U.S. patent application number 10/354805 was filed with the patent office on 2004-08-19 for methods and composition for modulating flavonoid content.
This patent application is currently assigned to Lipton, Division of Conopco, Inc.. Invention is credited to Bovy, Arnaud Guillaume, De Vos, Cornelis Henricus, Hughes, Stephen Glyn, Muir, Shelagh Rachael, Van Tunen, Adrianus Joannes, Verhoeyen, Martine Elisa.
Application Number | 20040163142 10/354805 |
Document ID | / |
Family ID | 8234935 |
Filed Date | 2004-08-19 |
United States Patent
Application |
20040163142 |
Kind Code |
A1 |
Bovy, Arnaud Guillaume ; et
al. |
August 19, 2004 |
Methods and composition for modulating flavonoid content
Abstract
A method for manipulating the production of flavonoids in
tomatoes by manipulating gene activity in the flavonoid
biosynthetic pathway by expressing genes encoding chalcone
isomerase, compositions for use in such a method and tomato plants
having altered flavonoid levels are disclosed.
Inventors: |
Bovy, Arnaud Guillaume; (De
Bilt, NL) ; Hughes, Stephen Glyn; (Essex, GB)
; Muir, Shelagh Rachael; (Bedford, GB) ; Van
Tunen, Adrianus Joannes; (Wageningen, NL) ;
Verhoeyen, Martine Elisa; (Bedford, GB) ; De Vos,
Cornelis Henricus; (Wageningen, NL) |
Correspondence
Address: |
UNILEVER
PATENT DEPARTMENT
45 RIVER ROAD
EDGEWATER
NJ
07020
US
|
Assignee: |
Lipton, Division of Conopco,
Inc.
|
Family ID: |
8234935 |
Appl. No.: |
10/354805 |
Filed: |
January 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10354805 |
Jan 30, 2003 |
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09353242 |
Jul 14, 1999 |
|
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6608246 |
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Current U.S.
Class: |
800/278 ;
800/282 |
Current CPC
Class: |
A23L 27/63 20160801;
C12N 15/8242 20130101; C12N 9/90 20130101; A23L 23/00 20160801;
A23L 19/00 20160801; C12N 15/8243 20130101; C12N 15/825
20130101 |
Class at
Publication: |
800/278 ;
800/282 |
International
Class: |
A01H 001/00; C12N
015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 14, 1998 |
EP |
98305570.8 |
Claims
1. A method for producing a plant capable of exhibiting altered
levels of flavonoids comprising incorporating into said plant one
or more gene sequences encoding a protein with chalcone isomerase
activity, or incorporating a nucleotide sequence encoding a protein
functionally equivalent thereto.
2. Method according to claim 1 characterised in that said gene or
genes encode chalcone isomerase, isolated from a species selected
from the group comprising tomato plant, petunia, maize,
arabidopsis, alfalfa, pea, bean, grape, apple.
3. Method according to claim 1 characterised in that said gene or
genes or nucleotide sequence encode the protein chalcone isomerase,
isolated from petunia.
4. A method according to claim 1 characterised in that said plant
is a tomato plant.
5. A method according to any one of claims 1 to 4 characterised in
that levels of "specific flavonoids" in said plant are increased
compared to untransformed plants.
6. A method according to any one of claims 1-5 characterised in
that the level of "specific flavonoids" in transformed plants is at
least 4 times higher than in similar untransformed plants, more
preferred 5-100, most preferred 10-40 times higher than in similar
untransformed plants.
7. A method according to any one of claims 1 to 6 characterised in
that the level of specific flavonoids in the peel of the fruit of
said plant is increased.
8. A method according to any one of claims 1 to 7 wherein said
flavonoid is a flavonol.
9. A method according to any of claims 1 to 7 characterised in that
the flavonoid is quercetin or kaempferol or their glycosides or any
other derivative thereof.
10. A method according to any one of claims 1 to 8 characterised in
that the introduced gene comprises a nucleotide sequence or
complementary nucleotide sequence selected from: (i) a nucleotide
sequence, encoding an amino acid sequence having at least 40%
similarity, to seq ID No1; (ii) a nucleotide sequence capable of
hybridising under low stringent conditions to a sequence selected
from the group of sequences set forth under (i) above; (iii) a
nucleotide sequence encoding a protein that is being functionally
equivalent to the protein encoded by seq ID no 1.
11. A method according to claim 10, characterised in that said gene
comprises a nucleotide sequence encoding an amino acid sequence
having at least 60% similarity, preferably at least 90%, more
preferred at least 95%, most preferred at least 95% similarity to
the sequence as set forth in seq ID No1.
12. A method according to claim 10, characterised in that said gene
comprises a nucleotide sequence, encoding an amino acid sequence
having 99-100% similarity, to seq ID No1.
13. A method according to any of claims 1-12, characterised in that
said nucleotide sequence comprises a sequence which has at least
50, more preferably at least 60% similarity, more preferred at
least 75%, even more preferred at least 80%, still more preferred
at least 90%, most preferred 95-100% similarity to seq ID no 2, and
whereby said sequence encodes a protein having chalcone isomerase
activity.
14. A method according to any one of claims 1-13 characterised in
that the gene encoding a protein with chalcone isomerase is
operably linked to a promoter.
15. A method according to claim 14 characterised in that the
promoter is selected from the group of: (a) constitutive promoters,
such as carnation edged ring virus, cauliflower mosaic virus 35 S
promoter, enhanced cauliflower mosaic virus 35 S promoter; (b)
fruit specific promoters; (c) any other suitable promoter such as
GBSS (granular bound starch synthase) promoter.
16. A method according to claim 15 characterised in that the
promoter is a fruit specific promoter selected from the group of
PG, 2A11, E8, E4, and fpb11.
17. A method according to any of claims 1-16 comprising the
additional step increasing phenylalanine biosynthesis.
18. A plant having one or more transgenes, each encoding a protein
with chalcone isomerase activity or a protein functionally
equivalent thereto, incorporated into its genome such that its
ability to produce flavonoids is altered.
19. A plant according to claim 18, whereby said plant is a tomato
plant, prepared according to the method of any one of claims 1 to
17.
20. A DNA construct suitable for use to overexpress the encoded
protein, comprising sequences coding for a protein with chalcone
isomerase activity, or a functionally equivalent sequence thereof,
operably linked to a promoter.
21. A plant comprising a DNA construct according to claim 20.
22. A transformed plant having enhanced levels of specific
flavonoids compared to similar, untransformed plants.
23. A tomato plant having enhanced levels of specific flavonoids in
the peel of the fruit compared to the level of flavonoids in the
peel of the fruit of untransformed plants.
24. Seeds, fruits, progeny and hybrids of a plant according to any
one of claims 18, 19, 21, 22, or 23.
25. A food product comprising at least part of a plant according to
any one of claims 18, 19, 21, 22, or 23.
26. A food product according to claim 25 characterised in that the
food product is sauce, dressing, ketchup or soup.
27. A skin or hair protective product comprising at least part of a
plant according to any one of claims 18, 19, 21, 22, or 23.
28. A pharmaceutical product comprising at least part of a plant
according to any one of claims 18, 19, 21, 22, or 23.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to methods for
manipulating the production of flavonoids in plants by manipulating
endogeneous and incorporated gene activity in the flavonoid
biosynthetic pathway and compositions for use in such methods. In
particular, it relates to methods for increasing flavonoid levels
by altering the level of chalcone isomerase activity. Chalcone
isomerase is an enzyme involved in the biosynthetic pathway of
flavonoids.
BACKGROUND OF THE INVENTION
[0002] Flavonoids form a large group of polyphenolic compounds,
based on a common diphenylpropane skeleton, which occur naturally
in plants. Included within this class of compounds are flavonols,
flavones, flavanones, catechins, anthocyanins, isoflavonoids,
dihydroflavonols and stilbenes. The flavonoids are mostly present
as glycosides.
[0003] In tomato fruits, the main flavonoid found is naringenin
chalcone (Hunt et al, Phytochemistry, 19, (1980), 1415-1419). It is
known to accumulate almost exclusively in the peel and is
simultaneously formed with colouring of the fruit. In addition to
naringenin chalcone, glycosides of quercetin and, to a lesser
extent, kaempferol are also found in tomato peel.
[0004] Reports in the literature suggest that there is increasing
evidence that flavonoids are potentially health-protecting
components in the human diet. Epidemiological studies suggest a
direct relationship between cardioprotection and increased
consumption of flavonoids, in particular flavonols of the quercetin
and kaempferol type, from dietary sources such as onion, apples and
tea (see, for example, Hertog et al, Lancet, 342 (1993),
1007-1011).
[0005] Flavonoids have been reported to exhibit a wide range of
biological activities in vitro including anti-inflammatory,
anti-allergic and vasodilatory activity (Cook et al, Nutritional
Biochemistry, 7, (1996), 66-76). Such activity has been attributed
in part to their ability to act as antioxidants, capable of
scavenging free radicals and preventing free radical production.
Within this group of compounds, those having the most potent
antioxidant activity are the flavonols (Rice-Evans et al, Free
Radical Research, 22, (1995), 375-383). In addition, flavonoids can
also inhibit the activity of key processes such as lipid
peroxidation, platelet aggregation and capillary permeability (see
Rice-Evans et al, Trends in Plant Science, 2, (1997), 152-159).
[0006] Based on studies of this type, there is presently
considerable interest in the development of food products from
plants rich in such protective flavonoids.
[0007] It would be desirable to produce plants which intrinsically
possess elevated levels of health protecting compounds such as
flavonoids in order to develop food products with enhanced
protective properties. Traditionally, the approach to improving
plant varieties has been based on conventional cross-breeding
techniques, but these are slow as they require time for breeding
and growing successive plant generations. More recently,
recombinant DNA technology has been applied to the general problem
of modifying plant genomes to produce plants with desired
phenotypic traits. Whilst reference has been made in the literature
to the use of genetic manipulation techniques in modifying the
flavonoid biosynthetic pathway, as discussed beneath, it is notable
that these attempts have been directed in general towards modifying
pigmentary anthocyanin production.
