U.S. patent application number 13/585675 was filed with the patent office on 2013-03-07 for genetically modified plants which synthesize a low amylose starch with increased swelling power.
This patent application is currently assigned to Bayer CropScience AG. The applicant listed for this patent is Claus Frohberg, Ralf-Christian Schmidt. Invention is credited to Claus Frohberg, Ralf-Christian Schmidt.
Application Number | 20130060016 13/585675 |
Document ID | / |
Family ID | 38001903 |
Filed Date | 2013-03-07 |
United States Patent
Application |
20130060016 |
Kind Code |
A1 |
Frohberg; Claus ; et
al. |
March 7, 2013 |
GENETICALLY MODIFIED PLANTS WHICH SYNTHESIZE A LOW AMYLOSE STARCH
WITH INCREASED SWELLING POWER
Abstract
The present invention relates to genetically modified
monocotyledonous plant cells and plants whose starch has an
apparent amylose content of less than 5% by weight and an increased
activity of a protein with the activity of a starch synthase II and
an increased activity of a protein with the activity of a glucan,
water dikinase. Such plants synthesize starch with an increased
hot-water swelling power. Methods and processes for the
generation/preparation of these plant cells, plants, starches and
flours are likewise subject matter of the present invention.
Inventors: |
Frohberg; Claus;
(Kleinmachnow, DE) ; Schmidt; Ralf-Christian;
(Stahnsdorf, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Frohberg; Claus
Schmidt; Ralf-Christian |
Kleinmachnow
Stahnsdorf |
|
DE
DE |
|
|
Assignee: |
Bayer CropScience AG
Monheim am Rhein
DE
|
Family ID: |
38001903 |
Appl. No.: |
13/585675 |
Filed: |
August 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12524542 |
Jul 24, 2009 |
8252975 |
|
|
PCT/EP2008/000614 |
Jan 23, 2008 |
|
|
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13585675 |
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60898427 |
Jan 30, 2007 |
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Current U.S.
Class: |
536/102 |
Current CPC
Class: |
C12N 9/1051 20130101;
C12N 9/1294 20130101; A23L 29/212 20160801; C12N 15/8245
20130101 |
Class at
Publication: |
536/102 |
International
Class: |
C08B 31/00 20060101
C08B031/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 26, 2007 |
EP |
EP 07090009.7 |
Claims
1-11. (canceled)
12. A modified starch comprising an apparent amylose content of
below 5% by weight and a hot water swelling power of at least 60
g/g.
13. The modified starch of claim 12, wherein said starch has a
hot-swelling power of between 60-100 g/g.
14. A process for the preparation of a derivatized starch,
comprising derivatizing the modified starch of claim 12.
15. A derivatized starch, produced by the process of claim 14.
16. (canceled)
17. A flour comprising the modified starch of claim 12.
18. (canceled)
19. (canceled)
20. A product comprising the starch of claim 12.
21. A product comprising the flour of claim 17.
22. The modified starch of claim 13, wherein said starch has a
hot-swelling power of between 70-95 g/g.
23. The modified starch of claim 13, wherein said starch has a
hot-swelling power of between 80-95 g/g.
24. The modified starch of claim 13, wherein said starch has a
hot-swelling power of between 80-90 g/g.
25. A modified rice starch comprising an apparent amylose content
of below 5% by weight and a hot water swelling power of at least 60
g/g.
26. The modified rice starch of claim 25, wherein said starch has a
hot-swelling power of between 60-100 g/g.
27. The modified rice starch of claim 26, wherein said starch has a
hot-swelling power of between 70-95 g/g.
28. The modified rice starch of claim 26, wherein said starch has a
hot-swelling power of between 80-95 g/g.
29. The modified rice starch of claim 26, wherein said starch has a
hot-swelling power of between 80-90 g/g.
30. A process for the preparation of a derivatized starch,
comprising derivatizing the modified rice starch of claim 25.
31. A derivatized starch, produced by the process of claim 30.
32. A flour comprising the modified rice starch of claim 25.
33. A product comprising the rice starch of claim 25.
34. A product comprising the flour of claim 32.
Description
[0001] The present invention relates to genetically modified
monocotyledonous plant cells and plants whose starch has an
apparent amylose content of less than 5% by weight and an increased
activity of a protein with the activity of a starch synthase II and
an increased activity of a protein with the activity of a glucan,
water dikinase. Such plants synthesize starch with an increased
hot-water swelling power. Methods and processes for the
generation/preparation of these plant cells, plants, starches and
flours are likewise subject matter of the present invention.
[0002] Besides oils, fats and proteins, polysaccharides are the
most important renewable resources from plants. Starch, which is
one of the most important reserve materials in Higher Plants, plays
a central role in the polysaccharides, besides cellulose.
[0003] Furthermore, starch is a nutritionally essential component
of human and animal food. The structural features of the starch
which is present in foodstuffs may have an effect on the functional
properties (for example water-binding capacity, swelling power),
the nutritional characteristics (for example digestibility, effect
of the foodstuff on the glycemic index) or the structural
characteristics (for example sliceability, texture, stickiness,
processability) of a very wide range of foodstuffs. Food products
therefore frequently comprise a starch with specific structural
features which bring about the desired characteristics of the
foodstuff in question. Also, the starch which is present in the
plant tissues may affect the characteristics of foodstuffs which
comprise starch-storing plant tissues (for example grains, fruits,
flours).
[0004] The polysaccharide starch is a polymer made up of chemically
uniform units, the glucose molecules. However, it constitutes a
highly complex mixture of different forms of molecules which differ
with regard to their degree of polymerization, the occurrence of
branches of the glucose chains and their chain lengths and which,
moreover, may be modified, for example phosphorylated. Starch
therefore does not constitute a uniform raw material. In
particular, one differentiates between amylose and amylopectin. In
typical plants used for industrial starch production or as
foodstuffs such as, for example, maize, rice, wheat or potato,
amylose accounts for approximately 20%-25% and amylopectin for
approximately 70%-80% of the synthesized starch.
[0005] The functional, nutritional or structure-imparting
characteristics of starch such as, for example, solubility, the
retrogradation behavior, the water-binding capacity, the
film-forming properties, the viscosity, the gelatination
properties, the freeze-thaw stability, the stability to acid, the
gel strength, the swelling power, the digestibility, the size of
the starch grains of starches are affected, inter alia, by the
structural features of the starch, such as the amylose/amylopectin
ratio, the molecular weight of the glucose polymers, the side-chain
distribution pattern, the ion content, the lipid and protein
content and/or the starch grain morphology.
[0006] Methods based on plant breeding may be used to modify
selected structural characteristics of the starch and therefore
functional, nutritional or structure-imparting characteristics of
starch in plant cells. However, at present this is only possible
for selected structural features of starch (for example
amylopectin/amylose content, U.S. Pat. No. 5,300,145). It is not
possible currently for example to influence the starch phosphate
content in plants by plant breeding methods alone.
[0007] An alternative to plant breeding methods is the targeted
modification of starch-producing plants by means of recombinant
methods. However, a prerequisite for doing so is the identification
and characterization of the enzymes involved in starch synthesis
and/or starch modification, and their subsequent functional
analysis in transgenic plants.
[0008] A variety of enzymes which characterize different reactions
are involved in the synthesis of starch in plant cells. Starch
synthases (EC2.4.1.21, ADP-glucose:1,4-alpha-D-glucan
4-alpha-D-glucosyltransferase) catalyze a polymerization reaction
by transferring a glucosyl residue from ADP-glucose to
alpha-1,4-glucans, where the glucosyl residue transferred is linked
with the alpha-1,4-glucan by generating an alpha-1,4-linkage.
Several isoforms of starch synthases have been identified in each
of the plants studied to date. Two classes of starch synthases can
be distinguished: the granule-bound starch synthases (GBSS) and the
soluble starch synthases (in the context of the present invention
also abbreviated to "SS"). Granule-bound starch synthases catalyze
the synthesis of amylose, while soluble starch synthases are
involved in the synthesis of amylopectin (Ball and Morell, 2003,
Annu. Rev, Plant Biol. 54, 207-233; Teltow et al., 2004, J. Expt.
Bot. 55(406), 2131-2145). The group of the soluble starch synthases
has several isoforms which are referred to the specialist
literature as SSI, SSII, SSIII, SSIV and SSV. The association of
starch synthases to the individual isoforms (SSI, SSII, SSIII,
SSIV, SSV) is made with the sequence homologies of the respective
protein sequences of the enzymes in question (Ball and Morell,
2003, Annu. Rev, Plant Biol. 54, 207-233). Each individual isoform
of the soluble starch synthases has, in accordance with current
teaching, allocated to it a specific function in the synthesis of
starch. While only one isoform of SSI proteins has been detected in
dicotyledonous plants, two different classes of SSII proteins have
been detected in some monocotyledonous plants (for example maize),
which are referred to as SSIIa and SSIIb, respectively. In
monocotyledonous plants, SSIIa is expressed preferentially in the
endosperm, and SSIIb preferentially in the leaf tissue (Teltow et
al., 2004, J. Expt. Bot. 55(406): 2131-2145). The specific
function, in particular of the individual soluble starch synthases,
in the synthesis of the starch is currently not fully explained
(Ball and Morell, 2003, Annu. Rev, Plant Biol. 54: 207-233).
[0009] The functional, nutritional or structure-parting
characteristics of starch are also affected by the phosphate
content, a noncarbon component. Here, one has to distinguish
between phosphate which is bonded covalently to starch glucose
molecules in the form of monoesters (referred to as starch
phosphate in the context of the present invention) and phosphate in
the form of starch-associated phospholipids.
[0010] The starch phosphate content varies with the plant cultivar.
Thus, for example, certain maize mutants synthesize a starch with
an increased starch phosphate content (waxy maize at 0.002% and
high-amylose maize at 0.013%), while traditional maize varieties
only contain traces of starch phosphate. Likewise, small amounts of
starch phosphate are found in wheat (0.001%), while no starch
phosphate was detected in oats and Sorghum. In waxy rice mutants,
less starch phosphate (0.003%) was found than in traditional rice
varieties (0.013%). Significant amounts of starch phosphate were
detected in plants which synthesize tuber or root storage starch,
such as, for example, tapioca (0.008%), sweet potato (0.011%),
arrow root (0.021%) or potato (0.089%). The above-cited percentages
for the starch phosphate content refer in each case to the dry
weight of the starch and have been determined by Jane et al. (1996,
Cereal Foods World 41 (11): 827-832).
[0011] Starch phosphate may be present in the form of monoesters at
the C2, C3 or C6 position of the polymerized glucose monomers
(Takeda and Hizukuri, 1971, Starch/Starke 23: 267-272). The
phosphate distribution of the phosphate in starch synthesized by
plants is generally distinguished by the fact that approximately
30% to 40% of the phosphate residues are bonded covalently in the
C3 position and approximately 60% to 70% of the phosphate residues
in the C6 position of the glucose molecules (Blennow et al., 2000,
Int. J. of Biological Macromolecules 27: 211-218). Blennow et al.
(2000, Carbohydrate Polymers 41: 163-174) determined a starch
phosphate content which is bonded in the C6 position of the glucose
molecules for a variety of starches such as, for example, potato
starch (between 7.8 and 33.5 nmol per mg starch, depending on
variety), starch from various Curcuma species (between 1.8 and 63
nmol per mg starch), tapioca starch (2.5 nmol per mg starch), rice
starch (1.0 nmol per mg starch), mungbean starch (3.5 nmol per mg
starch) and sorghum starch (0.9 nmol per mg starch). These authors
did not detect any starch phosphate bonded in the C6 position in
barley starch and starch from various waxy mutants of maize. No
relationship between the genotype of a plant and the starch
phosphate content has been established as yet (Jane et al., 1996,
Cereal Foods World 41 (11): 827-832).
[0012] To date there have been described two proteins which mediate
the introduction of covalent bonds of phosphate residues to
starch's glucose molecules. The first protein has the enzymatic
activity of an alpha-glucan, water dikinase (GWD, E.C.: 2.7.9.4)
(Ritte et al., 2002, PNAS 99: 7166-7171), is frequently referred to
as R1 in particular in the earlier scientific literature and is
bound to the starch granules of storage starch in potato tubers
(Lorberth et al., 1998, Nature Biotechnology 16: 473-477). The
second protein described in the literature which catalyzes the
introduction of starch phosphate into starch has the enzymatic
activity of a phosphor-glucan, water dikinase (PWD, E.C.: 2.7.9.5)
(Kotting et al., 2005, Plant Physiol. 137: 2424-252, Baunsgaard et
al., 2005, Plant Journal 41: 595-605).
[0013] One essential difference between GWD and PWD is that GWD is
capable of utilizing unphosphorylated starch as its substrate, i.e.
a de novo phosphorylation of unphosphorylated starch can be
catalyzed by GWD, while PWD requires already phosphorylated starch
as its substrate, i.e. introduces additional phosphate into
already-phosphorylated starch (Kotting et al., 2005, Plant Physiol.
137: 2424-252, Baunsgaard et al., 2005, Plant Journal 41: 595-605).
A further essential difference between GWD and PWD is that GWD
introduces phosphate groups exclusively in the C6 position of the
starch's glucose molecules, while PWD exclusively phosphorylates
the C3 position of starch's glucose molecules (Ritte et al., 2006,
FEBS Letters 580: 4872-4876). In the reaction which is catalyzed by
GWD, or PWD, the starting materials alpha-1,4-glucan (in the case
of GWD) and phosphorylated alpha-1,4-glucan (in the case of PWD),
respectively, adenosin triphosphate (ATP) and water are converted
into the products glucan phosphate (starch phosphate), inorganic
phosphate and adenosin monophosphate (Kotting et al., 2005, Plant
Physiol. 137: 2424-252; Ritte et al., 2002, PNAS 99:
7166-7171).
[0014] Wheat plants which have an elevated activity of GWD proteins
as the result of the expression of a GWD-encoding gene from potato
are described in WO 02/34923. In comparison with corresponding
wild-type plants in which no starch phosphate could be detected,
these plants synthesize starch with significant amounts of starch
phosphate in the C6 position of the glucose molecules.
[0015] WO 05/002359 describes the expression, in maize plants, of a
potato GWD which has been optimized for the codon usage in maize
plants. By means of .sup.31P NMR, a total phosphate content of
0.0736% phosphate based on the amount of glucose (bonded at the C6,
C3 and C2 position of the glucose molecules) of the maize starch in
question was determined. If a molecular weight 98 is assumed for
phosphate (H.sub.3PO.sub.4), a total phosphate content of
approximately 7.5 nmol of phosphate per mg of starch results for
the total phosphate content of 0.0736%--which has been determined
in WO 05/002359 --for starch isolated from transgenic maize plants.
Plants which as the result of the expression of a PWD-encoding gene
from Arabidopsis thaliana show an increased activity of a PWD
protein are described in WO 05/095617. In comparison to
corresponding untransformed wild type plants, these plants have an
increased starch phosphate content.
[0016] An important functional characteristic, for example when
processing starches in the food industry, is the swelling power.
Various structural characteristics of starches, such as the
amylose-/amylopectin ratio, the side chain length, the molecular
weight distribution of the starch polymers, the number of branches
and the amount of starch phosphate have an effect on functional
characteristics, in particular on the swelling power of the
starches in question (Narayana and Moorthy, 2002, Starch/Starke 54:
559-592).
[0017] Amylose has long been regarded as a linear polymer
consisting of .alpha.-1,4-glycosidically linked .alpha.-D-glucose
monomers. However, more recent studies have demonstrated the
presence of .alpha.-1,6-glycosidic branch points (approx. 0.1%)
(Hizukuri and Takagi, 1984, Carbohydr. Res. 134: 1-10; Takeda et
al., 1984, Carbohydr. Res. 132: 83-92).
[0018] Amylopectin constitutes a complex mixture of glucose chains
with different branching patterns. In contrast to amylose,
amylopectin comprises more branches. Side chains are linked via
.alpha.-1,6-glycosidic linkages to the main chain of
.alpha.-D-Glucose monomers, which are .alpha.-1,4-glycosidically
linked. According to the literature (Voet and Voet, 1990.
Biochemistry, John Wiley & Sons), the .alpha.-1,6-branches
occur on average every 24 to 30 glucose residues. This corresponds
to a degree of branching of approx. 3%-4%. The data on the degree
of branching vary and depend on the origin of the starch in
question (for example plant species, plant variety and the like).
In typical plants used for the industrial production of starch,
such as, for example, maize, wheat or potato, amylose starch
accounts for approximately 20%-30% and amylopectin starch for
approximately 70%-80% of the starch synthesized.
[0019] Another important difference between amylose and amylopectin
is their molecular weight. While amylose, depending on the origin
of the starch, has a molecular weight of 5.times.10.sup.5-10.sup.6
Da, the molecular weight of amylopectin is between 10.sup.7 and
10.sup.8 Da. The two macromolecules can be distinguished on the
basis of their molecular weight and their different
physical-chemical characteristics, and the simplest way of
visualizing this is through their different iodine-binding
characteristics.
[0020] A large number of technical applications only require
amylopectin since amylopectin has a thickening action. Amylose has
a gelling action and is therefore rather undesired for a number of
uses. Pure amylopectin starch makes possible a very uniform surface
structure combined with high viscosity, stability and transparency.
Possible applications for these starches are in papermaking, in the
adhesives industry, the textiles industry, the building industry
and the cosmetics industry. Furthermore, amylopectin starch is the
preferred starting material for the preparation of maltodextrins as
the result of their increased solubility in water, stability to
dissolution and transparency in comparison with maltodextrins which
are prepared from amylose-comprising starches.
[0021] In the food industry, amylopectin starches are frequently
employed as stabilizers, binders and for improving texture.
Amylopectin starches are particularly advantageous in the case of
those processing steps at which large temperature variations occur
during processing and finishing (for example
freeze-thaw-stability). The use of amylopectin starches in the food
industry is growing, in particular taking into consideration the
increasing demand for (semi-)finished products.
[0022] GBSSI ("granule-bound starch synthase I") is involved in
amylose formation. To date, plants have been described in which the
activity of the granule-bound starch synthase GBSSI is reduced
(Shure et al., 1983, Cell 35: 225-233; Hovenkamp-Hermelink et al.,
1987, Theoretical and Applied Genetics 75: 217-221; Visser et al.,
1991, Mol. Gen. Genet. 225: 289-296; Hergersberg, 1988, Thesis,
University of Cologne; WO 92/11376). Furthermore, there are known
mutants which lack a functional GBSSI gene and which therefore
synthesize an amylose-free (=amylopectin) starch (Kossmann and
Lloyd 2000, Critical Reviews in Plant Sciences, 19(3): 171-226).
The endosperm of corresponding GBSSI mutant of maize is waxy in
appearance, which is why the term "waxy" endosperm has been
introduced as a synonym for amylose-free starches.
[0023] When describing the swelling power of starch, one must
distinguish between swelling power in cold water (for example room
temperature) and swelling power in warm or hot water. The swelling
power of native starches in cold water is negligible, if not
nonexistent, while physically modified (pregellatized, dried)
starches are capable of swelling even in cold water. Preparation
methods for cold water swelling starches are described for example
in U.S. Pat. No. 4,280,851. In the context of the present
invention, the term "swelling power" refers to the behavior of
starch in warm/hot aqueous suspensions. The swelling power is
routinely determined by warming starch granules in the presence of
an excess of water, removing unbound water after centrifugation of
the suspension and forming the quotient from the weight of the
residue obtained and that of the amount of starch weighed in. When
carrying out this procedure, warming the starch suspension causes
crystalline regions of the starch granules to dissolve and the
water molecules to intercalate into the starch granules without
dissolving the structure of the starch granule itself, i.e. only a
swelling of the individual starch granules takes place.
[0024] In comparison with starches from cereals, starches isolated
from tubers or tuber-like tissues have a considerably higher
hot-water swelling power.
[0025] For potato starches isolated from various varieties, a
maximum swelling power of 74.15 g/g (variety Kufri Jyoti) at
85.degree. C. has been determined (Singh et al., 2002, Journal of
the Science of Food and Agriculture 82: 1376-1383), using the
method of Leach et al. (1959, Cereal Chemistry 36: 534-544).
Takizawa et al. (2004, Brazilian Archives of Biology and Technology
47(6): 921-931) determined a swelling power of 100 g/g for potato
starch (90.degree. C., using the method of Leach et al., above).
Wheat starch isolated from various cultivars has a swelling power
of 16.6 g/g to 26.0 g/g (temperature: boiling aqueous 0.1%
AgNO.sub.3 suspension) (Yamamori and Quynh, 2000, Theor Appl Genet.
100: 23-38). Starch isolated from various cultivars of hull-less
barley has a swelling power of 16.5 g/g or 19.3 g/g, and waxy, or
amylose-free starch of various cultivars of said barley has a
swelling power of 36.0 g/g to 55.7 g/g (temperature: 70.degree. C.,
aqueous 0.1% AgNO.sub.3, Yasui et al., 2002, Starch/Starke 54:
179-184). For maize starch, a swelling power of 22.3 g/g has been
determined, and for high-amylose maize starches a swelling power of
9.6 g/g (Nylon V), 6.1 g/g (Nylon VII) or 3.9 g/g (LAPS=Low
AmyloPectin Starch) (90.degree. C., Shi et al., 1998, J. Cereal
Sci. 27: 289-299). U.S. Pat. No. 6,299,907 states a swelling power
of 35.4 g/g for waxy maize starch. For starch isolated from various
rice cultivars, a swelling power of 26.0 g/g to 33.2 g/g has been
determined (Sodhi and Singh, 2003, Food Chemistry 80: 99-108),
using the method of Leach et al. (above). Chen et al. (2003,
Starch/Starke 55: 203-212) determined a swelling power of
approximately 25 g/g to approximately 49 g/g (95.degree. C.,
aqueous suspension) for various mixtures of waxy rice starches with
high-amylose rice starches. Yasui et al. (2002, Starch/Starke 54:
179-184) determined a swelling power of 55.7 g/g (measured in
boiling water in 0.1% aqueous silver nitrate solution) for an
amylase-free rice starch. By producing derivatives of native
starches, it is possible to modify functional characteristics of
the starches. Cross-linked wheat starches have a swelling power of
from 6.8 g/g to 8.9 g/g, depending on the degree of crosslinking,
acetylated wheat starches have a swelling power of a maximum of
10.3 g/g, and simultaneously crosslinked and acetylated wheat
starches have a swelling power of 9.4 g/g, while the corresponding
non-derivatized starches have a swelling power of 8.8 g/g (measured
at 90.degree. C.; Van Hung and Morita, 2005, Starch/Starke 57:
413-420).
[0026] For acetylated waxy rice starches, a swelling power of
approximately 30 g/g has been determined and for crosslinked waxy
rice starch a swelling power of approximately 15 g/g, while
corresponding non-derivatized waxy rice starch had a swelling power
of approximately 41 g/g. Acetylated rice starch had a swelling
power of approximately 20 g/g and crosslinked rice starch a
swelling power of approximately 13 g/g, while corresponding
non-derivatized rice starch had a swelling power of approximately
14 g/g (measured at 90.degree. C., Liu et al., 1999, Starch/Starke
52: 249-252). U.S. Pat. No. 6,299,907 describes crosslinked
starches, where the crosslinking reaction had been carried out
after preswelling the starches in question in a sodium
hydroxide/sulfate solution. Depending on the degree of
crosslinking, wheat starch was found to have a swelling power of
from 6.8 g/g to 7.3 g/g (corresponding non-derivatized wheat starch
14.7 g/g), wheat hydroxypropyl starch a swelling power of 9.7 g/g
(corresponding non-derivatized wheat starch 22.9 g/g), crosslinked
maize starch a swelling power of 5.9 g/g (corresponding
non-derivatized maize starch 16.7 g/g), crosslinked waxy maize
starch a swelling power of 8.3 g/g (corresponding non-derivatized
waxy maize starch 35.4 g/g), and crosslinked potato starch a
swelling power of 6.7 g/g (corresponding non-derivatized potato
starch was not specified in detail) (measurements at 95.degree.
C.). This reveals that the swelling power of starch cannot be
increased substantially, if at all, by current derivatization
methods.
[0027] The object of the present invention is to provide modified
waxy starches with altered functional characteristics, and novel
plant cells and plants which synthesize a waxy starch with altered
functional characteristics, as well as methods and means for
generating said plants and/or plant cells.
[0028] In particular, the altered functional characteristics
consist in the fact that the modified starches have an increased
hot-water swelling power.
[0029] Thus, the present invention relates to genetically modified
monocotyledonous plant cells or genetically modified
monocotyledonous plants whose starch has an apparent amylose
content of less than 5% by weight, and which additionally have an
increased activity of a protein with the enzymatic activity of a
starch synthase II and additionally an increased activity of a
protein with the enzymatic activity of a glucan, water dikinase in
comparison with corresponding genetically not modified wild-type
plant cells, or corresponding genetically not modified wild-type
plants.
[0030] In this context, the genetic modification may be any genetic
modification which leads to the synthesis of a starch with less
than 5% by weight amylose and simultaneously to an increase in the
activity of at least one protein with the activity of a starch
synthase II and (simultaneously) of at least one protein with the
activity of a glucan, water dikinase in genetically modified plant
cells or genetically modified plants in comparison with
corresponding not genetically modified wild-type plant cells or
wild-type plants.
[0031] In the context of the present invention, the term "wild-type
plant cell" means plant cells which act as starting material for
the generation of the plant cells according to the invention, i.e.
whose genetic information, with the exception of the introduced
genetic modification, corresponds to that of a plant cell according
to the invention.
[0032] In the context of the present invention, the term "wild-type
plant" means plants which acted as starting material for the
generation of the plants according to the invention, i.e. whose
genetic information, with the exception of the introduced genetic
modification, corresponds to that of a plant according to the
invention.
[0033] In the context of the present invention, the term
"corresponding" means that, when comparing several objects, the
objects in question which are compared with one another are
maintained under identical conditions. In the context of the
present invention, the term "corresponding" in the context of
wild-type plant cell or wild-type plant means that the plant cells
or plants which are compared with one another were grown under
identical culture conditions and have an identical (culture)
age.
[0034] The term "monocotyledonous plants" refers to the monocots.
Botanically, they belong to one of the three classes of the
angiosperms (Magnoliophyta). In contrast to dicots,
monocotyledonous plants are distinguished by the fact that the
embryo typically has only one cotyledon primordium (Greek:
monos="single" and kotyledon="cotyledon"). Moreover, they have
sheathed vascular bundles, i.e. phloem and xylem are not separated
by a meristem, which is why no secondary thickening of the stem is
possible. This class of plants includes, inter alia, the grasses
with the orders Cyperales and Poales, and a large number of other
families.
[0035] In the context of the present invention, the term "increased
activity of at least one protein with the (enzymatic) activity of a
starch synthase II" means an increase in the expression of
endogenous genes which code for proteins with the activity of a
starch synthase II and/or an increase in the amount of proteins
with the activity of a starch synthase II in the cells and/or an
increase in the activity of proteins with the activity of a starch
synthase II in the cells.
[0036] In the context of the present invention, the term "increased
activity of a protein with the (enzymatic) activity of a glucan,
water dikinase" means an increase in the expression of endogenous
genes which code for proteins with the activity of a glucan, water
dikinase and/or an increase in the amount of proteins with the
activity of a glucan, water dikinase in the cells and/or an
increase in the activity of proteins with the activity of a glucan,
water dikinase in the cells.
[0037] The increase in expression can be determined, for example,
by measuring the amount of transcripts which code for proteins with
the activity of a starch synthase II or proteins with the activity
of a glucan, water dikinase. This can be done for example by
northern blot analysis or by Q-PCR (quantitative transcription
polymerase chain reaction).
[0038] An increase in the amount of a protein with the activity of
a glucan, water dikinase means, in this context, preferably an
increase in the amount of the protein in question by at least 50%,
in particular by at least 70%, preferably by at least 85% and
especially preferably by at least 100% in comparison to
corresponding, not genetically modified cells.
[0039] An increase in the amount of protein with the activity of a
glucan, water dikinase also means that plants or plant cells which
contain no detectable amount of proteins with the activity of a
glucan, water dikinase will, following genetic modification
according to the invention, contain a detectable amount of protein
with the activity of a glucan, water dikinase.
[0040] Methods for raising antibodies which specifically react to a
certain protein, i.e. which specifically bind to said protein, are
known to the skilled worker (see, for example, Lottspeich and
Zorbas (Eds.), 1998, Bioanalytik, Spektrum akad, Verlag,
Heidelberg, Berlin, ISBN 3-8274-0041-4). The raising of such
antibodies can be commissioned from some companies (for example
Eurogentec, Belgian). Antibodies by means of which an increase in
the amount of protein with the activity of a glucan, water dikinase
can be determined by means of immunological methods are described
by Lorberth et al. (1998, Nature Biotechnology 16: 473-477) and
Ritte et al. (2000, Plant Journal 21: 387-391). Antibodies by means
of which an increase in the amount of protein with the activity of
a starch synthase II can be determined by means of immunological
methods are described by Walter ("Untersuchungen der Expression and
Funktion der Starkesynthase II (SSII) aus Weizen (Triticum
aestivum) [Studies into the expression and function of starch
synthase II (SSII) from wheat (Triticum aestivum)]", PhD Thesis at
the Faculty of Biology, University of Hamburg, ISBN
3-8265-8212-8).
[0041] The amount of the activity of a protein with the activity of
a glucan, water dikinase can be detected for example as described
in the literature (Mikkelsen et al., 2004, Biochemical Journal 377:
525-532; Ritte et al., 2002, PNAS 99: 7166-7171).
[0042] The amount of the activity of a protein with the activity of
a starch synthase II can be determined for example as described in
the literature (Nishi et al., 2001, Plant Physiology 127: 459-472).
A preferred method for determining the amount of the activity of a
protein with the activity of a starch synthase II is described
under "General Methods".
[0043] Preferably, plant cells according to the invention or plants
according to the invention have an activity of a protein with the
activity of a starch synthase II which is increased by at least a
factor of 2, preferably by at least a factor of 6, in comparison
with corresponding genetically not modified wild-type plant cells,
or wild-type plants.
[0044] The construction of proteins with the activity of a starch
synthase II (ADP-glucose:1,4-alpha-D-glucan
4-alpha-D-glucosyltransferase; EC 2.4.1.21) shows a sequence of
certain domains. At the N terminus, they have a signal peptide for
the transport into plastids. From the N terminus toward the C
terminus, there follow an N-terminal region and a catalytic domain
(Li et al., 2003, Funct Integr Genomics 3, 76-85). Further analyses
based on amino acid sequence alignments
(http://hits.isb-sib.ch/cgi-bin/PFSCAN) of various proteins with
the activity of a starch synthase II revealed that these proteins
have three specific domains. In the amino acid sequence shown as
SEQ ID NO 4, the amino acids 322 to 351 represent domain 1, the
amino acids 423 to 462 domain 2 and the amino acids 641 to 705 the
domain 3. Domain 1 is encoded by the nucleotides 1190 to 1279,
domain 2 by the nucleotides 1493 to 1612 and domain 3 by the
nucleotides 2147 to 2350 of the nucleic acid sequence shown as SEQ
ID NO 3.
[0045] In the context of the present invention, the term "protein
with the activity of a starch synthase II" is understood as meaning
a protein which catalyzes a glucosylation reaction in which glucose
residues of the substrate ADP-glucose are transferred to
alpha-1,4-linked glucan chains, with formation of an
alpha-1,4-linkage
(ADP-Glucose+{(1,4)-alpha-D-glucosyl}(N)<=>ADP+{(1,4)-alpha-D-gluco-
syl}(N+1)), where the amino acid sequence of the protein with the
activity of a protein of a starch synthase II has at least 86%,
preferably at least 93%, particularly preferably at least 95%,
especially preferably at least 98% identity with the amino acids
322 to 351 (domain 1) of the amino acid sequence shown as SEQ ID NO
4, and at least 83%, preferably at least 86%, particularly
preferably at least 95%, especially preferably at least 98%
identity with the amino acids 423 to 462 (domain 2) of the amino
acid sequence shown as SEQ ID NO 4 and at least 70%, preferably at
least 82%, preferably 86%, particularly preferably 95%, especially
preferably at least 98% identity with the amino acids 641 to 705
(domain 3) of the amino acid sequence shown as SEQ ID NO 4.
[0046] Nucleic acid sequences and the corresponding amino acid
sequences which have said identity with domains 1, 2 and 3 and
which code for a protein with the activity of a starch synthase II
are known to the skilled worker and published for example as
Accession No AY133249 (Hordeum vulgare), Accession No AY133248
(Aegilops tauschii), Accession Nos XP467757, AAK64284 (Oryza
sativa), Accession No AAK81729 (Oryza sativa) Accession Nos
AAD13341, AAS77569, No AAD13342 (Zea mays), Accession No AAF13168
(Manihut esculenta), Accession No BAD18846 (Phaseolus vulgaris),
Accession No CAA61241 (Solanum tuberosum), Accession No CAA61269
(Pisum sativum), Accession No AAC19119 (Ipomea batatas), Accession
No AAF 26156 (Arabidopsis thaliana), Accession No AAP41030
(Colocasia esculenta), Accession No AAS88880 (Ostraeococcus tauri)
or Accession No AAC17970 (Chlamydomonas reinhardii). The
abovementioned nucleic acid sequences and amino acid sequences
coding for a protein with the activity of a starch synthase II are
accessible via NCBI (http://www.ncbi.nlm.nih.gov/entrez/) and are
expressly incorporated into the description of the present
application by reference.
[0047] For the purposes of the present invention, the term "protein
with the activity of a glucan, water dikinase" is understood as
meaning a protein which transfers a beta-phosphate residue from ATP
to starch. Starches isolated from leaves of an Arabidopsis thaliana
sex1-3 mutant contain no detectable amounts of covalently bonded
phosphate residues, but are phosphorylated in vitro by a protein
with the activity of a glucan, water dikinase. This means that
unphosphorylated starch, for example isolated from leaves of an
Arabidopsis thaliana sex1-3 mutant is used as the substrate in a
phosphorylation reaction which is catalyzed by a protein with the
activity of a glucan, water dikinase. A protein with the activity
of a glucan, water dikinase transfers the beta-phosphate residue of
ATP to starch in the C6 position of glucose, and the
gamma-phosphate residue of ATP to water. Another reaction product
which is generated is AMP (adenosin monophosphate). A protein with
the activity of a glucan, water dikinase is therefore also referred
to as [alpha-1,4-glucan], water dikinase, or else starch: water
dikinase (E.C.: 2.7.9.4; Ritte et al., 2002, PNAS 99:
7166-7171).
[0048] The phosphorylation of starch which is catalyzed by a
protein with the activity of a glucan, water dikinase gives rise to
additional phosphate monoester bonds exclusively in the C6 position
of the glucose molecules (Ritte et al., 2006, FEBS Letters 580:
4872-4876). The catalysis of the phosphorylation reaction of a
starch by a protein with the activity of a glucan, water dikinase
gives rise to an intermediate phosphorylated protein in which the
beta-phosphate residue of ATP is bonded covalently to an amino acid
of the protein with the activity of a glucan, water dikinase (Ritte
et al., 2002, PNAS 99, 7166-7171). The intermediate is formed by
autophosphorylation of the protein with the activity of a glucan,
water dikinase, i.e. the protein with the activity of a glucan,
water dikinase itself catalyzes the reaction which leads to the
intermediate. Amino acid sequences which code for proteins with the
activity of a glucan, water dikinase contain a phosphohistidine
domain. Phosphohistidine domains are described for example by
Tien-Shin Yu et al. (2001, Plant Cell 13, 1907-1918). In the
autophosphorylation of a protein with the activity of a glucan,
water dikinase, a histidine residue in the phosphohistidine domain
of the amino acid sequence, coding for a protein with the activity
of a glucan, water dikinase, is phosphorylated (Mikkelsen et al.,
2004, Biochemical Journal 377: 525-532). In the protein sequence,
shown for example as SEQ ID NO 2, of a protein with the activity of
a glucan, water dikinase from Solanum tuberosum, the amino acids
1064 to 1075 constitute the phosphohistidine domain. If another
amino acid is substituted for the conserved histidine residue
(amino acid 1069 in the protein sequence shown for example as SEQ
ID NO 2) of the phosphohistidine domain, autophosphorylation, and
thus phosphorylation, of glucans by the mutagenized protein no
longer takes place (Mikkelsen et al., 2004, Biochemical Journal
377: 525-532). Furthermore, a protein with the activity of a
glucan, water dikinase is distinguished by the fact that it has a
C-terminal nucleotide binding domain which is comprised by the
amino acids 1121 to 1464 in the amino acids sequence shown for
example as SEQ ID NO 2. A deletion of the nucleotide binding domain
leads to inactivation of a protein with the activity of a glucan,
water dikinase (Mikkelsen and Blennow, 2005, Biochemical Journal
385, 355-361). Proteins with the activity of a glucan, water
dikinase have on their N terminals a carbohydrate binding domain
(CBM) which is comprised by the amino acids 78 to 362 in the amino
acid sequence shown as SEQ ID NO 2. Carbohydrate binding domains
are distinguished inter alia by the fact that their amino acid
sequences contain conserved tryptophan residues. If other amino
acids are substituted for these conserved amino acid residues, the
carbohydrate binding domains lose their ability of binding glucans.
Thus, for example, a substitution of amino acids W139 or W194 in
the amino acid sequence shown as SEQ ID NO 2 leads to a loss of
function of this carbohydrate binding domain. If, however, the
carbohydrate binding domain of a glucan, water dikinase is deleted
(for example a deletion of amino acids 1 to 362, where the amino
acids 1 to 77 in the amino acid sequence shown as SEQ ID NO 2
constitute a plastidal signal peptide), this does not lead to the
inactivation of the phosphorylating activity of the enzyme
(Mikkelsen et al., 2006, Biochemistry 45: 4674-4682).
[0049] Nucleic acid sequences and their corresponding amino acid
sequences coding for a protein with the activity of a glucan, water
dikinase are described from different species such as, for example,
potato (WO 97/11188, GenBank Acc.: AY027522, Y09533), wheat (WO
00/77229, U.S. Pat. No. 6,462,256, GenBank Acc.: AAN93923, GenBank
Acc.: AR236165), rice (GenBank Acc.: AAR61445, GenBank Acc.:
AR400814), maize (GenBank Acc.: AAR61444, GenBank Acc.: AR400813),
Soybean (GenBank Acc.: AAR61446, GenBank Acc.: AR400815; citrus
(GenBank Acc.: AY094062), Arabidopsis (GenBank Acc.: AF312027) and
the green algae Ostreococcus tauri (GenBank Acc.: AY570720.1). The
abovementioned nucleic acid sequences and amino acid sequences
coding for a protein with the activity of a glucan, water dikinase
are published inter alia by the NCBI
(http://www.ncbi.nlm.nih.gov/entrez/) and are expressly
incorporated into the description of the present application by
reference.
[0050] In the context of the present invention, the term "GBSS I"
is to be understood to mean any enzyme which belongs to the group
of the granule-bound starch synthase of isoform I (EC
2.4.1.21).
[0051] In the context of the present invention, the term
"GBSSI-Gen" is understood as meaning a nucleic acid molecule or
polynucleotide (cDNA, DNA) which codes for a granule-bound starch
synthase I (GBSS I). Seq ID No 7-12 comprise nucleic acid sequences
or amino acid sequences which code in each case for a protein with
the activity of a GBSS I from rice, wheat and maize.
[0052] Polynucleotides coding for GBSS I are described for a
variety of monocotyledonous plant species such as, for example, for
maize (Genbank Acc. Nos. AF079260, AF079261), wheat (Genbank Acc.
Nos. AB019622, AB019623, AB019624), rice (Genbank Acc. Nos.
AF092443, AF092444, AF031162), barley (Genbank Acc. Nos. X07931,
X07932), Sorghum bicolor (Genbank Acc. No U23945) and durum wheat
(Genbank Acc. No AB029063). The abovementioned nucleic acid
sequences and amino acid sequences coding for a protein with the
activity of a GBSS I are published inter alia by NCBI
(http://www.ncbi.nlm.nih.gov/entrez/) and are expressly
incorporated into the description of the present application by
reference.
[0053] Mutants which lack a functional GBSS I gene synthesize an
amylose-free starch (=waxy starch). Such mutants are described for
a series of crops such as, for example, for maize (for example by
Sprague et al, 1943, J. Am. Soc. Agron. 35:817-822; Shure et al.
1983, Cell 35: 225-233), rice (Sano 1984, Theor. Appl. Genet. 68:
467-473; Villareal and Juliano 1986, Starch/Staerke 38:118-119),
barley (Rohde et al 1988, Nucleic Acids Res 16: 7185-7186), wheat
(Nakamura et al 1995, Mol. Gen. Genet. 248: 253-259), potato
(Hovenkamp-Hermelink et al. 1987, Theor. Appl. Genet. 75: 217-221)
and millet (Okuno and Sakaguchi 1982, J. Hered 73: 467). The term
"waxy mutant" is used synonymously, owing to the fact that, in
maize, the endosperm has a waxy appearance. The GBSS I protein is
also frequently referred to as "waxy protein" (Kossmann and Lloyd
2000 "Understanding and Influencing Starch Biochemistry", Critical
Reviews in Plant Sciences, 19(3): 171-226).
[0054] Suitable plant cells or plants for the generation of the
plant cells and plants according to the invention are those which
show a reduction of the apparent amylose content in the starch
synthesized by them to less than 5% by weight.
[0055] In one embodiment of the present invention, a genetic
modification of the plant cells according to the invention or of
the plants according to the invention is brought about by
mutagenesis of one or more GBSS I genes. The nature of the mutation
is of no consequence as long as it brings about a reduction, or
complete diminishment, of the GBSSI activity, and thus a reduction
of the apparent amylose content of the starch present in the plants
according to the invention to less than 5% by weight.
[0056] A mutation which leads to the reduction of the GBSSI
activity and to the diminishment of the apparent amylose content of
the starch to less than 5% by weight in the plant cells and plants
according to the invention may occur spontaneously, and the plants
in question can be selected and propagated with the aid of the
methods described hereinbelow.
[0057] For the purposes of the present invention, a "waxy mutant"
is understood as meaning a plant whose starch has an apparent
amylose content of less than 5% by weight. Equally, "waxy starch"
refers to a starch with an apparent amylose content of less than 5%
by weight.
[0058] In the context of the present invention, the term
"mutagenesis" is understood as meaning any type of introduced
mutation such as, for example, deletions, point mutations
(nucleotide substitutions), insertions, inversions, gene
conversions or chromosomal translocations.
[0059] Agents which can be employed for generating chemically
induced mutations, and the types of mutation obtained thereby as
the result of the effect of the mutagens in question are described,
for example, by Ehrenberg and Husain (1981, Mutation Research 86:
1-113) and Muller (1972, Biologisches Zentralblatt 91 (1): 31-48).
The generation of rice mutants using gamma rays,
ethylmethanesulfonate (EMS), N-methyl-N-nitrosourea or sodium azide
(NaN.sub.3) is described for example, by Jauhar and Siddiq (1999,
Indian Journal of Genetics, 59 (1): 23-28), Rao (1977, Cytologica
42: 443-450), Gupta and Sharma (1990, Oryza 27: 217-219) and Satoh
and Omura (1981, Japanese Journal of Breeding 31 (3): 316-326). The
generation of wheat mutants using NaN.sub.3 or maleic anhydrazide
is described by Arora et al. (1992, Annals of Biology 8 (1):
65-69). An review of the generation of wheat mutants using various
types of high-energy radiation and chemical agents is described by
Scarascia-Mugnozza et al. (1993, Mutation Breeding Review 10:1-28).
Svec et al. (1998, Cereal Research Communications 26 (4): 391-396)
describe the use of N-ethyl-N-nitrosourea for the generation of
mutants in triticale. The use of MMS (methylmethanesulfonic acid)
and gamma radiation for the generation of millet mutants is
described by Shashidhara et al. (1990, Journal of Maharashtra
Agricultural Universities 15 (1): 20-23).
[0060] Monocotyledonous plant cells and plants which synthesize a
starch with an apparent amylose content of less than 5% by weight
(=waxy plants, or waxy plant cells) can also be generated by using
what is known as insertion mutagenesis (review: Thorneycroft et
al., 2001, Journal of Experimental Botany 52 (361): 1593-1601).
"Insertion mutagenesis" is understood as meaning in particular the
insertion of transposons, or what is known as transfer DNA (T-DNA)
into a gene.
[0061] The transposons may take the form of transposons which occur
naturally in a (wild-type) plant cell (endogenous transposons) or
else those which do not occur naturally in said cell but have been
introduced into the cell by means of recombinant methods, such as,
for example, by transforming the cell (heterologous transposons).
Modifying the expression of genes by means of transposons is known
to the skilled worker. A review of the utilization of endogenous
and heterologous transposons as tools in plant biotechnology can be
found in Ramachandran and Sundaresan (2001, Plant Physiology and
Biochemistry 39, 234-252). The possibility of identifying mutants
in which specific genes have been inactivated by transposon
insertion mutagenesis can be found in a review by Maes et al.
(1999, Trends in Plant Science 4 (3), 90-96). The generation of
rice mutants with the aid of endogenous transposons is described by
Hirochika (2001, Current Opinion in Plant Biology 4, 118-122). The
identification of maize genes with the aid of endogenous
retrotransposons is shown, for example, in Hanley et al. (2000, The
Plant Journal 22 (4), 557-566). The possibility of generating
mutants with the aid of retrotransposons and methods for
identifying mutants are described by Kumar and Hirochika (2001,
Trends in Plant Science 6 (3), 127-134). The activity of
heterologous transposons in different species has been described
both for dicotyledonous and for monocotyledonous plants, for
example for rice (Greco et al., 2001, Plant Physiology 125,
1175-1177; Liu et al., 1999, Molecular and General Genetics 262,
413-420; Hiroyuki et al., 1999, The Plant Journal 19 (5), 605-613;
Jeon and Gynheung, 2001, Plant Science 161, 211-219), barley
(Koprek et al., 2000, The Plant Journal 24 (2), 253-263),
Arabidopsis thaliana (Aarts et al., 1993, Nature 363, 715-717,
Schmidt and Willmitzer, 1989, Molecular and General Genetics 220,
17-24; Altmann et al., 1992, Theoretical and Applied Genetics 84,
371-383; Tissier et al., 1999, The Plant Cell 11, 1841-1852),
tomato (Belzile and Yoder, 1992, The Plant Journal 2 (2), 173-179)
and potato (Frey et al., 1989, Molecular and General Genetics 217,
172-177; Knapp et al., 1988, Molecular and General Genetics 213,
285-290).
[0062] In principle, monocotyledonous "waxy" plant cells and plants
can be generated, with the aid of both homologous and heterologous
transposons, the use of homologous transposons also including those
transposons which are already naturally present in the plant
genome. In principle, T-DNA mutagenesis is likewise suitable for
producing "waxy" plant cells and plants.
[0063] T-DNA insertion mutagenesis is based on the fact that
certain segments (T-DNA) of Ti plasmids from Agrobacterium are
capable of integrating into the genome of plant cells. The site of
integration into the plant chromosome is not fixed but may take
place at any position. If the T-DNA integrates in a segment of the
chromosome which constitutes a gene function, this may lead to a
modification of the gene expression and thus also to an altered
activity of a protein encoded by the gene in question. In
particular, the integration of a T-DNA into the coding region of a
gene frequently means that the protein in question can no longer be
synthesized in active form, or not at all, by the cell in question.
The use of T-DNA insertions for the generation of mutants is
described, for example, for Arabidopsis thaliana (Krysan et al.,
1999, The Plant Cell 11, 2283-2290; Atipiroz-Leehan and Feldmann,
1997, Trends in Genetics 13 (4), 152-156; Parinov and Sundaresan,
2000, Current Opinion in Biotechnology 11, 157-161) and rice (Jeon
and An, 2001, Plant Science 161, 211-219; Jeon et al., 2000, The
Plant Journal 22 (6), 561-570). Methods for identifying mutants
which have been generated with the aid of T-DNA insertion
mutagenesis are described, inter alia, by Young et al., (2001,
Plant Physiology 125, 513-518), Parinov et al. (1999, The Plant
cell 11, 2263-2270), Thorneycroft et al. (2001, Journal of
Experimental Botany 52, 1593-1601), and McKinney et al. (1995, The
Plant Journal 8 (4), 613-622).
