U.S. patent application number 10/524652 was filed with the patent office on 2006-11-09 for method for the production of zeaxanthin and/or biosynthetic intermediates and/or subsequent products thereof.
This patent application is currently assigned to SunGene GmbH & Co. KGaA. Invention is credited to Ralf Flachmann, Karin Herbers, Martin Klebsattel, Irene Kunze, Matt Sauer, Christel Renate Schopfer.
Application Number | 20060253927 10/524652 |
Document ID | / |
Family ID | 31950810 |
Filed Date | 2006-11-09 |
United States Patent
Application |
20060253927 |
Kind Code |
A1 |
Schopfer; Christel Renate ;
et al. |
November 9, 2006 |
Method for the production of zeaxanthin and/or biosynthetic
intermediates and/or subsequent products thereof
Abstract
The present invention relates to a process for preparing
zeaxanthin and/or biosynthetic intermediates and/or secondary
products thereof by culturing genetically modified plants which,
compared to the wild type, have a reduced .epsilon.-cyclase
activity caused by double-stranded .epsilon.-cyclase ribonucleic
acid sequences, to the genetically modified plants and to the use
thereof as foodstuffs and feedstuffs and for producing carotenoid
extracts.
Inventors: |
Schopfer; Christel Renate;
(Quedlinburg, DE) ; Flachmann; Ralf; (Quedlinburg,
DE) ; Herbers; Karin; (Quedlinburg, DE) ;
Kunze; Irene; (Quedlinburg, DE) ; Sauer; Matt;
(Quedlinburg, DE) ; Klebsattel; Martin;
(Quedlinburg, DE) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Assignee: |
SunGene GmbH & Co. KGaA
Corrensstr. 3
Gatersleben
DE
06466
|
Family ID: |
31950810 |
Appl. No.: |
10/524652 |
Filed: |
August 18, 2003 |
PCT Filed: |
August 18, 2003 |
PCT NO: |
PCT/EP03/09105 |
371 Date: |
March 28, 2005 |
Current U.S.
Class: |
800/282 ;
435/419; 435/468; 435/67; 800/323 |
Current CPC
Class: |
Y02A 40/818 20180101;
C12N 15/825 20130101; C09B 61/00 20130101; A23L 33/155 20160801;
A23K 20/179 20160501; C12N 9/0069 20130101; C12N 15/8243 20130101;
C12N 9/0004 20130101; A23L 5/44 20160801; A23K 10/30 20160501; C12P
23/00 20130101; A23K 50/80 20160501; C07H 21/00 20130101; C12N
15/823 20130101; A23V 2002/00 20130101; A23V 2300/21 20130101; A23V
2002/00 20130101 |
Class at
Publication: |
800/282 ;
435/067; 435/419; 435/468; 800/323 |
International
Class: |
A01H 1/00 20060101
A01H001/00; A01H 5/00 20060101 A01H005/00; C12P 23/00 20060101
C12P023/00; C12N 15/82 20060101 C12N015/82; C12N 5/04 20060101
C12N005/04 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 20, 2002 |
DE |
102 38 980.2 |
Aug 20, 2002 |
DE |
102 38 978.0 |
Aug 20, 2002 |
DE |
102 38 979.9 |
Nov 13, 2002 |
DE |
102 53 112.9 |
Dec 16, 2002 |
DE |
102 58 971.2 |
Claims
1. A process for preparing zeaxanthin and/or biosynthetic
intermediates and/or secondary products thereof by culturing
genetically modified plants which, compared to the wild type, have
a reduced .epsilon.-cyclase activity caused by double-stranded
.epsilon.-cyclase ribonucleic acid sequences.
2. The process according to claim 1, wherein an RNA is introduced
into the plant, which has a double-stranded structural region and
comprises, in said region, a nucleic acid sequence which a) is
identical to at least part of the .epsilon.-cyclase transcript
intrinsic to said plant and/or b) is identical to at least part of
the .epsilon.-cyclase-promoter sequence intrinsic to said
plant.
3. The process according to claim 2, wherein the double-stranded
structural region comprises a nucleic acid sequence which is
identical to at least part of the .epsilon.-cyclase transcript
intrinsic to the plant and which comprises the 5' end or the 3' end
of the nucleic acids coding for a .epsilon.-cyclase and intrinsic
to the plant.
4. The process according to claim 2, wherein the double-stranded
structural region comprises in each case a sense-RNA strand
comprising at least one ribonucleotide sequence which is
essentially identical to at least part of the sense-RNA
.epsilon.-cyclase transcript, and comprises an antisense-RNA strand
which is essentially complementary to the sense-RNA strand.
5. The process according to claim 1, wherein genetically modified
plants are used whose flowers have the lowest rate of expression of
an .epsilon.-cyclase.
6. The process according to claim 5, wherein the double-stranded
.epsilon.-cyclase ribonucleic acid sequence is transcribed under
the control of a flower-specific promoter.
7. The process according to claim 1, wherein the plant used is a
plant selected from the families Ranunculaceae, Berberidaceae,
Papaveraceae, Cannabaceae, Rosaceae, Fabaceae, Linaceae, Vitaceae,
Brassicaceae, Cucurbitaceae, Primulaceae, Caryophyllaceae,
Amaranthaceae, Gentianaceae, Geraniaceae, Caprifoliaceae, Oleaceae,
Tropaeolaceae, Solanaceae, Scrophulariaceae, Asteraceae, Liliaceae,
Amaryllidaceae, Poaceae, Orchidaceae, Malvaceae, Illiaceae or
Lamiaceae.
8. The process according to claim 7, wherein the plant used is a
plant selected from the plant genera Marigold, Tagetes, Acacia,
Aconitum, Adonis, Arnica, Aquilegia, Aster, Astragalus, Bignonia,
Calendula, Caltha, Campanula, Canna, Centaurea, Cheiranthus,
Chrysanthemum, Citrus, Crepis, Crocus, Curcurbita, Cytisus,
Delonia, Delphinium, Dianthus, Dimorphotheca, Doronicum,
Eschscholtzia, Forsythia, Fremontia, Gazania, Gelsemium, Genista,
Gentiana, Geranium, Gerbera, Geum, Grevilla, Helenium, Helianthus,
Hepatica, Heracleum, Hibiscus, Heliopsis, Hypericum, Hypochoeris,
Impatiens, Iris, Jacaranda, Kerria, Laburnum, Lathyrus, Leontodon,
Lilium, Linum, Lotus, Lycopersicon, Lysimachia, Maratia, Medicago,
Mimulus, Narcissus, Oenothera, Osmanthus, Petunia, Photinia,
Physalis, Phyteuma, Potentilla, Pyracantha, Ranunculus,
Rhododendron, Rosa, Rudbeckia, Senecio, Silene, Silphium, Sinapsis,
Sorbus, Spartium, Tecoma, Torenia, Tragopogon, Trollius,
Tropaeolum, Tulipa, Tussilago, Ulex, Viola or Zinnia.
9. The process according to claim 8, wherein the plant used is a
plant selected from the plant species Marigold, Tagetes erecta or
Tagetes patula.
10. The process according to claim 1, wherein the genetically
modified plants are harvested after cultivation and subsequently
zeaxanthin and/or its biosynthetic intermediates and/or secondary
products are isolated from said plants.
11. The process according to claim 1, wherein the biosynthetic
intermediates and/or secondary products are selected from the group
consisting of lycopene, .beta.-carotene, astaxanthin,
canthaxanthin, echinenone, 3-hydroxyechinenone,
3'-hydroxyechinenone, adonirubin adonixanthin, antheraxanthin,
violaxanthin, neoxanthin, capsorubin, and capsanthin.
12. A ribonucleic acid construct, comprising RNA which has a
double-stranded structural region and comprises, in said region, a
nucleic acid sequence which a) is identical to at least part of the
.epsilon.-cyclase transcript intrinsic to said plant and/or b) is
identical to at least part of the .epsilon.-cyclase-promoter
sequence intrinsic to said plant.
13. A nucleic acid construct, transcribable into a) a sense-RNA
strand comprising at least one ribonucleotide sequence which is
essentially identical to at least part of the sense-RNA
.epsilon.-cyclase transcript, and b) an antisense-RNA strand which
is essentially, preferably fully, complementary to the RNA sense
strand under a).
14. A nucleic acid construct, comprising a) a sense-DNA strand
which is essentially identical to at least part of the promoter
region of an .epsilon.-cyclase gene, and b) an antisense-DNA strand
which is essentially, preferably fully, complementary to the DNA
sense strand under a).
15. The nucleic acid construct according to claim 13, wherein SEQ
ID NO: 4 describes the cDNA sequence deducible from the
.epsilon.-cyclase transcript.
16. The nucleic acid construct according to claim 14, wherein SEQ
ID NO: 13 describes the nucleic acid sequence of the promoter
region of the .epsilon.-cyclase gene.
17. The nucleic acid construct according to claim 13, wherein the
sense-RNA and antisense-RNA strands are covalently connected to one
another in the form of an inverted repeat.
18. The nucleic acid construct according to claim 12, wherein the
nucleic acid construct additionally comprises a promoter in a
functionally linked manner.
19. The nucleic acid construct according to claim 18, wherein a
flower-specific promoter is used.
20. A process for preparing genetically modified plants, wherein
expression cassettes comprising a nucleic acid construct according
to claim 12 are introduced into a parent plant.
21. A genetically modified plant which, compared to the wild type,
has a reduced .epsilon.-cyclase activity caused by double-stranded
.epsilon.-cyclase ribonucleic acid sequences.
22. The genetically modified plant according to claim 21, wherein
said genetically modified plant comprises an RNA which has a
double-stranded structural region and comprises, in said region, a
nucleic acid sequence which, a) is identical to at least part of
the .epsilon.-cyclase transcript intrinsic to said plant and/or b)
is identical to at least part of the .epsilon.-cyclase-promoter
sequence intrinsic to said plant.
23. The genetically modified plant according to claim 21, wherein
the plant is selected from the families Ranunculaceae,
Berberidaceae, Papaveraceae, Cannabaceae, Rosaceae, Fabaceae,
Linaceae, Vitaceae, Brassicaceae, Cucurbitaceae, Primulaceae,
Caryophyllaceae, Amaranthaceae, Gentianaceae, Geraniaceae,
Caprifoliaceae, Oleaceae, Tropaeolaceae, Solanaceae,
Scrophulariaceae, Asteraceae, Liliaceae, Amaryllidaceae, Poaceae,
Orchidaceae, Malvaceae, Illiaceae or Lamiaceae.
24. The genetically modified plant according to claim 23, wherein
the plant is selected from the plant genera Marigold, Tagetes,
Acacia, Aconitum, Adonis, Arnica, Aquilegia, Aster, Astragalus,
Bignonia, Calendula, Caltha, Campanula, Canna, Centaurea,
Cheiranthus, Chrysanthemum, Citrus, Crepis, Crocus, Curcurbita,
Cytisus, Delonia, Delphinium, Dianthus, Dimorphoteca, Doronicum,
Escholtzia, Forsythia, Fremontia, Gazania, Gelsemium, Genista,
Gentiana, Geranium, Gerbera, Geum, Grevilla, Helenium, Helianthus,
Hepatica, Heracleum, Hisbiscus, Heliopsis, Hyperricum, Hypochoeris,
Impatiens, Iris, Jacaranda, Kerria, Laburnum, Lathyrus, Leontodon,
Lilium, Linum, Lotus, Lycopersicon, Lysimachia, Maratia, Medicago,
Mimulus, Narcissus, Oenothera, Osmanthus, Petunia, Photinia,
Physalis, Phyteuma, Potentilla, Pyracantha, Ranunculus,
Rhododendron, Rosa, Rudbeckia, Senecio, Silene, Silphium, Sinapsis,
Sorbus, Spartium, Tecoma, Torenia, Tragopogon, Trollius,
Tropaeolum, Tulipa, Tussilago, Ulex, Viola or Zinnia.
25. The genetically modified plant according to claim 24, wherein
the plant is selected from the plant species Marigold, Tagetes
erecta or Tagetes patula.
26. A method of producing the genetically modified plants according
to claim 21 wherein the plants are used as ornamental plants or as
feedstuffs and foodstuffs.
27. A method of producing the genetically modified plants according
to claim 21 wherein the plants are used for preparing
carotenoid-containing extracts or for preparing feed and food
supplements.
Description
[0001] The present invention relates to a process for preparing
zeaxanthin and/or biosynthetic intermediates and/or secondary
products thereof by culturing genetically modified plants which,
compared to the wild type, have a reduced .epsilon.-cyclase
activity caused by double-stranded .epsilon.-cyclase ribonucleic
acid sequences, to the genetically modified plants and to the use
thereof as foodstuffs and feedstuffs and for producing carotenoid
extracts.
[0002] Carotenoids such as, for example, lycopene, lutein,
.beta.-carotene or zeaxanthin, are synthesized de novo in bacteria,
algae, fungi and plants. Ketocarotenoids, i.e. carotenoids,
containing at least one keto group, such as, for example,
astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone,
3'-hydroxyechinenone, adonirubin and adonixanthin are natural
antioxidants and pigments produced as secondary metabolites by some
algae and microorganisms.
[0003] Owing to their coloring properties, the carotenoids are used
as pigmenting agents and pigmenting aids. Zeaxanthin and lutein,
for example, are used in yolk pigmentation, .beta.-carotene serves
as an orange pigment in food and beverages, astaxanthin is used as
a pigmenting aid in livestock nutrition, especially in trout,
salmon and shrimp rearing.
[0004] In addition, the carotenoids such as, for example, lutein,
zeaxanthin, lycopene, .beta.-carotene and astaxanthin are used in
supplementing human and livestock nutrition for the therapy and
prevention of diseases, owing to their antioxidant properties.
[0005] An economical, biotechnological process for preparing
natural carotenoids is of great importance.
[0006] WO 00/32788 discloses influencing particular carotenoid
ratios in tagetes petals by a combination of overexpression of
carotenoid biosynthesis genes and antisense processes.
[0007] Although the process disclosed in WO 00/32788 provides
genetically modified plants which, compared to the wild type, have
an altered carotenoid content, said process has the disadvantage
that the content level of carotenoids of the ".beta.-carotenoid
pathway", such as, for example, .beta.-carotene or zeaxanthin, and
the purity of said carotenoids, and thus the ratio of carotenoids
of the ".beta.-carotenoid pathway", such as, for example
.beta.-carotene or zeaxanthin, to the carotenoids of the
".alpha.-carotenoid pathway", such as .alpha.-carotene or luteine,
for example, are not yet satisfactory.
[0008] It was therefore the object of the invention to provide an
alternative process for preparing zeaxanthin and/or biosynthetic
intermediates and/or secondary products thereof by cultivation of
plants and, respectively, provide further transgenic plants which
produce zeaxanthin and/or biosynthetic intermediates and/or
secondary products thereof and which have optimized properties such
as, for example, a higher content of zeaxanthin and/or biosynthetic
intermediates and/or secondary products thereof in comparison with
carotenoids of the ".alpha.-carotenoid pathway" and do not have the
reported disadvantage of the prior art.
[0009] Accordingly, a process for preparing zeaxanthin and/or
biosynthetic intermediates and/or secondary products thereof by
culturing genetically modified plants which, compared to the wild
type, have a reduced .epsilon.-cyclase activity caused by
double-stranded .epsilon.-cyclase ribonucleic acid sequences, was
found.
[0010] .epsilon.-Cyclase activity means the enzyme activity of an
.epsilon.-cyclase.
[0011] .epsilon.-Cyclase means a protein which has the enzymic
activity of converting a terminal, linear lycopene radical into an
.epsilon.-ionone ring.
[0012] .epsilon.-Cyclase, therefore, means in particular a protein
which has the enzymic activity of converting lycopene to
.delta.-carotene.
[0013] Consequently, .epsilon.-cyclase activity means the amount of
lycopene converted or the amount of .delta.-carotene produced in a
particular time by the .epsilon.-cyclase protein.
[0014] Thus, when an .epsilon.-cyclase activity is reduced compared
with the wild type, the amount of lycopene converted or the amount
of .delta.-carotene produced in a particular time is reduced by the
.epsilon.-cyclase protein, in comparison with the wild type.
