Flavonoid 4'-O-methyltransferase gene and its use

Abstract

Provided is: a transgenic plant that has a modified flower color; a self-fertilized or cross-fertilized progeny of the transgenic plant; or a propagule, a portion of a plant body, a tissue, or a cell of the transgenic plant or said progeny.　According to the present invention, both a delphinidin-type anthocyanin and a flavone C-glycoside in which hydroxyl groups at positions 7 and 4' are methylated, are caused to coexist in cells of a plant.

Description

Technical Field

[0001] The present invention relates to a novel polynucleotide encoding a protein having an activity of transferring a methyl group to the 4'-hydroxyl group of flavone C-glycoside, and its use.

Background Art

[0002] Roses, petunias, chrysanthemums, carnations, etc. are industrially important flowers worldwide. In particular, roses are the most popular flower plants, with records of cultivation dating back to before the Christian era, and have been artificially improved over hundreds of years. However, due to problems such as the absence of wild species with blue flower colors among closely related species that can crossbreed, it has been difficult to create rose varieties with blue flower colors through conventional crossbreeding and mutation breeding. The creation of completely new blue flower colors can evoke new demands associated with the expansion of the utilization scenarios of flower plants, leading to the expansion of production and consumption. Therefore, attempts have been made to create roses with blue flower colors by genetic engineering methods.

[0003] For example, it is known that purple to blue flowers contain a large amount of delphinidin-type anthocyanins with delphinidin, petunidin, and malvidin as skeletons. However, flower plants such as roses cannot produce such delphinidin-type anthocyanins. Therefore, research has been conducted to artificially produce delphinidin by expressing the flavonoid 3',5'-hydroxylase gene required for their synthesis (Non-Patent Document 1). However, even if the plant metabolism is artificially modified to express the enzyme gene for the target substance in recombinant plants, often due to competition with endogenous enzymes possessed by the plant itself, the accumulation of the target substance rarely or hardly occurs.

[0004] Furthermore, in addition to the structure of anthocyanin itself, the color of flowers also changes depending on coexisting flavonoids (referred to as copigments), metal ions, the pH of vacuoles, etc. Flavones and flavonols are typical copigments, and by stacking on anthocyanin in a sandwich-like manner, they have the effect of increasing the blue color, darkening the color, or enhancing the color tone (Non-Patent Document 2). This is known as the copigment effect. In particular, flavones are known to exhibit a strong copigment effect. For example, in the analysis of genetically modified carnations, it has been reported that flavones exhibit a significant copigment effect (Non-Patent Document 3). Also, in Dutch iris, it has been reported that the higher the ratio of the total flavone amount to the total delphinidin amount, the stronger the copigment effect and the bluer the color (Non-Patent Document 4). Furthermore, in Commelina communis, it has been reported that the color turns blue by forming commelinin (a metal complex of malonylcyanidin, flavocommelin, and magnesium ions) (Non-Patent Document 9).

[0005] However, not all plants can produce flavones. Roses, petunias, etc. do not accumulate flavones. Therefore, attempts have been made to modify flower colors by expressing genes encoding proteins with the activity to synthesize flavones from flavanones in such plants (Patent Document 1).

[0006] In plants, flavones are distributed not only in the free form but also as glycosides, and mainly flavone O-glycosides and flavone C-glycosides are produced. In particular, flavone C-glycosides are known to exhibit a strong copigmentation effect. For example, isovitexin, a type of flavone C-glycoside, has been reported to show a copigmentation effect on anthocyanins in Iris ensata Thunb. and stabilize blue flower colors by stabilizing anthocyanins (Non-Patent Document 5). Regarding flavone C-glycosides, two biosynthetic pathways have been reported so far. One is synthesized from flavanone by the reactions catalyzed by flavanone 2-hydroxylase, flavone C-glycosyltransferase, and dehydratase. The other is synthesized from flavanone by the reactions catalyzed by flavone synthase and flavone C-glycosyltransferase (Non-Patent Document 6).

[0007] Furthermore, the copigmentation effect is considered to be affected by the quantitative ratio of anthocyanins and flavones, and the modifications by sugars, methyl groups, acyl groups, etc. in anthocyanins and flavones. Simply expressing the flavone synthase gene and accumulating flavones does not necessarily result in blue flower colors. When the flavone synthase gene of Torenia is expressed in Petunia, the blue-violet flower color becomes lighter (Non-Patent Document 7). Also, when the flavone synthase gene derived from Gentiana is expressed in tobacco, flavones are synthesized (Non-Patent Document 8), but the flower color also becomes lighter. In addition, a flavone C-glycosyltransferase involved in the biosynthesis of isosaponarin in Wasabi has been identified (Non-Patent Document 12). Furthermore, attempts have been made to modify the flower color of roses by artificially containing flavones and malvidin (Patent Document 2), but no success has been achieved in creating roses with blue-based flower colors.

[0008] In recent years, the inventors of the present application have obtained a flavone 7-O-methyltransferase gene that transfers a methyl group to the 7-position hydroxyl group of flavone C-glycoside from Commelina communis var. hortensis, which is known as a variety (cultivated variety) of Commelina communis. Using this gene, they have succeeded in obtaining roses with bluer flower colors than before by coexisting swertisin and delphinidin-type anthocyanins in the petals of plants (Patent Document 3). However, the development of a blue expression control technology that enables the creation of roses with truly blue flower colors is still desired.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Non - Patent Document 11

Non - Patent Document 12

Summary of the Invention

Problems to be Solved by the Invention

[0011] The problem to be solved by the present invention is to provide a transformed plant with modified flower color, or its self - propagated or cross - propagated progeny, or their vegetative propagules, a part of the plant body, tissue, or cell.

Means for Solving the Problems

[0012] In order to solve the above problems, the inventors of the present application conducted intensive studies and repeated experiments. As a result, when delphinidin-type anthocyanin and flavone C-glycoside coexist in the petals of plants, a transformed plant having a flower color that has not been obtained conventionally, particularly a rose plant having a blue-based flower color (RHS Color Chart, 5th Edition: Violet-Blue Group / Blue Group and / or hue angle: 339.7° to 270.0°) can be obtained. Furthermore, surprisingly, the inventors of the present application have now found that among various flavone C-glycosides, a combination with enigenin in which not only the hydroxyl group at the 7-position but also the hydroxyl group at the 4'-position is methylated results in a bluer color, and they have successfully obtained a novel flavone 4'-O-methyltransferase gene that transfers a methyl group to the hydroxyl group at the 4'-position of the flavone C-glycoside from Iris japonica. Based on such findings, the present invention has been completed.

[0013] The present invention is as follows. [1] The following (A) to (E): (A) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO: 21; (B) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO: 21, and encodes a protein having an activity of transferring a methyl group to the hydroxyl group at the 4'-position of a flavone C-glycoside; (C) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 20; (D) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 20, and encodes a protein having an activity of transferring a methyl group to the hydroxyl group at the 4'-position of a flavone C-glycoside; and (E) A polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 20, and encodes a protein having an activity of transferring a methyl group to the hydroxyl group at the 4'-position of a flavone C-glycoside; A polynucleotide selected from the group consisting of. [2] The polynucleotide according to 1, which is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO: 21. [3] The polynucleotide according to 1, which is a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 20. [4] A protein encoded by the polynucleotide according to any one of 1 to 3. [5] A vector containing the polynucleotide according to any one of 1 to 3. [6] The vector according to 5, further comprising a flavone 7-O-methyltransferase (Fn-7OMT) gene or a homolog thereof. [7] The Fn-7OMT gene or a homolog thereof is (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1; (b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, and encodes a protein having an activity of transferring a methyl group to the 7-position hydroxyl group of a flavone C-glycoside. (c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2; (d) In the amino acid sequence of SEQ ID NO: 2, a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added, and encodes a protein having an activity of transferring a methyl group to the 7-position hydroxyl group of a flavone C-glycoside; and (e) A polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and encodes a protein having an activity of transferring a methyl group to the 7-position hydroxyl group of a flavone C-glycoside. The vector according to 6, selected from the group consisting of [8] The vector according to any one of 5 to 7, further comprising a flavone synthase (FNS) gene or a homolog thereof, and a flavone C-glycosyltransferase (CGT) gene or a homolog thereof. [9] The FNS gene or a homolog thereof is (1-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3; (1-b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (1-a); (1-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4; (1-d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 4, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (1-c); (1-e) A polynucleotide selected from the group consisting of polynucleotides having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (1-c), and wherein the flavone CGT gene or its homolog (2-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 22; (2-b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 22, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (2-a); (2-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23; (2-d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (2-c); A polynucleotide selected from the group consisting of a polynucleotide encoding a protein having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23 and having the same activity as the protein encoded by the polynucleotide described in (2-c).

