U.S. patent application number 11/630012 was filed with the patent office on 2008-11-27 for method for modifying plant morphology, biochemistry and physiology comprising expression of cytokinin oxydase in the seeds.
Invention is credited to Thomas Schmulling, Tomas Werner.
Application Number | 20080295197 11/630012 |
Document ID | / |
Family ID | 31496961 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080295197 |
Kind Code |
A1 |
Schmulling; Thomas ; et
al. |
November 27, 2008 |
Method for Modifying Plant Morphology, Biochemistry and Physiology
Comprising Expression of Cytokinin Oxydase in the Seeds
Abstract
The present invention relates to methods and compositions for
increasing seed yield of a plant. The methods comprise expression
of a cytokinin oxidase in the aleurone and/or embryo of a seed. The
invention also relates to vectors comprising a nucleic acid
encoding a cytokinin oxidase that is operably linked to a promoter
capable of driving expression in the aleurone and/or embryo of a
seed, and to host cells, transgenic cells and plants comprising
such sequences. The use of these sequences for increasing yield is
also provided.
Inventors: |
Schmulling; Thomas; (Berlin,
DE) ; Werner; Tomas; (Berlin, DE) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Family ID: |
31496961 |
Appl. No.: |
11/630012 |
Filed: |
June 20, 2005 |
PCT Filed: |
June 20, 2005 |
PCT NO: |
PCT/EP05/06620 |
371 Date: |
February 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10871304 |
Jun 18, 2004 |
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11630012 |
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10014101 |
Dec 10, 2001 |
7259296 |
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10871304 |
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PCT/EP01/06833 |
Jun 18, 2001 |
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10014101 |
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60258415 |
Dec 27, 2000 |
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Current U.S.
Class: |
800/278 ;
435/252.3; 435/254.11; 435/320.1; 435/419; 536/23.2; 800/298;
800/320; 800/320.1; 800/320.2; 800/320.3 |
Current CPC
Class: |
C12N 9/0032 20130101;
C12N 15/8287 20130101; Y02A 40/146 20180101; C12N 15/8261 20130101;
C12N 15/8271 20130101; C12N 15/8295 20130101 |
Class at
Publication: |
800/278 ;
536/23.2; 435/419; 435/254.11; 435/252.3; 435/320.1; 800/298;
800/320; 800/320.1; 800/320.2; 800/320.3 |
International
Class: |
C12N 15/52 20060101
C12N015/52; C12N 15/82 20060101 C12N015/82; C12N 5/10 20060101
C12N005/10; C12N 1/15 20060101 C12N001/15; C12N 1/21 20060101
C12N001/21; A01H 5/00 20060101 A01H005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 16, 2000 |
EP |
00870132.8 |
Mar 16, 2001 |
EP |
01870053.4 |
Dec 10, 2002 |
EP |
PCT/EP02/13990 |
Claims
1. A method for increasing seed yield in a plant, said method
comprising increasing the level and/or activity of a cytokinin
oxidase (CKX) in the embryo and/or aleurone of a plant seed,
wherein said embryo and/or aleurone have increased levels and/or
activity of cytokinin oxidase relative to other parts of said
seed.
2. The method according to claim 1, wherein said increased
expression in the embryo and/or aleurone is effected by introducing
a genetic modification.
3. The method according to claim 2, wherein said genetic
modification is effected by one of: T-DNA activation, TILLING,
site-directed mutagenesis or directed evolution.
4. A method for increasing seed yield in a plant, said method
comprising introducing into a plant cell a genetic construct
comprising an isolated nucleic acid molecule encoding a cytokinin
oxidase wherein said isolated nucleic acid molecule is operably
linked to a promoter capable of driving expression in the embryo
and/or aleurone of a seed.
5. A method for producing a plant having increased seed yield and
increased expression of a cytokinin oxidase in the embryo and/or
aleurone of a seed, said method comprising introducing into a plant
cell a genetic construct comprising an isolated nucleic acid
molecule encoding a cytokinin oxidase wherein said isolated nucleic
acid molecule is operably linked to a promoter capable of driving
expression in the embryo and/or aleurone of a seed.
6. A method for producing a plant having increased seed yield and
increased expression of a cytokinin oxidase in the embryo and/or
aleurone of a seed, said method comprising: (a) introducing into a
plant cell a genetic construct comprising an isolated nucleic acid
molecule encoding a cytokinin oxidase, wherein said isolated
nucleic acid molecule is operably linked to a promoter capable of
driving expression in the embryo and/or aleurone of a seed; (b)
regenerating a plant therefrom; (c) growing the regenerated plant
to seed set; and (d) selecting a plant with increased seed yield
compared to a corresponding wild type plant.
7. The method of any of claim 4 wherein the promoter capable of
driving expression in the embryo and/or aleurone comprises
nucleotides 1-1256 of SEQ ID NO: 41.
8. The method of claim 2 wherein the nucleic acid molecule encoding
a cytokinin oxidase is selected from the group consisting of: (a)
nucleic acids comprising a DNA sequence as given in any of SEQ ID
NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or
the complement thereof, (b) nucleic acids comprising the RNA
sequences corresponding to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5,
7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the complement thereof,
(c) nucleic acids specifically hybridizing to the complement of any
of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33
or 34, (d) nucleic acids encoding a protein comprising the amino
acid sequence as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12,
35, 36 or 38, or the complement thereof, (e) nucleic acids as
defined in any of (a) to (d) characterized in that said nucleic
acid is DNA, genomic DNA, cDNA, synthetic DNA or RNA wherein T is
replaced by U, (f) nucleic acid which is degenerate compared to a
nucleic acid as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7,
9, 11, 25, 26, 28 to 30, 33 or 34, or which is degenerate compared
to a nucleic acid as defined in any of (a) to (e) as a result of
the genetic code, (g) nucleic acids which are divergent from a
nucleic acid encoding a protein as given in any of SEQ ID NOs: 2,
4, 6, 8, 10, 12, 35, 36 or 38, or which is divergent from a nucleic
acid as defined in any of (a) to (e), due to the differences in
codon usage between the organisms, (h) nucleic acids encoding a
protein as given in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38,
or nucleic acids as defined in (a) to (e) which are divergent due
to the differences between alleles, (i) nucleic acids encoding a
protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36
or 38, (j) functional fragments of nucleic acids as defined in any
of (a) to (i) having the biological activity of a cytokinin
oxidase, and (k) nucleic acids encoding a plant cytokinin oxidase,
comprising the consensus sequence hTDYLhholGGTLSssG and
cLFxushGsLGQFGIIstA or comprising expression in seeds of a nucleic
acid encoding a protein that reduces the level of active cytokinins
in plants or plant parts.
9. The method of claim 4, wherein the isolated nucleic acid
molecule operably linked to a promoter capable of driving
expression in the embryo and/or aleurone comprises the nucleotide
sequence as set forth in SEQ ID NO: 41.
10. The method of claim 1 wherein the increase in seed yield
comprises at least one of: total weight of seeds, total number of
seeds, total number of filled seeds, harvest index, and thousand
kernel weight.
11. An isolated nucleic acid molecule encoding a cytokinin oxidase
operably linked to a promoter capable of driving overexpression of
said nucleic acid molecule in the embryo and/or aleurone of a seed
relative to other parts of said seed.
12. The isolated nucleic acid molecule of claim 11 wherein the
nucleic acid molecule encoding a cytokinin oxidase is selected from
the group consisting of: (a) nucleic acids comprising a DNA
sequence as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9,
11, 25, 26, 28 to 30, 33 or 34, or the complement thereof, (b)
nucleic acids comprising the RNA sequences corresponding to any of
SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or
34, or the complement thereof, (c) nucleic acids specifically
hybridizing to the complement of any of SEQ ID NOs: 42, 37, 27, 1,
3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, (d) nucleic acids
encoding a protein comprising the amino acid sequence as given in
any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or the
complement thereof, (e) nucleic acids as defined in any of (a) to
(d) characterized in that said nucleic acid is DNA, genomic DNA,
cDNA, synthetic DNA or RNA wherein T is replaced by U, (f) nucleic
acid which is degenerate compared to a nucleic acid as given in any
of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33
or 34, or which is degenerate compared to a nucleic acid as defined
in any of (a) to (e) as a result of the genetic code, (g) nucleic
acids which are divergent from a nucleic acid encoding a protein as
given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or
which is divergent from a nucleic acid as defined in any of (a) to
(e), due to the differences in codon usage, (h) nucleic acids
encoding a protein as given in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35,
36 or 38 or nucleic acids as defined in (a) to (e) which are
divergent due to the differences between alleles, (i) nucleic acids
encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10,
12, 35, 36 or 38, (j) functional fragments of nucleic acids as
defined in any of (a) to (i) having the biological activity of a
cytokinin oxidase, and (k) nucleic acids encoding a plant cytokinin
oxidase, comprising the consensus sequence hTDYLhholGGTLSssG and
cLFxushGsLGQFGIIstA or comprising expression, preferably in seeds,
of a nucleic acid encoding a protein that reduces the level of
active cytokinins in plants or plant parts.
13. A vector comprising the isolated nucleic acid molecule of claim
11.
14. A vector comprising the isolated nucleic acid molecule having
the sequence set forth in SEQ ID NO: 41.
15. A host cell comprising the isolated nucleic acid molecule of
claim 11.
16. A host cell comprising a vector of claims 13.
17. The host cell of claim 16, which is a bacterial, fungal, or
plant cell.
18. Plant having increased seed yield, comprising a genetic
modification, which genetic modification results in increased
expression of cytokinin oxidase in the aleurone and/or embryo of a
seed, relative to other parts of said seed.
19. A transgenic plant, plant part, or plant tissue comprising
plant cells of claim 17.
20. The transgenic plant of claim 19 which is a dicotyledonous or
monocotyledonous plant.
21. Transgenic plant according to claim 20, wherein said
monocotyledonous plant is selected from a group consisting of
sugarcane, rice, maize, wheat, barley, millet, rye, oats, and
sorghum.
22. A transgenic harvestable part, or a product directly derived
from a transgenic harvestable part of the transgenic plant of claim
19.
23. A transgenic harvestable part of the transgenic plant of claim
22 selected from the group consisting of seeds, leaves, fruits,
stem cultures, rhizomes, roots, tubers and bulbs.
24. Transgenic progeny of the plant or plant part of claim 19.
25. Harvestable parts of the plant according to claim 18, or
products directly derived from said harvestable parts.
26-29. (canceled)
30. A method for increasing seed yield in a plant, said method
comprises introducing into a plant cell the vector according to
claim 13.
31. The method according to claim 1, wherein said increased
expression in the embryo and/or aleurone is effected by introducing
a genetic modification in the locus of the cytokinin
oxidase-encoding gene.
Description
[0001] The present invention generally relates to methods for
modifying plant morphological properties or characteristics, such
as developmental processes, and in particular seed development
and/or seed yield. The methods comprise expressing a cytokinin
degradation control protein, in particular cytokinin oxidase, in
the plant, operably under the control of a regulatable promoter
sequence, wherein the regulatable promoter is an aleurone and/or
embryo-specific promoter sequence. The present invention extends to
genetic constructs which are useful in performing the method of the
invention and to plants transformed therewith, the plants having
altered morphological properties compared to their otherwise
isogenic counterparts.
[0002] Seeds are the reproduction unit of higher plants. Seeds
contain reserve compounds to ensure nutrition of the embryo after
germination. These storage compounds contribute significantly to
human nutrition as well as to cattle feeding. Seeds consist of
three major parts, namely the embryo, the endosperm and the seed
coat. Reserve compounds are deposited in storage tissue, which is
either the endosperm (resulting from double fertilisation; e.g. in
all cereals), the so-called perisperm (derived from the nucellus
tissue) or the cotyledons (e.g. bean varieties). The endosperm is
covered by a layer of dense cytoplasmic cells, known as the
aleurone. Storage compounds include lipids (oil seed rape),
proteins (e.g. in the aleuron of cereals) or carbohydrates (starch,
oligosaccharides like raffinose).
[0003] Starch is the storage compound in the seeds of cereals. The
most important cereal species are maize (yearly production ca. 570
million tonnes), rice (540 million tonnes per annum) and wheat (530
million tonnes per annum). Protein-rich seeds include different
kinds of beans (Phaseolus spec., Vicia faba, Vigna spec.; ca. 20
million tonnes per annum), pea (Pisum sativum; million tonnes per
annum) and soybean (Glycine max; 136 million tonnes per annum).
Soybean seeds are also an important source of lipids, as are the
seeds of different Brassica species (app. 30 million tonnes per
annum), cotton, oriental sesame, flax, poppy, castor bean,
sunflower, peanut, coconut, oilpalm and some other plants of lesser
economic importance.
[0004] After fertilization, the developing seed becomes a sink
organ that attracts nutritional compounds from source organs of the
plant and uses them to produce the reserve compounds in storage
organs, such as seeds, bulbs or tubers. The common concept predicts
that cytokinins are a positive regulator of sink strength.
[0005] Numerous reports ascribe a stimulatory or inhibitory
function to cytokinins in different developmental processes such as
root growth and branching, control of apical dominance in the
shoot, chloroplast development, and leaf senescence (Mok M. C.
(1994) in Cytokines: Chemistry, Activity and Function, eds., Mok,
D. W. S. & Mok, M. C. (CRC Boca Raton, Fla.), pp. 155-166).
Conclusions about the biological functions of cytokinins have
mainly been derived from studies on the consequences of exogenous
cytokinin application or endogenously enhanced cytokinin levels
(Klee, H. J. & Lanehon, M. B. (1995) in Plant Hormones:
Physiology, Biochemisry and Molecular Biology, ed. Davies, P. J.
(Kluwer, Dordrecht, the Netherlands), pp. 340-353, Schmulling, T.,
Rupp, H. M. Frank, M. & Schafer, S. (1999) in Advances in
Regulation of Plant Growth and Development, eds. Sumad, M. Pac P.
& Beck, E. (Peres, Prague), pp. 85-96).
[0006] The cloning of cytokinin oxidase allowed the study of the
relevance of iP- and Z-type cytokinins during the whole life cycle
of higher plants. The catabolic enzyme cytokinin oxidase (CKX)
plays a principal role in controlling cytokinin levels in plant
tissues. CKX activity has been found in a great number of higher
plants and in different plant tissues. The enzyme is a
FAD-containing oxidoreductase that catalyzes the degradation of
cytokinins bearing unsaturated isoprenoid side chains and is
sometimes referred to as a cytokinin dehydrogenase (Frebortova et
al., Biochem. J. 380, 121-130, 2004). The free bases iP and Z, and
their respective ribosides are the preferred substrates. The
reaction products of iP catabolism are adenine and the unsaturated
aldehyde 3-methyl-2-butonal (Armstrong, D. J. (1994) in Cytokinins:
Chemistry, Activity and Functions, eds. Mok. D. W. S & Mok, M.
C. (CRC Boca Raton, Fla.), pp. 139-154).
[0007] Cytokinin oxidase genes from various plant species have been
isolated: Zea mays (Morris, R. O., Bilyeu, K. D., Laskey, J. G.
& Chemch, N. N. (1999) Biochem. Biophys. Res. Commun. 255,
328-333, Houba-Herin, N., Pethe, C., d'Alayer, J. & Laloue, M.
(1999) Plant J. 17, 615-626), Arabidopsis thaliana (WO 01/96580, WO
03/050287), and Dendrobium sp. (Yang et al., J. Exp. Bot. 53,
1899-1907). The manipulation of CKX gene expression is used as a
powerful tool to study the relevance of iP- and Z-type cytokinins
during the life cycle of higher plants; in particular the
manipulation of CKX expression allowed the influence of cytokinin
on root growth and shoot development and morphology, on seed size,
and on leaf senescence to be determined (WO 01/96580, WO 03/050287,
Werner et al. Plant Cell 15, 2632-2550, 2003). Transformants
showing constitutive AtCKX mRNA expression and increased cytokinin
oxidase activity manifested enhanced formation and growth of roots.
Negative effects on shoot growth were also observed.
[0008] It was postulated that cytokinins play a fundamental role in
maize for establishing seed size, decreasing tip kernel abortion
and increased seed setting during unfavourable environmental
conditions (WO 00/63401). Smigocki et al. (Proc Natl Acad Sci USA
85, 5131-5135, 1988) observed increased shoot organogenesis upon
infection of stems, leaf pieces and seedling with Agrabacterium
tumefaciens overexppessing isopentenyltransferase (ipt). It was
therefore also postulated that enhanced levels of cytokinins in the
seed would provide for improved seed size and increased seed set
(WO 00/63401).
[0009] It is known in the art that constitutive CKX expression in a
plant leads to increased root growth but decreased shoot growth (WO
01/96580). It has furthermore been demonstrated that expression of
CKX under the control of a strong constitutive promoter in a plant
results in increased seed size, increased embryo size and increased
cotyledon size (WO 03/050287). However, it was not known which
parts of the seeds would be best suited for CKX expression so as to
achieve increased seed yield. The inventors have now shown for the
first time that increased seed yield may be obtained relative to
control plants when CKX is overexpressed in certain parts of the
seed, namely the aleurone and/or embryo. The inventors have also
shown that aleurone- and/or embryo-specific overexpression of CKX
leads to a larger increase in seed yield compared to
endosperm-specific CKX overexpression.
[0010] In accordance with the present invention, it has been
surprisingly discovered that transgenic plants overexpressing a
cytokinin oxidase gene in a confined part of the seed, such
confined part being the aleurone and/or embryo, have an increased
seed yield compared to corresponding wild type plants. These
results are surprising, as a reduced cytokinin content would have
been expected to be associated with a reduced organ growth and
hence with reduced yield.
[0011] The present invention provides a method for modifying plant
morphological properties, in particular for increasing seed yield
of a plant, comprising expressing a cytokinin oxidase under the
control of a regulatable promoter capable of driving expression in
the aleurone and/or embryo of a seed. The invention also provides
compositions for modifying plant morphological properties; in
particular for increasing seed yield of a plant.
[0012] "Plant morphology" or "plant morphological characteristic"
or similar term will, when used herein, be understood by those
skilled in the art to refer to the external appearance of a plant.
More particularly the plant morphological characteristic that is
improved by using the methods according to the present invention is
seed yield.
[0013] As used herein, the term "control plant" refers to plants
that are, except for the modified or improved morphological
characterisitics, as similar as possible or identical to the
modified plant. Such control plants are also known as "wild type
plants/lines" or "otherwise isogenic plants/lines". The inventors
have furthermore shown that the increase in seed yield is higher
for plants wherein CKX is overexpressed in the aleurone and/or
embryo, compared to plants wherein CKX is overexpressed in the
endosperm. The term "control plants" therefore also includes plants
wherein CKX is overexpressed in the endosperm.
[0014] There are several well known parameters which may be used
for measuring increased seed yield which include but are not
limited to: total weight of seeds, total number of seeds, total
number of filled seeds, harvest index, and Thousand Kernel Weight.
As described in the examples herein, the total weight of seeds may
be measured by weighing all filled seeds harvested from a plant.
The total number of seeds may be measured by counting the number of
seeds harvested from a plant. The total number of filled seeds may
be measured by counting the number of filled seeds harvested from a
plant. The term "harvest index" as used herein is defined as the
ratio between the total seed weight and the above ground area
(mm.sup.2), multiplied by a factor 10.sup.6. Thousand Kernel Weight
may be derived from the number of filled seeds counted and their
total weight.
[0015] Advantageously, performance of the methods according to the
present invention results in plants having increased yield or
biomass, relative to corresponding control plants.
[0016] By "yield" is meant the amount of harvested material per
area of production. The term "increased yield" encompasses an
increase in biomass in one or more parts of a plant relative to the
biomass of corresponding control plants. Depending on the crop, the
harvested part of the plant may be different, for example it may be
seed (e.g. rice, sorghum or corn when grown for seed); total
above-ground biomass (e.g. corn, when used as silage, sugarcane),
root (e.g. sugar beet), fruit (e.g. tomato), cotton fibres, or any
other part of the plant which is of economic value. For example,
the methods of the present invention are used to increase seed
yield in rice and in corn. The increase in yield encompasses an
increase in seed yield, which includes an increase in the total
biomass of the seed (total seed weight), total number of seeds
and/or an increase in the number of (filled) seeds. The increase in
yield is also reflected as an increase in the Harvest Index, which
is expressed as a ratio of the yield of harvestable parts, such as
seeds, over the total biomass and is also reflected as an increased
Thousand Kernel Weight (derived from the number of filled seeds
counted and their total weight).
[0017] Thus, there is provided a method for increasing seed yield
of a plant, increasing the level and/or activity of a cytokinin
oxidase (CKX) in the embryo and/or aleurone of a plant seed,
wherein said embryo and/or aleurone have increased levels and/or
activity of cytokinin oxidase relative to other parts of said seed,
and wherein the increase of seed yield comprises at least one of
increased total weight of seeds, increased total number of seeds,
increased number of filled seeds, increased harvest index or
increased thousand kernel weight, each relative to corresponding
control plants.
[0018] Yield is by its nature a complex parameter where total yield
depends on a number of yield components. The parameters for
increased yield of a crop are well known by a person skilled in the
art. By way of example, key yield components for corn include
number of plants per hectare or acre, number of ears per plant,
number of rows (of seeds) per ear, number of kernels per row, and
Thousand Kernel Weight. The improvement in yield as obtained in
accordance with the methods of the invention, may be obtained as a
result of one or more of these yield components. By way of example,
key yield components for rice include number of plants per hectare
or acre, number of panicles per plant, number of spikelets per
panicle, seed filling rate and thousand kernel weight. The
improvement in yield as obtained in accordance with the methods of
the invention may be obtained as a result in one or more of these
yield components, preferentially the improvement in yield is
obtained primarily on the basis of an increased number of flowers
per panicle and an increased seed filling rate.
[0019] Performance of the methods according to the present
invention result in plants having modified seed yield. The modified
yield includes at least an increase in any one or more of total
weight of seeds, total seed number, number of filled seeds,
thousand kernel weight and harvest index, each relative to control
plants. Therefore, according to the present invention, there is
provided a method for increasing one or more of: total seed number,
total weight of seeds, number of filled seeds, thousand kernel
weight and harvest index of plants, which method comprises
modulating expression of a nucleic acid molecule encoding a CKX
protein and/or modulating activity of the CKX itself in a plant in
a aleurone- and/or embryo-preferred way, preferably wherein the CKX
protein is encoded by a nucleic acid sequence represented by SEQ ID
NO: 26, SEQ ID NO: 42, or a portion thereof or by sequences capable
of hybridising therewith or wherein the CKX is represented by SEQ
ID NO: 4, SEQ ID NO:36, or a homologue, derivative or active
fragment thereof. Alternatively, the CKX may be encoded by a
nucleic acid sequence represented by SEQ ID NO: 37, or by a portion
thereof or by sequences capable of hybridising therewith, or
wherein the CKX is represented by SEQ ID NO: 38, or a homologue,
derivative or active fragment of any thereof.
[0020] The present invention is applicable to any plant, in
particular a monocotyledonous plants and dicotyledonous plants
including a fodder or forage legume, ornamental plant, food crop,
tree, or shrub selected from the list comprising Acacia spp., Acer
spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia
amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca
catechu, Astelia fragrans, Astragalus cicer, Avena sativa, Baikiaea
plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza,
Burkea africana, Butea frondosa, Cadaba fannosa, Calliandra spp,
Camellia sinensis, Canna indica, Capsicum spp., Cassia spp.,
Centroema pubescens, Chaenomeles spp., Cinnamomum cassia, Coffea
arabica, Colophospermum mopane, Coronillia vana, Cotoneaster
serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea
dealbata, Cydonia oblonga, Clyptomeria japonica, Cymbopogon spp.,
Cynthea dealbata, Cydonia ob/onga, Dalbergia monetaria, Davallia
divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon
amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum,
Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana,
Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi,
Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp.,
Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo
biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum,
Grevillea spp., Guibourtia coleosperna, Hedysarum spp., Hemarthia
altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia
rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incamata,
Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp.,
Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus
spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago
sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum
spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum
africanum, Pennisetum spp., Persea gratissima, Petunia spp.,
Phaseolus spp., Phoenix canariensis, Phornium cookianum, Photinia
spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara,
Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp.,
Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum,
Pyrus communis, Quercus spp., Rhaphiolepsis umbellata,
Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes
spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp.,
Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia
sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia
spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos
humilis, Tadehagi spp, Taxodium distichum, Themeda triandra,
Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp.,
Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschia
aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli,
brussel sprout, cabbage, canola, carrot, cauliflower, celery,
collard greens, flax, kale, lentil, oilseed rape, okra, onion,
potato, rice, soybean, straw, sugarbeet, sugar cane, sunflower,
tomato, squash, and tea, amongst others, or the seeds of any plant
specifically named above or a tissue, cell or organ culture of any
of the above species.
[0021] According to a preferred embodiment of the present
invention, the plant is a crop plant such as soybean, sunflower,
canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco.
Further preferably, the plant is a monocotyledonous plant, such as
sugarcane. More preferably the plant is a cereal, such as rice,
maize, wheat, barley, millet, rye, sorghum or oats.
[0022] The term "plant" as used herein encompasses whole plants,
ancestors and progeny of the plants and plant parts, including
seeds, shoots, stems, leaves, roots (including tubers), flowers,
and tissues and organs, wherein each of the aforementioned comprise
the gene/nucleic acid of interest. The term "plant" also
encompasses suspension cultures, callus tissue, embryos,
meristematic regions, gametophytes, sporophytes, pollen and
microspores, again wherein each of the aforementioned comprise the
gene/nucleic acid of interest. The terms "plant" and "plant part"
are used interchangeably with the terms "plants" and "plant
parts".
[0023] It should be clear that, although the invention is supported
in the examples section by AtCKX genes and proteins, the inventive
concept also relates to the use of cytokinin oxidases isolated from
other plants and expressed in the aleurone and/or embryo of a seed
of these other plants to obtain similar effects in plants as
described in the examples section.
[0024] The cytokinin oxidase gene family contains at least six
members from Arabidopsis. It is anticipated that functional
homologs of the described Arabidopsis cytokinin oxidases can be
isolated from other organisms, given the evidence for the presence
of cytokinin oxidase activity in many green plants (Hare and van
Staden, Physiol Plant 91:128-136, 1994; Jones and Schreiber, Plant
Growth Reg 23:123-134, 1997), as well as in other organisms
(Armstrong, in Cytokinins: Chemistry, Activity and Function. Eds
Mok and Mok, CRC Press, pp 139-154, 1994). Therefore, the sequence
of the cytokinin oxidase, functional in the invention, need not to
be identical to those described herein. This invention is
particularly useful for cereal crops and monocot crops in general
and cytokinin oxidase genes from for example wheat or maize may be
used as well (Morris et al., 1999; Rinaldi and Comandini, 1999). It
is envisaged that other genes with cytokinin oxidase activity or
with any other cytokinin metabolizing activity (see Za{hacek over
(z)}imalova et al., Biochemistry and Molecular Biology of Plant
Hormones, Hooykaas, Hall and Libbenga (Eds.), Elsevier Science, pp
141-160, 1997) can also be used for the purpose of this invention.
Similarly, genes encoding proteins that would increase endogenous
cytokinin-metabolizing activity can also be used for the purpose of
this invention. In principle, similar phenotypes could also be
obtained by interfering with genes that function downstream of
cytokinin such as receptors or proteins involved in signal
transduction pathways of cytokinin.
