JP2006115861A - Cyclamen flower color synthase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a DNA encoding an enzyme involved in flower color synthesis of cyclamen, particularly chalcone synthase and dihydroflavonol-4-reductase.
CDNA is synthesized from mRNA prepared from cyclamen, DNA encoding chalcone synthase and a part of dihydroflavonol-4-reductase is amplified by reverse transcription PCR, and the obtained DNA fragment is used as a probe. DNA encoding the amino acid sequences shown in SEQ ID NOs: 2 and 4 is isolated from a cyclamen cDNA library.
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Description

  The present invention relates to two novel DNAs encoding proteins of cyclamen flower color synthase. Specifically, the present invention relates to two novel DNAs encoding novel proteins having the activity of cyclamen chalcone synthase and dihydroflavonol-4-reductase.

  In general, anthocyanins, which are a kind of flavonoid, are widely known as red, purple and blue pigments in plant flower colors. In the biosynthetic pathway of anthocyanins in plant petals, chalcone synthase (hereinafter abbreviated as “CHS”) is known to be involved in the biosynthetic system that condenses coumaroyl CoA and malonyl CoA to produce tetrahydroxychalcone.

  Dihydro-4-flavonol reductase (hereinafter abbreviated as “DFR”) is involved in a biosynthetic system that reduces dihydroflavonols such as dihydrokaempferol, dihydroquercetin, dihydromyricetin and the like to produce the corresponding leucoanthocyanidins. It is known.

  CHS and DFR involved in these biosynthetic systems are considered to play an especially important role in recent researches on improving flower color of flowering plants using genetic engineering techniques.

  The following are known base sequences of genes encoding CHS of flowering plants so far.

(1) Petunia hybrida (Nucleic Acids Res. Vol. 14, pages 5229-5239, 1986).
(2) Stock (Matthiola incana) (Plant Mol. Biol. 14, 1061-1063, 1990).
(3) Gerbera hybrida (Plant Mol. Biol. 28, 47-60, 1995).
(4) Carnation (Dianthus caryophyllus) (GenBank accession Z67982 (1995)).
(5) Gentian (genus Gentiana) (Japanese Patent Laid-Open No. 8-89251 (1996)).
(6) Hydrangea macrophylla (GenBank accession AB011467 (1999)).
Moreover, the following is known about the base sequence of the gene which codes DFR of a flowering plant so far.

(1) Petunia hybrida (Plant Mol. Biol. Vol. 13, 491-502 (1989)).
(2) Gerbera hybrida (Plant Mol. Biol. 22: 183-193 (1993)).
(3) Carnation (Dianthus caryophyllus) (GenBank accession Z67983 (1995)).
(4) Rose (Rosa hybrida) (Plant Cell Physiol. 36, 1023-1031 (1995)).
(5) Gentiana triflora (Plant Cell Physiol. 37, 711-716 (1996)).
(6) Forsythia intermedia (Plant Mol. Biol. 35, 303-311, 1997).
(7) Morning Glory (Ipomoea purpurea) (GenBank accession U90432 (1997)).
(8) Cymbidium hybrida (GenBank accession AF017451 (1998)).
JP-A-8-89251 (1996) Nucleic Acids Res. Volume 14, pp. 5229-5239, 1986 Plant Mol. Biol. 14: 1061-1063, 1990 Plant Mol. Biol. 28, 47-60, 1995 GenBank accession Z67982 (1995) GenBank accession AB011467 (1999) Plant Mol. Biol. 13, 491-502 (1989) Plant Mol. Biol. Vol. 22, 183-193 (1993) GenBank accession Z67983 (1995) Plant Cell Physiol. 36, 1023-1031 (1995) Plant Cell Physiol. 37, 711-716 (1996) Plant Mol. Biol. 35, 303-311, 1997 GenBank accession U90432 (1997) GenBank accession AF017451 (1998)

  However, the genes encoding CHS and DFR from cyclamen plants are not known.

  The first object of the present invention is to obtain DNA encoding CHS of a novel cyclamen from cyclamen.

  The second object of the present invention is to obtain DNA encoding DFR of a novel cyclamen from cyclamen.

The present inventors diligently studied to solve the above problems. As a result, the present invention has been completed.
That is, the gist of the present invention is the following DNA.