[10] The vector according to 8 or 9, wherein the vector further comprises a flavonoid F3'5' hydroxylase (F3'5'H) gene or a homolog thereof, and a methyltransferase (MT) gene or a homolog thereof.

[11] The F3'5'H gene or a homolog thereof is (3-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7; (3-b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 7 and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (3-a); (3-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8; (3-d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 8 and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (3-c); (3-e) Selected from the group consisting of a polynucleotide having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 8 and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (3-c), and The MT gene or a homolog thereof is (4-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9; A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9, and that encodes a protein having an activity similar to the protein encoded by the polynucleotide described in (4-a); (4-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10; (4-d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 10, and that encodes a protein having an activity similar to the protein encoded by the polynucleotide described in (4-c); (4-e) A polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10, and that encodes a protein having an activity similar to the protein encoded by the polynucleotide described in (4-c), wherein the vector according to 10 is selected from the group consisting of;

[12] The vector according to any one of 9 to 11, wherein a 5'untranslated region (5'-UTR) (SEQ ID NO: 11) derived from the Arabidopsis thaliana alcohol dehydrogenase (ADH) gene is added to the flavone CGT gene or its homolog.

[13] The vector according to any one of 5 to 7, further comprising a flavanone 2-hydroxylase (F2H) gene or its homolog, a flavone C-glycosyltransferase (CGT) gene or its homolog, and a dehydrogenase (FDH) gene or its homolog.

[14] The vector according to 13, further comprising a flavonoid F3'5'hydroxylase (F3'5'H) gene or its homolog, and a methyltransferase (MT) gene or its homolog.

[15] The F2H gene or its homolog is (5-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12; A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (5-a); (5-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 13; (5-d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 13, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (5-c); (5-e) A polynucleotide selected from the group consisting of a polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 13, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (5-c); The flavone CGT gene or its homolog is (6-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (6-b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 16, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (6-a); (6-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 17; (6-d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 17, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (6-c); (6-e) A polynucleotide selected from the group consisting of a polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 17, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (6-c); the FDH gene or its homolog is (7-a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14; (7-b) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 14, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (7-a); (7-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 15; (7-d) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 15, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (7-c); (7-e) a polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 15, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (7-c), and is selected from the group consisting of the F3'5'H gene or its homolog is (8-a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7; (8-b) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 7, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (8-a); (8-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8; (8-d) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 8, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (8-c); A polynucleotide selected from the group consisting of a polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8 and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (8-c), and wherein the MT gene or its homolog is (9-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9; (9-b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9 and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (9-a); (9-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10; (9-d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 10 and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (9-c); (9-e) A polynucleotide selected from the group consisting of a polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (9-c), the vector according to 14.

[16] The vector according to 15, wherein a non-translated region (5'-UTR) (SEQ ID NO: 18) derived from the Arabidopsis thaliana HSPRO gene is added to the flavone CGT gene.

[17] A transformed plant containing the polynucleotide according to any one of 1 to 3, or its self-propagated or cross-propagated progeny.

[18] The transformed plant according to 17, or its self-propagated or cross-propagated progeny, wherein the plant is selected from rose, petunia, chrysanthemum, carnation or lily.

[19] The transformed plant according to 18, or its self-propagated or cross-propagated progeny, wherein the plant is rose.

[20] A transformed plant according to any one of 17 to 19, or a vegetative propagule, a part of a plant body, a tissue, or a cell of its self-propagated or cross-propagated progeny.

[21] A cut flower of a transformed plant according to any one of 17 to 19, or a processed product made from the cut flower, or its self-propagated or cross-propagated progeny.

[22] A method for producing a transformed plant with a modified flower color, comprising the step of co-existing a delphinidin-type anthocyanin and a flavone C-glycoside in a plant cell, wherein the hydroxyl groups at the 7-position and 4'-position of the flavone C-glycoside are methylated.

[23] The method according to 22, wherein the flavone C-glycoside is envinin.

[24] The method according to 22 or 23, wherein the delphinidin-type anthocyanin is selected from the group consisting of malvidin 3,5-diglucoside (malvin), delphinidin 3,5-diglucoside (delphin), petunidin 3,5-diglucoside, acylated delphin, acylated malvin, and combinations thereof.

[25] The method according to any one of 22 to 24, comprising the step of introducing the vector according to any one of 5 to 16 into a plant cell.

[26] The method according to 25, wherein the plant is selected from rose, petunia, chrysanthemum, carnation, or lily.

[27] The method according to 26, wherein the plant is rose.

Advantages of the Invention

[0014] According to the present invention, a plant variety having a flower color that has not been obtained conventionally can be produced.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0016] Anthocyanins are a group of pigments widely present in plants and are known to exhibit red, blue, and purple flower colors. Depending on the number of hydroxy groups in the B ring of the anthocyanidin moiety, which is the aglycone, they are classified into three groups: pelargonidin, cyanidin, and delphinidin. The chromophore is the aglycone part, and pelargonidin-type anthocyanins exhibit orange, cyanidin-type anthocyanins exhibit red, and delphinidin-type anthocyanins exhibit purple to blue. In this specification, for example, "delphinidin-type anthocyanins" include those derivatives having delphinidin, malvidin, or petunidin as the skeleton, and preferably malvidin.

[0017] Delphinidin-type anthocyanins may coexist with substances such as flavones, flavonols, organic acid esters, and tannins, and may exhibit a bluish color by intermolecular interaction with them. This phenomenon is called copigmentation, and the substances that cause such a phenomenon are called copigments (auxiliary pigments). Copigmentation has not only a darkening effect that causes the expression of blue color, but also a concentration effect and an effect of improving color stability. The inventors of the present invention confirmed that the petals of roses exhibit blue color due to the copigmentation between delphinidin-type anthocyanins and flavone C-glycosides.

[0018] Flavone is a kind of organic compound and is a cyclic ketone of a flavan derivative. In plants, it mainly exists as a glycoside. Flavone, in a narrow sense, has the chemical formula C 15 H10 Refers to O2, a compound with a molecular weight of 222.24, and 2,3-didehydroflavan-4-one. Flavonoids in a broad sense are one category of flavonoids. Among flavonoids, those with a flavone structure as the basic skeleton and no hydroxyl group at the 3-position are classified as "flavones". In the specification of this application, "flavone C-glycoside" means a glycoside in which the aglycone is directly bonded to the anomeric carbon of an aldose among the glycosides of derivatives belonging to flavonoids in a broad sense, i.e., flavonoids. Examples of flavone C-glycosides include, but are not limited to, luteolin C-glycosides, tricetin C-glycosides, apigenin C-glycosides, and acacetin C-glycosides. Flavone C-glycosides also include glycosides of apigenin, luteolin, tricetin, and acacetin derivatives. In plants, two biosynthetic pathways of flavone C-glycosides are known (Figure 1). In Pathway 1, flavone 6-C-glucoside and flavone 8-C-glucoside are produced through the action of flavanone 2-hydroxylase (F2H), flavone C-glycosyltransferase (CGT), and dehydratase (FDH). On the other hand, in Pathway 2, flavone 6-C-glucoside is produced through the action of flavone synthase (FNS) and flavone C-glycosyltransferase (CGT). The flavone C-glycoside is preferably selected from the group consisting of flavone 6-C-glucoside, flavone 8-C-glucoside, and combinations thereof. For example, apigenin 6-C-glucoside (isovitexin), apigenin 8-C-glucoside (vitexin), luteolin 6-C-glucoside (isoorientin), luteolin 8-C-glucoside (orientin), tricetin 6-C-glucoside, tricetin 8-C-glucoside, or derivatives thereof can be mentioned.

[0019] The accumulation of flavone C-glycosides in plant cells can be achieved by transforming a host plant with a vector containing essential genes in Pathway 1 (i.e., flavanone 2-hydroxylase (F2H) gene, flavone C-glycosyltransferase (CGT) gene, and dehydratase (FDH) gene) or their homologs, or a vector containing essential genes in Pathway 2 (i.e., flavone synthase (FNS) gene, and flavone C-glycosyltransferase (CGT) gene) or their homologs.