[0064] Mutations in the corresponding gene can be found with the
aid of methods with which the skilled worker is familiar. For
example, it is possible to employ molecular analyzes based on
hybridizations with probes ("Southern blot"), on the amplification
by means of polymerized chain reaction (PCR), on the sequencing of
suitable genomic nucleic acid fragments and the search for
individual nucleotides substitutions. An example of a method of
identifying mutations with the aid of hybridization patterns is the
search for restriction fragment length polymorphisms (RFLP) (Nam et
al., 1989, The Plant Cell 1: 699-705; Leister and Dean, 1993, The
Plant Journal 4 (4): 745-750). A PCR based method is, for example,
the analysis of amplified fragment length polymorphisms (AFLP)
(Castiglioni et al., 1998, Genetics 149: 2039-2056; Meksem et al.,
2001, Molecular Genetics and Genomics 265: 207-214; Meyer et al.
1998, Molecular and General Genetics 259: 150-160). The use of
amplified fragments which have been cleaved with restriction
endonucleases ("cleaved amplified polymorphic sequences", CAPS) is
a further possibility of identifying mutations (Konieczny and
Ausubel, 1993, The Plant Journal 4: 403-410; Jarvis et al., 1994,
Plant Mol. Biol. 24: 685-687; Bachem et al., 1996, The Plant
Journal 9 (5): 745-753). Methods of determining SNPs have been
described by, inter alia, Qi et al. (2001, Nucleic Acids Research
29 (22): 116), Drenkard et al. (2000, Plant Physiology 124:
1483-1492) and Cho et al. (1999, Nature Genetics 23: 203-207).
Particularly suitable methods are those which permit a large number
of plants to be studied for mutations in certain genes within a
short period of time. Such a method, known as TILLING ("targeting
induced local lesions in genomes") has been described by McCallum
et al. (2000, Plant Physiology 123: 439-442).
[0065] The skilled worker knows that the above-described mutations
are, as a rule, recessive mutations. To manifest the waxy
phenotype, it is therefore necessary to generate true-breeding
(homozygous) plant cells or plants. Methods of generating
true-breeding plants are known to the skilled worker.
[0066] Homozygous "waxy" mutants can be identified by staining the
starch with iodine. To this end, starch-comprising tissue samples
(for example endosperm, pollen) are stained with iodine solution
and studied for example under the microscope. Waxy starches stain
brown (in comparison with the blue staining of the wild type).
[0067] In a further embodiment of the present invention, the
introduction of one or more foreign nucleic acid
molecules/polynucleotides, their presence and/or the expression of
one or more foreign nucleic acid molecules/polynucleotides lead to
the inhibition of the expression of endogenous genes which code for
the GBSS1 protein and to a reduction of the apparent amylose
content of the starch present in the plant cell according to the
invention, or plant according to the invention, to less than 5% by
weight.
[0068] This can be done by various methods with which the skilled
worker is familiar. These methods include, for example, the
expression of a suitable antisense RNA, or of a double-stranded
RNA, the provision of molecules or vectors which confer a
cosuppression effect, the expression of a suitably constructed
ribozyme which specifically cleaves transcripts which code for
GBSSI, or what is known as "in-vivo mutagenesis". Furthermore, the
reduction of the GBSSI activity/activities and/or the reduction of
the gene expression of the GBSSI gene in the plant cells can also
be brought about by the simultaneous expression of sense and
antisense RNA molecules of the specific target gene to be
repressed, preferably the GBSSI gene. These methods are known to
the skilled worker.
[0069] In addition, it is known that the formation of
double-stranded RNA of promoter sequences in trans can bring about
methylation and transcriptional inactivation of homologous copies
of this promoter in planta (Mette et al., 2000, EMBO J. 19:
5194-5201).
[0070] To inhibit the gene expression by means of antisense or
cosuppression technology, for example, it is possible to employ a
DNA molecule which comprises all of the GBSSI coding sequence
including any flanking sequences present, or else DNA molecules
which only comprise parts of the coding sequence, where these parts
must be long enough to bring about an antisense effect, or
cosuppression effect, in the cells. Generally suitable are
sequences with a minimum length of 15 bp, preferably with a minimum
length of 20-30 bp, especially preferably with a length of 100-500
bp, and, for highly efficient antisense or cosuppression
inhibition, in particular sequences with a length of more than 500
bp.
[0071] Also suitable for antisense or cosuppression approaches is
the use of polynucleotide sequences with a high degree of identity
with the endogenous sequences which are present in the plant cell
and which encode GBSSI. The minimum identity should be greater than
approximately 65%. The use of sequences with identities of at least
90%, in particular between 95% and 100%, is to be preferred.
[0072] To achieve an antisense effect, or a cosuppression effect,
it is furthermore also feasible to use introns, i.e. from noncoding
regions of genes which code for GBSSI.
[0073] The use of intron sequences for inhibiting the expression of
genes which code for starch biosynthesis proteins has been
described in WO 97/04112, WO 97/04113, WO 98/37213, WO
98/37214.
[0074] The skilled worker knows how to achieve an antisense effect
and a cosuppression effect. The method of cosuppression inhibition
has been described, for example, by Jorgensen (1990, Trends
Biotechnol. 8: 340-344), Niebel et al. (1995, Top. Microbiol.
Immunol. 197: 91-103), Flavell et al. (1995, Curr. Top. Microbiol.
Immunol. 197: 43-46), Palauqui and Vaucheret (1995, Plant Mol.
Biol. 29: 149-159), Vaucheret et al. (1995, Mol. Gen. Genet. 248:
311-317), de Borne et al. (1994, Mol. Gen. Genet. 243:
613-621).
[0075] Furthermore, a reduction of the GBSSI activity in the plant
cells can also be brought about by the simultaneous expression of
sense and antisense RNA molecule of the specific target gene to be
repressed, preferably the GBSSI gene.
[0076] This can be achieved for example by using chimeric
constructs which comprise "inverted repeats" of the target gene in
question, or parts of the target gene. The chimeric constructs code
for sense and antisense RNA molecules of the target gene in
question. Sense and antisense RNA are synthesized simultaneously in
planta as one RNA molecule, it being possible for sense and
antisense RNA to be separated from each other by a spacer, to form
a double-stranded RNA molecule (RNAi technology).
[0077] It has been demonstrated that the introduction of
inverted-repeat DNA constructs into the genome of plants is a
highly effective method for repressing the genes corresponding to
the inverted-repeat DNA constructs (Waterhouse et al., 1998, Proc.
Natl. Acad. Sci. USA 95, 13959-13964; Wang and Waterhouse, 2000,
Plant Mol. Biol. 43, 67-82; Singh et al., 2000, Biochemical Society
Transactions 28 (6), 925-927; Liu et al., 2000, Biochemical Society
Transactions 28 (6), 927-929; Smith et al., 2000, Nature 407,
319-320; WO 99/53050). Sense and antisense sequences of the target
gene, or target genes, may also be expressed separately from one
another by means of identical or different promoters (Nap et al,
6.sup.th International Congress of Plant Molecular Biology, 18-24
Jun. 2000, Quebec, Poster S7-27, Lecture Session S7).
[0078] The expression of ribozymes for reducing the activity of
specific enzymes in cells is also known to the skilled worker and
described, for example, in EP-B1 0321201. The expression of
ribozymes in plant cells has been described for example by Feyter
et al. (1996, Mol. Gen. Genet. 250: 329-338).
[0079] Moreover, the reduction of the GBSSI activity and/or the
reduction of the apparent amylose content of the starch present in
the plant cells to less than 5% by weight may also be achieved by
what is known as "in-vivo" mutagenesis, where an RNA-DNA
oligonucleotide hybrid ("chimeroplast") is introduced into cells by
means of transforming cells (Kipp et al., Poster Session at the
5.sup.th International Congress of Plant Molecular Biology, 21-27
Sep. 1997, Singapore; R. A. Dixon and C. J. Arntzen, Meeting report
regarding Metabolic Engineering in Transgenic Plants, Keystone
Symposia, Copper Mountain, Colo., USA, 1997, TIBTECH 15: 441-447;
WO 95/15972; Kren et al., 1997, Hepatology 25: 1462-1468;
Cole-Strauss et al., 1996, Science 273: 1386-1389; Beetham et al.,
1999, PNAS 96: 8774-8778).
[0080] Part of the DNA component of the RNA-DNA oligonucleotide is
homologous with a polynucleotide sequence of an endogenous GBSSI
gene, but comprises a mutation in comparison with the
polynucleotide acid sequence of an endogenous GBSSI gene or
comprises a heterologous region which is surrounded by the
homologous regions. Owing to base pairing of the homologous regions
of the RNA-DNA oligonucleotide and of the endogenous
polynucleotide, followed by homologous recombination, the mutation
or heterologous region present in the DNA component of the RNA-DNA
oligonucleotide can be transferred into the genome of a plant
cell.
[0081] Thus, the reduction of the GBSSI activity in the plant cells
can also be achieved by generating double-stranded RNA molecules of
GBSSI genes. To this end, it is preferred to introduce, into the
genome of plants, inverted repeats of DNA molecules which are
derived from nucleotide sequences formed by GBSSI genes or cDNAs
formed by such genes, where the DNA molecules to be transcribed are
under the control of a promoter which governs the expression of
said RNA molecules.
[0082] A further possibility of reducing the activity of proteins
in plant cells or plants is the method of what is known as
immunomodulation. It is known that an expression in planta of
antibodies which specifically recognize a plant protein results in
a reduction of the activity of said proteins in corresponding plant
cells or plants as the result of the formation of a
protein/antibody complex (Conrad and Manteufel, 2001, Trends in
Plant Science 6: 399-402; De Jaeger et al., 2000, Plant Molecular
Biology 43: 419-428; Jobling et al., 2003, Nature Biotechnology 21:
77-80).
[0083] All the abovementioned methods are based on the introduction
of one or more foreign nucleic acid molecules into the genome of
plant cells or plants and are therefore suitable in principle for
the generation of plant cells according to the invention and plants
according to the invention.
[0084] The reduction of the expression can be determined for
example by measuring the amount of transcripts which code for the
enzymes in question, for example by means of Northern blot analysis
or quantitative RT-PCR.
[0085] The reduction of the amount of GBSS1 protein can be
determined for example by immunological methods such as Western
blot analysis, ELISA ("enzyme linked immuno sorbent assay") or RIA
("radio immune assay").
[0086] A reduction in the GBSSI activity in the plant cells, or
plants, according to the invention can also be detected indirectly
via quantifying of the reaction product of the GBSSI protein,
amylose. The skilled worker knows a multiplicity of methods for
determining the amylose content in plant starches. For cereals, in
particular rice, the apparent amylose content is preferably
determined by a method similar to that of Juliano (1971, Cereal
Science Today 16 (10): 334-340), as described further below in the
chapter "Materials and Methods".
[0087] In a further embodiment for generating the plant cells
according to the invention or the plants according to the
invention, it is possible to use, instead of a wild-type plant cell
or wild-type plant, a mutant which is distinguished by the fact
that it already synthesizes a starch with an apparent amylose
content of less than 5% by weight and/or which has an increased
activity of a protein with the activity of a glucan, water dikinase
and/or an increased activity of a protein with the activity of a
starch synthase II. These mutants may be either spontaneously
occurring mutants or else those which have been generated by the
targeted use of mutagens. Possibilities of generating such mutants
have been described hereinabove.
[0088] The present invention furthermore comprises a genetically
modified monocotyledonous plant cell, or plant, according to the
invention whose genetic modification consists in the introduction
of at least one foreign nucleic acid molecule into the genome of
the plant used for the transformation.
[0089] As the result of the introduction of a foreign nucleic acid
molecule, the genetic information of the plant cells according to
the invention or plants according to the invention are altered. The
presence of at least one foreign nucleic acid molecule leads to an
altered "phenotype". Here, "altered phenotype" means a measurable
alteration of one or more cellular functions. For example, the
genetically modified plant cells according to the invention and the
genetically modified plants according to the invention show, as the
result of the presence or, in the case of expression of introduced
foreign nucleic acid molecules, an increase in the activity of a
protein with the activity of a glucan, water dikinase and an
increase in the activity of a protein with the activity of a starch
synthase II and/or a reduction of the activity of a protein with
the activity of a GBSSI.
[0090] In the context of the present invention, the term "foreign
nucleic acid molecule" is understood as meaning a molecule which
either does not occur naturally in the plant cells used for the
transformation, or which does not occur naturally in the specific
spatial arrangement in the plant cells used for the transformation,
or which is located at a locus in the genome of the plant cell used
for the transformation at which it does not occur naturally. The
foreign nucleic acid molecule is preferably a recombinant molecule
which consists of various elements whose combination or specific
spatial arrangement does not occur naturally in plant cells. Thus,
recombinant nucleic acid molecules may, for example, besides
nucleic acid molecules which code for a protein with the activity
of a glucan, water dikinase and/or a protein with the activity of a
starch synthase II and/or a nucleic acid which brings about a
reduction in the activity of a GBSSI, have additional nucleic acid
sequences which are not naturally present in combination with the
abovementioned nucleic acid molecules. The abovementioned
additional nucleic acid sequences which are present on a
recombinant nucleic acid molecule in combination with a nucleic
acid molecule coding for protein with the activity of a glucan,
water dikinase and/or protein with the activity of a starch
synthase II and/or with a nucleic acid which is suitable for
mediating a reduction in the activity of a protein with the
activity of a GBSSI may be any sequences. They may be for example
genomic and/or plant nucleic acid sequences. Preferably, these
additional nucleic acid sequences are regulatory sequences
(promoters, termination signals, enhancers), particularly
preferably regulatory sequences which are active in plant tissue;
especially preferably tissue-specific regulatory sequences.
[0091] Methods of generating recombinant nucleic acid molecules are
known to the skilled worker and comprise genetic engineering
methods such as, for example, the linking of nucleic acid molecules
by ligation, genetic recombination or the de-novo synthesis of
nucleic acid molecules (see, for example, Sambrok et al., Molecular
Cloning, A Laboratory Manual, 3rd edition (2001) Cold Spring
Harbour Laboratory Press, Cold Spring Harbour, N.Y., ISBN:
0879695773; Ausubel et al., Short Protocols in Molecular Biology,
John Wiley & Sons; 5th edition (2002), ISBN: 0471250929).
[0092] In the context of the present invention, the term "genome"
is understood as meaning the totality of the hereditary material
present in a plant cell. The skilled worker knows that not only the
nucleus, but other compartments too (for example plastids,
mitochondria) comprise hereditary material.
[0093] In principle, a foreign nucleic acid molecule can be any
nucleic acid molecule which brings about, in the plant cell or
plant, an increase in the activity of a protein with the activity
of a glucan, water dikinase and of a protein with the activity of a
starch synthase II and a reduction in the activity of a protein
with the activity of a GBSSI.
[0094] In a preferred embodiment, the foreign nucleic acid
molecules coding for a protein with the activity of a glucan, water
dikinase take the form of the already-mentioned nucleic acid
molecules from the various plant species, which nucleic acid
molecules are known to the skilled worker. Particularly preferred
in this context are nucleic acid molecules coding for a protein
with the activity of a glucan, water dikinase from potato,
especially preferred is a protein with the activity of a glucan,
water dikinase which has the amino acid sequence shown in SEQ ID NO
2 or is encoded by the nucleic acid sequence shown in SEQ ID NO
1.
[0095] In a further preferred embodiment, the foreign nucleic acid
molecules coding for a protein with the activity of a starch
synthase II take the form of the already-mentioned nucleic acid
molecules from the various plant species, which nucleic acid
molecules are known to the skilled worker. Particularly preferred
in this context are nucleic acid molecules coding for a protein
with the activity of a starch synthase II from wheat, especially
preferred is a protein with the activity of a starch synthase II
which has the amino acid sequence shown in SEQ ID NO 4 or is
encoded by the nucleic acid sequence shown in SEQ ID NO 3.
[0096] A further preferred embodiment takes the form of nucleic
acid molecules coding for a protein with the activity of a starch
synthase II from rice, especially preferably a protein with the
activity of a starch synthase II which has the amino acid sequence
shown in SEQ ID NO 6 or is encoded by the nucleic acid sequence
shown in SEQ ID NO 5.
[0097] In a further preferred embodiment, the foreign nucleic acid
molecules coding for a protein with the activity of a GBSSI take
the form of the already-mentioned nucleic acid molecules from the
various plant species, which nucleic acid molecules are known to
the skilled worker. Particularly preferred in this context are
nucleic acid molecules coding for a protein with the activity of a
GBSSI from rice, especially preferred is a protein with the
activity of a GBSSI which has the amino acid sequence shown in SEQ
ID NO 8 or is encoded by the nucleic acid sequence shown in SEQ ID
NO 7.
[0098] A further preferred embodiment takes the form of nucleic
acid molecules coding for a protein with the activity of a GBSSI
from wheat, especially preferably a protein with the activity of a
GBSSI which has the amino acid sequence shown in SEQ ID NO 10 or is
encoded by the nucleic acid sequence shown in SEQ ID NO 9.
[0099] A further preferred embodiment takes the form of nucleic
acid molecules coding for a protein with the activity of the GBSSI
from maize, especially preferably a protein with the activity of a
GBSSI which has the amino acid sequence shown in SEQ ID NO 12 or is
encoded by the nucleic acid sequence shown in SEQ ID NO 11.
[0100] In a further embodiment, the plant cells and plants
according to the invention are homozygous for the waxy mutation(s)
and thus synthesize a starch whose apparent amylose content is less
than 5% by weight.
[0101] In the context of the present invention, the term
"homozygous for the waxy mutation(s)" is understood as meaning that
the plant breeds true for the non-functional GBSSI genes. To the
skilled worker, homozygosis means that, within the hereditary
material of a cell, all alleles regarding a particular trait are
identical, that is to say two or more identical copies of a certain
gene are present on the two chromatids of a chromosome, which
chromatids comprise the gene. They are homozygous (=breed true) for
this gene and, when selfed, pass on the trait in question to all
progeny. The skilled worker knows that polyploid plants such as,
for example, wheat may, under certain circumstances, require three
non-functional GBSSI alleles (on the subgenomes A, B and D) in
homozygous form in order to manifest the waxy phenotype.
[0102] The foreign nucleic acid molecules introduced, for the
purposes of genetic modification, into the plant cells or plant
which manifest the waxy phenotype may take the form of a single
nucleic acid molecule or more nucleic acid molecules. They may take
the form of nucleic acid molecules which comprise nucleic acid
sequences which code for a protein with the activity of a glucan,
water dikinase and nucleic acid sequences which code for a protein
with the activity of a starch synthase II, but also nucleic acid
molecules in which the nucleic acid sequences which code for a
protein with the activity of a glucan, water dikinase and the
nucleic acid sequences which code for a protein with the activity
of a starch synthase II are present on different nucleic acid
molecules. For example, the nucleic acid sequences which code for a
protein with the activity of a glucan, water dikinase and the
nucleic acid sequences which code for a protein with the activity
of a starch synthase II may be present simultaneously in a vector,
plasmid or in linear nucleic acid molecules ("dual construct") or
else be components of two vectors, plasmids or linear nucleic acid
molecules which are separate in each case.
[0103] If the nucleic acid sequences which code for a protein with
the activity of a glucan, water dikinase and the nucleic acid
sequences which code for a protein with the activity of a starch
synthase II are present in two separate nucleic acid molecules,
they can be introduced into the genome of the plant cell or plant
either simultaneously ("cotransformation") or else one after the
other, i.e. with a chronological interval ("supertransformation").
The separate nucleic acid molecules may also be introduced into
different individual plant cells or plants of a species. Thereby it
is possible to generate plant cells or plants in which the activity
of either at least one protein with the activity of a glucan, water
dikinase or else at least one protein with the activity of a starch
synthase II is elevated. Plants according to the invention can then
be generated by subsequently hybridizing those plants in which the
activity of a protein with the activity of a glucan, water dikinase
is elevated with those in which the activity of a protein with the
activity of a starch synthase II is elevated. The parameters for
the selection of plants which are used for the process steps in
question are defined further below.
[0104] In a further embodiment, the waxy phenotype of the plant
cells or plants according to the invention is brought about by
introducing one or more recombinant nucleic acid molecules suitable
for reducing the GBSSI activity.
[0105] The foreign nucleic acid molecules introduced, for the
purposes of genetic modification, into the wild-type plant cell or
plant may take the form of a single nucleic acid molecule or more
nucleic acid molecules. They may therefore take the form of nucleic
acid molecules which comprise nucleic acid sequences which code for
a protein with the activity of a glucan, water dikinase and the
nucleic acid sequences which code for a protein with the activity
of a starch synthase II and additionally to nucleic acid sequences
which are suitable for inhibiting the activity of the GBSSI
activity (triple construct). Equally, they may also take the form
of nucleic acid molecules in which the nucleic acid sequences which
code for a protein with the activity of a glucan, water dikinase
and the nucleic acid sequences which code for a protein with the
activity of a starch synthase II are present on different nucleic
acid molecules, where one or the other of these two nucleic acid
molecules additionally comprises nucleic acid sequences which are
suitable for inhibiting the activity of the GBSSI activity.
Alternatively, they may also take the form of nucleic acid
molecules in which the nucleic acid sequences which code for a
protein with the activity of a glucan, water dikinase and the
nucleic acid sequences which code for a protein with the activity
of a starch synthase II are present on one nucleic acid molecule
and the nucleic acid molecules which are suitable for inhibiting
the GBSSI activity are present on a different nucleic acid molecule
(3 variants of one dual construct and one simple construct).
[0106] In a further embodiment, they may also take the form of
three different nucleic acid molecules, where one comprises nucleic
acid sequences which code for a glucan, water dikinase protein,
another one comprises nucleic acid sequences coding for a starch
synthase II and a further one comprises nucleic acid sequences
which are suitable for inhibiting the GBSSI activity (3 simple
constructs).
[0107] The nucleic acid molecules which are suitable for generating
the plant cells or plants according to the invention may be present
for example in a vector, plasmid or in linear nucleic acid
molecules.
[0108] If the constructs to be used for the generation of plant
cells or plants according to the invention are present in two or
three separate nucleic acid molecules, they can be introduced into
the genome of the plant cell or plant either simultaneously
("cotransformation") or else one after the other, i.e. with a
chronological interval ("supertransformation"). The separate
nucleic acid molecules may also be introduced into different
individual plant cells or plants of a species. Thereby it is
possible to generate plant cells or plants in which the activity of
either at least one protein with the activity of a glucan, water
dikinase and/or at least one protein with the activity of a starch
synthase II is elevated and/or at least one protein with the
activity of a GBSSI activity is reduced to such an extent that the
starch synthetized by the plant cells or plants has apparent
amylose content of less than 5% by weight. Plants according to the
invention can then be generated by subsequently hybridizing the
plants.
[0109] Furthermore, it is also possible to generate plants in which
the activity of at least one protein with the (enzymatic) activity
of a GBSSI is reduced to such an extent that the starch synthetized
by the plant cells or plants has an apparent amylose content of
less than 5% by weight and which, in a further step, by crossing
with plants in which the activity of at least one protein with the
activity of a starch synthase II is elevated, leads to plant cells
or plants according to the invention.
[0110] In the event that one or more nucleic acid molecules which
comprise nucleic acid sequences suitable for increasing the
activity of at least one protein with the activity of a glucan,
water dikinase and/or of a starch synthase II in the plant cells
and reducing the activity of a GBSSI in the plant cells to such an
extent that the starch synthetized by the cells has an apparent
amylose content of less than 5%, are introduced into the genome of
the plant cells in one methodological step/simultaneously, the
plants according to the invention may be selected directly among
the plants to which the transformation gives rise.
[0111] In a further embodiment, the plant cells according to the
invention and the plants according to the invention comprise that
at least one foreign nucleic acid molecule codes for a protein with
the activity of a starch synthase II and a second foreign nucleic
acid molecule codes for a protein with the activity of a glucan,
water dikinase. In a further embodiment, the plant cells according
to the invention of the plants according to the invention comprise
that a first foreign nucleic acid molecule codes for a protein with
the activity of a glucan, water dikinase and a second foreign
nucleic acid molecule codes for a protein with the activity of a
starch synthase II.
[0112] A multiplicity of techniques is available for introducing
DNA into a plant host cell. These techniques comprise the
transformation of plant cells with T-DNA using Agrobacterium
tumefaciens or Agrobacterium rhizogenes as transformation agent,
the fusion of protoplasts, the injection, the electroporation of
DNA, the introduction of the DNA by means of the biolistic
approach, and other possibilities.
[0113] The use of the agrobacteria-mediated transformation of plant
cells has been studied intensively and has been described, inter
alia, in EP 120516; Hoekema (In: The Binary Plant Vector System,
Offsetdrukkerij Kanters B.V. Alblasserdam (1985), Chapter V);
Fraley et al., Crit. Rev. Plant Sci. 4: 1-46) and by An et al.
(1985, EMBO J. 4: 277-287).
[0114] The transformation of monocotyledonous plants by means of
vectors based on Agrobacterium transformation has also been
described (Chan et al. 1993, Plant Mol. Biol. 22: 491-506; Hiei et
al., 1994, Plant J. 6, 271-282; Deng et al, 1990, Science in China
33: 28-34; Wilmink et al., 1992, Plant Cell Reports 11: 76-80; May
et al., 1995, Bio/Technology 13: 486-492; Conner and Domisse, 1992,
Int. J. Plant Sci. 153: 550-555; Ritchie et al, 1993, Transgenic
Res. 2: 252-265). Alternative methods for the transformation of
monocotyledonous plants are the transformation by means of the
biolistic approach (Wan and Lemaux, 1994, Plant Physiol. 104:
37-48; Vasil et al., 1993, Bio/Technology 11: 1553-1558; Ritala et
al., 1994, Plant Mol. Biol. 24: 317-325; Spencer et al., 1990,
Theor. Appl. Genet. 79: 625-631), the transformation of
protoplasts, the electroporation of partially permeabilized cells
or the introduction of DNA by means of glass fibers. The
transformation of maize, in particular, is described repeatedly in
the literature (cf., for example, WO95/06128, EP0513849,
EPO.sub.465875, EP0292435; Fromm et al., 1990, Biotechnology 8:
833-844; Gordon-Kamm et al., 1990, Plant Cell 2: 603-618; Koziel et
al., 1993, Biotechnology 11: 194-200; Moroc et al., 1990, Theor.
Appl. Genet. 80: 721-726).
[0115] This successful transformation of other cereal species has
also been described, for example in the case of barley (Wan and
Lemaux, s.o.; Ritala et al., s.o.; Krens et al., 1982, Nature 296:
72-74) and wheat (Nehra et al., 1994, Plant J. 5: 285-297; Becker
et al., 1994, Plant Journal 5: 299-307). All the above methods are
suitable within the scope of the present invention.
[0116] Plant cells and plants whose starch has an amylose content
of less than 5% by weight and which are genetically modified as the
result of the introduction of a gene coding for a protein with the
activity of a glucan, water dikinase and/or a gene coding for a
protein with the activity of a starch synthase II can be
distinguished from wild-type plant cells, or wild-type plants,
inter alia by the fact that they comprise at least one foreign
nucleic acid molecule which does not occur naturally in wild-type
plant cells, or wild-type plants, or by the fact that such a
molecule is present at a location in the genome of the plant cell
according to the invention or in the genome of the plant according
to the invention at which it does not occur in wild-type plant
cells, or wild-type plants, i.e. in a different genomic
environment. Furthermore, such plant cells according to the
invention or plants according to the invention can be distinguished
from wild-type plant cells, or wild-type plants, by the fact that
they comprise at least one copy of the foreign nucleic acid
molecule stably integrated in their genome, if appropriate
additionally to copies of such a molecule which are naturally
present in the wild-type plant cells, or wild-type plants. If the
foreign nucleic acid molecule(s) which has been introduced into the
plant cells according to the invention or plants according to the
invention takes the form of additional copies, besides molecules
which naturally occur in the wild-type plant cells, or wild-type
plants, the plant cells according to the invention and the plants
according to the invention can be distinguished from wild-type
plant cells, or wild-type plants, in particular by the fact that
this additional copy, or these additional copies, is/are located at
locations in the genome where it does not occur, or they do not
occur, in wild-type plant cells or wild-type plants. This can be
verified for example with the aid of a Southern blot analysis.
[0117] The plant cells according to the invention or the plants
according to the invention can furthermore be preferably
distinguished from wild-type plant cells, or wild-type plants, by
at least one of the following features: if a foreign nucleic acid
molecule which has been introduced is heterologous with regard to
the plant cell or plant, then the plant cells according to the
invention, or plants according to the invention, comprise
transcripts of the nucleic acid molecules which have been
introduced. These transcripts can be detected for example by
Northern blot analysis or by RT-PCR (reverse transcription
polymerase chain reaction).
[0118] Plant cells according to the invention or plants according
to the invention which express an antisense transcript and/or an
RNAi transcript can be detected for example with the aid of
specific nucleic acid probes which are complementary to the RNA
which codes for the protein (and which occurs naturally in the
plant cell). Preferably, the plant cells according to the invention
and the plants according to the invention comprise a protein which
is encoded by a nucleic acid molecule which has been introduced.
This protein can be detected for example by immunological methods,
in particular by Western blot analysis.
[0119] Preferably, the plant cells according to the invention or
the plants according to the invention comprise a protein which is
encoded by a nucleic acid molecule which has been introduced. This
protein can be detected for example by immunological methods, in
particular by Western blot analysis.
[0120] If a foreign nucleic acid molecule which has been introduced
is homologous with regard to the plant cell or plant, then the
plant cells according to the invention, or the plants according to
the invention, can be distinguished from wild-type plant cells, or
wild-type plants, for example on the basis of the additional
expression of the foreign nucleic acid molecules which have been
introduced. The plant cells according to the invention and the
plants according to the invention preferably comprise transcripts
of the foreign nucleic acid molecules. This can be detected for
example by Northern blot analysis or with the aid of what is known
as quantitative PCR.
[0121] A further subject matter of the present invention relates to
genetically modified monocotyledonous plant cells or genetically
modified monocotyledonous plants which synthetize a modified starch
in comparison with starch isolated from corresponding, not
genetically modified wild-type plant cells, or isolated from
corresponding not genetically modified wild-type plants.
[0122] The invention furthermore relates to genetically modified
monocotyledonous plants which comprise plant cells according to the
invention. Such plants can be generated from plant cells according
to the invention by means of regeneration.
[0123] The plants according to the invention may, in principle,
take the form of any monocotyledonous plants. Preferably, they take
the form of monocotyledonous crop plants, i.e. plants which are
grown by man for the purposes of nutrition or for technical, in
particular industrial, purposes.
[0124] In a further embodiment, the plants according to the
invention take the form of starch-storing monocotyledonous plants,
or the plant cells according to the invention are derived from a
starch-storing plant.
[0125] In the context of the present invention, the term
"starch-storing plant" means all plants with plant parts which
comprise a storage starch such as, for example, maize, rice, wheat,
rye, oats, barley, sago, taro and millet/sorghum.
[0126] In a preferred embodiment, the present invention relates to
monocotyledonous plants of the (systematic) family Poaceae. These
plants particularly preferably take the form of rice, maize or
wheat plants. These plants very particularly preferably take the
form of rice plants.
[0127] In the context of the present invention, the term "wheat
plants" means plant species of the genus Triticum or plants which
have originated from crosses with plants of the genus Triticum,
particularly plant species of the genus Triticum which are grown in
agriculture for commercial purposes, or plants which have
originated from crosses with plants of genus Triticum, with
Triticum aestivum being especially preferred.
[0128] In the context of the present invention, the term "maize
plants" means plant species of the genus Zea, particularly plant
species of the genus Zea, which are grown in agriculture for
commercial purposes, particularly preferably Zea mays.
[0129] In the context of the present invention, the term "rice
plant" means plant species of the genus Oryza, particularly plant
species of the genus Oryza, which are grown in agriculture for
commercial purposes, particularly preferably Oryza sativa.
[0130] The present invention also relates to propagation material
of monocotyledonous plants comprising genetically modified plant
cells.
[0131] Here, the term "propagation material" comprises those parts
of the plant which are suitable for generating progeny via the
vegetative or sexual route. Examples which are suitable for
vegetative propagation are cuttings, callus cultures, rhizomes or
tubers. Other propagation material comprises for example fruits,
seeds, seedlings, protoplasts, cell cultures and the like.
[0132] In a further embodiment, the present invention relates to
plant parts capable of being harvested of plants according to the
invention such as fruits, storage roots, roots, flowers, buds,
shoots or stems, preferably seeds or kernels, these parts which are
capable of being harvested comprising plant cells according to the
invention.
[0133] In a further embodiment, the genetically modified
monocotyledonous plant cells according to the invention are
distinguished by the fact that they synthesize a (waxy) starch with
elevated hot-water swelling power and an amylose content of less
than 5% by weight.
[0134] In a preferred embodiment, the genetically modified
monocotyledonous plant cell is distinguished by the fact that it
comprises a waxy starch with an elevated hot-water swelling power
of between 60 to 100 g/g. Particularly preferred in this context is
a hot-water swelling power of between 70 and 95 g/g, very
particularly preferred of between 80 and 95 g/g and extraordinarily
preferred of between 80 and 90 g/g.
[0135] A further subject matter of the present invention relates to
a method of generating a genetically modified monocotyledonous
plant, where
a) a plant cell is genetically modified, the genetic modification
comprising the following steps i to iii: [0136] i) introduction,
into the plant cell, of a genetic modification, where the genetic
modification leads to an increase in the activity of a protein with
the activity of a starch synthase II in comparison with
corresponding not genetically modified wild-type plant cells,
[0137] ii) introduction, into the plant cell, of a genetic
modification, where the genetic modification leads to an increase
in the activity of a protein with the activity of a glucan, water
dikinase in comparison with corresponding not genetically modified
wild-type plant cells, [0138] iii) introduction, into the plant
cell, of a genetic modification, where the genetic modification
leads to a reduction in the activity of a protein with the activity
of a GBSSI in comparison with corresponding not genetically
modified wild-type plant cells, where steps i to iii can be carried
out in any desired sequence, individually or simultaneously as any
desired combination of steps i to iii, b) a plant is regenerated
from plant cells of step a); c) if appropriate, further plants are
generated with the aid of the plants of step b), where, if
appropriate, plant cells are isolated from plants in accordance
with steps b) or c) and the method steps a) to c) are repeated
until a plant has been generated which has an increased activity of
a protein with the activity of a starch synthase II in comparison
with corresponding not genetically modified wild-type plant cells
and reduced activity of a protein with the activity of a glucan,
water dikinase in comparison with corresponding not genetically
modified wild-type plant cells and reduced activity of a protein
with the activity of a GBSSI in comparison with corresponding not
genetically modified wild-type plant cells.
[0139] The present invention furthermore also relates to a method
of generating a genetically modified plant, in which a plant cell
whose starch has an amylose content of less than 5% by weight is
genetically modified, where genetic modification comprises the
following steps a) and b) in any desired sequence, individually or
simultaneously: [0140] a) introduction, into the plant cell, of a
genetic modification, where the genetic modification leads to an
increase in the activity of a protein with the activity of a starch
synthase II in comparison with corresponding not genetically
modified wild-type plant cells, [0141] b) introduction, into the
plant cell, of a genetic modification, where the genetic
modification leads to an increase in the activity of a protein with
the activity of a glucan, water dikinase in comparison with
corresponding not genetically modified wild-type plant cells, and
[0142] c) a plant is regenerated from plant cells of step a) and
b); [0143] d) if appropriate, further plants are generated with the
aid of the plants from steps a) and b), where, if appropriate,
plant cells are isolated from plants according to step a) or b) and
the method steps a) to c) are repeated until a plant has been
generated which comprises a foreign nucleic acid molecule coding
for a protein with the activity of a starch synthase II and a
foreign nucleic acid molecule coding for a protein with the
activity of a glucan, water dikinase.
[0144] A preferred subject matter of the present invention relates
to methods of generating a monocotyledonous plant, wherein
a) a plant cell is genetically modified, where the genetic
modification comprises the following steps i to iii in any desired
sequence, or any desired combinations of the following steps i to
iii are carried out individually or simultaneously [0145] i)
introduction, into the plant cell, of a genetic modification, where
the genetic modification leads to an increase in the activity of a
protein with the activity of a starch synthase II in comparison
with corresponding not genetically modified wild-type plant cells,
[0146] ii) introduction, into the plant cell, of a genetic
modification, where the genetic modification leads to an increase
in the activity of a protein with the activity of a glucan, water
dikinase in comparison with corresponding not genetically modified
wild-type plant cells, [0147] iii) introduction, into the plant
cell, of a genetic modification, where the genetic modification
leads to a reduction in the activity of a protein with the activity
of a GBSSI in comparison with corresponding not genetically
modified wild-type plant cells, b) a plant is regenerated from
plant cells comprising the genetic modification in accordance with
steps [0148] i) a) i [0149] ii) a) ii [0150] iii) a) iii [0151] iv)
a) i and a) ii, [0152] v) a) i and a) iii, [0153] vi) a) ii and a)
iii, or [0154] vii) a) i and a) ii and a) iii c) there is
introduced, into plant cells from plants in accordance with step
[0155] i) b) i, a genetic modification in accordance with step a)
ii, [0156] ii) b) i, a genetic modification in accordance with step
a) iii, [0157] iii) b) i, a genetic modification in accordance with
step a) ii and simultaneously a genetic modification in accordance
with step a) iii, [0158] iv) b) ii, a genetic modification in
accordance with step a) i, [0159] v) b) ii, a genetic modification
in accordance with step a) iii, [0160] vi) b) ii, a genetic
modification in accordance with step a) i and simultaneously a
genetic modification in accordance with step a) iii, [0161] vii) b)
iii, a genetic modification in accordance with step a) i, [0162]
viii) b) iii, a genetic modification in accordance with step a) ii,
[0163] ix) b) iii, a genetic modification in accordance with step
a) i and simultaneously a genetic modification in accordance with
step a) ii, [0164] x) b) iv, a genetic modification in accordance
with step a) iii, [0165] xi) b) v, a genetic modification in
accordance with step a) ii, or [0166] xii) b) vi, a genetic
modification in accordance with step a) i and the plant is
regenerated, d) there is introduced, into plant cells of plants in
accordance with step [0167] i) c) i, a genetic modification in
accordance with step a) iii, [0168] ii) c) ii, a genetic
modification in accordance with step a) ii, [0169] iii) c) iv, a
genetic modification in accordance with step a) iii, [0170] iv) c)
v, a genetic modification in accordance with step a) ii, [0171] v)
c) vii, a genetic modification in accordance with step a) ii,
[0172] vi) c) vii, a genetic modification in accordance with step
a) i, or [0173] vii) c) ix, a genetic modification in accordance
with step a) ii and a plant is regenerated, if appropriate, further
plants are generated with the aid of the plants in accordance with
one of steps b) vii, c) iii, c) vi, c) x, c) xi, c) xii or in
accordance with any of steps d) i to d) vii.
[0174] The genetic modifications introduced in accordance with step
a) into the plant cell may, in principle, take the form of any type
of modification which leads to an increase in the activity of a
protein with the activity of a starch synthase II and/or which
leads to the increase in the activity of a protein with the
activity of a glucan, water dikinase and/or which leads to the
reduction in the activity of a protein with the activity of a
GBSSI.
[0175] The regeneration of the plants in accordance with steps b)
to e) of the methods according to the invention can be accomplished
by methods known to the skilled worker (for example described in
"Plant Cell Culture Protocols", 1999, ed. by R. D. Hall, Humana
Press, ISBN 0-89603-549-2).
[0176] The generation of further plants of the methods according to
the invention can be accomplished for example by vegetative
propagation (for example via cuttings, tubers or via callus culture
and regeneration of intact plants) or by generative propagation.
Generative propagation preferably takes place in a controlled
manner, i.e. selected plants with specific properties are crossed
with each other and propagated. The selection is preferably
accomplished in such a way that the further plants (which are
generated, depending on the method, in accordance with step c) or
step d) or step e)) have the modifications introduced in the
preceding steps.
[0177] The parameters for the selection of the plant cells or
plants according to the invention which can be generated by
crossing or by transformation are detailed hereinbelow: in the case
where exclusively at least one protein with the activity of a
glucan, water dikinase is increased, suitable plants or plant cells
are those which have a phosphate content in the C6 position of the
starch of at least 2.5 nmol per mg starch. In the case where
exclusively at least one protein with the activity of a starch
synthase II is increased, suitable plants or plant cells are those
which have an SSII activity which is increased by at least a factor
of 2 over the SSII activity in the plant cells or plants which are
used for introducing the nucleic acid molecule(s) according to the
invention or used for crossing.
[0178] In the case where at least one protein with the activity of
a glucan, water dikinase and at least one protein with the activity
of a starch synthase II are increased, suitable plants or plant
cells are those which have a phosphate content in the C6 position
of the starch of at least 2.5 nmol per mg starch and additionally
an SSII activity which is increased by at least a factor of 2 over
the SSII activity in the plant cells or plants which are used for
introducing the nucleic acid molecule(s) according to the invention
or used for crossing. in the case where the GBSSI activity is
reduced, or waxy mutants are employed, suitable plants are those
which have an apparent amylose content of less than 5% by weight
when the mutation is present in homozygous form.
[0179] Another suitable selection criterion is the level of the
starch phosphate content in the C6 position. Plants which are
preferably selected are those which comprise the genetic
modification in accordance with step a) and b) and whose starch
phosphate content is at least 2.5 nmol C6P/mg starch and whose
starch has an apparent amylose content of less than 5% by
weight.
[0180] In the method according to the invention for the generation
of genetically modified plants, the genetic modifications for
generating the genetically modified plant cells according to the
invention can be effected simultaneously or in successive steps. In
this context, it is not critical whether the same method is used
for successive genetic modifications which lead to an increased
activity of a protein with the activity of a starch synthase II as
for the genetic modification which leads to an increased activity
in a protein with the activity of a glucan, water dikinase and/or
for the genetic modification which leads to a reduced activity of a
protein with the activity of a GBSSI.
[0181] Various selection criteria may be chosen for selecting the
plants according to the invention, or those plants which are used
for further modifications.
[0182] In a further embodiment of the method according to the
invention for the generation of a genetically modified plant, step
c) is followed by a method step c)-1, in which plants are selected
whose starch has an apparent amylose content of less than 5% by
weight and an increased activity in a protein with the activity of
a starch synthase II in accordance with step a)i) and/or has an
increased activity of a protein with the activity of a glucan,
water dikinase in accordance with step a)ii). The selected plants
are then used for the further method steps.
[0183] In a further embodiment of the method according to the
invention for the generation of a genetically modified plant
according to the invention, at least one foreign nucleic acid
molecule codes for a protein with the activity of a glucan, water
dikinase from potato, wheat, rice, maize, soybean, citrus, Curcuma
or Arabidopsis. Preferably, at least one foreign nucleic acid
molecule codes for a protein with the activity of a glucan, water
dikinase from potato and especially preferably for a protein which
has the amino acid sequence shown in SEQ ID NO 2 or which is
encoded by the nucleic acid sequence shown in SEQ ID NO 1.
References for nucleic acid sequences coding for proteins with the
activity of a glucan, water dikinase from the abovementioned plants
have already been detailed further above.
[0184] In a further embodiment of the method according to the
invention for generating a genetically modified plant according to
the invention, at least one foreign nucleic acid molecule codes for
a protein with the activity of a starch synthase II from wheat,
barley, Aegilops, rice, maize, cassava, bean, potato, pea, sweet
potato, Arabidopsis, taro, Ostreococcus or Chlamydomonas.
Preferably, at least one foreign nucleic acid molecule codes for a
protein with the activity of a starch synthase II from wheat, in
particular Seq ID No 3. References for nucleic acid sequences
coding for proteins with the activity of a starch synthase II from
the abovementioned plants have already been detailed further
above.
[0185] As already described above for foreign nucleic acid
molecules introduced into a plant cell or plant for the purposes of
genetic modification, the nucleic acid molecule(s) in step a) of
the method according to the invention for the generation of a
genetically modified plant whose starch has an amylose content of
less than 5% by weight may take the form of a single nucleic acid
molecule or a plurality of nucleic acid molecules. Thus, the
foreign nucleic acid molecules coding for a protein with the
activity of a starch synthase II, or coding for a protein with the
activity of a glucan, water dikinase, may be present together on a
single nucleic acid molecule or else they may be present in
separate nucleic acid molecules. If the nucleic acid molecules
coding for a protein with the activity of a starch synthase II and
coding for a protein with the activity of a glucan, water dikinase
are present in a plurality of nucleic acid molecules, these nucleic
acid molecules may be introduced into a plant cell either
simultaneously or in successive steps.
[0186] In a further embodiment of the method according to the
invention for the generation of a genetically modified plant
according to the invention, at least one foreign nucleic acid
molecule codes for a protein with the activity of a GBSSI from a
monocotyledonous plant, preferably from rice, wheat, barley, maize,
Aegilops, sorghum or oats.
[0187] References for the abovementioned nucleic acid sequences
coding for proteins with the activity of a GBSS1 from the
abovementioned plants have already been detailed further above.
[0188] Preferably, at least one foreign nucleic acid molecule codes
for a protein with the activity of a GBSS1 from rice and especially
preferably for a protein which is encoded by the nucleic acid
sequence shown in SEQ ID NO 7 or by the amino acid sequence shown
in SEQ ID NO 8.
[0189] In a further preferred embodiment, at least one foreign
nucleic acid molecule codes for a protein with the activity of a
GBSS1 from wheat and especially preferably for a protein which is
encoded by the amino acid sequence shown in SEQ ID NO 9 or shown in
SEQ ID NO 10.
[0190] In a further preferred embodiment, at least one foreign
nucleic acid molecule codes for a protein with the activity of a
GBSS1 from maize and especially preferably for a protein which is
encoded by the nucleic acid sequence shown in SEQ ID NO 11 or by
the amino acid sequence shown in SEQ ID NO 12.
[0191] Here, the foreign nucleic acid molecule brings about the
inhibition of the activity of a GBSS I and thus the synthesis of a
starch with an amylose content of less than 5% by weight. What has
been said above regarding the use of the nucleic acids in question
for the generation of plant cells or plants according to the
invention also applies here analogously.
[0192] The foreign nucleic acid molecule(s) used for the genetic
modification may take the form of one combined or of a plurality of
separate nucleic acid constructs, in particular of what are known
as simple, dual or triple constructs. Thus, the foreign nucleic
acid molecule may be what is known as a "triple construct", which
is understood as meaning a single vector for the transformation of
plants which comprises not only the genetic information for
inhibiting the expression of an endogenous GBSSI gene, but also the
information for the overexpression of one or more SSII genes and
for the overexpression of one or more GWD genes.
[0193] A basic principle in the construction of the foreign nucleic
acid molecules for inhibiting the GBSSI activity is the use of
antisense, cosuppression, ribozym and double-stranded RNA
constructs and of sense constructs, which use leads to a reduction
in the expression of endogenous genes which code for GBSSI and
which leads to a simultaneous increase in the activity of the
proteins with the activities of an SSII and/or of a GWD.
[0194] In this context, the foreign nucleic acid molecules may be
introduced into the genome of the plant cell either simultaneously
("cotransformation") or else one after the other, i.e. in
chronological succession ("supertransformation").
[0195] The foreign nucleic acid molecules may also be introduced
into different individual plants of one species. In this way, it is
possible to generate plants in which the activity of a protein with
the activity of a GBSSI is reduced and/or the activity of a protein
with the activity of an SSII or GWD is increased. Subsequently,
crosses may then be made to generate plants in which the activity
of a protein with the activity of a GBSSI is reduced and the
activity of a protein with the activity of an SSII and a GWD is
increased.
[0196] In the context of the present invention, the term "identity"
is understood as meaning the number of amino acids/nucleotides
which agree (identity) with other proteins/nucleic acids, expressed
in percent.
[0197] Preferably, the identity regarding a protein with the
activity of a starch synthase II is determined by comparing the
amino acid sequences detailed under SEQ ID NO 4 or SEQ ID NO 6, or
the identity regarding a nucleic acid molecule coding for a protein
with the activity of a starch synthase II by comparing the nucleic
acid sequences detailed under SEQ ID NO 3 or SEQ ID NO 5, and the
identity regarding a protein with the activity of a glucan, water
dikinase by comparing the amino acid sequence detailed in SEQ ID NO
2, or the identity regarding a nucleic acid molecule coding for a
protein with the activity of a glucan, water dikinase by comparing
the nucleic acid sequence detailed in SEQ ID NO 1, and the identity
regarding a nucleic acid molecule coding for a protein with the
activity of a GBSSI by comparing the nucleic acid sequences
detailed in SEQ ID NO 7 or SEQ ID NO 9 or SEQ ID NO 11, or the
amino acid sequences detailed in SEQ ID NO 8 or SEQ ID NO 10 or SEQ
ID NO 12, with other proteins/nucleic acids with the aid of
computer programs.
