antisense orientations. The antisense construct was used for the production of transgenic plants.

FIG. 3: Fragment A (529 bp) contains the CaMV 35S promoter (cauliflower mosaic virus nucleotides 6909-7437). Fragment B contains a fragment of the F3H gene in the antisense orientation. Fragment C (192 bp) contains the termination signal for the octopine synthase gene.

[0040] Example 2 flavanone 3-hydroxylase transgenic tomatoes expressing partial fragments in antisense orientation; method used for manufacturing a (Lycopersicon esculentum Mill cultivar Moneymaker) is, Ling et al., Plant Cell Report 17, 843- 847 (1998). Cultivation was carried out at about 22 ° C. under the control system of 16 hours-light / 8 hours-dark.

Tomato seeds (Lycopersicon esculentum Mill; variety Moneymaker) were sterilized by incubation in a 4% strength sodium hypochlorite solution for 10 minutes,
Subsequently, it was washed 3 to 4 times with sterile distilled water, and 3% sucrose was added for germination.
It was placed on MS medium (pH 6.1). After a germination period of 7-10 days, cotyledons were prepared for use in transformation.

Day 1: A Petri dish containing "MSBN" medium was overlaid with 1.5 ml of approximately 10 day old tobacco suspension culture. The plate was covered with film and incubated at room temperature until the next day.

Day 2: Sterile filter paper was placed on the plate overlaid with the tobacco suspension culture to prevent air bubbles from forming. The cotyledons cut laterally were placed upside down on the filter paper. The dish was incubated in the culture chamber for 3 days.

Day 5: Agrobacterium culture (LBA4404) was precipitated by centrifugation at about 3000 g for 10 minutes and resuspended in MS medium to an OD of 0.3.
Cotyledon sections were placed on this suspension and incubated for 30 minutes at room temperature with gentle shaking. Then, the cotyledon pieces were dried to some extent on sterile filter paper, returned to the original plate, and co-culture was continued in the culture chamber for 3 days.

Day 8: Co-cultured cotyledonary sections were mounted on MSZ2K50 + β and incubated in the culture chamber for an additional 4 weeks. Then, they were subcultured.

[0046]   The formed shoots were transferred to root induction medium.

[0047]   Once rooted successfully, the plants were tested and transferred to the greenhouse.

Example 3 Inhibition of Cholesterol Biosynthesis in Cultures of Primary Rat Hepatocytes Preparation of Stock Solution A) Containing only the flavanone 3-hydroxylase gene inherently present (control)
And B) 10-20 mg of ripe tomato (variety: "Moneym", further comprising a flavanone 3-hydroxylase partial fragment in the antisense orientation, as described in Example 2.
aker ') lyophilisate was accurately weighed and treated with DMSO in an amount such that a stock solution of 10 mM total flavonoids was obtained. Immediately before starting the test, dilutions of these stock solutions with medium were prepared. ^Serial 10-fold dilutions between 10 ^{-4 and} 10 ^-8 M were performed.

Preparation of Hepatocyte Cultures Primary hepatocytes were isolated from the liver of male Sprague-Dawley rats (240-290 g) by collagenase perfusion [Gebhardt et al., Arzneimittel-Forschung / Drug Res. 41: 800-804 (1991).
1990]. 125 the hepatocytes in a collagen-coated dish (6 well plate, Greiner, Nurtingen) in Williams Medium E supplemented with 10% bovine serum.
The cells were cultured at a cell density of 1,000 cells / cm ² . For more details, see G
ebhardt et al., Cell Biol. Toxicol. 6: 369-372 (1990) and Mewes et al., Cancer Res.
. 53: 5135-5142 (1993). After 2 hours, the culture was transferred to serum-free medium supplemented with 0.1 μM insulin. After a further 20 hours they were used in the test. The test substances were tested in each case in 3 independent cultures from 2-3 rats.

Incubation of hepatocyte cultures with test substances A and B In order to demonstrate that cholesterol biosynthesis is influenced by test substances A and B, hepatocyte cultures were kept for a total of 22 hours. They were then incubated with serum-free Williams Medium E supplemented with ¹⁴ C acetate (tracer amount only) for 2 hours with the indicated concentrations of test substances. A control was included in each test system. The methodology is described in more detail by Gebhardt (1991) and Gebhardt, Lipids 28: 613-619 (1993). A tracer amount of ¹⁴ C-acetate rapidly displaces the intracellular acetyl-CoA pool, thereby facilitating uptake of ¹⁴ C-acetate into the sterol fraction (over 90% of which is composed of cholesterol). Be able to measure (Gebhardt, 1993).

Method for Analyzing Effects on Cholesterol Biosynthesis Incorporation of ¹⁴ C-acetate into sterol fractions (non-hydrolyzable lipids) was determined by Gebhar
It was measured by the method of dt (1991). Extraction used [Extrelut® column (Merc
k, Darmstadt)], more than 90% of ¹⁴ C acetate (and small amounts of other low molecular weight metabolites produced therefrom) were removed. This test makes it possible to compare the relative synthesis rates between cholesterol and the precursor sterols under the influence of test substances (Gebhardt, 1993).