[0020] The F2H gene, which is an essential gene in Pathway 1, or its homolog is not particularly limited as to its origin as long as it has the desired function, but is preferably the F2H gene derived from licorice or its homolog, and is the following polynucleotide: (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12; (b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (a); (c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 13; (d) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 13, and having the same activity as the protein encoded by the polynucleotide described in (c); (e) A polynucleotide encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 13, and having the same activity as the protein encoded by the polynucleotide described in (c), and is selected from the group consisting of.

[0021] The flavone CGT gene or its homolog, which is an essential gene in Pathway 1, is not particularly limited with respect to its origin as long as it has the desired function, but is preferably a codon usage-modified flavone CGT gene or its homolog derived from buckwheat, and is the following polynucleotide: (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 16; (b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 16, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (a); (c) A polynucleotide that encodes a protein consisting of the amino acid sequence of SEQ ID NO: 17; (d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 17, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (c); (e) A polynucleotide that has an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 17, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (c), and is selected from the group consisting of:

[0022] The FDH gene or its homolog, which is an essential gene in Pathway 1, is not particularly limited with respect to its origin as long as it has the desired function, but is preferably an FDH gene or its homolog derived from Miyakogusa, and is the following polynucleotide: (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14; (b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 14, and that encodes a protein having the same activity as the protein encoded by the polynucleotide described in (a); (c) A polynucleotide that encodes a protein consisting of the amino acid sequence of SEQ ID NO: 15; (d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 15, and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (c); (e) A polynucleotide selected from the group consisting of a polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 15 and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (c).

[0023] The FNS gene or its homolog, which is an essential gene in Pathway 2, is not particularly limited with respect to its origin as long as it has the desired function, but is preferably the FNS gene derived from Torenia or its homolog. (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3; (b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (a). (c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4; (d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 4 and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (c). (e) A polynucleotide selected from the group consisting of a polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4 and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (c).

[0024] The flavone CGT gene or its homolog, which is an essential gene in Pathway 2, is not particularly limited with respect to its origin as long as it has the desired function, but is preferably a flavone CGT gene or its homolog derived from Gentiana or Wasabi, (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 22; (b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 22, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (2-a); (c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23; (d) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23, and having the same activity as the protein encoded by the polynucleotide described in (2-c); (e) A polynucleotide encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23, and having the same activity as the protein encoded by the polynucleotide described in (c), and is selected from the group consisting of.

[0025] It is preferable that the essential gene flavone CGT gene or its homolog in Pathway 2 has added thereto a 5'-untranslated region (5'-UTR) (SEQ ID NO: 11) derived from the Arabidopsis thaliana alcohol dehydrogenase (ADH) gene.

[0026] The inventors of the present application have found that a combination with swertisin, which is a flavone C-glycoside in which the hydroxyl group at the 7-position is methylated, becomes bluer, and a flavone 7-O-methyltransferase gene that transfers a methyl group to the hydroxyl group at the 7-position of the flavone C-glycoside obtained by Route 1 or 2 has already been successfully obtained from Commelina communis var. hortensis, which is known as a variety (cultivar) of Commelina communis (Patent Document 3).

[0027] The flavone 7-O-methyltransferase (CcFn-7OMT) gene derived from Commelina communis or its homolog has the following (a) to (e): (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1; (b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and encodes a protein having an activity of transferring a methyl group to the hydroxyl group at the 7-position of a flavone C-glycoside; (c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2; (d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2 and encodes a protein having an activity of transferring a methyl group to the hydroxyl group at the 7-position of a flavone C-glycoside; and (e) A polynucleotide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and encodes a protein having an activity of transferring a methyl group to the hydroxyl group at the 7-position of a flavone C-glycoside, and is selected from the group consisting of these.

[0028] Surprisingly, the inventors of the present application have now found that, among flavone C-glycosides, a combination with envinin, which is a flavone C-glycoside in which not only the 7-position hydroxyl group but also the 4'-position hydroxyl group is methylated, turns bluer. They have successfully obtained a novel flavone 4'-O-methyltransferase gene that transfers a methyl group to the 4'-position hydroxyl group of the flavone C-glycoside obtained by Route 1 or 2 from Solanum lycopersicum.

[0029] The Solanum lycopersicum-derived flavone 4'-O-methyltransferase (IjFn-4'OMT) gene or its homolog has the following (A) to (E): (A) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO: 21; (B) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO: 21, and that encodes a protein having an activity of transferring a methyl group to the 4'-position hydroxyl group of a flavone C-glycoside; (C) A polynucleotide that encodes a protein consisting of the amino acid sequence of SEQ ID NO: 20; (D) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 20, and that encodes a protein having an activity of transferring a methyl group to the 4'-position hydroxyl group of a flavone C-glycoside; and (E) A polynucleotide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 20, and that encodes a protein having an activity of transferring a methyl group to the 4'-position hydroxyl group of a flavone C-glycoside, and is selected from the group consisting of these.

[0030] The accumulation of delphinidin-type anthocyanins in plant cells can be achieved by incorporating a flavonoid F3’5’ hydroxylase (F3’5’H) gene or its homolog, and a methyltransferase (MT) gene or its homolog into a host plant (Patent Document 2). Therefore, in addition to the essential genes in Pathway 1 or their homologs, or the essential genes in Pathway 2 or their homologs, by transforming a host plant with a vector further containing an F3’5’H gene or its homolog, and an MT gene or its homolog, delphinidin-type anthocyanins and flavone C-glycosides can coexist in the cells of the host plant.

[0031] The F3’5’H gene or its homolog is not particularly limited as to its origin as long as it has the desired function, but is preferably a F3’5’H gene or its homolog derived from Campanula, (a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7; (b) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 7, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (a); (c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8; (d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 8, and having the same activity as the protein encoded by the polynucleotide described in (c); (e) a polynucleotide encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8, and having the same activity as the protein encoded by the polynucleotide described in (c), and is selected from the group consisting of.

[0032] The MT gene or its homolog is not particularly limited as to its origin as long as it has the desired function, but is preferably a MT gene derived from Trinia or its homolog. (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9; (b) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9 and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (a); (c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10; (d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 10 and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (c); (e) A polynucleotide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (c), and is selected from the group consisting of.

[0033] As used herein, the term "polynucleotide" means DNA or RNA. As used herein, the term "stringent conditions" refers to conditions that enable selective and detectable specific binding between a polynucleotide or oligonucleotide and genomic DNA. Stringent conditions are defined by an appropriate combination of salt concentration, organic solvent (e.g., formamide), temperature, and other known conditions. That is, stringency increases by reducing the salt concentration, increasing the organic solvent concentration, or raising the hybridization temperature. Furthermore, the washing conditions after hybridization also affect stringency. These washing conditions are also defined by salt concentration and temperature, and the stringency of washing increases by decreasing the salt concentration and raising the temperature. Therefore, the term "stringent conditions" means conditions under which specific hybridization occurs only between nucleotide sequences having high identity such that the degree of "identity" between each base sequence is, for example, about 80% or more, preferably about 90% or more, more preferably about 95% or more, still more preferably 97% or more, and most preferably 98% or more on average over the whole. Examples of "stringent conditions" include conditions such as a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, at a temperature of 60°C to 68°C and a pH of 6 to 8. Specific examples include performing hybridization under the conditions of 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 1% SDS, 5×Denhardt's solution, 50% formamide, and 42°C, and performing washing under the conditions of 0.1×SSC (15 mM NaCl, 1.5 mM trisodium citrate), 0.1% SDS, and 55°C.

[0034] Hybridization can be carried out according to methods known in the art, such as the methods described in Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987), or methods analogous thereto. When using a commercially available library, it can be carried out according to the methods described in the attached instruction manual. The genes selected by such hybridization may be of natural origin, for example, of plant origin or other than plant origin. Also, the genes selected by hybridization may be cDNA, genomic DNA, or chemically synthesized DNA.

