U.S. patent application number 13/286808 was filed with the patent office on 2012-04-05 for rice blast susceptibility gene pi21, resistance gene pi21, and uses thereof.
This patent application is currently assigned to National Institute of Agrobiological Sciences. Invention is credited to Shuichi Fukuoka, Makoto Kawase, Kazutoshi Okuno.
Application Number | 20120083595 13/286808 |
Document ID | / |
Family ID | 37595134 |
Filed Date | 2012-04-05 |
United States Patent
Application |
20120083595 |
Kind Code |
A1 |
Fukuoka; Shuichi ; et
al. |
April 5, 2012 |
RICE BLAST SUSCEPTIBILITY GENE Pi21, RESISTANCE GENE pi21, AND USES
THEREOF
Abstract
The present inventors succeeded in isolating the rice field
resistance gene pi21 by linkage analysis, and found that field
resistance to blast in plants could be modified by introducing or
controlling the expression of the gene. Thus, it became possible to
efficiently confer plants with field resistance. It also became
possible to select, at an early stage, rice plants having field
resistance to blast. Moreover, by changing the tissue specificity
of expression and the expression level of the gene involved in
field resistance, varieties having resistance as well as high
practical use can be grown.
Inventors: |
Fukuoka; Shuichi; (Ibaraki,
JP) ; Okuno; Kazutoshi; (Ibaraki, JP) ;
Kawase; Makoto; (Ibaraki, JP) |
Assignee: |
National Institute of
Agrobiological Sciences
Tsukuba-shi
JP
|
Family ID: |
37595134 |
Appl. No.: |
13/286808 |
Filed: |
November 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12881685 |
Sep 14, 2010 |
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13286808 |
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11922994 |
Apr 24, 2008 |
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PCT/JP2006/311341 |
Jun 6, 2006 |
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12881685 |
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Current U.S.
Class: |
536/23.6 ;
536/24.33 |
Current CPC
Class: |
C12Q 2600/13 20130101;
C12Q 1/6895 20130101; C12Q 1/6895 20130101; C12N 15/8282 20130101;
C12Q 2600/158 20130101; C12Q 2531/113 20130101; C07K 14/415
20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
536/23.6 ;
536/24.33 |
International
Class: |
C12N 15/113 20100101
C12N015/113; C07H 21/04 20060101 C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 28, 2005 |
JP |
2005-187867 |
Claims
1. A set of nucleic acids capable of amplifying all or a part of
the nucleotide sequence of SEQ ID NO: 1, 4, or 20.
2. The set of claim 1, comprising at least one of the following
sets of nucleic acids selected from the group consisting of: (a) a
nucleic acid comprising the nucleotide sequence of SEQ ID NO: 8,
and a nucleic acid comprising the nucleotide sequence of SEQ ID NO:
9; (b) a nucleic acid comprising the nucleotide sequence of SEQ ID
NO: 16, and a nucleic acid comprising the nucleotide sequence of
SEQ ID NO: 17; and (c) a nucleic acid comprising the nucleotide
sequence of SEQ ID NO: 26, and a nucleic acid comprising the
nucleotide sequence of SEQ ID NO: 27.
3. An isolated nucleic acid comprising the nucleotide sequence of
SEQ ID NO: 7, 10, 18, 19, 23, or 25.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 12/881,685, filed Sep. 14, 2010, which is a
divisional application of U.S. application Ser. No. 11/922,994,
filed Apr. 24, 2008, which is a 35 U.S.C. 371 national stage filing
of International Application No. PCT/JP2006/0311341, filed Jun. 6,
2006, which claims priority to Japanese Application No.
2005-187867, filed Jun. 28, 2005. The entire contents of each of
these applications are hereby incorporated by reference herein.
TECHNICAL FIELD
[0002] The present invention relates to pi21, a gene conferring
rice with blast field resistance, and methods for modifying field
resistance to blast in plants by using the gene.
BACKGROUND ART
[0003] Resistance of rice to blast fungi is classified into two
types: true resistance and field resistance (Non-patent Document
1). The former is based on hypersensitive reactions, and is a very
effective and qualitative resistance highly specific to race. It
has been known by experience that a variety introduced with a
single resistance gene loses its effect in a few years due to the
appearance of fungi compatible with the gene. On the other hand,
field resistance is defined as the difference of resistance among
varieties that is observed under conditions where true resistance
is not functioning. Although the effect of field resistance is
smaller compared to true resistance, it is practically useful
because it has low race specificity and can confer continuous
resistance to varieties.
[0004] Thirty or more kinds of genes associated with true
resistance are known, and of these, Pib and Pita genes have been
isolated (Non-patent Document 2). It has been found that these
genes are NBS-LRR class genes having nucleotide binding sites (NBS)
and leucine-rich repeats (LRRs), with structures similar to
previously reported plant disease resistance genes. Like other
disease resistance genes, products of plant resistance genes are
considered to have a receptor function, directly or indirectly
recognizing products of nonpathogenic genes in pathogens
corresponding to the diseases. It has been actually revealed that
Pita physically and directly binds to a nonpathogenic gene
product.
[0005] As for field resistance, Japanese upland rice varieties are
known to have excellent traits, and chromosomal positions of
multiple gene loci involved in field resistance have been
identified (Non-patent Document 3). However, the structure and
expression mechanisms of the genes have not been elucidated, and
thus field resistance cannot yet be efficiently used for breeding
selection compared to true resistance. Multiple chromosome regions
in the West Africa upland rice variety Moroberekan have been
reported to play a role in incomplete resistance, which is a
concept similar to field resistance (Non-patent Document 4);
however, no genes have been identified. [0006] [Patent Document 1]
Japanese Patent Application Kokai Publication No. (JP-A) 2000-93028
(unexamined, published Japanese patent application) [0007] [Patent
Document 2] JP-A 2000-342262 [0008] [Patent Document 3]
(Granted/Registered) Japanese Patent No.3376453 (P3376453) [0009]
[Patent Document 4] JP-A 2003-88379 (P2003-88379A) [0010] [Patent
Document 5] JP-A 2003-199577 (P2003-199577A) [0011] [Patent
Document 6] JP-A 2003-199448 (P2003-199448A) [0012] [Patent
Document 7] JP-A 2004-329215 (P2004-329215A) [0013] [Non-patent
Document 1] Rice Blast and Breeding for its resistance. Kousaka and
Yamazaki eds., p175-186, 1980, Hakuyusha [0014] [Non-patent
Document 2] Wang et al., Plant J 19:55-64, 1999; Bryan et al. Plant
Cell. 12: 2033-46, 2000 [0015] [Non-patent Document 3] Fukuoka and
Okuno, Theor Appl Genet. 03:185-190, 2001 [0016] [Non-patent
Document 4] Wang et al., Genet 136:1421-1434, 1994
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0017] The present invention was accomplished under such
circumstances. The problems to be solved by the present invention
are to isolate and identify genes involved in field resistance to
blast by map-based cloning, and to provide methods for modifying
blast field resistance in plants using the genes.
Means for Solving the Problems
[0018] The present invention relates to genes controlling blast
resistance in plants. The allele pi21 of the Pi21 gene was known to
confer rice (Oryza sativa L.) with field resistance to blast and to
exist at some position in the vast region of rice chromosome 4. The
present inventors aimed to elucidate the existence region and to
isolate the gene as a single gene.
[0019] First, the present inventors performed a detailed linkage
analysis of the pi21 region using a large-scale segregating
population indispensable for map-based cloning, in order to create
a genetic map of the pi21 region. The inventors obtained a
backcross population by continuously backcrossing a paddy rice
variety, Nipponbare or Aichi Asahi, comprising a susceptibility
allele Pi21 which does not suppress blotch progression, with a
Japanese upland rice variety, Owarihatamochi comprising a
resistance allele pi21 which suppresses blotch progression. When a
linkage analysis with RFLP markers was performed for the obtained
backcross population, it was confirmed that the pi21 gene locus is
located between the RFLP markers G271 and G317.
[0020] Next, using the RFLP markers RA3591 and 13S1, which are
located on each side of the pi21 locus, the present inventors
selected organisms with chromosomal recombination near the pi21
locus in order to create a more accurate genetic map of the pi21
region. The present inventors also selected organisms with
chromosomal recombination near the pi21 locus, by searching an F2
population obtained by crossing a line having the resistance allele
from Owarihatamochi with a line having the genetic background of a
Japanese paddy rice variety and the susceptibility allele from the
Indian paddy rice variety Kasalath. As a result of creating a
detailed linkage map using these organisms and the produced DNA
markers, the pi21 gene locus turned out to be located in the
genomic region of about 25 kb sandwiched between the SSCP marker
Pa102484 and the SNP marker P702D3.sub.--#12. Further, the
nucleotide sequence of PAC clone P702D03 that was considered to
include the pi21 gene was determined. Moreover, by analyzing the
nucleotide sequence of the 25-kb candidate genomic region in the
resistant variety Owarihatamochi and the susceptibility varieties
Aichi Asahi and Kasalath, it was found that the pi21 gene is
located in the genomic region of about 1.8 kb sandwiched between
the SNP markers P702D03.sub.--#38 and P702D03.sub.--#80.
[0021] Thus, the present inventors designed primers that could
amplify a corresponding portion using the already obtained
nucleotide sequence information of Nipponbare, and then compared
the nucleotide sequences of the genomic PCR and RT-PCR products
between the susceptibility varieties Nipponbare and Aichi Asahi and
the resistant variety Owarihatamochi. As a result, it was found
that there are two DNA mutations in the exon region of the gene in
the resistant variety compared to the susceptibility varieties. It
was demonstrated that in contrast to the susceptibility varieties,
the resistant variety has deletions of 7 amino acids and 16 amino
acids, and that these mutations are related to blotch progression
caused by blast infection. That is, the present invention relates
to the pi21 gene that controls plant resistance to blast, and
specifically provides the following inventions: [0022] [1] a DNA of
any one of the following (a) to (h):
[0023] (a) a DNA that encodes a protein comprising the amino acid
sequence of SEQ ID NO: 3 or 22,
[0024] (b) a DNA comprising a coding region of the nucleotide
sequence of SEQ ID NO: 1, 2, 20, or 21,
[0025] (c) a DNA encoding a protein which comprises an amino acid
sequence with a substitution, deletion, addition, and/or insertion
of one or more amino acids in the amino acid sequence of SEQ ID NO:
3 or 22, and which has a function equivalent to that of a protein
comprising the amino acid sequence of SEQ ID NO: 3 or 22,
[0026] (d) a DNA which hybridizes under stringent conditions to a
DNA comprising the nucleotide sequence of SEQ ID NO: 1, 2, 20, or
21, and which encodes a protein having a function equivalent to
that of a protein comprising the amino acid sequence of SEQ ID NO:
3 or 22,
[0027] (e) a DNA that encodes a protein comprising the amino acid
sequence of SEQ ID NO: 6,
[0028] (f) a DNA comprising a coding region of the nucleotide
sequence of SEQ ID NO: 4 or 5,
[0029] (g) a DNA encoding a protein which comprises an amino acid
sequence with a substitution, deletion, addition, and/or insertion
of one or more amino acids in the amino acid sequence of SEQ ID NO:
6, and which has a function equivalent to that of a protein
comprising the amino acid sequence of SEQ ID NO: 6, and
[0030] (h) a DNA which hybridizes under stringent conditions to a
DNA comprising the nucleotide sequence of SEQ ID NO: 4 or 5, and
which encodes a protein having a function equivalent to that of a
protein comprising the amino acid sequence of SEQ ID NO: 6; [0031]
[2] a DNA of any one of the following (i) to (iv), having an
ability to confer plants with field resistance to blast:
[0032] (i) a DNA that encodes an RNA complementary to a
transcription product of the DNA of any one of (a) to (d) in
[1],
[0033] (ii) a DNA that encodes an RNA having the ribozyme activity
to specifically cleave a transcription product of the DNA of any
one of (a) to (d) in [1],
[0034] (iii) a DNA that encodes an RNA which inhibits expression of
the DNA of any one of (a) to (d) in [1] by a co-suppression effect,
and
[0035] (iv) a DNA that encodes an RNA having RNAi activity to
specifically cleave a transcription product of the DNA of any one
of (a) to (d) in [1]; [0036] [3] the DNA of [2], wherein the plant
is rice, wheat, barley, oat, corn, Job's tears, Italian ryegrass,
perennial ryegrass, timothy, meadow fescue, millet, foxtail millet,
or sugarcane; [0037] [4] a vector comprising the DNA of any one of
[1] to [3]; [0038] [5] a transformed cell that maintains the DNA of
any one of [1] to [3] in an expressible state; [0039] [6] a
transformed plant cell into which the DNA of any one of (a) to (d)
in [1] has been introduced; [0040] [7] a transformed plant cell
into which the DNA of [2] or [3] has been introduced; [0041] [8]
the transformed plant cell of [6] or [7], wherein the plant is
rice, wheat, barley, oat, corn, Job's tears, Italian ryegrass,
perennial ryegrass, timothy, meadow fescue, millet, foxtail millet,
or sugarcane; [0042] [9] a transformed plant comprising the
transformed cell of any one of [6] to [8]; [0043] [10] a
transformed plant that is a progeny or clone of the transformed
plant of [8]; [0044] [11] a propagation material of the transformed
plant of [9] or [10]; [0045] [12] a method for producing the
transformed plant of [9] or [10], which comprises the step of
introducing into a plant cell the DNA of any one of (a) to (d) in
[1] or the DNA of [2] or [3], and then regenerating a plant from
the plant cell; [0046] [13] a method for conferring a plant with
field resistance to blast, which comprises the step of expressing
the DNA of [2] or [3] in a cell of the plant; [0047] [14] the
method of [13], wherein the plant is rice, wheat, barley, oat,
corn, Job's tears, Italian ryegrass, perennial ryegrass, timothy,
meadow fescue, millet, foxtail millet, or sugarcane; [0048] [15] a
protein encoded by the DNA of any one of (a) to (d) in [1]; [0049]
[16] a method for producing the protein of [15], which comprises
the step of culturing a transformed cell comprising a vector that
comprises the DNA of any one of (a) to (d) in [1], and then
collecting a recombinant protein from the cell or its culture
supernatant; [0050] [17] an antibody that binds to the protein of
[15]; [0051] [18] a DNA comprising at least 15 consecutive
nucleotides complementary to the DNA of [1] or a complementary
sequence thereof; [0052] [19] an agent that increases field
resistance to blast in a plant, which comprises any one of the DNA
of [2] or [3] or the vector comprising the DNA; [0053] [20] a
primer set that amplifies all or a part of the nucleotide sequence
of SEQ ID NO: 1, 4, or 20; [0054] [21] a primer set, that is at
least any one of the following (a) to (c):
[0055] (a) a DNA comprising the nucleotide sequence of SEQ ID NO:
8, and a DNA comprising the nucleotide sequence of SEQ ID NO:
9,
[0056] (b) a DNA comprising the nucleotide sequence of SEQ ID NO:
16, and a DNA comprising the nucleotide sequence of SEQ ID NO: 17,
and
[0057] (c) a DNA comprising the nucleotide sequence of SEQ ID NO:
26, and a DNA comprising the nucleotide sequence of SEQ ID NO: 27;
[0058] [22] a DNA comprising the nucleotide sequence of SEQ ID NO:
7, 10, 18, 19, 23, or 25; [0059] [23] a method comprising the
following steps (a) to (c):
[0060] (a) preparing a DNA sample from a test plant,
[0061] (b) amplifying the DNA region described in [1] from the DNA
sample, and
[0062] (c) comparing the molecular weight or the nucleotide
sequence of the amplified DNA fragment with that of the DNA of (e)
or (f) in [1],
which is a method that judges the test plant to have field
resistance to blast when the molecular weight or nucleotide
sequence is consistent with that of the DNA of (e) or (f) in [1];
[0063] [24] a method comprising the following steps (a) to (d):
[0064] (a) preparing a DNA sample from a test plant,
[0065] (b) amplifying the DNA region described in [1] from the DNA
sample,
[0066] (c) separating the amplified double-stranded DNA on a
non-denaturating gel, and
[0067] (d) comparing the mobility of the separated double-stranded
DNA on the gel with that of the DNA of (e) or (f) in [1],
which is a method that judges the test plant to have field
resistance to blast when the mobility on the gel is consistent with
that of the DNA of (e) or (f) in [1]; [0068] [25] a method
comprising the following steps (a) to (e):
[0069] (a) preparing a DNA sample from a test plant,
[0070] (b) amplifying the DNA region described in [1] from the DNA
sample,
[0071] (c) dissociating the amplified DNA into single-stranded
DNAs,
[0072] (d) separating the dissociated single-stranded DNAs on a
non-denaturating gel, and
[0073] (e) comparing the mobility of the separated single-stranded
DNAs on the gel with that of the DNA of (e) or (f) in [1],
which is a method that judges the test plant to have field
resistance to blast when the mobility on the gel is consistent with
the DNA of (e) or (f) in [1]; [0074] [26] a method comprising the
following steps (a) to (d):
[0075] (a) preparing a DNA sample from a test plant,
[0076] (b) amplifying the DNA region described in [1] from the DNA
sample,
[0077] (c) separating the amplified DNA on a gel with a gradually
increasing concentration of a DNA denaturant, and
[0078] (d) comparing the mobility of the separated DNA on the gel,
with that of the DNA of (e) or (f) in [1],
which is the method that judges the test plant to have field
resistance to blast when the mobility on the gel is consistent with
that of the DNA of (e) or (f) in [1]; [0079] [27] a method for
selecting a plant having field resistance to blast, which comprises
the following steps (a) and (b):
[0080] (a) producing a hybrid variety by crossing a plant having
field resistance to blast with a plant having an arbitrary
function, and
[0081] (b) judging whether the plant produced in step (a) has field
resistance to blast by the method of any one of [23] to [26];
[0082] [28] a method for judging a test rice plant to have field
resistance to blast when a molecular marker linked to the DNA of
[1] shows the same genotype as that in a rice plant having field
resistance to blast; [0083] [29] the method of [28], wherein the
molecular marker comprises the DNA of SEQ ID NO: 10; [0084] [30] a
method for selecting a rice plant having field resistance to blast,
wherein the method comprises the following steps (a) and (b):
[0085] (a) producing a hybrid variety by crossing a rice plant
having field resistance to blast with a rice plant having an
arbitrary function, and
[0086] (b) judging whether the rice plant produced in step (a) has
field resistance to blast using the method of [28] or [29]; [0087]
[31] a method of screening for an agent that prevents or
ameliorates blast in a plant, wherein the method comprises the
following steps (a) to (c):
[0088] (a) contacting a test compound with a transcription product
of the DNA of any one of (a) to (d) in [1],
[0089] (b) detecting the binding of the transcription product of
the DNA of any one of (a) to (d) in [1] to the test compound,
and
[0090] (c) selecting a test compound that binds to the
transcription product of the DNA of any one of (a) to (d) in [1];
[0091] [32] a method of screening for an agent that prevents or
ameliorates blast in a plant, wherein the method comprises the
following steps (a) to (c):
[0092] (a) contacting a test compound with a cell collected from a
plant,
[0093] (b) measuring the expression level of a transcription
product of the DNA of any one of (a) to (d) in [1], and
[0094] (c) selecting a test compound that decreases the expression
level of the transcription product as compared to when the test
compound is not contacted; [0095] [33] a method of screening for an
agent that prevents or ameliorates blast in a plant, which
comprises the following steps (a) to (d):
[0096] (a) providing a cell or cell extract comprising a DNA in
which a reporter gene is operably linked downstream of a promoter
region of the DNA of any one of (a) to (d) in [1],
[0097] (b) contacting a test compound with the cell or cell
extract,
[0098] (c) measuring the expression level of the reporter gene in
the cell or cell extract, and
[0099] (d) selecting a test compound that decreases the expression
level of the reporter gene as compared to when the test compound is
not contacted; [0100] [34] a method of screening for an agent that
prevents or ameliorates blast in a plant, which comprises the
following steps (a) to (d):
[0101] (a) regenerating a transformed plant from the transformed
plant cell of [6],
[0102] (b) contacting the blast fungus and a test compound with the
transformed plant, and
[0103] (c) selecting a test compound that suppresses blast in the
transformed plant as compared to when the test compound is not
contacted; and [0104] [35] a kit for use in the screening method of
any one of [31] to [34].
