Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR PRODUCING PLANTS WITH INCREASED
FLAVONOID AND PHENOLIC COMPOUND CONTENT
The present invention re:Lates to a method of increasing the
content of flavonoids and phenolic constituents in plants,
wherein a plant is generated, by methods of molecular genetics,
in which the activity of the enzyme flavanone 3-hydroxylase is
reduced.
Moreover, in the method according to the invention, the activity
of the enzyme flavanone 3-hydroxylase is reduced fully or
partially, permanently or. transiently, in the entire plant or in
parts of the plant by met; hods of molecular biology (for example
antisense constructs, cosuppression, the expression of specific
antibodies or the expression of specific inhibitors).
The invention furthermorES relates to plants with an elevated
content of flavonoids and phenolic constituents, whose enzymatic
activity of the enzyme flavanone 3-hydroxylase is reduced.
Moreover, the invention relates to the use of plants, generated
by the method according t:o the invention, or parts of these
plants as foodstuffs, supplements or for producing curative
compositions, health-pronnoting compositions or tonics (juices,
infusions, extracts, fermentation products) for humans and
animals, and for the production of cosmetics.
A variety of phenolic substances are found in plants, for examplc
caffeic acid, ferulic acid, chlorogenic acid, gallic acid,
eugenol, lignans, coumarins, lignin, stilbenes (polydatin,
resveratrol), flavonoids (flavones, catechines, flavanones,
anthocyanidines, isoflavones), and polymethoxylated flavones.
Accordingly, phenols are also generally a constituent of a number
of plant-derived foodstuffs and stimulants. Certain phenolic
substances are of particular importance since, after having been
taken up together with trie food, they can exert an antioxidant
effect in the human or animal metabolism (Baum, B. O.; Perun, A.
L. Antioxidant efficiency versus structure. Soc. Plast. Engrs
Traps 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.
Sci. 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 Radic. Eiol. Med. 20:
933-956, (1996); Salah, N.; Miller, N. J.; Paganga, G.; Tijburg,
L.; Bolwell, G. P.; Rice-Evans, C. Polyphenolic flavonoids as
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2
scavenger of aqueous phase radicals and as chain-breaking
antioxidants. Arch Biochem Biophys 322: 339-346, (1995); Stryer,
L. Biochemistry S. Francisco: Freeman, (1975); Vieira, O.;
Escargueil-Blanc, I.; Meilhac, O.; Basile, 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)). Moreover, pol.yphenols also have a multiplicity of
effects on the cell metabolism. Inter olio, they modulate signal
transduction enzymes such as protein kinase C, tyrosine protein
kinase and phosphatidylinositol 3-kinase (Agullo, G.;
Garnet-payrastre, L.; Mfanenti, S.; Viola, C.; Remesy, C.; Chap,
H.; Payrastre, B. Relationship between flavonoid structure and
inhibition of phosphat.idylinositol 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, D. L.; Geahlen, R. L. Synthesis and protein-tyrosine kinase
inhibitory activity of flavonoids analogues. J Meed Chem 34:
798-806, (1991); Hagiwara, M.; Inoue, S.; Tanaka, T.; Nunoki, R.;
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
down-regulates inducible NO synthase (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)) and
regulates the gene expression of, for example, interleucins and
adhesion molecules (IC.AM-1, VCAM-1) (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. B.iochem 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 transcriptional factor
NF-kB. Atherioscler Th:romb Vasc Bio1 16: 1501-1508, (1996)). It
is now accepted that these effects are beneficial for preventing
infarctions, cardiovascular diseases, diabetes, a variety of
certain cancers, tumors and other chronic diseases (Bertuglia,
S.; Malandrino, S.; Co:Lantuoni, A. Effects of the natural
flavonoid delphinidin on diabetic microangiopathy.
Arznei-Forsch/Drug Res 45: 481-485, (1995); Griffiths, K.;
Adlercreutz, H.; Boyle,, P.; Denis, L.; Nicholson, R.I.; Morton,
M.S. Nutrition and Cancer Oxford: Isis Medical Media, (1996);
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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 elderly study. The Lancet
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 Vasc Bio1 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
disease 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 Sci 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 receptor-deficient mice. J Nutr 128:
954-9, (1998) - references - ). A series of curative
compositions, health-promoting compositions or tonics whose
effect is based on their content of phenolic substances is
therefore already being obtained from suitable plants (Gerritsen,
M. E.; Carley, W. W.; :Ranges, G. E.; Shen, C. P.; Phan, S. A.;
Ligon, G. F.; Perry, C. A. Flavonoids inhibit cytokine-induced
endothelial cell adhesion protein gene expression. Am J Pathol
147: 278-292, (1995); :Gin, J. K.; Chen, Y. C.; Huang, Y. T.;
Lin-Shiau, S. Y. 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
chemopreventive effects of a flavonoid antioxidant silymarin:
inhibition of mRNA expression of an endogenous tumor promoter TNF
alpha. Biochem Biophys Res Comm 239: 334-339, 1997. Furthermore,
it is known that certain plant-derived foodstuffs or stimulants
prepared from them havE~ a positive effect on various diseases.
