JP5447806B2 - Methylase isolated from sweet pea - Google Patents

Description

  The present invention relates to an enzyme that methylates the hydroxyl group of the B ring of a dye precursor in the flavonoid biosynthesis pathway of Lathyrus odoratus.

  Sweet pea (Lathyrus odoratus) belongs to the leguminous family and is an annual herb that originates from Sicily in the Mediterranean to Crete. This species is a climbing plant with a height of 1 to 2 m, a flower width of 3 to 5 cm, and abundant flower color of horticultural varieties.

  For example, there is a report that multiple alleles are involved in flower color inheritance related to flower color expression (Patent Document 1). Specifically, Patent Document 1 “Sweet pea (Leguminosae) petal pigments were analyzed and petal pigment genotypes of each cultivar line were examined. As a result, petal pigment genotypes of each cultivar line were clarified. The pigment phenotypes include methylated anthocyanidins, malvidin (Mv) and petunidin (Pt), both of which are included in the pigment genotype that produces Dpn. The type included peonidin (Pn), which is a methylated anthocyanidin, and was included in the pigment genotype that produces Cyn. "

  On the other hand, plant O-methylation converting enzyme (OMT) is an important enzyme that methylates lignin and various secondary metabolites. OMT is an enzyme that catalyzes the conversion of a hydroxyl group in a receptor molecule to a methyl group via S-adenosyl-methionine, resulting in a methyl ether derivative. Flavonoids involved in sweet pea flower color expression, particularly anthocyanidins, are known to contain methylated anthocyanidins such as peonidin, petunidin, and malvidin (Non-patent Document 1). No gene has been reported that encodes.

  So far, as the genes for enzymes involved in anthocyanin methylation in the anthocyanin biosynthetic pathway, the feline F3 ′, 5′MT gene (Non-patent Document 2) and the periwinkle F3 ′, 5′MT gene (Non-patent Document 3). ), F3 ′, 5′MT gene of cyclamen (Patent Document 2) and the like are known.

  Patent Document 3 states that “the tea catechin methylation enzyme is allowed to act on the tea catechin extract extracted from tea leaves to convert the tea catechins in the tea catechin extract into tea methylated catechins. There is a description that a tea methylated catechins-containing composition containing tea methylated catechins at a high concentration can be produced efficiently and inexpensively. Furthermore, as the tea catechin methylase used in the present invention, as long as it is an enzyme capable of synthesizing and converting tea catechins to tea methylated catechins, the origin and the like are not particularly limited, For example, it can be derived from plants, animals, microorganisms, etc. Specific examples of plants include tea leaves and the like, and specific examples of animals include rat hepatocytes and the like, and specific examples of microorganisms. Examples of such tea catechin methylating enzymes include catechol O-methyl transferase (Catechol O-methyl transferase, EC 2.1.1.6, hereinafter abbreviated COMT). ) Etc. "can be used.

International publication WO2004 / 103065 pamphlet JP 2005-312388 JP 2007-306806

Hashimoto F. et al., Biosci. Biotechnol. Biochem., 2003, Vol. 67, P.396-401. Plant Molecular Biology, (Netherlands), 1996, 32, p.1163-1169 Phytochemistry, (Netherlands), 2003, 62, p.127-137

  As mentioned above, it is known that the sweet pea petal pigment is controlled by the petal pigment genotype and that the sweet pea petal contains methylated anthocyanidins such as peonidin, petunidin, and malvidin. No gene encoding an O-methylation converting enzyme has been reported.

  Therefore, an object of the present invention is to provide an O-methylation converting enzyme involved in flower color expression of sweet pea, and a method using the enzyme.

  In order to solve the above problems, the present inventors specify a relatively highly conserved region from the sequence information of O-methylation converting enzyme already known in existing plants, and sweet pea based on the region. An attempt was made to identify a full-length O-methylation converting enzyme. In the process, it was found that a polypeptide encoded by a single polynucleotide having similarity to the ipomea and medicalogo O-methylation converting enzyme genes has an activity of catalyzing the production of various methylated flavonoids, The present invention has been completed.