[0025] Any nucleotide sequence encoding a polypeptide with
cytokinin oxidase activity may be used in the methods of the
invention. For example, any of the various sequences provided
herein encoding a polypeptide with cytokinin oxidase activity may
be used in the methods of increasing seed yield.
[0026] The terms "protein(s)", "peptide(s)", "polypeptide(s)" or
"oligopeptide(s)" when used herein refer to amino acids in a
polymeric form of any length. These terms also include known amino
acid modifications as well as non-naturally occurring amino acid
residues, L-amino acid residues and D-amino acid residues.
[0027] The CKX proteins useful in the methods according to the
invention are defined herein as having cytokinin
oxidase/dehydrogenase activity and comprising at least 2 sequences
of 17 and 19 consecutive amino acid residues respectively with a
consensus sequence as shown below:
[0028] Consensus sequence 1 (17 amino acids): hTDYLhhoIGGTLSssG,
(SEQ ID NO: 44)
[0029] Consensus sequence 2 (19 amino acids): cLFxushGsLGQFGIIstA,
(SEQ ID NO: 45) wherein the capital letters are the standard single
letter IUPAC codes for the various amino acids and the other
letters symbolise the nature of the amino acids, as shown in Table
1. The right column lists for each class the particular amino acids
that are allowed in the consensus sequences. This classification is
based on the amino acid grouping as defined in the SMART database
(Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864;
Letunic et al. (2002) Nucleic Acids Res 30, 242-244).
TABLE-US-00001 TABLE 1 Class Key Allowed amino acids h hydrophobic
G, H, L, R, T, W, Y o alcohol S, T l aliphatic V, I s small A, D,
G, N, T, V c charged D, E, R x any Y, R, N, F, D, H u tiny A, G, S
t turnlike R, N
[0030] The crystal structure of maize CKX was elucidated by Malito
et al. (J. Mol. Biol. 341, 1237-1249, 2004), which allowed a
structure-function analysis and the identification of amino acids
that were important for the catalytic activity of maize CKX. These
results may be extrapolated to other CKX proteins by comparison of
protein sequences.
[0031] Methods for measuring cytokinin oxidase/dehydrogenase
activity are well known in the art. Suitable methods are based on
the conversion of [2.sup.-3H]iP to adenine (Motyka et al., Plant
Physiology 112, 1035-1043, 1996), on calorimetric assays
(Libreros-Minotta and Tipton, Anal. Biochem. 231, 339-341, 1995) or
on the measurement of reduced electron acceptors (Bilyeu et al.,
Plant Physiol. 125, 378-386, 2001).
[0032] The present invention relates to methods for increasing seed
yield. In particular, the methods comprise expression in the
aleurone and/or embryo of a seed, of a nucleic acid encoding a
cytokinin oxidase selected from the group consisting of: [0033] (a)
nucleic acids comprising a DNA sequence as given in any of SEQ ID
NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or
the complement thereof, [0034] (b) nucleic acids comprising the RNA
sequences corresponding to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5,
7, 9, 11, 25, 26, 28 to 31, 33 or 34, or the complement thereof,
[0035] (c) nucleic acids specifically hybridizing to any of SEQ ID
NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or
to the complement thereof, [0036] (d) nucleic acids encoding a
protein comprising the amino acid sequence as given in any of SEQ
ID NOs: 2, 4, 6, 8, 10, 12, 32, 35, 36 or 38, or the complement
thereof, [0037] (e) nucleic acids as defined in any of (a) to (d)
characterized in that said nucleic acid is DNA, genomic DNA, cDNA,
synthetic DNA or RNA wherein T is replaced by U, [0038] (f) nucleic
acid which is degenerate compared to a nucleic acid as given in any
of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33
or 34, or which is degenerate compared to a nucleic acid as defined
in any of (a) to (e) as a result of the genetic code, [0039] (g)
nucleic acids which are divergent from a nucleic acid encoding a
protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36
or 38, or which is divergent from a nucleic acid as defined in any
of (a) to (e), due to the differences in codon usage between the
organisms, [0040] (h) nucleic acids encoding a protein as given in
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or nucleic acids as
defined in (a) to (e) which are divergent due to the differences
between alleles, [0041] (i) nucleic acids encoding a protein as
given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38,
[0042] (j) functional fragments of nucleic acids as defined in any
of (a) to (i) having the biological activity of a cytokinin
oxidase, and [0043] (k) nucleic acids encoding a plant cytokinin
oxidase, comprising the consensus sequence hTDYLhhoIGGTLSssG and
cLFxushGsLGQFGIIstA or comprising expression in seeds of a nucleic
acid encoding a protein that reduces the level of active cytokinins
in plants or plant parts.
[0044] The expression of a nucleic acid encoding a CKX polypeptide
or a homologue thereof may also be increased by introducing a
genetic modification (preferably in the locus of a CKX gene).
[0045] In addition to the cytokinin oxidase genes and corresponding
proteins described above, a cytokinin oxidase 2 gene (CKX2) is
particularly suited for use in increasing seed yield in a plant. In
addition to the Arabidopsis thaliana CKX2 set forth in SEQ ID
NO:36, other CKX proteins from Arabidopsis thaliana, such as the
ones represented in GenBank Accessions NP.sub.--181682,
NP.sub.--200507, NP.sub.--849470, NP.sub.--194703, NP.sub.--850863
or AAG30909 may be used. CKX proteins from other species like Zea
mays (for example GenBank Accessions CAE55202, CAE55200 or
AAC27500), Dendrobium (GenBank CAC17752), Hordeum vulgare (GenBank
AAN16383, M050082, AAM08400), or rice (GenBank NP.sub.--913145,
NP.sub.--916348, NP.sub.--922039) are also available for use in the
methods and compositions of the present invention. A prokaryotic
homologue of SEQ ID NO: 36 is represented by GenBank Accession
P46377.
[0046] Therefore, the present invention more generally relates to
method for increasing seed yield in a plant, said method comprising
increasing the level and/or activity of a cytokinin oxidase (CKX)
in the embryo and/or aleurone of a plant seed, wherein said embryo
and/or aleurone have increased levels and/or activity of cytokinin
oxidase relative to other parts of said seed. Preferred cytokinin
oxidases to be used are encoded by the nucleic acids encoding the
cytokinin oxidases as defined above. More preferably, the cyokinin
oxidases to be used are from Arabidopsis thaliana, most preferably
the cyokinin oxidase to be used is AtCKX2 encoded by one of SEQ ID
NO: 3, 26, 37 or 42.
[0047] The terms "gene(s)", "nucleic acid(s)", "nucleic acid
sequence(s)", "nucleotide sequence(s)", or "nucleic acid
molecule(s)", when used herein refer to nucleotides, either
ribonucleotides or deoxyribonucleotides or a combination of both,
in a polymeric form of any length. The terms furthermore include
double-stranded and single-stranded DNA and RNA. The terms also
include known nucleotide modifications such as methylation,
cyclization and `caps` and substitution of one or more of the
naturally occurring nucleotides with an analog such as inosine. The
terms also encompass peptide nucleic acids (PNAs), a DNA analogue
in which the backbone is a pseudopeptide consisting of
N-(2-aminoethyl)-glycine units rather than a sugar.
[0048] A "coding sequence" or "open reading frame" or "ORF" is
defined as a nucleotide sequence that can be transcribed into mRNA
and/or translated into a polypeptide when placed under the control
of appropriate control sequences or regulatory sequences, i.e. when
the coding sequence or ORF is present in an expressible format.
This coding sequence or ORF is bound by a 5' translation start
codon and a 3' translation stop codon. A coding sequence or ORF can
include, but is not limited to RNA, mRNA, cDNA, recombinant
nucleotide sequences, synthetically manufactured nucleotide
sequences or genomic DNA. Such coding sequence or ORF can be
interrupted by intervening nucleic acid sequences.
[0049] Genes and coding sequences essentially encoding the same
protein but isolated from different sources can consist of
substantially divergent nucleic acid sequences. Reciprocally,
substantially divergent nucleic acid sequences can be designed to
effect expression of essentially the same protein. These nucleic
acid sequences are the result of e.g. the existence of different
alleles of a given gene, of the degeneracy of the genetic code or
of differences in codon usage. Thus, amino acids such as methionine
and tryptophan are encoded by a single codon whereas other amino
acids such as arginine, leucine and serine can each be translated
from up to six different codons. Differences in preferred codon
usage are known in the art. For example, the codon GGC (for
glycine) is the most frequently used codon in Agrobacterium
tumefaciens (36.2.Salinity.), is the second most frequently used
codon in Oryza sativa but is used at much lower frequencies in A.
thaliana and Medicago sativa (9.Salinity. and 8.4.Salinity.,
respectively). Of the four possible codons encoding glycine, this
GGC codon is most preferably used in A. tumefaciens and O. sativa.
However, in A. thaliana this is the GGA (and GGU) codon whereas in
M. sativa this is the GGU (and GGA) codon.
[0050] Alleles exist in nature, and encompassed within the methods
of the present invention is the use of these natural allelic
variants. Allelic variants encompass Single Nucleotide
Polymorphisms (SNPs), as well as Small Insertion/Deletion
Polymorphisms (INDELs). The size of INDELs is usually less than 100
bp. SNPs and INDELs form the largest set of sequence variants in
naturally occurring polymorphic strains of most organisms.
[0051] DNA sequences as defined in the current invention can be
interrupted by intervening sequences. With "intervening sequences"
is meant any nucleic acid sequence which disrupts a coding sequence
comprising such inventive DNA sequence or which disrupts the
expressible format of a DNA sequence comprising the inventive DNA
sequence. Removal of the intervening sequence restores the coding
sequence or the expressible format. Examples of intervening
sequences include introns and mobilizable DNA sequences such as
transposons. With "mobilizable DNA sequence" is meant any DNA
sequence that can be mobilized as the result of a recombination
event.
[0052] The methods according to the present invention may also be
practised using an alternative splice variant of a nucleic acid
molecule encoding a CKX protein. The term "alternative splice
variant" as used herein encompasses variants of a nucleic acid
molecule in which selected introns and/or exons have been excised,
replaced or added. Such variants will be ones in which the
biological activity of the protein remains unaffected, which can be
achieved by selectively retaining functional segments of the
protein. Such splice variants may be found in nature or can be
manmade. Methods for making such splice variants are well known in
the art. Therefore according to another aspect of the present
invention, there is provided a method for modifying the
morphological characteristics of plants, and in particular seed
yield, comprising expression in the aleurone and/or embryo of a
seed of an alternative splice variant of a nucleic acid molecule
encoding a CKX. Preferably, the splice variant is a splice variant
of a sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11 or 33. A
preferred splice variant of AtCKX2 is as represented by SEQ ID NO:
38.
[0053] Also useful in the methods of in the present invention are
nucleic acids capable of hybridising under reduced stringency
conditions, preferably under stringent conditions, with a CKX
encoding nucleic acid. Preferred is a nucleic acid capable of
hybridising to a nucleic acid represented by SEQ ID NO: 42 or
26.
[0054] "Hybridization" is the process wherein substantially
homologous complementary nucleotide sequences anneal to each other.
The hybridization process can occur entirely in solution, i.e. both
complementary nucleic acids are in solution. Tools in molecular
biology relying on such a process include PCR, subtractive
hybridization and DNA sequence determination. The hybridization
process can also occur with one of the complementary nucleic acids
immobilized to a matrix such as magnetic beads, Sepharose beads or
any other resin. Tools in molecular biology relying on such a
process include the isolation of poly (A+) mRNA. The hybridization
process can furthermore occur with one of the complementary nucleic
acids immobilized to a solid support such as a nitrocellulose or
nylon membrane or immobilized by e.g. photolithography to e.g. a
silicious glass support (the latter known as nucleic acid arrays or
microarrays or as nucleic acid chips). Tools in molecular biology
relying on such a process include RNA and DNA gel blot analysis,
colony hybridization, plaque hybridization and microarray
hybridization. In order to allow hybridization to occur, the
nucleic acid molecules are generally thermally or chemically (e.g.
by NaOH) denatured to melt a double strand into two single strands
and/or to remove hairpins or other secondary structures from single
stranded nucleic acids. The stringency of hybridization is
influenced by conditions such as temperature, salt concentration
and hybridization buffer composition. High stringency conditions
for hybridization include high temperature and/or low salt
concentration (salts include NaCl and Na.sub.3-citrate) and/or the
inclusion of formamide in the hybridization buffer and/or lowering
the concentration of compounds such as SDS (detergent) in the
hybridization buffer and/or exclusion of compounds such as dextran
sulfate or polyethylene glycol (promoting molecular crowding) from
the hybridization buffer. Conventional hybridization conditions are
described in e.g. Sambrook et al. (1989) but the skilled craftsman
will appreciate that numerous different hybridization conditions
can be designed in function of the known or the expected homology
and/or length of the nucleic acid sequence. Sufficiently low
stringency hybridization conditions are particularly preferred to
isolate nucleic acids heterologous to the DNA sequences of the
invention defined supra. Elements contributing to heterology
include allelism, degeneration of the genetic code and differences
in preferred codon usage as discussed supra.
[0055] The term "specifically hybridizing" or "hybridizing
specifically" refers to the binding, duplexing, or hybridizing of a
molecule to a particular nucleotide sequence under medium to
stringent conditions when that sequence is presented in a complex
mixture e.g., total cellular DNA or RNA.
[0056] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and Northern
hybridizations are sequence dependent and are different under
different environmental parameters. For example, longer sequences
hybridize specifically at higher temperatures. The T.sub.m is the
temperature under defined ionic strength and pH, at which 50% of
the target sequence hybridizes to a perfectly matched probe.
Specificity is typically the function of post-hybridization washes.
Critical factors of such washes include the ionic strength and
temperature of the final wash solution.
[0057] Generally, stringent conditions are selected to be about
50.degree. C. lower than the thermal melting point (T.sub.m) for
the specific sequence at a defined ionic strength and pH. The
T.sub.m is the temperature (under defined ionic strength and pH) at
which 50% of the target sequence hybridizes to a perfectly matched
probe. The T.sub.m is dependent upon the solution conditions and
the base composition of the probe, and may be calculated using the
following equation:
T m = 79.8 .degree. C . + ( 18.5 .times. Log [ Na + ] ) + ( 58.4
.degree. C . .times. % [ G + C ] ) - ( 820 / # bp in duplex ) - (
0.5 .times. % formamide ) ##EQU00001##
[0058] More preferred stringent conditions are when the temperature
is 20.degree. C. below T.sub.m, and the most preferred stringent
conditions are when the temperature is 10.degree. C. below T.sub.m.
Nonspecific binding may also be controlled using any one of a
number of known techniques such as, for example, blocking the
membrane with protein-containing solutions, addition of
heterologous RNA, DNA, and SDS to the hybridization buffer, and
treatment with RNase.
[0059] Wash conditions are typically performed at or below
stringency. Generally, suitable stringent conditions for nucleic
acid hybridization assays or gene amplification detection
procedures are as set forth above. More or less stringent
conditions may also be selected.
[0060] For the purposes of defining the level of stringency,
reference can conveniently be made to Sambrook, J., E. F. Fritsch,
et al. 1989 "Molecular Cloning: a Laboratory Manual, 2.sup.nd
Edition, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory
Press. An example of low stringency conditions is
4-6.times.SSC/0.1-0.5% w/v SDS at 37.degree.-45.degree. C. for 2-3
hours. Depending on the source and concentration of the nucleic
acid involved in the hybridization, alternative conditions of
stringency may be employed such as medium stringent conditions.
Examples of medium stringent conditions include 14.times.SSC/0.25%
w/v SDS at .gtoreq.45.degree. C. for 2-3 hours. An example of high
stringency conditions includes 0.1-1.times.SSC/0.1% w/v SDS at 60 C
for 1-3 hours. The skilled artisan is aware of various parameters
which may be altered during hybridization and washing and which
will either maintain or change the stringency conditions. For
example, another stringent hibridization condition is hybridization
at 4.times.SSC at 65.degree. C., followed by a washing in
0.1.times.SSC at 65.degree. C. for about one hour. Alternatively,
an exemplary stringent hybridization condition is in 50% formamide,
4.times.SSC, at 42.degree. C. Still another example of stringent
conditions include hybridization at 62.degree. C. in 6.times.SSC,
0.05.times. BLOTTO, and washing at 2.times.SSC, 0.1% SDS at
62.degree. C.
[0061] Clearly, the current invention embodies the use of DNA
sequences hybridising to DNA sequences encoding a cytokinin
oxidase, homologue, derivative or immunologically active and/or
functional fragment thereof as defined below in methods for
increasing seed yield comprising expression of these DNA sequences
in the aleurone and/or embryo of a seed. Preferably the cytokinin
oxidase is a plant cytokinin oxidase, more specifically the
Arabidopsis thaliana (At)CKX.
[0062] "Homologues" of a CKX polypeptide may also be useful in the
present invention.
[0063] "Homologues" of a CKX useful in the methods of the invention
are those peptides, oligopeptides, polypeptides, proteins and
enzymes which contain amino acid substitutions, deletions and/or
additions relative to the protein with respect to which they are a
homologue, without altering one or more of its functional
properties, in particular without reducing the activity of the
resulting protein. For example, a homologue of a CKX will consist
of a bioactive amino acid sequence variant of this CKX.
[0064] Two special forms of homology, orthologous and paralogous
homology, are evolutionary concepts used to describe ancestral
relationships of genes. The term "paralogous" relates to homologous
genes that result from one or more gene duplications within the
genome of a species. The term "orthologous" relates to homologous
genes in different organisms due to ancestral relationship of these
genes. The term "homologues" as used herein also encompasses
paralogues and orthologues of the proteins useful in the methods
according to the invention. Orthologous genes can be identified by
querying one or more gene databases with a query gene of interest,
using for example, the BLAST program. The highest-ranking subject
genes that result from the search are then again subjected to a
BLAST analysis, and only those subject genes that match again with
the query gene are retained as true orthologous genes. For example,
to find a rice orthologue of an Arabidopsis thaliana gene, one may
perform a BLASTN or TBLASTX analysis on a rice database (such as
(but not limited to) the Oryza sativa Nipponbare database available
at the NCBI (http://www.ncbi.nim.nih.gov) or the genomic sequences
of rice (cultivars indica or japonica)). In a next step, the
obtained rice sequences are used in a reverse BLAST analysis using
an Arabidopsis database. The results may be further refined when
the resulting sequences are analysed with ClustalW and visualised
in a neighbour joining tree. The method can be used to identify
orthologues from many different species.
[0065] BLAST (Basic Local Alignment Search Tool) is a family of
programs (http://www.ncbi.nlm.nih.gov/BLAST/) aiming to identify
regions of optimal local alignment, i.e. the alignment of some
portion of two nucleic acid or protein sequences, and to detect
relationships among sequences which share only isolated regions of
similarity (Altschul et al., Nucleic Acids Res. 25: 3389-3402
(1997)).
[0066] To produce such homologues, amino acids present in the
protein can be replaced by other amino acids having similar
properties, for example hydrophobicity, hydrophilicity, hydrophobic
moment, antigenicity, propensity to form or break .alpha.-helical
structures or .beta.-sheet structures, and so on. An overview of
physical and chemical properties of amino acids is given in Table
2.
TABLE-US-00002 TABLE 2 Properties of naturally occurring amino
acids. Charge properties/ hydrophobicity Side group Amino Acid
Nonpolar Aliphatic ala, ile, leu, val hydrophobic aliphatic,
S-containing met aromatic phe, trp imino pro polar uncharged
Aliphatic gly Amide asn, gln Aromatic tyr Hydroxyl ser, thr
Sulfhydryl cys Positively charged Basic arg, his, lys Negatively
charged Acidic asp, glu
[0067] The homologues useful in the methods according to the
invention preferably have cytokinin oxidase/dehydrogenase activity
and comprise at least 2 sequences of 17 and 19 consecutive amino
acid residues respectively with a consensus sequence as shown
below:
[0068] Consensus sequence 1 (17 amino acids): hTDYLhhoIGGTLSssG,
(SEQ ID NO: 44)
[0069] Consensus sequence 2 (19 amino acids): cLFxushGsLGQFGIIstA,
(SEQ ID NO: 45) wherein the capital letters are the standard single
letter IUPAC codes for the various amino acids and the other
letters symbolise the nature of the amino acids, as shown in table
2 above. The right column lists for each class the particular amino
acids that are allowed in the consensus sequences.
[0070] Substitutional variants of a protein of the invention are
those in which at least one residue in the protein or amino acid
sequence has been removed and a different residue inserted in its
place. Amino acid substitutions are typically of single residues,
but may be clustered depending upon functional constraints placed
upon the polypeptide; insertions will usually be of the order of
about 1-10 amino acid residues and deletions will range from about
1-20 residues. Preferably, amino acid substitutions will comprise
conservative amino acid substitutions, such as those described
supra.
[0071] Insertional amino acid sequence variants of a protein of the
invention are those in which one or more amino acid residues are
introduced into a predetermined site in the protein. Insertions can
comprise amino-terminal and/or carboxy-terminal fusions as well as
intra-sequence insertions of single or multiple amino acids.
Generally, insertions within the amino acid sequence will be
smaller than amino or carboxyl terminal fusions, of the order of
about 1 to 10 residues. Examples of amino- or carboxy-terminal
fusion proteins or peptides include the binding domain or
activation domain of a transcriptional activator as used in a
two-hybrid system, phage coat proteins, (histidine).sub.6-tag,
glutathione S-transferase, protein A, maltose-binding protein,
dihydrofolate reductase, Tag 100 epitope (EETARFQPGYRS), c-myc
epitope (EQKLISEEDL), FLAG.RTM.-epitope (DYKDDDK), lacZ, CMP
(calmodulin-binding peptide), HA epitope (YPYDVPDYA), protein C
epitope (EDQVDPRLIDGK) and VSV epitope (YTDIEMNRLGK).
[0072] Deletional variants of a protein of the invention are
characterized by the removal of one or more amino acids from the
amino acid sequence of the protein.
[0073] Amino acid variants of a protein of the invention may
readily be made using peptide synthetic techniques well known in
the art, such as solid phase peptide synthesis and the like, or by
recombinant DNA manipulations. The manipulation of DNA sequences to
produce variant proteins which manifest as substitutional,
insertional or deletional variants are well known in the art. For
example, techniques for making substitution mutations at
predetermined sites in DNA having known sequence are well known to
those skilled in the art, such as by M13 mutagenesis, T7-Gen in
vitro mutagenesis kit (USB, Cleveland, Ohio), QuickChange Site
Directed mutagenesis kit (Stratagene, San Diego, Calif.),
PCR-mediated site-directed mutagenesis or other site-directed
mutagenesis protocols.
[0074] Derivatives of a CKX protein may also be useful in the
methods of the present invention. "Derivatives" of a protein of the
invention are those peptides, oligopeptides, polypeptides, proteins
and enzymes which comprise at least about five contiguous amino
acid residues of the polypeptide but which retain the biological
activity of this protein. A "derivative" may further comprise
additional naturally-occurring, altered glycosylated, acylated or
non-naturally occurring amino acid residues compared to the amino
acid sequence of a naturally-occurring form of the polypeptide.
Alternatively or in addition, a derivative may comprise one or more
non-amino acid substituents compared to the amino acid sequence of
a naturally-occurring form of the polypeptide, for example a
reporter molecule or other ligand, covalently or non-covalently
bound to the amino acid sequence such as, for example, a reporter
molecule which is bound thereto to facilitate its detection.
[0075] With "immunologically active" is meant that a molecule or
specific fragments thereof such as specific epitopes or haptens are
recognized by, i.e. bind to antibodies. Specific epitopes may be
determined using, for example, peptide scanning techniques as
described in Geysen et al. (1996) (Geysen, H. M., Rodda, S. J. and
Mason, T. J. (1986). A priori delineation of a peptide which mimics
a discontinuous antigenic determinant. Mol. Immunol. 23,
709-715.).
[0076] The term "fragment of a sequence" or "part of a sequence"
means a truncated sequence of the original sequence referred to.
The truncated sequence (nucleic acid or protein sequence) can vary
widely in length; the minimum size being a sequence of sufficient
size to provide a sequence with at least a comparable function
and/or activity or the original sequence referred to (e.g.
"functional fragment"), while the maximum size is not critical. In
some applications, the maximum size usually is not substantially
greater than that required to provide the desired activity and/or
function(s) of the original sequence. Typically, the truncated
amino acid sequence will range from about 5 to about 60 amino acids
in length. More typically, however, the sequence will be a maximum
of about 50 amino acids in length, preferably a maximum of about 60
amino acids. It is usually desirable to select sequences of at
least about 10, 12 or 15 amino acids, up to a maximum of about 20
or 25 amino acids. Functional fragments comprise at least 50 amino
acids, include an FAD binding domain (as defined in Pfam (version
14.0, june 2004) accession number 1565, Bateman et al., Nucleic
Acids Research Database Issue 32, D138-D141, 2004) and exhibit
cytokinin oxidase/dehydrogenase activity. Most preferably,
functional fragments exhibit cytokinin oxidase/dehydrogenase
activity and comprise an amino acid sequence corresponding to the
sequence spanning from Leu 94 to Leu 461 of SEQ ID NO: 26.
Functional fragments can also include those comprising an epitope
which is specific for the CKX proteins.
[0077] It should thus be understood that functional fragments may
also be immunologically active fragments.
[0078] In the methods of the invention use of homologues,
derivatives and/or immunologically active and/or functional
fragments of the cytokinin oxidases as defined supra is
contemplated. Particularly preferred homologues, derivatives and/or
immunologically active and/or functional fragments of the cytokinin
oxidase proteins which are contemplated for use in the methods of
the current invention are derived from plants, more specifically
from Arabidopsis thaliana, even more specifically the cytokinin
oxidases are the Arabidopsis thaliana (At)CKX, or are capable of
being expressed in the aleurone and/or embryo of a seed of cereals,
preferably of rice or corn. The present invention clearly
contemplates the use of functional homologues or derivatives and/or
immunologically active fragments of the AtCKX proteins and is not
to be limited in application to the use of a nucleotide sequence
encoding one of these AtCKX proteins in the methods of the present
invention.
[0079] To effect expression of a CKX protein in a cell, tissue or
organ, preferably of plant origin, either the protein may be
introduced directly to the cell, such as by microinjection or
ballistic means or alternatively, an isolated nucleic acid molecule
encoding the CKX protein may be introduced into the cell, tissue or
organ in an expressible format.
[0080] In the context of the present invention it should be
understood that the term "expression" and/or `overexpression` are
used interchangeably and both relate to an "enhanced and/or ectopic
expression" of a plant cytokinin oxidase. It should be clear that
herewith an enhanced expression of the plant cytokinin oxidase as
well as "de novo" expression of plant cytokinin oxidases is meant.
Methods for increasing expression of genes or proteins are well
documented in the art and include, for example, overexpression
driven by appropriate promoters, the use of transcription enhancers
or translation enhancers. Isolated nucleic acids which serve as
promoter or enhancer elements may be introduced in an appropriate
position (typically upstream) of a non-heterologous form of a
polynucleotide so as to upregulate expression of a CKX-encoding
nucleic acid or variant thereof. For example, endogenous promoters
may be altered in vivo by mutation, deletion, and/or substitution
(see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,
PCT/US93/03868), or isolated promoters may be introduced into a
plant cell in the proper orientation and distance from a gene of
the present invention so as to control the expression of the gene.