[0008] The flavonoid biosynthetic pathway is well established and
has been widely studied in a number of different plant species
(see, for example, Koes et al, BioEssays, 16, (1994), 123-132).
Briefly, three molecules of malonyl-CoA are condensed with one
molecule of Coumaroyl-CoA, catalysed by the enzyme chalcone
synthase, to give naringenin chalcone which rapidly isomerises,
catalysed by chalcone isomerase, to naringenin. Subsequent
hydroxylation of naringenin catalysed by flavanone 3-hydroxylase
leads to dihydrokaempferol. Dihydrokaempferol itself can be
hydroxylated to produce either dihydroquercetin or
dihydromyricetin. All three dihydroflavonols subsequently can be
converted to anthocyanins (by the action of dihydroflavonol
reductase and flavonoid glucosyltransferase) or alternatively
converted to flavonols such as kaempferol, quercetin and myricetin
by the action of flavonol synthase.
[0009] A schematic overview of the flavonoid biosynthetic pathway
is presented in appendix 1, FIG. 1.1.
[0010] The manipulation of flavonoid levels in plants by altering
the expression of a single flavonoid biosynthetic gene is disclosed
by Napoli (1990, Plant Cell, 2:279-289). Napoli discloses the
introduction of a chimeric chalcone synthase (CHS) gene into
Petunia. Said introduction is described to result in a block in the
anthocyanin biosynthesis. The resulting transformed petunia plants
therefore contained lower levels of flavonoids than untransformed
plants, presumably due to co-suppression of the endogeneous CHS
activity.
[0011] Que (1997, Plant Cell, 9: 1357-1368) discloses a comparison
of the effect of strong and weak promoters that drive sense
chalcone synthase transgenes in large populations of independently
transformed plants. It is shown that a strong transgene promoter is
required for high frequency cosuppression of CHS genes and for the
production of a full range of phenotypes.
[0012] Howles (1996, Plant Physiol. 112: 1617-1624) discloses the
stable genetic transfer of the flavonoid biosynthetic gene
phenylalanine ammonia-lyase (PAL) from french bean into tobacco. A
proportion of the obtained transgenic tobacco plants is shown to
display overexpression of PAL activity. According to Howles PAL
overexpressing plants do not contain altered levels of
flavonoids.
[0013] It has been disclosed by Tanaka et al (1395, Plant and Cell
Physiology 36: 6, 1023-1031) that heterologous transformation or
dihydroflavonol reductase (DFR) can be used for the production of
plants with altered levels of anthocyanins.
[0014] There is no disclosure in the literature of the manipulation
of flavonoids in plants by means of overexpression of chalcone
isomerase.
[0015] Accordingly, there remains a continuing need for the
development of methods for enhancing the levels of flavonoids, in
particular flavonols, in plants.
SUMMARY OF THE INVENTION
[0016] Therefore, in a first aspect, the invention provides a
method for producing a plant capable of exhibiting altered levels
of flavonoids comprising incorporating into said plant one or more
gene sequences encoding a protein with chalcone isomerase activity,
or incorporating a nucleotide sequence encoding a protein
functionally equivalent thereto.
[0017] The invention also provides a plant having one or more
transgenes each encoding a protein with chalcone isomerase
activity, or a protein functionally equivalent thereto,
incorporated into its genome such that its ability to produce
flavonoids is altered.
[0018] According to a highly preferred embodiment, the invention
further provides a tomato plant having one or more transgenes each
encoding a protein with chalcone isomerase activity, or a protein
functionally equivalent thereto, incorporated into its genome such
that its ability to produce flavonoids is altered.
[0019] Also provided is a transformed plant having enhanced
flavonoid levels, not being chalcones, particularly enhanced
flavonol levels compared to similar untransformed plants.
Preferably the level of said flavonoids, not being chalcones, in
transformed plants is least 4 times higher than in similar
untransformed plants, more preferred 5-100, most preferred 10-40
times higher than in similar untransformed plants.
[0020] Further provided is a fruit-bearing plant, particularly a
tomato plant, having flavonoids, particularly flavonols, in the
peel of the fruit.
[0021] Seeds, fruits and progeny of such plants and hybrids are
also included within the invention.
[0022] The invention further provides DNA constructs coding for a
protein with chalcone isomerase activity, or a functionally
equivalent sequence of said DNA construct, operably linked to a
promoter. When transformed into a plant cell, these constructs are
useful for overexpressing genes encoding proteins with chalcone
isomerase activity, thereby altering the ability of the plant to
produce flavonoids. The invention also provides for plants
comprising these constructs together with seeds, fruits and progeny
thereof.
[0023] Food products such as sauces, dressings, ketchups and soups,
comprising at least part of a plant prepared according to the
invention are also provided.
[0024] Also provided are skin and hair protective products
comprising at least part of a plant according to the invention.
[0025] Also provided are pharmaceuticals comprising at least part
of a plant according to the invention.
[0026] Definition of Terms
[0027] As used herein, "plant" means a whole plant or part thereof,
or a plant cell or group of plant cells. It will be appreciated
that also extracts are comprised in the invention.
[0028] A "flavonoid" or a "flavonol" may suitably be an aglycon or
a conjugate thereof, such as a glycoside, or a methyl, acyl,
sulfate derivative.
[0029] A "protein with chalcone isomerase activity" is a protein
being capable of enzymatically catalysing the conversion of a
chalcone into a flavanone, for example narichalcone into
naringenin.
[0030] A "gene" is a DNA sequence encoding a protein, including
modified or synthetic DNA sequences or naturally occurring
sequences encoding a protein, and excluding the 5' sequence which
drives the initiation of transcription.
[0031] A "DNA sequence functionally equivalent thereto" is any
sequence which encodes a protein which has similar functional
properties.
[0032] According to another embodiment, a functionally equivalent
DNA sequence shows at least 50% similarity to the respective DNA
sequence. More preferably a functionally equivalent DNA sequence
shows at least 60%, more preferred at least 75%, even more
preferred at least 80%, even more preferred at least 90%, most
preferred 95-100% similarity, to the respective DNA sequence.
[0033] According to the most preferred embodiment a functionally
equivalent DNA sequence shows not more than 5 base pairs difference
to the respective DNA sequence, more preferred less than 3, e.g.
only 1 or 2 base pairs different.
[0034] According to another embodiment a functionally equivalent
sequence is preferably capable of hybridising under low stringent
conditions to the respective sequence.
[0035] "Breaker" is the ripening stage corresponding to the
appearance of the first flush of colour on the green fruit.
[0036] "Operably linked to one or more promoters" means the gene,
or DNA sequence, is positioned or connected to the promoter in such
a way to ensure its functioning. The promoter is any sequence
sufficient to allow the DNA to be transcribed. After the gene and
promoter sequences are joined, upon activation of the promoter, the
gene will be expressed.
[0037] A "construct" is a polynucleotide comprising nucleic acid
sequences not normally associated in nature.
[0038] An "altered" level of flavonoids is used throughout this
specification to express that the level of specific flavonoids in
the transformed plants differs from the level of flavonoids present
in untransformed plants. Preferably the difference is between 0.1
and 100 fold. It will be appreciated that the specific flavonoids
as meant here are flavonoids other than chalcones as said specific
flavonoids are formed at the expense of chalcones.
[0039] Therefore in the specification where these flavonoids are
meant reference will be made to "specific flavonoids".
[0040] An "increased" level of flavonoids is used to indicate that
the level of is preferably at least 4 times higher than in similar
untransformed plants, more preferred 5-100, most preferred 10-40
times higher than in similar untransformed plants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The present invention may be more fully understood by
reference to the following description, when read together with the
accompanying drawings in which:
[0042] FIG. 1 shows the levels of the two dominant flavonoids,
rutin (A.) and narichalcone (B.) in FM6203 tomato peel during
ripening. Results represent the means of three independent
samples.
[0043] FIG. 2 shows the northern analysis of tomato fruit harvested
at different developmental stages, denoted as: green (G), breaker
(B), turning (T) and red (R), and separated into peel and flesh.
Leaves (L) were harvested from young tomato plants. RNA was
isolated from the samples, separated on formaldehyde-agarose gels,
blotted and hybridised with petunia chs-a, chi and fls probes.
[0044] FIG. 3 shows the restriction maps of pFLAP10 and
pFLAP50.
[0045] FIG. 4 shows the restriction maps of pBBC3, pBBC50 and
pSJ89.
[0046] FIG. 5 shows the Southern blot of chromosomal DNA from
tomato. Chromosomal DNA was isolated from young leaves of
transgenic and non-transgenic tomato plants. 10 .mu.g DNA was
digested with EcoRI, separated on an agarose gel and blotted onto a
nylon filter. The DNA was hybridised with a .sup.32P-labelled nptII
specific probe and autoradiographed.
[0047] FIG. 6 shows typical HPLC chromatograms, recorded at 370 nm,
of hydrolysed extracts of (A.) peel and (B.) flesh tissue of plants
transformed with the control plasmid pSJ89. Peaks corresponding to
the quercetin and kaempferol aglycons are indicated.
[0048] FIG. 7 shows a typical HPLC chromatogram, recorded at 360
nm, of a non-hydrolysed extract of peel tissue of a tomato plant
transformed with the control plasmid pSJ89. Peaks corresponding to
rutin, quercetin trisaccharide and narichalcone are indicated.
[0049] FIG. 8 shows levels of quercetin in hydrolysed extracts of
flesh of tomatoes transformed with either the control pSJ89 (G
series of transformed plants) or the pBBC50 (C series of
transformed plants) gene constructs.
[0050] FIG. 9 shows a typical HPLC chromatogram, recorded at 360
nm, of a non-hydrolysed extract of peel tissue of a tomato plant
transformed with pBBC50 (plant number C87). The major peaks
correspond to rutin (R), isoquercitrin (IQ), kaempferol
rutinoside/quercetin glycoside (KR/QG) (co-eluting compounds) and a
putative kaempferol glycoside (KG) are marked.