[0015] A reduced .epsilon.-cyclase activity means, preferably, to
partially or essentially completely stop or block, based on
different cell-biological mechanisms, the functionality of an
.epsilon.-cyclase in a plant cell, plant or part, tissue, organ,
cells or seeds derived therefrom.
[0016] The .epsilon.-cyclase activity in plants may be reduced,
compared with the wild type, for example by reducing the amount of
.epsilon.-cyclase protein or the amount of .epsilon.-cyclase mRNA
in the plant. Consequently, a reduced .epsilon.-cyclase activity,
compared with the wild type, may be determined directly or via
determining the amount of .epsilon.-cyclase protein or the amount
of .epsilon.-cyclase mRNA of the plant of the invention, in
comparison with the wild type.
[0017] A reduction in the .epsilon.-cyclase activity comprises
reducing the amount of an .epsilon.-cyclase down to an essentially
complete absence of said .epsilon.-cyclase (i.e. lack of
detectability of .epsilon.-cyclase activity or lack of
immunological detectability of said .epsilon.-cyclase). The
.epsilon.-cyclase activity (or the amount of .epsilon.-cyclase
protein or the amount of .epsilon.-cyclase mRNA) in the plant,
particularly preferably in flowers, is reduced, in comparison with
the wild type, preferably by at least 5%, more preferably by at
least 20%, more preferably by at least 50%, more preferably by
100%. "Reduction" means in particular also the complete absence of
.epsilon.-cyclase activity (or of the .epsilon.-cyclase protein or
.epsilon.-cyclase mRNA).
[0018] Preference is given to determining the .epsilon.-cyclase
activity in genetically modified plants of the invention and in
wild-type or reference plants under the following conditions:
[0019] The .epsilon.-cyclase activity may be determined in vitro
according to Fraser and Sandmann (Biochem. Biophys. Res. Comm.
185(1) (1992) 9-15), when the buffer potassium phosphate (pH 7.6),
the substrate lycopene, paprika stromal protein, NADP+, NADPH and
ATP are added to a particular amount of plant extract.
[0020] Particular preference is given to determining the
.epsilon.-cyclase activity in genetically modified plants of the
invention and in wild-type or reference plants according to
Bouvier, d'Harlingue and Camara (Molecular Analysis of carotenoid
cyclase inhibition; Arch. Biochem. Biophys. 346(1) (1997)
53-64):
[0021] The in-vitro assay is carried out in a volume of 0.25 ml.
The reaction mixture comprises 50 mM potassium phosphate (pH 7.6),
different amounts of plant extract, 20 nM lycopene, 0.25 mg of
paprika chromoplastid stromal protein, 0.2 mM NADP+, 0.2 mM NADPH
and 1 mM ATP. NADP/NADPH and ATP are dissolved in 0.01 ml of
ethanol with 1 mg of Tween 80 immediately before addition to the
incubation medium. After a reaction time of 60 minutes at
30.degree. C., the reaction is stopped by adding
chloroform/methanol (2:1). The reaction products extracted into
chloroform are analyzed by means of HPLC.
[0022] An alternative assay with radioactive substrate is described
in Fraser and Sandmann (Biochem. Biophys. Res. Comm. 185(1) (1992)
9-15). Another analytical method is described in Beyer, Kroncke and
Nievelstein (On the mechanism of the lycopene isomerase/cyclase
reaction in Narcissus pseudonarcissus L. chromoplast; J. Biol.
Chem. 266(26) (1991) 17072-17078).
[0023] Depending on the context, the term "plant" may mean the
parent plant (wild type) or a genetically modified plant of the
invention or both.
[0024] Preferably, and in particular in cases in which the plant or
the wild type cannot be classified unambiguously, "wild type" for
the reduction in .epsilon.-cyclase activity and the increase in the
content of zeaxanthin and/or biosynthetic intermediates and/or
secondary products thereof means a reference plant.
[0025] Said reference plant is Tagetes erecta, Tagetes patula,
Tagetes lucida, Tagetes pringlei, Tagetes palmeri, Tagetes minuta
or Tagetes campanulata, particularly preferably Tagetes erecta.
[0026] In the process of the invention, the .epsilon.-cyclase
activity is reduced by introducing at least one double-stranded
.epsilon.-cyclase ribonucleic acid sequence, also referred to as
.epsilon.-cyclase dsRNA hereinbelow, or of an expression cassette
or expression cassettes ensuring expression thereof, into
plants.
[0027] Included are those processes in which said .epsilon.-cyclase
dsRNA is directed against an .epsilon.-cyclase gene (i.e. genomic
DNA sequences such as the promoter sequence) or an
.epsilon.-cyclase transcript (i.e. mRNA sequences).
[0028] Genetically modified plants which, in comparison with the
wild type, have a reduced .epsilon.-cyclase activity caused by
double-stranded .epsilon.-cyclase ribonucleic acid sequences mean,
according to the invention, that the .epsilon.-cyclase activity is
reduced by using double-stranded .epsilon.-cyclase ribonucleic acid
sequences. This process of gene regulation by means of
double-stranded RNA ("double-stranded RNA interference", also
referred to as dsRNA process) is known per se and described, for
example, in Matzke M A et al. (2000) Plant Mol Biol 43:401-415;
Fire A. et al (1998) Nature 391:806-811; WO 99/32619; WO 99/53050;
WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035 or WO 00/63364.
Express reference is made hereby to the processes and methods
described in the citations indicated.
[0029] According to the invention, "double-stranded ribonucleic
acid sequence" means one or more ribonucleic acid sequences which,
either theoretically, owing to complementary sequences, for example
according to the base pair rules by Watson and Crick, and/or
practically, for example owing to hybridization experiments, are
capable of forming double-stranded RNA structures in vitro and/or
in vivo.
[0030] The skilled worker appreciates that the formation of
double-stranded RNA structures represents a state of equilibrium.
The ratio of double-stranded molecules to corresponding dissociated
forms is preferably at least 1 to 10, preferably 1:1, particularly
preferably 5:1, most preferably 10:1.
[0031] A double-stranded .epsilon.-cyclase ribonucleic acid
sequence or else .epsilon.-cyclase dsRNA, preferably means an RNA
molecule which has a double-stranded structural region and
comprises, in said region, a nucleic acid sequence which [0032] a)
is identical to at least part of the .epsilon.-cyclase transcript
intrinsic to said plant and/or [0033] b) is identical to at least
part of the .epsilon.-cyclase-promoter sequence intrinsic to said
plant.
[0034] In the process of the invention, therefore, the
.epsilon.-cyclase activity is preferably reduced by introducing
into the plant an RNA which has a double-stranded structural region
and comprises, in said region, a nucleic acid sequence which [0035]
a) is identical to at least part of the .epsilon.-cyclase
transcript intrinsic to said plant and/or [0036] b) is identical to
at least part of the .epsilon.-cyclase-promoter sequence intrinsic
to said plant.
[0037] The term ".epsilon.-cyclase transcript" means the
transcribed part of an .epsilon.-cyclase gene, which comprises, in
addition to the .epsilon.-cyclase-encoding sequence, also noncoding
sequences such as, for example, also UTRs.
[0038] An RNA which "is identical to at least part of the
.epsilon.-cyclase promoter sequence intrinsic to said plant"
preferably means that the RNA sequence is identical to at least
part of the theoretical transcript of the .epsilon.-cyclase
promoter sequence, i.e. the corresponding RNA sequence.
[0039] "Part" of the .epsilon.-cyclase transcript intrinsic to the
plant or of the .epsilon.-cyclase promoter sequence intrinsic to
the plant means partial sequences which may range from a few base
pairs up to complete sequences of the transcript or of the promoter
sequence. The optimal length of said partial sequences may be
determined readily by the skilled worker by means of routine
experiments.
[0040] The length of the partial sequences is usually at least 10
bases and no more than 2 kb, preferably at least 25 bases and no
more than 1.5 kb, particularly preferably at least 50 bases and no
more than 600 bases, very particularly preferably at least 100
bases and no more than 500, most preferably at least 200 bases or
at least 300 bases and no more than 400 bases.
[0041] The partial sequences are preferably selected so as to
achieve a specificity as high as possible and not to reduce
activities of other enzymes, whose reduction is not desired. It is
therefore advantageous to choose for the .epsilon.-cyclase dsRNA
partial sequences parts of the .epsilon.-cyclase transcript and/or
partial sequences of the .epsilon.-cyclase promoter sequences which
do not occur in other sequences.
[0042] In a particularly preferred embodiment, therefore, the
.epsilon.-cyclase dsRNA comprises a sequence which is identical to
part of the .epsilon.-cyclase transcript intrinsic to the plant and
which comprises the 5' end or the 3' end of the nucleic acids
coding for a .epsilon.-cyclase and intrinsic to the plant.
Untranslated regions in the 5' or 3' of the transcript are
particularly suitable for preparing selective double-stranded
structures.
[0043] The invention further relates to double-stranded RNA
molecules (dsRNA molecules) which cause a decrease in an
.epsilon.-cyclase when introduced into a plant organism (or a cell,
tissue, organ or propagation material derived therefrom).
[0044] In this context, a double-stranded RNA molecule for reducing
expression of an .epsilon.-cyclase (.epsilon.-cyclase dsRNA)
preferably comprises [0045] a) a sense-RNA strand comprising at
least one ribonucleotide sequence which is essentially identical to
at least part of a sense-RNA .epsilon.-cyclase transcript, and
[0046] b) an antisense-RNA strand which is essentially, preferably
fully, complementary to the RNA sense strand under a).
[0047] With respect to the dsRNA molecules, .epsilon.-cyclase
nucleic acid sequence or the corresponding transcript preferably
means the sequence according to SEQ ID No. 4 or a part thereof.
[0048] "Essentially identical" means that the dsRNA sequence may
also have insertions, deletions and single point mutations, in
comparison with the .epsilon.-cyclase target sequence, and
nevertheless causes an efficient reduction in expression. The
homology between the sense strand of an inhibitory dsRNA and at
least part of the sense-RNA transcript of an .epsilon.-cyclase gene
or between the antisense strand the complementary strand of an
.epsilon.-cyclase gene is preferably at least 75%, particularly
preferably 80%, very particularly preferably at least 90%, most
preferably 100%.
[0049] A sequence identity of 100% between dsRNA and an
.epsilon.-cyclase gene transcript is not absolutely necessary in
order to cause an efficient reduction in .epsilon.-cyclase
expression. As a consequence, it is advantageous that the process
is tolerant to sequence deviations as may be present due to genetic
mutations, polymorphisms or evolutionary divergences. Thus it is
possible, for example, by using the dsRNA generated starting from
the .epsilon.-cyclase sequence of the first organism, to suppress
.epsilon.-cyclase expression in a second organism. For this
purpose, the dsRNA preferably comprises sequence regions of
.epsilon.-cyclase gene transcripts corresponding to conserved
regions. Said conserved regions may be readily derived from
sequence comparisons.
[0050] Alternatively, an "essential identical" dsRNA may also be
defined as a nucleic acid sequence which is capable of hybridizing
with part of an .epsilon.-cyclase gene transcript, for example in
400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50.degree. C. or
70.degree. C. for 12 to 16 h).
[0051] "Essentially complementary" means that the antisense-RNA
strand may also have insertions, deletions and single point
mutations, in comparison with the complement of the sense-RNA
strand. The homology between the antisense-RNA strand and the
complement of the sense-RNA strand is preferably at least 80%,
particularly preferably at least 90%, very particularly preferably
at least 95%, most preferably 100%.
[0052] Preference is given to transforming the plant with an
.epsilon.-cyclase dsRNA by using a nucleic acid construct which is
introduced into said plant and which is transcribed in said plant
into the .epsilon.-cyclase dsRNA.
[0053] The present invention therefore also relates to a nucleic
acid construct transcribable into [0054] a) a sense-RNA strand
comprising at least one ribonucleotide sequence which is
essentially identical to at least part of the sense-RNA
.epsilon.-cyclase transcript, and [0055] b) an antisense-RNA strand
which is essentially, preferably fully, complementary to the RNA
sense strand under a).
[0056] These nucleic acid constructs are also referred to as
expression cassettes or expression vectors hereinbelow.
[0057] In a further embodiment, the .epsilon.-cyclase dsRNA
comprises [0058] a) a sense-RNA strand comprising at least one
ribonucleotide sequence which is essentially identical to at least
part of the sense-RNA transcript of the promoter region of an
.epsilon.-cyclase gene, and [0059] b) an antisense-RNA strand which
is essentially, preferably fully, complementary to the RNA sense
strand under a).
[0060] Preferably, the promoter region of an .epsilon.-cyclase
means a sequence according to SEQ ID No. 13 or a part thereof.
[0061] The corresponding nucleic acid construct to be used
preferably for transformation of the plants comprises [0062] a) a
sense-DNA strand which is essentially identical to at least part of
the promoter region of an .epsilon.-cyclase gene, and [0063] b) an
antisense-DNA strand which is essentially, preferably fully,
complementary to the DNA sense strand under a).
[0064] The .epsilon.-cyclase dsRNA sequences and in particular
expression cassettes thereof for reducing the .epsilon.-cyclase
activity, in particular for Tagetes erecta, are prepared by using
particularly preferably the following partial sequences:
[0065] SEQ ID No. 6: sense fragment of the 5'-terminal region of
.epsilon.-cyclase
[0066] SEQ ID No. 7: antisense fragment of the 5'-terminal region
of .epsilon.-cyclase
[0067] SEQ ID No. 8: sense fragment of the 3'-terminal region of
.epsilon.-cyclase
[0068] SEQ ID No. 9: antisense fragment of the 3'-terminal region
of .epsilon.-cyclase
[0069] SEQ ID No. 13: sense fragment of the .epsilon.-cyclase
promoter
[0070] SEQ ID No. 14: antisense fragment of the .epsilon.-cyclase
promoter
[0071] The dsRNA may consist of one or more strands of
polyribonucleotides. In order to achieve the same purpose, it is of
course also possible to introduce a plurality of individual dsRNA
molecules which in each case comprise one of the ribonucleotide
sequence sections defined above into the cell or the organism.
[0072] The double-stranded dsRNA structure may be formed starting
from two complementary, separate RNA strands or, preferably,
starting from a single, self-complementary RNA strand. In the
latter case, the sense-RNA and antisense-RNA strands are preferably
covalently connected to one another in the form of an inverted
repeat.
[0073] As described, for example, in WO 99/53050, the dsRNA may
also comprise a hairpin structure through connection of sense and
antisense strands by a connecting sequence ("linker"; for example
an intron). The self-complementary dsRNA structures are preferred,
because they require merely expression of one RNA sequence and
comprise the complementary RNA strands always in an equimolar
ratio; The connecting sequence is preferably an intron (e.g. an
intron of the potato ST-LS1 gene; Vancanneyt G F et al. (1990) Mol
Gen Genet 220(2):245-250).
[0074] The nucleic acid sequence coding for a dsRNA may include
further elements such as, for example, transcription termination
signals or polyadenylation signals.
[0075] However, if the dsRNA is directed against the promoter
sequence of an .epsilon.-cyclase, it preferably does not comprise
any transcription termination signals or polyadenylation signals.
This enables the dsRNA to be retained in the nucleus of the cell
and prevents the dsRNA from spreading throughout the plant.
[0076] If the two strands of the dsRNA are to be assembled in a
cell or plant, this may take place in the following way, for
example: [0077] a) transformation of the cell or plant with a
vector which comprises both expression cassettes, [0078] b)
cotransformation of the cell or plant with two vectors, one
including the expression cassettes with the sense strand, the other
one including the expression cassettes with the antisense strand.
[0079] c) crossing of two individual plant lines, one comprising
the expression cassettes with the sense strand, the other one
comprising the expression cassettes with the antisense strand.
[0080] Formation of the RNA duplex may be initiated either outside
or inside the cell.
[0081] The dsRNA may be synthesized either in vivo or in vitro. For
this purpose, it is possible to put a DNA sequence coding for a
dsRNA into an expression cassette under the control of at least one
genetic control element (such as, for example, a promoter).
Polyadenylation is unnecessary, nor need any elements be present to
initiate translation. The expression cassette for the MP dsRNA is
preferably present on the transformation construct or the
transformation vector.
