U.S. patent application number 15/455294 was filed with the patent office on 2017-08-31 for trichome-specific transcription factor modulating terpene biosynthesis.
This patent application is currently assigned to Keygene N.V.. The applicant listed for this patent is Keygene N.V.. Invention is credited to Michael Albertus HARING, Robert Cornelis SCHUURINK, Eleni SPYROPOULOU.
Application Number | 20170247713 15/455294 |
Document ID | / |
Family ID | 46456980 |
Filed Date | 2017-08-31 |
United States Patent
Application |
20170247713 |
Kind Code |
A1 |
SCHUURINK; Robert Cornelis ;
et al. |
August 31, 2017 |
TRICHOME-SPECIFIC TRANSCRIPTION FACTOR MODULATING TERPENE
BIOSYNTHESIS
Abstract
The present invention relates to identification and isolation of
zinc finger transcription factor in tomato that specifically
expresses in glandular trichomes of Solanum lycopersicum cultivar
Moneymaker and binds to the promoters of the genes encoding Terpene
Synthase 5 (also known as Monoterpene Synthase1) and Terpene
Synthase 11 (also known as Sesquiterpene Synthase 1). The invention
provides the isolated, recombinant or synthetic polynucleotides
encoding the polypeptide sequences of SEQ ID NO:2 and variants and
fragments thereof. The invention also provides constructs, vectors,
host cells and plants genetically modified to contain the
polynucleotides of the invention. The methods for producing plants
with altered levels of terpenes, including transformed and mutant
plants, are also provided.
Inventors: |
SCHUURINK; Robert Cornelis;
(Wageningen, NL) ; HARING; Michael Albertus;
(Wageningen, NL) ; SPYROPOULOU; Eleni;
(Wageningen, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Keygene N.V. |
Wageningen |
|
NL |
|
|
Assignee: |
Keygene N.V.
Wageningen
NL
|
Family ID: |
46456980 |
Appl. No.: |
15/455294 |
Filed: |
March 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14124629 |
Mar 6, 2014 |
9598473 |
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PCT/NL2012/050403 |
Jun 8, 2012 |
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15455294 |
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61495399 |
Jun 10, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 5/007 20130101;
A01H 5/12 20130101; C07K 14/415 20130101; A01H 1/04 20130101; Y02A
40/146 20180101; C12N 15/8286 20130101; Y02A 40/162 20180101; A01H
5/00 20130101; C12N 15/8243 20130101; A01H 1/02 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415; A01H 1/02 20060101
A01H001/02; C12P 5/00 20060101 C12P005/00; A01H 1/04 20060101
A01H001/04; A01H 5/00 20060101 A01H005/00 |
Claims
1. An isolated, synthetic or recombinant nucleic acid sequence
selected from the group comprising: a) a nucleic acid sequence of
SEQ ID NO: 1; b) a nucleic acid sequence that encodes a polypeptide
comprising an amino acid sequence of SEQ ID NO: 2 or an amino acid
sequence that is at least 60% identical to the amino acid sequence
of SEQ ID NO:2; c) a nucleic acid sequence that is at least 60%
identical to the nucleic acid sequences of (a) or (b), and encodes
a transcription factor that regulates terpene biosynthesis; d) a
nucleic acid sequence encoding a polypeptide comprising an amino
acid sequence which has been derived, by way of one or more amino
acid substitutions, deletions or insertions, from the polypeptide
having the amino acid sequence of SEQ ID NO:2 and which polypeptide
is functionally equivalent to the polypeptide consisting of the
amino acid sequence of SEQ ID NO: 2; and e) a nucleic acid sequence
that hybridizes under stringent conditions to the nucleic acid
sequences of (a), (b), (c), or (d), or f) a nucleic acid sequence
that hybridizes under stringent conditions to the complement of the
nucleic acid sequences of (a), (b), (c), or (d) and encodes a
polypeptide capable of regulating terpene biosynthesis in a
plant.
2. A chimeric gene comprising a nucleic acid sequence according to
claim 1.
3. A vector comprising a chimeric gene according to claim 2.
4. A host cell comprising a vector according to claim 3.
5. A polypeptide with DNA binding activity that regulates terpene
biosynthesis in a plant having an amino acid sequence selected from
the group of: (a) an amino acid sequence of SEQ ID NO: 2; (b) an
amino acid sequence which has been derived, by way of one or more
amino acid substitutions, deletions or insertions, from the
polypeptide having the amino acid sequence of SEQ ID NO:2 and which
polypeptide is functionally equivalent to the polypeptide
consisting of the amino acid sequence of SEQ ID NO: 2; and (c) an
amino acid sequence that is at least 60% identical to the amino
acid sequence according to (a).
6. The polypeptide according to claim 5 capable of binding a
nucleic acid sequence of a promoter that is operably linked to at
least one gene involved in terpene biosynthesis in the plant.
7. The polypeptide according to claim 6 wherein the gene is
selected from the group comprising a Terpene Synthase 5 (TPS5) and
a Terpene Synthase 11 (TPS11).
8. The polypeptide according to claim 6 wherein the promoter
comprises a trichome-specific promoter.
9. A method for increasing production of at least one terpene in a
plant, comprising modifying the plant to have an increased copy
number of a nucleic acid sequence of SEQ ID NO:1, or a nucleic acid
sequence with at least 60% identity to the nucleic acid sequence of
SEQ ID NO: 1, compared to a non-modified plant of the same genetic
background, thereby increasing the production of the at least one
terpene in the modified plant
10. A method for increasing production of at least one terpene in a
plant comprising: (a) contacting a plant cell with a composition
comprising a vector comprising a nucleic acid sequence having at
least 60% identity to SEQ ID NO:1 or a functional fragment thereof;
(b) selecting the plant cell transformed with the vector wherein
the plant cell overexpresses the nucleic acid sequence or the
fragment wherein overexpression results in an increased level of
the at least one terpene in the cell compared to a non-transformed
plant cell; and (c) regenerating the plant from the transformed
cell of (b) wherein the plant has the increased level of the at
least one terpene compared to a non-transformed plant of the same
genetic background.
11. A method for increasing production of at least one terpene in a
population of plants by selectively breeding the plant of claim 8
to produce the population having the increased level of the at
least one terpene.
12. A method for reducing production of at least one terpene in a
plant, comprising the step of impairing expression of functional
TF19(6) protein in a plant via genetic modification, and/or the
step of reducing binding of functional TF19(6) protein to the
promoter sequence of the nucleic acid sequence coding for Terpene
Synthase 5 (TPS5) and/or Terpene Synthase 11 (TPS11).
13. A method according to claim 12, said method comprising the step
of modifying the plant to have at least one mutation in a nucleic
acid sequence of SEQ ID NO: 1 or a sequence with at least 60%
identity to the nucleic acid sequence of SEQ ID NO: 1, or a nucleic
acid sequence of SEQ ID NO: 3 or a sequence with at least 60%
identity to the nucleic acid sequence of SEQ ID NO: 3, wherein the
mutation results in a decrease of expression of the amino acid
sequence of SEQ ID NO:2 or a variant thereof having at least 60%
sequence identity to the amino acid sequence of SEQ ID NO:2, and/or
a loss of function of the amino acid sequence of SEQ ID NO:2 or a
variant thereof having at least 60% sequence identity to the amino
acid sequence of SEQ ID NO:2 compared to a non-modified plant of
the same genetic background, thereby reducing the level of the at
least one terpene in the modified plant.
14. The method according to claim 13 wherein the mutation comprises
a substitution, a deletion, an insertion or an addition of at least
one nucleotide.
15. The method according to claim 12 wherein the step of impairing
expression of functional TF19(6) protein in a plant via genetic
modification comprises modifying the plant to have an increased
level of RNA having a nucleic acid sequence at least in portion
complementary to a nucleic acid sequence of SEQ ID NO: 1, or a
nucleic acid sequence with at least 60% identity to the nucleic
acid sequence of SEQ ID NO: 1, or a nucleic acid sequence of SEQ ID
NO: 3 or a nucleic acid sequence with at least 60% identity to the
nucleic acid sequence of SEQ ID NO: 3, compared to a non-modified
plant of the same genetic background, thereby decreasing production
of the at least one terpene in the modified plant.
16. A method for reducing terpene levels in a population of plants,
comprising: (a) providing at least one plants within a population
of plants comprising a mutation in a gene having a nucleic acid
sequence of SEQ ID NO: 1 or a sequence with at least 60% identity
to the nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid
sequence of SEQ ID NO: 3 or a sequence with at least 60% identity
to the nucleic acid sequence of SEQ ID NO: 3; and (b) selectively
breeding the at least one mutant plant of (a) to produce the
population of plants having the reduced terpene levels.
17. The method according to claim 9 wherein the terpene comprises
at least one of a monoterpene and a sesquiterpene that repel
insects.
18. The method according to claim 17 wherein the monoterpene
comprises at least one compound selected from the group of:
linalool, .beta.-myrcene, para-cymene, .gamma.-terpinene,
.alpha.-terpinene, and .alpha.-phellandrene.
19. The method according to claim 17 wherein the sesquiterpene
comprises at least one compound selected from the group of:
neralidol, germacrene, R-curcumine, S-curcumine and
7-epizingiberene.
20. The method according to claim 9 wherein the terpene comprises
at least one of a monoterpene and a sesquiterpene that attract
insects.
21. The method according to claim 20 wherein the monoterpene
comprises at least one compound selected from the group of:
.beta.-phellandrene, limonene and 2-carene.
22. The method according to claim 20 wherein the sesquiterpene
comprises at least .beta.-caryophyllene.
23. The method according to claim 17 wherein the insects comprise
sap-sucking insects and blood-sucking insects.
24. The method according to claim 23 wherein the sap-sucking
insects comprise psyllids, whiteflies, aphids, mealybugs, plant
hoppers and scale insects.
25. The method according to claim 24, wherein the sap-sucking
insects further comprise thrips, mites and leaf hoppers.
26. The method according to claim 23 wherein the blood sucking
insects comprise mosquito, ticks and midges.
27. The method according to claim 9 wherein the plant is at least
one crop plant selected from the group of: tomato, pepper,
eggplant, lettuce, oilseed rape, broccoli, cauliflower, cabbage
crops, cucumber, melon, pumpkin, squash, peanut, soybeans, corn,
cotton, beans, cassava, potatoes, sweet potatoes and okra.
28. The method according to claim 27 wherein the plant comprises at
least one plant selected from a Solanaceae family.
29. The method according to claim 9 wherein the plant is at least
one ornamental plant selected from the group of: hibiscus,
poinsettia, lilies, iris, rose and petunia.
30. A plant obtainable by a method according to claim 9.
31. A plant comprising a chimeric gene according to claim 2.
32. The plant according to claim 30 wherein the plant belongs to
the Solanaceae family.
33. A tissue culture obtained from the plant of claim 30 wherein
the culture has enhanced production or secretion of at least one
terpene, terpene isomer, or terpene derivative.
34. A method for producing a terpene, terpene isomer, or terpene
analog comprising isolating the terpene, terpene isomer or terpene
analog from the tissue culture of claim 33.
35. A method for a marker-assisted introgression of production of
at least one terpene into a plant comprising the steps of: (a)
identifying a difference in a gene encoding a polypeptide having an
amino acid sequence of SEQ ID NO:2 between a plant from the
Solanaceae family and a sexually compatible plant wherein the gene
with the difference comprises a molecular marker associated with
the presence of the at least one terpene in the plant; (b) making a
cross between the plant having the molecular marker and the
sexually compatible plant of (a); (c) screening a progeny resulting
from the cross for the presence of the molecular marker; (d)
identifying the plant within the progeny having the molecular
marker and thereby identifying the plant producing the at least one
terpene.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 14/124,629, filed Mar. 6, 2014, which is the
National Phase of International Application No. PCT/NL2012/050403
filed Jun. 8, 2012, published as WO 2012/169893, which claims
benefit of priority of U.S. Provisional Application 61/495,399,
filed Jun. 10, 2011. The contents of these applications are herein
incorporated by reference in their entirety.
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-WEB and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 9, 2017, is named 085342-1600SequenceListing.txt and is 24
KB.
FIELD OF THE INVENTION
[0003] The present invention relates to a zinc finger transcription
factor that regulates terpene biosynthesis in plants, a nucleic
acid molecule that encodes the transcription factor and methods of
use of the transcription factor for producing plants with altered
terpene content.
BACKGROUND OF THE INVENTION
[0004] Terpenes constitute a large group of structurally diverse
molecules synthesized naturally by organisms as diverse as
bacteria, fungi, plants and animals. Much research has been
concentrated on the biochemistry and biological functions of
terpenes or their derivatives for potential commercial exploitation
(Gershenzon and Dudareva 2007 Nat Chem Biol 3:408). The result of
these studies is a variety of terpene-based products ranging from
pharmaceuticals, such as anti-cancer drug paclitaxel and
anti-malaria drug artemisin, to fragrances and aroma ingredients,
such as menthol and patchuol.
[0005] In the field of plant breeding, there is an interest in
terpene secondary metabolites produced by many plant species to
resist pathogens, to repel or kill pests or to attract beneficial
organisms, e.g., predators or parasitoids of pest insects or plant
pollinators, or other organisms. Wild plant species frequently
produce beneficial secondary metabolites lacking in their
cultivated relatives, and therefore are an important source of
traits for introgression into cultivated varieties. It is known
that secondary metabolites, such as volatile terpenoid compounds,
can directly influence insect behavior (Bruce et al., 2005 Trends
Plant Sci 10:269-274). For instance, methyl ketones and
sesquiterpene carboxylic acids identified in Solanum habrochaites
and acyl-glucose esters from Solanum pennellii were found to be
toxic to different insect classes, such as Lepidoptera, mites, and
aphids (Williams et al., 1980 Science 20:888; Goffreda et al., 1990
Plant Cell 2:643; Juvik et al., 1994 J Econ Entomol 87:482;
Frelichowski and Juvik, 2001 J Econ Entomol 94:1249). Often mono-
and sesquiterpene hydrocarbons, sesquiterpene acids, methylketones
and sugar esters are accumulated in plants in specialized organs
such as glandular trichomes on stems and leaves. Several studies
correlated the density of glandular trichomes with levels of
resistance to pest insects, e.g., maize earworm Heliothis zea and
Colorado potato beetle (Kauffman and Kennedy, 1989 J Chem Ecol
15:1919-1930; Antonius et al., 2001 J Environ Sci Health B
36:835-848; Antonius et al., 2005 J Environ Sci Health B
40:619-631). The methylketones 2-undecanone and 2-tridecanone
accumulated in glandular trichomes of S. habrochaites were shown to
be toxic to larvae of Colorado potato beetle and adult whiteflies
B. tabaci, respectively (Antonius et al., 2005 J Environ Sci Health
B 40:619-631). The myrtle oil, including the monoterpene linalool
among its essential components, was shown to have an insecticidal
effect on bean weevils, Acanthoscelides obtectus Say (Coleoptera:
Bruchidae) (Ayvaz et al., 2010 J Insect Sci 10: 1536-2442). The
sesquiterpenes zingiberene and curcumene, and the monoterpenes
.rho.-cymene, .alpha.-terpinene, and .alpha.-phellandrene from wild
tomato S. habrochaites and S. pennellii, respectively, were shown
to have insecticidal properties (Bleeker et al., 2009 Plant
Physiol. 151:925). Bio-assays have demonstrated that the
sesquiterpenes 7-epizingiberene and its derivative R-curcumene
repelled adult whiteflies from landing on tomato plants (Bleeker et
al., 2011 Phytochemistry 72:68), and that plants with endogenous
production of zingiberene showed resistance to Tuta absoluta. (De
Azavedo et al., 2003 Euphitica 134:247-251).
[0006] Genetic inheritance of the genes associated with development
of different types of glandular trichomes and production of
zingiberene was studied in interspecific crosses between S.
lycopersicum, a cultivated tomato which does not produce
zingiberene, and S. habrochaites, a wild species with high
zingiberene production. In F2 plants from these crosses,
zingiberene content correlated with resistance to B. tabaci. This
study suggested feasibility of breeding plants with high levels of
zingiberene, 2-tridecanone, and/or acylsugars, which would lead to
high levels of resistance to whiteflies (Freitas et al., 2002
Euphytica 127: 275-287). However, programs on introgression of
useful traits into cultivated varieties are time consuming and
costly, therefore production of secondary metabolites in plants
lacking them or elevating levels of these metabolites in plants
synthesizing them--albeit in insufficient levels--is an attractive
goal.
[0007] The biosynthesis of terpenes in plants has been extensively
studied and many genes coding for the pathways steps from
precursors to final products were discovered (Wither and Keeling,
2007 Agro Microbial Biotechnology 73:980-990; Sallaud et al., 2009
Plant Cell 31:301).
[0008] Due to the widespread infestation of crop and ornamental
plant species with pest insects such as B. tabaci and the
greenhouse whitefly Trialeurodes vaporarium, resulting in great
economic losses, means of regulating plant natural defense
molecules to repel pests has received a renewed interest of
scientists and plant breeders.
[0009] It is known that manipulation of transcription factors can
regulate complex pathways in animals and plants involving numerous
target genes. This may result in increased expression of useful
compounds. Alternatively, blocking transcription factors may lead
to decreased or completely suppressed production of undesirable
compounds and/or removal of unwanted traits.
[0010] Several transcription factors controlling genes involved in
plant secondary metabolism were identified, cloned and showed high
efficiency in regulating complex metabolic pathways. For instance,
the transcription factor WRKY was shown to regulate
.delta.-Cadenine Synthase A, a sesquiterpene synthase that
catalyzes the first step of pathway leading to production of
gossypol in cotton (Xu et al., 2004 Plant Physiol 135:507-515).
