U.S. patent application number 12/922432 was filed with the patent office on 2011-03-31 for gene for increasing the production of plant biomass and/or seeds and method for use thereof.
Invention is credited to Satoshi Kondo, Norihiro Mitsukawa, Nobuhiko Muramoto, Kenichi Ogawa, Chikara Ohto, Hiroki Sugimoto, Tomoko Tanaka, Madoka Yonekura.
Application Number | 20110078818 12/922432 |
Document ID | / |
Family ID | 41065341 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110078818 |
Kind Code |
A1 |
Kondo; Satoshi ; et
al. |
March 31, 2011 |
GENE FOR INCREASING THE PRODUCTION OF PLANT BIOMASS AND/OR SEEDS
AND METHOD FOR USE THEREOF
Abstract
A technique by which the production of plant biomass can be
significantly increased is provided. A protein phosphatase 2C gene
having 3 consensus sequences comprising the amino acid sequences
shown in SEQ ID NOS: 1-3 from the N-terminal side in such order is
over-expressed.
Inventors: |
Kondo; Satoshi;
(Miyoshi-shi, JP) ; Ohto; Chikara; (Toyota-shi,
JP) ; Mitsukawa; Norihiro; (Miyoshi-shi, JP) ;
Muramoto; Nobuhiko; (Ichinomiya-shi, JP) ; Ogawa;
Kenichi; (Kyoto-shi, JP) ; Sugimoto; Hiroki;
(Aichi-gun, JP) ; Tanaka; Tomoko; (Nagoya-shi,
JP) ; Yonekura; Madoka; (Toyota-shi, JP) |
Family ID: |
41065341 |
Appl. No.: |
12/922432 |
Filed: |
March 13, 2009 |
PCT Filed: |
March 13, 2009 |
PCT NO: |
PCT/JP2009/054953 |
371 Date: |
December 3, 2010 |
Current U.S.
Class: |
800/278 ;
800/298 |
Current CPC
Class: |
C12N 15/8261 20130101;
Y02A 40/146 20180101; C12Y 301/03016 20130101; C12N 9/16
20130101 |
Class at
Publication: |
800/278 ;
800/298 |
International
Class: |
C12N 15/87 20060101
C12N015/87; A01H 5/00 20060101 A01H005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 14, 2008 |
JP |
2008-066460 |
Claims
1. A plant, in which a gene encoding protein phosphatase 2C having
3 consensus sequences comprising the amino acid sequences shown in
SEQ ID NOS: 1-3 in such order from the N-terminal side is
over-expressed.
2. The plant according to claim 1, wherein the gene encoding
protein phosphatase 2C is at least one gene selected from the group
consisting of At1g03590-AtPP2C6-6, At1g16220, At1g79630, At5g01700,
At3g02750, At5g36250, At5g26010, At4g32950, At3g16800, At3g05640,
At5g27930-AtPP2C6-7, At2g20050, and At3g06270, or a gene
functionally equivalent to the gene.
3. The plant according to claim 1, wherein the gene encoding
protein phosphatase 2C encodes any one of the following proteins
(a) to (c): (a) a protein comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 5, 7, 36, 42, and
48; (b) a protein comprising an amino acid sequence that has a
deletion, a substitution, an addition, or an insertion of one or a
plurality of amino acids with respect to an amino acid sequence
selected from the group consisting of SEQ ID NOS: 5, 7, 36, 42, and
48 and having protein phosphatase 2C activity; and (c) a protein
that is encoded by a polynucleotide hybridizing under stringent
conditions to a polynucleotide comprising a nucleotide sequence
complementary to a nucleotide sequence selected from the group
consisting of SEQ ID NOS: 4, 6, 35, 41, and 47 and has protein
phosphatase 2C activity.
4. The plant according to claim 2, wherein the functionally
equivalent gene is a protein phosphatase 2C gene from an organism
other than Arabidopsis thaliana.
5. The plant according to claim 4, wherein an organism other than
Arabidopsis thaliana is at least one type of organism selected from
the group consisting of rice (Oryza sativa), black cottonwood
(Populus trichocarpa), european grape (Vitis vinerfera), Medicago
truncatula (Medicago truncatula), alfalfa (Medicago sativa),
Physcomitrella patens (Physcomitrella patens), ice plant
(Mesembryanthemum crystallinum), Chlamydomonas reinhardtii
(Chlamydomonas reinhardtii), corn (Zea mays), rapeseed (Brassica
rapa), tomato (Solanum lycopersicum), monkey flower (Mimulus
guttatus), and monocellular red alga (Cyanidioschyzon merolae).
6-13. (canceled)
14. A method for increasing the production of biomass and/or seeds,
by which a gene encoding protein phosphatase 2C having 3 consensus
sequences comprising the amino acid sequences shown in SEQ ID NOS:
1-3 from the N-terminal side in such order is over-expressed by a
plant.
15. The method according to claim 14, wherein the gene encoding
protein phosphatase 2C is at least one gene selected from the group
consisting of At1g03590-AtPP2C6-6, At1g16220, At1g79630, At5g01700,
At3g02750, At5g36250, At5g26010, At4g32950, At3g16800, At3g05640,
At5g27930-AtPP2C6-7, At2g20050, and At3g06270, or a gene
functionally equivalent to the gene.
16. The method according to claim 14, wherein the gene encoding
protein phosphatase 2C encodes any one of the following proteins
(a) to (c): (a) a protein comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 5, 7, 36, 42, and
48; (b) a protein comprising an amino acid sequence that has a
deletion, a substitution, an addition, or an insertion of one or a
plurality of amino acids with respect to an amino acid sequence
selected from the group consisting of SEQ ID NOS: 5, 7, 36, 42, and
48 and having protein phosphatase 2C activity; and (c) a protein
that is encoded by a polynucleotide hybridizing under stringent
conditions to a polynucleotide comprising a nucleotide sequence
complementary to a nucleotide sequence selected from the group
consisting of SEQ ID NOS: 4, 6, 35, 41, and 47 and has protein
phosphatase 2C activity.
17. The method according to claim 15, wherein the functionally
equivalent gene is a protein phosphatase 2C gene from an organism
other than Arabidopsis thaliana.
18. The method according to claim 17, wherein an organism other
than Arabidopsis thaliana is at least one type of organism selected
from the group consisting of rice (Oryza sativa), black cottonwood
(Populus trichocarpa), european grape (Vitis vinerfera), Medicago
truncatula (Medicago truncatula), alfalfa (Medicago sativa),
Physcomitrella patens (Physcomitrella patens), ice plant
(Mesembryanthemum crystallinum), Chlamydomonas reinhardtii
(Chlamydomonas reinhardtii), corn (Zea mays), rapeseed (Brassica
rapa), tomato (Solanum lycopersicum), monkey flower (Mimulus
guttatus), and monocellular red alga (Cyanidioschyzon merolae).
19-26. (canceled)
27. A method for producing a plant, comprising the steps of:
preparing a transformed plant in which a gene encoding protein
phosphatase 2C having 3 consensus sequences comprising the amino
acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in
such order is over-expressed; and measuring the amount of biomass
and/or seeds of a progeny plant of the transformed plant and then
selecting a line with significantly improved production of biomass
and/or seeds.
28. The production method according to claim 27, wherein the gene
encoding protein phosphatase 2C is at least one gene selected from
the group consisting of At1g03590-AtPP2C6-6, At1g16220, At1g79630,
At5g01700, At3g02750, At5g36250, At5g26010, At4g32950, At3g16800,
At3g05640, At5g27930-AtPP2C6-7, At2g20050, and At3g06270, or a gene
functionally equivalent to the gene.
29. The production method according to claim 27, wherein the gene
encoding protein phosphatase 2C encodes any one of the following
proteins (a) to (c): (a) a protein comprising an amino acid
sequence selected from the group consisting of SEQ ID NOS: 5, 7,
36, 42, and 48; (b) a protein comprising an amino acid sequence
that has a deletion, a substitution, an addition, or an insertion
of one or a plurality of amino acids with respect to an amino acid
sequence selected from the group consisting of SEQ ID NOS: 5, 7,
36, 42, and 48 and having protein phosphatase 2C activity; and (c)
a protein that is encoded by a polynucleotide hybridizing under
stringent conditions to a polynucleotide comprising a nucleotide
sequence complementary to a nucleotide sequence selected from the
group consisting of SEQ ID NOS: 4, 6, 35, 41, and 47 and has
protein phosphatase 2C activity.
30. The production method according to claim 28, wherein the
functionally equivalent gene is a protein phosphatase 2C gene from
an organism other than Arabidopsis thaliana.
31. The production method according to claim 30, wherein an
organism other than Arabidopsis thaliana is at least one type of
organism selected from the group consisting of rice (Oryza sativa),
black cottonwood (Populus trichocarpa), european grape (Vitis
vinerfera), Medicago truncatula (Medicago truncatula), alfalfa
(Medicago sativa), Physcomitrella patens (Physcomitrella patens),
ice plant (Mesembryanthemum crystallinum), Chlamydomonas
reinhardtii (Chlamydomonas reinhardtii), corn (Zea mays), rapeseed
(Brassica rapa), tomato (Solanum lycopersicum), monkey flower
(Mimulus guttatus), and monocellular red alga (Cyanidioschyzon
merolae).
32-39. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to: a plant in which a given
gene is over-expressed; a method for increasing the production of
biomass and/or seeds through overexpression of a given gene; and a
method for producing such plant capable of producing an increased
amount of biomass and/or seeds.
BACKGROUND ART
[0002] The term "biomass" generally refers to the total amount of
organisms that inhabit or exist in a given area. When such term is
used with regard to plants, in particular, it refers to dry weight
per unit area. Biomass units are quantified in terms of mass or
energy. The expression "biomass" is synonymous with
"Seibutsutairyo" or "Seibutsuryo." In the case of plant biomass,
the term "standing crop" is occasionally used for "biomass." Since
plant biomass is generated by fixing atmospheric carbon dioxide
with the use of solar energy, it can be regarded as so-called
"carbon-neutral energy." Accordingly, an increase of plant biomass
is effective for global environmental preservation, the prevention
of global warming, and mitigation of greenhouse gas emissions.
Thus, technologies for increasing the production of plant biomass
have been industrially significant.
[0003] Plants are cultivated for the purpose of using some tissues
thereof (e.g., seeds, roots, leaves, or stems) or for the purpose
of producing various materials, such as fats and oils. Examples of
fats and oils produced from plants that have been heretofore known
include soybean oil, sesame oil, olive oil, coconut oil, rice oil,
cottonseed oil, sunflower oil, corn oil, safflower oil, palm oil,
and rapeseed oil. Such fats and oils are extensively used for
household and industrial applications. Also, fats and oils produced
from plants are used as raw materials for biodiesel fuel or
bioplastic, and the applicability thereof is increasing for
alternative energy to petroleum.
[0004] In particular, an energy crop such as sugar cane can be used
as a raw material for biofuel. Hence, the increased production of
the total mass of a plant itself (the amount of plant biomass) is
expected. Under such circumstances, improvement in productivity per
unit of cultivation area is required in order to increase the
production of the amount of plant biomass. It has been found that
if the number of cultivated plants is assumed to be constant per
unit of cultivation area, improvement in the amount of biomass per
plant would be necessary.
[0005] However, it is thought that since many genes are involved in
the amount of plant biomass (a so-called "kind of quantitative
trait"), individual gene introduction or individual genetic
modification cannot lead to an effective increase in production.
Meanwhile, a great deal of difficulties are associated with
introduction of many genes in a desired state into a plant. Such
gene introduction is also problematic in that if successful
introduction takes place, desirable traits cannot always be
acquired.
[0006] Various gene introduction techniques are known as techniques
for increasing the production of plant biomass, as disclosed in
Patent Documents 1-7, for example. However, none of these
techniques can be said to exert sufficient effects of increasing
the production of biomass.
Patent Documents
[0007] Patent Document 1: JP Patent Publication (Kohyo) No.
2001-505410 A [0008] Patent Document 2: JP Patent Publication
(Kohyo) No. 2001-519659 A [0009] Patent Document 3: JP Patent
Publication (Kohyo) No. 2007-530063 A [0010] Patent Document 4: JP
Patent Publication (Kokai) No. 2005-130770 A [0011] Patent Document
5: JP Patent Publication (Kohyo) No. 2000-515020 A [0012] Patent
Document 6: JP Patent Publication (Kohyo) No. 9-503389 A [0013]
Patent Document 7: JP Patent Publication (Kokai) No. 2005-52114
A
DISCLOSURE OF THE INVENTION
Object to be Achieved by the Invention
[0014] In view of the above circumstances, an object of the present
invention is to search for genes having novel functions of
drastically improving the amount of plant biomass and thus to
provide a technique with which the production of plant biomass can
be drastically increased.
Means to Achieve the Object
[0015] As a result of intensive studies to achieve the above
object, the present inventors have made the novel finding that the
production of plant biomass can be drastically increased by causing
overexpression of a gene encoding protein phosphatase 2C having
characteristic consensus sequences. Thus, they have completed the
present invention.
[0016] Specifically, the plant according to the present invention
is a plant in which a gene encoding protein phosphatase 2C having 3
consensus sequences comprising the amino acid sequences shown in
SEQ ID NOS: 1-3 from the N-terminal side in such order is
over-expressed.
[0017] Also, the method for increasing the production of biomass
according to the present invention comprises causing the
overexpression of a gene encoding protein phosphatase 2C having 3
consensus sequences comprising the amino acid sequences shown in
SEQ ID NOS: 1-3 from the N-terminal side in such order.
[0018] Furthermore, the method for producing a plant according to
the present invention comprises the steps of: [0019] preparing a
transformed plant in which a gene encoding protein phosphatase 2C
having 3 consensus sequences comprising the amino acid sequences
shown in SEQ ID NOS: 1-3 from the N-terminal side in such order is
over-expressed; and [0020] measuring the amount of biomass of a
progeny plant of the transformed plant and then selecting a line in
which the amount of biomass is significantly improved.
[0021] In the present invention, the above gene encoding protein
phosphatase 2C can be at least one type of gene selected from the
group consisting of At1g03590-AtPP2C6-6, At1g16220, At1g79630,
At5g01700, At3g02750, At5g36250, At5g26010, At4g32950, At3g16800,
At3g05640, At5g27930-AtPP2C6-7, At2g20050, and At3g06270, or a gene
functionally equivalent thereto.
[0022] In the present invention, the gene encoding protein
phosphatase 2C preferably encodes any one of the following proteins
(a) to (c): [0023] (a) a protein comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 5, 7, 36, 42, and
48; [0024] (b) a protein comprising an amino acid sequence that has
a deletion, a substitution, an addition, or an insertion of one or
a plurality of amino acids with respect to an amino acid sequence
selected from the group consisting of SEQ ID NOS: 5, 7, 36, 42, and
48 and having protein phosphatase 2C activity; and [0025] (c) a
protein that is encoded by a polynucleotide hybridizing under
stringent conditions to a polynucleotide comprising a nucleotide
sequence complementary to a nucleotide sequence selected from the
group consisting of SEQ ID NOS: 4, 6, 35, 41, and 47 and has
protein phosphatase 2C activity.
[0026] Also, in the present invention, an example of the above
functionally equivalent gene is a protein phosphatase 2C gene from
an organism other than Arabidopsis thaliana. Another example of an
organism other than Arabidopsis thaliana is an organism selected
from the group consisting of rice (Oryza sativa), black cottonwood
(Populus trichocarpa), european grape (Vitis vinifera), Medicago
truncatula (Medicago truncatula), alfalfa (Medicago sativa),
Physcomitrella patens (Physcomitrella patens), ice plant
(Mesembryanthemum crystallinum), Chlamydomonas reinhardtii
(Chlamydomonas reinhardtii), corn (Zea mays), rapeseed (Brassica
rapa), tomato (Solanum lycopersicum), monkey flower (Mimulus
guttatus), and monocellular red alga (Cyanidioschyzon merolae).
[0027] Examples of plants to be subjected to the present invention
include dicotyledons such as plants of the family Brassicaceae.
Examples of plants of the family Brassicaceae include Arabidopsis
thaliana and rapeseed. Other examples of plants to be subjected to
the present invention include monocotyledons such as plants of the
family Gramineae. Examples of plants of the family Gramineae
include rice and sugarcane.
Effect of the Invention
[0028] The plant according to the present invention is a plant
capable of producing significantly improved amount of biomass
and/or seeds compared with wild-type plants. Also, the method for
increasing the production of biomass and/or seeds according to the
present invention can realize the significantly increased
production of biomass and/or seeds compared with target wild-type
plants. Furthermore, the method for producing a plant according to
the present invention makes it possible to produce a plant capable
of producing significantly improved amount of biomass and/or seeds
compared with wild-type plants. Therefore, through application of
the present invention, for example, productivity can be improved
when the plant itself is a product and this can be achieved at
lower cost. Also, through application of the present invention, for
example, the productivity can be improved when seeds are directly
products or ingredients contained in seeds are directly products
and this can be achieved at lower cost.
[0029] This description hereby incorporates the entire content of
the description and/or the drawings of Japanese Patent Application
No. 2008-066460, which is the basis of the priority claim of this
application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a characteristic diagram showing the results of
alignment analysis using a CLUSTAL W (1.83) multiple sequence
alignment program for amino acid sequences encoded by
At1g03590-AtPP2C6-6, At1g16220, At1g79630, At5g01700, At3g02750,
At5g36250, At5g26010, At4g32950, At3g16800, At3g05640,
At5g27930-AtPP2C6-7, At2g20050, and At3g06270.
[0031] FIG. 2 is a characteristic diagram showing the results of
alignment analysis using a CLUSTAL W (1.83) multiple sequence
alignment program for amino acid sequences encoded by
At1g03590-AtPP2C6-6, At1g16220, At1g79630, At5g01700, At3g02750,
At5g36250, At5g26010, At4g32950, At3g16800, At3g05640, and
At5g27930-AtPP2C6-7.
[0032] FIG. 3 is a photo showing the above-ground parts of
wild-type plants and transformed plants into which a fragment
containing ORF of the PP2C (protein phosphatase 2C) gene
(At3g05640) was introduced.
[0033] FIG. 4 is a characteristic diagram showing the results of
measuring the amounts of biomass of the above-ground parts of
wild-type plants and transformed plants into which a fragment
containing ORF of the PP2C (protein phosphatase 2C) gene
(At3g05640) was introduced. The result for the wild-type plants is
the average value for 12 individual wild-type plants and each
result for the transformed plants is the average value for 5
individual transformed plants.
[0034] FIG. 5 is a characteristic diagram showing the results of
measuring the amounts of seeds of wild-type plants and transformed
plants into which a fragment containing ORF of the PP2C (protein
phosphatase 2C) gene (At3g05640) was introduced. The result for
wild-type plants is the average value for 12 individual wild-type
plants and each result for the transformed plants is the average
value for 5 individual transformed plants.
[0035] FIG. 6 shows photos showing the above-ground parts of a rice
control plant into which a plant expression vector (constructed by
ligating 3 multiple cloning sites of a pST-Bluel vector to
pBI-sGFP-R4R3) was introduced and a transformed rice plant into
which the coding region of PP2C (protein phosphatase 2C)
(At3g05640) was introduced.
[0036] FIG. 7 shows photos showing the above-ground parts of a
control rice plant into which no gene was introduced and a
transformed rice plant into which the coding region of rice-derived
PP2C (protein phosphatase 2C) (Os05g0358500) was introduced.
[0037] FIG. 8 is a photo showing the above-ground parts of a wild
type plant and a transformed plant into which a fragment containing
ORF of a PP2C (protein phosphatase 2C) gene (At5g27930) was
introduced.
[0038] FIG. 9 is a photo showing the above-ground parts of a
wild-type plant and a transformed plant into which a fragment
containing ORF of a PP2C (protein phosphatase 2C) gene (At5g27930)
was introduced.
[0039] FIG. 10 is a photo showing the above-ground parts of a
wild-type plant and transformed plants into which a fragment
containing ORF of a PP2C (protein phosphatase 2C) gene (At3g02750)
was introduced.
[0040] FIG. 11 is a photo showing the above-ground parts of a
wild-type plant and transformed plants into which a fragment
containing ORF of a PP2C (protein phosphatase 2C) gene (At3g16800)
was introduced.
[0041] FIG. 12 is a photo showing the above-ground parts of a
wild-type plant and transformed plants into which the coding region
of a rice-derived PP2C (protein phosphatase 2C) gene (Os05g0358500)
was introduced.
BEST MODE FOR CARRYING OUT THE INVENTION
[0042] The present invention will be described in detail as
follows.
[0043] The plant according to the present invention is a plant in
which: a gene encoding protein phosphatase 2C having characteristic
consensus sequences is over-expressed; and the amount of biomass is
significantly improved compared with wild-type plants. The plant
according to the present invention may be a plant in which the
protein phosphatase 2C gene is over-expressed in all plant tissues
or at least some plant tissues. Here, the term "plant tissue(s)"
refers to plant organ(s) such as leaves, stems, seeds, roots, and
flowers.
[0044] Here, the term "overexpression" refers to an expression
level that can be confirmed as a transcript as a result of
transcription of the protein phosphatase 2C gene introduced into a
plant.
Protein Phosphatase 2C Gene
[0045] The protein phosphatase 2C gene to be over-expressed in a
plant encodes protein phosphatase 2C that has 3 consensus sequences
comprising the amino acid sequences shown in SEQ ID NOS: 1-3 in
such order from the N-terminal side. In addition, a gene group
classified as Group E as in FIG. 1 of Topographic cladogram (on
page 237 of Reference: TRENDS in Plant Science Vol. 9 No. 5 May
2004 pages 236-243) encodes protein phosphatase 2C having 3
consensus sequences comprising the amino acid sequences shown in
SEQ ID NOS: 1-3 in such order from the N-terminal side. In
addition, the reference predicts the presence of 76 protein
phosphatase 2C genes in Arabidopsis thaliana and discloses the
results of producing a phylogenetic tree of these genes using
T-Coffee software (reference; Notredame, C. et al. 2000 T-Coffee: a
novel method for fast and accurate multiple sequence alignment. J.
Mol. Biol. 302, 205-247) as in FIG. 1. In this phylogenetic tree,
protein phosphatase 2C genes classified as members of Group E
encode protein phosphatase 2C that has 3 consensus sequences
comprising the amino acid sequences shown in SEQ ID NOS: 1-3 in
such order from the N-terminal side. The 3 consensus sequences
comprising the amino acid sequences shown in SEQ ID NOS: 1-3 are
characteristic sequences in Group E in the above-mentioned
classification and serve as a basis for clear differentiation from
other groups.
[0046] Group E in the above classification includes protein
phosphatase 2C genes specified by Arabidopsis thaliana-derived
At1g03590-AtPP2C6-6, At1g16220, At1g79630, At5g01700, At3g02750,
At5g36250, At5g26010, At4g32950, At3g16800, At3g05640,
At5g27930-AtPP2C6-7, At2g20050, and At3g06270. FIG. 1 shows the
results of alignment analysis using a CLUSTAL W (1.83) multiple
sequence alignment program (which can be used with the DDBJ of the
National Institute of Genetics
(http://clustalw.ddbj.nig.ac.jp/top-j.html)) for the amino acid
sequences encoded by these Arabidopsis thaliana-derived protein
phosphatase 2C genes, At1g03590-AtPP2C6-6, At1g16220, At1g79630,
At5g01700, At3g02750, At5g36250, At5g26010, At4g32950, At3g16800,
At3g05640, At5g27930-AtPP2C6-7, At2g20050, and At3g06270 (with the
amino acid (sequence) substitution matrix used herein being a
default matrix known as BLOSUM (Blocks of Amino Acid Substitution
Matrix)). As shown in FIG. 1, these protein phosphatase 2C genes
classified as members of Group E have consensus sequences
characteristic in the regions denoted as I to III. These regions
denoted as Ito III are subjected with a rice-derived protein
phosphatase 2C gene (described later) to alignment analysis, so
that the 3 consensus sequences comprising the amino acid sequences
shown in SEQ ID NOS: 1-3 can be defined.
[0047] Herein, in the amino acid sequence shown in SEQ ID NO: 1,
which is an amino acid residue denoted as "Xaa," may be any amino
acid, and it is not limited to any particular amino acid. However,
the 1.sup.st amino acid residue from the N-terminal side in the
amino acid sequence shown in SEQ ID NO: 1 is preferably leucine
(three character code: Leu and single character code: L; the same
applies to the following) or phenylalanine (Phe, F). The 4.sup.th
amino acid residue from the N-terminal side in the amino acid
sequence shown in SEQ ID NO: 1 is preferably valine (Val, V),
isoleucine (Ile, I), or methionine (Met, M). The 16.sup.th amino
acid residue from the N-terminal side in the amino acid sequence
shown in SEQ ID NO: 1 is preferably serine (Ser, S) or alanine
(Ala, A). The 17.sup.th amino acid residue from the N-terminal side
in the amino acid sequence shown in SEQ ID NO: 1 is preferably
lysine (Lys, K), arginine (Arg, R), glutamine (Gln, Q), or
asparagine (Asn, N). More specifically, a consensus sequence
comprising the amino acid sequence shown in SEQ ID NO: 1 is
preferably (L/F)XG(V/I/M)FDGHGXXGXXX(S/A)(K/R/Q/N)XV. In such amino
acid sequence, pluralities of amino acids in parentheses represent
possible variations of amino acid residues at the relevant
positions. Also, in the following amino acid sequences, "X" means
that any amino acid residue may be present at the relevant
position.
[0048] Also, such a consensus sequence may be a sequence containing
the following 3 amino acid residues on the N-terminal side of
Region I in FIG. 1: (D/E/N)XX.
[0049] Here, in the amino acid sequence shown in SEQ ID NO: 2, an
amino acid residue denoted as "Xaa," may be any amino acid, and it
is not limited to any particular amino acid. However, the 5.sup.th
amino acid residue from the N-terminal side in the amino acid
sequence shown in SEQ ID NO: 2 is preferably glycine (Gly, G),
alanine (Ala, A), or serine (Ser, S). The 6.sup.th amino acid
residue from the N-terminal side in the amino acid sequence shown
in SEQ ID NO: 2 is preferably valine (Val, V), leucine (Leu, L), or
isoleucine (Ile, I). The 9.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is
preferably isoleucine (Ile, I), valine (Val, V), phenylalanine
(Phe, F), methionine (Met, M), or leucine (Leu, L). The 12.sup.th
amino acid residue from the N-terminal side in the amino acid
sequence shown in SEQ ID NO: 2 is preferably glycine (Gly, G) or
alanine (Ala, A). The 15.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is
preferably leucine (Leu, L), valine (Val, V), or isoleucine (Ile,
I). The 17.sup.th amino acid residue from the N-terminal side in
the amino acid sequence shown in SEQ ID NO: 2 is preferably
isoleucine (Ile, I), valine (Val, V), or methionine (Met, M). The
18.sup.th amino acid residue from the N-terminal side in the amino
acid sequence shown in SEQ ID NO: 2 is preferably glycine (Gly, G)
or alanine (Ala, A). The 22.sup.nd amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is
preferably aspartic acid (Asp, D) or histidine (His, H). The
26.sup.th amino acid residue from the N-terminal side in the amino
acid sequence shown in SEQ ID NO: 2 is preferably valine (Val, V)
or isoleucine (Ile, I). The 27.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is
preferably leucine (Leu, L), methionine (Met, M), or isoleucine
(Ile, I). More specifically, a consensus sequence comprising the
amino acid sequence shown in SEQ ID NO: 2 is preferably
SGXT(G/A/S)(V/L/I)XX(I/V/F/M/L)XX(G/A)XX(L/V/I)X(I/V/M)(A/G)NXG(D/H)SRA(V-
/I)(L/M/I). In such amino acid sequence, pluralities of amino acids
in parentheses represent possible variations of amino acid residues
at the relevant positions. Also, in the following amino acid
sequences, "X" means that any amino acid residue may be present at
the relevant position.
[0050] Here, the amino acid sequence shown in SEQ ID NO: 3, an
amino acid residue denoted as "Xaa," may be any amino acid, and it
is not limited to any particular amino acid. However, the 4.sup.th
amino acid residue from the N-terminal side in the amino acid
sequence shown in SEQ ID NO: 3 is preferably methionine (Met, M),
valine (Val, V), or phenylalanine (Phe, F). The 5.sup.th amino acid
residue from the N-terminal side in the amino acid sequence shown
in SEQ ID NO: 3 is preferably serine (Ser, S), alanine (Ala, A), or
threonine (Thr, T). The 7.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is
preferably alanine (Ala, A) or serine (Ser, S). The 8.sup.th amino
acid residue from the N-terminal side in the amino acid sequence
shown in SEQ ID NO: 3 is preferably phenylalanine (Phe, F),
isoleucine (Ile, I), or valine (Val, V). The 14.sup.th amino acid
residue from the N-terminal side in the amino acid sequence shown
in SEQ ID NO: 3 is preferably lysine (Lys, K) or glutamic acid
(Glu, E). The 18.sup.th amino acid residue from the N-terminal side
in the amino acid sequence shown in SEQ ID NO: 3 is preferably
valine (Val, V) or leucine (Leu, L). The 19.sup.th amino acid
residue from the N-terminal side in the amino acid sequence shown
in SEQ ID NO: 3 is preferably isoleucine (Ile, I) or valine (Val,
V). The 23.sup.rd amino acid residue from the N-terminal side in
the amino acid sequence shown in SEQ ID NO: 3 is preferably
glutamic acid (Glu, E), glutamine (Gln, Q), or aspartic acid (Asp,
D). The 24.sup.th amino acid residue from the N-terminal side in
the amino acid sequence shown in SEQ ID NO: 3 is preferably
isoleucine (Ile, I), valine (Val, V), or phenylalanine (Phe, F).
The 29.sup.th amino acid residue from the N-terminal side in the
amino acid sequence shown in SEQ ID NO: 3 is preferably isoleucine
(Ile, I), leucine (Leu, L), or valine (Val, V). The 30.sup.th amino
acid residue from the N-terminal side in the amino acid sequence
shown in SEQ ID NO: 3 is preferably serine (Ser, S), threonine
(Thr, T), or asparagine (Asn, N). The 33.sup.rd amino acid residue
from the N-terminal side in the amino acid sequence shown in SEQ ID
NO: 3 is preferably aspartic acid (Asp, D), asparagine (Asn, N), or
histidine (His, H). The 35.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is
preferably phenylalanine (Phe, F) or tyrosine (Tyr, Y). The
36.sup.th amino acid residue from the N-terminal side in the amino
acid sequence shown in SEQ ID NO: 3 is preferably leucine (Leu, L),
isoleucine (Ile, I), valine (Val, V), phenylalanine (Phe, F), or
methionine (Met, M). The 37.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is
preferably valine (Val, V), leucine (Leu, L), or isoleucine (Ile,
I). The 38.sup.th amino acid residue from the N-terminal side in
the amino acid sequence shown in SEQ ID NO: 3 is preferably leucine
(Leu, L) or valine (Val, V). The 40.sup.th amino acid residue from
the N-terminal side in the amino acid sequence shown in SEQ ID NO:
3 is preferably threonine (Thr, T) or serine (Ser, S). The
43.sup.rd amino acid residue from the N-terminal side in the amino
acid sequence shown in SEQ ID NO: 3 is preferably valine (Val, V),
isoleucine (Ile, I), or methionine (Met, M). The 44.sup.th amino
acid residue from the N-terminal side in the amino acid sequence
shown in SEQ ID NO: 3 is preferably tryptophan (Trp, W) or
phenylalanine (Phe, F). The 45.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is
preferably aspartic acid (Asp, D) or glutamic acid (Glu, E). The
47.sup.th amino acid residue from the N-terminal side in the amino
acid sequence shown in SEQ ID NO: 3 is preferably leucine (Leu, L),
isoleucine (Ile, I), or methionine (Met, M). The 48.sup.th amino
acid residue from the N-terminal side in the amino acid sequence
shown in SEQ ID NO: 3 is preferably serine (Ser, S), threonine
(Thr, T), or proline (Pro, P). The 49.sup.th amino acid residue
from the N-terminal side in the amino acid sequence shown in SEQ ID
NO: 3 is preferably asparagine (Asn, N) or serine (Ser, S). The
52'' amino acid residue from the N-terminal side in the amino acid
sequence shown in SEQ ID NO: 3 is preferably valine (Val, V) or
alanine (Ala, A). The 55.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is
preferably leucine (Leu, L), valine (Val, V), isoleucine (Ile, I),
or methionine (Met, M). The 56.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is
preferably isoleucine (Ile, I) or valine (Val, V). Preferably, an
example of the consensus sequence comprising the amino acid
sequence shown in SEQ ID NO: 3 is more specifically
GXA(M/V/F)(S/A/T)R(A/S)(F/I/V)GDXXX(K/E)XXG(V/L)(I/V)XXP(E/Q/D)(I/V/F)X
XXX(I/L/V)(T/S)XX(D/N/H)X(F/Y)(L/I/V/F)(V/L/I)(L/V)A(T/S)DG(V/I/M)(W/F)(D-
/E)X(L/I/M)(S/T/P)(N/S)XX(V/A)XX(L/V/I/M)(I/V). In such amino acid
sequence, pluralities of amino acids in parentheses represent
possible variations of amino acid residues at the relevant
positions. Also, in the following amino acid sequences, "X" means
that any amino acid residue may be present at the relevant
position.
[0051] Here, the 20.sup.th amino acid residue from the N-terminal
side in the amino acid sequence shown in SEQ ID NO: 3 is more
preferably alanine (Ala, A), serine (Ser, S), or cysteine (Cys, C).
Also, the 50.sup.th amino acid residue from the N-terminal side in
the amino acid sequence shown in SEQ ID NO: 3 is more preferably
aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K),
glutamine (Gln, Q), or asparagine (Asn, N).
[0052] Variations of amino acid residues that can be present at
given positions are determined based on the following reasons. As
described in Reference (1) ("McKee Biochemistry," 3.sup.rd ed.,
Chapter 5 Amino Acid.cndot.Peptide.cndot.Protein 5.1 Amino Acid;
editorial supervisor: Atsushi Ichikawa; translation supervisor:
Shinichi Fukuoka; publisher: Ryosuke Sone; publishing office:
Kagaku-Dojin Publishing Company, INC, ISBN4-7598-0944-9), it is
well known that amino acids are classified based on side chains
having similar properties (e.g., chemical properties and physical
sizes). Also, it is well known that molecular evolutionary
substitution frequently takes place among amino acid residues
classified in a given group, while retaining protein activity.