[0035] The above-mentioned "amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added" means, for example, an amino acid sequence in which any number of 1 to 20, preferably 1 to 5, more preferably 1 to 3 amino acids are deleted, substituted, inserted, and / or added. Site-directed mutagenesis, which is one of the genetic engineering techniques, is useful because it can introduce specific mutations at specific positions, and can be carried out according to the methods described in Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, etc. By expressing this mutant DNA using an appropriate expression system, a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added can be obtained. In addition, polynucleotides can be obtained by methods known to those skilled in the art, for example, by chemical synthesis using the phosphoramidite method, etc., or by nucleic acid amplification methods using plant nucleic acid samples as templates and primers designed based on the nucleotide sequence of the target gene.

[0036] As used herein, the term "identity" refers to the amount (number) of residues or bases that can be determined to be identical in the pairing relationship between each amino acid residue or each base constituting the strands between two strands in a polypeptide sequence (or amino acid sequence) or a polynucleotide sequence (or nucleotide sequence), and means the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences, and "identity" can be easily calculated. Many methods for measuring the identity between two polynucleotide sequences or polypeptide sequences are known, and the term "identity" is well-known to those skilled in the art (see, for example, Lesk, A.M. (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, D.W. (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin, A.M. & Grifin, H.G. (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (1987); Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991), etc.).

[0037] Also, the numerical value of "identity" described in this specification may be a numerical value calculated using an identity search program known to those skilled in the art, unless otherwise specified. Preferably, it is a numerical value calculated using the ClustalW program of the MacVector application (version 9.5, Oxford Molecular Ltd., Oxford, England). In the present invention, the degree of "identity" between each amino acid sequence is, for example, about 90% or more, preferably about 95% or more, more preferably about 97% or more, and most preferably about 98% or more.

[0038] The polynucleotide (nucleic acid, gene) of the present invention "encodes" the protein of interest. Here, "encodes" means expressing the protein of interest in a state having its activity. Also, "encodes" includes both the meaning of encoding the protein of interest as a continuous structural sequence (exon) and encoding it via an intervening sequence (intron).

[0039] A gene having a native nucleotide sequence can be obtained, for example, by analysis using a DNA sequencer. Also, DNA encoding an enzyme having a modified amino acid sequence can be synthesized based on DNA having a native nucleotide sequence using common site-directed mutagenesis or PCR methods. For example, a DNA fragment to be modified is obtained by restriction enzyme treatment of native cDNA or genomic DNA, and using this as a template, site-directed mutagenesis or PCR is performed using a primer into which a desired mutation has been introduced to obtain a desired modified DNA fragment. Then, this DNA fragment into which the mutation has been introduced may be ligated to a DNA fragment encoding other parts of the target enzyme. Alternatively, to obtain DNA encoding an enzyme consisting of a shortened amino acid sequence, for example, DNA encoding an amino acid sequence longer than the target amino acid sequence, such as DNA encoding the full-length amino acid sequence, is cleaved with a desired restriction enzyme. If the resulting DNA fragment does not encode the entire target amino acid sequence, a DNA fragment consisting of the missing sequence may be synthesized and ligated.

[0040] In addition, by expressing the obtained polynucleotide using gene expression systems in Escherichia coli and yeast and measuring the enzyme activity, it can be confirmed that the obtained polynucleotide encodes a protein having the desired activity.

[0041] The present invention also relates to a (recombinant) vector containing the above polynucleotide, particularly an expression vector, and further to a plant transformed by the vector.

[0042] In addition, the vectors of the present invention contain expression control regions, such as promoters, terminators, origins of replication, etc., depending on the type of host plant into which they are introduced. Examples of promoters for constitutively expressing polynucleotides in plant cells include the 35S promoter of cauliflower mosaic virus, the El235S promoter in which two enhancer regions of the 35S promoter are linked, the rd29A gene promoter, the rbcS promoter, the mac-1 promoter, etc. Also, for tissue-specific gene expression, a promoter of a gene specifically expressed in that tissue can be used.

[0043] The production of the vector can be carried out according to a conventional method using restriction enzymes, ligases, etc. Also, the transformation of host plants with the expression vector can be carried out according to a conventional method.

[0044] Under the current state of the art, techniques for introducing a polynucleotide into a plant and constitutively or tissue-specifically expressing the polynucleotide can be utilized. The introduction of DNA into plants can be carried out by methods known to those skilled in the art, such as the Agrobacterium method, the binary vector method, the electroporation method, the PEG method, the particle gun method, etc.

[0045] In the present invention, the plants that can be used as hosts are not particularly limited, but plants of the genus Rosa in the Rosaceae family, the genus Petunia in the Solanaceae family, the genus Chrysanthemum in the Asteraceae family, the genus Dianthus in the Caryophyllaceae family (such as carnation), and the genus Lily in the Liliaceae family can be used. Particularly preferably, it is a cultivated rose (scientific name: Rosa hybrida) of the genus Rosa in the Rosaceae family. The term "rose plant" used in this specification refers to a cultivated rose (scientific name: Rosa hybrida) of the genus Rosa in the taxonomic position. Roses are mainly classified into Hybrid Tea type, Floribunda type, Polyantha type, etc. according to the tree form and flower size. However, the main pigments (anthocyanins) contained in the petals are only two types, cyanidin type and pelargonidin type. In the present invention, the type of rose plant used as a host is not particularly limited, and it can be preferably used for these varieties and strains. For example, rose varieties that can be used as hosts include Ocean Song, Noblesse, Rita Perfumer, Cool Water, Fame, Topless, Peach Avalanche, etc.

[0046] According to the present invention, a transformed plant with modified flower color in which delphinidin-type anthocyanin and flavone C-glycoside coexist intracellularly, preferably a plant of the genus Rosa in the Rosaceae family, the genus Petunia in the Solanaceae family, the genus Chrysanthemum in the Asteraceae family, or the genus Dianthus in the Caryophyllaceae family (such as carnation), particularly preferably a rose plant, can be obtained. In particular, when the obtained transformed plant is a rose plant, it shows a flower color with a hue angle of 339.7° to 270.0° in the RHS Color Chart and / or CIEL * a * b * color system.

[0047] Furthermore, the present invention also relates to cut flowers of the transformed plant obtained above or its self-propagated or cross-propagated progeny, their vegetative propagules, parts of the plant body, tissues, or cells, or processed products (particularly cut flower processed products) made from cut flowers. Here, the cut flower processed products include, but are not limited to, pressed flowers, preserved flowers, dried flowers, resin-sealed products, etc. using the cut flowers.

[0048] Hereinafter, the present invention will be specifically described by way of examples.

Example

[0049] [Example 1: Simulation of the Copigmentation Effect of Flavonoid C-Glycosides on Anthocyanin (Malvin)] To simulate the copigmentation effect of flavonoid C-glycosides on anthocyanin (malvin), malvin and flavonoid C-glycosides were prepared. Malvin (malvidin 3,5-diglucoside) and flavonoid C-glycosides (isovitexin (apigenin 6-C-glucoside), isoorientin (luteolin 6-C-glucoside), swertisin (gentiwanin 6-C-glucoside)) used in this experiment were purchased from Nacalai Tesque, Inc. In addition, embigenin (7,4'-dimethoxyisovitexin) was artificially prepared by organic synthesis from isovitexin. For malvin thus obtained, each flavonoid C-glycoside (isovitexin, isoorientin, swertisin, embigenin) was added at a molar concentration ratio of 10 equivalents in a buffer solution at pH 5.0, and the absorption spectrum was measured. The concentration of malvin was 0.5 mM. Due to the addition of flavonoid C-glycosides, the absorbance of the malvin aqueous solution increased, and the absorption maximum (λmax) shifted to the long wavelength side (blue direction). Among them, when embigenin was added, the absorption maximum shifted the most to the long wavelength side, indicating that embigenin shows the highest copigmentation effect on malvin.