Resveratrol, which is found in white wine, but in particular in
red wine (in addition to other components), for example, acts
against infarctions, cardiovascular diseases and cancer (Gehm,
B.D.; McAndrews, J.M.; Chien, P.-Y.; Jameson, J.L. Resveratrol, a
polyphenolic compound mound in grapes and wine, is an agonist for
estrogen receptor. Pro<: Natl Acad Sci 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; Farnsworth, 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. Science 275: 218-220, (1997). A similar
action is also found in substances such as catechin,
epicatechin-3-gallate, epigallocatechin and
0050/50062
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epigallocatechin-3-gallate, all of which are found in the leaves
of tea (Camellia sinensis). Beverages made with, in particular,
unfermented tea leave; (green tea) are health-promoting (Hu, G.;
Han, C.; Chen, J. Inhibition of oncogene expression by green tea
and (-)-epigallocatechin gallate in mice. Nvtr Cancer 24:
203-209; (1995); Schol.z, E; Bertram, B. Camellia sinensis (L.) O.
Kuntze. Der Teestrauch [the tea shrub]. Z. Phytotherapie 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
ZO kinases by green tea p~olyphenols: 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. Nature 387: 561, (1997). Moreover,
polymethoxylated flavones from citrus fruit, also show a potential
antitumor action (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)).
It is an object of the present invention to find a simple,
inexpensive method of increasing the content of flavonoids and
phenolic constituents in crop plants.
~ 25 We have found that this object is achieved, starting from
physiological studies on growth regulators from the
acylcyclohexanedione group, by genetic engineering methods which
are now available, surprisingly, and with the aid of which plants
can be generated which are characterized by an elevated content
of curative, health-promoting or tonifying constituents.
Acylcyclohexanediones ;such as prohexadione-calcium and
trinexapac-ethyl (earl:ier name: cimectacarb) are employed as
bioregulators for inhibiting the longitudinal growth of plants.
The reason for their b:ioregulatory action is that they block the
biosynthesis of gibberellins, which promote longitudinal growth.
Owing to their structural relationship with 2-oxoglutaric acid,
they inhibit certain dioxygenases which require 2-oxoglutaric
acid as co-substrate (Rademacher, W, Biochemical effects of plant
growth retardants, in: Plant Biochemical Regulators, Gausman, HW
(ed.), Marcel Dekker, 7Cnc., New York, pp. 169-200 (1991)). It is
known that such compounds also engage in the phenol metabolism
and are therefore capable of causing, in a variety of plant
species, the production of anthocyanins to be inhibited
(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,
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Vreugdenhil, D (eds.),, Kluwer Academic Publishers, Dordrecht
(1992)). Such effects on the balance of phenolic constituents are
considered to be the cause for the side effect of
prohexadione-calcium against fire blight (Rademacher, W et al.,
5 prohexadione-calcium -- a new plant growth regulator for apple
with interesting biochemical features, Poster presented at the
25th Annual Meeting of the Plant Growth Regulation Society of
America, 7.-10. Juli 1998, Chicago). A. Lux-Endrich (PhD thesis
at the Technical University of Munich at Weihenstephan, 1998)
finds in the course oi: her studies on the mechanism of action of
prohexadione-calcium against fire blight that
prohexadione-calcium causes, in apple cell cultures, the content
of phenolic substances to be increased several times over, during
which process a seriees of phenols are found which are usually not
present. It was also found within these studies that
prohexadione-calcium causes relatively high amounts of
luteoliflavan and eriodictyol to occur in the shoot tissue of
apples. Luteoliflavan does not normally occur in apple tissue,
and eriodictyol occur~~ in small amounts only as intermediate of
the flavonoid metabolism. However, the expected flavonoids
catechin and cyanidin were not detectable in the treated tissue,
or occurred in markedly reduced quantities only (S. Rommelt et
al, paper presented at. the 8th International Workshop on Fire
Blight, Kusadasi, Turkey, 12-15 October 1998).