  That is, the present invention includes the following features.

(1) The polypeptide according to any one of the following (a) to (d).
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a polypeptide comprising one or more amino acid substitutions, additions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 2 and having an activity of catalyzing the production of methylated flavonoids,
(c) a polypeptide comprising an amino acid sequence having at least 70% homology to the amino acid sequence shown in SEQ ID NO: 2 and having an activity of catalyzing methylation of flavonoids
(d) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a complementary strand of the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 and has an activity of catalyzing the methylation of flavonoids
(2) A polynucleotide encoding the polypeptide according to (1) above.
(3) The polynucleotide according to (2) above, comprising the nucleotide sequence shown in SEQ ID NO: 1.
(4) A recombinant vector comprising the polynucleotide according to (2) or (3) above.
(5) A transformed host cell transformed with the recombinant vector according to (4) above.

(6) The method includes culturing the transformed host cell according to (5) above under conditions that allow expression of the polypeptide encoded by the recombinant vector, and recovering the polypeptide. (1) A method for producing the polypeptide according to (1).
(7) A method for producing a methylated flavonoid, comprising treating a solution containing an unmethylated flavonoid with an enzyme using the polypeptide according to (1) above.
(8) The production method according to (7), wherein the solution containing unmethylated flavonoid is an extract derived from tea, apple, blueberry, grape, oyster or strawberry.
(9) A method for producing a mutant plant, comprising introducing the polynucleotide according to (2) or (3) above into a cell or tissue culture of a flowering plant.
(10) The production method according to the above (9), wherein the flowering plant is sweet pea.

  According to the present invention, an O-methylation converting enzyme involved in flower color expression of sweet pea is provided.

  Since the enzyme of the present invention can catalyze the production of methylated forms of various flavonoids including anthocyanidins and catechins, it is useful for breeding sweet peas having a new flower color and for producing methylated flavonoids.

Figure 1 shows A: Dp (delphinidin), Cy (cyanidine), My (myricetin), Qu (quercetin) and Ka (kaempferol), and B: epicatechin (ECG) and epigallocatechin gallate (EGCG) as substrates The figure which confirmed the production | generation of the methylated body by performing an enzyme reaction as follows. FIG. 2 shows the contents of Cy (cyanidine) and Pn (peonidin) measured at each flowering stage (upper figure) of sweet pea red petals (lower figure). FIG. 3 shows the contents of Dp (delphinidin), Pt (petunidin) and Mv (malvidin) measured at each flowering stage (upper figure) of sweet pea purple petals (lower figure). FIG. 4 shows OMT expression in stages 4, 6, 8, and 10 (a) red petals and (b) purple petals.

  The present invention relates to a sweet pea-derived methylation converting enzyme (hereinafter also referred to as OMT polypeptide) and a method of using the same.

  An example of the nucleotide sequence of the sweet pea OMT gene (hereinafter also referred to as OMT polynucleotide) is shown in SEQ ID NO: 1, and the amino acid sequence of the OMT polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2. The OMT polypeptide encoded by the OMT polynucleotide is not limited to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, but is substituted or added with one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2. A polypeptide comprising a deletion or insertion and having an activity of catalyzing methylation of flavonoids, or a homology of 70% or more, preferably 80% or more, more preferably to the amino acid sequence shown in SEQ ID NO: 2 A polypeptide comprising a polypeptide having a homology of 90% or more, most preferably 95% or more and having an activity of catalyzing methylation of flavonoids (hereinafter referred to as “mutant polypeptide”). There may be. Here, the number of amino acids to be substituted, deleted, added or inserted may be, for example, 1 to 20, preferably 1 to 10, and more preferably 1 to 5. The homology value means a value calculated with default settings using software (for example, FASTA, DANASYS, BLAST) that calculates homology between a plurality of amino acid sequences.

  The OMT polynucleotide is not limited to the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, but is a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1. Moreover, a polynucleotide encoding a polypeptide having an activity of catalyzing methylation of flavonoids (hereinafter also referred to as a mutant polynucleotide) is also included. The mutant polynucleotide is a polypeptide having an amino acid sequence different from the amino acid sequence shown in SEQ ID NO: 2, and encodes a mutant polypeptide having an activity of catalyzing flavonoid methylation.

  Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. As stringent conditions, for example, polynucleotides having sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more hybridize to each other, and homology is higher than that. A condition in which polynucleotides having a low pH do not hybridize with each other can be exemplified. Such conditions are obvious to those skilled in the art, for example by reference to J. Sambrook, et al., “Molecular Cloning”, Cold Spring Harbor Laboratory Press, 1989.

Whether or not the above-mentioned mutant polypeptide has the activity of catalyzing the production of methylated flavonoids is determined by detecting the presence or absence of the production of methylated flavonoids by acting the mutant polypeptide using an unmethylated flavonoid as a substrate Can be done by. Non-methylated flavonoids that can be used as a substrate include, but are not limited to, anthocyanidins (e.g., pelargonidin, cyanidin, delphinidin), quercetin, and glycosides thereof, and myricetin, catechin (e.g., epithelium). Polyphenols such as catechin (ECG) and epigallocatechin gallate (EGCG)) can be used. Specifically, whether or not a mutant polypeptide has an activity of catalyzing the production of methylated flavonoids can be tested as follows. Specifically, a 1.5 ml centrifuge tube was prepared, and 1M Tris-HCl (pH 8, 15 μl), S-adenosyl-L-methionine (10 mM, 10 μl) (Sigma-Aldrich, Inc., St. Louis, USA) ), 10 mM substrate (10 μl) and a solution containing the mutant polypeptide (10 μl) are added to make a total volume of 50 μl of reaction solution. Then, after reacting the reaction solution at 30 ° C. for 30 minutes, 50 μl of chloroform mixed solution (CHCl ₃ : methanol / 1% HCl, 2: 1) is added to the reaction solution, and the reaction is stopped by vigorously stirring. Thereafter, centrifugation (10,000 g, 3 minutes) is performed, the upper layer (50 μl) is transferred to a new tube, and the presence or absence of the activity can be evaluated by identifying the dye by high performance liquid chromatography.

  The OMT polynucleotide of the present invention is designed based on the nucleotide sequence information shown in SEQ ID NO: 1 by designing a primer set that can specifically amplify the sequence, and using the total DNA extracted from the sweet pea petals as a template for PCR amplification Can be cloned.

  Alternatively, the polynucleotide of the present invention can be obtained by methods known to those skilled in the art based on the nucleotide sequence information shown in SEQ ID NO: 1, for example, cloning of appropriate sequences and cleavage with restriction enzymes, phosphotriester method (for example, Narang et al., 1979). , Meth. Enzymol., 68, p90-99), phosphodiester method (see, for example, Brown et al., 1979, Meth. Enzymol., 68, p109-151), ethyl phosphoramidite method (see, for example, Beaucage). Et al., 1981, Tetrahedron Lett., Vol. 22, pp. 1859 to 1862). Moreover, you may synthesize | combine by using a commercially available automatic DNA synthesizer.

  The OMT polypeptide of the present invention can be produced according to a conventional method using the OMT polynucleotide cloned as described above. For example, the OMT polypeptide of the present invention can be produced by expressing an OMT polynucleotide in a suitable host cell and recovering the OMT polypeptide from the culture.

  Examples of host cells that can be used in the present invention include, but are not limited to, prokaryotic cells such as E. coli, yeast, insect cultured cells (eg, silkworm cultured cells), animal cell cells (eg, mouse cultured cells), plant cultures. Examples include eukaryotic cells such as cells (eg, tobacco cultured cells).

  Specifically, a recombinant vector in which the OMT polynucleotide is incorporated downstream of a promoter that induces expression in the host cell is produced, and this is introduced into the host cell to transform the host cell. The transformed host cell can be cultured under conditions where the expression regulatory sequence functions, and the OMT polypeptide of the present invention can be recovered from the culture.