[0198] If sequences which are compared with each other are
different in length, the identity is to be determined in such a way
that the number of amino acids/nucleotides which the shorter
sequence shares with the longer sequence determines the percentage
identity. The identity is preferably determined by means of known
computer programmes which are publicly available such as, for
example, ClustalW (Thompson et al., Nucleic Acids Research 22
(1994), 4673-4680). ClustalW is made publicly available by Julie
Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson
(Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory,
Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can
likewise be downloaded from various internet pages, inter alia the
IGBMC (Institut de Genetique et de Biologie Moleculaire et
Cellulaire, B.P. 163, 67404 Illkirch Cedex, France;
ftp://ftp-igbmc.u-strasbg.fr/pub/) and the EBI
(ftp://ftp.ebi.ac.uk/pub/software/) and all mirrored EBI internet
pages (European Bioinformatics Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge CB10 1SD, UK).
[0199] To determine the identity between proteins described within
the scope of the present invention and other proteins, it is
preferred to employ the ClustalW computer program version 1.8. The
following parameters are to be set: KTUPLE=1, TOPDIAG=5, WINDOW=5,
PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40,
MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.
[0200] To determine the identity between for example the nucleotide
sequence of the nucleic acid molecules described within the scope
of the present invention and the nucleotide sequence of other
nucleic acid molecules, it is preferred to employ the ClustalW
computer program version 1.8. The following parameters are to be
set: KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=10,
GAPEXT=5, MAXDIV=40, TRANSITIONS: unweighted.
[0201] Identity furthermore means that functional and/or structural
equivalence exists between the nucleic acid molecules in question
or the proteins encoded by them. The nucleic acid molecules which
are homologous to the above-described molecules and which are
derivatives of these molecules will, as a rule, take the form of
variations to these molecules which are modifications with the same
biological function. They may take the form of naturally occurring
variations, for example sequences from other species or else of
mutations, where it is possible that these mutations have occurred
naturally or else have been introduced by specific mutagenesis.
Furthermore, the variations may take the form of synthetically
generated sequences. The allelic variants may take the form of
naturally occurring variants or else of synthetically generated
variants or variants which have been generated by recombinant DNA
technology. A specific form of derivatives are for example nucleic
acid molecules which deviate from the nucleic acid molecules
described within the scope of the present invention as the result
of the degeneracy of the genetic code.
[0202] Within the scope of the present invention, the term
"hybridization" means hybridization under traditional hybridization
conditions, preferably under stringent conditions as are described
for example in Sambrook et al., (Molecular Cloning, A Laboratory
Manual, 3rd edition (2001) Cold Spring Harbour Laboratory Press,
Cold Spring Harbour, N.Y.; ISBN: 0879695773). Particularly
preferably, "to hybridize" means hybridization under the following
conditions: hybridization buffer:
[0203] 2.times.SSC; 10.times.Denhardt solution (Ficoll
400+PEG-FBSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250
.mu.g/ml herring sperm DNA; 50 .mu.g/ml tRNA; or 25 M sodium
phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS hybridization
temperature:
[0204] T=65 to 68.degree. C.
[0205] Wash buffer: 0.1.times.SSC; 0.1% SDS
[0206] Wash temperature: T=65 to 68.degree. C.
[0207] Nucleic acid molecules which hybridize with the
abovementioned molecules can be isolated for example from genomic
libraries or from cDNA libraries. The identification and isolation
of such nucleic acid molecules may be accomplished using the
abovementioned nucleic acid molecules or parts of these molecules,
or using the reverse complements of these molecules, for example by
means of hybridization by standard methods, or by amplification by
means of PCR.
[0208] Hybridization probes which can be used for isolating a
nucleic acid sequence coding for a protein with the activity of a
starch synthase II or with the activity of a glucan, water dikinase
or with the activity of a GBSSI are, for example, nucleic acid
molecules with exactly the nucleotide sequences, or essentially the
nucleotide sequences, detailed in SEQ ID NO 3 or SEQ ID NO 5
(starch synthase II) or in SEQ ID NO 1 (glucan, water dikinase) or
in SEQ ID NO 7, 9 or 11 (GBSSI), or parts of these sequences.
[0209] The fragments used as hybridization probe may also take the
form of synthetic fragments or oligonucleotides which have been
generated with the aid of the customary synthetic techniques and
whose sequence agrees essentially with that of a nucleic acid
molecule described within the scope of the present invention. When
genes which hybridize with the nucleic acid sequences described
within the scope of the present invention have been identified and
isolated, a determination of the sequence and an analysis of the
characteristics of the proteins encoded by this sequence should be
carried out to verify that they are proteins with the activity of a
starch synthase II or the activity of a glucan, water dikinase or
the activity of a GBSSI, respectively.
[0210] The molecules which hybridize with the nucleic acid
molecules described within the scope of the present invention
comprise in particular fragments, derivatives and allelic variants
of the abovementioned nucleic acid molecules. In the context of the
present invention, the term "derivative" means that the sequences
of these molecules differ from the sequences of the above-described
nucleic acid molecules at one or more positions and that they have
a high degree of identity with these sequences. The deviations from
the above-described nucleic acid molecules may have been generated
for example by deletion, addition, substitution, insertion or
recombination.
[0211] To express nucleic acid molecules according to the invention
which code for a protein with the activity of starch synthase II
and/or a protein with the activity of a glucan, water dikinase
and/or a protein with the activity of a GBSSI, these molecules are
preferably linked with regulatory DNA sequences which ensure
transcription in plant cells. These include in particular
promoters. In general, any promoter which is active in plant cells
is suitable for expression.
[0212] The promoter may be selected in such a way that expression
takes place constitutively or else only in a certain tissue, at a
certain point in time of plant development or at a point in time
determined by external factors. The promoter may be homologous or
heterologous both with regard to the plant and with regard to the
nucleic acid molecule.
[0213] Examples of suitable promoters are the 35S RNA promoter of
the Cauliflower Mosaic Virus and the maize ubiquitin promoter, the
rice ubiquitin promoter (Liu et al., Plant Science 165, (2003), the
rice actin promoter (Zhang, et al., Plant Cell 3:1150-1160, 1991),
the Cassava Vein Mosaic Virus (CVMV) promoter (Verdaguer et. al.,
Plant Mol. Biol. 31: 1129-1139), the maize histone H.sub.3C4
promoter (U.S. Pat. No. 6,750,378) or the Cestrum YLCV promoter
(Yellow Leaf Curling Virus; WO 01 73087; Stavolone et al., 2003,
Plant Mol. Biol. 53, 703-713) for the purposes of constitutive
expression. A promoter which ensures expression only in
photosynthetically active tissues may also be used, for example the
ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 1987,
84: 7943-7947; Stockhaus et al., EMBO J. 1989, 8: 2445-2451), or
for endosperm-specific expression, the wheat HMW promoter, the
Vicia faba USP promoter (Fiedler et al., 1993, Plant Mol. Biol. 22:
669-679; Baumlein et al., 1991, Mol. Gen. Genet. 225: 459-467), the
bean phaseolin promoter, promoters of zein genes from maize
(Pedersen et al., 1982, Cell 29: 1015-1026; Quatroccio et al.,
1990, Plant Mol. Biol. 15: 81-93), a glutelin promoter (Leisy et
al., 1990, Plant Mol. Biol. 14: 41-50; Zheng et al., 1993, Plant J.
4: 357-366; Yoshihara et al., 1996, FEBS Lett. 383: 213-218), a
globulin promoter (Nakase et al., 1996, Gene 170(2): 223-226), a
prolamin promoter (Qu and Takaiwa, 2004, Plant Biotechnology
Journal 2(2): 113-125). However, it is also possible to use
promoters which are activated only at a point in time which is
determined by external factors (see, for example, WO 93/07279).
Promoters which are also of interest may be promoters of heat-shock
proteins, which can make simple induction possible. Furthermore, it
is possible to use seed-specific promoters, such as, for example,
the Vicia faba USP promoter (see above).
[0214] A termination sequence (polyandenylation signal) may also be
present; this serves to add a poly-A tail to the transcript. The
poly-A tail is assumed to have a function in the stabilization of
the transcripts. Such elements are described in the literature (cf.
Gielen et al., 1989, EMBO J. 8: 23-29) and may be exchanged as
desired.
[0215] It is also possible for intron sequences to be present
between the promoter and the coding region. Such intron sequences
may lead to the stability of the expression and to an increased
expression in plants (Callis et al., 1987, Genes Devel. 1:
1183-1200; Luehrsen and Walbot 1991, Mol. Gen. Genet. 225: 81-93;
Rethmeier et al. 1997, Plant Journal. 12(4): 895-899; Rose and
Beliakoff 2000, Plant Physiol. 122 (2): 535-542; Vasil et al.,
1989, Plant Physiol. 91: 1575-1579; Xu et al. 2003, Science in
China Series C Vol. 46(6): 561-569). Examples of suitable intron
sequences are the first intron of the maize sh1 gene, the first
intron of the maize poly-ubiquitin gene 1, the first intron of the
rice EPSPS gene, or one of the first two introns of the Arabidopsis
PAT1 gene.
[0216] A further embodiment of the present invention relates to a
method of generating a genetically modified monocotyledonous plant
according to the invention, wherein a plant cell whose starch has
an apparent amylose content of less than 5% by weight
a) is genetically modified, where the genetic modification leads to
an increase in the activity of a protein with the activity of a
starch synthase II in comparison with corresponding not genetically
modified wild-type plant cells; b) a plant is regenerated from
plant cells of step a); c) if appropriate, further plants are
generated with the aid of the plants in accordance with step b),
and d) plants obtained in accordance with step b) or c) are crossed
with a plant which shows an increase in the activity of a protein
with the activity of a glucan, water dikinase in comparison with
corresponding not genetically modified wild-type plant cells.
[0217] A further embodiment of the present invention relates to a
method of generating a genetically modified monocotyledonous plant
according to the invention, wherein a plant cell whose starch has
an apparent amylose content of less than 5% by weight
a) is genetically modified, where the genetic modification leads to
an increase in the activity of a protein with the activity of a
glucan, water dikinase in comparison with corresponding not
genetically modified wild-type plant cells; b) a plant is
regenerated from plant cells of step a); c) if appropriate, further
plants are generated with the aid of the plants in accordance with
step b), and d) plants obtained in accordance with step b) or c)
are crossed with a plant which shows an increase in the enzymatic
activity of a protein with the activity of a starch synthase II in
comparison with corresponding not genetically modified wild-type
plant cells.
[0218] A further embodiment of the present invention relates to a
method of generating a genetically modified monocotyledonous plant
according to the invention, wherein a plant cell is genetic
modified, where
a) i) the genetic modification leads to an increase in the activity
of a protein with the activity of a glucan, water dikinase; a) ii)
a further genetical modification is carried out which leads to an
increase in the activity of a protein with the activity of a starch
synthase II [0219] in comparison with corresponding not genetically
modified wild-type plant cells; where steps a) i) and ii) can be
carried out in any desired sequence, b) a plant is regenerated from
plant cells of step a) i) and ii); c) if appropriate, further
plants are generated with the aid of the plants in accordance with
step b), and d) plants obtained in accordance with steps a) to c)
are crossed with a plant whose starch thus has an amylose content
of less than 5% by weight in comparison with corresponding not
genetically modified wild-type plant cells.
[0220] In the three last-mentioned methods of generating a
genetically modified plant, the plants may be genetically modified
in accordance with step a), as already described above. The
regeneration of plants in accordance with step b) and the
generation of further plants in accordance with steps c) and d)
have also been detailed further above.
[0221] A plant which is crossed in accordance with step d) of the
first two embodiments with plants or progeny of the plants obtained
from step b) or c) may be any plant which shows an increase in the
activity of a protein with the activity of a starch synthase II or
an increase in the activity of a protein with the activity of a
glucan, water dikinase in comparison with corresponding wild-type
plants. The increase in the activity of a protein with the activity
of a starch synthase II, or a protein with the activity of a
glucan, water dikinase, may have been brought about by any
modification which leads to an increase in the activity of the
proteins in question in the corresponding plants. These plants may
take the form of mutants or of plants which have been modified by
recombinant methods. The mutants may take the form of spontaneously
(naturally) occurring mutants or else of those which have been
generated by the targeted use of mutagens (such as, for example,
chemical agents, ionizing radiation) or recombinant methods (for
example transposon activation tagging, T-DNA activation tagging, in
vivo mutagenesis).
[0222] Plants which are preferably used for crosses in the two
last-mentioned methods according to the invention are those with an
activity of a protein with the activity of a starch synthase II
which is increased by at least 3-fold, preferably 6-fold,
preferably at least 8-fold and particularly preferably at least
10-fold in comparison with corresponding genetically not modified
wild-type plants.
[0223] Such plants in question with an increased activity of a
protein with the activity of a glucan, water dikinase are used for
crosses in the two last-mentioned methods according to the
invention are preferably plants which synthesize a starch with a
starch phosphate content of at least 2.5 nmol C6P/mg starch.
[0224] In a preferred embodiment, methods according to the
invention are used for generating a genetically modified plant for
generating plants according to the invention or for generating
plants which have the characteristics of plants according to the
invention.
[0225] The present invention also relates to plants obtainable by
methods according to the invention.
[0226] Surprisingly, it has been found that plant cells according
to the invention and plants according to the invention whose starch
has an apparent amylose content of less than 5% by weight and an
increase in the activity of a protein with the activity of a starch
synthase II and an increase in the activity of a protein with the
activity of a glucan, water dikinase synthetize a modified starch.
The fact that starch synthetized by plant cells according to the
invention or plants according to the invention has an increased
hot-water swelling power was particularly surprisingly. The
increased hot-water swelling power of starches which can be
isolated from plant cells according to the invention and plants
according to the invention imparts to the starches according to the
invention properties which make them better suited to certain
applications than traditional starches. If starch is employed for
example as a thickener, the increased hot-water swelling power of
the starch means that considerably less starch is required for
achieving the same thickening power.
[0227] A further subject matter of the present invention relates to
modified starch with an apparent amylose content of less than 5% by
weight and an increased hot-water swelling power. The hot-water
swelling power of modified starch according to the invention is
increased preferably by at least the factor 1.5, particularly
preferably by at least the factor 2, especially preferably by at
least the factor 2.5 and very particularly preferably by at least
the factor 3 in comparison with starch isolated from corresponding
not genetically modified wild-type plant cells or isolated from
corresponding not genetically modified wild-type plants.
[0228] Methods for determining the hot-water swelling power are
known to the skilled worker and described in the literature (for
example Leach et al., 1959, Cereal Chemistry 36: 534-544). A method
to be used by preference in connection with the present invention
for determining the hot-water swelling power is described further
below in "General Methods".
[0229] A further subject matter of the present invention relates to
modified starch, isolated from a monocotyledonous plant cell or
from a monocotyledonous plant, with an apparent amylose content of
5% by weight and which has a hot-water swelling power of from at
least 60 g/g, preferably of from 60 to 100 g/g, particularly
preferably of from 70 to 95 g/g, especially preferably of from 80
to 95 g/g and specifically preferably of from 80 to 90 g/g.
[0230] A further subject matter of the present invention relates to
modified starch, isolated from rice plant cells or rice plants,
with an apparent amylose content of 5% by weight and a hot-water
swelling power of from at least 60 g/g, preferably of from 60 to
100 g/g, particularly preferably of from 70 to 95 g/g, especially
preferably of from 80 to 95 g/g and specifically preferably of from
80 to 90 g/g.
[0231] Starch synthetized by genetically modified plant cells
according to the invention or genetically modified plants according
to the invention preferably has an increased content of phosphate
in the C6 position of the starch. Here, the starch phosphate
content of starch isolated from plant cells according to the
invention and plants according to the invention is markedly higher
than the starch phosphate content which would be expected after
making crosses on the basis of the total of the starch phosphate
contents of the parent plants in question.
[0232] The amount of the starch phosphate bound in the C6 position
of the glucose molecules can be determined by methods known to the
skilled worker, such as, for example, photometrically by means of
coupled enzyme assays or by means of .sup.31P NMR, following the
method described by Kasemusuwan and Jane (1996, Cereal Chemistry
73: 702-707). In the context of the present invention, the amount
of starch phosphate bound in the C6 position of the glucose
molecules is preferably determined as described in "General
Methods".
[0233] A further preferred subject matter of the present invention
relates to modified starch according to the invention which has
been isolated from a monocotyledonous plant cell or from a
monocotyledonous plant and which has a starch phosphate content
bound in the C6 position of the glucose molecules of the starch of
at least 1.5 nmol per mg starch, particularly preferably of at
least 2.5 nmol per mg starch. This modified starch according to the
invention particularly preferably takes the form of maize, rice or
wheat starch.
[0234] In a further embodiment of the present invention, the
modified starches according to the invention take the form of
native starches.
[0235] In the context of the present invention, the term "native
starch" means that the starch is isolated by methods known to the
skilled worker from plants according to the invention, harvestable
plant parts according to the invention, starch-storing parts
according to the invention or plant propagation material according
to the invention.
[0236] The present invention also relates to modified starch
according to the invention obtainable from plant cells according to
the invention or plants according to the invention, from
propagation material according to the invention or from harvestable
plant parts according to the invention, or obtainable from plants
which have been generated using a method according to the invention
for generating a genetically modified plant.
[0237] Plant cells or plants which synthetize a modified starch
according to the invention are likewise subject matter of the
present invention.
[0238] The present invention furthermore relates to a method of
generating a modified starch comprising the step of extracting the
starch from a plant cell according to the invention or a plant
according to the invention, from propagation material according to
the invention of such a plant and/or from harvestable plant parts
according to the invention of such a plant, preferably from
starch-storing parts according to the invention of such a plant.
Preferably, such a method also comprises the step of harvesting the
plants or plant parts which have been grown and/or the propagation
material of these plants before extracting the starch, and
particularly preferably furthermore the step of growing plants
according to the invention before harvesting.
[0239] Methods for extracting the starch from plants, or from
starch-storing parts of plants, are known to the skilled worker.
Furthermore, methods for extracting the starch from various
starch-storing plants have been described, for example in Starch:
Chemistry and Technology (Ed.: Whistler, BeMiller and Paschall
(1994), 2nd edition, Academic Press Inc. London Ltd; ISBN
0-12-746270-8; see, for example, chapter XII, page 412-468: Mais
and sorghum starches: production; by Watson; chapter XIII, page
469-479: Tapioca, Arrowroot and Sago starches: production; by
Corbishley and Miller; chapter XIV, page 479-490: potato starch:
production and uses; by Mitch; chapter XV, page 491 to 506: wheat
starch: production, modification and uses; by Knight and Oson; and
chapter XVI, page 507 to 528: rice starch: production and uses; by
Rohmer and Klem; maize starch: Eckhoff et al., 1996, Cereal Chem.
73: 54-57, the extraction of maize starch on the industrial scale
is generally accomplished by what is known as wet milling). Devices
which are usually employed in processes for extracting starch from
plant material are separators, decanters, hydrocyclones, spray
dryers and fluidized-bed dryers.
[0240] In the context of the present invention, the term
"starch-storing parts" are understood as meaning those parts of a
plant in which starch, in contrast to transitory leaf starch, is
stored as a reserve for surviving for longer periods. Preferred
starch-storing plant parts are, for example, tubers, storage roots
and grains, particularly preferred are grains comprising an
endosperm, especially preferred are grains comprising an endosperm
from maize, rice or wheat plants.
[0241] In a preferred embodiment, methods according to the
invention for preparing a modified starch are used for preparing a
starch according to the invention.
[0242] Modified starch obtainable by a process according to the
invention for preparing modified starch is also a subject matter of
the present invention.
[0243] The use of plant cells according to the invention or plants
according to the invention for preparing a modified starch is also
subject matter of the present invention. The skilled worker knows
that the properties of starch can be altered for example via
thermal, chemical, enzymatic or mechanical derivatization.
Derivatized starches are particularly suitable for a variety of
uses in the food and/or nonfood sector. The starches according to
the invention are better suited as starting material for the
preparation of derivatized starches than conventional starches
since they comprise a higher proportion of reactive functional
groups, for example as a result of the higher starch phosphate
content. As the result of the increased hot-water swelling power of
starches according to the invention, the derivatization processes
can furthermore be carried out at higher temperatures without the
starch granule structure being damaged to a substantial degree.
[0244] The present invention therefore also relates to processes
for preparing a derivatized starch, wherein modified starch
according to the invention is subsequently derivatized. The present
invention furthermore relates to a derivatized starch prepared by
one of the known processes.
[0245] In the context of the present invention, the term
"derivatized starch" is understood as meaning a modified starch
according to the invention whose properties have been altered with
the aid of chemical, enzymatic, thermal or mechanical processes
after the starch has been isolated from plant cells.
[0246] In another embodiment of the present invention, the
derivatized starch according to the invention is heat- and/or
acid-treated starch.
[0247] In a further embodiment, the derivatized starches take the
form of starch ethers, in particular starch alkyl ethers, O-allyl
ethers, hydroxyl alkyl ethers, O-carboxylmethyl ethers,
nitrogen-containing starch ethers, phosphate-containing starch
ethers or sulfur-containing starch ethers.
[0248] In a further embodiment, the derivatized starches take the
form of crosslinked starches.
[0249] In a further embodiment, the derivatized starches take the
form of starch graft polymers.
[0250] In a further embodiment, the derivatized starches take the
form of oxidized starches.
[0251] In a further embodiment, the derivatized starches take the
form of starch esters, in particular starch esters which have been
introduced into the starch using organic acids. They particularly
preferably take the form of what are known as phosphate starches,
nitrate starches, sulfate starches, xanthate starches, acetate
starches or citrate starches.
[0252] The derivatized starches according to the invention are
suitable for a variety of uses in the pharmaceutical industry, in
the food sector and/or in the nonfood sector. Methods of preparing
derivatized starches according to the invention are known to the
skilled worker and extensively described in the general literature.
A review of the preparation of derivatized starches is found for
example in Orthoefer (in Corn, Chemistry and Technology, 1987, eds.
Watson and Ramstad, Chapter 16: 479-499).
[0253] Derivatized starch obtainable by the process according to
the invention for preparing a derivatized starch is likewise
subject matter of the present invention.
[0254] The use of modified starches according to the invention for
the preparation of derivatized starch is furthermore subject matter
of the present invention.
[0255] The present invention also comprises products comprising a
starch according to the invention.
[0256] The present invention also comprises mixtures comprising the
starch according to the invention.
[0257] Starch-storing parts of plants are frequently processed into
flours. Examples of parts of plants from which flours are prepared
are, for example, tubers of potato plants and grains of cereal
plants. To prepare flours from cereal plants, the
endosperm-containing grains of these plants are ground and sieved.
Starch is a main constituent of the endosperm. In other plants
which comprise no endosperm, but other starch-storing parts such
as, for example, tubers or roots, flour is frequently prepared by
comminuting, drying and subsequently grinding the storage organs in
question. The starch of the endosperm or present in starch-storing
parts of plants accounts for a considerable proportion of the flour
which is prepared from the plant parts in question. The properties
of flours are therefore also influenced by the starch present in
the flour in question. Plant cells according to the invention and
plants according to the invention synthesize an altered starch in
comparison with corresponding not genetically modified wild-type
plant cells, or not genetically modified wild-type plants. Flours
prepared from plant cells according to the invention, plants
according to the invention, propagation material according to the
invention or harvestable parts according to the invention therefore
have altered properties. The properties of flours may also be
influenced by mixing starch with flours or by mixing flours with
different properties.
[0258] A further subject matter of the present invention therefore
relates to flours comprising a starch according to the
invention.
[0259] A further subject matter of the present invention relates to
flours which can be prepared from plant cells according to the
invention, plants according to the invention, starch-storing parts
of plants according to the invention, from propagation material
according to the invention or from harvestable plant parts
according to the invention. Preferred starch-storing parts of
plants according to the invention for the preparation of flours are
tubers, storage roots and grains which comprise an endosperm.
Particularly preferred in the context of the present invention are
grains from plants of the (systematic) family Poaceae; especially
preferably, grains are obtained from maize, rice or wheat
plants.
[0260] In the context of the present invention, the term "flour" is
understood as meaning a powder which can be obtained by grinding
plant parts. If appropriate, plant parts are dried and sieved prior
to grinding.
[0261] On account of the starch according to the invention present
in them, flours according to the invention are distinguished by the
fact that they have an increased hot-water swelling power. This is
desirable for example in the processing of flours in the food
industry for a multiplicity of applications, in particular in the
production of baked good.
[0262] A preferred subject matter of the present invention relates
to flours prepared from grains of a monocotyledonous waxy plant,
which flours have a hot-water swelling power of at least 25 g/g,
preferably of from 25 to 50 g/g, particularly preferably of from 30
to 45 g/g and especially preferably of from 35 to 45 g/g.
[0263] In this context, the determination of the hot-water swelling
power of flours is effected analogously to the above-described
method for determining the hot-water swelling power for starch,
with the difference that flours are employed in place of starch. A
preferred method of determining the hot-water swelling power of
flours is described in "General Methods".
[0264] A further subject matter of the present invention is a
process for the preparation of flours, comprising the step of
grinding plant cells according to the invention, plants according
to the invention, parts of plants according to the invention,
starch-storing parts of plants according to the invention,
propagation material according to the invention or harvestable
material according to the invention.
[0265] Flours can be produced by grinding starch-storing parts of
plants according to the invention. The skilled worker knows how to
produce flours. Preferably, a process for the production of flours
also comprises the step of harvesting the plants or plant parts
which are grown and/or the propagation material and/or the
starch-storing parts of these plants before grinding, and
particularly preferably furthermore the step of growing plants
according to the invention before harvesting.
[0266] Products comprising a flour according to the invention are
likewise subject matter of the present invention.
[0267] In a further embodiment of the present invention, the
process for the production of flours comprises the processing of
plants according to the invention, of starch-storing parts of
plants according to the invention, of propagation material
according to the invention or of harvestable material according to
the invention prior to grinding.
[0268] In this context, processing may be a heat treatment and/or a
drying step. A heat treatment followed by the drying of the
heat-treated material is employed for example in the production of
flours from storage roots or tubers such as, for example, from
potato tubers, before grinding takes place. The comminution of
plants according to the invention, of starch-storing parts of
plants according to the invention, of propagation material
according to the invention or of harvestable material according to
the invention before grinding may likewise constitute processing
within the meaning of the present invention. The removal of plant
tissue before grinding, such as, for example, hulling the grains,
also constitutes processing before grinding within the meaning of
the present invention.
[0269] In a further embodiment of the present invention, the
process for the preparation of flours comprises processing the mill
base after grinding. In this context, the mill base may be sieved
after grinding in order to prepare various types of flours.
[0270] The present invention also comprises mixtures comprising a
flour according to the invention.
[0271] A further subject matter of the present invention is the use
of genetically modified plant cells according to the invention, of
plants according to the invention, of parts of plants according to
the invention, of starch-storing parts of plants according to the
invention, of propagation material according to the invention or of
harvestable material according to the invention for the preparation
of flours.
[0272] The disclosure of all documents cited in the patent
application is intended to be incorporated in the disclosure of the
present description of the invention.
DESCRIPTION OF THE SEQUENCES
[0273] SEQ ID NO 1: Nucleic acid sequence coding for a protein with
the activity of a glucan, water dikinase from Solanum tuberosum.
[0274] SEQ ID NO 2: Amino acid sequence of the protein encoded by
SEQ ID NO 1 with the activity of a glucan, water dikinase from
Solanum tuberosum. [0275] SEQ ID NO 3: Nucleic acid sequence coding
for a protein with the activity of a starch synthase II from
Triticum aestivum. [0276] SEQ ID NO 4: Amino acid sequence of the
protein encoded by SEQ ID NO 3 with the activity of a starch
synthase II from Triticum aestivum. [0277] SEQ ID NO 5: Nucleic
acid sequence coding for a protein with the activity of a starch
synthase II from Oryza sativa. [0278] SEQ ID NO 6: Amino acid
sequence of the protein encoded by SEQ ID NO 5 with the activity of
a starch synthase II from Oryza sativa. [0279] SEQ ID NO 7: Nucleic
acid sequence coding for a protein with the activity of a GBSS I
from Oryza sativa. [0280] SEQ ID NO 8: Amino acid sequence of the
protein encoded by SEQ ID NO 7 with the activity of a GBSS I from
Oryza sativa. [0281] SEQ ID NO 9: Nucleic acid sequence coding for
a protein with the activity of a GBSS I from Triticum aestivum.
[0282] SEQ ID NO 10: Amino acid sequence of the protein encoded by
SEQ ID NO 9 with the activity of a GBSS I from Triticum aestivum
[0283] SEQ ID NO 11: Nucleic acid sequence coding for a protein
with the activity of a GBSS I from Zea mays. [0284] SEQ ID NO 12:
Amino acid sequence of the protein encoded by SEQ ID NO 11 with the
activity of a GBSS I from Zea mays
General Methods
[0285] In the following text, methods will be described which can
be used for carrying out the methods/processes according to the
invention. These methods are specific embodiments of the present
invention, but do not limit the present invention to these methods.
The skilled worker knows that he can carry out the invention in the
same manner by modifying the methods described and/or by replacing
individual parts of the methods by alternative parts of methods.
The content of all cited publications is incorporated into the
description of the application by reference.
1. Transformation and Regeneration of Rice Plants
[0286] Rice plants were transformed by the method described by Hiei
et al. (1994, Plant Journal 6(2), 271-282).
[0287] The regimen of the rice plants in the greenhouse involved
the following conditions:
[0288] sowing: substrate: mixture of 100% sphagnum peat and 1001
sand/m.sup.2 and clay: 180 kg/m.sup.2 in 1.6 I rose pots
(manufacturer: H. Meyer, Germany), pH: 5.4-6.2; green manure:
Hakaphos (Compo, Germany) 14% N-16% P-18% K+2% Mg; 2 kg/m.sup.2;
fertilization: 3.5 g/plant until flowering: NH.sub.4NO.sub.3 (1.75
g) and Flory 2 basic mixture (manufacturer: Euflor, Germany): 1.75
g; 3% N-16% P-15% K+5% Mg.
[0289] Temperature: day 28.degree. C./night: 24.degree. C. (16 h/8
h); relative atmospheric humidity: 85-95%; Light: 16 h, 350
pEinstein/s.times.m.sup.2
2. Origin of the Sequences and Constructs Used for the
Transformation
[0290] The sequence T.a.-SSIIa from wheat was used for the
transformation of rice. It was isolated and cloned as described in
WO 97-45545 (under its then name "pTaSS1"). The transformation
vector used, AH32-191, is described in example 2.
[0291] The sequence of a glucan, water dikinase from potato (R1St)
was furthermore used. It was isolated and cloned as described in
example 5. The transformation vector used, pML82, is described in
WO 05/095619.
[0292] The waxy trait was introduced via a suitable mutant which is
explained in example 1.
3. Analysis of the Expression Level of a Gene by Means of Northern
Blot
[0293] The expression of a nucleic acid which codes for a protein
was studied by means of Northern blot analysis. To this end, three
immature rice grains (approximately 15 days after anthesis) were
harvested for each individual plant obtained by means of
transformation and frozen in liquid nitrogen. To homogenize the
material, the frozen rice grains were comminuted for 30 seconds in
a Retsch mill (model MM300) in a 96-well microtiter plate using a
4.5 mm steel ball at a frequency of 30 Hertz. Thereafter, the RNA
was isolated by means of the Promega RNA extraction kit following
the manufacturer's instructions (SV 96 Total RNA Isolation System,
Order No. Z3505, Promega, Mannheim). The concentration of the RNA
in the individual samples was determined by photometrically
measuring the absorption at 260 nm.
[0294] For each sample, 2 .mu.g of RNA were brought to a uniform
volume and treated with an identical volume of RNA sample buffer
(65% (v/v) formamide, 8% formaldehyde, 13% (v/v) gel buffer (see
above), 50 .mu.g/ml ethidium bromide). After heating (10 min,
65.degree. C.) and immediate cooling on ice, the RNA was separated
for approximately 2 hours using a 1.2% (w/v) agarose gel (20 mM
MOPS pH 8.0, 5 mM sodium acetate, 1 mM EDTA, 6% (v/v) formaldehyde)
using RNA running buffer (20 mM MOPS pH 8.0, 5 mM sodium acetate, 1
mM EDTA) at a constant amperage of 50-80 mA.
[0295] Thereafter, the RNA was transferred to a Hybond N membrane
by means of diffusion blot using 10.times.SSC (1.5 M NaCl, 150 mM
sodium citrate pH 7.0) and immobilized on the membrane by means of
UV irradiation.
[0296] The hybridization of the Northern blot for detecting the
expression of a nucleic acid molecule which codes for a protein
with the activity of a starch synthase II from wheat employed an
approx. 1 kb SpeI/BspHI fragment of the plasmid AH32-191 (bp
4568-5686), which encompasses the 5' region of the cDNA. The DNA
fragment was radiolabeled by means of the Random Primed DNA
Labeling Kit from Roche (Order No. 1004 760) using
.sup.32P-alpha-dCTP and following the manufacturer's instructions.
The nylon membrane comprising the transferred RNA was incubated for
4 hours at 60.degree. C. in a water bath with hybridization buffer
(250 mM sodium phosphate buffer pH 7.2, 1 mM EDTA, 6% (w/v) SDS, 1%
(w/v) BSA), with gentle shaking, whereupon the radiolabel DNA was
added to the hybridization buffer. After incubation for 16 hours,
the hybridization buffer was removed, and the membrane was washed
in succession once with 3.times.SSC and once with 2.times.SSC (see
above) at 60.degree. C., with gentle shaking, to remove
unspecifically bound DNA molecules.
[0297] To detect labeled RNA, the nylon membrane was
autoradiographed for one to three days at -70.degree. C. on an
x-ray film.
4. Determination of the Activity of a Protein with the Activity of
a Starch Synthase
[0298] II by Means of Activity Gels (Zymogramm)
[0299] The detection of the activity of proteins with the activity
of a starch synthase in immature rice grains was performed by means
of activity gels (zymogramms), in which protein extracts are
separated in a polyacrylamide gel under native conditions and
subsequently incubated with suitable substrates. The reaction
product formed (alpha-glucan) was stained in the gel using Lugol's
solution.
[0300] Individual immature rice grains (approx. 15 days after
anthesis) were frozen in liquid nitrogen and homogenized in 150-200
.mu.l of cold extraction buffer (50 mM Tris/HCl pH 7.6, 2.5 mM
EDTA, 2 mM DTT, 4 mM PMSF, 0.1% (w/v) glycogen, 10% (v/v)
glycerol). After centrifugation (15 min, 13 000 g, 4.degree. C.),
the clear supernatant was transferred into a fresh reaction vessel,
and an aliquot of the extract was used for determining the protein
content by the method of Bradford (1976, Anal Biochem 72:
248-254).
[0301] The protein extracts were separated by means of continuous
7.5% strength polyacrylamide gel (7.5% acrylamide: bisacrylamide
37.5:1; 25 mM Tris/HCl pH 7.6, 192 mM glycine, 0.1% (w/v) APS,
0.05% (v/v) TEMED) using running buffer in single concentration (25
mM Tris/HCl, 192 mM glycine). For each sample, amounts
corresponding to 15 .mu.g of protein were applied in each case, and
the electrophoresis was run for 2 to 2.5 hours at 4.degree. C.
[0302] Thereafter, the gels were incubated overnight at room
temperature in 15 ml of incubation buffer (0.5 mM sodium citrate pH
7.0, 25 mM potassium acetate, 2 mM EDTA, 2 mM DTT, 0.1% (w/v)
amylopectin, 50 mM tricine/NaOH pH 8.5, 1 mM ADP-glucose), with
constant shaking. The starch formed was stained by means of Lugol's
solution.
[0303] To determine by how many times the activity of a protein
with the activity of a starch synthase II is increased in
comparison with corresponding not genetically modified wild-type
plants, protein extracts from the genetically modified lines were
in each case subjected to sequential dilution and separated by
electrophoresis in accordance with the above-described method. The
remaining steps were carried out as already described above. After
the zymogramms had been stained with Lugol's solution, the
intensity of the stained products produced by a protein with the
activity of a starch synthase II (identified by an arrow in FIG. 1)
for the different dilutions of the protein extracts from
genetically modified plants were compared visually with the
relevant products of the undiluted wild-type protein extract. Since
the intensity of the coloration of the products correlates directly
with the activity of a protein with the activity of a starch
synthase II, product bands with the same intensities have the same
activity. If the band of the product of a protein with the activity
of a starch synthase II in the dilute protein extract has the same
intensity as the corresponding band of the product from
corresponding undiluted protein extract from wild-type plants, the
dilution factor corresponds to the degree of the increase in the
activity in the corresponding genetically modified plant (for
comparisons, see FIG. 1).
5. Generation of Plants from Isolated Rice Embryos (Embryo
Rescue)
[0304] Seeds are removed from the panicle, and the shells are
removed. The endosperm is dissected from the embryo using a
surgical blade and used for suitable analyses. To improve the
wettability, the embryo is briefly treated with 70% ethanol and
subsequently incubated for 20 minutes in a solution comprising 2%
NaOCl and one drop of commercially available washing-up liquid to
sterilize it.
[0305] Thereafter, as much as possible of the sterilization
solution is removed, and the embryo is washed with sterile
demineralized water, once for a minute and thereafter twice for in
each case 10 minutes. The seeds are plated out in Petri dishes on
agar solidified medium comprising in each case a quarter of the
salt concentration of MS medium (Murashige-Skoog medium) and 4%
sucrose. Thereafter, the Petri dishes are sealed using Parafilm and
incubated in the dark at 23.degree. C. After germination (approx.
5-7 days after plating out the embryos), the Petri dishes are
transferred into the light. When the hypocotyls of the seedlings
have reached a length of approx. 2 cm, the plants are transferred
into jars comprising agar-solidified MS medium with 2% sucrose.
After sufficient roots have developed, the plants can be potted in
compost.
6. Processing of Rice Grains, and Preparation of Rice Flours
[0306] To prepare sufficient amounts of test material, rice plants
were grown in the greenhouse and harvested when fully mature. The
mature rice grains were stored for 3-7 days at 37.degree. C. to dry
them further.
[0307] Thereafter, the grains were freed from the shells by means
of a sheller (Laboratory Paddy sheller, Grainman, Miami, Fla.,
USA), and the brown rice obtained was processed by polishing for 1
minute (Pearlest Rice Polisher, Kett, Villa Park, Calif., USA) to
give white rice. For grain composition studies and starch property
studies, the white grains were ground by means of a laboratory mill
(Cyclotec, Sample mill, Foss, Denmark) to give what is known as
rice flour.
7. Extraction of Rice Starch from Rice Flour
[0308] Rice starch was extracted from rice flour by a method
similar to the method described by Wang and Wang (2004; Journal of
Cereal Science 39: 291-296).
[0309] Approx. 10 g of rice flour were incubated for 16-18 hours
with 40 ml of 0.05% (w/v) NaOH at room temperature on a shaker.
Thereafter, the suspension was transferred into a Waring blender to
complete the digestion and mixed for 15 seconds at low speed and
subsequently for 45 seconds at high speed. To remove coarse
constituents (for example cell wall), the suspension was poured in
succession through sieves with a mesh size of 125 .mu.m and of 63
.mu.m. After centrifugation at 1500 rpm for 15 minutes (Microfuge
3.OR; Heraeus), the supernatant was decanted off, and the protein
layer at the top of the sediment was removed using a spatula. The
remainder of the sediment was resuspended in 0.05% (w/v) NaOH, and
the procedure described above was repeated. Thereafter, the
sediment was resuspended in water and the pH of the suspension was
brought to 6.5 to 7 using HCl. The rice starch obtained was washed
in total three times with water, where each wash step comprised a
sedimentation (centrifugation at 1500 rpm, 15 min, RT), discarding
the supernatant and resuspending the sediment in fresh water.
Before the last wash step, the pH was rechecked and, if necessary,
brought to pH 7 with HCl. The sediment of the last wash step was
resuspended in acetone, sedimented and the supernatant was
discarded. After resuspending the sediment again in acetone, the
suspension was poured into a Petri dish and dried in a fume hood at
room temperature for at least 18 hours.
[0310] In a last step, the resulting rice starch was made into a
fine powder by comminuting in a pestel and mortar, and this powder
can be employed directly for further studies.
8. Determination of the Hot-Water Swelling Power (SP)
[0311] 100 mg of sample (starch or flour) are suspended in 10 ml of
water and subsequently swelled for 20 minutes at 92.5.degree. C.
During the incubation of the sample of 92.5.degree. C., the
suspension is mixed repeatedly (continuously during the first 2
minutes, then after 3, 4, 5, 10, 15 and 25 minutes) by carefully
turning the sample containers by 360.degree.. After incubation for
a total of 30 minutes at 92.5.degree. C., the suspension is cooled
for approx. 1 minute in ice-water before carrying out an incubation
at 25.degree. C. for 5 minutes. After centrifugation (room
temperature, 1000.times.g, 15 minutes), the supernatant obtained is
removed carefully from the gel-like sediment and the sediment
weight is determined. The hot-water swelling power is calculated
using the following formula:
SP=(weight of the gel-like sediments)/(weight of the weighed-in
sample(flour or starch))
9. Determination of the Starch Phosphate Content in the C6 Position
of the Glucose Molecules
[0312] In starch, the positions C2, C3 and C6 of the glucose units
may be phosphorylated. To determine the C6-P content of the starch
or the flour (modified method of Nielsen et al., 1994, Plant
Physiol. 105: 111-117), 50 mg of rice flour or rice starch were
hydrolyzed for 4 hours in 500 .mu.l of 0.7 M HCl at 95.degree. C.,
with continuous shaking. Thereafter, the mixtures were centrifuged
for 10 minutes at 15.500.times.g, and the supernatants were freed
from suspended matter and cloudiness by means of a filter membrane
(0.45 .mu.M). 20 .mu.l of the clear hydrolyzate were mixed with 180
.mu.l of imidazol buffer (300 mM imidazol, pH 7.4; 7.5 mM MgCl2, 1
mM EDTA and 0.4 mM NADP), and the samples were measured in a
photometer at 340 nm. After recording the basic absorption, an
enzyme reaction was started by addition of 2 units of glucose
6-phosphate dehydrogenase (from Leuconostoc mesenteroides,
Boehringer Mannheim). The measured change (OD) is based on an
equimolar conversion of glucose 6-phosphate and NADP to give
6-phosphogluconate and NADPH, where the formation of NADPH is
recorded at the abovementioned wavelength. The reaction was
monitored until an end point had been reached. The result of this
measurement can be used for calculating the glucose 6-phosphate
content in the hydrolyzate:
nmol glucose 6 - phosphate / mg FW = OD .times. measuring volume (
200 .mu. l ) .times. hydrolyzate volume ( 500 .mu. l ) extinction
coefficient .times. sample volume ( 20 .mu. l ) .times. mg material
weighed in ( 50 mg ) ##EQU00001##
[0313] To avoid erroneous results caused by incomplete hydrolysis
of the starch in the material weighed in (flour or starch), the
degree of hydrolysis was subsequently determined. To this end, 10
.mu.l of hydrolyzate was removed from the respective hydrolyzates
which were measured by their glucose 6-phosphate content,
neutralized with 10 .mu.l of 0.7 M NaOH and brought to a final
volume of 2 ml with water (dilution 1:200). 4 .mu.l of this
dilution were treated with 196 .mu.l of measuring buffer (100 mM
imidazole pH 6.9; 5 mM MgCl2, 1 mM ATP, 0.4 mM NADP) and used for
the photometric determination of the glucose content. After
determining the basic absorption at 340 nm, the reaction was
monitored until the end point was reached in the photometer (340
nm) by addition of 2 .mu.l of enzyme mix (hexokinase 1:10; glucose
6-phosphate dehydrogenase from yeast 1:10 in measuring buffer). The
principle of the measurement corresponds to that of the first
reaction. Using the data obtained, the amount of glucose can be
calculated for the sample in question:
mmol Glucose / g FW = OD .times. measuring volume ( 200 .mu. l )
.times. hydrolyzate volume ( 500 .mu. l ) .times. total volume of
the dilution ( 2 ml ) extinction coefficient .times. sample volume
( 20 .mu. l ) .times. volume employed for the dilution ( 10 .mu. l
) .times. mg material weighed in ( 50 mg ) ##EQU00002##
[0314] The amount of glucose detected in the individual samples
corresponds to the amount of starch which is available for the
C6-phosphate determination. To simplify the further calculation,
the glucose content is converted into starch content.
starch content ( % ) = glucose content ( mmol / g FW ) .times.
molecular weight of glucose in starch ( 162 g / mol ) .times.
conversion factor ( % = 100 ) conversion factor ( mmol to mol =
1000 ) ##EQU00003##
[0315] In what follows, the result of the glucose 6-phosphate
measurement is related to the starch content of the sample in
question in order to express, in this manner, the glucose
6-phosphate content per mg of hydrolyzed starch:
nmol Glc - 6 P / mg starch = nmol glucose 6 - phosphate / mg
material weighed in .times. starch content ( mg starch / 100 mg
material weighed in ) ##EQU00004##
[0316] In contrast to when relating the amount of glucose
6-phosphate to the weighed-in weight of the sample (flour or
starch), this type of calculation relates the amount of glucose
6-phosphate only to the amount of starch which has been completely
hydrolyzed to give glucose.
10. Determination of the Apparent Amylose Content
[0317] The determination of the apparent amylose content was
carried out by a method similar to that of Juliano (1971, Cereal
Science Today 16 (10): 334-340).
[0318] For each sample, 50 mg of rice flour were weighed, in
duplicate, in 100 ml Erlenmeyer flasks and consecutively moistened
with 1 ml of 95% strength ethanol and 9 ml of 1M NaOH.
[0319] In parallel, flasks with defined amounts of pure amylose
from potato starch are treated in the same manner as the flour
samples, in order to establish a calibration curve. The flasks were
swirled briefly to mix the contents and subsequently incubated for
20 minutes in a boiling water bath, with gentle shaking. After 5-10
minutes cooling at RT, the volume was made up to 100 ml with
water.
[0320] A 100 .mu.l aliquot was treated with 1 ml measuring solution
(10 mM acetic acid, 0.004% (w/v) I.sub.2; 0.04% (w/v) KI), mixed
thoroughly, and the absorption was determined at 620 nm against a
suitable blank. The calculation of the amylose content was carried
out with the aid of the amylose standards used for establishing a
calibration curve.
11. Quantitative PCR
[0321] RNA was prepared from individual immature rice seeds (10-12
days after anthesis). After the seeds, which had been frozen in
liquid nitrogen, had been homogenized using a 4 mm steel ball
(Retsch mill, 30 Hz, 45 sec), the RNA was prepared using the "SV 96
Total RNA Isolation System" by Promega, following protocol No. 294
(Promega). The RNA was treated with in each case 10 .mu.l of "RQ1
RNase-Free DNase" (Promega), following the manufacturer's
instructions.
[0322] Identical amounts of RNA from in each case four seeds of one
plant were combined. The quantitative RT-PCR was carried out with
reagents of the "Access RT-PCR System" by Promega.
[0323] The reaction conditions for the RT-PCR were: 30 min at
55.degree. C., 2 min at 94.degree. C., 40.times.(15 sec 94.degree.
C., 1 min 60.degree. C.). The fluorescent signal was recorded using
an ABI Prism 7700 apparatus (Applied Biosystems), in each case
during the combined annealing/extension phase.
[0324] The controls which were employed in this approach were in
each case mixtures without reverse transcriptases.
[0325] The relative expression was calculated as described by M. W.
Pfaffl (2001, A new mathematical model for relative quantification
in real-time RT-PCR, Nucleic Acids Research 29, No 9 00).
EXAMPLES
1. Generation and Selection of the Waxy (GBSSI Knock Out)
Mutant
[0326] The waxy mutant originated from an agrobacteria-mediated
transformation of rice. An analysis of the progeny revealed that
the waxy phenotype of the rice grains is inherited independently of
the phosphinotricine resistance introduced with the transformation.
A sequence analysis of the GBSSI (waxy) gene revealed that the
manifestation of the waxy phenotype gene can be attributed to the
exchange of two nucleotides, as a result of which a premature stop
codon is generated, which leads to a truncated and probably
inactive protein. The RFLP analysis of the apparent amylose content
of the starch present in the rice grains confirmed a value of less
than 5% by weight, which means that the mutant identified is a
"waxy" mutant. As a consequence, the term "waxy phenotype" is
understood as meaning waxy mutants whose starch has an apparent
amylose content of less than 5%.
[0327] Lines 738-104 and 738-106, which are homozygous for the
abovementioned mutation, were used for the combination with the
transgenic approaches.