It is now accepted that prohexadione-calcium, trinexapac-ethyl
and other acylcyclohex:anediones inhibit
2-oxoglutaric-acid-deF~endent hydroxylases which play an important
role in the metabolism, of phenolic substances. These are,
primarily, chalcone synthetase (CHS) and flavanone 3-hydroxylase
(F3H) (W. Heller and G. Forkmann, Biosynthesis, in: The
Flavonoids, Harborne, JB (ed.), Chapman and Hall, New York,
1988). However, it cannot be excluded that acylcyclohexanediones
also inhibit other 2-oxoglutaric-acid-dependent hydroxylases
which are not known as yet. Furthermore, it seems to be obvious
that a lack of catechin, cyanidin and other end products of
flavonoid synthesis is registered by the plant, and that the
activity of the key enzyme phenylalanine ammonium lyase (PAL) is
raised via a feedback .mechanism. owing to the fact that CHS and
F3H continue to be inhibited, however, these flavonoid end
products cannot be formed, and the result is an increased
formation of luteoliflavan, eriodictyol and other phenols
(Fig. 1).
Because the enzyme activity of the enzyme flavanone 3-hydroxylase
(F3H) is reduced, the flavonoids eriodictyol, proanthocyanidins,
which are substituted on the C atom by hydrogen, for example
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luteoforol, luteoliflavan, apigeniflavan and tricetiflavan, and
homogeneous and heterogeneous oligomers and polymers of the
abovementioned structurally related substances are produced in
higher quantities.
Elevated concentrations of the phenols hydroxycinnamic acid
(p-coumaric acid, ferulic acid, sinapic acid), salicylic acid or
umbelliferone, including the homogeneous and heterogeneous
oligomers and polymer: formed from them, are found in plants
after the enzyme activity of the enzyme flavanone 3-hydroxylase
(F3H) has been reducect. Equally the concentration of the
chalcones such as, for example, phloretin, and of the stilbenes,
such as, for example, resveratrol, increases.
Since the enzyme activity of the enzyme flavanone 3-hydroxylase
is reduced, the concentration of the glycosides of the
flavonoids, of the phe~nolic compounds, of the chalcones and of
the stilbenes is also increased.
Starting from these findings and the hypotheses derived
therefrom, genetically modified crop plants were generated in
which the activity of F3H was reduced fully or partially,
permanently or transiently, in the entire plant or in individual
plant organs or plant tissues, by means of antisense constructs,
so that the content of curative, health-promoting or tonifying
substituents was improved in terms of quantity and quality.
The method according to the invention for increasing the content
of flavonoids and phen.olic compounds by expressing flavanone
3-hydroxylase in antisense orientation can be applied
successfully to the following crop plants, but the method is not
restricted to the plants mentioned: grapevines, cherries,
tomatoes, plums, sloes, blueberries, strawberries, citrus fruit
(such as oranges, grapefruit), pawpaw, red cabbage, broccoli,
Brussel sprouts, cacao, kale, carrots, parsley, celeriac/celery,
onions, garlic, tea, coffee, hops, Soya, oilseed rape, oats,
wheat, rye, Aronia melanocarpa, Ginkga biloba.
Moreover, the invention relates to plants with an elevated
content of flavonoids and phenolic constituents, generated by the
method according to the invention and with a reduced enzymatic
activity of the enzyme flavanone 3-hydroxylase.
As an alternative to generating plants whose flavanone
3-hydroxylase activity is reduced with the aid of antisense
technology, it is also possible to use other methods of molecular
genetics which are known from the literature, such as
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cosuppression or the expression of specific antibodies, in order
to achieve this effect.
In addition, the invention relates to the use of plants,
generated by the method according to the invention, or of parts
of these plants as foodstuffs, food supplements or for producing
curative compositions,, health-promoting compositions or tonics
(juices, infusions, a};tracts, fermentation products) for humans
and animals, and for t:he production of cosmetics.
Surprisingly, it has now been found that plants which have been
generated in accordance with the invention, or parts of these
plants or products produced from them (teas, extracts,
fermentation products, juices and the like) have the following
effects:
(1) the antioxidant capacity in vitro (Electron Spin Resonance
(ESR), LDL oxidation, total antioxidant capacity, NO
scavenging) is improved;
(2) a modulating effects on enzymes, especially signal
transduction enzymes (protein kinase C, tyrosine protein
kinase, phosphatidylinositol 3-kinase) is observed;
(3) a modulation of redox-sensitive transcriptional factors
(NF-kB, AP-1) is induced in endothelial cells, lymphocytes
and smooth muscle cells;
(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) an antiaggregatory effect is induced;
(6) the cholesterol synthesis in hepatocytes is inhibited;
(7) antiproliferative/antineoplastic effects exist.