[0082] In a preferred embodiment, genetically modified plants are
used whose flowers have the lowest rate of expression of an
.epsilon.-cyclase.
[0083] This is preferably achieved by reducing the
.epsilon.-cyclase activity in a flower-specific, particularly
preferably petal-specific, manner.
[0084] In the particularly preferred embodiment described above,
this is achieved by the .epsilon.-cyclase dsRNA sequences being
transcribed under the control of a flower-specific promoter or,
even more preferably, under the control of a petal-specific
promoter.
[0085] Therefore, in a particularly preferred embodiment,
expression of the dsRNA is carried out starting from an expression
construct under the functional control of a flower-specific
promoter, particularly preferably under the control of the promoter
described by SEQ ID No. 10 or a functionally equivalent part
thereof.
[0086] The expression cassettes coding for the antisense strand
and/or the sense strand of an .epsilon.-cyclase dsRNA or for the
self-complementary strand of said dsRNA are for this purpose
preferably inserted into a transformation vector and introduced
into the plant cell using the processes described below. Stable
insertion into the genome is advantageous for the process of the
invention.
[0087] The dsRNA may be introduced in an amount which makes at
least one copy possible per cell. Larger amounts (e.g. at least 5,
10, 100, 500 or 1000 copies per cell) may, if appropriate, cause a
more efficient reduction.
[0088] The methods of dsRNA, cosuppression by means of sense RNA
and VIGS (virus-induced gene silencing) are also referred to as
post-transcriptional gene silencing (PTGS) or transcriptional gene
silencing (TGS).
[0089] In a preferred embodiment of the process of the invention,
the plant used is a plant selected from the families Ranunculaceae,
Berberidaceae, Papaveraceae, Cannabaceae, Rosaceae, Fabaceae,
Linaceae, Vitaceae, Brassicaceae, Cucurbitaceae, Primulaceae,
Caryophyllaceae, Amaranthaceae, Gentianaceae, Geraniaceae.
Caprifoliaceae, Oleaceae, Tropaeolaceae, Solanaceae,
Scrophulariaceae, Asteraceae, Liliaceae, Amaryllidaceae, Poaceae,
Orchidaceae, Malvaceae, Illiaceae or Lamiaceae.
[0090] Particular preference is given to using as plant a plant
selected from the plant genera Marigold, Tagetes, Acacia, Aconitum,
Adonis, Arnica, Aquilegia, Aster, Astragalus, Bignonia, Calendula,
Caltha, Campanula, Canna, Centaurea; Cheiranthus, Chrysanthemum,
Citrus, Crepis, Crocus, Curcurbita, Cytisus, Delonia, Delphinium,
Dianthus, Dimorphotheca, Doronicum, Eschscholtzia, Forsythia,
Fremontia, Gazania, Gelsemium, Genista, Gentiana, Geranium,
Gerbera, Geum, Grevilla, Helenium, Helianthus, Hepatica, Heracleum,
Hibiscus, Heliopsis, Hypericum, Hypochoeris, Impatiens, Iris,
Jacaranda, Kerria, Laburnum, Lathyrus, Leontodon, Lilium, Linum,
Lotus, Lycopersicon, Lysimachia, Maratia, Medicago, Mimulus,
Narcissus, Oenothera, Osmanthus, Petunia, Photinia, Physalis,
Phyteuma, Potentilla, Pyracantha, Ranunculus, Rhododendron, Rosa,
Rudbeckia, Senecio, Silene, Silphium, Sinapsis, Sorbus, Spartium,
Tecoma, Torenia, Tragopogon, Trollius, Tropaeolum, Tulipa,
Tussilago, Ulex, Viola or Zinnia.
[0091] Very particular preference is given to using as plant a
plant selected from the plant species Marigold, Tagetes erecta or
Tagetes patula.
[0092] In the process of the invention for preparing zeaxanthin
and/or biosynthetic intermediates and/or secondary products
thereof, the step of culturing the genetically modified plants,
also referred to as transgenic plants hereinbelow, is preferably
followed by harvesting the plants and isolating zeaxanthin and/or
biosynthetic intermediates and/or secondary products thereof from
the plant, particularly preferably from the petals of the
plants.
[0093] The transgenic plants are grown on nutrient media in a
manner known per se and harvested accordingly.
[0094] Zeaxanthin and/or biosynthetic intermediates and/or
secondary products thereof are isolated from the harvested petals
in a manner known per se, for example by drying and subsequent
extraction and, if appropriate, further chemical or physical
purification processes such as, for example, precipitation methods,
crystallography, thermal separation processes such as rectification
processes or physical separation processes such as chromatography,
for example. For example, zeaxanthin and/or biosynthetic
intermediates and/or secondary products thereof are preferably
isolated from the petals by organic solvents such as acetone,
hexane, ether or methyl tert-butyl ether.
[0095] Further processes for isolating ketocarotenoids, in
particular from petals, are described, for example, in Egger and
Kleinig (Phytochemistry (1967) 6, 437-440) and Egger
(Phytochemistry (1965) 4, 609-618).
[0096] The biosynthetic intermediates and/or secondary products of
zeaxanthin are preferably selected from the group consisting of
lycopene, .beta.-carotene, astaxanthin, canthaxanthin, echinenone,
3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin and
adonixanthin, violaxanthin, antheraxanthin, neoxanthin, capsorubin,
capsanthin.
[0097] Biosynthetic intermediates of zeaxanthin mean carotenoids
which, in the biosynthesis diagram, are on the biochemical pathway
to zeaxanthin. Said intermediates are preferably lycopene and/or
.beta.-carotene.
[0098] Biosynthetic secondary products of zeaxanthin mean
carotenoids which, in the biosynthesis diagram, derive from
zeaxanthin, such as, for example, antheraxanthin, violaxanthin and
neoxanthin. However, biosynthetic secondary products of zeaxanthin
also mean, in particular, those carotenoids which can be derived
from zeaxanthin and its intermediates biosynthetically by
introducing further enzymic activities into the plant, for
example.
[0099] For example, by bringing about a ketolase activity in
genetically modified plants, for example by introducing nucleic
acids encoding a ketolase into a parent plant, it is possible for
the genetically modified plant to be enabled to produce, starting
from carotenoids of the .beta.-carotenoid pathway, such as, for
example, .beta.-carotene or zeaxanthin, ketocarotenoids such as,
for example, astaxanthin, canthaxanthin, echinenone,
3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin or
adonixanthin.
[0100] Therefore, biosynthetic secondary products of zeaxanthin
also mean in particular astaxanthin, canthaxanthin, echinenone,
3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin or
adonixanthin.
[0101] A particularly preferred zeaxanthin secondary product is
astaxanthin.
[0102] The present invention also refers to a process for preparing
genetically modified plants, wherein expression cassettes
comprising a nucleic acid construct described above are introduced
into a parent plant.
[0103] The expression cassettes comprise regulatory signals, i.e.
regulating nucleic acid sequences which control the expression of
the coding sequence in the host cell. In a preferred embodiment, an
expression cassette comprises a promoter upstream, i.e. at the 5'
end of the coding sequence, and a polyadenylation signal
downstream, i.e. at the 3' end, and, where appropriate, further
regulatory elements which are operatively linked to the coding
sequence, located in between, for at least one of the genes
described above. Operative linkage means the sequential arrangement
of promoter, coding sequence, terminator and, where appropriate,
further regulatory elements in such a way that each of the
regulatory elements is able to carry out its function as intended
in the expression of the coding sequence.
[0104] The preferred nucleic acid constructs, expression cassettes
and vectors for plants and processes for producing transgenic
plants, and the transgenic plants themselves, are described by way
of example below.
[0105] The sequences which are preferred for the operative linkage,
but are not restricted thereto, are targeting sequences to ensure
the subcellular localization in the apoplast, in the vacuole, in
plastids, in the mitochondrion, in the endoplasmic reticulum (ER),
in the cell nucleus, in elaioplasts or other compartments and
translation enhancers such as the 5' leader sequence from tobacco
mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987),
8693-8711).
[0106] A suitable promoter for the expression cassette is in
principle any promoter able to control the expression of foreign
genes in plants.
[0107] "Constitutive" promoter means promoters which ensure
expression in numerous, preferably all, tissues over a relatively
wide period during development of the plant, preferably at all
times during development of the plant.
[0108] Preferably used is, in particular, a plant promoter or a
promoter derived from a plant virus. Particular preference is given
to the CaMV promoter of the 35S transcript of cauliflower mosaic
virus (Franck et al. (1980) Cell 21:285-294; Odell et al. (1985)
Nature 313:810-812; Shewmaker et al. (1985) Virology 140:281-288;
Gardner et al. (1986) Plant Mol Biol 6:221-228), or the 19S CaMV
promoter (U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et
al.-(1989) EMBO J 8:2195-2202).
[0109] A further suitable constitutive promoter is the pds promoter
(Pecker et al. (1992) Proc. Natl. Acad. Sci USA 89: 4962-4966) or
the rubisco small subunit (SSU) promoter (U.S. Pat. No. 4,962,028),
the legumin B promoter (GenBank Acc. No. X03677), the agrobacterium
nopaline synthase promoter, the TR dual promoter, the agrobacterium
OCS (octopine synthase) promoter, the ubiquitin promoter (Holtorf S
et al. (1995) Plant Mol Biol 29:637-649), the ubiquitin 1 promoter
(Christensen et al. (1992) Plant Mol Biol 18:675-689; Bruce et al.
(1989) Proc Natl Acad Sci USA 86:9692-9696), the Smas promoter, the
cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),
the promoters of the vacuolar ATPase subunits or the promoter of a
proline-rich protein from wheat (WO 91/13991), the Pnit promoter
(Y07648.L, Hillebrand et al. (1998), Plant. Mol. Biol. 36, 89-99,
Hillebrand et al. (1996), Gene, 170, 197-200) and further promoters
of genes whose constitutive expression in plants is known to the
skilled worker.
[0110] The expression cassettes may also comprise a chemically
inducible promoter (review article: Gatz et al. (1997) Annu Rev
Plant Physiol Plant Mol Biol 48:89-108) by which expression of the
ketolase gene in the plant can be controlled at a particular time.
Promoters of this type, such as, for example, the PRP1 promoter
(Ward et al. (1993) Plant Mol Biol 22:361-366), a salicylic
acid-inducible promoter (WO 95/19443), a
benzenesulfonamide-inducible promoter (EP 0 388 186), a
tetracycline-inducible promoter (Gatz et al. (1992) Plant J
2:397-404), an abscisic acid-inducible promoter (EP 0 335 528) or
an ethanol- or cyclohexanone-inducible promoter (WO 93/21334), can
likewise be used.
[0111] Promoters which are further preferred are those induced by
biotic or abiotic stress, such as, for example, the
pathogen-inducible promoter of the PRP1 gene (Ward et al. (1993)
Plant Mol Biol 22:361-366), the heat-inducible tomato hsp70 or
hsp80 promoter (U.S. Pat. No. 5,187,267), the cold-inducible potato
alpha-amylase promoter (WO 96/12814), the light-inducible PPDK
promoter or the wound-induced pinII promoter (EP375091).
[0112] Pathogen-inducible promoters include those of genes which
are induced as a result of pathogen attack, such as, for example,
genes of PR proteins, SAR proteins, .beta.-1,3-glucanase, chitinase
etc. (for example Redolfi et al. (1983) Neth J Plant Pathol
89:245-254; Uknes, et al. (1992) The Plant Cell 4:645-656; Van Loon
(1985) Plant Mol Viral 4:111-116; Marineau et al. (1987) Plant Mol
Biol 9:335-342; Matton et al. (1987) Molecular Plant-Microbe
Interactions 2:325-342; Somssich et al. (1986) Proc Natl Acad Sci
USA 83:2427-2430; Somssich et al. (1988) Mol Gen Genetics 2:93-98;
Chen et al. (1996) Plant J 10:955-966; Zhang and Sing (1994) Proc
Natl Acad Sci USA 91:2507-2511; Warner, et al. (1993) Plant J
3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968 (1989).
[0113] Also included as wound-inducible promoters such as that of
the pinII gene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan et
al. (1996) Nat Biotech 14:494-498), of the wun1 and wun2 genes
(U.S. Pat. No. 5,428,148), of the win1 and win2 genes (Stanford et
al. (1989) Mol Gen Genet 215:200-208), of systemin (McGurl et al.
(1992) Science 255:1570-1573), of the WIP1 gene (Rohmeier et al.
(1993) Plant Mol Biol 22:783-792; Ekelkamp et al. (1993) FEBS
Letters 323:73-76), of the MPI gene (Corderok et al. (1994) The
Plant J 6(2):141-150) and the like.
[0114] Examples of further suitable promoters are fruit
ripening-specific promoters such as, for example, the tomato fruit
ripening-specific promoter (WO 94/21794, EP 409 625).
Development-dependent promoters include some of the tissue-specific
promoters because the formation of some tissues naturally depends
on development.
[0115] Further particularly preferred promoters are those which
ensure expression in tissues or parts of plants in which, for
example, the biosynthesis of ketocarotenoids or precursors thereof
takes place. Preferred examples are promoters having specificities
for anthers, ovaries, petals, sepals, flowers, leaves, stalks and
roots and combinations thereof.
[0116] Examples of promoters specific for tubers, storage roots or
roots are the patatin promoter class I (B33) or the potato
cathepsin D inhibitor promoter.
[0117] Examples of leaf-specific promoters are the promoter of the
potato cytosolic FBPase (WO 97/05900), the rubisco
(ribulose-1,5-bisphosphate carboxylase) SSU promoter (small
subunit) or the potato ST-LSI promoter (Stockhaus et al. (1989)
EMBO J 8:2445-2451).
[0118] Examples of flower-specific promoters are the phytoene
synthase promoter (WO 92/16635), the promoter of the P-rr gene (WO
98/22593) or, particularly preferably, the modified version, AP3P,
of the flower-specific Arabidopsis thaliana AP3 promoter (AL132971:
nucleotide region 9298-10200; Hill et al. (1998) Development 125:
1711-1721).
[0119] Examples of anther-specific promoters are the 5126 promoter
(U.S. Pat. No. 5,689,049, U.S. Pat. No. 5,689,051), the glob-1
promoter or the g-zein promoter.
[0120] Further promoters suitable for expression in plants are
described in Rogers et al. (1987) Meth in Enzymol 153:253-277;
Schardl et al. (1987) Gene 61:1-11 and Berger et al. (1989) Proc
Natl Acad Sci USA 86:8402-8406.
[0121] All of the promoters described in the present application
usually enable the double-stranded .epsilon.-cyclase ribonucleic
acid sequences to be expressed in the plants of the invention.
[0122] Particularly preferred in the process of the invention and
in the genetically modified plants of the invention are
flower-specific promoters.
[0123] An expression cassette is preferably produced by fusing a
suitable promoter to a nucleic acid sequence, described above,
transcribing a double-stranded .epsilon.-cyclase ribonucleic acid
sequence, and preferably to a nucleic acid which is inserted
between promoter and nucleic acid sequence and which codes for a
plastid-specific transit peptide, and to a polyadenylation signal
by conventional recombination and cloning techniques as described,
for example in T. Maniatis, E. F. Fritsch and J. Sambrook,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J. Silhavy,
M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
Greene Publishing Assoc. and Wiley-Interscience (1987).
[0124] The preferably inserted nucleic acids encoding a plastid
transit peptide ensure localization in plastids and, in particular,
in chromoplasts.
[0125] The particularly preferred transit peptide is derived from
the Nicotiana tabacum plastid transketolase or another transit
peptide (e.g. the transit peptide of the small subunit of rubisco
(rbcS) or of the ferredoxin NADP oxidoreductase, as well as the
isopentenyl-pyrophosphate isomerase 2) or its functional
equivalent.
