JP2001252079A - New polynucleotide and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and efficient detection method of the resistance of rice plants to an harmful insect, Nilaparvata lugens (which sucks the juice from rice plants). SOLUTION: In this examination process for detecting the resistance of rice plant to Nilaparvata lugens, it is judged whether the RNA having the base sequence encoding a specific amino acid or a partial base sequence thereof is present or not in the rice plant examined, through the polymerase chain reactions using a specifically sequneced primer or the hybridization using a specific probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel polynucleotide and a method for assaying a rice plant resistant to brown planthopper using the polynucleotide.

[0002]

BACKGROUND OF THE INVENTION The brown planthopper (Nilaparvata lugens Stal) is a major pest of rice and sucks rice leaves and stems to kill the rice. Damage by brown planthopper is a major problem in rice cultivation. In recent years, indica-type rice showing resistance to brown planthopper, for example, IR1154-243
(Kaneda C. et. Al., Bull. Natl. Agric. Res. Cent.
6,19-32) have been found, and research has been conducted to breed Japonica-type rice varieties exhibiting the same resistance to brown planthopper by crossing with these indica-type planthopper-resistant rice plants. It's getting better. To breed brown planthopper resistant rice, continuous backcrossing or selfing is generally performed. At that time, an assay method for selecting a resistant strain from the obtained population is required. As such an assay method, a method of assaying the damage of a test strain due to sucking of brown planthopper is known.However, in order to obtain a reliable result, the brown planthopper used for the assay must be bred uniformly and in large quantities. And a great deal of labor and cost are required. Therefore, development of a simple and efficient test method for brown rice planthopper-resistant rice has been required.

[0003]

Under such circumstances, the present inventors have conducted intensive studies and as a result, have detected the presence of a specific RNA in a test rice using a specific polynucleotide, thereby obtaining resistance to brown planthopper. The present inventors have found that sex rice can be assayed easily and efficiently, and completed the present invention.

That is, the present invention provides: (1) a polynucleotide having a base sequence described in any of the following (a) to (e) (hereinafter, referred to as the polynucleotide of the present invention): (a) SEQ ID NO: 2 (B) a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 (c) a nucleotide sequence represented by SEQ ID NO: 1 (d) a nucleotide sequence represented by SEQ ID NO: 1 (E) a base sequence complementary to the base sequence shown, (e) a partial base sequence of 10 or more bases in the base sequence described in any one of (a) to (d); A method for detecting a rice planthopper-resistant rice plant, characterized by detecting the presence of an RNA having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial nucleotide sequence thereof in a test rice (3) Polymerase chain reaction (hereinafter referred to as PCR) using the polynucleotide described in (1) as a primer and cDNA prepared from a test rice as a template.
And detecting the amplification of a polynucleotide having the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a polynucleotide having a partial nucleotide sequence thereof. (4) (1) Labeling the described polynucleotide, hybridizing it to RNA or cDNA prepared from a test rice, and detecting an RNA or cDNA having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial nucleotide sequence thereof A method for testing brown planthopper-resistant rice (hereinafter referred to as (2),
(3) and (4) are collectively referred to as the assay method of the present invention. ), Is provided.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The genetic engineering technique used in the present invention is described in, for example, Molecular Cloning 2nd Edition (Molecular Cloning 2nd Edition) by J., Sambrook, E., F., Frisch, T., Maniatis.
edition), Cold Spring Harbor Laboratory press (Cold Spring Harbor Laboratory press), 1
989, or D., M., Glover, DNA cloning (DN
A Cloning), IRL publication, 1985 and the like.

The polynucleotide of the present invention includes a polynucleotide having a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 and a polynucleotide having a base sequence complementary to the base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 Examples include a nucleotide, a polynucleotide having a base sequence represented by SEQ ID NO: 1, and a polynucleotide having a base sequence complementary to the base sequence represented by SEQ ID NO: 1. Furthermore, a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2, a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2, a nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence represented by SEQ ID NO: 1 A partial base sequence of any base sequence of the base sequence complementary to the base sequence to be prepared, for example, about 10 bases or more, preferably about 1 base
A polynucleotide having a base sequence of 5 bases or more, more preferably about 20 bases or more, can be mentioned.
Of a polynucleotide having such a partial base sequence,
As more specific examples, a polynucleotide having the nucleotide sequence of any of SEQ ID NOS: 3 to 7, a polynucleotide having the nucleotide sequence of any of SEQ ID NOs: 10 to 18, a SEQ ID NO: Base number 31 of 1
Polynucleotides having the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 123, and polynucleotides having the nucleotide sequence represented by nucleotide numbers 1 to 123 of SEQ ID NO: 1 can be exemplified. The polynucleotide of the present invention may contain a base sequence of about one to several tens of bases such as a restriction enzyme recognition sequence and a linker sequence at its terminal.

[0007] The polynucleotide of the present invention includes, for example, "DN
A / RNA synthesizer ”(edited by Shigenori Iwai and Eiko Otsuka Protein Nucleic Acid
Enzyme Vol39 No11 (1994) Kyoritsu Shuppan), “Molecular Genetics Experiments” (edited by Haruo Koseki, Kyoritsu Shuppan) and the like, and can be prepared by chemical synthesis.
Applied Biosystems (ABI) DNA
It can also be synthesized using an automatic synthesizer such as / RNA synthesizer.

[0008] The polynucleotide of the present invention may be a cDNA prepared from a brown planthopper-resistant rice such as IR1154-243, Kanto PL5 (registered in Agriculture, Forestry and Fisheries Gene Bank) and Norin-PL4 (registered in Agriculture, Forestry and Fisheries Gene Bank). Can also be prepared using PCR. First, from the rice planthopper resistant rice described above, for example, J., Sambrook,
Total RNA is prepared according to the method described in Molecular Cloning 2nd edition by E., F., Frisch, T., Maniatis. Specifically, for example, after freezing a part of the tissue such as leaves in liquid nitrogen, physically grind it with a mortar or the like, and then add a solution containing guanidine hydrochloride and phenol or SDS and phenol. A method of recovering total RNA by adding a solution containing the same, a method of adding a solution containing guanidine thiocyanate to the above-mentioned triturated material, further adding CsCl, and centrifuging to precipitate and collect the total RNA may be used. The preparation operation includes, for example, ISOGEN (manufactured by Nippon Gene), RNeasy Total RNA P
A commercially available kit such as a urification Kit (manufactured by QIAGEN) can also be used. Next, mRNA is prepared from the total RNA. For example, a method utilizing hybridization between oligo dT chain bound to cellulose or latex and poly A chain of mRNA can be used. The operation includes
Commercially available kits such as mRNA Purification Kit (Amersham Pharmacia) and Oligotex ^™ -dT30 <Super> (Takara Shuzo) can be used. Furthermore, using the thus-purified mRNA having a poly A chain, a single-stranded cDNA
Is prepared. A single-stranded cDNA can be produced, for example, by annealing an oligo dT chain or a random primer to mRNA and then allowing reverse transcriptase to act. In addition, for example, RNaseH, DNA Polyme
A double-stranded cDNA may be produced by the action of raseI. For the operation, a SMART ^™ PCR cDNA Synthesis Kit (manufactured by Clontech), a cDNA Synthesis Kit (manufactured by Takara Shuzo), and a cDNA Synthesis Kit (manufactured by Amersham Pharmacia) can be used. The polynucleotide of the present invention can be amplified by performing PCR using the obtained cDNA as a template and, for example, primers designed based on the nucleotide sequence of SEQ ID NO: 1. Specifically, for example, using the polynucleotide of the present invention consisting of the nucleotide sequence of SEQ ID NO: 4 and the polynucleotide of the present invention consisting of the nucleotide sequence of SEQ ID NO: 5 as primers, for example, at 94 ° C. for 5 seconds and then at 72 ° C. Keep warm for 4 minutes at ℃
Do this for 5 to 10 cycles as one cycle, and
By performing PCR in which the temperature is maintained at 94 ° C. for 5 seconds and then at 70 ° C. for 4 minutes as one cycle, and this is performed for 20 to 40 cycles, the base numbers 1 to 34 of the base sequence represented by SEQ ID NO: 1 are obtained.
The polynucleotide of the present invention consisting of the nucleotide sequence represented by No. 13 can be amplified. Similarly, using the cDNA prepared as described above as a template, for example, a polynucleotide of the present invention consisting of the nucleotide sequence of SEQ ID NO: 4 and a polynucleotide of the present invention consisting of the nucleotide sequence of SEQ ID NO: 18 Used as a primer, e.g.
This is followed by one cycle of incubation at 72 ° C for 4 minutes followed by 4 minutes.
Perform 5 to 10 cycles, then at 94 ° C for 5 seconds then at 70 ° C
The PCR of 20 to 40 cycles was carried out with the incubation for 4 minutes as one cycle for 4 minutes to amplify the polynucleotide of the present invention consisting of the nucleotide sequence represented by nucleotide numbers 1 to 363 of the nucleotide sequence represented by SEQ ID NO: 1. can do.
Such PCR is performed, for example, using the DNA polymerase, TAKARA, contained in the Advantage cDNA PCR Kit (Clontech).
Ex Taq (Takara Shuzo), PLATINUM ^™ PCR SUPER Mix
(Manufactured by Life Tech Oriental) according to the attached protocol. The polynucleotide of the present invention amplified as described above is referred to as “Molecula
r Cloning: A Laboratory Manual 2nd edition '' (1989),
Cold Spring Harbor Laboratory Press, `` Current Pro
tocols In Molecular Biology '' (1987), John Wiley &
Sons, Inc. ISBN0-471-50338-X and the like can be used for cloning into a vector according to the usual method. Examples of the vector to be used include a vector having an origin of replication and a selectable marker gene that can function in a microorganism suitable for genetic engineering techniques such as Escherichia coli, and specifically, pBlueScriptII vector (Stratagene), pUC
18/19 vector (Takara Shuzo), TA cloning vector (Invitrogen) and the like. Further, the nucleotide sequence of the cloned polynucleotide of the present invention is F.Sa
Proceedings of Natio, by nger, S. Nicklen, ARCoulson
nalAcademy of Science USA (1977), 74, 5463-54
It can be analyzed by the dideoxy terminating method described on page 67 and the like. At this time, for example, commercially available Termo Seqenase II dye terminator cycle sequencin
g kit (Amersham Pharmacia), Dye Terminato
r Cycle Sequencing FS Ready Reaction Kit (PE Biosystems Japan) can be used.