Methods for reducing the expression of genes or gene products are
well documented in the art.
[0081] The expression of a nucleic acid encoding a CKX polypeptide
or a homologue thereof may also be increased in the aleurone and/or
embryo of a seed by introducing a genetic modification (preferably
in the locus of a CKX gene). The locus of a gene as defined herein
is taken to mean a genomic region, which includes the gene of
interest and 10 kb up- or down stream of the coding region.
[0082] The genetic modification may be introduced, for example, by
any one (or more) of the following methods: T-DNA activation,
TILLING, site-directed mutagenesis, directed evolution and
homologous recombination or by introducing and expressing in a
plant a nucleic acid encoding a CKX polypeptide or a homologue
thereof. Following introduction of the genetic modification, there
follows a step of selecting for modified expression of a nucleic
acid encoding a CKX polypeptide or a homologue thereof, which
modification in expression gives plants having increased yield.
[0083] T-DNA activation tagging (Hayashi et al. Science (1992)
1350-1353) involves insertion of T-DNA, usually containing a
promoter (may also be a translation enhancer or an intron), in the
genomic region of the gene of interest or 10 kb up- or down stream
of the coding region of a gene in a configuration such that the
promoter directs expression of the targeted gene. Typically,
regulation of expression of the targeted gene by its natural
promoter is disrupted and the gene falls under the control of the
newly introduced promoter. The promoter is typically embedded in a
T-DNA. This T-DNA is randomly inserted into the plant genome, for
example, through Agrobacterium infection and leads to
overexpression of genes near the inserted T-DNA. The resulting
transgenic plants show dominant phenotypes due to overexpression of
genes close to the introduced promoter. According to the present
invention, the promoter to be introduced is a promoter capable of
driving expression in the aleurone and/or embryo of a seed.
[0084] A genetic modification may also be introduced in the locus
of a CKX gene using the technique of TILLING (Targeted Induced
Local Lesions In Genomes). This is a mutagenesis technology useful
to generate and/or identify, and to eventually isolate mutagenised
variants of a CKX nucleic acid capable of exhibiting CKX activity.
TILLING also allows selection of plants carrying such mutant
variants. These mutant variants may even exhibit higher CKX
activity than that exhibited by the gene in its natural form.
TILLNG combines high-density mutagenesis with high-throughput
screening methods. The steps typically followed in TILLING are: (a)
EMS mutagenesis (Redei G P and Koncz C (1992) In Methods in
Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore,
World Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994)
In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172;
Lightner J and Caspar T (1998) In J Martinez-Zapater, J Salinas,
eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa,
N.J., pp 91-104); (b) DNA preparation and pooling of individuals;
(c) PCR amplification of a region of interest; (d) denaturation and
annealing to allow formation of heteroduplexes; (e) DHPLC, where
the presence of a heteroduplex in a pool is detected as an extra
peak in the chromatogram; (e) identification of the mutant
individual; and (g) sequencing of the mutant PCR product. Methods
for TILLING are well known in the art (McCallum et al., (2000) Nat
Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet.
5(2): 145-50).
[0085] Site-directed mutagenesis may be used to generate variants
of CKX nucleic acids. Several methods are available to achieve
site-directed mutagenesis, the most common being PCR based methods
(current protocols in molecular biology. Wiley Eds.
http://www.4ulr.com/products/currentprotocols/index.html).
[0086] Directed evolution may also be used to generate variants of
CKK nucleic acids. This consists of iterations of DNA shuffling
followed by appropriate screening and/or selection to generate
variants of CKX nucleic acids or portions thereof encoding CKX
polypeptides or homologues or portions thereof having an modified
biological activity (Castle et al., (2004) Science 304(5674):
1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
[0087] T-DNA activation, TILLING, site-directed mutagenesis and
directed evolution are examples of technologies that enable the
generation of novel alleles and CKX variants.
[0088] Homologous recombination allows introduction in a genome of
a selected nucleic acid at a defined selected position. Homologous
recombination is a standard technology used routinely in biological
sciences for lower organisms such as yeast or the moss
Physcomitrella. Methods for performing homologous recombination in
plants have been described not only for model plants (Offring a et
al. (1990) EMBO J. 9(10): 3077-84) but also for crop plants, for
example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; Iida
and Terada (2004) Curr opin Biotech 15(2):132-8). The nucleic acid
to be targeted (which may be a CKX nucleic acid or variant thereof
as hereinbefore defined) need not be targeted to the locus of a CKX
gene, but may be introduced in, for example, regions of high
expression. The nucleic acid to be targeted may be an improved
allele used to replace the endogenous gene or may be introduced in
addition to the endogenous gene.
[0089] The invention also relates to a method for the production of
modified plants having increased seed yield, comprising the
introduction of a genetic modification, which genetic modification
results in increased expression of cytokinin oxidase in the
aleurone and/or embryo of a seed.
[0090] The invention furthermore relates to a method for increasing
seed yield in a plant comprising the introduction of a nucleic acid
molecule encoding a CKX as defined above, operably linked to one or
more control sequences or a vector stably integrated into the
genome of a plant cell capable of driving expression in the
aleurone and/or embryo of a seed.
[0091] Therefore, the invention also relates to a vector comprising
a nucleic acid encoding a CKX as defined above, wherein the vector
is an expression vector and wherein the nucleic acid encoding a CKX
is operably linked to one or more control sequences allowing the
expression in the aleurone and/or embryo of a seed.
[0092] In the present invention, the inventors have surprisingly
shown that the expression of cytokinin oxidases in the aleurone
and/or embryo of a seed resulted in the above-mentioned
seed-related features. Examples of seed-specific promoters include
but are not limited to those listed in Table 3.
TABLE-US-00003 TABLE 3 Examples of plant-expressible promoters
capable of driving expression in the seed EXPRESSION GENE SOURCE
PATTERN REFERENCE .alpha.-amylase (Amy32b) aleurone Lanahan, M. B.,
et al., Plant Cell 4: 203-211, 1992; Skriver, K., et al. Proc.
Natl. Acad. Sci. (USA) 88: 7266-7270, 1991 cathepsin .beta.-like
gene aleurone Cejudo, F. J., et al. Plant Molecular Biology 20:
849-856, 1992. seed-specific genes seed Simon, et al., Plant Mol.
Biol. 5: 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202,
1987.; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990. Brazil
Nut albumin seed Pearson, et al., Plant Mol. Biol. 18: 235-245,
1992. Legumin seed Ellis, et al., Plant Mol. Biol. 10: 203- 214,
1988. glutelin (rice) seed Takaiwa, et al., Mol. Gen. Genet. 208:
15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987. Zein
seed Matzke et al Plant Mol Biol, 14(3): 323-32 1990 NapA seed
Stalberg, et al, Planta 199: 515-519, 1996. wheat LMW and HMW
endosperm Mol Gen Genet 216: 81-90, 1989; glutenin-1 NAR 17: 461-2,
1989 wheat SPA seed Albani et al, Plant Cell, 9: 171-184, 1997
wheat .alpha., .beta., .gamma.-gliadins endosperm EMBO 3: 1409-15,
1984 barley ltr1 promoter endosperm barley B1, C, D, endosperm
Theor Appl Gen 98: 1253-62, 1999; hordein Plant J 4: 343-55, 1993;
Mol Gen Genet 250: 750-60, 1996 barley DOF endosperm Mena et al,
The Plant Journal, 116(1): 53-62, 1998 blz2 endosperm EP99106056.7
synthetic promoter endosperm Vicente-Carbajosa et al., Plant J. 13:
629-640, 1998. rice prolamin NRP33 endosperm Wu et al, Plant Cell
Physiology 39(8) 885-889, 1998 rice .alpha.-globulin Glb-1
endosperm Wu et al, Plant Cell Physiology 39(8) 885-889, 1998 rice
OSH1 embryo Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122,
1996 rice .alpha.-globulin endosperm Nakase et al. Plant Mol. Biol.
33: 513- REB/OHP-1 522, 1997 rice ADP-glucose PP endosperm Trans
Res 6: 157-68, 1997 maize ESR gene family endosperm Plant J 12:
235-46, 1997 sorgum .gamma.-kafirin endosperm PMB 32: 1029-35, 1996
KNOX embryo Postma-Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999
rice oleosin embryo and aleuron Wu et at, J. Biochem., 123: 386,
1998 sunflower oleosin seed (embryo and Cummins, et al., Plant Mol.
Biol. 19: dry seed) 873-876, 1992
[0093] Further examples of promoters suitable for seed specific
expression in a plant may be found in the examples section, Table
4.
[0094] In accordance with the present invention, there are provided
methods and compositions for increasing seed yield in a plant. Seed
yield may be increased by increasing the expression of a cytokinin
oxidase gene in the embryo and/or aleurone of a plant seed. Thus,
an embryo and/or aleurone preferred promoter may be utilized to
drive expression of a cytokinin oxidase in these particular
components of a seed.
[0095] In accordance with the present invention, a cytokinin
oxidase gene may be placed in a genetic construct such as a vector,
under control of a embryo and/or aleurone-preferred promoter. An
example of such a promoter capable of driving expression in the
embryo and/or aleurone is the sequence as represented in GenBank
under accession number AF019212 (sequence from nucleotide 1 to
1256, hereafter named PRO0218).
[0096] For example, the AtCKX2 gene (SEQ ID NO: 42) may be placed
under control of a promoter capable of driving expression in the
seed, more particularly a promoter capable of driving expression in
the embryo and/or aleurone, for example PRO0218.
[0097] Therefore, there is provided a vector comprising the
isolated nucleic acid molecule having the sequence set forth in SEQ
ID NO: 41 or 43.
[0098] These constructs may then be used to transform plants,
either dicotyledonous or monocotyledonous. In accordance with the
present invention, plants transformed with a vector according to
the present invention have an increased seed yield, when compared
to nullizygous control plants.
[0099] Preferably, the vector of the invention comprises a coding
sequence or open reading frame (ORF) encoding a cytokinin oxidase
protein or a homologue or derivative thereof or an immunologically
active and/or functional fragment thereof as defined supra.
Preferably the cytokinin oxidase is a plant cytokinin oxidase and
more specifically an Arabidopsis thaliana (At)CKX. Most preferably
the CKX is as represented by one of SEQ ID NO: 4, 36 or 38.
[0100] With "vector" or "vector sequence" or "genetic construct" is
meant a DNA sequence which can be introduced in an organism by
transformation and can be stably maintained in this organism.
Vector maintenance is possible in e.g. cultures of Escherichia
coli, A. tumefaciens, Saccharomyces cerevisiae or
Schizosaccharomyces pombe. Other vectors such as phagemids and
cosmid vectors can be maintained and multiplied in bacteria and/or
viruses. Vector sequences generally comprise a set of unique sites
recognized by restriction enzymes, the multiple cloning site (MCS),
wherein one or more non-vector sequence(s) can be inserted. As an
alternative to multiple cloning sites, the vector may also comprise
recombination sites. Gene cloning through recombination is well
known in the art.
[0101] With "non-vector sequence" is accordingly meant a DNA
sequence which is integrated in one or more of the sites of the MCS
comprised within a vector.
[0102] "Expression vectors" form a subset of vectors which, by
virtue of comprising the appropriate regulatory or control
sequences enable the creation of an expressible format for the
inserted non-vector sequence(s), thus allowing expression of the
protein encoded by this non-vector sequence(s). Expression vectors
are known in the art enabling protein expression in organisms
including bacteria (e.g. E. coli), fungi (e.g. S. cerevisiae, S.
pombe, Pichia pastoris), insect cells (e.g. baculoviral expression
vectors), animal cells (e.g. COS or CHO cells) and plant cells
(e.g. potato virus X-based expression vectors).
[0103] By "expressible format" is meant that the isolated nucleic
acid molecule is in a form suitable for being transcribed into mRNA
and/or translated to produce a protein, either constitutively or
following induction by an intracellular or extracellular signal,
such as an environmental stimulus or stress (mitogens, anoxia,
hypoxia, temperature, salt, light, dehydration, etc) or a chemical
compound such as IPTG (isopropyl-.beta.-D-thiogalactopyranoside) or
such as an antibiotic (tetracycline, ampicillin, rifampicin,
kanamycin), hormone (e.g. gibberellin, auxin, cytokinin,
glucocorticoid, brassinosteroid, ethylene, abscisic acid etc),
hormone analogue (indoleacetic acid (IAA), 2,4-D, etc), metal
(zinc, copper, iron, etc), or dexamethasone, amongst others. As
will be known to those skilled in the art, expression of a
functional protein may also require one or more post-translational
modifications, such as glycosylation, phosphorylation,
dephosphorylation, or one or more protein-protein interactions,
amongst others. All such processes are included within the scope of
the term "expressible format".
[0104] It should be understood that for expression in monocots of
the cytokinin oxidase genes according to the methods of the
invention, a nucleic acid sequence corresponding to the cDNA
sequence should be used to avoid mis-splicing of introns in
monocots. Preferred cDNA sequences to be expressed in monocots have
a nucleic acid sequence as represented in any of SEQ ID NOs: 25 to
30, SEQ ID NO: 34, SEQ ID NO: 37, or SEQ ID NO: 42.
[0105] Constitutive expression of the cytokinin oxidase gene in
plants resulted in increased root growth and decreased shoot
growth, illustrating the importance of confined expression of the
cytokinin oxidase gene for general plant growth properties.
Containment of cytokinin oxidase activity can be achieved by using
cell-, tissue- or organ-specific promoters, since cytokinin
degradation is a process limited to the tissues or cells that
express the CKX protein, this in contrast to approaches relying on
hormone synthesis.
[0106] Preferably, expression of a protein in a specific cell,
tissue, or organ, preferably of plant origin, is effected by
introducing and expressing an isolated nucleic acid molecule
encoding the protein, such as a cDNA molecule, genomic gene,
synthetic oligonucleotide molecule, mRNA molecule or open reading
frame, into this cell, tissue or organ, wherein the nucleic acid
molecule is placed operably in connection with suitable regulatory
or control sequences including a promoter, preferably a
plant-expressible promoter, and a terminator sequence. In
particular, and according to the methods of the present invention,
a nucleic acid sequence encoding a CKX is operably linked to a
promoter capable of driving expression in the aleurone and/or
embryo of a seed.
[0107] Reference herein to a "promoter" is to be taken in its
broadest context and includes the transcriptional regulatory
sequences derived from a classical eukaryotic genomic gene,
including the TATA box which is required for accurate transcription
initiation, with or without a CCAAT box sequence and additional
regulatory or control elements (i.e. upstream activating sequences,
enhancers and silencers) which alter gene expression in response to
developmental and/or external stimuli, or in a tissue-specific
manner.
[0108] The term "promoter" also includes the transcriptional
regulatory sequences of a classical prokaryotic gene, in which case
it may include a -35 box sequence and/or a -10 box transcriptional
regulatory sequences.
[0109] The term "promoter" is also used to describe a synthetic or
fusion molecule, or derivative which confers, activates or enhances
expression of a nucleic acid molecule in a cell, tissue or
organ.
[0110] Promoters may contain additional copies of one or more
specific regulatory elements, to further enhance expression and/or
to alter the spatial expression and/or temporal expression of a
nucleic acid molecule to which it is operably linked. Such
regulatory elements may be placed adjacent to a heterologous
promoter sequence to drive expression of a nucleic acid molecule in
response to e.g. copper, glucocorticoids, dexamethasone,
tetracycline, gibberellin, cAMP, abscisic acid, auxin, wounding,
ethylene, jasmonate or salicylic acid or to confer expression of a
nucleic acid molecule to specific cells, tissues or organs such as
meristems, leaves, roots, embryo, flowers, seeds or fruits.
[0111] In the context of the present invention, the promoter is a
plant-expressible promoter sequence, capable of driving expression
in the aleurone and/or embryo of a seed. By "plant-expressible" is
meant that the promoter sequence, including any additional
regulatory elements added thereto or contained therein, is at least
capable of inducing, conferring, activating or enhancing expression
in a plant cell, tissue or organ, preferably a monocotyledonous or
dicotyledonous plant cell, tissue, or organ, in casu the aleurone
and/or embryo.
[0112] The terms "plant-operable" and "operable in a plant" when
used herein, in respect of a promoter sequence, shall be taken to
be equivalent to a plant-expressible promoter sequence.
[0113] Regulatable promoters as part of a binary viral plant
expression system are also known to the skilled artisan (Yadav
1999-WO9922003; Yadav 2000-WO0017365).
[0114] In the present context, a "regulatable promoter sequence" is
a promoter that is capable of conferring expression on a structural
gene in a particular cell, tissue, or organ or group of cells,
tissues or organs of a plant, optionally under specific conditions,
however does generally not confer expression throughout the plant
under all conditions. Accordingly, a regulatable promoter sequence
may be a promoter sequence that confers expression on a gene to
which it is operably linked in a particular location within the
plant or alternatively, throughout the plant under a specific set
of conditions, such as following induction of gene expression by a
chemical compound or other elicitor.
[0115] Preferably, the regulatable promoter used in the methods of
the present invention confers expression in a specific location
within the plant, either constitutively or following induction,
however not in the whole plant under any circumstances. In
particular, the regulatable promoter for use in the present
invention is a promoter capable of driving expression in the
aleurone and/or embryo of a seed. Other types of such promoters are
cell-specific promoter sequences, tissue-specific promoter
sequences, organ-specific promoter sequences, cell cycle specific
gene promoter sequences, inducible promoter sequences and
constitutive promoter sequences that have been modified to confer
expression in a particular part of the plant at any one time, such
as by integration of such constitutive promoter within a
transposable genetic element (Ac, Ds, Spm, En, or other
transposon).
[0116] The term "cell-specific" shall be taken to indicate that
expression is predominantly in a particular cell or cell-type,
preferably of plant origin, albeit not necessarily exclusively in
this cell or cell-type. The term "tissue-specific" shall be taken
to indicate that expression is predominantly in a particular tissue
or tissue-type, preferably of plant origin, albeit not necessarily
exclusively in this tissue or tissue-type. Similarly, the term
"organ-specific" shall be taken to indicate that expression is
predominantly in a particular organ, preferably of plant origin,
albeit not necessarily exclusively in this organ. Similarly, the
term "cell cycle specific" shall be taken to indicate that
expression is predominantly cyclic and occurring in one or more,
not necessarily consecutive phases of the cell cycle albeit not
necessarily exclusively in cycling cells, preferably of plant
origin.
[0117] Those skilled in the art will be aware that an "inducible
promoter" is a promoter the transcriptional activity of which is
increased or induced in response to a developmental, chemical,
environmental, or physical stimulus. Similarly, the skilled
craftsman will understand that a "constitutive promoter" is a
promoter that is transcriptionally active throughout most, but not
necessarily all parts of an organism, preferably a plant, during
most, but not necessarily all phases of its growth and
development.
[0118] Those skilled in the art will readily be capable of
selecting appropriate promoter sequences for use according to the
present invention from publicly-available or readily-available
sources, without undue experimentation.
[0119] Placing a nucleic acid molecule under the regulatory control
of a promoter sequence, or operably linking with a promoter
sequence, means positioning the nucleic acid molecule such that
expression is controlled by the promoter sequence. A promoter is
usually, but not necessarily, positioned upstream, or at the
5'-end, and within 2 kb of the start site of transcription, of the
nucleic acid molecule which it regulates. In the construction of
heterologous promoter/structural gene combinations it is generally
preferred to position the promoter at a distance from the gene
transcription start site that is approximately the same as the
distance between that promoter and the gene it controls in its
natural setting (i.e., the gene from which the promoter is
derived). As is known in the art, some variation in this distance
can be accommodated without loss of promoter function. Similarly,
the preferred positioning of a regulatory sequence element with
respect to a heterologous gene to be placed under its control is
defined by the positioning of the element in its natural setting
(i.e., the gene from which it is derived). Again, as is known in
the art, some variation in this distance can also occur.
[0120] The term "terminator" refers to a DNA sequence at the end of
a transcriptional unit which signals termination of transcription.
Terminators are 3'-non-translated DNA sequences containing a
polyadenylation signal, which facilitates the addition of
polyadenylate sequences to the 3'-end of a primary transcript.
Terminators active in cells derived from viruses, yeasts, molds,
bacteria, insects, birds, mammals and plants are known and
described in the literature. They may be isolated from bacteria,
fungi, viruses, animals and/or plants.
[0121] Examples of terminators particularly suitable for use in the
gene constructs of the present invention include the Agrobacterium
tumefaciens nopaline synthase (NOS) gene terminator, the
Agrobacterium tumefaciens octopine synthase (OCS) gene terminator
sequence, the Cauliflower mosaic virus (CaMV) 35S gene terminator
sequence, the Oryza sativa ADP-glucose pyrophosphorylase terminator
sequence (t3'Bt2), the Zea mays zein gene terminator sequence, the
rbcs-1A gene terminator, and the rbcs-3A gene terminator sequences,
amongst others.
[0122] Those skilled in the art will be aware of additional
promoter sequences and terminator sequences which may be suitable
for use in performing the invention. Such sequences may readily be
used without any undue experimentation.
[0123] In the context of the current invention, the terms "ectopic
expression" or "ectopic overexpression" of a gene or a protein are
conferring to expression patterns and/or expression levels of this
gene or protein normally not occurring under natural conditions,
more specifically is meant increased expression and/or increased
expression levels in one or more of: aleurone and/or embryo of a
seed.
[0124] Preferably, the promoter sequence used in the context of the
present invention is operably linked to a coding sequence or open
reading frame (ORF) encoding a cytokinin oxidase protein or a
homologue, derivative or an immunologically active and/or
functional fragment thereof as defined supra.
[0125] The vector may optionally comprise a selectable marker gene.
As used herein, the term "selectable marker gene" or "selectable
marker" or "marker for selection" or "screenable marker" includes
any gene which confers a phenotype on a cell in which it is
expressed to facilitate the identification and/or selection of
cells which are transfected or transformed with a gene construct of
the invention or a derivative thereof. Suitable selectable marker
genes contemplated herein include the ampicillin resistance
(Amp.sup.r), tetracycline resistance gene (Tc.sup.r), bacterial
kanamycin resistance gene (Kan.sup.r), phosphinothricin resistance
gene, neomycin phosphotransferase gene (nptII), hygromycin
resistance gene, .beta.-glucuronidase (GUS) gene, chloramphenicol
acetyltransferase (CAT) gene, green fluorescent protein (gfp) gene
(Haseloff et al, 1997), and luciferase gene, amongst others.
[0126] The invention also relates to a host cell containing any of
the nucleic acid molecules or vectors of the invention. This host
cell is chosen from the group comprising bacterial, insect, fungal,
plant or animal cells.
[0127] The invention further relates to a method for the production
of transgenic plants, plant cells or plant tissues comprising the
introduction of a nucleic acid molecule of the invention in an
expressible format or a vector of the invention in this plant,
plant cell or plant tissue.
[0128] Therefore, there is provided a method for producing a plant
having increased seed yield, such method comprising: [0129] (a)
introducing into a plant cell a genetic construct comprising an
isolated nucleic acid molecule encoding a cytokinin oxidase wherein
the isolated nucleic acid molecule is operably linked to a promoter
capable of driving expression in the aleurone and/or embryo,
endosperm of a seed; [0130] (b) regenerating a plant therefrom;
[0131] (c) growing the regenerated plant to seed set; and [0132]
(d) selecting a plant with increased seed yield compared to a
corresponding control plant.
[0133] Generally after transformation, plant cells or cell
groupings are selected for the presence of one or more markers
which are encoded by plant-expressible genes co-transferred with
the gene of interest, following which the transformed material is
regenerated into a whole plant.
[0134] Following DNA transfer and regeneration, putatively
transformed plants may be evaluated, for instance using Southern
analysis, for the presence of the gene of interest, copy number
and/or genomic organisation. Alternatively or additionally,
expression levels of the newly introduced DNA may be monitored
using Northern and/or Western analysis, both techniques being well
known to persons having ordinary skill in the art.
[0135] The generated transformed plants may be propagated by a
variety of means, such as by clonal propagation or classical
breeding techniques. For example, a first generation (or T1)
transformed plant may be selfed to give homozygous second
generation (or T2) transformants, and the T2 plants further
propagated through classical breeding techniques.
[0136] The generated transformed organisms may take a variety of
forms. For example, they may be chimeras of transformed cells and
non-transformed cells; clonal transformants (e.g., all cells
transformed to contain the expression cassette); grafts of
transformed and untransformed tissues (e.g., in plants, a
transformed rootstock grafted to an untransformed scion).
[0137] The invention also relates to a transgenic plant cell
comprising a nucleic acid sequence of the invention which is
operably linked to regulatory elements allowing transcription
and/or expression of the nucleic acid in the aleurone and/or embryo
of a seed.
[0138] According to another preferred embodiment, the invention
relates to a transgenic plant cell as described hereinabove wherein
the nucleic acid of the invention is stably integrated into the
genome of this plant cell.
[0139] The invention further relates to a transgenic plant or plant
tissue comprising plant cells as herein described and also to a
harvestable part of such transgenic plant, preferably selected from
the group consisting of seeds, leaves, fruits, stem cultures,
roots, tubers, rhizomes and bulbs. The present invention
furthermore relates to products directly derived from a harvestable
part of a transgenic plant according to the invention, such as dry
pellets or powders, oil, fat and fatty acids, starch, or proteins.
The invention also relates to the progeny derived from any of the
transgenic plants according to the invention.
[0140] Preferably, transgenic plants are produced which express in
the aleurone and/or embryo of a seed a nucleic acid encoding a CKX,
preferably an Arabidopsis CKX, most preferably a CKX encoded by a
nucleic acid as set forth in any of SEQ ID NOs: 3, 26, 37, or 42 or
an ortholog of such nucleic acid. Preferably, the ortholog is
derived from a related species of the transgenic plant. Even more
preferably, the ortholog is specific (native or endogenous) to the
species of the transgenic plant.
[0141] Means for introducing recombinant DNA into plant tissue or
cells include, but are not limited to, transformation using
CaCl.sub.2 and variations thereof, in particular the method
described by Hanahan (1983), direct DNA uptake into protoplasts
(Krens et al., 1982; Paszkowski et al, 1984), PEG-mediated uptake
to protoplasts (Armstrong et al, 1990) microparticle bombardment,
electroporation (Fromm et al., 1985), microinjection of DNA
(Crossway et al., 1986), microparticle bombardment of tissue
explants or cells (Christou et al, 1988; Sanford, 1988),
vacuum-infiltration of tissue with nucleic acid, or in the case of
plants, T-DNA-mediated transfer from Agrobacterium to the plant
tissue as described essentially by An et al. (1985), Dodds et al.,
(1985), Herrera-Estrella et al. (1983a, 1983b, 1985). Methods for
transformation of monocotyledonous plants are well known in the art
and include Agrobacterium-mediated transformation (Cheng et al.,
1997-WO9748814; Hansen 1998-WO9854961; Hiei et al., 1994-WO9400977;
Hiei et al., 1998-WO9817813; Rikiishi et al., 1999-WO9904618; Saito
et al., 1995-WO9506722), microprojectile bombardment (Adams et al.,
1999-U.S. Pat. No. 5,969,213; Bowen et al., 1998-U.S. Pat. No.
5,736,369; Chang et al., 1994-WO9413822; Lundquist et al.,
1999-U.S. Pat. No. 5,874,265/U.S. Pat. No. 5,990,390; Vasil and
Vasil, 1995-U.S. Pat. No. 5,405,765. Walker et al., 1999-U.S. Pat.