[0126] Particular preference is given to nucleic acid sequences of
three cassettes of the plastid transit peptide of the tobacco
plastid transketolase in three reading frames as KpnI/BamHI
fragments with an ATG codon in the NcoI cleavage site:
TABLE-US-00001 pTP09
KpnI_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATC
CTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTC
CCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCA
CCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGG
TCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGA
GACTGCGGGATCC_BamHI pTP10
KpnI_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATC
CTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTC
CCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCA
CCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGG
TCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGA
GACTGCGCTGGATCC_BamHI pTP11
KpnI_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATC
CTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTC
CCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCA
CCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGG
TCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGA
GACTGCGGGGATCC_BamHI
[0127] Further examples of a plastid transit peptide are the
transit peptide of the Arabidopsis thaliana plastid
isopentenyl-pyrophosphate isomerase 2 (IPP-2) and the transit
peptide of the small subunit of ribulose-bisphosphate carboxylase
(rbcS) from pea (Guerineau, F, Woolston, S, Brooks, L, Mullineaux,
P (1988) An expression cassette for targeting foreign proteins into
the chloroplasts. Nucl. Acids Res. 16: 11380).
[0128] The nucleic acids of the invention can be prepared
synthetically or obtained naturally or comprise a mixture of
synthetic and natural nucleic acid constituents, and consist of
various heterologous gene sections from different organisms.
[0129] Examples of a terminator are the 35S terminator (Guerineau
et al. (1988) Nucl Acids Res. 16: 11380), the nos terminator
(Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman H M.
Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl
Genet. 1982;1(6):561-73) or the ocs terminator (Gielen, J, de
Beuckeleer, M, Seurinck, J, Debroek, H, de Greve, H, Lemmers, M,
van Montagu, M, Schell, J (1984) The complete sequence of the
TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5. EMBO J. 3:
835-846).
[0130] It is furthermore possible to employ manipulations which
provide appropriate restriction cleavage sites or delete the
redundant DNA or restriction cleavage sites. It is possible in
relation to insertions, deletions or substitutions, such as, for
example, transitions and transversions, to use in vitro
mutagenesis, primer repair, restriction or ligation.
[0131] It is possible with suitable manipulations, such as, for
example, restriction, chewing back or filling in of overhangs for
blunt ends, to provide complementary ends of the fragments for
ligation.
[0132] Preferred polyadenylation signals are plant polyadenylation
signals, preferably those which essentially correspond to T-DNA
polyadenylation signals from Agrobacterium tumefaciens, especially
of gene 3 of the T-DNA (octopine synthase) of the Ti plasmid
pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 ff) or functional
equivalents.
[0133] The transfer of nucleic acid sequences into the genome of a
plant is referred to as transformation.
[0134] It is possible to use for this purpose methods known per se
for the transformation and regeneration of plants from plant
tissues or plant cells for transient or stable transformation.
[0135] Suitable methods for transforming plants are protoplast
transformation by polyethylene glycol-induced DNA uptake, the
biolistic method using the gene gun--called the particle
bombardment method, electroporation, incubation of dry embryos in
DNA-containing solution, microinjection and gene transfer mediated
by Agrobacterium described above. Said processes are described, for
example, in B. Jenes et al., Techniques for Gene Transfer, in:
Transgenic Plants, Vol. 1, Engineering and Utilization, edited by
S. D. Kung and R. Wu, Academic Press (1993), 128-143 and in
Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991),
205-225.
[0136] The construct to be expressed is preferably cloned into a
vector which is suitable for transforming Agrobacterium
tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12
(1984), 8711) or particularly preferably, pSUN2, pSUN3, pSUN4 or
pSUN5 (WO 02/00900).
[0137] Agrobacteria transformed with an expression cassette can be
used in a known manner for transforming plants, e.g. bathing
wounded leaves or pieces of leaf in a solution of agrobacteria and
subsequently cultivating in suitable media.
[0138] For the preferred production of genetically modified plants,
also referred to as transgenic plants hereinafter, the fused
expression cassette is cloned into a vector, for example pBin19 or,
in particular, pSUN5, which is suitable for transforming
Agrobacterium tumefaciens.
[0139] Agrobacteria transformed with such a vector can then be used
in a known manner for transforming plants, in particular crop
plants, for example by bathing wounded leaves or pieces of leaf in
a solution of agrobacteria and subsequently cultivating in suitable
media.
[0140] The transformation of plants by agrobacteria is disclosed
inter alia in F. F. White, Vectors for Gene Transfer in Higher
Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization,
edited by S. D. Kung and R. Wu, Academic Press, 1993, pages 15-38.
Transgenic plants which comprise a gene, integrated into the
expression cassette for expression of a nucleic acid encoding a
ketolase can be regenerated in a known manner from the transformed
cells of the wounded leaves or pieces of leaf.
[0141] To transform a host cell with a double-stranded
.epsilon.-cyclase ribonucleic acid sequence, an expression cassette
is incorporated and inserted into a recombinant vector whose vector
DNA comprises additional functional regulatory signals, for example
sequences for replication or integration. Suitable vectors are
described inter alia in "Methods in Plant Molecular Biology and
Biotechnology" (CRC Press), chapters 6/7, pages 71-119 (1993).
[0142] Using the recombination and cloning techniques quoted above,
the expression cassettes can be cloned into suitable vectors which
make replication thereof possible for example in E. coli. Suitable
cloning vectors are, inter alia, pJIT117 (Guerineau et al. (1988)
Nucl. Acids Res. 16:11380), pBR322, pUC series, M13 mp series and
pACYC184. Binary vectors which are able to replicate both in E.
coli and in agrobacteria are particularly suitable.
[0143] The invention further relates to the genetically modified
plants which, in comparison with the wild type, have a reduced
.epsilon.-cyclase activity caused by double-stranded
.epsilon.-cyclase ribonucleic acid sequences.
[0144] As mentioned above, the genetically modified plant
comprises, in a particular embodiment, an RNA which has a
double-stranded structural region and comprises, in said region, a
nucleic acid sequence which [0145] a) is identical to at least part
of the .epsilon.-cyclase transcript intrinsic to said plant and/or
[0146] b) is identical to at least part of the
.epsilon.-cyclase-promoter sequence intrinsic to said plant.
[0147] Preference is given to genetically modified plants selected
from the families Ranunculaceae, Berberidaceae, Papaveraceae,
Cannabaceae, Rosaceae, Fabaceae, Linaceae, Vitaceae, Brassicaeae,
Cucurbitaceae, Primulaceae, Caryophyllaceae, Amaranthaceae,
Gentianaceae, Geraniaceae, Caprifoliaceae, Oleaceae, Tropaeolaceae,
Solanaceae, Scrophulariaceae, Asteraceae, Liliaceae,
Amaryllidaceae, Poaceae, Orchidaceae, Malvaceae, Illiaceae or
Lamiaceae.
[0148] Particular preference is given to genetically modified
plants selected from the plant genera Marigold, Tagetes, Acacia,
Aconitum, Adonis, Arnica, Aquilegia, Aster, Astragalus, Bignonia,
Calendula, Caltha, Campanula, Canna, Centaurea, Cheiranthus,
Chrysanthemum, Citrus, Crepis, Crocus, Curcurbita, Cytisus,
Delonia, Delphinium, Dianthus, Dimorphoteca, Doronicum, Escholtzia,
Forsythia, Fremontia, Gazania, Gelsemium, Genista, Gentiana,
Geranium, Gerbera, Geum, Grevilla, Helenium, Helianthus, Hepatica,
Heracleum, Hisbiscus, Heliopsis, Hyperricum, Hypochoeris,
Impatiens, Iris, Jacaranda, Kerria, Laburnum, Lathyrus, Leontodon,
Lilium, Linum, Lotus, Lycopersicon, Lysimachia, Maratia, Medicago,
Mimulus, Narcissus, Oenothera, Osmanthus, Petunia, Photinia,
Physalis, Phyteuma, Potentilla, Pyracantha, Ranunculus,
Rhododendron, Rosa, Rudbeckia, Senecio, Silene, Silphium, Sinapsis,
Sorbus, Spartium, Tecoma, Torenia, Tragopogon, Trollius,
Tropaeolum, Tulipa, Tussilago, Ulex, Viola or Zinnia.
[0149] Very particular preference is given to genetically modified
plants selected from the plant genera Marigold, Tagetes erecta or
Tagetes patula.
[0150] The present invention furthermore relates to the transgenic
plants, to the propagation material thereof and also to the plant
cells, tissues or parts thereof, in particular to the petals
thereof.
[0151] The genetically modified plants may, as described above, be
used for preparing zeaxanthin and/or biosynthetic intermediates
and/or secondary products thereof, in particular for preparing
lycopene, .epsilon.-carotene, astaxanthin, canthaxanthin,
echinenone 3-hydroxyechinenone, 3'-hydroxyechinenone, adonirubin or
adonixanthin, and in particular for preparing astaxanthin.
[0152] The genetically modified plants of the invention have, in
comparison with the wild type, an increased content of at least one
carotenoid selected from the group consisting of zeaxanthin and/or
biosynthetic intermediates and/or secondary products thereof.
[0153] In this case, an increased content also means a
ketocarotenoid, or astaxanthin, content which has been brought
about.
[0154] Genetically modified plants of the invention, which have an
increased content of zeaxanthin and/or biosynthetic intermediates
and/or secondary products thereof and which are consumable by
humans and animals, may also be used, for example, directly or
after processing known per se as foodstuffs or feedstuffs or as
feed supplements-and-food supplements. The genetically modified
plants may also be used for preparing carotenoid-containing
extracts of said plants and/or for preparing feed supplements and
food supplements.
[0155] The genetically modified plants may also be used as
ornamental plants in the field of horticulture.
[0156] The invention will now be illustrated by the following
examples, without being limited thereto:
General Experimental Conditions:
Sequence Analysis of Recombinant DNA
[0157] Recombinant DNA molecules were sequenced using a laser
fluorescence DNA sequencer from Licor (sold by MWG Biotech,
Ebersbach, Germany), according to the method of Sanger (Sanger et
al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).
EXAMPLE 1
Preparation of a Cloning Vector for Preparing Double-Stranded
.epsilon.-Cyclase Ribonucleic Acid Sequence Expression Cassettes
for Flower-Specific Expression of Epsilon-Cyclase dsRNAs in Tagetes
erecta
[0158] Inverted repeat transcripts consisting of epsilon-cyclase
fragments were expressed in Tagetes erecta under the control of a
modified version, AP3P, of the flower-specific Arabidopsis thaliana
promoter AP3 (AL132971: Nucleotide region 9298-10200; Hill et al.
(1998) Development 125: 1711-1721).
[0159] The inverted repeat transcript includes in each case a
fragment in the correct orientation (sense fragment) and a
sequence-identical fragment in the opposite orientation (antisense
fragment) which are connected to one another by a functional
intron, the PIV2 intron of the potato ST-LH1 gene (Vancanneyt G. et
al. (1990) Mol Gen Genet 220: 245-50).
[0160] The cDNA coding for the Arabidopsis thaliana AP3 promoter
(-902 to +15) was prepared by means of PCR using genomic DNA
(isolated from Arabidopsis thaliana by a standard method) and the
primers PR7 (SEQ ID No. 15) and PR10 (SEQ ID No. 18).
[0161] The PCR conditions were as follows:
[0162] The PCR for amplification of the DNA encoding the AP3
promoter fragment (-902 to +15) was carried out in a 50 .mu.l
reaction mixture containing: TABLE-US-00002 1 .mu.l A. thaliana
genomic DNA (diluted 1:100, prepared as described above) 0.25 mM
dNTPs 0.2 mM PR7 (SEQ ID No. 15) 0.2 mM PR10 (SEQ ID No. 18) 5
.mu.l 10.times. PCR buffer (Stratagene) 0.25 .mu.l Pfu polymerase
(Stratagene) 28.8 .mu.l distilled water
[0163] The PCR was carried out under the following cycle
conditions: TABLE-US-00003 1.times. 94.degree. C. 2 minutes
35.times. 94.degree. C. 1 minute 50.degree. C. 1 minute 72.degree.
C. 1 minute 1.times. 72.degree. C. 10 minutes
[0164] The 922 bp amplicon was cloned into the PCR cloning vector
pCR 2.1 (Invitrogen) by using standard methods, resulting in the
plasmid pTAP3. Sequencing of the pTAP3 clone confirmed a sequence
which differs from the published AP3 sequence (AL132971, nucleotide
region 9298-10200) merely in an insertion (a G in position 9765 of
the AL132971 sequence) and a base substitution (G for A in position
9726 of the AL132971 sequence) (position 33: T for G, position 55:
T for G). These nucleotide differences were reproduced in an
independent amplification experiment and thus represent the
nucleotide sequence in the Arabidopsis thaliana plant used.
[0165] The modified version, AP3P, was prepared by means of
recombinant PCR using the pTAP3 plasmid. The region 10200-9771 was
amplified using the primers PR7 (SEQ ID No. 15) and PR9 (SEQ ID No.
17) (amplicon A7/9), the region 9526-9285 was amplified using PR8
(SEQ ID No. 16) and PR10 (SEQ ID No. 18) (amplicon A8/10).
The PCR conditions were as follows:
[0166] The PCR reactions for amplification of the DNA fragments
coding for the regions 10200-9771 and 9526-9285 of the AP3 promoter
were carried out in 50 .mu.l reaction mixtures containing:
TABLE-US-00004 100 ng AP3 amplicon (described above) 0.25 mM dNTPs
0.2 mM PR7 (SEQ ID No. 15) or PR8 (SEQ ID No. 16) 0.2 mM PR9 (SEQ
ID No. 17) or PR10 (SEQ ID No. 18) 5 .mu.l 10.times. PCR buffer
(Stratagene) 0.25 .mu.l Pfu Taq polymerase (Stratagene) 28.8 .mu.l
distilled water
[0167] The PCR was carried out under the following cycle
conditions: TABLE-US-00005 1.times. 94.degree. C. 2 minutes
35.times. 94.degree. C. 1 minute 50.degree. C. 2 minutes 72.degree.
C. 3 minutes 1.times. 72.degree. C. 10 minutes
[0168] The recombinant PCR includes annealing of the amplicons A7/9
and A8/10 which overlap over a sequence of 25 nucleotides,
completion to give a double strand and subsequent amplification.
This results in a modified version of the AP3 promoter, AP3P, in
which positions 9670-9526 have been deleted. The two amplicons A7/9
and A8/10 were denatured (5 min at 95.degree. C.) and annealed
(slowly cooling to 40.degree. C. at room temperature) in a 17.6
.mu.l reaction mixture containing: TABLE-US-00006 0.5 .mu.g A7/9
0.25 .mu.g A8/10
[0169] The 3' ends were filled in (30 min at 30.degree. C.) in a 20
.mu.l reaction mixture containing: TABLE-US-00007 17.6 .mu.l A7/9
and A8/10 annealing reactions (prepared as described above) 50
.mu.M dNTPs 2 .mu.l 1.times. Klenow buffer 2 U Klenow enzyme
[0170] The nucleic acid coding for the modified promoter version,
AP3P, was amplified by means of PCR using a sense-specific primer
(PR7 SEQ ID No. 15) and an antisense-specific primer (PR10 SEQ ID
No. 18).
[0171] The PCR conditions were as follows:
[0172] The PCR for amplification of the AP3P fragment was carried
out in a 50 .mu.l reaction mixture containing: TABLE-US-00008 1
.mu.l annealing reaction (prepared as described above) 0.25 mM
dNTPs 0.2 mM PR7 (SEQ ID No. 15) 0.2 mM PR10 (SEQ ID No. 18) 5
.mu.l 10.times. PCR buffer (Stratagene) 0.25 .mu.l Pfu Taq
polymerase (Stratagene) 28.8 .mu.l distilled water
[0173] The PCR was carried out under the following cycle
conditions: TABLE-US-00009 1.times. 94.degree. C. 2 minutes
35.times. 94.degree. C. 1 minute 50.degree. C. 1 minute 72.degree.
C. 1 minute 1.times. 72.degree. C. 10 minutes
[0174] The PCR amplification with PR7, SEQ ID No. 15 and PR10 SEQ
ID No. 18 resulted in a 778 bp fragment coding for the modified
promoter version, AP3P. The amplicon was cloned into the cloning
vector pCR2.1 (Invitrogen). Sequencing reactions using the primers
T7 and M13 confirmed a sequence identical to the sequence AL132971,
region 10200-9298, with the internal region 9285-9526 having been
deleted. This clone was therefore used for cloning into the
expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res.
16: 11380).