[0009] The polynucleotide of the present invention is, for example, IR11
54-243, Kanto PL5, Norin-PL4, etc., can also be prepared using hybridization methods from cDNA obtained from brown planthopper resistant rice. First, cD derived from brown planthopper resistant rice prepared as described above
A cDNA library is prepared by linking NA to a vector derived from λ phage and packaging the phage particles. The cDNA library can be prepared, for example, according to the method described in Cloning and Sequence: Plant Biotechnology Experiment Manual (edited by Watanabe and Sugiura, Rural Culture Company, 1989). Next, the cDNA
A clone containing the polynucleotide of the present invention is selected from the library. For example, the polynucleotide of the present invention containing at least a part of the nucleotide sequence of SEQ ID NO: 1, specifically, the nucleotide sequence of SEQ ID NO: 1
The polynucleotide of the present invention having the nucleotide sequence represented by any one of Nos. 27 to 3449 and the like are chemically synthesized and labeled, and plaque hybridization is performed using this as a probe. For labeling the probe with a radioisotope,
For example, Random Primed DNA Labeling Kit (Boehringer), Random Primer DNA Labeling Kit Ver.2
(Manufactured by Takara Shuzo) or the like can be used. In addition, for example, ECL Direct Nucleic Acid
Labeling and Ditection System (Amersham Pharmacia) can be used. Hybridization is performed, for example, in "DNA cloning, a practice" edited by DMGlover.
al approach "IRL PRESS (1985) ISBN 0-947946-18-
7. Cloning and sequencing: Plant biotechnology experiment manual (edited by Watanabe and Sugiura, Rural Bunkasha 1989)
Year) or Molecular Cloning 2nd edition (J.Sambr
ook, EFFrisch, T. Maniatis, Cold Spring Harbor L
aboratory Press 1989). Specifically, hybridization conditions vary depending on the probe used, for example, 450 to 900 mM.
Sodium chloride, 45-90 mM sodium citrate, 0.
1-1.0% sodium dodecyl sulfate (SDS) and 0-20
Prehybridization solution containing 0 μg / ml denatured calf thymus DNA, and optionally containing albumin, ficoll, polyvinylpyrrolidone, etc. at a concentration of 0 to 0.2% each, preferably 900 mM sodium chloride, 90 mM sodium citrate, 1% SDS and 100
Prehybridization solution containing denatured calf thymus DNA at μg / ml was applied to the nylon filter per cm ²
Prepare the solution at a rate of 50 to 200 μl, immerse the nylon filter in the solution, and at 42 to 68 ° C. for 1 to 4 hours, preferably 6
Incubate at 5 ° C for 2 hours. Then, for example, 450-900 mM sodium chloride, 45-90 mM sodium citrate, 0.1
~ 1.0% SDS and 0-200 μg / ml denatured calf thymus
DNA, and in some cases albumin, ficoll,
A hybridization solution that may contain polyvinylpyrrolidone or the like at a concentration of 0 to 0.2%, preferably 900 m
M sodium chloride, 90 mM sodium citrate, 1% S
A solution obtained by mixing a hybridization solution containing DS and 100 μg / ml denatured calf thymus DNA with the above-mentioned labeled probe (equivalent to 1.0 × 10 ^{4 to} 2.0 × 10 ⁶ cpm per 1 cm ^{2 of} nylon filter) was used as a nylon filter. 50 per 1cm ²
200200 μl, prepare a nylon filter in the solution, immerse in a nylon filter at 42-68 ° C. for 4-20 hours, preferably at 65 ° C.
Incubate for a certain time to perform the hybridization reaction. After the hybridization, the nylon filter is removed, and a washing solution containing 42 to 68 ° C. containing 15 to 300 mM sodium chloride, 1.5 to 30 mM sodium citrate, 0.1 to 1.0% SDS, or the like, preferably 300 mM chloride is used. Nylon filter washing is performed 1 to 4 times for 10 to 60 minutes, preferably twice for 15 minutes with a washing solution of 55 ° C. containing sodium, 30 mM sodium citrate and 1% SDS. Further, the nylon filter is rinsed lightly with 2 × SSC solution (300 mM sodium chloride, 30 mM sodium citrate) and then dried. This nylon filter may be subjected to, for example, autoradiography, an image analyzer (manufactured by Fuji Film Co., Ltd.) or the like to detect a positive signal indicating that the probe used has hybridized. A clone containing the polynucleotide of the present invention that showed a positive signal in the above hybridization, for example, Molecular Cloning 2nd
edition (by J. Sambrook, EFFrisch, T. Maniatis, C
old Spring Harbor Laboratory Press 1989) Cultured in NZYM liquid medium according to the method described in 2.60 to 2.65, etc.
From the culture broth, commercially available Lambda-TRAP PLUS DNA Isolation K
The vector possessed by the clone is extracted using it (manufactured by Clontech) or the like. Next, the polynucleotide inserted into the vector is excised, and this is excised from the vector for easy preparation and analysis of the polynucleotide, for example, commercially available pUC18 vector (Takara Shuzo), pUC19 vector (Takara Shuzo), pBlue
Subcloning into ScriptII vector (Stratagene) or the like. The nucleotide sequence of the polynucleotide subcloned in the vector can be determined according to the method described above, and it can be confirmed that the polynucleotide is the polynucleotide of the present invention.

[0010] For example, the polynucleotide of the present invention can be obtained by PCR from the genomic DNA of the brown planthopper resistant rice.
Can also be prepared. For example, a tissue such as a leaf portion of the rice planthopper-resistant rice is collected, and the obtained tissue is frozen in liquid nitrogen, and then physically ground in a mortar or the like to obtain a fine powdery tissue. Make a piece. From the tissue pieces, conventional methods such as M. Shure et.
al, Cell, 35: 225 (1983) and the like,
SORogers and AJBendich, Plant.Mol.Biol., 5:69
(1985) and the like, and genomic DNA is extracted according to the urea-phenol method and the like. Also, for example, commercially available ISOPLAN
Genomic DNA can also be extracted using T (Nippon Gene) or the like. Using the obtained genomic DNA as a template,
For example, the polynucleotide of the present invention can be amplified by performing PCR using primers designed based on the nucleotide sequence shown in SEQ ID NO: 1. In particular,
For example, using the polynucleotide of the present invention consisting of the nucleotide sequence of SEQ ID NO: 4 and the polynucleotide of the present invention consisting of the nucleotide sequence of SEQ ID NO: 5 as primers, for example, at 94 ° C. for 5 seconds and then at 72 ° C. for 4 minutes 5 to 10 cycles with 1 cycle of heat retention, 94 ° C for 5 seconds, then 70
One cycle of 4 minutes of incubation at 4 ° C was followed by 5 to 10 cycles, followed by 1 second of 94 ° C for 5 seconds and then 68 ° C for 4 minutes.
By performing PCR in which the cycle is performed for 25 to 35 cycles, the polynucleotide of the present invention derived from the genome can be amplified. Such PCR is, for example, Advant
DN included in age cDNA PCR Kit (Clontech)
A polymerase, TAKARA Ex Taq (Takara Shuzo), PLATI
It can be performed using NUM ^™ PCR SUPER Mix (manufactured by Life Tech Oriental) according to the attached protocol. The polynucleotide of the present invention amplified as described above can be cloned and its nucleotide sequence determined according to the method described above.

The polynucleotide of the present invention can also be prepared by a hybridization method from genomic DNA derived from the above-described rice plant resistance to brown planthopper. Genomic DNA derived from the brown planthopper-resistant rice was prepared according to the method described above, and the genomic DNA was partially digested with a restriction enzyme, and the resultant was subjected to “Molecular Cloning: A Laboratory Manual 2nd edit”.
ion '' (1989), Cold Spring Harbor Laboratory Press,
`` Current Protocols In Molecular Biology '' (1987),
According to the genomic library production method described in John Wiley & Sons, Inc. ISBN 0-471-50338-X, etc., for example, λFI
XII (STRATAGENE), λEMBL3 (STRATAGENE),
A genomic library is prepared by ligating to a vector derived from λ phage such as λEMBL4 (manufactured by STRATAGENE) and λDASHII (manufactured by STRATAGENE) and packaging the phage into phage particles. For the above-mentioned packaging, for example, Gigapack packaging Extracts (manufactured by STRATAGENE) or the like can be used. In order to select a clone containing the polynucleotide of the present invention from the genomic library, for example, a polynucleotide having at least a part of the nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 1 is chemically synthesized, and this is labeled. Plaque hybridization is performed using the probe. The plaque hybridization is performed as described above, for example, DMGlov
er "DNA cloning, a practical approach" IRL PRESS
(1985) ISBN 0-947946-18-7, Cloning and sequencing: Plant Biotechnology Experiment Manual (edited by Watanabe and Sugiura, Rural Culture Company 1989), or Molecular
Cloning 2nd edition (J.Sambrook, EFFrisch, TM
aniatis, Cold Spring Harbor Laboratory Press 198
9 years). In addition, labeling of the probe with a radioisotope includes Rando
m Primed DNA Labeling Kit (Boehringer), Ra
ndom Primer DNA Labeling Kit Ver.2 (Takara Shuzo)
Etc. can be used. In addition, for example, ECL Direct Nucleic Acid Labeling and
Ditection System (Amersham Pharmacia) can be used. A clone containing the polynucleotide of the present invention, which showed a positive signal in the above hybridization, was cloned into, for example, Molecular Cloning 2nd edition (J. Samb.
rook, EFFrisch, T. Maniatis, Cold Spring Harbor
Laboratory Press 1989) Culture in NZYM liquid medium according to the method described in 2.60 to 2.65, etc.
Lambda-TRAP PLUS DNA Isolation Kit (Clontech)
The vector possessed by the clone is extracted using the method described above. Next, the polynucleotide inserted into the vector is cut out, and this is excised from the vector for easy preparation and analysis of the polynucleotide, for example, commercially available pUC18 vector (manufactured by Takara Shuzo), pUC19
Vector (Takara Shuzo), pBlueScriptII vector (Str
atagene). The nucleotide sequence of the subcloned polynucleotide can be determined according to the method described above, and it can be confirmed that the polynucleotide is the polynucleotide of the present invention.

The assay method of the present invention comprises detecting the presence of an RNA having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial sequence thereof in a test rice plant by using the polynucleotide of the present invention, thereby detecting resistance to brown planthopper. This is a method for testing rice. That is, SEQ ID NO: 2
When an RNA having a base sequence encoding the amino acid sequence represented by or a partial sequence thereof is detected from a test rice, the test rice can be assayed as a rice planthopper-resistant rice. Therefore, by using the assay method of the present invention, a test rice population, for example, a rice population obtained in various mating processes, is easily and efficiently obtained from a brown planthopper-resistant rice plant, for example, a biotype 2 brown planthopper. Can be selected. Specific examples of rice having resistance to the brown planthopper of biotype 2 include IR1154-243, Kanto PL5,
Cultivars such as Norin-PL4, or rice plant resistant to brown planthoppers produced by crossing using these varieties can be mentioned. The presence of an RNA having the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial sequence thereof in a test rice can be determined, for example, by (1) using the polynucleotide of the present invention as a primer and cDNA prepared from the test rice as a template PCR was performed using SEQ ID NO: 2
By detecting the amplification of a polynucleotide having the nucleotide sequence encoding the amino acid sequence represented by or a partial nucleotide sequence thereof, or (2) labeling the polynucleotide of the present invention and subjecting it to RNA prepared from test rice or It can be examined by hybridizing to the cDNA and detecting RNA or cDNA having the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial nucleotide sequence thereof. A method for examining the presence of RNA having a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial sequence thereof in a test rice plant will be specifically described below.