Example 1
Cloning the gene of a f:lavanone 3-hydroxylase from Lycopersicon
esculentum Mill.cv. Moneymaker.
Ripe tomato fruits of I,ycopersicon esculentum Mill.cv. Moneymaker
were washed, dried and, using a sterile blade, the pericarp was
freed from seeds, central columnella and woody parts. The
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pericarp (approx. 50 g) was frozen in liquid nitrogen. Then, the
material was comminuted in a blender. In a pre-cooled mortar, the
comminuted material was treated with 100 ml of homogenization
medium and mixed. Then, the suspension was transferred into a
centrifuge flask by squeezing it through sterile gauze. Then,
1/10 volume 10~ SDS was added and the material was mixed
thoroughly. After 10 minutes on ice, one volume of
phenol/chloroform was added, and the centrifuge flask was sealed
and the contents mixed thoroughly. After centrifugation for 15
minutes at 4000 rpm, t=he supernatant was transferred into a fresh
reaction vessel. This was followed by three more
phenol/chloroform extractions and one chloroform extraction.
Then, 1 volume of 3 M NaAc and 2.5 volumes of ethanol were added.
The nucleic acids were precipitated overnight at -20°C. The next
morning, the nucleic acids were pelleted for 15 minutes in the
refrigerated centrifuge (4°C) at 10,000 rpm. The supernatant was
discarded, and the pellet was resuspended in 5-10 ml of cold 3 M
NaAc. This washing step was repeated twice. The pellet was washed
with 80~ ethanol. When. completely dry, the pellet was taken up in
approx. 0.5 ml of sterile DEPC water, and the RNA concentration
was determined photometrically.
20 ~g of total RNA were treated first with 3.3 ~1 of 3M sodium
acetate solution, 2 ~1 of 1M magnesium sulfate solution, and the
mixture was made up to a final volume of 100 ~1 with DEPC water.
A microliter of RNase-free DNase (Boehringer Mannheim) was added
to this, and the mixture was incubated for 45 minutes at 37°C.
After the enzyme had been removed by extraction by shaking with
phenol/chloroform/isoamyl alcohol, the RNA was precipitated with
ethanol, and the pellet was taken up in 100 ~1 DEPC water. 2.5 ~g
of RNA from this solution were transcribed into cDNA using a cDNA
kit (Gibco BRL).
Using amino acid sequences which were derived from cDNA clones
encoding flavanone 3-hydroxylase, conserved regions in the
primary sequence were :identified {Britsch et al., Eur. J.
Biochem. 217, 745-754 (1993), and these acted as the basis for
designing degenerated PCR oligonucleotides. Using the peptide
sequence SRWPDK (amino acid 147-152 in the Petunia hybrida
sequence FL3H PETHY), i:he 5' oligonucleotide was determined and
had the following seque=nce:
5'-TCI (A/C) G (A/G) TC~G CC{A/C/G) GA (C/T) AA (A/G) CC-3.
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The sequence of the oligonucleotide deduced by using the peptide
sequence DHQAW (amino acid 276-281 in the Petunia hybrids
sequence FL3H PETHY) '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 tTth polymerase by
Perkin-Elmer, following the manufacturer's instructions. The
template used was 1/8 of the cDNA (corresponds to 0.3 ~g of RNA).
The PCR program was:
30 cycles
94 degrees 4 sec
40 degrees 30 sec
72 degrees 2 min
72 degrees 10 min
The fragment was cloned into Promega's vector pGEM-T following
the manufacturer's instructions.
The correctness of the' fragment was checked by sequencing. Using
the restriction cleavage sites NcoI and PstI, which are present
in the polylinker of t:he vector pGEM-T, the PCR fragment was
isolated, and the overhangs were made blunt-ended using
T4-polymerase. This fragment was cloned into an SmaI-(blunt-)cut
vector pBinAR (Hofgen and Willmitzer, Plant Sci. 66: 221 - 230
(1990)) (see Figure 2). This vector contains the CaMV
(cauliflower mosaic virus) 35S promoter (Franck et al., Cell 21:
285 - 294 (1980)) and the termination signal from the octopine
synthase gene (Gielen et al., EMBO J. 3: 835 - 846 (1984)). This
vector mediates, in plants, a resistance to the antibiotic
kanamycin. The resulting DNA constructs contained the PCR
fragment in sense and antisense orientation. The antisense
construct was employed. for generating transgenic plants.