No. 5,955,362), DNA uptake (Eyal et al., 1993-WO9318168),
microinjection of Agrobacterium cells (von Holt, 1994-DE4309203)
and sonication (Finer et al., 1997-U.S. Pat. No. 5,693,512).
[0142] A whole plant may be regenerated from the transformed or
transfected cell, in accordance with procedures well known in the
art. Plant tissue capable of subsequent clonal propagation, whether
by organogenesis or embryogenesis, may be transformed with a gene
construct of the present invention and a whole plant regenerated
therefrom. The particular tissue chosen will vary depending on the
clonal propagation systems available for, and best suited to, the
particular species being transformed. Exemplary tissue targets
include leaf disks, pollen, embryos, cotyledons, hypocotyls,
megagametophytes, callus tissue, existing meristematic tissue
(e.g., apical meristem, axillary buds, and root meristems), and
induced meristem tissue (e.g., cotyledon meristem and hypocotyl
meristem).
[0143] The term "organogenesis", as used herein, means a process by
which shoots and roots are developed sequentially from meristematic
centers.
[0144] The term "embryogenesis", as used herein, means a process by
which shoots and roots develop together in a concerted fashion (not
sequentially), whether from somatic cells or gametes.
[0145] Preferably, the transgenic plant produced according to the
inventive method is transfected or transformed with a genetic
sequence by any art-recognized means, such as microprojectile
bombardment, microinjection, Agrobacterium-mediated transformation
(including in planta transformation), protoplast fusion, or
electroporation, amongst others. Most preferably such plant is
produced by Agrobacterium-mediated transformation.
[0146] Agrobacterium-mediated transformation or agrolistic
transformation of plants, yeast, molds or filamentous fungi is
based on the transfer of part of the transformation vector
sequences, called the T-DNA, to the nucleus and on integration of
this T-DNA in the genome of the eukaryote.
[0147] With "T-DNA", or transferred DNA, is meant that part of the
transformation vector flanked by T-DNA borders which is, after
activation of the Agrobacterium vir genes, nicked at the T-DNA
borders and is transferred as a single stranded DNA to the nucleus
of an eukaryotic cell.
[0148] With "agrolistic transformation" is meant a transformation
method combining features of Agrobacterium-mediated transformation
and of biolistic DNA delivery. As such, a T-DNA containing target
plasmid is co-delivered with DNA/RNA enabling in planta production
of VirD1 and VirD2 with or without VirE2 (Hansen and Chilton 1996;
Hansen et al. 1997; Hansen and Chilton 1997-WO9712046).
[0149] With "foreign DNA" is meant any DNA sequence that is
introduced in the host's genome by recombinant techniques. The
foreign DNA includes e.g. a T-DNA sequence or a part thereof such
as the T-DNA sequence comprising the selectable marker in an
expressible format. Foreign DNA furthermore include intervening DNA
sequences as defined supra.
[0150] The present invention also encompasses use of CKX-encoding
nucleic acids and use of CKX polypeptides operably linked to a
promoter capable of driving expression in the aleurone and/or
embryo of a seed. One such use relates to increasing plant,
especially seed yield. The seed yield may include one or more of
the following: increased number of flowers per panicle, increased
total seed weight, increased number of filled seeds, increased
thousand kernel weight and increased harvest index, each relative
to corresponding wild type plants.
[0151] In particular, the present invention provides use of
cytokinin oxidase-encoding nucleic acids operably linked to a
promoter capable of driving overexpression in the aleurone and/or
embryo relative to other parts of the seed for increasing seed
yield of a plant. One such use is wherein the cytokinin
oxidase-encoding nucleic acid is comprised in a genetic construct
that is introduced into a plant cell. In particular, use of
cytokinin oxidase-encoding nucleic acids operably linked to a
promoter capable of driving overexpression in the aleurone and/or
embryo relative to other parts of the seed is envisaged for
increasing seed yield of a plant, wherein the cytokinin
oxidase-encoding nucleic acid is selected from the group consisting
of: [0152] (a) nucleic acids comprising a DNA sequence as given in
any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30,
33 or 34, or the complement thereof, [0153] (b) nucleic acids
comprising the RNA sequences corresponding to any of SEQ ID NOs:
42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the
complement thereof, [0154] (c) nucleic acids specifically
hybridizing to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11,
25, 26, 28 to 30, 33 or 34, or to the complement thereof, [0155]
(d) nucleic acids encoding a protein comprising the amino acid
sequence as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36
or 38, or the complement thereof, [0156] (e) nucleic acids as
defined in any of (a) to (d) characterized in that said nucleic
acid is DNA, genomic DNA, cDNA, synthetic DNA or RNA wherein T is
replaced by U, [0157] (f) nucleic acid which is degenerate compared
to a nucleic acid as given in any of SEQ ID NOs: 42, 37, 27, 1, 3,
5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or which is degenerate
compared to a nucleic acid as defined in any of (a) to (e) as a
result of the genetic code, [0158] (g) nucleic acids which are
divergent from a nucleic acid encoding a protein as given in any of
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or which is divergent
from a nucleic acid as defined in any of (a) to (e), due to the
differences in codon usage between the organisms, [0159] (h)
nucleic acids encoding a protein as given in SEQ ID NOs: 2, 4, 6,
8, 10, 12, 35, 36 or 38 or nucleic acids as defined in (a) to (e)
which are divergent due to the differences between alleles, [0160]
(i) nucleic acids encoding a protein as given in any of SEQ ID NOs:
2, 4, 6, 8, 10, 12, 35, 36 or 38, [0161] (j) functional fragments
of nucleic acids as defined in any of (a) to (i) having the
biological activity of a cytokinin oxidase, and [0162] (k) nucleic
acids encoding a plant cytokinin oxidase, comprising the consensus
sequence hTDYLhhoIGGTLSssG and cLFxushGsLGQFGIIstA.
[0163] Furthermore, use of SEQ ID NO: 41 and the use of a vector
comprising a cytokinin oxidase as defined in (a) to (k) above for
increasing seed yield in a plant is provided.
[0164] Those skilled in the art will be aware that the invention
described herein is subject to variations and modifications other
than those specifically described. It is to be understood that the
invention described herein includes all such variations and
modifications. The invention also includes all such steps,
features, compositions and compounds referred to or indicated in
this specification, individually or collectively, and any and all
combinations of any or more of these steps or features.
[0165] Throughout this specification, unless the context requires
otherwise the word "comprise", and variations such as "comprises"
and "comprising", will be understood to imply the inclusion of a
stated integer or step or group of integers or steps but not the
exclusion of any other integer or step or group of integers or
steps.
[0166] As used herein, the term "derived from" shall be taken to
indicate that a particular integer or group of integers has
originated from the species specified, but has not necessarily been
obtained directly from the specified source.
[0167] The following examples are given by means of illustration of
the present invention and are in no way limiting. The contents of
all references included in this application are incorporated by
reference herein as if fully set forth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0168] FIG. 1. Schematic representation of plant cytokinin oxidase
genes. Shown are the structures of different cytokinin oxidase
genes isolated from maize (ZmCKX1, accession number AF044603,
Biochem. Biophys. Res. Com. 255:328-333, 1999) and Arabidopsis
(AtCKX1 to AtCKX4). Exons are denominated with `E` and represented
by shaded boxes. Introns are represented by white boxes. Further
indicated are the gene sizes (in kb, on top of each structure), the
gene accession numbers (under the names) and a size bar
representing 0.5 kb.
[0169] FIG. 2. Alignment of plant cytokinin oxidase amino acid
sequences. The amino acid sequences from cytokinin oxidases from
maize (ZmCKX1) and Arabidopsis (AtCKX1 to AtCKX4) are aligned.
Identical amino acid residues are marked by a black box, similar
amino acid residues are in a grey box. Amino acid similarity
groups: (M,I,L,V), (F,W,Y), (G,A), (S,T), (R,K,H), (E,D),
(N,Q),
[0170] FIG. 3: Schematic presentation of the entry clone p41,
containing CDS0427-2 within the AttL1 and AttL2 sites for
Gateway.RTM. cloning in the pDONR201 backbone. CDS0427.sub.--2 is
the internal code for the Arabidopsis thaliana CKX2 coding sequence
(SEQ ID NO: 26). This vector contains also a bacterial
kanamycin-resistance cassette and a bacterial origin of
replication.
[0171] FIG. 4: Binary vector p37 for the expression in Oryza sativa
of the Arabidopsis thaliana CKX2 gene under the control of the
PRO0218 promoter. This vector contains a T-DNA derived from the Ti
plasmid, limited by a left border (LB repeat, LB Ti C58) and a
right border (RB repeat, RB Ti C58)). From the left border to the
right border, this T-DNA contains: a selectable and a screenable
marker for selection of transformed plants, each under control of a
constitutive promoter; the PRO0218_CDS0427.sub.--2-zein and
rbcS-deltaGA double terminator cassette for expression of the
Arabidopsis thaliana CKX2 gene. This vector also contains an origin
of replication from pBR322 for bacterial replication and a
selectable marker (Spe/SmeR) for bacterial selection with
spectinomycin and streptomycin.
[0172] FIG. 5: Binary vector p35 for the expression in Oryza sativa
of the Arabidopsis thaliana CKX2 gene under the control of the
PRO0090 promoter. This vector contains a T-DNA derived from the Ti
plasmid, limited by a left border (LB repeat, LB Ti C58) and a
right border (RB repeat, RBTi C58)). From the left border to the
right border, this T-DNA contains: a selectable and a screenable
marker for selection of transformed plants, each under control of a
constitutive promoter; the PRO0090--CDS0427.sub.--2-zein and
rbcS-deltaGA double terminator cassette for expression of the
Arabidopsis thaliana CKX2 gene. This vector also contains an origin
of replication from pBR322 for bacterial replication and a
selectable marker (Spe/SmeR) for bacterial selection with
spectinomycin and streptomycin.
EXAMPLES
Example 1
Brief Description of the Sequences of the Invention and Examples of
Seed Specific Promoters
TABLE-US-00004 [0173] SEQ ID NO: DESCRIPTION 1 AtCKX1 genomic 2
AtCKX1 protein 3 AtCKX2 genomic 4 AtCKX2 protein 5 AtCKX3 genomic 6
AtCKX3 protein 7 AtCKX4 genomic 8 AtCKX4 protein 9 AtCKX5 genomic
(short version) 10 AtCKX5 protein (short version) 11 AtCKX6 genomic
12 AtCKX6 protein 13 5'primer AtCKX1 14 3'primer AtCKX1 15 5'primer
AtCKX2 16 3'primer AtCKX2 17 5'primer AtCKX3 18 3'primer AtCKX3 19
5'primer AtCKX4 20 3'primer AtCKX4 21 5'primer AtCKX5 22 3'primer
AtCKX5 23 5'primer AtCKX6 24 3'primer AtCKX6 25 AtCKX1 cDNA 26
AtCKX2 cDNA 27 AtCKX3 cDNA 28 AtCKX4 cDNA 29 AtCKX5 cDNA (short
version) 30 AtCKX6 cDNA 31 AtCKX2 cDNA fragment 32 AtCKX2 peptide
fragment 33 AtCKX5 genomic (long version) 34 AtCKX5 cDNA (long
version) 35 AtCKX5 protein (long version) 36 AtCKX2, CDS0427_2
deduced protein sequence 37 AtCKX2 splice variant, DNA sequence 38
AtCKX2 splice variant, deduced protein sequence 39 PRM3769 (sense,
start codon at positions 35 to 37) 40 PRM1526 (reverse,
complementary stop codon at positions 30-32) 41 Expression cassette
with PRO0218 - CDS0427_2 - zein and rbcS-deltaGA double terminator
42 AtCKX2, CDS0427_2 cDNA 43 Expression cassette with PRO0090 -
CDS0427_2 - zein and rbcS-deltaGA double terminator 44
HTDYLhholGGTLSssG signature 45 cLFxushGsLGQFGllstA signature
TABLE-US-00005 TABLE 4 Examples of promoters suitable for seed
specific or seedling preferred expression Gene name Expression
Metallothionein Mte embryo/scutellum + calli putative beta-amylase
embryo/scutellum unknown scutellum proteinase inhibitor Rgpi9 seed
structural protein young tissues, calli, embryo/scutellum prolamine
10 Kda strong in endosperm allergen RA2 seed prolamine RP7
endosperm Metallothioneine-like ML2 embryo/scutellum + calli
prolamine RM9 strong in endosperm prolamine RP5 strong in endosperm
putative methionine embryo aminopeptidase putative 40S ribosomal
protein weak in endosperm alpha-globulin strong in endosperm
alanine aminotransferase aleurone, endosperm cyclophyllin 2 shoot,
embryo/endosperm sucrose synthase SS1 (barley) medium constitutive,
shoot endosperm/aleurone trypsin inhibitor ITR1 (barley) weak in
endosperm WSI18 embryo/aleurone aquaporine seedlings RAB21
embryo/aleurone OSH1 seedling Arceline 5A seed Cruciferine seed
Albumine 2S3 seed Albumine 2S2 seed FAE1 embryo Phaseolin Beta
subunit seed Lec1 embryo Gamma zein seed lipid Transfer Protein
seed
Example 2
Seed-Preferred or Seedling-Preferred Expression of a CKX2 Gene
Results in Increased Seed Yield
A) DNA Manipulation and Cloning of AtCKX2
[0174] Unless otherwise stated, recombinant DNA techniques are
performed according to standard protocols described in (Sambrook
& Russell (2001) Molecular Cloning: a laboratory manual, 3rd
Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in
Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in
Molecular Biology, Current Protocols. Standard materials and
methods for plant molecular work are described in Plant Molecular
Biology Labfax (1993) by R. D. D. Croy, published by BIOS
Scientific Publications Ltd (UK) and Blackwell Scientific
Publications (UK).
[0175] The Arabidopsis CKX2 gene (corresponding to SEQ ID NO: 42)
was amplified by PCR using as template an Arabidopsis thaliana
seedling cDNA library (Invitrogen, Paisley, UK). After reverse
transcription of RNA extracted from seedlings, the cDNAs were
cloned into pCMV Sport 6.0. Average insert size of the bank was 1.5
kb, and original number of clones was 1.59.times.10.sup.7 cfu. The
original titer was determined to be 9.6.times.10.sup.5 cfu/ml, and
became after a first amplification 6.times.10.sup.11 cfu/ml. After
plasmid extraction, 200 ng of template was used in a 50 .mu.l PCR
mix. Primers prm3769 (SEQ ID NO: 39) and prm1526 (SEQ ID NO: 40),
which include the AttB sites for Gateway recombination, were used
for PCR amplification.
[0176] PCR was performed using Hifi Taq DNA polymerase in standard
conditions. A PCR fragment of 1506 bp was amplified and purified
also using standard methods. The first step of the Gateway
procedure, the BP reaction, was then performed, during which the
PCR fragment recombines in vivo with the pDONR plasmid to produce,
according to the Gateway terminology, an "entry clone", p41 (FIG.
3). pDONR was purchased from Invitrogen, as part of the Gateway
technology.
B) Vector Construction
[0177] The entry clone p41 was subsequently used in an LR reaction
with p831 or p830, both destination vectors according to the
Gateway.TM. terminology, used for rice transformation.
[0178] p831 contains as functional elements within the T-DNA
borders a plant selectable marker, a screenable marker and a
Gateway cassette intended for LR in vivo recombination with the
sequence of interest already cloned in the donor vector. The
PRO0218 promoter for embryo and aleurone preferred expression is
located upstream of this Gateway cassette.
[0179] Similarly, p830 contains as functional elements within the
T-DNA borders: a plant selectable marker; a screenable marker; and
a Gateway cassette intended for LR in vivo recombination with the
sequence of interest already cloned in the donor vector. The
PRO0090 promoter for endosperm-preferred expression is located
upstream of this Gateway cassette.
[0180] After the recombination step, the resulting expression
vectors p37 (originating from p831, FIG. 4) and p35 (originating
from p830, FIG. 5) were transformed into Agrobacterium strain
LBA4404 and subsequently into Oryza sativa plants.
C) Transformation of Rice
[0181] Mature dry seeds of the rice japonica cultivar Nipponbare
were dehusked. Sterilization was done by incubating the seeds for
one minute in 70% ethanol, followed by 30 minutes in 0.2%
HgCl.sub.2 and by 6 washes of 15 minutes with sterile distilled
water. The sterile seeds were then germinated on a medium
containing 2,4-D (callus induction medium). After a 4-week
incubation in the dark, embryogenic, scutellum-derived calli were
excised and propagated on the same medium. Two weeks later, the
calli were multiplied or propagated by subculture on the same
medium for another 2 weeks. 3 days before co-cultivation,
embryogenic callus pieces were sub-cultured on fresh medium to
boost cell division activity. The Agrobacterium strain LBA4404,
harbouring T-DNA vectors comprising a suitable selection marker,
was used for co-cultivation. Agrobacterium was cultured for 3 days
at 28.degree. C. on AB medium with the appropriate antibiotics. The
bacteria were then collected and suspended in liquid co-cultivation
medium at an OD6% of about 1. The suspension was transferred to a
petri dish and the calli were immersed in the suspension during 15
minutes. Next, the callus tissues were blotted dry on a filter
paper, transferred to solidified co-cultivation medium and
incubated for 3 days in the dark at 25.degree. C.
[0182] Hereafter, co-cultivated callus was grown on
2,4-D-containing medium for 4 weeks in the dark at 28.degree. C. in
the presence of a selective agent at a suitable concentration.
During this period, rapidly growing resistant callus islands
developed. Upon transfer of this material to a regeneration medium
and incubation in the light, the embryogenic potential was released
and shoots developed in the next four to five weeks. Shoots were
excised from the callus and incubated for 2 to 3 weeks on an
auxin-containing medium from which they were transferred to soil.
Hardened shoots were grown under high humidity and short days in a
greenhouse. Finally seeds were harvested three to five months after
transplanting. The method yielded single locus transformants at a
rate of over 50% (Aldemita and Hodges, 1996, Chan et al., 1993,
Hiei et al., 1994).
D) Evaluation of Transformants: Vegetative Growth Measurements
[0183] Approximately 15 to 20 independent T0 transformants were
generated. The primary transformants were transferred from tissue
culture chambers to a greenhouse for growing and harvest of T1
seed. Four events (for p37 transformants, PRO0218 promoter) or five
events (for p35 transformants, PRO0090 promoter) of which the T1
progeny segregated 3:1 for presence/absence of the transgene were
retained. For each of these events, 10 T1 seedlings containing the
transgene (hetero- and homo-zygotes), and 10 T1 seedlings lacking
the transgene (nullizygotes), were selected by monitoring visual
marker expression. The selected T1 plants were transferred to a
greenhouse. Each plant received a unique barcode label to link
unambiguously the phenotyping data to the corresponding plant. The
selected T1 plants were grown on soil in 10 cm diameter pots under
the following environmental settings: photoperiod: 11.5 h, daylight
intensity: 30,000 lux or more, daytime temperature: 28.degree. C.
or higher, night time temperature: 22.degree. C., relative
humidity: 60-70%. Transgenic plants and the corresponding
nullizygotes were grown side-by-side at random positions. From the
stage of sowing until the stage of maturity the plants were passed
several times through a digital imaging cabinet. At each time point
digital images (2048.times.1536 pixels, 16 million colours) were
taken of each plant from at least 6 different angles.
[0184] In a next step, the mature primary panicles were harvested,
bagged, barcode-labelled and then dried for three days in the oven
at 37.degree. C. The panicles were then threshed and all the seeds
were collected and counted. The filled husks were separated from
the empty ones using an air-blowing device. The empty husks were
discarded and the remaining fraction was counted again. The filled
husks were weighed on an analytical balance and the cross-sectional
area of the seeds was measured using digital imaging. This
procedure allows deriving a set of seed-related parameters.
[0185] The parameters described below were derived in an automated
way from the digital images using image analysis software and were
analysed statistically.
[0186] A two factor ANOVA (ANalysis Of VAriance) corrected for the
unbalanced design was used as statistical model for the overall
evaluation of plant phenotypic characteristics. An F-test was
carried out on all the parameters measured in all the plants and of
all the events transformed with that gene. The F-test was carried
out to check for an effect of the gene over all the transformation
events and to verify for an overall effect of the gene, also named
herein "global gene effect". If the value of the F test showed that
the data are significant, than it was concluded that there is a
"gene" effect, meaning that not only presence or the position of
the gene is causing the effect. The threshold for significance for
a true global gene effect was set at 5% probability level for the F
test.
[0187] To check for an effect of the genes within an event, i.e.,
for a line-specific effect, a t-test was performed within each
event using data sets from the transgenic plants and the
corresponding null plants. "Null plants" or "Null segregants" or
"Nullizygotes" are the plants treated in the same way as the
transgenic plant, but from which the transgene has segregated. Null
plants can also be described as the homozygous negative transformed
plants. The threshold for significance for the t-test was set at a
10% probability level. The results for some events can be under or
below this threshold. This is based on the hypothesis that a gene
might only have an effect in certain positions in the genome, and
that the occurrence of this position-dependent effect is not
uncommon. This kind of gene effect is also named herein a "line
effect of the gene". The p value was obtained by comparing the t
value to the t distribution or alternatively, by comparing the F
value to the F distribution. The p value gives the probability of
the null hypothesis (i.e., that there is no effect of the
transgene) is correct. The threshold for significance was set at a
5% p-value for the F test and a 10% p-value for the t-test.
[0188] Vegetative growth and seed yield was measured according to
the methods as described above. The inventors surprisingly found
that the seed yield of transgenic plants was increased for both the
endosperm-preferred and embryo and/or aleurone-preferred promoter
constructs (expressed as total weight of seeds, number of (filled)
seeds and harvest index), when compared to null plants, and that
transgenic plants with the embryo and/or aleurone-preferred
promoter constructs additionally had an increased Thousand Kernel
Weight compared to transgenics with the endosperm-preferred
promoter construct. The inventors furthermore observed that the
yield increase was higher for plants transformed with the embryo
and/or aleurone-preferred promoter constructs than for plants with
the endosperm-preferred promoter construct. Details are given in
paragraphs E and F.
[0189] The data obtained in the experiment with T1 plants were then
confirmed in a further experiment with T2 plants. Seed batches from
the positive plants (both hetero- and homozygotes) in T1, were
screened by monitoring marker expression. For each chosen event,
the heterozygote seed batches were then retained for T2 evaluation.
Within each seed batch an equal number of positive and negative
plants were grown in the greenhouse for evaluation. In particular,
four events of p37 T2 transformants and three events of p35 T2
transformants were selected for further analysis. For both p37 T2
transformants and p35 T2 transformants, a total of 120 plants were
tested, evenly distributed over each event.
E) Evaluation of P37 Transformants: Measurement of Seed-Related
Parameters
[0190] Upon analysis of the seeds as described above, the inventors
found that plants transformed with the AtCKX2 gene under control of
the embryo and/or aleurone-preferred promoter had a higher total
weight of seeds, a higher number of filled seeds, a higher harvest
index and a higher Thousand Kernel Weight than plants lacking the
CKX2 transgene. These findings were consistent over 2 independent
experiments with T1 plants as well as in an experiment with T2
plants, as shown in table 5. In addition to these yield parameters,
3 lines in T1 scored also positive for the total number of seeds.
This increase in total seed number was confirmed in T2, where the
effect was shown to be a significant global gene effect (mean
increase +24%, p-value from the F-test 0.0032).
TABLE-US-00006 TABLE 5 Analysis of seed related parameters for p37
transformants T1 generation, 1.sup.st T1 generation, 2.sup.nd
experiment experiment T2 generation Difference over null Difference
over null Difference over Parameter plants plants null plants
p-value Total weight of +22% +22% +51% 0.0000 seeds Number of
filled +22% +17% +46 0.0000 seeds Harvest Index +25% +20% +37%
0.0025 Thousand +1% +5% +3% 0.0000 Kernel Weight
[0191] The total seed weight was measured by weighing all filled
seeds harvested from a transformed rice plant. The number of filled
seeds was determined by counting the number of filled seeds
harvested from a transformed rice plant. The total seed number was
determined by counting the number of seeds harvested from a plant.
The harvest index is defined as the ratio between the total seed
weight and the above ground area (mm.sup.2), multiplied by a factor
106. Thousand Kernel Weight (TKW) was derived from the number of
filled seeds that were counted, and their total weight. The figures
gave the mean increase (in %) of each parameter calculated from
transgenes versus corresponding nullizygotes of 4 independent
events in T1 generation, each event comprising 10 plants carrying
the transgene and 10 nullizygotes, and of 4 independent events in
the T2 generation, each event comprising 20 plants carrying the
transgene and 20 nullizygotes. The p-values of the F-test listed
for the data of the T2 generation demonstrate that the obtained
increases for the various seed yield parameters are all significant
and that there is clearly an overall gene effect.
F) Evaluation of p35 Transformants: Measurement of Seed-Related
Parameters
[0192] Plants transformed with the AtCKX2 gene under control of the
endosperm-preferred promoter also had a better yield compared to
the control nullizygous plants, in particular for total seed
weight, number of filled seeds and harvest index. The total seed
weight, number of filled seeds and harvest index are defined as
above.
[0193] In a first experiment, plants of the T1 generation of five
independent events were compared, for each event 10 T1 plants
carrying the transgene versus 10 corresponding control T1 plants.
For the parameter "total seed weight", two out of the five events
had a significant increase (58% and 67%, with a p-value of the
t-test of 0.0551 and 0.0211 respectively). Similar results were
obtained for the number of filled seeds, for which these two lines
showed in increase of 47% and 68% with a p-value of respectively
0.0846 and 0.0166. The two lines also scored positive for Harvest
Index (increases of 41% (p-value of 0.0223) and 31% respectively).
Besides these two lines, a third line also scored significantly
higher than the corresponding nullizygous control plants (+41%,
p-value of 0.035).
[0194] The positive data for seed yield observed in the T1
generation were confirmed in the T2 generation. Data are given in
Table 6.
TABLE-US-00007 TABLE 6 Analysis of seed related parameters for p37
transformants T2 generation Parameter Difference over null plants
p-value Total seed weight +24% 0.0484 Number of filled seeds +26%
0.0254 Harvest index +19% 0.0277
[0195] The figures give the mean increase (in %) of each parameter
calculated from transgenes versus corresponding nullizygotes of 3
independent events in the T2 generation, each event comprising 20
plants carrying the transgene and 20 nullizygotes. The p-values of
the F-test listed for the data of the T2 generation demonstrate
that the obtained increases for the various seed yield parameters
are all significant and that there is clearly an overall gene
effect.