[0051] FIG. 10 shows the proposed biosynthetic pathway for the
production of flavonoids.
[0052] FIG. 11 shows the graph of the data represented in table
2.
[0053] FIG. 12 shows restriction maps of plasmids pUCAP and
pUCM2.
[0054] FIG. 13 shows the restriction map of plasmid pFLAP10.
[0055] FIG. 14. shows the multiple cloning site as altered in pUCM2
from AscI to PacI in the 5' to 3' orientation.
DETAILED DESCRIPTION OF THE INVENTION
[0056] The present invention is based on the unexpected finding
that chalcone isomerase may be a rate limiting step in the
production of flavonoids in tomato fruit.
[0057] We have surprisingly found that upon incorporation of a gene
sequence encoding for a protein with chalcone isomerase activity in
plants, the subsequent overexpression of this protein leads to very
high (sometimes even 50-100 fold) increase in the amount of
flavonoids in the fruit of said plant.
[0058] Applicants have found that in ripening tomato fruit two
dominant flavonoids can be detected: flavonol rutin and
narichalcone, which both accumulate in the peel of tomato fruit. At
no developmental stage were significant amounts of flavonoids
detected in the flesh of fruit. Without wishing to be bound by any
theory applicants believe that the accumulation of narichalcone in
the peel of fruit before declining through the red and over ripe
stages, is indicative that chalcone isomerase represents a rate
limiting step in the formation of flavonoids.
[0059] A method for elucidation of the rate limiting step in
flavonoid biosynthesis is further illustrated in the examples.
[0060] Advantageously, by means of the invention, levels of
specific flavonoids, more particularly flavonols, in plants,
particularly tomatoes, may be altered. Preferably in the method
according to the invention the levels of flavonoids, more
particularly flavonols, in plants, particularly tomatoes, are
increased. Moreover, it has been found that the level of
flavonoids, in particular the level of specific flavonols, may be
increased specifically in the peel of tomato fruit, thereby
producing tomatoes with enhanced nutritional, preservative and
flavour characteristics.
[0061] Most preferred in the method according to the invention the
transformed plant exhibits increased levels of kaempferol and/or
quercetin, or their glycosides or derivatives thereof.
[0062] It will be appreciated that the invention furthermore
relates to a method for producing a plant capable of exhibiting
altered levels of flavonoids, comprising incorporating into said
plant a gene sequence encoding for chalcone isomerase, thereby
increasing the level of flavonoids by overexpression of said
chalcone isomerase. Therefore it will be understood that the
invention encompasses said gene sequence encoding for chalcone
isomerase and any sequence functionally equivalent thereto. This
group of sequences is in the course of this application also
referred to as "a gene comprising a nucleotide sequence encoding an
enzyme with chalcone isomerase activity".
[0063] Therefore according to a further embodiment the invention
relates to a method for producing a plant capable of exhibiting
altered levels of flavonoids comprising incorporating into said
plant a gene comprising a nucleotide sequence encoding an enzyme
with chalcone isomerase activity.
[0064] According to a preferred embodiment said gene comprises a
nucleotide selected from:
[0065] (i) a nucleotide sequence, encoding an amino acid sequence
having at least 40% similarity, to seq ID No1;
[0066] (ii) a nucleotide sequence capable of hybridising under low
stringent conditions to a sequence selected from the group of
sequences set forth under (i) above;
[0067] (iii) a nucleotide sequence encoding a protein that is
functionally equivalent to the protein encoded by seq ID no 1.
[0068] Seq, ID 1 is an amino acid sequence obtainable from PIR
database, accession number SO4725, as published by van Tunen et al,
EMBO J. 7, 1257-63 1988.
[0069] More preferred said gene comprises the nucleotide sequence
encoding an amino acid sequence having at least 60% similarity
preferably at least 90%, more preferred at least 95% or even 98%,
similarity to the sequence as set forth in seq ID No1 (amino acid
sequence of chalcone isomerase).
[0070] According to a highly desired embodiment, the gene which is
incorporated into the plant in the method according to the
invention encodes the amino acid sequence of chalcone isomerase
from petunia as set forth in seq ID No 1.
[0071] According to a preferred embodiment said nucleotide sequence
comprises a sequence which has at least 50% similarity, more
preferred at least 60%, even more preferred at least 75%, even more
preferred at least 80%, still more preferred at least 90%, most
preferred at least 95% or even 98-100% similarity to Sequence ID no
2, and whereby said sequence encodes a protein having chalcone
isomerase activity.
[0072] Although the percentage similarity referred to above assumes
an overall comparison between the sequence set forth in at least
one of the sequences of Seq ID 1, Seq ID 2, it is clear that there
may be specific regions within molecules being compared, having
less than 60% similarity.
[0073] It will be appreciated that the invention extends to any
plant which is amenable to transformation.
[0074] Therefore, according to another embodiment, the invention
repaties to a plant having one or more transgenes, each encoding a
protein with chalcone isomerase activity or a protein functionally
equivalent thereto, incorporated into its genome such that its
ability to produce flavonoids is altered.
[0075] Preferably the plants according to the invention are
suitable for human consumption. Suitable plants are for example
vegetables, fruits, nuts, herbs, spices, infusion materials.
Suitable vegetables are for example from the Pisum family such as
peas, family of Brassicae, such as green cabbage, Brussel sprouts,
cauliflower, the family of Phaseolus such as barlotti beans, green
beans, kidney beans, the family of Spinacea such as spinach, the
family of Solanaceae such as potato and tomato, the family of
Daucus, such as carrots, family of Capsicum such as green and red
pepper, and berries for example from the family of Ribesiaceae,
Pomaceae, Rosaceae, for example strawberries, black berries,
raspberries, black current and edible grasses from the family of
Gramineae such as maize, and citrus fruit for example from the
family of Rutaceae such as lemon, orange, tangerine. Also preferred
are plants which can form the basis of an infusion such as black
tea leaves, green tea leaves, jasmin tea leaves. Also preferred is
the tobacco plant.
[0076] A particularly preferred plant for use in the method
according to the invention is the tomato plant.
[0077] It will furthermore be appreciated that the sequence
encoding a protein with chalcone isomerase activity may be a
genomic or cDNA clone, or a sequence which in proper reading frame
encodes an amino acid sequence which is functionally equivalent to
the amino acid sequence of the protein encoded by the genomic or
cDNA clone. By "functionally equivalent" is meant any DNA sequence
which is capable of similar biological activity. A functional
derivative can be characterised by an insertion, deletion or a
substitution of one or more bases of the DNA sequence, prepared by
known mutagenic techniques such as site-directed mutagenesis. The
functionality can be evaluated by routine screening assays, for
example, by assaying the flavonoid content of the resulting
transgenic plant. An in vitro assay to determine chalcone isomerase
activity has been described by van Weely (1983, Planta 159:
226-230).
[0078] Gene sequences encoding a gene sequence for proteins with
chalcone isomerase activity for use according to the present
invention may suitably be obtained from plants, in particular
higher plants as these generally possess a flavonoid biosynthetic
pathway. Suitable gene sequences can for example be obtained from
petunia, maize, arabidopsis, alfalfa, pea, bean, grape, apple.
[0079] The gene sequences of interest are preferably operably
linked (that is, positioned to ensure the functioning of) to one or
more suitable promoters which allow the DNA to be transcribed. Said
promoters are preferably promoters useful to obtain overexpression
of the protein with chalcone isomerase activity in said host plant.
Suitable promoters, which may be homologous or heterologous to the
gene (that is, not naturally operably linked to the expressed gene
encoding a chalcone isomerase protein or a functional equivalent
thereof) useful for expression in plants are well known in art, as
described, for example, in Weising et al, (1988), Ann. Rev.
Genetics, 22, 421-477). Promoters for use according to the
invention may be inducible, constitutive or tissue-specific or have
various combinations of such characteristics. Useful promoters
include, but are not limited to, constitutive promoters such as
carnation etched ring virus (CERV), cauliflower mosaic virus (CaMV)
.sup.35S promoter, or more particularly the enhanced cauliflower
mosaic virus promoter, comprising two CaMV 35S promoters in tandem
(referred to as "Double 35S"), or the GBSS (granular bound starch
synthase) promoter.
[0080] According to a preferred embodiment fruit specific promoters
are used. Suitable fruit-specific promoters include the tomato E8
promoter (Deikman et al, (1988), EMBO J, 7, 3315-3320), 2A11 (Van
Haaren et al, Plant Mol Biol, 21, 625-640), E4 (Cordes et al,
(1989), Plant Cell, 1, 1025-1034) and PG (Bird et al, (1988), Plant
Mol. Biol., 11, 651-662) Nicholass et al, (1995), Plant Molecular
Biology, 28, 423-435, pTOM96 (ref), fpb11(WO-A-91/05054).
[0081] In another preferred embodiment, the promoter is a
constitutive enhanced 35S CaMV promoter.
[0082] It will be appreciated that accumulation of flavonoids may
be inhibited by the rate of production of the amino acid
phenylalanine, the primary substrate in the synthesis of
phenylpropanoids and subsequent flavonoids. In order to increase
phenylalanine biosynthesis, genes encoding enzymes of the
phenylalanine pathway that are insensitive to feed-back regulation
may be introduced as an optional additional step.
[0083] Preferably the desired gene sequences, operably linked to
respective suitable promoters, are fused to appropriate expression
sequences to provide an expression cassette functional in a plant
cell which can be introduced into a plant cell by any conventional
plant transformation method.
[0084] Therefore the invention also relates to a DNA construct
comprising sequences encoding for a protein with chalcone isomerase
activity, or a functionally equivalent sequence thereof, operably
linked to a promoter; and relates to plants, preferably tomato
plants comprising said DNA construct.