BRIEF DESCRIPTION OF THE DRAWINGS
[0105] FIG. 1 shows photographs indicating blotches of blast on the
line AA-pi21 having the pi21 gene with the genetic background of
Aichi Asahi (left), and those on Aichi Asahi (right).
[0106] FIG. 2 shows detailed linkage maps of the pi21 gene region,
and an alignment map of genomic clones. FIGS. 2A and 2B show the
genetic maps created using segregating populations of 72 samples
and 1014 samples. FIG. 2C shows the alignment map with PAC clones
of Nipponbare. FIG. 2D shows a detailed genetic map of the pi21
gene region, and indicates a candidate genomic region.
[0107] FIG. 3 shows a structure of a pi21 candidate gene, and a
comparison between genomic nucleotide sequences of Nipponbare and
Aichi Asahi and that of Owarihatamochi.
[0108] FIG. 4 shows photographs indicating blotches that appeared
on the transformants of the resistant line AApi21 into which the
Pi21 gene from Nipponbare was introduced. A: The vector alone was
introduced. B: One copy of the Pi21 gene was introduced. C: Three
or more copies of the Pi21 gene were introduced.
BEST MODE FOR CARRYING OUT THE INVENTION
[0109] The pi21 gene, an allele of the susceptibility gene Pi21
which does not suppress the progression of rice blast, was until
now known to be located somewhere in the vast region of rice
chromosome 4, as a gene that confers rice with field resistance to
blast. By using the map-based cloning technique, the present
inventors narrowed down the pi21 gene region on rice chromosome 4,
and finally succeeded in identifying it as a single gene. Moreover,
they also succeeded in isolating the Pi21 gene, an allele of the
pi21 gene.
[0110] As used herein, the term "blast" means the discoloring or
necrotization of a plant, or part of a plant infected with a blast
fungus, or a pathological feature (blotch) recognized thereby. The
blotches of blast appear in every part of the plant, and blast is
called seedling blast, leaf blast, panicle blast, spikelet blast,
node blast, and leaf node (ligule) blast and such, according to the
part where the blotches appear. "Blast" in the present invention
includes blast occurring on any of these parts. A "rice blast
fungus" which causes blast in rice is called Magnaporthe grisea or
Magnaporthe oryzae, although there is no unified scientific name at
present. Moreover, the blast fungus has a teleomorph name,
Magnaporthe oryzae, and a corresponding anamorph name, Pyricularia
oryzae, which are used depending on the situation. The blast fungus
in the present invention includes all these blast fungi, regardless
of their names.
[0111] As used herein, the term "blast susceptibility" means the
property of a plant to be infected with blast (sometimes means that
the symptoms are significant). The term "field resistance to blast"
means the difference in symptoms or the property of suppressing the
number or size of blotches, which are recognized as the difference
in the number or size of blotches between varieties or lines
(within the same plant species) when plants are infected with the
blast fungus. The term "true resistance" means the property of a
plant to cause cell death by a hypersensitive reaction in cells
invaded by the blast fungus to prevent infection.
[0112] The present invention provides the blast susceptibility gene
Pi21, involved in blast of plants, and pi21, a gene conferring
field resistance to blast.
[0113] More specifically, the Pi21 gene of the present invention
comprises the following:
[0114] (a) a DNA encoding a protein comprising the amino acid
sequence of SEQ ID NO: 3 or 22;
[0115] (b) a DNA comprising the coding region of the nucleotide
sequence of SEQ ID NO: 1, 2, 20, or 21;
[0116] (c) a DNA encoding a protein which comprises an amino acid
sequence with a substitution, deletion, addition, and/or insertion
of one or more amino acids in the amino acid sequence of SEQ ID NO:
3 or 22, and which has a function equivalent to that of a protein
comprising the amino acid sequence of SEQ ID NO: 3 or 22; and
[0117] (d) a DNA which hybridizes with a DNA comprising the
nucleotide sequence of SEQ ID NO: 1, 2, 20, or 21 under a stringent
condition, and which encodes a protein having the function
equivalent to a protein comprising the amino acid sequence of SEQ
ID NO: 3 or 22.
[0118] Furthermore, the pi21 gene of the present invention
specifically comprises the following:
[0119] (a) a DNA encoding a protein comprising the amino acid
sequence of SEQ ID NO: 6;
[0120] (b) a DNA comprising the coding region of the nucleotide
sequence of SEQ ID NO: 4 or 5;
[0121] (c) a DNA encoding a protein which comprises an amino acid
sequence with a substitution, deletion, addition, and/or insertion
of one or more amino acids in the amino acid sequence of SEQ ID NO:
6, and which has the function equivalent to that of a protein
comprising the amino acid sequence of SEQ ID NO: 6; and
[0122] (d) a DNA which hybridizes with a DNA comprising the
nucleotide sequence of SEQ ID NO: 4 or 5 under a stringent
condition, and which encodes a protein having the function
equivalent to a protein comprising the amino acid sequence of SEQ
ID NO: 6.
[0123] By using the Pi21 gene or the pi21 gene of the present
invention, it becomes possible, for example, to prepare recombinant
proteins or generate transformed plants with modified field
resistance to blast.
[0124] In the present invention, plants from which the genes of the
present invention are derived include, but are not particularly
limited to, for example, monocotyledons such as rice, corn, wheat,
barley, oat, Job's tears, Italian ryegrass, perennial ryegrass,
timothy, meadow fescue, millet, foxtail millet, sugarcane, and
pearl millet; and dicotyledons such as rapeseed, soybean, cotton,
tomato, and potato. They also include flowering plants such as
chrysanthemum, rose, carnation, and cyclamen, but are not
particularly limited thereto.
[0125] There is no particular restriction on the forms of the "Pi21
gene" and the "pi21 gene" of the present invention, as long as they
can encode the "Pi21 protein" and "pi21 protein", respectively; and
the "Pi21 gene" and the "pi21 gene" each comprises a genomic DNA,
chemically synthesized DNA and so on as well as a cDNA. Moreover,
the Pi21 gene and the pi21 gene comprise a DNA with any nucleotide
sequence based on genetic code degeneracy, as long as they encode
the Pi21 protein and the pi21 protein, respectively.
[0126] One skilled in the art can prepare genomic DNAs and cDNAs by
using conventional means. Genomic DNAs can be prepared, for
example, by extracting genomic DNAs from a plant; constructing a
genomic library (a plasmid, phage, cosmid, BAC, PAC or the like can
be used as a vector); developing it; and performing colony
hybridization or plaque hybridization using a probe prepared based
on the Pi21 gene or the pi21 gene (for example, the DNA of any one
of SEQ ID NO: 1, 2, 4, 5, 20, or 21). Alternatively, genomic DNAs
can be prepared by preparing primers specific for the Pi21 gene or
the pi21 gene and performing PCR by using these primers. cDNAs can
be prepared, for example, by synthesizing cDNAs based on mRNAs
extracted from a plant; inserting them into vectors such as XZAP to
create a cDNA library; developing it; and performing colony
hybridization or plaque hybridization as described above. They can
also be prepared by performing PCR.
[0127] Further, since the Pi21 gene or the pi21 gene is considered
to be widely present in the plant kingdom, the Pi21 gene or the
pi21 gene also includes not only genes in rice but also homologous
genes present in various plants. Herein, the term "homologous gene"
refers to a gene in various plants that encodes a protein having a
physiological function (for example, blast susceptibility or field
resistance to blast) similar to that of the Pi21 gene product or
the pi21 gene product in rice.
[0128] Methods for isolating homologous genes well known to one
skilled in the art include the hybridization technique (Southern E.
M., Journal of Molecular Biology, Vol. 98, 503, 1975) and the
polymerase chain reaction (PCR) technique (Saiki, R. K., et al.
Science, vol. 230, 1350-1354, 1985; Saiki, R. K. et al. Science,
vol. 239, 487-491, 1988). Specifically, one skilled in the art can
usually isolate homologous genes of the Pi21 gene or the pi21 gene
from various plants, by using as a probe the nucleotide sequences
(for example, the sequence of any one of SEQ ID NO: 1, 2, 4, 5, 20,
or 21) of the rice Pi21 gene or pi21 gene, or a part of it, or by
using as primers oligonucleotides which specifically hybridize to
the Pi21 gene or the pi21 gene.
[0129] In order to isolate DNAs encoding such homologous genes, the
hybridization reaction is usually performed under stringent
conditions. Examples of stringent hybridization conditions include
the conditions of 6 M urea, 0.4% SDS, and 0.5.times.SSC, or
hybridization conditions of equivalent stringency. Isolation of
DNAs with higher homology can be expected by using conditions with
higher stringency, for example, 6 M urea, 0.4% SDS, and
0.1.times.SSC. The sequences of the isolated DNAs can be determined
by a known method. Homology of isolated DNAs indicates a sequence
identity of at least 50% or more, more preferably 70% or more,
still more preferably 90% or more (for example, 95%, 96%, 97%, 98%,
99% or more) over the entire amino acid sequence. Sequence homology
can be determined using the programs of BLASTN (nucleic acid level)
or BLASTX (amino acid level) (Altschul et al. J. Mol. Biol. 215:
403-410, 1990). The programs are based on the algorithm BLAST by
Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268,
1990; Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). When
analyzing a nucleotide sequence by BLASTN, parameters are set to,
for example, score=100 and wordlength=12. When analyzing an amino
acid sequence by BLASTX, parameters are, for example, set to
score=50 and wordlength=3. Alternatively, an amino acid sequence
can be analyzed using Gapped BLAST program as indicated by Altschul
et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When BLAST and
Gapped BLAST programs are used, the default parameters of each
program are used. The specific procedures of these analysis methods
are known.
[0130] The present invention also provides the following DNAs that
are used to suppress the plant endogenous Pi21 gene expression:
[0131] (a) a DNA encoding an RNA complementary to a transcription
product of the Pi21 gene,
[0132] (b) a DNA encoding an RNA that has the ribozyme activity to
specifically cleave a transcription product of the Pi21 gene,
[0133] (c) a DNA encoding an RNA which inhibits Pi21 gene
expression by a co-suppression effect, and
[0134] (d) a DNA encoding an RNA that has the RNAi activity to
specifically cut a transcription product of the Pi21 gene.
[0135] These DNAs can suppress blotch progression by blast in
plants.
[0136] In the present invention, plants in which the Pi21 gene
expression is suppressed are not particularly limited, and any
plant desired to be conferred field resistance to blast can be
used; however, agricultural crops and ornamental plants are
suitable from an industrial viewpoint. Useful agricultural crops
include, but are not particularly limited to, monocotyledons such
as rice, corn, wheat, barley, oats, Job's tears, Italian ryegrass,
perennial ryegrass, timothy, meadow fescue, millet, foxtail millet,
sugarcane, and pearl millet; and dicotyledons such as rapeseed,
soybean, cotton, tomato, and potato. Ornamental plants include
flowering plants such as chrysanthemum, rose, carnation, and
cyclamen, but are not limited thereto. Plants susceptible to the
rice blast fungus include pasture grasses such as barley, Italian
ryegrass, and meadow fescue; and corn. In addition, many plants
have been reported as those which the blast fungus separated from
rice can parasitize, including tribe Oryzeae such as Leersia
oryzoides and wild rice; tribe Poeae; tribe Triticeae; tribe
Aveneae such as oat; tribe Chloridinae such as Eragrostis curvula;
and tribe Paniceae such as foxtail millet and crabgrass. These
plants are also included in the plants on which field resistance to
blast can be conferred.
[0137] As used herein, "suppression of Pi21 gene expression"
includes suppression of gene transcription and suppression of
translation to a protein. Moreover, it includes not only the
complete arrest of DNA expression, but also reduction of
expression.
[0138] One embodiment of "a DNA used to suppress Pi21 gene
expression" is a DNA which encodes an antisense RNA complementary
to the Pi21 gene. Using the temporal gene expression method, the
antisense effect in a plant cell was demonstrated for the first
time through the fact that an antisense RNA introduced by
electroporation exhibited an antisense effect in a plant (Ecker and
Davis, Proc. Natl. Acad. USA, 83: 5372, 1986). Thereafter,
expression of antisense RNAs in tobacco and petunia have also been
reported to reduce target gene expression (Krol et. al., Nature
333: 866, 1988). At present, it is established as a means to
suppress gene expression in plants.
[0139] There are a number of factors involved in the action of
antisense nucleic acids in suppressing target gene expression, as
indicated as follows: inhibiting transcription initiation by
forming triple strands; suppressing transcription by hybridizing
with a site where RNA polymerase has formed a local open loop
structure; inhibiting transcription by hybridizing with the RNA
being synthesized; suppressing splicing by hybridizing with an
intron-exon junction; suppressing splicing by hybridizing with the
site of spliceosome formation; suppressing transfer from the
nucleus to the cytoplasm by hybridizing with an mRNA; suppressing
splicing by hybridizing with a poly(A) addition site or capping
site; suppressing translation initiation by hybridizing with a
translation initiation factor binding site; suppressing translation
by hybridizing with a ribosome binding site near the initiation
codon; preventing peptide chain elongation by hybridizing with an
mRNA translation region or polysome binding site; and suppressing
gene expression by hybridizing with a nucleic acid-protein
interaction site. Antisense nucleic acids suppress target gene
expression by inhibiting transcription, splicing, or translation
process (Hirashima and Inoue, 1993, "Shin Seikagaku Jikken Kouza
(New Biochemistry Experimentation Lectures) 2, Kakusan (Nucleic
Acids) IV, Idenshi No Fukusei To Hatsugen (Replication and
Expression of Genes)", The Japanese Biochemical Society Ed., Tokyo
Kagaku Dojin, pp. 319-347).
[0140] The antisense sequences used in the present invention can
suppress the expression of a target gene by any of the above
actions. As one embodiment, an antisense sequence designed to be
complementary to an untranslated region close to the 5' end of the
mRNA of a gene will be effective in inhibiting translation of that
gene. However, a sequence complementary to a coding region, or to a
3'-end untranslated region can also be used. In this way, DNAs
comprising antisense sequences of a gene's translated regions as
well untranslated regions are included in the antisense DNAs that
can be used in the present invention. An antisense DNA to be used
herein is ligated downstream of an appropriate promoter, and a
sequence comprising a transcription termination signal is
preferably ligated to the 3' side of the DNA.
[0141] Antisense DNAs can be prepared, for example, based on the
DNA sequence of SEQ ID NO: 1, 2, 20, or 21 by using the
phosphorothioate method (Stein, Nucleic Acids Res., 16: 3209-3221,
1988) and such. DNAs thus prepared can be transformed into a
desired plant using known methods. Antisense DNA sequences are
preferably sequences complementary to a transcription product of an
endogenous gene of the plant to be transformed, but need not be
perfectly complementary as long as they can effectively inhibit
gene expression. The transcribed RNAs are preferably 90% or more
(for example, 95%, 96%, 97%, 98%, 99% or more) complementary to the
transcription products of the target genes. In order to effectively
inhibit target gene expression using an antisense sequence, an
antisense DNA should comprise at least 15 nucleotides or more,
preferably 100 nucleotides or more, and even more preferably 500
nucleotides or more. Antisense DNAs to be used are generally less
than 5 kb, and preferably less than 2.5 kb long.
[0142] Suppression of the endogenous Pi21 gene expression can also
be carried out using DNAs encoding ribozymes. The term "ribozyme"
refers to an RNA molecule having catalytic activity. Some ribozymes
have many different activities. Among them, research on ribozymes
as RNA-cleaving enzymes has enabled designing ribozymes to cleave
RNAs at specific sites. Ribozymes include those of 400 nucleotides
or more, such as M1RNA in RNaseP, or the group 1 intron type
ribozymes. In contrast, there are also hammerhead-type or
hairpin-type ribozymes that comprise an active domain of about 40
nucleotides (Koizumi, M. and Ohtsuka, E., 1990, Protein, Nucleic
acid and Enzyme, 35: 2191-2200).
[0143] For example, the self-cleaving domain of a hammerhead type
ribozyme cleaves at the 3' side of C15 in G13U14C15. Base pairing
between U14 and A9 is important for ribozyme activity. It has been
shown that cleavage can occur if A or U instead of C is at the 15th
position (Koizumi, M. et al., 1988, FEBS Lett. 228: 228-230). If
the substrate-binding site of the ribozyme is designed to be
complementary to the RNA sequences adjacent to the target site, a
restriction enzyme-like RNA-cleaving ribozyme can be created that
recognizes the sequence UC, UU, or UA within the target RNA
(Koizumi et al., 1988, FEBS Lett. 239: 285; Koizumi, M. and
Ohtsuka, E., 1990, Protein, Nucleic acid and Enzyme, 35: 2191;
Koizumi et al., 1989, Nucleic Acids Res. 17: 7059).
[0144] Hairpin type ribozymes are also useful for objectives of the
present invention. A hairpin type ribozyme can be found, for
example, in the minus strand of tobacco ringspot virus satellite
RNA (Buzayan, Nature 323: 349, 1986). It has also been shown that
this ribozyme can be designed to target-specifically cleave an RNA
(Kikuchi and Sasaki, Nucleic Acids Res. 19: 6751, 1992; Kikuchi,
H., Kagaku to Seibutsu (Chemistry and Biology) 30: 112, 1992).