(1) Assay method of the present invention using PCR The PCR using the polynucleotide of the present invention as a primer and the cDNA of a test rice as a template is described, for example, in "Plant PCR Experimental Protocol: Nucleic Acid Isolation and Genome / Gene Amplification of a polynucleotide having the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial nucleotide sequence thereof according to the method described in "Latest Expression Analysis Method (Shimamoto, Sasaki, Shujunsha)" etc. SEQ ID NO: 2 by detecting
Can be detected in test rice with RNA having a base sequence encoding the amino acid sequence represented by or a partial sequence thereof. Examples of the primer used here include the polynucleotide of the present invention having a chain length of about 10 to 50 bases, preferably about 20 to 30 bases. Such a polynucleotide of the present invention can be designed based on, for example, the nucleotide sequence shown in SEQ ID NO: 1. Specifically, for example, the base sequence represented by base numbers 1 to 363 of SEQ ID NO: 1, the base sequence represented by base numbers 1942 to 2166 of SEQ ID NO: 1, the base sequence represented by base numbers 3055 to 3449 of SEQ ID NO: 1 A partial base sequence is selected from a base sequence such as a base sequence complementary to these base sequences, and a polynucleotide of the present invention having the selected base sequence is designed and synthesized. Preferably, a base sequence represented by base numbers 1 to 123 of SEQ ID NO: 1, a base sequence represented by base numbers 3127 to 3449 of SEQ ID NO: 1, a partial base sequence from a base sequence complementary to these base sequences, or the like And designing and synthesizing the polynucleotide of the present invention having the selected base sequence. These polynucleotides of the present invention may contain a base sequence such as a restriction enzyme recognition sequence or a linker sequence at the 5 ′ end as long as the function as a primer is not hindered. Preferred examples of the primer include the polynucleotide of the present invention having the base sequence shown in any one of SEQ ID NOs: 12 to 17. As the conditions for PCR, conditions suitable for the primers used are selected. As the reaction solution, for example, 0.
1 to 100 mM Tris-HCl (pH 8.3), 0.1 to 500 mM KCl, 0.1 to 15
mM MgCl ₂ , containing a mixture of ₂₀ to 2000 μM dNTP (equimolar mixture of deoxyadenosine triphosphate, deoxycytosine triphosphate, deoxyguanine triphosphate, and deoxythymine triphosphate), and containing the primer of 0.02 to 2 μM. Containing 0.25-25 units / 100 μl of thermostable polymerase, 0.01 p
A reaction solution containing g to 10 μg of the cDNA of the template can be mentioned, preferably 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.
A reaction solution containing a mixed solution of 5 mM MgCl ₂ and 200 μM dNTP, containing 0.2 μM of the primer, containing 2.5 units / 100 μl of a thermostable polymerase, and containing 5 ng of cDNA of a template can be given. Also, as the heat retention condition, 90 ~ 100 ℃
0.5 seconds to 10 minutes, then 30 to 75 ° C. for 0.5 seconds to 10 minutes, and further 50 to 80 ° C. for 0.5 seconds to 15 minutes as a single cycle, which can be carried out for 20 to 45 cycles. At 94 ° C for 1 minute, then at 55 ° C for 2 minutes and at 72 ° C
A condition in which the heat retention for 3 minutes is defined as one cycle and the cycle is performed for 30 cycles can be given. Also, at 90-100 ° C for 0.5 seconds ~
The conditions in which this is carried out for 20 to 45 cycles, with 10 minutes followed by 30 to 80 ° C. for 0.5 seconds to 15 minutes as one cycle, and preferably 94 ° C. for 0.5 seconds and then 70 ° C. for 4 minutes for 1 cycle. As a cycle, conditions for performing this for 30 cycles can be cited. Further, the reaction may be performed by combining these conditions. Examples of the thermostable polymerase include TAKARA Taq (Takara Shuzo), TAKARA Ex Taq (Takara Shuzo), Pyrobest DNA Polymerase (Takara Shuzo),
Advantage cDNA polymerase (manufactured by Clontech) and the like can be mentioned. For the cDNA of the test rice as a template, total RNA was extracted using, for example, ISOGEN (manufactured by Nippon Gene), and then mRNA Purification Kit (manufactured by Amersham Pharmacia) and Oligotex ^™ -dT30 <Super> (manufactured by Takara Shuzo) were commercially available. Purify mRNA having a poly A chain using the kit of the above, using the resulting mRNA as a template, for example, SMAR
^TTM PCR cDNA Synthesis Kit (Clontech), cD
NA Synthesis Kit (Takara Shuzo), cDNA Synthesis Kit
(Manufactured by Amersham Pharmacia) or the like to carry out the reaction. The polynucleotide obtained by the PCR is fractionated by an electrophoresis method. In general, electrophoresis can be performed using a 3 to 20% polyacrylamide gel or a 2 to 8% agarose gel such as NusieveAgarose (manufactured by FMC) to fractionate short polynucleotides of about 1000 bases or less. For the fractionation of long polynucleotides of about 1000 bases or more, 0.6-2% agarose gel can be used. Electrophoresis conditions include, for example, 100 V for about 30 minutes, and 50 V for about 1 hour. Buffers used for electrophoresis include Tris-phosphate (pH 7.5 to 8.0), Tris-acetic acid (pH 7.5 to 8.0), T
ris-boric acid type (pH 7.5 to 8.0) and the like, and preferably
Tris-acetic acid system (pH 7.5 to 8.0) may be mentioned. If necessary, EDTA or the like can be added to the buffer. Examples of the method for detecting the polynucleotide in the electrophoresis gel include a staining method using a dye that is a phenanthridine-based dye and interacts with a nucleic acid, such as ethidium bromide. Specifically, for example, a final concentration of 0.5μ
After dipping the gel after electrophoresis in an aqueous solution of g / ml ethidium bromide, the red band of the complex of polynucleotide and ethidium bromide can be detected by irradiating the gel with ultraviolet light such as 254 nm or 365 nm in a dark place. it can. By comparing the mobility of the polynucleotide amplified by the PCR on electrophoresis with a size marker, it is determined whether the polynucleotide is a polynucleotide having a size corresponding to the combination of the primers used in the PCR. As a result, it is possible to detect the presence of RNA having a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial sequence thereof in a test rice. Further, the polynucleotide amplified by the PCR is used, for example, in a TA cloning vector (manufactured by Invitrogen), a pUC18 vector (manufactured by Takara Shuzo), a pUC19 vector (manufactured by Takara Shuzo), pBlueScriptII.
After subcloning into a vector (manufactured by Stratagene) or the like, the nucleotide sequence is determined according to a conventional method, and the amplified polynucleotide has a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial sequence thereof. You may check what you are doing. At the same time, as a negative control, using a cDNA prepared from a rice plant that does not show resistance to brown planthopper such as Tsukushibare or Nipponbare, PCR was performed under the same conditions as above using the same primers used in the case of brown planthopper-resistant rice. A test may be performed to determine that the polynucleotide that is amplified in the case of brown planthopper resistant rice is not amplified.

(2) Assay Method of the Present Invention Using Hybridization Method The polynucleotide of the present invention is labeled and hybridized with RNA or cDNA prepared from a test rice, and the amino acid sequence represented by SEQ ID NO: 2 is synthesized. By detecting RNA or cDNA having the encoding nucleotide sequence or a partial nucleotide sequence thereof, the presence of the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or the RNA having the partial nucleotide sequence thereof in a test rice can be detected. it can. Here, a labeled polynucleotide used as a probe is, for example, about 10 bases to about 3000 bases.
The polynucleotide of the present invention having a base chain length can be mentioned. Specific examples include the polynucleotide of the present invention designed based on, for example, the nucleotide sequence represented by SEQ ID NO: 1, and more specifically, the nucleotide sequence represented by SEQ ID NO: 1,
Nucleotide sequence represented by nucleotide numbers 1 to 363 of SEQ ID NO: 1, nucleotide sequence represented by nucleotide numbers 1942 to 2166 of SEQ ID NO: 1, SEQ ID NO: 1
The base sequence represented by base numbers 3055 to 3449, SEQ ID NO: 1
The base sequence represented by base numbers 1 to 123, the base sequence represented by base numbers 3127 to 3449 of SEQ ID NO: 1, and the polynucleotide of the present invention having a base sequence such as a base sequence complementary to these base sequences. it can. These polynucleotides of the present invention may have one end, such as a restriction enzyme recognition sequence or a linker sequence,
It may contain a base sequence of about one to several tens of bases. The label of the polynucleotide can be a radioisotope label or a fluorescent label. For labeling with a radioisotope, for example, Random Primed DNA Labeling Ki
t (Boehringer), Random Primer DNA Labelling
Kit Ver.2 (Takara Shuzo) or the like can be used. In addition, fluorescent labels include, for example, ECL DirectNucleic Acid Lab
elling and Ditection System (manufactured by Amersham Pharmacia) or the like can be used.

Hybridization using RNA can be performed, for example, in accordance with the method described in Cloning and Sequence: Plant Biotechnology Experiment Manual (edited by Watanabe and Sugiura, Rural Cultural Company, 1989).
For example, total RNA was extracted from test rice using ISOGEN (manufactured by Nippon Gene), and 5 μg to 5 μg thereof was extracted.
0 μg, preferably 20 μg, is fractionated on a 1.2% denaturing gel, and blotted on a nylon filter such as Hybond-N + (Amersham Pharmacia). Hybridization is performed using the nylon filter and the labeled probe. Hybridization conditions vary depending on the probe used. For example, 450 to 900 mM sodium chloride, 45
-90 mM sodium citrate, 0.1-1.0% sodium dodecyl sulfate (SDS) and 0-200 [mu]
g / ml of denatured calf thymus DNA, optionally containing albumin, ficoll, polyvinylpyrrolidone, etc. at a concentration of 0-0.2%, preferably 900 m
M sodium chloride, 90 mM sodium citrate,
A pre-hybridization solution containing 1% SDS and 100 μg / ml denatured calf thymus DNA was
50-200 μl per 1 cm ^{2 of the} nylon filter
, And the nylon filter is immersed in the solution and incubated at 42 to 68 ° C for 1 to 4 hours, preferably at 55 ° C for 2 hours. Then, for example, 450-900 mM
Sodium chloride, 45-90 mM sodium citrate, 0.1-1.0% SDS and 0-200 μg /
ml of denatured calf thymus DNA, optionally containing albumin, ficoll, polyvinylpyrrolidone, etc. at a concentration of 0-0.2%, preferably 900 mM sodium chloride, 90 mM A hybridization solution containing sodium citrate, 1% SDS, and 100 μg / ml denatured calf thymus DNA and the labeled probe (1.0 × 10 ⁴ -2.cm/cm ² nylon filter).
0 × 10 ⁶ cpm) is prepared at a rate of 50 to 200 μl per 1 cm ² of nylon filter, and the nylon filter is immersed in the solution, and the solution is immersed at 42 to 68 ° C. for 4 to 20 hours, preferably 55 ° C. For 16 hours to carry out a hybridization reaction. After the hybridization, the nylon filter was removed and 15 to 3
42 including 00 mM sodium chloride, 1.5-30 mM sodium citrate, 0.1-1.0% SDS, etc.
Wash solution at ~ 68 ° C, preferably 300 mM sodium chloride, 30 mM sodium citrate and 1%
The nylon filter is washed 1 to 4 times for 10 to 60 minutes, preferably twice for 15 minutes using a washing solution at 55 ° C. containing SDS. In addition, two nylon filters
× SSC solution (300 mM sodium chloride, 30 mM
Rinse briefly with sodium citrate) and dry. The nylon filter is subjected to, for example, autoradiography, an image analyzer (manufactured by Fuji Film Co., Ltd.), and the like, and a signal indicating that the probe used has hybridized with RNA on the nylon filter is detected. Can be detected in a test rice plant with an RNA having a nucleotide sequence encoding the amino acid sequence represented by or a partial nucleotide sequence thereof.

Using the probe described above, hybridization was carried out using the cDNA of the test rice prepared according to the method described above as a template, and a signal indicating that the probe used hybridized with the cDNA on the nylon filter. Can be detected to detect the presence of RNA having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial nucleotide sequence thereof in a test rice. Hybridization is performed for all R
Hybridization conditions when NA or mRNA is used as a template may be the same.

At the same time as performing hybridization using RNA or cDNA prepared from brown rice planthopper-resistant rice as described above, RNA or cDNA prepared from rice that does not show brown planthopper resistance, such as Tsukushibare or Nipponbare, is used as a negative control. cDNA
Using the same probe as in the case of brown planthopper-resistant rice, hybridization was performed under the same conditions, and it was determined that no signal indicating that the probe used hybridized with RNA or cDNA on the nylon filter was detected. You may.

[0018]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the present invention is not limited thereto.