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Sequence CWU 1
1
4512236DNAArabidopsis thaliana 1atgggattga cctcatcctt acggttccat
agacaaaaca acaagacttt cctcggaatc 60ttcatgatct tagttctaag ctgtatacca
ggtagaacca atctttgttc caatcattct 120gttagtaccc caaaagaatt
accttcttca aatccttcag atattcgttc ctcattagtt 180tcactagatt
tggagggtta tataagcttc gacgatgtcc acaatgtggc caaggacttt
240ggcaacagat accagttacc acctttggca attctacatc caaggtcagt
ttttgatatt 300tcatcgatga tgaagcatat agtacatctg ggctccacct
caaatcttac agtagcagct 360agaggccatg gtcactcgct tcaaggacaa
gctctagctc atcaaggtgt tgtcatcaaa 420atggagtcac ttcgaagtcc
tgatatcagg atttataagg ggaagcaacc atatgttgat 480gtctcaggtg
gtgaaatatg gataaacatt ctacgcgaga ctctaaaata cggtctttca
540ccaaagtcct ggacagacta ccttcatttg accgttggag gtacactatc
taatgctgga 600atcagcggtc aagcattcaa gcatggaccc caaatcaaca
acgtctacca gctagagatt 660gttacaggta tttcattcat gctttatctc
tgcggtagtc tcaaaaaaat atgcacctgt 720aaagaatatc catctcttca
tgagcaaaaa cactgacgac tttaaataat ttttgactat 780aaaacaagag
tgcataggca caaatgtgaa atatgcaaca cacaattgta acttgcacca
840agaaaaaagt tataaaaaca aacaactgat aagcaatata tttccaatat
ttaatcaggg 900aaaggagaag tcgtaacctg ttctgagaag cggaattctg
aacttttctt cagtgttctt 960ggcgggcttg gacagtttgg cataatcacc
cgggcacgga tctctcttga accagcaccg 1020catatggtaa agttctatct
tgaacaaagt tcaaacaata tacgctatga ttctaagaac 1080cactttcctg
acacagtcaa ataactttta ataggttaaa tggatcaggg tactctactc
1140tgacttttct gcattttcaa gggaccaaga atatctgatt tcgaaggaga
aaacttttga 1200ttacgttgaa ggatttgtga taatcaatag aacagacctt
ctcaataatt ggcgatcgtc 1260attcagtccc aacgattcca cacaggcaag
cagattcaag tcagatggga aaactcttta 1320ttgcctagaa gtggtcaaat
atttcaaccc agaagaagct agctctatgg atcaggtaag 1380atgtgaaagc
aatatataac tagacttagt ttccacagag agctccaaat caaccgttgg
1440ctactagcct actaacataa tgaatggttg ccgtgcagga aactggcaag
ttactttcag 1500agttaaatta tattccatcc actttgtttt catctgaagt
gccatatatc gagtttctgg 1560atcgcgtgca tatcgcagag agaaaactaa
gagcaaaggg tttatgggag gttccacatc 1620cctggctgaa tctcctgatt
cctaagagca gcatatacca atttgctaca gaagttttca 1680acaacattct
cacaagcaac aacaacggtc ctatccttat ttatccagtc aatcaatcca
1740agtaagtgag caaaatgcca aaagcaaatg cgtccagtga ttctgaaaca
taaattacta 1800accatatcca acattttgtg gtttcaggtg gaagaaacat
acatctttga taactccaaa 1860tgaagatata ttctatctcg tagcctttct
cccctctgca gtgccaaatt cctcagggaa 1920aaacgatcta gagtaccttt
tgaaacaaaa ccaaagagtt atgaacttct gcgcagcagc 1980aaacctcaac
gtgaagcagt atttgcccca ttatgaaact caaaaagagt ggaaatcaca
2040ctttggcaaa agatgggaaa catttgcaca gaggaaacaa gcctacgacc
ctctagcgat 2100tctagcacct ggccaaagaa tattccaaaa gacaacagga
aaattatctc ccatccaact 2160cgcaaagtca aaggcaacag gaagtcctca
aaggtaccat tacgcatcaa tactgccgaa 2220acctagaact gtataa
22362575PRTArabidopsis thaliana 2Met Gly Leu Thr Ser Ser Leu Arg
Phe His Arg Gln Asn Asn Lys Thr1 5 10 15Phe Leu Gly Ile Phe Met Ile
Leu Val Leu Ser Cys Ile Pro Gly Arg 20 25 30Thr Asn Leu Cys Ser Asn
His Ser Val Ser Thr Pro Lys Glu Leu Pro 35 40 45Ser Ser Asn Pro Ser
Asp Ile Arg Ser Ser Leu Val Ser Leu Asp Leu 50 55 60Glu Gly Tyr Ile
Ser Phe Asp Asp Val His Asn Val Ala Lys Asp Phe65 70 75 80Gly Asn
Arg Tyr Gln Leu Pro Pro Leu Ala Ile Leu His Pro Arg Ser 85 90 95Val
Phe Asp Ile Ser Ser Met Met Lys His Ile Val His Leu Gly Ser 100 105
110Thr Ser Asn Leu Thr Val Ala Ala Arg Gly His Gly His Ser Leu Gln
115 120 125Gly Gln Ala Leu Ala His Gln Gly Val Val Ile Lys Met Glu
Ser Leu 130 135 140Arg Ser Pro Asp Ile Arg Ile Tyr Lys Gly Lys Gln
Pro Tyr Val Asp145 150 155 160Val Ser Gly Gly Glu Ile Trp Ile Asn
Ile Leu Arg Glu Thr Leu Lys 165 170 175Tyr Gly Leu Ser Pro Lys Ser
Trp Thr Asp Tyr Leu His Leu Thr Val 180 185 190Gly Gly Thr Leu Ser
Asn Ala Gly Ile Ser Gly Gln Ala Phe Lys His 195 200 205Gly Pro Gln
Ile Asn Asn Val Tyr Gln Leu Glu Ile Val Thr Gly Lys 210 215 220Gly
Glu Val Val Thr Cys Ser Glu Lys Arg Asn Ser Glu Leu Phe Phe225 230
235 240Ser Val Leu Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala
Arg 245 250 255Ile Ser Leu Glu Pro Ala Pro His Met Val Lys Trp Ile
Arg Val Leu 260 265 270Tyr Ser Asp Phe Ser Ala Phe Ser Arg Asp Gln
Glu Tyr Leu Ile Ser 275 280 285Lys Glu Lys Thr Phe Asp Tyr Val Glu
Gly Phe Val Ile Ile Asn Arg 290 295 300Thr Asp Leu Leu Asn Asn Trp
Arg Ser Ser Phe Ser Pro Asn Asp Ser305 310 315 320Thr Gln Ala Ser
Arg Phe Lys Ser Asp Gly Lys Thr Leu Tyr Cys Leu 325 330 335Glu Val
Val Lys Tyr Phe Asn Pro Glu Glu Ala Ser Ser Met Asp Gln 340 345
350Glu Thr Gly Lys Leu Leu Ser Glu Leu Asn Tyr Ile Pro Ser Thr Leu
355 360 365Phe Ser Ser Glu Val Pro Tyr Ile Glu Phe Leu Asp Arg Val
His Ile 370 375 380Ala Glu Arg Lys Leu Arg Ala Lys Gly Leu Trp Glu
Val Pro His Pro385 390 395 400Trp Leu Asn Leu Leu Ile Pro Lys Ser
Ser Ile Tyr Gln Phe Ala Thr 405 410 415Glu Val Phe Asn Asn Ile Leu
Thr Ser Asn Asn Asn Gly Pro Ile Leu 420 425 430Ile Tyr Pro Val Asn
Gln Ser Lys Trp Lys Lys His Thr Ser Leu Ile 435 440 445Thr Pro Asn
Glu Asp Ile Phe Tyr Leu Val Ala Phe Leu Pro Ser Ala 450 455 460Val
Pro Asn Ser Ser Gly Lys Asn Asp Leu Glu Tyr Leu Leu Lys Gln465 470
475 480Asn Gln Arg Val Met Asn Phe Cys Ala Ala Ala Asn Leu Asn Val
Lys 485 490 495Gln Tyr Leu Pro His Tyr Glu Thr Gln Lys Glu Trp Lys
Ser His Phe 500 505 510Gly Lys Arg Trp Glu Thr Phe Ala Gln Arg Lys
Gln Ala Tyr Asp Pro 515 520 525Leu Ala Ile Leu Ala Pro Gly Gln Arg
Ile Phe Gln Lys Thr Thr Gly 530 535 540Lys Leu Ser Pro Ile Gln Leu
Ala Lys Ser Lys Ala Thr Gly Ser Pro545 550 555 560Gln Arg Tyr His
Tyr Ala Ser Ile Leu Pro Lys Pro Arg Thr Val 565 570
57532991DNAArabidopsis thaliana 3atggctaatc ttcgtttaat gatcacttta
atcacggttt taatgatcac caaatcatca 60aacggtatta aaattgattt acctaaatcc
cttaacctca ccctctctac cgatccttcc 120atcatctccg cagcctctca
tgacttcgga aacataacca ccgtgacccc cggcggcgta 180atctgcccct
cctccaccgc tgatatctct cgtctcctcc aatacgccgc aaacggaaaa
240agtacattcc aagtagcggc tcgtggccaa ggccactcct taaacggcca
agcctcggtc 300tccggcggag taatcgtcaa catgacgtgt atcactgacg
tggtggtttc aaaagacaag 360aagtacgctg acgtggcggc cgggacgtta
tgggtggatg tgcttaagaa gacggcggag 420aaaggggtgt cgccggtttc
ttggacggat tatttgcata taaccgtcgg aggaacgttg 480tcgaatggtg
gaattggtgg tcaagtgttt cgaaacggtc ctcttgttag taacgtcctt
540gaattggacg ttattactgg tacgcatctt ctaaactttg atgtacatac
aacaacaaaa 600actgtttttg ttttatagta tttttcattt tttgtaccat
aggttttatg ttttatagtt 660gtgctaaact tcttgcacca cacgtaagtc
ttcgaaacac aaaatgcgta acgcatctat 720atgttttttg tacatattga
atgttgttca tgagaaataa agtaattaca tatacacaca 780tttattgtcg
tacatatata aataattaaa gacaaatttt cacaattggt agcgtgttaa
840tttgggattt ttgtaatgta catgcatgac gcatgcatat ggagcttttc
ggttttctta 900gatttgtgta gtatttcaaa tatatcattt attttctttc
gaataaagag gtggtatatt 960tttaaaatag caacatttca gaatttttct
ttgaatttac actttttaaa ttgttattgt 1020taatatggat tttgaataaa
taatttcagg gaaaggtgaa atgttgacat gctcgcgaca 1080gctaaaccca
gaattgttct atggagtgtt aggaggtttg ggtcaatttg gaattataac
1140gagagccaga attgttttgg accatgcacc taaacgggta cgtatcatca
tattttacca 1200tttgttttag tcagcattca tttttcatta gtaattccgt
ttcaatttct aaattttttt 1260agtcaataga aaatgattct tatgtcagag
cttgattatt tagtgatttt tattgagata 1320aaataaaata taacctaacg
gaaataatta ttttactaat cggataatgt ctgattaaaa 1380cattttatga
tattacacta agagagttag agacgtatgg atcacaaaac atgaagcttt
1440cttagatggt atcctaaaac taaagttagg tacaagtttg gaatttaggt
caaatgctta 1500agttgcatta atttgaacaa aatctatgca ttgaataaaa
aaaagatatg gattatttta 1560taaagtatag tccttgtaat cctaggactt
gttgtctaat cttgtcttat gcgtgcaaat 1620ctttttgatg tcaatatata
atccttgttt attagagtca agctctttca ttagtcaact 1680actcaaatat
actccaaagt ttagaatata gtcttctgac taattagaat cttacaaccg
1740ataaacgtta caatttggtt atcattttaa aaaacagatt tggtcataat
atacgatgac 1800gttctgtttt agtttcatct attcacaaat tttatataat
tattttcaag aaaatattga 1860aatactatac tgtaatatgg tttctttata
tatgtgtgta taaattaaat gggattgttt 1920tctctaaatg aaattgtgta
ggccaaatgg tttcggatgc tctacagtga tttcacaact 1980tttacaaagg
accaagaacg tttgatatca atggcaaacg atattggagt cgactattta
2040gaaggtcaaa tatttctatc aaacggtgtc gttgacacct cttttttccc
accttcagat 2100caatctaaag tcgctgatct agtcaagcaa cacggtatca
tctatgttct tgaagtagcc 2160aagtattatg atgatcccaa tctccccatc
atcagcaagg tactacacat ttacattttc 2220atcatcgttt ttatcatacc
ataagatatt taaatgattc atcattgcac cacattaaga 2280tattcatcat
catcatcgtt acattttttt ttgcatctta tgcttctcat aatctactat
2340tgtgtaggtt attgacacat taacgaaaac attaagttac ttgcccgggt
tcatatcaat 2400gcacgacgtg gcctacttcg atttcttgaa ccgtgtacat
gtcgaagaaa ataaactcag 2460atctttggga ttatgggaac ttcctcatcc
ttggcttaac ctctacgttc ctaaatctcg 2520gattctcgat tttcataacg
gtgttgtcaa agacattctt cttaagcaaa aatcagcttc 2580gggactcgct
cttctctatc caacaaaccg gaataagtac atacttctct tcattcatat
2640ttatcttcaa gaaccaaagt aaataaattt ctatgaactg attatgctgt
tattgttaga 2700tgggacaatc gtatgtcggc gatgatacca gagatcgatg
aagatgttat atatattatc 2760ggactactac aatccgctac cccaaaggat
cttccagaag tggagagcgt taacgagaag 2820ataattaggt tttgcaagga
ttcaggtatt aagattaagc aatatctaat gcattatact 2880agtaaagaag
attggattga gcattttgga tcaaaatggg atgatttttc gaagaggaaa
2940gatctatttg atcccaagaa actgttatct ccagggcaag acatcttttg a
29914501PRTArabidopsis thaliana 4Met Ala Asn Leu Arg Leu Met Ile
Thr Leu Ile Thr Val Leu Met Ile1 5 10 15Thr Lys Ser Ser Asn Gly Ile
Lys Ile Asp Leu Pro Lys Ser Leu Asn 20 25 30Leu Thr Leu Ser Thr Asp
Pro Ser Ile Ile Ser Ala Ala Ser His Asp 35 40 45Phe Gly Asn Ile Thr
Thr Val Thr Pro Gly Gly Val Ile Cys Pro Ser 50 55 60Ser Thr Ala Asp
Ile Ser Arg Leu Leu Gln Tyr Ala Ala Asn Gly Lys65 70 75 80Ser Thr
Phe Gln Val Ala Ala Arg Gly Gln Gly His Ser Leu Asn Gly 85 90 95Gln
Ala Ser Val Ser Gly Gly Val Ile Val Asn Met Thr Cys Ile Thr 100 105
110Asp Val Val Val Ser Lys Asp Lys Lys Tyr Ala Asp Val Ala Ala Gly
115 120 125Thr Leu Trp Val Asp Val Leu Lys Lys Thr Ala Glu Lys Gly
Val Ser 130 135 140Pro Val Ser Trp Thr Asp Tyr Leu His Ile Thr Val
Gly Gly Thr Leu145 150 155 160Ser Asn Gly Gly Ile Gly Gly Gln Val
Phe Arg Asn Gly Pro Leu Val 165 170 175Ser Asn Val Leu Glu Leu Asp
Val Ile Thr Gly Lys Gly Glu Met Leu 180 185 190Thr Cys Ser Arg Gln
Leu Asn Pro Glu Leu Phe Tyr Gly Val Leu Gly 195 200 205Gly Leu Gly
Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile Val Leu Asp 210 215 220His
Ala Pro Lys Arg Ala Lys Trp Phe Arg Met Leu Tyr Ser Asp Phe225 230
235 240Thr Thr Phe Thr Lys Asp Gln Glu Arg Leu Ile Ser Met Ala Asn
Asp 245 250 255Ile Gly Val Asp Tyr Leu Glu Gly Gln Ile Phe Leu Ser
Asn Gly Val 260 265 270Val Asp Thr Ser Phe Phe Pro Pro Ser Asp Gln
Ser Lys Val Ala Asp 275 280 285Leu Val Lys Gln His Gly Ile Ile Tyr
Val Leu Glu Val Ala Lys Tyr 290 295 300Tyr Asp Asp Pro Asn Leu Pro
Ile Ile Ser Lys Val Ile Asp Thr Leu305 310 315 320Thr Lys Thr Leu
Ser Tyr Leu Pro Gly Phe Ile Ser Met His Asp Val 325 330 335Ala Tyr
Phe Asp Phe Leu Asn Arg Val His Val Glu Glu Asn Lys Leu 340 345
350Arg Ser Leu Gly Leu Trp Glu Leu Pro His Pro Trp Leu Asn Leu Tyr
355 360 365Val Pro Lys Ser Arg Ile Leu Asp Phe His Asn Gly Val Val
Lys Asp 370 375 380Ile Leu Leu Lys Gln Lys Ser Ala Ser Gly Leu Ala
Leu Leu Tyr Pro385 390 395 400Thr Asn Arg Asn Lys Trp Asp Asn Arg
Met Ser Ala Met Ile Pro Glu 405 410 415Ile Asp Glu Asp Val Ile Tyr
Ile Ile Gly Leu Leu Gln Ser Ala Thr 420 425 430Pro Lys Asp Leu Pro
Glu Val Glu Ser Val Asn Glu Lys Ile Ile Arg 435 440 445Phe Cys Lys
Asp Ser Gly Ile Lys Ile Lys Gln Tyr Leu Met His Tyr 450 455 460Thr
Ser Lys Glu Asp Trp Ile Glu His Phe Gly Ser Lys Trp Asp Asp465 470
475 480Phe Ser Lys Arg Lys Asp Leu Phe Asp Pro Lys Lys Leu Leu Ser
Pro 485 490 495Gly Gln Asp Ile Phe 50053302DNAArabidopsis thaliana
5atggcgagtt ataatcttcg ttcacaagtt cgtcttatag caataacaat agtaatcatc
60attactctct caactccgat cacaaccaac acatcaccac aaccatggaa tatcctttca
120cacaacgaat tcgccggaaa actcacctcc tcctcctcct ccgtcgaatc
agccgccaca 180gatttcggcc acgtcaccaa aatcttccct tccgccgtct
taatcccttc ctccgttgaa 240gacatcacag atctcataaa actctctttt
gactctcaac tgtcttttcc tttagccgct 300cgtggtcacg gacacagcca
ccgtggccaa gcctcggcta aagacggagt tgtggtcaac 360atgcggtcca
tggtaaaccg ggatcgaggt atcaaggtgt ctaggacctg tttatatgtt
420gacgtggacg ctgcgtggct atggattgag gtgttgaata aaactttgga
gttagggtta 480acgccggttt cttggacgga ttatttgtat ttaacagtcg
gtgggacgtt atcaaacggc 540ggaattagtg gacaaacgtt tcggtacggt
ccacagatca ctaatgttct agagatggat 600gttattactg gtacgtacca
cgatcttttt cacacagaga ttaaaaaaaa cagtaatagt 660gattttaact
tcgtacgttt ctgatagaca acaaagaact tcgtacgttt ttcgaagttt
720tttcgtcttt ttcattttag atctgcgcgg ccatttttgg ttatgctatt
gtttgtttgt 780attgtttgtc tctgtttatt tatttctcga acttgttgat
agcttttctt cttttcacac 840atcaatctaa tcaccttttt tggtcttaag
attagaaaga agatacggac taggtaaaaa 900taggtggttg taaacgtaga
cgcattaaaa aaatattggt ttttttattt tttgataagc 960aaaattggtg
gttggtctaa gattataaac ttgatattaa tgcaaaggtc gatctagcaa
1020tagaagatta atcaatattc ttggtgtttt aacaacagat tatttcatca
ttaaaatcgt 1080gaaacaaaga aattttggta gtatacatta cgtgtagttt
tgttagttta ttaaaaaaaa 1140tagtatatag ttttgttaaa acgcgattta
tttagtaaca cattagtata ttacacgttt 1200aaccaactaa actttttttt
ttgaataatt atgttctata tttcttactc aaattatgca 1260aatttcgtgg
attcgaagtc aaatttctgc gaaatttaca tggtcatata ttataaaact
1320gttcatataa cccggtgaac aaacagacaa ttaagggttt gaatggttac
ggcggttggg 1380gcggacacaa ccgtcaatag atcagaccgt tttttattta
ccattcatca attatattcc 1440gcagtggttt ggggtaaaaa aaatagaaga
aaaccgcagc ggaccaattc cataccgttt 1500ttacatacaa ataaacatgg
tgcgcaacgg tttattgtcc gcctcaaaaa tgaaatggac 1560taaaccgcag
ataaattaga ccgctttgtc cgctgcctcc attcatagac taaaaaaaaa
1620caaccaaaaa aaaaatggtc ccacgcccat gattttacac gaggtttctt
gtggcgtaag 1680gacaaaactc aaaagttcat aacgtttggt cctaaccagg
tgtaatggat taagtaacag 1740tcaattttct tattatagct gtatccatta
tgtccacata tgcatccata tacattacac 1800tgttggtctc aagtgtagtt
agattacgaa gactttcaag ttccattttt tggttaggag 1860ataaacataa
tttaatgata ccgactttag cactctaggc tcaaaacaag tacagaagag
1920aatagtttta tttcaaactc gttgcattgt tgtatcaatt aattgtgtta
gtctttgtat 1980attcttacat aacggtccaa gtttgttgaa atagtttact
tactaaactt ttcctaatgg 2040ggtcaaattt tattttatag gaaaaggaga
gattgcaact tgttccaagg acatgaactc 2100ggatcttttc ttcgcggtgt
taggaggttt gggtcaattc ggcattataa caagagccag 2160aattaaactt
gaagtagctc cgaaaagggt atgttaaatt tgtaaattat gcaactacag
2220aaaattctat gaaatttatg aatgaacata tatgcatttt tggatttttg
taggccaagt 2280ggttaaggtt tctatacata gatttctccg aattcacaag
agatcaagaa cgagtgatat 2340cgaaaacgga cggtgtagat ttcttagaag
gttccattat ggtggaccat ggcccaccgg 2400ataactggag atccacgtat
tatccaccgt ccgatcactt gaggatcgcc tcaatggtca 2460aacgacatcg
tgtcatctac tgccttgaag tcgtcaagta ttacgacgaa acttctcaat
2520acacagtcaa cgaggtccgt acatacatac aatcataaat catacatgta
taattgggag 2580atctttatgc attattcaat tatattaatt tactttagtt
atttaactta tgcaggaaat 2640ggaggagtta agcgatagtt taaaccatgt
aagagggttt atgtacgaga aagatgtgac 2700gtatatggat ttcctaaacc
gagttcgaac cggagagcta aacctgaaat ccaaaggcca 2760atgggatgtt
ccacatccat ggcttaatct cttcgtacca aaaactcaaa tctccaaatt
2820tgatgatggt gtttttaagg gtattatcct aagaaataac atcactagcg
gtcctgttct 2880tgtttatcct atgaatcgca acaagtaagt ttaactcgat
attgcaaaat ttactatcta 2940cattttcgtt ttggaatccg aaatattctt
acaagctaat tttatgcggc gtttttaggt 3000ggaatgatcg gatgtctgcc
gctatacccg aggaagatgt attttatgcg gtagggtttt 3060taagatccgc
gggttttgac aattgggagg
cttttgatca agaaaacatg gaaatactga 3120agttttgtga ggatgctaat
atgggggtta tacaatatct tccttatcat tcatcacaag 3180aaggatgggt
tagacatttt ggtccgaggt ggaatatttt cgtagagaga aaatataaat
3240atgatcccaa aatgatatta tcaccgggac aaaatatatt tcaaaaaata
aactcgagtt 3300ag 33026523PRTArabidopsis thaliana 6Met Ala Ser Tyr
Asn Leu Arg Ser Gln Val Arg Leu Ile Ala Ile Thr1 5 10 15Ile Val Ile
Ile Ile Thr Leu Ser Thr Pro Ile Thr Thr Asn Thr Ser 20 25 30Pro Gln
Pro Trp Asn Ile Leu Ser His Asn Glu Phe Ala Gly Lys Leu 35 40 45Thr
Ser Ser Ser Ser Ser Val Glu Ser Ala Ala Thr Asp Phe Gly His 50 55
60Val Thr Lys Ile Phe Pro Ser Ala Val Leu Ile Pro Ser Ser Val Glu65
70 75 80Asp Ile Thr Asp Leu Ile Lys Leu Ser Phe Asp Ser Gln Leu Ser
Phe 85 90 95Pro Leu Ala Ala Arg Gly His Gly His Ser His Arg Gly Gln
Ala Ser 100 105 110Ala Lys Asp Gly Val Val Val Asn Met Arg Ser Met
Val Asn Arg Asp 115 120 125Arg Gly Ile Lys Val Ser Arg Thr Cys Leu
Tyr Val Asp Val Asp Ala 130 135 140Ala Trp Leu Trp Ile Glu Val Leu
Asn Lys Thr Leu Glu Leu Gly Leu145 150 155 160Thr Pro Val Ser Trp
Thr Asp Tyr Leu Tyr Leu Thr Val Gly Gly Thr 165 170 175Leu Ser Asn
Gly Gly Ile Ser Gly Gln Thr Phe Arg Tyr Gly Pro Gln 180 185 190Ile
Thr Asn Val Leu Glu Met Asp Val Ile Thr Gly Lys Gly Glu Ile 195 200
205Ala Thr Cys Ser Lys Asp Met Asn Ser Asp Leu Phe Phe Ala Val Leu
210 215 220Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile
Lys Leu225 230 235 240Glu Val Ala Pro Lys Arg Ala Lys Trp Leu Arg
Phe Leu Tyr Ile Asp 245 250 255Phe Ser Glu Phe Thr Arg Asp Gln Glu
Arg Val Ile Ser Lys Thr Asp 260 265 270Gly Val Asp Phe Leu Glu Gly
Ser Ile Met Val Asp His Gly Pro Pro 275 280 285Asp Asn Trp Arg Ser
Thr Tyr Tyr Pro Pro Ser Asp His Leu Arg Ile 290 295 300Ala Ser Met
Val Lys Arg His Arg Val Ile Tyr Cys Leu Glu Val Val305 310 315
320Lys Tyr Tyr Asp Glu Thr Ser Gln Tyr Thr Val Asn Glu Glu Met Glu
325 330 335Glu Leu Ser Asp Ser Leu Asn His Val Arg Gly Phe Met Tyr
Glu Lys 340 345 350Asp Val Thr Tyr Met Asp Phe Leu Asn Arg Val Arg
Thr Gly Glu Leu 355 360 365Asn Leu Lys Ser Lys Gly Gln Trp Asp Val
Pro His Pro Trp Leu Asn 370 375 380Leu Phe Val Pro Lys Thr Gln Ile
Ser Lys Phe Asp Asp Gly Val Phe385 390 395 400Lys Gly Ile Ile Leu
Arg Asn Asn Ile Thr Ser Gly Pro Val Leu Val 405 410 415Tyr Pro Met
Asn Arg Asn Lys Trp Asn Asp Arg Met Ser Ala Ala Ile 420 425 430Pro
Glu Glu Asp Val Phe Tyr Ala Val Gly Phe Leu Arg Ser Ala Gly 435 440
445Phe Asp Asn Trp Glu Ala Phe Asp Gln Glu Asn Met Glu Ile Leu Lys
450 455 460Phe Cys Glu Asp Ala Asn Met Gly Val Ile Gln Tyr Leu Pro
Tyr His465 470 475 480Ser Ser Gln Glu Gly Trp Val Arg His Phe Gly