Figure 2: Fragment A (529 bp) contains the CaMV 35S promoter
(nucleotides 6909 to 7437 of the cauliflower mosaic virus).
Fragment B contains the fragment of the F3H gene in antisense
orientation. Fragment C (192 bp) contains the termination signal
of the octopine synthase gene.
Cloning a larger cDNA fragment of the flavanone 3-hydroxylase
from Zycopersicon esculentum Mill.cv. Moneymaker using the 5'RACE
system.
To exclude that the generation of plants with a reduced mRNA flow
equilibrium quantity of F3H is not successful due to the small
size of the F3H PCR fragment used in the antisense construct, a
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second antisense consi:ruct using a larger F3H fragment should be
generated.
The 5'RACE method (system for rapid amplification of cDNA ends)
5 was employed for cloning a larger F3H fragment.
Extension of the F3H PCR fragment by means of the 5'RACE method
using the 5'RACE system for rapid amplification of cDNA ends,
Version 2-0 by Life TechnologiesTM.
Total RNA was isolated from ripe tomato fruits of Lycopersicon
esculentum Mill.cv. Moneymaker (see above).
The cDNA first strand synthesis was carried out using the GSP-1
(gene-specific primer) 5'-TTCACCACTGCCTGGTGGTCC-3' following the
manufacturer's instructions. Following RNase digestion, the cDNA
was purified using the GlassMAX spin system by Life TechnologiesTM
following the manufact.urer's instructions.
A cytosine homopolymer was added onto the 3' end of the purified
simplex F3H cDNA using the terminal deoxynucleotidyl transferase,
following the manufacturer's instructions.
The 5'-extended F3H cDNA was amplified using a second
gene-specific primer (GSP-2), which binds in the 3' region
upstream of the GSP-1 recognition sequence and thus allowed a
"nested" PCR to be performed. The 5' primer used was the "5'RACE
abridged anchor primer", which was provided by the manufacturer
and which is complementary to the homopolymeric dC tail of the
cDNA.
The eDNA thus amplified, which was termed F3Hextended. was cloned
into Promega's vector ;pGEM-T following the manufacturer's
instructions.
The identity of the cDNA was confirmed by sequencing.
The F3Hextended cDNA fragment was isolated using the restriction
cleavage sites NcoI and Pstl which are present in the polylinker
of vector pGEM-T, and the overhangs were made blunt-ended using
T4-polymerase. This fragment was cloned into an SmaI-(blunt-) cut
vector pBinAR (Hofgen and Willmitzer, 1990) (see Figure 3). This
vector contains the CaMV (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, in plants, a resistance to the antibiotic kanamycin.
The resulting DNA constructs contained the PCR fragment in sense
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11
and antisense orientation. The antisense construct was employed
for generating transge~nic plants.
Figure 3: Fragment A (529 bp) contains the CaMV 35S promoter
(nucleotides 6909 to i'437 of the cauliflower mosaic virus).
Fragment B contains the fragment of the F3H gene in antisense
orientation. Fragment C (192 bp) contains the termination signal
of the octopine synthase gene.
Example 2
Generation of transgenic Lycopersicon esculentum Mill.cv.
Moneymaker which express a subfragment of flavanone 3-hydroxylase
in antisense orientation.
The method used was that of Ling et al., Plant Cell Report 17,
843 - 847 (1998). Cultivation was carried out at approx. 22°C
under a 16-hour-light~8-hour-dark regime.
Tomato seeds (Lycopers,icon esculentum Mill. cv. Moneymaker) were
sterilized by incubation for 10 minutes in 4~ strength sodium
hypochlorite solution, and subsequently washed 3-4 times with
sterile distilled water and placed on MS medium supplemented with
3~ sucrose, pH 6.1, for germination. After a germination time of
7-10 days, the cotyledons were ready for use in transformation.
Day 1: Petri dishes containing "MSBN" medium were overlaid with
1.5 ml of an approximately 10-day-old tobacco suspension culture.
The plates were covered with film and incubated at room
temperature until the next day.
Day 2: Sterile filter paper was placed onto the plates overlaid
with the tobacco suspension culture in such a way that no air
bubbles were formed. The cotyledons, which had been cut
crosswise, were placed on the filter paper upside down. The Petri
dishes were incubated for 3 days in a culture chamber.