Based on these concepts, a substitution (mutation) score matrix for
amino acid residues (BLOSUM: Blocks of Amino Acid Substitution
Matrix) is proposed in FIG. 2 of Reference (2): Henikoff S.,
Henikoff J. G., Amino-acid substitution matrices from protein
blocks, Proc. Natl. Acad. Sci. U.S.A., 89, 10915-10919 (1992) and
is broadly used. Reference (2) is based on a finding that amino
acid substitutions that take place among amino acids with side
chains having similar chemical properties result in less structural
or functional changes in the entire protein. According to
References (1) and (2) above, amino acid side chain groups to be
used in multiple alignment can be considered based on indices such
as chemical properties and physical sizes. They are shown as amino
acid groups with a score of 0 or higher and preferably as amino
acid groups with a score of 1 or higher through the use of the
score matrix (BLOSUM) disclosed in Reference (2). Typical groups
are the following 8 groups. Further precisely grouped amino acid
groups may be amino acid groups with a score of 0 or higher,
preferably a score of 1 or higher, and further preferably a score
of 2 or higher.
1) Aliphatic Hydrophobic Amino Acid Group (ILMV Group)
[0053] This group is a group of amino acids having aliphatic
hydrophobic side chains, among neutral nonpolar amino acids
disclosed in Reference (1) above, which is composed of V (Val,
valine), L (Leu, leucine), I (Ile, isoleucine), and M (Met,
methionine). Among amino acids classified as neutral nonpolar amino
acids according to Reference (1), FGACWP is not included in this
"aliphatic hydrophobic amino acid group" because of the following
reasons: G (Gly, glycine) and A (Ala, alanine) are the same size as
that of or smaller in size than a methyl group and have weak non
polar effects; C (Cys, cysteine) may play an important role in S--S
bonds and has a property of forming a hydrogen bond with an oxygen
atom or a nitrogen atom; F (Phe, phenylalanine) and W (Trp,
tryptophan) have side chains with significantly large molecular
weights and have strong aromatic effects; P (Pro, proline) has
strong imino acid effects, so as to fix the angle of the main chain
of the polypeptide.
2) Group Having Hydroxymethylene Group (ST Group)
[0054] This group is a group of amino acids (from among neutral
polar amino acids) having hydroxymethylene groups in side chains,
which is composed of S (Ser, serine) and T (Thr, threonine).
Hydroxy groups existing in the side chains of S and T constitute
sugar-binding sites, so that these sites are often important for a
polypeptide (protein) to have specific activity.
3) Acidic Amino Acid (DE Group)
[0055] This group is a group of amino acids having acidic carboxyl
groups in side chains, which is composed of D (Asp, aspartic acid)
and E (Glu, glutamic acid).
4) Basic Amino Acid (KR Group)
[0056] This group is a group of basic amino acids, which is
composed of K (Lys, lysine) and R (Arg, arginine). These K and R
are positively charged within a wide pH range and have basic
properties. On the other hand, H (His, histidine) classified in
basic amino acids is almost never ionized at pH 7, so that H is not
classified in this group.
5) Methylene group=Polar Group (DHN Group)
[0057] This group is characterized in that: in all cases, a
methylene group as a side chain binds to an .alpha.-carbon element
beyond which a polar group is present; and the physical sizes of
methylene groups (nonpolar groups) closely resemble from each
other. This group is composed of N (Asn, asparagine; polar group is
an amide group), D (Asp, aspartic acid; polar groups are carboxyl
groups), and H (His, histidine; polar groups are imidazole
groups).
6) Dimethylene Group=Polar Group (EKQR Group)
[0058] This group is characterized in that: in all cases, linear
hydrocarbon having a length longer than that of a dimethylene group
binds as a side chain to an .alpha.-carbon element, beyond which a
polar group is present; and the physical sizes of dimethylene
groups that are nonpolar groups closely resemble from each other.
This group is composed of E (Glu, glutamic acid, polar group is a
carboxyl group), K (Lys, lysine; polar groups are amino groups), Q
(Gln, glutamine; polar groups are amide groups), and R (Arg,
arginine; polar groups are imino groups and amino groups).
7) Aromatic Series (FYW Group)
[0059] This group is a group of aromatic amino acids having benzene
nuclei in the side chains and characterized by having chemical
properties unique in aromatic series. This group is composed of F
(Phe, phenylalanine), Y (Tyr, tyrosine), and W (Trp,
tryptophan).
8) Ring & Polar (HY Group)
[0060] This group is a group of amino acids having both ring
structures in the side chains and polarity, which is composed of H
(H, histidine; Both ring structures and polar groups are imidazole
groups), and Y (Tyr, tyrosine; Ring structures are benzene nuclei
and polar groups are hydroxy groups).
[0061] As described above, it is understood that: in the given
amino acid sequences shown in SEQ ID NOS: 1-3, an amino acid
residue denoted as Xaa may be any amino acid; or amino acid
residues denoted as Xaa may be substituted with each other within
the above groups 1)-8). Hence, in the present invention, the
protein phosphatase 2C gene to be over-expressed in a plant may be
a protein phosphatase 2C gene from any plant, as long as it has the
3 consensus sequences comprising the amino acid sequences shown in
SEQ ID NOS: 1-3 in such order from the N-terminal side.
[0062] More specifically, examples of an Arabidopsis thaliana
protein phosphatase 2C-coding gene having the 3 consensus sequences
(comprising the amino acid sequences shown in SEQ ID NOS: 1-3) in
such order from the N-terminal side include At1g03590-AtPP2C6-6,
At1g16220, At1g79630, At5g01700, At3g02750, At5g36250, At5g26010,
At4g32950, At3g16800, At3g05640, At5g27930-AtPP2C6-7, At2g20050,
and At3g06270. In the present invention, at least one type of gene
selected from the gene group is over-expressed. Particularly in the
present invention, it is preferable to cause overexpression of at
least one type of gene selected from among At1g03590-AtPP2C6-6,
At1g16220, At1g79630, At5g01700, At3g02750, At5g36250, At5g26010,
At4g32950, At3g16800, At3g05640, and At5g27930-AtPP2C6-7.
Particularly, in the present invention, it is more preferable to
cause overexpression of at least one type of gene selected from
among At3g16800, At3g05640, and At5g27930-AtPP2C6-7 and it is most
preferable to cause overexpression of a gene specified by
At3g05640.
[0063] In addition, FIG. 2 shows the results of alignment analysis
using a CLUSTAL W (1.83) multiple sequence alignment program (that
can be used with the DDBJ of the National Institute of Genetics
(http://clustalw.ddbj.nig.ac.jp/top-j.html)) for amino acid
sequences encoded by At1g03590-AtPP2C6-6, At1g16220, At1g79630,
At5g01700, At3g02750, At5g36250, At5g26010, At4g32950, At3g16800,
At3g05640, and At5g27930-AtPP2C6-7 (amino acid (sequence)
substitution matrix used herein is default matrix, BLOSUM (Blocks
of Amino Acid Substitution Matrix)).
[0064] That is, FIG. 2 shows the 3 consensus sequences in protein
phosphatase 2C encoded by At1g03590-AtPP2C6-6, At1g16220,
At1g79630, At5g01700, At3g02750, At5g36250, At5g26010, At4g32950,
At3g16800, At3g05640, and At5g27930-AtPP2C6-7. Regions denoted as
I-III in FIG. 2 are subjected with an ortholog of a rice-derived
protein phosphatase 2C gene (described later) to alignment
analysis, so that the 3 consensus sequences comprising the amino
acid sequences shown in SEQ ID NOS: 1-3 above can be defined as the
3 consensus sequences comprising the amino acid sequences shown in
SEQ ID NOS: 53-55, respectively.
[0065] The consensus sequence shown in SEQ ID NO: 53 is more
specifically (L/F)CG(V/I/M)FDGHGXXGXX(V/I)(S/A)(K/R)XV. The
consensus sequence shown in SEQ ID NO: 54 is more specifically
SGXT(G/A/S)(V/L)XX(I/V/F/L)XX(G/A)XX(L/V/I)X(I/V/M)(A/G)NXG(D/H)SRA(V/I)(-
L/M/I). The consensus sequence shown in SEQ ID NO: 55 is more
specifically
GLA(M/V)(S/A)R(A/S)(F/L)GDXX(L/I/V)KX(Y/F/H)G(V/L)(I/V)XXP(E/Q/D)(I/V/F)
XXXX(I/L/V)(T/S)XXDX(F/Y)(L/I/V/M)(V/L/I)LA(T/S)DG(V/I/M)WDX(L/I/M/V)(S/T-
)NX(E/D)(V/A)XX(L/V/I)(I/V).
[0066] In addition, in such amino acid sequences, pluralities of
amino acids in parentheses represent possible variations of amino
acid residues at the relevant positions. Also, in these amino acid
sequences, "X" means that any amino acid residue may be present at
the relevant position.
[0067] Here, the 9.sup.th amino acid residue from the N-terminal
side in the amino acid sequence shown in SEQ ID NO: 54 is more
preferably isoleucine (Ile, I), valine (Val, V), or phenylalanine
(Phe, F). Also, the 11.sup.th amino acid residue from the
N-terminal side in the amino acid sequence shown in SEQ ID NO: 54
is more preferably glutamine (Gln, Q) or histidine (His, H).
Moreover, the 13.sup.th amino acid residue from the N-terminal side
in the amino acid sequence shown in SEQ ID NO: 54 is more
preferably lysine (Lys, K), glutamic acid (Glu, E), serine (Ser,
S), glutamine (Gln, Q), aspartic acid (Asp, D), or asparagine (Asn,
N).
[0068] Here, the 7.sup.th amino acid residue from the N-terminal
side in the amino acid sequence shown in SEQ ID NO: 55 is more
preferably alanine (Ala, A). Also, the 8.sup.th amino acid residue
from the N-terminal side in the amino acid sequence shown in SEQ ID
NO: 55 is more preferably phenylalanine (Phe, F). Moreover, the
11.sup.th amino acid residue from the N-terminal side in the amino
acid sequence shown in SEQ ID NO: 55 is more preferably
phenylalanine (Phe, F) or tyrosine (Tyr, Y). Furthermore, the
13.sup.th amino acid residue from the N-terminal side in the amino
acid sequence shown in SEQ ID NO: 55 is more preferably leucine
(Leu, L) or isoleucine (Ile, I). Moreover, the 15.sup.th amino acid
residue from the N-terminal side in the amino acid sequence shown
in SEQ. ID NO: 55 is more preferably aspartic acid (Asp, D), serine
(Ser, S), or glutamic acid (Glu, E). Furthermore, the 20.sup.th
amino acid residue from. the N-terminal side in the amino acid
sequence shown in SEQ ID NO: 55 is more preferably serine (Ser, S),
alanine (Ala, A), or cysteine (Cys, C). Moreover, the 27.sup.th
amino acid residue from the N-terminal side in the amino acid
sequence shown in SEQ ID NO: 55 is more preferably histidine (His,
H) or arginine (Arg, R). Furthermore, the 34.sup.th amino acid
residue from the N-terminal side in the amino acid sequence shown
in SEQ ID NO: 55 is more preferably glutamine (Gln, Q), glutamic
acid (Glu, E), or histidine (His, H). Furthermore, the 36.sup.th
amino acid residue from the N-terminal side in the amino acid
sequence shown in SEQ ID NO: 55 is more preferably leucine (Leu,
L), isoleucine (Ile, I), or valine (Val, V). Furthermore, the
47.sup.th amino acid residue from the N-terminal side in the amino
acid sequence shown in SEQ ID NO: 55 is more preferably leucine
(Leu, L), isoleucine (Ile, I), or valine (Val, V). Furthermore, the
50.sup.th amino acid residue from the N-terminal side in the amino
acid sequence shown in SEQ ID NO: 55 is more preferably lysine
(Lys, K), glutamic acid (Glu, E), glutamine (Gln, Q), aspartic acid
(Asp, D), or asparagine (Asn, N).
[0069] As examples, the nucleotide sequence of the coding region in
the gene specified by At3g05640 is shown in SEQ ID NO: 4 and the
amino acid sequence of protein phosphatase 2C encoded by the gene
specified by At3g05640 is shown in SEQ ID NO: 5. Also, the
nucleotide sequence of the coding region in the gene specified by
At5g27930 is shown in SEQ ID NO: 35 and the amino acid sequence of
protein phosphatase 2C encoded by the gene specified by At5g27930
is shown in SEQ ID NO: 36. Moreover, the nucleotide sequence of the
coding region in the gene specified by At3g02750 is shown in SEQ ID
NO: 41 and the amino acid sequence of protein phosphatase 2C
encoded by the gene specified by At3g02750 is shown in SEQ ID NO:
42. Furthermore, the nucleotide sequence of the coding region in
the gene specified by At3g16800 is shown in SEQ ID NO: 47 and the
amino acid sequence of protein phosphatase 2C encoded by the gene
specified by At3g16800 is shown in SEQ ID NO: 48.
[0070] Also, in the present invention, genes functionally
equivalent to genes listed above may also be over-expressed. Here,
the term "functionally equivalent gene" refers to, for example, a
gene (from an organism other than Arabidopsis thaliana) that: has
the 3 consensus sequences (preferably, the 3 consensus sequences
comprising the amino acid sequences shown in SEQ ID NOS: 53-55. The
same applies to the following) comprising the amino acid sequences
shown in SEQ ID NOS: 1-3 in such order from the N-terminal side;
and encodes protein phosphatase 2C. Also, the term "functionally
equivalent gene" refers to a gene that encodes a protein having
protein phosphatase 2C activity. The term "protein phosphatase 2C
activity" refers to Mg.sup.2+- or Mn.sup.2+-dependent
serine/threonine phosphatase (Ser/Thr phosphatase) activity.
Therefore, whether or not a gene encodes a protein having protein
phosphatase 2C activity can be confirmed by examining whether or
not the gene product has serine/threonine phosphatase activity in
the presence of Mg.sup.2+ or Mn.sup.2+. Conventionally known
techniques can be appropriately employed for determining
serine/threonine phosphatase activity. For example, a commercially
available activity determination kit ProFluor (registered
trademark) Ser/Thr Phosphatase Assay (Promega) can be used.
[0071] Here, example of organisms is not limited to Arabidopsis
thaliana. For example, rice (Oryza sativa) is included.
Specifically, an example of a functionally equivalent gene is a
rice Os05g0358500 gene. The nucleotide sequence of a coding region
of the Os05g0358500 gene is shown in SEQ ID NO: 6 and the amino
acid sequence of the protein encoded by the gene is shown in SEQ ID
NO: 7. Also, examples of the above-mentioned rice-derived
functionally equivalent gene include Os11g0109000 (the nucleotide
sequence and the amino acid sequence are shown in SEQ ID NOS: 8 and
9, respectively), Os12g0108600 (the nucleotide sequence and the
amino acid sequence are shown in SEQ ID NOS: 10 and 11,
respectively), Os02g0471500 (the nucleotide sequence and the amino
acid sequence are shown in SEQ ID NOS: 12 and 13, respectively),
Os04g0321800 (the nucleotide sequence and the amino acid sequence
are shown in SEQ ID NOS: 14 and 15, respectively), Os11g0417400
(the nucleotide sequence and the amino acid sequence are shown in
SEQ ID NOS: 16 and 17, respectively), Os07g0566200 (the nucleotide
sequence and the amino acid sequence are shown in SEQ ID NOS: 18
and 19, respectively), Os08g0500300 (the nucleotide sequence and
the amino acid sequence are shown in SEQ ID NOS: 20 and 21,
respectively), Os02g0224100 (the nucleotide sequence and the amino
acid sequence are shown in SEQ ID NOS: 22 and 23, respectively),
and Os02g0281000 (the nucleotide sequence and the amino acid
sequence are shown in SEQ ID NOS: 56 and 57, respectively).
[0072] Moreover, examples of the above-mentioned functionally
equivalent genes from plants other than Arabidopsis thaliana and
rice include genes (UniProt data base Accession Nos. A9P973,
A9PFSO, and A9P7U4) from black cottonwood (Populus trichocarpa),
genes (UniProt data base Accession Nos. A7PRZ8, A7Q8H4, A7PV59,
A5C3B0, A5BF43, A7QFG6, A7P4H7, A5C0C9, A5AP53, A7QQF9, and A5BDP5)
from european grape (Vitis vinerfera), genes (UniProt data base
Accession Nos. Q2HW33 and Q4L0F8) from Medicago truncatula
(Medicago truncatula), a gene (GenBank data base Accession No.
AY651248) from alfalfa (Medicago sativa), genes (UniProt data base
Accession Nos. A9SE70, A9SE69, and A9RFU1) from Physcomitrella
patens (Physcomitrella patens), a gene (UniProt data base Accession
No. 2511453C) from ice plant (Mesembryanthemum crystallinum), a
gene (UniProt data base Accession No. A8HQG8) from Chlamydomonas
reinhardtii (Chlamydomonas reinhardtii), genes (GenBank data base
Accession Nos. BT024031, BT017414, and BT024134) from corn (Zea
mays), genes (GenBank data base Accession Nos. AC189312 and
AC189579) from rapeseed (Brassica rapa), genes (GenBank data base
Accession Nos. AP009550, AP009302, and AP009278) from tomato
(Solanum lycopersicum), a gene (GenBank data base Accession No.
AC182571) from monkey flower (Mimulus guttatus), and a gene
(GenBank data base Accession No. AP006489) from monocellular red
alga (Cyanidioschyzon merolae).
[0073] In these plants other than Arabidopsis thaliana, which are
represented by the above examples, a gene encoding protein
phosphatase 2C that has the 3 consensus sequences comprising the
amino acid sequences shown in SEQ ID NOS: 1-3 in such order from
the N-terminal side can be easily searched for and/or identified
from a known database such as GenBank based on the above-listed
nucleotide sequence of Arabidopsis thaliana-derived protein
phosphatase 2C gene or amino acid sequence of protein phosphatase
2C.
[0074] In addition, a protein phosphatase 2C gene to be
over-expressed in the present invention is not limited to the above
described protein phosphatase 2C genes comprising the nucleotide
sequences and the amino acid sequences shown in SEQ ID NOS: 4-23,
35, 36, 41, 42, 47, and 48. Hence, the protein phosphatase 2C gene
may be a gene that contains an amino acid sequence having a
deletion, a substitution, an addition, or an insertion of one or a
plurality of amino acid sequences with respect to the amino acid
sequences shown in odd numbers of SEQ ID NOS: 4-23 or the amino
acid sequence shown in SEQ ID NO: 36, 42, or 48, and, has protein
phosphatase 2C activity. Here the term "a plurality of amino acids"
refers to 1 to 20, preferably 1 to 10, more preferably 1 to 7,
further preferably 1 to 5, and particularly preferably 1 to 3 amino
acids, for example. In addition, amino acid deletion, substitution,
or addition can be performed by altering a nucleotide sequence
encoding the above protein phosphatase 2C gene by a technique known
in the art. Mutation can be introduced into a nucleotide sequence
by a known technique such as the Kunkel method or the Gapped duplex
method or a method based thereof. For example, mutation is
introduced with a mutagenesis kit using site-directed mutagenesis
(e.g., Mutant-K or Mutant-G (both are trade names of Takara Bio))
or the like, or a LA PCR in vitro Mutagenesis series kit (trade
name, Takara Bio). Also, a mutagenesis method may be: a method
using a chemical mutation agent represented by EMS (ethyl
methanesulfonate), 5-bromouracil, 2-aminopurine, hydroxylamine,
N-methyl-N'-nitro-N nitrosoguanidine, or other carcinogenic
compounds; or a method that involves radiation treatment or
ultraviolet [UV] treatment typically using X-rays, alpha rays, beta
rays, gamma rays, an ion beam, or the like.
[0075] Also, protein phosphatase 2C genes to be over-expressed
herein may be genes homologous to the protein phosphatase 2C genes
comprising the nucleotide sequences and the amino acid sequences
shown in SEQ ID NOS: 4-23. Here, the term "homologous gene"
generally refers to a gene that has evolutionarily branched off
from a common ancestor gene, including a homologous gene (ortholog)
of 2 types of species and a homologous gene (paralog) generated by
overlapping branching that takes place within the same species. In
other words, the above term "functionally equivalent gene" refers
to a homologous gene such as an ortholog or a paralog. Furthermore,
the above term "functionally equivalent gene" may also refer to a
gene that does not evolve from a common gene, but simply has
analogous functions.
[0076] Examples of genes analogous to the protein phosphatase 2C
genes comprising the nucleotide sequences and the amino acid
sequences shown in SEQ ID NOS: 4-23, 35, 36, 41, 42, 47, and 48
include genes encoding proteins having: amino acid sequences that
have 70% or more, preferably 80% or more, more preferably 90% or
more, and most preferably 95% or more similarity to these amino
acid sequences; the 3 consensus sequences comprising the amino acid
sequences shown in SEQ ID NOS: 1-3 in such order from the
N-terminal side; and protein phosphatase 2C activity. Here, the
value of similarity refers to a value that can be found based on
default setting using a computer mounted with a BLAST (Basic Local
Alignment Search Tool) program and a database containing gene
sequence information.
[0077] Also, genes analogous to protein phosphatase 2C genes
comprising the nucleotide sequences and the amino acid sequences
shown in SEQ ID NOS: 4-23, 35, 36, 41, 42, 47, and 48 can be
identified by, when the plant genome information remains
unclarified, extracting the genome from a target plant or
constructing a cDNA library for a target plant and then isolating a
genomic region or cDNA hybridizing under stringent conditions to at
least a portion of the protein phosphatase 2C genes comprising the
nucleotide sequences shown in even numbers of SEQ ID NOS: 4-23 or
the nucleotide sequence shown in 35, 41, or 47. Here, the term
"stringent conditions" refers to conditions under which namely a
specific hybrid is formed, but a non-specific hybrid is never
formed. For example, such conditions comprise hybridization at
45.degree. C. with 6.times.SSC (sodium chloride/sodium citrate),
followed by washing at 50.degree. C. to 65.degree. C. with
0.2-1.times.SSC and 0.1% SDS. Alternatively, such conditions
comprise hybridization at 65.degree. C. to 70.degree. C. with
1.times.SSC, followed by washing at 65.degree. C. to 70.degree. C.
with 0.3.times.SSC. Hybridization can be performed by a
conventionally known method such as a method described in J.
Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed.,
Cold Spring Harbor Laboratory (1989).
[0078] The plant according to the present invention exerts
significantly improved production of biomass and/or seeds compared
with wild-type plants, as a result of overexpression of a protein
phosphatase 2C gene having the above described 3 consensus
sequences that comprise the amino acid sequences shown in SEQ ID
NOS: 1-3 from the N-terminal side in such order. Examples of a
technique for causing the overexpression of such protein
phosphatase 2C gene include a technique for modifying a promoter of
an endogenous protein phosphatase 2C gene in a target plant, a
technique for introducing an expression vector in which an
exogenous protein phosphatase 2C gene is arranged under control of
a promoter that enables overexpression, and a technique by which
the two above techniques are performed simultaneously.
[0079] A preferred example is a technique for introducing an
expression vector in which the above protein phosphatase 2C gene is
arranged under control of a promoter that enables overexpression
into a target plant.
Expression Vector
[0080] An expression vector is constructed to contain a promoter
that enables overexpression and the above described protein
phosphatase 2C gene. As a vector serving as a mother body for an
expression vector, various conventionally known vectors can be
used. For example, plasmids, phages, cosmids, or the like can be
used and such vector can be appropriately selected depending on
plant cells into which it is introduced and introduction methods.
Specific examples of such vector include pBR322, pBR325, pUC19,
pUC119, pBluescript, pBluescriptSK, and pBI vectors. Particularly,
when a method for introduction of a vector into a plant uses
Agrobacterium, a pBI binary vector is preferably used. Specific
examples of such pBI binary vector include pBIG, pBIN19, pBI101,
pBI121, and pBI221.
[0081] A promoter to be used herein is not particularly limited, as
long as it enables overexpression of a protein phosphatase 2C gene
within a plant. Any known promoter can be appropriately used.
Examples of such promoter include a cauliflower mosaic virus 35S
promoter (CaMV35S), various actin gene promoters, various ubiquitin
gene promoters, a nopaline synthase gene promoter, a tobacco PR1a
gene promoter, a tomato ribulose 1,5-bisphosphate
carboxylase.oxidase small subunit gene promoter, a napin gene
promoter, and an oleosin gene promoter. Of these, a cauliflower
mosaic virus 35S promoter, an actin gene promoter, or a ubiquitin
gene promoter can be more preferably used. The use of each of the
above promoters enables strong expression of any gene when it is
introduced into plant cells.
[0082] Also, a promoter having functions of causing site-specific
overexpression in a plant can also be used herein. As such
promoter, any conventionally known promoter can be used. When the
above described protein phosphatase 2C gene is site-specifically
over-expressed using such promoter, a plant organ in which the gene
is over-expressed can be more increased than wild-type plant
organs.
[0083] In addition, an expression vector may further contain other
DNA segments in addition to a promoter and the above protein
phosphatase 2C gene. Such other DNA segments are not particularly
limited and examples thereof include a terminator, a selection
marker, an enhancer, and a nucleotide sequence for enhancing
translation efficiency. Also, the above recombinant expression
vector may further have a T-DNA region. A T-DNA region can enhance
efficiency for gene introduction particularly when the above
recombinant expression vector is introduced into a plant using
Agrobacterium.
[0084] A transcription terminator is not particularly limited, as
long as it has functions as a transcription termination site and
may be any known transcription terminator. For example,
specifically, a transcription termination region (Nos terminator)
of a nopaline synthase gene, a transcription termination region
(CaMV35S terminator) of cauliflower mosaic virus 35S, or the like
can be preferably used. Of them, the Nos terminator can be more
preferably used. In the case of the above recombinant vector, a
phenomenon such that an unnecessarily long transcript is
synthesized and that a strong promoter decreases the number of
copies of a plasmid after introduction into plant cells can be
prevented by arranging a transcription terminator at an appropriate
position.
[0085] As a transformant selection marker, a drug resistance gene
can be used, for example. Specific examples of such drug resistance
gene include drug resistance genes against hygromycin, bleomycin,
kanamycin, gentamicin, chloramphenicol, and the like. Transformed
plants can be easily selected by selecting plants that can grow in
medium containing the above antibiotics.
[0086] An example of a nucleotide sequence for increasing
translation efficiency is an omega sequence from tobacco mosaic
virus. This omega sequence is arranged in an untranslated region
(5'UTR) of a promoter, so that the translation efficiency of the
fusion gene can be increased. As such, the recombinant expression
vector may contain various DNA segments depending on purposes.
[0087] A method for constructing a recombinant expression vector is
not particularly limited. To an appropriately selected vector
serving as a mother body, the above promoter and the above protein
phosphatase 2C gene, and if necessary, the above other DNA segments
may be introduced in an predetermined order. For example, the above
protein phosphatase 2C gene and a promoter (and, if necessary, a
transcription terminator or the like) are linked to construct an
expression cassette and then the cassette may be introduced into a
vector. In construction of an expression cassette, for example,
cleavage sites of DNA segments are prepared to have protruding ends
complementary to each other and then performing a reaction with a
ligation enzyme, making it possible to specify the order of the DNA
segments. In addition, when an expression cassette contains a
terminator, DNA segments may be arranged in the following order
from upstream: a promoter, the above protein phosphatase 2C gene,
and a terminator. Also, reagents for construction of an expression
vector (that is, types of restriction enzymes, ligation enzymes,
and the like) are also not particularly limited. Hence,
commercially available reagents can be appropriately selected and
used.
[0088] Also, a method for replicating (a method for producing) the
above expression vector is not particularly limited and
conventionally known replication methods can be used herein. In
general, such expression vector may be replicated within
Escherichia coli as a host. At this time, preferred types of
Escherichia coli may be selected depending on the types of
vector.
Transformation
[0089] The above-described expression vector is introduced into a
target plant by a general transformation method. A method for
introducing an expression vector into plant cells (transformation
method) is not particularly limited. Conventionally known
appropriate introduction methods can be used depending on plant
cells. Specifically, a method using Agrobacterium or a method that
involves direct introduction into plant cells can be used, for
example. As a method using Agrobacterium, a method described in
Bechtold, E., Ellis, J. and Pelletier, G. (1993) In Planta
Agrobacterium-mediated gene transfer by infiltration of adult
Arabidopsis plants. C.R. Acad. Sci. Paris Sci. Vie, 316,
1194-1199., or a method described in Zyprian E, Kado Cl,
Agrobacterium-mediated plant transformation by novel mini-T vectors
in conjunction with a high-copy vir region helper plasmid. Plant
Molecular Biology, 1990, 15(2), 245-256. can be employed, for
example:
[0090] As a method for directly introducing an expression vector
into plant cells, microinjection, electroporation, a polyethylene
glycol method, a particle gun method, protoplast fusion, a calcium
phosphate method, or the like can be employed.
[0091] Also, when a method for directly introducing DNA into plant
cells is employed, DNA that can be used herein contains
transcriptional units required for the expression of a target gene,
such as a promoter and a transcription terminator, and a target
gene. Vector functions are not essential in such case. Moreover, a
DNA that contains a protein coding region alone of a target gene
having no transcriptional unit may be used herein, as long as it is
integrated into a host's transcriptional unit and then the target
gene can be expressed.
[0092] Examples of plant cells into which the above expression
vector or an expression cassette containing no expression vector,
but a target gene is introduced include cells of each tissue of
plant organs such as flowers, leaves, and roots, calluses, and
suspension-cultured cells. At this time, an appropriate expression
vector may be constructed according to the types of plant to be
produced or a versatile expression vector may be constructed in
advance and then introduced into plant cells.
[0093] Plants into which an expression vector is introduced or in
other words, plants required to increase the production of biomass
are not particularly limited. Specifically, through overexpression
of the above-described protein phosphatase 2C gene, effects of
increasing the production of biomass can be expected for all
plants. Examples of target plants include, but are not limited to,
dicotyledons and monocotyledons, such as plants (see below)
belonging to the families Brassicaceae, Gramineae, Solanaceae,
Leguminosae, Salicaceae, and the like. [0094] Family Brassicaceae:
Arabidopsis thaliana (Arabidopsis thaliana), rapeseed (Brassica
rapa, Brassica napus, Brassica campestris), cabbage (Brassica
oleracea var capitata), napa (Brassica rapa var. pekinensis),
ging-geng-cai (Brassica rapa var. chinensis), turnip (Brassica rapa
var. rapa), turnip greens (Brassica rapa var. hakabura), potherb
mustard (Brassica rapa var. lancinifolia), komatsuna (Brassica rapa
var. peruviridis), pak choi (Brassica rapa var. chinensis), daikon
(Raphanus sativus), Japanese horseradish (Wasabia japonica), and
the like. [0095] Family Solanaceae: tobacco (Nicotiana tabacum),
eggplant (Solanum melongena), potato (Solaneum tuberosum), tomato
(Lycopersicon lycopersicum), chile pepper (Capsicum annuum),
petunia, and the like. [0096] Family Leguminosae: soy (Glycine
max), pea (Pisum sativum), broad bean (Vicia faba), Wisteria
(Wisteria floribunda), peanuts (Arachis hypogaea), bird's foot
trefoil (Lotus corniculatus var. japonicus), common bean (Phaseolus
vulgaris), azuki bean (Vigna angularis); Acacia, and the like.
[0097] Family Asteraceae: florists' daisy (Chrysanthemum
morifolium), sunflower (Helianthus annuus), and the like. [0098]
Family Arecaceae: oil palm (Elaeis guineensis, Elaeis oleifera),
coconut (Cocos nucifera), date palm (Phoenix dactylifera),
copernicia, and the like. [0099] Family Anacardiaceae: wax tree
(Rhus succedanea), cashew nut (Anacardium occidentale), lacquer
tree (Toxicodendron vernicifluum), mango (Mangifera indica),
pistachio (Pistacia vera), and the like. [0100] Family
Cucurbitaceae: pumpkin (Cucurbita maxima, Cucurbita moschata,
Cucurbita pepo), cucumber (Cucumis sativus), snake gourd
(Trichosanthes cucumeroides), gourd (Lagenaria siceraria var.
gourda), and the like. [0101] Family Rosaceae: almond (Amygdalus
communis), rose (Rosa), strawberry (Fragaria), cherry (Prunus),
apple (Malus pumila var domestica), and the like. [0102] Family
Caryophyllaceae: carnation (Dianthus caryophyllus) and the like.
[0103] Family Salicaceae: poplar (Populus trichocarpa, Populus
nigra, or Populus tremula) and the like. [0104] Family Gramineae:
corn (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare),
wheat (Triticum aestivum), bamboo (Phyllostachys), sugarcane
(Saccharum officinarum), napier grass (Pennisetum pupureum),
erianthus (Erianthus ravenae), miscanthus (Japanese silver grass)
(Miscanthus virgatum), sorghum (Sorghum) and switch grass
(Panicum), and the like. [0105] Family Liliaceae: tulip (Tulipa),
lily (Lilium), and the like.
[0106] Of these examples, energy crops such as sugarcane, corn,
rapeseed, and sunflower, which can serve as raw materials for
biofuel, may be preferable targets. This is because the costs for
biofuel such as bioethanol, biodiesel, biomethanol, bioDME, bioGTL
(BTL), and biobutanol can be reduced by increasing the production
of biomass using energy crops.
[0107] Also, as described above, protein phosphatase 2C genes that
can be used in the present invention can be isolated from various
plants and used. Such protein phosphatase 2C genes can be
appropriately selected and used, depending on the types of target
plant required to increase the biomass production. Specifically,
when a plant required to increase the biomass production is a
monocotyledon, a protein phosphatase 2C gene that is isolated from
a monocotyledon is preferably over-expressed. In particular, when a
plant required to increase the biomass production is rice, the
rice-derived protein phosphatase 2C gene (SEQ ID NO: 6) is
preferably over-expressed.
[0108] In addition, in the present invention, even when a plant
required to increase the biomass production is a monocotyledon, a
dicotyledon-derived protein phosphatase 2C gene may be
over-expressed. Specifically, for example, the Arabidopsis
thaliana-derived protein phosphatase 2C gene (SEQ ID NO: 4) may be
introduced into not only dicotyledons, but also a variety of plants
that are classified as monocotyledons, so that the gene is
over-expressed.
Other steps and methods
[0109] After the above transformation, a step of selecting proper
transformants from plants can be performed by a conventionally
known method. Such selection method is not particularly limited.
For example, selection can be made based on drug resistance such as
hygromycin resistance. Alternatively, after the growth of
transformants, plants are directly weighed or the any organs or
tissues thereof are weighed, the weights are compared with those of
wild-type plants, and then plants with significantly increased
weights thereof may be selected.
[0110] Also, progeny plants can be obtained from transformed plants
obtained by transformation according to a conventional method.
Progeny plants retaining a trait such that the protein phosphatase
2C gene is over-expressed are selected based on the amount of
biomass. Therefore, a stable plant line capable of producing an
increased amount of biomass because of having the above trait can
be produced. Also, plant cells or reproductive materials, such as
seeds, fruits, stocks, calluses, tubers, cut ears, or lumps, may be
obtained from a transformed plant or an offspring plant thereof. A
stable plant line capable of producing an increased amount of
biomass because of having the above trait can be mass-produced
therefrom based on such materials.
