WO2000008176A1 - Gene de la barnase mutant et vegetal transforme par ce gene - Google Patents
Gene de la barnase mutant et vegetal transforme par ce gene Download PDFInfo
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- WO2000008176A1 WO2000008176A1 PCT/JP1999/004167 JP9904167W WO0008176A1 WO 2000008176 A1 WO2000008176 A1 WO 2000008176A1 JP 9904167 W JP9904167 W JP 9904167W WO 0008176 A1 WO0008176 A1 WO 0008176A1
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- WO
- WIPO (PCT)
- Prior art keywords
- gene
- plant
- barnase
- mutant
- anther
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
Definitions
- the present invention relates to a mutant barnidase gene that can efficiently obtain a male sterile transformant by specifically expressing it at a specific site of a plant, particularly at an anther.
- the present invention also relates to a recombinant vector capable of expressing the mutant barnase gene of the present invention in a host cell, a plant transformed with the vector, and a method for producing a transformed plant.
- barnase is an RNAse enzyme (RNase) derived from Bacillus' amyloliguifaciens (ci_ll sa myloliguifaciens) (S. Nishimura and M. Nomura, Biochem. Biophys. Acta 30, 430-431: 1958 RW Hartley, J. Mol. Biol., 202, 913-915: 1988).
- RNase RNAse enzyme
- This enzyme has 110 amino acid residues and hydrolyzes RNA.
- This enzyme When this enzyme is expressed in cells, its strong RNA-degrading activity inhibits cell function and in many cases kills cells. If the property of barnase can be expressed in a predetermined site of a plant by utilizing this property, the function of the site can be selectively suppressed.
- PCT application WO 8910396 discloses that a male non-human gene created by binding the above-mentioned barnase gene downstream of an expression motor specific to tumor cells in anther tissue is introduced into plants. Techniques for obtaining male sterile plants have been reported. Such a male sterility technique is very useful in the development of efficient F1 hybrid varieties.
- WO 9626283 also discloses the property of strong reflex mosaic virus 35S promoter-1 (hereinafter referred to as CaMV35S promoter-1) that it is strongly expressed in tissues other than anthers.
- CaMV35S promoter-1 strong reflex mosaic virus 35S promoter-1
- this method uses barstar, an inhibitory protein for barnase, and simultaneously introduces the Burster gene linked to the CaMV35S promoter into a plant to construct the Burster gene constitutively in tissues other than anthers. By expressing it, the effect of non-lunase other than ⁇ is eliminated.
- the barnase gene but also the buster gene must be introduced in this method, and this technology makes it possible to apply this technology to F1 varieties of crops that use seeds such as rice and corn.
- Gene silencing problems can occur when applied to breeding. Gene silencing is a phenomenon in which the expression of a foreign gene is suppressed by introducing multiple copies of the gene into a plant. At present, although the mechanism is unclear, it is said that this problem is likely to occur particularly when a foreign gene is expressed by the 35S promoter (RB Flavel, Proc. Natl. Acad. Sci. USA 91, 3490). -3496: 1994; J. Finnegan, Bio.Technology 12, 883-888: 1994; MA Matzke and AJM Matzke, Plant Physiol., 107, 679-685: 1995).
- the present invention provides a method for producing a male-sterile plant using a barnase gene without using a barthase gene.
- the present invention also provides a mutant barnase gene used in the above method and a method for producing the same.
- At least a part of the barnase gene DNA sequence (RW Hartley, J. Mol. Biol. 202, 913-915: 1988) is mutated, and the mutated gene is specifically expressed in anthers of plants.
- the plant can be substantially male-sterilized without substantially adversely affecting tissues other than anthers.
- Mutation can be performed by a known method such as site-directed mutagenesis, deletion of a partial fragment with a restriction enzyme, and Low Fidelity PCE.
- a preferred mutation method is the Low Fidelity PCK method. For details, see D. Leung, E. Chen and D.
- PCR can be performed under conditions where multiple errors are likely to occur during the amplification reaction, so that random mutations can be efficiently introduced into the target DNA fragment.
- primers used for the Low Fidelity PCK method are selected in the same manner as in the ordinary PCR method.
- the length of the primer is preferably the same as the number of bases in the ordinary PCR method.
- the present inventors used DNA containing the sequence of SEQ ID NO: 1 for type I, and primer 1 (5 ′ -CGTTCGGCTC GATGGTACCG GTTATCAACA CGTTTGA-3 ', SEQ ID NO: 6), Primer 2 (5'-CCTCTAGATT ATCTGATTTT TGTAAAGGTC TGATAATG-3', SEQ ID NO: 7)
- the mutant barnase gene having the DM sequence shown in 3 was able to be isolated.