[0050]

Table 1

[0051] [Example 2: Detection of Embigenin in Mushroom] It has already been reported that enbinin (engenin 2”-rhamnoside) is contained in the petals of Iris japonica (https: / / www.jstage.jst.go.jp / article / yakushi1947 / 93 / 12 / 93_12_1655 / _pdf). Therefore, pigment analysis was carried out to confirm whether engenin, a precursor of enbinin, could be detected from the petals and leaves of Iris japonica. After freezing the petal and leaf samples of Iris japonica, they were dried overnight using a vacuum freeze dryer, VirTis sentry2.0 (SP SCIENTIFIC), and then gently crushed with a spatula. To this, 4 mL of 50% acetonitrile containing 0.1% trifluoroacetic acid (TFA) was added per 10 mg of dry weight, and after treatment under ultrasonic waves for 20 minutes, centrifugation (3,600 rpm, 4°C, 10 minutes) was performed, and the supernatant was collected. The obtained supernatant was filtered through a 0.45 μm filter (Cosmonice filter (aqueous system), 0.45 μm, 13 mm). Of this, 200 μL was dried, β-glucosidase and naringinase were added, and after treatment at 30°C overnight, 200 μL of 90% acetonitrile containing 0.1% TFA was added to stop the reaction. Next, after treatment under ultrasonic waves for 2 minutes, centrifugation (15,000 rpm, 4°C, 5 minutes) was performed, and the supernatant obtained by filtering through a 0.45 μm filter (Milex-LH 0.45 μm, Millipore) was subjected to high performance liquid chromatography. The analysis conditions are as follows. <Analysis conditions> Apparatus: Prominence HPLC system (Shimadzu Corporation) Detector: SPD-M20A (250 - 450 nm) Column: Shim-pack FC-ODS 150 x 4.6 mm, 3 μm (Shimadzu GLC Corporation) Eluent A: 0.1% TFA aqueous solution Eluent B: 90% acetonitrile containing 0.1% TFA Flow rate: 0.6 mL / min

[0052] For dissolution, Solution A (0.1% aqueous TFA solution) and Solution B (90% aqueous acetonitrile solution containing 0.1% TFA) were used. A linear concentration gradient for 20 minutes from a 9:1 mixture of the two to an 8:2 mixture, a linear concentration gradient for 15 minutes from an 8:2 mixture of the two to a 2:8 mixture, a linear concentration gradient for 5 minutes from a 2:8 mixture of the two to a 0:10 mixture, and subsequent elution with a 0:10 mixture for 1 minute were performed. The flow rate was 0.6 mL / min. As a result of analysis by the above method, emigenin was detected only from saffron petals (6.6 mg per 1 g of dry weight).

[0053]

Table 2

[0054] [Example 3: Acquisition of candidate genes for genes encoding proteins having the activity of transferring a methyl group to the 4'-hydroxyl group of flavone C-glycoside] <Isolation of total RNA> Total RNA was isolated from saffron petals and leaves using the RNeasy Plant Mini Kit (QIAGEN) according to the method recommended by the manufacturer.

[0055] <Analysis of the expression level of saffron-derived cDNA> Using the SureSelect Strand-Specific RNA Library Preparation Kit (Agilent Technologies) from the total RNA prepared above, a library for the NextSeq 500 next-generation sequencer was prepared according to the steps recommended by the manufacturer. After determining the nucleotide sequence of the prepared library using the NextSeq 500 (Illumina), the obtained reads were inspected. Next, the reads of all samples were mixed and assembled using Trinity v2.8.5 to obtain contig sequences. Furthermore, for the obtained contig sequences, the paired reads of each sample were mapped using RSEM 1.3.0, and the expression level was determined by calculating the FPKM value.

[0056] <Estimation of gene function> For the contig sequences obtained above, BLAST searches against NCBI NR and Araport11 were performed for functional annotation (estimation of gene function).

[0057] <Obtaining full-length cDNA of candidate genes> Among the obtained contig sequences, a search was conducted using "Methyltransferase" as a keyword, and 612 candidate genes were obtained. Furthermore, a BLAST search was performed using the sequence of the flavone 7-O-methyltransferase gene from *Ranunculus asiaticus*, and an additional 25 candidate genes were obtained. Among these, contig sequences with high expression levels and mainly expressed in petals were selected, and the candidates were narrowed down to 9. By creating a phylogenetic tree by adding 37 previously reported methyltransferase genes such as the flavonoid 7-O-methyltransferase gene (F1-OMT, Non-Patent Document 10) of barley and the isoflavone 7-O-methyltransferase gene (MtIOMT2, Non-Patent Document 11) of castor bean, the candidate gene DN144 was selected. Primers were designed based on the sequence of the full-length cDNA obtained by assembly, and full-length cDNA clones were obtained by the following method.

[0058] Using the total RNA of *R. asiaticus* petals isolated above as a template, cDNA was synthesized according to the method recommended by the manufacturer using the SuperScript First-Strand Synthesis System for RT-PCR (ThermoFisher SCIENTIFIC). Using the obtained *R. asiaticus* petal cDNA as a template, a PCR reaction was performed in a reaction volume of 50 μL according to the method recommended by the manufacturer using PrimeSTAR Max (Takara Bio Inc.) (cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 15 seconds were repeated 30 times and then held at 4°C). The nucleotide sequence of DN144 thus obtained was determined using a DNA sequencer 3500 Genetic Analyzer (Applied Biosystems).

[0059] When the amino acid sequence homology between DN144 and known methyltransferase genes was examined, the homology with flavonoid 7-O-methyltransferase from Hordeum vulgare was 33%, the homology with isoflavone 7-O-methyltransferase (MtIOMT1) from Medicago tructula was 37%, the homology with isoflavone / isoflavone 7-O-methyltransferase (MtIOMT2) from Medicago tructula was 36%, the homology with isoflavone O-methyltransferase from Medicago sativa was 37%, and the homology with daidzein 7-O-methyltransferase from Glycyrrhiza echinata was 36%. Therefore, DN144 was clearly distinguishable from known methyltransferase genes.

[0060] [Example 4: Measurement of Enzyme Activity in Escherichia coli of a Protein Having Activity to Transfer a Methyl Group to the 4'-Hydroxyl Group of Flavone C-Glycoside] <Preparation of Escherichia coli Expression Vector> DN144 was used as a protein candidate having activity to transfer a methyl group to the 4'-hydroxyl group of flavone C-glycoside, and using pET15b (Novagen), an Escherichia coli expression vector pSPB7942 containing the full length of DN144 was prepared according to the method recommended by the manufacturer.

[0061] <Expression of Methyltransferase in Escherichia coli> pSPB7942 was introduced into Escherichia coli strain BL21(DE3) (New England Biolabs Japan Inc) according to the method recommended by the manufacturer to obtain transformed Escherichia coli. This Escherichia coli was cultured using the Overnight Express Autoinduction System1 (Novagen) according to the method recommended by the manufacturer. With 2 mL of the prepared culture solution, the transformed Escherichia coli was cultured at 37°C until the OD600 value reached 0.5 (about 4 hours). Using this Escherichia coli solution as a preculture solution, it was added to 50 mL of the culture solution and subjected to main culture at 16°C for two nights. The Escherichia coli solution after two nights of main culture was centrifuged (3000 rpm, 4°C, 15 minutes), and the collected cells were suspended in sonic buffer (composition; KPB (pH 7.5): 40 mM, dithiothreitol: 1 mM, amidinophenylmethanesulfonyl fluoride hydrochloride: 50 μM, ethylenediaminetetraacetic acid: 500 μM, MgCl2: 2 mM, S-adenosylmethionine (SAM): 1 μM). 5 mL of sonic buffer was added per 1 g of Escherichia coli. After the suspended Escherichia coli was disrupted by sonication, it was centrifuged (15000 rpm, 4°C, 10 minutes) to recover the supernatant. The supernatant was used as a protein solution roughly extracted from Escherichia coli expressing DN144. For centrifugation, TOMY MX-307 (rotor: AR015-24) was used (Tomy Seiko Co., Ltd.).