Reference Example 1 (C33-31-67-29)
Breeding) Norin-PL4 is crossed with Tsukushibare to obtain seeds. The obtained seeds are sown on acid soil, and genomic DNA is prepared from rice grown from various offspring using, for example, a commercially available ISOPLANT (manufactured by Nippon Gene) according to the attached protocol. The genomic DNA was transferred to EcoRI,
After digestion with HindIII and BamHI respectively,
The mixture is fractionated on a 0.8% agarose gel and blotted on a nylon filter Hybond-N + (manufactured by Amersham Pharmacia). An RFLP marker (Murata) mapped on the chromosome of Norin-PL4
K. et. al. Genes & Genet.
Syst. 73: (6) 359-364 (199
8)) Random Labeling Kit
[Boehringer Mannheim] [α-32
[P] dCTP (manufactured by Amersham Pharmacia) is used as a probe to perform hybridization with the above nylon filter. At this time, thymus DNA of calf denatured to 6 × SSC / 1% SDS was used as a hybridization solution at a final concentration of 100 μg / m 2.
l is used. The reaction was performed at 65 ° C. overnight, and the nylon filter was washed with 2 × SSC / 1% SD.
In S, the operation of shaking at 65 ° C. for 30 minutes is repeated twice. Hybridization using genomic DNA and RFLP markers is performed for Norin-PL4 in the same manner as described above. After exposing each of the washed nylon filters to an imaging plate, the imaging plate is subjected to BAS2000 (manufactured by Fuji Film Co., Ltd.).
Analyze with. From the results, Genes & Gene
t. Syst. 73: (6) 359-364 (19
98) A clone of planthopper-resistant rice is selected in accordance with the method described in 98). The selected clones (one generation of self-breeding) are further self-bred to obtain the next generation, and the same analysis and selection as above are performed. By repeating such self-breeding, C33-
31-67-29 is obtained.

Example 1 (Preparation of the Polynucleotide of the Present Invention) (1) Preparation of mRNA Tsukushibare and C33-31-67-29 seeds were
Each was sown on an acid soil Bonsol (manufactured by Sumitomo Chemical), and cultivated at 26 ° C. under conditions of repeating a light place for 16 hours and a dark place for 8 hours. Ten leaves of the plant body 2 months after sowing were sampled, and total RNA was extracted using ISOGEN (manufactured by Nippon Gene) according to the attached protocol. From 500 μg of this total RNA, Oligote
mRNA was purified using x-dT30 (manufactured by Takara Shuzo) according to the attached protocol.

(2) The present invention by the subtraction method
Selection of Renucleotide mRNA prepared from horsetail barley in Example 1 (1)
Using CLONTECH PCR-Select^TM 
cDNA Subtraction Kit (Clonte
Product) and follow the attached protocol
Traction. First obtained in Example 1 (1)
For 3.5 μg of each mRNA, 2 μl of 5 × 1^st 
Strand buffer (250 mM Tris-HCl
Buffer (pH 8.5), 40 mM MgCl_Two, 150
mM KCl, 5 mM DTT), 1 μl of 10 mM
dNTPs and 1 μl of 10 μM c provided with the kit
DNA synthesis primer was mixed
AMV reverse tracescript in solution
Add 20 units of ase and further add distilled water
To a final volume of 10 μl.
For 1.5 hours. To this, 16 μl of 5 × 2^nd
 Strand buffer (500 mM KCl, 5
0 mM ammonium sulfate, 25 mM MgCl_Two, 0.75 mM β
-Nicotinamide nucleotide, 100 mM Tris-salt
Acid buffer (pH 7.5), 0.25 mg / ml BS
A) 1.6 μl of 10 mM dNTP, 4 μl of 20
x2 ^nd strand enzyme cockta
il (DNA Polymerase I 6 units
/ Μl, RNaseH 0.25 units / μl, E.C.
coli DNA ligase 1.2 units / μ
1), and further by adding distilled water.
After adjusting the final volume to 80 μl, the mixture was incubated at 16 ° C. for 2 hours.
Was. In addition, 6 units of T4 DNA polymerase
and further warmed at 16 ° C. for 30 minutes,
μl of 20 × EDTA / glycogenix (0.
2M EDTA, 1 mg / ml glycogen)
After addition, 80 μl of phenol / chloroform /
Add and mix isoamyl alcohol (25: 24: 1)
Then, centrifuge at 14000 rpm for 5 minutes at room temperature.
The layers were separated. Add 40 μl of 4M ammonium acetate
After adding 300 μl of 95% ethanol and mixing,
Centrifuge at 4000 rpm for 10 minutes at 4 ° C.
Precipitated. The precipitated polynucleotide is treated with 80% ethanol
After rinsing with sterile water, suspend in 50 μl of sterile water.
15 units of RsaI was added to the mixture and incubated for 1.5 hours.
Was. 2.5 μl of 20 × EDTA / glycog
After adding en mix, phenol /
Chloroform / isoamyl alcohol (25:24:
1) treatment and ethanol precipitation
Rinsed with 80% ethanol
Thereafter, the suspension was suspended in 5.5 μl of sterilized water (hereinafter referred to as “suspension”).
The contained polynucleotide is designated as driver cDNA.
Describe. ). This driver cDNA suspension 1
2 μl of a solution obtained by adding 5 μl of sterile water to μl, attached to the kit
Genus 10 μM Adapter 12 μl and T4 DN
Mix 400 units of A ligase
The final volume was adjusted to 10 μl by adding bacterial water.
Was. After keeping this at 16 ° C. overnight, 1 μl of 20 × E
DTA / glycogen mix was added at 72 ° C. for 5 minutes.
Incubated for a minute. Hereinafter, the cDNA contained in the reaction solution is referred to as t
Ester cDNA1. In addition, adapter 1
A similar reaction was performed using adapter 2R instead.
The cDNA contained in the obtained reaction solution was tested c
Described as DNA 2R. Similarly, C33-31-67
-29 using mRNA prepared from
As in the case of using mRNA,
er cDNA, tester cDNA 1 and te cDNA
Ster cDNA2R was obtained. Then, subtraction
Was performed. That is, C33- prepared as described above.
Driver cD from 31-67-29 cDNA
1.5 μl NA solution, te from barnacle cDNA
1.5 μl of ster cDNA1 solution and kit
4x hybridization buffer of the genus
 1 μl mixed solution (solution A) and C33-31
Driver cDNA solution derived from 67-29 cDNA
 1.5 μl, tester derived from Tsukushibare cDNA
 1.5 µl cDNA 2R solution and kit included
4x hybridization buffer
A solution (solution B) mixed with 1 μl was prepared.
Each of them was placed at 98 ° C. for 1.5 minutes and then at 68 ° C. for 8 minutes.
Incubated for hours. On the other hand, C33-31-67-29 cD
1 μl of NA derived driver cDNA solution, kit
4x hybridization buff attached to
er and 1 μl of sterile water (solution)
C) was prepared and kept at 98 ° C. for 1.5 minutes. Obtained above
4 μl of solution A, 4 μl of solution B and 4 μl of solution C
And kept at 68 ° C. overnight before adding 200 μl
l dilution buffer (20 mM HE
PES-HCl (pH 8.3), 50 mM NaCl,
0.2 mM EDTA (pH 8.0) was added and 68
Incubated at ℃ for 7 minutes. To 1 μl of the solution, add a PCR reaction solution
(40 mM Tricine-KOH (pH 9.2),
15 mM KOAc, 3.5 mM Mg (OAc)_Two,
3.75 μg / ml BSA, 0.2 mM dNTP)
23.5 μl, 50 × Advantage cDN
A polymerase mix (KlenTaq-
1 DNA polymerase 1.1 μg / μ
1, 50% Glycerol, 40 mM Tris-HCl buffer
Buffer (pH 7.5), 50 mM KCl, 25 mM
(NH_Four)_TwoSO_Four, 0.1 mM EDTA, 5 mM β-
Mercaptoethanol, 0.25% Thesit)
(Clontech) 0.5 μl and 10 μM
1 μl of PCR Primer 1 was added, and PCR (75
5 minutes at 94 ° C and 25 seconds at 94 ° C,
10 seconds, then 66 ° C for 30 seconds, 72 ° C for 1.5 minutes
This is done for 30 cycles with the heat retention of 1 cycle)
went. Next, 1 μl of a 10-fold dilution of the reaction solution was added
1 μm of 0 μM nested PCR Primer 1
l and 10 μM Nested PCR Primer2
Add 1 μl of R as a primer and have the same composition as above.
PCR (94 ° C. for 10 seconds, then
Incubate at 68 ° C for 30 seconds and 72 ° C for 1.5 minutes for one cycle.
This was carried out for 15 cycles). As above
The subtraction made in REVERSE S
Described as UBTRACTION. Also, drive
rcDNA from Tsukushibare-derived cDNA
er cDNA1 and tester cDNA2R
Using cDNA derived from C33-31-67-29,
Same conditions as REVERSE SUBTRACTION
Subtraction was performed. The subtraction
Hereinafter, referred to as FORWARD SUBTRACTION
Put on. FORWARD SUBTRACTION
The polynucleotide in the reaction solution obtained as described above is S-400
Using a column (Pharmacia), attach the attached protocol
Purified according to the rules. 9 μl of this purified fraction and TAC
loaning Vector (Invitrogen)
Ligation kit (Takara Shuzo)
The reaction was performed using Half of the reaction solution was used for E. coli JM1.
09 competent cells (Takara Shuzo)
The formation was performed. The E. coli is transformed into ampicillin.
100 μg / ml and 5-bromo-4-chloro-3-
Indolyl-β-D-galactopyranoside 40 μg / m
and cultivated at 37 ° C. overnight.
A clone that formed a white colony is represented by SEQ ID NO: 6.
Consisting of the base sequence shown in SEQ ID NO: 7
And a polynucleotide consisting of the nucleotide sequence represented by
PCR (94 ° C. for 30 seconds followed by 68 ° C.
1 cycle of heat retention for 1 minute and 23 cycles of this)
went. A part of this reaction solution was added to 4% Nusieve Ag.
Fractionation by gel electrophoresis using arose (FMC)
And a polynucleotide of 100 bp to 1.4 kb is detected.
223 clones thus selected were selected.

(3) Selection of the Polynucleotide of the Present Invention by the Dot Hybridization Method From the 223 clones selected in Example 1 (2), the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6 and the polynucleotide Mixing an equivalent amount of 0.6N NaOH with a reaction solution obtained by PCR using a polynucleotide consisting of the nucleotide sequence represented by 7 as a primer,
1 μl was spotted at two locations on a nylon filter Hybond-N + (Amersham). As a negative control, spots were similarly spotted on four clones in which amplification of the polynucleotide was not confirmed. Filter the nylon filter with 1.5N NaCl
/0.5 Tris · HCl (pH 7.5), shaken for 5 minutes, shaken in 2 × SSC for 5 minutes, and air-dried. This was irradiated with UV for 3 minutes to fix the polynucleotide. Two such nylon filters
A set was made. FORWARD SUBTRACTI
The polynucleotide was recovered from the PCR reaction solution obtained by ON by performing ethanol precipitation. After completely digesting this polynucleotide with RsaI, S-4
Purification was carried out using a No. 00 column (manufactured by Pharmacia) according to the attached protocol, and a polynucleotide obtained by performing ethanol precipitation was further added with 20 μl of TE.
(10 mM Tris · HCl (pH 8.0), 1 mM
About 30 ng of the polynucleotide.
The above-mentioned suspension containing Random Labeling
Using Kit (manufactured by Boehringer) according to the attached protocol, a probe labeled with [α- ³² P] dCTP (manufactured by Amersham) was prepared. REVE
PC obtained by RSE SUBTRACTION
A probe was similarly prepared from the R reaction solution. The nylon filters prepared as described above were placed in a hybrid bag, one set at a time, and the hybridization solution (1
% SDS and 100 μg / ml denatured calf thymus D
(6 × SSC containing NA) was added thereto, and the mixture was kept at 68 ° C. for 1 hour to perform prehybridization. Then, FORWARDSUBTRACTI prepared as described above
3 million cpm of the probe derived from ON was enclosed in a hybrid bag together with a hybridization solution (20 ml) and a set of nylon filters, and kept at 68 ° C. overnight. Similarly, 3 million cpm of the probe derived from REVERSE SUBTRACTION was sealed in a hybrid bag together with 20 ml of the hybridization solution and the remaining one set of nylon filters, and kept at 68 ° C. overnight. After incubation, 72% using 2 × SSC containing 1% SDS.
Washed at C for 1 hour. This washing was repeated twice. After exposing the washed nylon filter to the imaging plate for 2 hours, the imaging plate was exposed to BAS20.
00 (manufactured by Fuji Film Co., Ltd.). The strength of the obtained signal was measured, and REVERSE SUBTR was measured.
Calculate the ratio of the signal obtained with the FORWARD SUBTRACTION-derived probe to the signal obtained with the ACTION-derived probe, and identify 66 polynucleotides having a large ratio (that is, many polynucleotides present in the PCR solution obtained by FORWARD SUBTRACTION). did.