  Examples of vectors that can be used in the present invention include plasmid vectors, Ti plasmid vectors, phage vectors, phagemid vectors, YAC vectors, and viral vectors. Depending on the type of host cell used for subsequent transformation, these vectors may contain additional sequences that regulate and assist in the expression of the OMT polypeptide in the host cell. Such sequences include, but are not limited to, enhancers, terminators, operators, SD sequences and the like. In addition, the vector may contain a selection marker (for example, an ampicillin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a neomycin resistance gene, etc.) for selecting transformed host cells.

  Transformation of host cells with the above expression construct can be carried out using methods commonly used by those skilled in the art, for example, protoplast method, competent cell method, electroporation method, particle gun method, and Agrobacterium method.

  The culture of the transformed host cell may be substantially the same as the culture method of the host cell used, for example, using a medium containing an assimilating carbon source, nitrogen source, metal salt, vitamin, etc. under appropriate conditions. It can be cultured under. From the culture broth obtained in this way, fractionation and purification are performed by a general method, and if necessary, the OMT polymorph is retained in such a way as to retain its activity by ultrafiltration concentration, freeze drying, spray drying, crystallization, etc. Peptides can be obtained.

  Alternatively, the OMT polypeptide of the present invention can also be produced using a cell-free expression system, that is, the above expression construct in vitro. Cell-free expression systems that can be used in the present invention include Cell Free Science's WEPRO (registered trademark) series and the like.

  The OMT polypeptide of the present invention is a methylated form of a flavonoid such as methylated myricetin, a methylated catechin ((-)-epigallocatechin3-O-gallate, (-)-epicatechin3-O-gallate, etc.) ) Have the activity of catalyzing the formation of (see FIG. 1).

  Therefore, the OMT polypeptide of the present invention can be used for the production of methylated flavonoids.

  The method for producing methylated flavonoids of the present invention includes enzymatic treatment of a solution containing unmethylated flavonoids using the OMT polypeptide of the present invention.

  The solution to be subjected to the above method is not particularly limited as long as it is a solution containing unmethylated flavonoids, and may be an extract from a natural product or a prepared solution. Among methylated flavonoids, methylated catechin has recently attracted attention as a compound having an antiallergic action, and is considered to be a useful compound as an active ingredient for functional foods and pharmaceuticals. Therefore, preferably, the solution containing unmethylated flavonoids is a solution containing unmethylated catechins. Examples of the solution containing catechin include, but are not limited to, tea, apple, blueberry, grape, oyster, strawberry-derived extract, and the like.

  The form of the OMT polypeptide to be used is not particularly limited. In addition to the purified polypeptide, a crude polypeptide (eg, an extract from a host cell that expresses the polypeptide) can be used. Expression host cells may be used. When using a crude enzyme, it should be noted that it does not contain a factor that inhibits the enzyme activity of the OMT polypeptide of the present invention.

  In the present invention, the polypeptide can be used by immobilizing it on a carrier. Examples of the method for preparing an immobilized polypeptide that can be used in the present invention include carrier binding methods (physical adsorption method, ion binding method, covalent bonding method, biochemical specific binding method, etc.), cross-linking methods, and inclusion methods. Methods well known to those skilled in the art are included. The carrier binding method is a method of binding an enzyme to a water-insoluble carrier. In this case, a polysaccharide derivative such as cellulose, dextran, or agarose, a synthetic polymer such as polyacrylamide gel, polystyrene resin, or ion exchange resin, porous Inorganic materials such as porous glass and metal oxides can be used as the carrier. The cross-linking method is a method of immobilizing by reacting a reagent having two or more functional groups with an enzyme and cross-linking between the enzymes. As the cross-linking reagent, glutaraldehyde, isocyanate derivatives, N, N -Ethylene bismaleimide, bisdiazobenzidine, N, N-polymethylenebisiodoacetamide, etc. can be used. The inclusion method includes a lattice type that traps the enzyme in a gel matrix of a natural polymer or a synthetic polymer, and polyacrylamide gel, polyvinyl alcohol, photocurable resin, starch, konjac powder, etc. as the polymer compound used in that case , Gelatin, alginic acid, carrageenan and the like.