[0085] Accordingly, the invention provides in a further aspect an
expression cassette comprising as operably linked components in the
5'-3' direction of transcription at least one unit, comprising a
promoter functional in a plant cell, a gene sequence encoding a
protein with chalcone isomerase activity and a transcriptional
termination regulatory region functional in a plant cell.
[0086] The promoter and termination regulatory regions will be
functional in the host plant cell and may be heterologous (that is,
not naturally occurring) or homologous (derived from the plant host
species) to the plant cell and the gene. Suitable promoters which
may be used are described above.
[0087] The termination regulatory region may be derived from the 3'
region of the gene from which the promoter was obtained or from
another gene. Suitable termination regions which may be used are
well known in the art and include Agrobacterium tumefaciens
nopaline synthase terminator (Tnos), Agrobacterium tumefaciens
mannopine synthase terminator (Tmas) and the CaMN 35S terminator
(T35S). Particularly preferred termination regions for use
according to the invention include the tobacco ribulose
bisphosphate carboxylase small subunit termination region (TrbcS)
or the Tnos termination region.
[0088] Such gene constructs may suitably be screened for activity
by transformation into a host plant via Agrobacterium and screening
for flavonoid levels.
[0089] Conveniently, the expression cassette according to the
invention may be prepared by cloning the individual
promoter/gene/terminator unit into a suitable cloning vector.
Suitable cloning vectors are well known in the art, including such
vectors as pUC (Norrander et al, (1983, Gene 26, 101-106), pEMBL
(Dente et al (1983), Nucleic Acids Research, 11, 1645-1699),
pBLUESCRIPT (available from Stratagene), pGEM (available from
Promega) and pBR322 (Bolivar et al, (1977), Gene, 2, 95-113).
Particularly useful cloning vectors are those based on the pUC
series. The cloning vector allows the DNA to be amplified or
manipulated, for example, by adding sequences. The cloning sites
are preferably in the form of a polylinker, that is a sequence
containing multiple adjacent restriction sites, so as to allow
flexibility in cloning.
[0090] In a particularly preferred embodiment, the individual
promoter/gene/terminator units are cloned into adjacent pairs of
restriction sites in a suitable cloning vector.
[0091] Suitably, the nucleotide sequences for the genes may be
extracted from any nucleotide database and searched for restriction
enzymes that do not cut. These restriction sites may be added to
the genes by conventional methods such as incorporating these sites
in PCR primers or by sub-cloning.
[0092] Preferably the DNA construct according to the invention is
comprised within a vector, most suitably an expression vector
adapted for expression in an appropriate host (plant) cell. It will
be appreciated that any vector which is capable of producing a
plant comprising the introduced DNA sequence will be
sufficient.
[0093] Suitable vectors are well known to those skilled in the art
and are described in general technical references such as Pouwels
et al, Cloning Vectors. A laboratory manual, Elsevier, Amsterdam
(1986). Particularly suitable vectors include the Ti plasmid
vectors.
[0094] Transformation techniques for introducing the DNA constructs
according to the invention into host cells are well known in the
art and include such methods as micro-injection, using polyethylene
glycol, electroporation, or high velocity ballistic penetration. A
preferred method for use according to the present invention relies
on agrobacterium--mediated transformation.
[0095] After transformation of the plant cells or plant, those
plant cells or plants into which the desired DNA has been
incorporated may be selected by such methods as antibiotic
resistance, herbicide resistance, tolerance to amino-acid analogues
or using phenotypic markers.
[0096] Various assays may be used to determine whether the plant
cell shows an increase in gene expression, for example, Northern
blotting or quantitative reverse transcriptase PCR (RT-PCR). Whole
transgenic plants may be regenerated from the transformed cell by
conventional methods. Such transgenic plants having improved
flavonoid levels may be propagated and crossed to produce
homozygous lines. Such plants produce seeds containing the genes
for the introduced trait and can be grown to produce plants that
will produce the selected phenotype.
[0097] In accordance with a particular embodiment of the invention,
the cloning vectors plasmid pUCM2 and pUCM3 were prepared by
modifying the cloning vector pUCAP (Van Engelen et al, (1995),
Transgenic Research, 4, 288-290).
[0098] The invention furthermore relates to a plant having one or
more transgenes each encoding a protein with chalcone isomerase
activity, or a sequence functionally equivalent thereto,
incorporated into its genome such that its ability to produce
flavonoids is altered.
[0099] The invention also encompasses a tomato plant prepared
according to the method of the invention.
[0100] The following examples are provided by way of illustration
only.
[0101] DNA manipulations were performed using standard procedures
well known in the art, as described, for example, in Sambrook et
al, Molecular Cloning, A Laboratory Manual, Second Edition, Cold
Spring Harbour Laboratory Press, 1989 (hereinafter "Sambrook").
[0102] The following literature references are mentioned in the
Examples:
[0103] Becker, D. et al. (1992) Plant Mol. Biol. 20: 1195-1197
[0104] Bovy, A. G. et al. (1995) Acta Hortic. 405: 179-189.
[0105] Fulton, T. M. et al. (1995) Plant Mol. Biol. Rep. 13:
225-227
[0106] Hanahan, D. (1983) J. Mol. Biol. 166: 557-580.
[0107] Hertog, M. G. L. et al. (1992) J. Agric. Food Chem. 40:
1591-1598.
[0108] Hoekema, A. et al. (1985) Plant Mol. Biol. 5: 85-89
[0109] Jefferson, R. et al. (1987) Embo J. 6: 3901-3907
[0110] Loyd, A. et al (1992), Science 258, 1773-1775
[0111] Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15:
73-97
[0112] Sambrook, J. et al. (1989) Molecular Cloning. A laboratory
manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.
[0113] Saul, M. W. et al. (1988) Plant Mol. Biol. Man. Al: 1-16
(Eds.
[0114] Gelvin S. B. and Schilperoort, R. A.) Kluwer Academic Pubs.,
London Symmans et al (1990) Biotechnology 8, 217-221
[0115] Vancanneyt, G. et al (1990). Mol. Gen. Gen. 220,
245-250.
[0116] Van Engelen, F. et al. (1995) Transgenic R. 4: 288-290
[0117] VanTunen, A. J. et al. (1988) EMBO J. 7: 1257-1263
EXAMPLES
Example 1
Plant Material
[0118] Plants of tomato line FM6203 and transformants are grown in
soil in a glasshouse with a 16 hour photoperiod and a 23/18.degree.
C. day/night temperature.
Example 2
Bacterial Strains
[0119] The Escherichia coli strain used is:
[0120] DH5.alpha. supE44, (lac ZYA-ArgF)U169, 80lacZM15, hsdR17
(rk-, mk+), recA1, encA1, gyrA96, thi-1, relA1, deoR (Hanahan,
1983).
[0121] The Agrobacterium strain used is LBA4404 (Hoekema,
1985).
[0122] Transformation of E. Coli DH5.alpha. is performed using the
method of Hanahan, (1983).
[0123] Transformation of Agrobacterium LBA4404 is performed using a
freeze/thaw method according to Saul et al, (1988).
Example 3
Elucidation of the Rate-Limiting Step in Flavonol Production in
Tomato Fruit
[0124] The rate-limiting step in flavonol production in tomato
fruit is determined using two complimentary approaches; high
performance liquid chromatography (HPLC) analysis of flavonoids in
ripening tomato fruit and northern analysis using probes for the
flavonoid biosynthetic genes chalcone synthase (chs), chalcone
isomerase (chi) and flavonol synthase (fls).
[0125] 3.1 Analysis of Flavonoids in Ripening Tomato Fruit by
HPLC
[0126] 3.1.1 Harvest of Tomato Fruit
[0127] Tomato fruit are harvested at five stages of ripening
(green, breaker (the ripening stage corresponding to the appearance
of the first flush of color on the green fruit), turning, red and
over-ripe; corresponding to approximately 21, 28, 31, 46 and 55
days post anthesis respectively). For discrimination between
flavonoids in peel and flesh tissues, the outer layer of
approximately 2 mm thick (i.e. cuticula, epidermal layer plus some
sub-epidermal tissue) is separated from the fruit using a scalpel
and classified as peel. The jelly and seeds are then removed and
the remainder of the fruit is classified as flesh tissue. After
separation, tissues are quickly cut into pieces and frozen in
liquid nitrogen before being ground into a fine powder using a
pre-cooled coffee grinder. Peel and flesh tissues are lyophilised
for 24 hr and then stored under desiccating conditions at 4.degree.
C. until use.
[0128] 3.1.2 Extraction of Flavonoids from Tomato Tissues
[0129] Determination of flavonoid glycosides and narichalcone
(2',4',6',4-tetrahydroxychalcone) in tomato fruit is carried out
using a non-hydrolysing method as follows: 40 mg of freeze-dried
tomato tissue is weighed and transferred to a 10 ml Pyrex glass
tube. To each tube 4 ml of 75% aqueous methanol acidified with HCl
to pH 2 is added. The tubes are closed with screw tops containing a
Teflon inlay and incubated at room temperature (20-25.degree. C.)
for 1 hr with continuous mixing on a roller band.
[0130] 3.1.3 High Performance Liquid Chromatography (HPLC)
Conditions for Flavonoid Analysis
[0131] 1 ml of each tomato fruit extract is taken using a
disposable syringe and filtered through a 0.2 .mu.m PTFE disposable
filter (Inacom Instruments BV, The Netherlands) before injection
into the HPLC system.
[0132] The HPLC system consisted of a Waters 600E Multisolvent
Delivery System (Waters Chromatography), a Promis autoinjector
(Separations Analytical Instruments BV) with a fixed 10 .mu.l loop,
and a Nova-Pak C.sub.18 (3.9.times.150 mm, particle size 4 .mu.m)
analytical column (Waters Chromatography) protected by a Guard-Pak
Nova-Pak C18 insert. Both columns are placed in a LKB 2155 HPLC
column oven (Pharmacia Biotech) set at 30.degree. C. A photodiode
array detector (Waters 996) is used to record spectra of compounds
eluting from the column on-line. The detector is set at recording
absorbance spectra from 240 to 600 nm with a resolution of 4.8 nm,
at a time interval of 1 sec. Millennium 2010 Chromatography Manager
(Waters Chromatography BV) is used to control the solvent delivery
system and the photodiode array detector.