[0111] In addition, examples of the term "plant(s)" in the present
invention include at least any of grown plants, plant cells, plant
tissues, calluses, and seeds. Specifically, in the present
invention, any forms of plants that can be finally grown to mature
plants are regarded as "plants." Also, examples of such plant cells
include various forms of plant cells, such as suspended culture
cells, protoplasts, and leaf sections. Plants can be obtained
through the growth and differentiation of these plant cells. In
addition, regeneration of plants from plant cells can be performed
using a conventionally known method depending on the type of plant
cells.
[0112] As explained above, according to the present invention,
plants capable of exerting the significantly increased production
of biomass and/or seeds per plant compared with wild-type plants
can be provided through overexpression of the above described
protein phosphatase 2C gene. Here, the term "significantly
increased production of biomass" refers to a situation in which the
total weight of each plant is statistically significantly increased
compared with the same of a wild-type plant. In this case, even
when some plant tissues become specifically large and the sizes of
the other tissues are equivalent to those of a wild-type plant, it
is concluded that the production of biomass is increased if the
total weight of the entire plant is large. Also, the term
"significantly increased production of seeds" refers to a situation
in which the total amount and/or total number of seeds harvested
from a plant is statistically significantly high compared with
wild-type plants. That is, the term "significantly increased
production of seeds" may refer to any of: a case in which the size
of each seed is improved; a case where the size per seed is
equivalent but the number of seeds is improved; or a case in which
the size per seed is improved and the number of seeds is also
improved.
[0113] According to the present invention, the production of
biomass and/or seeds by plants is increased. Hence, improvement in
productivity can be achieved in both of the following cases: a case
in which a purpose is to produce the whole plant; and a case in
which a purpose is to produce some plant tissues (e.g., seeds) or
components contained in plants. For example, when a purpose is to
produce fats and oils contained in plant seeds, the amounts of fats
and oils that can be harvested per area under cultivation can be
greatly improved. Here, examples of fats and oils include, but are
not particularly limited to, plant-derived fats and oils such as
soybean oil, sesame oil, olive oil, coconut oil, rice oil,
cottonseed oil, sunflower oil, corn oil, safflower oil, and
rapeseed oil. Also, the thus produced fats and oils can be broadly
used for household uses or industrial uses and can be further used
as raw materials for biodiesel fuel. Hence, according to the
present invention, the above fats and oils for household uses or
industrial uses, biodiesel fuel, and the like can be produced at
low cost with the use of plants over-expressing the above protein
phosphatase 2C gene.
EXAMPLES
[0114] The present invention will be specifically described in the
following reference examples and examples. However, the examples
are not intended to limit the technical scope of the present
invention.
Example 1
[0115] Preparation of Transformants (Arabidopsis thaliana) Through
Introduction of the PP2C (Protein Phosphatase 2C) Gene
(At3g05640)
1. Materials and Methods
1-1. Experimental Materials
[0116] As experimental materials, seeds of Arabidopsis thaliana
mutants (Activation-tag T-DNA lines: Weigel T-DNS lines, Total of
20072 lines) were used. In addition, the seeds were purchased from
the Nottingham Arabidopsis Stock Centre (NASC). Regarding the seeds
used as experimental materials, Weigel, D. et al., 2000, Plant
Physiol. 122, 1003-1013 can be referred to.
1-2. Methods
1-2-1. Selection of Salt-Resistant Mutants
[0117] Seeds of Weigel T-DNA lines were aseptically sowed on 125 mM
or 150 mM NaCl-containing modified MS agar (1%) medium [vitamins in
B5 medium, 10 g/l sucrose, and 8 g/L agar (for bacterial medium;
Wako Pure Chemical Industries, Ltd.)] and then cultured at
22.degree. C. under 30-100 .mu.mol/m.sup.2/sec illumination (a
cycle of 16 hours in the light/8 hours in the dark). Two to 4 weeks
after sowing, salt-resistant mutant candidates were selected. In
addition, regarding MS medium, see Murashige, T. et al., 1962,
Physiol. Plant. 15, 473-497. Also, regarding the B5 medium, see
Gamborg, O. L. et al., 1968, Experimental Cell Research 50,
151-158.
1-2-2. DNA Preparation
[0118] A site for insertion of T-DNA into the genome of the thus
selected salt-resistant Arabidopsis thaliana line was determined by
a TAIL-PCR method. First, young leaves were harvested from the
cultivated Arabidopsis thaliana plants and then crushed under
liquid nitrogen freezing. DNA was prepared using a DNA preparation
kit (DNeasy Plant Mini Kit, QIAGEN) according to the standard
protocols included with the kit.
1-2-3. TAIL-PCR Method and Presumption of T-DNA Insertion Site
[0119] Three (3) types of specific primer, TL1, TL2, and TL3, were
determined to be located near the left T-DNA sequence (T-DNA left
border) of an activation-tagging vector (pSKI015: GenBank accession
No. AF187951) used in Weigel T-DNA lines. With the use of an
arbitrary primer P1 and the following PCR reaction solutions and
reaction conditions, TAIL-PCR (supervisors, Isao Shimamoto and
Takuji Sasaki, New Edition, Plant PCR Experimental Protocols, 2000,
pp. 83-89, Shujunsha, Tokyo, Japan; Liu, Y. G. and Whttier, R. F.,
1995, Genomics 25, 674-681; Liu, Y.G. et al., Plant J., 8, 457-463,
1995) was performed, so that genomic DNA adjacent to T-DNA was
amplified. The specific sequences of the primers TL1, TL2, TL3, and
P1 are as follows.
TABLE-US-00001 (SEQ ID NO: 24) TL1: 5'-TGC TTT CGC CAT TAA ATA GCG
ACG G-3' (SEQ ID NO: 25) TL2: 5'-CGC TGC GGA CAT CTA CAT TTT TG-3'
(SEQ ID NO: 26) TL3: 5'-TCC CGG ACA TGA AGC CAT TTA C-3' (SEQ ID
NO: 27) P1: 5'-NGT CGA SWG ANA WGA A-3'
[0120] In addition, in SEQ ID NO: 25, "n" represents "a," "g," "c,"
or "t" (location: 1 and 11), "s" represents "g" or "c" (location:
7), and "w" represents "a" or "t" (location: 8 and 13).
[0121] The 1.sup.st PCR reaction solution composition and reaction
conditions are shown in Table 1 and Table 2, respectively.
TABLE-US-00002 TABLE 1 Template (genomic DNA) 10 ng 10 x PCR buffer
(Takara Bio) 2 .mu.l 2.5 mM dNTPs (Takara Bio) 1.6 .mu.l 1.sup.st
specific primer (TL1: SEQ ID NO: 24) 0.5 pmol Arbitrary primer 1
(SEQ ID NO: 27) 100 pmol TaKaRa Ex Taq (Takara Bio) 1.0 unit Total
20 .mu.l
TABLE-US-00003 TABLE 2 #1: 94.degree. C. (30 seconds)/95.degree. C.
(30 seconds) #2: 5 cycles of 94.degree. C. (30 seconds)/65.degree.
C. (30 seconds)/72.degree. C. (1 minute) #3: 1 cycle of 94.degree.
C. (30 seconds)/25.degree. C. (1 minute).fwdarw.raised to
72.degree. C. within 3 minutes/72.degree. C. (3 minutes) #4:
94.degree. C. (15 seconds)/65.degree. C. (30 seconds)/72.degree. C.
(1 minute), 94.degree. C. (15 seconds)/68.degree. C. (30
seconds)/72.degree. C. (1 minute), and 15 cycles of 94.degree. C.
(15 seconds)/44.degree. C. (30 seconds)/72.degree. C. (1 minute)
#5: 72.degree. C. (3 minutes)
[0122] The 2.sup.nd PCR reaction solution composition and reaction
conditions are shown in Table 3 and Table 4, respectively.
TABLE-US-00004 TABLE 3 Template (50-fold dilution of the 1.sup.st
PCR product) 1 .mu.l 10 x PCR buffer (Takara Bio) 2 .mu.l 2.5 mM
dNTPs (Takara Bio) 1.5 .mu.l 2.sup.nd specific primer (TL2: SEQ ID
NO: 25) 5 pmol Arbitrary primer 1 (SEQ ID NO: 27) 100 pmol TaKaRa
Ex Taq (Takara Bio) 0.8 unit Total 20 .mu.l
TABLE-US-00005 TABLE 4 #6: 94.degree. C. (15 seconds)/64.degree. C.
(30 seconds)/72.degree. C. (1 minute), 94.degree. C. (15
seconds)/64.degree. C. (30 seconds)/72.degree. C. (1 minute), and
12 cycles of 94.degree. C. (15 seconds)/44.degree. C. (30
seconds)/72.degree. C. (1 minute) #5: 72.degree. C. (5 minutes)
[0123] The 3.sup.rd PCR reaction solution composition and reaction
conditions are shown in Table 5 and Table 6, respectively.
TABLE-US-00006 TABLE 5 Template (50-fold dilution of the 2.sup.nd
PCR product) 1 .mu.l 10 x PCR buffer (Takara Bio) 5 .mu.l 2.5 mM
dNTPs (Takara Bio) 0.5 .mu.l 3.sup.rd specific primer (TL3: SEQ ID
NO: 26) 10 pmol Arbitrary primer 1 (SEQ ID NO: 27) 100 pmol TaKaRa
Ex Taq (Takara Bio) 1.5 unit Total 50 .mu.l
TABLE-US-00007 TABLE 6 #7: 20 cycles of 94.degree. C. (30
seconds)/44.degree. C. (30 seconds)/72.degree. C. (1 minute) #5:
72.degree. C. (3 minutes)
[0124] Subsequently, the 2.sup.nd and the 3.sup.rd reaction
products were subjected to agarose gel electrophoresis and then the
presence or the absence of amplification and the specificity of
reaction products were confirmed. Also, the 3.sup.rd amplification
products were subjected to a sequencing reaction directly using a
BigDye Terminator Cycle Sequencing Kit Ver. 3. 1 (Applied
Biosystems) and the specific primer TL3. Thus, a nucleotide
sequence was determined using an ABI PRISM 3100 Genetic Analyzer
(Applied Biosystems). As a result, 498-bp sequence information was
obtained (SEQ ID NO: 28).
[0125] The Arabidopsis Information Resource (TAIR:
http://www.arabidopsis.org/) was subjected to a BLAST search for
the thus obtained sequence. Thus, the insertion site was found to
be the gene of [AGI (Arabidopsis Genome Initiative gene code) code:
At3g05630] of Arabidopsis thaliana chromosome 3.
1-2-4. Prediction of Activated Genes
[0126] Activated genes were predicted from the sequence of a
presumed open reading frame (ORF) gene existing within a 10-Kb
range near the T-DNA insertion site (At3g05630) revealed in
1-2-3.
1-2-5. Obtainment of Predicted Genes
[0127] For amplification of a fragment containing the ORF region of
PP2C (protein phosphatase 2C) gene (At3g05640) predicted to be
activated in 1-2-4, PCR primers 5640PF1 and 5640PR1 were designed
and synthesized based on the sequence information disclosed at the
TAIR (http://www.arabidopsis.org/home.html). In addition, these
primers were designed, so that a restriction enzyme site (BsrG I or
Sal. I) required for introduction into expression vectors was added
to the terminus of each primer.
TABLE-US-00008 5640PF1 (SEQ ID NO: 29): 5'-ACG CGT CGA CAT GGG ACA
TTT CTC TTC CAT GTT CAA CGG-3' 5640PR1 (SEQ ID NO: 30): 5'-TGT ACA
TGT ACA CTA TAG AGA TGG CGA CGA CGA TGA AGA ATG G-3'
[0128] According to the method described in 1-2-2, a template DNA
was prepared from wild-type Arabidopsis thaliana (ecotype Col-0).
Phusion High-Fidelity DNA Polymerase (New England BioLabs: NEB) was
used as an enzyme and the above 5640PF1 and 5640PR1 were used as
primers. The relevant PCR reaction solution composition and
reaction conditions are shown in Table 7 and Table 8,
respectively.
TABLE-US-00009 TABLE 7 Template (genomic DNA) 60 ng 10 x HF buffer
(NEB) 5 .mu.l 10 mM dNTPs (NEB) 1.0 .mu.l Each primer 20 pmol
Phusion High-Fidelity DNA Polymerase 1.0 unit Total 50 .mu.l
TABLE-US-00010 TABLE 8 #1: 98.degree. C. (30 seconds) #2: 15 cycles
of 98.degree. C. (10 seconds)/55.degree. C. (30 seconds)/72.degree.
C. (30 seconds) #5: 72.degree. C. (10 minutes)
[0129] PCR amplification products were subjected to electrophoresis
with 2% agarose gel (TAE buffer) and then fragments were stained
with ethidium bromide. A gel containing target fragments was
excised using a scalpel. Target DNA fragments were eluted and
purified using GFX PCR DNA and a GEL Band Purification Kit
(Amersham). Adenin was added to the thus obtained DNA fragment
using an A-Addition Kit (QIAGEN). The amplified DNA to which
adenine had been added was ligated to a TA-Cloning pCR2.1 vector
using a TOPO TA Cloning Kit (Invitrogen) and then transformed into
competent cells (E. coli TOP 10) included with the kit. After
transformation, cells were cultured in LB medium supplemented with
50 .mu.l/ml kanamycin and then transformants were selected.
Colonies that had appeared were subjected to liquid culture in LB
medium supplemented with 50 .mu.l/ml kanamycin. Plasmid DNA was
prepared from the thus obtained microorganisms using a Plasmid Mini
Kit (QIAGEN). The thus obtained fragment containing ORF of the PP2C
(protein phosphatase 2C) gene (At3g05640) was cloned into a vector,
followed by determination of the nucleotide sequence and sequence
analysis.
1-2-6. Construction of Plant Expression Vector
[0130] A fragment containing ORF of the PP2C (protein phosphatase
2C) gene (At3g05640) was inserted into a plant expression vector
pBI121 containing an omega sequence from tobacco mosaic virus.
Thus, a construct was prepared.
[0131] First, the pCR2.1 vector, in which a fragment containing ORF
of the PP2C (protein phosphatase 2C) gene (At3g05640) had been
cloned in 1-2-5, was treated with restriction enzymes Sal I and
BsrG I.
[0132] Next, similarly pBI121 containing an omega sequence was
treated with restriction enzymes Sal I and BsrG I. The products
digested with these restriction enzymes were subjected to 0.8%
agarose gel electrophoresis. A fragment of about 2700 by containing
ORF of the PP2C (protein phosphatase 2C) gene (At3g05640) and
pBI121 containing the omega sequence were each fractioned and
purified from the gel using GFX PCR DNA and a GEL Band Purification
Kit (Amersham).
[0133] For introduction of a fragment containing ORF of the PP2C
(protein phosphatase 2C) gene (At3g05640) using a pBI121 fragment
containing the omega sequence as a vector, the vector and the
insert were mixed at a ratio of 1 : 10, followed by an overnight
ligation reaction at 16.degree. C. using an equivalent amount of a
TaKaRa Ligation kit ver. 2 (Takara Bio Inc.).
[0134] The total amount of the reaction solution was added to 100
.mu.l of competent cells (E. coli strain DH5.alpha., TOYOBO), so
that transformation was performed according to protocols included
with the kit. Cells were applied to LB agar medium containing 50
.mu.g/ml kanamycin and then cultured overnight. Colonies that had
appeared were subjected to liquid culture in LB medium supplemented
with 50 .mu.g/ml kanamycin. Plasmid DNA was prepared from the thus
obtained microorganisms using a Plasmid Mini Kit (QIAGEN).
[0135] The thus obtained fragment containing ORF of the PP2C
(protein phosphatase 2C) gene (At3g05640) was subcloned into an
expression vector, followed by determination of the nucleotide
sequence and sequence analysis.
1-2-7. Gene Introduction into Arabidopsis thaliana Using
Agrobacterium Method
[0136] The plant expression vector constructed in 1-2-6 was
introduced into Agrobacterium tumefaciens C58C1 strain by
electroporation (Plant Molecular Biology Mannal, Second Edition, B.
G. Stanton and A. S. Robbert, Kluwer Acdemic Publishers 1994).
Subsequently, Agrobacterium tumefaciens in which the plant
expression vector had been introduced was introduced into wild-type
Arabidopsis thaliana (ecotype Col-0) by an infiltration method
described by Clough et al. (Steven J. Clough and Andrew F. Bent,
1998, The Plant Journal 16, 735-743).
[0137] Transformants were selected using kanamycin-containing
medium. T1 generation plants were produced by self-pollination from
the transformants, so that T2 seeds were obtained.
1-2-8. Confirmation of the Phenotype of Transformant
[0138] T2 seeds produced in 1-2-7 were aseptically sowed and then
the resulting plants were transplanted into pots (each with a
diameter of 50 mm) containing vermiculite mixed soil. As control
plants for comparison, Arabidopsis plants that had not undergone
recombination were transplanted. They were cultivated under
conditions of 22.degree. C. and 16 hours in the light/8 hours in
the dark, and with a light intensity ranging from about 30 to 45
.mu.mol/m.sup.-2/s.sup.-1, for a total of 11 weeks after
transplantation. After cultivation, above-ground parts of the
plants were placed in paper bags and dried under conditions of
22.degree. C. and humidity of 60% for 2 weeks. The total amounts of
biomass and seeds were weighed using an electronic balance.
1-3. Results
[0139] Regarding the results of 1-2-8, FIG. 3 shows a photo of the
above-ground parts of wild-type plants and transformed plants into
which a fragment containing ORF of the PP2C (protein phosphatase
2C) gene (At3g05640) had been introduced. Also, FIG. 4 and FIG. 5
show the results of measuring the total amounts of biomass and
seeds of the above-ground parts of the plants.
[0140] As shown in FIGS. 3, 4, and 5, it was revealed that in the
case of transformed plants into which the fragment containing ORF
of the PP2C (protein phosphatase 2C) gene (At3g05640) had been
introduced, the total amounts of biomass of the above-ground parts
were much higher (about 1.9 to 2.1 times) than the amounts of the
same in the cases of wild-type plants. In addition, the amounts of
seeds were much greater (by about 1.7 to 1.8 times) than the same
in the cases of wild-type plants.
Example 2
[0141] Preparation of Transformants (Rice) Through Introduction of
the PP2C (Protein Phosphatase 2C) cDNA (At3g05640)
2. Materials and Methods
2-1. Experimental Materials
[0142] As experimental materials, Arabidopsis transformants into
which a fragment containing ORF of the PP2C gene (At3g05640)
prepared in 1 was introduced into Arabidopsis thaliana and rice
(Oryza sativa L. ssp. japonica (cv. Nipponbare)) to make
transformants as experimental materials.
2-2. Methods
[0143] 2-2-1. Obtainment of PP2C (Protein Phosphatase 2C) cDNA
(At3g05640)
[0144] Arabidopsis transformants prepared by introduction of a
fragment containing ORF of the PP2C gene (At3g05640) prepared in 1
were grown until the plants reached 4 weeks of age. Total RNA was
isolated from the above-ground parts, then RT-PCR was performed
using TaqMan Reverse Transcription Reagents (Applied Biosystems),
so that cDNA was prepared.
[0145] PCR was performed using the following primers that had been
designed based on the nucleotide sequence (SEQ ID NO: 4) of the
coding region of PP2C (At3g05640) and PrimeSTAR HS DNA Polymerease
(Takara Bio). The thus amplified fragment was TA-cloned into a
pCR-Blunt II-TOPO vector (Invitrogen).
TABLE-US-00011 (SEQ ID NO: 31) AP041-F:
5'-AGGATCCATGGGACATTTCTCTTCCATGT-3' (SEQ ID NO: 32) AP041-R:
5'-AGAGCTCCTATAGAGATGGCGACGACG-3'
2-2-2. Construction of Plant Expression Vector
[0146] A GUS (.beta.-Glucuronidase) portion of pIG121-Hm (Ohat, S.
et al., 1990, Plant Cell Physiol. 31, 805-813) was substituted with
sGFP (S65T) having an intron fragment of Ricinus communis-derived
catalase, so that a plant expression vector, pBIsGFP, was
constructed. Furthermore, a sequence containing pDEST R4-R3
recombination sites (attR4 and attR3) included in a MultiSite
Gateway Three-Fragement Vector Construction Kit (Invitrogen) was
inserted, so that a destination vector, pBI-sGFP-R4R3, was
constructed.
[0147] A corn-derived ubiquitin gene promoter (SEQ ID NO: 33:
Christensen, A. H. and Quail, P. H., Transgenic Research 1996, 5,
213-218), PP2C cDNA (At3g05640) obtained in 2-2-1, and an
Agrobacterium tumefaciens Ti plasmid-derived nopaline synthase gene
(NOS) terminator (obtained from SEQ ID NO: 34: pIG121-Hm) were
cloned by BP reaction to result in donor clones, pDONR P4-P1R,
pDONR 221, and DONR P2R-P3, respectively, included in a MultiSite
Gateway Three-Fragement Vector Construction Kit (Invitrogen), so
that entry clones were prepared.
[0148] An LR reaction was performed for each of the thus prepared
entry clones and a destination vector, pBI-sGFP-R4R3, so that a
plant expression vector containing the corn-derived ubiquitin gene
promoter, PP2C cDNA (At3g05640), and the nopaline synthase gene
(NOS) terminator, in such order, was constructed. The nucleotide
sequences of the thus obtained expression vectors were determined
and sequence analysis was conducted.
2-2-3. Gene Introduction into Rice Using Agrobacterium Method
[0149] The plant expression vectors constructed in 2-2-2 were
introduced into an Agrobacterium tumefaciens EHA101 strain. Then
Agrobacterium tumefaciens in which the plant expression vector had
been introduced was introduced into rice (Oryza sativa L. ssp.
japonica (cv. Nipponbare)). Specifically, the experiment was
conducted under conditions in accordance with the method disclosed
in JP Patent No. 3141084.
[0150] Transformed rice plants that had grown in
hygromycin-containing medium were selected and then T1 seedlings
(about 12 cm) were aseptically prepared.
2-2-4. Confirmation of Phenotype of Transformant
[0151] T1 plants prepared in 2-2-3 were transplanted in pots with a
diameter of about 10 cm containing Kumiai Hitetsu culture soil No.
2 (JA Aichi Keizairen (economic federation)). After
acclimatization, the plants were transplanted into 1/5000a Wagner
pots containing the same culture soil and then cultivated under
conditions of 30.degree. C., 16 hours in the light/8 hours in the
dark, and a light intensity of about 100 .mu.mol
m.sup.-2s.sup.-1.
[0152] As control plants, T1 plants into which a plant expression
vector (constructed by ligating 3 multiple cloning sites of a
pST-Blue1 vector (Novagen) to a destination vector pBI-sGFP-R4R3)
had been introduced were similarly cultivated.
2-3. Results
[0153] Regarding the results of 2-2-4 above, FIG. 6 shows photos
showing a control rice plant and a transformed rice plant prepared
by introduction of the coding region of PP2C (protein phosphatase
2C) (At3g05640). As shown in FIG. 6, in the above-ground parts of
the transformed rice plant into which the coding region of PP2C
(protein phosphatase 2C) (At3g05640) had been introduced, the total
amount of biomass was improved compared with the control rice
plant. It was revealed through the above results that when the
Arabidopsis thaliana-derived PP2C gene is expressed at a high level
in a plant other than Arabidopsis thaliana, the production of plant
biomass can be increased.
Example 3
[0154] Preparation of Transformant (Rice) Through Introduction of
PP2C (Protein Phosphatase 2C) cDNA (Os05g0358500)
3. Materials and Methods
3-1. Experimental Materials
[0155] As experimental materials, rice (Oryza sativa L. ssp.
japonica (cv. Nipponbare)) was used.
3-2. Methods
[0156] 3-2-1. Obtainment of Rice PP2C (Protein Phosphatase 2C) cDNA
(Os05g0358500)
[0157] In this Example, a rice homologous gene (PP2C gene
(Os05g0358500)) homologous to PP2C (protein phosphatase 2C)
(At3g05640) used in Examples 1 and 2 was used. The entire sequence
was chemically synthesized based on the nucleotide sequence (SEQ ID
NO: 6) of the coding region of rice PP2C (Os05g0358500). A fragment
of the chemically synthesized entire sequence was cloned into pDONR
221 that was a donor clone of a MultiSite Gateway Three-Fragement
Vector Construction Kit (Invitro gen).
3-2-2. Construction of Plant Expression Vector
[0158] A corn-derived ubiquitin gene promoter (SEQ ID NO: 33:
Christensen, A. H. and Quail, P. H., Transgenic Research 1996, 5,
213-218) and an Agrobacterium tumefaciens Ti plasmid-derived
nopaline synthase gene (NOS) terminator (SEQ ID NO: 34: obtained
from pIG121-Hm) were cloned by BP reaction to result in donor
clones, pDONR P4-P1R and DONR P2R-P3, respectively, included in a
MultiSite Gateway Three-Fragement Vector Construction Kit
(Invitrogen), so that entry clones were prepared.
[0159] An LR reaction was performed for pDONR 221 into which the
rice PP2C (Os05g0358500) cDNA sequence prepared in 3-2-1 had been
cloned, pDONR P4-P1R into which the above prepared corn-derived
ubiquitin gene promoter sequence had been cloned, DONR P2R-P3 into
which the nopaline synthase gene (NOS) terminator sequence had been
cloned, and the destination vector pBI-sGFP-R4R3 constructed in
2-2-2. Thus, a plant expression vector containing the corn-derived
ubiquitin gene promoter, the rice PP2C cDNA (Os05g0358500), and the
nopaline synthase gene (NOS) terminator, in such order, was
constructed. The nucleotide sequence of the thus obtained
expression vector was determined and then sequence analysis was
conducted.
3-2-3. Gene Introduction into Rice Using Agrobacterium Method
[0160] The plant expression vector constructed in 2-2-2 was
introduced into an Agrobacterium tumefaciens EHA101 strain. Then
Agrobacterium tumefaciens in which the plant expression vector had
been introduced was introduced into rice (Oryza sativa L. ssp.
japonica (cv. Nipponbare)). Specifically, the experiment was
conducted under conditions according to the method disclosed in JP
Patent No. 3141084.
[0161] Transformed rice plants that had grown in
hygromycin-containing medium were selected and then T1 seedlings
(about 12 cm) were aseptically prepared.
3-2-4. Confirmation of the Phenotype of Transformant
[0162] T1 plants prepared in 3-2-3 were transplanted in pots with a
diameter of about 10 cm containing Kumiai Hitetsu culture soil No.
2 (JA Aichi Keizairen (economic federation)). After
acclimatization, the plants were transplanted into 1/5000a Wagner
pots containing the same culture soil and then cultivated under
conditions of 30.degree. C., 16 hours in the light/8 hours in the
dark, and a light intensity of about 100 .mu.mol
[0163] As control plants, rice plants, into which no gene had been
introduced, were regulated to be at almost the same growth stage as
that of transformants, transplanted simultaneously with the
transplantation of transformants, and then cultivated
similarly.
3-3. Results
[0164] As the results of 3-2-4 above, FIG. 7 shows photos showing
control rice plants and transformed rice plants into which the
coding region of rice-derived plant PP2C (protein phosphatase 2C)
(Os05g0358500) had been introduced. As shown in FIG. 7, in the
above-ground parts of the transformed rice plants into which the
coding region of the rice-derived PP2C (protein phosphatase 2C)
(Os05g0358500) had been introduced, the total amount of biomass was
improved compared with the control rice plants. It was revealed by
the above results that when the rice-derived PP2C gene is expressed
at a high level in a rice plant, the production of rice plant
biomass can be increased.
Example 4
[0165] Preparation of Transformant (Arabidopsis thaliana) Through
Introduction of PP2C (Protein Phosphatase 2C) gene (At5g27930)
4. Materials and Methods
4-1. Experimental Materials
[0166] As experimental materials, wild-type Arabidopsis thaliana
(ecotype Col-0) was used.
4-2. Methods
[0167] 4-2-1. Obtainment of Arabidopsis thaliana PP2C (Protein
Phosphatase 2C) Gene (At5g27930)
[0168] In this Example, a PP2C (protein phosphatase 2C) gene
(At5g27930) having 3 consensus sequences comprising the amino acid
sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such
order was used, instead of PP2C (protein phosphatase 2C)
(At3g05640) used in Examples 1 and 2. For amplification of a
fragment containing an ORF region of Arabidopsis PP2C (protein
phosphatase 2C) gene (At5g27930), PCR primers, AP042-F5 and
AP042-R, were designed and synthesized based on the sequence
information disclosed in TAIR
(http://www.arabidopsis.org/home.html). Also, PCR primers
SalI-AP042-F2 and AP042-BsrGI-R2 were also designed and synthesized
so as to add a sequence (restriction enzyme site: BsrG I or Sal I)
on the vector side required upon cloning of the thus amplified
fragment into the vector using an In-Fusion cloning system
(Clontech). The nucleotide sequence of the coding region in the
PP2C gene (At5g27930) is shown in SEQ ID NO: 35 and the amino acid
sequence of the protein encoded by the PP2C gene (At5g27930) is
shown in SEQ ID NO: 36.
TABLE-US-00012 (SEQ ID NO: 37) AP042-F5:
5'-ATGGGACATTTCTCATCGATGTTC-3' (SEQ ID NO: 38) AP042-R:
5'-TTACTTTAAAATCGTCATGGCATGATG-3' (SEQ ID NO: 39) SalI-AP042-F2:
5'-AATTACTATTTACAATTACAGTCGACATGGGACATTTCTCATCGATG TTCAATGGA-3'
(SEQ ID NO: 40) AP042-BsrGI-R2:
5'-AGCCGGGCGGCCGCTTTACTTGTACATTACTTTAAAATCGTCATGGC
ATGATGATGTTG-3'
[0169] PCR was performed using template DNA prepared from wild-type
Arabidopsis thaliana (ecotype Col-0) according to the method of
1-2-2, the above primers, AP042-F5 and AP042-R, and PrimeSTAR HS
DNA Polymerase (Takara Bio), so that a fragment containing ORF of
the PP2C (protein phosphatase 2C) gene (At5g27930) was obtained.
4-2-2. Construction of plant expression vector
[0170] A fragment containing ORF of the PP2C (protein phosphatase
2C) gene (At5g27930) was inserted into a plant expression vector
pBI121 containing a tobacco mosaic virus-derived omega sequence, so
that a construct was prepared.
[0171] A fragment containing the PP2C gene (At5g27930) obtained in
4-2-1 was cloned into a vector using an In-Fusion cloning system
(Clontech), so that a construct was prepared. The nucleotide
sequence of the thus obtained expression vector in which the
fragment containing the PP2C gene (At5g27930) had been subcloned
was determined and sequence analysis was conducted.
4-2-3. Gene Introduction into Arabidopsis thaliana Using
Agrobacterium Method
[0172] The plant expression vector constructed in 4-2-2 was
introduced by electroporation (Plant Molecular Biology Mannal,
Second Edition, B. G. Stanton A. S. Robbert, Kluwer Acdemic
Publishers 1994) into an Agrobacterium tumefaciens C58C1-strain.
Subsequently, Agrobacterium tumefaciens into which the plant
expression vector had been introduced was introduced into wild-type
Arabidopsis thaliana (ecotype Col-0) by an infiltration method
described by Clough et al. (1998, The Plant Journal 16: 735-743).
T1 seeds were obtained by self-pollination.
4-2-4. Confirmation of the Phenotype of Transformant
[0173] T1 seeds obtained in 4-2-3 were aseptically sowed in
kanamycin-containing medium, so that T1 plants were prepared.
Seedlings selected using kanamycin-containing medium were
transplanted in pots with a diameter of 50 mm containing
vermiculite-mixed soil. As control plants, non-recombinant
Arabidopsis plants were transplanted. They were cultivated under
conditions of 22.degree. C., 16 hours in the light/8 hours in the
dark, and a light intensity of about 30-45 .mu.mol
m.sup.-2s.sup.-1.
4-3. Results
[0174] As the results of 4-2-4 above, FIGS. 8 and 9 show photos
showing the above-ground parts of wild-type plants and transformed
plants into which a fragment containing ORF of the PP2C (protein
phosphatase 2C) gene (At5g27930) had been introduced. As shown in
FIGS. 8 and 9, in the above-ground parts of the transformed plants
into which the fragment containing ORF of the PP2C gene (At5g27930)
had been introduced, the total amount of biomass was improved
compared with the wild-type plants. It was revealed by the above
results that when the PP2C (protein phosphatase 2C) gene having 3
consensus sequences comprising the amino acid sequences shown in
SEQ ID NOS: 1-3 from the N-terminal side in such order is expressed
at a high level, the production of the plant biomass can be
increased.
Example 5
[0175] Preparation of Transformant (Arabidopsis thaliana) Through
Introduction of PP2C (Protein Phosphatase 2C) Gene (At3g02750)
5. Materials and Methods
5-1. Experimental Materials
[0176] As experimental materials, wild-type Arabidopsis thaliana
(ecotype Col-0) was used.
5-2. Methods
[0177] 5-2-1. Obtainment of Arabidopsis thaliana PP2C (Protein
Phosphatase 2C) Gene (At3g02750)
[0178] In this Example, a PP2C (protein phosphatase 2C) gene
(At3g02750) having 3 consensus sequences comprising the amino acid
sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such
order was used, instead of PP2C (protein phosphatase 2C)
(At3g05640) used in Examples 1 and 2. For amplification of a
fragment containing an ORF region of the PP2C (protein phosphatase
2C) gene (At3g02750), PCR primers, AP036-F4 and AP036-R, were
designed and synthesized based on the sequence information
disclosed in TAIR (http://www.arabidopsis.org/home.html).
[0179] Also, PCR primers, SalI-AP036-F2 and AP036-BsrGI-R2, were
designed and synthesized so as to add a sequence (restriction
enzyme site: BsrG I or Sal I) on the vector side required upon
cloning of the thus amplified fragment into the vector using an
In-Fusion cloning system (Clontech). The nucleotide sequence of the
coding region in the PP2C gene (At3g02750) is shown in SEQ ID NO:
41 and the amino acid sequence of the protein encoded by the PP2C
gene (At3g02750) is shown in SEQ ID NO: 42.
TABLE-US-00013 (SEQ ID NO: 43) AP036-F4:
5'-ATGGGGTCCTGTTTATCTGCAG-3' (SEQ ID NO: 44) AP036-R:
5'-TCACTTTCCAGGCACAAATCTTG-3' (SEQ ID NO: 45) SalI-AP036-F2:
5'-AATTACTATTTACAATTACAGTCGACATGGGGTCCTGTTTATCTGCA GAGAGCAGG-3'
(SEQ ID NO: 46) AP036-BsrGI-R2:
5'-AGCCGGGCGGCCGCTTTACTTGTACATCACTTTCCAGGCACAAATCT TGGTAAGTT-3'
[0180] PCR was performed using template DNA prepared from wild-type
Arabidopsis thaliana (ecotype Col-0) according to the method of
1-2-2, the above primers, and PrimeSTAR HS DNA Polymerase (Takara
Bio), so that a fragment containing ORF of the PP2C (protein
phosphatase 2C) gene (At3g02750) was obtained.