  The conditions for the enzymatic reaction are not particularly limited. For example, 1-10 units of the OMT polypeptide of the present invention may be used per 10 mM of the flavonoid substrate, and the reaction may be performed for 5-30 minutes. As the reaction temperature and pH, the optimum temperature of the OMT polypeptide of the present invention and the value close to the optimum pH are adopted. Specifically, the temperature is 25 to 30 ° C., and the conditions are pH 6.5 to 8.5. Use it. The OMT polypeptide includes 1M Tris-HCl (pH 8, 15 μl), S-adenosyl-L-methionine (10 mM, 10 μl) (Sigma-Aldrich, Inc., St. Louis, USA), 10 mM cyanidin (10 μl) and In a reaction solution consisting of a polypeptide solution (10 μl) and a total volume of 50 μl, the amount of 1 mM mol of peonidin per minute is defined as 1 unit.

  The OMT polypeptide of the present invention has an activity to produce methylated forms of flavonoids, for example, methylated anthocyanidins (petunidin, malvidin, peonidin, etc.) involved in sweet pea flower color, isorhamnetin, and methylated quercetin ( refer graph1).

  Therefore, the OMT polynucleotide of the present invention can be used for production of mutant plants. The “mutant plant” in the present invention is a flowering plant having a flower color different from that of the wild type as a result of the expression of the externally introduced OMT polynucleotide. In addition, the flowering plant which is the production target of the mutant plant in the present invention is not particularly limited as long as it is a plant in which methylation of a flavonoid is involved in flower color expression. In the present invention, the flowering plant that is the production target of the mutant plant is preferably sweet pea.

  Specifically, the production of mutant plants can be achieved by using transgenic techniques known to those skilled in the art to convert the OMT polynucleotide of the present invention into a flowering plant in the form of a plant expression vector containing the polynucleotide, if necessary. In a cell or tissue culture.

  As a transgenic technique that can be used in the present invention, for example, transformation using Agrobacterium can be mentioned. Briefly, a plasmid is removed from Agrobacterium which is infectious to plants, the T-DNA gene of the plasmid is replaced with the OMT polynucleotide of the present invention, and this is returned to Agrobacterium for use in infecting plant tissues. To do. In that case, the plant into which the OMT polynucleotide is introduced can be selected by inserting a selection marker gene known to those skilled in the art together with the OMT polynucleotide. As such a selectable marker gene, for example, a gene conferring resistance to antibiotics such as kanamycin, tetracycline, rifampicin, spectinomycin, carbenicillin, gentamicin and the like can be used. Examples of transformation using Agrobacterium include experimental protocols for model plants, edited by rice and Arabidopsis, cell engineering separate volume, plant cell engineering series 4, supervised by Isao Shimamoto and Kiyotaka Okada (1996), US Patent No. 5,188,958, etc. It is described in.

  Other transgenic techniques that can be used in the present invention include, but are not limited to, transformation via viral vectors, electroporation and particle gun. Examples of transformation using a particle gun are described in, for example, US Pat. No. 5,204,253.

  The cell or tissue culture transformed with the OMT polynucleotide of the present invention can then be regenerated into plants by techniques known to those skilled in the art. The regeneration of the plant body may be performed according to a method known to those skilled in the art. In order to efficiently produce mutant plants, it is preferable that the target tissue used for transformation has a high regenerative capacity, such as cotyledons, hypocotyl tissues, leaves, stems, floral organs, Roots, embryos, immature embryos, pupae, ovary, ovules, endosperm, pistils, stamens and the like can be mentioned.

  Hereinafter, although an example of the present invention will be described in detail as an example, the present invention is not limited to this example.

As a material sweet pea, a horticultural cultivar “Cuthbertson MIX” was used (Takagi Seed Co., Kyoto). Seeds that were sown in December 2005 and flowered in the spring of 2006 were collected. The collected seeds were sown again in December 2006, and petals having various flower colors of the self-bred lines that flowered in the spring of 2007 were used for flower color measurement and pigment analysis. The seeds of a red inbred line that mainly contains cyanidin and peonidin were collected from the inbred line that blossomed in the spring of 2007. In addition, seeds of a purple inbred line mainly containing delphinidin, peonidin, and malvidin were collected from the inbred line that bloomed in the spring of 2007. The collected seeds were sown in December 2007 and flowered in the spring of 2008. The flower petals were used for flower color measurement and pigment analysis.