(1) A protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and encoding a cyclamen chalcone synthase protein.
(2) The DNA of (1), which is a DNA comprising at least the base numbers 57 to 1223 described in SEQ ID NO: 1 in the sequence listing.
(3) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and having a chalcone synthase activity DNA encoding
(4) DNA having a base sequence partially different from the base sequence described in SEQ ID NO: 1 in the sequence listing, but having homology to the base sequence described in SEQ ID NO: 1, The DNA according to (3), which is a DNA that is capable of hybridizing under stringent conditions to the DNA having the base sequence described in SEQ ID NO: 1 and that encodes a protein having chalcone synthase activity.
(5) A DNA having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, which encodes a protein of cyclamen dihydroflavonol-4-reductase.
(6) DNA of (5) which is DNA containing at least base numbers 35 to 1063 described in SEQ ID NO: 3 of the sequence listing.
(7) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, wherein dihydroflavonol-4-reductase DNA encoding a protein having activity.
(8) A DNA having a base sequence partially different from the base sequence described in SEQ ID NO: 3 in the sequence listing, but having homology to the base sequence described in SEQ ID NO: 3, The DNA according to (7), which is capable of hybridizing under stringent conditions to DNA having the base sequence set forth in SEQ ID NO: 3 and which encodes a protein having dihydroflavonol-4-reductase activity DNA.
(9) A cyclamen chalcone synthase which is a protein of the following (A) or (B).
(A) A protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a chalcone synthase activity.
(10) Dihydroflavonol-4-reductase of cyclamen, which is a protein of the following (C) or (D).
(C) A protein having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
(D) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4 and having the activity of dihydroflavonol-4-reductase Protein.

  In the present invention, “CHS activity” refers to the activity of a protein that catalyzes a reaction in which one molecule of coumaroyl CoA and three molecules of malonyl CoA are condensed to form tetrahydroxychalcone. The “DFR activity” refers to the activity of a protein that reduces dihydroflavonols such as dihydrokaempferol, dihydroquercetin, dihydromyricetin and the like and biosynthesizes the corresponding leucoanthocyanidins.

  Hereinafter, DNA encoding CHS or DNA encoding DFR, or these may be collectively referred to as “DNA of the present invention”.

  The genes encoding cyclamen CHS and DFR provided by the present invention are introduced into homologous or heterologous plants alone or in combination to regulate the enzyme reaction involved in anthocyanin synthesis in the plants. It can be used for plant breeding development, such as the diversity of plant flower colors.

  The DNA of the present invention is a DNA encoding CHS and DFR proteins. In completing the present invention, these DNAs were isolated from a cyclamen cDNA library as described later. However, since the base sequences were clarified, they are shown in SEQ ID NO: 1 or 3. It can also be obtained by chemical synthesis based on the sequence. In addition, it can also be obtained from a DNA library by a known method by hybridization or polymerase chain reaction (PCR) using a synthetic oligonucleotide probe or oligonucleotide primer prepared based on the sequence.

  The following is a method for obtaining a full length of the DNA of the present invention by obtaining a part of the DNA of the present invention from cyclamen RNA by RT-PCR (reverse transcription PCR) and further using the probe as a probe. Illustrate.

(1) Preparation of mRNA and construction of cDNA library After extracting total RNA from tissues such as cyclamen (Cyclamen persicum) petals, foliage, roots and callus, preferably petals, protein, polysaccharides, etc. MRNA can be obtained by removing Poly (A) ⁺ RNA using a column packed with oligo dT cellulose.

  Next, cDNA is synthesized from the mRNA and incorporated into a phage vector such as λgt11 or λZAPII to obtain a recombinant phage. A large number of plaques can be obtained by infecting these cells with Escherichia coli as a host. Since a cDNA cloning kit is commercially available, these series of operations may be used.

(2) Preparation of cloning probe and screening of cDNA library
In order to isolate the cDNA encoding the CHS and DFR of the target cyclamen from the cDNA library, the base sequence conserved in the gene encoding the known CHS and DFR encoding genes such as petunia is used. Using the cyclamen RNA extracted above as a template, using this as a primer, a cDNA fragment serving as a probe encoding a part of cyclamen CHS and DFR is obtained by RT-PCR. As the base sequence of the primer for amplifying a part of the CHS gene, the base sequence shown in SEQ ID NOs: 5 and 6 is used, and as the base sequence of the primer for amplifying a part of the DFR gene, SEQ ID NO: 7 And the base sequences shown in 8, respectively.