5-2-2. Construction of Plant Expression Vector
[0181] A fragment containing ORF of the PP2C (protein phosphatase
2C) gene (At3g02750) was inserted into a plant expression vector
pBI121 containing a tobacco mosaic virus-derived omega sequence, so
that a construct was prepared.
[0182] A fragment containing the PP2C gene (At3g02750) obtained in
5-2-1 was cloned into a vector using an In-Fusion cloning system
(Clontech), so that a construct was prepared. The nucleotide
sequence of the thus obtained expression vector in which the
fragment containing the PP2C gene (At3g02750) had been subcloned
was determined and sequence analysis was conducted.
5-2-3. Gene Introduction into Arabidopsis thaliana Using
Agrobacterium Method
[0183] The plant expression vector constructed in 5-2-2 was
introduced by electroporation (Plant Molecular Biology Mannal,
Second Edition, B. G. Stanton A. S. Robbert, Kluwer Acdemic
Publishers 1994) into an Agrobacterium tumefaciens C58C1 strain.
Subsequently, Agrobacterium tumefaciens into which the plant
expression vector had been introduced was introduced into wild-type
Arabidopsis thaliana (ecotype Col-0) by an infiltration method
described by Clough et al. (1998, The Plant Journal 16: 735-743).
T1 seeds were obtained by self-pollination.
5-2-4. Confirmation of the Phenotype of Transformant
[0184] T1 seeds obtained in 5-2-3 were aseptically sowed in
kanamycin-containing medium, so that T1 plants were prepared.
Seedlings selected using kanamycin-containing medium were
transplanted in pots with a diameter of 50 mm containing
vermiculite-mixed soil. As control plants, non-recombinant
Arabidopsis plants were transplanted. They were cultivated under
conditions of 22.degree. C., 16 hours in the light/8 hours in the
dark, and a light intensity of about 30-45 .mu.mol
m.sup.-2s.sup.-1.
5-3. Results
[0185] As the results of 5-2-4 above, FIG. 10 shows a photo showing
the above-ground parts of wild-type plants and transformed plants
into which a fragment containing ORF of the PP2C (protein
phosphatase 2C) gene (At3g02750) had been introduced. As shown in
FIG. 10, in the above-ground parts of the transformed plants into
which the fragment containing ORF of the PP2C gene (At3g02750) had
been introduced, the total amount of biomass was improved compared
with the wild-type plants. It was revealed by the above results
that when the PP2C (protein phosphatase 2C) gene having 3 consensus
sequences comprising the amino acid sequences shown in SEQ ID NOS:
1-3 from the N-terminal side in such order is expressed at a high
level, the production of the plant biomass can be increased.
Example 6
[0186] Preparation of Transformants (Arabidopsis thaliana) Through
Introduction of PP2C (Protein Phosphatase 2C) Gene (At3g16800)
6. Materials and Methods
6-1. Experimental Materials
[0187] As experimental materials, wild-type Arabidopsis thaliana
(ecotype Col-0) was used.
6-2. Methods
[0188] 6-2-1. Obtainment of Arabidopsis thaliana PP2C (Protein
Phosphatase 2C) Gene (At3g16800)
[0189] In this Example, a PP2C (protein phosphatase 2C) gene
(At3g16800) having 3 consensus sequences comprising the amino acid
sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such
order was used, instead of PP2C (protein phosphatase 2C)
(At3g05640) used in Examples 1 and 2. For amplification of a
fragment containing an ORF region of the PP2C (protein phosphatase
2C) gene (At3g16800), PCR primers, AP040-F4 and AP040-R, were
designed and synthesized based on the sequence information
disclosed in TAIR (http://www.arabidopsis.org/home.html). Also, PCR
primers, SalI-AP040-F2 and AP040-BsrGI-R2, were synthesized and
designed so as to add a sequence (restriction enzyme site: BsrG I
or Sal I) on the vector side required upon cloning of the thus
amplified fragment into the vector using an In-Fusion cloning
system (Clontech). The nucleotide sequence of the coding region in
the PP2C gene (At3g16800) is shown in SEQ ID NO: 47 and the amino
acid sequence of the protein encoded by the PP2C gene (At3g16800)
is shown in SEQ ID NO: 48.
TABLE-US-00014 (SEQ ID NO: 49) AP040-F4:
5'-ATGGTGCTTTTACCAGCGTTTTTG-3' (SEQ ID NO: 50) AP040-R:
5'-CTAAGAAGGACGAAAGAAGAGAC-3' (SEQ ID NO: 51) SalI-AP040-F2:
5'-AATTACTATTTACAATTACAGTCGACATGGTGCTTTTACCAGCGTTT TTGGACGGATTAG-3'
(SEQ ID NO: 52) AP040-BsrGI-R2:
5'-AGCCGGGCGGCCGCTTTACTTGTACACTAAGAAGGACGAAAGAAGAG ACAGAGAAC-3'
[0190] PCR was performed using template DNA prepared from wild-type
Arabidopsis thaliana (ecotype Col-0) according to the method of
1-2-2, the above primers, and PrimeSTAR HS DNA Polymerase (Takara
Bio), so that a fragment containing ORF of the PP2C (protein
phosphatase 2C) gene (At3g16800) was obtained.
6-2-2. Construction of Plant Expression Vector
[0191] The fragment containing ORF of the PP2C (protein phosphatase
2C) gene (At3g16800) was inserted to a plant expression vector
pBI121 containing a tobacco mosaic virus-derived omega sequence, so
that a construct was prepared.
[0192] The fragment containing the PP2C gene (At3g16800) obtained
in 6-2-.1 was cloned into a vector using an In-Fusion cloning
system (Clontech), so that a construct was prepared. The nucleotide
sequence of the thus obtained expression vector into which the
fragment containing the PP2C gene (At3g16800) had been subcloned
was determined and sequence analysis was conducted.
6-2-3. Gene Introduction into Arabidopsis thaliana Using
Agrobacterium Method
[0193] The plant expression vector constructed in 6-2-2 was
introduced by electroporation (Plant Molecular Biology Mannal,
Second Edition, B. G. Stanton A. S. Robbert, Kluwer Acdemic
Publishers 1994) into an Agrobacterium tumefaciens C58C1 strain.
Subsequently, Agrobacterium tumefaciens into which the plant
expression vector had been introduced was introduced into wild-type
Arabidopsis thaliana (ecotype Col-0) by an infiltration method
described by Clough et al. (1998, The Plant Journal 16: 735-743).
T1 seeds were obtained by self-pollination.
6-2-4. Confirmation of the Phenotype of Transformant
[0194] T1 seeds obtained in 6-2-3 were aseptically sowed in
kanamycin-containing medium, so that T1 plants were prepared.
Seedlings selected using kanamycin-containing medium were
transplanted in pots with a diameter of 50 mm containing
vermiculite-mixed soil. As control plants, non-recombinant
Arabidopsis plants were transplanted. They were cultivated under
conditions of 22.degree. C., 16 hours in the light/8 hours in the
dark, and a light intensity of about 30-45 .mu.mol
m.sup.-2s.sup.-1.
6-3. Results
[0195] As the results of 6-2-4 above, FIG. 11 shows a photo showing
the above-ground parts of a wild-type plant and transformed plants
into which a fragment containing ORF of the PP2C (protein
phosphatase 2C) gene (At3g16800) had been introduced. As shown in
FIG. 11, in the above-ground parts of the transformed plants into
which the fragment containing ORF of the PP2C gene (At3g16800) had
been introduced, the total amount of biomass was improved compared
with the wild-type plant. It was revealed by the above results that
when the PP2C (protein phosphatase 2C) gene having 3 consensus
sequences comprising the amino acid sequences shown in SEQ ID NOS:
1-3 from the N-terminal side in such order is expressed at a high
level, the production of the plant biomass can be increased.
Example 7
[0196] Preparation of Transformant (Arabidopsis thaliana) Through
Introduction of PP2C (Protein Phosphatase 2C) cDNA
(Os05g0358500)
7. Materials and Methods 7-1. Experimental Materials
[0197] An experimental material, wild-type Arabidopsis thaliana
(ecotype Col-0) was used.
7-2. Methods
[0198] 7-2-1. Obtainment of Rice PP2C (Protein Phosphatase 2C) cDNA
(Os05g0358500)
[0199] In this Example, a rice homologous gene (PP2C gene
(Os05g0358500)) homologous to PP2C (protein phosphatase 2C)
(At3g05640) used in Examples 1 and 2 was used. The entire sequence
was chemically synthesized based on the nucleotide sequence (SEQ ID
NO: 6) of the coding region of rice PP2C (Os05g0358500). A fragment
of the chemically synthesized entire sequence was cloned into pDONR
221 that was a donor clone of a MultiSite Gateway Three-Fragement
Vector Construction Kit (Invitrogen).
7-2-2. Construction of Plant Expression Vector
[0200] A cauliflower mosaic virus-derived 35S (CaMV35S .OMEGA.)
promoter (SEQ ID NOS: 58) containing a tobacco mosaic virus-derived
omega sequence and an Agrobacterium tumefaciens Ti plasmid-derived
nopaline synthase gene (NOS) terminator (SEQ ID NOS: 34: obtained
from pIG121-Hm) were cloned by BP reaction to result in donor
clones, pDONR P4-P1R and DONR P2R-P3, respectively, included in a
MultiSite Gateway Three-Fragement Vector Construction Kit
(Invitrogen), so that entry clones were prepared.
[0201] An LR reaction was performed for pDONR 221 into which the
rice PP2C (Os05g0358500) cDNA sequence prepared in 7-2-1 had been
cloned, pDONR P4-P1R into which the above prepared CaMV35S .OMEGA.
promoter sequence had been cloned, DONR P2R-P3 into which the
nopaline synthase gene (NOS) terminator sequence had been cloned,
and the destination vector pBI-sGFP-R4R3 constructed in 2-2-2.
Thus, a plant expression vector containing the CaMV35S .OMEGA.
promoter, the rice PP2C cDNA (Os05g0358500), and the nopaline
synthase gene (NOS) terminator in such order was constructed. The
nucleotide sequence of the thus obtained expression vector was
determined and then sequence analysis was conducted.
7-2-3. Gene Introduction into Arabidopsis thaliana Using
Agrobacterium Method
[0202] The plant expression vector constructed in 7-2-2 was
introduced by electroporation (Plant Molecular Biology Mannal,
Second Edition, B. G. Stanton A. S. Robbert, Kluwer Acdemic
Publishers 1994) into an Agrobacterium tumefaciens C58C1 strain.
Subsequently, Agrobacterium tumefaciens into which the plant
expression vector had been introduced was introduced into wild-type
Arabidopsis thaliana (ecotype Col-0) by an infiltration method
described by Clough et al. (1998, The Plant Journal 16: 735-743).
T1 seeds were obtained by self-pollination.
7-2-4. Confirmation of the Phenotype of Transformant
[0203] T1 seeds obtained in 7-2-3 were aseptically sowed in
kanamycin-containing medium, so that T1 plants were prepared.
Seedlings selected using kanamycin-containing medium were
transplanted in pots with a diameter of 50 mm containing
vermiculite-mixed soil. As control plants, non-recombinant
Arabidopsis plants were transplanted. They were cultivated under
conditions of 22.degree. C., 16 hours in the light/8 hours in the
dark, and a light intensity of about 30-45 .mu.mol
m.sup.-2s.sup.-1.
7-3. Results
[0204] As the results of 7-2-4 above, FIG. 12 shows a photo showing
the above-ground parts of a wild-type plant and transformed plants
into which the coding region of the rice-derived PP2C (protein
phosphatase 2C) gene (Os05g0358500) had been introduced. As shown
in FIG. 12, in the above-ground parts of the transformed plants
into which the coding region of the rice-derived PP2C gene
(Os05g0358500) had been introduced, the total amount of biomass was
improved compared with the wild-type plant. It was revealed by the
above results that when the rice-derived PP2C gene is expressed at
a high level in Arabidopsis thaliana, the production of Arabidopsis
biomass can be increased.
[0205] All publications, patents, and patent applications cited
herein are incorporated herein by reference in their entirety.
Sequence CWU 1
1
58119PRTArtificialA consensus sequence in a protein phosphatase
1Xaa Xaa Gly Xaa Phe Asp Gly His Gly Xaa Xaa Gly Xaa Xaa Xaa Xaa1 5
10 15Xaa Xaa Val227PRTArtificialA consensus sequence in a protein
phosphatase 2Ser Gly Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 10 15Xaa Xaa Asn Xaa Gly Xaa Ser Arg Ala Xaa Xaa 20
25356PRTArtificialA consensus sequence in a protein phosphatase
3Gly Xaa Ala Xaa Xaa Arg Xaa Xaa Gly Asp Xaa Xaa Xaa Xaa Xaa Xaa1 5
10 15Gly Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Asp Gly Xaa Xaa Xaa Xaa
Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50
5541077DNAArabidopsis thalianaCDS(1)..(1077) 4atg gga cat ttc tct
tcc atg ttc aac ggt ata gct aga tcc ttc tcg 48Met Gly His Phe Ser
Ser Met Phe Asn Gly Ile Ala Arg Ser Phe Ser1 5 10 15atc aag aaa gcg
aag aac atc aac agc agc aaa agc tac gct aag gaa 96Ile Lys Lys Ala
Lys Asn Ile Asn Ser Ser Lys Ser Tyr Ala Lys Glu 20 25 30gcc aca gat
gaa atg gcg aga gag gcg aag aag aag gaa ctt att ttg 144Ala Thr Asp
Glu Met Ala Arg Glu Ala Lys Lys Lys Glu Leu Ile Leu 35 40 45aga tcc
tct ggt tgc att aat gca gat gga tct aat aac ttg gct tct 192Arg Ser
Ser Gly Cys Ile Asn Ala Asp Gly Ser Asn Asn Leu Ala Ser 50 55 60gtt
ttc tct aga cgc ggt gag aaa ggc gtt aat cag gac tgt gcc atc 240Val
Phe Ser Arg Arg Gly Glu Lys Gly Val Asn Gln Asp Cys Ala Ile65 70 75
80gtc tgg gag gga tat ggg tgt caa gaa gac atg ata ttc tgt ggg ata
288Val Trp Glu Gly Tyr Gly Cys Gln Glu Asp Met Ile Phe Cys Gly Ile
85 90 95ttc gat gga cat ggt ccc tgg gga cac ttt gtt tct aaa caa gtc
aga 336Phe Asp Gly His Gly Pro Trp Gly His Phe Val Ser Lys Gln Val
Arg 100 105 110aac tca atg cct ata tct ttg ctc tgt aac tgg aaa gag
act ctt tct 384Asn Ser Met Pro Ile Ser Leu Leu Cys Asn Trp Lys Glu
Thr Leu Ser 115 120 125cag acc aca ata gca gaa ccc gat aaa gag cta
cag cgg ttt gca atc 432Gln Thr Thr Ile Ala Glu Pro Asp Lys Glu Leu
Gln Arg Phe Ala Ile 130 135 140tgg aaa tac tca ttc ctc aaa acc tgt
gaa gct gtt gat ctg gag ctt 480Trp Lys Tyr Ser Phe Leu Lys Thr Cys
Glu Ala Val Asp Leu Glu Leu145 150 155 160gag cat cac cga aag ata
gat tct ttc aac agc ggt acg acc gct cta 528Glu His His Arg Lys Ile
Asp Ser Phe Asn Ser Gly Thr Thr Ala Leu 165 170 175acc att gtg aga
cag ggt gat gtt att tat ata gca aac gtc ggg gat 576Thr Ile Val Arg
Gln Gly Asp Val Ile Tyr Ile Ala Asn Val Gly Asp 180 185 190tca cgt
gcg gta ttg gcc aca gtt tca gac gaa gga agc ttg gtc gcg 624Ser Arg
Ala Val Leu Ala Thr Val Ser Asp Glu Gly Ser Leu Val Ala 195 200
205gtt cag ctc acc gta gat ttc aag cca aac ctg cct cag gag gaa gag
672Val Gln Leu Thr Val Asp Phe Lys Pro Asn Leu Pro Gln Glu Glu Glu
210 215 220cgg ata atc gga tgc aac ggg aga gta ttt tgc ctt caa gat
gag cca 720Arg Ile Ile Gly Cys Asn Gly Arg Val Phe Cys Leu Gln Asp
Glu Pro225 230 235 240ggg gtc cac cgt gta tgg caa cca gta gat gaa
tct ccg ggg ctc gca 768Gly Val His Arg Val Trp Gln Pro Val Asp Glu
Ser Pro Gly Leu Ala 245 250 255atg tca aga gca ttc gga gac tat tgt
atc aaa gat tac gga ttg gtc 816Met Ser Arg Ala Phe Gly Asp Tyr Cys
Ile Lys Asp Tyr Gly Leu Val 260 265 270tca gtg cct gaa gtc act cag
agg cat ata tcc att aga gac cag ttt 864Ser Val Pro Glu Val Thr Gln
Arg His Ile Ser Ile Arg Asp Gln Phe 275 280 285ata atc ttg gcc act
gat ggg gta tgg gat gtg ata tca aac caa gag 912Ile Ile Leu Ala Thr
Asp Gly Val Trp Asp Val Ile Ser Asn Gln Glu 290 295 300gcc ata gat
att gtt tcc tcg acg gcg gag cgg gca aaa gct gcc aag 960Ala Ile Asp
Ile Val Ser Ser Thr Ala Glu Arg Ala Lys Ala Ala Lys305 310 315
320cga ctg gta cag caa gca gtt agg gct tgg aat aga aag aga cgc gga
1008Arg Leu Val Gln Gln Ala Val Arg Ala Trp Asn Arg Lys Arg Arg Gly
325 330 335atc gcc atg gat gat atc tct gcc gtg tgc ctc ttc ttc cat
tct tca 1056Ile Ala Met Asp Asp Ile Ser Ala Val Cys Leu Phe Phe His
Ser Ser 340 345 350tcg tcg tcg cca tct cta tag 1077Ser Ser Ser Pro
Ser Leu 3555358PRTArabidopsis thaliana 5Met Gly His Phe Ser Ser Met
Phe Asn Gly Ile Ala Arg Ser Phe Ser1 5 10 15Ile Lys Lys Ala Lys Asn
Ile Asn Ser Ser Lys Ser Tyr Ala Lys Glu 20 25 30Ala Thr Asp Glu Met
Ala Arg Glu Ala Lys Lys Lys Glu Leu Ile Leu 35 40 45Arg Ser Ser Gly
Cys Ile Asn Ala Asp Gly Ser Asn Asn Leu Ala Ser 50 55 60Val Phe Ser
Arg Arg Gly Glu Lys Gly Val Asn Gln Asp Cys Ala Ile65 70 75 80Val
Trp Glu Gly Tyr Gly Cys Gln Glu Asp Met Ile Phe Cys Gly Ile 85 90
95Phe Asp Gly His Gly Pro Trp Gly His Phe Val Ser Lys Gln Val Arg
100 105 110Asn Ser Met Pro Ile Ser Leu Leu Cys Asn Trp Lys Glu Thr
Leu Ser 115 120 125Gln Thr Thr Ile Ala Glu Pro Asp Lys Glu Leu Gln
Arg Phe Ala Ile 130 135 140Trp Lys Tyr Ser Phe Leu Lys Thr Cys Glu
Ala Val Asp Leu Glu Leu145 150 155 160Glu His His Arg Lys Ile Asp
Ser Phe Asn Ser Gly Thr Thr Ala Leu 165 170 175Thr Ile Val Arg Gln
Gly Asp Val Ile Tyr Ile Ala Asn Val Gly Asp 180 185 190Ser Arg Ala
Val Leu Ala Thr Val Ser Asp Glu Gly Ser Leu Val Ala 195 200 205Val
Gln Leu Thr Val Asp Phe Lys Pro Asn Leu Pro Gln Glu Glu Glu 210 215
220Arg Ile Ile Gly Cys Asn Gly Arg Val Phe Cys Leu Gln Asp Glu
Pro225 230 235 240Gly Val His Arg Val Trp Gln Pro Val Asp Glu Ser
Pro Gly Leu Ala 245 250 255Met Ser Arg Ala Phe Gly Asp Tyr Cys Ile
Lys Asp Tyr Gly Leu Val 260 265 270Ser Val Pro Glu Val Thr Gln Arg
His Ile Ser Ile Arg Asp Gln Phe 275 280 285Ile Ile Leu Ala Thr Asp
Gly Val Trp Asp Val Ile Ser Asn Gln Glu 290 295 300Ala Ile Asp Ile
Val Ser Ser Thr Ala Glu Arg Ala Lys Ala Ala Lys305 310 315 320Arg
Leu Val Gln Gln Ala Val Arg Ala Trp Asn Arg Lys Arg Arg Gly 325 330
335Ile Ala Met Asp Asp Ile Ser Ala Val Cys Leu Phe Phe His Ser Ser
340 345 350Ser Ser Ser Pro Ser Leu 35561176DNAOryza
sativaCDS(1)..(1176) 6atg cgg cac atc tcg tcg ctg ctg cag ggg ctg
gcg cgc tcg ctg tcg 48Met Arg His Ile Ser Ser Leu Leu Gln Gly Leu
Ala Arg Ser Leu Ser1 5 10 15gtg ggg aag gag agg aag ggc ggc gac ggc
gac gac ggg aag gcg gcg 96Val Gly Lys Glu Arg Lys Gly Gly Asp Gly
Asp Asp Gly Lys Ala Ala 20 25 30gcg gcg acg gcg acg gcg gtg ctg agg
aca tcg ggg acg ctg tgg ggc 144Ala Ala Thr Ala Thr Ala Val Leu Arg
Thr Ser Gly Thr Leu Trp Gly 35 40 45gag ggc tct gag acg ttc gcc gcc
gtc tgc tcc cgc cgc ggc gag aag 192Glu Gly Ser Glu Thr Phe Ala Ala
Val Cys Ser Arg Arg Gly Glu Lys 50 55 60ggc atc aac cag gac tgc tcc
atc gtc tgc gag gga ttc ggg tgc gag 240Gly Ile Asn Gln Asp Cys Ser
Ile Val Cys Glu Gly Phe Gly Cys Glu65 70 75 80gag ggg agc gtg ttg
tgc ggc atc ttc gac ggg cac ggg cag tgg ggc 288Glu Gly Ser Val Leu
Cys Gly Ile Phe Asp Gly His Gly Gln Trp Gly 85 90 95cac tac gtg gcg
aag gcg gtg agg gag tcg ctg ccg ccg gcg ctg ctc 336His Tyr Val Ala
Lys Ala Val Arg Glu Ser Leu Pro Pro Ala Leu Leu 100 105 110cgg cgg
tgg cgg gag gcc gtg acg ctg gcg gcg ctc atc gac ggc ggc 384Arg Arg
Trp Arg Glu Ala Val Thr Leu Ala Ala Leu Ile Asp Gly Gly 115 120
125gag aag cgg ctc tgc gag tgc cgg ccc gac ctg tgg cgc cag tcc tac
432Glu Lys Arg Leu Cys Glu Cys Arg Pro Asp Leu Trp Arg Gln Ser Tyr
130 135 140ctg gcc gcc tgc gcc gcc gtc gac gcc gag ctc cgc gcc agc
cgc cgc 480Leu Ala Ala Cys Ala Ala Val Asp Ala Glu Leu Arg Ala Ser
Arg Arg145 150 155 160ctc gac gcc gtc cac agc ggc tgc acc gcg ctg
tcc ctc gtc aag cac 528Leu Asp Ala Val His Ser Gly Cys Thr Ala Leu
Ser Leu Val Lys His 165 170 175ggc gac ctc ctc gtc gtc gcc aac gtc
ggc gac tcg cgc gcc gtc ctg 576Gly Asp Leu Leu Val Val Ala Asn Val
Gly Asp Ser Arg Ala Val Leu 180 185 190gcc acc gcc tcc ccc gac gac
ggt ggc ggc gcc cgc ctc gcc gcc gtg 624Ala Thr Ala Ser Pro Asp Asp
Gly Gly Gly Ala Arg Leu Ala Ala Val 195 200 205cag ctc acc gtc gac
ttc aag ccc aac ctg ccc cag gag agg gag agg 672Gln Leu Thr Val Asp
Phe Lys Pro Asn Leu Pro Gln Glu Arg Glu Arg 210 215 220atc atg gag
tgc aac ggg agg gtg cag tgc ctc gcc gac gag ccc ggg 720Ile Met Glu
Cys Asn Gly Arg Val Gln Cys Leu Ala Asp Glu Pro Gly225 230 235
240gtg cac cgg gtg tgg cgg ccg gac agg gag ggc cca ggc ctc gcc atg
768Val His Arg Val Trp Arg Pro Asp Arg Glu Gly Pro Gly Leu Ala Met
245 250 255tcg cgc gcc ttc ggc gac tac tgc gtc aag gat tac ggc gtc
atc tcg 816Ser Arg Ala Phe Gly Asp Tyr Cys Val Lys Asp Tyr Gly Val
Ile Ser 260 265 270gcg ccg gag gtg acg cac cgc cgg atc acc gcc cag
gac cac ttc gtc 864Ala Pro Glu Val Thr His Arg Arg Ile Thr Ala Gln
Asp His Phe Val 275 280 285atc ctc gcc acc gac ggg gac aaa cat ctc
aac ttg ttc gtc ttc gtc 912Ile Leu Ala Thr Asp Gly Asp Lys His Leu
Asn Leu Phe Val Phe Val 290 295 300tgc gcg gca ggt gtg gga cgt ggt
gtc gaa cga gga ggc ggt gca gat 960Cys Ala Ala Gly Val Gly Arg Gly
Val Glu Arg Gly Gly Gly Ala Asp305 310 315 320cgt ggc gtc ggc gcc
gga gag gga gaa ggc ggc gaa gcg gct cgt cga 1008Arg Gly Val Gly Ala
Gly Glu Gly Glu Gly Gly Glu Ala Ala Arg Arg 325 330 335gtt cgc cgt
ccg ggc atg gag gcg caa gcg ccg ggg cat cgc cgt cga 1056Val Arg Arg
Pro Gly Met Glu Ala Gln Ala Pro Gly His Arg Arg Arg 340 345 350cga
ctg ctc ggc gat ctg cct ctt ctt cca ctc gcc gcc gtc cta aac 1104Arg
Leu Leu Gly Asp Leu Pro Leu Leu Pro Leu Ala Ala Val Leu Asn 355 360
365aac aca cac gct gac acg cac gca gcc aac aaa aac cgc aca cgc cga
1152Asn Thr His Ala Asp Thr His Ala Ala Asn Lys Asn Arg Thr Arg Arg
370 375 380cga caa tgt cgc cgt cgt cgt tga 1176Arg Gln Cys Arg Arg
Arg Arg385 3907391PRTOryza sativa 7Met Arg His Ile Ser Ser Leu Leu
Gln Gly Leu Ala Arg Ser Leu Ser1 5 10 15Val Gly Lys Glu Arg Lys Gly
Gly Asp Gly Asp Asp Gly Lys Ala Ala 20 25 30Ala Ala Thr Ala Thr Ala
Val Leu Arg Thr Ser Gly Thr Leu Trp Gly 35 40 45Glu Gly Ser Glu Thr
Phe Ala Ala Val Cys Ser Arg Arg Gly Glu Lys 50 55 60Gly Ile Asn Gln
Asp Cys Ser Ile Val Cys Glu Gly Phe Gly Cys Glu65 70 75 80Glu Gly
Ser Val Leu Cys Gly Ile Phe Asp Gly His Gly Gln Trp Gly 85 90 95His
Tyr Val Ala Lys Ala Val Arg Glu Ser Leu Pro Pro Ala Leu Leu 100 105
110Arg Arg Trp Arg Glu Ala Val Thr Leu Ala Ala Leu Ile Asp Gly Gly
115 120 125Glu Lys Arg Leu Cys Glu Cys Arg Pro Asp Leu Trp Arg Gln
Ser Tyr 130 135 140Leu Ala Ala Cys Ala Ala Val Asp Ala Glu Leu Arg
Ala Ser Arg Arg145 150 155 160Leu Asp Ala Val His Ser Gly Cys Thr
Ala Leu Ser Leu Val Lys His 165 170 175Gly Asp Leu Leu Val Val Ala
Asn Val Gly Asp Ser Arg Ala Val Leu 180 185 190Ala Thr Ala Ser Pro
Asp Asp Gly Gly Gly Ala Arg Leu Ala Ala Val 195 200 205Gln Leu Thr
Val Asp Phe Lys Pro Asn Leu Pro Gln Glu Arg Glu Arg 210 215 220Ile
Met Glu Cys Asn Gly Arg Val Gln Cys Leu Ala Asp Glu Pro Gly225 230
235 240Val His Arg Val Trp Arg Pro Asp Arg Glu Gly Pro Gly Leu Ala
Met 245 250 255Ser Arg Ala Phe Gly Asp Tyr Cys Val Lys Asp Tyr Gly
Val Ile Ser 260 265 270Ala Pro Glu Val Thr His Arg Arg Ile Thr Ala
Gln Asp His Phe Val 275 280 285Ile Leu Ala Thr Asp Gly Asp Lys His
Leu Asn Leu Phe Val Phe Val 290 295 300Cys Ala Ala Gly Val Gly Arg
Gly Val Glu Arg Gly Gly Gly Ala Asp305 310 315 320Arg Gly Val Gly
Ala Gly Glu Gly Glu Gly Gly Glu Ala Ala Arg Arg 325 330 335Val Arg
Arg Pro Gly Met Glu Ala Gln Ala Pro Gly His Arg Arg Arg 340 345
350Arg Leu Leu Gly Asp Leu Pro Leu Leu Pro Leu Ala Ala Val Leu Asn
355 360 365Asn Thr His Ala Asp Thr His Ala Ala Asn Lys Asn Arg Thr
Arg Arg 370 375 380Arg Gln Cys Arg Arg Arg Arg385 39081263DNAOryza