[0011] Moreover, while overexpression of individual genes of the
biosynthetic pathways was shown to provide limited success,
perhaps, due to poor substrate availability, genome-wide expression
of the flavonol-specific transcription factor, AtMYB12, in tobacco
not only regulated the phenylpropanoid pathway, but also modulated
other metabolic pathways that led to increased flux availability to
this pathway, and eventually to an increased resistance against
Spodopter lituralis and Helicoverpa armigera insects (Misra et al.,
2010 Plant Physiol 152: 2258-2268).
[0012] MYB transcription factors have been indicated also to
activate multiple enzymes required for production of
glucosinolates, crucifer-specific secondary metabolites, in
Arabidopsis. MYB51 was shown to activate the indolic glucosinolates
biosynthesis and confer enhanced resistance to the herbivorous pest
Spodoptera exigua in plants overexpressing it (Gigolashvili et al.,
2007 Plant J 50: 886-901). Other MYB transcription factors, such as
MYB76, MYB28 and MYB29, are shown to regulate enzymes involved in
the production of aliphatic glucosinolates (Gigolashvili et al.,
2007 Plant J 51: 247-261; Gigolashvili et al., 2008 New Phytol
177:627-642).
[0013] There is a need in the art to provide transcription factors
regulating terpene biosynthesis, in particular transcription
factors that have an effect on the biosynthesis of mono- or
sesquiterpenes in plants or other organisms leading to the
production of terpene compounds that repel or attract insects, or
other organisms.
SUMMARY OF THE INVENTION
[0014] An embodiment of the invention herein provides an isolated,
synthetic or recombinant nucleic acid sequence selected from the
group including: a) a nucleic acid sequence of SEQ ID NO: 1; b) a
nucleic acid sequence that encodes a polypeptide having an amino
acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at
least 60% identical to the amino acid sequence of SEQ ID NO:2; c) a
nucleic acid sequence that is at least 60% identical to the nucleic
acid sequences of (a) or (b), and encodes a transcription factor
that regulates terpene biosynthesis; d) a nucleic acid sequence
encoding a polypeptide comprising which has been derived, by way of
one or more amino acid substitutions, deletions or insertions, from
the polypeptide having the amino acid sequence of SEQ ID NO: 2 and
which polypeptide is functionally equivalent to the polypeptide
consisting of the amino acid sequence of SEQ ID NO: 2; e) a nucleic
acid sequence that hybridizes under stringent conditions to the
nucleic acid sequences of (a), (b), (c), or (d); and f) a nucleic
acid sequence that hybridizes under stringent conditions to the
(optionally reverse) complement of the nucleic acid sequences of
(a), (b), (c), or (d) and that encodes the transcription factor
that regulates terpene biosynthesis. Further embodiments include a
chimeric gene comprising such nucleic acid sequence, a vector
comprising such nucleic acid sequence or such chimeric gene, and a
host cell comprising such chimeric gene or such vector.
[0015] A related embodiment of the invention provides a polypeptide
with DNA binding activity that regulates terpene biosynthesis in a
plant, such polypeptide having an amino acid sequence selected from
the group of: (a) an amino acid sequence of SEQ ID NO: 2; (b) the
amino acid sequence according to (a) in which at least one amino
acid is substituted, deleted, inserted or added and wherein the
polypeptide is functionally equivalent to the polypeptide
consisting of the amino acid sequence of SEQ ID NO: 2; and (c) an
amino acid sequence that is at least 60% identical to the amino
acid sequence according to (a).
[0016] The polypeptide (i.e., transcription factor) of the
invention herein is capable of binding a nucleic acid sequence of a
promoter that is operably linked to at least one gene involved in
terpene biosynthesis in the plant. For example, the gene is
selected from the group comprising a Terpene Synthase 5 (TPS5) and
a Terpene Synthase 11 (TPS11). For example, the promoter comprises
a trichome-specific promoter.
[0017] An alternative embodiment of the invention provides a method
for increasing the production of at least one terpene in a plant,
involving up-regulating a transcription factor that positively
regulates at least one gene involved in terpene biosynthesis in the
plant, such gene preferably being selected from the group
comprising TPS5 and TPS11. For example, up-regulating the
transcription factor involves modifying the plant to have an
increased copy number of a nucleic acid sequence of SEQ ID NO:1, or
a sequence with at least 60% identity to the nucleic acid sequence
of SEQ ID NO: 1, compared to a non-modified plant of the same
genetic background, thereby increasing the level of the at least
one terpene in the modified plant.
[0018] In a preferred embodiment, a method is provided for
increasing the level of at least one terpene in a plant involving:
(a) contacting a plant cell or plant protoplast with a composition
that includes a vector having a nucleic acid sequence of SEQ ID
NO:1 or a fragment thereof with at least 60% identity to the
sequence of SEQ ID NO:1; (b) selecting the plant cell or plant
protoplast transformed with the vector wherein the plant cell or
plant protoplast overexpresses the nucleic acid sequence or the
fragment thereof so that overexpression results in an increased
level of the at least one terpene in the cell compared to a
non-transformed plant cell or plant protoplast; and (c)
regenerating the plant from the transformed cell or protoplast
wherein the plant has an increased level of the at least one
terpene compared to a non-transformed plant of the same genetic
background. Within the scope of the invention is also a method for
increasing production of at least one terpene in a population of
plants by selectively breeding the transformed plant to produce the
population of transformed plants having the increased level of the
at least one terpene compared to a non-transformed plants of the
same genetic background.
[0019] In yet another embodiment, a method is provided for reducing
production of at least one terpene in a plant that involves
down-regulating a transcription factor that positively regulates at
least one gene involved in terpene biosynthesis in the plant,
wherein the gene preferably is selected from the group comprising
TPS5 and TPS11. For example, down-regulating the transcription
factor may involve modifying the plant to have a mutation in a
nucleic acid sequence of SEQ ID NO: 1 or a sequence with at least
60% identity to the nucleic acid sequence of SEQ ID NO: 1, or a
nucleic acid sequence of SEQ ID NO: 3 or a sequence with at least
60% identity to the nucleic acid sequence of SEQ ID NO: 3, wherein
the mutation results in a decrease of the level of the
transcription factor or a loss of function of the transcription
factor, compared to a non-modified plant of the same genetic
background, thereby reducing the level of the at least one terpene
in the modified plant. For example, the mutation may include a
substitution, a deletion, an insertion or an addition of at least
one nucleotide. In a related embodiment of the method,
down-regulating the transcription factor may involve modifying the
plant to have an increased level of RNA having a nucleic acid
sequence at least in portion complementary to a nucleic acid
sequence of SEQ ID NO: 1, or a sequence with at least 60% identity
to the nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid
sequence of SEQ ID NO: 3 or a sequence with at least 60% identity
to the nucleic acid sequence of SEQ ID NO: 3, compared to a
non-modified plant of the same genetic background, thereby
decreasing the level of the at least one terpene in the modified
plant.
[0020] In a particularly preferred embodiment, a method is provided
for reducing terpene levels in a population of plants, comprising
steps of: (a) providing at least one plant within a population of
plants comprising a mutation in a gene comprising a nucleic acid
sequence of SEQ ID NO: 1 or a nucleic acid sequence with at least
60% identity to the nucleic acid sequence of SEQ ID NO: 1, or a
nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence
with at least 60% identity to the nucleic acid sequence of SEQ ID
NO: 3; and (b) selectively breeding the at least one mutant plant
of (a) to produce the population of plants having the reduced
terpene levels.
[0021] In particular, the terpene of the method includes at least
one of a monoterpene and a sesquiterpene that repel insects. For
example, the monoterpene includes at least one compound selected
from the group of: linalool, .beta.-myrcene, para-cymene,
.gamma.-terpinene, .alpha.-terpinene, and .alpha.-phellandrene. For
example, the sesquiterpene comprises at least one compound selected
from the group of: nerolidol, germacrene, R-curcumene, S-curcumene
and 7-epizingiberene.
[0022] Alternatively, the terpene of the methods herein includes at
least a monoterpene and a sesquiterpene that attract insects. For
example, the monoterpene includes at least one compound selected
from the group of: .beta.-phellandrene, limonene and 2-carene. For
example, the sesquiterpene comprises at least
.beta.-caryophyllene.
[0023] Generally, the invention herein is concerned with attracting
or repelling insects including sap-sucking insects and
blood-sucking insects. For example, the sap-sucking insects include
psyllids, whiteflies, aphids, mealybugs, plant hoppers and scale
insects. For example, the blood sucking insects comprise mosquito,
ticks and midges. Insects including thrips, mites and leaf hoppers
are also within the scope of the invention.
[0024] Generally, the plant of the methods of the present invention
is at least one crop plant selected from the group of: tomato,
pepper, eggplant, lettuce, oilseed rape, broccoli, cauliflower,
cabbage crops, cucumber, melon, pumpkin, squash, peanut, soybeans,
corn, cotton, beans, cassava, potatoes, sweet potatoes and okra.
The plant also includes at least one plant selected from a
Solanaceae family. The methods herein may also be directed at at
least one ornamental plant selected from the group of: hibiscus,
poinsettia, lily, iris, rose and petunia.
[0025] An embodiment of the invention also provides a plant
obtainable or obtained by the methods described herein.
Additionally, the invention pertains to a plant comprising a
chimeric gene as provided herein. Such plant may be a genetically
engineered plant comprising a nucleic acid sequence of SEQ ID NO:1
and variants and fragments thereof. For example, the plant may
belong to the Solanaceae family.
[0026] A tissue culture initiated from the plants described herein,
e.g., a transformed or modified plant, also is within the scope of
the invention. Such tissue culture has enhanced production or
secretion of at least one terpene, terpene isomer, or terpene
analog.
[0027] A related embodiment provides a method for producing a
terpene, terpene isomer, or terpene analog involving isolating the
terpene, terpene isomer or terpene analog from the tissue culture
resulting from the transformed plant.
[0028] A final embodiment of the invention provides a method for a
marker-assisted introgression of a terpene into a plant including
the steps of: (a) identifying a difference in a gene encoding an
amino acid sequence of SEQ ID NO:2 between a plant from the
Solanaceae family and a sexually compatible plant wherein the gene
with the difference comprises a molecular marker associated with
presence of the terpene in the plant; (b) making a cross between
the plant having the molecular marker and the sexually compatible
plant; (c) screening a progeny resulting from the cross for the
presence of the molecular marker; and (d) identifying the plant
within the progeny having the molecular marker and thereby
identifying the plant producing the terpene.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a schematic drawing of a PJVII-GUS-sYFP1, a
pMON999 vector with a modified multiple cloning site. The S.
lycopersicum Terpene Synthase 5 (SITPS5), also referred to as
Monoterpene Synthase 1 (SIMTS1), promoter was cloned between SacI
and XbaI sites and .beta.-glucuronidase (GUS) fused to a yellow
fluorescent protein (sYFP1) was cloned between XbaI and BamHI
sites.
[0030] FIG. 2 is a set of bar graphs showing quantification of GUS
expression in isolated trichomes from stems of transgenic 5' SITPS5
promoter deletion plants and controls. Average relative GUS
activity for each promoter construct (full length (fl), 1045 bp,
805 bp, 612 bp, 408 bp, 207 bp), empty vector control and
untransformed Moneymaker plant. Trichome-specific activity is lost
at 408 bp and then partially restored at 207 bp. The 207 bp
fragment was chosen for the Y1H assay.
[0031] FIGS. 3A and 3B are a set of bar graphs showing results of
Quantitative Real Time PCR for a candidate gene 19(6).
[0032] FIG. 3A, panel A shows tissue-specific expression of the
gene in tomato Money maker leaves, whole stem including trichomes,
bald stem and isolated trichomes. The highest level of expression
was observed in the isolated trichomes.
[0033] FIG. 3B, panel B shows trichome expression of the gene in
control and jasmonic acid-sprayed plants (JA), (values corrected
for actin).
[0034] FIG. 4 is set of bar graphs showing average relative GUS
activity in N. benthamiana plants. Five week old N. benthamiana
leaves were co-infiltrated with A. tumefaciens GV3101 cultures
carrying various promoter:GUS reporter and the 35S:19(6) effector
construct. Average relative GUS activity normalized for
35S:luciferase activity is shown. Activities of these promoter:GUS
reporter constructs in N. benthamiana with a control 35S:RFP
effector construct were approximately 0.05. Enzymatic GUS activity
of the crude extract was determined spectrophotometrically using
4-methylumbelliferyl .beta.-D-glucuronide (MUG) as a substrate.
[0035] FIGS. 5A, 5B, 5C, 5D, 5E, and 5F show a genomic nucleic acid
sequence of SEQ ID NO: 3 encoding the transcription factor TF
19(6). The underlined capital letters herein denote exons and the
underlined small letters indicate introns. The capital letters in
bold show start and stop codons. The capital letters which are not
underlined indicate the putative TF19(6) 4 kb promoter region
upstream of the start codon and 2 kb '3 UTR region downstream of
the stop codon. The position of the sequences encoding the putative
zinc finger motifs are indicated in a light shade of gray.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention relates to a transcription factor,
referred to herein as TF19(6), that regulates terpene biosynthesis
in plants. The TF19(6) cDNA comprises a nucleic acid sequence of
1056 bp (SEQ ID NO:1) with a single open reading frame encoding a
polypeptide of 351 amino acids (SEQ ID NO:2).
[0037] Accordingly, the skilled artisan would understand that by
expressing the sequences of the present invention in a plant, one
may change the expression of one or more genes involved in terpene
biosynthesis. By affecting the expression of the genes, one may
alter a plant phenotype to include plants with an improved
resistance to insects or other pests and pathogens, or plants
attracting beneficial organisms, e.g., predators or parasitoids of
pest insects or plant pollinators, or other organisms.
[0038] The sequences of the present invention may originate from
any species, especially from plant species, or from any other
sources including recombinant or synthetic.
[0039] The transcription factors of the invention may also include
a DNA regulatory sequence that regulates expression of one or more
genes in a plant when a transcription factor having one or more
domain binding sites binds to the regulatory sequence of one or
more genes in the pathway.
[0040] The present invention relates also to methods for modifying
a plant phenotype by employing one or more polynucleotides or
polypeptides of the invention for altering the expression of one or
more genes of the terpene biosynthesis pathway. Alternatively, the
polynucleotides and peptides of the invention have a variety of
additional uses including, without limitation, use as substrates
for further reactions such as inducing mutations, performing PCR
reactions, use as substrates for cloning including digestion and
ligation reactions, identifying exogenous or endogenous modulators
of the transcription factors, use in the production of recombinant
protein, use as diagnostic probes for the presence of complementary
or partially complementary nucleic acids, or the like.
[0041] The terpene biosynthesis pathway refers to the pathways
leading to the formation of various terpene molecules. The terms
"terpenes" and "terpenoids" are used herein interchangeably, and
refer to hydrocarbons having a carbon skeleton derived from
isoprene units (C.sub.5H.sub.8). Terpenes are subdivided into
groups based on their carbon number and may be cyclic or acyclic
molecules. The five-, ten-, fifteen-, twenty- and thirty-carbon
terpenes are referred to as hemi-, mono-, sesqui-, di-, and
triterpenes, respectively.
[0042] For example, the term "monoterpenes" refers to a class of
terpenes that consists of two isoprene units and have the molecular
formula (C.sub.10H.sub.16). Monoterpenes include but are not
limited to cyclic monoterpenes, including myrcene, (Z)- and
(E)-ocimene, linalool, geraniol, nerol, citronellol, myrcenol,
geranial, citral a, neral, citral b, citronellal; monocyclic
monoterpenes, including limonene, .alpha.- and .gamma.-terpinene,
.alpha.- and .beta. phellandrene, terpinolene, menthol, carveol;
bicyclic monoterpenes including .alpha.-pinene, .beta.-pinene,
myrtenol, myrtenal, verbanol, verbanon, pinocarveol; and tricyclic
monoterpenes, including tricyclene.
[0043] As used herein, the term "sesquiterpenes" refers to a class
of terpenes that consists of three isoprene units and has the
molecular formula C.sub.15H.sub.24 Sesquiterpenes include but are
not limited to cyclic sesquiterpenes, including farnesene;
monocyclic, including zingiberene and humulene; bicyclic, including
caryophyllene, vetivazulene, guaizulene; tricyclic, including
longifolene, copaene, patchoulol.
[0044] As used herein the term "diterpenes" refers to terpenes
consisting of four isoprene units and have the molecular formula
C.sub.20H.sub.32. Known diterpenes include, for instance,
taxol.
[0045] As used herein, the term terpenes refers also to terpene
analogs, such as alcohols, aldehydes, ketons and esters, and
isomers, including stereoisomers and tautomers. Reference to
specific isomers herein such as .alpha.- and/or .beta.-isomers does
not preclude the skilled artisan to use other isomers and
appreciate that the other isomers or mixtures of isomers can
substitute for the isomer specifically mentioned, as long as these
are functional. For example, it was observed that transformed
potato and Arabidopsis plants overexpressing the Nerolidol Synthase
1 gene from strawberry emitted not only linalool but also linalool
derivatives including E-8-hydroxy linalool, Z-8-hydroxy-linalool
and E-8-hydroxy-6,7-dihydrolinalool (Aharoni et al., 2006
Phytochemistry Review 5:49-58). Metabolic engineering of terpenoids
in plants by overexpressing enzymes catalyzing steps in the terpene
biosynthesis pathway was shown to be successful to generate
substantial levels of terpenoids (Lewinson et al., 2001 Plant
Physiol 127:1256-1264; Aharoni et al., 2003 Plant Cell
15:2866-2884; Lucker et al., 2004 Plant J 39: 135-145; Lucker et
al., 2004 Plant Physiol 134: 510-519; Aharoni et al., 2006
Phytochemistry Review 5:49-58). However, manipulation of the
expression of the individual genes of the pathways sometimes shows
limited success due to lack or poor availability of essential
precursors. Regulation of the expression of genes involved in
metabolic pathways using transcription factors was shown to
modulate additional pathways and lead to availability of precursors
(Misra et al., 2010 Plant Physiol 152:2258-2268).