[0175] The cloning was carried out by isolating the 771 bp
SacI-HindIII fragment from pTAP3P and ligation into the
SacI-HindIII-cut pJIT117 vector. The clone which contains the
promoter AP3P instead of the original promoter d35S is denoted
pJAP3P.
[0176] A DNA fragment containing the PIV2 intron of the ST-LS1 gene
was prepared by means of PCR using p35SGUS INT plasmid DNA
(Vancanneyt G. et al. (1990) Mol Gen Genet 220: 245-50) and the
primers PR40 (Seq ID No. 20) and PR41 (Seq ID No. 21).
[0177] The PCR conditions were as follows:
[0178] The PCR for amplification of the PIV2 intron sequence of the
ST-LS1 gene was carried out in a 50 .mu.l reaction mixture
containing: TABLE-US-00010 1 .mu.l p35SGUS INT 0.25 mM dNTPs 0.2
.mu.M PR40 (SEQ ID No. 20) 0.2 .mu.M PR41 (SEQ ID No. 21) 5 .mu.l
10.times. PCR buffer (TAKARA) 0.25 .mu.l R Taq polymerase (TAKARA)
28.8 .mu.l distilled water
[0179] The PCR was carried out under the following cycle
conditions: TABLE-US-00011 1.times. 94.degree. C. 2 minutes
35.times. 94.degree. C. 1 minute 53.degree. C. 1 minute 72.degree.
C. 1 minute 1.times. 72.degree. C. 10 minutes
[0180] PCR amplification using PR40 and PR41 resulted in a 206 bp
fragment. Using standard methods, the amplicon was cloned into the
PCR cloning vector pBluntII (Invitrogen), resulting in the clone
pBluntII-40-41. Sequencing reactions of this clone, using the
primer SP6, confirmed a sequence which is identical to the
corresponding sequence of the p35SGUS INT vector.
[0181] This clone was therefore for cloning into the pJAP3P vector
(described above).
[0182] The cloning was carried out by isolating the 206 bp
SalI-BamHI fragment from pBluntII-40-41 and ligation with the
SalI-BamHI-cut pJAP3P vector. The clone which contains the PIV2
intron of the ST-LS1 gene in the correct orientation, downstream of
the 3' end of the rbcs transit peptide, is denoted pJAI1 and is
suitable for preparation of expression cassettes for
flower-specific expression of inverted repeat transcripts.
[0183] In FIG. 2, the AP3P fragment includes the modified AP3P
promoter (771 bp), the rbcs fragment includes the pea rbcS transit
peptide (204 bp), the intron fragment includes the PIV2 intron of
the potato ST-LS1 gene, and the term fragment (761 bp) includes the
CaMV polyadenylation signal.
EXAMPLE 2
Preparation of Inverted Repeat Expression Cassettes for
Flower-Specific Expression of Epsilon-Cyclase dsRNAs in Tagetes
erecta (Directed Against the 5' Region of Epsilon-Cyclase cDNA)
[0184] The nucleic acid containing the 5'-terminal 435 bp region of
epsilon-cyclase cDNA (GenBank accession no. AF251016) was amplified
by means of polymerase chain reaction (PCR) from Tagetes erecta
cDNA by using a sense-specific primer (PR42 SEQ ID No. 22) and an
antisense-specific primer (PR43 SEQ ID No. 23). The 5'-terminal 435
bp region of the Tagetes erecta epsilon-cyclase cDNA is composed of
138 bp of 5'-untranslated sequence (5'UTR) and 297 bp of the coding
region corresponding to the N terminus.
[0185] To prepare total RNA from Tagetes flowers, 100 mg of the
frozen, pulverized flowers were transferred to a reaction vessel
and taken up in 0.8 ml of Trizol buffer (LifeTechnologies). The
suspension was extracted with 0.2 ml of chloroform. After
centrifugation at 12 000 g for 15 minutes, the aqueous supernatant
was removed and transferred to a new reaction vessel and extracted
with one volume of ethanol. The RNA was precipitated with one
volume of isopropanol, washed with 75% ethanol and the pellet was
dissolved in DEPC water (overnight incubation of water with 1/1000
volume of diethyl pyrocarbonate at room temperature, with
subsequent autoclaving). The RNA concentration was determined
photometrically. For cDNA synthesis, 2.5 .mu.g of total RNA were
denatured at 60.degree. C. for 10 min, cooled on ice for 2 min and
transcribed into cDNA by means of a cDNA kit
(Ready-to-go-you-prime-beads, Pharmacia Biotech) according to the
manufacturer's information, using an antisense-specific primer
(PR17 SEQ ID No. 19).
[0186] The conditions of the subsequent PCR reactions were as
follows:
[0187] The PCR for amplification of the PR42-PR43 DNA fragment
containing the 5'-terminal 435 bp region of epsilon-cyclase was
carried out in a 50 .mu.l reaction mixture containing:
TABLE-US-00012 1 .mu.l cDNA (prepared as described above) 0.25 mM
dNTPs 0.2 .mu.M PR42 (SEQ ID No. 22) 0.2 .mu.M PR43 (SEQ ID No. 23)
5 .mu.l 10.times. PCR buffer (TAKARA) 0.25 .mu.l R Taq polymerase
(TAKARA) 28.8 .mu.l distilled water
[0188] The PCR for amplification of the PR44-PR45 DNA fragment
containing the 5'-terminal 435 bp region of epsilon-cyclase was
carried out in a 50 .mu.l reaction mixture containing:
TABLE-US-00013 1 .mu.l cDNA (prepared as described above) 0.25 mM
dNTPs 0.2 .mu.M PR44 (SEQ ID No. 24) 0.2 .mu.M PR45 (SEQ ID No. 25)
5 .mu.l 10.times. PCR buffer (TAKARA) 0.25 .mu.l R Taq polymerase
(TAKARA) 28.8 .mu.l distilled water
[0189] The PCR reactions were carried out under the following cycle
conditions: TABLE-US-00014 1.times. 94.degree. C. 2 minutes
35.times. 94.degree. C. 1 minute 58.degree. C. 1 minute 72.degree.
C. 1 minute 1.times. 72.degree. C. 10 minutes
[0190] PCR amplification using primers PR42 and PR43 resulted in a
443 bp fragment, and PCR amplification using primers PR44 and PR45
resulted in a 444 bp fragment.
[0191] The two amplicons, the PR42-PR43 (HindIII-SalI sense)
fragment and the PR44-PR45 (EcoRI-BamHI antisense) fragment, were
cloned into the PCR-cloning vector pCR-BluntII (Invitrogen), using
standard methods. Sequence reactions using the SP6 primer confirmed
in each case a sequence identical to the published AF251016
sequence (SEQ ID No. 4), apart from the introduced restriction
sites. These clones were therefore used for preparing an inverted
repeat construct in the pJAI1 cloning vector (see Example 1).
[0192] The first cloning step was carried out by isolating the 444
bp PR44-PR45 BamHI-EcoRI fragment from the pCR-BluntII cloning
vector (Invitrogen) and ligation with the BamHI-EcoRI-cut pJAI1
vector. The clone which contains the 5'-terminal epsilon-cyclase
region in the antisense orientation is denoted pJAI2. The ligation
results in a transcriptional fusion between the antisense fragment
of the 5'-terminal epsilon-cyclase region and the CaMV
polyadenylation signal.
[0193] The second cloning step is carried out by isolating the 443
bp PR42-PR43 HindIII-SalI fragment from the pCR-BluntII cloning
vector (Invitrogen) and ligation with the HindIII-SalI-cut PJAI2
vector. The clone which contains the 435 bp 5'-terminal region of
epsilon-cyclase cDNA in the sense orientation is denoted pJAI3. The
ligation results in a transcriptional fusion between the AP3P and
the sense fragment of the 5'-terminal epsilon-cyclase region.
[0194] An inverted repeat expression cassette under the control of
the CHRC promoter was prepared by amplifying an CHRC promoter
fragment, using petunia genomic DNA (prepared according to standard
methods) and the primers PRCHRC5 (SEQ ID No. 42) and PRCHRC3 (SEQ
ID No. 43). The amplicon was cloned into the pCR2.1 cloning vector
(Invitrogen). Sequencing reactions of the resulting clone
pCR2.1-CHRC, using the primers M13 and T7, confirmed a sequence
identical to the AF099501 sequence. This clone was therefore used
for cloning into the pJAI3 expression vector.
[0195] The cloning was carried out by isolating the 1537 bp
SacI-HindIII fragment from pCR2.1-CHRC and ligation into the
SacI-HindIII-cut pJAI3 vector. The clone which contains the CHRC
promoter instead of the original AP3P promoter is denoted
pJCI3.
[0196] The expression vectors for Agrobacterium-mediated
transformation of the AP3P- or CHRC-controlled inverted repeat
transcript in Tagetes erecta were prepared using the binary vector
pSUN5 (WO02/00900).
[0197] The expression vector pS5AI3 was prepared by ligating the
2622 bp SacI-XhoI fragment of pJAI3 with the SacI-XhoI-cut pSUN5
vector (FIG. 3, construct map).
[0198] In FIG. 3, the AP3P fragment includes the modified AP3P
promoter (771 bp), the 5sense fragment includes the 5' region of
Tagetes erecta epsilon-cyclase (435 bp) in the sense orientation,
the intron fragment includes the PIV2 intron of the potato ST-LS1
gene, the 5anti fragment includes the 5' region of Tagetes erecta
epsilon-cyclase (435 bp) in the antisense orientation, and the term
fragment (761 bp) includes the CaMV polyadenylation signal. The
expression vector pS5CI3 was prepared by ligating the 3394 bp
SacI-XhoI fragment of pJCI3 with the SacI-XhoI-cut pSUN5 vector
(FIG. 4, construct map).
[0199] In FIG. 4, the CHRC fragment includes the promoter (1537
bp), the 5sense fragment includes the 5' region of Tagetes erecta
epsilon-cyclase (435 bp) in the sense orientation, the intron
fragment includes the PIV2 intron of the potato ST-LS1 gene, the
5anti fragment includes the 5' region of Tagetes erecta
epsilon-cyclase (435 bp) in the antisense orientation, and the term
fragment (761 bp) includes the CaMV polyadenylation signal.
EXAMPLE 3
Preparation of an Inverted Repeat Expression Cassette for
Flower-Specific Expression of Epsilon-Cyclase dsRNAs in Tagetes
erecta (Directed Against the 3' Region of Epsilon-Cyclase cDNA)
[0200] The nucleic acid containing the 3'-terminal region (384 bp)
of epsilon-cyclase cDNA (GenBank accession no. AF251016) was
amplified by means of polymerase chain reaction (PCR) from Tagetes
erecta cDNA, using a sense-specific primer (PR46 SEQ ID No. 26) and
an antisense-specific primer (PR47 SEQ ID No. 27). The 3'-terminal
region (384 bp) of Tagetes erecta epsilon-cyclase cDNA is composed
of 140 bp of 3'-untranslated sequence (3'UTR) and 244 bp of the
coding region corresponding to the C terminus.
[0201] Total RNA was prepared from Tagetes flowers as described in
Example 2.
[0202] The cDNA synthesis was carried out as described in Example
1, using the antisense-specific primer PR17 (SEQ ID No. 19).
[0203] The conditions of the subsequent PCR reactions were as
follows:
[0204] The PCR for amplification of the PR46-PR457 DNA fragment
containing the 3'-terminal 384 bp region of epsilon-cyclase was
carried out in a 50 .mu.l reaction mixture containing:
TABLE-US-00015 1 .mu.l cDNA (prepared as described above) 0.25 mM
dNTPs 0.2 .mu.M PR46 (SEQ ID No. 26) 0.2 .mu.M PR47 (SEQ ID No. 27)
5 .mu.l 10.times. PCR buffer (TAKARA) 0.25 .mu.l R Taq polymerase
(TAKARA) 28.8 .mu.l distilled water
[0205] The PCR for amplification of the PR48-PR49 DNA fragment
containing the 3'-terminal 384 bp region of epsilon-cyclase was
carried out in a 50 .mu.l reaction mixture containing:
TABLE-US-00016 1 .mu.l cDNA (prepared as described above) 0.25 mM
dNTPs 0.2 .mu.M PR48 (SEQ ID No. 28) 0.2 .mu.M PR49 (SEQ ID No. 29)
5 .mu.l 10.times. PCR buffer (TAKARA) 0.25 .mu.l R Taq polymerase
(TAKARA) 28.8 .mu.l distilled water
[0206] The PCR reactions were carried out under the following cycle
conditions: TABLE-US-00017 1.times. 94.degree. C. 2 minutes
35.times. 94.degree. C. 1 minute 58.degree. C. 1 minute 72.degree.
C. 1 minute 1.times. 72.degree. C. 10 minutes
[0207] PCR amplification using SEQ ID No. 26 and SEQ ID No. 27
resulted in a 392 bp fragment, and PCR amplification using SEQ ID
No. 28 and SEQ ID No. 29 resulted in a 396 bp fragment.
[0208] The two amplicons, the PR46-PR47 fragment and the PR48-PR49
fragment, were cloned into the PCR-cloning vector pCR-BluntII
(Invitrogen), using standard methods. Sequence reactions using the
SP6 primer confirmed in each case a sequence identical to the
published AF251016 sequence (SEQ ID No. 4), apart from the
introduced restriction sites. These clones were therefore used for
preparing an inverted repeat construct in the pJAI1 cloning vector
(see Example 1).
[0209] The first cloning step was carried out by isolating the 396
bp PR48-PR49 BamHI-EcoRI fragment from the pCR-BluntII cloning
vector (Invitrogen) and ligation with the BamHI-EcoRI-cut pJAI1
vector. The clone which contains the 3'-terminal epsilon-cyclase
region in the antisense orientation is denoted pJAI4. The ligation
results in a transcriptional fusion between the antisense fragment
of the 3'-terminal epsilon-cyclase region and the CaMV
polyadenylation signal.
[0210] The second cloning step is carried out by isolating the 392
bp PR46-PR47 HindIII-SalI fragment from the pCR-BluntII cloning
vector (Invitrogen) and ligation with the HindIII-SalI-cut pJAI4
vector. The clone which contains the 392 bp 3'-terminal region of
epsilon-cyclase cDNA in the sense orientation is denoted pJAI5. The
ligation results in a transcriptional fusion between the AP3P and
the sense fragment of the 3'-terminal epsilon-cyclase region.
[0211] An expression vector for Agrobacterium-mediated
transformation of the AP3P-controlled inverted repeat transcript in
Tagetes erecta was prepared using the binary pSUN5 vector
(WO02/00900). The expression vector pS5AI5 was prepared by ligating
the 2523 bp SacI-XhoI fragment of pJAI5 with the SacI-XhoI-cut
pSUN5 vector (FIG. 5, construct map).
[0212] In FIG. 5, the AP3P fragment includes the modified AP3P
promoter (771 bp), the sense fragment includes the 3' region of
Tagetes erecta epsilon-cyclase (435 bp) in the sense orientation,
the intron fragment includes the IV2 intron of the potato ST-LS1
gene, the anti fragment includes the 3' region of Tagetes erecta
epsilon-cyclase (435 bp) in the antisense orientation, and the term
fragment (761 bp) includes the CaMV polyadenylation signal.
EXAMPLE 4
Cloning of the Epsilon-Cyclase Promoter
[0213] A 199 bp fragment and, respectively, the 312 bp fragment of
the epsilon-cyclase promoter were isolated by two independent
cloning strategies, inverse PCR (adapted from Long et al. Proc.
Natl. Acad. Sci USA 90: 10370) and TAIL-PCR (Liu Y-G. et al. (1995)
Plant J. 8: 457-463), using genomic DNA (isolated by a standard
method from Tagetes erecta, line "Orangenprinz").
[0214] For the inverse PCR approach, 2 .mu.g of genomic DNA were
digested with EcoRV and RsaI in a 25 .mu.l reaction mixture, then
diluted to 300 .mu.l and religated at 16.degree. C. overnight,
using 3U of ligase. PCR amplification using the primers PR50 (SEQ
ID No. 30) and PR51 (SEQ ID No. 31) produced a fragment which
contains, in each case in the sense orientation, 354 bp of
epsilon-cyclase cDNA (GenBank Accession AF251016), ligated to 300
bp of the epsilon-cyclase promoter and 70 bp of the 5'-terminal
region of epsilon-cyclase cDNA (see FIG. 6).