[0145] In order to be transcribed in plant cells, a ribozyme
designed to cleave a target is linked to a transcription
termination sequence or a promoter such as the cauliflower mosaic
virus 35S promoter. However, if extra sequences are added to the
5'- or the 3'-end of the transcribed RNA, the ribozyme activity can
be lost. In this case, another cis-acting trimming ribozyme can be
placed in the 5' or 3' side of the ribozyme portion to precisely
trim only the ribozyme portion from the transcribed RNA comprising
the ribozyme (Taira et al., Protein Eng. 3: 733, 1990; Dzianott and
Bujarski, Proc. Natl. Acad. Sci. USA 86: 4823, 1989; Grosshans and
Cech, Nucleic Acids Res. 19: 3875, 1991; Taira et al., Nucleic Acid
Res. 19: 5125, 1991).
[0146] In addition, these structural units can be arranged in
tandem to cleave multiple sites within a target gene, thus
achieving greater effects (Yuyama et al., Biochem. Biophys.
Res.
[0147] Commun. 186: 1271, 1992). By using these kinds of ribozymes,
the transcription products of the target genes of the present
invention can be specifically cleaved, and the gene expression can
be suppressed.
[0148] Suppression of endogenous gene expression can also be
achieved by "co-suppression" resulting from transformation with a
DNA comprising a sequence identical or similar to a target gene
sequence. The term "co-suppression" refers to the phenomenon in
which, when a gene comprising a sequence identical or similar to
that of the target endogenous gene is introduced into plants by
transformation, expression of both the introduced exogenous gene
and the target endogenous gene is suppressed. The detailed
mechanism of co-suppression is unknown, but it is frequently
observed in plants (Curr. Biol., 7: R793, 1997; Curr. Biol. 6: 810,
1996).
[0149] For example, to obtain a plant in which the Pi21 gene is
co-suppressed, plants of interest are transformed with a vector DNA
constructed to express the Pi21 gene or a DNA comprising a similar
sequence, and plants with a characteristic of a mutant Pi21, i.e.,
plants with field resistance to blast, are selected from the plants
thus obtained. Genes to be used for co-suppression do not have to
be completely identical to the target gene; however, they have
sequence identity of at least 70% or more, preferably 80% or more,
and more preferably 90% or more (for example, 95%, 96%, 97%, 98%,
99% or more).
[0150] In addition, suppression of endogenous gene expression in
the present invention can also be achieved by transforming a plant
with a gene comprising a characteristic that is dominant-negative
to the target gene. A "gene comprising a dominant-negative
characteristic" refers to a gene that, when expressed, has the
function of eliminating or reducing the activity of an original
endogenous wild-type gene of the plant.
[0151] Another embodiment of "a DNA used to suppress Pi21 gene
expression" is a DNA which encodes a double-stranded RNA (dsRNA)
complementary to a transcription product of an endogenous Pi21
gene. By introducing a dsRNA comprising a sequence identical or
similar to a target gene sequence into a cell, a phenomenon called
RNAi (RNA interference) can be caused, where expression of both the
introduced foreign gene and the target endogenous gene are
suppressed. When a dsRNA of about 40 to several hundred base pairs
is introduced into a cell, an RNase III-like nuclease comprising a
helicase domain, called Dicer, cuts out about 21 to 23 base pair
portions from the 3'-terminus of the dsRNA in the presence of ATP,
thereby producing an siRNA (short interference RNA). This siRNA
binds to a specific protein to form a nuclease complex (RISC:
RNA-induced silencing complex). This complex recognizes and binds
to a sequence identical to the siRNA, and cuts the transcription
product (mRNA) of the target gene at the central part of the siRNA
with the RNaseIII-like enzymatic activity. Apart from this pathway,
an antisense strand of siRNA binds to an mRNA to act as a primer of
an RNA-dependent RNA polymerase (RsRP), thereby synthesizing a
dsRNA. Another pathway is also considered in which this dsRNA
serves again as a substrate of Dicer, produces a new siRNA, and
amplifies its effect.
[0152] RNAi was first discovered in nematodes (Fire, A. et al.,
Potent and specific genetic interference by double-stranded RNA in
Caenorhabditis elegans. Nature 391, 806-811, 1998). At present, it
is observed not only in nematodes, but also in various organisms
such as plants, Nemathelminthes, Drosophila, and protozoa (Fire, A.
RNA-triggered gene silencing. Trends Genet. 15, 358-363 (1999);
Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001);
Hammond, S. M., Caudy, A. A. & Hannon, G. J.
Post-transcriptional gene silencing by double-stranded RNA. Nature
Rev. Genet. 2, 110-119 (2001); Zamore, P. D. RNA interference:
listening to the sound of silence. Nat Struct Biol. 8, 746-750
(2001)). In these organisms, it was confirmed that target gene
expression was actually suppressed by externally introducing a
dsRNA. Further, RNAi is now being used as a method for creating
knockout organisms.
[0153] When RNAi was initially found, only a dsRNA of a certain
length (40 bases) or more was thought to be effective. However,
Tuschl et al. of Rockefeller University, United States, reported
that by introducing a short dsRNA (siRNA) of about 21 base pairs
into a cell, an RNAi effect was obtained in a mammalian cell
without causing an antiviral reaction by PKR (Tuschl, Nature, 411,
494-498 (2001)). Thus, RNAi has suddenly attracted attention as a
technique applicable to differentiated mammalian cells such as
human cells.
[0154] The DNAs of the present invention comprise an antisense code
DNA encoding an antisense RNA corresponding to any region of a
transcription product (mRNA) of a target gene, and a sense code DNA
encoding a sense RNA corresponding to any region of the mRNA. The
above-mentioned antisense RNA and sense RNA can be expressed from
the above-mentioned antisense code DNA and sense code DNA. A dsRNA
can also be produced from these antisense RNA and sense RNA. A
target sequence in the present invention is not particularly
limited, as long as the Pi21 gene expression is suppressed by
introducing into a cell a dsRNA comprising a sequence identical or
similar to the target sequence. An example of the target sequence
includes a sequence of 3'-untranslated region of the Pi21 gene. A
sequence of 3'-untranslated region of the Pi21 gene is shown in SEQ
ID NOs: 11 and 24.
[0155] An expression system of dsRNAs of the present invention is
maintained as follows in a vector or the like: an antisense RNA and
a sense RNA are expressed from the same vector; or an antisense RNA
and a sense RNA are expressed from different vectors, respectively.
For example, when expressing an antisense RNA and a sense RNA from
the same vector, an antisense RNA expression cassette and sense RNA
expression cassette are each constructed, in which a promoter like
the pol III system that can express a short RNA is connected
upstream of the antisense code DNA and sense code DNA,
respectively, and these cassettes are then inserted into a vector
in the same direction or in the opposite direction.
[0156] An expression system can also be constructed in which an
antisense code DNA and a sense code DNA are arranged in opposite
directions on different strands so that they face each other. This
construct can carry one double-stranded DNA (siRNA code DNA) in
which an antisense RNA-encoding strand and a sense RNA-encoding
strand are paired, and promoters which are oppositely oriented on
both sides so that the antisense RNA and the sense RNA can be
expressed from each strand. In this case, in order to prevent
addition of an excess sequence downstream of the sense RNA and the
antisense RNA, a terminator is preferably placed at the 3'-terminus
of each strand (the antisense RNA-encoding strand and the sense
RNA-encoding strand). A sequence of four or more consecutive A
(adenine) bases can be used for this terminator. Moreover, in this
palindrome type expression system, the kinds of two promoters are
preferably different to each other.
[0157] When expressing an antisense RNA and sense RNA from
different vectors, for example, the following procedures are
performed: An antisense RNA expression cassette and a sense RNA
expression cassette are constructed, in each of which a promoter
such as the pol III system that can express a short RNA, is
connected upstream of the antisense code DNA or the sense code DNA;
and then these cassettes are maintained in different vectors.
[0158] As for RNAi, an siRNA may be used as a dsRNA. The term
"siRNA" means a double-stranded RNA including short strands that
exhibit no toxicity within a cell, and is not limited to the full
length of 21 to 23 base pairs reported by Tuschl et al. (ibid.);
and is not particularly limited, as long as the length is in such a
range that it exhibits no toxicity. For example, an siRNA can be 15
to 49 base pairs, preferably 15 to 35 base pairs, and still more
preferably 21 to 30 base pairs in length. Alternatively, length of
the final double-stranded RNA portion that results from
transcription of an siRNA to be expressed, can be 15 to 49 base
pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30
base pairs, for example.
[0159] As a DNA of the present invention, such a construct that is
produced by inserting a suitable sequence (an intron sequence is
preferable) between the inverted repeats of a target sequence
(Smith, N. A., et al. Nature, 407: 319, 2000; Wesley, S. V. et al.
Plant J. 27: 581, 2001; Piccin, A. et al. Nucleic Acids Res. 29:
E55, 2001) and yields a double-stranded RNA having a hairpin
structure (self-complementary `hairpin` RNA (hpRNA)), can also be
used.
[0160] Although a DNA used for RNAi is not required to be
completely the same as a target gene, it has a sequence identity of
at least 70% or more, preferably 80% or more, still more preferably
90% or more (for example, 95%, 96%, 97%, 98%, 99% or more). The
sequence identity can be determined by using the above-mentioned
procedures.
[0161] The double-stranded RNA portions in dsRNAs, in which RNAs
are paired, are not necessarily completely paired, but may comprise
unpaired portions due to a mismatch (corresponding bases are not
complementary), a bulge (there is no corresponding base on one
strand) or the like. In the present invention, both bulges and
mismatches may be included in double-stranded RNA regions where
RNAs are paired with each other in dsRNAs.
[0162] The present invention also provides vectors and transformed
cells comprising any one of the Pi21 gene, the pi21 gene, and DNAs
that suppress Pi21 gene expression.
[0163] With regard to the above vectors, for example, when the host
is E. coli, as long as the vector has an "ori" for amplification in
E. coli, such that vectors are amplified and prepared in large
quantities in E. coli (for example, JM109, DH5.alpha., HB101, and
XL1Blue) or such, and further has a selection gene for transformed
E. coli (for example, a drug resistance gene that allows
discrimination using a certain drug (ampicillin, tetracycline,
kanamycin, or chloramphenicol)), the vectors are not particularly
limited. The vectors include, for example, M13 vectors, pUC
vectors, pBR322, pBluescript, and pCR-Script. In addition to the
above vectors, for example, pGEM-T, pDIRECT, and pT7 can also be
used for the subcloning and excision of cDNAs. When using vectors
to produce the Pi21 gene, the pi21 gene, and the DNAs that suppress
Pi21 gene expression, expression vectors are particularly useful.
When an expression vector is expressed in E. coli, for example, it
should have the above characteristics in order to be amplified in
E. coli. Additionally, when E. coli such as JM109, DH5.alpha.,
HB101, or XL1-Blue are used as the host, the vector must have a
promoter that allows efficient expression in E. coli, for example,
a lacZ promoter (Ward et al. Nature 341:544-546, 1989; FASEB J. 6:
2422-2427, 1992), araB promoter (Better et al. Science
240:1041-1043, 1988), or T7 promoter. Other examples of the vectors
include pGEX-5X-1 (Pharmacia), "QlAexpress system" (QIAGEN), pEGFP,
and pET.
[0164] Furthermore, the vector may comprise a signal sequence for
polypeptide secretion. When producing polypeptides into the
periplasm of E. coli, the pelB signal sequence (Lei, S. P. et al.
J. Bacteriol. 169:4379 (1987)) may be used as a signal sequence for
polypeptide secretion. For example, calcium chloride methods or
electroporation methods may be used to introduce the vector into a
host cell.
[0165] In addition to E. coli, expression vectors derived from
mammals (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids Res.
18(17): 5322 (1990)), pEF, and pCDM8), insect cells (e.g.,
"Bac-to-BAC baculovirus expression system" (GIBCO-BRL) and
pBacPAK8), plants (e.g., pMH1 and pMH2), animal viruses (e.g.,
pHSV, pMV, and pAdexLcw), retroviruses (e.g., pZIPneo), yeasts
(e.g., "Pichia Expression Kit" (Invitrogen), pNV11 and SP-Q01), and
Bacillus subtilis (e.g., pPL608 and pKTHSO) may also be used as
vectors for producing the Pi21 gene, the pi21 gene, and the DNAs
which suppress Pi21 gene expression.
[0166] For expression in animal cells such as CHO, COS, and NIH3T3
cells, the vector must have a promoter necessary for expression in
such cells, for example, an SV40 promoter (Mulligan et al. Nature
277: 108 (1979)), MMLV-LTR promoter, EF1.alpha. promoter (Mizushima
et al. Nucleic Acids Res. 18: 5322 (1990)), or CMV promoter. It is
even more preferable that the vector comprises a gene for selecting
transformants (for example, a drug-resistance gene enabling
discrimination by a drug (such as neomycin and G418)). Examples of
vectors with such characteristics include pMAM, pDR2, pBK-RSV,
pBK-CMV, pOPRSV, and pOP13.
[0167] Introduction of a DNA of the present invention into a cell
can be carried out by a method known to one skilled in the art, for
example, by an electroporation method.
[0168] Further, the present invention provides transformed plant
cells into which the DNA encoding the Pi21 protein or a DNA
suppressing the Pi21 gene expression has been introduced;
transformed plants derived from the cells; transformed plants which
are progenies or clones of the transformed plants; and propagation
materials of the transformed plants. Methods for producing the
above-mentioned transformants and propagation materials are also
provided.
[0169] The DNA encoding the Pi21 protein or DNAs suppressing Pi21
gene expression can be introduced into plant cells by the above
methods.
[0170] In addition, regeneration of plants is also possible using
methods known to those skilled in the art, according to the type of
plant cell (Toki et al., Plant Physiol., 100: 1503-1507, 1995). In
rice, for example, a number of techniques for producing transformed
plants are already established, and are widely used in the
technical field of the present invention. These methods include the
method for introducing genes into protoplasts using polyethylene
glycol and then regenerating plants (suitable for Indian varieties
of rice) (Datta et al., In Gene Transfer To Plants. Potrykus, I.
and Spangenberg, G. Eds., pp. 66-74, 1995), the method for
introducing genes into protoplasts using electric pulse and then
regenerating plants (suitable for Japanese varieties of rice) (Toki
et al., Plant Physiol. 100: 1503-1507, 1992), the method for
directly introducing genes into cells using the particle gun method
and then regenerating plants (Christou et al., Bio/technology, 9:
957-962, 1991), and the method for introducing genes via an
Agrobacterium, and then regenerating plants (Hiei et al., Plant J.,
6: 271-282, 1994). These methods can be appropriately used in the
present invention.
[0171] When using the above Agrobacterium method, the method of
Nagel et al. (Microbiol. Lett. 67: 325, 1990) is used, for example.
In this method, a recombinant vector is transformed into an
Agrobacterium, and subsequently the transformed Agrobacterium is
introduced into a cell by a known method such as the leaf disk
method. The above-mentioned vector comprises an expression promoter
so that, for example, the DNA encoding the Pi21 protein of the
present invention or a DNA suppressing the Pi21 gene expression is
expressed in a plant after introduction into the plant. Generally,
DNA encoding the Pi21 protein of the present invention or a DNA
suppressing the Pi21 gene expression is located downstream of the
promoter, and a terminator is located further downstream of such a
DNA. The recombinant vector used for this purpose is suitably
selected by one skilled in the art, depending on the type of plant
or method of introduction. The above-mentioned promoters include,
for example, the CaMV35S derived from cauliflower mosaic virus and
the ubiquitin promoter from corn (JP-A H2-79983).
[0172] Examples of the above-mentioned terminator can be a
terminator derived from cauliflower mosaic virus and the terminator
from the nopaline synthase gene; however, the promoter and
terminator are not limited thereto, as long as they function in a
plant.
[0173] The plants into which the DNA encoding the Pi21 protein of
the present invention or a DNA suppressing the Pi21 gene expression
is introduced, may be explants, or the DNA may be introduced into
the cultured cells prepared from these plants. "Plant cells" in the
present invention include, for example, plant cells of a leaf,
root, stem, flower, and scutellum in a seed; calluses; and
suspension-cultured cells.
[0174] In order to efficiently select the cells transformed by
introducing the DNA encoding the Pi21 protein of the present
invention or a DNA suppressing the Pi21 gene expression, the
above-mentioned recombinant vector is introduced into the plant
cells, preferably together with a suitable selection marker gene or
a plasmid vector comprising a selection marker gene. The selection
marker genes used for this purpose include, for example, the
hygromycin phosphotransferase gene resistant to the antibiotic
hygromycin, the neomycin phosphotransferase resistant to kanamycin
or gentamycin, and the acetyltransferase gene resistant to the
herbicide phosphinothricin.
[0175] The cells into which the recombinant vector has been
introduced are placed on a known selection medium containing a
suitable selection agent depending on the type of introduced
selection marker gene, and then cultured. In this way, the
transformed plant cultured cells can be obtained.
[0176] Next, plant bodies reproduced from the transformed cells are
cultured in an acclimation medium. The acclimated, regenerated
plant bodies are then grown under usual culture conditions to
obtain plant bodies having field resistance to blast, from which
seeds can be obtained once they mature and bear fruit.
[0177] The presence of the introduced foreign DNAs in the
transformed plants that are regenerated and grown in this manner
can be confirmed by the known PCR method or Southern hybridization
method, or by analyzing the nucleotide sequences of the DNAs in
plant bodies.
[0178] In this case, extraction of the DNAs from the transformed
plants can be carried out according to the known method by J.
Sambrook et al. (Molecular Cloning, the 2nd edition, Cold Spring
Harbor Laboratory Press, 1989).
[0179] When analyzing the foreign genes which are present in the
regenerated plant bodies and include the DNAs of the present
invention, using the PCR method, an amplification reaction is
carried out using as a template the DNAs extracted from the
regenerated plant bodies as mentioned above. An amplification
reaction can also be performed in a reaction mixture containing as
primers synthesized oligonucleotides which comprise nucleotide
sequences suitably selected according to the nucleotide sequences
of the DNAs of the present invention or the DNAs modified according
to the present invention. In the amplification reaction,
denaturation, annealing, and extension reactions of DNAs can be
repeated several tens of times to obtain amplified products of DNA
fragments comprising the DNA sequences of the present invention. By
subjecting the reaction mixture comprising the amplified products,
for example, to agarose electrophoresis, the various kinds of
amplified DNA fragments are fractionated, thereby enabling
confirmation of whether a certain DNA fragment corresponds to a DNA
of the present invention.
[0180] After obtaining a transformed plant in which a DNA encoding
the Pi21 protein of the present invention or a DNA suppressing the
Pi21 gene expression has been introduced into the chromosome,
progenies can be obtained by sexual or asexual reproduction from
the plant. Further, propagation materials (for example, seeds,
fruits, panicles, tubers, tuberous roots, stocks, calluses, and
protoplasts) can also be obtained from the plant or its progenies
or clones, and these materials can be used to mass-produce the
plants. The present invention comprises plant cells into which the
DNA encoding the Pi21 protein or a DNA suppressing the Pi21 gene
expression has been introduced; plants comprising the cells;
progenies and clones of the plants; and propagation materials of
the plants and their progenies and clones. Such plant cells, plants
comprising the cells, progenies and clones of the plants, and
propagation materials of the plants and their progenies and clones,
can be used in methods for conferring plants with field resistance
to blast.