sativaCDS(1)..(1263) 8atg ggg ata tgc tgc agc aag ggg aag gag gag
ctt gag gag gag gga 48Met Gly Ile Cys Cys Ser Lys Gly Lys Glu Glu
Leu Glu Glu Glu Gly1 5 10 15ttt cca tgg aag cac gac gcc ttc ttc cac
gac cag ctt tgg agc gct 96Phe Pro Trp Lys His Asp Ala Phe Phe His
Asp Gln Leu Trp Ser Ala 20 25 30ggc gtc tcc atg cac acc aag caa ggc
tgg aag ggc gcc aac cag gac 144Gly Val Ser Met His Thr Lys Gln Gly
Trp Lys Gly Ala Asn Gln Asp 35 40 45gcc atg act acc tgc cag gac ttt
gcg ggg cac aag ggc cag ata ttt 192Ala Met Thr Thr Cys Gln Asp Phe
Ala Gly His Lys Gly Gln Ile Phe 50 55 60tgt gga gtt ttt gat ggg cat
ggc cct ctc gga agg gaa gtt gct cgc 240Cys Gly Val Phe Asp Gly His
Gly Pro Leu Gly Arg Glu Val Ala Arg65 70 75 80cat gtc cgc gac gtc
ctt cca gtg aaa cta tcc tcc tct ttg gca ctg 288His Val Arg Asp Val
Leu Pro Val Lys Leu Ser Ser Ser Leu Ala Leu 85 90 95aag act gaa caa
gat cca tcc agc aac aca gat aag gaa acc ttg gaa 336Lys Thr Glu Gln
Asp Pro Ser Ser Asn Thr Asp Lys Glu Thr Leu Glu 100 105 110aag tca
gat tgc acc tca ttg agc gat aca agc aat gag aag caa ttg 384Lys Ser
Asp Cys Thr Ser Leu Ser Asp Thr Ser Asn Glu Lys Gln Leu 115 120
125tta tcc acc tgg aag aac ata ttt gtc aag aca ttt gag gat gtt gat
432Leu Ser Thr Trp Lys Asn Ile Phe Val Lys Thr Phe Glu Asp Val Asp
130 135 140gag gat ctg agg caa cat tct gga att gac tgc att tgt agt
ggc aca 480Glu Asp Leu Arg Gln His Ser Gly Ile Asp Cys Ile Cys Ser
Gly Thr145 150 155 160act gct gtc act gtc gtt agg cag ggt gat cac
ctg atc att gca aat 528Thr Ala Val Thr Val Val Arg Gln Gly Asp His
Leu Ile Ile Ala Asn 165 170 175ttg ggc gat tca cgt gcg gtt ctt tgc
acc cga gac agc aag gac cgc 576Leu Gly Asp Ser Arg Ala Val Leu Cys
Thr Arg Asp Ser Lys Asp Arg 180 185 190cca att tca gtc caa cta acc
act gac ctg aaa cca aat ctt cca agc 624Pro Ile Ser Val Gln Leu Thr
Thr Asp Leu Lys Pro Asn Leu Pro Ser 195 200 205gaa gct gag aga atc
ctg aat tcc aag ggg cgg gtt ttc gcc atg gac 672Glu Ala Glu Arg Ile
Leu Asn Ser Lys Gly Arg Val Phe Ala Met Asp 210 215 220gat gag ccg
gac gtg
cct agg atg tgg cta cca gac caa gac gcg ccg 720Asp Glu Pro Asp Val
Pro Arg Met Trp Leu Pro Asp Gln Asp Ala Pro225 230 235 240ggc ctc
gcc atg gca agg gca ttt gga gat ttc tgc ttg aag agt cat 768Gly Leu
Ala Met Ala Arg Ala Phe Gly Asp Phe Cys Leu Lys Ser His 245 250
255gga cta atc tgt aca cca gaa gtc tac tac agg aag cta tct gca aaa
816Gly Leu Ile Cys Thr Pro Glu Val Tyr Tyr Arg Lys Leu Ser Ala Lys
260 265 270gat gac ttc ttg gta ctt gct act gac ggg ata tgg gac gtg
ctg tcg 864Asp Asp Phe Leu Val Leu Ala Thr Asp Gly Ile Trp Asp Val
Leu Ser 275 280 285aac aag gag gtg atc aag atc gta tcg tcg gct act
gac cat tcc aag 912Asn Lys Glu Val Ile Lys Ile Val Ser Ser Ala Thr
Asp His Ser Lys 290 295 300gcc gcc aag cag ctc gtc gag cgg gcg gtg
cgc acg tgg cgg cgc aag 960Ala Ala Lys Gln Leu Val Glu Arg Ala Val
Arg Thr Trp Arg Arg Lys305 310 315 320ttc ccg acg tcg atg gtc gac
gac tgc gcc gtg gtg tgc ctc ttc ttg 1008Phe Pro Thr Ser Met Val Asp
Asp Cys Ala Val Val Cys Leu Phe Leu 325 330 335aag cct tca ccg tcg
tcg tcg gag agc acc ccc ggg gac gcg aaa cct 1056Lys Pro Ser Pro Ser
Ser Ser Glu Ser Thr Pro Gly Asp Ala Lys Pro 340 345 350cct cag gcc
gtg tcg ttc acg ggc agc ttc cga aag gtc ctg ggc ggc 1104Pro Gln Ala
Val Ser Phe Thr Gly Ser Phe Arg Lys Val Leu Gly Gly 355 360 365ggc
ggc ggc gag gcg gag gag ggg acg aat gta tgg aga gct ctg gag 1152Gly
Gly Gly Glu Ala Glu Glu Gly Thr Asn Val Trp Arg Ala Leu Glu 370 375
380ggg gtg gct cgg gtg aac tcg gtg gtg agg ctg ccg cgg atg ggc gcc
1200Gly Val Ala Arg Val Asn Ser Val Val Arg Leu Pro Arg Met Gly
Ala385 390 395 400gtg ctg agc tgg cgg cgg cgg tcg acg tcg ctg gag
gaa gac gac gag 1248Val Leu Ser Trp Arg Arg Arg Ser Thr Ser Leu Glu
Glu Asp Asp Glu 405 410 415gcg agg att gat tga 1263Ala Arg Ile Asp
4209420PRTOryza sativa 9Met Gly Ile Cys Cys Ser Lys Gly Lys Glu Glu
Leu Glu Glu Glu Gly1 5 10 15Phe Pro Trp Lys His Asp Ala Phe Phe His
Asp Gln Leu Trp Ser Ala 20 25 30Gly Val Ser Met His Thr Lys Gln Gly
Trp Lys Gly Ala Asn Gln Asp 35 40 45Ala Met Thr Thr Cys Gln Asp Phe
Ala Gly His Lys Gly Gln Ile Phe 50 55 60Cys Gly Val Phe Asp Gly His
Gly Pro Leu Gly Arg Glu Val Ala Arg65 70 75 80His Val Arg Asp Val
Leu Pro Val Lys Leu Ser Ser Ser Leu Ala Leu 85 90 95Lys Thr Glu Gln
Asp Pro Ser Ser Asn Thr Asp Lys Glu Thr Leu Glu 100 105 110Lys Ser
Asp Cys Thr Ser Leu Ser Asp Thr Ser Asn Glu Lys Gln Leu 115 120
125Leu Ser Thr Trp Lys Asn Ile Phe Val Lys Thr Phe Glu Asp Val Asp
130 135 140Glu Asp Leu Arg Gln His Ser Gly Ile Asp Cys Ile Cys Ser
Gly Thr145 150 155 160Thr Ala Val Thr Val Val Arg Gln Gly Asp His
Leu Ile Ile Ala Asn 165 170 175Leu Gly Asp Ser Arg Ala Val Leu Cys
Thr Arg Asp Ser Lys Asp Arg 180 185 190Pro Ile Ser Val Gln Leu Thr
Thr Asp Leu Lys Pro Asn Leu Pro Ser 195 200 205Glu Ala Glu Arg Ile
Leu Asn Ser Lys Gly Arg Val Phe Ala Met Asp 210 215 220Asp Glu Pro
Asp Val Pro Arg Met Trp Leu Pro Asp Gln Asp Ala Pro225 230 235
240Gly Leu Ala Met Ala Arg Ala Phe Gly Asp Phe Cys Leu Lys Ser His
245 250 255Gly Leu Ile Cys Thr Pro Glu Val Tyr Tyr Arg Lys Leu Ser
Ala Lys 260 265 270Asp Asp Phe Leu Val Leu Ala Thr Asp Gly Ile Trp
Asp Val Leu Ser 275 280 285Asn Lys Glu Val Ile Lys Ile Val Ser Ser
Ala Thr Asp His Ser Lys 290 295 300Ala Ala Lys Gln Leu Val Glu Arg
Ala Val Arg Thr Trp Arg Arg Lys305 310 315 320Phe Pro Thr Ser Met
Val Asp Asp Cys Ala Val Val Cys Leu Phe Leu 325 330 335Lys Pro Ser
Pro Ser Ser Ser Glu Ser Thr Pro Gly Asp Ala Lys Pro 340 345 350Pro
Gln Ala Val Ser Phe Thr Gly Ser Phe Arg Lys Val Leu Gly Gly 355 360
365Gly Gly Gly Glu Ala Glu Glu Gly Thr Asn Val Trp Arg Ala Leu Glu
370 375 380Gly Val Ala Arg Val Asn Ser Val Val Arg Leu Pro Arg Met
Gly Ala385 390 395 400Val Leu Ser Trp Arg Arg Arg Ser Thr Ser Leu
Glu Glu Asp Asp Glu 405 410 415Ala Arg Ile Asp 420101632DNAOryza
sativaCDS(1)..(1632) 10atg gat ggg gtg cct gat gcc caa cgc aca aca
tca cca tca atg ata 48Met Asp Gly Val Pro Asp Ala Gln Arg Thr Thr
Ser Pro Ser Met Ile1 5 10 15aaa caa caa aac tac ttc aac tac ccc tac
gca ttc aac tcc att cta 96Lys Gln Gln Asn Tyr Phe Asn Tyr Pro Tyr
Ala Phe Asn Ser Ile Leu 20 25 30ctc tct acc ccc tcc ttc ctt cct tcc
ttc ctt cct agc tac ctc tac 144Leu Ser Thr Pro Ser Phe Leu Pro Ser
Phe Leu Pro Ser Tyr Leu Tyr 35 40 45gaa gta cca gca gca gaa gaa gca
atg ggg atc tgc tgc agc aag ggg 192Glu Val Pro Ala Ala Glu Glu Ala
Met Gly Ile Cys Cys Ser Lys Gly 50 55 60aag gag gag ctt gag gag gga
ttt cca tgg aag cac gac gcc ttc ttc 240Lys Glu Glu Leu Glu Glu Gly
Phe Pro Trp Lys His Asp Ala Phe Phe65 70 75 80cac gac cag ctt tgg
agc gct ggc gtc tcc atg cac acc aag caa ggc 288His Asp Gln Leu Trp
Ser Ala Gly Val Ser Met His Thr Lys Gln Gly 85 90 95tgg aag ggc gct
aac cag gat gcc atg act acc tgc cag gac ttt gcg 336Trp Lys Gly Ala
Asn Gln Asp Ala Met Thr Thr Cys Gln Asp Phe Ala 100 105 110ggg cac
aag ggc cag ata ttt tgt gga gtt ttt gat ggg cat ggc cct 384Gly His
Lys Gly Gln Ile Phe Cys Gly Val Phe Asp Gly His Gly Pro 115 120
125ctc gga agg gaa gtt gct cgc cat gtc cgc gac gtc ctt cca atg aaa
432Leu Gly Arg Glu Val Ala Arg His Val Arg Asp Val Leu Pro Met Lys
130 135 140cta tcc tcc tct ttg gca ctg aaa act gaa caa gat cca tcc
agc aac 480Leu Ser Ser Ser Leu Ala Leu Lys Thr Glu Gln Asp Pro Ser
Ser Asn145 150 155 160aca gat aag gaa gcc ttg gaa aaa tca gat tgc
acc tca ttg agc gat 528Thr Asp Lys Glu Ala Leu Glu Lys Ser Asp Cys
Thr Ser Leu Ser Asp 165 170 175aca agc aat gag aag caa ttg tta tcc
acc tgg aag aac ata ttt gtc 576Thr Ser Asn Glu Lys Gln Leu Leu Ser
Thr Trp Lys Asn Ile Phe Val 180 185 190aag aca ttt gag gat gta gat
gat gat ctg aga caa aat tct gga att 624Lys Thr Phe Glu Asp Val Asp
Asp Asp Leu Arg Gln Asn Ser Gly Ile 195 200 205gac tgc att tgt agt
ggc aca act gct gtc act gtc gtc agg cag ggt 672Asp Cys Ile Cys Ser
Gly Thr Thr Ala Val Thr Val Val Arg Gln Gly 210 215 220gat cac ctg
atc att gca aat ttg ggc gat tca cgt gcg gtt ctt tgc 720Asp His Leu
Ile Ile Ala Asn Leu Gly Asp Ser Arg Ala Val Leu Cys225 230 235
240acc cga gat agc aag gac cgc cca att cca gtt caa cta acc act gac
768Thr Arg Asp Ser Lys Asp Arg Pro Ile Pro Val Gln Leu Thr Thr Asp
245 250 255ctg aaa cca aat ctt cca agc gaa gct gag aga atc ctg aat
tgt aag 816Leu Lys Pro Asn Leu Pro Ser Glu Ala Glu Arg Ile Leu Asn
Cys Lys 260 265 270ggg cgg gtt ttt gcc atg gac gac gag ccg gac gtg
tct agg atg tgg 864Gly Arg Val Phe Ala Met Asp Asp Glu Pro Asp Val
Ser Arg Met Trp 275 280 285cta cca gac caa gac gcg ccg ggc ctc gcc
atg gca agg gca ttt gga 912Leu Pro Asp Gln Asp Ala Pro Gly Leu Ala
Met Ala Arg Ala Phe Gly 290 295 300gat ttc tgc ttg aag agt cat gga
ctt atc tgt aca cca gaa gtc tat 960Asp Phe Cys Leu Lys Ser His Gly
Leu Ile Cys Thr Pro Glu Val Tyr305 310 315 320tac agg aag cta tcc
gaa aaa gat gaa ttc ttg gta ctt gct act gac 1008Tyr Arg Lys Leu Ser
Glu Lys Asp Glu Phe Leu Val Leu Ala Thr Asp 325 330 335ggg ata tgg
gac gtg cta tcg aac aag gaa gtg atc aag atc gta tcg 1056Gly Ile Trp
Asp Val Leu Ser Asn Lys Glu Val Ile Lys Ile Val Ser 340 345 350tcg
gct act gac cat tcc aag gcc gcc aag cag ctg gtc gag cgg gcg 1104Ser
Ala Thr Asp His Ser Lys Ala Ala Lys Gln Leu Val Glu Arg Ala 355 360
365gtg cgc gcg tgg cgg cgc aag ttc ccg acg tca atg gtc gac gac tgc
1152Val Arg Ala Trp Arg Arg Lys Phe Pro Thr Ser Met Val Asp Asp Cys
370 375 380gcc gtc gtc tgc ctc ttc ttg aag cct tct ccg tcg tcg gag
gag agc 1200Ala Val Val Cys Leu Phe Leu Lys Pro Ser Pro Ser Ser Glu
Glu Ser385 390 395 400acc cat gta gac gcg aag gcg cct cag gtc gtg
tcg ttc acg ggc agc 1248Thr His Val Asp Ala Lys Ala Pro Gln Val Val
Ser Phe Thr Gly Ser 405 410 415ttc cgc aag gcc ctg ggt ggt ggc ggc
ggc ggc gag gcg gag gag gtg 1296Phe Arg Lys Ala Leu Gly Gly Gly Gly
Gly Gly Glu Ala Glu Glu Val 420 425 430gaa aag att tat cga cga agt
atc cgc act gtc aca cgg gac att tgg 1344Glu Lys Ile Tyr Arg Arg Ser
Ile Arg Thr Val Thr Arg Asp Ile Trp 435 440 445gac aaa gta tct gca
aga ctc gac tgt gat cac ata tcc acg acg cac 1392Asp Lys Val Ser Ala
Arg Leu Asp Cys Asp His Ile Ser Thr Thr His 450 455 460aac cca gat
gaa acg ctg ctt gat tgg tgg gaa aga aga aca gag caa 1440Asn Pro Asp
Glu Thr Leu Leu Asp Trp Trp Glu Arg Arg Thr Glu Gln465 470 475
480aat gac aag gac aag acg aag gga acg cgc tcc att cac atg ctc ctt
1488Asn Asp Lys Asp Lys Thr Lys Gly Thr Arg Ser Ile His Met Leu Leu
485 490 495agc tgg gaa atc tgg tgt gaa agg aat agg cgc gtt ttc agg
aat aag 1536Ser Trp Glu Ile Trp Cys Glu Arg Asn Arg Arg Val Phe Arg
Asn Lys 500 505 510gag ctc gct atc tca caa ttg gtg acc aaa atc ctt
gat gaa atc aat 1584Glu Leu Ala Ile Ser Gln Leu Val Thr Lys Ile Leu
Asp Glu Ile Asn 515 520 525gtc tgg att gca tgc ggg gcg aag aat tta
gcg aga ata gtg ttg taa 1632Val Trp Ile Ala Cys Gly Ala Lys Asn Leu
Ala Arg Ile Val Leu 530 535 54011543PRTOryza sativa 11Met Asp Gly
Val Pro Asp Ala Gln Arg Thr Thr Ser Pro Ser Met Ile1 5 10 15Lys Gln
Gln Asn Tyr Phe Asn Tyr Pro Tyr Ala Phe Asn Ser Ile Leu 20 25 30Leu
Ser Thr Pro Ser Phe Leu Pro Ser Phe Leu Pro Ser Tyr Leu Tyr 35 40
45Glu Val Pro Ala Ala Glu Glu Ala Met Gly Ile Cys Cys Ser Lys Gly
50 55 60Lys Glu Glu Leu Glu Glu Gly Phe Pro Trp Lys His Asp Ala Phe
Phe65 70 75 80His Asp Gln Leu Trp Ser Ala Gly Val Ser Met His Thr
Lys Gln Gly 85 90 95Trp Lys Gly Ala Asn Gln Asp Ala Met Thr Thr Cys
Gln Asp Phe Ala 100 105 110Gly His Lys Gly Gln Ile Phe Cys Gly Val
Phe Asp Gly His Gly Pro 115 120 125Leu Gly Arg Glu Val Ala Arg His
Val Arg Asp Val Leu Pro Met Lys 130 135 140Leu Ser Ser Ser Leu Ala
Leu Lys Thr Glu Gln Asp Pro Ser Ser Asn145 150 155 160Thr Asp Lys
Glu Ala Leu Glu Lys Ser Asp Cys Thr Ser Leu Ser Asp 165 170 175Thr
Ser Asn Glu Lys Gln Leu Leu Ser Thr Trp Lys Asn Ile Phe Val 180 185
190Lys Thr Phe Glu Asp Val Asp Asp Asp Leu Arg Gln Asn Ser Gly Ile
195 200 205Asp Cys Ile Cys Ser Gly Thr Thr Ala Val Thr Val Val Arg
Gln Gly 210 215 220Asp His Leu Ile Ile Ala Asn Leu Gly Asp Ser Arg
Ala Val Leu Cys225 230 235 240Thr Arg Asp Ser Lys Asp Arg Pro Ile
Pro Val Gln Leu Thr Thr Asp 245 250 255Leu Lys Pro Asn Leu Pro Ser
Glu Ala Glu Arg Ile Leu Asn Cys Lys 260 265 270Gly Arg Val Phe Ala
Met Asp Asp Glu Pro Asp Val Ser Arg Met Trp 275 280 285Leu Pro Asp
Gln Asp Ala Pro Gly Leu Ala Met Ala Arg Ala Phe Gly 290 295 300Asp
Phe Cys Leu Lys Ser His Gly Leu Ile Cys Thr Pro Glu Val Tyr305 310
315 320Tyr Arg Lys Leu Ser Glu Lys Asp Glu Phe Leu Val Leu Ala Thr
Asp 325 330 335Gly Ile Trp Asp Val Leu Ser Asn Lys Glu Val Ile Lys
Ile Val Ser 340 345 350Ser Ala Thr Asp His Ser Lys Ala Ala Lys Gln
Leu Val Glu Arg Ala 355 360 365Val Arg Ala Trp Arg Arg Lys Phe Pro
Thr Ser Met Val Asp Asp Cys 370 375 380Ala Val Val Cys Leu Phe Leu
Lys Pro Ser Pro Ser Ser Glu Glu Ser385 390 395 400Thr His Val Asp
Ala Lys Ala Pro Gln Val Val Ser Phe Thr Gly Ser 405 410 415Phe Arg
Lys Ala Leu Gly Gly Gly Gly Gly Gly Glu Ala Glu Glu Val 420 425
430Glu Lys Ile Tyr Arg Arg Ser Ile Arg Thr Val Thr Arg Asp Ile Trp
435 440 445Asp Lys Val Ser Ala Arg Leu Asp Cys Asp His Ile Ser Thr
Thr His 450 455 460Asn Pro Asp Glu Thr Leu Leu Asp Trp Trp Glu Arg
Arg Thr Glu Gln465 470 475 480Asn Asp Lys Asp Lys Thr Lys Gly Thr
Arg Ser Ile His Met Leu Leu 485 490 495Ser Trp Glu Ile Trp Cys Glu
Arg Asn Arg Arg Val Phe Arg Asn Lys 500 505 510Glu Leu Ala Ile Ser
Gln Leu Val Thr Lys Ile Leu Asp Glu Ile Asn 515 520 525Val Trp Ile
Ala Cys Gly Ala Lys Asn Leu Ala Arg Ile Val Leu 530 535
540121557DNAOryza sativaCDS(1)..(1557) 12atg gtg gag gcc gcc gcg
ggg cgc cgg tcg ggg gcc aac cgt cgg cgg 48Met Val Glu Ala Ala Ala
Gly Arg Arg Ser Gly Ala Asn Arg Arg Arg1 5 10 15cct agc ggc ggg ggc
gag cgg cgg cgg cag cag cag cag cac cag cgc 96Pro Ser Gly Gly Gly
Glu Arg Arg Arg Gln Gln Gln Gln His Gln Arg 20 25 30ctc gtc gcg gtc
gcg gtg gcc gcg cgc gtc gtc atg gtg gcg ccc gcg 144Leu Val Ala Val
Ala Val Ala Ala Arg Val Val Met Val Ala Pro Ala 35 40 45gcc acg ccc
gcg ccc gcg gcg ggg ggt ggc ggg ggc tgc gtc gag gac 192Ala Thr Pro
Ala Pro Ala Ala Gly Gly Gly Gly Gly Cys Val Glu Asp 50 55 60atc ctc
ggg tgc ctc ctc ggc gtg ctg cgc gcg ctc ggc gtc acg tgg 240Ile Leu
Gly Cys Leu Leu Gly Val Leu Arg Ala Leu Gly Val Thr Trp65 70 75
80gcg gcg gcg gcg agg ccg cag agg cag cag ccg cgc ctg gcg gcg cag
288Ala Ala Ala Ala Arg Pro Gln Arg Gln Gln Pro Arg Leu Ala Ala Gln
85 90 95acg ccg cga ggg ccc gcg cct ggg gcg gat ggg cgc cgc gcc gcc
gcc 336Thr Pro Arg Gly Pro Ala Pro Gly Ala Asp Gly Arg Arg Ala Ala
Ala 100 105 110gag ctg agg ggg atc ccc ggc cgg atc gcg ggg aac ggg
gcc tgc gcc 384Glu Leu Arg Gly Ile Pro Gly Arg Ile Ala Gly Asn Gly
Ala Cys Ala 115 120 125gtc gcg tcg ctc tac acg ctg cag ggg aag aaa
ggc gtc aac caa gac 432Val Ala Ser Leu Tyr Thr Leu Gln Gly Lys Lys
Gly Val Asn Gln Asp 130 135 140gcc atg atc gtc tgg gag aat ttc tgt
tca aga gag gat acc att ttt 480Ala Met Ile Val Trp Glu Asn Phe Cys
Ser Arg Glu Asp Thr Ile Phe145 150 155 160tgt ggt gtt ttt gat ggc
cat gga cca aac ggc cat ttg gtt gct aag 528Cys Gly Val Phe Asp Gly
His Gly Pro Asn Gly His Leu Val Ala Lys 165 170 175agg gtg aga gat
ctt ctg ccc att aag ctt ggt gcg gat ttg ggg acg 576Arg Val Arg Asp
Leu Leu Pro Ile Lys Leu Gly Ala Asp Leu Gly Thr 180 185
190gat gaa gga cga cag aca tcc act agc agc atc aaa agc aat gga gat
624Asp Glu Gly Arg Gln Thr Ser Thr Ser Ser Ile Lys Ser Asn Gly Asp
195 200 205gaa aca gga tcc cct gga aac atg ggc aga gat gct gag cag
aac gga 672Glu Thr Gly Ser Pro Gly Asn Met Gly Arg Asp Ala Glu Gln
Asn Gly 210 215 220gag tac cca gag atc ttc aca gca ttg aga act tca
ttt ttg agg gcg 720Glu Tyr Pro Glu Ile Phe Thr Ala Leu Arg Thr Ser
Phe Leu Arg Ala225 230 235 240ttc aat gtc atg gat aga gat ctc aag
tta cat aaa agt ata gat tgt 768Phe Asn Val Met Asp Arg Asp Leu Lys
Leu His Lys Ser Ile Asp Cys 245 250 255ttt ttc agt gga aca aca gca
gtg gca gtg ctc aag cag gga cgg aat 816Phe Phe Ser Gly Thr Thr Ala
Val Ala Val Leu Lys Gln Gly Arg Asn 260 265 270ctt ata att ggt aac
ctc ggg gac tcg cgg gcc atc tta ggc aca aga 864Leu Ile Ile Gly Asn
Leu Gly Asp Ser Arg Ala Ile Leu Gly Thr Arg 275 280 285gat aaa gat
aat cag ctt atg gct gtc caa ttg aca gtt gat ctc aaa 912Asp Lys Asp
Asn Gln Leu Met Ala Val Gln Leu Thr Val Asp Leu Lys 290 295 300cct
aac att cca agt gaa gca cag cga atc agg caa cgc agg ggc agg 960Pro
Asn Ile Pro Ser Glu Ala Gln Arg Ile Arg Gln Arg Arg Gly Arg305 310
315 320ata ttt gca ctt cct gag gag cca gag gtt gct cgt gtt tgg ctt
ccg 1008Ile Phe Ala Leu Pro Glu Glu Pro Glu Val Ala Arg Val Trp Leu
Pro 325 330 335aag tac aac tcc cct gga ctg gcc atg gct agg gca ttt
gga gac ttc 1056Lys Tyr Asn Ser Pro Gly Leu Ala Met Ala Arg Ala Phe
Gly Asp Phe 340 345 350tgt ctc aag gat tat ggt cta atc tct atg cct
gaa gtc tcg tac cac 1104Cys Leu Lys Asp Tyr Gly Leu Ile Ser Met Pro
Glu Val Ser Tyr His 355 360 365cgt atc aca gaa aag gat gag ttt gtt
gta ttg gct act gat ggg gtt 1152Arg Ile Thr Glu Lys Asp Glu Phe Val
Val Leu Ala Thr Asp Gly Val 370 375 380tgg gat gtg ctg tca aac act
gaa gtt gtt agt att gtc aac aga gct 1200Trp Asp Val Leu Ser Asn Thr
Glu Val Val Ser Ile Val Asn Arg Ala385 390 395 400act tct cgg gcc
tct gca gca cga ttg cta gtc gaa tca gct cac cgt 1248Thr Ser Arg Ala
Ser Ala Ala Arg Leu Leu Val Glu Ser Ala His Arg 405 410 415gcc tgg
cgt gca cgt ttc ccc act tct aaa att gat gat tgt gct gtg 1296Ala Trp
Arg Ala Arg Phe Pro Thr Ser Lys Ile Asp Asp Cys Ala Val 420 425
430gtc tgc cta ttc ctg gat aca gac gaa tta agt gaa aca tcc agt tct
1344Val Cys Leu Phe Leu Asp Thr Asp Glu Leu Ser Glu Thr Ser Ser Ser
435 440 445atg gcc cgc gat atg aca aat gct gta gaa gtt agc agt ggg
cag cac 1392Met Ala Arg Asp Met Thr Asn Ala Val Glu Val Ser Ser Gly
Gln His 450 455 460tcc aat act atc caa ttg agc act gga gta tct tca
gat gtt gtt act 1440Ser Asn Thr Ile Gln Leu Ser Thr Gly Val Ser Ser
Asp Val Val Thr465 470 475 480gca gtt cta aca gat ggt gat gat ctg
tct gct gtt gat gca gtt gca 1488Ala Val Leu Thr Asp Gly Asp Asp Leu
Ser Ala Val Asp Ala Val Ala 485 490 495aag ctg gtt act ctc acg gat
ttg ccg aac aat gct tca ggc gca acg 1536Lys Leu Val Thr Leu Thr Asp
Leu Pro Asn Asn Ala Ser Gly Ala Thr 500 505 510caa agc atc acc acc
aag tga 1557Gln Ser Ile Thr Thr Lys 51513518PRTOryza sativa 13Met
Val Glu Ala Ala Ala Gly Arg Arg Ser Gly Ala Asn Arg Arg Arg1 5 10
15Pro Ser Gly Gly Gly Glu Arg Arg Arg Gln Gln Gln Gln His Gln Arg
20 25 30Leu Val Ala Val Ala Val Ala Ala Arg Val Val Met Val Ala Pro
Ala 35 40 45Ala Thr Pro Ala Pro Ala Ala Gly Gly Gly Gly Gly Cys Val
Glu Asp 50 55 60Ile Leu Gly Cys Leu Leu Gly Val Leu Arg Ala Leu Gly
Val Thr Trp65 70 75 80Ala Ala Ala Ala Arg Pro Gln Arg Gln Gln Pro
Arg Leu Ala Ala Gln 85 90 95Thr Pro Arg Gly Pro Ala Pro Gly Ala Asp
Gly Arg Arg Ala Ala Ala 100 105 110Glu Leu Arg Gly Ile Pro Gly Arg
Ile Ala Gly Asn Gly Ala Cys Ala 115 120 125Val Ala Ser Leu Tyr Thr
Leu Gln Gly Lys Lys Gly Val Asn Gln Asp 130 135 140Ala Met Ile Val
Trp Glu Asn Phe Cys Ser Arg Glu Asp Thr Ile Phe145 150 155 160Cys
Gly Val Phe Asp Gly His Gly Pro Asn Gly His Leu Val Ala Lys 165 170
175Arg Val Arg Asp Leu Leu Pro Ile Lys Leu Gly Ala Asp Leu Gly Thr
180 185 190Asp Glu Gly Arg Gln Thr Ser Thr Ser Ser Ile Lys Ser Asn
Gly Asp 195 200 205Glu Thr Gly Ser Pro Gly Asn Met Gly Arg Asp Ala
Glu Gln Asn Gly 210 215 220Glu Tyr Pro Glu Ile Phe Thr Ala Leu Arg
Thr Ser Phe Leu Arg Ala225 230 235 240Phe Asn Val Met Asp Arg Asp
Leu Lys Leu His Lys Ser Ile Asp Cys 245 250 255Phe Phe Ser Gly Thr
Thr Ala Val Ala Val Leu Lys Gln Gly Arg Asn 260 265 270Leu Ile Ile
Gly Asn Leu Gly Asp Ser Arg Ala Ile Leu Gly Thr Arg 275 280 285Asp
Lys Asp Asn Gln Leu Met Ala Val Gln Leu Thr Val Asp Leu Lys 290 295
300Pro Asn Ile Pro Ser Glu Ala Gln Arg Ile Arg Gln Arg Arg Gly
Arg305 310 315 320Ile Phe Ala Leu Pro Glu Glu Pro Glu Val Ala Arg
Val Trp Leu Pro 325 330 335Lys Tyr Asn Ser Pro Gly Leu Ala Met Ala
Arg Ala Phe Gly Asp Phe 340 345 350Cys Leu Lys Asp Tyr Gly Leu Ile
Ser Met Pro Glu Val Ser Tyr His 355 360 365Arg Ile Thr Glu Lys Asp
Glu Phe Val Val Leu Ala Thr Asp Gly Val 370 375 380Trp Asp Val Leu
Ser Asn Thr Glu Val Val Ser Ile Val Asn Arg Ala385 390 395 400Thr
Ser Arg Ala Ser Ala Ala Arg Leu Leu Val Glu Ser Ala His Arg 405 410
415Ala Trp Arg Ala Arg Phe Pro Thr Ser Lys Ile Asp Asp Cys Ala Val
420 425 430Val Cys Leu Phe Leu Asp Thr Asp Glu Leu Ser Glu Thr Ser
Ser Ser 435 440 445Met Ala Arg Asp Met Thr Asn Ala Val Glu Val Ser
Ser Gly Gln His 450 455 460Ser Asn Thr Ile Gln Leu Ser Thr Gly Val
Ser Ser Asp Val Val Thr465 470 475 480Ala Val Leu Thr Asp Gly Asp
Asp Leu Ser Ala Val Asp Ala Val Ala 485 490 495Lys Leu Val Thr Leu
Thr Asp Leu Pro Asn Asn Ala Ser Gly Ala Thr 500 505 510Gln Ser Ile
Thr Thr Lys 515141383DNAOryza sativaCDS(1)..(1383) 14atg gtg gcg
gtg acc ggg ggc agg ccc ccc ggc ctg cag gat gcg ccg 48Met Val Ala
Val Thr Gly Gly Arg Pro Pro Gly Leu Gln Asp Ala Pro1 5 10 15ggg gca
cca cca cca gca cca gca gca gag gct gtg ccg tcg cgc ccg 96Gly Ala
Pro Pro Pro Ala Pro Ala Ala Glu Ala Val Pro Ser Arg Pro 20 25 30ctc
gcg cgg gac gcg act tac gga ggc cgc gtg tac ggt ggc gta gga 144Leu
Ala Arg Asp Ala Thr Tyr Gly Gly Arg Val Tyr Gly Gly Val Gly 35 40
45gga gga gga tgc tgc ctc gag ttc ctc gac tgc gtg ctc cgg gcg atg
192Gly Gly Gly Cys Cys Leu Glu Phe Leu Asp Cys Val Leu Arg Ala Met
50 55 60ggc gtc gcc acc ccg gcc gag atc atg ccc ccc gcg gac ttc agg
tgg 240Gly Val Ala Thr Pro Ala Glu Ile Met Pro Pro Ala Asp Phe Arg
Trp65 70 75 80gcc gcg cgc ccg atg cgg cgg cgc cgc cgc ggg ggc tcc
tcg tcc tcc 288Ala Ala Arg Pro Met Arg Arg Arg Arg Arg Gly Gly Ser
Ser Ser Ser 85 90 95tcc tcc tcg ccg cgc gac cgc gag ccg agg gac ggc
cgg atc gcc gcc 336Ser Ser Ser Pro Arg Asp Arg Glu Pro Arg Asp Gly
Arg Ile Ala Ala 100 105 110aac ggc gcc tcc gct gcc gcc tcg ctc tac
acg atg cgg ggc aac aag 384Asn Gly Ala Ser Ala Ala Ala Ser Leu Tyr
Thr Met Arg Gly Asn Lys 115 120 125ggc gtc aac cag gac gcc atg ctt
gtc tgg gag aat ttc tgt tca aag 432Gly Val Asn Gln Asp Ala Met Leu
Val Trp Glu Asn Phe Cys Ser Lys 130 135 140gaa gat aca att ttt tgt
ggt gtt ttt gat ggc cat gga cca tat ggc 480Glu Asp Thr Ile Phe Cys
Gly Val Phe Asp Gly His Gly Pro Tyr Gly145 150 155 160cat ttg gtg
tcc aag agg gtc aga gat ctc ctc cct ata aag ttg agt 528His Leu Val
Ser Lys Arg Val Arg Asp Leu Leu Pro Ile Lys Leu Ser 165 170 175gca
aat tta gga aga gat gga cac aaa gaa act tca act aac att gtc 576Ala
Asn Leu Gly Arg Asp Gly His Lys Glu Thr Ser Thr Asn Ile Val 180 185
190aca agc agc atg act gaa ggt ggt ggc acc gaa cgc atg gat aga gat
624Thr Ser Ser Met Thr Glu Gly Gly Gly Thr Glu Arg Met Asp Arg Asp
195 200 205act gaa act ccc ctg gga acg gag gag aat gga gac tac ccc
gag atg 672Thr Glu Thr Pro Leu Gly Thr Glu Glu Asn Gly Asp Tyr Pro