[0133] HPLC separation of flavonoids in non-hydrolysed extracts is
performed using a gradient of acetonitril in 0.1% TFA, at a flow
rate of 1 ml/min: 12.5-17.5% linear in 3 min, then 17.5-25% in 32
min and 25-50% in 2 min, followed by a 3 min washing with 50%
acetonitril in 0.1% TFA. After washing, the eluent composition is
brought to the initial condition in 2 min, and the column is
equilibrated for 6 min before the next injection.
[0134] HPLC data are analysed using the software of the Millennium
2010 Chromatography Manager. Absorbance spectra (corrected for
baseline spectrum) and retention times of eluting peaks (with peak
purity better than purity threshold value) are compared with those
of commercially available flavonoid standards. Quercetin and
kaempferol glycosides and narichalcone are quantified based on
absorption at 360 nm. Dose-response curves (0 to 20 .mu.g/ml) were
established to quantify these compounds in the non-hydrolysed
tomato extracts. Flavonoid levels in tomatoes are calculated on a
dry weight basis for peel and flesh tissues. With the HPLC system
and software used, the lowest detection limit for flavonoids in
tomato extracts is about 0.1 .mu.g/ml, corresponding with 10 mg/kg
dry weight and 1 mg/kg fresh weight. Variation between replicate
injections is generally less than 5%.
[0135] 3.1.4 Characterisation of the Flavonoid Content in Ripening
Tomato Fruit
[0136] Two dominant flavonoids are detected in the peel of ripe
tomato fruit, the flavonol rutin (quercetin 3-rutinoside) and
narichalcone, which are identified by their retention time (RT) and
absorbance spectrum. At least four other flavonol glycosides are
also identified in the tomato peel extracts, albeit in much smaller
quantities than rutin or narichalcone. A full identification of
these minor flavonol glycoside species is described in Example
8.
[0137] In contrast, the flesh tissue from ripe tomato fruit
contains only traces of rutin, no other flavonoid species are
detectable.
[0138] The levels of rutin and narichalcone in the peel during
ripening of tomato fruit are shown in FIG. 1. Rutin levels increase
during tomato ripening reaching their highest levels in the
over-ripe stage (approximately 1 mg/g dry weight of peel).
Narichalcone is absent in the peel of green fruit but increases
sharply during coloring of the fruit, reaching levels of
approximately 10 mg/g dry weight in peel of turning fruit before
declining through the red and over-ripe stages. The enzyme chalcone
isomerase (CHI) is believed to be responsible for catalysing the
formation of naringenin from narichalcone in the flavonoid
biosynthetic pathway (FIG. 10). Applicants are of the opinion that
the accumulation of narichalcone suggests that in the peel of
ripening tomatoes CHI represents a rate limiting enzyme in the
formation of flavonols.
[0139] 3.2 Northern Analysis of Ripening Tomato Fruit
[0140] Northern analysis is used to determine the endogenous
expression of the flavonoid biosynthetic genes chs, chi and fls
during the development of FM6203 tomato fruit.
[0141] RNA is isolated from the peel and flesh of green, breaker,
turning and red fruit and also young leaves according to the
protocol of van Tunen (1988). For RNA gel blot analysis, 10 .mu.g
of RNA is loaded on formaldehyde agarose gels and electrophoresed
overnight at 25V. Separated RNA is then blotted overnight onto
Hybond N.sup.+ membrane (Amersham).
[0142] Petunia hybrida cDNA fragments encoding the following
flavonoid biosynthetic enzymes are used as probes: chalcone
synthase (CHS-A), chalcone isomerase (CHI) and flavonol synthase
(FLS). These fragments are obtained by RT-PCR on RNA extracted from
closed flowers of Petunia hybrida W115 with primer combinations
F15/F16 (chi-a), F13/F14 (chs-a) and F20/F21 (fls). The obtained
PCR products are checked by sequence analysis.
[0143] Probes are labelled with .sup.32P and purified according to
methods given in Gibco Life Technologies RadPrime Labelling system.
Blots are hybridised overnight at 55.degree. C. and washed three
times in 2.times.SSC, 0.1% SDS, 55.degree. C., 30 min, before being
exposed to X-ray film for 48 hr.
[0144] The results of the northern blot are shown in FIG. 2. Both
the chs and fls transcripts are abundantly present in the peel of
tomato fruit in all developmental stages tested. The level of these
two transcripts peaks during the breaker and turning stage of
development and subsequently decreased in the red stage. The chi
transcript level is very low in the peel of all developmental
stages. Without wishing to be bound by any theory, applicants
believe that this is indicative that one of the rate limiting steps
in flavonoid biosynthesis in the peel may lie at the level of chi
gene expression. This result is in agreement with the observation
that narichalcone (the substrate for CHI), accumulated to high
levels in breaker and turning stage fruit (Example 3.1).
[0145] In the flesh of tomato fruit, the levels of chs, chi and fls
transcripts are very low, in agreement with the HPLC data which
showed only trace amounts of rutin in this tissue (Example
3.1).
[0146] Chs, chi and fls transcripts are present in low but
detectable levels in tomato leaves.
Example 4
Gene Constructs
[0147] 4.1 Strategy to Overexpress a Rate-Liming Step of Flavonol
Production in Tomato Fruit
[0148] During the early stages of ripening of tomato fruit
narichalcone accumulates in the peel of the fruit (Example 3.1).
The enzyme responsible for converting narichalcone into naringenin
is CHI. The expression levels of the gene encoding CHI remain low
throughout ripening of the fruit (Example 3.2) suggesting CHI may
constitute a rate-liming step in the production of flavonols in the
peel of tomato fruit.
[0149] The strategy consists of increasing the production of
flavonols in tomato fruit by overexpression of the Petunia hybrida
gene encoding CHI. The introduced gene is expressed under the
control of the constitutive enhanced CaMV 35S promoter (also called
double P35s or Pd35S).
[0150] 4.1 Cloning the chi Gene from Petunia Hybrida
[0151] The chi-a gene is amplified from plasmid pMIP41, which
contains the complete chi-a cDNA from Petunia hybrida inbred line
V30 (Van Tunen et al. 1988), with primer combination F15/F16. These
primers contain a 5' extension with a unique BamHI (F15) and SalI
(F16) restriction site (Table 1). This results in a 0.73 kb chi-a
fragment.
[0152] 4.2 Construction of the chi Gene Fusion
[0153] The Pd35S-chi-Tnos gene construct is made as follows.
Plasmid pFLAP10, a pUC-derivative containing a fusion of the
consitutive enhanced CaMV 35S promoter (P35s), the maize cl gene
(cl) and the Agrobacterium tumefaciens nos terminator (Tnos), is
used as recipient of the chi-a gene (FIG. 3a). A description of the
properties of plasmid pFLAP10 is given in FIG. 3. The amplified
chi-a cDNA is digested with BamHI/SalI and the resulting 730 bp
fragment is ligated in plasmid pFLAP10 digested with the same
enzymes, thus replacing the cl gene with the chi-a gene. The
resulting plasmid is denoted pFLAP50 (FIG. 3b).
[0154] 4.2.1 Construction of pFLAP10
[0155] The C.sub.1 gene fusion was cloned in plasmid pUCM2, a
derivative of plasmid pUCAP (Van Engelen et al. 1995, Transgenic
Research 4, p. 288-290), in which the multiple-cloning-site was
altered (FIG. 12), in three major steps. Said altered multiple
cloning site is shown in FIG. 14.
[0156] Firstly, Tnos was amplified by PCR from pBI121 with primers
F12 and AB13 (see Table 1). The resulting 250 bp product was cloned
in pUCM2 as a SalI/ClaI fragment. This resulted in plasmid
pFLAP1.
[0157] Secondly, the cl gene was cloned as a BamHI/SalI fragment
upstream of Tnos in pFlap2 as follows. The cl gene was transferred
as a 2 kb EcoRI fragment from plasmid pAL77 (Loyd 1992) to
high-copy plasmid pBluescript SK-, resulting in plasmid pBlC1. The
cl gene was isolated from pBlC1 as a 1.6 kb EcoRI/PacI fragment and
adapters F7F8 and F9F10 (Table 1) were ligated to each end of the
fragment in order to add unique BamHI and SalI restriction sites on
both ends of the gene and to destroy the EcoRI and PacI sites. The
resulting BamHI/SalI cl fragment was cloned upstream of the nos
terminator, resulting in plasmid pFLAP2.
[0158] Thirdly, Pd35s was cloned as a KpnI/BamHT fragment upstream
of cl in pFLAP2 as follows. To create a unique BamHI site at the 3'
end of the d35s promoter, plasmid pMOG18 (Symans et al 1990,
Biotechnology 8, p. 217-221) was digested with EcoRV/BamHI, thus
removing the 3' part of the d35s promoter and the gusA gene. The 3'
part of the 35S promoter present in plasmid pAB80 (Bovy et al.
(1995)) was ligated as a 0.2 kb EcoRV/BamHI fragment in the pMOG18
vector, resulting in plasmid pMOG18B. To Create a unique KpnT site
at the 5' end of the d35s promoter plasmid pMOG18B was digested
with EcoRI, the ends were polished with Klenow polymerase, and a
subsequent digest with BamHI was done. The resulting 0.85 kb
blunt/BamHI d35s promoter fragment was cloned into plasmid pBlC1
followed by digestion with XhoI and polished with Klenow
polymerase/BamHI. This resulted in plasmid pBld35S. Finally the
d35s promoter was transferred as a KpnI/BamHI fragment from pBld35s
to plasmid pFLAP2. This resulted in plasmid pFLAP10 (FIG. 13).