[0215] The conditions of the PCR reactions were as follows:
[0216] The PCR for amplification of the PR50-PR51 DNA fragment
which contains, inter alia, the 312 bp promoter fragment of
epsilon-cyclase was carried out in a 50 .mu.l reaction mixture
containing: TABLE-US-00018 1 .mu.l ligation mixture (prepared as
described above) 0.25 mM dNTPs 0.2 .mu.M PR50 (SEQ ID No. 30) 0.2
.mu.M PR51 (SEQ ID No. 31) 5 .mu.l 10.times. PCR buffer (TAKARA)
0.25 .mu.l R Taq polymerase (TAKARA) 28.8 .mu.l distilled water
[0217] The PCR reactions were carried out under the following cycle
conditions: TABLE-US-00019 1.times. 94.degree. C. 2 minutes
35.times. 94.degree. C. 1 minute 53.degree. C. 1 minute 72.degree.
C. 1 minute 1.times. 72.degree. C. 10 minutes
[0218] PCR amplification using primers PR50 and PR51 resulted in a
734 bp fragment containing, inter alia, the 312 bp promoter
fragment of epsilon-cyclase (FIG. 6).
[0219] The amplicon was cloned into the PCR-cloning vector pCR2.1
(Invitrogen), using standard methods. Sequencing reactions using
the primers M13 and T7 produced the sequence SEQ ID No. 11. This
sequence was reproduced in an independent amplification experiment
and thus represents the nucleotide sequence in the Tagetes erecta
line "Orangenprinz" used.
[0220] For the TAIL-PCR approach, three successive PCR reactions
were carried out, using in each case different gene-specific
primers (nested primers).
[0221] The TAIL1-PCR was carried out in a 20 .mu.l reaction mixture
containing: TABLE-US-00020 1 ng genomic DNA (prepared as described
above) 0.2 mM each of dNTPs 0.2 .mu.M PR60 (SEQ ID No. 32) 0.2
.mu.M AD1 (SEQ ID No. 35) 2 .mu.l 10.times. PCR buffer (TAKARA) 0.5
.mu.l R Taq polymerase (TAKARA) ad 20 .mu.l distilled water
[0222] In this context, AD1 was initially a mixture of primers of
the sequences (a/c/g/t)tcga(g/c)t(a/t)t(g/c)g(a/t)gtt.
[0223] The PCR reaction TAIL1 was carried out under the following
cycle conditions: [0224] 1.times.93.degree. C.: 1 min., 95.degree.
C.: 1 min. [0225] 5.times.94.degree. C.: 30 sec., 62.degree. C.: 1
min., 72.degree. C.: 2.5 min. [0226] 1.times.94.degree. C.: 30
sec., 25.degree. C.: 3 min., ramp to 72.degree. C. in 3 min.
72.degree. C.: 2.5 min [0227] 15.times.94.degree. C.: 10 sec.,
68.degree. C.: 1 min., 72.degree. C.: 2.5 min.; 94.degree. C.: 10
sec., 68.degree. C.: 1 min., 72.degree. C.: 2.5 min.; 94.degree.
C.: 10 sec., 29.degree. C.: 1 min., 72.degree. C.: 2.5 min. [0228]
1.times.72.degree. C.: 5 min.
[0229] The TAIL2-PCR was carried out in a 21 .mu.l reaction mixture
containing: TABLE-US-00021 1 .mu.l 1:50 dilution of the TAIL1
reaction mixture (prepared as described above) 0.8 mM dNTP 0.2
.mu.M PR61 (SEQ ID No. 33) 0.2 .mu.M AD1 (SEQ ID No. 35) 2 .mu.l
10.times. PCR buffer (TAKARA) 0.5 .mu.l R Taq polymerase (TAKARA)
ad 21 .mu.l distilled water
[0230] The PCR reaction TAIL2 was carried out under the following
cycle conditions: [0231] 12.times.94.degree. C.: 10 seconds,
64.degree. C.: 1 minute, 72.degree. C.: 2.5 minutes; 94.degree. C.:
10 seconds, 64.degree. C.: 1 minute, 72.degree. C.: 2.5 minutes;
94.degree. C.: 10 seconds, 29.degree. C.: 1 minute, 72.degree. C.:
2.5 minutes; [0232] 1.times.72.degree. C.: 5 minutes
[0233] The TAIL3 PCR was carried out in a 100 .mu.l reaction
mixture containing: TABLE-US-00022 1 .mu.l 1:10 dilution of the
TAIL2 reaction mixture (prepared as described above) 0.8 mM dNTP
0.2 .mu.M PR63 (SEQ ID No. 34) 0.2 .mu.M AD1 (SEQ ID No. 35) 10
.mu.l 10.times. PCR buffer (TAKARA) 0.5 .mu.l R Taq polymerase
(TAKARA) ad 100 .mu.l distilled water
[0234] The PCR reaction TAIL3 was carried out under the following
cycle conditions: [0235] 20.times.94.degree. C.: 15 seconds,
29.degree. C.: 30 seconds, 72.degree. C.: 2 minutes [0236]
1.times.72.degree. C.: 5 minutes
[0237] PCR amplification using primers PR63 and AD1 resulted in a
280 bp fragment containing, inter alia, the 199 bp promoter
fragment of epsilon-cyclase (FIG. 7).
[0238] The amplicon was cloned into the PCR-cloning vector pCR2.1
(Invitrogen), using standard methods. Sequencing reactions using
the primers M13 and T7 produced the sequence SEQ ID No. 12. This
sequence is identical to the .epsilon.-cyclase region within the
sequence SEQ ID No. 11, isolated using the IPCR strategy, and thus
represents the nucleotide sequence in the Tagetes erecta line
"Orangenprinz" used.
[0239] The pCR2.1 clone which contains the 312 bp fragment (SEQ ID
No. 11) of the epsilon-cyclase promoter, isolated by the IPCR
strategy, is denoted pTA-ecycP and was used for preparing the IR
constructs.
EXAMPLE 5
Preparation of an Inverted Repeat Expression Cassette for
Flower-Specific Expression of Epsilon-Cyclase dsRNAs in Tagetes
erecta (Directed Against the Promoter Region of Epsilon-Cyclase
cDNA).
[0240] Inverted repeat transcripts consisting of promoter fragments
of epsilon-cyclase were expressed in Tagetes erecta under the
control of a modified version, AP3P, of the flower-specific
Arabidopsis promoter AP3 (see Example 1) or of the flower-specific
promoter CHRC (GenBank accession no. AF099501). The inverted repeat
transcript contains in each case an epsilon-cyclase promoter
fragment in the correct orientation (sense fragment) and a
sequence-identical epsilon-cyclase promoter fragment in the
opposite orientation (antisense fragment) which are connected to
one another by a functional intron (see Example 1).
[0241] The promoter fragments were prepared by means of PCR using
plasmid DNA (pTA-ecycP clone, see Example 4) and the primers PR124
(SEQ ID No. 36) and PR126 (SEQ ID No. 38) and, respectively, the
primers PR125 (SEQ ID No. 37) and PR127 (SEQ ID No. 39).
[0242] The conditions of the PCR reactions were as follows:
[0243] The PCR for amplification of the PR124-PR126 DNA fragment
containing the promoter fragment of epsilon-cyclase was carried out
in a 50 .mu.l reaction mixture containing: TABLE-US-00023 1 .mu.l
cDNA (prepared as described above) 0.25 mM dNTPs 0.2 .mu.M PR124
(SEQ ID No. 36) 0.2 .mu.M PR126 (SEQ ID No. 38) 5 .mu.l 10.times.
PCR buffer (TAKARA) 0.25 .mu.l R Taq polymerase (TAKARA) 28.8 .mu.l
distilled water
[0244] The PCR for amplification of the PR125-PR127 DNA fragment
containing the 312 bp promoter fragment of epsilon-cyclase was
carried out in a 50 .mu.l reaction mixture containing:
TABLE-US-00024 1 .mu.l cDNA (prepared as described above) 0.25 mM
dNTPs 0.2 .mu.M PR125 (SEQ ID No. 37) 0.2 .mu.M PR127 (SEQ ID No.
39) 5 .mu.l 10.times. PCR buffer (TAKARA) 0.25 .mu.l R Taq
polymerase (TAKARA) 28.8 .mu.l distilled water
[0245] The PCR reactions were carried out under the following cycle
conditions: TABLE-US-00025 1.times. 94.degree. C. 2 minutes
35.times. 94.degree. C. 1 minute 53.degree. C. 1 minute 72.degree.
C. 1 minute 1.times. 72.degree. C. 10 minutes
[0246] PCR amplification using the primers PR124 and PR126 resulted
in a 358 bp fragment, and PCR amplification using primers PR125 and
PR127 resulted in a 361 bp fragment.
[0247] The two amplicons, the PR124-PR126 (HindIII-SalI sense)
fragment and the PR125-PR127 (EcoRI-BamHI antisense) fragment, were
cloned into the PCR-cloning vector pCR-BluntII (Invitrogen), using
standard methods. Sequencing reactions using the SP6 primer
confirmed in each case a sequence which is identical to SEQ ID No.
11, apart from the introduced restriction sites. These clones were
therefore used for preparing an inverted repeat construct in the
pJAI1 cloning vector (see Example 1).
[0248] The first cloning step was carried out by isolating the 358
bp PR124-PR126 HindIII-SalI fragment from the pCR-BluntII cloning
vector (Invitrogen) and ligation with the BamHI-EcoRI-cut pJAI1
vector. The clone which contains the epsilon-cyclase promoter
fragment in the sense orientation is denoted cs43. The ligation
causes the sense fragment of the epsilon-cyclase promoter to be
inserted between the AP3P promoter and the intron.
[0249] The second cloning step was carried out by isolating the 361
bp PR125-PR127 BamHI-EcoRI fragment from the pCR-BluntII cloning
vector (Invitrogen) and ligation with the BamHI-EcoRI-cut cs43
vector. The clone which contains the epsilon-cyclase promoter
fragment in the antisense orientation is denoted cs44. The ligation
produces a transcriptional fusion between the intron and the
antisense fragment of the epsilon-cyclase promoter.
[0250] An inverted repeat expression cassette under the control of
the CHRC promoter was prepared by amplifying a CHRC promoter
fragment, using petunia genomic DNA (prepared by standard methods)
and the primers PRCHRC3' (SEQ ID No. 43) and PRCHRC5' (SEQ ID No.
42). The amplicon was cloned into the pCR2.1 cloning vector
(Invitrogen). Sequencing reactions of the resulting clone
pCR2.1-CHRC, using the M13 and T7 primers, confirmed a sequence
identical to the AF099501 sequence. This clone was therefore used
for cloning into the expression vector cs44.
[0251] The cloning was carried out by isolating the 1537 bp
SacI-HindIII fragment of pCR2.1-CHRC and ligation into the
SacI-HindIII-cut cs44 vector. The clone which contains the CHRC
promoter instead of the original AP3P promoter is denoted cs45.
[0252] An inverted repeat expression cassette under the control of
two promoters, the CHRC promoter and the AP3P promoter, was
prepared by cloning the AP3P promoter in antisense orientation to
the 3' terminus of the epsilon-cyclase antisense fragment in cs45.
The AP3P promoter fragment of pJAI1 was amplified using the primers
PR128 and PR129. The amplicon was cloned into the pCR2.1 cloning
vector (Invitrogen). Sequencing using the M13 and T7 primers
confirmed a sequence identical to the sequence SEQ ID No. 1. This
clone, pCR2.1-AP3PSX, was used for preparing an inverted repeat
expression cassette under the control of two promoters.
[0253] The cloning was carried out by isolating the 771 bp
SalI-XhoI fragment from pCR2.1-AP3PSX and ligation into the
XhoI-cut cs45 vector. The clone which contains, 3' of the inverted
repeat, the AP3P promoter in the antisense orientation is denoted
cs46.
[0254] The expression vectors for Agrobacterium-mediated
transformation of the AP3P-controlled inverted repeat transcript in
Tagetes erecta were prepared using the binary pSUN5 vector
(WO02/00900).
[0255] The expression vector pS5AI7 was prepared by ligating the
1685 bp SacI-XhoI fragment of cs44 with the SacI-XhoI-cut pSUN5
vector (FIG. 8, construct map).
[0256] In FIG. 8, the AP3P fragment includes the modified AP3P
promoter (771 bp), the P-sense fragment includes the 312 bp
promoter fragment of epsilon-cyclase in the sense orientation, the
intron fragment includes the IV2 intron of the potato ST-LS1 gene,
and the P-anti fragment includes the 312 bp promoter fragment of
epsilon-cyclase in antisense orientation.
[0257] The expression vector pS5CI7 was prepared by ligating the
2445 bp SacI-XhoI fragment of cs45 with the SacI-XhoI-cut pSUN5
vector (FIG. 9, construct map).
[0258] In FIG. 9, the CHRC fragment includes the CHRC promoter
(1537 bp), the P-sense fragment includes the 312 bp promoter
fragment of epsilon-cyclase in the sense orientation, the intron
fragment includes the IV2 intron of the potato ST-LS1 gene, and the
P-anti fragment includes the 312 bp promoter fragment of
epsilon-cyclase in antisense orientation.
[0259] The expression vector pS5CI7 was prepared by ligating the
3219 bp SacI-XhoI fragment of cs46 with the SacI-XhoI-cut pSUN5
vector (FIG. 10, construct map).
[0260] In FIG. 10, the CHRC fragment includes the CHRC promoter
(1537 bp), the P-sense fragment includes the 312 bp promoter
fragment of epsilon-cyclase in the sense orientation, the intron
fragment includes the IV2 intron of the potato ST-LS1 gene, the
P-anti fragment includes the 312 bp promoter fragment of
epsilon-cyclase in antisense orientation, and the AP3P fragment
includes the 771 bp AP3P promoter fragment in the antisense
orientation.
EXAMPLE 6
Preparation of Transgenic Tagetes Plants
[0261] Tagetes seeds are sterilized and placed on germination
medium (MS medium; Murashige and Skoog, Physiol. Plant. 15(1962),
473-497) pH 5.8, 2% sucrose). Germination takes place in a
temperature/light/time interval of 18 to 28.degree. C./20 to 200
.mu.E/3 to 16 weeks, but preferably at 21.degree. C., 20 to 70
.mu.E, for 4 to 8 weeks.
[0262] All the leaves of the plants which have developed in vitro
by then are harvested and cut perpendicular to the mid rib. The
leaf explants produced in this way with a size of 10 to 60 mm.sup.2
are stored during the preparation in liquid MS medium at room
temperature for a maximum of 2 h.
[0263] The Agrobacterium tumefaciens strain EHA105 was transformed
with the binary plasmid PS5AI3. The transformed A. tumefaciens
EHA105 strain was grown overnight under the following conditions: a
single colony was inoculated in YEB (0.1% yeast extract, 0.5% beef
extract, 0.5% peptone, 0.5% sucrose, 0.5% magnesium
sulfate.times.7H.sub.20) with 25 mg/l kanamycin and grown at
28.degree. C. for 16 to 20 h. The bacterial suspension was then
harvested by centrifugation at 6000 g for 10 min and resuspended in
liquid MS medium such that an OD.sub.600 of approx. 0.1 to 0.8 was
produced. This suspension was used for the cocultivation with the
leaf material.
[0264] Immediately before the cocultivation, the MS medium in which
the leaves have been stored is replaced by the bacterial
suspension. The leaves were incubated in the suspension of
agrobacteria for 30 min while shaking gently at room temperature.
The infected explants are placed on an MS medium with grown
regulators such as, for example, 3 mg/l benzylaminopurine (BAP) and
1 mg/l indolyl acetic acid (IAA), which has been solidified with
agar (e.g. 0.8% plant agar (Duchefa, NL)). The orientation of the
leaves on the medium has no significance. The explants are
cultivated for 1 to 8 days, but preferably for 6 days, during which
the following conditions can be used: light intensity: 30 to 80
.mu.mol/m.sup.2.times.s, temperature: 22 to 24.degree. C., 16/8
hours of light/dark alternation. The cocultivated explants are then
transferred to fresh MS medium, preferably with the same growth
regulators, this second medium additionally containing an
antibiotic to suppress bacterial growth. Timentin in a
concentration of from 200 to 500 mg/l is very suitable for this
purpose. The second selective component employed is one for
selecting for successful transformation. Phosphinothricin in a
concentration of from 1 to 5 mg/l selects very efficiently, but
other selective components are also conceivable according to the
process to be used.