Glu Met 210 215 220ttt gct gca tta aga act tca tta tta agg gca ttt
tat gta atg gac 720Phe Ala Ala Leu Arg Thr Ser Leu Leu Arg Ala Phe
Tyr Val Met Asp225 230 235 240agg gat ctt aag ttt cat aaa acc att
gac tct gtg ttc agt ggt act 768Arg Asp Leu Lys Phe His Lys Thr Ile
Asp Ser Val Phe Ser Gly Thr 245 250 255aca gca gtc aca gtg atc aag
cag gga cat gat ctc ctg att gga aac 816Thr Ala Val Thr Val Ile Lys
Gln Gly His Asp Leu Leu Ile Gly Asn 260 265 270ttg ggg gat tct aga
gct gtc ttg gga act aga gat gaa tat gac cag 864Leu Gly Asp Ser Arg
Ala Val Leu Gly Thr Arg Asp Glu Tyr Asp Gln 275 280 285ttt ttt gct
gta caa ttg aca gtt gac ctg aag cct acc att cca agt 912Phe Phe Ala
Val Gln Leu Thr Val Asp Leu Lys Pro Thr Ile Pro Ser 290 295 300gaa
gct gca cga att agg gaa cga agt ggc aga ata ttc tct ctg cca 960Glu
Ala Ala Arg Ile Arg Glu Arg Ser Gly Arg Ile Phe Ser Leu Pro305 310
315 320gat gag cca gat gtt gct cgt gtt tgg ctt ccg aag tac aac atg
cca 1008Asp Glu Pro Asp Val Ala Arg Val Trp Leu Pro Lys Tyr Asn Met
Pro 325 330 335ggg ttg gcc atg gca aga gca ttt gga gac ttt tgt cta
aag gat tat 1056Gly Leu Ala Met Ala Arg Ala Phe Gly Asp Phe Cys Leu
Lys Asp Tyr 340 345 350ggt cta att tct atg cct gat gtt tcc tac cac
cgc atc act gaa aag 1104Gly Leu Ile Ser Met Pro Asp Val Ser Tyr His
Arg Ile Thr Glu Lys 355 360 365gat gaa ttt gtt gtg ttg gca act gat
ggg gtg tgg gat gta ctt tcc 1152Asp Glu Phe Val Val Leu Ala Thr Asp
Gly Val Trp Asp Val Leu Ser 370 375 380aac tca gaa gtt gtt agc att
gtc agc caa gcc aag tca gaa gcc tca 1200Asn Ser Glu Val Val Ser Ile
Val Ser Gln Ala Lys Ser Glu Ala Ser385 390 395 400gcg gca cga ttt
gtt gtt gaa tcg gct caa cgt gca tgg aga aca cgg 1248Ala Ala Arg Phe
Val Val Glu Ser Ala Gln Arg Ala Trp Arg Thr Arg 405 410 415ttc ccc
aca tca aaa att gat gac tgc gct gtt gtc tgc ctg ttc ttg 1296Phe Pro
Thr Ser Lys Ile Asp Asp Cys Ala Val Val Cys Leu Phe Leu 420 425
430aat aca gat gct aga aat aaa ccc ccc ggt tca gga atc aaa gat ttg
1344Asn Thr Asp Ala Arg Asn Lys Pro Pro Gly Ser Gly Ile Lys Asp Leu
435 440 445gcc aat gcc ata gaa ctg ggt ggt ggt aat ctt tct tga
1383Ala Asn Ala Ile Glu Leu Gly Gly Gly Asn Leu Ser 450 455
46015460PRTOryza sativa 15Met Val Ala Val Thr Gly Gly Arg Pro Pro
Gly Leu Gln Asp Ala Pro1 5 10 15Gly Ala Pro Pro Pro Ala Pro Ala Ala
Glu Ala Val Pro Ser Arg Pro 20 25 30Leu Ala Arg Asp Ala Thr Tyr Gly
Gly Arg Val Tyr Gly Gly Val Gly 35 40 45Gly Gly Gly Cys Cys Leu Glu
Phe Leu Asp Cys Val Leu Arg Ala Met 50 55 60Gly Val Ala Thr Pro Ala
Glu Ile Met Pro Pro Ala Asp Phe Arg Trp65 70 75 80Ala Ala Arg Pro
Met Arg Arg Arg Arg Arg Gly Gly Ser Ser Ser Ser 85 90 95Ser Ser Ser
Pro Arg Asp Arg Glu Pro Arg Asp Gly Arg Ile Ala Ala 100 105 110Asn
Gly Ala Ser Ala Ala Ala Ser Leu Tyr Thr Met Arg Gly Asn Lys 115 120
125Gly Val Asn Gln Asp Ala Met Leu Val Trp Glu Asn Phe Cys Ser Lys
130 135 140Glu Asp Thr Ile Phe Cys Gly Val Phe Asp Gly His Gly Pro
Tyr Gly145 150 155 160His Leu Val Ser Lys Arg Val Arg Asp Leu Leu
Pro Ile Lys Leu Ser 165 170 175Ala Asn Leu Gly Arg Asp Gly His Lys
Glu Thr Ser Thr Asn Ile Val 180 185 190Thr Ser Ser Met Thr Glu Gly
Gly Gly Thr Glu Arg Met Asp Arg Asp 195 200 205Thr Glu Thr Pro Leu
Gly Thr Glu Glu Asn Gly Asp Tyr Pro Glu Met 210 215 220Phe Ala Ala
Leu Arg Thr Ser Leu Leu Arg Ala Phe Tyr Val Met Asp225 230 235
240Arg Asp Leu Lys Phe His Lys Thr Ile Asp Ser Val Phe Ser Gly Thr
245 250 255Thr Ala Val Thr Val Ile Lys Gln Gly His Asp Leu Leu Ile
Gly Asn 260 265 270Leu Gly Asp Ser Arg Ala Val Leu Gly Thr Arg Asp
Glu Tyr Asp Gln 275 280 285Phe Phe Ala Val Gln Leu Thr Val Asp Leu
Lys Pro Thr Ile Pro Ser 290 295 300Glu Ala Ala Arg Ile Arg Glu Arg
Ser Gly Arg Ile Phe Ser Leu Pro305 310 315 320Asp Glu Pro Asp Val
Ala Arg Val Trp Leu Pro Lys Tyr Asn Met Pro 325 330 335Gly Leu Ala
Met Ala Arg Ala Phe Gly Asp Phe Cys Leu Lys Asp Tyr 340 345 350Gly
Leu Ile Ser Met Pro Asp Val Ser Tyr His Arg Ile Thr Glu Lys 355 360
365Asp Glu Phe Val Val Leu Ala Thr Asp Gly Val Trp Asp Val Leu Ser
370 375 380Asn Ser Glu Val Val Ser Ile Val Ser Gln Ala Lys Ser Glu
Ala Ser385 390 395 400Ala Ala Arg Phe Val Val Glu Ser Ala Gln Arg
Ala Trp Arg Thr Arg 405 410 415Phe Pro Thr Ser Lys Ile Asp Asp Cys
Ala Val Val Cys Leu Phe Leu 420 425 430Asn Thr Asp Ala Arg Asn Lys
Pro Pro Gly Ser Gly Ile Lys Asp Leu 435 440 445Ala Asn Ala Ile Glu
Leu Gly Gly Gly Asn Leu Ser 450 455 460161299DNAOryza
sativaCDS(1)..(1299) 16atg ggg aca tgc ctt acg acg gcg gag cag cgg
gcc atg gag gtg ccg 48Met Gly Thr Cys Leu Thr Thr Ala Glu Gln Arg
Ala Met Glu Val Pro1 5 10 15gct gcg tcg gtg aag gga gga ggg ggc agg
agg agt gac gag gag gcg 96Ala Ala Ser Val Lys Gly Gly Gly Gly Arg
Arg Ser Asp Glu Glu Ala 20 25 30ccc ggc agg atc gcg ggt aac ggc gcg
ggg aat gtg gcc tgc ctg ttc 144Pro Gly Arg Ile Ala Gly Asn Gly Ala
Gly Asn Val Ala Cys Leu Phe 35 40 45act cgg cag ggg aag aag ggc acc
aac cag gat gcc atg gtc gcg tgg 192Thr Arg Gln Gly Lys Lys Gly Thr
Asn Gln Asp Ala Met Val Ala Trp 50 55 60gag aac tat aac gga aga tca
gac acg gta ttt tgt gga gtt ttt gat 240Glu Asn Tyr Asn Gly Arg Ser
Asp Thr Val Phe Cys Gly Val Phe Asp65 70 75 80ggc cac ggt cca cat
ggc cat ctc att gct agg aaa gta aga gat att 288Gly His Gly Pro His
Gly His Leu Ile Ala Arg Lys Val Arg Asp Ile 85 90 95ctc cct tcg aga
ctc tgt gat ttg ata tat gaa gac tgt ggg gat agt 336Leu Pro Ser Arg
Leu Cys Asp Leu Ile Tyr Glu Asp Cys Gly Asp Ser 100 105 110cca acc
agc aat tca gat gtc tca act ctg gaa gag aat tta tct ccg 384Pro Thr
Ser Asn Ser Asp Val Ser Thr Leu Glu Glu Asn Leu Ser Pro 115 120
125tat gca gat gca gag tgc aga tct ccc aca
ttg gct gga caa aaa gaa 432Tyr Ala Asp Ala Glu Cys Arg Ser Pro Thr
Leu Ala Gly Gln Lys Glu 130 135 140cat caa gaa ttc ttc aac gca atg
aaa gaa tct ttc aga aag gct ttt 480His Gln Glu Phe Phe Asn Ala Met
Lys Glu Ser Phe Arg Lys Ala Phe145 150 155 160aaa aat gtg gat aag
gag ctc aaa tta caa cgg aac att gat agc att 528Lys Asn Val Asp Lys
Glu Leu Lys Leu Gln Arg Asn Ile Asp Ser Ile 165 170 175tgc agt gga
act act gca gtt act tta atc aag caa ggt cat gat ctt 576Cys Ser Gly
Thr Thr Ala Val Thr Leu Ile Lys Gln Gly His Asp Leu 180 185 190att
gtt ggg aat cta ggt gac tct aga gct gta tta ggc acc aga gat 624Ile
Val Gly Asn Leu Gly Asp Ser Arg Ala Val Leu Gly Thr Arg Asp 195 200
205cag aac gat aag ttg gtt gct cat cag ttg act gtt gac ctg aaa cct
672Gln Asn Asp Lys Leu Val Ala His Gln Leu Thr Val Asp Leu Lys Pro
210 215 220gat cat cca agg gag gct agg agg atc aga cgg tgt aat ggg
agg gtc 720Asp His Pro Arg Glu Ala Arg Arg Ile Arg Arg Cys Asn Gly
Arg Val225 230 235 240ttt gct cat cag gat gaa cca gat gtg gct cgc
ctt tgg ctt cct aat 768Phe Ala His Gln Asp Glu Pro Asp Val Ala Arg
Leu Trp Leu Pro Asn 245 250 255tgc aac tct cct gga ctg gca atg gcc
cga gct ttt ggt gac ttt tgt 816Cys Asn Ser Pro Gly Leu Ala Met Ala
Arg Ala Phe Gly Asp Phe Cys 260 265 270cta aag gat ttt ggg ttg atc
tca gta cct gat gtc acc tat agg caa 864Leu Lys Asp Phe Gly Leu Ile
Ser Val Pro Asp Val Thr Tyr Arg Gln 275 280 285att act gaa aaa gac
gag ttt att gtc ctg gcg aca gat ggg gtg tgg 912Ile Thr Glu Lys Asp
Glu Phe Ile Val Leu Ala Thr Asp Gly Val Trp 290 295 300gat gtt ctc
tcc aac cag gaa gtg gtg gat gtt gtt gcc tca tgc tct 960Asp Val Leu
Ser Asn Gln Glu Val Val Asp Val Val Ala Ser Cys Ser305 310 315
320ggt cgt ttc gct gca gct cgt tct gtt gtt gat tta gca aat gag act
1008Gly Arg Phe Ala Ala Ala Arg Ser Val Val Asp Leu Ala Asn Glu Thr
325 330 335tgg agg ttc aaa tac cca acc tca aaa act gat gat tgt gca
gtg gtc 1056Trp Arg Phe Lys Tyr Pro Thr Ser Lys Thr Asp Asp Cys Ala
Val Val 340 345 350tgt ctt ttc ctg aac aag tat gaa gtt acc ggt ggt
tta tca ggg caa 1104Cys Leu Phe Leu Asn Lys Tyr Glu Val Thr Gly Gly
Leu Ser Gly Gln 355 360 365cct gga tat agt cca agg atg cct gcc cta
tca ggt att acc cgg ccc 1152Pro Gly Tyr Ser Pro Arg Met Pro Ala Leu
Ser Gly Ile Thr Arg Pro 370 375 380aat agt aaa agg gtt act cct gac
gac gtc gat gat ggt agt gac tca 1200Asn Ser Lys Arg Val Thr Pro Asp
Asp Val Asp Asp Gly Ser Asp Ser385 390 395 400aac gta agc gga gat
gag agg tcc ttg gat ggt ttc act cga ttg aac 1248Asn Val Ser Gly Asp
Glu Arg Ser Leu Asp Gly Phe Thr Arg Leu Asn 405 410 415aca ttg ttg
gca cta cca aag ttt ggt gac aca agt cca act aag aaa 1296Thr Leu Leu
Ala Leu Pro Lys Phe Gly Asp Thr Ser Pro Thr Lys Lys 420 425 430tga
1299 17432PRTOryza sativa 17Met Gly Thr Cys Leu Thr Thr Ala Glu Gln
Arg Ala Met Glu Val Pro1 5 10 15Ala Ala Ser Val Lys Gly Gly Gly Gly
Arg Arg Ser Asp Glu Glu Ala 20 25 30Pro Gly Arg Ile Ala Gly Asn Gly
Ala Gly Asn Val Ala Cys Leu Phe 35 40 45Thr Arg Gln Gly Lys Lys Gly
Thr Asn Gln Asp Ala Met Val Ala Trp 50 55 60Glu Asn Tyr Asn Gly Arg
Ser Asp Thr Val Phe Cys Gly Val Phe Asp65 70 75 80Gly His Gly Pro
His Gly His Leu Ile Ala Arg Lys Val Arg Asp Ile 85 90 95Leu Pro Ser
Arg Leu Cys Asp Leu Ile Tyr Glu Asp Cys Gly Asp Ser 100 105 110Pro
Thr Ser Asn Ser Asp Val Ser Thr Leu Glu Glu Asn Leu Ser Pro 115 120
125Tyr Ala Asp Ala Glu Cys Arg Ser Pro Thr Leu Ala Gly Gln Lys Glu
130 135 140His Gln Glu Phe Phe Asn Ala Met Lys Glu Ser Phe Arg Lys
Ala Phe145 150 155 160Lys Asn Val Asp Lys Glu Leu Lys Leu Gln Arg
Asn Ile Asp Ser Ile 165 170 175Cys Ser Gly Thr Thr Ala Val Thr Leu
Ile Lys Gln Gly His Asp Leu 180 185 190Ile Val Gly Asn Leu Gly Asp
Ser Arg Ala Val Leu Gly Thr Arg Asp 195 200 205Gln Asn Asp Lys Leu
Val Ala His Gln Leu Thr Val Asp Leu Lys Pro 210 215 220Asp His Pro
Arg Glu Ala Arg Arg Ile Arg Arg Cys Asn Gly Arg Val225 230 235
240Phe Ala His Gln Asp Glu Pro Asp Val Ala Arg Leu Trp Leu Pro Asn
245 250 255Cys Asn Ser Pro Gly Leu Ala Met Ala Arg Ala Phe Gly Asp
Phe Cys 260 265 270Leu Lys Asp Phe Gly Leu Ile Ser Val Pro Asp Val
Thr Tyr Arg Gln 275 280 285Ile Thr Glu Lys Asp Glu Phe Ile Val Leu
Ala Thr Asp Gly Val Trp 290 295 300Asp Val Leu Ser Asn Gln Glu Val
Val Asp Val Val Ala Ser Cys Ser305 310 315 320Gly Arg Phe Ala Ala
Ala Arg Ser Val Val Asp Leu Ala Asn Glu Thr 325 330 335Trp Arg Phe
Lys Tyr Pro Thr Ser Lys Thr Asp Asp Cys Ala Val Val 340 345 350Cys
Leu Phe Leu Asn Lys Tyr Glu Val Thr Gly Gly Leu Ser Gly Gln 355 360
365Pro Gly Tyr Ser Pro Arg Met Pro Ala Leu Ser Gly Ile Thr Arg Pro
370 375 380Asn Ser Lys Arg Val Thr Pro Asp Asp Val Asp Asp Gly Ser
Asp Ser385 390 395 400Asn Val Ser Gly Asp Glu Arg Ser Leu Asp Gly
Phe Thr Arg Leu Asn 405 410 415Thr Leu Leu Ala Leu Pro Lys Phe Gly
Asp Thr Ser Pro Thr Lys Lys 420 425 430181284DNAOryza
sativaCDS(1)..(1284) 18atg ggg aac tgc gtg gcg agg agc ggg acg gcg
gtg gat gcg ggt ggt 48Met Gly Asn Cys Val Ala Arg Ser Gly Thr Ala
Val Asp Ala Gly Gly1 5 10 15gat gga ggg gag gat ggg aag agg cgg agg
agg agg tgg aag gcg ccg 96Asp Gly Gly Glu Asp Gly Lys Arg Arg Arg
Arg Arg Trp Lys Ala Pro 20 25 30cgg gaa gat cag ctc ggg atg gtg ccc
ggc cgg atc ttc tcc aac gac 144Arg Glu Asp Gln Leu Gly Met Val Pro
Gly Arg Ile Phe Ser Asn Asp 35 40 45ggc cgc agc cgg acg gcg acg gtg
tac acg cag caa ggg cgc aag ggg 192Gly Arg Ser Arg Thr Ala Thr Val
Tyr Thr Gln Gln Gly Arg Lys Gly 50 55 60atc aac cag gac gcc atg ctc
gtc tgg gat ggg ttc ggc ggc gag gac 240Ile Asn Gln Asp Ala Met Leu
Val Trp Asp Gly Phe Gly Gly Glu Asp65 70 75 80gac ggc gtg ctg tgc
ggg gtg ttc gac ggg cac ggg ccg cac ggg cac 288Asp Gly Val Leu Cys
Gly Val Phe Asp Gly His Gly Pro His Gly His 85 90 95gtg gtg gcg cgg
agg gtc cgc gac tcg ctg ccg ctg agg ctc atg tcc 336Val Val Ala Arg
Arg Val Arg Asp Ser Leu Pro Leu Arg Leu Met Ser 100 105 110gcg gcg
cgc gac agc ggg gcg gac atg ccg gcc gcc gca tgg agg aag 384Ala Ala
Arg Asp Ser Gly Ala Asp Met Pro Ala Ala Ala Trp Arg Lys 115 120
125gcc ttc gcg cgc gcc tac aag gcc atg gac aag gac ctc cgg tcg cac
432Ala Phe Ala Arg Ala Tyr Lys Ala Met Asp Lys Asp Leu Arg Ser His
130 135 140cct tcc ctc gat tgc ttc tgc agc gga agc act gcc gtc acc
gtc ctc 480Pro Ser Leu Asp Cys Phe Cys Ser Gly Ser Thr Ala Val Thr
Val Leu145 150 155 160aag ctc ggc tcg gat ctc tac atg gcc aac att
ggg gac tcg cgc gcc 528Lys Leu Gly Ser Asp Leu Tyr Met Ala Asn Ile
Gly Asp Ser Arg Ala 165 170 175gtg ctc ggc tcc agg gag gcc acc ggc
ggc ggc atg gtc gcc gtg cag 576Val Leu Gly Ser Arg Glu Ala Thr Gly
Gly Gly Met Val Ala Val Gln 180 185 190ctc acc gtt gat ctc aag ccg
gat gtc ccc agc gaa gcg gag agg atc 624Leu Thr Val Asp Leu Lys Pro
Asp Val Pro Ser Glu Ala Glu Arg Ile 195 200 205aag aag tgc agg ggc
agg gtg ttc gcg ctg cag gac gag ccg gag gtg 672Lys Lys Cys Arg Gly
Arg Val Phe Ala Leu Gln Asp Glu Pro Glu Val 210 215 220cca agg gtc
tgg ctg ccg ttc gac gac gcg ccg ggc ctc gcg atg gcg 720Pro Arg Val
Trp Leu Pro Phe Asp Asp Ala Pro Gly Leu Ala Met Ala225 230 235
240cga gcg ttc ggg gac ttc tgc ctg aaa gat tac ggg gtc atc tcg gtg
768Arg Ala Phe Gly Asp Phe Cys Leu Lys Asp Tyr Gly Val Ile Ser Val
245 250 255ccg gaa ttc ttc cac tgg tct ctc aca gaa aag gac cag ttc
gtc att 816Pro Glu Phe Phe His Trp Ser Leu Thr Glu Lys Asp Gln Phe
Val Ile 260 265 270ctt gca tcg gat ggg gta tgg gat gtc ctc agc aat
caa gag gct gtt 864Leu Ala Ser Asp Gly Val Trp Asp Val Leu Ser Asn
Gln Glu Ala Val 275 280 285gat ata gtg tcc gcg tcc cca agc aga tca
aag gct gca aaa tcc ctt 912Asp Ile Val Ser Ala Ser Pro Ser Arg Ser
Lys Ala Ala Lys Ser Leu 290 295 300gtt gag gca gcc act cgt gaa tgg
aaa acc aaa tat cca aca tcc aaa 960Val Glu Ala Ala Thr Arg Glu Trp
Lys Thr Lys Tyr Pro Thr Ser Lys305 310 315 320atc gat gat tgc gcg
gtt gtt tgc tta tat ttg gat gga aaa atg gac 1008Ile Asp Asp Cys Ala
Val Val Cys Leu Tyr Leu Asp Gly Lys Met Asp 325 330 335cat gag cgt
gac tca act gcc tca ttg gac aac atc agt att gaa gag 1056His Glu Arg
Asp Ser Thr Ala Ser Leu Asp Asn Ile Ser Ile Glu Glu 340 345 350ggt
tca gtt gca gat cct aat gaa cct cag gag cag gag ccc acc tta 1104Gly
Ser Val Ala Asp Pro Asn Glu Pro Gln Glu Gln Glu Pro Thr Leu 355 360
365act cgg aat ttc aca gtt agg aca gtt gca ggc agc acg caa gag aag
1152Thr Arg Asn Phe Thr Val Arg Thr Val Ala Gly Ser Thr Gln Glu Lys
370 375 380acc tta gca ggg gtg gat gcg agg att gct ggt gta gcg aac
gac caa 1200Thr Leu Ala Gly Val Asp Ala Arg Ile Ala Gly Val Ala Asn
Asp Gln385 390 395 400aat tgg tca ggt ctc gat gga gtg aca cgg gta
aac tca ctt gtt cag 1248Asn Trp Ser Gly Leu Asp Gly Val Thr Arg Val
Asn Ser Leu Val Gln 405 410 415ctt cct agg ttt tct gaa gag agg gca
att ggc tga 1284Leu Pro Arg Phe Ser Glu Glu Arg Ala Ile Gly 420
42519427PRTOryza sativa 19Met Gly Asn Cys Val Ala Arg Ser Gly Thr
Ala Val Asp Ala Gly Gly1 5 10 15Asp Gly Gly Glu Asp Gly Lys Arg Arg
Arg Arg Arg Trp Lys Ala Pro 20 25 30Arg Glu Asp Gln Leu Gly Met Val
Pro Gly Arg Ile Phe Ser Asn Asp 35 40 45Gly Arg Ser Arg Thr Ala Thr
Val Tyr Thr Gln Gln Gly Arg Lys Gly 50 55 60Ile Asn Gln Asp Ala Met
Leu Val Trp Asp Gly Phe Gly Gly Glu Asp65 70 75 80Asp Gly Val Leu
Cys Gly Val Phe Asp Gly His Gly Pro His Gly His 85 90 95Val Val Ala
Arg Arg Val Arg Asp Ser Leu Pro Leu Arg Leu Met Ser 100 105 110Ala
Ala Arg Asp Ser Gly Ala Asp Met Pro Ala Ala Ala Trp Arg Lys 115 120
125Ala Phe Ala Arg Ala Tyr Lys Ala Met Asp Lys Asp Leu Arg Ser His
130 135 140Pro Ser Leu Asp Cys Phe Cys Ser Gly Ser Thr Ala Val Thr
Val Leu145 150 155 160Lys Leu Gly Ser Asp Leu Tyr Met Ala Asn Ile
Gly Asp Ser Arg Ala 165 170 175Val Leu Gly Ser Arg Glu Ala Thr Gly
Gly Gly Met Val Ala Val Gln 180 185 190Leu Thr Val Asp Leu Lys Pro
Asp Val Pro Ser Glu Ala Glu Arg Ile 195 200 205Lys Lys Cys Arg Gly
Arg Val Phe Ala Leu Gln Asp Glu Pro Glu Val 210 215 220Pro Arg Val
Trp Leu Pro Phe Asp Asp Ala Pro Gly Leu Ala Met Ala225 230 235
240Arg Ala Phe Gly Asp Phe Cys Leu Lys Asp Tyr Gly Val Ile Ser Val
245 250 255Pro Glu Phe Phe His Trp Ser Leu Thr Glu Lys Asp Gln Phe
Val Ile 260 265 270Leu Ala Ser Asp Gly Val Trp Asp Val Leu Ser Asn
Gln Glu Ala Val 275 280 285Asp Ile Val Ser Ala Ser Pro Ser Arg Ser
Lys Ala Ala Lys Ser Leu 290 295 300Val Glu Ala Ala Thr Arg Glu Trp
Lys Thr Lys Tyr Pro Thr Ser Lys305 310 315 320Ile Asp Asp Cys Ala
Val Val Cys Leu Tyr Leu Asp Gly Lys Met Asp 325 330 335His Glu Arg
Asp Ser Thr Ala Ser Leu Asp Asn Ile Ser Ile Glu Glu 340 345 350Gly
Ser Val Ala Asp Pro Asn Glu Pro Gln Glu Gln Glu Pro Thr Leu 355 360
365Thr Arg Asn Phe Thr Val Arg Thr Val Ala Gly Ser Thr Gln Glu Lys
370 375 380Thr Leu Ala Gly Val Asp Ala Arg Ile Ala Gly Val Ala Asn
Asp Gln385 390 395 400Asn Trp Ser Gly Leu Asp Gly Val Thr Arg Val
Asn Ser Leu Val Gln 405 410 415Leu Pro Arg Phe Ser Glu Glu Arg Ala
Ile Gly 420 425201596DNAOryza sativaCDS(1)..(1596) 20atg ggc tcc
tgc ctc tcc tcc gac ctg cct ccc cgc gcc ggc gcc ggc 48Met Gly Ser
Cys Leu Ser Ser Asp Leu Pro Pro Arg Ala Gly Ala Gly1 5 10 15gcg gga
gcg tca ccc ggg tgg ccg cag cgg tgg cgg agg agg agg cag 96Ala Gly
Ala Ser Pro Gly Trp Pro Gln Arg Trp Arg Arg Arg Arg Gln 20 25 30cgg
ggg gtg gag cgg ggc ggg gct gtt tcc ggc ggc ggc ggc ggc gtc 144Arg
Gly Val Glu Arg Gly Gly Ala Val Ser Gly Gly Gly Gly Gly Val 35 40
45ttc tcc atc ggc gtc ggc ggc aag aag ctg cac cac ggc ggc gga gga
192Phe Ser Ile Gly Val Gly Gly Lys Lys Leu His His Gly Gly Gly Gly
50 55 60gga ggg gag atg acg gag gag gag ctc gcg aag gtc gag ggg agg
gtg 240Gly Gly Glu Met Thr Glu Glu Glu Leu Ala Lys Val Glu Gly Arg
Val65 70 75 80tgc gtc aac ggc gcg agc gcg gcg gcg tgc ctg cac acg
cag cag ggg 288Cys Val Asn Gly Ala Ser Ala Ala Ala Cys Leu His Thr
Gln Gln Gly 85 90 95cgg aag ggc acc aac cag gac gcc atg gtc gtg tgg
gag aac ttt aat 336Arg Lys Gly Thr Asn Gln Asp Ala Met Val Val Trp
Glu Asn Phe Asn 100 105 110aca agt gat agt gtc ttc tgt ggt gtg ttt
gat ggt cat ggt cca tat 384Thr Ser Asp Ser Val Phe Cys Gly Val Phe
Asp Gly His Gly Pro Tyr 115 120 125ggt cat ttt gtt gcc aag aag gtc
aga gat tct ctt cct gtc aaa ata 432Gly His Phe Val Ala Lys Lys Val
Arg Asp Ser Leu Pro Val Lys Ile 130 135 140cgc aca cta tgg aaa acc
agt gcc aac gag gac act agt tcc cac caa 480Arg Thr Leu Trp Lys Thr
Ser Ala Asn Glu Asp Thr Ser Ser His Gln145 150 155 160aat gga agc
att tct gga agt gtt aat tca gaa gag tca cct gtt gtt 528Asn Gly Ser
Ile Ser Gly Ser Val Asn Ser Glu Glu Ser Pro Val Val 165 170 175gat
gat gaa tgg ggt gaa tat gct gat gac agc gag aag ctt cct gag 576Asp
Asp Glu Trp Gly Glu Tyr Ala Asp Asp Ser Glu Lys Leu Pro Glu 180 185
190atg ttt ctt cca ctt aag cag tct tat ttt aag gct ttc aaa ttg atg
624Met Phe Leu Pro Leu Lys Gln Ser Tyr Phe Lys Ala Phe Lys Leu Met
195 200 205gac aag gaa ctc aaa atg cac cct aca gtt gat tgc ttt tgc
agt gga 672Asp Lys Glu Leu Lys Met His Pro Thr Val Asp Cys Phe Cys
Ser Gly 210 215 220tca aca gca gtc acg tta gta aaa cag gga ttg gat
ctt gtg gtt ggg 720Ser Thr Ala Val Thr Leu Val Lys Gln Gly Leu Asp
Leu Val Val Gly225 230 235 240aac ctt ggg gac tcg aga gca ata atg
ggg aca cga gat gct gcc aat 768Asn Leu Gly Asp Ser Arg Ala Ile Met
Gly Thr Arg Asp Ala Ala Asn 245 250 255aat cta act gct gta caa ctc
aca gtt gat
ttg aag cct aac ctt cca 816Asn Leu Thr Ala Val Gln Leu Thr Val Asp
Leu Lys Pro Asn Leu Pro 260 265 270agg gaa gct gcg agg atc cag cag
tgt agg gga aga gtt ttt gct ctt 864Arg Glu Ala Ala Arg Ile Gln Gln
Cys Arg Gly Arg Val Phe Ala Leu 275 280 285cag gat gaa cca gaa gtt
gcc aga gta tgg ttg cca aat aat gac tct 912Gln Asp Glu Pro Glu Val
Ala Arg Val Trp Leu Pro Asn Asn Asp Ser 290 295 300cct gga ttg gca
atg gca aga gct ttt gga gac ttc tgc ctt aaa gat 960Pro Gly Leu Ala
Met Ala Arg Ala Phe Gly Asp Phe Cys Leu Lys Asp305 310 315 320tat
ggt tta ata tct gtt cca cag ata tcc tat cgt cgt ctt act gaa 1008Tyr
Gly Leu Ile Ser Val Pro Gln Ile Ser Tyr Arg Arg Leu Thr Glu 325 330
335aag gat gag ttc ata ata ctg gcc act gat ggg gtt tgg gac gtc ctc
1056Lys Asp Glu Phe Ile Ile Leu Ala Thr Asp Gly Val Trp Asp Val Leu
340 345 350tca aac aag gag gct gtt gac ata gta gcc gca gct cca tct
cgt gca 1104Ser Asn Lys Glu Ala Val Asp Ile Val Ala Ala Ala Pro Ser
Arg Ala 355 360 365acg gct gcc agg gct ctt gtc gac tgt gct gtc aga
tca tgg aga ttg 1152Thr Ala Ala Arg Ala Leu Val Asp Cys Ala Val Arg
Ser Trp Arg Leu 370 375 380aag ttc cca aca tcc aag agc gat gac tgc
gct gtt gtg tgc cta ttc 1200Lys Phe Pro Thr Ser Lys Ser Asp Asp Cys
Ala Val Val Cys Leu Phe385 390 395 400tta gac cat gca aag tca cct
gac ttg att caa gag aac gag agc gag 1248Leu Asp His Ala Lys Ser Pro
Asp Leu Ile Gln Glu Asn Glu Ser Glu 405 410 415gaa gaa act aca gag
gat gtt gca atc cca gac acc gtt gct aag gtt 1296Glu Glu Thr Thr Glu
Asp Val Ala Ile Pro Asp Thr Val Ala Lys Val 420 425 430gac caa gac
att gca caa gga gat gca cat atc tcc agt gaa gag caa 1344Asp Gln Asp
Ile Ala Gln Gly Asp Ala His Ile Ser Ser Glu Glu Gln 435 440 445atc
acc gag cca gca ttg cag cac tcc tac aca tta agg gat gtt gat 1392Ile
Thr Glu Pro Ala Leu Gln His Ser Tyr Thr Leu Arg Asp Val Asp 450 455
460gag att gta ccg gta gag gag cct cca gtc tca aag gaa cct gaa aga
1440Glu Ile Val Pro Val Glu Glu Pro Pro Val Ser Lys Glu Pro Glu
Arg465 470 475 480tgt gga tct gcc cgc agc ctt gct gat tgt ata tcc
aca aac gag gag 1488Cys Gly Ser Ala Arg Ser Leu Ala Asp Cys Ile Ser
Thr Asn Glu Glu 485 490 495gag gaa tgg tca gca ctc gaa ggt gtg acg
cgg gtc aat tcc ctc ttg 1536Glu Glu Trp Ser Ala Leu Glu Gly Val Thr
Arg Val Asn Ser Leu Leu 500 505 510aac ctt ccc aga ata ctt tca ggc
gag aag aga tca acc agc tgg agg 1584Asn Leu Pro Arg Ile Leu Ser Gly
Glu Lys Arg Ser Thr Ser Trp Arg 515 520 525aag cgg cga tga 1596Lys
Arg Arg 53021531PRTOryza sativa 21Met Gly Ser Cys Leu Ser Ser Asp
Leu Pro Pro Arg Ala Gly Ala Gly1 5 10 15Ala Gly Ala Ser Pro Gly Trp
Pro Gln Arg Trp Arg Arg Arg Arg Gln 20 25 30Arg Gly Val Glu Arg Gly
Gly Ala Val Ser Gly Gly Gly Gly Gly Val 35 40 45Phe Ser Ile Gly Val
Gly Gly Lys Lys Leu His His Gly Gly Gly Gly 50 55 60Gly Gly Glu Met
Thr Glu Glu Glu Leu Ala Lys Val Glu Gly Arg Val65 70 75 80Cys Val
Asn Gly Ala Ser Ala Ala Ala Cys Leu His Thr Gln Gln Gly 85 90 95Arg
Lys Gly Thr Asn Gln Asp Ala Met Val Val Trp Glu Asn Phe Asn 100 105
110Thr Ser Asp Ser Val Phe Cys Gly Val Phe Asp Gly His Gly Pro Tyr
115 120 125Gly His Phe Val Ala Lys Lys Val Arg Asp Ser Leu Pro Val
Lys Ile 130 135 140Arg Thr Leu Trp Lys Thr Ser Ala Asn Glu Asp Thr
Ser Ser His Gln145 150 155 160Asn Gly Ser Ile Ser Gly Ser Val Asn
Ser Glu Glu Ser Pro Val Val 165 170 175Asp Asp Glu Trp Gly Glu Tyr
Ala Asp Asp Ser Glu Lys Leu Pro Glu 180 185 190Met Phe Leu Pro Leu
Lys Gln Ser Tyr Phe Lys Ala Phe Lys Leu Met 195 200 205Asp Lys Glu
Leu Lys Met His Pro Thr Val Asp Cys Phe Cys Ser Gly 210 215 220Ser
Thr Ala Val Thr Leu Val Lys Gln Gly Leu Asp Leu Val Val Gly225 230
235 240Asn Leu Gly Asp Ser Arg Ala Ile Met Gly Thr Arg Asp Ala Ala
Asn 245 250 255Asn Leu Thr Ala Val Gln Leu Thr Val Asp Leu Lys Pro
Asn Leu Pro 260 265 270Arg Glu Ala Ala Arg Ile Gln Gln Cys Arg Gly
Arg Val Phe Ala Leu 275 280 285Gln Asp Glu Pro Glu Val Ala Arg Val
Trp Leu Pro Asn Asn Asp Ser 290 295 300Pro Gly Leu Ala Met Ala Arg