TABLE-US-00001 BamHI M202 GAG TGG GAT CCT AGC Waxy_Mutant GAG TGA
AAT CCT AGC Stop
2. Preparation of the Plant Expression Vector pAH32-191, which
Comprises a Coding Sequence for a Protein with the Activity of a
Starch Synthase II
[0328] The complete encoding sequence of the protein with the
activity of a starch synthase II from wheat (T.a.--SSII) was
excised from the plasmid pCF31 (described in WO 97/45545 under the
name pTaSS1) by means of the restriction endonucleases Ecl 13611
and Xho I and cloned into the plasmid pIR103-123 (described in WO
05/030941) which had been cleaved with the restriction
endonucleases Eco RV and Xho I. The expression vector obtained was
named pAH32-191. The plant expression vector pIR103-123 serves for
the endosperm-specific expression of the target gene under the
control of the endosperm-specific globulin promoter (Nakase et al.
(1996) Gene 170(2): 223-226) from rice. In addition, the plant
expression vector pIR103-123 comprises the bar gene under the
control of the CaMV 35S promoter, which gene was used as the
selection marker for the transformation of plants.
3. Generation of Rice Plants with an Increased Activity of a
Protein with the Activity of a Starch Synthase II
[0329] Rice plants (variety M202) were transformed by means of
agrobacteria comprising the plasmid pAH32-191 using the method
described by Hiei et al. (1994, Plant Journal 6(2), 271-282). The
resulting plants were named oe-SSII-O.s.-X, where X means
independent plants obtained from the transformation.
4. Analysis of the Rice Plants which Had been Transformed with the
Expression Vector pAH32-191
[0330] Rice plants (TO plants) of the lines named oe-SSII-O.s.-X
and which had originated from the transformation with the
expression vector pAH32-191 where grown in soil in the greenhouse.
RNA was isolated from immature grains (T1 seeds) of various lines,
and a Northern blot analysis was carried out in accordance with the
method described in "General Methods", using an SSII-specific
probe. A plurality of lines with an increased amount of transcript
of the wheat starch synthase II in comparison with corresponding
not genetically modified wild-type plants were identified (see
diagram shown by way of example in FIG. 2).
[0331] In addition, an increased activity of a protein with the
activity of a starch synthase II in protein extracts of immature T1
seeds from different lines of the above-mentioned transformation
was determined by means of zymograms (see diagram shown by way of
example in FIGS. 1 and 2). The analysis was carried out by means of
zymograms as described in "General Methods".
[0332] Based on the results of the analyses described, the
following line was selected for the combination with other
approaches: [0333] oe-SSII-O.s-01502 On the basis of a variety of
analyses, it was possible to demonstrate that this line is
homozygous for the integrations of the T-DNA(s) of the vector
pAH32-191.
5. Generation of Rice Plants with an Increased Activity of a
Protein with the Activity of a Glucan, Water Dikinase
[0334] Rice plants (variety M202) were transformed by means of
agrobacteria which comprise the plasmid pML82 (described in WO
05/095619), using the method described by Hiei et al. (1994, Plant
Journal 6(2), 271-282). The resulting plants were named
oe-GWD-O.s.-X, where X means independent plants obtained from the
transformation.
6. Analysis of the Rice Plants which Had been Transformed with the
Expression Vector pML82
[0335] Rice plants (TO plants) of the lines named oe-GWD-O.s.-X and
which had originated from the transformation with the expression
vector pML82 were grown in soil in the greenhouse. Individual,
mature grains (T1 seeds) from different lines were made into a
flour. To this end, individual grains were comminuted, in a ball
mill (from Retsch, Model MM300), for 30 seconds at a frequency of
30 Hertz in an Eppendorf reaction vessel using a tungsten carbide
ball. This was followed by a determination of the starch phosphate
content in the C6 position of glucose molecules of the starch
present in the flour as described in "General Methods".
[0336] The following results were obtained for selected plants:
TABLE-US-00002 TABLE 1 Starch phosphate content in the C6 position
of the glucose molecules of individual T1 seeds from different
lines with the name oe-GWD- O.s.-X in comparison with seeds of
corresponding not genetically modified wild-type plants (WT) of
variety M202. Line nmol C6P/mg material weighed oe-GWD-O.s.-2 1.68
oe-GWD-O.s.-4 1.70 oe-GWD-O.s.-9 1.47 WT 0.30
[0337] As can be seen from table 1, it was possible to identify
independent lines which are the result of the transformation with
the plant expression vector pML82 and which, in comparison with
corresponding not genetically modified wild-type plants have an
increased starch phosphate content in the C6 position of the
glucose molecules. It is known that plant cells with an increased
expression of a protein with the activity of a glucan, water
dikinase synthesize a starch with a higher starch phosphate content
in comparison with corresponding genetically not modified wild-type
plants (see, for example, WO 02/34923).
[0338] Based on the above-described analyses, the following lines
were selected for the combination with other approaches: [0339]
oe-GWD-O.s.-2 [0340] oe-GWD-O.s.-4 [0341] oe-GWD-O.s.-9
[0342] On the basis of various analyses, it was possible to
demonstrate that these lines are homozygous for the integrations of
the T-DNA(s) of vector pML82.
7. Generation of Plants with a Waxy Phenotype and an Increased
Activity of a Protein with the Activity of a Glucan, Water
Dikinase
[0343] The following crosses were made:
TABLE-US-00003 TABLE 2 Crosses of the combination of 738- 104/4
(M202 waxy) with oe-GWD-O.s. Name Plasmid Name Plasmid of female of
female of male of male Pedigree cross parent parent parent parent
XPOS0001 M202 waxy -- oe-GWD-O.s. pML82 -01 738-106 -- oe-GWD-O.s-2
pML82 -02 738-104 -- oe-GWD-O.s-2 pML82 -03 738-104 -- oe-GWD-O.s-4
pML82 -04 738-106 -- oe-GWD-O.s-4 pML82 -05 738-104 -- oe-GWD-O.s-9
pML82 -06 738-106 -- oe-GWD-O.s-9 pML82
[0344] The endosperm of the F1 seeds, which were the result of the
cross, was studied for the starch phosphate content in the C6
position of the glucose molecules (C6P). The embryos of those
grains whose starch phosphate content (C6P) was markedly increased
in comparison with the female parent were germinated by means of
tissue culture techniques. After a sufficient size had been
attained, relevant plants were transferred to the greenhouse in
order to produce F2 seeds.
[0345] Grains with waxy phenotype were selected from the mature F2
seeds by means of visual scoring and placed in the greenhouse.
After germination, the plants were sprayed with Basta.RTM. (Bayer
CropScience), and leaf samples were taken from Basta.RTM.-tolerant
plants. Plants which were homozygous for the integration of the
T-DNA of vector pML82 were identified by means of a copy number
determination using invader technology
(http://www.twt.com/invader_chemistry/invaderchem.htm; Ledford et
al (2000, J. of Mol. Diagnostics. 2(2): 97-104; Mein et al., 2000,
Genome Res. 10: 330-343) for the bar gene. The plants thus selected
were grown on in the greenhouse for the production of F3 seeds.
[0346] Some mature F3 seeds of the potentially doubly homozygous
plants were studied individually for their starch phosphate (C6P)
content. Those plants where all grains had an expectedly high
starch phosphate (C6P) content were retained.
[0347] The seed of all doubly homozygous plants of a parental
combination was pooled and used for further propagation and for
grain and flour property analyses.
[0348] For the combination with line oe-SSII-O.s, the event
XPOS0001-05, which is homozygous both for the waxy mutation and for
the T-DNA of the vector pML82, was selected.
8. Generation of Plants with a Waxy Phenotype and with an Increased
Activity of a Protein with the Activity of a Glucan, Water Dikinase
and with an Increased Activity of a Protein with the Activity of a
Starch Synthase II
[0349] The following crosses were made:
TABLE-US-00004 TABLE 3 Crosses of the combination of oe-SSII-O.s.
with XPOS0001-05 Plasmid Plasmid Pedigree Female of female Male of
male cross parent parent parent parent XPOS0025-01 oe-SSII-O.s.-
pAH31- XPOS0001-05 pML82 01502 191 XPOS0026-01 XPOS0001-05 pML82
oe-SSII-O.s.- pAH32- 01502 191
[0350] Successful events in crosses were identified by measuring
the starch phosphate content of the F1 endosperm, since the starch
phosphate content of the combination is markedly higher than that
of the parental lines.
9. Analysis of Plants with a Waxy Phenotype and with an Increased
Activity of a Protein with the Activity of a Glucan, Water Dikinase
and with an Increased Activity of a Protein with the Activity of a
Starch Synthase II
[0351] Embryos of F1 seeds whose endosperm has a starch phosphate
content of more than 5 nmol C6P/mg starch and is therefore markedly
above that of both parents (2.5 nmol/mg starch for oe-GWD-O.s. and
at least 0.8 nmol/mg starch for oe-SSII-O.s.) were germinated by
means of tissue culture techniques, and the plants in question,
once they had reached a suitable size, were transferred to the
greenhouse to produce F2 seeds.
[0352] To identify progeny which is homozygous for both transgenes
and for the waxy mutation, the above-described procedure was
repeated for F2 seeds which had been preselected visually with
regard to a "waxy phenotype", including the embryo rescue.
10. Selection and Analysis of the F2 Plants
[0353] Based on the results of the starch phosphate measurement, F2
seeds were selected
[0354] (C6P>8 nmol/mg starch), their embryos were germinated,
and the F2 plants in question were grown in the greenhouse.
[0355] Genomic DNA was extracted from leaf material of the F2
plants, and the copy number of the two transgenes and of the bar
gene (total of the values for the two transgenes) was determined by
means of quantitative PCR.
[0356] The proof that the waxy mutation was homozygous was carried
out using an RFLP (Bam HI) in the GBSSI gene (definition and/or
method) of the waxy mutant.
[0357] F2 plants which are potentially homozygous for the two
transgenes and homozygous for the waxy RFLP were grown on in the
greenhouse and used for the production of F3 seeds.
11. Selection of the F3 Plants/Analysis of F3 Seeds
[0358] To identify triply homozygous lines, some individual grains
of suitably selected plants were examined visually for a waxy
phenotype and subsequently studied for their starch phosphate
content. If all grains have a waxy phenotype, and if the starch
phosphate content for all grains of one plant is found to be
approximately equally high, it can be assumed that the plant is
homozygous for the waxy mutation and for the T-DNA of pML82 and
pAH32-191.
12. Generation of F4 Material
[0359] The following lines were found in the abovementioned
analysis to be triply homozygous: [0360] XPOS002501-1-37 [0361]
XPOS002501-1-13 [0362] XPOS002601-1-19
[0363] Plants from these lines were grown in the greenhouse, and
the F4 seeds produced were harvested and dried and then pooled as
one line for all progeny.
13. Functionalities and Analysis of the Constituents of the F4
Material
a) Grain Composition
Apparent Amylose Content:
TABLE-US-00005 [0364] TABLE 4 Apparent amylose content in rice
flours and rice starches for the single-gene approaches and the
triple combination Apparent amylose Apparent amylose content of
rice flours content of rice starches Sample name (% amylose/FW) (%
amylose/FW) Wild type 8.9 11.8 oe-GWD-O.s.-4 10.6 14.4
oe-GWD-O.s.-9 10.6 14.3 oe-SSII-O.s.-01502 6.6 9.2 738-104/6 2.3
2.2 XPOS025-01-1-37 3.7 3.5 XPOS025-01-1-13 3.7 3.7 XPOS026-01-1-19
3.9 4.1
[0365] It emerged that the combinations XPOS0025/6 have an amylase
content above that of the waxy mutant (738-104/6).
Starch Phosphate Content (C6P Contents)
TABLE-US-00006 [0366] TABLE 5 Starch phosphate content in the C6
position of rice flours or starches for the single-gene approaches
and for the triple combinations Starch phosphate content in the C6
Starch phosphate position of starches content in the C6 present in
rice flours position of rice starches Sample name (nmol C6P/mg
starch) (nmol C6P/mg starch) Wild type 0.46 0.37 oe-GWD-O.s.-4 2.85
2.65 oe-GWD-O.s.-9 3.27 2.56 oe-SSII-O.s.-01502 1.22 0.91 738-104/6
0.52 0.38 XPOS025-01-1-37 11.45 9.50 XPOS025-01-1-13 11.20 10.24
XPOS026-01-1-19 11.06 10.23
[0367] The starch phosphate content in the C6 position of the
triple combination is markedly higher than that of the single-gene
approaches.
b) Functionalities of Rice Flours and Rice Starches
Hot-Water Swelling Power
TABLE-US-00007 [0368] TABLE 6 Hot-water swelling power of rice
flours or rice starches of the single-gene approaches and of the
triple combination Hot-water swelling Hot-water swelling power of
rice power of rice Sample name flours (g/g) starches (g/g) Wild
type 15.7 31.9 oe-GWD-O.s.-4 21.6 38.6 oe-GWD-O.s.-9 21.3 39.9
oe-SSII-O.s.-01502 20.2 40.8 738-104/6 19.9 47.3 XPOS025-01-1-37
40.6 86.0 XPOS025-01-1-13 41.9 89.1 XPOS026-01-1-19 38.3 87.2
[0369] The determination of the hot-water swelling power of flours
or starches prepared from F4 seeds of the abovementioned lines and
from wild-type plants was accomplished as described in "General
Method".
[0370] The hot-water swelling power of the triple combination is
markedly above that of the single-gene approaches.
DESCRIPTION OF THE FIGURES
[0371] FIG. 1A-C shows zymograms for determining the activity of
proteins with the activity of a starch synthase II in comparison
with the wild type. The material used were total protein extracts
from immature grains (15 days after anthesis) of wild-type plants
(WT) and of the three independent genetically modified plants which
are the result of the transformations with the expression vector
AH32-191 (oe-SSII-O.s.-5 (FIG. 1A), oe-SSII-O.s.-12 (FIG. 1B),
oe-SSII-O.s.-19 (FIG. 1C)). In the lanes WT and pur, in each case
identical amounts of protein of the respective extracts are
applied. The protein extracts of the genetically modified plants
were subjected to serial dilution (1:2, 1:4, 1:6, 1:8, 1:10, 1:20,
1:50 or 1:100), and these dilutions were separated by
electrophoresis, also separately from one another. The increase in
the activity of a starch synthase II in comparison with wild-type
plants can be determined by comparing the intensity of the specific
products which are present in the zymogram after staining with
Lugol's solution and which have been synthesized by a protein with
the activity of a starch synthase II (identified by an arrow) of
protein extracts from wild-type plants with the intensity of the
corresponding bands of protein extracts from genetically modified
plants. Equal intensities mean equal activities.
[0372] FIG. 2 shows the autoradiogram of a Northern blot analysis
of immature T1 seeds of the rice lines oe-SSII-O.s.-19,
oe-SSII-O.s.-20, oe-SSII-O.s.-21, oe-SSII-O.s.-22, oe-SSII-O.s.-23
in comparison with not genetically modified wild-type plants (WT).
To this end, RNA was extracted from in each case three seeds of
lines which have independently originated from the transformation
with the expression vector AH32-191 and was analyzed in accordance
with the method described in General Methods, item 8. The band
which hybridizes with a labeled nucleic acid probe coding for a
protein with the activity of a starch synthase II from wheat is
identified as SSII.
[0373] FIG. 3 shows a zymogram of protein extracts from immature T1
seeds of the rice lines oe-SSII-O.s.-8, oe-SSII-O.s.-19,
oe-SSII-O.s.-23 in comparison with seeds of not genetically
modified wild-type plants (WT) after staining with Lugol's
solution. Protein extracts from two (oe-SSII-O.s.-8) or three
(oe-SSII-O.s.-19, oe-SSII-O.s.-23) different grains were analyzed
per line. The analysis by means of zymogram was performed following
the method described in General Methods, item 9. The band in the
zymogram which is specific for a protein with the activity of a
starch synthase II is identified as SSII.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 12 <210> SEQ ID NO 1 <211> LENGTH: 4851
<212> TYPE: DNA <213> ORGANISM: Solanum tuberosum
<220> FEATURE: <221> NAME/KEY: transit_peptide
<222> LOCATION: (1)..(77) <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (105)..(4499) <300>
PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:
EMBL / Y09533 <309> DATABASE ENTRY DATE: 1998-07-30
<313> RELEVANT RESIDUES IN SEQ ID NO: (1)..(4499) <400>
SEQUENCE: 1 catcttcatc gaatttctcg aagcttcttc gctaatttcc tggtttcttc
actcaaaatc 60 gacgtttcta gctgaacttg agtgaattaa gccagtggga ggat atg
agt aat tcc 116 Met Ser Asn Ser 1 tta ggg aat aac ttg ctg tac cag
gga ttc cta acc tca aca gtg ttg 164 Leu Gly Asn Asn Leu Leu Tyr Gln
Gly Phe Leu Thr Ser Thr Val Leu 5 10 15 20 gaa cat aaa agt aga atc
agt cct cct tgt gtt gga ggc aat tct ttg 212 Glu His Lys Ser Arg Ile
Ser Pro Pro Cys Val Gly Gly Asn Ser Leu 25 30 35 ttt caa caa caa
gtg atc tcg aaa tca cct tta tca act gag ttt cga 260 Phe Gln Gln Gln
Val Ile Ser Lys Ser Pro Leu Ser Thr Glu Phe Arg 40 45 50 ggt aac
agg tta aag gtg cag aaa aag aaa ata cct atg gaa aag aag 308 Gly Asn
Arg Leu Lys Val Gln Lys Lys Lys Ile Pro Met Glu Lys Lys 55 60 65
cgt gct ttt tct agt tct cct cat gct gta ctt acc act gat acc tct 356
Arg Ala Phe Ser Ser Ser Pro His Ala Val Leu Thr Thr Asp Thr Ser 70
75 80 tct gag cta gca gaa aag ttc agt cta ggg ggg aat att gag cta
cag 404 Ser Glu Leu Ala Glu Lys Phe Ser Leu Gly Gly Asn Ile Glu Leu
Gln 85 90 95 100 gtt gat gtt agg cct ccc act tca ggt gat gtg tcc
ttt gtg gat ttt 452 Val Asp Val Arg Pro Pro Thr Ser Gly Asp Val Ser
Phe Val Asp Phe 105 110 115 caa gta aca aat ggt agt gat aaa ctg ttt
ttg cac tgg ggg gca gta 500 Gln Val Thr Asn Gly Ser Asp Lys Leu Phe
Leu His Trp Gly Ala Val 120 125 130 aaa ttc ggg aaa gaa aca tgg tct
ctt ccg aat gat cgt cca gat ggg 548 Lys Phe Gly Lys Glu Thr Trp Ser
Leu Pro Asn Asp Arg Pro Asp Gly 135 140 145 acc aaa gtg tac aag aac
aaa gca ctt aga act cca ttt gtt aaa tct 596 Thr Lys Val Tyr Lys Asn
Lys Ala Leu Arg Thr Pro Phe Val Lys Ser 150 155 160 ggc tct aac tcc
atc ctg aga ctg gag ata cga gac act gct atc gaa 644 Gly Ser Asn Ser
Ile Leu Arg Leu Glu Ile Arg Asp Thr Ala Ile Glu 165 170 175 180 gct
att gag ttt ctc ata tac gat gaa gcc cac gat aaa tgg ata aag 692 Ala
Ile Glu Phe Leu Ile Tyr Asp Glu Ala His Asp Lys Trp Ile Lys 185 190
195 aat aat ggt ggt aat ttt cgt gtc aaa ttg tca aga aaa gag ata cga
740 Asn Asn Gly Gly Asn Phe Arg Val Lys Leu Ser Arg Lys Glu Ile Arg
200 205 210 ggc cca gat gtt tct gtt cct gag gag ctt gta cag atc caa
tca tat 788 Gly Pro Asp Val Ser Val Pro Glu Glu Leu Val Gln Ile Gln
Ser Tyr 215 220 225 ttg agg tgg gag agg aag gga aaa cag aat tac ccc
cct gag aaa gag 836 Leu Arg Trp Glu Arg Lys Gly Lys Gln Asn Tyr Pro
Pro Glu Lys Glu 230 235 240 aag gag gaa tat gag gct gct cga act gtg
cta cag gag gaa ata gct 884 Lys Glu Glu Tyr Glu Ala Ala Arg Thr Val
Leu Gln Glu Glu Ile Ala 245 250 255 260 cgt ggt gct tcc ata cag gac
att cga gca agg cta aca aaa act aat 932 Arg Gly Ala Ser Ile Gln Asp
Ile Arg Ala Arg Leu Thr Lys Thr Asn 265 270 275 gat aaa agt caa agc
aaa gaa gag cct ctt cat gta aca aag agt gat 980 Asp Lys Ser Gln Ser
Lys Glu Glu Pro Leu His Val Thr Lys Ser Asp 280 285 290 ata cct gat
gac ctt gcc caa gca caa gct tac att agg tgg gag aaa 1028 Ile Pro
Asp Asp Leu Ala Gln Ala Gln Ala Tyr Ile Arg Trp Glu Lys 295 300 305
gca gga aag ccg aac tat cct cca gaa aag caa att gaa gaa ctc gaa
1076 Ala Gly Lys Pro Asn Tyr Pro Pro Glu Lys Gln Ile Glu Glu Leu
Glu 310 315 320 gaa gca aga aga gaa ttg caa ctt gag ctt gag aaa ggc
att acc ctt 1124 Glu Ala Arg Arg Glu Leu Gln Leu Glu Leu Glu Lys
Gly Ile Thr Leu 325 330 335 340 gat gag ttg cgg aaa acg att aca aaa
ggg gag ata aaa act aag gtg 1172 Asp Glu Leu Arg Lys Thr Ile Thr
Lys Gly Glu Ile Lys Thr Lys Val 345 350 355 gaa aag cac ctg aaa aga
agt tct ttt gcc gtt gaa aga atc caa aga 1220 Glu Lys His Leu Lys
Arg Ser Ser Phe Ala Val Glu Arg Ile Gln Arg 360 365 370 aag aag aga
gac ttt ggg cat ctt att aat aag tat act tcc agt cct 1268 Lys Lys
Arg Asp Phe Gly His Leu Ile Asn Lys Tyr Thr Ser Ser Pro 375 380 385
gca gta caa gta caa aag gtc ttg gaa gaa cca cca gcc tta tct aaa
1316 Ala Val Gln Val Gln Lys Val Leu Glu Glu Pro Pro Ala Leu Ser
Lys 390 395 400 att aag ctg tat gcc aag gag aag gag gag cag att gat
gat ccg atc 1364 Ile Lys Leu Tyr Ala Lys Glu Lys Glu Glu Gln Ile
Asp Asp Pro Ile 405 410 415 420 cta aat aaa aag atc ttt aag gtc gat
gat ggg gag cta ctg gta ctg 1412 Leu Asn Lys Lys Ile Phe Lys Val
Asp Asp Gly Glu Leu Leu Val Leu 425 430 435 gta gca aag tcc tct ggg
aag aca aaa gta cat cta gct aca gat ctg 1460 Val Ala Lys Ser Ser
Gly Lys Thr Lys Val His Leu Ala Thr Asp Leu 440 445 450 aat cag cca
att act ctt cac tgg gca tta tcc aaa agt cct gga gag 1508 Asn Gln
Pro Ile Thr Leu His Trp Ala Leu Ser Lys Ser Pro Gly Glu 455 460 465
tgg atg gta cca cct tca agc ata ttg cct cct ggg tca att att tta
1556 Trp Met Val Pro Pro Ser Ser Ile Leu Pro Pro Gly Ser Ile Ile
Leu 470 475 480 gac aag gct gcc gaa aca cct ttt tca gcc agt tct tct
gat ggt cta 1604 Asp Lys Ala Ala Glu Thr Pro Phe Ser Ala Ser Ser
Ser Asp Gly Leu 485 490 495 500 act tct aag gta caa tct ttg gat ata
gta att gaa gat ggc aat ttt 1652 Thr Ser Lys Val Gln Ser Leu Asp
Ile Val Ile Glu Asp Gly Asn Phe 505 510 515 gtg ggg atg cca ttt gtt
ctt ttg tct ggt gaa aaa tgg att aag aac 1700 Val Gly Met Pro Phe
Val Leu Leu Ser Gly Glu Lys Trp Ile Lys Asn 520 525 530 caa ggg tcg
gat ttc tat gtt ggc ttc agt gct gca tcc aaa tta gca 1748 Gln Gly
Ser Asp Phe Tyr Val Gly Phe Ser Ala Ala Ser Lys Leu Ala 535 540 545
ctc aag gct gct ggg gat ggc agt gga act gca aag tct tta ctg gat
1796 Leu Lys Ala Ala Gly Asp Gly Ser Gly Thr Ala Lys Ser Leu Leu
Asp 550 555 560 aaa ata gca gat atg gaa agt gag gct cag aag tca ttt
atg cac cgg 1844 Lys Ile Ala Asp Met Glu Ser Glu Ala Gln Lys Ser
Phe Met His Arg 565 570 575 580 ttt aat att gca gct gac ttg ata gaa
gat gcc act agt gct ggt gaa 1892 Phe Asn Ile Ala Ala Asp Leu Ile
Glu Asp Ala Thr Ser Ala Gly Glu 585 590 595 ctt ggt ttt gct gga att
ctt gta tgg atg agg ttc atg gct aca agg 1940 Leu Gly Phe Ala Gly
Ile Leu Val Trp Met Arg Phe Met Ala Thr Arg 600 605 610 caa ctg ata
tgg aac aaa aac tat aac gta aaa cca cgt gaa ata agc 1988 Gln Leu
Ile Trp Asn Lys Asn Tyr Asn Val Lys Pro Arg Glu Ile Ser 615 620 625
aag gct cag gac aga ctt aca gac ttg ttg cag aat gct ttc acc agt
2036 Lys Ala Gln Asp Arg Leu Thr Asp Leu Leu Gln Asn Ala Phe Thr
Ser 630 635 640 cac cct cag tac cgt gaa att ttg cgg atg att atg tca
act gtt gga 2084 His Pro Gln Tyr Arg Glu Ile Leu Arg Met Ile Met
Ser Thr Val Gly 645 650 655 660 cgt gga ggt gaa ggg gat gta gga cag
cga att agg gat gaa att ttg 2132 Arg Gly Gly Glu Gly Asp Val Gly
Gln Arg Ile Arg Asp Glu Ile Leu 665 670 675 gtc atc cag agg aac aat
gac tgc aag ggt ggt atg atg caa gaa tgg 2180 Val Ile Gln Arg Asn
Asn Asp Cys Lys Gly Gly Met Met Gln Glu Trp 680 685 690 cat cag aaa
ttg cat aat aat act agt cct gat gat gtt gtg atc tgt 2228 His Gln
Lys Leu His Asn Asn Thr Ser Pro Asp Asp Val Val Ile Cys 695 700 705
cag gca tta att gac tac atc aag agt gat ttt gat ctt ggt gtt tat
2276 Gln Ala Leu Ile Asp Tyr Ile Lys Ser Asp Phe Asp Leu Gly Val
Tyr 710 715 720 tgg aaa acc ctg aat gag aac gga ata aca aaa gag cgt
ctt ttg agt 2324 Trp Lys Thr Leu Asn Glu Asn Gly Ile Thr Lys Glu
Arg Leu Leu Ser 725 730 735 740 tat gac cgt gct atc cat tct gaa cca
aat ttt aga gga gat caa aag 2372 Tyr Asp Arg Ala Ile His Ser Glu
Pro Asn Phe Arg Gly Asp Gln Lys 745 750 755 ggt ggt ctt ttg cgt gat
tta ggt cac tat atg aga aca ttg aag gca 2420 Gly Gly Leu Leu Arg
Asp Leu Gly His Tyr Met Arg Thr Leu Lys Ala 760 765 770 gtt cat tca
ggt gca gat ctt gag tct gct att gca aac tgc atg ggc 2468 Val His
Ser Gly Ala Asp Leu Glu Ser Ala Ile Ala Asn Cys Met Gly 775 780 785
tac aaa act gag gga gaa ggc ttt atg gtt gga gtc cag ata aat cct
2516 Tyr Lys Thr Glu Gly Glu Gly Phe Met Val Gly Val Gln Ile Asn
Pro 790 795 800 gta tca ggc ttg cca tct ggc ttt cag gac ctc ctc cat
ttt gtc tta 2564 Val Ser Gly Leu Pro Ser Gly Phe Gln Asp Leu Leu
His Phe Val Leu 805 810 815 820 gac cat gtg gaa gat aaa aat gtg gaa
act ctt ctt gag aga ttg cta 2612 Asp His Val Glu Asp Lys Asn Val
Glu Thr Leu Leu Glu Arg Leu Leu 825 830 835 gag gct cgt gag gag ctt
agg ccc ttg ctt ctc aaa cca aac aac cgt 2660 Glu Ala Arg Glu Glu
Leu Arg Pro Leu Leu Leu Lys Pro Asn Asn Arg 840 845 850 cta aag gat
ctg ctg ttt ttg gac ata gca ctt gat tct aca gtt aga 2708 Leu Lys
Asp Leu Leu Phe Leu Asp Ile Ala Leu Asp Ser Thr Val Arg 855 860 865
aca gca gta gaa agg gga tat gaa gaa ttg aac aac gct aat cct gag
2756 Thr Ala Val Glu Arg Gly Tyr Glu Glu Leu Asn Asn Ala Asn Pro
Glu 870 875 880 aaa atc atg tac ttc atc tcc ctc gtt ctt gaa aat ctc
gca ctc tct 2804 Lys Ile Met Tyr Phe Ile Ser Leu Val Leu Glu Asn
Leu Ala Leu Ser 885 890 895 900 gtg gac gat aat gaa gat ctt gtt tat
tgc ttg aag gga tgg aat caa 2852 Val Asp Asp Asn Glu Asp Leu Val
Tyr Cys Leu Lys Gly Trp Asn Gln 905 910 915 gct ctt tca atg tcc aat
ggt ggg gac aac cat tgg gct tta ttt gca 2900 Ala Leu Ser Met Ser
Asn Gly Gly Asp Asn His Trp Ala Leu Phe Ala 920 925 930 aaa gct gtg
ctt gac aga acc cgt ctt gca ctt gca agc aag gca gag 2948 Lys Ala
Val Leu Asp Arg Thr Arg Leu Ala Leu Ala Ser Lys Ala Glu 935 940 945
tgg tac cat cac tta ttg cag cca tct gcc gaa tat cta gga tca ata
2996 Trp Tyr His His Leu Leu Gln Pro Ser Ala Glu Tyr Leu Gly Ser
Ile 950 955 960 ctt ggg gtg gac caa tgg gct ttg aac ata ttt act gaa
gaa att ata 3044 Leu Gly Val Asp Gln Trp Ala Leu Asn Ile Phe Thr
Glu Glu Ile Ile 965 970 975 980 cgt gct gga tca gca gct tca tta tcc
tct ctt ctt aat aga ctc gat 3092 Arg Ala Gly Ser Ala Ala Ser Leu
Ser Ser Leu Leu Asn Arg Leu Asp 985 990 995 ccc gtg ctt cgg aaa act
gca aat cta gga agt tgg cag att atc 3137 Pro Val Leu Arg Lys Thr
Ala Asn Leu Gly Ser Trp Gln Ile Ile 1000 1005 1010 agt cca gtt gaa
gcc gtt gga tat gtt gtc gtt gtg gat gag ttg 3182 Ser Pro Val Glu
Ala Val Gly Tyr Val Val Val Val Asp Glu Leu 1015 1020 1025 ctt tca
gtt cag aat gaa atc tac gag aag ccc acg atc tta gta 3227 Leu Ser
Val Gln Asn Glu Ile Tyr Glu Lys Pro Thr Ile Leu Val 1030 1035 1040
gca aaa tct gtt aaa gga gag gag gaa att cct gat ggt gct gtt 3272
Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro Asp Gly Ala Val 1045
1050 1055 gcc ctg ata aca cca gac atg cca gat gtt ctt tca cat gtt
tct 3317 Ala Leu Ile Thr Pro Asp Met Pro Asp Val Leu Ser His Val
Ser 1060 1065 1070 gtt cga gct aga aat ggg aag gtt tgc ttt gct aca
tgc ttt gat 3362 Val Arg Ala Arg Asn Gly Lys Val Cys Phe Ala Thr
Cys Phe Asp 1075 1080 1085 ccc aat ata ttg gct gac ctc caa gca aag
gaa gga agg att ttg 3407 Pro Asn Ile Leu Ala Asp Leu Gln Ala Lys
Glu Gly Arg Ile Leu 1090 1095 1100 ctc tta aag cct aca cct tca gac
ata atc tat agt gag gtg aat 3452 Leu Leu Lys Pro Thr Pro Ser Asp
Ile Ile Tyr Ser Glu Val Asn 1105 1110 1115 gag att gag ctc caa agt
tca agt aac ttg gta gaa gct gaa act 3497 Glu Ile Glu Leu Gln Ser
Ser Ser Asn Leu Val Glu Ala Glu Thr 1120 1125 1130 tca gca aca ctt
aga ttg gtg aaa aag caa ttt ggt ggt tgt tac 3542 Ser Ala Thr Leu
Arg Leu Val Lys Lys Gln Phe Gly Gly Cys Tyr 1135 1140 1145 gca ata
tca gca gat gaa ttc aca agt gaa atg gtt gga gct aaa 3587 Ala Ile
Ser Ala Asp Glu Phe Thr Ser Glu Met Val Gly Ala Lys 1150 1155 1160
tca cgt aat att gca tat ctg aaa gga aaa gtg cct tcc tcg gtg 3632
Ser Arg Asn Ile Ala Tyr Leu Lys Gly Lys Val Pro Ser Ser Val 1165
1170 1175 gga att cct acg tca gta gct ctt cca ttt gga gtc ttt gag
aaa 3677 Gly Ile Pro Thr Ser Val Ala Leu Pro Phe Gly Val Phe Glu
Lys 1180 1185 1190 gta ctt tca gac gac ata aat cag gga gtg gca aaa
gag ttg caa 3722 Val Leu Ser Asp Asp Ile Asn Gln Gly Val Ala Lys
Glu Leu Gln 1195 1200 1205 att ctg atg aaa aaa cta tct gaa gga gac
ttc agc gct ctt ggt 3767 Ile Leu Met Lys Lys Leu Ser Glu Gly Asp
Phe Ser Ala Leu Gly 1210 1215 1220 gaa att cgc aca acg gtt tta gat
ctt tca gca cca gct caa ttg 3812 Glu Ile Arg Thr Thr Val Leu Asp
Leu Ser Ala Pro Ala Gln Leu 1225 1230 1235 gtc aaa gag ctg aag gag
aag atg cag ggt tct ggc atg cct tgg 3857 Val Lys Glu Leu Lys Glu
Lys Met Gln Gly Ser Gly Met Pro Trp 1240 1245 1250 cct ggt gat gaa
ggt cca aag cgg tgg gaa caa gca tgg atg gcc 3902 Pro Gly Asp Glu
Gly Pro Lys Arg Trp Glu Gln Ala Trp Met Ala 1255 1260 1265 ata aaa
aag gtg tgg gct tca aaa tgg aat gag aga gca tac ttc 3947 Ile Lys
Lys Val Trp Ala Ser Lys Trp Asn Glu Arg Ala Tyr Phe 1270 1275 1280
agc aca agg aag gtg aaa ctg gat cat gac tat ctg tgc atg gct 3992
Ser Thr Arg Lys Val Lys Leu Asp His Asp Tyr Leu Cys Met Ala 1285
1290 1295 gtc ctt gtt caa gaa ata ata aat gct gat tat gca ttt gtc
att 4037 Val Leu Val Gln Glu Ile Ile Asn Ala Asp Tyr Ala Phe Val
Ile 1300 1305 1310 cac aca acc aac cca tct tcc gga gac gac tca gaa
ata tat gcc 4082 His Thr Thr Asn Pro Ser Ser Gly Asp Asp Ser Glu
Ile Tyr Ala 1315 1320 1325 gag gtg gtc agg ggc ctt ggg gaa aca ctt
gtt gga gct tat cca 4127 Glu Val Val Arg Gly Leu Gly Glu Thr Leu
Val Gly Ala Tyr Pro 1330 1335 1340 gga cgt gct ttg agt ttt atc tgc
aag aaa aag gat ctc aac tct 4172 Gly Arg Ala Leu Ser Phe Ile Cys
Lys Lys Lys Asp Leu Asn Ser 1345 1350 1355 cct caa gtg tta ggt tac
cca agc aaa ccg atc ggc ctt ttc ata 4217 Pro Gln Val Leu Gly Tyr
Pro Ser Lys Pro Ile Gly Leu Phe Ile 1360 1365 1370 aaa aga tct atc
atc ttc cga tct gat tcc aat ggg gaa gat ttg 4262 Lys Arg Ser Ile
Ile Phe Arg Ser Asp Ser Asn Gly Glu Asp Leu 1375 1380 1385 gaa ggt
tat gcc ggt gct ggc ctc tac gac agt gta cca atg gat 4307 Glu Gly
Tyr Ala Gly Ala Gly Leu Tyr Asp Ser Val Pro Met Asp 1390 1395 1400
gag gag gaa aaa gtt gta att gat tac tct tcc gac cca ttg ata 4352
Glu Glu Glu Lys Val Val Ile Asp Tyr Ser Ser Asp Pro Leu Ile 1405
1410 1415 act gat ggt aac ttc cgc cag aca atc ctg tcc aac att gct
cgt 4397 Thr Asp Gly Asn Phe Arg Gln Thr Ile Leu Ser Asn Ile Ala
Arg 1420 1425 1430 gct gga cat gct atc gag gag cta tat ggc tct cct
caa gac att 4442 Ala Gly His Ala Ile Glu Glu Leu Tyr Gly Ser Pro
Gln Asp Ile 1435 1440 1445 gag ggt gta gtg agg gat gga aag att tat
gtc gtt cag aca aga 4487 Glu Gly Val Val Arg Asp Gly Lys Ile Tyr
Val Val Gln Thr Arg 1450 1455 1460 cca cag atg tga ttatattctc
gttgtatgtt gttcagagaa gaccacagat 4539 Pro Gln Met gtgatcatat
tctcattgta tcagatctgt gaccacttac ctgatacctc ccatgaagtt 4599
acctgtatga ttatacgtga tccaaagcca tcacatcatg ttcaccttca gctattggag
4659 gagaagtgag aagtaggaat tgcaatatga ggaataataa gaaaaacttt
gtaaaagcta 4719 aattagctgg gtatgatata gggagaaatg tgtaaacatt
gtactatata tagtatatac 4779 acacgcatta tgtattgcat tatgcactga
ataatatcgc agcatcaaag aagaaatcct 4839 ttgggtggtt tc 4851
<210> SEQ ID NO 2 <211> LENGTH: 1464 <212> TYPE:
PRT <213> ORGANISM: Solanum tuberosum <400> SEQUENCE: 2
Met Ser Asn Ser Leu Gly Asn Asn Leu Leu Tyr Gln Gly Phe Leu Thr 1 5
10 15 Ser Thr Val Leu Glu His Lys Ser Arg Ile Ser Pro Pro Cys Val
Gly 20 25 30 Gly Asn Ser Leu Phe Gln Gln Gln Val Ile Ser Lys Ser
Pro Leu Ser 35 40 45 Thr Glu Phe Arg Gly Asn Arg Leu Lys Val Gln
Lys Lys Lys Ile Pro 50 55 60 Met Glu Lys Lys Arg Ala Phe Ser Ser
Ser Pro His Ala Val Leu Thr 65 70 75 80 Thr Asp Thr Ser Ser Glu Leu
Ala Glu Lys Phe Ser Leu Gly Gly Asn 85 90 95 Ile Glu Leu Gln Val
Asp Val Arg Pro Pro Thr Ser Gly Asp Val Ser 100 105 110 Phe Val Asp
Phe Gln Val Thr Asn Gly Ser Asp Lys Leu Phe Leu His 115 120 125 Trp
Gly Ala Val Lys Phe Gly Lys Glu Thr Trp Ser Leu Pro Asn Asp 130 135
140 Arg Pro Asp Gly Thr Lys Val Tyr Lys Asn Lys Ala Leu Arg Thr Pro
145 150 155 160 Phe Val Lys Ser Gly Ser Asn Ser Ile Leu Arg Leu Glu
Ile Arg Asp 165 170 175 Thr Ala Ile Glu Ala Ile Glu Phe Leu Ile Tyr
Asp Glu Ala His Asp 180 185 190 Lys Trp Ile Lys Asn Asn Gly Gly Asn
Phe Arg Val Lys Leu Ser Arg 195 200 205 Lys Glu Ile Arg Gly Pro Asp
Val Ser Val Pro Glu Glu Leu Val Gln 210 215 220 Ile Gln Ser Tyr Leu
Arg Trp Glu Arg Lys Gly Lys Gln Asn Tyr Pro 225 230 235 240 Pro Glu
Lys Glu Lys Glu Glu Tyr Glu Ala Ala Arg Thr Val Leu Gln 245 250 255