(4) Determination of Nucleotide Sequence of Selected Polynucleotides 66 Escherichia coli clones containing the polynucleotide selected in Example 1 (3) were shake-cultured overnight in 2 ml of LB medium containing 100 μg / ml of ampicillin. did. The obtained culture was centrifuged at 8000 rpm for 2 minutes to collect cells, and the obtained cells were Qia-pre.
The plasmid was treated with pspin (manufactured by QIAGEN) according to the attached protocol, and the respective plasmids were purified. Using these plasmids as templates, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 and a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 were used as primers, respectively, and Taq Dye D
eoxy Terminator Cycle Seq
The nucleotide sequences of the polynucleotides inserted into the plasmids were determined by using a uinging Kit (manufactured by PE Biosystems Japan) and a fluorescent sequencer (manufactured by PE Biosystems Japan), and the base sequences were compared. From the results, five types of polynucleotides commonly inserted into a plurality of clones were selected.

(5) Selection of the Polynucleotide of the Present Invention by Northern Hybridization Using 5 ng of each of the clones containing the five polynucleotides selected in Example 1 (4) as a template, the base sequence represented by SEQ ID NO: 6 PCR (using 94 ° C. for 30 seconds, then 55 ° C. for 1 minute, and further 72 ° C. for 1 minute) using the polynucleotide of the present invention and the polynucleotide of the present invention consisting of the nucleotide sequence represented by SEQ ID NO: 7 as primers. The cycle was 30 cycles). The reaction solution was fractionated on a 4% acrylamide gel, and the gel containing the amplified polynucleotide was cut out. Each of these gels was used in an amount equivalent to that of the gel.
onBuffer (0.5 M ammonium acetate, 1 mM
After immersion in EDTA (pH 8.0) and standing at room temperature overnight, the mixture was centrifuged at 15000 rpm for 10 minutes. After transferring the supernatant to a new tube, 1/2 volume of Elution Buffer was added to the precipitate and mixed.
Centrifuged at 5000 rpm for 10 minutes. The supernatant was transferred to the tube described above, 100% ethanol twice the amount in the tube was added and mixed, and the mixture was allowed to stand on ice for 30 minutes. 1
The precipitate was collected by centrifugation at 5000 rpm for 10 minutes. This is suspended in 200 μl of TE and 1/10 volume of 3
M sodium acetate, 2 volumes of 100% ethanol were added, mixed, and left on ice. 30 minutes later, 15000
The precipitate was collected by centrifugation at rpm for 10 minutes, washed with 70% ethanol, and air-dried. The precipitate was dissolved in sterile water, completely digested with RsaI, and then subjected to ethanol precipitation to recover a polynucleotide.
Dissolved in 0 μl TE. About 30 of this polynucleotide
ng of the solution is transferred to a Random Labeling Ki.
t (manufactured by Boehringer Mannheim) according to the attached protocol, and the five polynucleotides were labeled with [α- ³² P] dCTP (manufactured by Amersham Pharmacia). Tsukushibare and C33-31-67-2 prepared as described in Example 1 (1)
20 μg of total RNA was fractionated by 1.2% denaturing agarose gel electrophoresis, and the fractionated RNA was subjected to 10 × S
During the SC, the gel was filtered from the nylon filter Hybond-N + by the capillary blotting method (cloning and sequencing: Rural Bunkasha, supervised by Tadashi Watanabe, edited by Masahiro Sugiura).
(Manufactured by Amersham). After blotting, the nylon filter was shaken in 2 × SSC for 5 minutes, air-dried, and baked at 80 ° C. for 2 hours. Five such nylon filters were produced. The nylon filter prepared as described above was placed in a hybrid bag, a hybridization solution was placed therein, and the mixture was kept at 55 ° C. for 1 hour to perform pre-hybridization. Either of the above-mentioned five types of labeled polynucleotides, each in an amount of 3,000,000 cpm and a hybridization solution of 10 ml, were sealed in a hybrid bag together with the nylon filter, and kept at 55 ° C. overnight. After keeping the temperature, the nylon filter was washed at 55 ° C. for 20 minutes in a solution containing 2 × SSC and 1% SDS. After repeating this washing three times, the nylon filter was exposed to the imaging plate overnight. The imaging plate was analyzed using BAS2000 (manufactured by Fuji Film). As a result, in one of the five polynucleotides, all Rs of C33-31-67-29
A signal was detected at a position of about 3.5 kbp from NA,
On the other hand, no signal was detected from the total RNA of Tsukubabare. Therefore, the polynucleotide was found to be a polynucleotide specifically transcribed in brown planthopper resistant rice. Further, using a plasmid of a clone containing this polynucleotide as a template, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 as primers, respectively, and using Taq Dye
Deoxy Terminator Cycle Se
By using a quenching kit (manufactured by PE Biosystems Japan) and a fluorescent sequencer (manufactured by PE Biosystems Japan), the nucleotide sequence of the polynucleotide inserted into the plasmid was analyzed. The determined nucleotide sequence is shown in SEQ ID NO: 3 (corresponding to the nucleotide sequence represented by nucleotide numbers 2726 to 2956 in the nucleotide sequence represented by SEQ ID NO: 1).

(6) Preparation of polynucleotide of the present invention (a) Using 800 ng of mRNA prepared from C33-31-67-29 in Example 1 (1) as a template, SMART
^TM PCR cDNA Synthesis Kit
(Clontech) according to the attached protocol to prepare double-stranded cDNA. This double-stranded cDNA was converted to Marathon ^™ cDNA Ampli
It was treated according to the attached protocol using a Fission Kit (manufactured by Clontech) to obtain SEQ ID NO: 10.
PCR using a polynucleotide consisting of the nucleotide sequence represented by the formula (1) and a primer attached to the kit which can anneal to the adapter (5 cycles at 94 ° C. for 5 seconds followed by 72 ° C. for 4 minutes as one cycle, and C. for 5 seconds followed by 70 ° C. for 4 minutes as one cycle, followed by 5 cycles. Further, 94 ° C. for 5 seconds and then 68 ° C. for 4 minutes as one cycle.
Cycle). Part of the PCR reaction solution is 4% Nus
The fractionated sample was subjected to gel electrophoresis using ieAgarose (manufactured by FMC). As a result, it was found that a polynucleotide of about 900 bp was amplified. The remaining PCR reaction solution was purified using an S-400 column (Pharmacia) according to the attached protocol. 4 μl of purified fraction and TA Cloning Vec
A ligation reaction was performed with 25 ng of tor (manufactured by Invitrogen) using a ligation kit (manufactured by Takara Shuzo). 2 μl of the reaction solution was added to E. coli JM109 competent cells (manufactured by Takara Shuzo Co., Ltd.), transformation was performed, and 100 μg of the E. coli was added to ampicillin.
/ Ml, 5-bromo-4-chloro-3-indolyl-β
-D-galactopyranoside was seeded on an LB plate containing 40 µg / ml, and cultured at 37 ° C overnight. A part of the white colony was suspended in a PCR reaction solution containing a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 8 and a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 9, and subjected to PCR (at 94 ° C). A cycle of 5 seconds followed by 1 minute at 55 ° C. and further 3 minutes at 72 ° C.
Cycle). A part of this reaction solution is 4% Nusi
The resultant was subjected to gel electrophoresis using Eve Agarose (manufactured by FMC) to fractionate, and a clone into which a polynucleotide of about 900 bp was inserted was selected. The selected E. coli clone was transformed into LB2 containing 100 μg / ml of ampicillin.
The cells were cultured overnight with shaking in ml. From the culture, 8000
The cells were collected by centrifuging at 2 rpm for 2 minutes, and the obtained cells were Qia-prep spin (QIAGE)
(Manufactured by N. Co.) according to the attached protocol, and the plasmid was purified. Using this plasmid as a template, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 and a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 as primers,
Dye Deoxy Terminator Cycle
The nucleotide sequence of the polynucleotide inserted into the plasmid was analyzed by using eSequencing Kit (manufactured by PE Biosystems Japan) and a fluorescent sequencer (manufactured by PE Biosystems Japan). Furthermore, a polynucleotide was synthesized based on the revealed base sequence, and this was used as a primer to analyze the base sequence in the same manner as described above. As a result, SEQ ID NO: 1
The base sequence represented by base numbers 2734 to 3472 of the base sequence represented by was clarified.

(B) Using the double-stranded cDNA prepared from C33-31-67-29 as a template, a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 and Marat
hon ^™ cDNA Amplification K
it (Clontech) kit and PCR (94)
5 seconds at 72 ° C. and then 4 minutes at 72 ° C.
Five cycles were performed with one cycle of keeping the temperature at 0 ° C. for 4 minutes, and 25 cycles were performed with one cycle of keeping the temperature at 94 ° C. for five seconds and then at 68 ° C. for four minutes. P
As a result of fractionating a part of the CR reaction solution on a 0.8% agarose gel, it was found that a polynucleotide of about 3 kb had been amplified. The remaining PCR reaction solution was fractionated on a 0.8% agarose gel, a gel containing a polynucleotide of about 3 kb was cut out, and the attached protocol was prepared using a Prep-A-Gene DNA purification kit (Bio-Rad). And the polynucleotide contained in the gel was purified. 50 ng of this polynucleotide and TA Cloning Vector (Invitro
(Gen Co., Ltd.) and a ligation reaction using a ligation kit (Takara Shuzo Co., Ltd.). The total amount of the reaction solution was added to E. coli JM109 competent cells (manufactured by Takara Shuzo Co., Ltd.), and the E. coli was transformed with ampicillin 100 μg / ml, 5-bromo-4
-Chloro-3-indolyl-β-D-galactopyranoside was seeded on an LB plate containing 40 μg / ml and cultured at 37 ° C. overnight. A part of the white colony was suspended in a PCR reaction solution containing a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 8 and a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 9, and subjected to PCR (at 94 ° C). For 5 seconds, the temperature was kept at 55 ° C. for 1 minute, and further kept at 72 ° C. for 3 minutes as one cycle. A part of the reaction solution was fractionated by 0.8% agarose gel electrophoresis, and a clone into which a polynucleotide of about 3 kb was inserted was selected. E. coli clones
Shaking culture was performed overnight in 2 ml of LB medium containing 00 μg / ml. The cells were collected by centrifuging the culture at 8000 rpm for 2 minutes, and the obtained cells were separated by Qia-p
The plasmid was treated with reppin (manufactured by QIAGEN) according to the attached protocol, and the plasmid was purified.
Using this plasmid as a template, a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 as primers, respectively, were used as Taq Dye DeoxyTerm.
indicator Cycle Sequencing K
The nucleotide sequence of the polynucleotide inserted in the plasmid was determined using it (manufactured by PE Biosystems Japan) and a fluorescent sequencer (manufactured by PE Biosystems Japan). Furthermore, a polynucleotide was synthesized based on the revealed base sequence, and this was used as a primer to analyze the base sequence in the same manner as described above. As a result, base numbers 1-29 of the base sequence shown in SEQ ID NO: 1
The base sequence represented by 20 was revealed.

(C) From the nucleotide sequences obtained in (a) and (b) and the nucleotide sequence shown in SEQ ID NO: 3, the nucleotide sequence shown in SEQ ID NO: 1 was determined.