Ala Phe Gly Asp Phe Cys Leu Lys Asp305 310 315 320Tyr Gly Leu Ile
Ser Val Pro Gln Ile Ser Tyr Arg Arg Leu Thr Glu 325 330 335Lys Asp
Glu Phe Ile Ile Leu Ala Thr Asp Gly Val Trp Asp Val Leu 340 345
350Ser Asn Lys Glu Ala Val Asp Ile Val Ala Ala Ala Pro Ser Arg Ala
355 360 365Thr Ala Ala Arg Ala Leu Val Asp Cys Ala Val Arg Ser Trp
Arg Leu 370 375 380Lys Phe Pro Thr Ser Lys Ser Asp Asp Cys Ala Val
Val Cys Leu Phe385 390 395 400Leu Asp His Ala Lys Ser Pro Asp Leu
Ile Gln Glu Asn Glu Ser Glu 405 410 415Glu Glu Thr Thr Glu Asp Val
Ala Ile Pro Asp Thr Val Ala Lys Val 420 425 430Asp Gln Asp Ile Ala
Gln Gly Asp Ala His Ile Ser Ser Glu Glu Gln 435 440 445Ile Thr Glu
Pro Ala Leu Gln His Ser Tyr Thr Leu Arg Asp Val Asp 450 455 460Glu
Ile Val Pro Val Glu Glu Pro Pro Val Ser Lys Glu Pro Glu Arg465 470
475 480Cys Gly Ser Ala Arg Ser Leu Ala Asp Cys Ile Ser Thr Asn Glu
Glu 485 490 495Glu Glu Trp Ser Ala Leu Glu Gly Val Thr Arg Val Asn
Ser Leu Leu 500 505 510Asn Leu Pro Arg Ile Leu Ser Gly Glu Lys Arg
Ser Thr Ser Trp Arg 515 520 525Lys Arg Arg 530221170DNAOryza
sativaCDS(1)..(1170) 22atg ggg atc tgt gca tct tca gag cag ctg gag
cat gtt cat gag aca 48Met Gly Ile Cys Ala Ser Ser Glu Gln Leu Glu
His Val His Glu Thr1 5 10 15gat gag agc att gtg tat gtg aag gat gag
caa gga agg ggg ggt agg 96Asp Glu Ser Ile Val Tyr Val Lys Asp Glu
Gln Gly Arg Gly Gly Arg 20 25 30ggg gtg gag agt ggg ggg gct agg aag
gtg gcc tcc ctc ttc tcc cag 144Gly Val Glu Ser Gly Gly Ala Arg Lys
Val Ala Ser Leu Phe Ser Gln 35 40 45agg ggc aag aaa ggc ccc aac cag
gac tct gtc atc ctc tgc cag gga 192Arg Gly Lys Lys Gly Pro Asn Gln
Asp Ser Val Ile Leu Cys Gln Gly 50 55 60ttc ggc atg gag gac ggc gtg
ttc tgc ggc gtg ttc gac ggc cat ggc 240Phe Gly Met Glu Asp Gly Val
Phe Cys Gly Val Phe Asp Gly His Gly65 70 75 80cgg tgc ggg caa ttc
atc agc aag ctg gtg cgg gac tac ctc ccg ttc 288Arg Cys Gly Gln Phe
Ile Ser Lys Leu Val Arg Asp Tyr Leu Pro Phe 85 90 95atg atc ctg agc
cac cgg aac gcg ctg ctc ctg gcc gac gcc gcc gcc 336Met Ile Leu Ser
His Arg Asn Ala Leu Leu Leu Ala Asp Ala Ala Ala 100 105 110gac gac
gac gac gac gcc gcg ttc agc gac gac gcg gcg gcg tcg tcg 384Asp Asp
Asp Asp Asp Ala Ala Phe Ser Asp Asp Ala Ala Ala Ser Ser 115 120
125tcc gcg gac agc agc ggc aac tcg tcg ccg cag ccg tcg gcg tcg gcg
432Ser Ala Asp Ser Ser Gly Asn Ser Ser Pro Gln Pro Ser Ala Ser Ala
130 135 140tcg gcg cag atg ctg gag gag tgg agg cag gcg tgc gcc agc
gcg ttc 480Ser Ala Gln Met Leu Glu Glu Trp Arg Gln Ala Cys Ala Ser
Ala Phe145 150 155 160gcc gcc atg gac ggc gag ctc aag ctc cag ccg
aac ctc gac tgc gcg 528Ala Ala Met Asp Gly Glu Leu Lys Leu Gln Pro
Asn Leu Asp Cys Ala 165 170 175ttc agc ggc acg acg gcg gtg tgc gcc
atc aag cag ggc agg gac ctc 576Phe Ser Gly Thr Thr Ala Val Cys Ala
Ile Lys Gln Gly Arg Asp Leu 180 185 190atc atc gcc aac ctc ggc gac
tcg agg gcg gtg ctc gcc acc atg tcg 624Ile Ile Ala Asn Leu Gly Asp
Ser Arg Ala Val Leu Ala Thr Met Ser 195 200 205gac acc ggc tac ctc
cag gcg gtg cag ctg acg gtg gac cac aag ccg 672Asp Thr Gly Tyr Leu
Gln Ala Val Gln Leu Thr Val Asp His Lys Pro 210 215 220agc gtg ccg
gag gag gcg gcg agg atc aag cgg agc ggg ggg agg gtg 720Ser Val Pro
Glu Glu Ala Ala Arg Ile Lys Arg Ser Gly Gly Arg Val225 230 235
240ttc ggg ctg aag gac gag ccg ggg gtg atg cgg gtg tgg ctc ccc ggc
768Phe Gly Leu Lys Asp Glu Pro Gly Val Met Arg Val Trp Leu Pro Gly
245 250 255gag aac tcg ccg ggg ctc gcc atg gcg agg tcg ctg ggc gac
atg agg 816Glu Asn Ser Pro Gly Leu Ala Met Ala Arg Ser Leu Gly Asp
Met Arg 260 265 270ctg aag cgg cac ggc gtg atc ccg gcg ccg gag gtg
acg tcg cgg cgc 864Leu Lys Arg His Gly Val Ile Pro Ala Pro Glu Val
Thr Ser Arg Arg 275 280 285gtg acg ggc gcc gac ctg ttc atg gtg ctc
gcc acg gac ggg gtg tgg 912Val Thr Gly Ala Asp Leu Phe Met Val Leu
Ala Thr Asp Gly Val Trp 290 295 300gac gtg ctg agc aac gag gag gtg
gtg tcc atc gtg tgc gcg acg ccg 960Asp Val Leu Ser Asn Glu Glu Val
Val Ser Ile Val Cys Ala Thr Pro305 310 315 320cgg aag cag cac gcg
tcg aag gcg gtg gtg gag gcc gcc gtg cag cgg 1008Arg Lys Gln His Ala
Ser Lys Ala Val Val Glu Ala Ala Val Gln Arg 325 330 335tgg cgg gcc
aag ttc ccg acg tcc agg gtg gac gac tgc tcc gcc gtc 1056Trp Arg Ala
Lys Phe Pro Thr Ser Arg Val Asp Asp Cys Ser Ala Val 340 345 350tgc
ctc ttc ctc cac gac cac acc ctc ggc acg gcc gcc gcc gcc tcc 1104Cys
Leu Phe Leu His Asp His Thr Leu Gly Thr Ala Ala Ala Ala Ser 355 360
365gcc gca gcc gcc gcg gcc gcc aga aag gcg cgc agg gcc tcc acc gcc
1152Ala Ala Ala Ala Ala Ala Ala Arg Lys Ala Arg Arg Ala Ser Thr Ala
370 375 380acg ccg ccg gcg agc tga 1170Thr Pro Pro Ala
Ser38523389PRTOryza sativa 23Met Gly Ile Cys Ala Ser Ser Glu Gln
Leu Glu His Val His Glu Thr1 5 10 15Asp Glu Ser Ile Val Tyr Val Lys
Asp Glu Gln Gly Arg Gly Gly Arg 20 25 30Gly Val Glu Ser Gly Gly Ala
Arg Lys Val Ala Ser Leu Phe Ser Gln 35 40 45Arg Gly Lys Lys Gly Pro
Asn Gln Asp Ser Val Ile Leu Cys Gln Gly 50 55 60Phe Gly Met Glu Asp
Gly Val Phe Cys Gly Val Phe Asp Gly His Gly65 70 75 80Arg Cys Gly
Gln Phe Ile Ser Lys Leu Val Arg Asp Tyr Leu Pro Phe 85 90 95Met Ile
Leu Ser His Arg Asn Ala Leu Leu Leu Ala Asp Ala Ala Ala 100 105
110Asp Asp Asp Asp Asp Ala Ala Phe Ser Asp Asp Ala Ala Ala Ser Ser
115 120 125Ser Ala Asp Ser Ser Gly Asn Ser Ser Pro Gln Pro Ser Ala
Ser Ala 130 135 140Ser Ala Gln Met Leu Glu Glu Trp Arg Gln Ala Cys
Ala Ser Ala Phe145 150 155 160Ala Ala Met Asp Gly Glu Leu Lys Leu
Gln Pro Asn Leu Asp Cys Ala 165 170 175Phe Ser Gly Thr Thr Ala Val
Cys Ala Ile Lys Gln Gly Arg Asp Leu 180 185 190Ile Ile Ala Asn Leu
Gly Asp Ser Arg Ala Val Leu Ala Thr Met Ser 195 200 205Asp Thr Gly
Tyr Leu Gln Ala Val Gln Leu Thr Val Asp His Lys Pro 210 215 220Ser
Val Pro Glu Glu Ala Ala Arg Ile Lys Arg Ser Gly Gly Arg Val225 230
235 240Phe Gly Leu Lys Asp Glu Pro Gly Val Met Arg Val Trp Leu Pro
Gly 245 250 255Glu Asn Ser Pro Gly Leu Ala Met Ala Arg Ser Leu Gly
Asp Met Arg 260 265 270Leu Lys Arg His Gly Val Ile Pro Ala Pro Glu
Val Thr Ser Arg Arg 275 280 285Val Thr Gly Ala Asp Leu Phe Met Val
Leu Ala Thr Asp Gly Val Trp 290 295 300Asp Val Leu Ser Asn Glu Glu
Val Val Ser Ile Val Cys Ala Thr Pro305 310 315 320Arg Lys Gln His
Ala Ser Lys Ala Val Val Glu Ala Ala Val Gln Arg 325 330 335Trp Arg
Ala Lys Phe Pro Thr Ser Arg Val Asp Asp Cys Ser Ala Val 340 345
350Cys Leu Phe Leu His Asp His Thr Leu Gly Thr Ala Ala Ala Ala Ser
355 360 365Ala Ala Ala Ala Ala Ala Ala Arg Lys Ala Arg Arg Ala Ser
Thr Ala 370 375 380Thr Pro Pro Ala Ser3852425DNAArtificialSynthetic
DNA 24tgctttcgcc attaaatagc gacgg 252523DNAArtificialSynthetic DNA
25cgctgcggac atctacattt ttg 232622DNAArtificialSynthetic DNA
26tcccggacat gaagccattt ac 222716DNAArtificialSynthetic DNA
27ngtcgaswga nawgaa 1628498DNAArabidopsis
thalianamisc_feature(53)..(53)n is a, c, g, or t 28taaccttacg
ctttgctcgg tcccagacgc aagattacat ctctttctat ggnttgagat 60cgnacggacg
gctgtttgag gacggtccaa ttgccactag ccagatttac gtgcatagca
120agttaatgat tgttgatgac cggatcgcag tgatcggatc ttctaatata
aacgatagga 180gcttactagg ttcacgagac tctgaggtac tttcaaaaat
ccaattcatt ctttattgca 240gcaaaacaga gttatgtatt catttgaatc
aatcatgttt cagatcggtg ttgtgattga 300agacaaagaa ttcgtggaat
cttcgatgaa cggaatgaag tggatggccg ggaagttctc 360ttacagtctt
agatgttcct tgtggtcaga gcatctcggc cttcacgccg gagaggtaat
420tttaaaaaat ttctagaaac gcctactact atacattttt gacttcagaa
acctttattt 480tcatctcact cgaccaaa 4982939DNAArtificialSynthetic DNA
29acgcgtcgac atgggacatt tctcttccat gttcaacgg
393043DNAArtificialSynthetic DNA 30tgtacatgta cactatagag atggcgacga
cgatgaagaa tgg 433129DNAArtificialSynthetic DNA 31aggatccatg
ggacatttct cttccatgt 293227DNAArtificialSynthetic DNA 32agagctccta
tagagatggc gacgacg 27331992DNAZea mays 33ctgcagtgca gcgtgacccg
gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60agttataaaa aattaccaca
tatttttttt gtcacacttg tttgaagtgc agtttatcta 120tctttataca
tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa
180tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa
ggacaattga 240gtattttgac aacaggactc tacagtttta tctttttagt
gtgcatgtgt tctccttttt 300ttttgcaaat agcttcacct atataatact
tcatccattt tattagtaca tccatttagg 360gtttagggtt aatggttttt
atagactaat ttttttagta catctatttt attctatttt 420agcctctaaa
ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata
480taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag
aaattaaaaa 540aactaaggaa acatttttct tgtttcgagt agataatgcc
agcctgttaa acgccgtcga 600cgagtctaac ggacaccaac cagcgaacca
gcagcgtcgc gtcgggccaa gcgaagcaga 660cggcacggca tctctgtcgc
tgcctctgga cccctctcga gagttccgct ccaccgttgg 720acttgctccg
ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac
780ggcaggcggc ctcctcctcc tctcacggca cggcagctac gggggattcc
tttcccaccg 840ctccttcgct ttcccttcct cgcccgccgt aataaataga
caccccctcc acaccctctt 900tccccaacct cgtgttgttc ggagcgcaca
cacacacaac cagatctccc ccaaatccac 960ccgtcggcac ctccgcttca
aggtacgccg ctcgtcctcc cccccccccc ctctctacct 1020tctctagatc
ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt
1080tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac
ggatgcgacc 1140tgtacgtcag acacgttctg attgctaact tgccagtgtt
tctctttggg gaatcctggg 1200atggctctag ccgttccgca gacgggatcg
atttcatgat tttttttgtt tcgttgcata 1260gggtttggtt tgcccttttc
ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320tcttttcatg
cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct
1380agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt
ggatctgtat 1440gtgtgtgcca tacatattca tagttacgaa ttgaagatga
tggatggaaa tatcgatcta 1500ggataggtat acatgttgat gcgggtttta
ctgatgcata
tacagagatg ctttttgttc 1560gcttggttgt gatgatgtgg tgtggttggg
cggtcgttca ttcgttctag atcggagtag 1620aatactgttt caaactacct
ggtgtattta ttaattttgg aactgtatgt gtgtgtcata 1680catcttcata
gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg
1740ttgatgtggg ttttactgat gcatatacat gatggcatat gcagcatcta
ttcatatgct 1800ctaaccttga gtacctatct attataataa acaagtatgt
tttataatta ttttgatctt 1860gatatacttg gatgatggca tatgcagcag
ctatatgtgg atttttttag ccctgccttc 1920atacgctatt tatttgcttg
gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg 1980ttacttctgc ag
199234256DNAZea mays 34gatcgttcaa acatttggca ataaagtttc ttaagattga
atcctgttgc cggtcttgcg 60atgattatca tataatttct gttgaattac gttaagcatg
taataattaa catgtaatgc 120atgacgttat ttatgagatg ggtttttatg
attagagtcc cgcaattata catttaatac 180gcgatagaaa acaaaatata
gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240atgttactag atcggg
256351122DNAArabidopsis thalianaCDS(1)..(1122) 35atg gga cat ttc
tca tcg atg ttc aat gga tta gct cga tca ttt tct 48Met Gly His Phe
Ser Ser Met Phe Asn Gly Leu Ala Arg Ser Phe Ser1 5 10 15ata aag aaa
gtg aag aac aac aat gga aac tgc gac gca aag gaa gct 96Ile Lys Lys
Val Lys Asn Asn Asn Gly Asn Cys Asp Ala Lys Glu Ala 20 25 30gct gat
gag atg gca agc gag gct aag aaa aaa gaa ttg att ctg aaa 144Ala Asp
Glu Met Ala Ser Glu Ala Lys Lys Lys Glu Leu Ile Leu Lys 35 40 45tcc
tct ggt tat gtt aat gta caa gga tct aat aat tta gcc tct ctt 192Ser
Ser Gly Tyr Val Asn Val Gln Gly Ser Asn Asn Leu Ala Ser Leu 50 55
60ttc tcc aaa cgc ggc gaa aaa ggc gtt aat cag gat tgt gca ctc gtt
240Phe Ser Lys Arg Gly Glu Lys Gly Val Asn Gln Asp Cys Ala Leu
Val65 70 75 80tgg gag gga ttt ggg tgc caa gaa gac atg atc ttc tgc
ggg ata ttc 288Trp Glu Gly Phe Gly Cys Gln Glu Asp Met Ile Phe Cys
Gly Ile Phe 85 90 95gat gga cac ggt cca tgg ggt cac tat gta gcc aaa
caa gta aga aac 336Asp Gly His Gly Pro Trp Gly His Tyr Val Ala Lys
Gln Val Arg Asn 100 105 110tca atg cct ttg tcg ctt ctt tgc aac tgg
caa aag att ctt gct cag 384Ser Met Pro Leu Ser Leu Leu Cys Asn Trp
Gln Lys Ile Leu Ala Gln 115 120 125gcc act cta gaa ccc gag ctc gac
ctc gaa ggc tct aat aaa aaa atc 432Ala Thr Leu Glu Pro Glu Leu Asp
Leu Glu Gly Ser Asn Lys Lys Ile 130 135 140tca aga ttc gac ata tgg
aag caa tcc tat cta aaa acg tgt gca acg 480Ser Arg Phe Asp Ile Trp
Lys Gln Ser Tyr Leu Lys Thr Cys Ala Thr145 150 155 160gtt gat caa
gag ctt gaa cat cac cgc aag atc gat tct tac tat agc 528Val Asp Gln
Glu Leu Glu His His Arg Lys Ile Asp Ser Tyr Tyr Ser 165 170 175ggc
aca aca gct cta acc att gtg aga cag ggt gaa gtt att tat gta 576Gly
Thr Thr Ala Leu Thr Ile Val Arg Gln Gly Glu Val Ile Tyr Val 180 185
190gca aat gta ggc gat tca aga gcg gta cta gcc atg gag tcg gat gag
624Ala Asn Val Gly Asp Ser Arg Ala Val Leu Ala Met Glu Ser Asp Glu
195 200 205gga agc ttg gtt gcg gtt cag ctc acc ctc gat ttc aaa ccg
aat cta 672Gly Ser Leu Val Ala Val Gln Leu Thr Leu Asp Phe Lys Pro
Asn Leu 210 215 220cca cag gag aag gag cgg ata att ggc tgc aaa ggg
cgg gtt ttc tgt 720Pro Gln Glu Lys Glu Arg Ile Ile Gly Cys Lys Gly
Arg Val Phe Cys225 230 235 240cta gat gat gag ccg gga gtc cat cgt
gtg tgg cag cca gac gca gaa 768Leu Asp Asp Glu Pro Gly Val His Arg
Val Trp Gln Pro Asp Ala Glu 245 250 255aca ccg ggg ctc gca atg tca
aga gca ttc gga gac tac tgt att aaa 816Thr Pro Gly Leu Ala Met Ser
Arg Ala Phe Gly Asp Tyr Cys Ile Lys 260 265 270gag tat gga ttg gtc
tca gtc cct gaa gtc act caa aga cac atc tct 864Glu Tyr Gly Leu Val
Ser Val Pro Glu Val Thr Gln Arg His Ile Ser 275 280 285act aaa gac
cac ttc ata atc ttg gcc agt gat ggg ata tgg gat gtg 912Thr Lys Asp
His Phe Ile Ile Leu Ala Ser Asp Gly Ile Trp Asp Val 290 295 300atc
tct aac caa gag gct ata gag att gtc tcc tca acg gct gag cgg 960Ile
Ser Asn Gln Glu Ala Ile Glu Ile Val Ser Ser Thr Ala Glu Arg305 310
315 320cct aag gcg gct aag cga tta gta gag caa gcg gtt cgg gct tgg
aag 1008Pro Lys Ala Ala Lys Arg Leu Val Glu Gln Ala Val Arg Ala Trp
Lys 325 330 335aaa aag aga cga gga tac tcc atg gat gat atg tca gtc
gtc tgc ctc 1056Lys Lys Arg Arg Gly Tyr Ser Met Asp Asp Met Ser Val
Val Cys Leu 340 345 350ttc ctc cat tct tct tca tcg tca tct cta tca
caa cat cat cat gcc 1104Phe Leu His Ser Ser Ser Ser Ser Ser Leu Ser
Gln His His His Ala 355 360 365atg acg att tta aag taa 1122Met Thr
Ile Leu Lys 37036373PRTArabidopsis thaliana 36Met Gly His Phe Ser
Ser Met Phe Asn Gly Leu Ala Arg Ser Phe Ser1 5 10 15Ile Lys Lys Val
Lys Asn Asn Asn Gly Asn Cys Asp Ala Lys Glu Ala 20 25 30Ala Asp Glu
Met Ala Ser Glu Ala Lys Lys Lys Glu Leu Ile Leu Lys 35 40 45Ser Ser
Gly Tyr Val Asn Val Gln Gly Ser Asn Asn Leu Ala Ser Leu 50 55 60Phe
Ser Lys Arg Gly Glu Lys Gly Val Asn Gln Asp Cys Ala Leu Val65 70 75
80Trp Glu Gly Phe Gly Cys Gln Glu Asp Met Ile Phe Cys Gly Ile Phe
85 90 95Asp Gly His Gly Pro Trp Gly His Tyr Val Ala Lys Gln Val Arg
Asn 100 105 110Ser Met Pro Leu Ser Leu Leu Cys Asn Trp Gln Lys Ile
Leu Ala Gln 115 120 125Ala Thr Leu Glu Pro Glu Leu Asp Leu Glu Gly
Ser Asn Lys Lys Ile 130 135 140Ser Arg Phe Asp Ile Trp Lys Gln Ser
Tyr Leu Lys Thr Cys Ala Thr145 150 155 160Val Asp Gln Glu Leu Glu
His His Arg Lys Ile Asp Ser Tyr Tyr Ser 165 170 175Gly Thr Thr Ala
Leu Thr Ile Val Arg Gln Gly Glu Val Ile Tyr Val 180 185 190Ala Asn
Val Gly Asp Ser Arg Ala Val Leu Ala Met Glu Ser Asp Glu 195 200
205Gly Ser Leu Val Ala Val Gln Leu Thr Leu Asp Phe Lys Pro Asn Leu
210 215 220Pro Gln Glu Lys Glu Arg Ile Ile Gly Cys Lys Gly Arg Val
Phe Cys225 230 235 240Leu Asp Asp Glu Pro Gly Val His Arg Val Trp
Gln Pro Asp Ala Glu 245 250 255Thr Pro Gly Leu Ala Met Ser Arg Ala
Phe Gly Asp Tyr Cys Ile Lys 260 265 270Glu Tyr Gly Leu Val Ser Val
Pro Glu Val Thr Gln Arg His Ile Ser 275 280 285Thr Lys Asp His Phe
Ile Ile Leu Ala Ser Asp Gly Ile Trp Asp Val 290 295 300Ile Ser Asn
Gln Glu Ala Ile Glu Ile Val Ser Ser Thr Ala Glu Arg305 310 315
320Pro Lys Ala Ala Lys Arg Leu Val Glu Gln Ala Val Arg Ala Trp Lys
325 330 335Lys Lys Arg Arg Gly Tyr Ser Met Asp Asp Met Ser Val Val
Cys Leu 340 345 350Phe Leu His Ser Ser Ser Ser Ser Ser Leu Ser Gln
His His His Ala 355 360 365Met Thr Ile Leu Lys
3703724DNAArtificialSynthetic DNA 37atgggacatt tctcatcgat gttc
243827DNAArtificialSynthetic DNA 38ttactttaaa atcgtcatgg catgatg
273956DNAArtificialSynthetic DNA 39aattactatt tacaattaca gtcgacatgg
gacatttctc atcgatgttc aatgga 564059DNAArtificialSynthetic DNA
40agccgggcgg ccgctttact tgtacattac tttaaaatcg tcatggcatg atgatgttg
59411584DNAArabidopsis thalianaCDS(1)..(1584) 41atg ggg tcc tgt tta
tct gca gag agc agg agc cct aga ccg ggc tct 48Met Gly Ser Cys Leu
Ser Ala Glu Ser Arg Ser Pro Arg Pro Gly Ser1 5 10 15cct tgc tct cct
gct ttt agt gtg agg aag agg aag aac tct aag aag 96Pro Cys Ser Pro
Ala Phe Ser Val Arg Lys Arg Lys Asn Ser Lys Lys 20 25 30cga cct ggt
tct agg aac tct tcc ttt gat tac cgg aga gaa gaa ccg 144Arg Pro Gly
Ser Arg Asn Ser Ser Phe Asp Tyr Arg Arg Glu Glu Pro 35 40 45ttg aat
cag gtt ccg ggc cgg atg ttc ttg aat gga tca act gag gtt 192Leu Asn
Gln Val Pro Gly Arg Met Phe Leu Asn Gly Ser Thr Glu Val 50 55 60gct
tgt atc tac act caa caa ggc aag aaa ggg cct aat caa gat gcc 240Ala
Cys Ile Tyr Thr Gln Gln Gly Lys Lys Gly Pro Asn Gln Asp Ala65 70 75
80atg gtt gtt tgg gag aat ttt ggt tcg agg aca gat aca atc ttc tgt
288Met Val Val Trp Glu Asn Phe Gly Ser Arg Thr Asp Thr Ile Phe Cys
85 90 95gga gtg ttt gat gga cat ggt cca tat ggt cat atg gtt gca aag
aga 336Gly Val Phe Asp Gly His Gly Pro Tyr Gly His Met Val Ala Lys
Arg 100 105 110gtc aga gac aat ctt cct ctc aaa tta agt gct tat tgg
gaa gca aaa 384Val Arg Asp Asn Leu Pro Leu Lys Leu Ser Ala Tyr Trp
Glu Ala Lys 115 120 125gta cca gtt gaa ggt gtt ctt aag gca atc acc
acc gac act gtc aat 432Val Pro Val Glu Gly Val Leu Lys Ala Ile Thr
Thr Asp Thr Val Asn 130 135 140aat gta acc aac att aac aac cct gaa
gat gct gct gct gct gct gct 480Asn Val Thr Asn Ile Asn Asn Pro Glu
Asp Ala Ala Ala Ala Ala Ala145 150 155 160ttt gtc act gct gaa gaa
gaa cct agg aca tct gct gac atg gag gag 528Phe Val Thr Ala Glu Glu
Glu Pro Arg Thr Ser Ala Asp Met Glu Glu 165 170 175gag aac aca gaa
acc caa ccg gaa ttg ttt caa acg ctg aaa gag tcg 576Glu Asn Thr Glu
Thr Gln Pro Glu Leu Phe Gln Thr Leu Lys Glu Ser 180 185 190ttt ctt
aag gct ttt aaa gtt atg gat aga gag ctt aaa ttc cat gga 624Phe Leu
Lys Ala Phe Lys Val Met Asp Arg Glu Leu Lys Phe His Gly 195 200
205agt gtt gac tgt ttc tgc agt ggg aca aca gct gta acc ttg atc aag
672Ser Val Asp Cys Phe Cys Ser Gly Thr Thr Ala Val Thr Leu Ile Lys
210 215 220cag ggt cag tat ctc gtt gtt gga aat gtt ggg gat tcc aga
gct gta 720Gln Gly Gln Tyr Leu Val Val Gly Asn Val Gly Asp Ser Arg
Ala Val225 230 235 240atg ggt aca aga gac agt gaa aat act ctt gtc
gct gtt caa cta act 768Met Gly Thr Arg Asp Ser Glu Asn Thr Leu Val
Ala Val Gln Leu Thr 245 250 255gtg gat ctt aag cca aat ctc cca ggt
tgg att atc tta tgt gaa tgt 816Val Asp Leu Lys Pro Asn Leu Pro Gly
Trp Ile Ile Leu Cys Glu Cys 260 265 270atg atg ttg tcc tgt gga tgt
atg atg gat cca tta atc atg ttt att 864Met Met Leu Ser Cys Gly Cys
Met Met Asp Pro Leu Ile Met Phe Ile 275 280 285ggg ttt ttt ttt att
ccc tca att gaa ctt gca gct gag gca gag aga 912Gly Phe Phe Phe Ile
Pro Ser Ile Glu Leu Ala Ala Glu Ala Glu Arg 290 295 300ata aga aag
tgt cga gga cga gtg ttt gct ctt aga gat gaa cct gaa 960Ile Arg Lys
Cys Arg Gly Arg Val Phe Ala Leu Arg Asp Glu Pro Glu305 310 315
320gtt tgt aga gtt tgg ctg cca aat tgt gac tca cct gga ctt gct atg
1008Val Cys Arg Val Trp Leu Pro Asn Cys Asp Ser Pro Gly Leu Ala Met
325 330 335gca cgt gct ttt ggt gac ttt tgc ctt aaa gat ttt ggc cta
atc tct 1056Ala Arg Ala Phe Gly Asp Phe Cys Leu Lys Asp Phe Gly Leu
Ile Ser 340 345 350gtg cct gat gta tct ttc cgt cag tta acc gaa aaa
gat gag ttt ata 1104Val Pro Asp Val Ser Phe Arg Gln Leu Thr Glu Lys
Asp Glu Phe Ile 355 360 365gtg ttg gct aca gat ggg att tgg gat gtt
ctc tca aat gaa gat gta 1152Val Leu Ala Thr Asp Gly Ile Trp Asp Val
Leu Ser Asn Glu Asp Val 370 375 380gtg gcg att gta gct tca gct cca
tcg cgc tcc tct gca gca aga gct 1200Val Ala Ile Val Ala Ser Ala Pro
Ser Arg Ser Ser Ala Ala Arg Ala385 390 395 400tta gtc gag tct gcg
gtc aga gct tgg aga tac aaa tac ccg act tcc 1248Leu Val Glu Ser Ala
Val Arg Ala Trp Arg Tyr Lys Tyr Pro Thr Ser 405 410 415aaa gtc gat
gac tgt gcc gct gtt tgc ttg tat cta gac tcc agc aac 1296Lys Val Asp
Asp Cys Ala Ala Val Cys Leu Tyr Leu Asp Ser Ser Asn 420 425 430aca
aac gcc ata tct aca gct tct tcc atc tcc aaa ctt gaa gat gga 1344Thr
Asn Ala Ile Ser Thr Ala Ser Ser Ile Ser Lys Leu Glu Asp Gly 435 440
445gaa gaa gaa gaa cta aaa gcc acg act gag gat gat gat gca tca gga
1392Glu Glu Glu Glu Leu Lys Ala Thr Thr Glu Asp Asp Asp Ala Ser Gly
450 455 460cca agc ggt cta ggc cgt tcg agt act gtc agg tcg ggg aaa
gag att 1440Pro Ser Gly Leu Gly Arg Ser Ser Thr Val Arg Ser Gly Lys
Glu Ile465 470 475 480gct ctc gac gaa agt gaa act gag aag ctg ata
aaa gaa gcg gat aac 1488Ala Leu Asp Glu Ser Glu Thr Glu Lys Leu Ile
Lys Glu Ala Asp Asn 485 490 495ttg gat tca gaa cct gga aca gag tat
tct gca ctt gaa ggt gtt gca 1536Leu Asp Ser Glu Pro Gly Thr Glu Tyr
Ser Ala Leu Glu Gly Val Ala 500 505 510aga gtt aat aca ctt tta aac
tta cca aga ttt gtg cct gga aag tga 1584Arg Val Asn Thr Leu Leu Asn
Leu Pro Arg Phe Val Pro Gly Lys 515 520 52542527PRTArabidopsis
thaliana 42Met Gly Ser Cys Leu Ser Ala Glu Ser Arg Ser Pro Arg Pro
Gly Ser1 5 10 15Pro Cys Ser Pro Ala Phe Ser Val Arg Lys Arg Lys Asn
Ser Lys Lys 20 25 30Arg Pro Gly Ser Arg Asn Ser Ser Phe Asp Tyr Arg
Arg Glu Glu Pro 35 40 45Leu Asn Gln Val Pro Gly Arg Met Phe Leu Asn
Gly Ser Thr Glu Val 50 55 60Ala Cys Ile Tyr Thr Gln Gln Gly Lys Lys
Gly Pro Asn Gln Asp Ala65 70 75 80Met Val Val Trp Glu Asn Phe Gly
Ser Arg Thr Asp Thr Ile Phe Cys 85 90 95Gly Val Phe Asp Gly His Gly
Pro Tyr Gly His Met Val Ala Lys Arg 100 105 110Val Arg Asp Asn Leu
Pro Leu Lys Leu Ser Ala Tyr Trp Glu Ala Lys 115 120 125Val Pro Val
Glu Gly Val Leu Lys Ala Ile Thr Thr Asp Thr Val Asn 130 135 140Asn
Val Thr Asn Ile Asn Asn Pro Glu Asp Ala Ala Ala Ala Ala Ala145 150
155 160Phe Val Thr Ala Glu Glu Glu Pro Arg Thr Ser Ala Asp Met Glu
Glu 165 170 175Glu Asn Thr Glu Thr Gln Pro Glu Leu Phe Gln Thr Leu
Lys Glu Ser 180 185 190Phe Leu Lys Ala Phe Lys Val Met Asp Arg Glu
Leu Lys Phe His Gly 195 200 205Ser Val Asp Cys Phe Cys Ser Gly Thr
Thr Ala Val Thr Leu Ile Lys 210 215 220Gln Gly Gln Tyr Leu Val Val
Gly Asn Val Gly Asp Ser Arg Ala Val225 230 235 240Met Gly Thr Arg
Asp Ser Glu Asn Thr Leu Val Ala Val Gln Leu Thr 245 250 255Val Asp
Leu Lys Pro Asn Leu Pro Gly Trp Ile Ile Leu Cys Glu Cys 260 265
270Met Met Leu Ser Cys Gly Cys Met Met Asp Pro Leu Ile Met Phe Ile
275 280 285Gly Phe Phe Phe Ile Pro Ser Ile Glu Leu Ala Ala Glu Ala
Glu Arg 290 295 300Ile Arg Lys Cys Arg Gly Arg Val Phe Ala Leu Arg
Asp Glu Pro Glu305 310 315 320Val Cys Arg Val Trp Leu Pro Asn Cys
Asp Ser Pro Gly Leu Ala Met 325 330 335Ala Arg Ala Phe Gly Asp Phe
Cys Leu Lys Asp Phe Gly Leu Ile Ser 340 345 350Val Pro Asp Val Ser
Phe Arg Gln Leu Thr Glu Lys Asp Glu Phe Ile 355 360 365Val Leu Ala
Thr Asp Gly Ile Trp Asp Val Leu Ser Asn Glu Asp Val 370 375 380Val
Ala Ile Val Ala Ser Ala Pro Ser Arg Ser Ser Ala Ala Arg Ala385 390