[0159] 4.3 Construction of Binary Vector pBBC3
[0160] To obtain a binary vector with suitable cloning sites to
transfer the chi gene fusion into, plasmid pBBC3, a derivative of
pGPTV-KAN (Becker et al. (1992)) is constructed as follows.
Synthetic adapter F38F39 (Table 1) is ligated in plasmid pGPTV-KAN
digested with EcoRI/HinDIII. In this way the gusA-Tnos gene in
pGPTV-KAN is replaced by a small multiple-cloning-site consisting
of PacI/EcoRI/HinDIII/AscI restriction sites (FIG. 4a).
[0161] 4.4 Transfer of the chi Gene Fusion into pBBC3
[0162] The Pd35s-chi-Tnos insert is transferred from pFLAP50 as a
PacI/AscI fragment into binary vector pBBC3, digested with the same
enzymes. The resulting binary plasmid is denoted pBBC50 (FIG.
4b).
[0163] 4.5 GPTV Control Plasmid
[0164] A GPTV-based binary plasmid (pSJ89) containing the
.beta.-glucuronidase gene (with the st-ls1 intron; Vancanneyt et
al. 1990) under control of the CaMV .sup.35S promoter and the nos
poly(A) signal (P35s-gusA-Tnos) is used as a control plasmid to
transform FM6203 (FIG. 4c). This allows direct comparison between
gus transformed control plants and plants containing the chi
construct as both sets of plants have gone through the tissue
culture procedure.
[0165] Plasmid pSJ89 is constructed as follows: the CaMV 35S
promoter--gus-int fragment (Vancanneyt et al, 1990) is cloned as a
HindIII--SacI fragment into the same sites of plasmid pSJ34, a
derivative of the binary vector pGPTV-KAN (Becker et al, 1992) in
which the BamHI site between the NPTII selectable marker and the
gene 7 poly(A) signal is destroyed by filling in with klenow
polymerase.
Example 5
Stable Transformation of chi Construct into Tomato Line FM6203
[0166] 5.1 Agrobacterium Tumefaciens Transformations
[0167] Binary plasmids pBBC50 and pSJ89 are introduced into
Agrobacterium strain LBA4404 by adding 1 .mu.g of plasmid DNA to
100 .mu.l of competent Agrobacterium cells, prepared by inoculating
a 50 ml culture in YEP medium (Sambrook, 1989) and growing at
28.degree. C. until the culture reaches an OD.sub.600 of 0.5-1.0.
The cells are then pelleted, resuspended in 1 ml of CaCl.sub.2
solution and dispensed into 100 .mu.l aliquots. The
DNA-Agrobacterium mixture is frozen in liquid nitrogen and thawed
in a water bath at 37.degree. C. After the addition of 1 ml YEP
medium the bacteria are incubated at 28.degree. C. for 4 hours with
gentle shaking. Finally transformed bacteria are selected on
YEP-agar plates containing 50 .mu.g/ml kanamycin. The presence of
the plasmids is tested by PCR analysis using pBBC50 (chi 5 and nos
ant) or pSJ89 (300 35s and gus 2) specific primers respectively
(Table 1).
[0168] 5.2 Tomato Transformations
[0169] Seeds from tomato line FM6203 are sterilised by a 2 h
incubation in 1.5% hypochlorite, followed by three rinses of
sterile water. The seeds are germinated and seedlings are grown for
8 days on a 1:1 mixture of vermacolite and MS medium (Murashige and
Skoog, 1962; Duchefa) supplemented with 0.3% (w/v) sucrose, with a
photoperiod of 16 h (3000 lux) at 25.degree. C.
[0170] Eight-day old cotyledons are cut into 25 mm.sup.2 squares
and preincubated for 24 h on tobacco suspension feeder layer plates
at low light intensity (1000 lux). The tobacco leaf suspension
culture is grown on plates containing MS medium including vitamins,
supplemented with sucrose (3% w/v), agarose (6 g/l),
2,4-dichlorophenoxyacetic acid (2,4-D; 0.5 mg/l) and
benzylaminopurine (BAP; 0.5 mg/l).
[0171] A single colony from the Agrobacterium LBA4404 cultures
containing one of the binary vectors mentioned in Example 4.4 and
4.5 is grown for 48 h in liquid Minimal A medium (Sambrook, 1989)
supplemented with 50 .mu.g/ml kanamycin to an OD.sub.600 of
0.5-1.0. The bacteria are pelleted by centrifugation and
resuspended in MS medium including vitamins (Duchefa) and 3% (w/v)
sucrose at an OD.sub.600 of 0.5. The cotyledon explants are
incubated in the Agrobacterium suspension for 30 min, blotted dry
on filter paper and co-cultivated for 48 h on tobacco feeder layer
plates at 25.degree. C. and low light intensity.
[0172] After co-cultivation, the explants are transferred to
regeneration medium, consisting of MS medium supplemented with
Nitsch vitamins, sucrose (2% w/v), agargel (5 g/l), zeatin-riboside
(2 mg/l), kanamycin (100 mg/l) and cefotaxime (500 mg/l).
Regenerating explants are transferred to fresh medium every two
weeks. Regenerating kanamycin resistant shoots were transferred to
rooting medium, consisting of MS medium plus B5 vitamins,
supplemented with sucrose (0.5% w/v), gelrite (2 g/l), kanamvcin
(50 mg/l) and cefotaxime (250 mg/l). During regeneration and
rooting explants are incubated in a growth chamber at 25.degree. C.
with a 16 h photoperiod (3000 lux). After root formation, the
presence of the CHI insert is confirmed by PCR analysis of
cotyledon tissue using specific primers (chi 5 and nos ant), and
the presence of the GUS insert using 300 35s and gus2 specific
primers (Table 1). PCR positive plantlets are transferred to soil
and grown in the greenhouse.
[0173] Transgenic plants carrying the construct pBBC50 are numbered
from C6 onward. Control transgenic plants carrying the construct
pSJ89 are numbered from G2 onward.
Example 6
Southern Analysis of Transgenic Plants
[0174] The presence and the copy number of the transgenes is
determined in transgenic plants by southern hybridisation. Genomic
DNA is isolated from young leaves as described by Fulton et al.,
(1995). Aliquots of 10 .mu.g genomic DNA are digested for 16 h with
EcoRI and separated on a 0.8% TAE agarose gel. The DNA is denatured
in 0.5 M NaOH, 1.5M NaCl for 45 min before being transferred to a
Hybond N+ membrane (Amersham) in 20.times.SSC.
[0175] The blots are probed with a 700 base pair .sup.32P
radiolabeled nptII-specific PCR fragment, amplified from plasmid
pBBC3 with primers npt IIa and npt IIb (Table 1), under stringent
conditions (65.degree. C.) Prehybridisation is carried out for 2 h
at 65.degree. C. in a mix of 0.5 M Na.sub.2PO.sub.4 pH 7.2, 7% SDS
and 0.1 mg/ml denatured herring sperm DNA. Hybridisation is
performed by adding denatured probe DNA to the prehybridisation
medium and continuing the incubation at 65.degree. C. for 16 h. The
hybridised blots are washed once for 30' at 25.degree. C. in
2.times.SSC, 0.1% SDS and then once for 30' at 65.degree. C. in
2.times.SSC, 0.1% SDS before being autoradiographed.
[0176] The result of the southern analysis is shown in FIG. 5. The
control is an untransformed FM6203 plant. Southern analysis
confirms the initial screening of transgenics by PCR (see Example
5.2), every pBBC50 transformed plant hybridised with the npt II
probe. Transgenic plants contain either 1 or 2 copies of the
insert.
Example 7
Measurement of Flavonoids in Transformed Tomato Plants
[0177] 7.1 Growth and Harvest of Tomato Fruits
[0178] Transgenic tomato plants are grown in 10 l cots in a
glasshouse at standard growth conditions (day/night temperatures
23.degree. C./18.degree. C., 16 hr light)). Fruits are harvested
between 15-21 days post-breaker stage (corresponding to fully red
ripe fruit. For discrimination between flavonoids in peel and flesh
tissues, the outer layer of approximately 2 mm thick (i.e.
cuticula, epidermal layer plus some sub-epidermal tissue) is
separated from the fruit using a scalpel and classified as peel.
The jelly and seeds are then removed and the remainder of the fruit
was classified as flesh tissue. After separation, tissues are
quickly cut into pieces, frozen in liquid nitrogen and stored at
-80.degree. C. until use.
[0179] 7.2 Extraction of Flavonoids from Tomato Tissues
[0180] Flavonoids are determined as aglycons or as their glycosides
by preparing hydrolysed and non-hydrolysed extracts,
respectively.
[0181] Acid hydrolysis is used as an initial screen of
transformants in order to identify those lines containing high
amounts of flavonols as compared to the control. Acid hydrolysis
ensures that flavonoid glycosides such as rutin and kaempferol
rutinoside are hydrolysed to their respective aglycones i.e.
quercetin and kaempferol.
[0182] Preparation of hydrolysed extracts is performed according to
Hertog et al (1992) with some modifications. Frozen tissues are
ground into a fine powder using a pre-cooled coffee grinder. Peel
and flesh tissues are lyophilised for 24 h before flavonoid
extraction. 50 mg of this freeze-dried material was weighed and
transferred to a 6 ml Pyrex glass tube. To each tube 1.6 ml of
62.5% methanol (HPLC grade) in distilled water and 0.4 of 6 M HCl
are added. The tubes are closed with screw caps containing a Teflon
inlay and incubated for 60 mm at 90.degree. C. in a waterbath.
[0183] After hydrolysis, the tubes are cooled on ice, the extracts
are diluted with 2 ml of 100% methanol and sonicated for 5 min. 1
ml of the sample was then filtered over a 0.2 .mu.m PTFE disposable
filter into a standard 1.8 ml HPLC vial.