(D) Using double-stranded cDNA prepared from C33-31-67-29 as a template,
e cDNA polymerase mix (Clontech) according to the attached protocol.
Perform CR. Using a combination of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 and a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 as primers, one cycle of incubation at 94 ° C. for 5 seconds and then at 72 ° C. for 4 minutes. This was repeated for 5 cycles, and the temperature was maintained at 94 ° C. for 5 seconds and then at 70 ° C. for 4 minutes as one cycle.
The cycle is further maintained at 94 ° C. for 5 seconds and then at 68 ° C. for 4 minutes as one cycle, and 25 cycles are performed. A part of the PCR reaction solution is fractionated by 0.8% agarose gel electrophoresis, and it is confirmed that a polynucleotide of about 3.5 kbp is amplified. The remaining PCR reaction solution was applied to an S-400 column (manufactured by Pharmacia) to purify the polynucleotide in the PCR reaction solution according to the attached protocol, and a purified fraction containing 150 ng of the polynucleotide and a TA cloning vector.
(Invitrogen) and 50 ng were reacted using a ligation kit (Takara Shuzo). Half of the reaction solution was added to E. coli JM109 competent cells (Takara Shuzo), and the mixture was transformed.
The E. coli was treated with 100 μg / ml ampicillin and 40 μg / ml.
Plate on LB plate containing μg / ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside and incubate at 37 ° C. overnight. A part of the white colony was suspended in a PCR reaction solution containing a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 8 and a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 9,
(40 cycles of 1 cycle at 94 ° C. for 5 seconds followed by 55 ° C. for 1 minute and 72 ° C. for 3 minutes)
I do. A part of the reaction solution is fractionated by 0.8% agarose gel electrophoresis, and an E. coli clone in which amplification of a polynucleotide of about 3.5 kbp is detected is selected. The selected E. coli clone is shake-cultured overnight in 2 ml of LB containing 100 μg / ml of ampicillin. The cells are collected by centrifuging the culture at 8000 rpm for 2 minutes. The cells were transformed into Qia-prep spin (QIAGE
(Manufactured by N Company) according to the attached protocol to purify the plasmid. Using this plasmid as a template, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 and a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 as primers,
Dye Deoxy Terminator Cycle
e Sequencing Kit (manufactured by PE Biosystems Japan) and a fluorescent sequencer (manufactured by PE Biosystems Japan) are used to analyze the nucleotide sequence of the polynucleotide inserted into the plasmid.
Further, a polynucleotide was synthesized based on the revealed base sequence, the base sequence was analyzed in the same manner as described above using this as a primer, and the plasmid was analyzed using base numbers 1-3413 of the base sequence shown in SEQ ID NO: 1. Confirm that it contains a polynucleotide having the indicated base sequence.

Example 2 (Assay of Rice Planthopper-resistant Rice Plant by the Assay Method of the Present Invention Using PCR) Seeds of C33-31-67-29, Tsukushibare, and Nipponbare were each put on Bonsol (manufactured by Sumitomo Chemical Co., Ltd.), an acid soil. The plants were sown and cultivated at 26 ° C. for 16 hours in a bright place and then in a dark place for 8 hours. Ten leaves of the plant body 2 months after sowing were sampled. Total RNA was extracted from the obtained sample using ISOGEN (manufactured by Nippon Gene) in accordance with the attached protocol in the same manner as in Example 1 (1), and Oligotex-dT3 was extracted from the extracted total RNA.
0 (manufactured by Takara Shuzo) according to the attached protocol to purify the mRNA. MRNA purified from each rice
Using 800 ng as a template, SMART ^™ PCR c
Using DNA Synthesis Kit (manufactured by Clontech), operation was performed according to the attached protocol, and single-stranded cDNA was prepared for each. These single-stranded cDNs
Using A as a template, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 17 and SEQ ID NO: 1 to SEQ ID NO: 1
PCR using one of the polynucleotides consisting of the nucleotide sequence represented by any one of 5 as a primer (5 cycles at 94 ° C. for 5 seconds and 72 ° C. for 4 minutes as one cycle, Further 94
The temperature was kept at 5 ° C. for 5 seconds and then at 70 ° C. for 4 minutes as one cycle, and this was performed for 25 cycles. A part of this PCR reaction solution was fractionated by 0.8% agarose gel electrophoresis, and the amplified polynucleotide was analyzed. As a result, in PCR using any combination of primers, single-stranded c prepared from C33-31-67-29
Only when DNA was used as a template, it was revealed that a polynucleotide having a size of about 3 kb was amplified.
Similarly, a combination of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16 and one of the polynucleotides consisting of the nucleotide sequence of any of SEQ ID NO: 12 to SEQ ID NO: 15 as a primer,
R (5 cycles of 94 ° C. for 5 seconds followed by 72 ° C. for 4 minutes as one cycle, followed by 25 cycles of 94 ° C. for 5 seconds and 70 ° C. for 4 minutes as 1 cycle). . A part of this PCR reaction solution was fractionated by 0.8% agarose gel electrophoresis, and the amplified polynucleotide was analyzed. As a result, in PCR using any combination of primers, C3
Single-stranded cDNA prepared from 3-31-67-29
Only when was used as a template, it was found that a polynucleotide having a size of about 3 kb was amplified.

Example 3 (Assay of Rice Planthopper-resistant Rice Plant by the Assay Method of the Present Invention Using Hybridization Method) Seeds of C33-31-67-29 were sown on Bonsol (manufactured by Sumitomo Chemical Co., Ltd.) which is an acidic soil, and Cultivation is carried out under the condition of repeating a light place for 16 hours and then a dark place for 8 hours. Two weeks after seeding, a part of the leaf is sampled, and total RNA is prepared using ISOGEN (Nippon Gene). All R
20 μg of NA was fractionated by 1.2% denaturing agarose gel electrophoresis, and the fractionated RNA was subjected to capillary blotting in 10 × SSC (cloning and sequencing:
Rural culture company: Watanabe Tadashi, edited by Masahiro Sugiura) Blot from gel to nylon filter Hybond-N + (Amersham). After blotting, the nylon filter is shaken in 2 × SSC for 5 minutes, air-dried, and baked at 80 ° C. for 2 hours. Using a polynucleotide having a base sequence represented by base numbers 3127 to 3449 of SEQ ID NO: 1 as a template, Random Label
A probe labeled with [α-32P] dCTP (manufactured by Amersham Pharmacia) is prepared using an lling Kit (manufactured by Boehringer Mannheim). Hybridization is performed using the prepared probe and the above-mentioned nylon filter. At this time, the thymic DNA of calf denatured to a solution containing 6 × SSC and 1% SDS was used as a hybridization solution at a final concentration of 100 μg / m 2.
Incubate overnight at 55 ° C. using the solution added in l. To wash the nylon filter, remove the nylon filter by 2
30 ° C. in a solution containing XSSC and 1% SDS at 55 ° C.
The operation of shaking for 2 minutes is repeated twice.
Expose the washed nylon filter to the imaging plate. The exposed imaging plate is replaced with BAS2
000 (manufactured by Fuji Film Co., Ltd.), and a signal indicating that the probe used has hybridized with RNA on the nylon filter is detected. The same procedure as for C33-31-67-29 was carried out on Tsukushibare, and it was confirmed that no signal indicating that the probe used hybridized with RNA on the nylon filter was detected on the nylon filter.

The method for preparing the LB medium used in the examples is described below. 10 g of bactotripton (manufactured by Difco) and Bacto yeast extract (Difc)
o) (5 g) and 10 g of NaCl in 1 L of distilled water.
, Adjusted to pH 7.0 with 5M NaOH, and sterilized in an autoclave. In the case of an LB plate, 10 g of bactotryptone, 5 g of bactoeast extract, 10 g of NaCl and 15 g of agar are dissolved in 1 L of distilled water, and the pH is adjusted with 5 M NaOH.
Adjusted to 7.0 and autoclaved.

[0032]

According to the present invention, it is possible to provide a simple and efficient assay method for brown rice planthopper-resistant rice.

"Sequence List Free Text" SEQ ID NO: 4 A primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 5 A primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 6 Amplify the polynucleotide of the present invention SEQ ID NO: 7 A primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 8 A primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 9 SEQ ID NO: 10 Primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 11 Primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 12 Designed to amplify the polynucleotide of the present invention Primer SEQ ID NO: 13 Primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 14 Primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 15 Primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 16 A primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 17 A primer designed to amplify the polynucleotide of the present invention SEQ ID NO: 18 A primer designed to amplify the polynucleotide of the present invention

[0034]