[0046] Terpenes are synthesized from the common precursor
isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl
diphosphate (DMAPP) through two distinct biosynthesis pathways: the
mevalonate pathway found in the plants cytosol and in eukaryotes
and the deoxyxylulose-5-phosphate (DXP) pathway found in the plant
plastids and in prokaryotes. In the mevalonate pathway, the
biosynthesis of IPP starts from the conversion of three molecules
of acetyl-CoA to mevalonate followed by subsequent sequential
phosphorylation of mevalonate to diphosphomevalonate followed by
decarboxylation to produce IPP. In the DXP pathway, the production
of DXP starts from one molecule each of pyruvate and
glyceraldehyde-3-phosphate catalyzed by
1-deoxy-D-xylulose-5-phosphate synthase and results in the
production of IPP and DMAPP in contrast to the mevalonate pathway
where IPP is the sole product. Generally, sesquiterpenes are
synthesized from the relevant precursors through the mevalonate
pathway in the cytosol, and monoterpenes and diterpens are produced
through the DXP pathway in plastids. Exchange of precursors between
plastids and cytosol was also observed.
[0047] In both pathways, the IPP is further isomerized to DMAPP by
the IPP isomerase with subsequent formation of the higher molecular
weight acyclic polyprenyl pyrophosphate precursors by prenyl
transferases to form the acyclic pyrophosphate terpene precursors.
For example, these reactions produce ten-, fifteen-, and
twenty-carbon precursors geranyl-pyrophosphate (GPP),
farnesyl-pyrophosphate (FPP), geranylgeranyl-pyrophosphate (GGPP),
respectively. Prenyltransferases are divided into two main classes
based on stereochemistry of the double bond formed at each
elongation cycle. Prenyltransferases leading to the formation of
double bonds in cis-configuration are called cis- or
Z-prenyltransferases and prenyltransferases leading to the
formation of double bonds in trans-configuration are called trans-,
or E-prenyltransferases.
[0048] Terpene synthases (TPSs) are the enzymes catalyzing the
cyclisation of the acyclic precursors in the multi-step reactions
producing the carbon skeleton of terpene, monoterpene or
sesquiterpene compounds. For example, the initial step of the
catalyzed cyclisation may be the ionization of the diphosphate
group to form an allylic cation. The substrate then undergoes
isomerizations and rearrangements which can be controlled by the
active site of an enzyme. The product, for example, may be an
acyclic, mono-, di or tricyclic terpene.
[0049] It is known in the art that GPP and neryl diphospate (NPP),
the cis-isomer of GPP, are the substrates for monoterpene
biosynthesis, and that FPP and GGPP are the respective substrates
for sesquiterpene synthases and diterpene synthases (Chen et al.,
2011 Plant J 66:212-229; Schilmiller et al., 2009 Proc Natl Acad
Sci 106:10865-10870; Tholl 2006 Curr Opin Plant Biol 9:297-304;
Wang and Ohnuma, 2000 Biochim Biophys Acta 1529:33-48). Some TPSs
produce a single product, but many produce multiple products from
the same precursor, or can produce multiple compounds depending on
the precursor supplied (Van Schie et al., 2007 Plant Mol Biol
64:251-263).
[0050] Induced terpene biosynthesis was observed to correlate with
induced expression of terpene synthases (Navia-Gine et al., 2009
Plant Phys Biochem 47: 416-425; Herde et al., 2008 Plant Cell 20:
1152-1168).
[0051] Plants emit volatiles consisting of a mixture of mono- and
sesquiterpenes to repel pest insects or to attract beneficial
insects, e.g., predators or parasitoids of pest insects or plant
pollinators. Two terpene synthases TPS5 (formerly Monoterpene
Synthase 1, MTS1) and TPS4 (formerly Monoterpene Synthase 2, MTS2)
were identified in tomato (Van Schie et al., 2007 Plant Mol Biol
64:251-263; Falahara et al., manuscript in preparation; Bleeker et
al., manuscript in preparation). The study shows that TPS5 is
expressed in trichomes and that TPS5 protein catalyzes formation of
linalool from GPP in vitro. TPS5 expression in tomato trichomes was
observed to be low under normal condition and elevated after
induction with jasmonic acid (Van Schie et al., 2007 Plant Mol Biol
64:251-263; Kharel and Koyama, 2003 Nat Prod Rep 20:11-118). Van
Schie's study also found that TPS4 is expressed in stems, roots and
in trichomes and that this gene encodes an enzyme leading to the
formation of .beta.-myrcene, .beta.-phellandrene and sabinene from
GPP (Van Schie et al., 2007 Plant Mol Biol 64:251-263).
[0052] Although monoterpenes appear to dominate terpenes identified
in S. lycopersicum, majority of TPS genes mined from tomato genome
are sesquiterpene synthases (Falahara et al., manuscript in
preparation). Sequencing of cDNAs derived from trichomes of S.
lycopersicum and S. habrochaites resulted in identification of
multiple TPS sequences having similarities to known sesquiterpene
synthases (Bleeker et al., manuscript in preparation). For
instance, in S. lycopersicum database, transcripts for TPS9
(formerly germacrene C synthase; van Der Hoeven et al., 2000 Plant
Cell 12: 2283-2294), TPS12 (formerly
.beta.-caryophyIlene/.alpha.-humulene synthase; van Der Hoeven et
al., 2000 Plant Cell 12: 2283-2294), TPS15, TPS16, TPS17 and TPS31
(formerly LeVS2 because of similarity to vetispiradiene synthase
from potato) were identified. In S. habrochaites, cDNA sequences
were identified to have similarities to the TPS9, TPS12, TPS14,
TPS15 and TPS17.
[0053] The compounds which can be regulated by the transcription
factor of the present invention include, without limitation,
terpene compounds that attract insects such as .beta.-phellandrene,
limonene and 2-carene, referred to herein as "attractants". The
compounds regulated by the transcription factor of the present
invention also include, but are not limited, to terpenes that repel
insects such as R-curcumene, S-curcumene, .beta.-myrcene,
para-cymene, .gamma.-terpinene, zingiberene, 7-epizingiberene,
.alpha.-terpinene and .alpha.-phellandrene, referred to herein as
"repellents". These compounds are described in the patent
application WO 2010/099985 which is incorporated herein by
reference.
[0054] Additionally, sesquiterpenes present in trichomes of S.
lycopersicum cv. Moneymaker and S. hacbrochaites P1127826 can also
be regulated by the transcription factor herein (See Table below;
Bleeker et al., manuscript in preparation).
TABLE-US-00001 TABLE Sesquiterpenes present in trichomes of S.
lycopersicum cv. Moneymaker and S. habrochaites PI127826.
sesquiterpene S. lycopersicum S. habrochaites Azulene x
.alpha.-copaene x .beta.-elemene x caryophyllene x x
.gamma.-elemene x x Alpha humulene x x .beta.-farnesene x
.beta.-acoradiene x Curcumene x germacrene D x x Zingiberene x
Cuparene x .beta.-bisabolene x .beta.-sesquiphellandrene x
Valencene x germacrene C x selina-3.7(11)-diene x germacrene B
x
[0055] Trichome-specific promoters were identified in tomato that
regulate expression of the TPS5 and TPS11 genes. (The TPS5 promoter
was referred to as the MTS1 promoter and the TPS11 promoter was
referred to as the STS1 promoter in the international patent
application WO 09/082208 incorporated herein by reference).
[0056] As used herein, the term "promoter" refers to a nucleic acid
sequence that is capable of initiating transcription of a nucleic
acid sequence to which it is operably linked. The promoter controls
transcription of one or more genes, located upstream of the
transcription site of the gene. Structurally the promoter is
characterized by the presence of a binding site for DNA-dependent
RNA polymerase, transcription initiation sites and any other DNA
domains (cis regulatory elements), including, but not limited to
transcription factor binding sites, repressor and activator protein
binding sites, and any other sequences of nucleotides known to one
of skill in the art to act directly or indirectly to regulate the
rate of transcription from the promoter. Examples of eukaryotic cis
regulatory elements include the TATA box located approximately 25
base pairs upstream of the transcription site, the CAAT box located
75-80 base pairs upstream of the initial transcription site,
enhancer or silencer elements. The promoters of the invention
herein include constitutive promoters that are active in all
tissues and organs of the organism but preferably tissue-specific
promoters that are active mainly in specific tissues such as
trichomes. Especially included are the promoters of Terpene
Synthase 5, i.e., linalool synthase, or Monoterepene Synthase 1,
and Terpene Synthase 11 found in plants belonging to the genus
Solanum or other plant species.
[0057] Polynucleotides of the Invention
[0058] The present invention provides an isolated, recombinant, or
synthetic polynucleotide encoding the polypeptide or variant
polypeptides of the transcription factor provided herein. An
embodiment of the invention provides an isolated, recombinant or
synthetic nucleic acid sequence of SEQ ID NO:1, or a variant
thereof which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%,
95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence of
SEQ ID NO:1 and which encodes a transcription factor having an
amino acid sequence as shown in SEQ ID NO:2, or a fragment or
variant thereof that is capable of regulating terpene
biosynthesis.
[0059] In a further aspect of this embodiment, the nucleic acid
sequence encodes a peptide portion of TF19(6) polypeptide having
DNA binding activity. Such DNA binding domains binds to a specific
target DNA sequence, and have an amino acid sequence that is
different from those of known zinc finger domain DNA binding
proteins.
[0060] In another embodiment, a full length genomic nucleic acid
sequence of SEQ ID:NO: 3 is provided that contains coding and
non-coding nucleic acid sequences.
[0061] The terms "nucleic acid sequence," "nucleotide sequence",
"nucleic acid," and "polynucleotides" are used interchangeably
meaning a sequence of nucleotides. A nucleic acid sequence may be a
single-stranded or double-stranded deoxyribonucleotide, or
ribonucleotide of any length, and include coding and non-coding
sequences of a gene, exons, introns, sense and anti-sense
complimentary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA,
rRNA, tRNA, recombinant nucleic acid sequences, isolated and
purified naturally occurring DNA and/or RNA sequences, synthetic
DNA and RNA sequences, fragments, primers and nucleic acid probes.
Due to the degeneracy of the genetic code various nucleic acid
sequences may encode the same amino acid sequence. Any nucleic acid
sequence encoding TF19(6) or variants thereof is referred herein as
TF19(6) encoding sequence.
[0062] "Isolated" refers to a nucleic acid sequence that is removed
from its natural environment and which is substantially free from
other nucleic acid sequences, and the nucleic acid sequence does
not contain portions of unrelated sequences such as functional
genes or polypeptide coding regions. An isolated molecule may be
obtained by any methods or combination of methods including
molecular biology, biochemical and synthetic techniques. This
limitation does not pertain to nucleic acid sequences encoding
genes or coding regions artificially added to the nucleic acid
sequence after isolation.
[0063] "Recombinant nucleic acid sequence" refers to a combination
of nucleic acid sequences that are joined together using
recombinant DNA technology.
[0064] "Recombinant DNA technology" refers to molecular biology
procedures to join together nucleic acid sequences as described for
instance in Laboratory manuals edited by Weigel and Glazebrook,
2002 Cold Spring Harbor Lab Press; and Sambrook et al., 1989 Cold
Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.
[0065] A fragment of a polynucleotide of SEQ ID NO:1 refers to a
nucleic acid sequence comprising contiguous nucleotides of the
polynucleotide of the invention herein that is preferably at least
15 bp, at least 30 bp, at least 40 bp, at least 50 bp and/or at
least 60 bp in length. Preferably the fragment of a polynucleotide
comprises at least 25, at least 50, at least, 75, at least 75, at
least 100, at least 150, at least 200, at least 300, at least 400,
at least 500, at least 600, at least 700, at least 800, at least
900, at least 1000, at least 1100 contiguous nucleotides of the
polynucleotide of the invention. Without being limited, the
fragment of the polynucleotides herein may be used as a PCR primer,
and/or as a probe, or for anti-sense gene silencing. The "primer"
refers to a short nucleic acid sequence that is hybridized to a
template nucleic acid sequence and is used for polymerization of a
nucleic acid sequence complimentary to the template.
[0066] In a related embodiment of the invention, PCR primers and/or
probes for detecting nucleic acid sequences encoding TF19(6) are
provided. The skilled artisan would be aware of methods to
synthesize degenerate or specific PCR primer pairs to amplify a
nucleic acid sequence encoding TF19(6) or fragments thereof, based
on SEQ ID NO:1 (see Dieffenbach and Dveksler, 1995 PCR Primer: A
Laboratory Manual, Cold Spring Harbor Laboratory Press; McPherson
et al., 2000 PCR Basics: From Background to Bench, 1.sup.st ed.,
Springer Verlag, Germany). A detection kit for nucleic acid
sequences encoding TF19(6) may include primers and/or probes
specific for nucleic acid sequences encoding TF19(6), and an
associated protocol to use the primers and/or probes to detect
nucleic acid sequences encoding TF19(6) in a sample. Such detection
kits may be used to determine whether a plant has been modified,
i.e., transformed with a sequence encoding TF19(6).
[0067] It is clear to the skilled artisan that mutations,
deletions, insertions, and/or substitutions of one or more
nucleotides can be introduced into the DNA sequence of SEQ ID NO:1
or shorter fragments thereof. Generally, a mutation is a change in
the DNA sequence of a gene that can alter the amino acid sequence
of the protein encoded by the gene.
[0068] To test a function of a variant DNA sequences according to
the invention, the sequence of interest is operably linked to a
selectable or screenable marker gene and expression of the reporter
gene is tested in transient expression assays with protoplasts or
in stably transformed plants. It is known to the skilled artisan
that DNA sequences capable of driving expression are built as
modules. Accordingly, expression levels from shorter DNA fragments
may be different than the one from the longest fragment and may be
different from each other. Embraced by the present invention are
also functional equivalents of the nucleic acid sequence coding the
transcription factor of the present invention, i.e., nucleotide
sequences that hybridize under stringent conditions to the nucleic
acid sequence of SEQ ID NO:1. A stringent hybridization is
performed at a temperature 65.degree. C. and most preferably at
55.degree. C. in double strength (2.times.) citrate buffered saline
(SSC) containing 0.1% SDS followed by rinsing of the support at the
same temperature but with a buffer having reduced SSC
concentration. Such reduced concentration buffers are typically one
tenth strength SSC (0.1.times.SSC) containing 0.1% SDS, preferably
0.2.times.SSC containing 0.1% SSC and most preferably half strength
SSC (0.5.times.SSC) containing 0.1% SDS. Functional equivalents of
the transcription factor from other organisms can be found by
hybridizing a nucleic acid sequence with SEQ ID NO:1 with genomic
DNA isolated from other organisms. The skilled artisan knows
methods to identify homologous sequences in other organisms. Such
newly identified DNA molecules then can be sequenced and the
sequence can be compared with the nucleic acid sequence of SEQ ID
NO:1 and tested for functional equivalence. Within the scope of the
present invention are DNA molecules having at least 60%, 70%, or
75%, preferably 80%, more preferably 90% and most preferably 95%,
96%, 97%, 98%, 99% or more sequence identity to the nucleotide
sequence of SEQ ID NO:1.
[0069] The percentage of sequence identity between two sequences is
determined using computer programs that are based on standard
alignment algorithms. Sequences are substantially identical when
they share at least a certain minimal percentage of sequence
identity as identified by standard computer programs. Preferably,
the sequence identity refers to the sequence identity over the
entire length of the sequence.
[0070] A related embodiment of the invention provides a nucleic
acid sequence which is complementary or reverse complementary to
the nucleic acid sequence according to SEQ ID NO:1, such as
inhibitory RNAs, or a nucleic acid sequence which hybridizes under
stringent conditions to at least part of the sequence according to
SEQ ID NO:1 or the reverse complementary sequence to SEQ ID NO:1
(e.g., the non-coding DNA strand).
[0071] The polynucleotides of the invention may be overexpressed in
plant cells and the changes in the expression levels of a number of
genes and/or proteins of the plant cells may be observed.
Therefore, polynucleotides and polypeptides of the invention may be
employed to change the expression of the genes and/or protein in
plants, especially the genes and/or proteins of the terpene
biosynthesis. Alternatively, polynucleotides or polypeptides may be
employed in knockout plants that lead to changes in the expression
levels of one or more genes to improve characteristics or traits of
the plants especially traits associated with insect resistance.
[0072] The term "gene" means a DNA sequence comprising a region,
which is transcribed into a RNA molecule, e.g., a mRNA in a cell,
operably linked to suitable regulatory regions, e.g., a promoter. A
gene may thus comprise several operably linked sequences, such as a
promoter, a 5' leader sequence comprising e.g. sequences involved
in translation initiation, a coding region of cDNA or genomic DNA,
introns, and a 3'non-translated sequence comprising, e.g.,
transcription termination sites.
[0073] A "chimeric gene" or "recombinant gene" refers to any gene,
which is not normally found in nature in a species, in particular,
a gene in which one or more parts of the nucleic acid sequence are
present that are not associated with each other in nature. For
example the promoter is not associated in nature with part or all
of the transcribed region or with another regulatory region. The
term "chimeric gene" is understood to include expression constructs
in which a promoter or transcription regulatory sequence is
operably linked to one or more coding sequences or to an antisense,
i.e., reverse complement of the sense strand, or inverted repeat
sequence (sense and antisense, whereby the RNA transcript forms
double stranded RNA upon transcription).
[0074] A "3' UTR" or "3' non-translated sequence" (also referred to
as 3' untranslated region, or 3'end) refers to the nucleic acid
sequence found downstream of the coding sequence of a gene, which
comprises for example a transcription termination site and (in
most, but not all eukaryotic mRNAs) a polyadenylation signal (such
as AAUAAA or variants thereof). After termination of transcription,
the mRNA transcript may be cleaved downstream of the
polyadenylation signal and a poly(A) tail may be added, which is
involved in the transport of the mRNA to the site of translation,
e.g., cytoplasm.