[0184] Preparation of non-hydrolysed extracts is performed as
follows: Frozen tissues are ground to a fine powder using a
pre-cooled coffee grinder. Peel and flesh tissues are lyophilised
for 24 h before flavonoid extraction. 50 mg of freeze dried
material is weighed and transferred to a 6 ml Pyrex glass tube. 4
ml of 70% methanol (HPLC grade) in distilled water is added to each
tube. The tubes are closed with screw top caps containing a Teflon
inlay and placed in a sonicating water bath at room temperature for
30 min. After sonication 1 ml of the sample is filtered over a 0.2
.mu.m PTFE disposable filter into a standard 1.8 ml HPLC vial.
[0185] 7.3 HPLC Conditions for Flavonoid Analysis
[0186] Chromatography of samples is performed using a
chromatography station equipped with a dual pump system and
automated gradient controller (model 1100; Hewlett Packard), a
Waters auto-injector (model 717) with a variable 20 .mu.l loop and
a Nova-Pak C.sub.18 (3.9.times.150 mm, particle size 4 .mu.m)
analytical column (Waters Chromatography) protected by a Guard-Pak
Nova-Pak C18 insert. Both columns are placed in a LKB 2155 HPLC
column oven (Pharmacia Biotech) set at 30.degree. C. A photodiode
array detector (model 1040M, Hewlett Packard) is used to record
spectra of compounds eluting from the column on-line. The detector
is set at recording absorbance spectra from 240 to 600 nm with a
resolution of 4.8=m, at a time interval of 1 second. Peak purity,
identification and integration were carried out on Hewlett Packard
Chemstations software version A.04.02.
[0187] HPLC separation of flavonoids present in hydrolyzed extracts
(flavonols and naringenin) is carried out under isocratic
conditions of 25% acetonitril (for HPLC far UV) in 0.1%
trifluoroacetic acid (TFA) at a flow rate of 0.9 ml/min.
[0188] HPLC separation of flavonoids in non-hydrolysed extracts
(flavonoid-glycosides and narichalcone) is performed using a
gradient of acetonitril in 0.1% TFA, at a flow rate of 1.0 ml/min:
5-25% linear in 30 min, then 25-30% in 5 min and 30-50% in 2 min
followed by a 3 min washing with 50% acetonitril in 0.1% TFA. After
washing, the eluent composition is brought to the initial condition
in 2 min, and the column is equilibrated for 6 min before next
injection.
[0189] HPLC data are analysed using the software of the Hewlett
Packard Chemstations software version A.04.02. Absorbance spectra
(corrected for baseline spectrum) and retention times of eluting
peaks (with peak purity better than purity threshold value) are
compared with those of commercially available flavonoid standards.
Quercetin and kaempferol aglycons are detected and calculated from
their absorbance at 370 nm, naringenin at 280 nm and
flavonol-glycosides as well as narichalcone at 360 nm. Flavonoid
levels in tomatoes are calculated on a dry weight basis. With the
HPLC system and software used, the lowest detection limit for
flavonoids in tomato extracts is about 0.1 .mu.g/ml, corresponding
with 10 mg/kg dry weight and 1 mg/kg fresh weight.
[0190] Using flavonoid standards (obtained from Apin Chemicals Ltd,
Abingdon, UK) it is established that during the hydrolysis step,
aglycons are released from their respective glycosides for 100%,
while chemically converted into naringenin for more than 95%.
Recoveries of quercetin, kaempferol and naringenin standards added
to peel or flesh extracts just before hydrolysis are more than
90%.
Example 8
Characterisation of the Flavonoid Content in Transgenic Tomato
Fruit
[0191] 8.1 Flavonoids in Peel and Flesh of Control Tomatoes
[0192] In hydrolysed extracts of control red fruit of variety
FM6203 transformed with pSJ89, both quercetin and kaempferol are
present in peel tissue (FIG. 6a). In contrast, the hydrolysed
extracts of flesh tissue from this fruit contain only traces of
quercetin with no detectable levels of kaempferol (FIG. 6b).
Without wishing to be bound by any theory, applicants believe that
the small amount of quercetin detected in the hydrolysed extracts
of flesh originates from the vascular tissue in the flesh.
Chromatograms obtained at 280 nm (not shown) of the same extracts
reveal a large peak of naringenin in the peel, but not in the
flesh. There is no significant difference in the identity and
quantity of flavonoids found in the control pSJ89-transformed
tomatoes and those found in untransformed tomatoes (data not
shown).
[0193] In non-hydrolysed extracts of control tomatoes transformed
with pSJ89, at least 5 different flavonol-glycosides as well as
narichalcone are detected in the peel (FIG. 7). NMR-studies (not
shown) prove that the peak at RT=16.9 min is rutin while the peak
at 15.2 min is a quercetin-3-trisaccharide: rutin with apiose
linked to the glucose of the rutinoside. The retention time and
absorbance spectrum of the minor peak at 17.3 min correspond with
those of quercetin-3-glucoside, while those of the peak at 19.7 min
correspond with kaempferol-3-rutinoside. The small peak at 20.4 min
has an absorbance spectrum comparable to kaempferol-3-rutinoside,
but its higher RT value indicates a yet unknown
kaempferol-glycoside. The large peak at 33.2 min is narichalcone.
Aglycons of quercetin and kaempferol, as well as naringenin (all
present in hydrolysed peel extracts) are not detectable in any of
the non-hydrolysed extracts. In the non-hydrolysed flesh sample
only a small peak corresponding to rutin is detected (data not
shown).
[0194] After comparing the flavonoid species in hydrolysed extracts
with those in non-hydrolysed extracts of the same tissue, we
conclude that the presence of quercetin and kaempferol aglycons in
the hydrolysed extracts results from hydrolysis of their respective
glycosides; the presence of naringenin in hydrolysed peel extracts
results from isomerization of narichalcone during the hydrolysis
step (cf. Example 7.3).
[0195] 8.2 Flavonoids in Fruits of Transformed Tomato Plants
[0196] To determine whether the pBBC50 construct was able to
overcome the suspected rate limiting step in flavonol production in
tomato fruit, transformants are analysed for the presence of
flavonoids in the flesh and peel of their fruits. This screening is
performed by HPLC using hydrolysed extracts. Thirty six independent
plants transformed with pBBC50, as well as six control plants
transformed with pSJ89 are analysed.
[0197] Analysis of hydrolysed extracts of flesh samples from pBBC50
transformed fruit reveals no significant increase in flavonoids
compared to the control pSJ89 fruit (FIG. 8). The differences in
quercetin concentration in tomato flesh that are shown in FIG. 8
are believed to be within the experimental error. Said experimental
error is believed to be relatively high when working at low
concentration near the detection limit of 20 .mu.g/g DW flesh.
[0198] In contrast, analysis of the hydrolysed extracts of peel of
the tomato fruit reveals that the presence of the pBBC50 construct
results in a significant increase in the levels of both quercetin
and kaempferol type flavonols in a proportion of transformed plants
(Table 2, FIG. 11). Hydrolysed extracts of pBBC50 transformed
plants display a range of peel quercetin concentrations with one
line expressing a 69 fold increase over the pSJ89 transformed
control lines (plant C20). The amount of kaempferol present in the
hydrolysed extracts of pBBC50 transformed plants correlates with
their quercetin concentrations--lines with higher concentrations of
quercetin seem also to possess higher concentrations of kaempferol
in hydrolysed extracts of their peel. Applicants wish to point out
that the variety in concentrations of quercetin, kaempferol and
naringenin as measured for the transformed plants is believed to
represent the common representation of a transgenic population.
Applicants however wish to stress that the currently obtained data
clearly show an increase in the level of quercetin and kaempferol
in the peel of transformed plants.
[0199] The pBBC50 transformed plants also display a range of peel
naringenin concentrations (note that these measurements were
carried out on hydrolysed extracts, therefore the naringenin was
originally derived from narichalcone as can be deduced from
analysis of non-hydrolysed extracts cf. Example 8.1). In general,
those transformants possessing increased concentrations of
flavonols in their peel also possess decreased concentrations of
naringenin when compared to the control fruit (Table 2). That the
decrease in naringenin concentrations correlates with an increase
in flavonol concentrations in the peel of the pBBC50 transformed
fruits strongly suggests that CHI no longer represents a
rate-limiting step in these plants.
[0200] The significant increase in fruit flavonol levels seen in
the pBBC50 transformed plants seems to reveal that, as suggested in
Example 2, CHI represents a major rate limiting step in the
production of flavonols in tomato peel which has now been overcome
by the heterologous expression of the petunia chi gene.
[0201] Using non-hydrolysed extracts, subsequently it is analysed
in which form the flavonoids accumulated in the tomato peel of
pBBC50 transformed plants. FIG. 9 shows an example of HPLC
chromatograms obtained with non-hydrolysed peel extracts from a
pBBC50 transformed tomato. As with the control FM6203 peel, rutin
(RT=16.0 min) represents the major quercetin glycoside which
accumulates in the peel of the pBBC50 transformed tomato. In
addition, significant amounts of isoquercitrin (quercetin
3-glucoside) (RT=17.4 min) also accumulate in the pBBC50 peel. The
peak at 19.7 min appeared to contain a mixture of two compounds:
the retention time and absorbance spectra of the major component
corresponded to that of kaempferol rutinoside, whilst that of the
minor component has an absorbance spectrum comparable to that of a
quercetin glycoside. The small peak at 20.3 min has an absorbance
spectrum comparable to kaempferol-3-rutinoside, but its higher RT
value indicates a yet unknown kaempferol-glycoside.
[0202] Quercetin and kaempferol aglycons, all clearly present in
the hydrolysed extracts, are not detectable in the non-hydrolysed
peel extracts of pBBC50 transformed tomatoes. Therefore, applicants
believe that these compounds are fully derived from hydrolysis of
their respective glycosides. No anthocyanins accumulate in the
transformed red tomatoes, as is obvious from the absence of any
peak in the chromatograms recorded at 520 nm (not shown).