Visual and microbial qualification test of hepatocyte cultures Before and after incubation, all cultures used were visually checked under the microscope for microbial contamination and cell monolayer integrity. No discernible changes in cell morphology (especially at high concentrations) were seen in any of the samples. This largely excludes the possibility that the test results were affected by the cytotoxic effect of the test compound.

Routine nonviability testing on all cultures did not show any signs of microbial contamination.

Results Sample A) from a genetically unmodified tomato (control) showed no effect on cholesterol biosynthesis. In contrast, cholesterol synthesis was significantly inhibited by sample B from tomato containing a partial fragment of flavanone 3-hydroxylase in the antisense orientation.

[Brief description of drawings]

FIG. 1 shows that inhibition of flavanone 3-hydroxylase does not produce flavonoid end products such as catechin and cyanidin, but increases phenol production such as luteoliflavan.

FIG. 2: Fragment A (529 bp) contains the CaMV 35S promoter (nucleotides 6909-7437 of the cauliflower mosaic virus). Fragment B contains a fragment of the F3H gene in the antisense orientation. Fragment C (192 bp) contains the termination signal for the octopine synthase gene.

FIG. 3: Fragment A (529 bp) contains the CaMV 35S promoter (cauliflower mosaic virus nucleotides 6909-7437). Fragment B contains a fragment of the F3H gene in the antisense orientation. Fragment C (192 bp) contains the termination signal for the octopine synthase gene.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) A61K 35/78 A61K 35/78 H 4C083 J 4C088 K N R U V X A61P 1/14 A61P 1/14 3 / 06 3/06 29/00 29/00 35/00 35/00 39/06 39/06 43/00 111 43/00 111 C12N 5/10 C12N 5/00 C // C12N 15/09 ZNA 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Schweden, Jurgen German Federation Republic D-67433 Neustadt, Heinrich-Stree Ehrler-Strasse 19 (72) Inventor Harbors, Karin Germany D-06484 Quedlinburg, Amheinge 6 F Term (reference) 2B030 AA02 AB03 AD08 CA17 CA19 CB02 CD02 CD03 CD07 CD09 CD13 CD21 2B150 AB03 AC37 CE01 CE16 CE18 CE23 CE25 DD36 DD37 DD40 DD45 4B018 MD42 MD48 ME02 4B024 AA08 BA08 CA04 DA01 DA05 EA01 EA04 FA02 GA11 4B065 AA88X AB01 BA01 BA24 CA41 CA44 4C083 AA111 CC01 4C088 AB02 AB12 AB12 AB14 AB15 AB40 AB05 AB02 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB51 AB52 AB52 AB51 AB52 AB52 AB52 AB52 AB52 AB51 AB52 AB52 AB52 AB51 AB52 AB52 AB52 AB51 AB52 AB52 AB52 AB51 AB52 AB52 AB52 AB51 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB51 AB52 AB52 AB52 AB51 AB52 AB52 AB52 AB51 AB52 AB52 AB51 AB52 AB52 AB52 AB51 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AB52 AC11 AC12 AC13 BA07 BA08 CA01 CA03 CA25 NA05 NA14 ZA66 ZB01 ZB11 ZB21 ZB26 ZC02 ZC19 ZC21 ZC33

Claims (5)

[Claims] 1. A method for increasing the content of flavonoid and phenol components in a plant, which comprises producing a plant in which the activity of the enzyme flavanone 3-hydroxylase is reduced by a method of molecular genetics. 2. The method according to claim 1, wherein the content of flavonoids and phenolic components in a plant is increased, wherein the activity of the enzyme flavanone 3-hydroxylase is increased in whole or part of the plant body of the plant. Method of molecular biology (
For example, the method as described above, wherein the antisense construct, co-suppression, expression of a specific antibody, or expression of a specific inhibitor) is permanently or temporarily reduced, either completely or partially. 3. The plant is grape vine, cherry, tomato, plum, limpet, cowberry, strawberry, citrus fruit (eg, orange, grapefruit), popow, red cabbage, broccoli, mecha cabbage, cacao, kale, carrot. , Parsley, celeriac / celery, onion, garlic, tea, coffee, hops, soybeans, rape, oats, wheat, rye, caidou (
Aronia melanocarpa) and Ginkgo biloba).
The method described in. 4. A plant having an increased content of flavonoids and phenol components produced by the method according to any one of claims 1 to 3, wherein the enzyme flavanone 3-hydroxylase has an enzymatic activity. The above plants that are declining. 5. Human and animal foods, dietary supplements, or therapeutic compositions, health promoting compositions or tonics (squeezed juices, teas, extracts,
Use of plants or parts of these plants produced by the method according to any one of claims 1 to 3 for the production of fermentation products) as well as for the production of cosmetics.