[0075] As used herein, a molecular marker refers to any
morphological, biochemical or nucleic acid based phenotypic
difference that reveals a DNA polymorphism. Examples of molecular
markers include, but are not limited to, AFLPs (amplification
fragment length polymorphisms), RFLPs (restriction fragment length
polymorphisms), SNPs (single nucleotide polymorphisms), SSRs
(single sequence repeats), and alike. For instance, the skilled
artisan would understand how to detect a genomic polymorphism
between Solanum lycopersicum and sexually compatible species
comprising identifying a difference in a gene encoding an amino
acid sequence of SEQ ID NO:2 and fragments thereof between the
species and thus identifying a molecular marker. For example, a
molecular marker can be identified in at least one of a nucleic
acid sequence of SEQ ID NO: 1 and a nucleic acid sequence of SEQ ID
NO: 3. The molecular marker so identified can be used in
marker-assisted selection of plants having the desired composition
of terpenes that repel or, alternatively, attract insects, or other
organisms.
[0076] Polypeptides of the Invention
[0077] An embodiment of the present invention provides a
transcription factor, transcription factor homologous polypeptides,
and variants thereof.
[0078] The phrase "transcription factor" refers to a protein that
regulates expression of one or more genes involved in terpene
biosynthesis in an organism. The transcription factor possesses one
or more domains for binding DNA regulatory sequences and at least
one conserved domains characteristic of a particular family of
transcription factors. Transcription factors encompass
transcription factors-activators stimulating expression of one or
more genes involved in terpene biosynthesis and transcription
factors-suppressors inhibiting transcription of the genes by
binding to their regulatory sequences.
[0079] "Zinc finger protein transcription factor" refers to an
activator or a repressor composed of a zinc finger protein domain
and any of a variety of transcription factor effectors domains
which effect or modulate expression of nucleic acid sequences in
the vicinity of zinc finger protein binding site. Zinc finger
domains generally are about 25 to 30 amino acid residues in length,
and contain high number of cystein residues in a
C-Xn-C-Xn-C-Xn-C-Xn-C-type motif where X denotes a variable amino
acid and n indicates the number of X residues. X residues are
generally polar and basic, and implicate the region as involving in
binding nucleic acids. Zinc ions are essential components of zinc
finger domain structure designed to interact and bind nucleotides
of a nucleic acid molecule.
[0080] A protein is an amino acid sequence of any length linked by
covalent peptide bonds, and includes oligopeptide, peptide,
polypeptide and full length protein whether naturally occurring or
synthetic. A polypeptide means an amino acid sequence of
consecutively polymerized amino acid residues, for instance, at
least 15 residues, at least 30 residues, at least 50 residues.
[0081] The term "isolated" polypeptide refers to an amino acid
sequence that is removed from its natural environment by any method
or combination of methods known in the art and includes
recombinant, biochemical and synthetic methods.
[0082] An embodiment of the invention provides an isolated or
recombinant polypeptide which has an amino acid sequence set forth
in SEQ ID NO:2 or fragments, or variants or derivatives
thereof.
[0083] The term "fragment" of the polypeptide of SEQ ID NO: 2
refers to a subsequence of the polypeptide of the invention that
retains its biological function and capacity to alter transcript
levels of the genes of the terpene biosynthesis pathways, and is
preferably capable of binding a nucleic acid sequence of a promoter
that is operably linked to at least one gene selected from the
group comprising a Terpene Synthase 5 (TPS5) and a Terpene Synthase
11 (TPS11). The term may refer to a recombinant polypeptide and/or
an aggregate polypeptide such as a dimer or multimer.
[0084] As used herein, the terms "variant" or "derivative" of the
polypeptide set forth in SEQ ID NO:2 refers to polypeptides with
substantial similarity of amino acid sequences to the polypeptide
herein. The amino acid sequences of the polypeptide of the
invention and variants thereof may differ by one or more deletions,
additions, and/or substitutions of amino acids while retaining
functional equivalence to the polypeptide. In one embodiment,
variants of TF19(6) include, for example, proteins having at least
60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or
more amino acid sequence identity over the entire length to SEQ ID
NO:2. Amino acid sequence identity may be determined by pairwise
alignment using the Needleman and Wunsch algorithm and GAP default
parameters. Variants also include proteins capable of binding a
promoter sequence operably linked to another nucleic acid sequence
preferably of a gene that regulates terpene biosynthesis, such as
TPS5 and/or TPS11, which have been derived, by way of one or more
amino acid substitutions, deletions or insertions, from the
polypeptide having the amino acid sequence of SEQ ID NO:2.
Preferably, such proteins comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25,
20, 15 amino acid substitutions, deletions or insertions. For
instance, amino acids of the polypeptide of the invention may be
modified based on similarity in hydrophobicity, hydrophilicity,
solubility, polarity of amino acid residues, as long as the variant
polypeptide remains functionally equivalent to the polypeptide of
the invention. Alternatively, a variant may differ from the
polypeptide of the invention by attachment of modifying groups
which are covalently or non-covalently linked to the polypeptide
backbone. The variant also includes a polypeptide which differs
from the polypeptide of the present invention by introduced
N-linked or O-linked glycosylation sites, and/or an addition of
cysteine residues. The skilled artisan would understand how to
modify an amino acid sequence and preserve biological activity
using computer programs such as DNASTAR (Madison, Wis., USA). A
variant or derivative of zinc finger polypeptide of the invention
retains a capacity to alter transcript levels of the genes of the
terpene biosynthesis pathways, including or in particular the
transcript levels of the genes encoding a Terpene Synthase 5 (TPS5)
and/or Terpene Synthase 11 (TPS11).
[0085] The polypeptide of the invention is capable of binding a
promoter sequence operably linked to another nucleic acid sequence
preferably of a gene that regulates terpene biosynthesis, such as
TPS5 and/or TPS11.
[0086] A nucleic acid sequence encoding the polypeptide of the
invention is "operably linked" to another nucleic acid sequence,
typically a coding gene, when the gene so linked is transcribed.
Operably linked DNA sequences form contiguous reading frames to
produce a "fusion protein," i.e., a protein composed of various
protein "domains" or "motifs." The nucleotide sequence associated
with the promoter sequence may be of homologous or heterologous
origin with respect to the plant to be transformed. The sequence
may be also entirely or partially synthetic. Regardless of the
origin, the nucleic acid sequence associated with the promoter
sequence will be expressed or silenced in accordance with promoter
properties to which it is linked after binding to the polypeptide
of the invention. In a preferred embodiment of the invention, the
associated nucleic acid may code for a protein that is desired to
be expressed or suppressed throughout the plant at all times or,
alternatively, in specific cells and tissues. Such nucleotide
sequences preferably encode proteins conferring desirable
phenotypic traits to the plant modified or transformed therewith.
More preferably, the associated nucleotide sequence leads to the
production of terpenes in the plant. Preferably, the nucleotide
sequence encodes TPS5 or TPS11 and terpenes that confer insect
resistance, disease resistance, resistances to other pests and/or
attraction of beneficial organisms, e.g., predators or parasitoids
of pest insects or plant pollinators.
[0087] Homologous Polynucleotides and Polypeptides
[0088] Homologs, paralogs, orthologs and any other variants of the
sequences herein are expected to function in a similar manner by
making plants that repel or resist insects, or alternatively,
attract beneficial organisms, e.g., predators or parasitoids of
pest insects or plant pollinators. Homologous sequences are
sequences that share substantial sequence identity or similarity to
the nucleic acid sequence of SEQ ID NO:1. Homologous sequences may
be derived from any plants including monocots or dicots, and
especially crops including but not limited to tomato, pepper,
eggplant, lettuce, sunflower, oilseed rape, broccoli, cauliflower
and cabbage crops, cucumber, melon, watermelon, pumpkin, squash,
peanut, soybeans, cotton, beans, avocado, onion, endive, leek,
roots such as arrowroot, carrot, beet, turnip, radish, yam,
cassava, potatoes, sweet potatoes and okra. Homologous sequences
may also be derived from crop species including maize, barley,
pearl millet, wheat, rye, sorghum, rice, tobacco and forage
grasses. Homologous sequences may be derived from tree species and
fleshy fruit species such as lemons, tangerines, oranges, grapes,
peaches, plums, currant, cherries, melons, strawberry, and mango,
or from ornamental plant species such as hibiscus, poinsettia,
lily, iris, rose and petunia, and the like. Additionally,
homologous sequences may be derived from plant species that are
wild relatives of crop plant species. For example, homologous
sequences may be derived from nightshade Atropa belladonna which is
a wild relative of a cultivated tomato Solanum lycopersicum, or
teosinte species related to maize. Homologous sequences include
orthologous or paralogous sequences. Methods of identifying
orthologs or paralogs including phylogenetic methods, sequence
similarity and hybridization methods are known in the art and are
described herein.
[0089] Paralogs result from gene duplication that gives rise to two
or more genes with similar sequences and similar functions.
Paralogs typically cluster together and are formed by duplications
of genes within related plant species. Paralogs are found in groups
of similar genes using pair-wise BLAST.RTM. (Basic Local Alignment
Search Tool) analysis (Feng and Dollitle, 1987 J Mol Evol:
25:351-360) or during phylogenetic analysis of gene families using
programs such as CLUSTAL (Thompson et al., 1994 Nucl. Acid Res
22:4573-4680; Higgins et al., 1996 Methods Enzymol 266:383-402). In
paralogs, consensus sequences can be identified characteristic to
sequences within related genes and having similar functions of the
genes.
[0090] Genes encoding regulatory elements and transcription factors
are conserved in eukaryotes. For instance, plant species that have
common ancestors are known to contain many transcription factors
that have similar sequences and functions. These sequences are
referred to as orthologs. The skilled artisan can identify
orthologous sequences and predict the functions of the orthologs,
for example, by constructing a polygenic tree for a gene family of
one species using CLUSTAL or BLAST.RTM. (Basic Local Alignment
Search Tool) programs. A method for identifying or confirming
similar functions among homologous sequences is by comparing of the
transcript profiles in plants overexpressing or lacking (in
knockouts/knockdowns) related polypeptides. The skilled artisan
will understand that genes having a similar transcript profiles,
with greater than 50% regulated transcripts in common, or with
greater than 70% regulated transcripts in common, or greater than
90% regulated transcripts in common will have similar
functions.
[0091] Homology refers to a sequence similarity, or identity
between a polypeptide or a fragment thereof and a references
sequence. A homology of polypeptide sequences are determined based
on the number of amino acid sequences in the positions shared by
the polypeptides. Homologous sequences encompass amino acid
sequences of polypeptide of the present invention modified by
chemical or enzymatic means known in the art. See Ausubel et al.
(eds) 2000 Current Protocols Mol Biol, Willey & Sons, New
York.
[0092] Polypeptides with "substantial identity" refers to sequences
of sufficient similarity to the transcription factor of SEQ ID NO:2
which retains biological function of the transcription factor when
overexpressed, ectopically expressed or knocked out in a plant.
Polypeptide sequences that are at least 50% identical to the
polypeptide of the present invention are considered sufficiently
identical.
[0093] Regulation of Terpene Biosynthesis
[0094] An embodiment of the invention provides a polypeptide, a
polynucleotide, a fragment thereof or a chimeric gene or vector
that may be used to up- or down regulate expression of genes
involved in terpene biosynthesis, preferably selected from at least
one of Terpene Synthase 5 (TPS5) and Terpene Synthase 11 (TPS11),
and thereby modify downstream products of the pathway.
[0095] For example, a polypeptide, a polynucleotide, a fragment
thereof or a vector of the invention is used to regulate volatiles
emitted by plants to repel pest insects or to attract beneficial
insects, e.g., predators or parasitoids of pest insects or plant
pollinators, or to regulate the transcription level of the genes
involved in the terpene biosynthesis and to alter transcript
profiles of such genes. An altered transcript profile of the gene
refers to the transcript profile that is substantially different
from the transcript profile of the correspondent gene in a
reference state. Differences and similarities between expression
levels are evaluated by statistical methods known in the art.
[0096] An embodiment of the invention provides methods to modify
terpene levels in a plant by up- and down regulating expression of
TPS5 and TPS11 genes and thereby modifying levels of at least one
terpene form a group of: linalool, neralidol, germacrene,
.alpha.-humulene, .beta.-caryophyllene, .beta.-elemene,
.beta.-phellandrene, limonene, 2-carene and zingiberene (7S
configuration), .beta.-curcumene, .beta.-myrcene, para-cymene,
.gamma.-terpinene, 7-epizingiberene, .alpha.-terpinene and
.alpha.-phellandrene.
[0097] A related embodiment of the invention provides a method for
increasing a terpene in a plant by up-regulating a transcription
factor of the invention that positively regulates terpene
biosynthesis. For instance, up-regulating may increase the level of
terpenes which are part of chemical defense of the plants and
thereby repel insects or pathogenic organisms.
[0098] In yet another embodiment, the invention provides a method
for reducing a terpene in a plant by down-regulating a
transcription factor that positively regulates terpene
biosynthesis. For instance, down-regulation may change the profile
of the volatile terpene compounds emitted by the plants thereby
making the plants less attractive to insects or other pests
[0099] Generally, methods for up- and down-regulation of expression
of transcription factors in plant or animal systems are well known
to the skilled artisans. Up-regulation may result from
overexpression of a protein or polypeptide in a whole plant, plant
cells or specialized plant tissues, such as trichomes.
Alternatively, the promoter may be altered to up-regulate
expression of transcription factors of the invention.
[0100] Down-regulation occurs at DNA level by interfering with
transcription of the genes thereby decreasing expression of the
genes. Alternatively, down-regulation occurs at RNA level by
interfering with protein translation from RNA molecules, or by
interfering with RNA splicing to produce mRNA species.
Down-regulation at RNA level is achieved through RNA interference
(RNAi) approach using double stranded RNAs (dsRNAs), small hairpin
RNAs (shRNAs), micro RNAs (miRNAs) or small interfering RNA
(siRNAs). Phenomenon of RNA interference is also known in the art
as cosuppression, post transcriptional gene silencing, and
quelling. See Hamilton and Baulcombe, 1999 Science 286: 950-952;
Hammond and Hannon, 2001 Nature Rev Gen 2: 110-119; Baulcombe, 2007
Science 315:199-200.
[0101] An embodiment of the invention provides a method for
reducing terpene levels in a population of plants by providing
plants mutagenized by either chemical or physical methods,
detecting a mutated plant within a population such that the plant
has decreased expression of the transcription factor of the
invention that positively regulates terpene biosynthesis and
selectively breeding the mutated plant to produce the population of
mutated plants thereby reducing terpene levels in the plants. An
alternative method is provided for increasing terpene levels in a
plant population by detecting and selecting a mutated plant within
the population that has decreased expression of the transcription
factor that negatively regulates terpene biosynthesis.
[0102] For example, methods for the detection of a mutation in a
target sequence in a member of a mutagenized population is
disclosed in WO 2007/037678. The method involves isolating DNA of
mutagenized plants, pooling DNA, amplifying the target sequence,
i.e., the nucleic acid sequence of TF19(6) with a pair of primers
from the DNA pool, determining the nucleic acid sequences of the
amplification fragments using high throughput sequencing,
identifying mutations by clustering (aligning) the sequences of the
fragments, screening the identified mutations for modified
functions of the target sequence and identifying members carrying
the mutation.
[0103] The sequencing may be conducted by methods known in the art,
including the dideoxy chain termination method (Sanger sequencing),
and high-throughput sequencing methods, such as the methods
disclosed in WO 03/004690, WO 03/054142, WO 2004/069849, WO
20041070005, WO 2004/070007, and WO 2005/003375, by Seo et al.
(2004) Proc. Natl. Acad. Sci. USA 101:5488-93, and technologies of
Helios, Solexa, US Genomics, and the like, which are herein
incorporated by reference. It is most preferred that sequencing is
performed using the apparatus and/or method disclosed in WO
03/004690, WO 03/054142, WO 2004/069849, WO 2004/070005, WO
2004/070007, and WO 2005/003375, which are herein incorporated by
reference.
[0104] An alternative embodiment of the invention provides a method
to alter gene expression in a plant, plant tissue or plant cell.
For instance, the polynucleotide of the invention may be
overexpressed in a plant, cell or tissue. The term
"overexpresssion" refers to an increased expression of a gene in a
plant, tissue or a plant cell compared to expression in a
non-altered or wild type plant, tissue or cell, at any stage of
development or location of the gene. Overexpression occurs when
gene encoding transcription factor of the invention is under
control of a strong constitutive or a tissue specific promoter.
[0105] Alteration of expression of a polynucleotide of the present
invention also results in "ectopic expression" which is the
different expression pattern in a transgenic or mutant plant and in
a control or wild-type plant. Alteration of expression occurs from
interactions of polypeptide of the invention with exogenous or
endogenous modulators, or as a result of chemical modification of
polypeptide. The term also refers to an altered expression pattern
of the polynucleotide of the invention which is altered below the
detection level or completely suppressed activity.
[0106] An alternative embodiment of the invention provides a method
for increasing production or level of a terpene in a population of
plants including the following steps: contacting a plurality of
plant cells with a composition which includes a vector
incorporating a nucleic acid molecule set forth in SEQ ID NO:1;
detecting and selecting a transgenic plant cell within the
plurality of the cells such that the cell has an increased level of
a transcription factor that positively regulates terpene
biosynthesis compared to a control plant cell thereby increasing
the terpene in the cell; regenerating the cell into a plant and
selectively breeding the plant to produce the population of plants
with the increased terpene.
[0107] The vectors for inserting transgenes into the genome of host
cells are well known in the art. As used herein, the term "vector"
refers to a nucleic acid molecule engineered using molecular
biology methods and recombinant DNA technology for delivery of
foreign or exogenous DNA into a host cell. Typically the vector is
a DNA molecule that consists of a transgene insert and a nucleic
acid backbone. Vectors include plasmids, viruses, cosmids and
artificial chromosomes. Binary or co-integrated vectors into which
a chimeric gene is inserted may be used for transforming
plants.
[0108] The chimeric gene generally includes a promoter sequence
operably linked to a nucleic acid sequence of a coding gene to be
transcribed in the host cells. Alternatively, the promoter sequence
may already be present in a vector so that the nucleic acid
sequence which is to be transcribed is inserted into the vector
downstream of the promoter sequence. Vectors are typically
engineered to have an origin of replication, a multicloning site
and a selectable marker.