Pro Arg Trp Asn Ile 485 490 495Phe Val Glu Arg Lys Tyr Lys Tyr Asp
Pro Lys Met Ile Leu Ser Pro 500 505 510Gly Gln Asn Ile Phe Gln Lys
Ile Asn Ser Ser 515 52072782DNAArabidopsis thaliana 7atgactaata
ctctctgttt aagcctcatc accctaataa cgctttttat aagtttaacc 60ccaaccttaa
tcaaatcaga tgagggcatt gatgttttct tacccatatc actcaacctt
120acggtcctaa ccgatccctt ctccatctct gccgcttctc acgacttcgg
taacataacc 180gacgaaaatc ccggcgccgt cctctgccct tcctccacca
cggaggtggc tcgtctcctc 240cgtttcgcta acggaggatt ctcttacaat
aaaggctcaa ccagccccgc gtctactttc 300aaagtggctg ctcgaggcca
aggccactcc ctccgtggcc aagcctctgc acccggaggt 360gtcgtcgtga
acatgacgtg tctcgccatg gcggctaaac cagcggcggt tgttatctcg
420gcagacggga cttacgctga cgtggctgcc gggacgatgt gggtggatgt
tctgaaggcg 480gcggtggata gaggcgtctc gccggttaca tggacggatt
atttgtatct cagcgtcggc 540gggacgttgt cgaacgctgg aatcggtggt
cagacgttta gacacggccc tcagattagt 600aacgttcatg agcttgacgt
tattaccggt acgtaaatac caaaacttca ctaatctcgt 660tacaattttt
taattttttg gtaatataaa ttttgtacgg ctcaactctt aattaagaat
720gaaacagtat ctatgatctt ctagatgctc tttttttgtc tgcaagcttt
aattgtagta 780acatcagcga tatatatatc acatgcatgt gtattattga
tgataatata taatgtttta 840gttacaaatt tgattctcaa ggtaaaactc
acacgccata accagtataa aactccaaaa 900atcacgtttt ggtcagaaat
acatatcctt cattaacagt agttatgcta taatttgtga 960ttataaataa
ctccggagtt tgttcacaat actaaatttc aggaaaaggt gaaatgatga
1020cttgctctcc aaagttaaac cctgaattgt tctatggagt tttaggaggt
ttgggtcaat 1080tcggtattat aacgagggcc aggattgcgt tggatcatgc
acccacaagg gtatgtatca 1140tgcatctata gtgtaatcaa tttataattt
taatgtagtg gtcctaaatc caaaatttga 1200tttgatttgg ttggaacgta
cgtatatata ataagtcaaa aggctgattt tgaagacgaa 1260tttatatact
tttgttgaat taaatctgat tttgcttacg ttttattaga ttctgcgtaa
1320taaatcctag gacttgctcg agtgtaatct tgtcttatgc ttgcaaatct
tgttgatgtc 1380aatatctaat cttttttatt atatttccct acgtaagttt
tagatatagt tattttaaac 1440tgctataaat tgtgtacgta tagactttag
ataaaaagtt gtggtcgctt gcacctattt 1500gtttatcgct atagtgattc
aaaggtctat atatgattct tggtttttct ttttgaaaaa 1560aatagaccat
acaatccaag gaagatgatc ttaaatggac taatttatgg atataaattg
1620atatacaaat ctgcaggtga aatggtctcg catactctac agtgacttct
cggcttttaa 1680aagagaccaa gagcgtttaa tatcaatgac caatgatctc
ggagttgact ttttggaagg 1740tcaacttatg atgtcaaatg gcttcgtaga
cacctctttc ttcccactct ccgatcaaac 1800aagagtcgca tctcttgtga
atgaccaccg gatcatctat gttctcgaag tagccaagta 1860ttatgacaga
accacccttc ccattattga ccaggtacta aaatccatta ttcatgatga
1920ttatcttcac acaatcagta tcatcaccaa ttaccatcat cacttgtcat
atatgatcca 1980aagtaaatat atcacatgat ataaataaat cgttcaaatc
ttttttttta aagaataaaa 2040gaatcatttt caagcattac tcatacacat
ctacgaatca ccgtgaccat atataaccat 2100acgcttatta aataatcatt
tttgtttgta ggtgattgac acgttaagta gaactctagg 2160tttcgctcca
gggtttatgt tcgtacaaga tgttccgtat ttcgatttct tgaaccgtgt
2220ccgaaacgaa gaagataaac tcagatcttt aggactatgg gaagttcctc
atccatggct 2280taacatcttt gtcccggggt ctcgaatcca agattttcat
gatggtgtta ttaatggcct 2340tcttctaaac caaacctcaa cttctggtgt
tactctcttc tatcccacaa accgaaacaa 2400gtaaatattt actttttgat
tttgttttat ttgaaagtat atcccaataa tgtatgttaa 2460attgttaaca
agaatttatt ttattaatag atggaacaac cgcatgtcaa cgatgacacc
2520ggacgaagat gttttttatg tgatcggatt actgcaatca gctggtggat
ctcaaaattg 2580gcaagaactt gaaaatctca acgacaaggt tattcagttt
tgtgaaaact cgggaattaa 2640gattaaggaa tatttgatgc actatacaag
aaaagaagat tgggttaaac attttggacc 2700aaaatgggat gattttttaa
gaaagaaaat tatgtttgat cccaaaagac tattgtctcc 2760aggacaagac
atatttaatt aa 27828524PRTArabidopsis thaliana 8Met Thr Asn Thr Leu
Cys Leu Ser Leu Ile Thr Leu Ile Thr Leu Phe1 5 10 15Ile Ser Leu Thr
Pro Thr Leu Ile Lys Ser Asp Glu Gly Ile Asp Val 20 25 30Phe Leu Pro
Ile Ser Leu Asn Leu Thr Val Leu Thr Asp Pro Phe Ser 35 40 45Ile Ser
Ala Ala Ser His Asp Phe Gly Asn Ile Thr Asp Glu Asn Pro 50 55 60Gly
Ala Val Leu Cys Pro Ser Ser Thr Thr Glu Val Ala Arg Leu Leu65 70 75
80Arg Phe Ala Asn Gly Gly Phe Ser Tyr Asn Lys Gly Ser Thr Ser Pro
85 90 95Ala Ser Thr Phe Lys Val Ala Ala Arg Gly Gln Gly His Ser Leu
Arg 100 105 110Gly Gln Ala Ser Ala Pro Gly Gly Val Val Val Asn Met
Thr Cys Leu 115 120 125Ala Met Ala Ala Lys Pro Ala Ala Val Val Ile
Ser Ala Asp Gly Thr 130 135 140Tyr Ala Asp Val Ala Ala Gly Thr Met
Trp Val Asp Val Leu Lys Ala145 150 155 160Ala Val Asp Arg Gly Val
Ser Pro Val Thr Trp Thr Asp Tyr Leu Tyr 165 170 175Leu Ser Val Gly
Gly Thr Leu Ser Asn Ala Gly Ile Gly Gly Gln Thr 180 185 190Phe Arg
His Gly Pro Gln Ile Ser Asn Val His Glu Leu Asp Val Ile 195 200
205Thr Gly Lys Gly Glu Met Met Thr Cys Ser Pro Lys Leu Asn Pro Glu
210 215 220Leu Phe Tyr Gly Val Leu Gly Gly Leu Gly Gln Phe Gly Ile
Ile Thr225 230 235 240Arg Ala Arg Ile Ala Leu Asp His Ala Pro Thr
Arg Val Lys Trp Ser 245 250 255Arg Ile Leu Tyr Ser Asp Phe Ser Ala
Phe Lys Arg Asp Gln Glu Arg 260 265 270Leu Ile Ser Met Thr Asn Asp
Leu Gly Val Asp Phe Leu Glu Gly Gln 275 280 285Leu Met Met Ser Asn
Gly Phe Val Asp Thr Ser Phe Phe Pro Leu Ser 290 295 300Asp Gln Thr
Arg Val Ala Ser Leu Val Asn Asp His Arg Ile Ile Tyr305 310 315
320Val Leu Glu Val Ala Lys Tyr Tyr Asp Arg Thr Thr Leu Pro Ile Ile
325 330 335Asp Gln Val Ile Asp Thr Leu Ser Arg Thr Leu Gly Phe Ala
Pro Gly 340 345 350Phe Met Phe Val Gln Asp Val Pro Tyr Phe Asp Phe
Leu Asn Arg Val 355 360 365Arg Asn Glu Glu Asp Lys Leu Arg Ser Leu
Gly Leu Trp Glu Val Pro 370 375 380His Pro Trp Leu Asn Ile Phe Val
Pro Gly Ser Arg Ile Gln Asp Phe385 390 395 400His Asp Gly Val Ile
Asn Gly Leu Leu Leu Asn Gln Thr Ser Thr Ser 405 410 415Gly Val Thr
Leu Phe Tyr Pro Thr Asn Arg Asn Lys Trp Asn Asn Arg 420 425 430Met
Ser Thr Met Thr Pro Asp Glu Asp Val Phe Tyr Val Ile Gly Leu 435 440
445Leu Gln Ser Ala Gly Gly Ser Gln Asn Trp Gln Glu Leu Glu Asn Leu
450 455 460Asn Asp Lys Val Ile Gln Phe Cys Glu Asn Ser Gly Ile Lys
Ile Lys465 470 475 480Glu Tyr Leu Met His Tyr Thr Arg Lys Glu Asp
Trp Val Lys His Phe 485 490 495Gly Pro Lys Trp Asp Asp Phe Leu Arg
Lys Lys Ile Met Phe Asp Pro 500 505 510Lys Arg Leu Leu Ser Pro Gly
Gln Asp Ile Phe Asn 515 52092805DNAArabidopsis thaliana 9atgacgtcaa
gctttcttct cctgacgttc gccatatgta aactgatcat agccgtgggt 60ctaaacgtgg
gccccagtga gctcctccgc atcggagcca tagatgtcga cggccacttc
120accgtccacc cttccgactt agcctccgtc tcctcagact tcggtatgct
gaagtcacct 180gaagagccat tggccgtgct tcatccatca tcggccgaag
acgtggcacg actcgtcaga 240acagcttacg gttcagccac ggcgtttccg
gtctcagccc gaggccacgg ccattccata 300aacggacaag ccgcggcggg
gaggaacggt gtggtggttg aaatgaacca cggcgtaacc 360gggacgccca
agccactcgt ccgaccggat gaaatgtatg tggatgtatg gggtggagag
420ttatgggtcg atgtgttgaa gaaaacgttg gagcatggct tagcaccaaa
atcatggacg 480gattacttgt atctaaccgt tggaggtaca ctctccaatg
caggaatcag tggtcaagct 540tttcaccatg gtcctcaaat tagtaacgtc
cttgagctcg acgttgtaac tggttagtat 600taaaacattc aagttcatat
attttaaatg cttttgtctg aagttttact aataacaaga 660aattgatacc
aaaaagtagg gaaaggagag gtgatgagat gctcagaaga agagaacaca
720aggctattcc atggagttct tggtggatta ggtcaatttg ggatcatcac
tcgagcacga 780atctctctcg aaccagctcc ccaaagggta atattttttt
aatgactagc tatcaaaaat 840ccctggcggg tccatacgtt gtaatctttt
tagtttttac tgttgatggt attttttata 900tattttggat aataaaaccc
taaaatggta tattgtgatg acaggtgaga tggatacggg 960tattgtattc
gagcttcaaa gtgtttacgg aggaccaaga gtacttaatc tcaatgcatg
1020gtcaattaaa gtttgattac gtggaaggtt ttgtgattgt ggacgaagga
ctcgtcaaca 1080attggagatc ttctttcttc tctccacgta accccgtcaa
gatctcctct gttagttcca 1140acggctctgt tttgtattgc cttgagatca
ccaagaacta ccacgactcc gactccgaaa 1200tcgttgatca ggtcactttc
attattcact tagaaaaaag cgatattttc attttttata 1260ttgatgaata
tctggaagga tttaacgcta tgcgactatt gggaaatcat tatgaaaaaa
1320tatttagttt atatgattga aagtggtctc catagtattt ttgttgtgtc
gactttatta 1380taacttaaat ttggaagagg acatgaagaa gaagccagag
aggatctaca gagatctagc 1440ttttccacct gaacttaata atgcacattt
atataattat ttttcttctt ctaaagttta 1500gtttatcact agcgaattaa
tcatggttac taattaagta gtggacaggg tcatggacca 1560ctcactcacc
aaataatgat tcctctttac tcttaagttt aattttaata aaaccaactc
1620tactggaatc ttaacttatc cttggttttg gtaggctttt atagcaacac
ggttttttta 1680attttcctat tccagatttt gtatattaaa tgtcgatttt
ttttcttttt gtttcaggaa 1740gttgagattc tgatgaagaa attgaatttc
ataccgacat cggtctttac aacggattta 1800caatatgtgg actttctcga
ccgggtacac aaggccgaat tgaagctccg gtccaagaat 1860ttatgggagg
ttccacaccc atggctcaac ctcttcgtgc caaaatcaag aatctctgac
1920ttcgataaag gcgttttcaa gggcattttg ggaaataaaa caagtggccc
tattcttatc 1980taccccatga acaaagacaa gtaagtcttg acattaccat
tgattactac ttctaaattt 2040cttctctaga aaaaagaata aaacgagttt
tgcattgcat gcatgcaaag ttacacttgt 2100ggggattaat tagtggtcca
agaaaaaaag tttgtcaaaa ttgaaaaaaa ctagacacgt 2160ggtacatggg
attgtccgaa aaacgttgtc cacatgtgca tcgaaccagc taagattgac
2220aacaacactt cgtcggctcg tatttctctt tttgttttgt gaccaaatcc
gatggtccag 2280attgggttta tttgttttta agttcctaga actcatggtg
ggtgggtccc aatcagattc 2340tcctagacca aaccgatctc aacgaaccct
ccgcacatca ttgattatta cattaatata 2400gatattgtcg ttgctgacgt
gtcgtaattt gatgttattg tcagatggga cgagaggagc 2460tcagccgtga
cgccggatga ggaagttttc tatctggtgg ctctattgag atcagcttta
2520acggacggtg aagagacaca gaagctagag tatctgaaag atcagaaccg
tcggatcttg 2580gagttctgtg aacaagccaa gatcaatgtg aagcagtatc
ttcctcacca cgcaacacag 2640gaagagtggg tggctcattt tggggacaag
tgggatcggt tcagaagctt aaaggctgag 2700tttgatccgc gacacatact
cgctactggt cagagaatct ttcaaaaccc atctttgtct 2760ttgtttcctc
cgtcgtcgtc ttcttcgtca gcggcttcat ggtga 280510536PRTArabidopsis
thaliana 10Met Thr Ser Ser Phe Leu Leu Leu Thr Phe Ala Ile Cys Lys
Leu Ile1 5 10 15Ile Ala Val Gly Leu Asn Val Gly Pro Ser Glu Leu Leu
Arg Ile Gly 20 25 30Ala Ile Asp Val Asp Gly His Phe Thr Val His Pro
Ser Asp Leu Ala 35 40 45Ser Val Ser Ser Asp Phe Gly Met Leu Lys Ser
Pro Glu Glu Pro Leu 50 55 60Ala Val Leu His Pro Ser Ser Ala Glu Asp
Val Ala Arg Leu Val Arg65 70 75 80Thr Ala Tyr Gly Ser Ala Thr Ala
Phe Pro Val Ser Ala Arg Gly His 85 90 95Gly His Ser Ile Asn Gly Gln
Ala Ala Ala Gly Arg Asn Gly Val Val 100 105 110Val Glu Met Asn His
Gly Val Thr Gly Thr Pro Lys Pro Leu Val Arg 115 120 125Pro Asp Glu
Met Tyr Val Asp Val Trp Gly Gly Glu Leu Trp Val Asp 130 135 140Val
Leu Lys Lys Thr Leu Glu His Gly Leu Ala Pro Lys Ser Trp Thr145 150
155 160Asp Tyr Leu Tyr Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly
Ile 165 170 175Ser Gly Gln Ala Phe His His Gly Pro Gln Ile Ser Asn
Val Leu Glu 180 185 190Leu Asp Val Val Thr Gly Lys Gly Glu Val Met
Arg Cys Ser Glu Glu 195 200 205Glu Asn Thr Arg Leu Phe His Gly Val
Leu Gly Gly Leu Gly Gln Phe 210 215 220Gly Ile Ile Thr Arg Ala Arg
Ile Ser Leu Glu Pro Ala Pro Gln Arg225 230 235 240Val Arg Trp Ile
Arg Val Leu Tyr Ser Ser Phe Lys Val Phe Thr Glu 245 250 255Asp Gln
Glu Tyr Leu Ile Ser Met His Gly Gln Leu Lys Phe Asp Tyr 260 265
270Val Glu Gly Phe Val Ile Val Asp Glu Gly Leu Val Asn Asn Trp Arg
275 280 285Ser Ser Phe Phe Ser Pro Arg Asn Pro Val Lys Ile Ser Ser
Val Ser 290 295 300Ser Asn Gly Ser Val Leu Tyr Cys Leu Glu Ile Thr
Lys Asn Tyr His305 310 315 320Asp Ser Asp Ser Glu Ile Val Asp Gln
Glu Val Glu Ile Leu Met Lys 325 330 335Lys Leu Asn Phe Ile Pro Thr
Ser Val Phe Thr Thr Asp Leu Gln Tyr 340 345 350Val Asp Phe Leu Asp
Arg Val His Lys Ala Glu Leu Lys Leu Arg Ser 355 360 365Lys Asn Leu
Trp Glu Val Pro His Pro Trp Leu Asn Leu Phe Val Pro 370 375 380Lys
Ser Arg Ile Ser Asp Phe Asp Lys Gly Val Phe Lys Gly Ile Leu385 390
395 400Gly Asn Lys Thr Ser Gly Pro Ile Leu Ile Tyr Pro Met Asn Lys
Asp 405 410 415Lys Trp Asp Glu Arg Ser Ser Ala Val Thr Pro Asp Glu
Glu Val Phe 420 425 430Tyr Leu Val Ala Leu Leu Arg Ser Ala Leu Thr
Asp
Gly Glu Glu Thr 435 440 445Gln Lys Leu Glu Tyr Leu Lys Asp Gln Asn
Arg Arg Ile Leu Glu Phe 450 455 460Cys Glu Gln Ala Lys Ile Asn Val
Lys Gln Tyr Leu Pro His His Ala465 470 475 480Thr Gln Glu Glu Trp
Val Ala His Phe Gly Asp Lys Trp Asp Arg Phe 485 490 495Arg Ser Leu
Lys Ala Glu Phe Asp Pro Arg His Ile Leu Ala Thr Gly 500 505 510Gln
Arg Ile Phe Gln Asn Pro Ser Leu Ser Leu Phe Pro Pro Ser Ser 515 520
525Ser Ser Ser Ser Ala Ala Ser Trp 530 535111936DNAArabidopsis
thaliana 11atgcttatag taagaagttt caccatcttg cttctcagct gcatagcctt
taagttggct 60tgctgcttct ctagcagcat ttcttctttg aaggcgcttc ccctagtagg
ccatttggag 120tttgaacatg tccatcacgc ctccaaagat tttggaaatc
gataccagtt gatccctttg 180gcggtcttac atcccaaatc ggtaagcgac
atcgcctcaa cgatacgaca catctggatg 240atgggcactc attcacagct
tacagtggca gcgagaggtc gtggacattc actccaaggc 300caagctcaaa
caagacatgg aattgttata cacatggaat cactccatcc ccagaagctg
360caggtctaca gtgtggattc ccctgctcca tatgttgatg tgtctggtgg
tgagctgtgg 420ataaacattt tgcatgagac cctcaagtac gggcttgcac
caaaatcatg gacggattac 480ctgcatttaa ctgtaggtgg tactctgtcc
aatgctggaa taagcggcca ggcattccga 540catggaccac agatcagcaa
tgttcatcaa ctggagattg tcacaggtta gttcagagtt 600gcagtattcg
tgttttgaaa gcatagactc tatatggttg gtgactatta acaacatgaa
660gagattcccg agaatagcta cccactaatg tcatgcctat ttattgactg
caggaaaagg 720cgagatccta aactgtacaa agaggcagaa cagcgactta
tttaatggtg ttcttggtgg 780tttaggtcag tttggcatca taacgcgggc
aagaatagca ttggaaccag caccaaccat 840ggtaaacaat aaataaataa
aaaacttaaa aactgaacac gcgtgtgtcc tcctaactct 900gtataatgga
caggtaaaat ggataagagt gttatacctg gattttgcag cttttgccaa
960ggaccaagag caactaatat ctgcccaggg ccacaaattc gattacatag
aagggtttgt 1020gataataaac aggacaggcc tcctgaacag ctggaggttg
tctttcaccg cagaagagcc 1080tttagaagca agccaattca agtttgatgg
aaggactctg tattgtctgg agctagccaa 1140gtatttgaag caagataaca
aagacgtaat caaccaggtg agaaaacaga gtagaagcaa 1200tcggtagaat
cttctttggt agatgacatt cattggaact gaaaatatat atatatttgt
1260ccaatccagg aagtgaaaga aacattatca gagctaagct acgtgacgtc
gacactgttt 1320acaacggagg tagcatatga agcattcttg gacagggtac
atgtgtctga ggtaaaactc 1380cgatcgaaag ggcagtggga ggtgccacat
ccatggctga acctcctggt accaagaagc 1440aaaatcaatg aatttgcaag
aggtgtattt ggaaacatac taacggatac aagcaacggc 1500ccagtcatcg
tctacccagt gaacaaatca aagtaagaaa gaaagaaaga aagagctagt
1560catgattttg tttcttttca cttgttgaca aaacaaaagc atgttggtga
gcaggtggga 1620caatcaaaca tcagcagtaa caccggagga agaggtattc
tacctggtgg cgatcctaac 1680atcggcatct ccagggtcgg caggaaagga
tggagtagaa gagatcttga ggcggaacag 1740aagaatactg gaattcagtg
aagaagcagg gatagggttg aagcagtatc tgccacatta 1800cacgacaaga
gaagagtgga gatcccattt cggggacaag tggggagaat ttgtgaggag
1860gaaatccaga tatgatccat tggcaattct tgcgcctggc caccgaattt
ttcaaaaggc 1920agtctcatac tcatga 193612504PRTArabidopsis thaliana
12Met Leu Ile Val Arg Ser Phe Thr Ile Leu Leu Leu Ser Cys Ile Ala1
5 10 15Phe Lys Leu Ala Cys Cys Phe Ser Ser Ser Ile Ser Ser Leu Lys
Ala 20 25 30Leu Pro Leu Val Gly His Leu Glu Phe Glu His Val His His
Ala Ser 35 40 45Lys Asp Phe Gly Asn Arg Tyr Gln Leu Ile Pro Leu Ala
Val Leu His 50 55 60Pro Lys Ser Val Ser Asp Ile Ala Ser Thr Ile Arg
His Ile Trp Met65 70 75 80Met Gly Thr His Ser Gln Leu Thr Val Ala
Ala Arg Gly Arg Gly His 85 90 95Ser Leu Gln Gly Gln Ala Gln Thr Arg
His Gly Ile Val Ile His Met 100 105 110Glu Ser Leu His Pro Gln Lys
Leu Gln Val Tyr Ser Val Asp Ser Pro 115 120 125Ala Pro Tyr Val Asp
Val Ser Gly Gly Glu Leu Trp Ile Asn Ile Leu 130 135 140His Glu Thr
Leu Lys Tyr Gly Leu Ala Pro Lys Ser Trp Thr Asp Tyr145 150 155
160Leu His Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly Ile Ser Gly
165 170 175Gln Ala Phe Arg His Gly Pro Gln Ile Ser Asn Val His Gln
Leu Glu 180 185 190Ile Val Thr Gly Lys Gly Glu Ile Leu Asn Cys Thr
Lys Arg Gln Asn 195 200 205Ser Asp Leu Phe Asn Gly Val Leu Gly Gly
Leu Gly Gln Phe Gly Ile 210 215 220Ile Thr Arg Ala Arg Ile Ala Leu
Glu Pro Ala Pro Thr Met Asp Gln225 230 235 240Glu Gln Leu Ile Ser
Ala Gln Gly His Lys Phe Asp Tyr Ile Glu Gly 245 250 255Phe Val Ile
Ile Asn Arg Thr Gly Leu Leu Asn Ser Trp Arg Leu Ser 260 265 270Phe
Thr Ala Glu Glu Pro Leu Glu Ala Ser Gln Phe Lys Phe Asp Gly 275 280
285Arg Thr Leu Tyr Cys Leu Glu Leu Ala Lys Tyr Leu Lys Gln Asp Asn
290 295 300Lys Asp Val Ile Asn Gln Glu Val Lys Glu Thr Leu Ser Glu
Leu Ser305 310 315 320Tyr Val Thr Ser Thr Leu Phe Thr Thr Glu Val
Ala Tyr Glu Ala Phe 325 330 335Leu Asp Arg Val His Val Ser Glu Val
Lys Leu Arg Ser Lys Gly Gln 340 345 350Trp Glu Val Pro His Pro Trp
Leu Asn Leu Leu Val Pro Arg Ser Lys 355 360 365Ile Asn Glu Phe Ala
Arg Gly Val Phe Gly Asn Ile Leu Thr Asp Thr 370 375 380Ser Asn Gly
Pro Val Ile Val Tyr Pro Val Asn Lys Ser Lys Trp Asp385 390 395
400Asn Gln Thr Ser Ala Val Thr Pro Glu Glu Glu Val Phe Tyr Leu Val
405 410 415Ala Ile Leu Thr Ser Ala Ser Pro Gly Ser Ala Gly Lys Asp
Gly Val 420 425 430Glu Glu Ile Leu Arg Arg Asn Arg Arg Ile Leu Glu
Phe Ser Glu Glu 435 440 445Ala Gly Ile Gly Leu Lys Gln Tyr Leu Pro
His Tyr Thr Thr Arg Glu 450 455 460Glu Trp Arg Ser His Phe Gly Asp
Lys Trp Gly Glu Phe Val Arg Arg465 470 475 480Lys Ser Arg Tyr Asp
Pro Leu Ala Ile Leu Ala Pro Gly His Arg Ile 485 490 495Phe Gln Lys
Ala Val Ser Tyr Ser 5001331DNAArtificial Sequenceoligonucleotide
primer or probe 13cggtcgacat gggattgacc tcatccttac g
311435DNAArtificial Sequenceoligonucleotide primer or probe
14gcgtcgactt atacagttct aggtttcggc agtat 351533DNAArtificial
Sequenceoligonucleotide primer or probe 15gcggtaccag agagagaaac
ataaacaaat ggc 331631DNAArtificial Sequenceoligonucleotide primer
or probe 16gcggtaccca attttacttc caccaaaatg c 311734DNAArtificial
Sequenceoligonucleotide primer or probe 17gcggtacctt cattgataag
aatcaagcta ttca 341831DNAArtificial Sequenceoligonucleotide primer
or probe 18gcggtaccca aagtggtgag aacgactaac a 311928DNAArtificial
Sequenceoligonucleotide primer or probe 19gcggtacccc cattaaccta
cccgtttg 282032DNAArtificial Sequenceoligonucleotide primer or
probe 20gcggtaccag acgatgaacg tacttgtctg ta 322128DNAArtificial
Sequenceoligonucleotide primer or probe 21ggggtacctt gatgaatcgt
gaaatgac 282231DNAArtificial Sequenceoligonucleotide primer or
probe 22ggggtaccct ttcctcttgg ttttgtcctg t 312332DNAArtificial
Sequenceoligonucleotide primer or probe 23gctctagatc aggaaaagaa
ccatgcttat ag 322432DNAArtificial Sequenceoligonucleotide primer or
probe 24gctctagatc atgagtatga gactgccttt tg 32251728DNAArabidopsis