Glu Glu Ile Ala Arg Gly Ala Ser Ile Gln Asp Ile Arg Ala Arg Leu 260
265 270 Thr Lys Thr Asn Asp Lys Ser Gln Ser Lys Glu Glu Pro Leu His
Val 275 280 285 Thr Lys Ser Asp Ile Pro Asp Asp Leu Ala Gln Ala Gln
Ala Tyr Ile 290 295 300 Arg Trp Glu Lys Ala Gly Lys Pro Asn Tyr Pro
Pro Glu Lys Gln Ile 305 310 315 320 Glu Glu Leu Glu Glu Ala Arg Arg
Glu Leu Gln Leu Glu Leu Glu Lys 325 330 335 Gly Ile Thr Leu Asp Glu
Leu Arg Lys Thr Ile Thr Lys Gly Glu Ile 340 345 350 Lys Thr Lys Val
Glu Lys His Leu Lys Arg Ser Ser Phe Ala Val Glu 355 360 365 Arg Ile
Gln Arg Lys Lys Arg Asp Phe Gly His Leu Ile Asn Lys Tyr 370 375 380
Thr Ser Ser Pro Ala Val Gln Val Gln Lys Val Leu Glu Glu Pro Pro 385
390 395 400 Ala Leu Ser Lys Ile Lys Leu Tyr Ala Lys Glu Lys Glu Glu
Gln Ile 405 410 415 Asp Asp Pro Ile Leu Asn Lys Lys Ile Phe Lys Val
Asp Asp Gly Glu 420 425 430 Leu Leu Val Leu Val Ala Lys Ser Ser Gly
Lys Thr Lys Val His Leu 435 440 445 Ala Thr Asp Leu Asn Gln Pro Ile
Thr Leu His Trp Ala Leu Ser Lys 450 455 460 Ser Pro Gly Glu Trp Met
Val Pro Pro Ser Ser Ile Leu Pro Pro Gly 465 470 475 480 Ser Ile Ile
Leu Asp Lys Ala Ala Glu Thr Pro Phe Ser Ala Ser Ser 485 490 495 Ser
Asp Gly Leu Thr Ser Lys Val Gln Ser Leu Asp Ile Val Ile Glu 500 505
510 Asp Gly Asn Phe Val Gly Met Pro Phe Val Leu Leu Ser Gly Glu Lys
515 520 525 Trp Ile Lys Asn Gln Gly Ser Asp Phe Tyr Val Gly Phe Ser
Ala Ala 530 535 540 Ser Lys Leu Ala Leu Lys Ala Ala Gly Asp Gly Ser
Gly Thr Ala Lys 545 550 555 560 Ser Leu Leu Asp Lys Ile Ala Asp Met
Glu Ser Glu Ala Gln Lys Ser 565 570 575 Phe Met His Arg Phe Asn Ile
Ala Ala Asp Leu Ile Glu Asp Ala Thr 580 585 590 Ser Ala Gly Glu Leu
Gly Phe Ala Gly Ile Leu Val Trp Met Arg Phe 595 600 605 Met Ala Thr
Arg Gln Leu Ile Trp Asn Lys Asn Tyr Asn Val Lys Pro 610 615 620 Arg
Glu Ile Ser Lys Ala Gln Asp Arg Leu Thr Asp Leu Leu Gln Asn 625 630
635 640 Ala Phe Thr Ser His Pro Gln Tyr Arg Glu Ile Leu Arg Met Ile
Met 645 650 655 Ser Thr Val Gly Arg Gly Gly Glu Gly Asp Val Gly Gln
Arg Ile Arg 660 665 670 Asp Glu Ile Leu Val Ile Gln Arg Asn Asn Asp
Cys Lys Gly Gly Met 675 680 685 Met Gln Glu Trp His Gln Lys Leu His
Asn Asn Thr Ser Pro Asp Asp 690 695 700 Val Val Ile Cys Gln Ala Leu
Ile Asp Tyr Ile Lys Ser Asp Phe Asp 705 710 715 720 Leu Gly Val Tyr
Trp Lys Thr Leu Asn Glu Asn Gly Ile Thr Lys Glu 725 730 735 Arg Leu
Leu Ser Tyr Asp Arg Ala Ile His Ser Glu Pro Asn Phe Arg 740 745 750
Gly Asp Gln Lys Gly Gly Leu Leu Arg Asp Leu Gly His Tyr Met Arg 755
760 765 Thr Leu Lys Ala Val His Ser Gly Ala Asp Leu Glu Ser Ala Ile
Ala 770 775 780 Asn Cys Met Gly Tyr Lys Thr Glu Gly Glu Gly Phe Met
Val Gly Val 785 790 795 800 Gln Ile Asn Pro Val Ser Gly Leu Pro Ser
Gly Phe Gln Asp Leu Leu 805 810 815 His Phe Val Leu Asp His Val Glu
Asp Lys Asn Val Glu Thr Leu Leu 820 825 830 Glu Arg Leu Leu Glu Ala
Arg Glu Glu Leu Arg Pro Leu Leu Leu Lys 835 840 845 Pro Asn Asn Arg
Leu Lys Asp Leu Leu Phe Leu Asp Ile Ala Leu Asp 850 855 860 Ser Thr
Val Arg Thr Ala Val Glu Arg Gly Tyr Glu Glu Leu Asn Asn 865 870 875
880 Ala Asn Pro Glu Lys Ile Met Tyr Phe Ile Ser Leu Val Leu Glu Asn
885 890 895 Leu Ala Leu Ser Val Asp Asp Asn Glu Asp Leu Val Tyr Cys
Leu Lys 900 905 910 Gly Trp Asn Gln Ala Leu Ser Met Ser Asn Gly Gly
Asp Asn His Trp 915 920 925 Ala Leu Phe Ala Lys Ala Val Leu Asp Arg
Thr Arg Leu Ala Leu Ala 930 935 940 Ser Lys Ala Glu Trp Tyr His His
Leu Leu Gln Pro Ser Ala Glu Tyr 945 950 955 960 Leu Gly Ser Ile Leu
Gly Val Asp Gln Trp Ala Leu Asn Ile Phe Thr 965 970 975 Glu Glu Ile
Ile Arg Ala Gly Ser Ala Ala Ser Leu Ser Ser Leu Leu 980 985 990 Asn
Arg Leu Asp Pro Val Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp 995
1000 1005 Gln Ile Ile Ser Pro Val Glu Ala Val Gly Tyr Val Val Val
Val 1010 1015 1020 Asp Glu Leu Leu Ser Val Gln Asn Glu Ile Tyr Glu
Lys Pro Thr 1025 1030 1035 Ile Leu Val Ala Lys Ser Val Lys Gly Glu
Glu Glu Ile Pro Asp 1040 1045 1050 Gly Ala Val Ala Leu Ile Thr Pro
Asp Met Pro Asp Val Leu Ser 1055 1060 1065 His Val Ser Val Arg Ala
Arg Asn Gly Lys Val Cys Phe Ala Thr 1070 1075 1080 Cys Phe Asp Pro
Asn Ile Leu Ala Asp Leu Gln Ala Lys Glu Gly 1085 1090 1095 Arg Ile
Leu Leu Leu Lys Pro Thr Pro Ser Asp Ile Ile Tyr Ser 1100 1105 1110
Glu Val Asn Glu Ile Glu Leu Gln Ser Ser Ser Asn Leu Val Glu 1115
1120 1125 Ala Glu Thr Ser Ala Thr Leu Arg Leu Val Lys Lys Gln Phe
Gly 1130 1135 1140 Gly Cys Tyr Ala Ile Ser Ala Asp Glu Phe Thr Ser
Glu Met Val 1145 1150 1155 Gly Ala Lys Ser Arg Asn Ile Ala Tyr Leu
Lys Gly Lys Val Pro 1160 1165 1170 Ser Ser Val Gly Ile Pro Thr Ser
Val Ala Leu Pro Phe Gly Val 1175 1180 1185 Phe Glu Lys Val Leu Ser
Asp Asp Ile Asn Gln Gly Val Ala Lys 1190 1195 1200 Glu Leu Gln Ile
Leu Met Lys Lys Leu Ser Glu Gly Asp Phe Ser 1205 1210 1215 Ala Leu
Gly Glu Ile Arg Thr Thr Val Leu Asp Leu Ser Ala Pro 1220 1225 1230
Ala Gln Leu Val Lys Glu Leu Lys Glu Lys Met Gln Gly Ser Gly 1235
1240 1245 Met Pro Trp Pro Gly Asp Glu Gly Pro Lys Arg Trp Glu Gln
Ala 1250 1255 1260 Trp Met Ala Ile Lys Lys Val Trp Ala Ser Lys Trp
Asn Glu Arg 1265 1270 1275 Ala Tyr Phe Ser Thr Arg Lys Val Lys Leu
Asp His Asp Tyr Leu 1280 1285 1290 Cys Met Ala Val Leu Val Gln Glu
Ile Ile Asn Ala Asp Tyr Ala 1295 1300 1305 Phe Val Ile His Thr Thr
Asn Pro Ser Ser Gly Asp Asp Ser Glu 1310 1315 1320 Ile Tyr Ala Glu
Val Val Arg Gly Leu Gly Glu Thr Leu Val Gly 1325 1330 1335 Ala Tyr
Pro Gly Arg Ala Leu Ser Phe Ile Cys Lys Lys Lys Asp 1340 1345 1350
Leu Asn Ser Pro Gln Val Leu Gly Tyr Pro Ser Lys Pro Ile Gly 1355
1360 1365 Leu Phe Ile Lys Arg Ser Ile Ile Phe Arg Ser Asp Ser Asn
Gly 1370 1375 1380 Glu Asp Leu Glu Gly Tyr Ala Gly Ala Gly Leu Tyr
Asp Ser Val 1385 1390 1395 Pro Met Asp Glu Glu Glu Lys Val Val Ile
Asp Tyr Ser Ser Asp 1400 1405 1410 Pro Leu Ile Thr Asp Gly Asn Phe
Arg Gln Thr Ile Leu Ser Asn 1415 1420 1425 Ile Ala Arg Ala Gly His
Ala Ile Glu Glu Leu Tyr Gly Ser Pro 1430 1435 1440 Gln Asp Ile Glu
Gly Val Val Arg Asp Gly Lys Ile Tyr Val Val 1445 1450 1455 Gln Thr
Arg Pro Gln Met 1460 <210> SEQ ID NO 3 <211> LENGTH:
3000 <212> TYPE: DNA <213> ORGANISM: Triticum aestivum
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (227)..(2623) <400> SEQUENCE: 3 ctccaccgcg
gtggcggccg ctctagaact agtggatccc ccgggctgca ggaattcggc 60
acgagcttcg gcctgacccc gttcgtttac ccccacacag agcacactcc agtccagtcc
120 agcccactgc caccgcgcta ctctccactc ccactgccac cacctccgcc
tgcgccgcgc 180 tctgggcgga ccaacccgcg aaccgtacca tctcccgccc cgatcc
atg tcg tcg 235 Met Ser Ser 1 gcg gtc gcg tcc gcc gca tcc ttc ctc
gcg ctc gcg tca gcc tcc ccc 283 Ala Val Ala Ser Ala Ala Ser Phe Leu
Ala Leu Ala Ser Ala Ser Pro 5 10 15 ggg aga tca cgc agg cgg gcg agg
gtg agc gcg cag cca ccc cac gcc 331 Gly Arg Ser Arg Arg Arg Ala Arg
Val Ser Ala Gln Pro Pro His Ala 20 25 30 35 ggg gcc ggc agg ttg cac
tgg ccg ccg tgg ccg ccg cag cgc acg gct 379 Gly Ala Gly Arg Leu His
Trp Pro Pro Trp Pro Pro Gln Arg Thr Ala 40 45 50 cgc gac gga gct
gtg gcg gcg ctc gcc gcc ggg aag aag gac gcg ggg 427 Arg Asp Gly Ala
Val Ala Ala Leu Ala Ala Gly Lys Lys Asp Ala Gly 55 60 65 atc gac
gac gcc gcc gcg tcc gtg agg cag ccc cgc gca ctc cgc ggt 475 Ile Asp
Asp Ala Ala Ala Ser Val Arg Gln Pro Arg Ala Leu Arg Gly 70 75 80
ggc gcc gcc acc aag gtc gcg gag cga agg gat ccc gtc aag acg ctc 523
Gly Ala Ala Thr Lys Val Ala Glu Arg Arg Asp Pro Val Lys Thr Leu 85
90 95 gac cgc gac gcc gcg gaa ggc ggc ggg ccg tcc ccg ccg gca gcg
agg 571 Asp Arg Asp Ala Ala Glu Gly Gly Gly Pro Ser Pro Pro Ala Ala
Arg 100 105 110 115 cag gac gcc gcc cgt ccg ccg agt atg aac ggc atg
ccg gtg aac ggc 619 Gln Asp Ala Ala Arg Pro Pro Ser Met Asn Gly Met
Pro Val Asn Gly 120 125 130 gag aac aaa tct acc ggc ggc ggc ggc gcg
act aaa gac agc ggg ctg 667 Glu Asn Lys Ser Thr Gly Gly Gly Gly Ala
Thr Lys Asp Ser Gly Leu 135 140 145 ccc acg ccc gca cgc gcg ccc cat
ccg tcg acc cag aac aga gca ccg 715 Pro Thr Pro Ala Arg Ala Pro His
Pro Ser Thr Gln Asn Arg Ala Pro 150 155 160 gtg aac ggt gaa aac aaa
gct aac gtc gcc tcg ccg ccg acg agc ata 763 Val Asn Gly Glu Asn Lys
Ala Asn Val Ala Ser Pro Pro Thr Ser Ile 165 170 175 gcc gag gcc gcg
gct tcg gat tcc gca gct acc att tcc atc agc gac 811 Ala Glu Ala Ala
Ala Ser Asp Ser Ala Ala Thr Ile Ser Ile Ser Asp 180 185 190 195 aag
gcg ccg gag tcc gtt gtc cca gct gag aag acg ccg ccg tcg tcc 859 Lys
Ala Pro Glu Ser Val Val Pro Ala Glu Lys Thr Pro Pro Ser Ser 200 205
210 ggc tca aat ttc gag tcc tcg gcc tct gct ccc ggg tct gac act gtc
907 Gly Ser Asn Phe Glu Ser Ser Ala Ser Ala Pro Gly Ser Asp Thr Val
215 220 225 agc gac gtg gaa caa gaa ctg aag aag ggt gcg gtc gtt gtc
gaa gaa 955 Ser Asp Val Glu Gln Glu Leu Lys Lys Gly Ala Val Val Val
Glu Glu 230 235 240 gct cca aag cca aag gct ctt tcg ccg cct gca gcc
ccc gct gta caa 1003 Ala Pro Lys Pro Lys Ala Leu Ser Pro Pro Ala
Ala Pro Ala Val Gln 245 250 255 gaa gac ctt tgg gat ttc aag aaa tac
att ggt ttc gag gag ccc gtg 1051 Glu Asp Leu Trp Asp Phe Lys Lys
Tyr Ile Gly Phe Glu Glu Pro Val 260 265 270 275 gag gcc aag gat gat
ggc cgg gct gtc gca gat gat gcg ggc tcc ttt 1099 Glu Ala Lys Asp
Asp Gly Arg Ala Val Ala Asp Asp Ala Gly Ser Phe 280 285 290 gaa cac
cac cag aat cac gac tcc gga cct ttg gca ggg gag aat gtc 1147 Glu
His His Gln Asn His Asp Ser Gly Pro Leu Ala Gly Glu Asn Val 295 300
305 atg aac gtg gtc gtc gtg gct gct gag tgt tct ccc tgg tgc aaa aca
1195 Met Asn Val Val Val Val Ala Ala Glu Cys Ser Pro Trp Cys Lys
Thr 310 315 320 ggt ggt ctg gga gat gtt gcg ggt gct ctg ccc aag gct
ttg gca aag 1243 Gly Gly Leu Gly Asp Val Ala Gly Ala Leu Pro Lys
Ala Leu Ala Lys 325 330 335 aga gga cat cgt gtt atg gtt gtg gta cca
agg tat ggg gac tat gaa 1291 Arg Gly His Arg Val Met Val Val Val
Pro Arg Tyr Gly Asp Tyr Glu 340 345 350 355 gaa gcc tac gat gtc gga
gtc cga aaa tac tac aag gct gct gga cag 1339 Glu Ala Tyr Asp Val
Gly Val Arg Lys Tyr Tyr Lys Ala Ala Gly Gln 360 365 370 gat atg gaa
gtg aat tat ttc cat gct tat atc gat gga gtt gat ttt 1387 Asp Met
Glu Val Asn Tyr Phe His Ala Tyr Ile Asp Gly Val Asp Phe 375 380 385
gtg ttc att gac gct cct ctc ttc cga cac cgt cag gaa gac att tat
1435 Val Phe Ile Asp Ala Pro Leu Phe Arg His Arg Gln Glu Asp Ile
Tyr 390 395 400 ggg ggc agc aga cag gaa att atg aag cgc atg att ttg
ttc tgc aag 1483 Gly Gly Ser Arg Gln Glu Ile Met Lys Arg Met Ile
Leu Phe Cys Lys 405 410 415 gcc gct gtt gag gtt cca tgg cac gtt cca
tgc ggc ggt gtc cct tat 1531 Ala Ala Val Glu Val Pro Trp His Val
Pro Cys Gly Gly Val Pro Tyr 420 425 430 435 ggg gat gga aat ctg gtg
ttt att gca aat gat tgg cac acg gca ctc 1579 Gly Asp Gly Asn Leu
Val Phe Ile Ala Asn Asp Trp His Thr Ala Leu 440 445 450 ctg cct gtc
tat ctg aaa gca tat tac agg gac cat ggt ttg atg cag 1627 Leu Pro
Val Tyr Leu Lys Ala Tyr Tyr Arg Asp His Gly Leu Met Gln 455 460 465
tac act cgg tcc att atg gtg ata cat aac atc gct cac cag ggc cgt
1675 Tyr Thr Arg Ser Ile Met Val Ile His Asn Ile Ala His Gln Gly
Arg 470 475 480 ggc cct gta gat gaa ttc ccg ttc acc gag ttg cct gag
cac tac ctg 1723 Gly Pro Val Asp Glu Phe Pro Phe Thr Glu Leu Pro
Glu His Tyr Leu 485 490 495 gaa cac ttc aga ctg tac gac ccc gtg ggt
ggt gaa cac gcc aac tac 1771 Glu His Phe Arg Leu Tyr Asp Pro Val
Gly Gly Glu His Ala Asn Tyr 500 505 510 515 ttc gcc gcc ggc ctg aag
atg gcg gac cag gtt gtc gtg gtg agc ccc 1819 Phe Ala Ala Gly Leu
Lys Met Ala Asp Gln Val Val Val Val Ser Pro 520 525 530 ggg tac ctg
tgg gag ctg aag acg gtg gag ggc ggc tgg ggg ctt cac 1867 Gly Tyr
Leu Trp Glu Leu Lys Thr Val Glu Gly Gly Trp Gly Leu His 535 540 545
gac atc ata cgg cag aac gac tgg aag acc cgc ggc atc gtc aac ggc
1915 Asp Ile Ile Arg Gln Asn Asp Trp Lys Thr Arg Gly Ile Val Asn
Gly 550 555 560 atc gac aac atg gag tgg aac ccc gag gtg gac gcc cac
ctc aag tcg 1963 Ile Asp Asn Met Glu Trp Asn Pro Glu Val Asp Ala
His Leu Lys Ser 565 570 575 gac ggc tac acc aac ttc tcc ctg agg acg
ctg gac tcc ggc aag cgg 2011 Asp Gly Tyr Thr Asn Phe Ser Leu Arg
Thr Leu Asp Ser Gly Lys Arg 580 585 590 595 cag tgc aag gag gcc ctg
cag cgc gag ctg ggc ctg cag gtc cgc gcc 2059 Gln Cys Lys Glu Ala
Leu Gln Arg Glu Leu Gly Leu Gln Val Arg Ala 600 605 610 gac gtg ccg
ctg ctc ggc ttc atc ggc cgc ctg gac ggg cag aag ggc 2107 Asp Val
Pro Leu Leu Gly Phe Ile Gly Arg Leu Asp Gly Gln Lys Gly 615 620 625
gtg gag atc atc gcg gac gcc atg ccc tgg atc gtg agc cag gac gtg
2155 Val Glu Ile Ile Ala Asp Ala Met Pro Trp Ile Val Ser Gln Asp
Val 630 635 640 cag ctg gtg atg ctg ggc acc ggg cgc cac gac ctg gag
agc atg ctg 2203 Gln Leu Val Met Leu Gly Thr Gly Arg His Asp Leu
Glu Ser Met Leu 645 650 655 cag cac ttc gag cgg gag cac cac gac aag
gtg cgc ggg tgg gtg ggg 2251 Gln His Phe Glu Arg Glu His His Asp
Lys Val Arg Gly Trp Val Gly 660 665 670 675 ttc tcc gtg cgc ctg gcg
cac cgg atc acg gcg ggg gcg gac gcg ctc 2299 Phe Ser Val Arg Leu
Ala His Arg Ile Thr Ala Gly Ala Asp Ala Leu 680 685 690 ctc atg ccc
tcc cgg ttc gag ccg tgc ggg ctg aac cag ctc tac gcc 2347 Leu Met
Pro Ser Arg Phe Glu Pro Cys Gly Leu Asn Gln Leu Tyr Ala 695 700 705
atg gcc tac ggc acc gtc ccc gtc gtg cac gcc gtc ggc ggc ctc agg
2395 Met Ala Tyr Gly Thr Val Pro Val Val His Ala Val Gly Gly Leu
Arg 710 715 720 gac acc gtg ccg ccg ttc gac ccc ttc aac cac tcc ggg
ctc ggg tgg 2443 Asp Thr Val Pro Pro Phe Asp Pro Phe Asn His Ser
Gly Leu Gly Trp 725 730 735 acg ttc gac cgc gcc gag gcg cac aag ctg
atc gag gcg ctc ggg cac 2491 Thr Phe Asp Arg Ala Glu Ala His Lys
Leu Ile Glu Ala Leu Gly His 740 745 750 755 tgc ctc cgc acc tac cga
gac ttc aag gag agc tgg agg gcc ctc cag 2539 Cys Leu Arg Thr Tyr
Arg Asp Phe Lys Glu Ser Trp Arg Ala Leu Gln 760 765 770 gag cgc ggc
atg tcg cag gac ttc agc tgg gag cac gcc gcc aag ctc 2587 Glu Arg
Gly Met Ser Gln Asp Phe Ser Trp Glu His Ala Ala Lys Leu 775 780 785
tac gag gac gtc ctc gtc aag gcc aag tac cag tgg tgaacgctag 2633 Tyr
Glu Asp Val Leu Val Lys Ala Lys Tyr Gln Trp 790 795 ctgctagccg
ctccagcccc gcatgcgtgc atgacaggat ggaactgcat tgcgcacgca 2693
ggaaagtgcc atggagcgcc ggcatccgcg aagtacagtg acatgaggtg tgtgtggttg
2753 agacgctgat tccaatccgg cccgtagcag agtagagcgg aggtatatgg
gaatcttaac 2813 ttggtattgt aatttgttat gttgtgtgca ttattacaat
gttgttactt attcttgtta 2873 agtcggaggc caagggcgaa agctagctca
catgtctgat ggatgcacgt gccatggttg 2933 gtttggtagc gcagtgcaaa
cggcaagaat gggaagtgaa ttcctccctg cttgaaaaaa 2993 aaaaaaa 3000
<210> SEQ ID NO 4 <211> LENGTH: 799 <212> TYPE:
PRT <213> ORGANISM: Triticum aestivum <400> SEQUENCE: 4
Met Ser Ser Ala Val Ala Ser Ala Ala Ser Phe Leu Ala Leu Ala Ser 1 5
10 15 Ala Ser Pro Gly Arg Ser Arg Arg Arg Ala Arg Val Ser Ala Gln
Pro 20 25 30 Pro His Ala Gly Ala Gly Arg Leu His Trp Pro Pro Trp
Pro Pro Gln 35 40 45 Arg Thr Ala Arg Asp Gly Ala Val Ala Ala Leu
Ala Ala Gly Lys Lys 50 55 60 Asp Ala Gly Ile Asp Asp Ala Ala Ala
Ser Val Arg Gln Pro Arg Ala 65 70 75 80 Leu Arg Gly Gly Ala Ala Thr
Lys Val Ala Glu Arg Arg Asp Pro Val 85 90 95 Lys Thr Leu Asp Arg
Asp Ala Ala Glu Gly Gly Gly Pro Ser Pro Pro 100 105 110 Ala Ala Arg
Gln Asp Ala Ala Arg Pro Pro Ser Met Asn Gly Met Pro 115 120 125 Val
Asn Gly Glu Asn Lys Ser Thr Gly Gly Gly Gly Ala Thr Lys Asp 130 135
140 Ser Gly Leu Pro Thr Pro Ala Arg Ala Pro His Pro Ser Thr Gln Asn
145 150 155 160 Arg Ala Pro Val Asn Gly Glu Asn Lys Ala Asn Val Ala
Ser Pro Pro 165 170 175 Thr Ser Ile Ala Glu Ala Ala Ala Ser Asp Ser
Ala Ala Thr Ile Ser 180 185 190 Ile Ser Asp Lys Ala Pro Glu Ser Val
Val Pro Ala Glu Lys Thr Pro 195 200 205 Pro Ser Ser Gly Ser Asn Phe
Glu Ser Ser Ala Ser Ala Pro Gly Ser 210 215 220 Asp Thr Val Ser Asp
Val Glu Gln Glu Leu Lys Lys Gly Ala Val Val 225 230 235 240 Val Glu
Glu Ala Pro Lys Pro Lys Ala Leu Ser Pro Pro Ala Ala Pro 245 250 255
Ala Val Gln Glu Asp Leu Trp Asp Phe Lys Lys Tyr Ile Gly Phe Glu 260
265 270 Glu Pro Val Glu Ala Lys Asp Asp Gly Arg Ala Val Ala Asp Asp
Ala 275 280 285 Gly Ser Phe Glu His His Gln Asn His Asp Ser Gly Pro
Leu Ala Gly 290 295 300 Glu Asn Val Met Asn Val Val Val Val Ala Ala
Glu Cys Ser Pro Trp 305 310 315 320 Cys Lys Thr Gly Gly Leu Gly Asp
Val Ala Gly Ala Leu Pro Lys Ala 325 330 335 Leu Ala Lys Arg Gly His
Arg Val Met Val Val Val Pro Arg Tyr Gly 340 345 350 Asp Tyr Glu Glu
Ala Tyr Asp Val Gly Val Arg Lys Tyr Tyr Lys Ala 355 360 365 Ala Gly
Gln Asp Met Glu Val Asn Tyr Phe His Ala Tyr Ile Asp Gly 370 375 380
Val Asp Phe Val Phe Ile Asp Ala Pro Leu Phe Arg His Arg Gln Glu 385
390 395 400 Asp Ile Tyr Gly Gly Ser Arg Gln Glu Ile Met Lys Arg Met
Ile Leu 405 410 415 Phe Cys Lys Ala Ala Val Glu Val Pro Trp His Val
Pro Cys Gly Gly 420 425 430 Val Pro Tyr Gly Asp Gly Asn Leu Val Phe
Ile Ala Asn Asp Trp His 435 440 445 Thr Ala Leu Leu Pro Val Tyr Leu
Lys Ala Tyr Tyr Arg Asp His Gly 450 455 460 Leu Met Gln Tyr Thr Arg
Ser Ile Met Val Ile His Asn Ile Ala His 465 470 475 480 Gln Gly Arg
Gly Pro Val Asp Glu Phe Pro Phe Thr Glu Leu Pro Glu 485 490 495 His
Tyr Leu Glu His Phe Arg Leu Tyr Asp Pro Val Gly Gly Glu His 500 505
510 Ala Asn Tyr Phe Ala Ala Gly Leu Lys Met Ala Asp Gln Val Val Val
515 520 525 Val Ser Pro Gly Tyr Leu Trp Glu Leu Lys Thr Val Glu Gly
Gly Trp 530 535 540 Gly Leu His Asp Ile Ile Arg Gln Asn Asp Trp Lys
Thr Arg Gly Ile 545 550 555 560 Val Asn Gly Ile Asp Asn Met Glu Trp
Asn Pro Glu Val Asp Ala His 565 570 575 Leu Lys Ser Asp Gly Tyr Thr
Asn Phe Ser Leu Arg Thr Leu Asp Ser 580 585 590 Gly Lys Arg Gln Cys
Lys Glu Ala Leu Gln Arg Glu Leu Gly Leu Gln 595 600 605 Val Arg Ala
Asp Val Pro Leu Leu Gly Phe Ile Gly Arg Leu Asp Gly 610 615 620 Gln
Lys Gly Val Glu Ile Ile Ala Asp Ala Met Pro Trp Ile Val Ser 625 630
635 640 Gln Asp Val Gln Leu Val Met Leu Gly Thr Gly Arg His Asp Leu
Glu 645 650 655 Ser Met Leu Gln His Phe Glu Arg Glu His His Asp Lys
Val Arg Gly 660 665 670 Trp Val Gly Phe Ser Val Arg Leu Ala His Arg
Ile Thr Ala Gly Ala 675 680 685 Asp Ala Leu Leu Met Pro Ser Arg Phe
Glu Pro Cys Gly Leu Asn Gln 690 695 700 Leu Tyr Ala Met Ala Tyr Gly
Thr Val Pro Val Val His Ala Val Gly 705 710 715 720 Gly Leu Arg Asp
Thr Val Pro Pro Phe Asp Pro Phe Asn His Ser Gly 725 730 735 Leu Gly
Trp Thr Phe Asp Arg Ala Glu Ala His Lys Leu Ile Glu Ala 740 745 750
Leu Gly His Cys Leu Arg Thr Tyr Arg Asp Phe Lys Glu Ser Trp Arg 755
760 765 Ala Leu Gln Glu Arg Gly Met Ser Gln Asp Phe Ser Trp Glu His
Ala 770 775 780 Ala Lys Leu Tyr Glu Asp Val Leu Val Lys Ala Lys Tyr
Gln Trp 785 790 795 <210> SEQ ID NO 5 <211> LENGTH:
2433 <212> TYPE: DNA <213> ORGANISM: Oryza sativa
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(2430) <400> SEQUENCE: 5 atg tct agc gcg gtg
gtt gcg tcc agc aca act ttt ctc gtc gca ctt 48 Met Ser Ser Ala Val
Val Ala Ser Ser Thr Thr Phe Leu Val Ala Leu 1 5 10 15 gcc tct agc
gcg agc cgg ggc ggg cca cgt agg ggg cgc gtc gtg ggc 96 Ala Ser Ser
Ala Ser Arg Gly Gly Pro Arg Arg Gly Arg Val Val Gly 20 25 30 gtg
gcc gct ccc cca gcc ctc ctg tat gac ggg aga gct ggc agg cta 144 Val
Ala Ala Pro Pro Ala Leu Leu Tyr Asp Gly Arg Ala Gly Arg Leu 35 40
45 gcc ctg cgc gcc cct ccg cca ccc cgc cct aga cct agg cgc agg gat
192 Ala Leu Arg Ala Pro Pro Pro Pro Arg Pro Arg Pro Arg Arg Arg Asp
50 55 60 gcg ggt gtt gtc agg cgg gct gat gac ggg gag aac gag gcc
gca gtg 240 Ala Gly Val Val Arg Arg Ala Asp Asp Gly Glu Asn Glu Ala
Ala Val 65 70 75 80 gag cgg gcc ggc gag gac gat gac gag gag gag gag
ttc tcg tcc ggg 288 Glu Arg Ala Gly Glu Asp Asp Asp Glu Glu Glu Glu
Phe Ser Ser Gly 85 90 95 gcc tgg cag cca ccg cgt tca agg cgc ggt
gga gtt ggc aag gtc ctc 336 Ala Trp Gln Pro Pro Arg Ser Arg Arg Gly
Gly Val Gly Lys Val Leu 100 105 110 aaa cgt cgc ggt acc gtg ccg cca
gtc gga agg tac ggc tcc ggt gga 384 Lys Arg Arg Gly Thr Val Pro Pro
Val Gly Arg Tyr Gly Ser Gly Gly 115 120 125 gac gcc gct cgg gtg aga
gga gcc gcg gca ccc gct cca gca ccg acg 432 Asp Ala Ala Arg Val Arg
Gly Ala Ala Ala Pro Ala Pro Ala Pro Thr 130 135 140 caa gac gca gcg
tcg tct aag aat ggc gcg ctt ttg tca ggc agg gat 480 Gln Asp Ala Ala
Ser Ser Lys Asn Gly Ala Leu Leu Ser Gly Arg Asp 145 150 155 160 gac
gac aca cct gcc tca cgg aac gga tcg gtc gtt acc ggc gcc gac 528 Asp
Asp Thr Pro Ala Ser Arg Asn Gly Ser Val Val Thr Gly Ala Asp 165 170
175 aag cct gcc gcc gcc acg ccg ccg gtg acc ata acg aag ctc cca gcg
576 Lys Pro Ala Ala Ala Thr Pro Pro Val Thr Ile Thr Lys Leu Pro Ala
180 185 190 ccg gac tcc ccc gtg atc ctt cca tcc gta gac aag ccg cag
ccg gag 624 Pro Asp Ser Pro Val Ile Leu Pro Ser Val Asp Lys Pro Gln
Pro Glu 195 200 205 ttc gtc atc cca gac gcg acg gcg ccg gcg ccg cca
ccg ccc ggt tca 672 Phe Val Ile Pro Asp Ala Thr Ala Pro Ala Pro Pro
Pro Pro Gly Ser 210 215 220 aat ccc agg tcg tcc gct cct ctc ccc aag
cct gac aat tcg gaa ttt 720 Asn Pro Arg Ser Ser Ala Pro Leu Pro Lys
Pro Asp Asn Ser Glu Phe 225 230 235 240 gca gag gat aag agc gca aaa
gtt gtt gag agt gct ccg aag cca aag 768 Ala Glu Asp Lys Ser Ala Lys
Val Val Glu Ser Ala Pro Lys Pro Lys 245 250 255 gcg act aga tct tcc
cct att cct gcg gta gaa gag gag acg tgg gat 816 Ala Thr Arg Ser Ser
Pro Ile Pro Ala Val Glu Glu Glu Thr Trp Asp 260 265 270 ttc aag aaa
tat ttt gat ctg aac gaa ccg gac gcc gcg gag gat ggc 864 Phe Lys Lys
Tyr Phe Asp Leu Asn Glu Pro Asp Ala Ala Glu Asp Gly 275 280 285 gat
gac gat gat gac tgg gct gat tca gat gcg tca gat tct gag atc 912 Asp
Asp Asp Asp Asp Trp Ala Asp Ser Asp Ala Ser Asp Ser Glu Ile 290 295
300 gac cag gat gac gat tcg ggt cct ttg gct ggg gag aat gtc atg aac
960 Asp Gln Asp Asp Asp Ser Gly Pro Leu Ala Gly Glu Asn Val Met Asn
305 310 315 320 gtg atc gtg gtg gct gct gaa tgt tct ccc tgg tgc aaa
aca ggt ggg 1008 Val Ile Val Val Ala Ala Glu Cys Ser Pro Trp Cys
Lys Thr Gly Gly 325 330 335 ctt gga gat gtt gca ggt gct tta ccc aag
gct ttg gcg agg aga gga 1056 Leu Gly Asp Val Ala Gly Ala Leu Pro
Lys Ala Leu Ala Arg Arg Gly 340 345 350 cat cgt gtt atg gtt gtc gta
cca agg tac ggt gat tac gcg gaa gcc 1104 His Arg Val Met Val Val
Val Pro Arg Tyr Gly Asp Tyr Ala Glu Ala 355 360 365 cag gat gta gga
atc agg aaa tac tac aag gct gct gga cag gat ctg 1152 Gln Asp Val
Gly Ile Arg Lys Tyr Tyr Lys Ala Ala Gly Gln Asp Leu 370 375 380 gaa
gtg aaa tat ttc cat gca ttt atc gac gga gtt gat ttt gtg ttc 1200
Glu Val Lys Tyr Phe His Ala Phe Ile Asp Gly Val Asp Phe Val Phe 385
390 395 400 att gac gct cct ctc ttc cgt cac cgt cag gat gac atc tat
ggg ggg 1248 Ile Asp Ala Pro Leu Phe Arg His Arg Gln Asp Asp Ile
Tyr Gly Gly 405 410 415 aac aga cag gaa atc atg aag cgc atg att ctg
ttt tgt aag gct gct 1296 Asn Arg Gln Glu Ile Met Lys Arg Met Ile
Leu Phe Cys Lys Ala Ala 420 425 430 gtt gag gtt cct tgg cac gtt cca
tgc ggt ggt gtg ccc tat ggg gat 1344 Val Glu Val Pro Trp His Val
Pro Cys Gly Gly Val Pro Tyr Gly Asp 435 440 445 ggc aac ttg gtg ttc
ctt gca aac gat tgg cac act gca ctc ctg cct 1392 Gly Asn Leu Val
Phe Leu Ala Asn Asp Trp His Thr Ala Leu Leu Pro 450 455 460 gtt tat
ctg aag gca tat tac aga gac aat ggc atg atg cag tac act 1440 Val
Tyr Leu Lys Ala Tyr Tyr Arg Asp Asn Gly Met Met Gln Tyr Thr 465 470
475 480 cgc tct gtc ctt gtg ata cat aat atc gct tac cag ggc cgt ggc
cca 1488 Arg Ser Val Leu Val Ile His Asn Ile Ala Tyr Gln Gly Arg
Gly Pro 485 490 495 gta gat gaa ttc ccc tac atg gaa ttg ccg gag cac
tac ctg gat cac 1536 Val Asp Glu Phe Pro Tyr Met Glu Leu Pro Glu
His Tyr Leu Asp His 500 505 510 ttc aag ctg tac gac ccc gtc ggc ggc
gag cac gcc aac atc ttc ggc 1584 Phe Lys Leu Tyr Asp Pro Val Gly
Gly Glu His Ala Asn Ile Phe Gly 515 520 525 gcg ggc ctg aag atg gcg
gac cgg gtg gtg acc gtg agc ccc ggc tac 1632 Ala Gly Leu Lys Met
Ala Asp Arg Val Val Thr Val Ser Pro Gly Tyr 530 535 540 ctc tgg gag
ctg aag acg acg gag ggc ggc tgg ggc ctc cac gac atc 1680 Leu Trp
Glu Leu Lys Thr Thr Glu Gly Gly Trp Gly Leu His Asp Ile 545 550 555
560 ata cgg gag aac gac tgg aag atg aac ggc atc gtg aac ggc atc gac
1728 Ile Arg Glu Asn Asp Trp Lys Met Asn Gly Ile Val Asn Gly Ile
Asp 565 570 575 tac cgg gag tgg aac ccg gag gtg gac gtg cac ctg cag
tcc gac ggc 1776 Tyr Arg Glu Trp Asn Pro Glu Val Asp Val His Leu
Gln Ser Asp Gly 580 585 590 tac gcc aac tac acc gtg gcc tcg ctg gac
tcc agc aag ccg cgg tgc 1824 Tyr Ala Asn Tyr Thr Val Ala Ser Leu
Asp Ser Ser Lys Pro Arg Cys 595 600 605 aag gcg gcg ctg cag cgc gag
ctg ggg ctg gag gtg cgc gac gac gtg 1872 Lys Ala Ala Leu Gln Arg
Glu Leu Gly Leu Glu Val Arg Asp Asp Val 610 615 620 ccg ctg atc ggg
ttc atc ggg cgg ctc gac ggg cag aaa ggt gtg gac 1920 Pro Leu Ile
Gly Phe Ile Gly Arg Leu Asp Gly Gln Lys Gly Val Asp 625 630 635 640
atc atc ggc gac gcg atg ccg tgg atc gcc ggg cag gac gtg cag ctg
1968 Ile Ile Gly Asp Ala Met Pro Trp Ile Ala Gly Gln Asp Val Gln
Leu 645 650 655 gtg ctg ctg ggc tcc ggc cgc cgc gac ctg gag gtg atg
ctg cag cgg 2016 Val Leu Leu Gly Ser Gly Arg Arg Asp Leu Glu Val
Met Leu Gln Arg 660 665 670 ttc gag gcg cag cac aac agc aag gtg cgc
ggg tgg gtg ggg ttc tcg 2064 Phe Glu Ala Gln His Asn Ser Lys Val
Arg Gly Trp Val Gly Phe Ser 675 680 685 gtg aag atg gcg cac cgg atc
acg gcg ggc gcc gac gtg ctg gtc atg 2112 Val Lys Met Ala His Arg
Ile Thr Ala Gly Ala Asp Val Leu Val Met 690 695 700 ccg tcg cgg ttc
gag ccg tgc ggc ctc aac cag ctc tac gcc atg gcg 2160 Pro Ser Arg
Phe Glu Pro Cys Gly Leu Asn Gln Leu Tyr Ala Met Ala 705 710 715 720
tac ggc acc gtc ccc gtc gtg cac gcc gtc ggc ggg ctg agg gac acc
2208 Tyr Gly Thr Val Pro Val Val His Ala Val Gly Gly Leu Arg Asp
Thr 725 730 735 gtg tcg gcg ttc gac ccg ttc gag gac acc ggc ctc ggg
tgg acg ttc 2256 Val Ser Ala Phe Asp Pro Phe Glu Asp Thr Gly Leu
Gly Trp Thr Phe 740 745 750 gac cgc gcc gag ccg cac aag ctc atc gag
gcg ctc ggc cac tgc ctg 2304 Asp Arg Ala Glu Pro His Lys Leu Ile
Glu Ala Leu Gly His Cys Leu 755 760 765 gag acg tac cgc aag tac aag
gag agc tgg agg ggg ctc cag gtg cgc 2352 Glu Thr Tyr Arg Lys Tyr
Lys Glu Ser Trp Arg Gly Leu Gln Val Arg 770 775 780 ggc atg tcg cag
gac ctc agc tgg gac cac gcc gcc gag ctc tac gag 2400 Gly Met Ser
Gln Asp Leu Ser Trp Asp His Ala Ala Glu Leu Tyr Glu 785 790 795 800
gag gtc ctt gtc aag gcc aag tac caa tgg tga 2433 Glu Val Leu Val
Lys Ala Lys Tyr Gln Trp 805 810 <210> SEQ ID NO 6 <211>
LENGTH: 810 <212> TYPE: PRT <213> ORGANISM: Oryza
sativa <400> SEQUENCE: 6 Met Ser Ser Ala Val Val Ala Ser Ser
Thr Thr Phe Leu Val Ala Leu 1 5 10 15 Ala Ser Ser Ala Ser Arg Gly
Gly Pro Arg Arg Gly Arg Val Val Gly 20 25 30 Val Ala Ala Pro Pro
Ala Leu Leu Tyr Asp Gly Arg Ala Gly Arg Leu 35 40 45 Ala Leu Arg
Ala Pro Pro Pro Pro Arg Pro Arg Pro Arg Arg Arg Asp 50 55 60 Ala
Gly Val Val Arg Arg Ala Asp Asp Gly Glu Asn Glu Ala Ala Val 65 70
75 80 Glu Arg Ala Gly Glu Asp Asp Asp Glu Glu Glu Glu Phe Ser Ser
Gly 85 90 95 Ala Trp Gln Pro Pro Arg Ser Arg Arg Gly Gly Val Gly
Lys Val Leu 100 105 110 Lys Arg Arg Gly Thr Val Pro Pro Val Gly Arg
Tyr Gly Ser Gly Gly 115 120 125 Asp Ala Ala Arg Val Arg Gly Ala Ala
Ala Pro Ala Pro Ala Pro Thr 130 135 140 Gln Asp Ala Ala Ser Ser Lys
Asn Gly Ala Leu Leu Ser Gly Arg Asp 145 150 155 160 Asp Asp Thr Pro
Ala Ser Arg Asn Gly Ser Val Val Thr Gly Ala Asp 165 170 175 Lys Pro
Ala Ala Ala Thr Pro Pro Val Thr Ile Thr Lys Leu Pro Ala 180 185 190
Pro Asp Ser Pro Val Ile Leu Pro Ser Val Asp Lys Pro Gln Pro Glu 195
200 205 Phe Val Ile Pro Asp Ala Thr Ala Pro Ala Pro Pro Pro Pro Gly
Ser 210 215 220 Asn Pro Arg Ser Ser Ala Pro Leu Pro Lys Pro Asp Asn
Ser Glu Phe 225 230 235 240 Ala Glu Asp Lys Ser Ala Lys Val Val Glu
Ser Ala Pro Lys Pro Lys 245 250 255 Ala Thr Arg Ser Ser Pro Ile Pro
Ala Val Glu Glu Glu Thr Trp Asp 260 265 270 Phe Lys Lys Tyr Phe Asp
Leu Asn Glu Pro Asp Ala Ala Glu Asp Gly 275 280 285 Asp Asp Asp Asp
Asp Trp Ala Asp Ser Asp Ala Ser Asp Ser Glu Ile 290 295 300 Asp Gln
Asp Asp Asp Ser Gly Pro Leu Ala Gly Glu Asn Val Met Asn 305 310 315
320 Val Ile Val Val Ala Ala Glu Cys Ser Pro Trp Cys Lys Thr Gly Gly
325 330 335 Leu Gly Asp Val Ala Gly Ala Leu Pro Lys Ala Leu Ala Arg
Arg Gly 340 345 350 His Arg Val Met Val Val Val Pro Arg Tyr Gly Asp
Tyr Ala Glu Ala 355 360 365 Gln Asp Val Gly Ile Arg Lys Tyr Tyr Lys
Ala Ala Gly Gln Asp Leu 370 375 380 Glu Val Lys Tyr Phe His Ala Phe
Ile Asp Gly Val Asp Phe Val Phe 385 390 395 400 Ile Asp Ala Pro Leu
Phe Arg His Arg Gln Asp Asp Ile Tyr Gly Gly 405 410 415 Asn Arg Gln
Glu Ile Met Lys Arg Met Ile Leu Phe Cys Lys Ala Ala 420 425 430 Val
Glu Val Pro Trp His Val Pro Cys Gly Gly Val Pro Tyr Gly Asp 435 440
445 Gly Asn Leu Val Phe Leu Ala Asn Asp Trp His Thr Ala Leu Leu Pro
450 455 460 Val Tyr Leu Lys Ala Tyr Tyr Arg Asp Asn Gly Met Met Gln
Tyr Thr 465 470 475 480 Arg Ser Val Leu Val Ile His Asn Ile Ala Tyr
Gln Gly Arg Gly Pro 485 490 495 Val Asp Glu Phe Pro Tyr Met Glu Leu
Pro Glu His Tyr Leu Asp His 500 505 510 Phe Lys Leu Tyr Asp Pro Val
Gly Gly Glu His Ala Asn Ile Phe Gly 515 520 525 Ala Gly Leu Lys Met
Ala Asp Arg Val Val Thr Val Ser Pro Gly Tyr 530 535 540 Leu Trp Glu
Leu Lys Thr Thr Glu Gly Gly Trp Gly Leu His Asp Ile 545 550 555 560
Ile Arg Glu Asn Asp Trp Lys Met Asn Gly Ile Val Asn Gly Ile Asp 565
570 575 Tyr Arg Glu Trp Asn Pro Glu Val Asp Val His Leu Gln Ser Asp
Gly 580 585 590 Tyr Ala Asn Tyr Thr Val Ala Ser Leu Asp Ser Ser Lys
Pro Arg Cys 595 600 605 Lys Ala Ala Leu Gln Arg Glu Leu Gly Leu Glu
Val Arg Asp Asp Val 610 615 620 Pro Leu Ile Gly Phe Ile Gly Arg Leu
Asp Gly Gln Lys Gly Val Asp 625 630 635 640 Ile Ile Gly Asp Ala Met
Pro Trp Ile Ala Gly Gln Asp Val Gln Leu 645 650 655 Val Leu Leu Gly
Ser Gly Arg Arg Asp Leu Glu Val Met Leu Gln Arg 660 665 670 Phe Glu
Ala Gln His Asn Ser Lys Val Arg Gly Trp Val Gly Phe Ser 675 680 685
Val Lys Met Ala His Arg Ile Thr Ala Gly Ala Asp Val Leu Val Met 690
695 700 Pro Ser Arg Phe Glu Pro Cys Gly Leu Asn Gln Leu Tyr Ala Met
Ala 705 710 715 720 Tyr Gly Thr Val Pro Val Val His Ala Val Gly Gly
Leu Arg Asp Thr 725 730 735 Val Ser Ala Phe Asp Pro Phe Glu Asp Thr
Gly Leu Gly Trp Thr Phe 740 745 750 Asp Arg Ala Glu Pro His Lys Leu
Ile Glu Ala Leu Gly His Cys Leu 755 760 765 Glu Thr Tyr Arg Lys Tyr
Lys Glu Ser Trp Arg Gly Leu Gln Val Arg 770 775 780 Gly Met Ser Gln
Asp Leu Ser Trp Asp His Ala Ala Glu Leu Tyr Glu 785 790 795 800 Glu
Val Leu Val Lys Ala Lys Tyr Gln Trp 805 810 <210> SEQ ID NO 7
<211> LENGTH: 1830 <212> TYPE: DNA <213>
ORGANISM: Oryza sativa <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)..(1827) <400> SEQUENCE: 7 atg
tcg gct ctc acc acg tcc cag ctc gcc acc tcg gcc acc ggc ttc 48 Met
Ser Ala Leu Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe 1 5 10
15 ggc atc gcc gac agg tcg gcg ccg tcg tcg ctg ctc cgc cac ggg ttc
96 Gly Ile Ala Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe
20 25 30 cag ggc ctc aag ccc cgc agc ccc gcc ggc ggc gac gcg acg
tcg ctc 144 Gln Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr
Ser Leu 35 40 45 agc gtg acg acc agc gcg cgc gcg acg ccc aag cag
cag cgg tcg gtg 192 Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln
Gln Arg Ser Val 50 55 60 cag cgt ggc agc cgg agg ttc ccc tcc gtc
gtc gtg tac gcc acc ggc 240 Gln Arg Gly Ser Arg Arg Phe Pro Ser Val
Val Val Tyr Ala Thr Gly 65 70 75 80 gcc ggc atg aac gtc gtg ttc gtc
ggc gcc gag atg gcc ccc tgg agc 288 Ala Gly Met Asn Val Val Phe Val
Gly Ala Glu Met Ala Pro Trp Ser 85 90 95 aag acc ggc ggc ctc ggt
gac gtc ctc ggt ggc ctc ccc cct gcc atg 336 Lys Thr Gly Gly Leu Gly
Asp Val Leu Gly Gly Leu Pro Pro Ala Met 100 105 110 gct gcg aat ggc
cac agg gtc atg gtg atc tct cct cgg tac gac cag 384 Ala Ala Asn Gly
His Arg Val Met Val Ile Ser Pro Arg Tyr Asp Gln 115 120 125 tac aag
gac gct tgg gat acc agc gtt gtg gct gag atc aag gtt gca 432 Tyr Lys
Asp Ala Trp Asp Thr Ser Val Val Ala Glu Ile Lys Val Ala 130 135 140
gac agg tac gag agg gtg agg ttt ttc cat tgc tac aag cgt gga gtc 480
Asp Arg Tyr Glu Arg Val Arg Phe Phe His Cys Tyr Lys Arg Gly Val 145
150 155 160 gac cgt gtg ttc atc gac cat ccg tca ttc ctg gag aag gtt
tgg gga 528 Asp Arg Val Phe Ile Asp His Pro Ser Phe Leu Glu Lys Val
Trp Gly 165 170 175 aag acc ggt gag aag atc tac gga cct gac act gga
gtt gat tac aaa 576 Lys Thr Gly Glu Lys Ile Tyr Gly Pro Asp Thr Gly
Val Asp Tyr Lys 180 185 190 gac aac cag atg cgt ttc agc ctt ctt tgc
cag gca gca ctc gag gct 624 Asp Asn Gln Met Arg Phe Ser Leu Leu Cys
Gln Ala Ala Leu Glu Ala 195 200 205 cct agg atc cta aac ctc aac aac
aac cca tac ttc aaa gga act tat 672 Pro Arg Ile Leu Asn Leu Asn Asn
Asn Pro Tyr Phe Lys Gly Thr Tyr 210 215 220 ggt gag gat gtt gtg ttc
gtc tgc aac gac tgg cac act ggc cca ctg 720 Gly Glu Asp Val Val Phe
Val Cys Asn Asp Trp His Thr Gly Pro Leu 225 230 235 240 gcg agc tac
ctg aag aac aac tac cag ccc aat ggc atc tac agg aat 768 Ala Ser Tyr