[0109] Examples of selectable markers are described below. The
skilled artisan would know that different antibiotic or herbicide
selectable marker are applicable to different target species.
Selectable markers that are routinely used in plant transformation
include the npt II gene conferring resistance to kanamycin,
paromymycin, geneticin, and related antibiotics (Veira and Messing,
1982 Gene 19: 259-268; Bevan et al., 1983 Nature 304: 184-187) the
bacterial aadA gene encoding aminoglycoside 3'-adenyltransferase
conferring resistance to antibiotics streptomycin or spectinomycin
(Goldschmidt-Clermont, 1991 Nucl Acid Res 19: 4083-4089), the hph
gene conferring resistance to hygromycin (Blochlinger and
Diggelmann, 1984 Mol Cell Bio 14: 2929-2931). Other markers that
can be used include a mutant EPSP gene conferring resistance to
glyphosate (Hinchee et al., 1988 Biotechnology 6: 915-922), a
mutant acetolactate synthase (ALS) gene conferring resistance to
imidazoline or sulphonylurea herbicides (Lee at all., EMBO Journal
7: 1241-1248), a phospinothricin acetyltransferase gene which
confers resistance to herbicide phosphinothricin (White at al.,
1990 Nucl Acid Res 18: 1062; Spencer et al., 1990 Theor Appl Genet
79: 625-631). Selection markers resulting in positive selection
such as phosphomannose isomerase gene are also used (see WO
93/05163).
[0110] An embodiment of the invention provides recombinant
expression vectors comprising a nucleic acid sequence of the
invention fused to associated nucleic acid sequences such as, for
instance, promoter sequences. The vector is used to transform the
host cells and the chimeric gene is preferably inserted in the
nuclear genome or into the genome of cell organelles, i.e.,
mitochondria or plastids, such that the expression of the nucleic
acid sequence is driven by the activity of the promoter. See
Arabidopsis, A Laboratory Manual Eds. Weigel and Glazebrook, Cold
Spring Harbor Laboratory Press (2002) and Maniatis et al.,
Molecular Cloning, Cold Spring Harbor Laboratory Press (1982).
[0111] Methods for obtaining transgenic plant cells and plants are
well known in the art and include but are not limited to
Agrobacterium-mediated transformation of plant explants, particle
bombardment of plant explants, transformation of plant explants
using whiskers technology, transformation using viral vectors,
electroporation of plant protoplasts, direct uptake of DNA by
protoplasts using polyethylene glycol, microinjection of plant
explants and/or protoplasts. Agrobacterium-mediated transformation
is a preferred method to introduce the nucleic acid molecule of the
invention into plant explants. Agrobacterium tumefaciens harbors a
natural vector called Ti plasmid which was engineered to make it
suitable for introduction of exogenous nucleic acid molecules into
plant genomes. For genetic transformation, plant-derived explants
are incubated with suspension of Agrobacterium cells followed by
cultivation of the explants on the medium containing a selective
agent that promotes growth and regeneration of the transformed
cells only.
[0112] Methods for detecting transformed or modified plant include
without limitation, Southern Blot Analysis and PCR based methods.
Methods for analyzing terpene content in modified plants using gas
chromatography-mass spectrometry (GC-MS) are known in the art and
are described in Schilmiller et al., 2009 Proc Natl Acad Sci
106:10865-10870 and Adams, 2007 Identification of Essential Oil
Components by Gas Chromatography/Mass Spectrometry, 4.sup.th ed.,
Allured Pub Corp., Carol Stream, Ill. The resulting transformed or
modified plant may be used in a conventional breeding to produce
more transformed or modified plants with altered profile of terpene
compounds.
[0113] An embodiment of the invention provides antibodies specific
for polypeptides of the invention or variants thereof. The skilled
artisan would understand that the transcription factor or variants
thereof can be expressed and purified in a heterologous expression
system, for instance Escherichia coli, and used to raise monoclonal
or polyclonal antibodies specific for polypeptides of the
invention. Antibodies can be also raised against synthetic
polypeptides from the amino acid sequences of the transcription
factor or variants thereof. Methods of raising antibodies are known
in the art and described in Harlow and Lane, 1988 Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York. Such
antibodies are used to screen expression libraries from the plants
and may be used, for example, in a method for identifying plants
emitting volatiles that attract beneficial insects or alternatively
repel pest insects.
[0114] Host Cells, Plants and Tissue Cultures of the Invention
[0115] A phrase "host cell" or "transformed cell" refers to a
genetically engineered cell that includes at least one nucleic acid
molecule, especially a chimeric gene encoding a desired protein or
a nucleic acid sequence which upon transcription yields a terpene
useful to repel or attract insect pests. The host cell is
preferably a plant cell, but may also be a fungal cell, a yeast
cell or a bacterial cell. The host cell preferably includes the
chimeric gene integrated into the nuclear or organelle genomes, but
may also contain the gene extra-chromosomally. An embodiment of the
invention herein also provides a genetically engineered plant cell
that includes a nucleic acid sequence set forth in SEQ ID NO:1 and
a plant regenerated from the cell. A genetically engineered plant
of the invention includes the plant having an increased level of a
terpene compared to a non-genetically engineered plant of the same
genetic background. As used herein a genetic background refers to
the genotypic base of a breeding line or population of
organisms.
[0116] A related embodiment of the invention also provides for a
tissue culture obtained from the transgenic plant such that the
culture has enhanced production or secretion of terpene, terpene
isomer or terpene analog, and a method for isolating the same from
the tissue culture of the invention.
[0117] Suitable cells for expression of a polypeptide of the
invention include prokaryotic and eukaryotic cells such as plant
cells. Plants suitable for expression of a polypeptide of the
invention include, but are not limited to crop species, which are
natural hosts of insect pests such as tomato, pepper, eggplant,
lettuce, sunflower, oilseed rape, broccoli, cauliflower and cabbage
crops, cucumber, melon, watermelon, pumpkin, squash, peanut,
soybeans, cotton, beans, cassava, potatoes, sweet potato and okra.
Crop species also include maize, barley, pearl millet, wheat, rye,
sorghum, rice and forage grasses. Additionally, plant hosts include
tree species, fleshy fruit species such as grapes, peaches, plums,
strawberry and mango, and ornamental species such as hibiscus,
poinsettia, lilies, iris, rose and petunia. Especially preferred
are plants belonging to the family Solanaceae, including plants
that belongs to genera Solanum, Capsicum, Nicotiana, Petunia, and
the like. In a preferred embodiment, vegetable species especially
of genus Solanum are included, for example, tomato (S.
lycopersicum), eggplant (S. melongena), pepino (S. muricatum), and
the like.
[0118] As used herein, the phrase "crop species" refers to plants
cultivated for purposes of obtaining food, feed or plant derived
products including carbohydrates, oils and medicinal
ingredients.
[0119] Insects Controlled by Regulation of Terpene Levels in
Plants
[0120] An embodiment of the invention provides a method for
increasing resistance to a pest insect by down regulating or
alternatively up regulating the genes of the terpene biosynthesis
which would result in altering profiles of volatile terpenes
emitted by plants to attract beneficial insects or repel pest
insects.
[0121] As used herein, the phrases "plant insects" or "plant pests"
refer to insect species that infest and damage host crop and
ornamental plants. An "infestation" refers to presence of a large
number of pest organisms in a field or greenhouse, on the surface
of a host plant or on anything that might contact a host plant, or
in the soil. Insect pests include sap-sucking insect pests, such as
psyllids, whiteflies, aphids, mealybugs, plant hoppers and scale
insects and share a common property, namely the utilization of
plant sap as their food source. Insect pests also include thrips,
cicada, mites and leaf hoppers.
[0122] The term "whitefly" or "whiteflies" refers to species of the
genus Bemisia, especially B. tabaci, species of the genus
Trialeurodes, especially greenhouse whitefly T. vaporariorum and
banded winged whitefly T. abutinolea. All biotypes of B. tabaci
such as biotype Q and B, are also included as well as any
developmental stage, such as eggs, larvae, pupae and adults. As
used herein, the term "aphids" refers to plant insect pests
belonging to the family Aphididae, including but not limiting to
Aphis gossypii, A. fabae, A. glycines, A. nerii, A. nasturtii,
Myzus persicae, M. cerasi, M. ornatus, Nasonovia especially N.
ribisnigri, Macrosiphum, and Brevicoryne.
[0123] The term "insect pests" also refers herein to insects of the
order Diptera including but not limiting to blood sucking or biting
insects attacking animals, especially mammals. Blood sucking ticks
are also included. Such insects may act as vectors of human and/or
mammalian diseases such as malaria.
[0124] "Insect vectors" are insects that are capable of carrying
and transmitting viruses to plants. In the context of mammalian
disease vectors, insect vectors are insects which attack mammals
and can potentially transmit diseases to mammals, such as
mosquitoes, which are able to transmit the parasite Plasmodium to
humans or heartworm to canines.
[0125] Preferably, the modified plants of the invention develop
enhanced resistance to one or more pest insects.
[0126] "Insect pest resistance" is an enhanced ability of modified
plants of the present invention to withstand attacks of one or more
pests compared to wild type or control plants. Methods to assess
insect pest resistance in plants are known in the art. For example,
disease symptoms may be scored visually at one or more time points
after infestation or contact with an insect pest. Alternatively,
insect pests may be detected and optionally quantified during
infestation of the plants in an assay. A modified plant shows
enhanced pest resistance if the number of insect pests detected in
the tissue is significantly lower compared to the number of insects
detected in control. Preferably, a significant increase in average
yield of modified plants of the present invention compared to
control (e.g. at least 1%, 2%, 5%, 10% or more) when plants are
grown under insect pet pressure provides an indirect measurement of
enhanced resistance to pest insects. Statistical analyses are
employed to determine existence of significant differences.
[0127] The invention now having been fully described it is
exemplified in the Examples below and in the claims, which are not
to be construed as further limiting. References cited herein are
hereby incorporated by reference in their entireties.
[0128] Sequences Referred to Herein:
[0129] A nucleotide sequence of SEQ ID NO:1 encoding a
transcription factor TF19(6):
TABLE-US-00002 ATGGCTAATTTCTTTTCATTAGGTGGGAATCAAGAACAACAACATCAAGA
AATTAGCAGCAGCCAAGCATTAGTACCCACAGAGAGTAATAATTGGTTTT
TGTACAGAAATGAACATCATCATCATCATCATAATCAAGAAATACCCAAC
ACTTACAAAGGTTTTGAGTTATGGCAAAGTGGTAACACTCCACAACACCA
ACACCAACACCACCAACAACAACAACAGTTTCGTCATCCGATTTATCCTT
TGCAAGATCTTTATTCCACTGATGTTGGATTAGGGGTTGGGCCAAGCAGA
AGTGGCTTTGATATATCTGCAGGTGATCATGAGGCGTCGAGGTCGGGATT
CGTGATGATGAGGAGTGGTGGAGGAGGAATAAGTTGCCAAGATTGTGGGA
ACCAAGCTAAGAAAGATTGTCAACATATGAGGTGTAGGACTTGTTGTAAG
AGTAGAGGGTTTCAGTGTCAAACTCATGTGAAAAGTACTTGGGTTCCAGC
AGCTAAAAGGAGAGAAAGGCAACAACAACTTGCTGCTTTGCAACAACAAC
AACAAGGACATAATAATAATAATAATAATCATAAGAATAAAAGGCAAAGG
GAGGATCCAAGTGCTTCTTCTCTTGTGTCTACTCGTTTGCCTTCAAACAC
TAATGGGTTAGAAGTGGGAAAATTTCCATCAAAAGTACGTACAAGTGCTG
TATTTCAGTGTATTCAAATGAGTTCAATTGAGGATGATGAAGATCAATTA
GCATATCAAGCTGCTGTGAGCATTGGTGGACATGTTTTCAAAGGAATTTT
ATATGATCAAGGTCATGAAAGTCAGTACAATAACATGGTTGCAGCCGGAG
GCGATACGTCTTCCGGTGGTAGTGCTGGCGGAGTTCAGCACCACCACCAT
AATTCCGCTGCAGTAGCTACCGCCACCACTACAAGTGGTGGCGATGCTAC
TGCAGCGGGTCCATCGAATTTTCTAGATCCTTCTTTATTTCCAGCTCCCC
TTAGCACTTTTATGGTAGCTGGTACGCAATTTTTTCCACCTTCAAGATCT CCTTGA
[0130] An amino acid sequence of SEQ ID:2 of the transcription
factor TF19(6):
TABLE-US-00003 MANFFSLGGNQEQQHQEISSSQALVPTESNNWFLYRNEHHHHHHNQEIPN
TYKGFELWQSGNTPQHQHQHHQQQQQFRHPIYPLQDLYSTDVGLGVGPSR
SGFDISAGDHEASRSGFVMMRSGGGGISCQDCGNQAKKDCQHMRCRTCCK
SRGFQCQTHVKSTWVPAAKRRERQQQLAALQQQQQGHNNNNNNHKNKRQR
EDPSASSLVSTRLPSNTNGLEVGKFPSKVRTSAVFQCIQMSSIEDDEDQL
AYQAAVSIGGHVFKGILYDQGHESQYNNMVAAGGDTSSGGSAGGVQHHHH
NSAAVATATTTSGGDATAAGPSNFLDPSLFPAPLSTFMVAGTQFFPPSRS P
[0131] A genomic nucleic acid sequence of SEQ ID NO: 3 encoding the
transcription factor TF 19(6).