[Sequence List] <110> Sumitomo Chemical Co. Ltd. <120> New polynucleotides and their use <130> P151213 <160> 18 <210> 1 <211> 3472 <212> DNA <213> Oryza sativa <220> < 221> CDS <222> (124) ... (3129) <400> Met Gly Leu Thr Cys Asp Thr Gln Thr Ala Lys Leu Ala Ile Ile 1 5 10 15 cta cta gca ttc ata ctg cta tgt cat ggg att ggc aac gtc gat tgc 216 Leu Leu Ala Phe Ile Leu Leu Cys His Gly Ile Gly Asn Val Asp Cys 20 25 30 cgt ggg aac aga gcc gat cag ctc tca ctg ctt gat ttc aag aag ggc 264 Arg Gly Asn Arg Ala Asp Gln Leu Ser Leu Leu Asp Phe Lys Lys Gly 35 40 45 atc acc aac gat cca tat gga gcc ttg gcc act tgg aac acc agc aca 312 Ile Thr Asn Asp Pro Tyr Gly Ala Leu Ala Thr Trp Asn Thr Ser Thr 50 55 60 cat ttc tgt agg tgg caa ggt gtc aag tgc acc tcc act ggg cca tgg 360 His Phe Cys Arg Trp Gln Gly Val Lys Cys Thr Ser Thr Gly Pro Trp 65 70 75 cgc gtc atg gcg ctc aat ctc tcc agc caa agt ttg aca ggc caa ata 408 Arg Val Met Ala Leu Asn Leu Ser Ser Gln Ser Leu Thr Gly Gln Ile 80 85 90 95 agg tcc tct ctc gga aac cta tcc ttc ctt aat ata ctt gac ctc ggc 456 Arg Ser Ser Leu Gly Asn Leu Ser Phe Leu Asn Ile Leu Asp Leu Gly 100 105 110 gat aat aac tta ctt ggc tcc tta cct cgc ctt ggc atc caa 504 Asp Asn Asn Leu Leu Gly Ser Leu Pro Arg Leu Gly Asn Leu Lys Gln 115 120 125 ctt cag gca ctc tat ctg tat aaa aac aat ttg aca ggg ata att cct 552 Leu Gln Ala Leu Tyr Leu Tyr Lys Asn Asn Leu Thr Gly Ile Ile Pro 130 135 140 gat gaa ctt aca aat tgt tcc agc tta acg tac ata gac ctc tca gga 600 Asp Glu Leu Thr Asn Cys Ser Ser Leu Thr Tyr Ile Asp Leu Ser Gly 145 150 155 aat gcc cta act ggt gca ctt cct cca aat tta ggc tct ctg tcc aat 648 Asn Ala Leu Thr Gly Ala Leu Pro Pro Asn Leu Gly Ser Leu Ser Asn 160 165 170 175 cta gct tat ctt tat ctt tct gcg aac aag cta act gga acc atc ccg 696 Leu Ala Tyr Leu T yr Leu Ser Ala Asn Lys Leu Thr Gly Thr Ile Pro 180 185 190 cag gcc ctt ggc aac atc acc acc ttg gta gaa atc tat ctt gat aca 744 Gln Ala Leu Gly Asn Ile Thr Thr Leu Val Glu Ile Tyr Leu Asp Thr 195 200 205 aat cga ttc gaa ggg ggc att cca gac aag ctt tgg caa ttg ccg aac 792 Asn Arg Phe Glu Gly Gly Ile Pro Asp Lys Leu Trp Gln Leu Pro Asn 210 215 220 ttg aca att ttg gcc cta ggt caa tac ggt gat atc cca 840 Leu Thr Ile Leu Ala Leu Gly Gln Asn Met Leu Ser Gly Asp Ile Pro 225 230 235 ttt aac ttc tcc agc ctt tct ctt caa ctt tta agc ttg gaa tac aat 888 Phe Asn Phe Ser Ser Leu Ser Leu Gln Le Leu Ser Leu Glu Tyr Asn 240 245 250 255 atg ttc ggc aag gta ttg cca caa aac att agt gat atg gta cct aat 936 Met Phe Gly Lys Val Leu Pro Gln Asn Ile Ser Asp Met Val Pro Asn 260 265 270 270 ctc caa att ctt cgc ttg gat tac aat atg ttc caa ggt caa atc cca 984 Leu Gln Ile Leu Arg Leu Asp Tyr Asn Met Phe Gln Gly Gln Ile Pro 275 280 285 tct tct cta gga aat gct ttg cag cta aca gaa ata agt atgcaSer Leu Gly Asn Ala Leu Gln Leu Thr Glu Ile Ser Met Ala Asn 290 295 300 aac tac ttc acc ggg caa att cct agc tct ttc gga aag ctt tct aaa 1080 Asn Tyr Phe Thr Gly Gln Ile Pro Ser Ser Phe Gly Lys Leu Ser Lys 305 310 315 ctc tcc tat att agt ctc gaa aat aat agc cta gag gct tct gat ggc 1128 Leu Ser Tyr Ile Ser Leu Glu Asn Asn Ser Leu Glu Ala Ser Asp Gly 320 325 330 335 caa ggc tgg gag ttc ctag c aga aac tgt agt aat cta gaa 1176 Gln Gly Trp Glu Phe Leu His Ala Leu Arg Asn Cys Ser Asn Leu Glu 340 345 350 ctg ttg tca ctg gct caa aat cag cta caa gga gaa ata ccg aat tca 1224 Leu Leu Ser Le Asn Gln Leu Gln Gly Glu Ile Pro Asn Ser 355 360 365 att ggg gac ctt ccg ctc aaa ctt caa cag cta gtg ctg agt gaa aat 1272 Ile Gly Asp Leu Pro Leu Lys Leu Gln Gln Leu Val Leu Ser Glu Asn 370 375 380 ag cta tca gga gaa gtt cca gca agc ata gga aac ctt cag ggc ctg 1320 Lys Leu Ser Gly Glu Val Pro Ala Ser Ile Gly Asn Leu Gln Gly Leu 385 390 395 ttt agg tta agt cta gat ttg aac aat ctt act ggc aaa ta gat gag 1368 Phe Arg Leu Ser Leu Asp Leu Asn Asn Leu Thr Gly Lys Ile Asp Glu 400 405 410 415 tgg gtt cca aag ctt aca aaa ctg caa aaa tta ctt ctc cac agg aac 1416 Trp Val Pro Lys Leu Thr Lys Leu Gln Lys Leu Leu Leu His Arg Asn 420 425 430 aac ttt agc ggg tcg att cca tcg tct ata gca gag ctt cct cgt ttg 1464 Asn Phe Ser Gly Ser Ile Pro Ser Ser Ile Ala Glu Leu Pro Arg Leu 435 440 445 tca acg ctt tca cta gca tat aat gca ttt gat ggt ccc ata cca tcc 1512 Ser Thr Leu Ser Leu Ala Tyr Asn Ala Phe Asp Gly Pro Ile Pro Ser 450 455 460 tca ttg gga aac ctt tca gga ctc cag aag tta tat cta agt cat aat 1560 Ser Leu Gly Asn Leu Ser Gly Leu Gln Lys Leu Tyr Leu Ser His Asn 465 470 475 aat ctc gaa ggt gtc ata cct cca gag ctt agt tac ctt aaa caa cta 1608 Asn Leu Glu Gly Val Ile Pro Pro Glu Leu Ser Tyr Leu Lys Gln Leu 480 485 490 495 ata aat cta agt tta tca gaa aac aag ctt act ggg gag atc cct ggc 1656 Ile Asn Leu Ser Leu Ser Glu Asn Lys Leu Thr Gly Glu Ile Pro Gly 500 505 510 act ttg agc cag tgt aaa gac ct g gca aac atc caa atg ggc aat aac 1704 Thr Leu Ser Gln Cys Lys Asp Leu Ala Asn Ile Gln Met Gly Asn Asn 515 520 525 ttc ctt act ggc aat atc cca gta act ttt ggc gac cta aag agc ctg 1752 Phe Leu Asn Ile Pro Val Thr Phe Gly Asp Leu Lys Ser Leu 530 535 540 ggc gta ctc aat ctt tcc cat aac agc ttg tcg ggc aca att ccg act 1800 Gly Val Leu Asn Leu Ser His Asn Ser Leu Ser Gly Thr Ile Pro Thr 545 550 555 aca cta aat gat cta cca gtc atg agc aaa ctg gac ctt tca tac aat 1848 Thr Leu Asn Asp Leu Pro Val Met Ser Lys Leu Asp Leu Ser Tyr Asn 560 565 570 575 cgt ctc caa gga aaa ata cca atg act gga ata t gca aat cct act 1896 Arg Leu Gln Gly Lys Ile Pro Met Thr Gly Ile Phe Ala Asn Pro Thr 580 585 590 gtt gtt tcg gtc cag gga aac ata gga cta tgt gga gga gtg atg gat 1944 Val Val Ser Val Gln Gly Asn Ile Gly Leu Cys Gly Gly Val Met Asp 595 600 605 ctc cgt atg ccc cca tgc caa gtt gtt tcc cag aga aga aaa aca cag 1992 Leu Arg Met Pro Pro Cys Gln Val Val Ser Gln Arg Arg Lys Thr Gln 610 615 620 620 tac tat ttg att aga gta ttg atc cct ata ttt ggc ttc atg tca ctc 2040 Tyr Tyr Leu Ile Arg Val Leu Ile Pro Ile Phe Gly Phe Met Ser Leu 625 630 635 ata tta gtg gtc tac ttc tta ctt ctt gag aag atg aagca 20 Ile Leu Val Val Tyr Phe Leu Leu Leu Glu Lys Met Lys Pro Arg Glu 640 645 650 655 aaa tat ata tcc tcg caa tct ttt ggt gag aat ttc ctt aaa gtc tca 2136 Lys Tyr Ile Ser Ser Gln Ser Phe Gly Glu Asn Phe Leu Lys Val Ser 660 665 670 tat aat gat cta gct caa gcc aca aga aac ttc tcg gag gct aac ctt 2184 Tyr Asn Asp Leu Ala Gln Ala Thr Arg Asn Phe Ser Glu Ala Asn Leu 675 680 685 att ggc aaa gga agc gt ggt gtg tac aga ggt aag ttg aag gaa 2232 Ile Gly Lys Gly Ser Tyr Gly Thr Val Tyr Arg Gly Lys Leu Lys Glu 690 695 700 tgt aag ttg gaa gta gcc gtg aag gtt ttt gac ctt gag atg cga ggt 2280 Cys Vals Les Ala Val Lys Val Phe Asp Leu Glu Met Arg Gly 705 710 715 gcc gaa aga agc ttc ata tcg gaa tgt gaa gct ctc aga agc att caa 2328 Ala Glu Arg Ser Phe Ile Ser Glu Cys Glu Ala Leu Arg Ser Ile 720 725 730 735 cat aga aat ctt ctt cca atc ata act gca tgc tcg acc gtg gac agt 2376 His Arg Asn Leu Leu Pro Ile Ile Thr Ala Cys Ser Thr Val Asp Ser 740 745 750 acg ggc aac gtt ttc aaa gct tta gtt tac gag tac atg cct aat ggc 2424 Thr Gly Asn Val Phe Lys Ala Leu Val Tyr Glu Tyr Met Pro Asn Gly 755 760 765 aac ttg gac act tgg ata cat gac aaa gag ggt ggg aaa gct ccc gga 2472 Asn Leu Asp Thr Trp Ile His Asp Lys Glu Gly Gly Lys Ala Pro Gly 770 775 780 cgt ttg ggt tta aga caa aca att agc ata tgt gtt aac ata gcc gat 2520 Arg Leu Gly Leu Arg Gln Thr Ile Ser Ile Cys Val Asn Ile Ala Asp 785 790 790 gac ctg tat tta cac cat gaa tgc gga aga aca act atc cat tgt 2568 Ala Leu Asp Tyr Leu His His Glu Cys Gly Arg Thr Thr Ile His Cys 800 805 810 815 gat ttg aaa ccc agc aat ata ctt tta gct gat gac atg aat gct ctt 2616 Asp Leu Lys Pro Ser Asn Ile Leu Leu Ala Asp Asp Met Asn Ala Leu 820 825 830 ttg gga gat ttt ggc att gca aga ttt tat atc gac tct tgg tcg aca 2664 Leu Gly Asp Phe Gly Ile Ala Arg Phe Tyr Ile Asp Ser Trp Ser Thr 835 840 845 tca aca ggt tcg aat agt aca gtc ggt gtt aag ggt acc ata ggg tat 2712 Ser Thr Gly Ser Asn Ser Thr Val Gly Val Lys Gly Thr Ile Gly Tyr 850 855 860 att cct cca gag tac gcg gga ggt ggt cat cca tct act tct ggg gat 2760 Ile Pro Pro Glu Tyr Ala Gly Gly Gly His Pro Ser Thr Ser Gly Asp 865 870 875 gtt tat agt ttc ggg ata gtg atc ctg gag ctt ata aca gga aaa aga 2808 Val Tyr Ser Phe Gly Ile Val Ile Leu Glu Leu Ile Thr Gly Lys Arg 880 885 890 895 ccg act gat cct atg ttc aaa gac gga ctc gac att atc agc ttt gtc 2856 Pro Thr Asp Pro Met Phe Lys Asp Gly Leu Asp Ile Ile Ser Phe 900 905 910 gag agt aac ttt cca cac cag ata ttt caa gtg att gat gct cga ctc 2904 Glu Ser Asn Phe Pro His Gln Ile Phe Gln Val Ile Asp Ala Arg Leu 915 920 925 gca gaa aag tcc atg gac tcc aat caacaat atg aca ctg gaa aat 2952 Ala Glu Lys Ser Met Asp Ser Asn Gln Thr Asn Met Thr Leu Glu Asn 930 935 940 gct gtc cac cag tgc ttg ata tct ctg cta caa ctt gcg ctt tct tgt 3000 Ala Val His Gln Cys Leu Ile Ser Leu Leu Gln Leu Ala Leu Ser Cys 945 950 955 acg cgc aaa tta cca agt gat cgc atg aac atg aaa caa ata gcc aac 3048 Thr Arg Lys Leu Pro Ser Asp Arg Met Asn Met Lys Gln Ile Ala Asn 960 965 970 975 aag atg cat tca atc aag acg aca tat gtt gga ttg gaa gct aag aaa 3096 Lys Met His Ser Ile Lys Thr Thr Tyr Val Gly Leu Glu Ala Lys Lys 980 985 990 tat ggt ccg gca tgt aat gaa caa gat gca tga attaccacgc 3 Tyr Gly Pro Ala Cys Asn Glu Gln Asp Ala 995 1000 gcgaccctga gtcgcgcaca acaattgcaa catattgata agtgtatagt ccaacaagaa 3209 gaaaatggtt cttgtgtgtt agaagttaag ataattaagg tgataatatg aagcaccaaa 3269 gcgtaccttc taacggtgat aatatgaaga ttggtgattg tgactttatg tcatggttta 3329 tttataacaa tatattactt caataatgta atagtatatg tatacgtaga taatgatgtt 3389 gtacctagca tgatcttgta ttatatgcaa gttgaactaa taaaaaacca tttctgcaga 3449 aaaaaaaaaa aaaaaaaaaa aaa 3472 <210> 2 <211> 1001 <212> PRT <213> Oryza sativa <400> 2 Met Gly Leu Thr Cys Asp Thr Gln Thr Ala Lys Leu Ala Ile Ile Leu 1 5 10 15 Leu Ala Phe Ile Le u Leu Cys His Gly Ile Gly Asn Val Asp Cys Arg 20 25 30 Gly Asn Arg Ala Asp Gln Leu Ser Leu Leu Asp Phe Lys Lys Gly Ile 35 40 45 Thr Asn Asp Pro Tyr Gly Ala Leu Ala Thr Trp Asn Thr Ser Thr His 50 55 60 Phe Cys Arg Trp Gln Gly Val Lys Cys Thr Ser Thr Gly Pro Trp Arg 65 70 75 80 Val Met Ala Leu Asn Leu Ser Ser Gln Ser Leu Thr Gly Gln Ile Arg 85 90 95 Ser Ser Leu Gly Asn Leu Ser Phe Leu Asn Ile Leu Asp Leu Gly Asp 100 105 110 Asn Asn Leu Leu Gly Ser Leu Pro Arg Leu Gly Asn Leu Lys Gln Leu 115 120 125 Gln Ala Leu Tyr Leu Tyr Lys Asn Asn Leu Thr Gly Ile Ile Pro Asp 130 135 140 Glu Leu Thr Asn Cys Ser Ser Leu Thr Tyr Ile Asp Leu Ser Gly Asn 145 150 155 160 Ala Leu Thr Gly Ala Leu Pro Pro Asn Leu Gly Ser Leu Ser Asn Leu 165 170 175 Ala Tyr Leu Tyr Leu Ser Ala Asn Lys Leu Thr Gly Thr Ile Pro Gln 180 185 190 Ala Leu Gly Asn Ile Thr Thr Leu Val Glu Ile Tyr Leu Asp Thr Asn 195 200 205 Arg Phe Glu Gly Gly Ile Pro Asp Lys Leu Trp Gln Leu Pro Asn Leu 210 215 220 Thr Ile Leu Ala Leu Gly Gln Asn Met L eu Ser Gly Asp Ile Pro Phe 225 230 235 240 Asn Phe Ser Ser Leu Ser Leu Gln Leu Leu Ser Leu Glu Tyr Asn Met 245 250 255 Phe Gly Lys Val Leu Pro Gln Asn Ile Ser Asp Met Val Pro Asn Leu 260 265 270 Gln Ile Leu Arg Leu Asp Tyr Asn Met Phe Gln Gly Gln Ile Pro Ser 275 280 285 Ser Leu Gly Asn Ala Leu Gln Leu Thr Glu Ile Ser Met Ala Asn Asn 290 295 300 Tyr Phe Thr Gly Gln Ile Pro Ser Ser Phe Gly Lys Leu Ser Lys Leu 305 310 315 320 Ser Tyr Ile Ser Leu Glu Asn Asn Ser Leu Glu Ala Ser Asp Gly Gln 325 330 335 Gly Trp Glu Phe Leu His Ala Leu Arg Asn Cys Ser Asn Leu Glu Leu 340 345 345 350 Leu Ser Leu Ala Gln Asn Gln Leu Gln Gly Glu Ile Pro Asn Ser Ile 355 360 365 Gly Asp Leu Pro Leu Lys Leu Gln Gln Leu Val Leu Ser Glu Asn Lys 370 375 380 Leu Ser Gly Glu Val Pro Ala Ser Ile Gly Asn Leu Gln Gly Leu Phe 385 390 395 400 Arg Leu Ser Leu Asp Leu Asn Asn Leu Thr Gly Lys Ile Asp Glu Trp 405 410 415 Val Pro Lys Leu Thr Lys Leu Gln Lys Leu Leu Leu His Arg Asn Asn 420 425 430 Phe Ser Gly Ser Ile Pro Ser Ser Ile A la Glu Leu Pro Arg Leu Ser 435 440 445 Thr Leu Ser Leu Ala Tyr Asn Ala Phe Asp Gly Pro Ile Pro Ser Ser 450 455 460 Leu Gly Asn Leu Ser Gly Leu Gln Lys Leu Tyr Leu Ser His Asn Asn 465 470 475 480 Leu Glu Gly Val Ile Pro Pro Glu Leu Ser Tyr Leu Lys Gln Leu Ile 485 490 495 Asn Leu Ser Leu Ser Glu Asn Lys Leu Thr Gly Glu Ile Pro Gly Thr 500 505 510 Leu Ser Gln Cys Lys Asp Leu Ala Asn Ile Gln Met Gly Asn Asn Phe 515 520 525 Leu Thr Gly Asn Ile Pro Val Thr Phe Gly Asp Leu Lys Ser Leu Gly 530 535 540 Val Leu Asn Leu Ser His Asn Ser Leu Ser Gly Thr Ile Pro Thr Thr 545 550 555 560 Leu Asn Asp Leu Pro Val Met Ser Lys Leu Asp Leu Ser Tyr Asn Arg 565 570 575 Leu Gln Gly Lys Ile Pro Met Thr Gly Ile Phe Ala Asn Pro Thr Val 580 585 590 590 Val Ser Val Gln Gly Asn Ile Gly Leu Cys Gly Gly Val Met Asp Leu 595 600 605 Arg Met Pro Pro Cys Gln Val Val Ser Gln Arg Arg Lys Thr Gln Tyr 610 615 620 Tyr Leu Ile Arg Val Leu Ile Pro Ile Phe Gly Phe Met Ser Leu Ile 625 630 635 640 640 Leu Val Val Tyr Phe Leu Leu Leu GluLys Met Lys Pro Arg Glu Lys 645 650 655 Tyr Ile Ser Ser Gln Ser Phe Gly Glu Asn Phe Leu Lys Val Ser Tyr 660 665 670 Asn Asp Leu Ala Gln Ala Thr Arg Asn Phe Ser Glu Ala Asn Leu Ile 675 680 685 Gly Lys Gly Ser Tyr Gly Thr Val Tyr Arg Gly Lys Leu Lys Glu Cys 690 695 700 Lys Leu Glu Val Ala Val Lys Val Phe Asp Leu Glu Met Arg Gly Ala 705 710 710 715 720 Glu Arg Ser Phe Ile Ser Glu Cys Glu Ala Leu Arg Ser Ile Gln His 725 730 735 Arg Asn Leu Leu Pro Ile Ile Thr Ala Cys Ser Thr Val Asp Ser Thr 740 745 750 Gly Asn Val Phe Lys Ala Leu Val Tyr Glu Tyr Met Pro Asn Gly Asn 755 760 765 Leu Asp Thr Trp Ile His Asp Lys Glu Gly Gly Lys Ala Pro Gly Arg 770 775 780 Leu Gly Leu Arg Gln Thr Ile Ser Ile Cys Val Asn Ile Ala Asp Ala 785 790 795 800 Leu Asp Tyr Leu His His Glu Cys Gly Arg Thr Thr Ile His Cys Asp 805 810 815 Leu Lys Pro Ser Asn Ile Leu Leu Ala Asp Asp Met Asn Ala Leu Leu 820 825 830 Gly Asp Phe Gly Ile Ala Arg Phe Tyr Ile Asp Ser Trp Ser Thr Ser 835 840 845 Thr Gly Ser Asn Ser Thr Val Gly Val LysGly Thr Ile Gly Tyr Ile 850 855 860 Pro Pro Glu Tyr Ala Gly Gly Gly His Pro Ser Thr Ser Gly Asp Val 865 870 875 880 Tyr Ser Phe Gly Ile Val Ile Leu Glu Leu Ile Thr Gly Lys Arg Pro 885 890 895 Thr Asp Pro Met Phe Lys Asp Gly Leu Asp Ile Ile Ser Phe Val Glu 900 905 910 Ser Asn Phe Pro His Gln Ile Phe Gln Val Ile Asp Ala Arg Leu Ala 915 920 925 Glu Lys Ser Met Asp Ser Asn Gln Thr Asn Met Thr Leu Glu Asn Ala 930 935 940 Val His Gln Cys Leu Ile Ser Leu Leu Gln Leu Ala Leu Ser Cys Thr 945 950 955 960 Arg Lys Leu Pro Ser Asp Arg Met Asn Met Lys Gln Ile Ala Asn Lys 965 970 975 Met His Ser Ile Lys Thr Thr Tyr Val Gly Leu Glu Ala Lys Lys Tyr 980 985 990 Gly Pro Ala Cys Asn Glu Gln Asp Ala 995 1000 <210> 3 <211> 231 <212> DNA <213> Oryza sativa <400> 3 ac gcg gga ggt ggt cat cca tct act tct ggg gat gtt tat agt ttc 47 Ala Gly Gly Gly His Pro Ser Thr Ser Gly Asp Val Tyr Ser Phe 1 5 10 15 ggg ata gtg atc ctg gag ctt ata aca gga aaa aga ccg act gat cct 95 Gly Ile Val Ile Leu Glu Leu Ile Thr Gly Lys Arg Pro Thr Asp Pro 20 25 30 atg ttc aaa gac gga ctc gac att atc agc ttt gtc gag agt aac ttt 143 Met Phe Lys Asp Gly Leu Asp Ile Ile Ser Phe Val Glu Ser Asn Phe 35 40 45 cca cac cag ata ttt caa gtg att gat gct cga ctc gca gaa aag tcc 191 Pro His Gln Ile Phe Gln Val Ile Asp Ala Arg Leu Ala Glu Lys Ser 50 55 60 atg gac tcc aat caa aca aat atg aca ctg gaa aat gct g 231 Met Asp Ser Asn Gln Thr Asn Met Thr Leu Glu Asn Ala 65 70 75 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 4 agcagtggta acaacgcaga gtac 24 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 5 ataatacaag atcatgctag gtacaacatc 30 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 6 tcgagcggcc gcccgggcag gt 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 7 agcgtggtcg cggccgaggt 20 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 8 caggaaacag ctatgaccat gattacgcca 30 <210> 9 <211> 29 <212> DNA <213 > Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 9 gtaaaacgac ggccagtgaa ttgtaatac 29 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 10 ggtggtcatc catctacttc tgggggtg 28 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 11 ccatggactt ttctgcgagt cgagcatc 28 <210> 12 <211 > 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 12 accattttct tcttgttgga ctatacac 28 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 13 ttggtgcttc atattatcac cttaattatc 30 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for P CR <400> 14 ccatgacata aagtcacaat caccaatc 28 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 15 tcatgctagg tacaacatca ttatctac 28 <210> 16 < 211> 26 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 16 taacatgggc ctcacgtgtg atacac 26 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220 > <223> Designed oligonucleotide primer for PCR <400> 17 accaagcaga acaggctgct gagctc 26 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 18 gcgccatggc ccagtggagg tgcacttg 28