[0265] After one to three weeks in each case, the explants are
transferred to fresh medium until plumules and small shoots
develop, and these are then transferred to the same basal medium
including Timentin and PPT or alternative components with growth
regulators, namely, for example, 0.5 mg/l indolylbutyric acid (IBA)
and 0.5 mg/l gibberilic acid GA.sub.3, for rooting. Rooted shoots
can be transferred to a glasshouse.
[0266] In addition to the method described, the following
advantageous modifications are possible: [0267] before the explants
are infected with bacteria, they can be preincubated on the medium
described above for the cocultivation for 1 to 12 days, preferably
3 to 4. This is followed by infection, cocultivation and selective
regeneration as described above. [0268] the pH for the regeneration
(normally 5.8) can be lowered to pH 5.2. This improves control of
the growth of agrobacteria. [0269] addition of AgNO.sub.3 (3 to 10
mg/l) to the regeneration medium improves the condition of the
culture, including the regeneration itself. [0270] components which
reduce phenol formation and are known to the skilled worker, such
as, for example, citric acid, ascorbic acid, PVP and many others,
have beneficial effects on the culture. [0271] liquid culture
medium can also be used for the whole process. The culture can also
be incubated on commercially available supports which are
positioned on the liquid medium.
[0272] According to the transformation method described above, the
following lines were obtained using the following expression
constructs:
CS30-1, CS30-3 and CS30-4 were obtained with pS5AI3.
EXAMPLE 7
Characterization of the Transgenic Plants
[0273] The flower material of the transgenic Tagetes erecta plants
of Example 6 was crushed in liquid nitrogen and the powder (about
250 to 500 mg) was extracted with 100% acetone (three times, 500
.mu.l each). The solvent was evaporated and the carotenoids were
resuspended in 100 .mu.l of acetone.
[0274] Using a C30 reverse phase column it was possible to
distinguish between the carotenoid mono- and diesters. The HPLC run
conditions were virtually identical to a published method (Frazer
et al. (2000), Plant Journal 24(4): 551-558). It was possible to
identify the carotenoids on the basis of the UV-VIS spectra.
[0275] Table 1 depicts the carotenoid profile in Tagetes petals of
the transgenic Tagetes plants prepared according to the examples
described above and of control Tagetes plants. All of the
carotenoid quantities are given in [.mu.g/g] fresh weight, with
percentages of change compared to the control plant being indicated
in parentheses.
[0276] In comparison with the genetically unmodified control plant,
the genetically modified plants have a distinctly increased content
of carotenoids of the ".beta.-carotene pathway", such as, for
example, .beta.-carotene and zeaxanthin, and a distinctly reduced
content of carotenoids of the ".alpha.-carotene pathway", such as
lutein, for example. TABLE-US-00026 TABLE 1 Viola- Total Plant
Lutein .beta.-Carotene Zeaxanthin xanthin carotenoids Control 260
4.8 2.7 36 304 CS 30-1 35 13 4.4 59 111 (-86%) (+170%) (+62%)
(+63%) (-63%) Control 456 6.4 6.9 58 527 CS 30-3 62 13 8.9 75 159
(-86%) (+103%) (+29%) (+29%) (-70%) CS 30-4 68 9.1 5.7 61 144
(-85%) (+42%) (-17%) (+5%) (-73%) Control 280 4.1 2.6 42 329 CS
32-9 69 5.5 2.3 25 102 (-75%) (+34%) (-12%) (-38%) (-69%)
COMPARATIVE EXAMPLE 1
Reduction of .epsilon.-Cyclase Activity in Tagetes erecta by
Antisense
[0277] Using conventional methods known to the skilled worker, a
Tagetes erecta antisense line, CS32-9, in which the
.epsilon.-cyclase activity was reduced by antisense was prepared as
comparative example. The carotenoid profile of this line (CS32-9),
measured by the method described above, is likewise depicted in
Table 1.
Sequence CWU 1
1
43 1 777 DNA Arabidopsis thaliana promoter (1)..(777) 1 gagctcactc
actgatttcc attgcttgaa aattgatgat gaactaagat caatccatgt 60
tagtttcaaa acaacagtaa ctgtggccaa cttagttttg aaacaacact aactggtcga
120 agcaaaaaga aaaaagagtt tcatcatata tctgatttga tggactgttt
ggagttagga 180 ccaaacatta tctacaaaca aagacttttc tcctaacttg
tgattccttc ttaaacccta 240 ggggtaatat tctattttcc aaggatcttt
agttaaaggc aaatccggga aattattgta 300 atcatttggg gaaacatata
aaagatttga gttagatgga agtgacgatt aatccaaaca 360 tatatatctc
tttcttctta tttcccaaat taacagacaa aagtagaata ttggctttta 420
acaccaatat aaaaacttgc ttcacaccta aacacttttg tttactttag ggtaagtgca
480 aaaagccaac caaatccacc tgcactgatt tgacgtttac aaacgccgtt
aagtcgatgt 540 ccgttgattt aaacagtgtc ttgtaattaa aaaaatcagt
ttacataaat ggaaaattta 600 tcacttagtt ttcatcaact tctgaactta
cctttcatgg attaggcaat actttccatt 660 tttagtaact caagtggacc
ctttacttct tcaactccat ctctctcttt ctatttcact 720 tctttcttct
cattatatct cttgtcctct ccaccaaatc tcttcaacaa aaagctt 777 2 195 DNA
Potato Intron (1)..(195) 2 tacgtaagtt tctgcttcta cctttgatat
atatataata attatcatta attagtagta 60 atataatatt tcaaatattt
ttttcaaaat aaaagaatgt agtatatagc aattgctttt 120 ctgtagttta
taagtgtgta tattttaatt tataactttt ctaatatatg accaaaattt 180
gttgatgtgc agctg 195 3 212 DNA Artificial sequence Chemically
synthesized 3 gtcgactacg taagtttctg cttctacctt tgatatatat
ataataatta tcattaatta 60 gtagtaatat aatatttcaa atattttttt
caaaataaaa gaatgtagta tatagcaatt 120 gcttttctgt agtttataag
tgtgtatatt ttaatttata acttttctaa tatatgacca 180 aaatttgttg
atgtgcaggt atcaccggat cc 212 4 1830 DNA Tagetes erecta CDS
(141)..(1691) 4 ggcacgaggc aaagcaaagg ttgtttgttg ttgttgttga
gagacactcc aatccaaaca 60 gatacaaggc gtgactggat atttctctct
cgttcctaac aacagcaacg aagaagaaaa 120 agaatcatta ctaacaatca atg agt
atg aga gct gga cac atg acg gca aca 173 Met Ser Met Arg Ala Gly His
Met Thr Ala Thr 1 5 10 atg gcg gct ttt aca tgc cct agg ttt atg act
agc atc aga tac acg 221 Met Ala Ala Phe Thr Cys Pro Arg Phe Met Thr
Ser Ile Arg Tyr Thr 15 20 25 aag caa att aag tgc aac gct gct aaa
agc cag cta gtc gtt aaa caa 269 Lys Gln Ile Lys Cys Asn Ala Ala Lys
Ser Gln Leu Val Val Lys Gln 30 35 40 gag att gag gag gaa gaa gat
tat gtg aaa gcc ggt gga tcg gag ctg 317 Glu Ile Glu Glu Glu Glu Asp
Tyr Val Lys Ala Gly Gly Ser Glu Leu 45 50 55 ctt ttt gtt caa atg
caa cag aat aag tcc atg gat gca cag tct agc 365 Leu Phe Val Gln Met
Gln Gln Asn Lys Ser Met Asp Ala Gln Ser Ser 60 65 70 75 cta tcc caa
aag ctc cca agg gta cca ata gga gga gga gga gac agt 413 Leu Ser Gln
Lys Leu Pro Arg Val Pro Ile Gly Gly Gly Gly Asp Ser 80 85 90 aac
tgt ata ctg gat ttg gtt gta att ggt tgt ggt cct gct ggc ctt 461 Asn
Cys Ile Leu Asp Leu Val Val Ile Gly Cys Gly Pro Ala Gly Leu 95 100
105 gct ctt gct gga gaa tca gcc aag cta ggc ttg aat gtc gca ctt atc
509 Ala Leu Ala Gly Glu Ser Ala Lys Leu Gly Leu Asn Val Ala Leu Ile
110 115 120 ggc cct gat ctt cct ttt aca aat aac tat ggt gtt tgg gag
gat gaa 557 Gly Pro Asp Leu Pro Phe Thr Asn Asn Tyr Gly Val Trp Glu
Asp Glu 125 130 135 ttt ata ggt ctt gga ctt gag ggc tgt att gaa cat
gtt tgg cga gat 605 Phe Ile Gly Leu Gly Leu Glu Gly Cys Ile Glu His
Val Trp Arg Asp 140 145 150 155 act gta gta tat ctt gat gac aac gat
ccc att ctc ata ggt cgt gcc 653 Thr Val Val Tyr Leu Asp Asp Asn Asp
Pro Ile Leu Ile Gly Arg Ala 160 165 170 tat gga cga gtt agt cgt gat
tta ctt cac gag gag ttg ttg act agg 701 Tyr Gly Arg Val Ser Arg Asp
Leu Leu His Glu Glu Leu Leu Thr Arg 175 180 185 tgc atg gag tca ggc
gtt tca tat ctg agc tcc aaa gtg gaa cgg att 749 Cys Met Glu Ser Gly
Val Ser Tyr Leu Ser Ser Lys Val Glu Arg Ile 190 195 200 act gaa gct
cca aat ggc cta agt ctc ata gag tgt gaa ggc aat atc 797 Thr Glu Ala
Pro Asn Gly Leu Ser Leu Ile Glu Cys Glu Gly Asn Ile 205 210 215 aca
att cca tgc agg ctt gct act gtc gct tct gga gca gct tct gga 845 Thr
Ile Pro Cys Arg Leu Ala Thr Val Ala Ser Gly Ala Ala Ser Gly 220 225
230 235 aaa ctt ttg cag tat gaa ctt ggc ggt ccc cgt gtt tgc gtt caa
aca 893 Lys Leu Leu Gln Tyr Glu Leu Gly Gly Pro Arg Val Cys Val Gln
Thr 240 245 250 gct tat ggt ata gag gtt gag gtt gaa agc ata ccc tat
gat cca agc 941 Ala Tyr Gly Ile Glu Val Glu Val Glu Ser Ile Pro Tyr
Asp Pro Ser 255 260 265 cta atg gtt ttc atg gat tat aga gac tac acc
aaa cat aaa tct caa 989 Leu Met Val Phe Met Asp Tyr Arg Asp Tyr Thr
Lys His Lys Ser Gln 270 275 280 tca cta gaa gca caa tat cca aca ttt
ttg tat gtc atg cca atg tct 1037 Ser Leu Glu Ala Gln Tyr Pro Thr
Phe Leu Tyr Val Met Pro Met Ser 285 290 295 cca act aaa gta ttc ttt
gag gaa act tgt ttg gct tca aaa gag gcc 1085 Pro Thr Lys Val Phe
Phe Glu Glu Thr Cys Leu Ala Ser Lys Glu Ala 300 305 310 315 atg cct
ttt gag tta ttg aag aca aaa ctc atg tca aga tta aag act 1133 Met
Pro Phe Glu Leu Leu Lys Thr Lys Leu Met Ser Arg Leu Lys Thr 320 325
330 atg ggg atc cga ata acc aaa act tat gaa gag gaa tgg tca tat att
1181 Met Gly Ile Arg Ile Thr Lys Thr Tyr Glu Glu Glu Trp Ser Tyr
Ile 335 340 345 cca gta ggt gga tcc tta cca aat acc gag caa aag aac
ctt gca ttt 1229 Pro Val Gly Gly Ser Leu Pro Asn Thr Glu Gln Lys
Asn Leu Ala Phe 350 355 360 ggt gct gct gct agc atg gtg cat cca gcc
aca gga tat tcg gtt gta 1277 Gly Ala Ala Ala Ser Met Val His Pro
Ala Thr Gly Tyr Ser Val Val 365 370 375 aga tca ctg tca gaa gct cct
aat tat gca gca gta att gca aag att 1325 Arg Ser Leu Ser Glu Ala
Pro Asn Tyr Ala Ala Val Ile Ala Lys Ile 380 385 390 395 tta ggg aaa
gga aat tca aaa cag atg ctt gat cat gga aga tac aca 1373 Leu Gly
Lys Gly Asn Ser Lys Gln Met Leu Asp His Gly Arg Tyr Thr 400 405 410
acc aac atc tca aag caa gct tgg gaa aca ctt tgg ccc ctt gaa agg
1421 Thr Asn Ile Ser Lys Gln Ala Trp Glu Thr Leu Trp Pro Leu Glu
Arg 415 420 425 aaa aga cag aga gca ttc ttt ctc ttt gga tta gca ctg
att gtc cag 1469 Lys Arg Gln Arg Ala Phe Phe Leu Phe Gly Leu Ala
Leu Ile Val Gln 430 435 440 atg gat att gag ggg acc cgc aca ttc ttc
cgg act ttc ttc cgc ttg 1517 Met Asp Ile Glu Gly Thr Arg Thr Phe
Phe Arg Thr Phe Phe Arg Leu 445 450 455 ccc aca tgg atg tgg tgg ggg
ttt ctt gga tct tcg tta tca tca act 1565 Pro Thr Trp Met Trp Trp
Gly Phe Leu Gly Ser Ser Leu Ser Ser Thr 460 465 470 475 gac ttg ata
ata ttt gcg ttt tac atg ttt atc ata gca ccg cat agc 1613 Asp Leu
Ile Ile Phe Ala Phe Tyr Met Phe Ile Ile Ala Pro His Ser 480 485 490
ctg aga atg ggt ctg gtt aga cat ttg ctt tct gac ccg aca gga gga
1661 Leu Arg Met Gly Leu Val Arg His Leu Leu Ser Asp Pro Thr Gly
Gly 495 500 505 aca atg tta aaa gcg tat ctc acg ata taa ataactctag
tcgcgatcag 1711 Thr Met Leu Lys Ala Tyr Leu Thr Ile 510 515
tttagattat aggcacatct tgcatatata tatgtataaa ccttatgtgt gctgtatcct
1771 tacatcaaca cagtcattaa ttgtatttct tggggtaatg ctgatgaagt
attttctgg 1830 5 516 PRT Tagetes erecta 5 Met Ser Met Arg Ala Gly
His Met Thr Ala Thr Met Ala Ala Phe Thr 1 5 10 15 Cys Pro Arg Phe
Met Thr Ser Ile Arg Tyr Thr Lys Gln Ile Lys Cys 20 25 30 Asn Ala
Ala Lys Ser Gln Leu Val Val Lys Gln Glu Ile Glu Glu Glu 35 40 45
Glu Asp Tyr Val Lys Ala Gly Gly Ser Glu Leu Leu Phe Val Gln Met 50
55 60 Gln Gln Asn Lys Ser Met Asp Ala Gln Ser Ser Leu Ser Gln Lys
Leu 65 70 75 80 Pro Arg Val Pro Ile Gly Gly Gly Gly Asp Ser Asn Cys
Ile Leu Asp 85 90 95 Leu Val Val Ile Gly Cys Gly Pro Ala Gly Leu
Ala Leu Ala Gly Glu 100 105 110 Ser Ala Lys Leu Gly Leu Asn Val Ala
Leu Ile Gly Pro Asp Leu Pro 115 120 125 Phe Thr Asn Asn Tyr Gly Val
Trp Glu Asp Glu Phe Ile Gly Leu Gly 130 135 140 Leu Glu Gly Cys Ile
Glu His Val Trp Arg Asp Thr Val Val Tyr Leu 145 150 155 160 Asp Asp