[0062] <Enzyme Activity Measurement> For the activity measurement, apigenin, apigenin 7-glucoside, isovitexin, isovitexin 2”-rhamnoside, saponarin (isovitexin 4’-glucoside), swertisin, luteolin, luteolin 7-glucoside, isoorientin, swertiajaponin (isoorientin 7-methyl ether), delphinidin 3-glucoside, delphinidin 3,5-diglucoside, and malvidin 3,5-diglucoside were used as substrates. A mixed solution was prepared by mixing 8 μL of 1 mM various substrates (dissolved in a 50% aqueous acetonitrile solution containing 0.1% TFA), 20 μL of 10 mM SAM, 10 μL of 1 M KPB (pH 7.5), and 20 μL of 10 mM MgCl2 to make a total volume of 58 μL, and the mixture was held at 30 °C for 10 minutes. Then, 42 μL of a protein solution, which was crudely extracted from Escherichia coli expressing DN144, was added to carry out the enzyme reaction (at 30 °C for 30 minutes). Thereafter, 100 μL of a stop buffer (90% aqueous acetonitrile solution containing 0.1% TFA) was added to stop the enzyme reaction, and the enzyme reaction solution was analyzed by high-performance liquid chromatography (LC-2030C, Shimadzu Corporation). The detector used was the Shimadzu PDA SPD-M20A, and detection was performed at 330 nm. The column used was Shim-Pack FC-ODS 150 mm * 4.6 mm (Shimadzu GLC Corporation). For elution, solution A (0.1% aqueous TFA solution) and solution B (90% aqueous acetonitrile solution containing 0.1% TFA) were used. A linear concentration gradient was carried out for 20 minutes from a 9:1 mixture of the two to an 8:2 mixture, a linear concentration gradient was carried out for 15 minutes from an 8:2 mixture of the two to a 2:8 mixture, a linear concentration gradient was carried out for 5 minutes from a 2:8 mixture of the two to a 0:10 mixture, and then elution was carried out with a 0:10 mixture for 1 minute. The flow rate was set at 0.6 mL / min. As a control, the same experiment was carried out using a protein solution crudely extracted from Escherichia coli introduced with a pET15b vector without inserting an insert. As a result, a generated compound was detected from the enzyme reaction solution obtained by reacting the protein solution crudely extracted from Escherichia coli expressing DN144 with each of the 10 flavones. In particular, high activity was shown against isovitexin 2”-rhamnoside, isoorientin, and swertiajaponin, and 75 - 100% was converted into the generated compound.Furthermore, using the standard product, it was confirmed that when isovitexin was used as the substrate, it was converted to isosytiside, and when swertisin was used as the substrate, it was converted to enbigenin (see Table 3, Figures 2 and 3). On the other hand, in the reaction with the three anthocyanins, no peaks other than the substrate were detected in the enzyme reaction solution. When the enzyme reaction was carried out using anthocyanin as the substrate, the enzyme reaction conditions were 30 °C for 15 minutes, and a 90% acetonitrile aqueous solution containing 0.1% TFA and 0.24 N hydrochloric acid was used as the stop buffer. When analyzing the enzyme reaction solution by high-performance liquid chromatography (Prominence (Shimadzu Corporation)), the detector was the Shimadzu PDA SPD-M20A, and detection was carried out at 520 nm. The column used was Shodex RSpak DE-413L (Showa Denko K.K.). For elution, Solution A (0.5% TFA aqueous solution) and Solution B (50% acetonitrile aqueous solution containing 0.5% TFA) were used. A 15-minute linear concentration gradient from an 8:2 mixture of the two to a 0:10 mixture and subsequent elution with a 0:10 mixture for 5 minutes were performed. The flow rate was 0.6 mL / min.

[0063]

Table 3

[0064] From these results, it became clear that DN144 exhibits the activity of specifically transferring a methyl group to the 4'-hydroxyl group of flavone C-glycoside, and it was shown that DN144 is a gene encoding a protein having the activity of transferring a methyl group to the 4'-hydroxyl group of flavone C-glycoside. In addition, DN144 showed strong methyltransferase activity against isovitexin 2”-rhamnoside, isoorientin, and swertiajaponin. From the above results, this gene was identified as a gene encoding a protein having the activity of transferring a methyl group to the 4'-hydroxyl group of flavone C-glycoside and named IjFn-4’OMT.

[0065] [Example 5: Introduction of campanula-derived F3’5’H gene, torenia-derived MT gene, glycyrrhiza-derived F2H gene, buckwheat-derived codon Usage-modified flavone CGT gene, myagrum-derived FDH gene, oenothera-derived F7OMT gene, and dahlia-derived F4’OMT gene into the rose cultivar “Ocean Song” (Path 1)] pSPB7964 uses pBINPLUS as the basic backbone and contains the following seven expression cassettes. (1) El235S promoter, campanula-derived F3’5’H full-length cDNA (SEQ ID NO: 7), and D8 terminator (2) El235S promoter, torenia-derived MT full-length cDNA (SEQ ID NO: 9), and arabidopsis-derived HSP terminator (3) El235S promoter, glycyrrhiza-derived F2H full-length cDNA (SEQ ID NO: 12), and perilla-derived AT terminator (4) El235S promoter, buckwheat-derived codon Usage-modified flavone CGT full-length cDNA (SEQ ID NO: 16) (5’-UTR (SEQ ID NO: 18) derived from the arabidopsis HSPRO gene added to the 5’ side), and arabidopsis-derived HSP terminator (5) 35S promoter, myagrum-derived FDH full-length cDNA (SEQ ID NO: 14), and arabidopsis-derived HSP terminator (6) El235S promoter, oenothera-derived F7OMT full-length cDNA (SEQ ID NO: 1), and arabidopsis-derived HSP terminator (7) El235S promoter, dahlia-derived F4’OMT full-length cDNA (SEQ ID NO: 19), and arabidopsis-derived HSP terminator

[0066] In plants, this plasmid constitutively expresses the campanula F3',5'H gene, torenia MT gene, glycyrrhiza F2H gene, buckwheat codon Usage-modified flavone CGT gene, myagrum FDH gene, oenothera F7OMT gene, and dahlia F4’OMT gene. The thus-prepared pSPB7964 was introduced into the blue rose cultivar “Ocean Song”.

[0067] [Example 6: (Path 1) Introduction of campanula-derived F3'5'H gene, torenia-derived MT gene, glycyrrhiza-derived F2H gene, buckwheat-derived codon Usage-modified flavone CGT gene, myosoton-derived FDH gene, coreopsis-derived F7OMT gene, and chaga-derived codon Usage-modified F4'OMT gene into rose cultivar "Ocean Song"] pSPB7965 is based on pBINPLUS as the basic backbone and contains the following seven expression cassettes. (1) El235S promoter, campanula-derived F3'5'H full-length cDNA (SEQ ID NO: 7), and D8 terminator (2) El235S promoter, torenia-derived MT full-length cDNA (SEQ ID NO: 9), and arabidopsis-derived HSP terminator (3) El235S promoter, glycyrrhiza-derived F2H full-length cDNA (SEQ ID NO: 12), and perilla-derived AT terminator (4) El235S promoter, buckwheat-derived codon Usage-modified flavone CGT full-length cDNA (SEQ ID NO: 16) (with a 5'-UTR (SEQ ID NO: 18) derived from the arabidopsis HSPRO gene added to the 5'-end), and arabidopsis-derived HSP terminator (5) 35S promoter, myosoton-derived FDH full-length cDNA (SEQ ID NO: 14), and arabidopsis-derived HSP terminator (6) El235S promoter, coreopsis-derived F7OMT full-length cDNA (SEQ ID NO: 1), and arabidopsis-derived HSP terminator (7) El235S promoter, chaga-derived codon Usage-modified F4'OMT full-length cDNA (SEQ ID NO: 21), and arabidopsis-derived HSP terminator

[0068] In plants, this plasmid constitutively expresses the campanula F3',5'H gene, torenia MT gene, glycyrrhiza F2H gene, buckwheat codon Usage-modified flavone CGT gene, myosoton FDH gene, coreopsis F7OMT gene, and chaga codon Usage-modified F4'OMT gene. The thus-prepared pSPB7965 was introduced into the blue rose cultivar "Ocean Song".

[0069] [Example 7: (Route 2) Introduction of the campanula-derived F3’5’H gene, torenia-derived MT gene, torenia-derived FNS gene, gentian-derived flavone CGT gene, chrysanthemum indicum-derived F7OMT gene, and scutellaria-derived F4’OMT gene into the rose cultivar "Ocean Song"] pSPB7960 has pBINPLUS as its basic backbone and contains the following six expression cassettes. (1) El235S promoter, campanula-derived F3’5’H full-length cDNA (SEQ ID NO: 7), and D8 terminator (2) El235S promoter, torenia-derived MT full-length cDNA (SEQ ID NO: 9), and Arabidopsis thaliana-derived HSP terminator (3) El235S promoter, torenia-derived FNS full-length cDNA (SEQ ID NO: 3), and D8 terminator (4) El235S promoter, gentian-derived flavone CGT full-length cDNA (SEQ ID NO: 5) (with a 5’-UTR (SEQ ID NO: 11) derived from the Arabidopsis thaliana alcohol dehydrogenase (ADH) gene added to the 5’-side), and Arabidopsis thaliana-derived HSP terminator (5) El235S promoter, chrysanthemum indicum-derived F7OMT full-length cDNA (SEQ ID NO: 1), and Arabidopsis thaliana-derived HSP terminator (6) El235S promoter, scutellaria-derived F4’OMT full-length cDNA (SEQ ID NO: 19), and Arabidopsis thaliana-derived HSP terminator

[0070] In plants, this plasmid constitutively expresses the campanula F3',5'H gene, torenia MT gene, torenia FNS gene, gentian flavone CGT gene, chrysanthemum indicum F7OMT gene, and scutellaria F4’OMT gene. The thus-prepared pSPB7960 was introduced into the blue rose cultivar "Ocean Song".