  Next, by hybridizing the probe and the plaque prepared in (1), the cDNA encoding the target CHS and the cDNA encoding DFR can be selected from the cDNA library.

  Phages are collected from the plaques selected by the above hybridization, and phage DNA is collected from the phages. The base sequence of the inserted DNA fragment can be determined by the dideoxy method or the like. The cDNA corresponding to CHS and DFR of the target cyclamen is confirmed by comparing the amino acid sequence defined from the protein coding region (open reading frame) in the base sequence with the amino acid sequence of known petunia etc. It can be identified as a fragment.

  In general, in the amino acid sequence encoding a protein having a specific function or physiological activity, even when one or more amino acids are added, deleted or substituted, the function or physiological activity is maintained. It is widely recognized by those skilled in the art. The present invention also includes a DNA fragment encoding a protein having such modifications and having CHS and DFR activities. That is, DNA encoding a protein consisting of amino acids in which one or several amino acids are deleted, substituted, inserted or added in SEQ ID NOs: 2 and 4 in the sequence listing and having CHS and DFR activity is also included in the present invention. It is included in the range. Such a modified DNA can be obtained by modifying the base sequence of the DNA of the present invention so that an amino acid at a specific site is deleted, substituted or added, for example, by site-directed mutagenesis.

Further, the DNA of the present invention or a cell having the same is subjected to a mutation treatment, and from these DNAs or cells, for example, DNA having the base sequences described in SEQ ID NOS: 1 and 3 in the sequence listing or a part thereof are stringent. By selecting DNA that hybridizes under conditions,
Modified DNA fragments can be obtained. The “stringent conditions” referred to herein are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, nucleic acids with high homology, for example, DNAs with homology of 95% or more hybridize and nucleic acids with lower homology A condition where they do not hybridize with each other can be mentioned.

  Furthermore, DNA encoding CHS and DNA encoding DFR can be obtained from the cyclamen chromosome by conventional hybridization using the DNA of the present invention or a part thereof as a probe, but the CHS gene and DFR gene derived from the cyclamen chromosome Are expected to contain introns. Such DNAs separated by introns are also included in the DNA of the present invention as long as they encode CHS and DFR.

  The cyclamen CHS cDNA obtained in the examples below was subcloned into the pBluescriptSK (-) plasmid vector, and Escherichia coli SOLR (CPCHS-1) introduced therein was named Escherichia coli SOLR (CPCHS-1). Deposited at the Institute on June 29, 1999 under the deposit number FERM P-17440. The DFR cDNA was subcloned into the pUC119 plasmid vector, and Escherichia coli introduced with this was named Escherichia coli JM109 (CPDFR-1). Deposited under the deposit number FERM P-17439.

  CHS can be produced by expressing the DNA encoding the CHS of the present invention in a suitable host. Furthermore, DFR can be produced by expressing DNA encoding the DFR of the present invention in a suitable host. Examples of the host include bacteria such as Escherichia coli and Bacillus subtilis, yeasts such as Saccharomyces cerevisiae, insect cultured cells, animal cultured cells, and plant cultured cells. Into these host cells, a DNA encoding the CHS or DFR of the present invention, which is operably linked downstream of an expression regulatory sequence such as a promoter that functions in the host cell, is introduced. When introducing these DNAs, these DNAs are inserted into a plasmid to construct a recombinant plasmid, and the host is transformed with the obtained recombinant plasmid, or the DNA is converted into a chromosomal DNA by homologous recombination or the like. Transformation by incorporation. CHS or DFR is produced by culturing the resulting transformed cells under conditions where the expression regulatory sequence functions.