Leu Lys Asn Asn Tyr Gln Pro Asn Gly Ile Tyr Arg Asn 245 250 255 gca
aag gtt gct ttc tgc atc cac aac atc tcc tac cag ggc cgt ttc 816 Ala
Lys Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe 260 265
270 gct ttc gag gat tac cct gag ctg aac ctc tcc gag agg ttc agg tca
864 Ala Phe Glu Asp Tyr Pro Glu Leu Asn Leu Ser Glu Arg Phe Arg Ser
275 280 285 tcc ttc gat ttc atc gac ggg tat gac acg ccg gtg gag ggc
agg aag 912 Ser Phe Asp Phe Ile Asp Gly Tyr Asp Thr Pro Val Glu Gly
Arg Lys 290 295 300 atc aac tgg atg aag gcc gga atc ctg gaa gcc gac
agg gtg ctc acc 960 Ile Asn Trp Met Lys Ala Gly Ile Leu Glu Ala Asp
Arg Val Leu Thr 305 310 315 320 gtg agc ccg tac tac gcc gag gag ctc
atc tcc ggc atc gcc agg gga 1008 Val Ser Pro Tyr Tyr Ala Glu Glu
Leu Ile Ser Gly Ile Ala Arg Gly 325 330 335 tgc gag ctc gac aac atc
atg cgg ctc acc ggc atc acc ggc atc gtc 1056 Cys Glu Leu Asp Asn
Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val 340 345 350 aac ggc atg
gac gtc agc gag tgg gat ccc agc aag gac aag tac atc 1104 Asn Gly
Met Asp Val Ser Glu Trp Asp Pro Ser Lys Asp Lys Tyr Ile 355 360 365
acc gcc aag tac gac gca acc acg gca atc gag gcg aag gcg ctg aac
1152 Thr Ala Lys Tyr Asp Ala Thr Thr Ala Ile Glu Ala Lys Ala Leu
Asn 370 375 380 aag gag gcg ttg cag gcg gag gcg ggt ctt ccg gtc gac
agg aaa atc 1200 Lys Glu Ala Leu Gln Ala Glu Ala Gly Leu Pro Val
Asp Arg Lys Ile 385 390 395 400 cca ctg atc gcg ttc atc ggc agg ctg
gag gaa cag aag ggc tct gac 1248 Pro Leu Ile Ala Phe Ile Gly Arg
Leu Glu Glu Gln Lys Gly Ser Asp 405 410 415 gtc atg gcc gcc gcc atc
ccg gag ctc atg cag gag gac gtc cag atc 1296 Val Met Ala Ala Ala
Ile Pro Glu Leu Met Gln Glu Asp Val Gln Ile 420 425 430 gtt ctt ctg
ggt act gga aag aag aag ttc gag aag ctg ctc aag agc 1344 Val Leu
Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Leu Leu Lys Ser 435 440 445
atg gag gag aag tat ccg ggc aag gtg agg gcc gtg gtg aag ttc aac
1392 Met Glu Glu Lys Tyr Pro Gly Lys Val Arg Ala Val Val Lys Phe
Asn 450 455 460 gcg ccg ctt gct cat ctc atc atg gcc gga gcc gac gtg
ctc gcc gtc 1440 Ala Pro Leu Ala His Leu Ile Met Ala Gly Ala Asp
Val Leu Ala Val 465 470 475 480 ccc agc cgc ttc gag ccc tgt gga ctc
atc cag ctg cag ggg atg aga 1488 Pro Ser Arg Phe Glu Pro Cys Gly
Leu Ile Gln Leu Gln Gly Met Arg 485 490 495 tac gga acg ccc tgt gct
tgc gcg tcc acc ggt ggg ctc gtg gac acg 1536 Tyr Gly Thr Pro Cys
Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr 500 505 510 gtc atc gaa
ggc aag act ggt ttc cac atg ggc cgt ctc agc gtc gac 1584 Val Ile
Glu Gly Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp 515 520 525
tgc aag gtg gtg gag cca agc gac gtg aag aag gtg gcg gcc acc ctg
1632 Cys Lys Val Val Glu Pro Ser Asp Val Lys Lys Val Ala Ala Thr
Leu 530 535 540 aag cgc gcc atc aag gtc gtc ggc acg ccg gcg tac gag
gag atg gtc 1680 Lys Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr
Glu Glu Met Val 545 550 555 560 agg aac tgc atg aac cag gac ctc tcc
tgg aag ggg cct gcg aag aac 1728 Arg Asn Cys Met Asn Gln Asp Leu
Ser Trp Lys Gly Pro Ala Lys Asn 565 570 575 tgg gag aat gtg ctc ctg
ggc ctg ggc gtc gcc ggc agc gcg ccg ggg 1776 Trp Glu Asn Val Leu
Leu Gly Leu Gly Val Ala Gly Ser Ala Pro Gly 580 585 590 atc gaa ggc
gac gag atc gcg ccg ctc gcc aag gag aac gtg gct gct 1824 Ile Glu
Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala 595 600 605
cct tga 1830 Pro <210> SEQ ID NO 8 <211> LENGTH: 609
<212> TYPE: PRT <213> ORGANISM: Oryza sativa
<400> SEQUENCE: 8 Met Ser Ala Leu Thr Thr Ser Gln Leu Ala Thr
Ser Ala Thr Gly Phe 1 5 10 15 Gly Ile Ala Asp Arg Ser Ala Pro Ser
Ser Leu Leu Arg His Gly Phe 20 25 30 Gln Gly Leu Lys Pro Arg Ser
Pro Ala Gly Gly Asp Ala Thr Ser Leu 35 40 45 Ser Val Thr Thr Ser
Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val 50 55 60 Gln Arg Gly
Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala Thr Gly 65 70 75 80 Ala
Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser 85 90
95 Lys Thr Gly Gly Leu Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met
100 105 110 Ala Ala Asn Gly His Arg Val Met Val Ile Ser Pro Arg Tyr
Asp Gln 115 120 125 Tyr Lys Asp Ala Trp Asp Thr Ser Val Val Ala Glu
Ile Lys Val Ala 130 135 140 Asp Arg Tyr Glu Arg Val Arg Phe Phe His
Cys Tyr Lys Arg Gly Val 145 150 155 160 Asp Arg Val Phe Ile Asp His
Pro Ser Phe Leu Glu Lys Val Trp Gly 165 170 175 Lys Thr Gly Glu Lys
Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys 180 185 190 Asp Asn Gln
Met Arg Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala 195 200 205 Pro
Arg Ile Leu Asn Leu Asn Asn Asn Pro Tyr Phe Lys Gly Thr Tyr 210 215
220 Gly Glu Asp Val Val Phe Val Cys Asn Asp Trp His Thr Gly Pro Leu
225 230 235 240 Ala Ser Tyr Leu Lys Asn Asn Tyr Gln Pro Asn Gly Ile
Tyr Arg Asn 245 250 255 Ala Lys Val Ala Phe Cys Ile His Asn Ile Ser
Tyr Gln Gly Arg Phe 260 265 270 Ala Phe Glu Asp Tyr Pro Glu Leu Asn
Leu Ser Glu Arg Phe Arg Ser 275 280 285 Ser Phe Asp Phe Ile Asp Gly
Tyr Asp Thr Pro Val Glu Gly Arg Lys 290 295 300 Ile Asn Trp Met Lys
Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr 305 310 315 320 Val Ser
Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly 325 330 335
Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val 340
345 350 Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser Lys Asp Lys Tyr
Ile 355 360 365 Thr Ala Lys Tyr Asp Ala Thr Thr Ala Ile Glu Ala Lys
Ala Leu Asn 370 375 380 Lys Glu Ala Leu Gln Ala Glu Ala Gly Leu Pro
Val Asp Arg Lys Ile 385 390 395 400 Pro Leu Ile Ala Phe Ile Gly Arg
Leu Glu Glu Gln Lys Gly Ser Asp 405 410 415 Val Met Ala Ala Ala Ile
Pro Glu Leu Met Gln Glu Asp Val Gln Ile 420 425 430 Val Leu Leu Gly
Thr Gly Lys Lys Lys Phe Glu Lys Leu Leu Lys Ser 435 440 445 Met Glu
Glu Lys Tyr Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn 450 455 460
Ala Pro Leu Ala His Leu Ile Met Ala Gly Ala Asp Val Leu Ala Val 465
470 475 480 Pro Ser Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly
Met Arg 485 490 495 Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly
Leu Val Asp Thr 500 505 510 Val Ile Glu Gly Lys Thr Gly Phe His Met
Gly Arg Leu Ser Val Asp 515 520 525 Cys Lys Val Val Glu Pro Ser Asp
Val Lys Lys Val Ala Ala Thr Leu 530 535 540 Lys Arg Ala Ile Lys Val
Val Gly Thr Pro Ala Tyr Glu Glu Met Val 545 550 555 560 Arg Asn Cys
Met Asn Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn 565 570 575 Trp
Glu Asn Val Leu Leu Gly Leu Gly Val Ala Gly Ser Ala Pro Gly 580 585
590 Ile Glu Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala
595 600 605 Pro <210> SEQ ID NO 9 <211> LENGTH: 1818
<212> TYPE: DNA <213> ORGANISM: Triticum aestivum
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(1815) <400> SEQUENCE: 9 atg gcg gct ctg gtc
acg tcg cag ctc gcc acc tcc ggc acc gtc ctc 48 Met Ala Ala Leu Val
Thr Ser Gln Leu Ala Thr Ser Gly Thr Val Leu 1 5 10 15 ggc atc acc
gac agg ttc cgg cgt gca ggt ttt cag ggt gtg agg ccc 96 Gly Ile Thr
Asp Arg Phe Arg Arg Ala Gly Phe Gln Gly Val Arg Pro 20 25 30 cgg
agc ccg gca gat gcg ccg ctc ggc atg agg act acc gga gcg agc 144 Arg
Ser Pro Ala Asp Ala Pro Leu Gly Met Arg Thr Thr Gly Ala Ser 35 40
45 gcc gcc ccg aag caa caa agc cgg aaa gcg cac cgc ggg acc cgg cgg
192 Ala Ala Pro Lys Gln Gln Ser Arg Lys Ala His Arg Gly Thr Arg Arg
50 55 60 tgc ctc tcc atg gtg gtg cgc gcc acg ggc agc gcc ggc atg
aac ctc 240 Cys Leu Ser Met Val Val Arg Ala Thr Gly Ser Ala Gly Met
Asn Leu 65 70 75 80 gtg ttc gtc ggc gcc gag atg gcg ccc tgg agc aag
acc ggc ggc ctc 288 Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser Lys
Thr Gly Gly Leu 85 90 95 ggc gac gtc ctc ggg ggc ctc ccc cca gcc
atg gcc gcc aac ggt cac 336 Gly Asp Val Leu Gly Gly Leu Pro Pro Ala
Met Ala Ala Asn Gly His 100 105 110 cgg gtc atg gtc atc tcc ccg cgc
tac gac cag tac aag gac gcc tgg 384 Arg Val Met Val Ile Ser Pro Arg
Tyr Asp Gln Tyr Lys Asp Ala Trp 115 120 125 gac acc agc gtc gtc tcc
gag atc aag gtc gcg gac gag tac gag agg 432 Asp Thr Ser Val Val Ser
Glu Ile Lys Val Ala Asp Glu Tyr Glu Arg 130 135 140 gtg agg tac ttc
cac tgc tac aag cgc ggg gtg gac cgc gtg ttc gtc 480 Val Arg Tyr Phe
His Cys Tyr Lys Arg Gly Val Asp Arg Val Phe Val 145 150 155 160 gac
cac ccg tgc ttc ctg gag aag gtc cgg ggc aag acc aag gag aag 528 Asp
His Pro Cys Phe Leu Glu Lys Val Arg Gly Lys Thr Lys Glu Lys 165 170
175 atc tac ggg ccc gat gcc ggc acg gac tac gag gac aac cag cta cgc
576 Ile Tyr Gly Pro Asp Ala Gly Thr Asp Tyr Glu Asp Asn Gln Leu Arg
180 185 190 ttc agc ctg ctc tgc cag gca gcg ctt gag gca ccc agg atc
ctc gac 624 Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala Pro Arg Ile
Leu Asp 195 200 205 ctc aac aac aac cca tac ttc tcc gga ccc tac ggg
gaa gac gtg gtg 672 Leu Asn Asn Asn Pro Tyr Phe Ser Gly Pro Tyr Gly
Glu Asp Val Val 210 215 220 ttc gtg tgc aac gac tgg cac acg ggc ctt
ctg gcc tgc tac ctc aag 720 Phe Val Cys Asn Asp Trp His Thr Gly Leu
Leu Ala Cys Tyr Leu Lys 225 230 235 240 agc aac tac cag tcc agt ggc
atc tat agg acg gcc aag gta gcg ttc 768 Ser Asn Tyr Gln Ser Ser Gly
Ile Tyr Arg Thr Ala Lys Val Ala Phe 245 250 255 tgc atc cac aac atc
tcg tat cag ggc cgc ttc tcc ttc gac gac ttc 816 Cys Ile His Asn Ile
Ser Tyr Gln Gly Arg Phe Ser Phe Asp Asp Phe 260 265 270 gcg cag ctc
aac ctg ccc gac agg ttc aag tcg tcc ttc gac ttc atc 864 Ala Gln Leu
Asn Leu Pro Asp Arg Phe Lys Ser Ser Phe Asp Phe Ile 275 280 285 gac
ggc tac gac aag ccg gtg gag ggg cgc aag atc aac tgg atg aag 912 Asp
Gly Tyr Asp Lys Pro Val Glu Gly Arg Lys Ile Asn Trp Met Lys 290 295
300 gcc ggg atc ctg cag gcc gac aag gtg ctc acg gtg agc ccc tac tac
960 Ala Gly Ile Leu Gln Ala Asp Lys Val Leu Thr Val Ser Pro Tyr Tyr
305 310 315 320 gcg gag gag ctc atc tcc ggc gaa gcc agg ggc tgc gag
ctc gac aac 1008 Ala Glu Glu Leu Ile Ser Gly Glu Ala Arg Gly Cys
Glu Leu Asp Asn 325 330 335 atc atg cgc ctc acg ggc atc acc ggc atc
gtc aac ggc atg gac gtc 1056 Ile Met Arg Leu Thr Gly Ile Thr Gly
Ile Val Asn Gly Met Asp Val 340 345 350 agc gag tgg gac ccc gcc aag
gac aag ttc ctc gcc gcc aac tac gac 1104 Ser Glu Trp Asp Pro Ala
Lys Asp Lys Phe Leu Ala Ala Asn Tyr Asp 355 360 365 gtc acc acc gcg
ttg gag ggg aag gcg ctg aac aag gag gcg ctg cag 1152 Val Thr Thr
Ala Leu Glu Gly Lys Ala Leu Asn Lys Glu Ala Leu Gln 370 375 380 gcc
gag gtg ggg ctg ccg gtg gac cgg aag gtg ccc ctg gtg gcc ttc 1200
Ala Glu Val Gly Leu Pro Val Asp Arg Lys Val Pro Leu Val Ala Phe 385
390 395 400 atc ggc agg ctg gag gag cag aag ggc ccc gac gtg atg atc
gcc gcc 1248 Ile Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp Val Met
Ile Ala Ala 405 410 415 atc ccg gag atc ttg aag gag gag gac gtc cag
atc gtt ctc ctg ggc 1296 Ile Pro Glu Ile Leu Lys Glu Glu Asp Val
Gln Ile Val Leu Leu Gly 420 425 430 acc ggg aag aag aag ttt gag cgg
ctg ctc aag agc gtg gag gag aag 1344 Thr Gly Lys Lys Lys Phe Glu
Arg Leu Leu Lys Ser Val Glu Glu Lys 435 440 445 ttc ccg agc aag gtg
agg gcc gtg gtc agg ttc aac gcg ccg ctg gct 1392 Phe Pro Ser Lys
Val Arg Ala Val Val Arg Phe Asn Ala Pro Leu Ala 450 455 460 cac cag
atg atg gcc ggc gcc gac gtg ctc gcc gtc acc agc cgc ttc 1440 His
Gln Met Met Ala Gly Ala Asp Val Leu Ala Val Thr Ser Arg Phe 465 470
475 480 gag ccc tgc ggc ctc atc cag ctc cag ggg atg cgc tac gga acg
ccg 1488 Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg Tyr Gly
Thr Pro 485 490 495 tgc gcg tgc gcg tcc acc ggc ggg ctc gtc gac acg
atc atg gag ggc 1536 Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp
Thr Ile Met Glu Gly 500 505 510 aag acc ggg ttc cac atg ggc cgc ctc
agc gtc gac tgc aac gtg gtg 1584 Lys Thr Gly Phe His Met Gly Arg
Leu Ser Val Asp Cys Asn Val Val 515 520 525 gag ccg gcc gac gtg aag
aag gtg gtg acc acc ctg aag cgc gcc gtc 1632 Glu Pro Ala Asp Val
Lys Lys Val Val Thr Thr Leu Lys Arg Ala Val 530 535 540 aag gtc gtc
ggc acg cca gcc tac cat gag atg gtc aag aac tgc atg 1680 Lys Val
Val Gly Thr Pro Ala Tyr His Glu Met Val Lys Asn Cys Met 545 550 555
560 atc cag gat ctc tcc tgg aag ggg cca gcc aag aac tgg gag gac gtg
1728 Ile Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn Trp Glu Asp
Val 565 570 575 ctt ctg gaa ctg ggg gtc gag ggg agc gag cca ggg gtc
atc ggc gag 1776 Leu Leu Glu Leu Gly Val Glu Gly Ser Glu Pro Gly
Val Ile Gly Glu 580 585 590 gag att gcg ccg ctc gcc atg gag aac gtc
gcc gct ccc tga 1818 Glu Ile Ala Pro Leu Ala Met Glu Asn Val Ala
Ala Pro 595 600 605 <210> SEQ ID NO 10 <211> LENGTH:
605 <212> TYPE: PRT <213> ORGANISM: Triticum aestivum
<400> SEQUENCE: 10 Met Ala Ala Leu Val Thr Ser Gln Leu Ala
Thr Ser Gly Thr Val Leu 1 5 10 15 Gly Ile Thr Asp Arg Phe Arg Arg
Ala Gly Phe Gln Gly Val Arg Pro 20 25 30 Arg Ser Pro Ala Asp Ala
Pro Leu Gly Met Arg Thr Thr Gly Ala Ser 35 40 45 Ala Ala Pro Lys
Gln Gln Ser Arg Lys Ala His Arg Gly Thr Arg Arg 50 55 60 Cys Leu
Ser Met Val Val Arg Ala Thr Gly Ser Ala Gly Met Asn Leu 65 70 75 80
Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser Lys Thr Gly Gly Leu 85
90 95 Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met Ala Ala Asn Gly
His 100 105 110 Arg Val Met Val Ile Ser Pro Arg Tyr Asp Gln Tyr Lys
Asp Ala Trp 115 120 125 Asp Thr Ser Val Val Ser Glu Ile Lys Val Ala
Asp Glu Tyr Glu Arg 130 135 140 Val Arg Tyr Phe His Cys Tyr Lys Arg
Gly Val Asp Arg Val Phe Val 145 150 155 160 Asp His Pro Cys Phe Leu
Glu Lys Val Arg Gly Lys Thr Lys Glu Lys 165 170 175 Ile Tyr Gly Pro
Asp Ala Gly Thr Asp Tyr Glu Asp Asn Gln Leu Arg 180 185 190 Phe Ser
Leu Leu Cys Gln Ala Ala Leu Glu Ala Pro Arg Ile Leu Asp 195 200 205
Leu Asn Asn Asn Pro Tyr Phe Ser Gly Pro Tyr Gly Glu Asp Val Val 210
215 220 Phe Val Cys Asn Asp Trp His Thr Gly Leu Leu Ala Cys Tyr Leu
Lys 225 230 235 240 Ser Asn Tyr Gln Ser Ser Gly Ile Tyr Arg Thr Ala
Lys Val Ala Phe 245 250 255 Cys Ile His Asn Ile Ser Tyr Gln Gly Arg
Phe Ser Phe Asp Asp Phe 260 265 270 Ala Gln Leu Asn Leu Pro Asp Arg
Phe Lys Ser Ser Phe Asp Phe Ile 275 280 285 Asp Gly Tyr Asp Lys Pro
Val Glu Gly Arg Lys Ile Asn Trp Met Lys 290 295 300 Ala Gly Ile Leu
Gln Ala Asp Lys Val Leu Thr Val Ser Pro Tyr Tyr 305 310 315 320 Ala
Glu Glu Leu Ile Ser Gly Glu Ala Arg Gly Cys Glu Leu Asp Asn 325 330
335 Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val Asn Gly Met Asp Val
340 345 350 Ser Glu Trp Asp Pro Ala Lys Asp Lys Phe Leu Ala Ala Asn
Tyr Asp 355 360 365 Val Thr Thr Ala Leu Glu Gly Lys Ala Leu Asn Lys
Glu Ala Leu Gln 370 375 380 Ala Glu Val Gly Leu Pro Val Asp Arg Lys
Val Pro Leu Val Ala Phe 385 390 395 400 Ile Gly Arg Leu Glu Glu Gln
Lys Gly Pro Asp Val Met Ile Ala Ala 405 410 415 Ile Pro Glu Ile Leu
Lys Glu Glu Asp Val Gln Ile Val Leu Leu Gly 420 425 430 Thr Gly Lys
Lys Lys Phe Glu Arg Leu Leu Lys Ser Val Glu Glu Lys 435 440 445 Phe
Pro Ser Lys Val Arg Ala Val Val Arg Phe Asn Ala Pro Leu Ala 450 455
460 His Gln Met Met Ala Gly Ala Asp Val Leu Ala Val Thr Ser Arg Phe
465 470 475 480 Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg Tyr
Gly Thr Pro 485 490 495 Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp
Thr Ile Met Glu Gly 500 505 510 Lys Thr Gly Phe His Met Gly Arg Leu
Ser Val Asp Cys Asn Val Val 515 520 525 Glu Pro Ala Asp Val Lys Lys
Val Val Thr Thr Leu Lys Arg Ala Val 530 535 540 Lys Val Val Gly Thr
Pro Ala Tyr His Glu Met Val Lys Asn Cys Met 545 550 555 560 Ile Gln
Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn Trp Glu Asp Val 565 570 575
Leu Leu Glu Leu Gly Val Glu Gly Ser Glu Pro Gly Val Ile Gly Glu 580
585 590 Glu Ile Ala Pro Leu Ala Met Glu Asn Val Ala Ala Pro 595 600
605 <210> SEQ ID NO 11 <211> LENGTH: 1818 <212>
TYPE: DNA <213> ORGANISM: Zea mays <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (1)..(1815)
<400> SEQUENCE: 11 atg gcg gct ctg gcc acg tcg cag ctc gtc
gca acg cgc gcc ggc ctg 48 Met Ala Ala Leu Ala Thr Ser Gln Leu Val
Ala Thr Arg Ala Gly Leu 1 5 10 15 ggc gtc ccg gac gcg tcc acg ttc
cgc cgc ggc gcc gcg cag ggc ctg 96 Gly Val Pro Asp Ala Ser Thr Phe
Arg Arg Gly Ala Ala Gln Gly Leu 20 25 30 agg ggg gcc cgg gcg tcg
gcg gcg gcg gac acg ctc agc atg cgg acc 144 Arg Gly Ala Arg Ala Ser
Ala Ala Ala Asp Thr Leu Ser Met Arg Thr 35 40 45 agc gcg cgc gcg
gcg ccc agg cac cag cag cag gcg cgc cgc ggg ggc 192 Ser Ala Arg Ala
Ala Pro Arg His Gln Gln Gln Ala Arg Arg Gly Gly 50 55 60 agg ttc
ccg tcg ctc gtc gtg tgc gcc agc gcc ggc atg aac gtc gtc 240 Arg Phe
Pro Ser Leu Val Val Cys Ala Ser Ala Gly Met Asn Val Val 65 70 75 80
ttc gtc ggc gcc gag atg gcg ccg tgg agc aag acc ggc ggc ctc ggc 288
Phe Val Gly Ala Glu Met Ala Pro Trp Ser Lys Thr Gly Gly Leu Gly 85
90 95 gac gtc ctc ggc ggc ctg ccg ccg gcc atg gcc gcg aac ggg cac
cgt 336 Asp Val Leu Gly Gly Leu Pro Pro Ala Met Ala Ala Asn Gly His
Arg 100 105 110 gtc atg gtc gtc tct ccc cgc tac gac cag tac aag gac
gcc tgg gac 384 Val Met Val Val Ser Pro Arg Tyr Asp Gln Tyr Lys Asp
Ala Trp Asp 115 120 125 acc agc gtc gtg tcc gag atc aag atg gga gac
ggg tac gag acg gtc 432 Thr Ser Val Val Ser Glu Ile Lys Met Gly Asp
Gly Tyr Glu Thr Val 130 135 140 agg ttc ttc cac tgc tac aag cgc gga
gtg gac cgc gtg ttc gtt gac 480 Arg Phe Phe His Cys Tyr Lys Arg Gly
Val Asp Arg Val Phe Val Asp 145 150 155 160 cac cca ctg ttc ctg gag
agg gtt tgg gga aag acc gag gag aag atc 528 His Pro Leu Phe Leu Glu
Arg Val Trp Gly Lys Thr Glu Glu Lys Ile 165 170 175 tac ggg cct gtc
gct gga acg gac tac agg gac aac cag ctg cgg ttc 576 Tyr Gly Pro Val
Ala Gly Thr Asp Tyr Arg Asp Asn Gln Leu Arg Phe 180 185 190 agc ctg
cta tgc cag gca gca ctt gaa gct cca agg atc ctg agc ctc 624 Ser Leu
Leu Cys Gln Ala Ala Leu Glu Ala Pro Arg Ile Leu Ser Leu 195 200 205
aac aac aac cca tac ttc tcc gga cca tac ggg gag gac gtc gtg ttc 672
Asn Asn Asn Pro Tyr Phe Ser Gly Pro Tyr Gly Glu Asp Val Val Phe 210
215 220 gtc tgc aac gac tgg cac acc ggc cct ctc tcg tgc tac ctc aag
agc 720 Val Cys Asn Asp Trp His Thr Gly Pro Leu Ser Cys Tyr Leu Lys
Ser 225 230 235 240 aac tac cag tcc cac ggc atc tac agg gac gca aag
acc gct ttc tgc 768 Asn Tyr Gln Ser His Gly Ile Tyr Arg Asp Ala Lys
Thr Ala Phe Cys 245 250 255 atc cac aac atc tcc tac cag ggc cgg ttc
gcc ttc tcc gac tac ccg 816 Ile His Asn Ile Ser Tyr Gln Gly Arg Phe
Ala Phe Ser Asp Tyr Pro 260 265 270 gag ctg aac ctc ccg gag aga ttc
aag tcg tcc ttc gat ttc atc gac 864 Glu Leu Asn Leu Pro Glu Arg Phe
Lys Ser Ser Phe Asp Phe Ile Asp 275 280 285 ggc tac gag aag ccc gtg
gaa ggc cgg aag atc aac tgg atg aag gcc 912 Gly Tyr Glu Lys Pro Val
Glu Gly Arg Lys Ile Asn Trp Met Lys Ala 290 295 300 ggg atc ctc gag
gcc gac agg gtc ctc acc gtc agc ccc tac tac gcc 960 Gly Ile Leu Glu
Ala Asp Arg Val Leu Thr Val Ser Pro Tyr Tyr Ala 305 310 315 320 gag
gag ctc atc tcc ggc atc gcc agg ggc tgc gag ctc gac aac atc 1008
Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly Cys Glu Leu Asp Asn Ile 325
330 335 atg cgc ctc acc ggc atc acc ggc atc gtc aac ggc atg gac gtc
agc 1056 Met Arg Leu Thr Gly Ile Thr Gly Ile Val Asn Gly Met Asp
Val Ser 340 345 350 gag tgg gac ccc agc agg gac aag tac atc gcc gtg
aag tac gac gtg 1104 Glu Trp Asp Pro Ser Arg Asp Lys Tyr Ile Ala
Val Lys Tyr Asp Val 355 360 365 tcg acg gcc gtg gag gcc aag gcg ctg
aac aag gag gcg ctg cag gcg 1152 Ser Thr Ala Val Glu Ala Lys Ala
Leu Asn Lys Glu Ala Leu Gln Ala 370 375 380 gag gtc ggg ctc ccg gtg
gac cgg aac atc ccg ctg gtg gcg ttc atc 1200 Glu Val Gly Leu Pro
Val Asp Arg Asn Ile Pro Leu Val Ala Phe Ile 385 390 395 400 ggc agg
ctg gaa gag cag aag ggc ccc gac gtc atg gcg gcc gcc atc 1248 Gly
Arg Leu Glu Glu Gln Lys Gly Pro Asp Val Met Ala Ala Ala Ile 405 410
415 ccg cag ctc atg gag atg gtg gag gac gtg cag atc gtt ctg ctg ggc
1296 Pro Gln Leu Met Glu Met Val Glu Asp Val Gln Ile Val Leu Leu
Gly 420 425 430 acg ggc aag aag aag ttc gag cgc atg ctc atg agc gcc
gag gag aag 1344 Thr Gly Lys Lys Lys Phe Glu Arg Met Leu Met Ser
Ala Glu Glu Lys 435 440 445 ttc cca ggc aag gtg cgc gcc gtg gtc aag
ttc aac gcg gcg ctg gcg 1392 Phe Pro Gly Lys Val Arg Ala Val Val
Lys Phe Asn Ala Ala Leu Ala 450 455 460 cac cac atc atg gcc ggc gcc
gac gtg ctc gcc gtc acc agc cgc ttc 1440 His His Ile Met Ala Gly
Ala Asp Val Leu Ala Val Thr Ser Arg Phe 465 470 475 480 gag ccc tgc
ggc ctc atc cag ctg cag ggg atg cga tac gga acg ccc 1488 Glu Pro
Cys Gly Leu Ile Gln Leu Gln Gly Met Arg Tyr Gly Thr Pro 485 490 495
tgc gcc tgc gcg tcc acc ggt gga ctc gtc gac acc atc atc gaa ggc
1536 Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr Ile Ile Glu
Gly 500 505 510 aag acc ggg ttc cac atg ggc cgc ctc agc gtc gac tgt
aac gtc gtg 1584 Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp
Cys Asn Val Val 515 520 525 gag ccg gcg gac gtc aag aag gtg gcc acc
aca ttg cag cgc gcc atc 1632 Glu Pro Ala Asp Val Lys Lys Val Ala
Thr Thr Leu Gln Arg Ala Ile 530 535 540 aag gtg gtc ggc acg ccg gcg
tac gag gag atg gtg agg aac tgc atg 1680 Lys Val Val Gly Thr Pro
Ala Tyr Glu Glu Met Val Arg Asn Cys Met 545 550 555 560 atc cag gat
ctc tcc tgg aag ggc cct gcc aag aac tgg gag aac gtg 1728 Ile Gln
Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn Trp Glu Asn Val 565 570 575
ctg ctc agc ctc ggg gtc gcc ggc ggc gag cca ggg gtc gaa ggc gag
1776 Leu Leu Ser Leu Gly Val Ala Gly Gly Glu Pro Gly Val Glu Gly
Glu 580 585 590 gag atc gcg ccg ctc gcc aag gag aac gtg gcc gcg ccc
tga 1818 Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala Pro 595
600 605 <210> SEQ ID NO 12 <211> LENGTH: 605
<212> TYPE: PRT <213> ORGANISM: Zea mays <400>
SEQUENCE: 12 Met Ala Ala Leu Ala Thr Ser Gln Leu Val Ala Thr Arg
Ala Gly Leu 1 5 10 15 Gly Val Pro Asp Ala Ser Thr Phe Arg Arg Gly
Ala Ala Gln Gly Leu 20 25 30 Arg Gly Ala Arg Ala Ser Ala Ala Ala
Asp Thr Leu Ser Met Arg Thr 35 40 45 Ser Ala Arg Ala Ala Pro Arg
His Gln Gln Gln Ala Arg Arg Gly Gly 50 55 60 Arg Phe Pro Ser Leu
Val Val Cys Ala Ser Ala Gly Met Asn Val Val 65 70 75 80 Phe Val Gly
Ala Glu Met Ala Pro Trp Ser Lys Thr Gly Gly Leu Gly 85 90 95 Asp
Val Leu Gly Gly Leu Pro Pro Ala Met Ala Ala Asn Gly His Arg 100 105
110 Val Met Val Val Ser Pro Arg Tyr Asp Gln Tyr Lys Asp Ala Trp Asp
115 120 125 Thr Ser Val Val Ser Glu Ile Lys Met Gly Asp Gly Tyr Glu
Thr Val 130 135 140 Arg Phe Phe His Cys Tyr Lys Arg Gly Val Asp Arg
Val Phe Val Asp 145 150 155 160 His Pro Leu Phe Leu Glu Arg Val Trp
Gly Lys Thr Glu Glu Lys Ile 165 170 175 Tyr Gly Pro Val Ala Gly Thr
Asp Tyr Arg Asp Asn Gln Leu Arg Phe 180 185 190 Ser Leu Leu Cys Gln
Ala Ala Leu Glu Ala Pro Arg Ile Leu Ser Leu 195 200 205 Asn Asn Asn
Pro Tyr Phe Ser Gly Pro Tyr Gly Glu Asp Val Val Phe 210 215 220 Val
Cys Asn Asp Trp His Thr Gly Pro Leu Ser Cys Tyr Leu Lys Ser 225 230
235 240 Asn Tyr Gln Ser His Gly Ile Tyr Arg Asp Ala Lys Thr Ala Phe
Cys 245 250 255 Ile His Asn Ile Ser Tyr Gln Gly Arg Phe Ala Phe Ser
Asp Tyr Pro 260 265 270 Glu Leu Asn Leu Pro Glu Arg Phe Lys Ser Ser
Phe Asp Phe Ile Asp 275 280 285 Gly Tyr Glu Lys Pro Val Glu Gly Arg
Lys Ile Asn Trp Met Lys Ala 290 295 300 Gly Ile Leu Glu Ala Asp Arg
Val Leu Thr Val Ser Pro Tyr Tyr Ala 305 310 315 320 Glu Glu Leu Ile
Ser Gly Ile Ala Arg Gly Cys Glu Leu Asp Asn Ile 325 330 335 Met Arg
Leu Thr Gly Ile Thr Gly Ile Val Asn Gly Met Asp Val Ser 340 345 350
Glu Trp Asp Pro Ser Arg Asp Lys Tyr Ile Ala Val Lys Tyr Asp Val 355
360 365 Ser Thr Ala Val Glu Ala Lys Ala Leu Asn Lys Glu Ala Leu Gln
Ala 370 375 380 Glu Val Gly Leu Pro Val Asp Arg Asn Ile Pro Leu Val
Ala Phe Ile 385 390 395 400 Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp
Val Met Ala Ala Ala Ile 405 410 415 Pro Gln Leu Met Glu Met Val Glu
Asp Val Gln Ile Val Leu Leu Gly 420 425 430 Thr Gly Lys Lys Lys Phe
Glu Arg Met Leu Met Ser Ala Glu Glu Lys 435 440 445 Phe Pro Gly Lys
Val Arg Ala Val Val Lys Phe Asn Ala Ala Leu Ala 450 455 460 His His
Ile Met Ala Gly Ala Asp Val Leu Ala Val Thr Ser Arg Phe 465 470 475
480 Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg Tyr Gly Thr Pro
485 490 495 Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr Ile Ile
Glu Gly 500 505 510 Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp
Cys Asn Val Val 515 520 525 Glu Pro Ala Asp Val Lys Lys Val Ala Thr
Thr Leu Gln Arg Ala Ile 530 535 540 Lys Val Val Gly Thr Pro Ala Tyr
Glu Glu Met Val Arg Asn Cys Met 545 550 555 560 Ile Gln Asp Leu Ser
Trp Lys Gly Pro Ala Lys Asn Trp Glu Asn Val 565 570 575 Leu Leu Ser
Leu Gly Val Ala Gly Gly Glu Pro Gly Val Glu Gly Glu 580 585 590 Glu
Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala Pro 595 600 605
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 12 <210>
SEQ ID NO 1 <211> LENGTH: 4851 <212> TYPE: DNA
<213> ORGANISM: Solanum tuberosum <220> FEATURE:
<221> NAME/KEY: transit_peptide <222> LOCATION:
(1)..(77) <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (105)..(4499) <300> PUBLICATION
INFORMATION: <308> DATABASE ACCESSION NUMBER: EMBL / Y09533
<309> DATABASE ENTRY DATE: 1998-07-30 <313> RELEVANT
RESIDUES IN SEQ ID NO: (1)..(4499) <400> SEQUENCE: 1
catcttcatc gaatttctcg aagcttcttc gctaatttcc tggtttcttc actcaaaatc
60 gacgtttcta gctgaacttg agtgaattaa gccagtggga ggat atg agt aat tcc
116 Met Ser Asn Ser 1 tta ggg aat aac ttg ctg tac cag gga ttc cta
acc tca aca gtg ttg 164 Leu Gly Asn Asn Leu Leu Tyr Gln Gly Phe Leu
Thr Ser Thr Val Leu 5 10 15 20 gaa cat aaa agt aga atc agt cct cct
tgt gtt gga ggc aat tct ttg 212 Glu His Lys Ser Arg Ile Ser Pro Pro
Cys Val Gly Gly Asn Ser Leu 25 30 35 ttt caa caa caa gtg atc tcg
aaa tca cct tta tca act gag ttt cga 260 Phe Gln Gln Gln Val Ile Ser
Lys Ser Pro Leu Ser Thr Glu Phe Arg 40 45 50 ggt aac agg tta aag
gtg cag aaa aag aaa ata cct atg gaa aag aag 308 Gly Asn Arg Leu Lys
Val Gln Lys Lys Lys Ile Pro Met Glu Lys Lys 55 60 65 cgt gct ttt
tct agt tct cct cat gct gta ctt acc act gat acc tct 356 Arg Ala Phe
Ser Ser Ser Pro His Ala Val Leu Thr Thr Asp Thr Ser 70 75 80 tct
gag cta gca gaa aag ttc agt cta ggg ggg aat att gag cta cag 404 Ser
Glu Leu Ala Glu Lys Phe Ser Leu Gly Gly Asn Ile Glu Leu Gln 85 90
95 100 gtt gat gtt agg cct ccc act tca ggt gat gtg tcc ttt gtg gat
ttt 452 Val Asp Val Arg Pro Pro Thr Ser Gly Asp Val Ser Phe Val Asp
Phe 105 110 115 caa gta aca aat ggt agt gat aaa ctg ttt ttg cac tgg
ggg gca gta 500 Gln Val Thr Asn Gly Ser Asp Lys Leu Phe Leu His Trp
Gly Ala Val 120 125 130 aaa ttc ggg aaa gaa aca tgg tct ctt ccg aat
gat cgt cca gat ggg 548 Lys Phe Gly Lys Glu Thr Trp Ser Leu Pro Asn
Asp Arg Pro Asp Gly 135 140 145 acc aaa gtg tac aag aac aaa gca ctt
aga act cca ttt gtt aaa tct 596 Thr Lys Val Tyr Lys Asn Lys Ala Leu
Arg Thr Pro Phe Val Lys Ser 150 155 160 ggc tct aac tcc atc ctg aga
ctg gag ata cga gac act gct atc gaa 644 Gly Ser Asn Ser Ile Leu Arg
Leu Glu Ile Arg Asp Thr Ala Ile Glu 165 170 175 180 gct att gag ttt
ctc ata tac gat gaa gcc cac gat aaa tgg ata aag 692 Ala Ile Glu Phe
Leu Ile Tyr Asp Glu Ala His Asp Lys Trp Ile Lys 185 190 195 aat aat
ggt ggt aat ttt cgt gtc aaa ttg tca aga aaa gag ata cga 740 Asn Asn
Gly Gly Asn Phe Arg Val Lys Leu Ser Arg Lys Glu Ile Arg 200 205 210
ggc cca gat gtt tct gtt cct gag gag ctt gta cag atc caa tca tat 788
Gly Pro Asp Val Ser Val Pro Glu Glu Leu Val Gln Ile Gln Ser Tyr 215
220 225 ttg agg tgg gag agg aag gga aaa cag aat tac ccc cct gag aaa
gag 836 Leu Arg Trp Glu Arg Lys Gly Lys Gln Asn Tyr Pro Pro Glu Lys
Glu 230 235 240 aag gag gaa tat gag gct gct cga act gtg cta cag gag
gaa ata gct 884 Lys Glu Glu Tyr Glu Ala Ala Arg Thr Val Leu Gln Glu
Glu Ile Ala 245 250 255 260 cgt ggt gct tcc ata cag gac att cga gca
agg cta aca aaa act aat 932 Arg Gly Ala Ser Ile Gln Asp Ile Arg Ala
Arg Leu Thr Lys Thr Asn 265 270 275 gat aaa agt caa agc aaa gaa gag
cct ctt cat gta aca aag agt gat 980 Asp Lys Ser Gln Ser Lys Glu Glu
Pro Leu His Val Thr Lys Ser Asp 280 285 290 ata cct gat gac ctt gcc
caa gca caa gct tac att agg tgg gag aaa 1028 Ile Pro Asp Asp Leu
Ala Gln Ala Gln Ala Tyr Ile Arg Trp Glu Lys 295 300 305 gca gga aag
ccg aac tat cct cca gaa aag caa att gaa gaa ctc gaa 1076 Ala Gly
Lys Pro Asn Tyr Pro Pro Glu Lys Gln Ile Glu Glu Leu Glu 310 315 320
gaa gca aga aga gaa ttg caa ctt gag ctt gag aaa ggc att acc ctt
1124 Glu Ala Arg Arg Glu Leu Gln Leu Glu Leu Glu Lys Gly Ile Thr
Leu 325 330 335 340 gat gag ttg cgg aaa acg att aca aaa ggg gag ata
aaa act aag gtg 1172 Asp Glu Leu Arg Lys Thr Ile Thr Lys Gly Glu
Ile Lys Thr Lys Val 345 350 355 gaa aag cac ctg aaa aga agt tct ttt
gcc gtt gaa aga atc caa aga 1220 Glu Lys His Leu Lys Arg Ser Ser
Phe Ala Val Glu Arg Ile Gln Arg 360 365 370 aag aag aga gac ttt ggg
cat ctt att aat aag tat act tcc agt cct 1268 Lys Lys Arg Asp Phe
Gly His Leu Ile Asn Lys Tyr Thr Ser Ser Pro 375 380 385 gca gta caa
gta caa aag gtc ttg gaa gaa cca cca gcc tta tct aaa 1316 Ala Val
Gln Val Gln Lys Val Leu Glu Glu Pro Pro Ala Leu Ser Lys 390 395 400
att aag ctg tat gcc aag gag aag gag gag cag att gat gat ccg atc
1364 Ile Lys Leu Tyr Ala Lys Glu Lys Glu Glu Gln Ile Asp Asp Pro
Ile 405 410 415 420 cta aat aaa aag atc ttt aag gtc gat gat ggg gag
cta ctg gta ctg 1412 Leu Asn Lys Lys Ile Phe Lys Val Asp Asp Gly
Glu Leu Leu Val Leu 425 430 435 gta gca aag tcc tct ggg aag aca aaa
gta cat cta gct aca gat ctg 1460 Val Ala Lys Ser Ser Gly Lys Thr
Lys Val His Leu Ala Thr Asp Leu 440 445 450 aat cag cca att act ctt
cac tgg gca tta tcc aaa agt cct gga gag 1508 Asn Gln Pro Ile Thr
Leu His Trp Ala Leu Ser Lys Ser Pro Gly Glu 455 460 465 tgg atg gta
cca cct tca agc ata ttg cct cct ggg tca att att tta 1556 Trp Met
Val Pro Pro Ser Ser Ile Leu Pro Pro Gly Ser Ile Ile Leu 470 475 480
gac aag gct gcc gaa aca cct ttt tca gcc agt tct tct gat ggt cta
1604 Asp Lys Ala Ala Glu Thr Pro Phe Ser Ala Ser Ser Ser Asp Gly
Leu 485 490 495 500 act tct aag gta caa tct ttg gat ata gta att gaa
gat ggc aat ttt 1652 Thr Ser Lys Val Gln Ser Leu Asp Ile Val Ile
Glu Asp Gly Asn Phe 505 510 515 gtg ggg atg cca ttt gtt ctt ttg tct
ggt gaa aaa tgg att aag aac 1700 Val Gly Met Pro Phe Val Leu Leu
Ser Gly Glu Lys Trp Ile Lys Asn 520 525 530 caa ggg tcg gat ttc tat
gtt ggc ttc agt gct gca tcc aaa tta gca 1748 Gln Gly Ser Asp Phe
Tyr Val Gly Phe Ser Ala Ala Ser Lys Leu Ala 535 540 545 ctc aag gct
gct ggg gat ggc agt gga act gca aag tct tta ctg gat 1796 Leu Lys
Ala Ala Gly Asp Gly Ser Gly Thr Ala Lys Ser Leu Leu Asp 550 555 560
aaa ata gca gat atg gaa agt gag gct cag aag tca ttt atg cac cgg
1844 Lys Ile Ala Asp Met Glu Ser Glu Ala Gln Lys Ser Phe Met His
Arg 565 570 575 580 ttt aat att gca gct gac ttg ata gaa gat gcc act
agt gct ggt gaa 1892 Phe Asn Ile Ala Ala Asp Leu Ile Glu Asp Ala
Thr Ser Ala Gly Glu 585 590 595 ctt ggt ttt gct gga att ctt gta tgg
atg agg ttc atg gct aca agg 1940 Leu Gly Phe Ala Gly Ile Leu Val
Trp Met Arg Phe Met Ala Thr Arg 600 605 610 caa ctg ata tgg aac aaa
aac tat aac gta aaa cca cgt gaa ata agc 1988 Gln Leu Ile Trp Asn
Lys Asn Tyr Asn Val Lys Pro Arg Glu Ile Ser 615 620 625 aag gct cag
gac aga ctt aca gac ttg ttg cag aat gct ttc acc agt 2036 Lys Ala
Gln Asp Arg Leu Thr Asp Leu Leu Gln Asn Ala Phe Thr Ser 630 635 640
cac cct cag tac cgt gaa att ttg cgg atg att atg tca act gtt gga
2084 His Pro Gln Tyr Arg Glu Ile Leu Arg Met Ile Met Ser Thr Val
Gly 645 650 655 660 cgt gga ggt gaa ggg gat gta gga cag cga att agg
gat gaa att ttg 2132 Arg Gly Gly Glu Gly Asp Val Gly Gln Arg Ile
Arg Asp Glu Ile Leu 665 670 675 gtc atc cag agg aac aat gac tgc aag
ggt ggt atg atg caa gaa tgg 2180 Val Ile Gln Arg Asn Asn Asp Cys
Lys Gly Gly Met Met Gln Glu Trp 680 685 690 cat cag aaa ttg cat aat
aat act agt cct gat gat gtt gtg atc tgt 2228 His Gln Lys Leu His
Asn Asn Thr Ser Pro Asp Asp Val Val Ile Cys 695 700 705 cag gca tta
att gac tac atc aag agt gat ttt gat ctt ggt gtt tat 2276 Gln Ala
Leu Ile Asp Tyr Ile Lys Ser Asp Phe Asp Leu Gly Val Tyr 710 715 720
tgg aaa acc ctg aat gag aac gga ata aca aaa gag cgt ctt ttg agt
2324 Trp Lys Thr Leu Asn Glu Asn Gly Ile Thr Lys Glu Arg Leu Leu
Ser 725 730 735 740 tat gac cgt gct atc cat tct gaa cca aat ttt aga
gga gat caa aag 2372 Tyr Asp Arg Ala Ile His Ser Glu Pro Asn Phe
Arg Gly Asp Gln Lys 745 750 755 ggt ggt ctt ttg cgt gat tta ggt cac
tat atg aga aca ttg aag gca 2420 Gly Gly Leu Leu Arg Asp Leu Gly
His Tyr Met Arg Thr Leu Lys Ala 760 765 770 gtt cat tca ggt gca gat
ctt gag tct gct att gca aac tgc atg ggc 2468 Val His Ser Gly Ala
Asp Leu Glu Ser Ala Ile Ala Asn Cys Met Gly 775 780 785 tac aaa act
gag gga gaa ggc ttt atg gtt gga gtc cag ata aat cct 2516 Tyr Lys
Thr Glu Gly Glu Gly Phe Met Val Gly Val Gln Ile Asn Pro 790 795 800