thaliana 25atgggattga cctcatcctt acggttccat agacaaaaca acaagacttt
cctcggaatc 60ttcatgatct tagttctaag ctgtatacca ggtagaacca atctttgttc
caatcattct 120gttagtaccc caaaagaatt accttcttca aatccttcag
atattcgttc ctcattagtt 180tcactagatt tggagggtta tataagcttc
gacgatgtcc acaatgtggc caaggacttt 240ggcaacagat accagttacc
acctttggca attctacatc caaggtcagt ttttgatatt 300tcatcgatga
tgaagcatat agtacatctg ggctccacct caaatcttac agtagcagct
360agaggccatg gtcactcgct tcaaggacaa gctctagctc atcaaggtgt
tgtcatcaaa 420atggagtcac ttcgaagtcc tgatatcagg atttataagg
ggaagcaacc atatgttgat 480gtctcaggtg gtgaaatatg gataaacatt
ctacgcgaga ctctaaaata cggtctttca 540ccaaagtcct ggacagacta
ccttcatttg accgttggag gtacactatc taatgctgga 600atcagcggtc
aagcattcaa gcatggaccc caaatcaaca acgtctacca gctagagatt
660gttacaggga aaggagaagt cgtaacctgt tctgagaagc ggaattctga
acttttcttc 720agtgttcttg gcgggcttgg acagtttggc ataatcaccc
gggcacggat ctctcttgaa 780ccagcaccgc atatggttaa atggatcagg
gtactctact ctgacttttc tgcattttca 840agggaccaag aatatctgat
ttcgaaggag aaaacttttg attacgttga aggatttgtg 900ataatcaata
gaacagacct tctcaataat tggcgatcgt cattcagtcc caacgattcc
960acacaggcaa gcagattcaa gtcagatggg aaaactcttt attgcctaga
agtggtcaaa 1020tatttcaacc cagaagaagc tagctctatg gatcaggaaa
ctggcaagtt actttcagag 1080ttaaattata ttccatccac tttgttttca
tctgaagtgc catatatcga gtttctggat 1140cgcgtgcata tcgcagagag
aaaactaaga gcaaagggtt tatgggaggt tccacatccc 1200tggctgaatc
tcctgattcc taagagcagc atataccaat ttgctacaga agttttcaac
1260aacattctca caagcaacaa caacggtcct atccttattt atccagtcaa
tcaatccaag 1320tggaagaaac atacatcttt gataactcca aatgaagata
tattctatct cgtagccttt 1380ctcccctctg cagtgccaaa ttcctcaggg
aaaaacgatc tagagtacct tttgaaacaa 1440aaccaaagag ttatgaactt
ctgcgcagca gcaaacctca acgtgaagca gtatttgccc 1500cattatgaaa
ctcaaaaaga gtggaaatca cactttggca aaagatggga aacatttgca
1560cagaggaaac aagcctacga ccctctagcg attctagcac ctggccaaag
aatattccaa 1620aagacaacag gaaaattatc tcccatccaa ctcgcaaagt
caaaggcaac aggaagtcct 1680caaaggtacc attacgcatc aatactgccg
aaacctagaa ctgtataa 1728261506DNAArabidopsis thaliana 26atggctaatc
ttcgtttaat gatcacttta atcacggttt taatgatcac caaatcatca 60aacggtatta
aaattgattt acctaaatcc cttaacctca ccctctctac cgatccttcc
120atcatctccg cagcctctca tgacttcgga aacataacca ccgtgacccc
cggcggcgta 180atctgcccct cctccaccgc tgatatctct cgtctcctcc
aatacgccgc aaacggaaaa 240agtacattcc aagtagcggc tcgtggccaa
ggccactcct taaacggcca agcctcggtc 300tccggcggag taatcgtcaa
catgacgtgt atcactgacg tggtggtttc aaaagacaag 360aagtacgctg
acgtggcggc cgggacgtta tgggtggatg tgcttaagaa gacggcggag
420aaaggggtgt cgccggtttc ttggacggat tatttgcata taaccgtcgg
aggaacgttg 480tcgaatggtg gaattggtgg tcaagtgttt cgaaacggtc
ctcttgttag taacgtcctt 540gaattggacg ttattactgg gaaaggtgaa
atgttgacat gctcgcgaca gctaaaccca 600gaattgttct atggagtgtt
aggaggtttg ggtcaatttg gaattataac gagagccaga 660attgttttgg
accatgcacc taaacgggcc aaatggtttc ggatgctcta cagtgatttc
720acaactttta caaaggacca agaacgtttg atatcaatgg caaacgatat
tggagtcgac 780tatttagaag gtcaaatatt tctatcaaac ggtgtcgttg
acacctcttt tttcccacct 840tcagatcaat ctaaagtcgc tgatctagtc
aagcaacacg gtatcatcta tgttcttgaa 900gtagccaagt attatgatga
tcccaatctc cccatcatca gcaaggttat tgacacatta 960acgaaaacat
taagttactt gcccgggttc atatcaatgc acgacgtggc ctacttcgat
1020ttcttgaacc gtgtacatgt cgaagaaaat aaactcagat ctttgggatt
atgggaactt 1080cctcatcctt ggcttaacct ctacgttcct aaatctcgga
ttctcgattt tcataacggt 1140gttgtcaaag acattcttct taagcaaaaa
tcagcttcgg gactcgctct tctctatcca 1200acaaaccgga ataaatggga
caatcgtatg tcggcgatga taccagagat cgatgaagat 1260gttatatata
ttatcggact actacaatcc gctaccccaa aggatcttcc agaagtggag
1320agcgttaacg agaagataat taggttttgc aaggattcag gtattaagat
taagcaatat 1380ctaatgcatt atactagtaa agaagattgg attgagcatt
ttggatcaaa atgggatgat 1440ttttcgaaga ggaaagatct atttgatccc
aagaaactgt tatctccagg gcaagacatc 1500ttttga
1506271572DNAArabidopsis thaliana 27atggcgagtt ataatcttcg
ttcacaagtt cgtcttatag caataacaat agtaatcatc 60attactctct caactccgat
cacaaccaac acatcaccac aaccatggaa tatcctttca 120cacaacgaat
tcgccggaaa actcacctcc tcctcctcct ccgtcgaatc agccgccaca
180gatttcggcc acgtcaccaa aatcttccct tccgccgtct taatcccttc
ctccgttgaa 240gacatcacag atctcataaa actctctttt gactctcaac
tgtcttttcc tttagccgct 300cgtggtcacg gacacagcca ccgtggccaa
gcctcggcta aagacggagt tgtggtcaac 360atgcggtcca tggtaaaccg
ggatcgaggt atcaaggtgt ctaggacctg tttatatgtt 420gacgtggacg
ctgcgtggct atggattgag gtgttgaata aaactttgga gttagggtta
480acgccggttt cttggacgga ttatttgtat ttaacagtcg gtgggacgtt
atcaaacggc 540ggaattagtg gacaaacgtt tcggtacggt ccacagatca
ctaatgttct agagatggat 600gttattactg gaaaaggaga gattgcaact
tgttccaagg acatgaactc ggatcttttc 660ttcgcggtgt taggaggttt
gggtcaattc ggcattataa caagagccag aattaaactt 720gaagtagctc
cgaaaagggc caagtggtta aggtttctat acatagattt ctccgaattc
780acaagagatc aagaacgagt gatatcgaaa acggacggtg tagatttctt
agaaggttcc 840attatggtgg accatggccc accggataac tggagatcca
cgtattatcc accgtccgat 900cacttgagga tcgcctcaat ggtcaaacga
catcgtgtca tctactgcct tgaagtcgtc 960aagtattacg acgaaacttc
tcaatacaca gtcaacgagg aaatggagga gttaagcgat 1020agtttaaacc
atgtaagagg gtttatgtac gagaaagatg tgacgtatat ggatttccta
1080aaccgagttc gaaccggaga gctaaacctg aaatccaaag gccaatggga
tgttccacat 1140ccatggctta atctcttcgt accaaaaact caaatctcca
aatttgatga tggtgttttt 1200aagggtatta tcctaagaaa taacatcact
agcggtcctg ttcttgttta tcctatgaat 1260cgcaacaagt ggaatgatcg
gatgtctgcc gctatacccg aggaagatgt attttatgcg 1320gtagggtttt
taagatccgc gggttttgac aattgggagg cttttgatca agaaaacatg
1380gaaatactga agttttgtga ggatgctaat atgggggtta tacaatatct
tccttatcat 1440tcatcacaag aaggatgggt tagacatttt ggtccgaggt
ggaatatttt cgtagagaga 1500aaatataaat atgatcccaa aatgatatta
tcaccgggac aaaatatatt tcaaaaaata 1560aactcgagtt ag
1572281575DNAArabidopsis thaliana 28atgactaata ctctctgttt
aagcctcatc accctaataa cgctttttat aagtttaacc 60ccaaccttaa tcaaatcaga
tgagggcatt gatgttttct tacccatatc actcaacctt 120acggtcctaa
ccgatccctt ctccatctct gccgcttctc acgacttcgg taacataacc
180gacgaaaatc ccggcgccgt cctctgccct tcctccacca cggaggtggc
tcgtctcctc 240cgtttcgcta acggaggatt ctcttacaat aaaggctcaa
ccagccccgc gtctactttc 300aaagtggctg ctcgaggcca aggccactcc
ctccgtggcc aagcctctgc acccggaggt 360gtcgtcgtga acatgacgtg
tctcgccatg gcggctaaac cagcggcggt tgttatctcg 420gcagacggga
cttacgctga cgtggctgcc gggacgatgt gggtggatgt tctgaaggcg
480gcggtggata gaggcgtctc gccggttaca tggacggatt atttgtatct
cagcgtcggc 540gggacgttgt cgaacgctgg aatcggtggt cagacgttta
gacacggccc tcagattagt 600aacgttcatg agcttgacgt tattaccgga
aaaggtgaaa tgatgacttg ctctccaaag 660ttaaaccctg aattgttcta
tggagtttta ggaggtttgg gtcaattcgg tattataacg 720agggccagga
ttgcgttgga tcatgcaccc acaagggtga aatggtctcg catactctac
780agtgacttct cggcttttaa aagagaccaa gagcgtttaa tatcaatgac
caatgatctc 840ggagttgact ttttggaagg tcaacttatg atgtcaaatg
gcttcgtaga cacctctttc 900ttcccactct ccgatcaaac aagagtcgca
tctcttgtga atgaccaccg gatcatctat 960gttctcgaag tagccaagta
ttatgacaga accacccttc ccattattga ccaggtgatt 1020gacacgttaa
gtagaactct aggtttcgct ccagggttta tgttcgtaca agatgttccg
1080tatttcgatt tcttgaaccg tgtccgaaac gaagaagata aactcagatc
tttaggacta 1140tgggaagttc ctcatccatg gcttaacatc tttgtcccgg
ggtctcgaat ccaagatttt 1200catgatggtg ttattaatgg ccttcttcta
aaccaaacct caacttctgg tgttactctc 1260ttctatccca caaaccgaaa
caaatggaac aaccgcatgt caacgatgac accggacgaa 1320gatgtttttt
atgtgatcgg attactgcaa tcagctggtg gatctcaaaa ttggcaagaa
1380cttgaaaatc tcaacgacaa ggttattcag ttttgtgaaa actcgggaat
taagattaag 1440gaatatttga tgcactatac aagaaaagaa gattgggtta
aacattttgg accaaaatgg 1500gatgattttt taagaaagaa aattatgttt
gatcccaaaa gactattgtc tccaggacaa 1560gacatattta attaa
1575291611DNAArabidopsis thaliana 29atgacgtcaa gctttcttct
cctgacgttc gccatatgta aactgatcat agccgtgggt 60ctaaacgtgg gccccagtga
gctcctccgc atcggagcca tagatgtcga cggccacttc 120accgtccacc
cttccgactt agcctccgtc tcctcagact tcggtatgct gaagtcacct
180gaagagccat tggccgtgct tcatccatca tcggccgaag acgtggcacg
actcgtcaga 240acagcttacg gttcagccac ggcgtttccg gtctcagccc
gaggccacgg ccattccata 300aacggacaag ccgcggcggg gaggaacggt
gtggtggttg aaatgaacca cggcgtaacc 360gggacgccca agccactcgt
ccgaccggat gaaatgtatg tggatgtatg gggtggagag 420ttatgggtcg
atgtgttgaa gaaaacgttg gagcatggct tagcaccaaa atcatggacg
480gattacttgt atctaaccgt tggaggtaca ctctccaatg caggaatcag
tggtcaagct 540tttcaccatg gtcctcaaat tagtaacgtc cttgagctcg
acgttgtaac tgggaaagga 600gaggtgatga gatgctcaga agaagagaac
acaaggctat tccatggagt tcttggtgga 660ttaggtcaat ttgggatcat
cactcgagca cgaatctctc tcgaaccagc tccccaaagg 720gtgagatgga
tacgggtatt gtattcgagc ttcaaagtgt ttacggagga ccaagagtac
780ttaatctcaa tgcatggtca attaaagttt gattacgtgg aaggttttgt
gattgtggac 840gaaggactcg tcaacaattg gagatcttct ttcttctctc
cacgtaaccc cgtcaagatc 900tcctctgtta gttccaacgg ctctgttttg
tattgccttg agatcaccaa gaactaccac 960gactccgact ccgaaatcgt
tgatcaggaa gttgagattc tgatgaagaa attgaatttc 1020ataccgacat
cggtctttac aacggattta caatatgtgg actttctcga ccgggtacac
1080aaggccgaat tgaagctccg gtccaagaat ttatgggagg ttccacaccc
atggctcaac 1140ctcttcgtgc caaaatcaag aatctctgac ttcgataaag
gcgttttcaa gggcattttg 1200ggaaataaaa caagtggccc tattcttatc
taccccatga acaaagacaa atgggacgag 1260aggagctcag ccgtgacgcc
ggatgaggaa gttttctatc tggtggctct attgagatca 1320gctttaacgg
acggtgaaga gacacagaag ctagagtatc tgaaagatca gaaccgtcgg
1380atcttggagt tctgtgaaca agccaagatc aatgtgaagc agtatcttcc
tcaccacgca 1440acacaggaag agtgggtggc tcattttggg gacaagtggg
atcggttcag aagcttaaag 1500gctgagtttg atccgcgaca catactcgct
actggtcaga gaatctttca aaacccatct 1560ttgtctttgt ttcctccgtc
gtcgtcttct tcgtcagcgg cttcatggtg a 1611301515DNAArabidopsis
thaliana 30atgcttatag taagaagttt caccatcttg cttctcagct gcatagcctt
taagttggct 60tgctgcttct ctagcagcat ttcttctttg aaggcgcttc ccctagtagg
ccatttggag 120tttgaacatg tccatcacgc ctccaaagat tttggaaatc
gataccagtt gatccctttg 180gcggtcttac atcccaaatc ggtaagcgac
atcgcctcaa cgatacgaca catctggatg 240atgggcactc attcacagct
tacagtggca gcgagaggtc gtggacattc actccaaggc 300caagctcaaa
caagacatgg aattgttata cacatggaat cactccatcc ccagaagctg
360caggtctaca gtgtggattc ccctgctcca tatgttgatg tgtctggtgg
tgagctgtgg 420ataaacattt tgcatgagac cctcaagtac gggcttgcac
caaaatcatg gacggattac 480ctgcatttaa ctgtaggtgg tactctgtcc
aatgctggaa taagcggcca ggcattccga 540catggaccac agatcagcaa
tgttcatcaa ctggagattg tcacaggaaa aggcgagatc 600ctaaactgta
caaagaggca gaacagcgac ttatttaatg gtgttcttgg tggtttaggt
660cagtttggca tcataacgcg ggcaagaata gcattggaac cagcaccaac
catggaccaa 720gagcaactaa tatctgccca gggccacaaa ttcgattaca
tagaagggtt tgtgataata 780aacaggacag gcctcctgaa cagctggagg
ttgtctttca ccgcagaaga gcctttagaa 840gcaagccaat tcaagtttga
tggaaggact ctgtattgtc tggagctagc caagtatttg 900aagcaagata
acaaagacgt aatcaaccag gaagtgaaag aaacattatc agagctaagc
960tacgtgacgt cgacactgtt tacaacggag gtagcatatg aagcattctt
ggacagggta 1020catgtgtctg aggtaaaact ccgatcgaaa gggcagtggg
aggtgccaca tccatggctg 1080aacctcctgg taccaagaag caaaatcaat
gaatttgcaa gaggtgtatt tggaaacata 1140ctaacggata caagcaacgg
cccagtcatc gtctacccag tgaacaaatc aaagtgggac 1200aatcaaacat
cagcagtaac accggaggaa gaggtattct acctggtggc gatcctaaca
1260tcggcatctc cagggtcggc aggaaaggat ggagtagaag agatcttgag
gcggaacaga 1320agaatactgg aattcagtga agaagcaggg atagggttga
agcagtatct gccacattac 1380acgacaagag aagagtggag atcccatttc
ggggacaagt ggggagaatt tgtgaggagg 1440aaatccagat atgatccatt
ggcaattctt gcgcctggcc accgaatttt tcaaaaggca 1500gtctcatact catga
15153184DNAArabidopsis thaliana 31tcagcttcgg gactcgctct tctctatcca
acaaaccgga ataaatggga caatcgtatg 60tcggcgatga taccagagat cgat
843228PRTArabidopsis thaliana 32Ser Ala Ser Gly Leu Ala Leu Leu Tyr
Pro Thr Asn Arg Asn Lys Trp1 5 10 15Asp Asn Arg Met Ser Ala Met Ile
Pro Glu Ile Asp 20 25332814DNAArabidopsis thaliana 33atgaatcgta
tgacgtcaag ctttcttctc ctgacgttcg ccatatgtaa actgatcata 60gccgtgggtc
taaacgtggg ccccagtgag ctcctccgca tcggagccat agatgtcgac
120ggccacttca ccgtccaccc ttccgactta gcctccgtct cctcagactt
cggtatgctg 180aagtcacctg aagagccatt ggccgtgctt catccatcat
cggccgaaga cgtggcacga 240ctcgtcagaa cagcttacgg ttcagccacg
gcgtttccgg tctcagcccg aggccacggc 300cattccataa acggacaagc
cgcggcgggg aggaacggtg tggtggttga aatgaaccac 360ggcgtaaccg
ggacgcccaa gccactcgtc cgaccggatg aaatgtatgt ggatgtatgg
420ggtggagagt tatgggtcga tgtgttgaag aaaacgttgg agcatggctt
agcaccaaaa 480tcatggacgg attacttgta tctaaccgtt ggaggtacac
tctccaatgc aggaatcagt 540ggtcaagctt ttcaccatgg tcctcaaatt
agtaacgtcc ttgagctcga cgttgtaact 600ggttagtatt aaaacattca
agttcatata ttttaaatgc ttttgtctga agttttacta 660ataacaagaa
attgatacca aaaagtaggg aaaggagagg tgatgagatg ctcagaagaa
720gagaacacaa ggctattcca tggagttctt ggtggattag gtcaatttgg
gatcatcact 780cgagcacgaa tctctctcga accagctccc caaagggtaa
tattttttta atgactagct 840atcaaaaatc cctggcgggt ccatacgttg
taatcttttt agtttttact gttgatggta 900ttttttatat attttggata
ataaaaccct aaaatggtat attgtgatga caggtgagat 960ggatacgggt
attgtattcg agcttcaaag tgtttacgga ggaccaagag tacttaatct
1020caatgcatgg tcaattaaag tttgattacg tggaaggttt tgtgattgtg
gacgaaggac 1080tcgtcaacaa ttggagatct tctttcttct ctccacgtaa
ccccgtcaag atctcctctg 1140ttagttccaa cggctctgtt ttgtattgcc
ttgagatcac caagaactac cacgactccg 1200actccgaaat cgttgatcag
gtcactttca ttattcactt agaaaaaagc gatattttca 1260ttttttatat
tgatgaatat ctggaaggat ttaacgctat gcgactattg ggaaatcatt
1320atgaaaaaat atttagttta tatgattgaa agtggtctcc atagtatttt
tgttgtgtcg 1380actttattat aacttaaatt tggaagagga catgaagaag
aagccagaga ggatctacag 1440agatctagct tttccacctg aacttaataa
tgcacattta tataattatt tttcttcttc 1500taaagtttag tttatcacta
gcgaattaat catggttact aattaagtag tggacagggt 1560catggaccac
tcactcacca aataatgatt cctctttact cttaagttta attttaataa
1620aaccaactct actggaatct taacttatcc ttggttttgg taggctttta
tagcaacacg 1680gtttttttaa ttttcctatt ccagattttg tatattaaat
gtcgattttt tttctttttg 1740tttcaggaag ttgagattct gatgaagaaa
ttgaatttca taccgacatc ggtctttaca 1800acggatttac aatatgtgga
ctttctcgac cgggtacaca aggccgaatt gaagctccgg 1860tccaagaatt
tatgggaggt tccacaccca tggctcaacc tcttcgtgcc aaaatcaaga
1920atctctgact tcgataaagg cgttttcaag ggcattttgg gaaataaaac
aagtggccct 1980attcttatct accccatgaa caaagacaag taagtcttga
cattaccatt gattactact 2040tctaaatttc ttctctagaa aaaagaataa
aacgagtttt gcattgcatg catgcaaagt 2100tacacttgtg gggattaatt
agtggtccaa gaaaaaaagt ttgtcaaaat tgaaaaaaac 2160tagacacgtg
gtacatggga ttgtccgaaa aacgttgtcc acatgtgcat cgaaccagct
2220aagattgaca acaacacttc gtcggctcgt atttctcttt ttgttttgtg
accaaatccg 2280atggtccaga ttgggtttat ttgtttttaa gttcctagaa
ctcatggtgg gtgggtccca 2340atcagattct cctagaccaa accgatctca
acgaaccctc cgcacatcat tgattattac 2400attaatatag atattgtcgt
tgctgacgtg tcgtaatttg atgttattgt cagatgggac 2460gagaggagct
cagccgtgac gccggatgag gaagttttct atctggtggc tctattgaga
2520tcagctttaa cggacggtga agagacacag aagctagagt atctgaaaga
tcagaaccgt 2580cggatcttgg agttctgtga acaagccaag atcaatgtga
agcagtatct tcctcaccac 2640gcaacacagg aagagtgggt ggctcatttt
ggggacaagt gggatcggtt cagaagctta 2700aaggctgagt ttgatccgcg
acacatactc gctactggtc agagaatctt tcaaaaccca 2760tctttgtctt
tgtttcctcc gtcgtcgtct tcttcgtcag cggcttcatg gtga
2814341620DNAArabidopsis thaliana 34atgaatcgta tgacgtcaag
ctttcttctc ctgacgttcg ccatatgtaa actgatcata 60gccgtgggtc taaacgtggg
ccccagtgag ctcctccgca tcggagccat agatgtcgac 120ggccacttca
ccgtccaccc ttccgactta gcctccgtct cctcagactt cggtatgctg
180aagtcacctg aagagccatt ggccgtgctt catccatcat cggccgaaga
cgtggcacga 240ctcgtcagaa cagcttacgg ttcagccacg gcgtttccgg
tctcagcccg aggccacggc 300cattccataa acggacaagc cgcggcgggg
aggaacggtg tggtggttga aatgaaccac 360ggcgtaaccg ggacgcccaa
gccactcgtc cgaccggatg aaatgtatgt ggatgtatgg 420ggtggagagt
tatgggtcga tgtgttgaag aaaacgttgg agcatggctt agcaccaaaa
480tcatggacgg attacttgta tctaaccgtt ggaggtacac tctccaatgc
aggaatcagt 540ggtcaagctt ttcaccatgg tcctcaaatt agtaacgtcc
ttgagctcga cgttgtaact 600gggaaaggag aggtgatgag atgctcagaa
gaagagaaca caaggctatt ccatggagtt 660cttggtggat taggtcaatt
tgggatcatc actcgagcac gaatctctct cgaaccagct 720ccccaaaggg
tgagatggat acgggtattg tattcgagct tcaaagtgtt tacggaggac
780caagagtact taatctcaat gcatggtcaa ttaaagtttg attacgtgga
aggttttgtg 840attgtggacg aaggactcgt caacaattgg agatcttctt
tcttctctcc acgtaacccc 900gtcaagatct cctctgttag ttccaacggc
tctgttttgt attgccttga gatcaccaag 960aactaccacg actccgactc
cgaaatcgtt gatcaggaag ttgagattct gatgaagaaa 1020ttgaatttca
taccgacatc ggtctttaca acggatttac aatatgtgga ctttctcgac
1080cgggtacaca aggccgaatt gaagctccgg tccaagaatt tatgggaggt
tccacaccca 1140tggctcaacc tcttcgtgcc aaaatcaaga atctctgact
tcgataaagg cgttttcaag 1200ggcattttgg gaaataaaac aagtggccct
attcttatct accccatgaa caaagacaaa 1260tgggacgaga ggagctcagc
cgtgacgccg gatgaggaag ttttctatct ggtggctcta 1320ttgagatcag
ctttaacgga cggtgaagag acacagaagc tagagtatct gaaagatcag
1380aaccgtcgga tcttggagtt ctgtgaacaa gccaagatca atgtgaagca
gtatcttcct 1440caccacgcaa cacaggaaga gtgggtggct cattttgggg
acaagtggga tcggttcaga 1500agcttaaagg ctgagtttga tccgcgacac
atactcgcta ctggtcagag aatctttcaa 1560aacccatctt tgtctttgtt
tcctccgtcg tcgtcttctt cgtcagcggc ttcatggtga 162035539PRTArabidopsis
thaliana 35Met Asn Arg Met Thr Ser Ser Phe Leu Leu Leu Thr Phe Ala
Ile Cys1 5 10 15Lys Leu Ile Ile Ala Val Gly Leu Asn Val Gly Pro Ser
Glu Leu Leu 20 25 30Arg Ile Gly Ala Ile Asp Val Asp Gly His Phe Thr
Val His Pro Ser 35 40 45Asp Leu Ala Ser Val Ser Ser Asp Phe Gly Met
Leu Lys Ser Pro Glu 50 55 60Glu Pro Leu Ala Val Leu His Pro Ser Ser
Ala Glu Asp Val Ala Arg65 70 75 80Leu Val Arg Thr Ala Tyr Gly Ser
Ala Thr Ala Phe Pro Val Ser Ala 85 90 95Arg Gly His Gly His Ser Ile
Asn Gly Gln Ala Ala Ala Gly Arg Asn 100 105 110Gly Val Val Val Glu
Met Asn His Gly Val Thr Gly Thr Pro Lys Pro 115 120 125Leu Val Arg
Pro Asp Glu Met Tyr Val Asp Val Trp Gly Gly Glu Leu 130 135 140Trp
Val Asp Val Leu Lys Lys Thr Leu Glu His Gly Leu Ala Pro Lys145 150
155 160Ser Trp Thr Asp Tyr Leu Tyr Leu Thr Val Gly Gly Thr Leu Ser
Asn 165 170 175Ala Gly Ile Ser Gly Gln Ala Phe His His Gly Pro Gln