Asn Asp Pro Ile Leu Ile Gly Arg Ala Tyr Gly Arg Val Ser 165 170 175
Arg Asp Leu Leu His Glu Glu Leu Leu Thr Arg Cys Met Glu Ser Gly 180
185 190 Val Ser Tyr Leu Ser Ser Lys Val Glu Arg Ile Thr Glu Ala Pro
Asn 195 200 205 Gly Leu Ser Leu Ile Glu Cys Glu Gly Asn Ile Thr Ile
Pro Cys Arg 210 215 220 Leu Ala Thr Val Ala Ser Gly Ala Ala Ser Gly
Lys Leu Leu Gln Tyr 225 230 235 240 Glu Leu Gly Gly Pro Arg Val Cys
Val Gln Thr Ala Tyr Gly Ile Glu 245 250 255 Val Glu Val Glu Ser Ile
Pro Tyr Asp Pro Ser Leu Met Val Phe Met 260 265 270 Asp Tyr Arg Asp
Tyr Thr Lys His Lys Ser Gln Ser Leu Glu Ala Gln 275 280 285 Tyr Pro
Thr Phe Leu Tyr Val Met Pro Met Ser Pro Thr Lys Val Phe 290 295 300
Phe Glu Glu Thr Cys Leu Ala Ser Lys Glu Ala Met Pro Phe Glu Leu 305
310 315 320 Leu Lys Thr Lys Leu Met Ser Arg Leu Lys Thr Met Gly Ile
Arg Ile 325 330 335 Thr Lys Thr Tyr Glu Glu Glu Trp Ser Tyr Ile Pro
Val Gly Gly Ser 340 345 350 Leu Pro Asn Thr Glu Gln Lys Asn Leu Ala
Phe Gly Ala Ala Ala Ser 355 360 365 Met Val His Pro Ala Thr Gly Tyr
Ser Val Val Arg Ser Leu Ser Glu 370 375 380 Ala Pro Asn Tyr Ala Ala
Val Ile Ala Lys Ile Leu Gly Lys Gly Asn 385 390 395 400 Ser Lys Gln
Met Leu Asp His Gly Arg Tyr Thr Thr Asn Ile Ser Lys 405 410 415 Gln
Ala Trp Glu Thr Leu Trp Pro Leu Glu Arg Lys Arg Gln Arg Ala 420 425
430 Phe Phe Leu Phe Gly Leu Ala Leu Ile Val Gln Met Asp Ile Glu Gly
435 440 445 Thr Arg Thr Phe Phe Arg Thr Phe Phe Arg Leu Pro Thr Trp
Met Trp 450 455 460 Trp Gly Phe Leu Gly Ser Ser Leu Ser Ser Thr Asp
Leu Ile Ile Phe 465 470 475 480 Ala Phe Tyr Met Phe Ile Ile Ala Pro
His Ser Leu Arg Met Gly Leu 485 490 495 Val Arg His Leu Leu Ser Asp
Pro Thr Gly Gly Thr Met Leu Lys Ala 500 505 510 Tyr Leu Thr Ile 515
6 445 DNA tagetes erecta sense fragment 6 aagcttgcac gaggcaaagc
aaaggttgtt tgttgttgtt gttgagagac actccaatcc 60 aaacagatac
aaggcgtgac tggatatttc tctctcgttc ctaacaacag caacgaagaa 120
gaaaaagaat cattactaac aatcaatgag tatgagagct ggacacatga cggcaacaat
180 ggcggctttt acatgcccta ggtttatgac tagcatcaga tacacgaagc
aaattaagtg 240 caacgctgct aaaagccagc tagtcgttaa acaagagatt
gaggaggaag aagattatgt 300 gaaagccggt ggatcggagc tgctttttgt
tcaaatgcaa cagaataagt ccatggatgc 360 acagtctagc ctatcccaaa
agctcccaag ggtaccaata ggaggaggag gagacagtaa 420 ctgtatactg
gatttggttg tcgac 445 7 446 DNA tagetes erecta antisense fragment 7
gaattcgcac gaggcaaagc aaaggttgtt tgttgttgtt gttgagagac actccaatcc
60 aaacagatac aaggcgtgac tggatatttc tctctcgttc ctaacaacag
caacgaagaa 120 gaaaaagaat cattactaac aatcaatgag tatgagagct
ggacacatga cggcaacaat 180 ggcggctttt acatgcccta ggtttatgac
tagcatcaga tacacgaagc aaattaagtg 240 caacgctgct aaaagccagc
tagtcgttaa acaagagatt gaggaggaag aagattatgt 300 gaaagccggt
ggatcggagc tgctttttgt tcaaatgcaa cagaataagt ccatggatgc 360
acagtctagc ctatcccaaa agctcccaag ggtaccaata ggaggaggag gagacagtaa
420 ctgtatactg gatttggttg gatcct 446 8 393 DNA Tagetes erecta sense
fragment 8 aagctttgga ttagcactga ttgtccagat ggatattgag gggacccgca
cattcttccg 60 gactttcttc cgcttgccca catggatgtg gtgggggttt
cttggatctt cgttatcatc 120 aactgacttg ataatatttg cgttttacat
gtttatcata gcaccgcata gcctgagaat 180 gggtctggtt agacatttgc
tttctgaccc gacaggagga acaatgttaa aagcgtatct 240 cacgatataa
ataactctag tcgcgatcag tttagattat aggcacatct tgcatatata 300
tatgtataaa ccttatgtgt gctgtatcct tacatcaaca cagtcattaa ttgtatttct
360 tggggtaatg ctgatgaagt attttctgtc gac 393 9 397 DNA Tagetes
erecta AntisenseFragment 9 gaattctctt tggattagca ctgattgtcc
agatggatat tgaggggacc cgcacattct 60 tccggacttt cttccgcttg
cccacatgga tgtggtgggg gtttcttgga tcttcgttat 120 catcaactga
cttgataata tttgcgtttt acatgtttat catagcaccg catagcctga 180
gaatgggtct ggttagacat ttgctttctg acccgacagg aggaacaatg ttaaaagcgt
240 atctcacgat ataaataact ctagtcgcga tcagtttaga ttataggcac
atcttgcata 300 tatatatgta taaaccttat gtgtgctgta tccttacatc
aacacagtca ttaattgtat 360 ttcttggggt aatgctgatg aagtattttc tggatcc
397 10 1537 DNA Artificial sequence Description of synthetic
sequence promoter 10 gagctctaca aattagggtt actttattca ttttcatcca
ttctctttat tgttaaattt 60 tgtacattta ttcaataata ttatatgttt
attacaaatt ctcactttct tattcatacc 120 tattcactca agcctttacc
atcttccttt tctatttcaa tactatttct acttcatttt 180 tcacgttttt
aacatctttc tttatttctt gtccacttcg tttagggatg cctaatgtcc 240
caaatttcat ctctcgtagt aacacaaaac caatgtaatg ctacttctct ctacattttt
300 aatacaaata aagtgaaaca aaatatctat aaataaacaa atatatatat
tttgttagac 360 gctgtctcaa cccatcaatt aaaaaatttt gttatatttc
tactttacct actaaatttg 420 tttctcatat ttacctttta acccccacaa
aaaaaaatta taaaaaagaa agaaaaaagc 480 taaaccctat ttaaatagct
aactataaga tcttaaaatt atcctcatca gtgtatagtt 540 taattggtta
ttaacttata acattatata tctatgacat atactctctc ctagctattt 600
ctcacatttt ttaacttaag aaaatagtca taacatagtc taaaattcaa acatccacat
660 gctctaattt gattaacaaa aagttagaaa tatttattta aataaaaaag
actaataaat 720 atataaaatg aatgttcata cgcagaccca tttagagatg
agtatgcttt cacatgctga 780 gattattttc aaaactaagg ttgtagcaat
attaaatcaa taaaattatt ataaataaca 840 aaattaacct gctcgtgttt
gctgtatatg ggaggctaca aaataaatta aactaaagat 900 gattatgttt
tagacatttt ttctatctgt attagtttat acatattaat tcaggagctg 960
cacaacccaa ttctattttc gttccttggt ggctgggttt ctcacaaggt tcaatagtca
1020 atattaggtt ttattggact tttaatagta tcaaacaaat ctatgtgtga
acttaaaaat 1080 tgtattaaat atttagggta acctgttgcc gtttttagaa
taatgtttct tcttaataca 1140 cgaaagcgta ttgtgtattc attcatttgg
cgcctcacat gcttcggttg gctcgcttta 1200 gtctctgcct tctttgtata
ttgtactccc cctcttccta tgccacgtgt tctgagctta 1260 acaagccacg
ttgcgtgcca ttgccaaaca agtcatttta acttcacaag gtccgatttg 1320
acctccaaaa caacgacaag tttccgaaca gtcgcgaaga tcaagggtat aatcgtcttt
1380 ttgaattcta tttctcttta tttaatagtc cctctcgtgt gatagttttt
aaaagatttt 1440 taaaacgtag ctgctgttta agtaaatccc agtccttcag
tttgtgcttt tgtgtgtttt 1500 gtttctctga tttacggaat ttggaaataa taagctt
1537 11 734 DNA artificial sequence Description of synthetic
sequence variation 11 ctaacaatca atgagtagag agctggacac atgacggcaa
caatggcggc ttttacatgc 60 cctaggttta tgactagcat cagatacacg
aagcaaatta agtgcaacgc tgctaaaagc 120 cagctagtcg ttaaacaaga
gattgaggag gaagaagatt atgtgaaagc cggtggatcg 180 gagctgcttt
ttgttcaaat gcaacagaat aagtccatgg atgcacagtc tagcctatcc 240
caaaaggtca ctccagactt aattgcttat aaataaataa atatgttttt taggaataat
300 gatatttaga tagattagct atcacctgtg ctgtggtgtg cagctcccaa
gggtcttacc 360 gatagtaaaa tcgttagtta tgattaatac ttgggaggtg
ggggattata ggctttgttg 420 tgagaatgtt gagaaagagg tttgacaaat
cggtgtttga atgaggttaa atggagttta 480 attaaaataa agagaagaga
aagattaaga gggtgatggg gatattaaag acggscaata 540 tagtgatgcc
acgtagaaaa aggtaagtga aaacatacaa cgtggcttta aaagatggct 600
tggctgctaa tcaactcaac tcaactcata tcctatccat tcaaattcaa ttcaattcta
660 ttgaatgcaa agcaaagcaa aggttgtttg ttgttgttgt tgagagacac
tccaatccaa 720 acagatacaa ggcg 734 12 280 DNA artificial sequence
Description of synthetic sequence variation 12 gtcgagtatg
gagttcaatt aaaataaaga gaagaraaag attaagaggg tgatggggat 60
attaaagacg gccaatrtag tgatgccacg taagaaaaag gtaagtgaaa acatacaacg
120 tggctttaaa agatggcttg gctgctaatc aactcaactc aactcatatc
ctatccattc 180 aaattcaatt caattctatt gaatgcaaag caaagcaaag
caaaggttgt ttgttgttgt 240 tgttgagaga cactccaatc caaacagata
caaggcgtga 280 13 358 DNA Tagetes erecta promoter (1)..(358)
(sense) promotor 13 aagcttaccg atagtaaaat cgttagttat gattaatact
tgggaggtgg gggattatag 60 gctttgttgt gagaatgttg agaaagaggt
ttgacaaatc ggtgtttgaa tgaggttaaa 120 tggagtttaa
ttaaaataaa gagaagagaa agattaagag ggtgatgggg atattaaaga 180
cggccaatat agtgatgcca cgtagaaaaa ggtaagtgaa aacatacaac gtggctttaa
240 aagatggctt ggctgctaat caactcaact caactcatat cctatccatt
caaattcaat 300 tcaattctat tgaatgcaaa gcaaagcaaa gcaaaggttg
tttgttgttg ttgtcgac 358 14 361 DNA Tagetes erecta promoter
(1)..(361) (antisense) promotor 14 ctcgagctta ccgatagtaa aatcgttagt
tatgattaat acttgggagg tgggggatta 60 taggctttgt tgtgagaatg
ttgagaaaga ggtttgacaa atcggtgttt gaatgaggtt 120 aaatggagtt
taattaaaat aaagagaaga gaaagattaa gagggtgatg gggatattaa 180
agacggccaa tatagtgatg ccacgtagaa aaaggtaagt gaaaacatac aacgtggctt
240 taaaagatgg cttggctgct aatcaactca actcaactca tatcctatcc
attcaaattc 300 aattcaattc tattgaatgc aaagcaaagc aaagcaaagg
ttgtttgttg ttgttggatc 360 c 361 15 28 DNA artificial sequence
Description of synthetic sequence Primer 15 gagctcactc actgatttcc
attgcttg 28 16 37 DNA artificial sequence Description of synthetic
sequence Primer 16 cgccgttaag tcgatgtccg ttgatttaaa cagtgtc 37 17
34 DNA artificial sequence Description of synthetic sequence Primer
17 atcaacggac atcgacttaa cggcgtttgt aaac 34 18 25 DNA artificial
sequence Description of synthetic sequence Primer 18 taagcttttt
gttgaagaga tttgg 25 19 23 DNA artificial sequence Description of
synthetic sequence Primer 19 gaaaatactt catcagcatt acc 23 20 28 DNA
artificial sequence Description of synthetic sequence Primer 20
gtcgactacg taagtttctg cttctacc 28 21 26 DNA artificial sequence
Description of synthetic sequence Primer 21 ggatccggtg atacctgcac
atcaac 26 22 28 DNA artificial sequence Description of synthetic
sequence Primer 22 aagcttgcac gaggcaaagc aaaggttg 28 23 29 DNA
artificial sequence Description of synthetic sequence Primer 23
gtcgacaacc aaatccagta tacagttac 29 24 30 DNA artificial sequence
Description of synthetic sequence Primer 24 aggatccaac caaatccagt
atacagttac 30 25 28 DNA artificial sequence Description of
synthetic sequence Primer 25 gaattcgcac gaggcaaagc aaaggttg 28 26
25 DNA artificial sequence Description of synthetic sequence Primer
26 aagctttgga ttagcactga ttgtc 25 27 29 DNA artificial sequence
Description of synthetic sequence Primer 27 gtcgacagaa aatacttcat
cagcattac 29 28 29 DNA artificial sequence Description of synthetic
sequence Primer 28 ggatccagaa aatacttcat cagcattac 29 29 27 DNA
artificial sequence Description of synthetic sequence Primer 29
gaattctctt tggattagca ctgattg 27 30 23 DNA artificial sequence
Description of synthetic sequence Primer 30 cgccttgtat ctgtttggat
tgg 23 31 24 DNA artificial sequence Description of synthetic
sequence Primer 31 ctaacaatca atgagtatga gagc 24 32 26 DNA
artificial sequence Description of synthetic sequence Primer 32
agagcaaggc cagcaggacc acaacc 26 33 26 DNA artificial sequence
Description of synthetic sequence Primer 33 ccttgggagc ttttgggata
ggctag 26 34 26 DNA artificial sequence Description of synthetic
sequence Primer 34 tcacgccttg tatctgtttg gattgg 26 35 15 DNA
artificial sequence Description of synthetic sequence Primer 35
gtcgagtatg gagtt 15 36 28 DNA artificial sequence Description of
synthetic sequence Primer 36 aagcttaccg atagtaaaat cgttagtt 28 37
31 DNA artificial sequence Description of synthetic sequence Primer
37 ctcgagctta ccgatagtaa aatcgttagt t 31 38 28 DNA artificial
sequence Description of synthetic sequence Primer 38 gtcgacaaca
acaacaaaca acctttgc 28 39 28 DNA artificial sequence Description of
synthetic sequence Primer 39 ggatccaaca acaacaaaca acctttgc 28 40
28 DNA artificial sequence Description of synthetic sequence Primer
40 gtcgactttt tgttgaagag atttggtg 28 41 28 DNA artificial sequence
Description of synthetic sequence Primer 41 ctcgagactc actgatttcc
attgcttg 28 42 22 DNA artificial sequence Description of synthetic
sequence Primer 42 gagctctaca aattagggtt ac 22 43 23 DNA artificial
sequence Description of synthetic sequence Primer 43 aagcttatta
tttccaaatt ccg 23
* * * * *