Further, by introducing the DNA encoding CHS or DFR of the present invention into cyclamen or other plants alone or in combination, the enzyme reaction involved in anthocyanin synthesis in those plants can be regulated. . In order to introduce the DNA of the present invention into a plant, the Agrobacterium method (edited by Draper, J. et al., “Plant Genetic Transformation and Gene Expression A Laboratory”
Manual ", Blackwell Scientific Publications, p. 69-160, 1988), whisker method (U.S. Pat. No. 5,302,523, U.S. Pat. No. 7,472,538, JP-A-10-179174, Kaepper, HF et al. 1992, “Theoretical and Applied Genetics”, 84, 560-566, Asano, Y. et al., 1991, “Plant Science”, 79, 247-252), ultrasound on plant cells. Irradiation to partially damage cells and introduce a gene through the damaged site (Jörsbo, M. et al., 1990, “Plant Cell Reports”, Vol. 9, pp. 207-210, Joersbo, M. et al. , 19 1992, “Physiologia Plantarum”, Vol. 85, pages 230 to 234).

Examples of the present invention will be described below in more detail, but the scope of the present invention is not limited thereto.

  Unless otherwise stated, the following experimental procedures were performed according to the method described in “Molecular Cloning 2nd edition” (J. Sambrook et al., Cold Spring Habor Laboratory press, 1989).

(1) Preparation of cyclamen mRNA 3 g of petals during flowering of cyclamen (variety: fragrance mini) were frozen in the presence of liquid nitrogen and then ground using a pestle mortar. 30 ml of 2 × CTAB solution (2% cetyltrimethylammonium bromide, 0.1 M Tris (pH 9.5), 20 mM EDTA, 1.4 M NaCl, 4% β-mercaptoethanol) was added to this pulverized product, stirred, and then 65 ° C. Incubated for 10 minutes. Next, chloroform extraction was performed twice, isopropanol precipitation was performed, and nucleic acid was precipitated. The nucleic acid is dissolved in 3 ml of TE buffer (10 mM Tris (pH 8.0), 1 mM EDTA), 750 μl of 10 M lithium chloride solution is added, and placed on ice for 2 hours, followed by centrifugation at 19000 g for 10 minutes at 4 ° C. RNA was precipitated. The RNA was dissolved in distilled water and further purified by phenol extraction and ethanol precipitation to obtain 0.6 mg of purified total RNA. Furthermore, from the total RNA obtained above, mRNA was isolated using an mRNA purification kit (manufactured by PerSeptive Biosystems, BioMag mRNA purification Kit) to obtain about 10 μg of cyclamen mRNA.

(2) Preparation of cDNA library Double-stranded cDNA was obtained from the mRNA obtained in (1) above using a cDNA synthesis kit (cDNA synthesis module, manufactured by Amersham Life Sciences). An adapter (EcoRI-NotI-BamHI Adapter manufactured by Takara Shuzo Co., Ltd.) was ligated to these cDNAs, and the adapter was phosphorylated. After ligating these cDNAs to λZAPII phage vector (Lambda ZAP Predigested EcoRI / CIAP-Treated Vector Kit, manufactured by STRATAGENE), packaged in lambda phage using in vitro packaging kit (STRATAGENE, manufactured by GigapackIII Gold Packaging Kit) Performed. A large number of recombinant phages were obtained by infecting this phage with E. coli XL1-Blue MRF ′, and these were used as a cyclamen cDNA library.

(3) Preparation of probe for cloning
In order to prepare probes for cloning cDNA fragments of CHS and DFR using RT-PCR (Reverse Transcription-Polymerase Chain Reaction), first, primers were designed.

a) Preparation of probe for CHS Two kinds of oligonucleotides having the base sequences shown below were prepared by chemical synthesis as primers No. 1 and No. 2.

(Primer No. 1: SEQ ID NO: 5)
5'-GAGAARTTYMAGCGCATGTG-3 '
(Primer No. 2: SEQ ID NO: 6)
5'-ACRCAMGCRCTYGACATGTT-3 '

  The above two types of oligonucleotides were prepared by synthesizing oligonucleotides using a DNA synthesizer (Model 391, Applied Biosystems) and further purifying the synthesized product by ion exchange HPLC. .

Next, using 1 μg of total RNA obtained in (1) above as a template, reverse transcription reaction mixture (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl ₂ , 1 mM dNTP (dATP, dGTP, dCTP, dTTP) mixture , 2.5μM Oligo (dT) (Oligo (dT) _12-18 primer, Lifetech Oriental Co., Ltd.), RNA inhibitor (RNase Inhibitor, Takara Shuzo Co., Ltd.) 20 units and reverse transcriptase (AMV Reverse Tra
nscriptase XL (Takara Shuzo Co., Ltd.) 5 units) 20 μl was prepared and subjected to reverse transcription reaction. The reverse transcription reaction was performed under the conditions of incubation at 30 ° C. for 10 minutes, 50 ° C. for 30 minutes, 99 ° C. for 5 minutes, 4 ° C. for 5 minutes.