Ile Ser Asn 180 185 190Val Leu Glu Leu Asp Val Val Thr Gly Lys Gly
Glu Val Met Arg Cys 195 200 205Ser Glu Glu Glu Asn Thr Arg Leu Phe
His Gly Val Leu Gly Gly Leu 210 215 220Gly Gln Phe Gly Ile Ile Thr
Arg Ala Arg Ile Ser Leu Glu Pro Ala225 230 235 240Pro Gln Arg Val
Arg Trp Ile Arg Val Leu Tyr Ser Ser Phe Lys Val 245 250 255Phe Thr
Glu Asp Gln Glu Tyr Leu Ile Ser Met His Gly Gln Leu Lys 260 265
270Phe Asp Tyr Val Glu Gly Phe Val Ile Val Asp Glu Gly Leu Val Asn
275 280 285Asn Trp Arg Ser Ser Phe Phe Ser Pro Arg Asn Pro Val Lys
Ile Ser 290 295 300Ser Val Ser Ser Asn Gly Ser Val Leu Tyr Cys Leu
Glu Ile Thr Lys305 310 315 320Asn Tyr His Asp Ser Asp Ser Glu Ile
Val Asp Gln Glu Val Glu Ile 325 330 335Leu Met Lys Lys Leu Asn Phe
Ile Pro Thr Ser Val Phe Thr Thr Asp 340 345 350Leu Gln Tyr Val Asp
Phe Leu Asp Arg Val His Lys Ala Glu Leu Lys 355 360 365Leu Arg Ser
Lys Asn Leu Trp Glu Val Pro His Pro Trp Leu Asn Leu 370 375 380Phe
Val Pro Lys Ser Arg Ile Ser Asp Phe Asp Lys Gly Val Phe Lys385 390
395 400Gly Ile Leu Gly Asn Lys Thr Ser Gly Pro Ile Leu Ile Tyr Pro
Met 405 410 415Asn Lys Asp Lys Trp Asp Glu Arg Ser Ser Ala Val Thr
Pro Asp Glu 420 425 430Glu Val Phe Tyr Leu Val Ala Leu Leu Arg Ser
Ala Leu Thr Asp Gly 435 440 445Glu Glu Thr Gln Lys Leu Glu Tyr Leu
Lys Asp Gln Asn Arg Arg Ile 450 455 460Leu Glu Phe Cys Glu Gln Ala
Lys Ile Asn Val Lys Gln Tyr Leu Pro465 470 475 480His His Ala Thr
Gln Glu Glu Trp Val Ala His Phe Gly Asp Lys Trp 485 490 495Asp Arg
Phe Arg Ser Leu Lys Ala Glu Phe Asp Pro Arg His Ile Leu 500 505
510Ala Thr Gly Gln Arg Ile Phe Gln Asn Pro Ser Leu Ser Leu Phe Pro
515 520 525Pro Ser Ser Ser Ser Ser Ser Ala Ala Ser Trp 530
53536501PRTArabidopsis thaliana 36Met Ala Asn Leu Arg Leu Met Ile
Thr Leu Ile Thr Val Leu Met Ile1 5 10 15Thr Lys Ser Ser Asn Gly Ile
Lys Ile Asp Leu Pro Lys Ser Leu Asn 20 25 30Leu Thr Leu Ser Thr Asp
Pro Ser Ile Ile Ser Ala Ala Ser His Asp 35 40 45Phe Gly Asn Ile Thr
Thr Val Thr Pro Gly Gly Val Ile Cys Pro Ser 50 55 60Ser Thr Ala Asp
Ile Ser Arg Leu Leu Gln Tyr Ala Ala Asn Gly Lys65 70 75 80Ser Thr
Phe Gln Val Ala Ala Arg Gly Gln Gly His Ser Leu Asn Gly 85 90 95Gln
Ala Ser Val Ser Gly Gly Val Ile Val Asn Met Thr Cys Ile Thr 100 105
110Asp Val Val Val Ser Lys Asp Lys Lys Tyr Ala Asp Val Ala Ala Gly
115 120 125Thr Leu Trp Val Asp Val Leu Lys Lys Thr Ala Glu Lys Gly
Val Ser 130 135 140Pro Val Ser Trp Thr Asp Tyr Leu His Ile Thr Val
Arg Gly Thr Leu145 150 155 160Ser Asn Gly Gly Ile Gly Gly Gln Val
Phe Arg Asn Gly Pro Leu Val 165 170 175Ser Asn Val Leu Glu Leu Asp
Val Ile Thr Gly Lys Gly Glu Met Leu 180 185 190Thr Cys Ser Arg Gln
Leu Asn Pro Glu Leu Phe Tyr Gly Val Leu Gly 195 200 205Gly Leu Gly
Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile Val Leu Asp 210 215 220His
Ala Pro Lys Arg Ala Lys Trp Phe Arg Met Leu Tyr Ser Asp Phe225 230
235 240Thr Thr Phe Thr Lys Asp Gln Glu Arg Leu Ile Ser Met Ala Asn
Asp 245 250 255Ile Gly Val Asp Tyr Leu Glu Gly Gln Ile Phe Leu Ser
Asn Gly Val 260 265 270Val Asp Thr Ser Phe Phe Pro Pro Ser Asp Gln
Ser Lys Val Ala Asp 275 280 285Leu Val Lys Gln His Gly Ile Ile Tyr
Val Leu Glu Val Ala Lys Tyr 290 295 300Tyr Asp Asp Pro Asn Leu Pro
Ile Ile Ser Lys Val Ile Asp Thr Leu305 310 315 320Thr Lys Thr Leu
Ser Tyr Leu Pro Gly Phe Ile Ser Met His Asp Val 325 330 335Ala Tyr
Phe Asp Phe Leu Asn Arg Val His Val Glu Glu Asn Lys Leu 340 345
350Arg Ser Leu Gly Leu Trp Glu Leu Pro His Pro Trp Leu Asn Leu Tyr
355 360 365Val Pro Lys Ser Arg Ile Leu Asp Phe His Asn Gly Val Val
Lys Asp 370 375 380Ile Leu Leu Lys Gln Lys Ser Ala Ser Gly Leu Ala
Leu Leu Tyr Pro385 390 395 400Thr Asn Arg Asn Lys Trp Asp Asn Arg
Met Ser Ala Met Ile Pro Glu 405 410 415Ile Asp Glu Asp Val Ile Tyr
Ile Ile Gly Leu Leu Gln Ser Ala Thr 420 425 430Pro Lys Asp Leu Pro
Glu Val Glu Ser Val Asn Glu Lys Ile Ile Arg 435 440 445Phe Cys Lys
Asp Ser Gly Ile Lys Ile Lys Gln Tyr Leu Met His Tyr 450 455 460Thr
Ser Lys Glu Asp Trp Ile Glu His Phe Gly Ser Lys Trp Asp Asp465 470
475 480Phe Ser Lys Arg Lys Asp Leu Phe Asp Pro Lys Lys Leu Leu Ser
Pro 485 490 495Gly Gln Asp Ile Phe 500371455DNAArabidopsis thaliana
37atggctaatc ttcgtttaat gatcacttta atcacggttt taatgatcac caaatcatca
60aacggtatta aaattgattt acctaaatcc cttaacctca ccctctctac cgatccttcc
120atcatctccg cagcctctca tgacttcgga aacataacca ccgtgacccc
cggcggcgta 180atctgcccct cctccaccgc tgatatctct cgtctcctcc
aatacgccgc aaacggaaaa 240agtacattcc aagtagcggc tcgtggccaa
ggccactcct taaacggcca agcctcggtc 300tccggcggag taatcgtcaa
catgacgtgt atcactgacg tggtggtttc aaaagacaag 360aagtacgctg
acgtggcggc cgggacgtta tgggtggatg tgcttaagaa gacggcggag
420aaaggggtgt cgccggtttc ttggacggat tatttgcata taaccgtccg
aggaacgttg 480tcgaatggtg gaattggtgg tcaagtgttt cgaaacggtc
ctcttgttag taacgtcctt 540gaattggacg ttattactgg gaaaggtgaa
atgttgacat gctcgcgaca gctaaaccca 600gaattgttct atggagtgtt
aggaggtttg ggtcaatttg gaattataac gagagccaga 660attgttttgg
accatgcacc taaacggcaa gaacgtttga tatcaatggc aaacgatatt
720ggagtcgact atttagaagg tcaaatattt ctatcaaacg gtgtcgttga
cacctctttt 780ttcccacctt cagatcaatc taaagtcgct gatctagtca
agcaacacgg tatcatctat 840gttcttgaag tagccaagta ttatgatgat
cccaatctcc ccatcatcag caaggttatt 900gacacattaa cgaaaacatt
aagttacttg cccgggttca tatcaatgca cgacgtggcc 960tacttcgatt
tcttgaaccg tgtacatgtc gaagaaaata aactcagatc tttgggatta
1020tgggaacttc ctcatccttg gcttaacctc tacgttccta aatctcggat
tctcgatttt 1080cataacggtg ttgtcaaaga cattcttctt aagcaaaaat
cagcttcggg actcgctctt 1140ctctatccaa caaaccggaa taaatgggac
aatcgtatgt cggcgatgat accagagatc 1200gatgaagatg ttatatatat
tatcggacta ctacaatccg ctaccccaaa ggatcttcca 1260gaagtggaga
gcgttaacga gaagataatt aggttttgca aggattcagg tattaagatt
1320aagcaatatc taatgcatta tactagtaaa gaagattgga ttgagcattt
tggatcaaaa 1380tgggatgatt tttcgaagag gaaagatcta tttgatccca
agaaactgtt atctccaggg 1440caagacatct tttga 145538484PRTArabidopsis
thaliana 38Met Ala Asn Leu Arg Leu Met Ile Thr Leu Ile Thr Val Leu
Met Ile1 5 10 15Thr Lys Ser Ser Asn Gly Ile Lys Ile Asp Leu Pro Lys
Ser Leu Asn 20 25 30Leu Thr Leu Ser Thr Asp Pro Ser Ile Ile Ser Ala
Ala Ser His Asp 35 40 45Phe Gly Asn Ile
Thr Thr Val Thr Pro Gly Gly Val Ile Cys Pro Ser 50 55 60Ser Thr Ala
Asp Ile Ser Arg Leu Leu Gln Tyr Ala Ala Asn Gly Lys65 70 75 80Ser
Thr Phe Gln Val Ala Ala Arg Gly Gln Gly His Ser Leu Asn Gly 85 90
95Gln Ala Ser Val Ser Gly Gly Val Ile Val Asn Met Thr Cys Ile Thr
100 105 110Asp Val Val Val Ser Lys Asp Lys Lys Tyr Ala Asp Val Ala
Ala Gly 115 120 125Thr Leu Trp Val Asp Val Leu Lys Lys Thr Ala Glu
Lys Gly Val Ser 130 135 140Pro Val Ser Trp Thr Asp Tyr Leu His Ile
Thr Val Arg Gly Thr Leu145 150 155 160Ser Asn Gly Gly Ile Gly Gly
Gln Val Phe Arg Asn Gly Pro Leu Val 165 170 175Ser Asn Val Leu Glu
Leu Asp Val Ile Thr Gly Lys Gly Glu Met Leu 180 185 190Thr Cys Ser
Arg Gln Leu Asn Pro Glu Leu Phe Tyr Gly Val Leu Gly 195 200 205Gly
Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile Val Leu Asp 210 215
220His Ala Pro Lys Arg Gln Glu Arg Leu Ile Ser Met Ala Asn Asp
Ile225 230 235 240Gly Val Asp Tyr Leu Glu Gly Gln Ile Phe Leu Ser
Asn Gly Val Val 245 250 255Asp Thr Ser Phe Phe Pro Pro Ser Asp Gln
Ser Lys Val Ala Asp Leu 260 265 270Val Lys Gln His Gly Ile Ile Tyr
Val Leu Glu Val Ala Lys Tyr Tyr 275 280 285Asp Asp Pro Asn Leu Pro
Ile Ile Ser Lys Val Ile Asp Thr Leu Thr 290 295 300Lys Thr Leu Ser
Tyr Leu Pro Gly Phe Ile Ser Met His Asp Val Ala305 310 315 320Tyr
Phe Asp Phe Leu Asn Arg Val His Val Glu Glu Asn Lys Leu Arg 325 330
335Ser Leu Gly Leu Trp Glu Leu Pro His Pro Trp Leu Asn Leu Tyr Val
340 345 350Pro Lys Ser Arg Ile Leu Asp Phe His Asn Gly Val Val Lys
Asp Ile 355 360 365Leu Leu Lys Gln Lys Ser Ala Ser Gly Leu Ala Leu
Leu Tyr Pro Thr 370 375 380Asn Arg Asn Lys Trp Asp Asn Arg Met Ser
Ala Met Ile Pro Glu Ile385 390 395 400Asp Glu Asp Val Ile Tyr Ile
Ile Gly Leu Leu Gln Ser Ala Thr Pro 405 410 415Lys Asp Leu Pro Glu
Val Glu Ser Val Asn Glu Lys Ile Ile Arg Phe 420 425 430Cys Lys Asp
Ser Gly Ile Lys Ile Lys Gln Tyr Leu Met His Tyr Thr 435 440 445Ser
Lys Glu Asp Trp Ile Glu His Phe Gly Ser Lys Trp Asp Asp Phe 450 455
460Ser Lys Arg Lys Asp Leu Phe Asp Pro Lys Lys Leu Leu Ser Pro
Gly465 470 475 480Gln Asp Ile Phe3960DNAArtificial
sequenceoligonucleotide primer or probe 39ggggacaagt ttgtacaaaa
aagcaggctt cacaatggct aatcttcgtt taatgatcac 604049DNAArtificial
sequenceoligonucleotide primer or probe 40ggggaccact ttgtacaaga
aagctgggtt caaaagatgt cttgccctg 49413328DNAArtificial
sequenceexpression cassette 41ggtcagccaa tacattgatc cgttgccaat
catgcaaagt attttggctg tggccgagtg 60ccggaattga taattgtgtt ctgactaaat
taaatgacca gaagtcgcta tcttccaatg 120tatccgaaac ctggattaaa
caatcctgtt ctgttctcta gcccctcctg catggccgga 180ttgttttttt
gacatgtttt cttgactgag gcctgtttgt tctaaacttt ttcttcaaac
240ttttaacttt ttcatcacat cagaactttt ctacacatat aaacttttaa
cttttccgtc 300acatcgttcc aatttcaatc aaactttcaa ttttggcgtg
aactaaacac accctgagtc 360ttttattgct cctccgtacg ggttggctgg
ttgagaatag gtattttcag agagaaaatc 420tagatattgg gaggaacttg
gcatgaatgg ccactatatt tagagcaatt ctacggtcct 480tgaggaggta
ccatgaggta ccaaaatttt agtgtaaatt ttagtatctc attataacta
540ggtattatga ggtaccaaat ttacaataga aaaaatagta cttcatggta
ctttcttaag 600taccgtaaaa ttgctcctat atttaagggg atgtttatat
ctatccatat ccataatttg 660attttgataa gaaaaaatgt gagcacacca
agcatgtcca tgaccttgca ctcttggctc 720actcgtcaac tgtgaagaac
ctcaaaaatg ctcaatatag ctacaggtgc ctgaaaaaat 780aactttaaag
ttttgaacat cgatttcact aaacaacaat tattatctcc ctctgaaaga
840tgatagttta gaactctaga atcattgtcg gcggagaaag taaattattt
tccccaaatt 900tccagctatg aaaaaaccct caccaaacac catcaaacaa
gagttcacca aaccgcccat 960gcggccatgc tgtcacgcaa cgcaccgcat
tgcctgatgg ccgctcgatg catgcatgct 1020tccccgtgca catatccgac
agacgcgccg tgtcagcgag ctcctcgacc gacctgtgta 1080gcccatgcaa
gcatccaccc ccgccacgta caccccctcc tcctccctac gtgtcaccgc
1140tctctccacc tatatatgcc cacctggccc ctctcctccc atctccactt
cacccgatcg 1200cttcttcttc ttcttcgttg cattcatctt gctagcattt
aaatcaacta gggatatcac 1260aagtttgtac aaaaaagcag gcttcacaat
ggctaatctt cgtttaatga tcactttaat 1320cacggtttta atgatcacca
aatcatcaaa cggtattaaa attgatttac ctaaatccct 1380taacctcacc
ctctctaccg atccttccat catctccgca gcctctcatg acttcggaaa
1440cataaccacc gtgacccccg gcggcgtaat ctgcccctcc tccaccgctg
atatctctcg 1500tctcctccaa tacgccgcaa acggaaaaag tacattccaa
gtagcggctc gtggccaagg 1560ccactcctta aacggccaag cctcggtctc
cggcggagta atcgtcaaca tgacgtgtat 1620cactgacgtg gtggtttcaa
aagacaagaa gtacgctgac gtggcggccg ggacgttatg 1680ggtggatgtg
cttaagaaga cggcggagaa aggggtgtcg ccggtttctt ggacggatta
1740tttgcatata accgtcggag gaacgttgtc gaatggtgga attggtggtc
aagtgtttcg 1800aaacggtcct cttgttagta acgtccttga attggacgtt
attactggga aaggtgaaat 1860gttgacatgc tcgcgacagc taaacccaga
attgttctat ggagtgttag gaggtttggg 1920tcaatttgga attataacga
gagccagaat tgttttggac catgcaccta aacggcaaga 1980acgtttgata
tcaatggcaa acgatattgg agtcgactat ttagaaggtc aaatatttct
2040atcaaacggt gtcgttgaca cctctttttt cccaccttca gatcaatcta
aagtcgctga 2100tctagtcaag caacacggta tcatctatgt tcttgaagta
gccaagtatt atgatgatcc 2160caatctcccc atcatcagca aggttattga
cacattaacg aaaacattaa gttacttgcc 2220cgggttcata tcaatgcacg
acgtggccta cttcgatttc ttgaaccgtg tacatgtcga 2280agaaaataaa
ctcagatctt tgggattatg ggaacttcct catccttggc ttaacctcta
2340cgttcctaaa tctcggattc tcgattttca taacggtgtt gtcaaagaca
ttcttcttaa 2400gcaaaaatca gcttcgggac tcgctcttct ctatccaaca
aaccggaata agtacatact 2460tctcttcatt catatttatc ttcaagaacc
aaagnnatgg gacaatcgta tgtcggcgat 2520gataccagag atcgatgaag
atgttatata tattatcgga ctactacaat ccgctacccc 2580aaaggatctt
ccagaagtgg agagcgttaa cgagaagata attaggtttt gcaaggattc
2640aggtattaag attaagcaat atctaatgca ttatactagt aaagaagatt
ggattgagca 2700ttttggatca aaatgggatg atttttcgaa gaggaaagat
ctatttgatc ccaagaaact 2760gttatctcca gggcaagaca tcttttgaac
ccagctttct tgtacaaagt ggtgatatca 2820caagcccggg cggtcttcta
gggataacag ggtaattata tccctctaga tcacaagccc 2880gggcggtctt
ctacgatgat tgagtaataa tgtgtcacgc atcaccatgg gtggcagtgt
2940cagtgtgagc aatgacctga atgaacaatt gaaatgaaaa gaaaaaaagt
actccatctg 3000ttccaaatta aaattcattt taacctttta ataggtttat
acaataattg atatatgttt 3060tctgtatatg tctaatttgt tatcatccgg
gcggtcttct agggataaca gggtaattat 3120atccctctag acaacacaca
acaaataaga gaaaaaacaa ataatattaa tttgagaatg 3180aacaaaagga
ccatatcatt cattaactct tctccatcca tttccatttc acagttcgat
3240agcgaaaacc gaataaaaaa cacagtaaat tacaagcaca acaaatggta
caagaaaaac 3300agttttccca atgccataat actcgaac
3328421506DNAArabidopsis thaliana 42atggctaatc ttcgtttaat
gatcacttta atcacggttt taatgatcac caaatcatca 60aacggtatta aaattgattt
acctaaatcc cttaacctca ccctctctac cgatccttcc 120atcatctccg
cagcctctca tgacttcgga aacataacca ccgtgacccc cggcggcgta
180atctgcccct cctccaccgc tgatatctct cgtctcctcc aatacgccgc
aaacggaaaa 240agtacattcc aagtagcggc tcgtggccaa ggccactcct
taaacggcca agcctcggtc 300tccggcggag taatcgtcaa catgacgtgt
atcactgacg tggtggtttc aaaagacaag 360aagtacgctg acgtggcggc
cgggacgtta tgggtggatg tgcttaagaa gacggcggag 420aaaggggtgt
cgccggtttc ttggacggat tatttgcata taaccgtccg aggaacgttg
480tcgaatggtg gaattggtgg tcaagtgttt cgaaacggtc ctcttgttag
taacgtcctt 540gaattggacg ttattactgg gaaaggtgaa atgttgacat
gctcgcgaca gctaaaccca 600gaattgttct atggagtgtt aggaggtttg
ggtcaatttg gaattataac gagagccaga 660attgttttgg accatgcacc
taaacgggcc aaatggtttc ggatgctcta cagtgatttc 720acaactttta
caaaggacca agaacgtttg atatcaatgg caaacgatat tggagtcgac
780tatttagaag gtcaaatatt tctatcaaac ggtgtcgttg acacctcttt
tttcccacct 840tcagatcaat ctaaagtcgc tgatctagtc aagcaacacg
gtatcatcta tgttcttgaa 900gtagccaagt attatgatga tcccaatctc
cccatcatca gcaaggttat tgacacatta 960acgaaaacat taagttactt
gcccgggttc atatcaatgc acgacgtggc ctacttcgat 1020ttcttgaacc
gtgtacatgt cgaagaaaat aaactcagat ctttgggatt atgggaactt
1080cctcatcctt ggcttaacct ctacgttcct aaatctcgga ttctcgattt
tcataacggt 1140gttgtcaaag acattcttct taagcaaaaa tcagcttcgg
gactcgctct tctctatcca 1200acaaaccgga ataaatggga caatcgtatg
tcggcgatga taccagagat cgatgaagat 1260gttatatata ttatcggact
actacaatcc gctaccccaa aggatcttcc agaagtggag 1320agcgttaacg
agaagataat taggttttgc aaggattcag gtattaagat taagcaatat
1380ctaatgcatt atactagtaa agaagattgg attgagcatt ttggatcaaa
atgggatgat 1440ttttcgaaga ggaaagatct atttgatccc aagaaactgt
tatctccagg gcaagacatc 1500ttttga 1506432746DNAArtificial
sequenceexpression cassette 43cttctacatc ggcttaggtg tagcaacacg
actttattat tattattatt attattatta 60ttattttaca aaaatataaa atagatcagt
ccctcaccac aagtagagca agttggtgag 120ttattgtaaa gttctacaaa
gctaatttaa aagttattgc attaacttat ttcatattac 180aaacaagagt
gtcaatggaa caatgaaaac catatgacat actataattt tgtttttatt
240attgaaatta tataattcaa agagaataaa tccacatagc cgtaaagttc
tacatgtggt 300gcattaccaa aatatatata gcttacaaaa catgacaagc
ttagtttgaa aaattgcaat 360ccttatcaca ttgacacata aagtgagtga
tgagtcataa tattattttc tttgctaccc 420atcatgtata tatgatagcc
acaaagttac tttgatgatg atatcaaaga acatttttag 480gtgcacctaa
cagaatatcc aaataatatg actcacttag atcataatag agcatcaagt
540aaaactaaca ctctaaagca accgatggga aagcatctat aaatagacaa
gcacaatgaa 600aatcctcatc atccttcacc acaattcaaa tattatagtt
gaagcatagt agtaatttaa 660atcaactagg gatatcacaa gtttgtacaa
aaaagcaggc ttcacaatgg ctaatcttcg 720tttaatgatc actttaatca
cggttttaat gatcaccaaa tcatcaaacg gtattaaaat 780tgatttacct
aaatccctta acctcaccct ctctaccgat ccttccatca tctccgcagc
840ctctcatgac ttcggaaaca taaccaccgt gacccccggc ggcgtaatct
gcccctcctc 900caccgctgat atctctcgtc tcctccaata cgccgcaaac
ggaaaaagta cattccaagt 960agcggctcgt ggccaaggcc actccttaaa
cggccaagcc tcggtctccg gcggagtaat 1020cgtcaacatg acgtgtatca
ctgacgtggt ggtttcaaaa gacaagaagt acgctgacgt 1080ggcggccggg
acgttatggg tggatgtgct taagaagacg gcggagaaag gggtgtcgcc
1140ggtttcttgg acggattatt tgcatataac cgtcggagga acgttgtcga
atggtggaat 1200tggtggtcaa gtgtttcgaa acggtcctct tgttagtaac
gtccttgaat tggacgttat 1260tactgggaaa ggtgaaatgt tgacatgctc
gcgacagcta aacccagaat tgttctatgg 1320agtgttagga ggtttgggtc
aatttggaat tataacgaga gccagaattg ttttggacca 1380tgcacctaaa
cggcaagaac gtttgatatc aatggcaaac gatattggag tcgactattt
1440agaaggtcaa atatttctat caaacggtgt cgttgacacc tcttttttcc
caccttcaga 1500tcaatctaaa gtcgctgatc tagtcaagca acacggtatc
atctatgttc ttgaagtagc 1560caagtattat gatgatccca atctccccat
catcagcaag gttattgaca cattaacgaa 1620aacattaagt tacttgcccg
ggttcatatc aatgcacgac gtggcctact tcgatttctt 1680gaaccgtgta
catgtcgaag aaaataaact cagatctttg ggattatggg aacttcctca
1740tccttggctt aacctctacg ttcctaaatc tcggattctc gattttcata
acggtgttgt 1800caaagacatt cttcttaagc aaaaatcagc ttcgggactc
gctcttctct atccaacaaa 1860ccggaataag tacatacttc tcttcattca
tatttatctt caagaaccaa agnnatggga 1920caatcgtatg tcggcgatga
taccagagat cgatgaagat gttatatata ttatcggact 1980actacaatcc
gctaccccaa aggatcttcc agaagtggag agcgttaacg agaagataat
2040taggttttgc aaggattcag gtattaagat taagcaatat ctaatgcatt
atactagtaa 2100agaagattgg attgagcatt ttggatcaaa atgggatgat
ttttcgaaga ggaaagatct 2160atttgatccc aagaaactgt tatctccagg
gcaagacatc ttttgaaccc agctttcttg 2220tacaaagtgg tgatatcaca
agcccgggcg gtcttctagg gataacaggg taattatatc 2280cctctagatc
acaagcccgg gcggtcttct acgatgattg agtaataatg tgtcacgcat
2340caccatgggt ggcagtgtca gtgtgagcaa tgacctgaat gaacaattga
aatgaaaaga 2400aaaaaagtac tccatctgtt ccaaattaaa attcatttta
accttttaat aggtttatac 2460aataattgat atatgttttc tgtatatgtc
taatttgtta tcatccgggc ggtcttctag 2520ggataacagg gtaattatat
ccctctagac aacacacaac aaataagaga aaaaacaaat 2580aatattaatt
tgagaatgaa caaaaggacc atatcattca ttaactcttc tccatccatt
2640tccatttcac agttcgatag cgaaaaccga ataaaaaaca cagtaaatta
caagcacaac 2700aaatggtaca agaaaaacag ttttcccaat gccataatac tcgaac
27464417PRTArtificial sequenceprotein signature 44His Thr Asp Tyr
Leu Xaa Xaa Xaa Xaa Gly Gly Thr Leu Ser Xaa Xaa1 5 10
15Gly4519PRTArtificial sequenceprotein signature 45Xaa Leu Phe Xaa
Xaa Xaa Xaa Gly Xaa Leu Gly Gln Phe Gly Xaa Xaa1 5 10 15Xaa Xaa
Ala
* * * * *
References