[0181] The present invention further provides methods for
conferring plants with field resistance to blast, comprising the
step of expressing a DNA which suppresses Pi21 gene expression in
cells of plant bodies. Field resistance to blast can be conferred
on plants by introducing into plant cells vectors which comprise,
in an expressible state, a DNA suppressing Pi21 gene expression,
using the above-mentioned methods, and by regenerating plants using
these cells.
[0182] As used herein, the term "'conferring' field resistance to
blast" means not only to provide a blast field resistance capacity
to plants having no blast field resistance capacity, but also to
further increase blast field resistance capacity of plants which
already have it. In the present invention, plants in which Pi21
gene expression is suppressed and on which field resistance to
blast is conferred, are not particularly limited; and such plants
include, for example, the above-mentioned plants.
[0183] The present invention also provides proteins encoded by the
Pi21 gene of the present invention, methods for producing the
proteins, and antibodies which bind to the proteins.
[0184] Recombinant proteins are typically prepared by inserting
DNAs encoding proteins of the present invention into appropriate
expression vectors, introducing the vectors into appropriate cells,
culturing the transformed cells, and purifying the expressed
proteins. Recombinant proteins can be expressed as fusion proteins
with other proteins to make purification easier, for example, as
fusion proteins with maltose-binding protein using Escherichia coli
as a host (New England Biolabs, USA, vector pMAL series), as fusion
proteins with glutathione S-transferase (GST) (Amersham Pharmacia
Biotech, vector pGEX series), or tagged with histidine (Novagen,
pET series). The host cells are not particularly limited, so long
as the cell is suitable for expressing the recombinant proteins. It
is possible to use, for example, yeast, various plant or animal
cells, insect cells or such in addition to the above-described E.
coli. Vectors can be introduced into host cells by a variety of
methods known to those skilled in the art. For example,
introduction methods using calcium ions can be used for
introduction into E. coli (Mandel, M. & Higa, A. Journal of
Molecular Biology, Vol. 53, 158-162 (1970); Hanahan, D. Journal of
Molecular Biology, Vol. 166, 557-580 (1983)). Recombinant proteins
expressed in the host cells can be purified and recovered from the
host cells or the culture supernatant thereof by known methods in
the art. When recombinant proteins are expressed as fusion proteins
with the aforementioned maltose-binding protein or such, affinity
purification can be carried out easily.
[0185] The obtained recombinant proteins can be used to prepare
antibodies which bind to the proteins. Polyclonal antibodies can be
obtained as follows, for example: Small animals such as rabbits are
immunized with the Pi21 protein a recombinant protein expressed as
a fusion protein with GST in microorganisms such as E. coli., or
its partial peptide to obtain sera. The antibodies are prepared by
purifying the sera using, for example, ammonium sulfate
precipitation, a protein A or protein G column, DEAE ion exchange
chromatography, or an affinity column coupled with the Pi21 protein
or a synthetic peptide. Monoclonal antibodies can be prepared as
follows: small animals such as mice are immunized with the Pi21
protein or its partial peptide; the spleen is harvested from the
mice and ground to separate cells; the cells and mouse myeloma
cells are fused using a reagent such as polyethylene glycol; and
from among the fused cells (hybridomas) thus obtained, clones which
produce antibodies binding to the Pi21 protein are selected.
Subsequently, the obtained hybridomas are transplanted into the
abdominal cavity of mice, ascites are collected from the mice to
prepare monoclonal antibodies, for example, by purifying using
ammonium sulfate precipitation, a protein A or protein G column,
DEAE ion exchange chromatography, an affinity column coupled with
the Pi21 protein or a synthetic peptide. The antibodies thus
obtained can be used for purification, detection and the like of
the proteins of the present invention. The present invention
comprises antibodies which bind to the proteins of the present
invention.
[0186] The present invention provides oligonucleotides comprising
at least 15 nucleotides which are complementary to the pi21 gene, a
DNA comprising the Pi21 gene, or the complementary strand
thereof.
[0187] A "complementary strand" herein refers to one strand
relative to the other strand in a double-stranded nucleic acid
comprising base pairs of A:T (U for RNA) and G:C. The term
"complementary" means not only that a sequence is completely
complementary in a region of at least 15 consecutive nucleotides,
but also that a sequence has a homology of at least 70%, preferably
at least 80%, more preferably 90%, still more preferably 95% or
more in the nucleotide sequence. Any algorithm known to one skilled
in the art may be used for determining homology.
[0188] The oligonucleotides of the present invention can be used as
probes or primers for detection and amplification of DNAs
comprising the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 20, or
21. Moreover, the oligonucleotides of the present invention can be
used in the form of a DNA array substrate.
[0189] When such an oligonucleotide is used as a primer, the length
is usually 15 by to 100 bp, and preferably 17 by to 30 bp. The
primer is not particularly limited, as long as it can amplify at
least a portion of a DNA of the present invention or its
complementary strand. When used as a primer, its 3' side region is
made to be complementary, and a restriction enzyme recognition
sequence, a tag or the like can be added to its 5' side.
[0190] When the above-mentioned oligonucleotide is used as a probe,
any oligonucleotide may be used without particular limitation, as
long as it can specifically hybridize at least a portion of a DNA
comprising the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 20, or
21 or its complementary strand. The probe may be a synthetic
oligonucleotide, and usually has a length of at least 15 bp or
more.
[0191] When an oligonucleotide of the present invention is used as
a probe, it is preferably labeled as appropriate. Labeling methods
include the following, for example: a method in which the 5' end of
an oligonucleotide is phosphorylated by .sup.32P using T4
polynucleotide kinase;
[0192] and a method (the random primed method or the like) in which
substrate bases labeled with an isotope such as .sup.32P, a
fluorescent dye, or biotin are incorporated into an
oligonucleotide, using a DNA polymerase such as Klenow enzyme and
using random hexamer oligonucleotides and the like as a primer.
[0193] The oligonucleotides of the present invention can be
produced, for example, with a commercially available
oligonucleotide synthesizer. The probes can also be produced as
double-stranded DNA fragments obtained by restriction enzyme
treatment or the like.
[0194] Further, the present invention provides uses of DNAs
suppressing Pi21 gene expression and vectors comprising the DNAs.
That is, the present invention relates to agents for increasing
field resistance to blast in plants, which comprise any one of a
DNA suppressing Pi21 gene expression and a vector comprising the
DNA as an active ingredient. Moreover, the present invention
relates to uses of DNAs suppressing Pi21 gene expression and
vectors comprising the DNAs, for preparing agents for increasing
field resistance to blast in plants.
[0195] The agents for increasing field resistance to blast in
plants of the present invention may include, for example,
sterilized water, physiological saline, vegetable oil, surfactants,
lipids, solubilizing agents, buffers, and preservatives, if needed,
in addition to active ingredients, i.e., the oligonucleotides.
[0196] The present invention also provides molecular markers linked
to the susceptibility gene Pi21 or the resistance gene pi21.
[0197] The term "molecular marker" in the present invention means a
DNA region which is genetically linked to the Pi21 gene or the pi21
gene and distinguishable from other DNA regions.
[0198] In general, when the map distance between a gene and a
molecular marker expressed with cM unit is shorter, the molecular
marker is located closer to the gene. Such a molecular marker is
highly useful because it will be inherited together with the gene.
pi21 was shown to be located between the marker "Pa102484" and the
marker "P702D3.sub.--#12" (FIG. 2c). Accordingly, in the methods of
the present invention, among the molecular markers described in
FIG. 2c, the above two markers and the markers located between the
two markers ("P702D03.sub.--#38", "P702D03.sub.--#79",
"P702D03.sub.--#80") are preferable. Among them,
""P702D03.sub.--#79'' is an especially preferable marker and can be
exemplified as the DNA of SEQ ID NO: 7 or 23 (linked to the
susceptibility gene Pi21), or SEQ ID NO: 10 (linked to the
resistance gene pi21).
[0199] The molecular markers of the present invention include
Sequence Tagged Site (STS) markers. The term "STS marker" refers to
a DNA region which can be used to judge the presence or absence of
polymorphism of a sequence tagged site (STS) on a DNA, and the term
"STS" refers to a specific sequence site at a particular position
of a DNA. The polymorphism of STS can be detected as the presence
or absence of bands or the difference in band position, by
amplifying a DNA region comprising a specific sequence site with a
nucleic acid amplification method such as the PCR method, and then
subjecting the amplification products to agarose or polyacrylamide
gel electrophoresis.
[0200] When a STS marker is used as a molecular marker of the
present invention, the identification methods of the present
invention can be carried out as follows, for example. First,
[0201] DNA samples are prepared by a well-known method from a test
rice plant and from a rice plant having field resistance to blast.
Next, a nucleic acid amplification reaction (for example, the PCR
method) is carried out by using the prepared DNAs as a template and
using primer DNAs. The sizes of the amplified DNA fragments are
compared between the test rice plant and a marker linked to the
gene for field resistance to blast (for example, the marker of SEQ
ID NO: 10) by electrophoresis or the like, and when they show the
same genotype, it is judged that the test plant has field
resistance to blast.
[0202] One skilled in the art can appropriately design optimal
primer DNAs used for the identification methods of the present
invention, considering the sequence information on various
molecular markers. Usually, the above-mentioned primers are a
primer set consisting of a pair of primers which are designed to
sandwich a nucleotide sequence that specifically exists in rice and
is linked to the Pi21 gene or the pi21 gene, for amplifying the
nucleotide sequence.
[0203] Specifically, primer sets for STS markers can include the
following, for example:
TABLE-US-00001 (a) a primer set consisting of primers (SEQ ID NO:
8) 5'-AGA AGG TGG AGT ACG ACG TGA AGA-3' and (SEQ ID NO: 9) 5'-AGT
TTA GTG AGC CTC TCC ACG ATT A-3', (b) a primer set consisting of
primers (SEQ ID NO: 16) 5'-GTA CGA CGT GAA GAA CAA CAG G-3' and
(SEQ ID NO: 17) 5'-GCT TGG GCT TGC AGT CC-3', and (c) a primer set
consisting of primers (SEQ ID NO: 26) 5'-GAT CCT CAT CGT CGA CGT
CTG GC-3' and (SEQ ID NO: 27) 5'-AGG GTA CGG CAC CAG CTT G-3'.
The presence of field resistance to blast in the test rice plant
can be judged by comparing the information characterized by the DNA
sequences amplified using these primer sets with the molecular
markers of the present invention
[0204] Besides the above-mentioned primers, one skilled in the art
can produce primer sets having a similar function utilizing the
nucleotide sequence of SEQ ID NO: 1, 4, or 20. The primers of the
present invention also comprise such primers.
[0205] One skilled in the art can produce PCR primers of the
present invention using, for example, an automatic oligonucleotide
synthesizer. One skilled in the art can also perform the methods of
the present invention by using a known polymorphism detection
method such as the below-mentioned PCR-SSCP method using the above
PCR primers, or the like.
[0206] When molecular markers of the present invention are located
in exons of a genomic DNA, it is also possible to utilize RT-PCR
using mRNAs as a template. By using the Taqman (a quantitative PCR
detection) system (Roche), the presence or absence of amplification
products can be detected by fluorescence. Since this system does
not need electrophoresis, it enables one to carry out the
identification methods of the present invention in a short
time.
[0207] The present invention further provides methods for judging
that the test plants have field resistance to blast when the
molecular weight or the nucleotide sequence is consistent with that
of the pi21 gene, which methods comprise the following steps (a) to
(c): [0208] (a) preparing DNA samples from test plants; [0209] (b)
amplifying the region of Pi21 gene or pi21 gene from the DNA
samples; and [0210] (c) comparing the molecular weight or the
nucleotide sequence of the amplified DNA fragments with that of the
pi21 gene.
[0211] One skilled in the art can prepare (extract) the
above-mentioned DNA samples of the present invention by known
methods. Preferable preparation methods include, for example, a
method for extracting DNAs using the CTAB method.
[0212] The DNA samples used in the identification methods of the
present invention are not particularly limited; however, genomic
DNAs extracted from a test plant, rice, are usually used.
[0213] The source for the genomic DNA extraction is not
particularly limited, and the DNAs can be extracted from any tissue
of rice. They can be extracted, for example, from a panicle, leaf,
root, stem, seed, endosperm portion, bran, or embryo.
[0214] In the methods of the present invention for identifying
field resistance to blast in plants, a nucleic acid amplification
reaction (for example, the PCR method) is subsequently carried out
by using the prepared DNAs as a template and using primer DNAs. The
amplified DNA fragments are cleaved by restriction enzymes, and the
sizes of the cleaved DNA fragments are compared between the test
plants and plants having field resistance to blast, by
electrophoresis or the like. When the molecular weight or the
nucleotide sequence is consistent with that of the compared plants,
the test plants are judged to have field resistance to blast.
"Plants having field resistance to blast" include Owarihatamochi
described in Examples, but are not limited thereto.
[0215] In the methods of the present invention for judging that
test plants have field resistance to blast, the term "consistent
with" means that the molecular weight or the nucleotide sequence of
both genes of an allele is consistent with that of a plant having
field resistance to blast, or that the deduced amino acid sequence
of such genes is consistent with that of the plant.
[0216] Accordingly, when the molecular weight, nucleotide sequence,
or deduced amino acid sequence of one gene of an allele differs
from that of a plant having field resistance to blast, but that of
the other gene of the allele is the same as that of the plant, such
a case is not included in the term "consistent with".
[0217] The above-mentioned electrophoresis analysis may be
conducted according to a conventional method. For example,
electrophoresis is carried out by applying voltage in an agarose or
polyacrylamide gel, and the separated DNA pattern is analyzed.
[0218] The present invention also provides methods for judging that
the test plants have field resistance to blast when the mobility on
the gel is consistent with that of the pi21 gene, which methods
comprise the following steps (a) to (d): [0219] (a) preparing DNA
samples from test plants; [0220] (b) amplifying the region of the
Pi21 gene or the pi21 gene from the DNA samples; [0221] (c)
separating the amplified double-stranded DNAs on a non-denaturating
gel; and [0222] (d) comparing the mobility of the separated
double-stranded DNAs on the gel with that of the pi21 gene.
[0223] The present invention further provides methods for judging
that the test plants have field resistance to blast when the
mobility on the gel is consistent with that of the pi21 gene, which
methods comprise the following steps (a) to (e): [0224] (a)
preparing DNA samples from test plants; [0225] (b) amplifying the
region of the Pi21 gene or the pi21 gene from the DNA samples;
[0226] (c) dissociating the amplified DNAs into single-stranded
DNAs; [0227] (d) separating the dissociated single-stranded DNAs on
a non-denaturating gel; and [0228] (e) comparing the mobility of
the separated single-stranded DNAs on the gel with that of the pi21
gene.
[0229] The above methods include the PCR-SSCP (single-strand
conformation polymorphism) method ("Cloning and polymerase chain
reaction-single-strand conformation polymorphism analysis of
anonymous Alu repeats on chromosome 11." Genomics 1992, Jan. 1,
12(1): 139-146; "Detection of p53 gene mutations in human brain
tumors by single-strand conformation polymorphism analysis of
polymerase chain reaction products." Oncogene 1991, Aug. 1; 6(8):
1313-1318; "Multiple fluorescence-based PCR-SSCP analysis with
postlabeling." PCR Methods Appl. 1995, Apr. 1; 4(5): 275-282). This
method is particularly preferable for screening many DNA samples,
since it has advantages such as comparative simplicity of operation
and a small amount of required test sample. The principle of the
method is as follows. A single-stranded DNA dissociated from a
double-stranded DNA fragment forms a unique higher conformation,
depending on the respective nucleotide sequence. After
electrophoresis on a polyacrylamide gel without a denaturant,
complementary single-stranded DNAs having the same chain length
shift to different positions in accordance with the difference of
the respective higher conformations. The conformation of a
single-stranded DNA changes even by a substitution of one base,
which change results in a different mobility on polyacrylamide gel
electrophoresis. Accordingly, the presence of a mutation in a DNA
fragment due to a point mutation, deletion, insertion and such can
be detected by detecting the changes in the mobility.
[0230] More specifically, a region comprising a target site of the
Pi21 gene or the pi21 gene is first amplified by the PCR method or
the like. Preferably, a region to be amplified is about 100 by to
600 bp in length. In amplifying gene fragments by PCR, DNA
fragments to be synthesized can be labeled by using primers labeled
with an isotope such as .sup.32P, a fluorescent dye, biotin and so
on, or by adding substrate nucleotides labeled with an isotope such
as .sup.32P, a fluorescent dye, biotin and so on, to the PCR
reaction solution. Alternatively, the synthesized DNA fragments can
be labeled after the PCR reaction by adding substrate nucleotides
labeled with an isotope such as .sup.32P, a fluorescent dye, biotin
and so on using the Klenow enzyme and such. The DNA fragments thus
obtained are electrophoresed in the form of a double strand on a
polyacrylamide gel without a denaturant such as urea.
Alternatively, such DNA fragments may be denatured by heating and
the like, and then subjected to electrophoresis on a polyacrylamide
gel without a denaturant such as urea. The conditions for
separating DNA fragments can be ameliorated by adding appropriate
amounts (about 5% to 10%) of glycerol to the polyacrylamide gel.
Further, although the electrophoresis conditions varies depending
on the properties of respective DNA fragments, it is usually
carried out at room temperature (20.degree. C. to 25.degree. C.).
When a preferable separation cannot be achieved, a temperature to
achieve the optimal mobility is selected from temperatures between
4.degree. C. and 30.degree. C. After the electrophoresis, the
mobility of the DNA fragments is detected by autoradiography using
X-ray films, a scanner for detecting fluorescence and the like, to
analyze the result. When bands with different mobility are
detected, the presence of a mutation can be confirmed by directly
excising the bands from the gel, amplifying them again by PCR, and
directly sequencing the amplified fragments. Even when labeled DNAs
are not used, the bands can also be detected by staining the gel
after electrophoresis with ethidium bromide, silver and such.
[0231] The present invention further provides methods for judging
that the test plants have field resistance to blast when the sizes
of the detected DNA fragments are consistent with that of the pi21
gene, which methods comprise the following steps (a) to (e): [0232]
(a) preparing DNA samples from test plants; [0233] (b) amplifying
the region of the Pi21 gene or the pi21 gene from the DNA samples;
[0234] (c) cleaving the prepared DNA samples with restriction
enzymes; [0235] (d) separating the DNA fragments according to their
sizes; and [0236] (e) comparing the sizes of the detected DNA
fragments with that of the pi21 gene.