  Furthermore, using primers No. 1 and No. 2 and 0.1 μM each as a primer, an amplification reaction solution for PCR containing the reverse transcription reaction solution obtained above was prepared, and DNA amplification reaction was performed. The amplification reaction solution used here was prepared with a PCR kit (manufactured by Takara Shuzo Co., Ltd., LA PCR Kit Ver.2.1).

The above-mentioned amplification reaction of DNA by the PCR method is performed using a PCR reaction apparatus (ASTEK, Program Temp Control
System PC-700) was used for denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 1 minute, and extension at 72 ° C. for 1 minute by repeating three reaction operations 35 times. .

  A part of the CHS cDNA amplified as described above was used as a probe DNA for screening a cDNA library.

b) Preparation of DFR probe Two kinds of oligonucleotides having the base sequences shown below were chemically synthesized as primers No. 3 and No. 4, and amplified by the same method as in (3) -a). A part of was used as a probe DNA for screening a cDNA library.

(Primer No. 3: SEQ ID NO: 7)
5'-GGSTTYRTCGGCTCATGGCTSGT-3 '
(Primer No. 4: SEQ ID NO: 8)
5'-AARTACATCCAWSCRGTCAT-3 '

(4) Cloning of cDNA library a) Screening of cDNA for CHS From the cyclamen cDNA prepared in (2) above, plaque hybridization was performed by plaque hybridization using the probe DNA prepared in (3) -a) above. Screening for cDNA of CHS was performed. The plaque hybridization method was performed as follows.

The probe DNA obtained in the above (3) -a) is labeled with digoxigenin (DIG) using a DNA labeling kit (Roche Diagnostics, DIG-ELISA DNA Labeling & Detection Kit). Probe DNA was obtained.
Next, plaques of recombinant lambda phage, which is the cyclamen cDNA library obtained in (2) above, were formed on a 1.5% agar medium. The phage plaques were transferred to a nylon membrane (Dupont) Gene Screen. Nylon membrane with transferred phage plaques was treated with alkali-denaturing solution (1.5M NaCl, 0.5M NaOH) and neutralizing solution (1M Tris-HCl (pH 7.5)) 2 × SSC (0.3M NaCl, 30 mM sodium citrate). After treatment for 10 minutes, air drying, and baking at 80 ° C. for 2 hours, the phage DNA contained in the phage plaques was immobilized on a nylon membrane.

  The nylon membrane on which the phage DNA was immobilized was immersed in a hybridization solution (500 mM NaPi buffer (pH 7.2), 7% SDS, 1 mM EDTA), and immersed at 65 ° C. for 2 hours. Next, the labeled probe DNA (10 ng / ml) labeled with DIG is incubated at 100 ° C. for 5 minutes, denatured, added to the hybridization solution, and dipped at 65 ° C. for 15 hours, whereby labeled probe DNA and nylon are added. A DNA-DNA hybrid was formed between the phage DNAs of interest immobilized on the membrane.

After completion of the reaction, the nylon membrane was washed three times for 20 minutes each with a washing solution (40 mM NaPi buffer (pH 7.2), 1% SDS) kept at 65 ° C. Then, above DIG-ELISA Labeling &
Using the Detection Kit, the target recombinant phage containing DNA encoding cyclamen CHS was detected.

  As a result, it was possible to isolate and select five phages that seem to have incorporated the CHS cDNA out of 45,000 phage plaques.

b) Screening of DFR cDNA Using the probe DNA prepared in (3) -b) above from the cyclamen cDNA prepared in (2) above, plaque hybridization was performed in the same manner as in (4) -a). Cyclamen DFR cDNA was screened.

  As a result, it was possible to isolate and select 6 phages from about 100,000 phage plaques that seem to have DFR cDNA incorporated therein.