Day 5: The agrobacterial culture (LBA4404) was sedimented by
centrifugation for 10 :minutes at approx. 3000 g and resuspended
in MS medium so that the OD was 0.3. The cotyledon sections were
placed into this suspension and incubated for 30 minutes at room
temperature with gentle shaking. Then, the cotyledon sections
were dried somewhat on sterile filter paper and returned to their
starting plates to continue cocultivation for 3 days in the
culture chamber.
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Day 8: The cocultured cotyledon sections were placed on MSZ2K50+13
and incubated for the next 4 weeks in the culture chamber. They
were then subcultured.
Shoots which formed were transferred to root induction medium
After successful rooting, the plants were tested and transferred
into the greenhouse.
Example 3
Inhibition of cholesterol biosynthesis in cultures of primary rat
hepatocytes
Preparation of the stock solutions
Between 10 and 20 mg of the lyophilisate of ripe tomatoes
cv."Moneymaker" containing A) only the native flavanone
3-hydroxylase gene (control) and B) as described in Example 2
additionally a subfragment of the flavanone 3-hydroxylase in
antisense orientation, was weighed exactly and treated with such
an amount of DMSO that a stock solution of 10 mM total flavonoids
resulted: Immediately before the tests were started, dilutions of
these stock solutions in the culture medium were prepared.
Ten-fold dilution steps were carried out between 10-4 and 10-$M.
Preparation of the hep,atocyte cultures
Primary hepatocytes were obtained from the livers of male
Sprague-Dawley rats (240-290 g) by means of collagenase perfusion
(Gebhardt et al., Arzneimittel-Forschung/Drug Res. 41: 800-804
(1991) 1990). The hepatocytes were cultured in collagen-coated
Petri dishes (6-well p:Lates, Greiner, Niirtingen) at a cell
density of 125,000 cel:Ls/cm2 in Williams Medium E supplemented
with 10~ calf serum. More details, in particular on the culture
medium, are found in Ge:bhardt et al., Cell Biol. Toxicol. 6: 369
- 372 (1990) and Mewes et al., Cancer Res. 53: 5135 - 5142
(1993). After 2 hours, the cultures were transferred to
serum-free medium supplemented with 0.1 ~M insulin. After a
further 20 hours, they were employed in the tests. The test
substances were tested in each case in three independent cultures
from 2-3 rats.
Incubation of the hepat:ocyte cultures with the test substances A
and B
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To demonstrate that the cholesterol biosynthesis is affected by
the test substances A and B, the hepatocyte cultures were kept
for a total of 22 hours. Then, they were incubated with
serum-free Williams Medium E supplemented with 14C acetate (only
tracer quantities) for 2 hours together with the test substances
at the concentrations indicated. In each test series, a control
was included. The methodology is described in detail by Gebhardt
(1991) and Gebhardt, Lipids 28: 613-619 (1993). The tracer
quantities of 14C acetate exchange rapidly with the intracellular
acetyl-CoA pool and therefore allow the incorporation of 14C
acetate into the sterol fraction, which consists of >900 of
cholesterol, to be determined in a trouble-free manner (Gebhardt,
1993).
Analytical methods fox' influencing the cholesterol biosynthesis
The incorporation of 14C acetate into the sterol fraction
(non-hydrolyzable lipids) was measured by the method of Gebhardt
(1991). In the extraction used, in which Extrelut~ columns
(Merck, Darmstadt) are employed, over 95~ of the 14C acetate (and
small amounts of other low-molecular-weight metabolites formed
from it) are removed. This test allows comparisons to be made
between the relative synthesis rate of cholesterol and precursor
sterols under the effect of test substances (Gebhardt, 1993).
Visual and microbial quality checks of the hepatocyte cultures
Before and after the test incubation, all the cultures used were
checked visually under the microscope for contamination with
microorganisms and for the integrity of the cell monolayer. A
recognizable change in cell morphology (in particular at the
higher concentrations) was not observed in any of the samples.
This largely excludes ithat the test results were affected by
cytotoxic effects of the test substances.
The routine sterility i:ests on all cultures revealed no signs of
contamination whatsoever with microorganisms.
Results
Samples A) from the tomatoes which were not genetically modified
(control) showed no effect whatsoever on cholesterol
biosynthesis. In contrast, the cholesterol synthesis was
inhibited significantly by samples B from the tomatoes which
contained a subfragment: of flavanone 3-hydroxylase in antisense
orientation.