[0237] The above methods include the RFLP method using Restriction
Fragment Length Polymorphism (RFLP) and the PCR-RFLP method.
Restriction enzymes are generally used as enzymes to cleave DNAs.
Specifically, when a nucleotide addition or deletion exists in the
recognition site of a restriction enzyme, or when a nucleotide
insertion or deletion exists in a DNA fragment generated by a
restriction enzyme treatment, the sizes of the fragments generated
after the restriction enzyme treatment differ between plants
susceptible to blast and plants having field resistance to blast.
The portion comprising such a mutation site is amplified by the PCR
method, and then treated with each restriction enzyme to detect the
polymorphic site as a difference in the mobility of bands by
electrophoresis. Alternatively, a polymorphic site can be detected
by treating chromosomal DNAs with such a restriction enzyme,
subjecting the fragments to electrophoresis, and then carrying out
Southern blotting with a probe DNA. The restriction enzymes to be
used can be appropriately selected in accordance with respective
mutation sites. In this method, Southern blotting can be performed
not only on genomic DNAs but also on cDNAs which are synthesized by
a reverse transcriptase from RNAs prepared from subjects and then
directly cleaved with restriction enzymes. Alternatively, a part or
whole of the Pi21 gene or the pi21 gene can be amplified by PCR
using such cDNAs as a template, and cleaved with restriction
enzymes, and then the difference in mobility can be examined.
[0238] The present invention provides methods for judging that the
test plants have field resistance to blast when the mobility on the
gel is consistent with that of the pi21 gene, which methods
comprise the following steps (a) to (d): [0239] (a) preparing DNA
samples from test plants; [0240] (b) amplifying the region of the
Pi21 gene or the pi21 gene from the DNA samples; [0241] (c)
separating the amplified DNAs on a gel with a gradually increasing
concentration of a DNA denaturant; and [0242] (d) comparing the
mobility of the separated DNAs on the gel with that of the pi21
gene.
[0243] The denaturant gradient gel electrophoresis method (DGGE
method) can be exemplified as one of such methods. A region
comprising a target site of the Pi21 gene or the pi21 gene is
amplified by the PCR method and the like using a primer of the
present invention and such; the resulting products are
electrophoresed on a polyacrylamide gel with a gradually increasing
concentration of a denaturant such as urea; and the result is
compared with that of a healthy subject. A polymorphism can be
identified by detecting the difference in mobility of the DNA
fragments, since the mobility rate of fragments with mutations
decreases drastically as the DNA fragments become single-stranded
DNAs at lower denaturant concentration points.
[0244] In addition to the above-mentioned methods, the Allele
Specific Oligonucleotide (ASO) hybridization method can be used. An
oligonucleotide comprising a nucleotide sequence where a
polymorphism is predicted to exist, is prepared, and is subjected
to hybridization with a DNA sample. When a polymorphic nucleotide
different from the oligonucleotide exists in the sample DNA used
for hybridization, the efficiency of hybridization is reduced. The
reduction of the hybridization efficiency can be detected by the
Southern blotting method; methods which utilize specific
fluorescent reagents that have a characteristic to quench by
intercalation into a gap of a hybrid; and the like.
[0245] Furthermore, the detection may be conducted by the
ribonuclease A mismatch truncation method. Specifically, a region
comprising a target site of the Pi21 gene or the pi21 gene is
amplified by the PCR method and the like, and the amplified
products are hybridized with labeled RNAs which are prepared from
healthy-type cDNAs and such incorporated into a plasmid vector and
the like. Since the hybrid forms a single strand conformation in a
portion comprising a nucleotide different from the healthy-type, a
polymorphism can be detected by cleaving this portion with
ribonuclease A and then performing autoradiography and the
like.
[0246] In the present invention, the term "test plant" is not
particularly limited, but includes all plants that can be infected
with the blast fungus. A preferable example is rice. Every variety
of rice can be used without any particular restriction, such as
Indica or Japonica rice varieties, and Indica-Japonica hybrid
varieties/lines, wild rice, or cultivar-wild rice hybrid or
crossbred varieties.
[0247] The present invention also provides methods for judging
field resistance to blast in rice by using as an indicator a
molecular marker which is linked to the pi21 gene and comprises at
least the DNA of SEQ ID NO: 7, 10, or 23. Preferable molecular
markers of the present invention include "P702D03.sub.--#38",
"P702D03.sub.--#79", and "P702D03.sub.--#80", as mentioned above.
Among them, "P702D03.sub.--#79" is an especially preferable marker,
and it may be the DNA of SEQ ID NO: 7 or 23 (linked to the
susceptibility gene Pi21) or SEQ ID NO: 10 (linked to the
resistance gene pi21), for example. The identification methods of
the present invention use as an indicator at least
"P702D03.sub.--#79" among these molecular markers. Therefore, in
the identification methods of the present invention,
"P702D03.sub.--#79" may be used alone or in combination with other
markers. The combinations of "P702D03.sub.--#79" with other markers
include the combination with "P702D03.sub.--#38", combination with
"P702D03.sub.--#80", and combinations with any other markers.
[0248] In the identification methods of the present invention,
field resistance to blast in test rice plants can be judged
specifically and efficiently by examining whether they comprise a
molecular marker linked to the pi21 gene. In the judging methods of
the present invention, when a desired rice plant to be judged for
having field resistance to blast or not comprises the nucleotide
sequence of SEQ ID NO: 10, the test rice plant is judged to have
field resistance to blast. When the test rice plant does not
comprise the nucleotide sequence of SEQ ID NO: 10 (when it
comprises the nucleotide sequence of SEQ ID NO: 7 or 23), it is
judged to be susceptible to blast.
[0249] Molecular markers in test rice plants can be compared with
those of the present invention not only for the DNA sequences of
molecular markers, but also for the information characterized by
the DNA sequences. The information characterized by the DNA
sequences of molecular markers includes information about the
molecular weight of the molecular markers and about the presence or
absence of a mutation site and polymorphic site comprised in the
molecular markers. One skilled in the art can identify polymorphic
sites (deletion sites and single base-substitution sites) by
comparing the nucleotide sequence of SEQ ID NO: 10 with that of SEQ
ID NO: 7 or 23 using known methods. The judging methods of the
present invention can also be performed by detecting such
information on the presence or absence of a mutation site or
polymorphic site comprised in molecular markers.
[0250] The above information on the presence or absence of a
mutation site or polymorphic site can be detected by using primers
which can amplify a region comprising a mutation site or
polymorphic site, or by using a probe (for example, the DNA
comprising the whole or a part of the nucleotide sequence of SEQ ID
NO: 18, 19, or 25) which can hybridize to a mutation site or
polymorphic site, as well as by directly determining the
sequences.
[0251] By using the judging methods of the present invention, it
becomes possible to select at an early stage plants (for example,
rice) to be identified as having field resistance to blast.
Specifically, the present invention provides methods for selecting
plants having field resistance to blast, which comprise the
following steps (a) and (b): [0252] (a) producing varieties in
which plants (for example, rice) having field resistance to blast
have been crossed with plants (for example, rice) having arbitrary
functions; [0253] (b) judging whether the plants obtained in step
(a) have field resistance to blast by the methods herein described
for judging whether test plants have field resistance to blast.
[0254] The term "plant" is not particularly limited in the present
invention, but preferably refers to rice. Specific examples of rice
are as mentioned above.
[0255] By using the selection methods of the present invention, it
becomes possible to select at an early stage plants (for example,
rice) to be identified as having field resistance to blast. The
present invention also provides such methods for selecting at an
early stage plants to be identified as having field resistance to
blast. As used herein, the term "early stage" refers to, for
example, the state before heading of rice, and preferably the state
immediately after germination. By using the selection methods of
the present invention, it becomes possible to breed varieties
having field resistance to blast in a shorter period than
before.
[0256] The present invention relates to methods of screening for
agents to prevent or ameliorate blast in plants. The first
embodiment of the screening methods of the present invention
includes methods of screening for agents to prevent or ameliorate
blast in plants, which comprise the following steps (a) to (c):
[0257] (a) contacting test compounds with a Pi21 gene transcription
product; [0258] (b) detecting the binding of the test compounds to
the Pi21 gene transcription product; and [0259] (c) selecting test
compounds that bind to the Pi21 gene transcription product.
[0260] In the first embodiment, test compounds are first contacted
with the Pi21 gene transcription product. "Pi21 gene transcription
product" in the screening methods of the present invention includes
not only the Pi21 gene transcription product, but also the
translation product translated from the transcription product.
[0261] The "test compounds" in the methods of the present invention
are not particularly limited, and include, for example, single
compounds such as natural compounds, organic compounds, inorganic
compounds, proteins, and peptides; as well as compound libraries,
expression products of gene libraries, cell extracts, cell culture
supernatants, products of fermentation microorganisms, marine
organism extracts, plant extracts, prokaryotic cell extracts,
unicellular eukaryote extracts, and animal cell extracts. If
needed, the above test compounds can be appropriately labeled
before use. Labels include, for example, radiolabels and
fluorescent labels.
[0262] In the present invention, "contacting" is carried out as
follows. For example, if the Pi21 gene transcription product is in
a purified state, the contact can be carried out by adding test
compounds to the purified preparation. If the transcription product
is in the state expressed in cells, or in the state expressed in
cell extracts, the contact can be carried out by adding test
compounds to the cell cultures or to the cell extracts,
respectively. The cells in the present invention are not
particularly limited, but cells derived from plants including rice
are preferable. When the test compounds are proteins, the contact
can also be carried out, for example, by introducing vectors
comprising the DNAs encoding the proteins into cells expressing the
Pi21 gene, or by adding the vectors to cell extracts in which the
Pi21 gene is expressed. Further, for example, two hybrid methods
using yeast or animal cells can be utilized.
[0263] In the first embodiment, the binding between the
above-mentioned Pi21 gene transcription product and test compounds
is subsequently detected. Detection or measurement of the binding
between proteins can be carried out by using, for example, labels
attached to the proteins. The types of labels include, fluorescent
labels and radiolabels, for example. The binding can also be
measured by known methods such as the yeast two hybrid method and
the method using BIACORE. In the present methods, the test
compounds bound to the above-mentioned biosynthesis enzyme are then
selected. Among the selected test compounds, agents for preventing
or ameliorating blast in plants are included. The selected test
compounds may be used as test compounds in the following
screenings.
[0264] In addition, the second embodiment of the screening methods
of the present invention provides methods of screening for agents
to prevent or ameliorate blast in plants, which comprise the
following steps (a) to (c): [0265] (a) contacting test compounds
with cells collected from plants; [0266] (b) measuring the
expression level of thePi21 gene transcription product; and [0267]
(c) selecting the test compounds that decrease the expression level
of the transcription product as compared to when the test compounds
are not contacted with the cells.
[0268] In the second embodiment, test compounds are first contacted
with cells collected from plants. As used herein, the term "cells
collected from a plant" may be an arbitrary plant clearly having a
blast susceptibility gene. The terms "test compound" and
"contacting" refer to the same as mentioned above.
[0269] In the second embodiment, the expression level of the "Pi21
protein" is subsequently measured. The expression level of the Pi21
protein can be measured by methods known to one skilled in the art.
For example, mRNA encoding the Pi21 protein is extracted according
to a conventional method, and the transcription level of the Pi21
gene can be measured by performing the Northern hybridization
method or the RT-PCR method using this mRNA as a template. Further,
the expression level of the Pi21 protein can be measured using DNA
array techniques.
[0270] The translation level of the gene can also be measured by
collecting fractions comprising the Pi21 protein in accordance with
a usual method, and detecting the expression of the Pi21 protein by
electrophoresis such as SDS-PAGE. The translation level of the gene
can also be measured by performing the Western blotting method
using an antibody against the Pi21 protein to detect the expression
of the Pi21 protein.
[0271] The antibodies used for detection of the Pi21 protein are
not particularly limited, as long as they can detect the Pi21
protein. Both monoclonal antibodies and polyclonal antibodies can
be used, for example. The antibodies can be prepared as mentioned
above, by methods known to one skilled in the art.
[0272] In the second embodiment, next, when the expression level of
the Pi21 protein decreases compared to when the test compounds are
not contacted, the test compounds are selected as agents to prevent
or ameliorate blast in plants.
[0273] The third embodiment of the screening methods of the present
invention provides methods of screening for agents to prevent or
ameliorate blast in plants, which comprise the following steps (a)
to (d): [0274] (a) providing cells or cell extracts comprising DNAs
in which a reporter gene is operably linked downstream of the
promoter region of the Pi21 gene; [0275] (b) contacting test
compounds with cells or the cell extracts; [0276] (c) measuring the
expression level of the reporter gene in the cells or the cell
extracts; and [0277] (d) selecting test compounds that decrease the
expression level of the reporter gene as compared to when the test
compounds are not contacted.
[0278] In the third embodiment, cells or cell extracts comprising
DNAs in which a reporter gene is operably linked downstream of the
promoter region of the Pi21 gene, are first provided.
[0279] In the third embodiment, the term "operably linked" means
that the promoter region of the Pi21 gene and a reporter gene are
connected to each other so that the reporter gene expression can be
induced by binding of a transcription factor to the promoter region
of the Pi21 gene. Therefore, the term "operably linked" also
inlcudes such cases where a reporter gene is connected to another
gene and produces a fused protein with another gene product, as
long as expression of the fused protein is induced by binding of a
transcription factor to the promoter region of the Pi21 gene.
[0280] The reporter gene is not particularly limited, so long as
its expression can be detected. For example, reporter genes
generally used by those skilled in the art include the CAT gene,
lacZ gene, luciferase gene, .beta.-glucuronidase gene (GUS), and
GFP gene.
[0281] In the third embodiment, the above-mentioned cells or cell
extracts are subsequently contacted with the test compounds. Then,
the expression level of the reporter gene in the cells or the cell
extracts is measured. The terms "test compound" and "contacting"
refer to the same as mentioned above.
[0282] The expression level of the reporter gene can be determined
using methods known to those skilled in the art, according to the
type of reporter gene. For example, when using the CAT gene as the
reporter gene, the CAT gene expression level can be determined by
measuring the acetylation of chloramphenicol, caused by the CAT
gene product. When the lacZ gene is used as the reporter gene, its
expression level can be determined by analyzing the colouring of a
dye compound due to the catalytic action of the gene expression
product. The expression level of the luciferase gene as a reporter
can be determined by measuring the fluorescence of a fluorescent
compound, caused by the catalytic action of the luciferase gene
expression product. The expression level of the
.beta.-glucuronidase (GUS) gene can be determined by analyzing the
coloring of 5-bromo-4-chloro-3-indolyl-.beta.-glucuronide (X-Gluc)
or the luminescence of Glucuron (ICN), caused by the catalytic
action of the GUS gene expression product. The expression level of
the GFP gene can be determined by measuring fluorescence due to the
GFP protein.
[0283] Next in the third embodiment, if the expression level of the
above-mentioned genes decrease compared to when the test compounds
are not contacted, the test compounds are selected as agents to
prevent or ameliorate blast in plants.
[0284] The fourth embodiment of the screening methods of the
present invention provides methods of screening for agents to
prevent or ameliorate blast in plants, which comprise the following
steps (a) to (c): [0285] (a) regenerating transformed plants from
transformed plant cells into which the Pi21 gene has been
introduced; [0286] (b) contacting the blast fungus and test
compounds with the transformed plants; and [0287] (c) selecting
test compounds that suppress blast in the transformed plants as
compared to when the test compounds are not contacted.
[0288] In the fourth embodiment, transformed plants are first
regenerated from transformed plant cells comprising the Pi21 gene.
The transformed plants can be regenerated as mentioned above, by a
method known to one skilled in the art.
[0289] In the fourth embodiment, next, the blast fungus and test
compounds are contacted with the transformed plants regenerated in
step (a). The terms "blast fungus" and "test compound" are the same
as mentioned above. An example of "contacting" is a method for
directly spraying a test compound on a plant using a sprayer.
However, "contacting" in the fourth embodiment is not limited
thereto, but includes any method, as long as plants and test
compounds can physically contact. The contact of the present
invention may be performed by contacting test compounds with
transformed plants infected with the blast fungus, or by infecting
with the blast fungus transformed plants which have contacted with
test compounds.
[0290] In the fourth embodiment, next, test compounds that suppress
blast in transformed plants as compared to when test compounds are
not contacted, are selected. Whether blast is suppressed or not can
be determined by using as an indicator a phenotype of the
transformed plants. The phenotypes of the transformed plants are
not particularly limited, but include discoloring and necrotizing
of an entire part of a plant, or a portion of it. Moreover,
suppression of blast in the transformed plants includes not only
complete suppression but also partial suppression.
[0291] The present invention also relates to kits for use in the
above-described screening methods. Such kits can comprise materials
used at the detection step and/or measurement step in the
above-described screening methods. For example, such materials can
include probes, primers, antibodies, and stain solutions, which are
necessary for measuring Pi21 gene expression level. In addition,
the kits may comprise distilled water, salts, buffer solutions,
protein stabilizers, preservatives and the like.
[0292] All prior art references cited in the present specification
are incorporated herein by reference.
EXAMPLES
[0293] Hereinafter, the present invention will be specifically
described with reference to Examples, but it is not construed as
being limited thereto.
Example 1
Genetic Mapping
[0294] A detailed linkage analysis of the pi21 region was conducted
using a large-scale segregating population indispensable for
map-based cloning. As the population for linkage analyses, 72
samples of the BC1F2 population were used. This BC1F2 population
was obtained by continuously backcrossing the paddy rice variety
Nipponbare or Aichi Asahi (FIG. 1) comprising the susceptibility
allele Pi21 that does not suppress blotch progression with the
Japanese upland rice variety Owarihatamochi comprising the
resistance allele pi21 that suppresses blotch progression. As a
result of linkage analysis with RFLP markers, it was found that the
pi21 gene locus is located between the RFLP markers G271 and G317
(FIG. 2A).
[0295] In order to create a more accurate genetic map of the pi21
region, a total of 1014 samples including the above-mentioned
crossbred 229 samples and 643 samples of the progeny BC1F4
population were used to select 27 samples with chromosomal
recombination near the pi21 locus, by using the RFLP markers RA3591
and 13S1 located on both sides of the pi21 locus (FIG. 2B).
Furthermore, a search was carried out using 2703 samples of F2
population, which was obtained by crossing a line having the
genetic background of Japanese paddy rice variety and the
susceptibility allele from the Indian paddy rice variety Kasalath
with a line having the resistance allele from Owarihatamochi. 24
samples with chromosome recombination near the pi21 locus were
selected using the PCR markers 14T1 and 4S1 located on both sides
of the pi21 locus. Furthermore, using those samples, a detailed
linkage map was created utilizing the DNA markers produced in the
following procedures.