Continued on the front page (72) Inventor Kenji Oeda 4-2-1 Takashi Takarazuka-shi, Hyogo F-term (reference) in Sumitomo Kagaku Kogyo Co., Ltd. 2B030 AA02 AB03 AD04 CA06 CA17 CA19 CB03 CD03 CD07 CD09 4B024 AA11 CA12 HA13 HA14 4B063 QA13 QA17 QA19 QQ04 QQ53 QR08 QR32 QR40 QR56 QR62 QS16 QS25 QS34 QX01 QX02

Claims (4)

[Claims] 1. A polynucleotide having a base sequence according to any one of the following (a) to (e): (A) a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 (b) a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 (c) a nucleotide sequence represented by SEQ ID NO: 1 ( d) a base sequence complementary to the base sequence represented by SEQ ID NO: 1 (e) a partial base sequence of 10 or more bases in the base sequence described in any one of (a) to (d) 2. The use of the polynucleotide according to claim 1, wherein the presence of an RNA having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial nucleotide sequence thereof in a test rice is detected. Test method of rice planthopper resistant rice. 3. A polymerase chain reaction is performed using the polynucleotide of claim 1 as a primer and cDNA prepared from a test rice as a template, and the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a nucleotide sequence thereof. A method for detecting a brown planthopper-resistant rice, comprising detecting amplification of a polynucleotide having a partial base sequence. 4. A polynucleotide according to claim 1, which is labeled with RNA or c prepared from test rice.
A method for detecting a rice planthopper-resistant rice plant, which comprises hybridizing to DNA and detecting an RNA or cDNA having a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or a partial nucleotide sequence thereof.