(5) cDNA subcloning a) CHS cDNA subcloning The cDNA contained in the phage isolated in the above (4) -a) was transformed into pBluescript SK (-) using the Lambda ZAPII Predigested EcoRI / CIAP-Treated Vector Kit. ) Subcloning into a plasmid vector (manufactured by STRATAGENE) and transforming E. coli SOLR cells. Each plasmid DNA was extracted using the alkaline SDS method, and 1 μl of each of these DNAs was separately digested with 5 units of restriction enzyme EcoRI in 10 μl of H buffer. As a result of analyzing the obtained digestion reaction solution by agarose gel electrophoresis, it was confirmed by the electrophoresis pattern of DNA that two types of DNA were present.

b) Subcloning of DFR cDNA Subcloning and DNA analysis of the cDNA contained in the phage isolated in the above (4) -b) by the same method as in (5) -a) It was confirmed to contain the same DNA.

(6) Sequencing of cDNA a) Sequencing of cDNA of CHS Of the two types of cDNA fragments subcloned in (5) -a) above, the larger fragment was selected and subjected to nucleotide sequence analysis. Base sequence analysis was performed using a plasmid purification kit (QIAfilter Plasmid Midi Kit (QIAGEN)) and a base sequence determination kit (TaKaRa Taq Cycle Sequensing Core Kit (Takara Shuzo Co., Ltd.)) using an automated DNA sequencer (PE- ABI model 377 Sequencer (manufactured by PE-ABI)). As a result, a 1446-base cDNA was determined. This cDNA contains a single open reading frame (ORF) of 1170 bases. Each ORF is sandwiched between a 56 base 5 ′ untranslated region and a 220 base 3 ′ untranslated region containing a poly (A) tail.

b) Sequencing of DFR cDNA After subcloning the cDNA fragment subcloned in (5) -b) above into pUC119, using this, E. coli JM109 was transformed, and the nucleotide sequence was determined in the same manner as in (6) -a). Analysis was performed. As a result, a 1220-base cDNA described in SEQ ID NO: 3 was determined. This cDNA contains a single open reading frame (ORF) of 1032 bases set forth in SEQ ID NO: 3. This ORF is sandwiched between a 34 base 5 ′ untranslated region and a 154 base 3 ′ untranslated region containing a poly (A) tail.

  Therefore, the base sequences of the genes encoding cyclamen CHS and DFR according to the present invention consist of 1446 bases and 1220 bases, respectively, of the single open reading frames described in SEQ ID NO: 1 and SEQ ID NO: 3. The base sequences of the genes encoding cyclamen CHS and DFR according to the present invention encode proteins consisting of 389 amino acid residues and 343 amino acid residues, respectively. When the amino acid sequence of SEQ ID NO: 2 is compared with Petunia's CHS (Nucleic Acids Res. 14 (13), 5229-5239 (1986)) that has been clarified so far, 90% homology is observed. This region is distinct from petunia's CHS and is unique to cyclamen. In addition, when the amino acid sequence of SEQ ID NO: 4 is compared with the amino acid sequence of Petunia DFR (Plant Mol. Biol. 13 (5), 491-502 (1989)) that has been revealed so far, 72% homology Although recognized, in other regions it is distinctly different from petunia DFR and is a unique sequence of cyclamen.

  The homology between the DNA encoding the CHS of the present invention and the petunia CHS gene was 75%, and the homology between the DNA encoding the DFR of the present invention and the petunia DFR gene was 73%.

Claims (5)

A DNA having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, which encodes a protein of cyclamen dihydroflavonol-4-reductase. The DNA according to claim 1, which is a DNA containing at least the base numbers 35 to 1063 described in SEQ ID NO: 3 in the sequence listing. A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, and having the activity of dihydroflavonol-4-reductase DNA encoding a protein. DNA having a base sequence partially different from the base sequence described in SEQ ID NO: 3 in the sequence listing, but having homology to the base sequence described in SEQ ID NO: 3, and SEQ ID NO: 3 in the sequence listing The DNA according to claim 3, which is a DNA encoding a protein capable of hybridizing under stringent conditions to the DNA having the base sequence described in claim 4 and having dihydroflavonol-4-reductase activity. . Cyclamen dihydroflavonol-4-reductase, which is a protein of the following (C) or (D):
(C) A protein having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
(D) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4 and having the activity of dihydroflavonol-4-reductase Protein.