395 400Leu Val Glu Ser Ala Val Arg Ala Trp Arg Tyr Lys Tyr Pro Thr
Ser 405 410 415Lys Val Asp Asp Cys Ala Ala Val Cys Leu Tyr Leu Asp
Ser Ser Asn 420 425 430Thr Asn Ala Ile Ser Thr Ala Ser Ser Ile
Ser
Lys Leu Glu Asp Gly 435 440 445Glu Glu Glu Glu Leu Lys Ala Thr Thr
Glu Asp Asp Asp Ala Ser Gly 450 455 460Pro Ser Gly Leu Gly Arg Ser
Ser Thr Val Arg Ser Gly Lys Glu Ile465 470 475 480Ala Leu Asp Glu
Ser Glu Thr Glu Lys Leu Ile Lys Glu Ala Asp Asn 485 490 495Leu Asp
Ser Glu Pro Gly Thr Glu Tyr Ser Ala Leu Glu Gly Val Ala 500 505
510Arg Val Asn Thr Leu Leu Asn Leu Pro Arg Phe Val Pro Gly Lys 515
520 5254322DNAArtificialSynthetic DNA 43atggggtcct gtttatctgc ag
224423DNAArtificialSynthetic DNA 44tcactttcca ggcacaaatc ttg
234556DNAArtificialSynthetic DNA 45aattactatt tacaattaca gtcgacatgg
ggtcctgttt atctgcagag agcagg 564656DNAArtificialSynthetic DNA
46agccgggcgg ccgctttact tgtacatcac tttccaggca caaatcttgg taagtt
56471056DNAArabidopsis thalianaCDS(1)..(1056) 47atg gtg ctt tta cca
gcg ttt ttg gac gga tta gcg aga act gta tcg 48Met Val Leu Leu Pro
Ala Phe Leu Asp Gly Leu Ala Arg Thr Val Ser1 5 10 15acg aag aaa ggt
aaa aaa cta tcg gaa gat gaa gat gga ggg aga gag 96Thr Lys Lys Gly
Lys Lys Leu Ser Glu Asp Glu Asp Gly Gly Arg Glu 20 25 30atc gca aaa
tcg atg att aaa gat tcg aag aag aac tcg acg ttg ctc 144Ile Ala Lys
Ser Met Ile Lys Asp Ser Lys Lys Asn Ser Thr Leu Leu 35 40 45ggt act
tca ggc ttt gtt agc tcc gaa agt tct aag agg ttt acc tct 192Gly Thr
Ser Gly Phe Val Ser Ser Glu Ser Ser Lys Arg Phe Thr Ser 50 55 60att
tgt tct aat aga ggt gag aaa gga atc aac caa gat cgt gca att 240Ile
Cys Ser Asn Arg Gly Glu Lys Gly Ile Asn Gln Asp Arg Ala Ile65 70 75
80gtt tgg gag gga ttt ggg tgc caa gaa gac ata aca ttt tgt ggg atg
288Val Trp Glu Gly Phe Gly Cys Gln Glu Asp Ile Thr Phe Cys Gly Met
85 90 95ttt gat gga cat gga cca tgg gga cat gtg ata gcc aaa aga gta
aaa 336Phe Asp Gly His Gly Pro Trp Gly His Val Ile Ala Lys Arg Val
Lys 100 105 110aaa tca ttt cca tct tct ctg ctt tgc caa tgg caa caa
act ctt gcc 384Lys Ser Phe Pro Ser Ser Leu Leu Cys Gln Trp Gln Gln
Thr Leu Ala 115 120 125tcc tta tca tcc tcg ccg gaa tgt tcc tct ccg
ttt gat ctt tgg aag 432Ser Leu Ser Ser Ser Pro Glu Cys Ser Ser Pro
Phe Asp Leu Trp Lys 130 135 140caa gct tgc ctg aaa aca ttc tcc atc
atc gat ctt gat ctc aag atc 480Gln Ala Cys Leu Lys Thr Phe Ser Ile
Ile Asp Leu Asp Leu Lys Ile145 150 155 160agt cct tcc att gat tct
tac tgt agc ggc tgc acc gct ctc acc gct 528Ser Pro Ser Ile Asp Ser
Tyr Cys Ser Gly Cys Thr Ala Leu Thr Ala 165 170 175gtt ttg cag ggt
gat cat ctc gtt ata gca aat gcg ggt gac tca cga 576Val Leu Gln Gly
Asp His Leu Val Ile Ala Asn Ala Gly Asp Ser Arg 180 185 190gca gta
ata gca aca act tct gat gat gga aac ggt tta gtc ccg gtt 624Ala Val
Ile Ala Thr Thr Ser Asp Asp Gly Asn Gly Leu Val Pro Val 195 200
205cag ctc tcg gta gac ttt aaa cca aac att ccc gag gaa gca gaa cgg
672Gln Leu Ser Val Asp Phe Lys Pro Asn Ile Pro Glu Glu Ala Glu Arg
210 215 220ata aaa caa tcg gat gga cga ttg ttc tgc cta gac gat gaa
ccg gga 720Ile Lys Gln Ser Asp Gly Arg Leu Phe Cys Leu Asp Asp Glu
Pro Gly225 230 235 240gtg tac cgg gtg ggt atg cct aat gga gga tca
ctc ggt tta gct gtt 768Val Tyr Arg Val Gly Met Pro Asn Gly Gly Ser
Leu Gly Leu Ala Val 245 250 255tca aga gcg ttt gga gat tac tgc ctt
aaa gac ttc ggt tta gtc tct 816Ser Arg Ala Phe Gly Asp Tyr Cys Leu
Lys Asp Phe Gly Leu Val Ser 260 265 270gaa ccg gaa gta aca tac cga
aag ata acc gac aag gac cag ttt cta 864Glu Pro Glu Val Thr Tyr Arg
Lys Ile Thr Asp Lys Asp Gln Phe Leu 275 280 285atc ttg gcc acc gat
ggg atg tgg gat gtg atg acg aat aat gag gca 912Ile Leu Ala Thr Asp
Gly Met Trp Asp Val Met Thr Asn Asn Glu Ala 290 295 300gtg gag ata
gta aga gga gtt aaa gag aga aga aag agc gca aag aga 960Val Glu Ile
Val Arg Gly Val Lys Glu Arg Arg Lys Ser Ala Lys Arg305 310 315
320ttg gta gag aga gct gtg acg ctt tgg cgt agg aag aga aga agc atc
1008Leu Val Glu Arg Ala Val Thr Leu Trp Arg Arg Lys Arg Arg Ser Ile
325 330 335gcc atg gat gat att tct gtt ctc tgt ctc ttc ttt cgt cct
tct tag 1056Ala Met Asp Asp Ile Ser Val Leu Cys Leu Phe Phe Arg Pro
Ser 340 345 35048351PRTArabidopsis thaliana 48Met Val Leu Leu Pro
Ala Phe Leu Asp Gly Leu Ala Arg Thr Val Ser1 5 10 15Thr Lys Lys Gly
Lys Lys Leu Ser Glu Asp Glu Asp Gly Gly Arg Glu 20 25 30Ile Ala Lys
Ser Met Ile Lys Asp Ser Lys Lys Asn Ser Thr Leu Leu 35 40 45Gly Thr
Ser Gly Phe Val Ser Ser Glu Ser Ser Lys Arg Phe Thr Ser 50 55 60Ile
Cys Ser Asn Arg Gly Glu Lys Gly Ile Asn Gln Asp Arg Ala Ile65 70 75
80Val Trp Glu Gly Phe Gly Cys Gln Glu Asp Ile Thr Phe Cys Gly Met
85 90 95Phe Asp Gly His Gly Pro Trp Gly His Val Ile Ala Lys Arg Val
Lys 100 105 110Lys Ser Phe Pro Ser Ser Leu Leu Cys Gln Trp Gln Gln
Thr Leu Ala 115 120 125Ser Leu Ser Ser Ser Pro Glu Cys Ser Ser Pro
Phe Asp Leu Trp Lys 130 135 140Gln Ala Cys Leu Lys Thr Phe Ser Ile
Ile Asp Leu Asp Leu Lys Ile145 150 155 160Ser Pro Ser Ile Asp Ser
Tyr Cys Ser Gly Cys Thr Ala Leu Thr Ala 165 170 175Val Leu Gln Gly
Asp His Leu Val Ile Ala Asn Ala Gly Asp Ser Arg 180 185 190Ala Val
Ile Ala Thr Thr Ser Asp Asp Gly Asn Gly Leu Val Pro Val 195 200
205Gln Leu Ser Val Asp Phe Lys Pro Asn Ile Pro Glu Glu Ala Glu Arg
210 215 220Ile Lys Gln Ser Asp Gly Arg Leu Phe Cys Leu Asp Asp Glu
Pro Gly225 230 235 240Val Tyr Arg Val Gly Met Pro Asn Gly Gly Ser
Leu Gly Leu Ala Val 245 250 255Ser Arg Ala Phe Gly Asp Tyr Cys Leu
Lys Asp Phe Gly Leu Val Ser 260 265 270Glu Pro Glu Val Thr Tyr Arg
Lys Ile Thr Asp Lys Asp Gln Phe Leu 275 280 285Ile Leu Ala Thr Asp
Gly Met Trp Asp Val Met Thr Asn Asn Glu Ala 290 295 300Val Glu Ile
Val Arg Gly Val Lys Glu Arg Arg Lys Ser Ala Lys Arg305 310 315
320Leu Val Glu Arg Ala Val Thr Leu Trp Arg Arg Lys Arg Arg Ser Ile
325 330 335Ala Met Asp Asp Ile Ser Val Leu Cys Leu Phe Phe Arg Pro
Ser 340 345 3504924DNAArtificialSynthetic DNA 49atggtgcttt
taccagcgtt tttg 245023DNAArtificialSynthetic DNA 50ctaagaagga
cgaaagaaga gac 235160DNAArtificialSynthetic DNA 51aattactatt
tacaattaca gtcgacatgg tgcttttacc agcgtttttg gacggattag
605256DNAArtificialSynthetic DNA 52agccgggcgg ccgctttact tgtacactaa
gaaggacgaa agaagagaca gagaac 565319PRTArtificialA consensus
sequence in a protein phosphatase 53Xaa Cys Gly Xaa Phe Asp Gly His
Gly Xaa Xaa Gly Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa
Val5427PRTArtificialA consensus sequence in a protein phosphatase
54Ser Gly Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Asn Xaa Gly Xaa Ser Arg Ala Xaa Xaa 20
255556PRTArtificialA consensus sequence in a protein phosphatase
55Gly Leu Ala Xaa Xaa Arg Xaa Xaa Gly Asp Xaa Xaa Xaa Lys Xaa Xaa1
5 10 15Gly Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30Asp Xaa Xaa Xaa Xaa Leu Ala Xaa Asp Gly Xaa Trp Asp Xaa
Xaa Xaa 35 40 45Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55563261DNAOryza
sativaCDS(1)..(3261) 56atg ggc tgc tca cct tct aag gtg tgt tca tgt
cca cat tat aag ggc 48Met Gly Cys Ser Pro Ser Lys Val Cys Ser Cys
Pro His Tyr Lys Gly1 5 10 15agt ttg tgc ttc tgt gac tgt gga tgc ttt
gga caa aca cct gac tcc 96Ser Leu Cys Phe Cys Asp Cys Gly Cys Phe
Gly Gln Thr Pro Asp Ser 20 25 30cca aga gag tca agg gga aaa tca aac
cgg gtt agg gga aag aca gat 144Pro Arg Glu Ser Arg Gly Lys Ser Asn
Arg Val Arg Gly Lys Thr Asp 35 40 45tct agt gct tca gat gct tct tct
gat gac cta gag gaa gat gat gat 192Ser Ser Ala Ser Asp Ala Ser Ser
Asp Asp Leu Glu Glu Asp Asp Asp 50 55 60gga ttg cac caa atg aac att
aca agg gac tct aat gtt ggt atc aat 240Gly Leu His Gln Met Asn Ile
Thr Arg Asp Ser Asn Val Gly Ile Asn65 70 75 80cga ctc tca agg gtc
tca tca caa ttt ctt cca cca gaa ggt tca cgt 288Arg Leu Ser Arg Val
Ser Ser Gln Phe Leu Pro Pro Glu Gly Ser Arg 85 90 95aaa gtt cga atc
cca ttg ggg aat tat gac ctg aga tat tcc tac ttg 336Lys Val Arg Ile
Pro Leu Gly Asn Tyr Asp Leu Arg Tyr Ser Tyr Leu 100 105 110tct caa
aga ggc tac tac cca gaa tca ttg gac aag cca aac caa gac 384Ser Gln
Arg Gly Tyr Tyr Pro Glu Ser Leu Asp Lys Pro Asn Gln Asp 115 120
125agt ttt tgt ata cat act cca ttt gga aca agc cct gat gac cat ttc
432Ser Phe Cys Ile His Thr Pro Phe Gly Thr Ser Pro Asp Asp His Phe
130 135 140ttt ggt gta ttt gat ggc cat gga gaa tat gga gct cag tgc
tca caa 480Phe Gly Val Phe Asp Gly His Gly Glu Tyr Gly Ala Gln Cys
Ser Gln145 150 155 160ttt gta aag cga aga cta tgc gaa aac ctg ctc
aga gat gac cgg ttc 528Phe Val Lys Arg Arg Leu Cys Glu Asn Leu Leu
Arg Asp Asp Arg Phe 165 170 175cgt act gat gtt gtt cag gct ctt cat
tct gct ttc ttg gca aca aat 576Arg Thr Asp Val Val Gln Ala Leu His
Ser Ala Phe Leu Ala Thr Asn 180 185 190tca cag ctt cat gca gac agc
tta gat gat tct atg agt ggt act act 624Ser Gln Leu His Ala Asp Ser
Leu Asp Asp Ser Met Ser Gly Thr Thr 195 200 205gca gtc act gtg ctg
gtg agg ggt aaa act att tac att gcg aat acg 672Ala Val Thr Val Leu
Val Arg Gly Lys Thr Ile Tyr Ile Ala Asn Thr 210 215 220ggt gat tca
cgt gct gtt att gcc gaa aaa aga ggg gaa gat gtt gtt 720Gly Asp Ser
Arg Ala Val Ile Ala Glu Lys Arg Gly Glu Asp Val Val225 230 235
240gct gtt gac ctg tcc ata gat caa aca ccc tac agg act gat gag ctt
768Ala Val Asp Leu Ser Ile Asp Gln Thr Pro Tyr Arg Thr Asp Glu Leu
245 250 255gaa agg gtc aag gag tgt ggt gct agg gtt atg acg ttg gat
cag ata 816Glu Arg Val Lys Glu Cys Gly Ala Arg Val Met Thr Leu Asp
Gln Ile 260 265 270gag ggg cta aag aac cca gat gta cag tgt tgg ggc
acc gag gaa agt 864Glu Gly Leu Lys Asn Pro Asp Val Gln Cys Trp Gly
Thr Glu Glu Ser 275 280 285gat gac ggt gat cct cca agg ttg tgg gtg
caa aat ggc atg tat cca 912Asp Asp Gly Asp Pro Pro Arg Leu Trp Val
Gln Asn Gly Met Tyr Pro 290 295 300gga act gct ttt act cgc agc att
gga gat tct gtc gct gaa tct atc 960Gly Thr Ala Phe Thr Arg Ser Ile
Gly Asp Ser Val Ala Glu Ser Ile305 310 315 320ggt gtt gtc gct aat
cct gag att ttt atc ctg gag ctc aat gcc aac 1008Gly Val Val Ala Asn
Pro Glu Ile Phe Ile Leu Glu Leu Asn Ala Asn 325 330 335cat cca ttc
ttt gtt ctt gct agt gat gga gtt ttt gag ttt ctt tct 1056His Pro Phe
Phe Val Leu Ala Ser Asp Gly Val Phe Glu Phe Leu Ser 340 345 350agt
caa act gtt gtc gac atg att gct aaa tac aag gat cct cgt gat 1104Ser
Gln Thr Val Val Asp Met Ile Ala Lys Tyr Lys Asp Pro Arg Asp 355 360
365gcg tgc gct gca att gtt gct gaa tcc tat cgc ctc tgg cta cag tat
1152Ala Cys Ala Ala Ile Val Ala Glu Ser Tyr Arg Leu Trp Leu Gln Tyr
370 375 380gaa act cgt aca gat gac att aca ata ata gtt gtt cat att
aac ggg 1200Glu Thr Arg Thr Asp Asp Ile Thr Ile Ile Val Val His Ile
Asn Gly385 390 395 400tta act gat atg gaa tgt act caa act gta atg
aaa gta tct tta caa 1248Leu Thr Asp Met Glu Cys Thr Gln Thr Val Met
Lys Val Ser Leu Gln 405 410 415cct tcc caa caa gtc gta gaa ttg gta
ggc tca gaa tca cca tcg aca 1296Pro Ser Gln Gln Val Val Glu Leu Val
Gly Ser Glu Ser Pro Ser Thr 420 425 430ata agt ttg aat ccc aag aac
cag cgt tcc agg caa gat cta tca cgt 1344Ile Ser Leu Asn Pro Lys Asn
Gln Arg Ser Arg Gln Asp Leu Ser Arg 435 440 445gct cgg ctg aga gca
ctt gaa agt tcc ctg gaa aat ggt cga cta tgg 1392Ala Arg Leu Arg Ala
Leu Glu Ser Ser Leu Glu Asn Gly Arg Leu Trp 450 455 460gtc cct cca
tcc cca tcg cat cgg aag aca tgg gaa gag caa gca cat 1440Val Pro Pro
Ser Pro Ser His Arg Lys Thr Trp Glu Glu Gln Ala His465 470 475
480att gag cga ata cta cac gac cat ttc ctc ttc agg aag ctc act gac
1488Ile Glu Arg Ile Leu His Asp His Phe Leu Phe Arg Lys Leu Thr Asp
485 490 495tca cag tgc cat gtt tta ctt gat tgc atg caa aga gtt gag
gtg aaa 1536Ser Gln Cys His Val Leu Leu Asp Cys Met Gln Arg Val Glu
Val Lys 500 505 510gct ggg gat ata gtg gtg cag cag ggc ggt gaa ggc
gag tgc ttc tat 1584Ala Gly Asp Ile Val Val Gln Gln Gly Gly Glu Gly
Glu Cys Phe Tyr 515 520 525gta gtt ggg agt ggt gag ttt gaa gtg cta
gcc att cag gaa gaa gat 1632Val Val Gly Ser Gly Glu Phe Glu Val Leu
Ala Ile Gln Glu Glu Asp 530 535 540gga aag gaa gtt aca aag gtt cta
cat cgg tat act gct gac aaa cta 1680Gly Lys Glu Val Thr Lys Val Leu
His Arg Tyr Thr Ala Asp Lys Leu545 550 555 560tct tct ttt ggg gag
cta gca cta atg tat aat aaa cca ctt caa gct 1728Ser Ser Phe Gly Glu
Leu Ala Leu Met Tyr Asn Lys Pro Leu Gln Ala 565 570 575tca gtc cgt
gct gtg act act gga act tta tgg gct cta aag cga gag 1776Ser Val Arg
Ala Val Thr Thr Gly Thr Leu Trp Ala Leu Lys Arg Glu 580 585 590gat
ttt cgg gga att ctg atg tca gag ttt tca aat ata cca tca tta 1824Asp
Phe Arg Gly Ile Leu Met Ser Glu Phe Ser Asn Ile Pro Ser Leu 595 600
605aag ttg ctc cga tca gtg gag ctg ttt acg aga ttg aca atg ctt caa
1872Lys Leu Leu Arg Ser Val Glu Leu Phe Thr Arg Leu Thr Met Leu Gln
610 615 620cta agt caa ctt gct gat tct ctt gtt gaa gta act ttt ggg
gat ggt 1920Leu Ser Gln Leu Ala Asp Ser Leu Val Glu Val Thr Phe Gly
Asp Gly625 630 635 640caa atg ata gta gac aag aat gat gat gca tct
tcc ttg tat att att 1968Gln Met Ile Val Asp Lys Asn Asp Asp Ala Ser
Ser Leu Tyr Ile Ile 645 650 655caa aga ggt cgt gtg aaa ctt aaa ttg
gct gca gat cag gta aat tca 2016Gln Arg Gly Arg Val Lys Leu Lys Leu
Ala Ala Asp Gln Val Asn Ser 660 665 670gat gcc tgg gat ctt ctt agt
tct caa aca aag gtg gcc caa tca agt 2064Asp Ala Trp Asp Leu Leu Ser
Ser Gln Thr Lys Val Ala Gln Ser Ser 675 680 685cga gaa gat ggt aat
tac gtg ttt gag ata gat gaa ggg gga cac ttt 2112Arg Glu Asp Gly Asn
Tyr Val Phe Glu Ile Asp Glu Gly Gly His Phe 690 695 700gga gag tgg
gct ctc ttt ggg gag aca att gct ttt act gct atg tca 2160Gly Glu Trp
Ala Leu Phe Gly Glu Thr Ile Ala Phe Thr Ala Met Ser705 710 715
720gtt ggt gat gtg act tgt tct act att gca aag gag aag ttt gac tca
2208Val Gly Asp Val Thr Cys Ser Thr Ile Ala Lys Glu Lys Phe Asp Ser
725 730 735att att ggg ccc ttg cca aaa gtt tcc cag tct gat tcc aag
ctc aaa 2256Ile Ile Gly Pro Leu Pro Lys Val Ser Gln Ser Asp Ser Lys
Leu Lys 740 745 750gat tcc ttg gtt cct aaa
ggg cat ggt gca gat gat agt tcc ttc agg 2304Asp Ser Leu Val Pro Lys
Gly His Gly Ala Asp Asp Ser Ser Phe Arg 755 760 765aag gcg cag cta
tct gat ttg gaa tgg aaa atg tgc ata tat gcc gct 2352Lys Ala Gln Leu
Ser Asp Leu Glu Trp Lys Met Cys Ile Tyr Ala Ala 770 775 780gat tgc
agt gag att ggt ctt gtc caa cta aga ggt tct gac aag atc 2400Asp Cys
Ser Glu Ile Gly Leu Val Gln Leu Arg Gly Ser Asp Lys Ile785 790 795
800aaa agc tta aag agg ttt tac atc aag aga gta aaa gac ctt cat aag
2448Lys Ser Leu Lys Arg Phe Tyr Ile Lys Arg Val Lys Asp Leu His Lys
805 810 815gaa aaa cac gta ttt gat gag aag gat ctc atg aaa tct ttg
agc caa 2496Glu Lys His Val Phe Asp Glu Lys Asp Leu Met Lys Ser Leu
Ser Gln 820 825 830tca act tgt gtg cca gaa gtt cta tgt act tgc gct
gat caa tcc tac 2544Ser Thr Cys Val Pro Glu Val Leu Cys Thr Cys Ala
Asp Gln Ser Tyr 835 840 845cta gga ata ctg ctg aat tgt tgc ctt tgt
tgc tca ctg gct tca ata 2592Leu Gly Ile Leu Leu Asn Cys Cys Leu Cys
Cys Ser Leu Ala Ser Ile 850 855 860ctt cat gca cca cta aat gag tcg
tct gca cga ttc tat gca gcc tct 2640Leu His Ala Pro Leu Asn Glu Ser
Ser Ala Arg Phe Tyr Ala Ala Ser865 870 875 880gtc gtc gta gcg cta
gaa aat ctc cat cag agg tcc att ctt tac aga 2688Val Val Val Ala Leu
Glu Asn Leu His Gln Arg Ser Ile Leu Tyr Arg 885 890 895ggt gtt tct
gca gac att ctt atg gtc gac cga tca ggg cat ctt caa 2736Gly Val Ser
Ala Asp Ile Leu Met Val Asp Arg Ser Gly His Leu Gln 900 905 910cta
gtt gac ttc agg ttt gca aag aag ttg caa ggt gaa agg act tac 2784Leu
Val Asp Phe Arg Phe Ala Lys Lys Leu Gln Gly Glu Arg Thr Tyr 915 920
925aca ata tgt ggg att gcc gac tct cta gct cca gag ata gtt ctt ggt
2832Thr Ile Cys Gly Ile Ala Asp Ser Leu Ala Pro Glu Ile Val Leu Gly
930 935 940agg ggc cat gga ttt tct gct gac tgg tgg gcg ctg gga gtg
ttg att 2880Arg Gly His Gly Phe Ser Ala Asp Trp Trp Ala Leu Gly Val
Leu Ile945 950 955 960tat ttc atg ctg caa tca gac atg cca ttt ggc
tct tgg agg gag agt 2928Tyr Phe Met Leu Gln Ser Asp Met Pro Phe Gly
Ser Trp Arg Glu Ser 965 970 975gaa ctg gaa cct ttt gca aag att gcc
aag ggt cac ctt gtc atg cca 2976Glu Leu Glu Pro Phe Ala Lys Ile Ala
Lys Gly His Leu Val Met Pro 980 985 990tca aca ttc agc atc gaa gtt
gtt gac ctt att aca aag cta ctc gag 3024Ser Thr Phe Ser Ile Glu Val
Val Asp Leu Ile Thr Lys Leu Leu Glu 995 1000 1005gta aac gaa aat
gcg cgc ctt ggg gcc aag gga gcg gaa tct gtg 3069Val Asn Glu Asn Ala
Arg Leu Gly Ala Lys Gly Ala Glu Ser Val 1010 1015 1020aaa aga cac
ccc tgg ttt gat ggc att gac tgg aaa caa ata gca 3114Lys Arg His Pro
Trp Phe Asp Gly Ile Asp Trp Lys Gln Ile Ala 1025 1030 1035gat ggt
act tat aca gta ccc caa gaa atc acc gat cgt gtc gac 3159Asp Gly Thr
Tyr Thr Val Pro Gln Glu Ile Thr Asp Arg Val Asp 1040 1045 1050agc
tat gta gaa act ctt aca gag gac ttg aca gca tcc cct tcc 3204Ser Tyr
Val Glu Thr Leu Thr Glu Asp Leu Thr Ala Ser Pro Ser 1055 1060
1065atg cca agt gaa gaa aca gct gat cag gct gct cca gaa tgg atc
3249Met Pro Ser Glu Glu Thr Ala Asp Gln Ala Ala Pro Glu Trp Ile
1070 1075 1080cag gat tgg tga 3261Gln Asp Trp 1085571086PRTOryza
sativa 57Met Gly Cys Ser Pro Ser Lys Val Cys Ser Cys Pro His Tyr
Lys Gly1 5 10 15Ser Leu Cys Phe Cys Asp Cys Gly Cys Phe Gly Gln Thr
Pro Asp Ser 20 25 30Pro Arg Glu Ser Arg Gly Lys Ser Asn Arg Val Arg
Gly Lys Thr Asp 35 40 45Ser Ser Ala Ser Asp Ala Ser Ser Asp Asp Leu
Glu Glu Asp Asp Asp 50 55 60Gly Leu His Gln Met Asn Ile Thr Arg Asp
Ser Asn Val Gly Ile Asn65 70 75 80Arg Leu Ser Arg Val Ser Ser Gln
Phe Leu Pro Pro Glu Gly Ser Arg 85 90 95Lys Val Arg Ile Pro Leu Gly
Asn Tyr Asp Leu Arg Tyr Ser Tyr Leu 100 105 110Ser Gln Arg Gly Tyr
Tyr Pro Glu Ser Leu Asp Lys Pro Asn Gln Asp 115 120 125Ser Phe Cys
Ile His Thr Pro Phe Gly Thr Ser Pro Asp Asp His Phe 130 135 140Phe
Gly Val Phe Asp Gly His Gly Glu Tyr Gly Ala Gln Cys Ser Gln145 150
155 160Phe Val Lys Arg Arg Leu Cys Glu Asn Leu Leu Arg Asp Asp Arg
Phe 165 170 175Arg Thr Asp Val Val Gln Ala Leu His Ser Ala Phe Leu
Ala Thr Asn 180 185 190Ser Gln Leu His Ala Asp Ser Leu Asp Asp Ser
Met Ser Gly Thr Thr 195 200 205Ala Val Thr Val Leu Val Arg Gly Lys
Thr Ile Tyr Ile Ala Asn Thr 210 215 220Gly Asp Ser Arg Ala Val Ile
Ala Glu Lys Arg Gly Glu Asp Val Val225 230 235 240Ala Val Asp Leu
Ser Ile Asp Gln Thr Pro Tyr Arg Thr Asp Glu Leu 245 250 255Glu Arg
Val Lys Glu Cys Gly Ala Arg Val Met Thr Leu Asp Gln Ile 260 265
270Glu Gly Leu Lys Asn Pro Asp Val Gln Cys Trp Gly Thr Glu Glu Ser
275 280 285Asp Asp Gly Asp Pro Pro Arg Leu Trp Val Gln Asn Gly Met
Tyr Pro 290 295 300Gly Thr Ala Phe Thr Arg Ser Ile Gly Asp Ser Val
Ala Glu Ser Ile305 310 315 320Gly Val Val Ala Asn Pro Glu Ile Phe
Ile Leu Glu Leu Asn Ala Asn 325 330 335His Pro Phe Phe Val Leu Ala
Ser Asp Gly Val Phe Glu Phe Leu Ser 340 345 350Ser Gln Thr Val Val
Asp Met Ile Ala Lys Tyr Lys Asp Pro Arg Asp 355 360 365Ala Cys Ala
Ala Ile Val Ala Glu Ser Tyr Arg Leu Trp Leu Gln Tyr 370 375 380Glu
Thr Arg Thr Asp Asp Ile Thr Ile Ile Val Val His Ile Asn Gly385 390
395 400Leu Thr Asp Met Glu Cys Thr Gln Thr Val Met Lys Val Ser Leu
Gln 405 410 415Pro Ser Gln Gln Val Val Glu Leu Val Gly Ser Glu Ser
Pro Ser Thr 420 425 430Ile Ser Leu Asn Pro Lys Asn Gln Arg Ser Arg
Gln Asp Leu Ser Arg 435 440 445Ala Arg Leu Arg Ala Leu Glu Ser Ser
Leu Glu Asn Gly Arg Leu Trp 450 455 460Val Pro Pro Ser Pro Ser His
Arg Lys Thr Trp Glu Glu Gln Ala His465 470 475 480Ile Glu Arg Ile
Leu His Asp His Phe Leu Phe Arg Lys Leu Thr Asp 485 490 495Ser Gln
Cys His Val Leu Leu Asp Cys Met Gln Arg Val Glu Val Lys 500 505
510Ala Gly Asp Ile Val Val Gln Gln Gly Gly Glu Gly Glu Cys Phe Tyr
515 520 525Val Val Gly Ser Gly Glu Phe Glu Val Leu Ala Ile Gln Glu
Glu Asp 530 535 540Gly Lys Glu Val Thr Lys Val Leu His Arg Tyr Thr
Ala Asp Lys Leu545 550 555 560Ser Ser Phe Gly Glu Leu Ala Leu Met
Tyr Asn Lys Pro Leu Gln Ala 565 570 575Ser Val Arg Ala Val Thr Thr
Gly Thr Leu Trp Ala Leu Lys Arg Glu 580 585 590Asp Phe Arg Gly Ile
Leu Met Ser Glu Phe Ser Asn Ile Pro Ser Leu 595 600 605Lys Leu Leu
Arg Ser Val Glu Leu Phe Thr Arg Leu Thr Met Leu Gln 610 615 620Leu
Ser Gln Leu Ala Asp Ser Leu Val Glu Val Thr Phe Gly Asp Gly625 630
635 640Gln Met Ile Val Asp Lys Asn Asp Asp Ala Ser Ser Leu Tyr Ile
Ile 645 650 655Gln Arg Gly Arg Val Lys Leu Lys Leu Ala Ala Asp Gln
Val Asn Ser 660 665 670Asp Ala Trp Asp Leu Leu Ser Ser Gln Thr Lys
Val Ala Gln Ser Ser 675 680 685Arg Glu Asp Gly Asn Tyr Val Phe Glu
Ile Asp Glu Gly Gly His Phe 690 695 700Gly Glu Trp Ala Leu Phe Gly
Glu Thr Ile Ala Phe Thr Ala Met Ser705 710 715 720Val Gly Asp Val
Thr Cys Ser Thr Ile Ala Lys Glu Lys Phe Asp Ser 725 730 735Ile Ile
Gly Pro Leu Pro Lys Val Ser Gln Ser Asp Ser Lys Leu Lys 740 745
750Asp Ser Leu Val Pro Lys Gly His Gly Ala Asp Asp Ser Ser Phe Arg
755 760 765Lys Ala Gln Leu Ser Asp Leu Glu Trp Lys Met Cys Ile Tyr
Ala Ala 770 775 780Asp Cys Ser Glu Ile Gly Leu Val Gln Leu Arg Gly
Ser Asp Lys Ile785 790 795 800Lys Ser Leu Lys Arg Phe Tyr Ile Lys
Arg Val Lys Asp Leu His Lys 805 810 815Glu Lys His Val Phe Asp Glu
Lys Asp Leu Met Lys Ser Leu Ser Gln 820 825 830Ser Thr Cys Val Pro
Glu Val Leu Cys Thr Cys Ala Asp Gln Ser Tyr 835 840 845Leu Gly Ile
Leu Leu Asn Cys Cys Leu Cys Cys Ser Leu Ala Ser Ile 850 855 860Leu
His Ala Pro Leu Asn Glu Ser Ser Ala Arg Phe Tyr Ala Ala Ser865 870
875 880Val Val Val Ala Leu Glu Asn Leu His Gln Arg Ser Ile Leu Tyr
Arg 885 890 895Gly Val Ser Ala Asp Ile Leu Met Val Asp Arg Ser Gly
His Leu Gln 900 905 910Leu Val Asp Phe Arg Phe Ala Lys Lys Leu Gln
Gly Glu Arg Thr Tyr 915 920 925Thr Ile Cys Gly Ile Ala Asp Ser Leu
Ala Pro Glu Ile Val Leu Gly 930 935 940Arg Gly His Gly Phe Ser Ala
Asp Trp Trp Ala Leu Gly Val Leu Ile945 950 955 960Tyr Phe Met Leu
Gln Ser Asp Met Pro Phe Gly Ser Trp Arg Glu Ser 965 970 975Glu Leu
Glu Pro Phe Ala Lys Ile Ala Lys Gly His Leu Val Met Pro 980 985
990Ser Thr Phe Ser Ile Glu Val Val Asp Leu Ile Thr Lys Leu Leu Glu
995 1000 1005Val Asn Glu Asn Ala Arg Leu Gly Ala Lys Gly Ala Glu
Ser Val 1010 1015 1020Lys Arg His Pro Trp Phe Asp Gly Ile Asp Trp
Lys Gln Ile Ala 1025 1030 1035Asp Gly Thr Tyr Thr Val Pro Gln Glu
Ile Thr Asp Arg Val Asp 1040 1045 1050Ser Tyr Val Glu Thr Leu Thr
Glu Asp Leu Thr Ala Ser Pro Ser 1055 1060 1065Met Pro Ser Glu Glu
Thr Ala Asp Gln Ala Ala Pro Glu Trp Ile 1070 1075 1080Gln Asp Trp
108558484DNAtobacco mosaic virus 58aagcttgcat gcctgcaggc tctagaggat
cccccctcag aagaccagag ggctattgag 60acttttcaac aaagggtaat atcgggaaac
ctcctcggat tccattgccc agctatctgt 120cacttcatcg aaaggacagt
agaaaaggaa ggtggctcct acaaatgcca tcattgcgat 180aaaggaaagg
ctatcgttca agatgcctct accgacagtg gtcccaaaga tggaccccca
240cccacgagga acatcgtgga aaaagaagac gttccaacca cgtcttcaaa
gcaagtggat 300tgatgtgata tctccactga cgtaagggat gacgcacaat
cccactatcc ttcgcaagac 360ccttcctcta tataaggaag ttcatttcat
ttggagagga caggcttctt gagatccttc 420aacaattacc aacaacaaca
aacaacaaac aacattacaa ttactattta caattacagt 480cgac 484
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References