TABLE-US-00004 GCAAGTCAAATTGTATATGCTCTTAATTAGGGTTTATGCACCTAGAAGTA
GTATTTTAATCGCATATTAGTTATCGATGTTCCTAAATTAATCGCCTATT
AGTTATCGATGTTCCTAAATATAATTGACCTAATTACAAAATTAAGATAG
AACTGATTATATTTTTTCAATTTTATCCTTACAAGGAGCAATTCTTTTTG
AAAGTATGAACCACTTTGTAAAGTTTTTTTTTAAAAAAAATCTTAAAGGA
GTAAATCAGTAAAACTACCTTTCATATTTATGATTTTTTAAGAAGCATGT
AAAGAAAAAATAGAAAATCAATATAGAACGAAAAAAGAATTTTATAAAAC
CTCATAACTTAATAAAAAGAATCATATTTATAAGAAATATTTTCTTCCCA
CATGGAATAATAAATTGCACAAACTGTAAATATTCTCTACTACAATATAA
TGTTATAACACACGTATACCGTTGGTTTTTCAGTATAAATATAATATCCA
TATTTTAGATATATTAGCTGTTAAAAACAATATAATATGGATGGAGACAA
GTTAAATGTATGTAATTTTACCTTGTAATCCCAATTCTCAATATATATAT
ATATATATATATATATATATATTCCTCCAAGACAAAACATTGGATTTTTA
TTCTAGGAACTTGAATTAAGAATTCAATTTACTCGTAAAAATTAAAAAGA
AATTTCTTATGATCTTATCAAATATTTAATAGGTGAATAGTTAAATTTGA
CAAGTTAAATTAAGATGTATGTCCATCACCTCATCATAATTCAAATTATT
TTAACAAATATCCTTAATCATGATCTTCTTTCTTTTTAGGTTGAAATAAT
TATCATTAGATTTGTACATAGTATAGGAATATATTAAGAGCTAATATATC
TAAAATGTGGATAAATAAAACAATTCTTGCTCAAAATTTTAAATAGTTTT
AATACTTTTACAATACTTGACACGCGGCATTATATAGCCAACAATTTTAC
GGGCTAAGACATAACATTTATCTTGGAAATTCTCTATTATTAATCATTAG
CTTAGATTGTCTGAGTTTTTGAAGGTCTTTTATTTAGTTATAGGCAATTT
TACCTAGTTTTATAGAATTAAAATTATTGTCCGTTGTTATATTTAGGTAA
GAAAAAAAGTTAATAAATCAGACAAGAAAATATAAAGAACCGAAATAATT
ATGTAATGCCTAAAATAGTTGGTTTTATATACATAAAGACTGTTGAAAAT
TGAAATTAATATTGCGGCTCCTTCATTTATTGGTATTACTGTTAATTACG
TGATTGAAAGGAAAAAAAAAGTTTTACCAAAAAAAAGTATAAAAATAAGT
TTTGTACTTTATGTCAACAGTTAGTCATCAATAGTTACTGCTATAATACT
AGGTGCCAACACTATGTATAATTCGAATGTGATAATAATTTCTGGAAAAA
AAAATTAAAGGATATTTGATTTGATATGGTCCTAGATAATGTAGATGATG
AAGGGGTGTTAATTAGTCGTTTCAAATTGATAGGTTATTTTGAAAAATTG
TGTCATATTAATTGTTTATATTTTCAATGATGTATGATTAAAATTAAAAT
TTTTGAATCTGTCTTAATCGTTTTGGTTTCTCTTTGATTTAGGTATAATT
CAAATTGATGGTTAATTTTTTTAAACGTCATCTAACAACTATAAAATTTG
ATAAAAAATATTTAAAATTTACAATAACATAAAATGAAAATATGTTTTCC
AACTATACCAATTTAGGAGGAGAAACATAGTTATTGTTTTACTATTATCG
CTAGTATTATGAATGAGATATGAAAATTTATATTAATTTATATTGGAATC
TATAATTGATTTTATTAAAAAAAATTAAGTGCGTGTACTTTGACATTTTT
TTTGTTTTTAACTCGGCATTCAAAGTTCATATTGAAGTTTTAACTAAATT
CGAATCGCACTCTTCAGAGCAATGCAGGGATGGGTCTCCCAACAACATTT
TGTCGATAGTCTATACCCAGAGCTCGAACTTAAGACCTCTGATTAAGAAT
AAAATACTTCATTTATAAACTGATCATCTTAATATTTTCAAAATTTAAAT
GTCACATATTTTCTAAGATATCCTCGAAACATAATAATAAGTTGAAATGT
ATAATGTTTGATTGAGACTAAACTGAGGCGTTTATATATACAATCGTAGA
ATTAAAATATTTAATTGCCATCTGAAAATTAATTTAAATATTTATCTATG
TACTATACCTTAATTAATTCTTTCATGACAAACTTTCTTGGACATTTTTT
CATGAAAAATGCATATAACTTAAACAAGGCCGATACCTTACACCCAAATT
GGACAGTATATTTAGAAGAGGGGGAATAATGGTAAAGAGGGCCGGTATCA
GGTTTACACAGAGATGAAAAGTTAGGTGGAGTTTATTTGTTCGGATGGAT
TTATCAGTTTTTTCGTAGATTTTATATTTATATTAGATTCTTTTTTTTAC
GTATATATTAAATTATAATCCCTAAACAAATTGATTTAGAATCTCAAACT
CATAATCTTAACTTCGCCCCTAACTTTTATATATATATATATATATATAA
TATTTTTAATATATCATTAGTTACACATTATTTTTTATATTATGTTGTGT
ATTACTGATGAATAATGATTTATGGAAATACAAAAAGCTCTTATTCAGTA
ATACATACATTAGTATGATCATCTTTTTTCACATTCTTTCCATCGCGATA
TATGTTTTTTTTTTAAACTATAACACAATAAACACTGCATTAAAAATAAT
TGTACATATTTTTTGTGTCTCAATTTATGTGATACCTTTTAGTTTTTTAA
GAGCTAAACAATTTAAATTTGAGCGAGAATTTACGCATGAAATTTTCGAA
AATTCTAAAAAGAAATTTATATATTAATAAAAACTACGTAAAAATACTAT
AAGACACAATAATTGACAATTCAAAATATTTAAAAGTCAAAGATATACTT
ATTTGAATTTCAAAATCTGAAAAGTATCACATAAATAGGAGGAGAGAGTA
ACGAATATCAATATTAATGATATATTATATTCACCACTAATATTCTTAAA
AATAAATATTAAAAACACCATTAAATTCGATGTGAATTATTAGTTTGATC
CCTGAACTATTGACAGTATTATAAACACTCCTCTACTGGGTTAGATGAAC
TTAAATACACACTCGATCTTGTCACAATGATGAAATACACCCTAATGAAA
ATCATATTTACTCTTCCTATTTCTAACCATCGGAAAGAGTCATCGTGGCT
AGGAAACTATACTAGCGACCTACCCAATTCATTATAGAAATTTTCGCGAT
CAATGATTGAAAATTTAGAATGTTTCCAACACTTTATCTGTCAACTTTTT
ATTAAGAGTTTCAAGCTCGTATAAGAATTTGAAATCACTTTTAGTATATC
ATGTAGTAGATCTAAATATATTTAAAATTATTATAAATTTTTTTTAAAAA
ACTAATAATTCACACTAAATTGACAAATATCTTCAATACTTAGCTTCTCA
CTTATTTTATACGACCTACCAAACAATCGCGAAACTTTTTAAGTTACTGC
AAACTGTAGCGGTAAAGAGAGGGGAGGGGGGGGGGGTAGTTGTGGTGCTT
TTTAGCGTTGGCGGCGTTTGCAGAGCTGTAATATATATAATATACCTTTT
CTATTAATGTACCCTCACTCACTCACTTCCTCTCCATAATTCTTTATACA
AACAATCATTTTTTCTTAAACTTGCTCTATTATAAATTCACATTTTTTCT
TTATATATACACATACATATAGAGCAAAAAAGAAGTTCTAATTTTGTAAA
CCCTTCAAAAAAAAGAAAAATAATTTTTTTTGAGATCATAAATGAAGAAA
TCCAAGGGATACAAACATCATATTTGTGTTATAAGTTGGTGCACTTTTGT
GGTATGGATTGTGATTAATCACTAATCATAATCAAGATTAACAACAAGTA
ATGGCTAATTTCTTTTCATTAGGTGGGAATCAAGAACAACAACATCAAGA
AATTAGCAGCAGCCAAGCATTAGTACCCACAGAGAGTAATAATTGGTTTT
TGTACAGAAATGAACATCATCATCATCATCATAATCAAGAAATACCCAAC
ACTTACAAAGGTTTTGAGTTATGGCAAAGTGGTAACACTCCACAACACCA
ACACCAACACCACCAACAACAACAACAGTTTCGTCATCCGATTTATCCTT
TGCAAGATCTTTATTCCACTGATGTTGGATTAGGGGTTGGGCCAAGCAGA
AGTGGCTTTGATATATCTGCAGGTGATCATCAGAAACAGATTTAGAATTT
AAACTTTATCTATTCAGTACTTTCTAAAGTACTTATAGATCTATAATTTA
AGTTTGATAAATTTAATATTTATGTTCTAAACAATTCACAACATTTTGCA
ATTAGGGATTTCGAAACGTATTTACTGAAGCATGTTAGAATTCCCAGCCT
CGAAAAGGCATGGGAATTTGGTCTATGGACTTGGGAAATTCTCCATTCAT
GAGCTAACTTTTGAGGTTAAATTAGGTTCATATGTCATATCTTTACATGA
TATCAGAGTAAGATTCATCTCAATTCTTTGTTCACCAATATTGGCCCCCC
ATATTATTGTGTCCACAATCTAGTTAACCTACGCTGGCCCCTCCATATTA
CAGTGTCCACGTTCTAGTTAACGAGATCTGGGCTTGCAGAAGAGTGTAAA
GAATTCAGAAAAAGGATGAGTATTTGGTCTCCTTGTATAAACTTGAGCAA
TCCTTCCTTCATGAGCTAGCTAGTTTTGGAATTAAGTTAGACTAGATGTC
ATATCTTTTAATATTTATGTTCTCACTGTAGAACCATATAGCAACGAAAC
TATAGTACTATTTGTTGCACCGCTCTCTCTATATATATCGTGCATATTAA
GTTCAATTGAATCTGTTGCTAAAAGGCGGGATGGGGATTATTATTGTGCA
GGTGATCATGAGGCGTCGAGGTCGGGATTCGTGATGATGAGGAGTGGTGG
AGGAGGAATAAGTTGCCAAGATTGTGGGAACCAAGCTAAGAAAGATTGTC
AACATATGAGGTGTAGGACTTGTTGTAAGAGTAGAGGGTTTCAGTGTCAA
ACTCATGTGAAAAGTACTTGGGTTCCAGCAGCTAAAAGGAGAGAAAGGCA
ACAACAACTTGCTGCTTTGCAACAACAACAACAAGGACATAATAATAATA
ATAATAATCATAAGAATAAAAGGCAAAGGGAGGATCCAAGTGCTTCTTCT
CTTGTGTCTACTCGTTTGCCTTCAAACACTAATGGTAAAGTACTTCATGT
TTTTCTTACCTTTTCATTGCTACGTCTGTTTTAATTTAAAGGTCTTAGTT
TGACTGAACATGAATATAAGATGTTGAAATTGAAAAACGTAGATAAATAT
TTAAATTGAAACGAGGGAATAATATTAATTTTTTTTGTATCACACAAAGA
CATAGAGTCTTGAGATCCATCATGTAAAGAAGATTAATTTGATCATTGCC
TAAATGAATTCTATATAAAGTAAGTCTATAGAGAAAAGAGACCCTATAGT
AAATTCGTCAGCTTTTTCTTTTTCTATTTGTCATTCTCTTCTTCCATCAT
CACTCTTCTTTTTTATTACTCTACAAAAGATTGACAAAAATTCGTAATGA
GATATATTCAAATTTTTGAGTTAATTATGAATTTTTAATTCTAGTTAATA
GAAAGTGTGAATAAATTATTTATATGTATTACTAACAAAATAGCAAAACT
AAAACTTTACTTGTACCCTTGCGCGTGTGTATGCACAATTTCTTTCTCTT
AGACCTACACATGATATTTATCTCGACCCTAAAAAGATCACCATTATTCT
TAATTTCAATTTTCGTCAATTTTTTTTTAAGATAATAACTATTATTTGAG
TAATAATATATGTGACTTACCCAAAAAACTGTTAGTGGAGTGAGTATTTG
AGAAACCAACTCTCTAATTCATGTATAATAATTGGTGTTATCATATATTG
TCATTAGTATTGGAATTAACTTATATATCTATTAGTAAATGTACTTTTGA
AATAATAACTATTATTTGAGTAATAATATATGTTGCTTACCAAAAAAATA
ACTATTAGTTGAGTGGCTATTAACTCTCCAAATATGTATAATAATTGGTG
TTATCATTTTCATTAGTATTGGAATTAACTTATATATATAGTAAATGCAC
TTGCATTTCAAATTTTTTTACCTGCTTTTCCTTTTAGTTCGATTAAAATA
AATTGACTATTTTTCAAGCAAGTGTTTATTCTAAACTTTTCAGATGAAAT
GTTTAAAAAAACCACAAGATTAAATAGTGTTTTGATACATTTGACATATT
TTTAGTTTTAGACCATAAAATTCAAATTGCTTTACTAAATTTCGTGTCAA
GTGATACTAGGTAAAAAAAAATATTTATTTGCAATACATTAGTCCAAATA
AACCTAATTTTGTATTATGGAATTTCATGTGTTATTTTTAGGGTTAGAAG
TGGGAAAATTTCCATCAAAAGTACGTAAAGTGCTGTATTTCAGTGTATTC
AAATGAGTTCAATTGAGGATGATGAAGATCAATTAGCATATCAAGCTGCT
GTGAGCATTGGTGGACATGTTTTCAAAGGAATTTTATATGATCAAGGTCA
TGAAAGTCAGTACAATAACATGGTTGCAGCCGGAGGCGATACGTCTTCCG
GTGGTAGTGCTGGCGGAGTTCAGCACCACCACCATAATTCCGCTGCAGTA
GCTACCGCCACCACTACAAGTGGTGGCGATGCTACTGCAGCGGGTCCATC
GAATTTTCTAGATCCTTCTTTATTTCCAGCTCCCCTTAGCACTTTTATGG
TAGCTGGTACGCAATTTTTTCCACCTTCAAGATCTCCTTGATCGTCCACA
TTGATAATATTGAGGTGTCTTTTTAATTTTTATGTCAAGAGATTTGTTTT
TAATTGAAGTATTGATGTTGAATTGAGTTGTTTACATTAATTCTCTTTGG
ATTCTACATGAAGTTGTTTTTTTTTCTCTAGTTCCTTATGGTTAATTATT
GGTATCATATAGATTTGCTTTTTTATTTCACGTTAAGATGATAATATAAG
ATAAGATGATAATATACTTAAATGTATATATGTTTTGGGTTGAGTCTTAC
GATTACTTATTATTAGAATTTTTTGTATGTGTATTCGGCTCATAATGTGC
CAAAAGATAACAAAAGCAAAATTTAAGAGCATTCACATAATATTATAAGT
TTGTGATGGACTGTAAGTATATTTTAGATTTTTTAATTAGAGTTTTTAAA
TTTAAACCTAAAAGAAATCGTATTTAAAAAGAGCAGTTTACCCTATAAGT
GATTTTTTTAAGAATAAATATGGATTAGTCGAACCCAATAGTCGGGCAAC
AGTTAGAAGCTAAAAAAGATTATAATTTTAAGAAAATACTTACTTTATAA
AATTGAGATATTTGGTTAAGTTTTTAGAGGGGGAAAAGAAATGTGCTTTT
GAATAATAGCATGAATTAATCTTTACAATTAGAAAAAAAGAAAATTAAAA
ATACAAAAAGTAATTGTGAAATTAGGTCAAGCACAAACTAAGGTTCTAAA
ACTGATTTTAAAAAAAAACTTTTAAATTAATTAATCAACACAAAATTATT
ACTCTCCAAAAATATTTTCTACATAATACTTATCAAAATAAATATATTTA
GAAAATTTGGCCAAACTAACATGACTCTTCTTGATTAAGCACATAAATCA
AGTTGTTAATAAAACTTTGGCTTTATAGCAATGACTCATTTGCTTTCAAA
ACATAAAAAAATGAACAAACATTAAATATATATTTAACGGAGTAAGATAT
ATTCCAAACTAGGACACTAGAAATGGTGAAAGCTTAGTACGTTTGGAACA
TCAATTCAATTAAACTCGAATGTCACTGTTTAACTTGTCTTAATATATGT
GATAATATTTGATGGATCTTAAATATTATTTCTTTAAAAAATAATTATTC
GTTAGAAGGACAATAAGTGCTACAATGACTTAAATTTCTAAATTTTCAAC
TAGGCATAATCCTTCAAAATAACTTTCATCATACTTTTGAATAATTAAAT
ATGATATTATTGAAGTTATGTAAATTTTCATGTTTCGGGCTTGTTCGGGT
TTTTTAAATATCAAATCAAATTATTCGTGTAAAATTTTTAAAATTATAAA
TCAGACCAAATTAATAAAATTCAGATTTTTTCGGGGTTTTCAACTCTGGG
TTGATTCGTATTTTTCAAGTACCAAACCAAACCATTGTGTCGAATTTTTA
AATTTTTAATCAAACCAAACTAATAAACTTCGGATTTTTCCAGATTTTTA
GATTTTTCGGGTAAAGTTTGCATACAAACATATAATTAACTTGTGCTCCA
AATATTTCTTTAGTCCAACCATAATATAATTATCTAAGGTATTTCTTGAA
AAAATTACACAAAAGATGAGATGAGTATTGATGACACAAAAATATTCAAT
AAAAAATAACAATAAATCATCATATAAAATAAATATTGTAAAGTCATAAT
GAAAATAATCATAATTTAAAATTTTTAAATCATGCTAAAATAAGTTTAAT
AAGTATTAGTTACATTATTAAATATTTAAGGAAAACAAAAATTAGATTAT
GTAAATAAATATAAAACTAAAGAACAAATATTCAATATTATTGTCATTTT
TAGTGTTGAATTGATTTTTTCTTTTTGCATTAGTATTAATTTAATTTTAA
TTTAAGCTTTATTATAATTATCAATCTATGAACTATAATCTATATTGGAC
CATTCCAAATTCTATATTTTAAACTTGAAACAATATATTAAAAGTTAAAA
ACTATGAAATAGTATAAGAAATATTTTAAAATAATATCAACGTAAATATT
TTATGTATAAAATAATATTTTACACATATAATATAAGGATTTTTTTCCCG
ATTTGATTCAATT
EXAMPLES
Example 1. Plant Material, Hormone Treatment and RNA Isolation
[0132] Tomato plants (Solanum lycopersicum cultivar Moneymaker)
were grown in soil in a greenhouse with day/night temperatures of
23.degree. C. to 18.degree. C. and a 16/8 hours light/dark regime
for 4 weeks. Trichomes were collected at the bottom of a 50 ml tube
by vortexing stem pieces frozen in liquid nitrogen. The remaining
cleaned stem segments were thoroughly brushed to remove all
remaining trichome material (bald stem sample). Whole stems
(including trichomes) and leaves were also frozen in liquid
nitrogen, the material was ground and total RNA was isolated using
TRIzol.RTM. (monophasic solution of phenol and guanidinium
isothiocyanate) (Invitrogen, Paisley, UK).
[0133] Jasmonic acid (JA) was applied to plants by spraying 1 mM
solution made with tap water containing 0.05% SilwetL-77. Control
plants were sprayed with tap water containing 0.05% SilwetL-77.
Trichomes were collected 24 hours later and total RNA was isolated
as described above. DNA was removed with DNase (Ambion, Huntingdon,
UK) according to the manufacturer and cDNA was synthesized from 1,5
.mu.g RNA using M-MuLV Reverse Transcriptase (Fermentas, St.
Leon-Rot, Germany) according to the manufacturer in 20 .mu.l volume
that was diluted to 50 .mu.l prior to using it for PCR.
Example 2. Constructs and Stable Plant Transformations
[0134] The full SITPS5 promoter and a series of 5' deletions of it
were generated using PCR. A SacI restriction enzyme site was
created at the 5' end of each forward primer and an XbaI
restriction enzyme site at the 3' end of the reverse primer. Fifty
ng of plasmid DNA pKG1662adp-SIMTS1p:GUS were used as template with
0.25 units of Phusion Hot Start polymerase (Finnzymes, Espoo,
Finland), each primer in a concentration of 0.4 mM and dNTPs in a
concentration of 0.2 mM in 25 .mu.l reaction volume. MgCl.sub.2 was
added to the PCR mix with a final concentration of 0.3 mM. The
cycling program was set to 1 min 98.degree. C., 30 cycles of 10 sec
98.degree. C., 30 sec 58.degree. C., 60 sec at 72.degree. C.,
followed by 5 min final extension at 72.degree. C. and cooling to
12.degree. C. until removed from the thermocycler.
[0135] Primers used (5'->3' sequence, numbers indicate the
position of the 5' nucleotide of each primer. The A of the start
codon ATG is assigned to +1):
TABLE-US-00005 (SEQ ID NO: 4) SI_TPS5p -18 R
GCTCTAGATTTATTTGTTCTGCTCAA (SEQ ID NO: 5) SI_TPS5p -1253 F2
CGAGCTCGTTTCATTCAAAGTAGTGG (SEQ ID NO: 6) SI_TPS5p -1045 F
CGAGCTCAGCTGAACCAAATCCCAA (SEQ ID NO: 7) SI_TPS5p -805 F2
CGAGCTCGTCCTATTTTTCCATATTG (SEQ ID NO: 8) SI_TPS5p -612 F2
CGAGCTCATCAACAGTATTAAATGTGCTTC (SEQ ID NO: 9) SI_TPS5p -408 F2
CGAGCTCAGTAATAATGAAAATCGCATCG (SEQ ID NO: 10) SI_TPS5p -207 F2
CGAGCTCACATGTGCTATTTTTATGCTA
[0136] The 6 PCR products were purified using an Invitek (Palm
Springs, Calif., USA) column according to the manufacturer's
protocol. They were subsequently double digested with SacI and XbaI
and ligated upstream of the ATG start codon of .beta.-glucuronidase
fused to yellow fluorescent protein (sYFP1) in the SacI and XbaI
sites of the vector pJVll, replacing CaMV 35S promoter (FIG. 1).