  The collected sweet pea petals, buds, and leaves were collected, weighed immediately, frozen in liquid nitrogen as it was, stored in a freezer at -80 ° C., taken out of the freezer for use, and used for experiments.

[Example 1]
Cloning of O-methylation converting enzyme (OMT) Total RNA was extracted from petals of relatively young sweet pea purple lines using the RNeasy maxi kit (Qiagen). Poly (A) ⁺ RNA was purified from total RNA using an Oligotex-dT30 <Super> (Takara Biotech) kit to obtain mRNA. cDNA was synthesized from mRNA using GeneRacer kit (Invitrogen). In each case, the experiment was performed according to the protocol attached to the product.

  Next, 5′RACE (Rapid amplification of cDNA ends) and 3 ′ were used with the following primers designed with reference to the sequence of each gene reported in Brugliera et al. (2003) using the synthesized cDNA as a template. A fragment containing the 5 'end and 3' end of the sweet pea OMT gene was PCR amplified by the 'RACE method: primer A, 5'-GTAGTTCACGTAGTTGCTCTTRTCNGCRTC-3'; primer B, 5'-CGCCGGCAGAAGGTGANNCCRTCNCC-3 '; primer C, 5 '-CCGGGAGCACGAGCACYTNAARGARYT-3'; Primer D, 5'-ACCATCGAGATCGGCGTNTTYCANGG-3 '. Specifically, the diluted cDNA containing 1 μl was reacted with 10 pmol of A or B and C or D primer in each combination. PCR reaction was performed by incubating a reaction solution containing 0.4 unit of KOD plus (TOYOBO) at 94 ° C for 1 minute, followed by heat denaturation (94 ° C for 30 seconds), annealing (50 ° C for 30 seconds), and extension reaction ( 1 cycle at 68 ° C.) was defined as one cycle, and 35 cycles were repeated. Subsequently, the amplified cDNA fragment was inserted into pBluscript SK plasmid to obtain a clone. The nucleotide sequence was determined using the DNA sequence service of the Kagoshima University Genetic Experiment Facility.

  A deduced amino acid sequence (SEQ ID NO: 2) encoded by this nucleotide sequence is obtained from the determined sequence information, and finally, a 5 ′ primer containing the ATG base sequence of the first methionine residue (primer SPMT1LIC5: GACGACGACAAGATGACCGCAAATAAAAAC) And a 3 ′ primer containing a stop codon (primer SPMT1LIC3: GAGGAGAAGCCCGGTTCAATGCGCACGCC), the full-length cDNA of the coding region of sweet pea OMT was amplified by PCR.

[Example 2]
Recombinant production and extraction of sweet pea OMT protein by E. coli The full-length cDNA clone of sweet pea OMT coding region was obtained by incorporating the full-length cDNA clone of sweet pea OMT into pET vector (Promega). This vector was then introduced into E. coli BL21 (DE3) (Novagen) and recombinant E. coli BL21 (DE3) expressing sweet pea OMT protein was incubated at 37 ° C. in 2YT liquid medium containing kanamycin (50 μg / ml). After overnight culture with IPTG, IPTG (400 μM) was subsequently added and cultured at 30 ° C. for 6 hours. E. coli cells were collected by centrifugation (10,000 g, 30 seconds), suspended in 50 mM K-Pi buffer (pH 7.0, 500 μl), and centrifuged again under the same conditions. The same buffer (300 μl) and 2-mercaptoethanol 14 mM were added to the pellet test tube, suspended, and E. coli cells were lysed using an ultrasonic disrupter (Brandson Sonifier Model 250, BRANDSON ULTRASONICS, Danbury, CT. USA). Cell debris was removed by centrifugation (12,000 g, 20 minutes, 4 ° C.) to obtain a crude protein solution. The obtained crude protein solution was stored at -20 ° C.