1TABLE 1 Overview of PCR primers and adapters used. primer *
sequence (5' to 3' ) gus2 GCATCACGCAGTTCAACGCTG (SeQ ID 3) 300 35S
CGCAAGACCCTTCCTCTATATAAG (SeQ ID 4) nos ant CCGGCAACAGGATTCAATCTT
(SeQ ID 5) chi5 GGTCGTGCCATTGAGAAGTT (SeQ ID 6) nptII a
GAGGCGATTCGGCTATGACTG (SeQ ID 7) npt IIb ATCGGGAGCGGCGATACCGTA (SeQ
ID 8) F7 AATTGCACCGGTCG (SeQ ID 9) F8 GATCCGACCG (SeQ ID 10) F9
TAGCCATGGG (SeQ ID 11) F10 TCGACCCATGGCTAAT (SeQ ID 12) F12
CCCGTCGACTTTCCCCGATCGTTCAAACATTTGC (SeQ ID 13) F13
CCCGGATCCAAAAATGGTGACAGTCGAGG (SeQ ID 14) F14
CCGGTCGACGCAAATACATTCATGGCAAACG (SeQ ID 15) F15
GGCGGATCCAAAAATGTCTCCTCCAGTGTC (SeQ ID 16) F16
CCCGTCGACCTAAACTAGACTCCAATCACT (SeQ ID 17) F20
CGGGGATCCAGAGGGCCTAACTTCTGTATAGAC (SeQ ID 18) F21
CCCGTCGACTCGCGAAGATATAGCTAATCG (SeQ ID 19) F38
AATTGGGCGCGCCAAGCTTCCGAATTCTTAATTAAG (SeQ ID 20) F39
AGCTCTTAATTAAGAATTCGGAAGCTTGGCGCGCCC (SeQ ID 21) AB13
CCCATCGATGCGTCTAGTAACATAGATGAC (SeQ ID 22) * Adapters are made by
combining two primers, heating to 95.degree. C. for 5' and anneal
both primers by cooling slowly to room temperature.
[0203]
2TABLE II Flavonoid level in peel of transformed plants Line
[Quercetin] .mu.g/g [Kaempferol] .mu.g/g [Naringenin] .mu.g/g
number dry weight peel dry weight peel dry weight peel G4 115 15
280 G2 206 32 1095 G95 210 45 980 G3 253 27 523 G96 265 45 625 G97
345 55 1150 C69 115 10 105 C102 190 20 280 C41 208 28 789 C22 215
40 235 C54 230 40 410 C73 250 35 930 C6 300 15 375 C10 301 37 383
C64 320 35 470 C24 325 35 545 C53 350 65 345 C33 388 55 2107 C120
530 45 230 C51 690 217 43 C57 750 130 830 C56 1490 100 270 C48 2530
215 60 C9 3155 95 80 C34 3375 175 75 C118 4355 615 65 C25 4445 455
70 C88 4850 570 115 C86 4980 510 130 C39 5540 325 80 C65 6203 596
80 C72 6372 951 20 C49 6970 735 50 C40 7244 1147 40 C67 7405 900 80
C38 7795 735 50 C11 7995 805 135 C103 8705 840 105 C104 10055 870
110 C66 10885 1370 70 C87 13410 1250 95 C20 16520 2048 80 Legend:
HPLC analysis of flavonoid aglycons in hydrolysed peel extracts of
transgenic tomatoes transformed with either PSJ89 (G series) or
pBBC 50 (C series)
[0204]
Sequence CWU 1
1
23 1 241 PRT PETUNIA HYBRIDA 1 Met Ser Pro Pro Val Ser Val Thr Lys
Met Gln Val Glu Asn Tyr Ala 1 5 10 15 Phe Ala Pro Thr Val Asn Pro
Ala Gly Ser Thr Asn Thr Leu Phe Leu 20 25 30 Ala Gly Ala Gly His
Arg Gly Leu Glu Ile Glu Gly Lys Phe Val Lys 35 40 45 Phe Thr Ala
Ile Gly Val Tyr Leu Glu Glu Ser Ala Ile Pro Phe Leu 50 55 60 Ala
Glu Lys Trp Lys Gly Lys Thr Pro Gln Glu Leu Thr Asp Ser Val 65 70
75 80 Glu Phe Phe Arg Asp Val Val Thr Gly Pro Phe Glu Lys Phe Thr
Arg 85 90 95 Val Thr Met Ile Leu Pro Leu Thr Gly Lys Gln Tyr Ser
Glu Lys Val 100 105 110 Ala Glu Asn Cys Val Ala His Trp Lys Gly Ile
Gly Thr Tyr Thr Asp 115 120 125 Asp Glu Gly Arg Ala Ile Glu Lys Phe
Leu Asp Val Phe Arg Ser Glu 130 135 140 Thr Phe Pro Pro Gly Ala Ser
Ile Met Phe Thr Gln Ser Pro Leu Gly 145 150 155 160 Leu Leu Thr Ile
Ser Phe Ala Lys Asp Asp Ser Val Thr Gly Thr Ala 165 170 175 Asn Ala
Val Ile Glu Asn Lys Gln Leu Ser Glu Ala Val Leu Glu Ser 180 185 190
Ile Ile Gly Lys His Gly Val Ser Pro Ala Ala Lys Cys Ser Val Ala 195
200 205 Glu Arg Val Ala Glu Leu Leu Lys Lys Ser Tyr Ala Glu Glu Ala
Ser 210 215 220 Val Phe Gly Lys Pro Glu Thr Glu Lys Ser Thr Ile Pro
Val Ile Gly 225 230 235 240 Val 2 729 DNA PETUNIA HYBRIDA 2
atgtctcctc cagtgtccgt tactaaaatg caggttgaga attacgcttt cgcaccgacc
60 gtgaaccctg ctggttccac caataccttg ttccttgctg gtgctgggca
tagaggtctg 120 gagatagaag ggaagtttgt taagtttacg gcgataggtg
tgtatctaga agagagtgct 180 attccttttc tggccgaaaa atggaaaggc
aaaacccccc aggagttgac tgactcggtc 240 gagttcttta gggatgttgt
tacaggtcca tttgagaaat ttactcgagt tactatgatc 300 ttgcccttga
cgggcaagca gtactcggag aaggtggcgg agaattgtgt tgcgcattgg 360
aaggggatag gaacgtatac tgatgatgag ggtcgtgcca ttgagaagtt tctagatgtt
420 ttccggagtg aaacttttcc acctggtgct tccatcatgt ttactcaatc
acccctaggg 480 ttgttgacga ttagcttcgc taaagatgat tcagtaactg
gcactgcgaa tgctgttata 540 gagaacaagc agttgtctga agcagtgctg
gaatcaataa ttgggaagca tggagtttct 600 cctgcggcaa agtgtagtgt
cgctgaaaga gtagcggaac tgctcaaaaa gagctatgct 660 gaagaggcat
ctgtttttgg aaaaccggag accgagaaat ctactattcc agtgattgga 720
gtctagttt 729 3 21 DNA Artificial Sequence Description of
Artificial SequencePRIMER 3 gcatcacgca gttcaacgct g 21 4 24 DNA
Artificial Sequence Description of Artificial SequencePRIMER 4
cgcaagaccc ttcctctata taag 24 5 21 DNA Artificial Sequence
Description of Artificial SequencePRIMER 5 ccggcaacag gattcaatct t
21 6 20 DNA Artificial Sequence Description of Artificial
SequencePRIMER 6 ggtcgtgcca ttgagaagtt 20 7 21 DNA Artificial
Sequence Description of Artificial SequencePRIMER 7 gaggcgattc
ggctatgact g 21 8 21 DNA Artificial Sequence Description of
Artificial SequencePRIMER 8 atcgggagcg gcgataccgt a 21 9 14 DNA
Artificial Sequence Description of Artificial SequenceADAPTER 9
aattgcaccg gtcg 14 10 10 DNA Artificial Sequence Description of
Artificial SequenceADAPTER 10 gatccgaccg 10 11 10 DNA Artificial
Sequence Description of Artificial SequenceADAPTER 11 tagccatggg 10
12 16 DNA Artificial Sequence Description of Artificial
SequenceADAPTER 12 tcgacccatg gctaat 16 13 35 DNA Artificial
Sequence Description of Artificial SequencePRIMER 13 cccgtcgact
ttccccgatc gttcaaacat ttggc 35 14 29 DNA Artificial Sequence
Description of Artificial SequencePRIMER 14 cccggatcca aaaatggtga
cagtcgagg 29 15 31 DNA Artificial Sequence Description of
Artificial SequencePRIMER 15 ccggtcgacg caaatacatt catggcaaac g 31
16 30 DNA Artificial Sequence Description of Artificial
SequencePRIMER 16 ggcggatcca aaaatgtctc ctccagtgtc 30 17 30 DNA
Artificial Sequence Description of Artificial SequencePRIMER 17
cccgtcgacc taaactagac tccaatcact 30 18 33 DNA Artificial Sequence
Description of Artificial SequencePRIMER 18 cggggatcca gagggcctaa
cttctgtata gac 33 19 30 DNA Artificial Sequence Description of
Artificial SequencePRIMER 19 cccgtcgact cgcgaagata tagctaatcg 30 20
36 DNA Artificial Sequence Description of Artificial
SequenceADAPTER 20 aattgggcgc gccaagcttc cgaattctta attaag 36 21 36
DNA Artificial Sequence Description of Artificial SequenceADAPTER
21 agctcttaat taagaattcg gaagcttggc gcgccc 36 22 30 DNA Artificial
Sequence Description of Artificial SequencePRIMER 22 cccatcgatg
cgtctagtaa catagatgac 30 23 91 DNA Artificial Sequence Description
of Artificial SequenceMULTIPLE CLONING SITE 23 ggcgcgccaa
gcttgcatgc atcgatatgg tcgactctag aggatccccg ggtaccgagc 60
tcgaattcca gatctgcggc cgcttaatta a 91
* * * * *