The PCR products were verified by sequencing and the expression
cassettes (promoter fragment+GUSsYFP1+NOS terminator) were
transferred to the binary vector pBINplus (van Engelen et al., 1995
Transgenic Res 4: 288-290) by digesting with restriction enzymes
SacI and SmaI and ligating in the multiple cloning site of pBINplus
at the same restriction sites. These 6 constructs and an empty
pBINplus vector were introduced to Agrobacterium tumefaciens strain
EHA105 and used to create transgenic plants using explants derived
from cotyledons of sterile seedlings of Solanum lycopersicum
cultivar Moneymaker (MM), as previously reported (Cortina and
Culianez-Macia, 2004, Plant Cell Tissue and Organ Culture 76:
269-275).
Example 3. Analysis of Transgenic Plants
[0137] One transgenic line was obtained from the empty pBINplus
vector, four independent transgenic lines from the full length
SITPS5 promoter construct, three from the 1045 bp SITPS5 promoter
construct, five for the 805 bp and 612 bp SITPS5 promoter
constructs, eight for the 408 bp SITPS5 promoter construct and nine
for the 207 bp SITPS5 promoter construct. Insertion of the
transgene was verified by PCR on genomic DNA isolated from leaves
of the different T0 lines.
[0138] Primers used (5'->3' sequence):
TABLE-US-00006 (SEQ ID NO: 11) pJVII_1182GUS_R CCACCAACGCTGATCAATTC
(SEQ ID NO: 12) pJVI1_6936_F ATGTGCTGCAAGGCGATTAAG
[0139] The PCR was performed with Taq DNA Polymerase (Fermentas,
St. Leon-Rot, Germany) in 25 .mu.l volume according to the
manufacturer and the cycling program used was set to 2 min
95.degree. C., 30 cycles of 30 sec 95.degree. C., 30 sec 58.degree.
C., 90 sec at 72.degree. C., followed by 5 min final extension at
72.degree. C. and cooling to 12.degree. C. until removed from the
thermocycler.
[0140] Initial YFP expression of the T0 plants was estimated under
a fluorescence stereoscope and it was determined to be specific for
the "head" of the type VI trichomes of the plants (data not shown).
Trichomes were isolated from T1 plants as mentioned above, crude
extracts were prepared according to Jefferson R. A. et al., 1987,
EMBO J 6: 3901-3907 and the enzymatic GUS activity was determined
spectrophotometrically using 4-methylumbelliferyl
.beta.-D-glucuronide (MUG) as a substrate (FIG. 2).
Example 4. Yeast One Hybrid and Identification of Clone 19(6)
[0141] The 207 bp SITPS5 promoter fragment showed trichome specific
activity, although less strong than that of the full length
promoter (FIG. 2), and therefore this fragment was used for the
yeast one hybrid (Y1H) assay.
[0142] An EcoRI restriction enzyme site was created at the 5' end
of the forward primer and the reverse primer SI_TPS5p-18 R with an
XbaI restriction enzyme site at the 3' end were used in a PCR to
generate the 207 bp fragment for cloning. PCR was performed with
Phusion Hot Start polymerase (Finnzymes, Espoo, Finland) as
mentioned above, except extension time was 30 sec at 72.degree. C.
[0143] Primer used (5'->3' sequence):
TABLE-US-00007 [0143] (SEQ ID NO: 13) SI_TPS5pEcoRI_207F
CGGAATTCACATGTGCTATTTTTATGCTA
[0144] The PCR fragment was purified using a Roche (Almere,
Netherlands) column according to the manufacturer's protocol. Then
it was double digested with EcoRI and XbaI and ligated in the
multiple cloning site of pHISi vector (Clontech, Mountain View,
Calif., USA) at the same sites. After verifying the sequence, the
construct was integrated in the yeast pj69-4a genome according to
the Clontech MATCHMAKER One-Hybrid System manual. A cDNA library
created with mRNA from Solanum lycopersicum cultivar Moneymaker
trichomes was screened 3 times according to the manufacturer's
protocol (Clontech, Mountain View, Calif., USA). Three Y1H screens
yielded 76 clones, among which one putative transcription factor
19(6), appearing 32 times. The clone was sequenced using primers
that fit on the library vector (pAD-GAL4-2.1, Stratagene, Santa
Clara, Calif., USA) and specific primers designed on the obtained
sequence to get the full length clone.
[0145] Primers used (5'->3' sequence):
TABLE-US-00008 (SEQ ID NO: 14) pActF TAATACCACTACAATGGATG (SEQ ID
NO: 15) pAct_seqR CAACTGTGCATCGTGCAC (SEQ ID NO: 16) 19(6)_seqF
TTATGGCAAAGTGGTAACA (SEQ ID NO: 17) 19(6)_seqF2 TCAGTGTCAAACTCATGTG
(SEQ ID NO: 18) 19(6)_seqF3 AAGTACGTACAAGTGCTG
[0146] The candidate transcription factor was checked for
JA-inducibility and trichome specificity by quantitative real-time
PCR (FIGS. 3A and 3B). The different tissue- and control and JA
treated trichome-cDNA was obtained as described above.
Example 5. Real Time Quantitative PCR
[0147] RT-Q-PCRs were performed in the ABI 7500 Real Time PCR
System (Applied Biosystems, Carlsbad, Calif., USA) using the
Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Paisley,
UK). Each 20 .mu.l reaction contained 0.25 .mu.M of each primer,
ROX reference dye and 1 .mu.l template cDNA. The cycling program
was set to 2 min 50.degree. C., 7 min 95.degree. C., 45 cycles of
15 sec at 95.degree. C. and 1 min at 60.degree. C. and a melting
curve analysis. Primer pairs were tested for specificity and for
linearity with a standard cDNA dilution curve. The tomato Actin
gene (ACT) was used as constitutively expressed control gene.
[0148] Primers used (5'->3' sequence):
TABLE-US-00009 (SEQ ID NO: 19) ACT_QF TTAGCACCTTCCAGCAGATGT (SEQ ID
NO: 20) ACT_QR2 AACAGACAGGACACTCGCACT (SEQ ID NO: 21) 19(6)_QF
TACAAGTGGTGGCGATGCTAC (SEQ ID NO: 22) 19(6)_QR
ACCTCAATATTATCAATGTGGACAATC
Example 6. Transactivation Assay
[0149] DNA binding activity was confirmed in transactivation
assays. A NcoI restriction enzyme site was created at the 5' end of
a forward primer and a SacI restriction enzyme site at the 3' end
of a reverse primer and a full length cDNA 19(6) was generated in a
PCR performed with Phusion Hot Start polymerase (Finnzymes, Espoo,
Finland) as mentioned above. Fifty ng of plasmid DNA
pAD-GAL4-2.1_clone19(6) were used as template.
[0150] Primers used (5'->3' sequence):
TABLE-US-00010 (SEQ ID NO: 23) NcoI_19(6)F
catgccATGGCTAATTTCTTTTCATTAGG (SEQ ID NO: 24) SacI_19(6)R
cgagctcTCAAGGAGATCTTGAAGGTG
[0151] The PCR fragment was purified using a Roche (Almere,
Netherlands) column according to the manufacturer's protocol. Then
it was double digested with NcoI and SacI and ligated downstream of
35S promoter in the same sites of the vector pKG1662-35S:GUS,
replacing .beta.-glucuronidase. The PCR product was verified by
sequencing and the expression cassette which included 35S promoter,
transcription factor 19(6) and nos terminator, was transferred to
the binary vector pBINplus (van Engelen et al., 1995 Transgenic Res
4: 288-290) by digesting with restriction enzymes HindIII and EheI
and ligating in the multiple cloning sites of pBINplus digested
with HindIII and SmaI. The construct was introduced to
Agrobacterium tumefaciens strain GV3101.
[0152] Five week old Nicotiana benthamiana leaves were
co-infiltrated with A. tumefaciens GV3101 cultures carrying various
promoter:GUS reporter and the 35S:19(6) effector constructs.
Specifically, the promoter constructs used were
pBINplus-ShMKS1p:GUS, pBINplus-SITPS11p:GUS and
pBINplus-SITPS5p:GUS. These constructs were made by cloning into a
PJVII-GUSSYFP1 vector each of SITPS5, SITPS11 or Solanum
habrochaites methylketone synthase 1 (ShMkS1; Fridman et al., 2005
Plant Cell 17:1252-1267, published on line Mar. 16, 20005) promoter
sequences between SacI and XbaI sites, and nucleic acid sequences
encoding .beta.-glucuronidase (GUS) fused to a yellow fluorescent
protein (sYFP1) between XbaI and BamHI sites (Schematic drawing of
PJVII-GUSSYFP1 is shown in FIG. 1). A control pGreen-35S:RFP
effector construct was also used (pGreen; Hellens et al., 2000,
Plant Molecular Biology 42: 819-832). In order to correct for
infiltration errors A. tumefaciens GV3101 culture carrying
pBINplus-35S:LUC was also added in each culture mix in a ratio of 5
(promoter:GUS): 5(35S:TF): 1(35S:LUC). Two days later leaf disks
from the infiltrated areas were collected, frozen in liquid
nitrogen and crude extracts were prepared in extraction buffer
containing 25 mM Tris phosphate pH 7.8, 2 mM DTT, 2 mM CDTA pH 7.8,
10% glycerol and 1% Triton X-100. The enzymatic GUS activity was
determined spectrophotometrically using 4-methylumbelliferyl
.beta.-D-glucuronide (MUG) as a substrate according to Jefferson R.
A. et al., 1987, EMBO J 6: 3901-3907. The luciferase assay was
performed using the same extraction buffer according to van Leeuwen
et al., 2000, Plant Molecular Biology Reporter 18: 143a-143t.
Enzymatic GUS activity was normalized for luciferase activity for
each sample and results are presented in FIG. 4.
[0153] As shown in FIG. 4, the 35S:19(6) effector construct
activated GUS activity in pBINplus-SITPS5p:GUS more than 10-fold
over the control pBINplus-ShMKS1p:GUS construct. Additionally, the
effector construct was also able to activate GUS in
pBINplus-SITPS11p:GUS 7-fold over the control.
[0154] These data confirmed TF 19(6) capability to activate SITPS5
and SITPS11 promoters and shows that TF19(6) may be used in
regulating expression levels of the TPS5 and TPS11, and alter
terpene content in a plant, tissue or cell.
Example 7. TF 19(6) Sequence Identity
[0155] Polypeptide sequence identity was determined using
BLAST.RTM. (Basic Local Alignment Search Tool) algorithm described
in Althsul et al. 1990 J Mol Biol 215:403-410. BLAST.RTM. (Basic
Local Alignment Search Tool) program is publicly available through
the National Center for Biotechnology Information (NCBI) at the web
site of the National Institute of Health, USA. A BLAST.RTM. (Basic
Local Alignment Search Tool) homology search identified that amino
acid sequence of TF 19(6) has 40.68% identity over the length of
the entire protein as compared to the Lateral Root Primordium
(LRP1) protein, a member of Arabidopsis thaliana SRS (short
internode related sequences)-type transcription factors with zinc
finger motifs that are induced by auxins. Two zinc finger-type
domains were found within TF 19(6): a zinc finger domain (amino
acids 128-170) in the N-terminal part and a small conserved
LRP1-type domain in the C terminus (amino acids 224-272). The
polypeptide of the invention also possess conserved DUF702 domain
of unknown function characteristic of SRS-type transcription
factors. The BLAST.RTM. (Basic Local Alignment Search Tool) search
also identified a tomato gene encoding a protein 45% homologous to
TF19(6) and containing one zinc finger-type domains and DUF702
domain. The function of this protein is unknown.
Example 8: Insect Bioassays
[0156] Insect bioassays are performed under controlled conditions
in the greenhouse. Plants are modified using the methods of the
present invention. For example: Solanum lycopersicum is modified by
means of Agrobacterium-mediated transformation with pBIN 35S-19(6)
(encoding the protein having the amino acid sequence of SEQ ID
NO:2). Alternatively, Solanum lycopersicum mutants are identified
within a mutagenized population so that the mutants carry a
mutation or mutations in a nucleic acid sequence of SEQ ID NO: 1 or
fragments thereof encoding the transcription factor of the present
invention. Insect pest resistance of modified plants is compared to
that of non-modified control plants in choice- and no-choice tests
as described in Bleeker et al., 2011 Phytochemistry 72: 8-73; and
patent application WO 2010/099,985. Resistance to the following
insect classes is determined: Lepidoptera; Coleoptera; Diptera;
Hemiptera; Acari; Thysanoptera.
[0157] Insect Preference Test.
[0158] A choice test is performed in which insects at different
life stages, e.g., larvae or adults, are allowed to choose between
plants that produce terpenes such as linalool or nerolidol (through
expression of a nucleic acid sequence encoding the amino acid
sequence of SEQ ID NO:2) and control plants. The test determines
the repellent activity of the terpenes produced because of the
activating effect of the protein of SEQ ID NO:2 Insect performance
test (no-choice test). A no-choice test is performed to determine
the toxic effects of the terpenes produced by activating expression
of a nucleic acid sequence encoding the amino acid sequence of SEQ
ID NO:2. In these experiments, insect pest species are forced to
eat from (transgenic) plants that have modified terpenes production
through expression of a nucleic acid sequence encoding the amino
acid sequence of SEQ ID NO:2 and control (or empty vector) plants.
Subsequently, insect performance, e.g., growth, development or
fitness, is determined as a measure of toxicity.