[Example 3]
Enzyme (crude protein) functional assay The enzyme activity assay of the crude protein solution was performed according to the following method. Prepare a 1.5 ml centrifuge tube to which 1 M Tris-HCl (pH 8, 15 μl), S-adenosyl-L-methionine (10 mM, 10 μl) (Sigma-Aldrich, Inc., St. Louis, USA), 10 mM substrate (10 μl) and crude protein solution (10 μl) were added to make a total volume of 50 μl. The solution was reacted at 30 ° C. for 30 minutes. After the reaction, 50 μl of a chloroform mixed solution (CHCl ₃ : methanol / 1% HCl, 2: 1) was added to the solution and stirred vigorously to stop the reaction. Centrifugation (10,000 g, 3 minutes) was performed, the upper layer (50 μl) was transferred to a new tube, and the dye was identified by high performance liquid chromatography. As standards, methylated flavonoids peonidin, petunidin, malvidin, and isorhamnetin were used. In addition, unmethylated flavonoids cyanidin, delphinidin, myricetin, quercetin, kaempferol (Extrasynthese, Genay, France), and polyphenols epicatechin (ECG) and epigallocatechin gallate (EGCG) were used as substrates.

  The results are shown in Figure 1. As is clear from FIG. 1A, Pt (petunidin) and Mv (malvidin) are produced from Dp (delphinidin), Pn (peonidin) is produced from Cy (cyanidine), and Mt-My (methylated myricetin) from My (myricetin). ) And Is (isoramnetin) and Mt-Qu (methylated quercetin) were confirmed to be produced from Qu (quercetin). As described above, it was proved that the crude protein (enzyme) obtained from sweet pea has a function of methylating flavonoids. In addition, as a result of reacting the crude protein with polyphenols such as ECG and EGCG, several peaks containing methylated polyphenol were observed as shown in FIG. The possibility is shown.

[Example 4]
Quantitative analysis of anthocyanin accumulation associated with the growth of sweet pea petals Only petals were excised from the vases, weighed and then frozen with liquid nitrogen. The frozen petals were pulverized with a pulverizer. The petal flavonoid of each sample was extracted with 1% hydrochloric acid-methanol solution (v / w), and this was repeated three times. The extracts were combined and centrifuged to obtain a supernatant. All sample solutions were adjusted to a concentration of 0.16 g / ml. 2 ml of each extracted solution was accurately collected, 2N aqueous hydrochloric acid (4 ml) was added, and the mixture was hydrolyzed at 100 ° C. (2 hours). Concentrate and dry the hydrolysis residue solution with an evaporator, add 1% hydrochloric acid-methanol (2 ml) to dissolve, filter with Dismic-25 _HP (0.45μm, ADVANTEC, Toyo Roshi Kaisha Ltd. Japan), and then add each sample solution. Was quantitatively analyzed by high performance liquid chromatography (20 μl, HPLC). The equipment and analysis conditions are as follows. HPLC system, Jasco HPLC System; liquid feeding system, 4 liquid gradient pump model PU-2089 plus; dynamic mixer, model Mx-2080-32; diode detector, multiwavelengthdetector model MD-2010 plus; autosampler, model AS-2051 plus; column oven, model CO-2080 plus; data system, ChromNAVchromatography Data Station (Jasco Co. Tokyo, Japan). The column used was TSK-Gel ODS-80Ts QA (5 μm, 4.6 mm x 15.0 cm, TOSHO Co. Tokyo, Japan. The liquid feed system was TFA: H ₂ O (5: 995), B solution. A linear gradient with acetonitrile: TFA: H ₂ O (500: 5: 495), the initial setting is 20% B, 25% B is 60%, and 15 minutes B is 100% An anthocyanin was detected simultaneously at a wavelength of 525 nm and flavonol was detected at a wavelength of 340 nm at a flow rate of 0.8 ml / min.The flavonoids were evaluated as delphinidin, cyanidin, peonidin, pelargonidin, Petunidin and malvidin were used, and kaempferol, quercetin, myricetin and isorhamnetin were used as flavonols.