Example 2
Alignment of P1-Derived Artificial Chromosome (PAC) Clones in the
pi21 Gene Region
[0296] Using the alignment map of Nipponbare PAC clones produced in
the rice genome research program, PAC clones comprising the DNA
markers RA3591 and C975 nucleotide sequences positioned near the
pi21 gene locus were identified (FIG. 2C). Furthermore, terminal
fragments of the identified PAC clones P479G02, P415D09, P473G08,
P703E11, P434F09, P702D03, P419B08, P472G09, and P502G01 were
isolated by the cassette method, and the identified PAC clones were
aligned. As a result, it was found that the PAC clones P032D02,
P678A02, P405D12, P689F04, P479G02, P415D09, P473G08, P703E11,
P434F09, and P702D03 comprise the pi21 gene region (FIG. 2C).
Example 3
Narrowing the Candidate Region of the pi21 Gene
[0297] Terminal fragments of the PAC clones aligned in the pi21
region were cloned, and the obtained clones are used as new RFLP
markers or CAPS markers to create a detailed genetic map. As a
result, it was found that the pi21 gene locus exists in the genomic
region sandwiched between the SSCP marker Pa102484 and the SNP
marker P702D3.sub.--#12. Accordingly, it was revealed that the pi21
gene locus is located in the genomic region of about 25 kb
sandwiched by the two markers (FIG. 2D).
[Example 4 ]
Identification of the Candidate Gene Region by Nucleotide Sequence
Analysis
[0298] The nucleotide sequence of the PAC clone P702D03 considered
to comprise the pi21 gene was determined, and the nucleotide
sequence of 25 kb candidate genomic region in the resistant variety
Owarihatamochi and the susceptible varieties Aichi Asahi and
Kasalath were analyzed. The nucleotide sequence was analyzed by
using the DNA fragments which were amplified from the
above-mentioned three varieties with primers designed utilizing the
sequence of the candidate region in Nipponbare and by using the
dye-terminator method. The candidate region was further narrowed
using the nucleotide polymorphism information in the candidate gene
region identified by linkage analysis. As a result, the pi21 gene
was shown to co-segregate with the STS marker P702D03.sub.--#79
(primers: 5'-AGA AGG TGG AGT ACG ACG TGA AGA-3' (SEQ ID NO: 8) and
AGT TTA GTG AGC CTC TCC ACG ATT A-3' (SEQ ID NO: 9)), and one
recombinant was detected between the SNP markers P702D03.sub.--#38
(primers: TTT TCC TGA GAA ATT TGT AAA GA-3' (SEQ ID NO: 12) and CGT
CGA CGA TGA GGA TCT-3' (SEQ ID NO: 13)) and P702D03.sub.--#80
(primers: 5'-CTC CCA ATG TGT TTA GCA TC-3'(SEQ ID NO: 14) and
5'-CAA CCA TAT GTC CCT AAG GAT-3' (SEQ ID NO: 15)), respectively.
These results showed that the pi21 gene is located in the genomic
region of about 1.8 kb sandwiched between the SNP markers
P702D03.sub.--#38 and P702D03.sub.--#80 (FIG. 2D).
[0299] The nucleotide sequence of the isolated Pi21 gene derived
from rice (Oryza sativa L, varieties Aichi Asahi and Nipponbare) is
shown in SEQ ID NO: 1, the nucleotide sequence of its cDNA is shown
in SEQ ID NO: 2, and the amino acid sequence of the protein ("the
Pi21 protein") encoded by the cDNA is shown in SEQ ID NO: 3. In
addition, the nucleotide sequence of the Pi21 gene derived from
Kasalath, corresponding to the Pi21 gene of Aichi Asahi and
Nipponbare, is shown in SEQ ID NO: 20, the nucleotide sequence of
its cDNA is shown in SEQ ID NO: 21, and the amino acid sequence of
the protein ("''the Pi21 protein") encoded by the cDNA is shown in
SEQ ID NO: 22.
Example 5
Nucleotide Sequence Analysis of the pi21 Candidate Gene
[0300] When a gene prediction and similarity search were carried
out for the sequence of the 1.8 kb candidate genomic region of the
variety Nipponbare, full-length cDNA clones of Nipponbare
(AK106153, AK070581, and AK072320) were discovered. However, no
similar genes were present in Arabidopsis or the like, and thus the
function of the gene could not be predicted from homology.
Nevertheless, a metal-binding site at a position about 10 amino
acids away from the predicted translation initiation site in this
gene brings to mind a gene reported in Arabidopsis (Hirayama et
al., 1999 Cell) with the function of a chaperone which carries a
metal in the ethylene signaling system. Since sensibility to
ethylene in the near-isogenic line AA-pi21 is actually changed
compared to Aichi Asahi, the site may have a similar function.
Primers which can amplify the corresponding part were designed
using the already obtained nucleotide sequence information of
Nipponbare, and the nucleotide sequences of the genomic PCR and
RT-PCR products of the susceptible varieties Nipponbare and Aichi
Asahi were compared with those of the resistant variety
Owarihatamochi. As a result, DNA mutations were found at two sites
in the exon region of the gene in the resistant variety compared to
the susceptible varieties. In the resistant variety, deletions of 7
amino acids and 16 amino acids were found relative to the
susceptible varieties, and these mutations were thought to be
associated with blotch progression in blast that had infected (FIG.
3).
[0301] The nucleotide sequence of the isolated pi21 gene is shown
in SEQ ID NO: 4, the nucleotide sequence of its cDNA is shown in
SEQ ID NO: 5, and the amino acid sequence of the protein encoded by
the cDNA ("the pi21 protein") is shown in SEQ ID NO: 6.
Example 6
Identification of the Function of the Candidate Gene by
Transformation
[0302] (1) Introduction of the Susceptibility Gene into AA-pi21
[0303] An XbaI 4.7 kb fragment of the genomic region including 5'
upstream predicted promoter region of the susceptible variety
Nipponbare, identified as a candidate of the pi21 gene, was
incorporated into the vector pPZP2H-lac that can be transformed
through Agrobacterium. Transformation was carried out by the method
of Toki (Plant Mol. Biol. Rep. 15: 16-21, 1997) using a vector into
which this fragment had been introduced and a vector alone. As the
line to be transformed, the pi21 near-isogenic line AA-pi21 was
used. 36 hygromycin-resistant organisms were obtained from the
vector into which the XbaI 4.7 kb fragment had been introduced, and
12 hygromycin-resistant organisms were obtained from the vector
alone. Whether the introduced region was incorporated or not, was
investigated by the PCR method using primers (sense strand: 5'-GTA
CGA CGT GAA GAA CAA CAG G-3' (SEQ ID NO: 16)) and (antisense
strand: 5'-GCT TGG GCT TGC AGT CC 3' (SEQ ID NO: 17)) that were
specific to the candidate gene. As a result, it was found that the
candidate gene was incorporated into all the transformants. These
organisms were grown in an isolated greenhouse, and the blast
fungus (race 007) was inoculated into the inbred line progenies. As
a result, in the organisms into which the vector alone was
introduced and the T1 organisms into which the introduced gene was
not delivered due to segregation, blotch progression caused by
blast was suppressed as shown in the near-isogenic line AA-pi21,
compared to the susceptible variety Aichi Asahi. In contrast, in
the T1 organisms into which the candidate gene had been introduced,
blotches progressed more extensively (FIG. 4). Especially, the
degree of sensibility was increased in the lines having a high copy
number of the introduced gene.
(2) Introduction of the Resistance Gene to a Susceptible
Variety
[0304] On the other hand, the XbaI 4.7 kb fragment of the resistant
variety Owarihatamochi was incorporated into the vector in the same
way, and the susceptible variety Aichi Asahi was transformed. 56
Hygromycin-resistant organisms were obtained from the vector into
which the XbaI 4.7 kb fragment had been introduced, and 24
hygromycin-resistant organisms were obtained from the vector alone.
Similarly to (1), whether the introduced region was incorporated or
not was investigated by the PCR method, and it was found that the
candidate gene was incorporated into all the transformants. These
organisms were grown in an isolated greenhouse in the same way as
(1), and the blast fungus (race 007) was inoculated into the inbred
line progenies. As a result, in all of the organisms into which the
vector alone had been introduced, T1 organisms to which the
introduced gene was not delivered, and T1 organisms into which the
candidate gene had been introduced, blast progression was observed
to the same degree as that in the susceptible variety Aichi
Asahi.
(3) Identification of the Function of the Candidate Gene
[0305] From the above results, it was found that the candidate gene
region from Nipponbare (XbaI 4.7 kb) has the function of promoting
blotch formation in the near-isogenic line AA-pi21, and thus the
candidate gene was judged to be the Pi21 gene.
Example 7
Mutations of the Candidate Gene in Rice
[0306] Mutations of the candidate gene were searched using 79 rice
varieties in the world. As a result, in addition to the mutation
types found in Nipponbare, Aichi Asahi, and Owarihatamochi, ten
types of mutations having insertions and/or deletions in the exon
region were found. These mutations are mainly defined by the
presence or absence and the size of an insertion/deletion at the
two deletion sites found in Owarihatamochi compared to Nipponbare
and Aichi Asahi. Because of the similarity to the metal molecule
chaperone proposed in the ethylene signaling system of Arabidopsis
thaliana, this region having no homology with known genes is
expected to bind to another molecule. Thus, the mutations in this
region may delicately control the signaling efficiency and bring
about functional alterations.
[0307] From the above results, the candidate gene narrowed down by
the map-based cloning method was found to be the pi21 gene which
suppresses blotch progression in rice blast disease. This
achievement is the first case to prove the biological function of a
quantitative resistance in a plant. The expression of the pi21 or
the Pi21 gene was investigated by RT-PCR analysis, and each gene
was found to be constitutively expressed in all the tissues of the
aerial part. Therefore, it is expected that these genes play a
fundamental role in the growth of plants. Since change of the copy
number leads to phenotype changes, alteration of the expression
level and the tissues where the genes are expressed by promoters
can be an important factor for functional modification. That is, it
may be possible to efficiently ameliorate disease resistance that
plants originally have, by utilizing the isolated pi21 gene or
other alleles found in the species.
INDUSTRIAL APPLICABILITY
[0308] The characteristics of the Pi21 gene are especially suitable
for producing varieties having field resistance to blast in plants.
Until now, in order to confer plants with field resistance to
blast, it was necessary to cross a variety that originally has
field resistance and inferior characteristics with a variety that
does not have field resistance but has many superior
characteristics, and to select from among their progenies, plants
having excellent field resistance as well as other excellent
characteristics. However, the precise evaluation of field
resistance needs a lot of effort. Moreover, when the exact position
on the chromosome of the gene that confers this resistance is
unclear, it is difficult to select this gene efficiently and
accurately and to introduce it into a variety with a high practical
use. In fact, this had not succeeded until now.
[0309] The present invention provides the chromosomal position and
the structure of the gene involved in field resistance. Thus it
became possible to efficiently confer plants with field resistance.
It also became possible to breed varieties having resistance and
highly practical characteristics by changing the tissue specificity
of expression and the expression level of the gene participating in
field resistance. Accordingly, the genes of the present invention
are useful for realizing very practical and highly safe
agriculture. Moreover, plants produced by the methods of the
present invention are expected, for example, to stably give a high
yield when it comes to useful agricultural plants, and also gain a
new aesthetic value when it comes to ornamental plants.
Sequence CWU 1
1
2714817DNAOryza sativa 1ctagatgatg ctttatgtga aggcaataaa tgataattaa
gtgtaatata atgtattttt 60taaacatcgt agtgaagtcc catttctagt acgtaatgga
tcgaaatggg gagggactgc 120ttctctttta gaaaaaaaaa atgtaaaatt
gagatgagct tttataagac atgtaaggcc 180atatgtctag tgtaagtatg
atgctagcaa gcacatttta gcatcttaaa ataaaaagca 240agcacattaa
tattttagga gttagcatgg atgcaataca gtagaagagg agatgccaac
300aaaaaagaat atggaaaatt tgtttcatac catcaaaagt tcctgtgttt
tgctttatac 360catcagcgtt tgtggtgtac tttaacacca tccaaagttt
gctccgttac ctatctactc 420cacgaaatgt gcttgctagc atggatgaaa
aagtaaccga aacaaacagt tgtggtatac 480tcctaagtaa tcctaagtag
ccaccggatt caatacttta ttctcaatcg tccttctagt 540ctattcccaa
ttatatgggt ttgacccgtt acaacgtacg gacatgttac tagcgtaagg
600aaagagatgg tttctgaaag agtttttttt ttccaacagt cctacccaat
ctatagcgct 660agacaagaaa gcgaaagcta aacccatatt tattaccgta
atgttgcaaa cttaatatta 720cctgcaatag acatgcccta acatggtttc
aggaccacaa accagagctc taaaattccg 780agcacatgtt cagaatatat
atacattttt aattcccttc tatactactc caacttacct 840ctatggaggt
aagaggctaa aattgaaact agataactga atgttcttat atttttccta
900ataaaaatat aattgctcct ttcatttaat acttatatca caacatgtag
ccatatgagc 960caaaactgta ttagccatat tttagcgaat aaggctcaat
gcgattctgt gtctgagctc 1020tggcctgaaa attcttgtaa ctttatctca
catatggtgt gtgaggaagc ttctcatgag 1080taatatattc cctatttccc
cgcaaaaaaa aaaacttata tcacaacaaa aatggaaaag 1140accaacactg
agtattgtag aagctagtac ttttggcaaa tgtgcccgaa gatccccaaa
1200acagtttgtg gcataattca gaagattgtt agctcagaaa tcatttcaga