Sequence CWU 1
1
2411056DNALycopersicon esculentum 1atggctaatt tcttttcatt aggtgggaat
caagaacaac aacatcaaga aattagcagc 60agccaagcat tagtacccac agagagtaat
aattggtttt tgtacagaaa tgaacatcat 120catcatcatc ataatcaaga
aatacccaac acttacaaag gttttgagtt atggcaaagt 180ggtaacactc
cacaacacca acaccaacac caccaacaac aacaacagtt tcgtcatccg
240atttatcctt tgcaagatct ttattccact gatgttggat taggggttgg
gccaagcaga 300agtggctttg atatatctgc aggtgatcat gaggcgtcga
ggtcgggatt cgtgatgatg 360aggagtggtg gaggaggaat aagttgccaa
gattgtggga accaagctaa gaaagattgt 420caacatatga ggtgtaggac
ttgttgtaag agtagagggt ttcagtgtca aactcatgtg 480aaaagtactt
gggttccagc agctaaaagg agagaaaggc aacaacaact tgctgctttg
540caacaacaac aacaaggaca taataataat aataataatc ataagaataa
aaggcaaagg 600gaggatccaa gtgcttcttc tcttgtgtct actcgtttgc
cttcaaacac taatgggtta 660gaagtgggaa aatttccatc aaaagtacgt
acaagtgctg tatttcagtg tattcaaatg 720agttcaattg aggatgatga
agatcaatta gcatatcaag ctgctgtgag cattggtgga 780catgttttca
aaggaatttt atatgatcaa ggtcatgaaa gtcagtacaa taacatggtt
840gcagccggag gcgatacgtc ttccggtggt agtgctggcg gagttcagca
ccaccaccat 900aattccgctg cagtagctac cgccaccact acaagtggtg
gcgatgctac tgcagcgggt 960ccatcgaatt ttctagatcc ttctttattt
ccagctcccc ttagcacttt tatggtagct 1020ggtacgcaat tttttccacc
ttcaagatct ccttga 10562351PRTLycopersicon esculentum 2Met Ala Asn
Phe Phe Ser Leu Gly Gly Asn Gln Glu Gln Gln His Gln 1 5 10 15 Glu
Ile Ser Ser Ser Gln Ala Leu Val Pro Thr Glu Ser Asn Asn Trp 20 25
30 Phe Leu Tyr Arg Asn Glu His His His His His His Asn Gln Glu Ile
35 40 45 Pro Asn Thr Tyr Lys Gly Phe Glu Leu Trp Gln Ser Gly Asn
Thr Pro 50 55 60 Gln His Gln His Gln His His Gln Gln Gln Gln Gln
Phe Arg His Pro 65 70 75 80 Ile Tyr Pro Leu Gln Asp Leu Tyr Ser Thr
Asp Val Gly Leu Gly Val 85 90 95 Gly Pro Ser Arg Ser Gly Phe Asp
Ile Ser Ala Gly Asp His Glu Ala 100 105 110 Ser Arg Ser Gly Phe Val
Met Met Arg Ser Gly Gly Gly Gly Ile Ser 115 120 125 Cys Gln Asp Cys
Gly Asn Gln Ala Lys Lys Asp Cys Gln His Met Arg 130 135 140 Cys Arg
Thr Cys Cys Lys Ser Arg Gly Phe Gln Cys Gln Thr His Val 145 150 155
160 Lys Ser Thr Trp Val Pro Ala Ala Lys Arg Arg Glu Arg Gln Gln Gln
165 170 175 Leu Ala Ala Leu Gln Gln Gln Gln Gln Gly His Asn Asn Asn
Asn Asn 180 185 190 Asn His Lys Asn Lys Arg Gln Arg Glu Asp Pro Ser
Ala Ser Ser Leu 195 200 205 Val Ser Thr Arg Leu Pro Ser Asn Thr Asn
Gly Leu Glu Val Gly Lys 210 215 220 Phe Pro Ser Lys Val Arg Thr Ser
Ala Val Phe Gln Cys Ile Gln Met 225 230 235 240 Ser Ser Ile Glu Asp
Asp Glu Asp Gln Leu Ala Tyr Gln Ala Ala Val 245 250 255 Ser Ile Gly
Gly His Val Phe Lys Gly Ile Leu Tyr Asp Gln Gly His 260 265 270 Glu
Ser Gln Tyr Asn Asn Met Val Ala Ala Gly Gly Asp Thr Ser Ser 275 280
285 Gly Gly Ser Ala Gly Gly Val Gln His His His His Asn Ser Ala Ala
290 295 300 Val Ala Thr Ala Thr Thr Thr Ser Gly Gly Asp Ala Thr Ala
Ala Gly 305 310 315 320 Pro Ser Asn Phe Leu Asp Pro Ser Leu Phe Pro
Ala Pro Leu Ser Thr 325 330 335 Phe Met Val Ala Gly Thr Gln Phe Phe
Pro Pro Ser Arg Ser Pro 340 345 350 39013DNALycopersicon esculentum
3gcaagtcaaa ttgtatatgc tcttaattag ggtttatgca cctagaagta gtattttaat
60cgcatattag ttatcgatgt tcctaaatta atcgcctatt agttatcgat gttcctaaat
120ataattgacc taattacaaa attaagatag aactgattat attttttcaa
ttttatcctt 180acaaggagca attctttttg aaagtatgaa ccactttgta
aagttttttt ttaaaaaaaa 240tcttaaagga gtaaatcagt aaaactacct
ttcatattta tgatttttta agaagcatgt 300aaagaaaaaa tagaaaatca
atatagaacg aaaaaagaat tttataaaac ctcataactt 360aataaaaaga
atcatattta taagaaatat tttcttccca catggaataa taaattgcac
420aaactgtaaa tattctctac tacaatataa tgttataaca cacgtatacc
gttggttttt 480cagtataaat ataatatcca tattttagat atattagctg
ttaaaaacaa tataatatgg 540atggagacaa gttaaatgta tgtaatttta
ccttgtaatc ccaattctca atatatatat 600atatatatat atatatatat
attcctccaa gacaaaacat tggattttta ttctaggaac 660ttgaattaag
aattcaattt actcgtaaaa attaaaaaga aatttcttat gatcttatca
720aatatttaat aggtgaatag ttaaatttga caagttaaat taagatgtat
gtccatcacc 780tcatcataat tcaaattatt ttaacaaata tccttaatca
tgatcttctt tctttttagg 840ttgaaataat tatcattaga tttgtacata
gtataggaat atattaagag ctaatatatc 900taaaatgtgg ataaataaaa
caattcttgc tcaaaatttt aaatagtttt aatactttta 960caatacttga
cacgcggcat tatatagcca acaattttac gggctaagac ataacattta
1020tcttggaaat tctctattat taatcattag cttagattgt ctgagttttt
gaaggtcttt 1080tatttagtta taggcaattt tacctagttt tatagaatta
aaattattgt ccgttgttat 1140atttaggtaa gaaaaaaagt taataaatca
gacaagaaaa tataaagaac cgaaataatt 1200atgtaatgcc taaaatagtt
ggttttatat acataaagac tgttgaaaat tgaaattaat 1260attgcggctc
cttcatttat tggtattact gttaattacg tgattgaaag gaaaaaaaaa
1320gttttaccaa aaaaaagtat aaaaataagt tttgtacttt atgtcaacag
ttagtcatca 1380atagttactg ctataatact aggtgccaac actatgtata
attcgaatgt gataataatt 1440tctggaaaaa aaaattaaag gatatttgat
ttgatatggt cctagataat gtagatgatg 1500aaggggtgtt aattagtcgt
ttcaaattga taggttattt tgaaaaattg tgtcatatta 1560attgtttata
ttttcaatga tgtatgatta aaattaaaat ttttgaatct gtcttaatcg
1620ttttggtttc tctttgattt aggtataatt caaattgatg gttaattttt
ttaaacgtca 1680tctaacaact ataaaatttg ataaaaaata tttaaaattt
acaataacat aaaatgaaaa 1740tatgttttcc aactatacca atttaggagg
agaaacatag ttattgtttt actattatcg 1800ctagtattat gaatgagata
tgaaaattta tattaattta tattggaatc tataattgat 1860tttattaaaa
aaaattaagt gcgtgtactt tgacattttt tttgttttta actcggcatt
1920caaagttcat attgaagttt taactaaatt cgaatcgcac tcttcagagc
aatgcaggga 1980tgggtctccc aacaacattt tgtcgatagt ctatacccag
agctcgaact taagacctct 2040gattaagaat aaaatacttc atttataaac
tgatcatctt aatattttca aaatttaaat 2100gtcacatatt ttctaagata
tcctcgaaac ataataataa gttgaaatgt ataatgtttg 2160attgagacta
aactgaggcg tttatatata caatcgtaga attaaaatat ttaattgcca
2220tctgaaaatt aatttaaata tttatctatg tactatacct taattaattc
tttcatgaca 2280aactttcttg gacatttttt catgaaaaat gcatataact
taaacaaggc cgatacctta 2340cacccaaatt ggacagtata tttagaagag
ggggaataat ggtaaagagg gccggtatca 2400ggtttacaca gagatgaaaa
gttaggtgga gtttatttgt tcggatggat ttatcagttt 2460tttcgtagat
tttatattta tattagattc ttttttttac gtatatatta aattataatc
2520cctaaacaaa ttgatttaga atctcaaact cataatctta acttcgcccc
taacttttat 2580atatatatat atatatataa tatttttaat atatcattag
ttacacatta ttttttatat 2640tatgttgtgt attactgatg aataatgatt
tatggaaata caaaaagctc ttattcagta 2700atacatacat tagtatgatc
atcttttttc acattctttc catcgcgata tatgtttttt 2760ttttaaacta
taacacaata aacactgcat taaaaataat tgtacatatt ttttgtgtct
2820caatttatgt gatacctttt agttttttaa gagctaaaca atttaaattt
gagcgagaat 2880ttacgcatga aattttcgaa aattctaaaa agaaatttat
atattaataa aaactacgta 2940aaaatactat aagacacaat aattgacaat
tcaaaatatt taaaagtcaa agatatactt 3000atttgaattt caaaatctga
aaagtatcac ataaatagga ggagagagta acgaatatca 3060atattaatga
tatattatat tcaccactaa tattcttaaa aataaatatt aaaaacacca
3120ttaaattcga tgtgaattat tagtttgatc cctgaactat tgacagtatt
ataaacactc 3180ctctactggg ttagatgaac ttaaatacac actcgatctt
gtcacaatga tgaaatacac 3240cctaatgaaa atcatattta ctcttcctat
ttctaaccat cggaaagagt catcgtggct 3300aggaaactat actagcgacc
tacccaattc attatagaaa ttttcgcgat caatgattga 3360aaatttagaa
tgtttccaac actttatctg tcaacttttt attaagagtt tcaagctcgt
3420ataagaattt gaaatcactt ttagtatatc atgtagtaga tctaaatata
tttaaaatta 3480ttataaattt tttttaaaaa actaataatt cacactaaat
tgacaaatat cttcaatact 3540tagcttctca cttattttat acgacctacc
aaacaatcgc gaaacttttt aagttactgc 3600aaactgtagc ggtaaagaga
ggggaggggg ggggggtagt tgtggtgctt tttagcgttg 3660gcggcgtttg
cagagctgta atatatataa tatacctttt ctattaatgt accctcactc
3720actcacttcc tctccataat tctttataca aacaatcatt ttttcttaaa
cttgctctat 3780tataaattca cattttttct ttatatatac acatacatat
agagcaaaaa agaagttcta 3840attttgtaaa cccttcaaaa aaaagaaaaa
taattttttt tgagatcata aatgaagaaa 3900tccaagggat acaaacatca
tatttgtgtt ataagttggt gcacttttgt ggtatggatt 3960gtgattaatc
actaatcata atcaagatta acaacaagta atggctaatt tcttttcatt
4020aggtgggaat caagaacaac aacatcaaga aattagcagc agccaagcat
tagtacccac 4080agagagtaat aattggtttt tgtacagaaa tgaacatcat
catcatcatc ataatcaaga 4140aatacccaac acttacaaag gttttgagtt
atggcaaagt ggtaacactc cacaacacca 4200acaccaacac caccaacaac
aacaacagtt tcgtcatccg atttatcctt tgcaagatct 4260ttattccact
gatgttggat taggggttgg gccaagcaga agtggctttg atatatctgc
4320aggtgatcat cagaaacaga tttagaattt aaactttatc tattcagtac
tttctaaagt 4380acttatagat ctataattta agtttgataa atttaatatt
tatgttctaa acaattcaca 4440acattttgca attagggatt tcgaaacgta
tttactgaag catgttagaa ttcccagcct 4500cgaaaaggca tgggaatttg
gtctatggac ttgggaaatt ctccattcat gagctaactt 4560ttgaggttaa
attaggttca tatgtcatat ctttacatga tatcagagta agattcatct
4620caattctttg ttcaccaata ttggcccccc atattattgt gtccacaatc
tagttaacct 4680acgctggccc ctccatatta cagtgtccac gttctagtta
acgagatctg ggcttgcaga 4740agagtgtaaa gaattcagaa aaaggatgag
tatttggtct ccttgtataa acttgagcaa 4800tccttccttc atgagctagc
tagttttgga attaagttag actagatgtc atatctttta 4860atatttatgt
tctcactgta gaaccatata gcaacgaaac tatagtacta tttgttgcac
4920cgctctctct atatatatcg tgcatattaa gttcaattga atctgttgct
aaaaggcggg 4980atggggatta ttattgtgca ggtgatcatg aggcgtcgag
gtcgggattc gtgatgatga 5040ggagtggtgg aggaggaata agttgccaag
attgtgggaa ccaagctaag aaagattgtc 5100aacatatgag gtgtaggact
tgttgtaaga gtagagggtt tcagtgtcaa actcatgtga 5160aaagtacttg
ggttccagca gctaaaagga gagaaaggca acaacaactt gctgctttgc
5220aacaacaaca acaaggacat aataataata ataataatca taagaataaa
aggcaaaggg 5280aggatccaag tgcttcttct cttgtgtcta ctcgtttgcc
ttcaaacact aatggtaaag 5340tacttcatgt ttttcttacc ttttcattgc
tacgtctgtt ttaatttaaa ggtcttagtt 5400tgactgaaca tgaatataag
atgttgaaat tgaaaaacgt agataaatat ttaaattgaa 5460acgagggaat
aatattaatt ttttttgtat cacacaaaga catagagtct tgagatccat
5520catgtaaaga agattaattt gatcattgcc taaatgaatt ctatataaag
taagtctata 5580gagaaaagag accctatagt aaattcgtca gctttttctt
tttctatttg tcattctctt 5640cttccatcat cactcttctt ttttattact
ctacaaaaga ttgacaaaaa ttcgtaatga 5700gatatattca aatttttgag
ttaattatga atttttaatt ctagttaata gaaagtgtga 5760ataaattatt
tatatgtatt actaacaaaa tagcaaaact aaaactttac ttgtaccctt
5820gcgcgtgtgt atgcacaatt tctttctctt agacctacac atgatattta
tctcgaccct 5880aaaaagatca ccattattct taatttcaat tttcgtcaat
ttttttttaa gataataact 5940attatttgag taataatata tgtgacttac
ccaaaaaact gttagtggag tgagtatttg 6000agaaaccaac tctctaattc
atgtataata attggtgtta tcatatattg tcattagtat 6060tggaattaac
ttatatatct attagtaaat gtacttttga aataataact attatttgag
6120taataatata tgttgcttac caaaaaaata actattagtt gagtggctat
taactctcca 6180aatatgtata ataattggtg ttatcatttt cattagtatt
ggaattaact tatatatata 6240gtaaatgcac ttgcatttca aattttttta
cctgcttttc cttttagttc gattaaaata 6300aattgactat ttttcaagca
agtgtttatt ctaaactttt cagatgaaat gtttaaaaaa 6360accacaagat
taaatagtgt tttgatacat ttgacatatt tttagtttta gaccataaaa
6420ttcaaattgc tttactaaat ttcgtgtcaa gtgatactag gtaaaaaaaa
atatttattt 6480gcaatacatt agtccaaata aacctaattt tgtattatgg
aatttcatgt gttattttta 6540gggttagaag tgggaaaatt tccatcaaaa
gtacgtaaag tgctgtattt cagtgtattc 6600aaatgagttc aattgaggat
gatgaagatc aattagcata tcaagctgct gtgagcattg 6660gtggacatgt
tttcaaagga attttatatg atcaaggtca tgaaagtcag tacaataaca
6720tggttgcagc cggaggcgat acgtcttccg gtggtagtgc tggcggagtt
cagcaccacc 6780accataattc cgctgcagta gctaccgcca ccactacaag
tggtggcgat gctactgcag 6840cgggtccatc gaattttcta gatccttctt
tatttccagc tccccttagc acttttatgg 6900tagctggtac gcaatttttt
ccaccttcaa gatctccttg atcgtccaca ttgataatat 6960tgaggtgtct
ttttaatttt tatgtcaaga gatttgtttt taattgaagt attgatgttg
7020aattgagttg tttacattaa ttctctttgg attctacatg aagttgtttt
tttttctcta 7080gttccttatg gttaattatt ggtatcatat agatttgctt
ttttatttca cgttaagatg 7140ataatataag ataagatgat aatatactta
aatgtatata tgttttgggt tgagtcttac 7200gattacttat tattagaatt
ttttgtatgt gtattcggct cataatgtgc caaaagataa 7260caaaagcaaa
atttaagagc attcacataa tattataagt ttgtgatgga ctgtaagtat
7320attttagatt ttttaattag agtttttaaa tttaaaccta aaagaaatcg
tatttaaaaa 7380gagcagttta ccctataagt gattttttta agaataaata
tggattagtc gaacccaata 7440gtcgggcaac agttagaagc taaaaaagat
tataatttta agaaaatact tactttataa 7500aattgagata tttggttaag
tttttagagg gggaaaagaa atgtgctttt gaataatagc 7560atgaattaat
ctttacaatt agaaaaaaag aaaattaaaa atacaaaaag taattgtgaa
7620attaggtcaa gcacaaacta aggttctaaa actgatttta aaaaaaaact
tttaaattaa 7680ttaatcaaca caaaattatt actctccaaa aatattttct
acataatact tatcaaaata 7740aatatattta gaaaatttgg ccaaactaac
atgactcttc ttgattaagc acataaatca 7800agttgttaat aaaactttgg
ctttatagca atgactcatt tgctttcaaa acataaaaaa 7860atgaacaaac
attaaatata tatttaacgg agtaagatat attccaaact aggacactag
7920aaatggtgaa agcttagtac gtttggaaca tcaattcaat taaactcgaa
tgtcactgtt 7980taacttgtct taatatatgt gataatattt gatggatctt
aaatattatt tctttaaaaa 8040ataattattc gttagaagga caataagtgc
tacaatgact taaatttcta aattttcaac 8100taggcataat ccttcaaaat
aactttcatc atacttttga ataattaaat atgatattat 8160tgaagttatg
taaattttca tgtttcgggc ttgttcgggt tttttaaata tcaaatcaaa
8220ttattcgtgt aaaattttta aaattataaa tcagaccaaa ttaataaaat
tcagattttt 8280tcggggtttt caactctggg ttgattcgta tttttcaagt
accaaaccaa accattgtgt 8340cgaattttta aatttttaat caaaccaaac
taataaactt cggatttttc cagattttta 8400gatttttcgg gtaaagtttg
catacaaaca tataattaac ttgtgctcca aatatttctt 8460tagtccaacc
ataatataat tatctaaggt atttcttgaa aaaattacac aaaagatgag
8520atgagtattg atgacacaaa aatattcaat aaaaaataac aataaatcat
catataaaat 8580aaatattgta aagtcataat gaaaataatc ataatttaaa
atttttaaat catgctaaaa 8640taagtttaat aagtattagt tacattatta
aatatttaag gaaaacaaaa attagattat 8700gtaaataaat ataaaactaa
agaacaaata ttcaatatta ttgtcatttt tagtgttgaa 8760ttgatttttt
ctttttgcat tagtattaat ttaattttaa tttaagcttt attataatta
8820tcaatctatg aactataatc tatattggac cattccaaat tctatatttt
aaacttgaaa 8880caatatatta aaagttaaaa actatgaaat agtataagaa
atattttaaa ataatatcaa 8940cgtaaatatt ttatgtataa aataatattt
tacacatata atataaggat ttttttcccg 9000atttgattca att
9013426DNAArtificial sequencePrimer 4gctctagatt tatttgttct gctcaa
26526DNAArtificial sequencePrimer 5cgagctcgtt tcattcaaag tagtgg
26625DNAArtificial SequencePrimer 6cgagctcagc tgaaccaaat cccaa
25726DNAArtificial SequencePrimer 7cgagctcgtc ctatttttcc atattg
26830DNAArtificial SequencePrimer 8cgagctcatc aacagtatta aatgtgcttc
30929DNAArtificial SequencePrimer 9cgagctcagt aataatgaaa atcgcatcg
291028DNAArtificial SequencePrimer 10cgagctcaca tgtgctattt ttatgcta
281120DNAArtificial SequencePrimer 11ccaccaacgc tgatcaattc
201221DNAArtificial SequencePrimer 12atgtgctgca aggcgattaa g
211329DNAArtificial SequencePrimer 13cggaattcac atgtgctatt
tttatgcta 291420DNAArtificial SequencePriemr 14taataccact
acaatggatg 201518DNAArtificial SequencePrimer 15caactgtgca tcgtgcac
181619DNAArtificial SequencePrimer 16ttatggcaaa gtggtaaca
191719DNAArtificial SequencePrimer 17tcagtgtcaa actcatgtg
191818DNAArtificial SequencePrimer 18aagtacgtac aagtgctg
181921DNAArtificial SequencePrimer 19ttagcacctt ccagcagatg t
212021DNAArtificial SequencePrimer 20aacagacagg acactcgcac t
212121DNAArtificial SequencePrimer 21tacaagtggt ggcgatgcta c
212227DNAArtificial SequencePrimer 22acctcaatat tatcaatgtg gacaatc
272329DNAArtificial SequencePrimer 23catgccatgg ctaatttctt
ttcattagg 292427DNAArtificial SequencePrimer 24cgagctctca
aggagatctt gaaggtg 27
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