  The results are shown in FIGS. It can be seen from FIG. 2 that Cy (cyanidine) and Pn (peonidin) are rapidly generated from the stage 7 in the sweet pea red petals. Further, FIG. 3 shows that Dp (delphinidin) and Mv (malvidin) are rapidly generated from stage 6 in the sweet pea purple petals, and Pt (petunidin) has the maximum content in stage 9.

  Thus, it was found that the methylated anthocyanin content increased in each stage in the red and purple petals.

[Example 5]
Quantitative analysis of mRNA transcription
mRNA was adjusted from the sweet pea bud and the trophic organ (leaf) at each stage shown in FIGS. MRNA (1 μg) of each material was obtained by reverse transcription from cDNA by the method described above using the following primer set: 5′-TTCATGGGCACTGCGCCTGATG-3 ′; and 5′-ATGCCACAGTTCCACCCCAAAG-3 ′. The latter primer was designed based on the sequence obtained by amplifying a fragment of up to 400 bp.

  Quantitative reverse transcription RT-PCR was performed using 7300 / Fast Real-Time PCR System (Applied Biosystems) to measure the mRNA changes in each stage of the sweet pea petals. The reaction was repeated three times and the conditions were as follows. Diluted cDNA (2 μl) was added to each reaction solution (20 μl), each primer was set to a concentration of 200 nM, and SYBER PermixEx Taq solution (10 μl) and ROX Referance Dye (0.4 μl) were further added. This solution was measured using a Fast Optical 96-well plate (BMBio, Tokyo, Japan) according to the handling instructions of SYBER Premix Ex Taq (TAKARA). The heat denaturation cycle conditions were 95 ° C for 10 seconds followed by 40 cycles of 95 ° C for 5 seconds and 60 ° C for 31 seconds, and 95 ° C for 15 seconds, 60 ° C for 1 minute and 95 ° C. One cycle of 15 seconds was performed. The results are shown in FIG.

Gene transcription was quantified by leveling 18S rRNA, compared to the cycle limit (C _T ) of the gene of interest. Looking at the results of RT-PCR, OMT is expressed in all stages, but expression in leaves (L) is not observed, so the OMT of the present invention is considered to be expressed in a large amount in flowers. . The real-time PCR results showed that OMT expression increased as the petal development stage progressed.

  According to the present invention, an O-methylation converting enzyme involved in flower color expression of sweet pea is provided.

  A DNA sequence encoding the O-methylation converting enzyme according to the present invention is introduced into a sweet pea species alone or in combination with other genes by a known method, or into other plant species alone or By introducing it in combination with other genes, the biosynthesis of flavonoids and polyphenols can be modified or controlled in the plant, thereby producing bioactive substances in the plant and increasing the diversity and newness of flower color. It becomes possible to develop plants with various functions.

  Further, the O-methylation converting enzyme gene according to the present invention is expressed in a suitable host such as Escherichia coli or expressed in a cell-free biological system by a known method to obtain a purified protein, which is used as a catalyst for flavonoids and It can be expected that the chemical structure of polyphenols can be changed to have new functions.

Claims (10)

A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.   A polynucleotide encoding the polypeptide of claim 1.   The polynucleotide according to claim 2, comprising the nucleotide sequence shown in SEQ ID NO: 1.   A recombinant vector comprising the polynucleotide according to claim 2 or 3.   A transformed host cell transformed with the recombinant vector according to claim 4.   The method according to claim 1, comprising culturing the transformed host cell according to claim 5 under conditions allowing expression of the polypeptide encoded by the recombinant vector, and recovering the polypeptide. A method for producing a polypeptide.   A method for producing a methylated flavonoid, comprising treating a solution containing an unmethylated flavonoid with an enzyme using the polypeptide according to claim 1.   8. The production method according to claim 7, wherein the solution containing an unmethylated flavonoid is an extract derived from tea, apple, blueberry, grape, oyster or strawberry.   A method for producing a mutant plant, comprising introducing the polynucleotide according to claim 2 or 3 into a cell or tissue culture of a flowering plant.   10. The production method according to claim 9, wherein the flowering plant is sweet pea.

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