ctaggatatt 1260gccgacacat cagagttctc gtgtaatcac gaagcaagga
actgtacagt gcagtgatga 1320tatacaacta taggagagga cgaactgata
ctgacggatg ctgtgtgtaa aatatctctg 1380taaaatcagg aatactggga
gactgattgg atgataggtt tatacggtca caatcatctc 1440gttaaacaca
cttttcatta aagaggaatt accaaataca gtaattgatt cgacggtgcc
1500gcgagaaatg tgctatatat aaatgctcac ctatatttgg gcagattagt
taatttcagg 1560aattttccac agagaaagct aaatgagtgc taaatgagtc
gcattgcgta ctactacaag 1620ggtccaaaca gaaattgata tctgaaatta
gcttttctta agataacaat gatatttttt 1680tttattgaag cacaaggtgt
gcctgaaatc gaattctgaa aggtattctt atttttcaga 1740ggaaaatgta
cagtggtacc attctgaaag ctaattgaga gatgacaagg cgtgagaagg
1800accaaactgt cctatataca tgtggtcatt ttccatctct tgcaataggt
tatcaagacc 1860cctcctgaaa catggaccgt ccagatgcga tccgacggac
gaaaaaaacc aatggcagaa 1920tatttcaggc tctggcatat ccaaacataa
ataagtaaca taaggttcag ctctgctcca 1980tccatgcatc gcctccatta
ttcatgcttc gattctccat gctttcctct actgctcatt 2040ggtaacattc
ggcaaatttg acaggtgagc tcagtatttt aaatcttaat gtagtacttt
2100ggtgtgctaa tctttgctct gttcaaaaga gaattctggt ttctttgcta
ttttgaaaag 2160agaattttcg ttacaggact tcaacttcca taatactttt
ttttataatt aaggcatgat 2220atatatcttc tttctgaatt ccacgggaat
tgcacttttt cctgagaaat ttgtaaagag 2280catgcctgtt aattgcaagg
ggcccctaac tctgttatga gaaaagagaa ctatataaga 2340tgctcaataa
gcacctcttt ttttttttct gttaactgac caaagcctgt ctatctgcat
2400ttttttttgt tttttgtttt tcttgtgtgc agatgggtat attggtcatc
ttggtggacc 2460tgcaatgctg ccgctgcgat gccaagatca ggaaggtcct
gggctgcctt gaaggtataa 2520taaattctgc ccgaatcgtc catgtttgat
tgaattttca aggctaatca gcagtgttcc 2580tgctcaattg ggagcaaaac
ctctgttaaa aagggtgtgt ttgaatgaat ataattgaat 2640atgaacgcag
aggagtactg catcgagaag gtggagtacg acgtgaagaa caacagggtg
2700atcgtgcgcg ggaagttcga cccggagaag ctgtgcaaga agatctggtg
caaggccggc 2760aagatcatca aggagatcct catcgtcgac gtctggccgc
cgccgctgcc gcagccgccg 2820ccgccgtgca agccgccgcc gtgcgagaag
cctccggagg actgcaagcc caagccctgc 2880cattgctgca gctgcgagaa
gcccaagccc aagcccaagc cctgccactg cgagaagccc 2940aagccctgtc
actgcgagaa gcccaagcca tgcgagaagc cgccgccgtg caagccggag
3000gagccgccga agccgccgcc ggagaagccg ccgccgaagc cggagtgcaa
gctggtgccg 3060tacccttacc cggtgccgta cccgtacgcc gggcagtggt
gctgcccaaa gcctgagccg 3120ccgaagccgc cgccggagcc accgaaggag
ccggagccgc cgaagccgtg cgggtgctcg 3180cacgccttcg tgtgcgtctg
caagccggcg ccgccgccgc cgccgccgtg cgggtgctcg 3240gggggccacg
ggaactgcgg ctgcggcatc aggccgtggc cgccgcaggt gtggccgccg
3300ccgcccgtct gcccgccgcc gccgtggtgc tacaccgagg acaacgccaa
cgcctgctcc 3360atcatgtgat ggccggccgg cggtcggcgt cgatcacgat
catctctgct gcttaatttc 3420cttgcttgct actacctctg ctcctttcct
tgcctcggaa atcggaataa attaaacacg 3480aggctgatcg atgtgtttgt
aattaatcca tggtgtttgt gttgtgtgct gtgtgggctg 3540tataataatt
aattacagta tgttcatgta aatttgtttg tttgtttgtt tatgttgttc
3600gatatgtata attatgtaca ataattaatc gtggagaggc tcactaaact
cataaactgt 3660agagtatctt ggctgtaaaa gtgtggcaat ttatcttttt
cttgtgttag cattggctac 3720aaatagtttt tggccgtctt ttctcttcgt
ttctcccctt ctttatgaga ttaattgtgt 3780gctgacctag atcaaattat
agcgcgctga cctagtttta ttgtaactgc tcttatggat 3840gtctgctaac
accatcaaac atgattaccg tggtatattt gtcttaatta ctactaacta
3900ggactaccta ggggcaccct tgcatatgtt tttttttcga acgaccagat
agatttgagt 3960catttgacta gcgttatatt aataggaggg aaaaaaaata
caaagtacaa atatccaaca 4020ggccgagaaa agagaaaaaa aactgtacat
gcctacgtgc aaacaagatt gcacgaagct 4080ctcctcactc ctcatggcaa
cattctccca atgtgtttag catcgaaaat accccaccaa 4140cctcgtcttg
gatgtgactg gtcatttggt gtattgtcgc ctccttttcc ctctgaagat
4200tcttacgttc cattccttcc aaatttccta ggcgatgagc aagaaaagag
atctgagccc 4260tttttgttta gttacttcca agtcgcttgt tccttgtgtc
caccaatgca agaggtttct 4320actgtcattc catttcagaa taagccaatt
gagaactcca aaccagattt acttcgaaac 4380caggcattcg aacataaggt
ggtcgacaat gtcgagattc cggattagca gagttatttg 4440gtttgagagt
cacattccta caaccaatag aaaaccatcc tatggaccat ttagatagtg
4500tcctagtatt tgcttttata tttgctataa atattcttcc tttattgttt
ttggagttca 4560caaacttaat aatcaaactt tgtgattttt taatttccat
taacgaattc aaggaaccac 4620ctttatctct catcttcatt gcacactact
gatttctttc atccttaggg acatatggtt 4680gatacggaga ctgtttttct
atcattatct aaaaaaaatc taaggggcat atatatattg 4740tgttttctct
cattcatgca tttcgcactt ttccctattc gtgaaatacc atttcccaca
4800tgagtgcaat gtttctt 48172801DNAOryza sativa 2atgggtatat
tggtcatctt ggtggacctg caatgctgcc gctgcgatgc caagatcagg 60aaggtcctgg
gctgccttga agaggagtac tgcatcgaga aggtggagta cgacgtgaag
120aacaacaggg tgatcgtgcg cgggaagttc gacccggaga agctgtgcaa
gaagatctgg 180tgcaaggccg gcaagatcat caaggagatc ctcatcgtcg
acgtctggcc gccgccgctg 240ccgcagccgc cgccgccgtg caagccgccg
ccgtgcgaga agcctccgga ggactgcaag 300cccaagccct gccattgctg
cagctgcgag aagcccaagc ccaagcccaa gccctgccac 360tgcgagaagc
ccaagccctg tcactgcgag aagcccaagc catgcgagaa gccgccgccg
420tgcaagccgg aggagccgcc gaagccgccg ccggagaagc cgccgccgaa
gccggagtgc 480aagctggtgc cgtaccctta cccggtgccg tacccgtacg
ccgggcagtg gtgctgccca 540aagcctgagc cgccgaagcc gccgccggag
ccaccgaagg agccggagcc gccgaagccg 600tgcgggtgct cgcacgcctt
cgtgtgcgtc tgcaagccgg cgccgccgcc gccgccgccg 660tgcgggtgct
cggggggcca cgggaactgc ggctgcggca tcaggccgtg gccgccgcag
720gtgtggccgc cgccgcccgt ctgcccgccg ccgccgtggt gctacaccga
ggacaacgcc 780aacgcctgct ccatcatgtg a 8013266PRTOryza sativa 3Met
Gly Ile Leu Val Ile Leu Val Asp Leu Gln Cys Cys Arg Cys Asp1 5 10
15Ala Lys Ile Arg Lys Val Leu Gly Cys Leu Glu Glu Glu Tyr Cys Ile
20 25 30Glu Lys Val Glu Tyr Asp Val Lys Asn Asn Arg Val Ile Val Arg
Gly 35 40 45Lys Phe Asp Pro Glu Lys Leu Cys Lys Lys Ile Trp Cys Lys
Ala Gly 50 55 60Lys Ile Ile Lys Glu Ile Leu Ile Val Asp Val Trp Pro
Pro Pro Leu65 70 75 80Pro Gln Pro Pro Pro Pro Cys Lys Pro Pro Pro
Cys Glu Lys Pro Pro 85 90 95Glu Asp Cys Lys Pro Lys Pro Cys His Cys
Cys Ser Cys Glu Lys Pro 100 105 110Lys Pro Lys Pro Lys Pro Cys His
Cys Glu Lys Pro Lys Pro Cys His 115 120 125Cys Glu Lys Pro Lys Pro
Cys Glu Lys Pro Pro Pro Cys Lys Pro Glu 130 135 140Glu Pro Pro Lys
Pro Pro Pro Glu Lys Pro Pro Pro Lys Pro Glu Cys145 150 155 160Lys
Leu Val Pro Tyr Pro Tyr Pro Val Pro Tyr Pro Tyr Ala Gly Gln 165 170
175Trp Cys Cys Pro Lys Pro Glu Pro Pro Lys Pro Pro Pro Glu Pro Pro
180 185 190Lys Glu Pro Glu Pro Pro Lys Pro Cys Gly Cys Ser His Ala
Phe Val 195 200 205Cys Val Cys Lys Pro Ala Pro Pro Pro Pro Pro Pro
Cys Gly Cys Ser 210 215 220Gly Gly His Gly Asn Cys Gly Cys Gly Ile
Arg Pro Trp Pro Pro Gln225 230 235 240Val Trp Pro Pro Pro Pro Val
Cys Pro Pro Pro Pro Trp Cys Tyr Thr 245 250 255Glu Asp Asn Ala Asn
Ala Cys Ser Ile Met 260 26544745DNAOryza sativa 4ctagatgatg
ctttatgtga aggcaataaa tgataattaa gtgtaatata atgtattttt 60taaacatcgt
agtgaagtcc catttctagt acgtaatgga tcgaaatggg gagggactgc
120ttctctttta gaaaaaaaat gtaaaattga gatgagcttt tataagacat
gtaaggccat 180atgtctagtg taagtatgat gctagcaagc acattttagc
atcttaaaat aaaaagcaag 240cacattaata ttttaggagt tagcatggat
gcaatacagt agaagaggag atgccaacaa 300aaaagaatat ggaaaatttg
tttcatacca tcaaaagttc ctgtgttttg ctttatacca 360tcagcgtttg
tggtgtactt taacaccatc caaagtttgc tccgttacct atctactcca
420cgaaatgtgc ttgctagcat ggatgaaaaa gtaaccgaaa caaacagttg
tggtatactc 480ctaagtaatc ctaagtagcc accggattca atactttatt
ctcaatcgtc cttctagtct 540attcccaatt atatgggttt gacccgttac
aacgtacgga catgttacta gcgtaaggaa 600agagatggtt tctgaaagag
tttttttttt ccaacagtcc tacccaatct atagcgctag 660acaagaaagc
gaaagctaaa cccatattta ttaccgtaat gttgcaaact taatattacc
720tgcaatagac atgccctaac atggtttcag gaccacaaac cagagctcta
aaattccgag 780cacatgttca gaatatatat acatttttaa ttcccttcta
tactactcca acttacctct 840atggaggtaa gaggctaaaa ttgaaactag
ataactgaat gttcttatat ttttcctaat 900aaaaatataa ttgctccttt
catttaatac ttatatcaca acatgtagcc atatgagcca 960aaactgtatt
agccatattt tagcgaataa ggctcaatgc gattctgtgt ctgagctctg
1020gcctgaaaat tcttgtaact ttatctcaca tatggtgtgt gaggaagctt
ctcatgagta 1080atatattccc tatttccccg caaaaaaaaa aacttatatc
acaacaaaaa tggaaaagac 1140caacactgag tattgtagaa gctagtactt
ttggcaaatg tgcccgaaga tccccaaaac 1200agtttgtggc ataattcaga
agattgttag ctcagaaatc atttcagact aggatattgc 1260cgacacatca
gagttctcgt gtaatcacga agcaaggaac tgtacagtgc agtgatgata
1320tacaactata ggagaggacg aactgatact gacggatgct gtgtgtaaaa
tatctctgta 1380aaatcaggaa tactgggaga ctgattggat gataggttta
tacggtcaca atcatctcgt 1440taaacacact tttcattaaa gaggaattac
caaatacagt aattgattcg acggtgccgc 1500gagaaatgtg ctatatataa
atgctcacct atatttgggc agattagtta atttcaggaa 1560ttttccacag
agaaagctaa atgagtgcta aatgagtcgc attgcgtact actacaaggg
1620tccaaacaga aattgatatc tgaaattagc ttttcttaag ataacaatga
tatttttttt 1680tattgaagca caaggtgtgc ctgaaatcga attctgaaag
gtattcttat ttttcagagg 1740aaaatgtaca gtggtaccat tctgaaagct
aattgagaga tgacaaggcg tgagaaggac 1800caaactgtcc tatatacatg
tggtcatttt ccatctcttg caataggtta tcaagacccc 1860tcctgaaaca
tggaccgtcc agatgcgatc cgacggacga aaaaaaccaa tggcagaata
1920tttcaggctc tggcatatcc aaacataaat aagtaacata aggttcagct
ctgctccatc 1980catgcatcgc ctccattatt catgcttcga ttctccatgc
tttcctctac tgctcattgg 2040taacattcgg caaatttgac aggtgagctc
agtattttaa atcttaatgt agtactttgg 2100tgtgctaatc tttgctctgt
tcaaaagaga attctggttt ctttgctatt ttgaaaagag 2160aattttcgtt
acaggacttc aacttccata atactttttt ttataattaa ggcatgatat
2220atatcttctt tctgaattcc acgggaattg cactttttcc tgagaaattt
gtaaagagca 2280tgcctgttaa ttgcaagggg cccctaactc tgttatgaga
aaagagaact atataagatg 2340ctcaataagc acctcttttt ttttttctgt
taactgacca aagcctgtct atctgcattt 2400ttttttgttt tttgtttttc
ttgtgtgcag atgggtatat tggtcatctt ggtggacctg 2460caatgctgcc
gctgcgatgc caagatcagg aaggtcctgg gctgccttga aggtataata
2520aattctgccc gaatcgtcca tgtttgattg aattttcaag gctaatcagc
agtgttcctg 2580ctcaattggg agcaaaacct ctgttaaaaa gggtgtgttt
gaatgaatat aattgaatat 2640gaacgcagag gagtactgca tcgagaaggt
ggagtacgac gtgaagaaca acagggtgat 2700cgtgcgcggg aagttcgacc
cggagaagct gtgcaagaag atctggtgca aggccggcaa 2760gatcatcaag
gagatcctca tcgtcgacgt ctggccgccg ccgtgcaagc cgccgccgtg
2820cgagaagcct ccggaggact gcaagcccaa gccctgccat tgctgcagct
gcgagaagcc 2880caagcccaag cccaagccct gccactgcga gaagcccaag
ccctgtcact gcgagaagcc 2940caagccatgc gagaagccgc cgccgaagcc
ggagtgcaag ctggtgccgt acccttaccc 3000ggtgccgtac ccgtacgccg
ggcagtggtg ctgcccaaag cctgagccgc cgaagccgcc 3060gccggagcca
ccgaaggagc cggagccgcc gaagccgtgc gggtgctcgc acgccttcgt
3120gtgcgtctgc aagccggcgc cgccgccgcc gccgccgtgc gggtgctcgg
ggggccacgg 3180gaactgcggc tgcggcatca ggccgtggcc gccgcaggtg
tggccgccgc cgcccgtctg 3240cccgccgccg ccgtggtgct acaccgagga
caacgccaac gcctgctcca tcatgtgatg 3300gccggccggc ggccggcgtc
gatcacgatc atctctgctg cttaatttcc ttgcttgcta 3360ctacctctgc
tcctttcctt gcctcggaaa tcggaataaa ttaaacacga ggctgatcga
3420tgtgtttgta attaatccat ggtgtttgtg ttgtgtgctg tgtgggctgt
ataataatta 3480attacagtat gttcatgtaa atttgtttgt ttgtttgttt
atgttgttcg atatgtataa 3540ttatgtacaa taattaatcg tggagaggct
cactaaactc ataaactgta gagtatcttg 3600gctgtaaaag tgtggcaatt
tatctttttc ttgtgttagc attggctaca aatagttttt 3660ggccgtcttt
tctcttcgtt tctccccttc tttatgagat taattgtgtg ctgacctaga
3720tcaaattata gcgcgctgac ctagttttat tgtaactgct cttatggatg
tctgctaaca 3780ccatcaaaca tgattaccgt ggtatatttg tcttaattac
tactaactag gactacctag 3840gggcaccctt gcatatgttt ttttttcgaa
cgaccagata gatttgagtc atttgactag 3900cgttatatta ataggaggga
aaaaaataca aagtacaaat atccaacagg ccgagaaaag 3960agaaaaaaaa
ctgtacatgc ctacgtgcaa acaagattgc acgaagctct cctcactcct
4020catggcaaca ttctcccaat gtgtttagca tcgaaaatac cccaccaacc
tcgtcttgga 4080tgtgactggt catttggtgt attgtcgcct ccttttccct
ctgaagattc ttacgttcca 4140ttccttccaa atttcctagg cgatgagcaa
gaaaagagat ctgagccctt tttgtttagt 4200tacttccaag tcgcttgttc
cttgtgtcca ccaatgcaag aggtttctac tgtcattcca 4260tttcagaata
agccaattga gaactccaaa ccagatttac ttcgaaacca ggcattcgaa
4320cataaggtgg tcgacagtgt cgagattccg gattagcaga gttatttggt
ttgagagtca 4380cattcctaca accaatagaa aaccatccta tggaccattt
agatagtgtc ctagtatttg 4440cttttatatt tgctataaat attcttcctt
tattgttttt ggagttcaca aacttaataa 4500tcaaactttg tgatttttta
atttccatta acgaattcaa ggaaccacct ttatctctca 4560tcttcattgc
acactactga tttctttcat ccttagggac atatggttga tacggagact
4620gtttttctat cattatctaa aaaaaatcta aggggcatat atatattgtg
ttttctctca 4680ttcatgcatt tcgcactttt ccctattcgt gaaataccat
ttcccacatg agtgcaatgt 4740ttctt 47455732DNAOryza sativa 5atgggtatat
tggtcatctt ggtggacctg caatgctgcc gctgcgatgc caagatcagg 60aaggtcctgg
gctgccttga agaggagtac tgcatcgaga aggtggagta cgacgtgaag
120aacaacaggg tgatcgtgcg cgggaagttc gacccggaga agctgtgcaa
gaagatctgg 180tgcaaggccg gcaagatcat caaggagatc ctcatcgtcg
acgtctggcc gccgccgtgc 240aagccgccgc cgtgcgagaa gcctccggag
gactgcaagc ccaagccctg ccattgctgc 300agctgcgaga agcccaagcc
caagcccaag ccctgccact gcgagaagcc caagccctgt 360cactgcgaga
agcccaagcc atgcgagaag ccgccgccga agccggagtg caagctggtg
420ccgtaccctt acccggtgcc gtacccgtac gccgggcagt ggtgctgccc
aaagcctgag 480ccgccgaagc cgccgccgga gccaccgaag gagccggagc
cgccgaagcc gtgcgggtgc 540tcgcacgcct tcgtgtgcgt ctgcaagccg
gcgccgccgc cgccgccgcc gtgcgggtgc 600tcggggggcc acgggaactg
cggctgcggc atcaggccgt ggccgccgca ggtgtggccg 660ccgccgcccg
tctgcccgcc gccgccgtgg tgctacaccg aggacaacgc caacgcctgc
720tccatcatgt ga 7326243PRTOryza sativa 6Met Gly Ile Leu Val Ile
Leu Val Asp Leu Gln Cys Cys Arg Cys Asp1 5 10 15Ala Lys Ile Arg Lys
Val Leu Gly Cys Leu Glu Glu Glu Tyr Cys Ile 20 25 30Glu Lys Val Glu
Tyr Asp Val Lys Asn Asn Arg Val Ile Val Arg Gly 35 40 45Lys Phe Asp
Pro Glu Lys Leu Cys Lys Lys Ile Trp Cys Lys Ala Gly 50 55 60Lys Ile
Ile Lys Glu Ile Leu Ile Val Asp Val Trp Pro Pro Pro Cys65 70 75
80Lys Pro Pro Pro Cys Glu Lys Pro Pro Glu Asp Cys Lys Pro Lys Pro
85 90 95Cys His Cys Cys Ser Cys Glu Lys Pro Lys Pro Lys Pro Lys Pro
Cys 100 105 110His Cys Glu Lys Pro Lys Pro Cys His Cys Glu Lys Pro
Lys Pro Cys 115 120 125Glu Lys Pro Pro Pro Lys Pro Glu Cys Lys Leu
Val Pro Tyr Pro Tyr 130 135 140Pro Val Pro Tyr Pro Tyr Ala Gly Gln
Trp Cys Cys Pro Lys Pro Glu145 150 155 160Pro Pro Lys Pro Pro Pro