gta tca ggc ttg cca tct ggc ttt cag gac ctc ctc cat ttt gtc tta
2564 Val Ser Gly Leu Pro Ser Gly Phe Gln Asp Leu Leu His Phe Val
Leu 805 810 815 820 gac cat gtg gaa gat aaa aat gtg gaa act ctt ctt
gag aga ttg cta 2612 Asp His Val Glu Asp Lys Asn Val Glu Thr Leu
Leu Glu Arg Leu Leu 825 830 835 gag gct cgt gag gag ctt agg ccc ttg
ctt ctc aaa cca aac aac cgt 2660 Glu Ala Arg Glu Glu Leu Arg Pro
Leu Leu Leu Lys Pro Asn Asn Arg 840 845 850 cta aag gat ctg ctg ttt
ttg gac ata gca ctt gat tct aca gtt aga 2708 Leu Lys Asp Leu Leu
Phe Leu Asp Ile Ala Leu Asp Ser Thr Val Arg 855 860 865 aca gca gta
gaa agg gga tat gaa gaa ttg aac aac gct aat cct gag 2756 Thr Ala
Val Glu Arg Gly Tyr Glu Glu Leu Asn Asn Ala Asn Pro Glu 870 875 880
aaa atc atg tac ttc atc tcc ctc gtt ctt gaa aat ctc gca ctc tct
2804
Lys Ile Met Tyr Phe Ile Ser Leu Val Leu Glu Asn Leu Ala Leu Ser 885
890 895 900 gtg gac gat aat gaa gat ctt gtt tat tgc ttg aag gga tgg
aat caa 2852 Val Asp Asp Asn Glu Asp Leu Val Tyr Cys Leu Lys Gly
Trp Asn Gln 905 910 915 gct ctt tca atg tcc aat ggt ggg gac aac cat
tgg gct tta ttt gca 2900 Ala Leu Ser Met Ser Asn Gly Gly Asp Asn
His Trp Ala Leu Phe Ala 920 925 930 aaa gct gtg ctt gac aga acc cgt
ctt gca ctt gca agc aag gca gag 2948 Lys Ala Val Leu Asp Arg Thr
Arg Leu Ala Leu Ala Ser Lys Ala Glu 935 940 945 tgg tac cat cac tta
ttg cag cca tct gcc gaa tat cta gga tca ata 2996 Trp Tyr His His
Leu Leu Gln Pro Ser Ala Glu Tyr Leu Gly Ser Ile 950 955 960 ctt ggg
gtg gac caa tgg gct ttg aac ata ttt act gaa gaa att ata 3044 Leu
Gly Val Asp Gln Trp Ala Leu Asn Ile Phe Thr Glu Glu Ile Ile 965 970
975 980 cgt gct gga tca gca gct tca tta tcc tct ctt ctt aat aga ctc
gat 3092 Arg Ala Gly Ser Ala Ala Ser Leu Ser Ser Leu Leu Asn Arg
Leu Asp 985 990 995 ccc gtg ctt cgg aaa act gca aat cta gga agt tgg
cag att atc 3137 Pro Val Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp
Gln Ile Ile 1000 1005 1010 agt cca gtt gaa gcc gtt gga tat gtt gtc
gtt gtg gat gag ttg 3182 Ser Pro Val Glu Ala Val Gly Tyr Val Val
Val Val Asp Glu Leu 1015 1020 1025 ctt tca gtt cag aat gaa atc tac
gag aag ccc acg atc tta gta 3227 Leu Ser Val Gln Asn Glu Ile Tyr
Glu Lys Pro Thr Ile Leu Val 1030 1035 1040 gca aaa tct gtt aaa gga
gag gag gaa att cct gat ggt gct gtt 3272 Ala Lys Ser Val Lys Gly
Glu Glu Glu Ile Pro Asp Gly Ala Val 1045 1050 1055 gcc ctg ata aca
cca gac atg cca gat gtt ctt tca cat gtt tct 3317 Ala Leu Ile Thr
Pro Asp Met Pro Asp Val Leu Ser His Val Ser 1060 1065 1070 gtt cga
gct aga aat ggg aag gtt tgc ttt gct aca tgc ttt gat 3362 Val Arg
Ala Arg Asn Gly Lys Val Cys Phe Ala Thr Cys Phe Asp 1075 1080 1085
ccc aat ata ttg gct gac ctc caa gca aag gaa gga agg att ttg 3407
Pro Asn Ile Leu Ala Asp Leu Gln Ala Lys Glu Gly Arg Ile Leu 1090
1095 1100 ctc tta aag cct aca cct tca gac ata atc tat agt gag gtg
aat 3452 Leu Leu Lys Pro Thr Pro Ser Asp Ile Ile Tyr Ser Glu Val
Asn 1105 1110 1115 gag att gag ctc caa agt tca agt aac ttg gta gaa
gct gaa act 3497 Glu Ile Glu Leu Gln Ser Ser Ser Asn Leu Val Glu
Ala Glu Thr 1120 1125 1130 tca gca aca ctt aga ttg gtg aaa aag caa
ttt ggt ggt tgt tac 3542 Ser Ala Thr Leu Arg Leu Val Lys Lys Gln
Phe Gly Gly Cys Tyr 1135 1140 1145 gca ata tca gca gat gaa ttc aca
agt gaa atg gtt gga gct aaa 3587 Ala Ile Ser Ala Asp Glu Phe Thr
Ser Glu Met Val Gly Ala Lys 1150 1155 1160 tca cgt aat att gca tat
ctg aaa gga aaa gtg cct tcc tcg gtg 3632 Ser Arg Asn Ile Ala Tyr
Leu Lys Gly Lys Val Pro Ser Ser Val 1165 1170 1175 gga att cct acg
tca gta gct ctt cca ttt gga gtc ttt gag aaa 3677 Gly Ile Pro Thr
Ser Val Ala Leu Pro Phe Gly Val Phe Glu Lys 1180 1185 1190 gta ctt
tca gac gac ata aat cag gga gtg gca aaa gag ttg caa 3722 Val Leu
Ser Asp Asp Ile Asn Gln Gly Val Ala Lys Glu Leu Gln 1195 1200 1205
att ctg atg aaa aaa cta tct gaa gga gac ttc agc gct ctt ggt 3767
Ile Leu Met Lys Lys Leu Ser Glu Gly Asp Phe Ser Ala Leu Gly 1210
1215 1220 gaa att cgc aca acg gtt tta gat ctt tca gca cca gct caa
ttg 3812 Glu Ile Arg Thr Thr Val Leu Asp Leu Ser Ala Pro Ala Gln
Leu 1225 1230 1235 gtc aaa gag ctg aag gag aag atg cag ggt tct ggc
atg cct tgg 3857 Val Lys Glu Leu Lys Glu Lys Met Gln Gly Ser Gly
Met Pro Trp 1240 1245 1250 cct ggt gat gaa ggt cca aag cgg tgg gaa
caa gca tgg atg gcc 3902 Pro Gly Asp Glu Gly Pro Lys Arg Trp Glu
Gln Ala Trp Met Ala 1255 1260 1265 ata aaa aag gtg tgg gct tca aaa
tgg aat gag aga gca tac ttc 3947 Ile Lys Lys Val Trp Ala Ser Lys
Trp Asn Glu Arg Ala Tyr Phe 1270 1275 1280 agc aca agg aag gtg aaa
ctg gat cat gac tat ctg tgc atg gct 3992 Ser Thr Arg Lys Val Lys
Leu Asp His Asp Tyr Leu Cys Met Ala 1285 1290 1295 gtc ctt gtt caa
gaa ata ata aat gct gat tat gca ttt gtc att 4037 Val Leu Val Gln
Glu Ile Ile Asn Ala Asp Tyr Ala Phe Val Ile 1300 1305 1310 cac aca
acc aac cca tct tcc gga gac gac tca gaa ata tat gcc 4082 His Thr
Thr Asn Pro Ser Ser Gly Asp Asp Ser Glu Ile Tyr Ala 1315 1320 1325
gag gtg gtc agg ggc ctt ggg gaa aca ctt gtt gga gct tat cca 4127
Glu Val Val Arg Gly Leu Gly Glu Thr Leu Val Gly Ala Tyr Pro 1330
1335 1340 gga cgt gct ttg agt ttt atc tgc aag aaa aag gat ctc aac
tct 4172 Gly Arg Ala Leu Ser Phe Ile Cys Lys Lys Lys Asp Leu Asn
Ser 1345 1350 1355 cct caa gtg tta ggt tac cca agc aaa ccg atc ggc
ctt ttc ata 4217 Pro Gln Val Leu Gly Tyr Pro Ser Lys Pro Ile Gly
Leu Phe Ile 1360 1365 1370 aaa aga tct atc atc ttc cga tct gat tcc
aat ggg gaa gat ttg 4262 Lys Arg Ser Ile Ile Phe Arg Ser Asp Ser
Asn Gly Glu Asp Leu 1375 1380 1385 gaa ggt tat gcc ggt gct ggc ctc
tac gac agt gta cca atg gat 4307 Glu Gly Tyr Ala Gly Ala Gly Leu
Tyr Asp Ser Val Pro Met Asp 1390 1395 1400 gag gag gaa aaa gtt gta
att gat tac tct tcc gac cca ttg ata 4352 Glu Glu Glu Lys Val Val
Ile Asp Tyr Ser Ser Asp Pro Leu Ile 1405 1410 1415 act gat ggt aac
ttc cgc cag aca atc ctg tcc aac att gct cgt 4397 Thr Asp Gly Asn
Phe Arg Gln Thr Ile Leu Ser Asn Ile Ala Arg 1420 1425 1430 gct gga
cat gct atc gag gag cta tat ggc tct cct caa gac att 4442 Ala Gly
His Ala Ile Glu Glu Leu Tyr Gly Ser Pro Gln Asp Ile 1435 1440 1445
gag ggt gta gtg agg gat gga aag att tat gtc gtt cag aca aga 4487
Glu Gly Val Val Arg Asp Gly Lys Ile Tyr Val Val Gln Thr Arg 1450
1455 1460 cca cag atg tga ttatattctc gttgtatgtt gttcagagaa
gaccacagat 4539 Pro Gln Met gtgatcatat tctcattgta tcagatctgt
gaccacttac ctgatacctc ccatgaagtt 4599 acctgtatga ttatacgtga
tccaaagcca tcacatcatg ttcaccttca gctattggag 4659 gagaagtgag
aagtaggaat tgcaatatga ggaataataa gaaaaacttt gtaaaagcta 4719
aattagctgg gtatgatata gggagaaatg tgtaaacatt gtactatata tagtatatac
4779 acacgcatta tgtattgcat tatgcactga ataatatcgc agcatcaaag
aagaaatcct 4839 ttgggtggtt tc 4851 <210> SEQ ID NO 2
<211> LENGTH: 1464 <212> TYPE: PRT <213>
ORGANISM: Solanum tuberosum <400> SEQUENCE: 2 Met Ser Asn Ser
Leu Gly Asn Asn Leu Leu Tyr Gln Gly Phe Leu Thr 1 5 10 15 Ser Thr
Val Leu Glu His Lys Ser Arg Ile Ser Pro Pro Cys Val Gly 20 25 30
Gly Asn Ser Leu Phe Gln Gln Gln Val Ile Ser Lys Ser Pro Leu Ser 35
40 45 Thr Glu Phe Arg Gly Asn Arg Leu Lys Val Gln Lys Lys Lys Ile
Pro 50 55 60 Met Glu Lys Lys Arg Ala Phe Ser Ser Ser Pro His Ala
Val Leu Thr 65 70 75 80 Thr Asp Thr Ser Ser Glu Leu Ala Glu Lys Phe
Ser Leu Gly Gly Asn 85 90 95 Ile Glu Leu Gln Val Asp Val Arg Pro
Pro Thr Ser Gly Asp Val Ser 100 105 110 Phe Val Asp Phe Gln Val Thr
Asn Gly Ser Asp Lys Leu Phe Leu His 115 120 125 Trp Gly Ala Val Lys
Phe Gly Lys Glu Thr Trp Ser Leu Pro Asn Asp 130 135 140 Arg Pro Asp
Gly Thr Lys Val Tyr Lys Asn Lys Ala Leu Arg Thr Pro 145 150 155 160
Phe Val Lys Ser Gly Ser Asn Ser Ile Leu Arg Leu Glu Ile Arg Asp 165
170 175 Thr Ala Ile Glu Ala Ile Glu Phe Leu Ile Tyr Asp Glu Ala His
Asp 180 185 190 Lys Trp Ile Lys Asn Asn Gly Gly Asn Phe Arg Val Lys
Leu Ser Arg 195 200 205 Lys Glu Ile Arg Gly Pro Asp Val Ser Val Pro
Glu Glu Leu Val Gln 210 215 220 Ile Gln Ser Tyr Leu Arg Trp Glu Arg
Lys Gly Lys Gln Asn Tyr Pro 225 230 235 240 Pro Glu Lys Glu Lys Glu
Glu Tyr Glu Ala Ala Arg Thr Val Leu Gln 245 250 255 Glu Glu Ile Ala
Arg Gly Ala Ser Ile Gln Asp Ile Arg Ala Arg Leu 260 265 270 Thr Lys
Thr Asn Asp Lys Ser Gln Ser Lys Glu Glu Pro Leu His Val 275 280 285
Thr Lys Ser Asp Ile Pro Asp Asp Leu Ala Gln Ala Gln Ala Tyr Ile 290
295 300 Arg Trp Glu Lys Ala Gly Lys Pro Asn Tyr Pro Pro Glu Lys Gln
Ile 305 310 315 320 Glu Glu Leu Glu Glu Ala Arg Arg Glu Leu Gln Leu
Glu Leu Glu Lys 325 330 335 Gly Ile Thr Leu Asp Glu Leu Arg Lys Thr
Ile Thr Lys Gly Glu Ile 340 345 350 Lys Thr Lys Val Glu Lys His Leu
Lys Arg Ser Ser Phe Ala Val Glu 355 360 365 Arg Ile Gln Arg Lys Lys
Arg Asp Phe Gly His Leu Ile Asn Lys Tyr 370 375 380 Thr Ser Ser Pro
Ala Val Gln Val Gln Lys Val Leu Glu Glu Pro Pro 385 390 395 400 Ala
Leu Ser Lys Ile Lys Leu Tyr Ala Lys Glu Lys Glu Glu Gln Ile 405 410
415
Asp Asp Pro Ile Leu Asn Lys Lys Ile Phe Lys Val Asp Asp Gly Glu 420
425 430 Leu Leu Val Leu Val Ala Lys Ser Ser Gly Lys Thr Lys Val His
Leu 435 440 445 Ala Thr Asp Leu Asn Gln Pro Ile Thr Leu His Trp Ala
Leu Ser Lys 450 455 460 Ser Pro Gly Glu Trp Met Val Pro Pro Ser Ser
Ile Leu Pro Pro Gly 465 470 475 480 Ser Ile Ile Leu Asp Lys Ala Ala
Glu Thr Pro Phe Ser Ala Ser Ser 485 490 495 Ser Asp Gly Leu Thr Ser
Lys Val Gln Ser Leu Asp Ile Val Ile Glu 500 505 510 Asp Gly Asn Phe
Val Gly Met Pro Phe Val Leu Leu Ser Gly Glu Lys 515 520 525 Trp Ile
Lys Asn Gln Gly Ser Asp Phe Tyr Val Gly Phe Ser Ala Ala 530 535 540
Ser Lys Leu Ala Leu Lys Ala Ala Gly Asp Gly Ser Gly Thr Ala Lys 545
550 555 560 Ser Leu Leu Asp Lys Ile Ala Asp Met Glu Ser Glu Ala Gln
Lys Ser 565 570 575 Phe Met His Arg Phe Asn Ile Ala Ala Asp Leu Ile
Glu Asp Ala Thr 580 585 590 Ser Ala Gly Glu Leu Gly Phe Ala Gly Ile
Leu Val Trp Met Arg Phe 595 600 605 Met Ala Thr Arg Gln Leu Ile Trp
Asn Lys Asn Tyr Asn Val Lys Pro 610 615 620 Arg Glu Ile Ser Lys Ala
Gln Asp Arg Leu Thr Asp Leu Leu Gln Asn 625 630 635 640 Ala Phe Thr
Ser His Pro Gln Tyr Arg Glu Ile Leu Arg Met Ile Met 645 650 655 Ser
Thr Val Gly Arg Gly Gly Glu Gly Asp Val Gly Gln Arg Ile Arg 660 665
670 Asp Glu Ile Leu Val Ile Gln Arg Asn Asn Asp Cys Lys Gly Gly Met
675 680 685 Met Gln Glu Trp His Gln Lys Leu His Asn Asn Thr Ser Pro
Asp Asp 690 695 700 Val Val Ile Cys Gln Ala Leu Ile Asp Tyr Ile Lys
Ser Asp Phe Asp 705 710 715 720 Leu Gly Val Tyr Trp Lys Thr Leu Asn
Glu Asn Gly Ile Thr Lys Glu 725 730 735 Arg Leu Leu Ser Tyr Asp Arg
Ala Ile His Ser Glu Pro Asn Phe Arg 740 745 750 Gly Asp Gln Lys Gly
Gly Leu Leu Arg Asp Leu Gly His Tyr Met Arg 755 760 765 Thr Leu Lys
Ala Val His Ser Gly Ala Asp Leu Glu Ser Ala Ile Ala 770 775 780 Asn
Cys Met Gly Tyr Lys Thr Glu Gly Glu Gly Phe Met Val Gly Val 785 790
795 800 Gln Ile Asn Pro Val Ser Gly Leu Pro Ser Gly Phe Gln Asp Leu
Leu 805 810 815 His Phe Val Leu Asp His Val Glu Asp Lys Asn Val Glu
Thr Leu Leu 820 825 830 Glu Arg Leu Leu Glu Ala Arg Glu Glu Leu Arg
Pro Leu Leu Leu Lys 835 840 845 Pro Asn Asn Arg Leu Lys Asp Leu Leu
Phe Leu Asp Ile Ala Leu Asp 850 855 860 Ser Thr Val Arg Thr Ala Val
Glu Arg Gly Tyr Glu Glu Leu Asn Asn 865 870 875 880 Ala Asn Pro Glu
Lys Ile Met Tyr Phe Ile Ser Leu Val Leu Glu Asn 885 890 895 Leu Ala
Leu Ser Val Asp Asp Asn Glu Asp Leu Val Tyr Cys Leu Lys 900 905 910
Gly Trp Asn Gln Ala Leu Ser Met Ser Asn Gly Gly Asp Asn His Trp 915
920 925 Ala Leu Phe Ala Lys Ala Val Leu Asp Arg Thr Arg Leu Ala Leu
Ala 930 935 940 Ser Lys Ala Glu Trp Tyr His His Leu Leu Gln Pro Ser
Ala Glu Tyr 945 950 955 960 Leu Gly Ser Ile Leu Gly Val Asp Gln Trp
Ala Leu Asn Ile Phe Thr 965 970 975 Glu Glu Ile Ile Arg Ala Gly Ser
Ala Ala Ser Leu Ser Ser Leu Leu 980 985 990 Asn Arg Leu Asp Pro Val
Leu Arg Lys Thr Ala Asn Leu Gly Ser Trp 995 1000 1005 Gln Ile Ile
Ser Pro Val Glu Ala Val Gly Tyr Val Val Val Val 1010 1015 1020 Asp
Glu Leu Leu Ser Val Gln Asn Glu Ile Tyr Glu Lys Pro Thr 1025 1030
1035 Ile Leu Val Ala Lys Ser Val Lys Gly Glu Glu Glu Ile Pro Asp
1040 1045 1050 Gly Ala Val Ala Leu Ile Thr Pro Asp Met Pro Asp Val
Leu Ser 1055 1060 1065 His Val Ser Val Arg Ala Arg Asn Gly Lys Val
Cys Phe Ala Thr 1070 1075 1080 Cys Phe Asp Pro Asn Ile Leu Ala Asp
Leu Gln Ala Lys Glu Gly 1085 1090 1095 Arg Ile Leu Leu Leu Lys Pro
Thr Pro Ser Asp Ile Ile Tyr Ser 1100 1105 1110 Glu Val Asn Glu Ile
Glu Leu Gln Ser Ser Ser Asn Leu Val Glu 1115 1120 1125 Ala Glu Thr
Ser Ala Thr Leu Arg Leu Val Lys Lys Gln Phe Gly 1130 1135 1140 Gly
Cys Tyr Ala Ile Ser Ala Asp Glu Phe Thr Ser Glu Met Val 1145 1150
1155 Gly Ala Lys Ser Arg Asn Ile Ala Tyr Leu Lys Gly Lys Val Pro
1160 1165 1170 Ser Ser Val Gly Ile Pro Thr Ser Val Ala Leu Pro Phe
Gly Val 1175 1180 1185 Phe Glu Lys Val Leu Ser Asp Asp Ile Asn Gln
Gly Val Ala Lys 1190 1195 1200 Glu Leu Gln Ile Leu Met Lys Lys Leu
Ser Glu Gly Asp Phe Ser 1205 1210 1215 Ala Leu Gly Glu Ile Arg Thr
Thr Val Leu Asp Leu Ser Ala Pro 1220 1225 1230 Ala Gln Leu Val Lys
Glu Leu Lys Glu Lys Met Gln Gly Ser Gly 1235 1240 1245 Met Pro Trp
Pro Gly Asp Glu Gly Pro Lys Arg Trp Glu Gln Ala 1250 1255 1260 Trp
Met Ala Ile Lys Lys Val Trp Ala Ser Lys Trp Asn Glu Arg 1265 1270
1275 Ala Tyr Phe Ser Thr Arg Lys Val Lys Leu Asp His Asp Tyr Leu
1280 1285 1290 Cys Met Ala Val Leu Val Gln Glu Ile Ile Asn Ala Asp
Tyr Ala 1295 1300 1305 Phe Val Ile His Thr Thr Asn Pro Ser Ser Gly
Asp Asp Ser Glu 1310 1315 1320 Ile Tyr Ala Glu Val Val Arg Gly Leu
Gly Glu Thr Leu Val Gly 1325 1330 1335 Ala Tyr Pro Gly Arg Ala Leu
Ser Phe Ile Cys Lys Lys Lys Asp 1340 1345 1350 Leu Asn Ser Pro Gln
Val Leu Gly Tyr Pro Ser Lys Pro Ile Gly 1355 1360 1365 Leu Phe Ile
Lys Arg Ser Ile Ile Phe Arg Ser Asp Ser Asn Gly 1370 1375 1380 Glu
Asp Leu Glu Gly Tyr Ala Gly Ala Gly Leu Tyr Asp Ser Val 1385 1390
1395 Pro Met Asp Glu Glu Glu Lys Val Val Ile Asp Tyr Ser Ser Asp
1400 1405 1410 Pro Leu Ile Thr Asp Gly Asn Phe Arg Gln Thr Ile Leu
Ser Asn 1415 1420 1425 Ile Ala Arg Ala Gly His Ala Ile Glu Glu Leu
Tyr Gly Ser Pro 1430 1435 1440 Gln Asp Ile Glu Gly Val Val Arg Asp
Gly Lys Ile Tyr Val Val 1445 1450 1455 Gln Thr Arg Pro Gln Met 1460
<210> SEQ ID NO 3 <211> LENGTH: 3000 <212> TYPE:
DNA <213> ORGANISM: Triticum aestivum <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (227)..(2623)
<400> SEQUENCE: 3 ctccaccgcg gtggcggccg ctctagaact agtggatccc
ccgggctgca ggaattcggc 60 acgagcttcg gcctgacccc gttcgtttac
ccccacacag agcacactcc agtccagtcc 120 agcccactgc caccgcgcta
ctctccactc ccactgccac cacctccgcc tgcgccgcgc 180 tctgggcgga
ccaacccgcg aaccgtacca tctcccgccc cgatcc atg tcg tcg 235 Met Ser Ser
1 gcg gtc gcg tcc gcc gca tcc ttc ctc gcg ctc gcg tca gcc tcc ccc
283 Ala Val Ala Ser Ala Ala Ser Phe Leu Ala Leu Ala Ser Ala Ser Pro
5 10 15 ggg aga tca cgc agg cgg gcg agg gtg agc gcg cag cca ccc cac
gcc 331 Gly Arg Ser Arg Arg Arg Ala Arg Val Ser Ala Gln Pro Pro His
Ala 20 25 30 35 ggg gcc ggc agg ttg cac tgg ccg ccg tgg ccg ccg cag
cgc acg gct 379 Gly Ala Gly Arg Leu His Trp Pro Pro Trp Pro Pro Gln
Arg Thr Ala 40 45 50 cgc gac gga gct gtg gcg gcg ctc gcc gcc ggg
aag aag gac gcg ggg 427 Arg Asp Gly Ala Val Ala Ala Leu Ala Ala Gly
Lys Lys Asp Ala Gly 55 60 65 atc gac gac gcc gcc gcg tcc gtg agg
cag ccc cgc gca ctc cgc ggt 475 Ile Asp Asp Ala Ala Ala Ser Val Arg
Gln Pro Arg Ala Leu Arg Gly 70 75 80 ggc gcc gcc acc aag gtc gcg
gag cga agg gat ccc gtc aag acg ctc 523 Gly Ala Ala Thr Lys Val Ala
Glu Arg Arg Asp Pro Val Lys Thr Leu 85 90 95 gac cgc gac gcc gcg
gaa ggc ggc ggg ccg tcc ccg ccg gca gcg agg 571 Asp Arg Asp Ala Ala
Glu Gly Gly Gly Pro Ser Pro Pro Ala Ala Arg
100 105 110 115 cag gac gcc gcc cgt ccg ccg agt atg aac ggc atg ccg
gtg aac ggc 619 Gln Asp Ala Ala Arg Pro Pro Ser Met Asn Gly Met Pro
Val Asn Gly 120 125 130 gag aac aaa tct acc ggc ggc ggc ggc gcg act
aaa gac agc ggg ctg 667 Glu Asn Lys Ser Thr Gly Gly Gly Gly Ala Thr
Lys Asp Ser Gly Leu 135 140 145 ccc acg ccc gca cgc gcg ccc cat ccg
tcg acc cag aac aga gca ccg 715 Pro Thr Pro Ala Arg Ala Pro His Pro
Ser Thr Gln Asn Arg Ala Pro 150 155 160 gtg aac ggt gaa aac aaa gct
aac gtc gcc tcg ccg ccg acg agc ata 763 Val Asn Gly Glu Asn Lys Ala
Asn Val Ala Ser Pro Pro Thr Ser Ile 165 170 175 gcc gag gcc gcg gct
tcg gat tcc gca gct acc att tcc atc agc gac 811 Ala Glu Ala Ala Ala
Ser Asp Ser Ala Ala Thr Ile Ser Ile Ser Asp 180 185 190 195 aag gcg
ccg gag tcc gtt gtc cca gct gag aag acg ccg ccg tcg tcc 859 Lys Ala
Pro Glu Ser Val Val Pro Ala Glu Lys Thr Pro Pro Ser Ser 200 205 210
ggc tca aat ttc gag tcc tcg gcc tct gct ccc ggg tct gac act gtc 907
Gly Ser Asn Phe Glu Ser Ser Ala Ser Ala Pro Gly Ser Asp Thr Val 215
220 225 agc gac gtg gaa caa gaa ctg aag aag ggt gcg gtc gtt gtc gaa
gaa 955 Ser Asp Val Glu Gln Glu Leu Lys Lys Gly Ala Val Val Val Glu
Glu 230 235 240 gct cca aag cca aag gct ctt tcg ccg cct gca gcc ccc
gct gta caa 1003 Ala Pro Lys Pro Lys Ala Leu Ser Pro Pro Ala Ala
Pro Ala Val Gln 245 250 255 gaa gac ctt tgg gat ttc aag aaa tac att
ggt ttc gag gag ccc gtg 1051 Glu Asp Leu Trp Asp Phe Lys Lys Tyr
Ile Gly Phe Glu Glu Pro Val 260 265 270 275 gag gcc aag gat gat ggc
cgg gct gtc gca gat gat gcg ggc tcc ttt 1099 Glu Ala Lys Asp Asp
Gly Arg Ala Val Ala Asp Asp Ala Gly Ser Phe 280 285 290 gaa cac cac
cag aat cac gac tcc gga cct ttg gca ggg gag aat gtc 1147 Glu His
His Gln Asn His Asp Ser Gly Pro Leu Ala Gly Glu Asn Val 295 300 305
atg aac gtg gtc gtc gtg gct gct gag tgt tct ccc tgg tgc aaa aca
1195 Met Asn Val Val Val Val Ala Ala Glu Cys Ser Pro Trp Cys Lys
Thr 310 315 320 ggt ggt ctg gga gat gtt gcg ggt gct ctg ccc aag gct
ttg gca aag 1243 Gly Gly Leu Gly Asp Val Ala Gly Ala Leu Pro Lys
Ala Leu Ala Lys 325 330 335 aga gga cat cgt gtt atg gtt gtg gta cca
agg tat ggg gac tat gaa 1291 Arg Gly His Arg Val Met Val Val Val
Pro Arg Tyr Gly Asp Tyr Glu 340 345 350 355 gaa gcc tac gat gtc gga
gtc cga aaa tac tac aag gct gct gga cag 1339 Glu Ala Tyr Asp Val
Gly Val Arg Lys Tyr Tyr Lys Ala Ala Gly Gln 360 365 370 gat atg gaa
gtg aat tat ttc cat gct tat atc gat gga gtt gat ttt 1387 Asp Met
Glu Val Asn Tyr Phe His Ala Tyr Ile Asp Gly Val Asp Phe 375 380 385
gtg ttc att gac gct cct ctc ttc cga cac cgt cag gaa gac att tat
1435 Val Phe Ile Asp Ala Pro Leu Phe Arg His Arg Gln Glu Asp Ile
Tyr 390 395 400 ggg ggc agc aga cag gaa att atg aag cgc atg att ttg
ttc tgc aag 1483 Gly Gly Ser Arg Gln Glu Ile Met Lys Arg Met Ile
Leu Phe Cys Lys 405 410 415 gcc gct gtt gag gtt cca tgg cac gtt cca
tgc ggc ggt gtc cct tat 1531 Ala Ala Val Glu Val Pro Trp His Val
Pro Cys Gly Gly Val Pro Tyr 420 425 430 435 ggg gat gga aat ctg gtg
ttt att gca aat gat tgg cac acg gca ctc 1579 Gly Asp Gly Asn Leu
Val Phe Ile Ala Asn Asp Trp His Thr Ala Leu 440 445 450 ctg cct gtc
tat ctg aaa gca tat tac agg gac cat ggt ttg atg cag 1627 Leu Pro
Val Tyr Leu Lys Ala Tyr Tyr Arg Asp His Gly Leu Met Gln 455 460 465
tac act cgg tcc att atg gtg ata cat aac atc gct cac cag ggc cgt
1675 Tyr Thr Arg Ser Ile Met Val Ile His Asn Ile Ala His Gln Gly
Arg 470 475 480 ggc cct gta gat gaa ttc ccg ttc acc gag ttg cct gag
cac tac ctg 1723 Gly Pro Val Asp Glu Phe Pro Phe Thr Glu Leu Pro
Glu His Tyr Leu 485 490 495 gaa cac ttc aga ctg tac gac ccc gtg ggt
ggt gaa cac gcc aac tac 1771 Glu His Phe Arg Leu Tyr Asp Pro Val
Gly Gly Glu His Ala Asn Tyr 500 505 510 515 ttc gcc gcc ggc ctg aag
atg gcg gac cag gtt gtc gtg gtg agc ccc 1819 Phe Ala Ala Gly Leu
Lys Met Ala Asp Gln Val Val Val Val Ser Pro 520 525 530 ggg tac ctg
tgg gag ctg aag acg gtg gag ggc ggc tgg ggg ctt cac 1867 Gly Tyr
Leu Trp Glu Leu Lys Thr Val Glu Gly Gly Trp Gly Leu His 535 540 545
gac atc ata cgg cag aac gac tgg aag acc cgc ggc atc gtc aac ggc
1915 Asp Ile Ile Arg Gln Asn Asp Trp Lys Thr Arg Gly Ile Val Asn
Gly 550 555 560 atc gac aac atg gag tgg aac ccc gag gtg gac gcc cac
ctc aag tcg 1963 Ile Asp Asn Met Glu Trp Asn Pro Glu Val Asp Ala
His Leu Lys Ser 565 570 575 gac ggc tac acc aac ttc tcc ctg agg acg
ctg gac tcc ggc aag cgg 2011 Asp Gly Tyr Thr Asn Phe Ser Leu Arg
Thr Leu Asp Ser Gly Lys Arg 580 585 590 595 cag tgc aag gag gcc ctg
cag cgc gag ctg ggc ctg cag gtc cgc gcc 2059 Gln Cys Lys Glu Ala
Leu Gln Arg Glu Leu Gly Leu Gln Val Arg Ala 600 605 610 gac gtg ccg
ctg ctc ggc ttc atc ggc cgc ctg gac ggg cag aag ggc 2107 Asp Val
Pro Leu Leu Gly Phe Ile Gly Arg Leu Asp Gly Gln Lys Gly 615 620 625
gtg gag atc atc gcg gac gcc atg ccc tgg atc gtg agc cag gac gtg
2155 Val Glu Ile Ile Ala Asp Ala Met Pro Trp Ile Val Ser Gln Asp
Val 630 635 640 cag ctg gtg atg ctg ggc acc ggg cgc cac gac ctg gag
agc atg ctg 2203 Gln Leu Val Met Leu Gly Thr Gly Arg His Asp Leu
Glu Ser Met Leu 645 650 655 cag cac ttc gag cgg gag cac cac gac aag
gtg cgc ggg tgg gtg ggg 2251 Gln His Phe Glu Arg Glu His His Asp
Lys Val Arg Gly Trp Val Gly 660 665 670 675 ttc tcc gtg cgc ctg gcg
cac cgg atc acg gcg ggg gcg gac gcg ctc 2299 Phe Ser Val Arg Leu
Ala His Arg Ile Thr Ala Gly Ala Asp Ala Leu 680 685 690 ctc atg ccc
tcc cgg ttc gag ccg tgc ggg ctg aac cag ctc tac gcc 2347 Leu Met
Pro Ser Arg Phe Glu Pro Cys Gly Leu Asn Gln Leu Tyr Ala 695 700 705
atg gcc tac ggc acc gtc ccc gtc gtg cac gcc gtc ggc ggc ctc agg
2395 Met Ala Tyr Gly Thr Val Pro Val Val His Ala Val Gly Gly Leu
Arg 710 715 720 gac acc gtg ccg ccg ttc gac ccc ttc aac cac tcc ggg
ctc ggg tgg 2443 Asp Thr Val Pro Pro Phe Asp Pro Phe Asn His Ser
Gly Leu Gly Trp 725 730 735 acg ttc gac cgc gcc gag gcg cac aag ctg
atc gag gcg ctc ggg cac 2491 Thr Phe Asp Arg Ala Glu Ala His Lys
Leu Ile Glu Ala Leu Gly His 740 745 750 755 tgc ctc cgc acc tac cga
gac ttc aag gag agc tgg agg gcc ctc cag 2539 Cys Leu Arg Thr Tyr
Arg Asp Phe Lys Glu Ser Trp Arg Ala Leu Gln 760 765 770 gag cgc ggc
atg tcg cag gac ttc agc tgg gag cac gcc gcc aag ctc 2587 Glu Arg
Gly Met Ser Gln Asp Phe Ser Trp Glu His Ala Ala Lys Leu 775 780 785
tac gag gac gtc ctc gtc aag gcc aag tac cag tgg tgaacgctag 2633 Tyr
Glu Asp Val Leu Val Lys Ala Lys Tyr Gln Trp 790 795 ctgctagccg
ctccagcccc gcatgcgtgc atgacaggat ggaactgcat tgcgcacgca 2693
ggaaagtgcc atggagcgcc ggcatccgcg aagtacagtg acatgaggtg tgtgtggttg
2753 agacgctgat tccaatccgg cccgtagcag agtagagcgg aggtatatgg
gaatcttaac 2813 ttggtattgt aatttgttat gttgtgtgca ttattacaat
gttgttactt attcttgtta 2873 agtcggaggc caagggcgaa agctagctca
catgtctgat ggatgcacgt gccatggttg 2933 gtttggtagc gcagtgcaaa
cggcaagaat gggaagtgaa ttcctccctg cttgaaaaaa 2993 aaaaaaa 3000
<210> SEQ ID NO 4 <211> LENGTH: 799 <212> TYPE:
PRT <213> ORGANISM: Triticum aestivum <400> SEQUENCE: 4
Met Ser Ser Ala Val Ala Ser Ala Ala Ser Phe Leu Ala Leu Ala Ser 1 5
10 15 Ala Ser Pro Gly Arg Ser Arg Arg Arg Ala Arg Val Ser Ala Gln
Pro 20 25 30 Pro His Ala Gly Ala Gly Arg Leu His Trp Pro Pro Trp
Pro Pro Gln 35 40 45 Arg Thr Ala Arg Asp Gly Ala Val Ala Ala Leu
Ala Ala Gly Lys Lys 50 55 60 Asp Ala Gly Ile Asp Asp Ala Ala Ala
Ser Val Arg Gln Pro Arg Ala 65 70 75 80 Leu Arg Gly Gly Ala Ala Thr
Lys Val Ala Glu Arg Arg Asp Pro Val 85 90 95 Lys Thr Leu Asp Arg
Asp Ala Ala Glu Gly Gly Gly Pro Ser Pro Pro 100 105 110 Ala Ala Arg
Gln Asp Ala Ala Arg Pro Pro Ser Met Asn Gly Met Pro 115 120 125 Val
Asn Gly Glu Asn Lys Ser Thr Gly Gly Gly Gly Ala Thr Lys Asp 130 135
140 Ser Gly Leu Pro Thr Pro Ala Arg Ala Pro His Pro Ser Thr Gln Asn
145 150 155 160 Arg Ala Pro Val Asn Gly Glu Asn Lys Ala Asn Val Ala
Ser Pro Pro 165 170 175 Thr Ser Ile Ala Glu Ala Ala Ala Ser Asp Ser
Ala Ala Thr Ile Ser 180 185 190 Ile Ser Asp Lys Ala Pro Glu Ser Val
Val Pro Ala Glu Lys Thr Pro 195 200 205 Pro Ser Ser Gly Ser Asn Phe
Glu Ser Ser Ala Ser Ala Pro Gly Ser 210 215 220 Asp Thr Val Ser Asp
Val Glu Gln Glu Leu Lys Lys Gly Ala Val Val 225 230 235 240 Val Glu
Glu Ala Pro Lys Pro Lys Ala Leu Ser Pro Pro Ala Ala Pro 245 250 255
Ala Val Gln Glu Asp Leu Trp Asp Phe Lys Lys Tyr Ile Gly Phe Glu 260
265 270 Glu Pro Val Glu Ala Lys Asp Asp Gly Arg Ala Val Ala Asp Asp
Ala 275 280 285 Gly Ser Phe Glu His His Gln Asn His Asp Ser Gly Pro
Leu Ala Gly
290 295 300 Glu Asn Val Met Asn Val Val Val Val Ala Ala Glu Cys Ser
Pro Trp 305 310 315 320 Cys Lys Thr Gly Gly Leu Gly Asp Val Ala Gly
Ala Leu Pro Lys Ala 325 330 335 Leu Ala Lys Arg Gly His Arg Val Met
Val Val Val Pro Arg Tyr Gly 340 345 350 Asp Tyr Glu Glu Ala Tyr Asp
Val Gly Val Arg Lys Tyr Tyr Lys Ala 355 360 365 Ala Gly Gln Asp Met
Glu Val Asn Tyr Phe His Ala Tyr Ile Asp Gly 370 375 380 Val Asp Phe
Val Phe Ile Asp Ala Pro Leu Phe Arg His Arg Gln Glu 385 390 395 400
Asp Ile Tyr Gly Gly Ser Arg Gln Glu Ile Met Lys Arg Met Ile Leu 405
410 415 Phe Cys Lys Ala Ala Val Glu Val Pro Trp His Val Pro Cys Gly
Gly 420 425 430 Val Pro Tyr Gly Asp Gly Asn Leu Val Phe Ile Ala Asn
Asp Trp His 435 440 445 Thr Ala Leu Leu Pro Val Tyr Leu Lys Ala Tyr
Tyr Arg Asp His Gly 450 455 460 Leu Met Gln Tyr Thr Arg Ser Ile Met
Val Ile His Asn Ile Ala His 465 470 475 480 Gln Gly Arg Gly Pro Val
Asp Glu Phe Pro Phe Thr Glu Leu Pro Glu 485 490 495 His Tyr Leu Glu
His Phe Arg Leu Tyr Asp Pro Val Gly Gly Glu His 500 505 510 Ala Asn
Tyr Phe Ala Ala Gly Leu Lys Met Ala Asp Gln Val Val Val 515 520 525
Val Ser Pro Gly Tyr Leu Trp Glu Leu Lys Thr Val Glu Gly Gly Trp 530
535 540 Gly Leu His Asp Ile Ile Arg Gln Asn Asp Trp Lys Thr Arg Gly
Ile 545 550 555 560 Val Asn Gly Ile Asp Asn Met Glu Trp Asn Pro Glu
Val Asp Ala His 565 570 575 Leu Lys Ser Asp Gly Tyr Thr Asn Phe Ser
Leu Arg Thr Leu Asp Ser 580 585 590 Gly Lys Arg Gln Cys Lys Glu Ala
Leu Gln Arg Glu Leu Gly Leu Gln 595 600 605 Val Arg Ala Asp Val Pro
Leu Leu Gly Phe Ile Gly Arg Leu Asp Gly 610 615 620 Gln Lys Gly Val
Glu Ile Ile Ala Asp Ala Met Pro Trp Ile Val Ser 625 630 635 640 Gln
Asp Val Gln Leu Val Met Leu Gly Thr Gly Arg His Asp Leu Glu 645 650
655 Ser Met Leu Gln His Phe Glu Arg Glu His His Asp Lys Val Arg Gly
660 665 670 Trp Val Gly Phe Ser Val Arg Leu Ala His Arg Ile Thr Ala
Gly Ala 675 680 685 Asp Ala Leu Leu Met Pro Ser Arg Phe Glu Pro Cys
Gly Leu Asn Gln 690 695 700 Leu Tyr Ala Met Ala Tyr Gly Thr Val Pro
Val Val His Ala Val Gly 705 710 715 720 Gly Leu Arg Asp Thr Val Pro
Pro Phe Asp Pro Phe Asn His Ser Gly 725 730 735 Leu Gly Trp Thr Phe
Asp Arg Ala Glu Ala His Lys Leu Ile Glu Ala 740 745 750 Leu Gly His
Cys Leu Arg Thr Tyr Arg Asp Phe Lys Glu Ser Trp Arg 755 760 765 Ala
Leu Gln Glu Arg Gly Met Ser Gln Asp Phe Ser Trp Glu His Ala 770 775
780 Ala Lys Leu Tyr Glu Asp Val Leu Val Lys Ala Lys Tyr Gln Trp 785
790 795 <210> SEQ ID NO 5 <211> LENGTH: 2433
<212> TYPE: DNA <213> ORGANISM: Oryza sativa
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(2430) <400> SEQUENCE: 5 atg tct agc gcg gtg
gtt gcg tcc agc aca act ttt ctc gtc gca ctt 48 Met Ser Ser Ala Val
Val Ala Ser Ser Thr Thr Phe Leu Val Ala Leu 1 5 10 15 gcc tct agc
gcg agc cgg ggc ggg cca cgt agg ggg cgc gtc gtg ggc 96 Ala Ser Ser
Ala Ser Arg Gly Gly Pro Arg Arg Gly Arg Val Val Gly 20 25 30 gtg
gcc gct ccc cca gcc ctc ctg tat gac ggg aga gct ggc agg cta 144 Val
Ala Ala Pro Pro Ala Leu Leu Tyr Asp Gly Arg Ala Gly Arg Leu 35 40
45 gcc ctg cgc gcc cct ccg cca ccc cgc cct aga cct agg cgc agg gat
192 Ala Leu Arg Ala Pro Pro Pro Pro Arg Pro Arg Pro Arg Arg Arg Asp
50 55 60 gcg ggt gtt gtc agg cgg gct gat gac ggg gag aac gag gcc
gca gtg 240 Ala Gly Val Val Arg Arg Ala Asp Asp Gly Glu Asn Glu Ala
Ala Val 65 70 75 80 gag cgg gcc ggc gag gac gat gac gag gag gag gag
ttc tcg tcc ggg 288 Glu Arg Ala Gly Glu Asp Asp Asp Glu Glu Glu Glu
Phe Ser Ser Gly 85 90 95 gcc tgg cag cca ccg cgt tca agg cgc ggt
gga gtt ggc aag gtc ctc 336 Ala Trp Gln Pro Pro Arg Ser Arg Arg Gly
Gly Val Gly Lys Val Leu 100 105 110 aaa cgt cgc ggt acc gtg ccg cca
gtc gga agg tac ggc tcc ggt gga 384 Lys Arg Arg Gly Thr Val Pro Pro
Val Gly Arg Tyr Gly Ser Gly Gly 115 120 125 gac gcc gct cgg gtg aga
gga gcc gcg gca ccc gct cca gca ccg acg 432 Asp Ala Ala Arg Val Arg
Gly Ala Ala Ala Pro Ala Pro Ala Pro Thr 130 135 140 caa gac gca gcg
tcg tct aag aat ggc gcg ctt ttg tca ggc agg gat 480 Gln Asp Ala Ala
Ser Ser Lys Asn Gly Ala Leu Leu Ser Gly Arg Asp 145 150 155 160 gac
gac aca cct gcc tca cgg aac gga tcg gtc gtt acc ggc gcc gac 528 Asp
Asp Thr Pro Ala Ser Arg Asn Gly Ser Val Val Thr Gly Ala Asp 165 170
175 aag cct gcc gcc gcc acg ccg ccg gtg acc ata acg aag ctc cca gcg
576 Lys Pro Ala Ala Ala Thr Pro Pro Val Thr Ile Thr Lys Leu Pro Ala
180 185 190 ccg gac tcc ccc gtg atc ctt cca tcc gta gac aag ccg cag
ccg gag 624 Pro Asp Ser Pro Val Ile Leu Pro Ser Val Asp Lys Pro Gln
Pro Glu 195 200 205 ttc gtc atc cca gac gcg acg gcg ccg gcg ccg cca
ccg ccc ggt tca 672 Phe Val Ile Pro Asp Ala Thr Ala Pro Ala Pro Pro
Pro Pro Gly Ser 210 215 220 aat ccc agg tcg tcc gct cct ctc ccc aag
cct gac aat tcg gaa ttt 720 Asn Pro Arg Ser Ser Ala Pro Leu Pro Lys
Pro Asp Asn Ser Glu Phe 225 230 235 240 gca gag gat aag agc gca aaa
gtt gtt gag agt gct ccg aag cca aag 768 Ala Glu Asp Lys Ser Ala Lys
Val Val Glu Ser Ala Pro Lys Pro Lys 245 250 255 gcg act aga tct tcc
cct att cct gcg gta gaa gag gag acg tgg gat 816 Ala Thr Arg Ser Ser
Pro Ile Pro Ala Val Glu Glu Glu Thr Trp Asp 260 265 270 ttc aag aaa
tat ttt gat ctg aac gaa ccg gac gcc gcg gag gat ggc 864 Phe Lys Lys
Tyr Phe Asp Leu Asn Glu Pro Asp Ala Ala Glu Asp Gly 275 280 285 gat
gac gat gat gac tgg gct gat tca gat gcg tca gat tct gag atc 912 Asp
Asp Asp Asp Asp Trp Ala Asp Ser Asp Ala Ser Asp Ser Glu Ile 290 295
300 gac cag gat gac gat tcg ggt cct ttg gct ggg gag aat gtc atg aac
960 Asp Gln Asp Asp Asp Ser Gly Pro Leu Ala Gly Glu Asn Val Met Asn
305 310 315 320 gtg atc gtg gtg gct gct gaa tgt tct ccc tgg tgc aaa
aca ggt ggg 1008 Val Ile Val Val Ala Ala Glu Cys Ser Pro Trp Cys
Lys Thr Gly Gly 325 330 335 ctt gga gat gtt gca ggt gct tta ccc aag
gct ttg gcg agg aga gga 1056 Leu Gly Asp Val Ala Gly Ala Leu Pro
Lys Ala Leu Ala Arg Arg Gly 340 345 350 cat cgt gtt atg gtt gtc gta
cca agg tac ggt gat tac gcg gaa gcc 1104 His Arg Val Met Val Val
Val Pro Arg Tyr Gly Asp Tyr Ala Glu Ala 355 360 365 cag gat gta gga
atc agg aaa tac tac aag gct gct gga cag gat ctg 1152 Gln Asp Val
Gly Ile Arg Lys Tyr Tyr Lys Ala Ala Gly Gln Asp Leu 370 375 380 gaa
gtg aaa tat ttc cat gca ttt atc gac gga gtt gat ttt gtg ttc 1200
Glu Val Lys Tyr Phe His Ala Phe Ile Asp Gly Val Asp Phe Val Phe 385
390 395 400 att gac gct cct ctc ttc cgt cac cgt cag gat gac atc tat
ggg ggg 1248 Ile Asp Ala Pro Leu Phe Arg His Arg Gln Asp Asp Ile
Tyr Gly Gly 405 410 415 aac aga cag gaa atc atg aag cgc atg att ctg
ttt tgt aag gct gct 1296 Asn Arg Gln Glu Ile Met Lys Arg Met Ile
Leu Phe Cys Lys Ala Ala 420 425 430 gtt gag gtt cct tgg cac gtt cca
tgc ggt ggt gtg ccc tat ggg gat 1344 Val Glu Val Pro Trp His Val
Pro Cys Gly Gly Val Pro Tyr Gly Asp 435 440 445 ggc aac ttg gtg ttc
ctt gca aac gat tgg cac act gca ctc ctg cct 1392 Gly Asn Leu Val
Phe Leu Ala Asn Asp Trp His Thr Ala Leu Leu Pro 450 455 460 gtt tat
ctg aag gca tat tac aga gac aat ggc atg atg cag tac act 1440 Val
Tyr Leu Lys Ala Tyr Tyr Arg Asp Asn Gly Met Met Gln Tyr Thr 465 470
475 480 cgc tct gtc ctt gtg ata cat aat atc gct tac cag ggc cgt ggc
cca 1488 Arg Ser Val Leu Val Ile His Asn Ile Ala Tyr Gln Gly Arg
Gly Pro 485 490 495 gta gat gaa ttc ccc tac atg gaa ttg ccg gag cac
tac ctg gat cac 1536 Val Asp Glu Phe Pro Tyr Met Glu Leu Pro Glu
His Tyr Leu Asp His 500 505 510 ttc aag ctg tac gac ccc gtc ggc ggc
gag cac gcc aac atc ttc ggc 1584 Phe Lys Leu Tyr Asp Pro Val Gly
Gly Glu His Ala Asn Ile Phe Gly 515 520 525 gcg ggc ctg aag atg gcg
gac cgg gtg gtg acc gtg agc ccc ggc tac 1632 Ala Gly Leu Lys Met
Ala Asp Arg Val Val Thr Val Ser Pro Gly Tyr 530 535 540 ctc tgg gag
ctg aag acg acg gag ggc ggc tgg ggc ctc cac gac atc 1680 Leu Trp
Glu Leu Lys Thr Thr Glu Gly Gly Trp Gly Leu His Asp Ile 545 550 555
560 ata cgg gag aac gac tgg aag atg aac ggc atc gtg aac ggc atc gac
1728 Ile Arg Glu Asn Asp Trp Lys Met Asn Gly Ile Val Asn Gly Ile
Asp 565 570 575 tac cgg gag tgg aac ccg gag gtg gac gtg cac ctg cag
tcc gac ggc 1776
Tyr Arg Glu Trp Asn Pro Glu Val Asp Val His Leu Gln Ser Asp Gly 580
585 590 tac gcc aac tac acc gtg gcc tcg ctg gac tcc agc aag ccg cgg
tgc 1824 Tyr Ala Asn Tyr Thr Val Ala Ser Leu Asp Ser Ser Lys Pro
Arg Cys 595 600 605 aag gcg gcg ctg cag cgc gag ctg ggg ctg gag gtg
cgc gac gac gtg 1872 Lys Ala Ala Leu Gln Arg Glu Leu Gly Leu Glu
Val Arg Asp Asp Val 610 615 620 ccg ctg atc ggg ttc atc ggg cgg ctc
gac ggg cag aaa ggt gtg gac 1920 Pro Leu Ile Gly Phe Ile Gly Arg
Leu Asp Gly Gln Lys Gly Val Asp 625 630 635 640 atc atc ggc gac gcg
atg ccg tgg atc gcc ggg cag gac gtg cag ctg 1968 Ile Ile Gly Asp