- the sequence of SEQ ID NO: 1 used here is the sequence of the coding region of the barnase gene present on the known plasmid pVE108 (W092 / 13956), and is N-terminal to the sequence of the original barnase gene. Unnecessary parts corresponding to secretory signals outside the cells are removed. In a similar manner, it is possible to obtain further different mutated barnase genes.
- the PCR amplification product is cloned in a host such as E. coli by a conventional technique, and an E. coli clone containing the mutant norenase gene is isolated.
- the clone having the barnase gene may be screened by measuring the RNase activity generated by the expression of the gene, but the growth of E. coli may be affected by the RNase activity of the renalase. It is convenient to carry out screening using the method comprising two steps described below.
- the first step first, a plasmid is prepared using the mutant barnase gene obtained as described above, and Escherichia coli is transformed thereby. Growth of the E. coli transformant thus obtained is suppressed by the activity of the introduced mutant barnase. Based on this fact, colonies with slower growth, that is, smaller colonies, are selected compared to Escherichia coli strains that have introduced the control vector without the mutant barnase. The E. coli strain selected in this manner is expected to contain the mutant barnase gene. Then, in order to confirm that growth suppression is caused by the expression of the mutant barnase gene, the second step is then performed. In the second step, the berth gene is used.
- Bath Yuichi is a barnase inhibitory protein as described above.
- Escherichia coli expressing Barth the enzyme activity of barnase is inhibited, and in the presence of barnase, the degradation of mRNA that should be degraded by the activity can be inhibited.
- Escherichia coli expressing the buster gene is transformed with the barnase gene, its growth is not suppressed, so that the transformation with the control plasmid containing no barnase gene is performed.
- the colony size is not expected to change significantly. Based on this principle, a plasmid is prepared from the E.
- the Escherichia coli that expresses the Burster gene constitutively, as used herein, can be prepared, for example, by the method described in Example 1 below.
- the details of the mutant barnase gene thus obtained can be examined by analyzing the DNA sequence by a conventional method, if necessary.
- mutant barnase gene of the present invention has the DNA sequence of SEQ ID NO: 3.
- the nucleotide sequence encoded by this gene has a T insertion at position 15 and a deletion at position 333 from A of ATG, which is the start codon. Have lost.
- the standard translation mode assuming that translation starts from the first ATG of the DNA sequence of SEQ ID NO: 3, the insertion of the 15th base T results in the termination of the ninth codon, where The translation power will be stopped.
- the likelihood of obtaining a monozyme gene is great. Furthermore, even if substitution or deletion, insertion, or addition of bases in the DNA sequence of SEQ ID NO: 3 is performed, insertion of the 15th T from A of the start codon, ATG, and / or 333th Due to the deletion of A, it is highly likely that the gene is a mutated lunase gene that causes a decrease in translation efficiency due to a shift in the reading frame as in the DNA sequence of SEQ ID NO: 3. All of these mutant genes are considered to be capable of substantially male sterilizing the plant when expressed specifically in a plant, as in the case of the gene of SEQ ID NO: 3.
- the present invention is included in the present invention similarly to the third embodiment.
- the plant that can be male-sterilized by the mutant barnase gene is not particularly limited as long as it is a plant into which the present gene has been introduced and can be substantially male-sterilized.
- the mutant ⁇ lunase gene is specifically expressed in the anther of the plant, and its RNase activity inhibits anther function.
- the method described in TO92 / 13957 can be used. Briefly, the mutant barnase gene is ligated downstream of an anther-specific promoter and incorporated into plant cells using an expression vector.
- agglomeration method for integration, there are the agglomeration method, the electroporation method, and the one-tickle gun method.
- regeneration is performed from the transformed plant cell callus.
- the method may be performed, for example, as in Y. Hiei et al. Plant J. 6 271-228 2: 1994.
- Primer 1 (CGTTCGGCTC GATGGTACCG GTTATCAACA CGTTTGA, SEQ ID NO: 6) and Primer 1 (CCTCTAGATT ATCTGATTTT TGTAAAGGTC TGATAATG, SEQ ID NO: 7) were described in SL Beaucage et al., Tetrahedron Lett., 22, 1859-1862: 1982.
- the DNA was synthesized using a DNA synthesizer (Applied Biosystems).
- PCK was carried out using the known plasmid pVE108 (W092 / 13956) in combination with Primer-1 and Primer-12.
- SEQ ID NO: 1 shows the sequence of the part corresponding to the coding region of the barnase gene of pVE108.
- the reaction conditions are as follows.
- the amplified product was separated by agarose gel electrophoresis (2% SeaKem GTG agarose, ⁇ ), purified by the DEAE-cellulose method (Masamatsu Muramatsu, “Lab Manual Genetic Engineering” Maruzen, 1988 pplll), converted into type II, The PCR reaction was performed again under the following conditions.