Glu Pro Pro Lys Glu Pro Glu Pro Pro Lys 165 170 175Pro Cys Gly Cys
Ser His Ala Phe Val Cys Val Cys Lys Pro Ala Pro 180 185 190Pro Pro
Pro Pro Pro Cys Gly Cys Ser Gly Gly His Gly Asn Cys Gly 195 200
205Cys Gly Ile Arg Pro Trp Pro Pro Gln Val Trp Pro Pro Pro Pro Val
210 215 220Cys Pro Pro Pro Pro Trp Cys Tyr Thr Glu Asp Asn Ala Asn
Ala Cys225 230 235 240Ser Ile Met7975DNAOryza sativa 7gtacgacgtg
aagaacaaca gggtgatcgt gcgcgggaag ttcgacccgg agaagctgtg 60caagaagatc
tggtgcaagg ccggcaagat catcaaggag atcctcatcg tcgacgtctg
120gccgccgccg ctgccgcagc cgccgccgcc gtgcaagccg ccgccgtgcg
agaagcctcc 180ggaggactgc aagcccaagc cctgccattg ctgcagctgc
gagaagccca agcccaagcc 240caagccctgc cactgcgaga agcccaagcc
ctgtcactgc gagaagccca agccatgcga 300gaagccgccg ccgtgcaagc
cggaggagcc gccgaagccg ccgccggaga agccgccgcc 360gaagccggag
tgcaagctgg tgccgtaccc ttacccggtg ccgtacccgt acgccgggca
420gtggtgctgc ccaaagcctg agccgccgaa gccgccgccg gagccaccga
aggagccgga 480gccgccgaag ccgtgcgggt gctcgcacgc cttcgtgtgc
gtctgcaagc cggcgccgcc 540gccgccgccg ccgtgcgggt
gctcgggggg ccacgggaac tgcggctgcg gcatcaggcc 600gtggccgccg
caggtgtggc cgccgccgcc cgtctgcccg ccgccgccgt ggtgctacac
660cgaggacaac gccaacgcct gctccatcat gtgatggccg gccggcggtc
ggcgtcgatc 720acgatcatct ctgctgctta atttccttgc ttgctactac
ctctgctcct ttccttgcct 780cggaaatcgg aataaattaa acacgaggct
gatcgatgtg tttgtaatta atccatggtg 840tttgtgttgt gtgctgtgtg
ggctgtataa taattaatta cagtatgttc atgtaaattt 900gtttgtttgt
ttgtttatgt tgttcgatat gtataattat gtacaataat taatcgtgga
960gaggctcact aaact 975824DNAArtificialan artificially synthesized
primer sequence 8agaaggtgga gtacgacgtg aaga 24925DNAArtificialan
artificially synthesized primer sequence 9agtttagtga gcctctccac
gatta 2510916DNAOryza sativa 10agaaggtgga gtacgacgtg aagaacaaca
gggtgatcgt gcgcgggaag ttcgacccgg 60agaagctgtg caagaagatc tggtgcaagg
ccggcaagat catcaaggag atcctcatcg 120tcgacgtctg gccgccgccg
tgcaagccgc cgccgtgcga gaagcctccg gaggactgca 180agcccaagcc
ctgccattgc tgcagctgcg agaagcccaa gcccaagccc aagccctgcc
240actgcgagaa gcccaagccc tgtcactgcg agaagcccaa gccatgcgag
aagccgccgc 300cgaagccgga gtgcaagctg gtgccgtacc cttacccggt
gccgtacccg tacgccgggc 360agtggtgctg cccaaagcct gagccgccga
agccgccgcc ggagccaccg aaggagccgg 420agccgccgaa gccgtgcggg
tgctcgcacg ccttcgtgtg cgtctgcaag ccggcgccgc 480cgccgccgcc
gccgtgcggg tgctcggggg gccacgggaa ctgcggctgc ggcatcaggc
540cgtggccgcc gcaggtgtgg ccgccgccgc ccgtctgccc gccgccgccg
tggtgctaca 600ccgaggacaa cgccaacgcc tgctccatca tgtgatggcc
ggccggcggc cggcgtcgat 660cacgatcatc tctgctgctt aatttccttg
cttgctacta cctctgctcc tttccttgcc 720tcggaaatcg gaataaatta
aacacgaggc tgatcgatgt gtttgtaatt aatccatggt 780gtttgtgttg
tgtgctgtgt gggctgtata ataattaatt acagtatgtt catgtaaatt
840tgtttgtttg tttgtttatg ttgttcgata tgtataatta tgtacaataa
ttaatcgtgg 900agaggctcac taaact 91611295DNAOryza sativa
11tggccggccg gcggtcggcg tcgatcacga tcatctctgc tgcttaattt ccttgcttgc
60tactacctct gctcctttcc ttgcctcgga aatcggaata aattaaacac gaggctgatc
120gatgtgtttg taattaatcc atggtgtttg tgttgtgtgc tgtgtgggct
gtataataat 180taattacagt atgttcatgt aaatttgttt gtttgtttgt
ttatgttgtt cgatatgtat 240aattatgtac aataattaat cgtggagagg
ctcactaaac tcataaactg tagag 2951223DNAArtificialan artificially
synthesized primer sequence 12ttttcctgag aaatttgtaa aga
231318DNAArtificialan artificially synthesized primer sequence
13cgtcgacgat gaggatct 181420DNAArtificialan artificially
synthesized primer sequence 14ctcccaatgt gtttagcatc
201521DNAArtificialan artificially synthesized primer sequence
15caaccatatg tccctaagga t 211622DNAArtificialan artificially
synthesized primer sequence 16gtacgacgtg aagaacaaca gg
221717DNAArtificialan artificially synthesized primer sequence
17gcttgggctt gcagtcc 171821DNAArtificialan artificially synthesized
probe sequence 18ctgccgcagc cgccgccgcc g 211948DNAArtificialan
artificially synthesized probe sequence 19tgcaagccgg aggagccgcc
gaagccgccg ccggagaagc cgccgccg 48204803DNAOryza sativa 20tctagatgat
gctttatgtg aaggcaataa atgataatta agtgtaatat aatgtatttt 60ttaaacatcg
tagtgaagtc ccatttctag tacgtaatgg atcgaaatgg ggagggactg
120cttctctttt agaaaaaaat gtaaaattga gatgagcttt tataagacat
gtaaggccat 180atgtctagtg taagtatgat gctagcaagc acattttagc
atcttaaaat aaaaggcaag 240cacattaata ttttaggagt tagcatggat
gcaatacagt agaagaggag atgccaacaa 300aaaagaatat ggaaaatttg
tttcatacca tcaaaagttc ctgtgttttg ctttatacca 360tcagcgtttg
tggtgtactt taacaccatc caaagtttgc tccgttacct atccactcca
420cgaaatgtgc ttgctagcat ggatgaaaaa gtaaccgaaa caaacagttg
tggtatactc 480ctaagtaatc ctaagtagcc accggattca atactttatt
ctcaatcgtc cttctagtct 540attcccaatt atatgggttt gacccgttac
aacgtacggg catgttacta gcgtaaggaa 600agagatggtt tctaaaagag
tttttttttt ccaacagtcc tacccaatct atagcgctag 660acaagaaagc
gaaagctaaa cccatattta ttaccgtaat gttgcaaact taatattacc
720tgcaatagac atgccctaac atggtttcag gaccacaaac cagagctcta
aaattccgag 780cacatgttca gaatatatat acatttttaa ttcccttcta
tactactcca acttacctct 840atggaggtaa gaggctaaaa ttgaaactag
ataactgaat gttcttatat ttttcctaat 900aaaaatataa ttgctccttt
catttaatac ttatatcaca acatgtagcc atatgagcca 960aaactgtatt
agccatattt tagcgaataa ggctcaatgc gattctgtgt ctgagctctg
1020gcctgaaaat tcttgtaact ttatctcaca tatgatgtgt gaggaagctt
ctcatgagta 1080atatattccc tatttccccg caaaaaaaaa acttatatca
caacaaaaat ggaaaagacc 1140aacactgagt attgtagaag ctagtacttt
tggcaaatgt gcccgaagat ccccaaaaca 1200gtttgtggca taattcagaa
gattgttagc tcagaaatca tttcagacta ggatattgcc 1260gacacatcag
agttctcgtg taatcaagaa gcaaggaact gtacagtgca gtgatgatat
1320acaactatag gagaggacga actgatactg acggatgctg tgtgtaaaat
atctctgtaa 1380aatcaggaat actgggagac tgattggatg atagatttat
acggtcacaa tcatctcgtt 1440aaacacactt ttcattaaag aggaattacc
aaatacagta attgattcga cggtgccgcg 1500agaaatgtgc tatatataaa
tgctcaccta tatttgggca gattagttaa tttcaggaat 1560tttccacaga
gaaagctaaa tgagtgctaa atgagtcgca ttgcgtacta ctacaagggt
1620ccaaacagaa attgatatct gaaattagct tttcttaaga taacaatgat
attttttttt 1680attgaagcac aaggtgtgcc tgaaatcgaa ttctgaaagg
tattcttatt tttcagagga 1740aaatgtacac tggtaccatt ctgaaagcta
attgagagat gacaaggcgt gagaaggacc 1800aaactgtcct atatacatgt
ggtcattttc catctcttgc aataggttat caagacccct 1860cctgaaacat
ggaccgtcca gatgcgatcc gacggacgaa aaaaaccaat ggcagaatat
1920ttcaggctct ggcatatcca aacataaata agtaacataa ggttcagctc
tgctccatcc 1980atgcattgcc tccattattc atgcttcgat tctccatgct
ttcctctact gctcattggt 2040aacattcggc aaatttgaca ggtgagctca
gtattttaaa tcttaatgta gtactttggt 2100gtgctaatcc ttgctctgtt
caaaagagaa ttctggtttc tttgctattt tgaaaagaga 2160attttcgtta
caggacttca acttccataa tacttttttt tataattaag gcatgatata
2220tatcttcttt ctgaattcca cgggaattgc actttttcct gagaaatttg
taaagagcat 2280gcctgttaat tgcaaggggc ccctaactct gttatgagaa
aagagaacta tataagatgc 2340tcaataagca cctctttttt tttttttgtt
aactgaccaa agcctgtcta tctgcatttt 2400tttttgtttt ttgtttttct
tgtgtgcaga tgggtatatt ggtcatctcg gtggacctgc 2460aatgctgccg
ctgcgatgcc aagatcagga aggtcctggg ctgccttgaa ggtataataa
2520attctgcccg aatcgtccat gtttgattga attttcaagg ctaatcagca
gtgttcctgc 2580tcaattggga gcaaaacctc tgttaaaaag ggtgtgtttg
aatgaatata attgaatatg 2640aacgcagagg agtactgcat cgagaaggtg
gagtacgacg tgaagaacaa cagggtgatc 2700gtgcgcggga agttcgaccc
ggagaagctg tgcaagaaga tctggtgcaa ggccggcaag 2760atcatcaagg
agatcctcat cgtcgacgtc tggccgccgc cgtcgccgcc gccgtgcaag
2820ccgccgccgt gcgagaagcc tccggaggac tgcaagccca agccctgcca
ttgctgcagc 2880tgcgagaagc ccaagcccaa gcccaagccc tgccactgcg
agaagcccaa gccctgtcac 2940tgcgagaagc ccaagccatg cgagaagccg
ccgccgtgca agccggagga gccgccgaag 3000ccgccgccgg agaagccgcc
gccgaagccg gagtgcaagc tggtgccgta cccttacccg 3060gtgccgtacc
cgtacgccgg gcagtggtgc tgcccaaagc ctgagccgcc gaagccgccg
3120ccggagccac cgaaggagcc ggagccgccg aagccgtgcg ggtgctcgca
cgccttcgtg 3180tgcgtctgca agccggcgcc gccgccgccg ccgccgtgcg
ggtgctcggg gggccacggg 3240aactgcggct gcggcatcag gccgtggccg
ccgcaggtgt ggccgccgcc gcccgtctgc 3300ccgccgccgc cgtggtgcta
caccgaggac aacgccaacg cctgctccat catgtgatgg 3360ccggccggcg
gccggcgtcg atcacgatca tctctgctgc ttaatttcct tgcttgctac
3420tacctctgct cctttccttg cctcggaaat cggaataaat taaacacgag
gctgatcgat 3480gtgtttgtaa ttaatccatg gtgtttgtgt tgtgtgctgt
gtgggctgta taataattaa 3540ttacagtatg ttcatgtaaa tttgtttgtt
tgtttgttta tgttgttcga tatgtataat 3600tatgtacaat aattaatcgt
ggagaggctc actaaactca taaactgtag agtatcttgg 3660ctgtaaaagt
gtggcaattt atctttttct tgtgttagca ttggctacaa atagtttttg
3720gccgtctttt ctcttcgttt ctccccttct ttatgagatt aattgtgtgc
tgacctagat 3780caaattatag cgcgctgacc tagttttatt gtaactgctc
ttatggatgt ctgctaacat 3840catcaaacat gattaccgtg gtatatttgt
cttaattact actaactagg actacctagg 3900ggcacccttg catatgtttt
ttttcgaacg accagataga tttgagtcat ttgactagcg 3960ttatattaat
aggagggaaa aaatacaaag tacaaatatc caacaggccg agaaaagaga
4020aaaaaaactg tacgtgccta cgtgcaaaca agattgcacg aagctctcct
cactcctcat 4080ggcaacattc tcccaatgtg tttagcatcg aaaatacccc
accaacctcg tcttggatgt 4140gattggtcat ttggtgtatt gtcgcctcct
tttccctctg aagattctta tgttccattc 4200cttccaaatt tcctaggcga
tgagcaagaa aagagatctg agcccttttt gtttagttac 4260ttccaagtcg
cttgttcctt gtgtccacca atgtaagagg tttctactgt cattccattt
4320cagaataagc caattgagaa ctccaaacca gatttacttc gaaaccaggc
attcgaatat 4380aaggtggtcg acagtttcga gattccggat tagcagagtt
atttggtttg agagtcacat 4440tcctacaacc aatagaaaac catcctatgg
accatttaga taatgtccta gtatttgctt 4500ttatatttgc tataaatatt
cttcctttat tgtttttgga gttcacaaac ttaataatca 4560aactttgtga
ttttttaatt tccattaacg aattcaagga accaccttta tctctcatct
4620tcattgcaca ctactgattt ctttcatcct tagggatata tggttgatac
ggagactgtt 4680tttctatcat tatctaaaaa aaaatctaag gggcatatat
atattgtgtt ttctctcatt 4740catgcatttc gcacttttcc ctattcgtga
aataccattt cccacatgag tgcaatgttt 4800ctt 4803211014DNAOryza sativa
21atgggtatat tggtcatctc ggtggacctg caatgctgcc gctgcgatgc caagatcagg
60aaggtcctgg gctgccttga agaggagtac tgcatcgaga aggtggagta cgacgtgaag
120aacaacaggg tgatcgtgcg cgggaagttc gacccggaga agctgtgcaa
gaagatctgg 180tgcaaggccg gcaagatcat caaggagatc ctcatcgtcg
acgtctggcc gccgccgtcg 240ccgccgccgt gcaagccgcc gccgtgcgag
aagcctccgg aggactgcaa gcccaagccc 300tgccattgct gcagctgcga
gaagcccaag cccaagccca agccctgcca ctgcgagaag 360cccaagccct
gtcactgcga gaagcccaag ccatgcgaga agccgccgcc gtgcaagccg
420gaggagccgc cgaagccgcc gccggagaag ccgccgccga agccggagtg
caagctggtg 480ccgtaccctt acccggtgcc gtacccgtac gccgggcagt
ggtgctgccc aaagcctgag 540ccgccgaagc cgccgccgga gccaccgaag
gagccggagc cgccgaagcc gtgcgggtgc 600tcgcacgcct tcgtgtgcgt
ctgcaagccg gcgccgccgc cgccgccgcc gtgcgggtgc 660tcggggggcc
acgggaactg cggctgcggc atcaggccgt ggccgccgca ggtgtggccg
720ccgccgcccg tctgcccgcc gccgccgtgg tgctacaccg aggacaacgc
caacgcctgc 780tccatcatgt gatggccggc cggcggccgg cgtcgatcac
gatcatctct gctgcttaat 840ttccttgctt gctactacct ctgctccttt
ccttgcctcg gaaatcggaa taaattaaac 900acgaggctga tcgatgtgtt
tgtaattaat ccatggtgtt tgtgttgtgt gctgtgtggg 960ctgtataata
attaattaca gtatgttcat gtaaatttgt ttgtttgttt gttt 101422263PRTOryza
sativa 22Met Gly Ile Leu Val Ile Leu Val Asp Leu Gln Cys Cys Arg
Cys Asp1 5 10 15Ala Lys Ile Arg Lys Val Leu Gly Cys Leu Glu Glu Glu
Tyr Cys Ile 20 25 30Glu Lys Val Glu Tyr Asp Val Lys Asn Asn Arg Val
Ile Val Arg Gly 35 40 45Lys Phe Asp Pro Glu Lys Leu Cys Lys Lys Ile
Trp Cys Lys Ala Gly 50 55 60Lys Ile Ile Lys Glu Ile Leu Ile Val Asp
Val Trp Pro Pro Pro Ser65 70 75 80Pro Pro Pro Cys Lys Pro Pro Pro
Cys Glu Lys Pro Pro Glu Asp Cys 85 90 95Lys Pro Lys Pro Cys His Cys
Cys Ser Cys Glu Lys Pro Lys Pro Lys 100 105 110Pro Lys Pro Cys His
Cys Glu Lys Pro Lys Pro Cys His Cys Glu Lys 115 120 125Pro Lys Pro
Cys Glu Lys Pro Pro Pro Cys Lys Pro Glu Glu Pro Pro 130 135 140Lys
Pro Pro Pro Glu Lys Pro Pro Pro Lys Pro Glu Cys Lys Leu Val145 150
155 160Pro Tyr Pro Tyr Pro Val Pro Tyr Pro Tyr Ala Gly Gln Trp Cys
Cys 165 170 175Pro Lys Pro Glu Pro Pro Lys Pro Pro Pro Glu Pro Pro
Lys Glu Pro 180 185 190Glu Pro Pro Lys Pro Cys Gly Cys Ser His Ala
Phe Val Cys Val Cys 195 200 205Lys Pro Ala Pro Pro Pro Pro Pro Pro
Cys Gly Cys Ser Gly Gly His 210 215 220Gly Asn Cys Gly Cys Gly Ile
Arg Pro Trp Pro Pro Gln Val Trp Pro225 230 235 240Pro Pro Pro Val
Cys Pro Pro Pro Pro Trp Cys Tyr Thr Glu Asp Asn 245 250 255Ala Asn
Ala Cys Ser Ile Met 26023976DNAOryza sativa 23agaaggtgga gtacgacgtg
aagaacaaca gggtgatcgt gcgcgggaag ttcgacccgg 60agaagctgtg caagaagatc
tggtgcaagg ccggcaagat catcaaggag atcctcatcg 120tcgacgtctg
gccgccgccg tcgccgccgc cgtgcaagcc gccgccgtgc gagaagcctc
180cggaggactg caagcccaag ccctgccatt gctgcagctg cgagaagccc
aagcccaagc 240ccaagccctg ccactgcgag aagcccaagc cctgtcactg
cgagaagccc aagccatgcg 300agaagccgcc gccgtgcaag ccggaggagc
cgccgaagcc gccgccggag aagccgccgc 360cgaagccgga gtgcaagctg
gtgccgtacc cttacccggt gccgtacccg tacgccgggc 420agtggtgctg
cccaaagcct gagccgccga agccgccgcc ggagccaccg aaggagccgg
480agccgccgaa gccgtgcggg tgctcgcacg ccttcgtgtg cgtctgcaag
ccggcgccgc 540cgccgccgcc gccgtgcggg tgctcggggg gccacgggaa
ctgcggctgc ggcatcaggc 600cgtggccgcc gcaggtgtgg ccgccgccgc
ccgtctgccc gccgccgccg tggtgctaca 660ccgaggacaa cgccaacgcc
tgctccatca tgtgatggcc ggccggcggc cggcgtcgat 720cacgatcatc
tctgctgctt aatttccttg cttgctacta cctctgctcc tttccttgcc
780tcggaaatcg gaataaatta aacacgaggc tgatcgatgt gtttgtaatt
aatccatggt 840gtttgtgttg tgtgctgtgt gggctgtata ataattaatt
acagtatgtt catgtaaatt 900tgtttgtttg tttgtttatg ttgttcgata
tgtataatta tgtacaataa ttaatcgtgg 960agaggctcac taaact
97624222DNAOryza sativa 24tggccggccg gcggccggcg tcgatcacga
tcatctctgc tgcttaattt ccttgcttgc 60tactacctct gctcctttcc ttgcctcgga
aatcggaata aattaaacac gaggctgatc 120gatgtgtttg taattaatcc
atggtgtttg tgttgtgtgc tgtgtgggct gtataataat 180taattacagt
atgttcatgt aaatttgttt gtttgtttgt tt 2222512DNAOryza sativa
25tcgccgccgc cg 122623DNAArtificialan artificially synthesized
primer sequence 26gatcctcatc gtcgacgtct ggc 232719DNAArtificialan
artificially synthesized primer sequence 27agggtacggc accagcttg
19
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