[0071] [Example 8: Introduction of the campanula-derived F3’5’H gene, torenia-derived MT gene, torenia-derived FNS gene, gentian-derived flavone CGT gene, greater plantain-derived F7OMT gene, and codon Usage-modified F4’OMT gene from Solanum lycopersicum into the rose cultivar “Ocean Song” (Pathway 2)] pSPB7961 uses pBINPLUS as the basic backbone and contains the following six expression cassettes. (1) El235S promoter, campanula-derived F3’5’H full-length cDNA (SEQ ID NO: 7), and D8 terminator (2) El235S promoter, torenia-derived MT full-length cDNA (SEQ ID NO: 9), and Arabidopsis thaliana-derived HSP terminator (3) El235S promoter, torenia-derived FNS full-length cDNA (SEQ ID NO: 3), and D8 terminator (4) El235S promoter, gentian-derived flavone CGT full-length cDNA (SEQ ID NO: 5) (with a 5’-UTR (SEQ ID NO: 11) derived from the Arabidopsis thaliana alcohol dehydrogenase (ADH) gene added to the 5’ side) and Arabidopsis thaliana-derived HSP terminator (5) El235S promoter, greater plantain-derived F7OMT full-length cDNA (SEQ ID NO: 1), and Arabidopsis thaliana-derived HSP terminator (6) El235S promoter, codon Usage-modified F4’OMT full-length cDNA from Solanum lycopersicum (SEQ ID NO: 21), and Arabidopsis thaliana-derived HSP terminator

[0072] In plants, this plasmid constitutively expresses the campanula F3',5'H gene, the torenia MT gene, the torenia FNS gene, the gentian flavone CGT gene, the greater plantain F7OMT gene, and the codon Usage-modified F4’OMT gene from Solanum lycopersicum. The thus-prepared pSPB7961 was introduced into the blue rose cultivar “Ocean Song”.

[0073] [Example 9: Expression 1 of a gene encoding a protein having the activity of transferring a methyl group to the 4’-hydroxyl group of flavone in petunia] To confirm whether the IjFn-4’OMT gene of the present invention has the activity of transferring a methyl group to the 4'-hydroxyl group of flavone in plants, a binary vector pSPB7993 into which the IjFn-4’OMT gene was introduced was constructed (as shown in FIG. 5 above). This vector has pBINPLUS as the basic backbone and contains the following four expression cassettes. (1) El235S promoter, full-length cDNA of FNS from Torenia (SEQ ID NO: 3), and D8 terminator (2) El235S promoter, full-length cDNA of CGT from Gentiana (SEQ ID NO: 5) (with the 5'-UTR (SEQ ID NO: 11) derived from the Arabidopsis thaliana ADH gene added), and HSP terminator from Arabidopsis thaliana (3) El235S promoter, full-length cDNA of CcFn-7OMT from Coreopsis grandiflora (SEQ ID NO: 1), and HSP terminator from Arabidopsis thaliana (4) El235S promoter, full-length cDNA of IjFn-4’OMT from Solanum tuberosum (SEQ ID NO: 19), and HSP terminator from Arabidopsis thaliana

[0074] This binary vector constitutively expresses the FNS gene of Torenia, the CGT gene of Gentiana, the CcFn-7OMT gene of Coreopsis grandiflora, and the IjFn-4’OMT gene of Solanum tuberosum in plants. pSPB7993 prepared in this way was introduced into the petunia variety "Saffinia Bouquet Red", and a total of 7 transgenic individuals were obtained. As a result of pigment analysis of these, the accumulation of embigenin (apigenin 7,4'-dimethyl-6-C-glucoside) in which the 7, 4'-positions of flavone C-glycoside were methylated was confirmed in one transgenic individual, and the content rate of the 7,4'-dimethylated form with respect to the total amount of flavone C-glycoside was 8.4% (Table 4).

[0075]

Table 4

[0076] In this strain, in addition to these 7,4'-dimethylated forms, swertisin (apigenin 7-methyl-6-C-glucoside), a flavone C-glycoside, was detected. On the other hand, none of the flavone C-glycosides were detected in the host. From the above, it became clear that IjFn-4'OMT has the activity of transferring a methyl group to the 4'-position of flavones in plants. By using this gene, it becomes possible to produce 4'-methylated forms of flavone C-glycosides in plants.

[0077] [Example 10: Expression of the gene encoding a protein having the activity of transferring a methyl group to the 4'-hydroxyl group of flavone in petunia 2] To confirm whether the IjFn-4'OMT gene of the present invention has the activity of transferring a methyl group to the 4'-hydroxyl group of flavones in plants, a binary vector pSPB7994 (bottom of Figure 5) into which the IjFn-4'OMT gene was introduced was constructed. This vector has pBINPLUS as the basic backbone and contains the following four expression cassettes. (1) El235S promoter, full-length cDNA of FNS derived from torenia (SEQ ID NO: 3), and D8 terminator (2) El235S promoter, full-length cDNA of CGT derived from wasabi (SEQ ID NO: 22) (with the 5'-UTR (SEQ ID NO: 11) derived from the Arabidopsis thaliana ADH gene added), and the HSP terminator derived from Arabidopsis thaliana (3) El235S promoter, full-length cDNA of CcFn-7OMT derived from Coreopsis grandiflora (SEQ ID NO: 1), and the HSP terminator derived from Arabidopsis thaliana (4) El235S promoter, full-length cDNA of IjFn-4'OMT derived from Solanum tuberosum (SEQ ID NO: 19), and the HSP terminator derived from Arabidopsis thaliana

[0078] This binary vector constitutively expresses the FNS gene of Torenia, the CGT gene of Wasabi, the CcFn-7OMT gene of Eustoma, and the IjFn-4’OMT gene of Gentiana in plants. pSPB7994 prepared in this way was introduced into the petunia cultivar "Saffinia Bouquet Red", and a total of 8 transformants were obtained. As a result of pigment analysis of these, accumulation of embigenin (apigenin 7,4'-dimethyl-6-C-glucoside) in which the 7- and 4'-positions of flavone C-glycoside were methylated was confirmed in 4 transformants, and the content rate of the 7,4'-dimethylated form with respect to the total amount of flavone C-glycoside was up to 9.5% (average content rate 8.0%) (Table 5).

[0079]

Table 5

[0080] In this strain, in addition to these 7,4'-dimethylated forms, two types of flavone C-glycosides, swertisin (apigenin 7-methyl-6-C-glucoside) and swertiajaponin (luteolin 7-methyl-6-C-glucoside), were detected. On the other hand, none of the flavone C-glycosides were detected in the host. From the above, it became clear that IjFn-4’OMT has the activity of transferring a methyl group to the 4'-position of flavone in plants. By using this gene, it becomes possible to produce 4'-methylated forms of flavone C-glycosides in plants.

Claims

1. The following (A) to (E): (A) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO: 21; (B) A polynucleotide that hybridizes specifically under stringent conditions that hybridize only between a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO: 21 and nucleotide sequences having an overall average identity of 90% or more, and that encodes a protein having an activity of transferring a methyl group to the 4'-hydroxyl group of flavone C-glycoside; (C) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 20; (D) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 20, and that encodes a protein having an activity of transferring a methyl group to the 4'-hydroxyl group of flavone C-glycoside; and (E) A polynucleotide having an amino acid sequence having an identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 20, and that encodes a protein having an activity of transferring a methyl group to the 4'-hydroxyl group of flavone C-glycoside; A polynucleotide selected from the group consisting of.

2. The polynucleotide according to claim 1, which is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19 or SEQ ID NO:

21.

3. The polynucleotide according to claim 1, which is a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO:

20.