- the reaction product was digested with Sacl and Xbal by a conventional method, and purified by agarose gel electrophoresis to prepare an inserted fragment.
- a plasmid for introducing the inserted fragment into Escherichia coli can be appropriately selected, for example, a plasmid pHMl can be used. Plasmid pH Ml is obtained by cleaving the EcoRI site of plasmid PBK322, blunting it with T4 DNA polymerase (Takara Shuzo), and using the lacZ expression cassette (322 bp) cut out from PUC18 with PvuII at the restriction enzyme site. This is a plush made by assembling after smoothing.
- Brassmid pHMl was digested with Sacl and Xbal and then dephosphorylated with calf intestine alkaline phosphstase (Takara Shuzo) to prepare a plasmid fragment treated with restriction enzymes.
- the insert fragment containing the mutant barnase gene was ligated into pHMl treated with a restriction enzyme using Takara Ligation Kit ver. 1 (Takara Shuzo).
- Escherichia coli into which the kinase gene has been introduced is selected, for example, as follows. That is, since mRNA is degraded in cells by the activity of barnase, protein synthesis is reduced, and as a result, the growth of Escherichia coli is suppressed. Therefore, it is possible to select for E. coli transformed with the barnase gene by utilizing the fact that the colony size is smaller than that of the E. coli colony transformed with the control plasmid not containing the mutant barnase gene. it can. When wild-type barnase or a mutant barnase having the same activity is incorporated into PHM1, Escherichia coli cannot form a colony, so select only clones with insufficient activity. It is possible.
- the plasmid ligated with the mutant barnase gene was precipitated with ethanol, and this plasmid was introduced into E. coli LE392 by electroporation using GenePulser (BioRad). The installation procedure was performed according to MoRad's manual. According to a similar procedure, a control plasmid containing no mutant barnase was also introduced into E. coli strain LE392.
- Bath is an enzyme having an antagonistic effect on barnase as described above.
- Escherichia coli expressing Barth the enzyme activity of barnase is inhibited, and in the presence of barnase, the activity of barnase can inhibit the degradation of mRNA which should be degraded.
- E. coli expressing Bursta-1 was transformed with the plasmid into which the barnase gene was inserted, its growth was not suppressed.
- the growth rate is compared with that obtained by transformation with the same, the growth rate is not significantly different between the two, and thus it is expected that the colony size does not change significantly.
- This E. coli was prepared as follows. R. Hartley, J. Mol. Biol. 202, 913-9 15: 1988, and the tac promoter-(de Boer et al., Proc. Natl. Acad. Sci. USA 80, 21-25: 1983). Binds, and is described in (Herrero et al., J. Bacteriol. 172, 6557-6567: 1990) along with the chloramphenicol resistance gene (NK Alton, and D. Vapenek, Nature 282, 864-869: 1979).
- the resulting plasmid is ligated into a transposon lacking a transferase (defective transposon), and then the obtained plasmid is introduced into Escherichia coli MC1061 strain, and this transposon (including the Burster gene cassette). ) was transferred to the E. coli chromosome. It can be strongly confirmed by the chloramphenicol resistance that the E. coli has maintained the Burster gene cassette stably. In the E. coli MC1061 strain, since the lacl gene is deleted, the tac promoter is always induced, and the Burster gene is constitutively expressed.
- a cloned mutant barnase gene suitable for the purpose of the present invention was prepared.
- clone # 4-31 selected in Example 1 was prepared.
- the mutant barnase gene fragment integrated into clone # 4-31 was cut out with Kpnl and Xbal, and ligated to the Kpnl and Xbal sites of pUC119 similarly cut with Kpnl and Xbal.
- the reaction was performed according to the protocol of the manufacturer using the cycle sequence method using Taq polymerase (Taq Dye Terminator Cycle Sequencing Kit, Applied Biosystems Inc.).
- the attenuated mutant barnase gene incorporated into PUC119 was The plasmid was cut with restriction enzymes Xbal and Kpnl and used to construct a plasmid vector shown in SEQ ID NO: 5—PTS431.
- PTS431 is powerful, with the following differences from the known plasmid pVE108 plasmid (PCT Application WO 9213956), with the exception of ⁇ specific promoters and mutated kinases. Is considered to be equivalent to
- the region corresponding to lacZ is removed from the portion derived from pUC19.
- the plasmid (PTS172) incorporating the conventional barnase gene, not the mutant, is shown in SEQ ID NO: 4.
- phosphinothricine concentration 10mg / L
- the phosphinothricine is used to select the calli into which the gene has been introduced.