Ala Met Pro Trp Ile Ala Gly Gln Asp Val Gln Leu 645 650 655 gtg ctg
ctg ggc tcc ggc cgc cgc gac ctg gag gtg atg ctg cag cgg 2016 Val
Leu Leu Gly Ser Gly Arg Arg Asp Leu Glu Val Met Leu Gln Arg 660 665
670 ttc gag gcg cag cac aac agc aag gtg cgc ggg tgg gtg ggg ttc tcg
2064 Phe Glu Ala Gln His Asn Ser Lys Val Arg Gly Trp Val Gly Phe
Ser 675 680 685 gtg aag atg gcg cac cgg atc acg gcg ggc gcc gac gtg
ctg gtc atg 2112 Val Lys Met Ala His Arg Ile Thr Ala Gly Ala Asp
Val Leu Val Met 690 695 700 ccg tcg cgg ttc gag ccg tgc ggc ctc aac
cag ctc tac gcc atg gcg 2160 Pro Ser Arg Phe Glu Pro Cys Gly Leu
Asn Gln Leu Tyr Ala Met Ala 705 710 715 720 tac ggc acc gtc ccc gtc
gtg cac gcc gtc ggc ggg ctg agg gac acc 2208 Tyr Gly Thr Val Pro
Val Val His Ala Val Gly Gly Leu Arg Asp Thr 725 730 735 gtg tcg gcg
ttc gac ccg ttc gag gac acc ggc ctc ggg tgg acg ttc 2256 Val Ser
Ala Phe Asp Pro Phe Glu Asp Thr Gly Leu Gly Trp Thr Phe 740 745 750
gac cgc gcc gag ccg cac aag ctc atc gag gcg ctc ggc cac tgc ctg
2304 Asp Arg Ala Glu Pro His Lys Leu Ile Glu Ala Leu Gly His Cys
Leu 755 760 765 gag acg tac cgc aag tac aag gag agc tgg agg ggg ctc
cag gtg cgc 2352 Glu Thr Tyr Arg Lys Tyr Lys Glu Ser Trp Arg Gly
Leu Gln Val Arg 770 775 780 ggc atg tcg cag gac ctc agc tgg gac cac
gcc gcc gag ctc tac gag 2400 Gly Met Ser Gln Asp Leu Ser Trp Asp
His Ala Ala Glu Leu Tyr Glu 785 790 795 800 gag gtc ctt gtc aag gcc
aag tac caa tgg tga 2433 Glu Val Leu Val Lys Ala Lys Tyr Gln Trp
805 810 <210> SEQ ID NO 6 <211> LENGTH: 810 <212>
TYPE: PRT <213> ORGANISM: Oryza sativa <400> SEQUENCE:
6 Met Ser Ser Ala Val Val Ala Ser Ser Thr Thr Phe Leu Val Ala Leu 1
5 10 15 Ala Ser Ser Ala Ser Arg Gly Gly Pro Arg Arg Gly Arg Val Val
Gly 20 25 30 Val Ala Ala Pro Pro Ala Leu Leu Tyr Asp Gly Arg Ala
Gly Arg Leu 35 40 45 Ala Leu Arg Ala Pro Pro Pro Pro Arg Pro Arg
Pro Arg Arg Arg Asp 50 55 60 Ala Gly Val Val Arg Arg Ala Asp Asp
Gly Glu Asn Glu Ala Ala Val 65 70 75 80 Glu Arg Ala Gly Glu Asp Asp
Asp Glu Glu Glu Glu Phe Ser Ser Gly 85 90 95 Ala Trp Gln Pro Pro
Arg Ser Arg Arg Gly Gly Val Gly Lys Val Leu 100 105 110 Lys Arg Arg
Gly Thr Val Pro Pro Val Gly Arg Tyr Gly Ser Gly Gly 115 120 125 Asp
Ala Ala Arg Val Arg Gly Ala Ala Ala Pro Ala Pro Ala Pro Thr 130 135
140 Gln Asp Ala Ala Ser Ser Lys Asn Gly Ala Leu Leu Ser Gly Arg Asp
145 150 155 160 Asp Asp Thr Pro Ala Ser Arg Asn Gly Ser Val Val Thr
Gly Ala Asp 165 170 175 Lys Pro Ala Ala Ala Thr Pro Pro Val Thr Ile
Thr Lys Leu Pro Ala 180 185 190 Pro Asp Ser Pro Val Ile Leu Pro Ser
Val Asp Lys Pro Gln Pro Glu 195 200 205 Phe Val Ile Pro Asp Ala Thr
Ala Pro Ala Pro Pro Pro Pro Gly Ser 210 215 220 Asn Pro Arg Ser Ser
Ala Pro Leu Pro Lys Pro Asp Asn Ser Glu Phe 225 230 235 240 Ala Glu
Asp Lys Ser Ala Lys Val Val Glu Ser Ala Pro Lys Pro Lys 245 250 255
Ala Thr Arg Ser Ser Pro Ile Pro Ala Val Glu Glu Glu Thr Trp Asp 260
265 270 Phe Lys Lys Tyr Phe Asp Leu Asn Glu Pro Asp Ala Ala Glu Asp
Gly 275 280 285 Asp Asp Asp Asp Asp Trp Ala Asp Ser Asp Ala Ser Asp
Ser Glu Ile 290 295 300 Asp Gln Asp Asp Asp Ser Gly Pro Leu Ala Gly
Glu Asn Val Met Asn 305 310 315 320 Val Ile Val Val Ala Ala Glu Cys
Ser Pro Trp Cys Lys Thr Gly Gly 325 330 335 Leu Gly Asp Val Ala Gly
Ala Leu Pro Lys Ala Leu Ala Arg Arg Gly 340 345 350 His Arg Val Met
Val Val Val Pro Arg Tyr Gly Asp Tyr Ala Glu Ala 355 360 365 Gln Asp
Val Gly Ile Arg Lys Tyr Tyr Lys Ala Ala Gly Gln Asp Leu 370 375 380
Glu Val Lys Tyr Phe His Ala Phe Ile Asp Gly Val Asp Phe Val Phe 385
390 395 400 Ile Asp Ala Pro Leu Phe Arg His Arg Gln Asp Asp Ile Tyr
Gly Gly 405 410 415 Asn Arg Gln Glu Ile Met Lys Arg Met Ile Leu Phe
Cys Lys Ala Ala 420 425 430 Val Glu Val Pro Trp His Val Pro Cys Gly
Gly Val Pro Tyr Gly Asp 435 440 445 Gly Asn Leu Val Phe Leu Ala Asn
Asp Trp His Thr Ala Leu Leu Pro 450 455 460 Val Tyr Leu Lys Ala Tyr
Tyr Arg Asp Asn Gly Met Met Gln Tyr Thr 465 470 475 480 Arg Ser Val
Leu Val Ile His Asn Ile Ala Tyr Gln Gly Arg Gly Pro 485 490 495 Val
Asp Glu Phe Pro Tyr Met Glu Leu Pro Glu His Tyr Leu Asp His 500 505
510 Phe Lys Leu Tyr Asp Pro Val Gly Gly Glu His Ala Asn Ile Phe Gly
515 520 525 Ala Gly Leu Lys Met Ala Asp Arg Val Val Thr Val Ser Pro
Gly Tyr 530 535 540 Leu Trp Glu Leu Lys Thr Thr Glu Gly Gly Trp Gly
Leu His Asp Ile 545 550 555 560 Ile Arg Glu Asn Asp Trp Lys Met Asn
Gly Ile Val Asn Gly Ile Asp 565 570 575 Tyr Arg Glu Trp Asn Pro Glu
Val Asp Val His Leu Gln Ser Asp Gly 580 585 590 Tyr Ala Asn Tyr Thr
Val Ala Ser Leu Asp Ser Ser Lys Pro Arg Cys 595 600 605 Lys Ala Ala
Leu Gln Arg Glu Leu Gly Leu Glu Val Arg Asp Asp Val 610 615 620 Pro
Leu Ile Gly Phe Ile Gly Arg Leu Asp Gly Gln Lys Gly Val Asp 625 630
635 640 Ile Ile Gly Asp Ala Met Pro Trp Ile Ala Gly Gln Asp Val Gln
Leu 645 650 655 Val Leu Leu Gly Ser Gly Arg Arg Asp Leu Glu Val Met
Leu Gln Arg 660 665 670 Phe Glu Ala Gln His Asn Ser Lys Val Arg Gly
Trp Val Gly Phe Ser 675 680 685 Val Lys Met Ala His Arg Ile Thr Ala
Gly Ala Asp Val Leu Val Met 690 695 700 Pro Ser Arg Phe Glu Pro Cys
Gly Leu Asn Gln Leu Tyr Ala Met Ala 705 710 715 720 Tyr Gly Thr Val
Pro Val Val His Ala Val Gly Gly Leu Arg Asp Thr 725 730 735 Val Ser
Ala Phe Asp Pro Phe Glu Asp Thr Gly Leu Gly Trp Thr Phe 740 745 750
Asp Arg Ala Glu Pro His Lys Leu Ile Glu Ala Leu Gly His Cys Leu 755
760 765 Glu Thr Tyr Arg Lys Tyr Lys Glu Ser Trp Arg Gly Leu Gln Val
Arg 770 775 780 Gly Met Ser Gln Asp Leu Ser Trp Asp His Ala Ala Glu
Leu Tyr Glu 785 790 795 800 Glu Val Leu Val Lys Ala Lys Tyr Gln Trp
805 810 <210> SEQ ID NO 7 <211> LENGTH: 1830
<212> TYPE: DNA <213> ORGANISM: Oryza sativa
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(1827) <400> SEQUENCE: 7 atg tcg gct ctc acc
acg tcc cag ctc gcc acc tcg gcc acc ggc ttc 48 Met Ser Ala Leu Thr
Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe 1 5 10 15 ggc atc gcc
gac agg tcg gcg ccg tcg tcg ctg ctc cgc cac ggg ttc 96 Gly Ile Ala
Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe 20 25 30 cag
ggc ctc aag ccc cgc agc ccc gcc ggc ggc gac gcg acg tcg ctc 144 Gln
Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu 35 40
45 agc gtg acg acc agc gcg cgc gcg acg ccc aag cag cag cgg tcg gtg
192 Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val
50 55 60 cag cgt ggc agc cgg agg ttc ccc tcc gtc gtc gtg tac gcc
acc ggc 240 Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala
Thr Gly 65 70 75 80
gcc ggc atg aac gtc gtg ttc gtc ggc gcc gag atg gcc ccc tgg agc 288
Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser 85
90 95 aag acc ggc ggc ctc ggt gac gtc ctc ggt ggc ctc ccc cct gcc
atg 336 Lys Thr Gly Gly Leu Gly Asp Val Leu Gly Gly Leu Pro Pro Ala
Met 100 105 110 gct gcg aat ggc cac agg gtc atg gtg atc tct cct cgg
tac gac cag 384 Ala Ala Asn Gly His Arg Val Met Val Ile Ser Pro Arg
Tyr Asp Gln 115 120 125 tac aag gac gct tgg gat acc agc gtt gtg gct
gag atc aag gtt gca 432 Tyr Lys Asp Ala Trp Asp Thr Ser Val Val Ala
Glu Ile Lys Val Ala 130 135 140 gac agg tac gag agg gtg agg ttt ttc
cat tgc tac aag cgt gga gtc 480 Asp Arg Tyr Glu Arg Val Arg Phe Phe
His Cys Tyr Lys Arg Gly Val 145 150 155 160 gac cgt gtg ttc atc gac
cat ccg tca ttc ctg gag aag gtt tgg gga 528 Asp Arg Val Phe Ile Asp
His Pro Ser Phe Leu Glu Lys Val Trp Gly 165 170 175 aag acc ggt gag
aag atc tac gga cct gac act gga gtt gat tac aaa 576 Lys Thr Gly Glu
Lys Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys 180 185 190 gac aac
cag atg cgt ttc agc ctt ctt tgc cag gca gca ctc gag gct 624 Asp Asn
Gln Met Arg Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala 195 200 205
cct agg atc cta aac ctc aac aac aac cca tac ttc aaa gga act tat 672
Pro Arg Ile Leu Asn Leu Asn Asn Asn Pro Tyr Phe Lys Gly Thr Tyr 210
215 220 ggt gag gat gtt gtg ttc gtc tgc aac gac tgg cac act ggc cca
ctg 720 Gly Glu Asp Val Val Phe Val Cys Asn Asp Trp His Thr Gly Pro
Leu 225 230 235 240 gcg agc tac ctg aag aac aac tac cag ccc aat ggc
atc tac agg aat 768 Ala Ser Tyr Leu Lys Asn Asn Tyr Gln Pro Asn Gly
Ile Tyr Arg Asn 245 250 255 gca aag gtt gct ttc tgc atc cac aac atc
tcc tac cag ggc cgt ttc 816 Ala Lys Val Ala Phe Cys Ile His Asn Ile
Ser Tyr Gln Gly Arg Phe 260 265 270 gct ttc gag gat tac cct gag ctg
aac ctc tcc gag agg ttc agg tca 864 Ala Phe Glu Asp Tyr Pro Glu Leu
Asn Leu Ser Glu Arg Phe Arg Ser 275 280 285 tcc ttc gat ttc atc gac
ggg tat gac acg ccg gtg gag ggc agg aag 912 Ser Phe Asp Phe Ile Asp
Gly Tyr Asp Thr Pro Val Glu Gly Arg Lys 290 295 300 atc aac tgg atg
aag gcc gga atc ctg gaa gcc gac agg gtg ctc acc 960 Ile Asn Trp Met
Lys Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr 305 310 315 320 gtg
agc ccg tac tac gcc gag gag ctc atc tcc ggc atc gcc agg gga 1008
Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly 325
330 335 tgc gag ctc gac aac atc atg cgg ctc acc ggc atc acc ggc atc
gtc 1056 Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly
Ile Val 340 345 350 aac ggc atg gac gtc agc gag tgg gat ccc agc aag
gac aag tac atc 1104 Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser
Lys Asp Lys Tyr Ile 355 360 365 acc gcc aag tac gac gca acc acg gca
atc gag gcg aag gcg ctg aac 1152 Thr Ala Lys Tyr Asp Ala Thr Thr
Ala Ile Glu Ala Lys Ala Leu Asn 370 375 380 aag gag gcg ttg cag gcg
gag gcg ggt ctt ccg gtc gac agg aaa atc 1200 Lys Glu Ala Leu Gln
Ala Glu Ala Gly Leu Pro Val Asp Arg Lys Ile 385 390 395 400 cca ctg
atc gcg ttc atc ggc agg ctg gag gaa cag aag ggc tct gac 1248 Pro
Leu Ile Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp 405 410
415 gtc atg gcc gcc gcc atc ccg gag ctc atg cag gag gac gtc cag atc
1296 Val Met Ala Ala Ala Ile Pro Glu Leu Met Gln Glu Asp Val Gln
Ile 420 425 430 gtt ctt ctg ggt act gga aag aag aag ttc gag aag ctg
ctc aag agc 1344 Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys
Leu Leu Lys Ser 435 440 445 atg gag gag aag tat ccg ggc aag gtg agg
gcc gtg gtg aag ttc aac 1392 Met Glu Glu Lys Tyr Pro Gly Lys Val
Arg Ala Val Val Lys Phe Asn 450 455 460 gcg ccg ctt gct cat ctc atc
atg gcc gga gcc gac gtg ctc gcc gtc 1440 Ala Pro Leu Ala His Leu
Ile Met Ala Gly Ala Asp Val Leu Ala Val 465 470 475 480 ccc agc cgc
ttc gag ccc tgt gga ctc atc cag ctg cag ggg atg aga 1488 Pro Ser
Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg 485 490 495
tac gga acg ccc tgt gct tgc gcg tcc acc ggt ggg ctc gtg gac acg
1536 Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp
Thr 500 505 510 gtc atc gaa ggc aag act ggt ttc cac atg ggc cgt ctc
agc gtc gac 1584 Val Ile Glu Gly Lys Thr Gly Phe His Met Gly Arg
Leu Ser Val Asp 515 520 525 tgc aag gtg gtg gag cca agc gac gtg aag
aag gtg gcg gcc acc ctg 1632 Cys Lys Val Val Glu Pro Ser Asp Val
Lys Lys Val Ala Ala Thr Leu 530 535 540 aag cgc gcc atc aag gtc gtc
ggc acg ccg gcg tac gag gag atg gtc 1680 Lys Arg Ala Ile Lys Val
Val Gly Thr Pro Ala Tyr Glu Glu Met Val 545 550 555 560 agg aac tgc
atg aac cag gac ctc tcc tgg aag ggg cct gcg aag aac 1728 Arg Asn
Cys Met Asn Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn 565 570 575
tgg gag aat gtg ctc ctg ggc ctg ggc gtc gcc ggc agc gcg ccg ggg
1776 Trp Glu Asn Val Leu Leu Gly Leu Gly Val Ala Gly Ser Ala Pro
Gly 580 585 590 atc gaa ggc gac gag atc gcg ccg ctc gcc aag gag aac
gtg gct gct 1824 Ile Glu Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu
Asn Val Ala Ala 595 600 605 cct tga 1830 Pro <210> SEQ ID NO
8 <211> LENGTH: 609 <212> TYPE: PRT <213>
ORGANISM: Oryza sativa <400> SEQUENCE: 8 Met Ser Ala Leu Thr
Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe 1 5 10 15 Gly Ile Ala
Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe 20 25 30 Gln
Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu 35 40
45 Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val
50 55 60 Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala
Thr Gly 65 70 75 80 Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met
Ala Pro Trp Ser 85 90 95 Lys Thr Gly Gly Leu Gly Asp Val Leu Gly
Gly Leu Pro Pro Ala Met 100 105 110 Ala Ala Asn Gly His Arg Val Met
Val Ile Ser Pro Arg Tyr Asp Gln 115 120 125 Tyr Lys Asp Ala Trp Asp
Thr Ser Val Val Ala Glu Ile Lys Val Ala 130 135 140 Asp Arg Tyr Glu
Arg Val Arg Phe Phe His Cys Tyr Lys Arg Gly Val 145 150 155 160 Asp
Arg Val Phe Ile Asp His Pro Ser Phe Leu Glu Lys Val Trp Gly 165 170
175 Lys Thr Gly Glu Lys Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys
180 185 190 Asp Asn Gln Met Arg Phe Ser Leu Leu Cys Gln Ala Ala Leu
Glu Ala 195 200 205 Pro Arg Ile Leu Asn Leu Asn Asn Asn Pro Tyr Phe
Lys Gly Thr Tyr 210 215 220 Gly Glu Asp Val Val Phe Val Cys Asn Asp
Trp His Thr Gly Pro Leu 225 230 235 240 Ala Ser Tyr Leu Lys Asn Asn
Tyr Gln Pro Asn Gly Ile Tyr Arg Asn 245 250 255 Ala Lys Val Ala Phe
Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe 260 265 270 Ala Phe Glu
Asp Tyr Pro Glu Leu Asn Leu Ser Glu Arg Phe Arg Ser 275 280 285 Ser
Phe Asp Phe Ile Asp Gly Tyr Asp Thr Pro Val Glu Gly Arg Lys 290 295
300 Ile Asn Trp Met Lys Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr
305 310 315 320 Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile
Ala Arg Gly 325 330 335 Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly
Ile Thr Gly Ile Val 340 345 350 Asn Gly Met Asp Val Ser Glu Trp Asp
Pro Ser Lys Asp Lys Tyr Ile 355 360 365 Thr Ala Lys Tyr Asp Ala Thr
Thr Ala Ile Glu Ala Lys Ala Leu Asn 370 375 380 Lys Glu Ala Leu Gln
Ala Glu Ala Gly Leu Pro Val Asp Arg Lys Ile 385 390 395 400 Pro Leu
Ile Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Ser Asp 405 410 415
Val Met Ala Ala Ala Ile Pro Glu Leu Met Gln Glu Asp Val Gln Ile 420
425 430 Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Leu Leu Lys
Ser 435 440 445 Met Glu Glu Lys Tyr Pro Gly Lys Val Arg Ala Val Val
Lys Phe Asn 450 455 460 Ala Pro Leu Ala His Leu Ile Met Ala Gly Ala
Asp Val Leu Ala Val 465 470 475 480 Pro Ser Arg Phe Glu Pro Cys Gly
Leu Ile Gln Leu Gln Gly Met Arg 485 490 495 Tyr Gly Thr Pro Cys Ala
Cys Ala Ser Thr Gly Gly Leu Val Asp Thr 500 505 510 Val Ile Glu Gly
Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp 515 520 525 Cys Lys
Val Val Glu Pro Ser Asp Val Lys Lys Val Ala Ala Thr Leu 530 535 540
Lys Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr Glu Glu Met Val 545
550 555 560 Arg Asn Cys Met Asn Gln Asp Leu Ser Trp Lys Gly Pro Ala
Lys Asn 565 570 575 Trp Glu Asn Val Leu Leu Gly Leu Gly Val Ala Gly
Ser Ala Pro Gly
580 585 590 Ile Glu Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu Asn Val
Ala Ala 595 600 605 Pro <210> SEQ ID NO 9 <211> LENGTH:
1818 <212> TYPE: DNA <213> ORGANISM: Triticum aestivum
<220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(1815) <400> SEQUENCE: 9 atg gcg gct ctg gtc
acg tcg cag ctc gcc acc tcc ggc acc gtc ctc 48 Met Ala Ala Leu Val
Thr Ser Gln Leu Ala Thr Ser Gly Thr Val Leu 1 5 10 15 ggc atc acc
gac agg ttc cgg cgt gca ggt ttt cag ggt gtg agg ccc 96 Gly Ile Thr
Asp Arg Phe Arg Arg Ala Gly Phe Gln Gly Val Arg Pro 20 25 30 cgg
agc ccg gca gat gcg ccg ctc ggc atg agg act acc gga gcg agc 144 Arg
Ser Pro Ala Asp Ala Pro Leu Gly Met Arg Thr Thr Gly Ala Ser 35 40
45 gcc gcc ccg aag caa caa agc cgg aaa gcg cac cgc ggg acc cgg cgg
192 Ala Ala Pro Lys Gln Gln Ser Arg Lys Ala His Arg Gly Thr Arg Arg
50 55 60 tgc ctc tcc atg gtg gtg cgc gcc acg ggc agc gcc ggc atg
aac ctc 240 Cys Leu Ser Met Val Val Arg Ala Thr Gly Ser Ala Gly Met
Asn Leu 65 70 75 80 gtg ttc gtc ggc gcc gag atg gcg ccc tgg agc aag
acc ggc ggc ctc 288 Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser Lys
Thr Gly Gly Leu 85 90 95 ggc gac gtc ctc ggg ggc ctc ccc cca gcc
atg gcc gcc aac ggt cac 336 Gly Asp Val Leu Gly Gly Leu Pro Pro Ala
Met Ala Ala Asn Gly His 100 105 110 cgg gtc atg gtc atc tcc ccg cgc
tac gac cag tac aag gac gcc tgg 384 Arg Val Met Val Ile Ser Pro Arg
Tyr Asp Gln Tyr Lys Asp Ala Trp 115 120 125 gac acc agc gtc gtc tcc
gag atc aag gtc gcg gac gag tac gag agg 432 Asp Thr Ser Val Val Ser
Glu Ile Lys Val Ala Asp Glu Tyr Glu Arg 130 135 140 gtg agg tac ttc
cac tgc tac aag cgc ggg gtg gac cgc gtg ttc gtc 480 Val Arg Tyr Phe
His Cys Tyr Lys Arg Gly Val Asp Arg Val Phe Val 145 150 155 160 gac
cac ccg tgc ttc ctg gag aag gtc cgg ggc aag acc aag gag aag 528 Asp
His Pro Cys Phe Leu Glu Lys Val Arg Gly Lys Thr Lys Glu Lys 165 170
175 atc tac ggg ccc gat gcc ggc acg gac tac gag gac aac cag cta cgc
576 Ile Tyr Gly Pro Asp Ala Gly Thr Asp Tyr Glu Asp Asn Gln Leu Arg
180 185 190 ttc agc ctg ctc tgc cag gca gcg ctt gag gca ccc agg atc
ctc gac 624 Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala Pro Arg Ile
Leu Asp 195 200 205 ctc aac aac aac cca tac ttc tcc gga ccc tac ggg
gaa gac gtg gtg 672 Leu Asn Asn Asn Pro Tyr Phe Ser Gly Pro Tyr Gly
Glu Asp Val Val 210 215 220 ttc gtg tgc aac gac tgg cac acg ggc ctt
ctg gcc tgc tac ctc aag 720 Phe Val Cys Asn Asp Trp His Thr Gly Leu
Leu Ala Cys Tyr Leu Lys 225 230 235 240 agc aac tac cag tcc agt ggc
atc tat agg acg gcc aag gta gcg ttc 768 Ser Asn Tyr Gln Ser Ser Gly
Ile Tyr Arg Thr Ala Lys Val Ala Phe 245 250 255 tgc atc cac aac atc
tcg tat cag ggc cgc ttc tcc ttc gac gac ttc 816 Cys Ile His Asn Ile
Ser Tyr Gln Gly Arg Phe Ser Phe Asp Asp Phe 260 265 270 gcg cag ctc
aac ctg ccc gac agg ttc aag tcg tcc ttc gac ttc atc 864 Ala Gln Leu
Asn Leu Pro Asp Arg Phe Lys Ser Ser Phe Asp Phe Ile 275 280 285 gac
ggc tac gac aag ccg gtg gag ggg cgc aag atc aac tgg atg aag 912 Asp
Gly Tyr Asp Lys Pro Val Glu Gly Arg Lys Ile Asn Trp Met Lys 290 295
300 gcc ggg atc ctg cag gcc gac aag gtg ctc acg gtg agc ccc tac tac
960 Ala Gly Ile Leu Gln Ala Asp Lys Val Leu Thr Val Ser Pro Tyr Tyr
305 310 315 320 gcg gag gag ctc atc tcc ggc gaa gcc agg ggc tgc gag
ctc gac aac 1008 Ala Glu Glu Leu Ile Ser Gly Glu Ala Arg Gly Cys
Glu Leu Asp Asn 325 330 335 atc atg cgc ctc acg ggc atc acc ggc atc
gtc aac ggc atg gac gtc 1056 Ile Met Arg Leu Thr Gly Ile Thr Gly
Ile Val Asn Gly Met Asp Val 340 345 350 agc gag tgg gac ccc gcc aag
gac aag ttc ctc gcc gcc aac tac gac 1104 Ser Glu Trp Asp Pro Ala
Lys Asp Lys Phe Leu Ala Ala Asn Tyr Asp 355 360 365 gtc acc acc gcg
ttg gag ggg aag gcg ctg aac aag gag gcg ctg cag 1152 Val Thr Thr
Ala Leu Glu Gly Lys Ala Leu Asn Lys Glu Ala Leu Gln 370 375 380 gcc
gag gtg ggg ctg ccg gtg gac cgg aag gtg ccc ctg gtg gcc ttc 1200
Ala Glu Val Gly Leu Pro Val Asp Arg Lys Val Pro Leu Val Ala Phe 385
390 395 400 atc ggc agg ctg gag gag cag aag ggc ccc gac gtg atg atc
gcc gcc 1248 Ile Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp Val Met
Ile Ala Ala 405 410 415 atc ccg gag atc ttg aag gag gag gac gtc cag
atc gtt ctc ctg ggc 1296 Ile Pro Glu Ile Leu Lys Glu Glu Asp Val
Gln Ile Val Leu Leu Gly 420 425 430 acc ggg aag aag aag ttt gag cgg
ctg ctc aag agc gtg gag gag aag 1344 Thr Gly Lys Lys Lys Phe Glu
Arg Leu Leu Lys Ser Val Glu Glu Lys 435 440 445 ttc ccg agc aag gtg
agg gcc gtg gtc agg ttc aac gcg ccg ctg gct 1392 Phe Pro Ser Lys
Val Arg Ala Val Val Arg Phe Asn Ala Pro Leu Ala 450 455 460 cac cag
atg atg gcc ggc gcc gac gtg ctc gcc gtc acc agc cgc ttc 1440 His
Gln Met Met Ala Gly Ala Asp Val Leu Ala Val Thr Ser Arg Phe 465 470
475 480 gag ccc tgc ggc ctc atc cag ctc cag ggg atg cgc tac gga acg
ccg 1488 Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg Tyr Gly
Thr Pro 485 490 495 tgc gcg tgc gcg tcc acc ggc ggg ctc gtc gac acg
atc atg gag ggc 1536 Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp
Thr Ile Met Glu Gly 500 505 510 aag acc ggg ttc cac atg ggc cgc ctc
agc gtc gac tgc aac gtg gtg 1584 Lys Thr Gly Phe His Met Gly Arg
Leu Ser Val Asp Cys Asn Val Val 515 520 525 gag ccg gcc gac gtg aag
aag gtg gtg acc acc ctg aag cgc gcc gtc 1632 Glu Pro Ala Asp Val
Lys Lys Val Val Thr Thr Leu Lys Arg Ala Val 530 535 540 aag gtc gtc
ggc acg cca gcc tac cat gag atg gtc aag aac tgc atg 1680 Lys Val
Val Gly Thr Pro Ala Tyr His Glu Met Val Lys Asn Cys Met 545 550 555
560 atc cag gat ctc tcc tgg aag ggg cca gcc aag aac tgg gag gac gtg
1728 Ile Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn Trp Glu Asp
Val 565 570 575 ctt ctg gaa ctg ggg gtc gag ggg agc gag cca ggg gtc
atc ggc gag 1776 Leu Leu Glu Leu Gly Val Glu Gly Ser Glu Pro Gly
Val Ile Gly Glu 580 585 590 gag att gcg ccg ctc gcc atg gag aac gtc
gcc gct ccc tga 1818 Glu Ile Ala Pro Leu Ala Met Glu Asn Val Ala
Ala Pro 595 600 605 <210> SEQ ID NO 10 <211> LENGTH:
605 <212> TYPE: PRT <213> ORGANISM: Triticum aestivum
<400> SEQUENCE: 10 Met Ala Ala Leu Val Thr Ser Gln Leu Ala
Thr Ser Gly Thr Val Leu 1 5 10 15 Gly Ile Thr Asp Arg Phe Arg Arg
Ala Gly Phe Gln Gly Val Arg Pro 20 25 30 Arg Ser Pro Ala Asp Ala
Pro Leu Gly Met Arg Thr Thr Gly Ala Ser 35 40 45 Ala Ala Pro Lys
Gln Gln Ser Arg Lys Ala His Arg Gly Thr Arg Arg 50 55 60 Cys Leu
Ser Met Val Val Arg Ala Thr Gly Ser Ala Gly Met Asn Leu 65 70 75 80
Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser Lys Thr Gly Gly Leu 85
90 95 Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met Ala Ala Asn Gly
His 100 105 110 Arg Val Met Val Ile Ser Pro Arg Tyr Asp Gln Tyr Lys
Asp Ala Trp 115 120 125 Asp Thr Ser Val Val Ser Glu Ile Lys Val Ala
Asp Glu Tyr Glu Arg 130 135 140 Val Arg Tyr Phe His Cys Tyr Lys Arg
Gly Val Asp Arg Val Phe Val 145 150 155 160 Asp His Pro Cys Phe Leu
Glu Lys Val Arg Gly Lys Thr Lys Glu Lys 165 170 175 Ile Tyr Gly Pro
Asp Ala Gly Thr Asp Tyr Glu Asp Asn Gln Leu Arg 180 185 190 Phe Ser
Leu Leu Cys Gln Ala Ala Leu Glu Ala Pro Arg Ile Leu Asp 195 200 205
Leu Asn Asn Asn Pro Tyr Phe Ser Gly Pro Tyr Gly Glu Asp Val Val 210
215 220 Phe Val Cys Asn Asp Trp His Thr Gly Leu Leu Ala Cys Tyr Leu
Lys 225 230 235 240 Ser Asn Tyr Gln Ser Ser Gly Ile Tyr Arg Thr Ala
Lys Val Ala Phe 245 250 255 Cys Ile His Asn Ile Ser Tyr Gln Gly Arg
Phe Ser Phe Asp Asp Phe 260 265 270 Ala Gln Leu Asn Leu Pro Asp Arg
Phe Lys Ser Ser Phe Asp Phe Ile 275 280 285 Asp Gly Tyr Asp Lys Pro
Val Glu Gly Arg Lys Ile Asn Trp Met Lys 290 295 300 Ala Gly Ile Leu
Gln Ala Asp Lys Val Leu Thr Val Ser Pro Tyr Tyr 305 310 315 320 Ala
Glu Glu Leu Ile Ser Gly Glu Ala Arg Gly Cys Glu Leu Asp Asn 325 330
335 Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val Asn Gly Met Asp Val
340 345 350 Ser Glu Trp Asp Pro Ala Lys Asp Lys Phe Leu Ala Ala Asn
Tyr Asp 355 360 365 Val Thr Thr Ala Leu Glu Gly Lys Ala Leu Asn Lys
Glu Ala Leu Gln 370 375 380 Ala Glu Val Gly Leu Pro Val Asp Arg Lys
Val Pro Leu Val Ala Phe 385 390 395 400
Ile Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp Val Met Ile Ala Ala 405
410 415 Ile Pro Glu Ile Leu Lys Glu Glu Asp Val Gln Ile Val Leu Leu
Gly 420 425 430 Thr Gly Lys Lys Lys Phe Glu Arg Leu Leu Lys Ser Val
Glu Glu Lys 435 440 445 Phe Pro Ser Lys Val Arg Ala Val Val Arg Phe
Asn Ala Pro Leu Ala 450 455 460 His Gln Met Met Ala Gly Ala Asp Val
Leu Ala Val Thr Ser Arg Phe 465 470 475 480 Glu Pro Cys Gly Leu Ile
Gln Leu Gln Gly Met Arg Tyr Gly Thr Pro 485 490 495 Cys Ala Cys Ala
Ser Thr Gly Gly Leu Val Asp Thr Ile Met Glu Gly 500 505 510 Lys Thr
Gly Phe His Met Gly Arg Leu Ser Val Asp Cys Asn Val Val 515 520 525
Glu Pro Ala Asp Val Lys Lys Val Val Thr Thr Leu Lys Arg Ala Val 530
535 540 Lys Val Val Gly Thr Pro Ala Tyr His Glu Met Val Lys Asn Cys
Met 545 550 555 560 Ile Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn
Trp Glu Asp Val 565 570 575 Leu Leu Glu Leu Gly Val Glu Gly Ser Glu
Pro Gly Val Ile Gly Glu 580 585 590 Glu Ile Ala Pro Leu Ala Met Glu
Asn Val Ala Ala Pro 595 600 605 <210> SEQ ID NO 11
<211> LENGTH: 1818 <212> TYPE: DNA <213>
ORGANISM: Zea mays <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (1)..(1815) <400> SEQUENCE: 11 atg gcg
gct ctg gcc acg tcg cag ctc gtc gca acg cgc gcc ggc ctg 48 Met Ala
Ala Leu Ala Thr Ser Gln Leu Val Ala Thr Arg Ala Gly Leu 1 5 10 15
ggc gtc ccg gac gcg tcc acg ttc cgc cgc ggc gcc gcg cag ggc ctg 96
Gly Val Pro Asp Ala Ser Thr Phe Arg Arg Gly Ala Ala Gln Gly Leu 20
25 30 agg ggg gcc cgg gcg tcg gcg gcg gcg gac acg ctc agc atg cgg
acc 144 Arg Gly Ala Arg Ala Ser Ala Ala Ala Asp Thr Leu Ser Met Arg
Thr 35 40 45 agc gcg cgc gcg gcg ccc agg cac cag cag cag gcg cgc
cgc ggg ggc 192 Ser Ala Arg Ala Ala Pro Arg His Gln Gln Gln Ala Arg
Arg Gly Gly 50 55 60 agg ttc ccg tcg ctc gtc gtg tgc gcc agc gcc
ggc atg aac gtc gtc 240 Arg Phe Pro Ser Leu Val Val Cys Ala Ser Ala
Gly Met Asn Val Val 65 70 75 80 ttc gtc ggc gcc gag atg gcg ccg tgg
agc aag acc ggc ggc ctc ggc 288 Phe Val Gly Ala Glu Met Ala Pro Trp
Ser Lys Thr Gly Gly Leu Gly 85 90 95 gac gtc ctc ggc ggc ctg ccg
ccg gcc atg gcc gcg aac ggg cac cgt 336 Asp Val Leu Gly Gly Leu Pro
Pro Ala Met Ala Ala Asn Gly His Arg 100 105 110 gtc atg gtc gtc tct
ccc cgc tac gac cag tac aag gac gcc tgg gac 384 Val Met Val Val Ser
Pro Arg Tyr Asp Gln Tyr Lys Asp Ala Trp Asp 115 120 125 acc agc gtc
gtg tcc gag atc aag atg gga gac ggg tac gag acg gtc 432 Thr Ser Val
Val Ser Glu Ile Lys Met Gly Asp Gly Tyr Glu Thr Val 130 135 140 agg
ttc ttc cac tgc tac aag cgc gga gtg gac cgc gtg ttc gtt gac 480 Arg
Phe Phe His Cys Tyr Lys Arg Gly Val Asp Arg Val Phe Val Asp 145 150
155 160 cac cca ctg ttc ctg gag agg gtt tgg gga aag acc gag gag aag
atc 528 His Pro Leu Phe Leu Glu Arg Val Trp Gly Lys Thr Glu Glu Lys
Ile 165 170 175 tac ggg cct gtc gct gga acg gac tac agg gac aac cag
ctg cgg ttc 576 Tyr Gly Pro Val Ala Gly Thr Asp Tyr Arg Asp Asn Gln
Leu Arg Phe 180 185 190 agc ctg cta tgc cag gca gca ctt gaa gct cca
agg atc ctg agc ctc 624 Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala Pro
Arg Ile Leu Ser Leu 195 200 205 aac aac aac cca tac ttc tcc gga cca
tac ggg gag gac gtc gtg ttc 672 Asn Asn Asn Pro Tyr Phe Ser Gly Pro
Tyr Gly Glu Asp Val Val Phe 210 215 220 gtc tgc aac gac tgg cac acc
ggc cct ctc tcg tgc tac ctc aag agc 720 Val Cys Asn Asp Trp His Thr
Gly Pro Leu Ser Cys Tyr Leu Lys Ser 225 230 235 240 aac tac cag tcc
cac ggc atc tac agg gac gca aag acc gct ttc tgc 768 Asn Tyr Gln Ser
His Gly Ile Tyr Arg Asp Ala Lys Thr Ala Phe Cys 245 250 255 atc cac
aac atc tcc tac cag ggc cgg ttc gcc ttc tcc gac tac ccg 816 Ile His
Asn Ile Ser Tyr Gln Gly Arg Phe Ala Phe Ser Asp Tyr Pro 260 265 270
gag ctg aac ctc ccg gag aga ttc aag tcg tcc ttc gat ttc atc gac 864
Glu Leu Asn Leu Pro Glu Arg Phe Lys Ser Ser Phe Asp Phe Ile Asp 275
280 285 ggc tac gag aag ccc gtg gaa ggc cgg aag atc aac tgg atg aag
gcc 912 Gly Tyr Glu Lys Pro Val Glu Gly Arg Lys Ile Asn Trp Met Lys
Ala 290 295 300 ggg atc ctc gag gcc gac agg gtc ctc acc gtc agc ccc
tac tac gcc 960 Gly Ile Leu Glu Ala Asp Arg Val Leu Thr Val Ser Pro
Tyr Tyr Ala 305 310 315 320 gag gag ctc atc tcc ggc atc gcc agg ggc
tgc gag ctc gac aac atc 1008 Glu Glu Leu Ile Ser Gly Ile Ala Arg
Gly Cys Glu Leu Asp Asn Ile 325 330 335 atg cgc ctc acc ggc atc acc
ggc atc gtc aac ggc atg gac gtc agc 1056 Met Arg Leu Thr Gly Ile
Thr Gly Ile Val Asn Gly Met Asp Val Ser 340 345 350 gag tgg gac ccc
agc agg gac aag tac atc gcc gtg aag tac gac gtg 1104 Glu Trp Asp
Pro Ser Arg Asp Lys Tyr Ile Ala Val Lys Tyr Asp Val 355 360 365 tcg
acg gcc gtg gag gcc aag gcg ctg aac aag gag gcg ctg cag gcg 1152
Ser Thr Ala Val Glu Ala Lys Ala Leu Asn Lys Glu Ala Leu Gln Ala 370
375 380 gag gtc ggg ctc ccg gtg gac cgg aac atc ccg ctg gtg gcg ttc
atc 1200 Glu Val Gly Leu Pro Val Asp Arg Asn Ile Pro Leu Val Ala
Phe Ile 385 390 395 400 ggc agg ctg gaa gag cag aag ggc ccc gac gtc
atg gcg gcc gcc atc 1248 Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp
Val Met Ala Ala Ala Ile 405 410 415 ccg cag ctc atg gag atg gtg gag
gac gtg cag atc gtt ctg ctg ggc 1296 Pro Gln Leu Met Glu Met Val
Glu Asp Val Gln Ile Val Leu Leu Gly 420 425 430 acg ggc aag aag aag
ttc gag cgc atg ctc atg agc gcc gag gag aag 1344 Thr Gly Lys Lys
Lys Phe Glu Arg Met Leu Met Ser Ala Glu Glu Lys 435 440 445 ttc cca
ggc aag gtg cgc gcc gtg gtc aag ttc aac gcg gcg ctg gcg 1392 Phe
Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn Ala Ala Leu Ala 450 455
460 cac cac atc atg gcc ggc gcc gac gtg ctc gcc gtc acc agc cgc ttc
1440 His His Ile Met Ala Gly Ala Asp Val Leu Ala Val Thr Ser Arg
Phe 465 470 475 480 gag ccc tgc ggc ctc atc cag ctg cag ggg atg cga
tac gga acg ccc 1488 Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met
Arg Tyr Gly Thr Pro 485 490 495 tgc gcc tgc gcg tcc acc ggt gga ctc
gtc gac acc atc atc gaa ggc 1536 Cys Ala Cys Ala Ser Thr Gly Gly
Leu Val Asp Thr Ile Ile Glu Gly 500 505 510 aag acc ggg ttc cac atg
ggc cgc ctc agc gtc gac tgt aac gtc gtg 1584 Lys Thr Gly Phe His
Met Gly Arg Leu Ser Val Asp Cys Asn Val Val 515 520 525 gag ccg gcg
gac gtc aag aag gtg gcc acc aca ttg cag cgc gcc atc 1632 Glu Pro
Ala Asp Val Lys Lys Val Ala Thr Thr Leu Gln Arg Ala Ile 530 535 540
aag gtg gtc ggc acg ccg gcg tac gag gag atg gtg agg aac tgc atg
1680 Lys Val Val Gly Thr Pro Ala Tyr Glu Glu Met Val Arg Asn Cys
Met 545 550 555 560 atc cag gat ctc tcc tgg aag ggc cct gcc aag aac
tgg gag aac gtg 1728 Ile Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys
Asn Trp Glu Asn Val 565 570 575 ctg ctc agc ctc ggg gtc gcc ggc ggc
gag cca ggg gtc gaa ggc gag 1776 Leu Leu Ser Leu Gly Val Ala Gly
Gly Glu Pro Gly Val Glu Gly Glu 580 585 590 gag atc gcg ccg ctc gcc
aag gag aac gtg gcc gcg ccc tga 1818 Glu Ile Ala Pro Leu Ala Lys
Glu Asn Val Ala Ala Pro 595 600 605 <210> SEQ ID NO 12
<211> LENGTH: 605 <212> TYPE: PRT <213> ORGANISM:
Zea mays <400> SEQUENCE: 12 Met Ala Ala Leu Ala Thr Ser Gln
Leu Val Ala Thr Arg Ala Gly Leu 1 5 10 15 Gly Val Pro Asp Ala Ser
Thr Phe Arg Arg Gly Ala Ala Gln Gly Leu 20 25 30 Arg Gly Ala Arg
Ala Ser Ala Ala Ala Asp Thr Leu Ser Met Arg Thr 35 40 45 Ser Ala
Arg Ala Ala Pro Arg His Gln Gln Gln Ala Arg Arg Gly Gly 50 55 60
Arg Phe Pro Ser Leu Val Val Cys Ala Ser Ala Gly Met Asn Val Val 65
70 75 80 Phe Val Gly Ala Glu Met Ala Pro Trp Ser Lys Thr Gly Gly
Leu Gly 85 90 95 Asp Val Leu Gly Gly Leu Pro Pro Ala Met Ala Ala
Asn Gly His Arg 100 105 110 Val Met Val Val Ser Pro Arg Tyr Asp Gln
Tyr Lys Asp Ala Trp Asp 115 120 125 Thr Ser Val Val Ser Glu Ile Lys
Met Gly Asp Gly Tyr Glu Thr Val 130 135 140 Arg Phe Phe His Cys Tyr
Lys Arg Gly Val Asp Arg Val Phe Val Asp 145 150 155 160 His Pro Leu
Phe Leu Glu Arg Val Trp Gly Lys Thr Glu Glu Lys Ile 165 170 175 Tyr
Gly Pro Val Ala Gly Thr Asp Tyr Arg Asp Asn Gln Leu Arg Phe 180 185
190 Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala Pro Arg Ile Leu Ser Leu
195 200 205 Asn Asn Asn Pro Tyr Phe Ser Gly Pro Tyr Gly Glu Asp Val
Val Phe
210 215 220 Val Cys Asn Asp Trp His Thr Gly Pro Leu Ser Cys Tyr Leu
Lys Ser 225 230 235 240 Asn Tyr Gln Ser His Gly Ile Tyr Arg Asp Ala
Lys Thr Ala Phe Cys 245 250 255 Ile His Asn Ile Ser Tyr Gln Gly Arg
Phe Ala Phe Ser Asp Tyr Pro 260 265 270 Glu Leu Asn Leu Pro Glu Arg
Phe Lys Ser Ser Phe Asp Phe Ile Asp 275 280 285 Gly Tyr Glu Lys Pro
Val Glu Gly Arg Lys Ile Asn Trp Met Lys Ala 290 295 300 Gly Ile Leu
Glu Ala Asp Arg Val Leu Thr Val Ser Pro Tyr Tyr Ala 305 310 315 320
Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly Cys Glu Leu Asp Asn Ile 325
330 335 Met Arg Leu Thr Gly Ile Thr Gly Ile Val Asn Gly Met Asp Val
Ser 340 345 350 Glu Trp Asp Pro Ser Arg Asp Lys Tyr Ile Ala Val Lys
Tyr Asp Val 355 360 365 Ser Thr Ala Val Glu Ala Lys Ala Leu Asn Lys
Glu Ala Leu Gln Ala 370 375 380 Glu Val Gly Leu Pro Val Asp Arg Asn
Ile Pro Leu Val Ala Phe Ile 385 390 395 400 Gly Arg Leu Glu Glu Gln
Lys Gly Pro Asp Val Met Ala Ala Ala Ile 405 410 415 Pro Gln Leu Met
Glu Met Val Glu Asp Val Gln Ile Val Leu Leu Gly 420 425 430 Thr Gly
Lys Lys Lys Phe Glu Arg Met Leu Met Ser Ala Glu Glu Lys 435 440 445
Phe Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn Ala Ala Leu Ala 450
455 460 His His Ile Met Ala Gly Ala Asp Val Leu Ala Val Thr Ser Arg
Phe 465 470 475 480 Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg
Tyr Gly Thr Pro 485 490 495 Cys Ala Cys Ala Ser Thr Gly Gly Leu Val
Asp Thr Ile Ile Glu Gly 500 505 510 Lys Thr Gly Phe His Met Gly Arg
Leu Ser Val Asp Cys Asn Val Val 515 520 525 Glu Pro Ala Asp Val Lys
Lys Val Ala Thr Thr Leu Gln Arg Ala Ile 530 535 540 Lys Val Val Gly
Thr Pro Ala Tyr Glu Glu Met Val Arg Asn Cys Met 545 550 555 560 Ile
Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn Trp Glu Asn Val 565 570
575 Leu Leu Ser Leu Gly Val Ala Gly Gly Glu Pro Gly Val Glu Gly Glu
580 585 590 Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala Pro 595
600 605
* * * * *
References