4. A protein encoded by the polynucleotide according to any one of claims 1 to 3.

5. A vector containing the polynucleotide according to any one of claims 1 to 3.

6. The vector according to claim 5, further comprising a flavone 7-O-methyltransferase (Fn-7OMT) gene.

7. The Fn-7OMT gene is (a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1; (b) A polynucleotide that hybridizes specifically under stringent conditions that hybridize only between a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and nucleotide sequences having an overall average identity of 90% or more, and that encodes a protein having an activity of transferring a methyl group to the 7-hydroxyl group of flavone C-glycoside; (c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2; (d) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2, and having an activity of transferring a methyl group to the 7-position hydroxyl group of flavone C-glycoside; and (e) A polynucleotide encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and having an activity of transferring a methyl group to the 7-position hydroxyl group of flavone C-glycoside; The vector according to claim 6, selected from the group consisting of.

8. The vector according to any one of claims 5 to 7, further comprising a flavone synthase (FNS) gene and a flavone C-glycosyltransferase (CGT) gene.

9. The FNS gene is (1-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3; (1-b) A polynucleotide that hybridizes specifically under stringent conditions that hybridize only between a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 and a nucleotide sequence having 90% or more identity on average as a whole, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (1-a); (1-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4; (1-d) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 4, and having the same activity as the protein encoded by the polynucleotide described in (1-c); (1-e) A polynucleotide encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4 and having the same activity as the protein encoded by the polynucleotide described in (1-c), selected from the group consisting of, and The flavone CGT gene is (2-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 22; A polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 5 or SEQ ID NO: 22, which hybridizes specifically under stringent conditions that hybridize only between base sequences having an identity of 90% or more on average over the whole, and which encodes a protein having the same activity as the protein encoded by the polynucleotide described in (2-a); A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23; In the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23, a polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added, and having the same activity as the protein encoded by the polynucleotide described in (2-c); The vector according to claim 8, selected from the group consisting of a polynucleotide encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 23, and having the same activity as the protein encoded by the polynucleotide described in (2-c).

10. The vector according to claim 8 or 9, wherein the vector further comprises a flavonoid F3'5'hydroxylase (F3'5'H) gene and a methyltransferase (MT) gene.

11. The F3'5'H gene is A polynucleotide consisting of the base sequence of SEQ ID NO: 7; A polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 7, which hybridizes specifically under stringent conditions that hybridize only between base sequences having an identity of 90% or more on average over the whole, and which encodes a protein having the same activity as the protein encoded by the polynucleotide described in (3-a); A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8; In the amino acid sequence of SEQ ID NO: 8, a polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added, and having the same activity as the protein encoded by the polynucleotide described in (3-c); A polynucleotide selected from the group consisting of a polynucleotide having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 8 and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (3-c), and wherein the MT gene is (4-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9; (4-b) A polynucleotide that hybridizes specifically under stringent conditions only between nucleotide sequences having an overall average identity of 90% or more with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (4-a); (4-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10; (4-d) A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 10, and encodes a protein having the same activity as the protein encoded by the polynucleotide described in (4-c); (4-e) A polynucleotide selected from the group consisting of a polynucleotide having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and encoding a protein having the same activity as the protein encoded by the polynucleotide described in (4-c), the vector according to claim 10.

12. The vector according to any one of claims 9 to 11, wherein a 5' untranslated region (5'-UTR) (SEQ ID NO: 11) derived from the Arabidopsis thaliana alcohol dehydrogenase (ADH) gene is added to the flavone CGT gene.

13. The vector according to any one of claims 5 to 7, further comprising a flavanone 2-hydroxylase (F2H) gene, a flavone C-glycosyltransferase (CGT) gene, and a dehydrogenase (FDH) gene.

14. The vector according to claim 13, further comprising a flavonoid F3'5'hydroxylase (F3'5'H) gene and a methyltransferase (MT) gene.

15. wherein the F2H gene is (5-a) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12; A polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 12, which hybridizes specifically under stringent conditions that hybridize only between base sequences having an identity of 90% or more on average over the whole, and which encodes a protein having the same activity as the protein encoded by the polynucleotide described in (5-a); A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 13; A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 13, and which encodes a protein having the same activity as the protein encoded by the polynucleotide described in (5-c); Selected from the group consisting of polynucleotides encoding a protein having an amino acid sequence having an identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 13 and having the same activity as the protein encoded by the polynucleotide described in (5-c); The flavone CGT gene is A polynucleotide consisting of the base sequence of SEQ ID NO: 16; A polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 16, which hybridizes specifically under stringent conditions that hybridize only between base sequences having an identity of 90% or more on average over the whole, and which encodes a protein having the same activity as the protein encoded by the polynucleotide described in (6-a); A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 17; A polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 17, and which encodes a protein having the same activity as the protein encoded by the polynucleotide described in (6-c); Selected from the group consisting of polynucleotides encoding a protein having an amino acid sequence having an identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 17 and having the same activity as the protein encoded by the polynucleotide described in (6-c); The FDH gene is A polynucleotide consisting of the base sequence of SEQ ID NO: 14; A polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 14, which hybridizes specifically under stringent conditions that hybridize only between base sequences having an identity of 90% or more on average as a whole, and which encodes a protein having the same activity as the protein encoded by the polynucleotide described in (7-a); A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 15; A polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 15, and having the same activity as the protein encoded by the polynucleotide described in (7-c); Selected from the group consisting of polynucleotides encoding a protein having an amino acid sequence having an identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 15 and having the same activity as the protein encoded by the polynucleotide described in (7-c), wherein the F3'5'H gene is A polynucleotide consisting of the base sequence of SEQ ID NO: 7; A polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 7, which hybridizes specifically under stringent conditions that hybridize only between base sequences having an identity of 90% or more on average as a whole, and which encodes a protein having the same activity as the protein encoded by the polynucleotide described in (8-a); A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8; A polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 8, and having the same activity as the protein encoded by the polynucleotide described in (8-c); Selected from the group consisting of polynucleotides encoding a protein having an amino acid sequence having an identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 8 and having the same activity as the protein encoded by the polynucleotide described in (8-c), and wherein the MT gene is A polynucleotide consisting of the base sequence of SEQ ID NO: 9; A polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 9, which hybridizes specifically under stringent conditions that hybridize only between base sequences having an identity of 90% or more on average over the whole, and which encodes a protein having the same activity as the protein encoded by the polynucleotide described in (9-a); A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10; A polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 10, and having the same activity as the protein encoded by the polynucleotide described in (9-c); The vector according to claim 14, selected from the group consisting of a polynucleotide encoding a protein having an amino acid sequence having an identity of 90% or more with respect to the amino acid sequence of SEQ ID NO: 10, and having the same activity as the protein encoded by the polynucleotide described in (9-c).

16. The vector according to claim 15, wherein an untranslated region (5'-UTR) (SEQ ID NO: 18) derived from the Arabidopsis thaliana HSPRO gene is added to the flavone CGT gene.

17. A transformed plant into which the polynucleotide according to any one of claims 1 to 3 has been introduced, or its self-propagated or cross-propagated progeny.

18. The transformed plant according to claim 17, or its self-propagated or cross-propagated progeny, wherein the plant is selected from rose, petunia, chrysanthemum, carnation or lily.

19. The transformed plant according to claim 18, or its self-propagated or cross-propagated progeny, wherein the plant is a rose.

20. A vegetative propagule, a part of a plant body, a tissue, or a cell of the transformed plant according to any one of claims 17 to 19, or its self-propagated or cross-propagated progeny.

21. A cut flower of the transformed plant according to any one of claims 17 to 19, or its self-propagated or cross-propagated progeny, or a processed product made from the cut flower.

22. A method for producing a transformed plant with modified flower color, comprising the step of co-existing delphinidin-type anthocyanin and flavone C-glycoside in a plant cell by introducing the vector according to any one of claims 7 to 16 into the plant cell, wherein the hydroxyl groups at the 7-position and 4'-position of the flavone C-glycoside are methylated.

23. The method according to claim 22, wherein the flavone C-glycoside is envinin.

24. The method according to claim 22 or 23, wherein the delphinidin-type anthocyanin is selected from the group consisting of malvidin 3,5-diglucoside, delphinidin 3,5-diglucoside, petunidin 3,5-diglucoside, acylated delphin, acylated malvin, and combinations thereof.

25. The method according to claim 22, wherein the plant is selected from rose, petunia, chrysanthemum, carnation or lily.

26. The method according to claim 25, wherein the plant is a rose.

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