- a barnase gene whose effect has been weakened by mutation is produced and used to introduce a male sterile gene into a plant.
- a male sterile plant having no undesirable traits was successfully obtained.
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Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU49333/99A AU756092B2 (en) | 1998-08-04 | 1999-08-03 | Mutant barnase gene and transgenic plant transformed by said gene |
CA002305784A CA2305784A1 (en) | 1998-08-04 | 1999-08-03 | Mutated barnase gene and plant transformed by the same |
KR1020007002963A KR20010024190A (ko) | 1998-08-04 | 1999-08-03 | 변이 바르나제 유전자 및 그의 형질전환 식물 |
EP99933237A EP1020527A1 (en) | 1998-08-04 | 1999-08-03 | Mutated barnase gene and plant transformed by the same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP22006098A JP3478975B2 (ja) | 1998-08-04 | 1998-08-04 | 変異バルナーゼ遺伝子およびその形質転換植物 |
JP10/220060 | 1998-08-04 |
Publications (1)
Publication Number | Publication Date |
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WO2000008176A1 true WO2000008176A1 (fr) | 2000-02-17 |
Family
ID=16745319
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/JP1999/004167 WO2000008176A1 (fr) | 1998-08-04 | 1999-08-03 | Gene de la barnase mutant et vegetal transforme par ce gene |
Country Status (8)
Country | Link |
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US (1) | US20020166140A1 (ja) |
EP (1) | EP1020527A1 (ja) |
JP (1) | JP3478975B2 (ja) |
KR (1) | KR20010024190A (ja) |
CN (1) | CN1274388A (ja) |
AU (1) | AU756092B2 (ja) |
CA (1) | CA2305784A1 (ja) |
WO (1) | WO2000008176A1 (ja) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8293503B2 (en) | 2003-10-03 | 2012-10-23 | Promega Corporation | Vectors for directional cloning |
JP4583051B2 (ja) * | 2004-03-02 | 2010-11-17 | 岩手県 | 新規な植物細胞死誘導因子NbCD3 |
JP4583050B2 (ja) * | 2004-03-02 | 2010-11-17 | 岩手県 | 新規な植物細胞死誘導因子NbCD2 |
US7453025B2 (en) | 2004-09-22 | 2008-11-18 | Arborgen, Llc | Reproductive ablation constructs |
WO2016166776A1 (en) | 2015-04-16 | 2016-10-20 | Council Of Scientific & Industrial Research | Novel reversible expression system for transgene expression in plants |
WO2020197558A1 (en) * | 2019-03-28 | 2020-10-01 | Bioceres Llc | Soybean transgenic event ind-øø41ø-5 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996026283A1 (en) * | 1995-02-21 | 1996-08-29 | Plant Genetic Systems, N.V. | Method to obtain male-sterile plants |
-
1998
- 1998-08-04 JP JP22006098A patent/JP3478975B2/ja not_active Expired - Fee Related
-
1999
- 1999-08-03 EP EP99933237A patent/EP1020527A1/en not_active Withdrawn
- 1999-08-03 WO PCT/JP1999/004167 patent/WO2000008176A1/ja not_active Application Discontinuation
- 1999-08-03 AU AU49333/99A patent/AU756092B2/en not_active Ceased
- 1999-08-03 US US09/509,945 patent/US20020166140A1/en not_active Abandoned
- 1999-08-03 KR KR1020007002963A patent/KR20010024190A/ko not_active Application Discontinuation
- 1999-08-03 CN CN99801291A patent/CN1274388A/zh active Pending
- 1999-08-03 CA CA002305784A patent/CA2305784A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996026283A1 (en) * | 1995-02-21 | 1996-08-29 | Plant Genetic Systems, N.V. | Method to obtain male-sterile plants |
Non-Patent Citations (1)
Title |
---|
FETTEN J.V. ET AL: "A Frameshift Mutation at the NH2 Terminus of the Nucleoprotein Gene Does not Affect Generation of Cytotoxic T Lymphocyte Epitopes", JOURNAL OF IMMUNOLOGY,, vol. 147, no. 8, 15 October 1991 (1991-10-15), USA, pages 2697 - 2705, XP002925170 * |
Also Published As
Publication number | Publication date |
---|---|
US20020166140A1 (en) | 2002-11-07 |
CA2305784A1 (en) | 2000-02-17 |
EP1020527A1 (en) | 2000-07-19 |
AU756092B2 (en) | 2003-01-02 |
KR20010024190A (ko) | 2001-03-26 |
AU4933399A (en) | 2000-02-28 |
CN1274388A (zh) | 2000-11-22 |
JP3478975B2 (ja) | 2003-12-15 |
JP2000041682A (ja) | 2000-02-15 |
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