C-glycosidase gene from buckwheat and its application

Abstract

The present invention provides a transformed plant with blue flower color or its self-propagated or hybridized offspring, or their vegetative propagation bodies, plant body parts, tissues or cells. The present invention introduces the C-glycosidase (CGT) gene or its homologous gene from buckwheat into the host plant, so that the delphinidin-type anthocyanin and the flavonoid mono-C-glycoside coexist in the cells of the plant.

Description

Technical Field

[0001] The present invention relates to a C-glycosidase (CGT) gene or a homologous gene thereof from buckwheat, and a method for producing a transformed plant having a blue flower color, comprising a step of using the gene to allow delphinidin-type anthocyanins (anthocyanins) and flavonoid mono-C-glycosides to coexist in plant cells. Background Art

[0002] Roses, chrysanthemums, carnations, etc. are important flowers in the world industry. In particular, roses are the most popular flower plants. There are records of them being cultivated since BC, and they have undergone artificial variety improvement over hundreds of years. However, due to the problem that there are no wild species with blue flowers among the closely related species that can be hybridized, it is difficult to produce rose varieties with blue flowers in the previous hybrid breeding and mutation breeding. The creation of new blue flowers has aroused new needs with the expansion of flower utilization occasions, and is related to the expansion of production or consumption. Therefore, attempts have been made to create roses with blue flowers through genetic engineering methods.

[0003] For example, it is known that flowers ranging from purple to blue are rich in delphinidin-type anthocyanins with delphinidin, petunidin and dimethicone as the skeleton, but since flowers such as roses cannot produce such delphinidin-type anthocyanins, research is being conducted on artificially producing delphinidin by expressing the flavonoid 3',5'-hydroxylase gene necessary for their synthesis (Non-Patent Document 1). However, even if the metabolism of plants is artificially changed in order to express the enzyme gene for producing the target substance in transgenic plants, the accumulation of the target substance does not occur at all or almost does not occur in most cases.

[0004] Further, flower color varies according to the coexisting flavonoids (called co-pigments) or metal ions, pH of vacuoles, etc., in addition to the structure of anthocyanins themselves. Flavonoids or flavonols are representative co-pigments, which have the effect of making anthocyanins blue and look thick by stacking into a sandwich shape with anthocyanins (non-patent literature 2). It is known that this is a co-pigment effect. It is especially known that flavonoids show a strong co-pigment effect, for example, it is reported that flavonoids show a significant co-pigment effect in the analysis of genetically modified carnations (non-patent literature 3). In addition, it is reported that in Dutch iris, the higher the ratio of the total flavonoid amount relative to the total delphinidin amount, the more the co-pigment effect is shown, and the bluer the color is (non-patent literature 4).

[0005] However, not all plants can produce flavonoids, and roses and petunias do not accumulate flavonoids. Therefore, attempts have been made to express genes encoding proteins having an activity of synthesizing flavonoids from flavanones in such plants to change flower color (Patent Document 1).

[0006] In addition, it is known that in plants, flavonoids are distributed in the form of glycosides except free state, mainly generating flavonoid O-glycosides and flavonoid C-glycosides, and particularly flavonoid C-glycosides show strong co-pigment effect. For example, it is reported that isovitexin, a kind of flavonoid C-glycosides, shows co-pigment effect for anthocyanins in iris ensata Thunb., and by stabilizing anthocyanins, flower color can be blued (non-patent literature 5). As one of the biosynthetic pathways of flavonoid C-glycosides, it is known that the reaction catalyzed by flavanone 2-hydroxylase (F2H) and C-glycosidase (CGT), dehydratase (FDH) is synthesized by flavonoids (non-patent literature 6).

[0007] So far, it has been reported that the F3'5'H gene from Campanula, the MT gene from Torenia, the F2H gene from Licorice, the CGT gene from Rice, and the FDH gene from Lotus japonicus are introduced to produce roses with blue flower colors by allowing delphinidin-type anthocyanins and flavone C-glycosides to coexist in plant cells (Patent Document 2). However, the flower colors of roses produced in this way tend to be strongly red, and thus it is still desired to develop a blue expression control technology for producing roses with uniform and stable blue flower colors.

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Publication No. 2000-279182

[0010] Patent Document 2: International Publication No. 2019 / 069946

[0011] Patent Document 3: International Publication No. 2008 / 156206

[0012] Non-patent literature

[0013] Non-patent literature 1: Phytochemistry Reviews 5, 283-291

[0014] Non-patent document 2: Prog. Chem. Org. Natl. Prod. 52

[0015] Non-patent document 3: Phytochemistry, 63, 15-23 (2003)

[0016] Non-patent document 4: Plant Physiol. Bioch. 72, 116-124 (2013)

[0017] Non-patent document 5: Euphytica 115, 1-5 (2000)

[0018] Non-patent document 6: FEBS Lett. 589, 182-187 (2015) Summary of the invention

[0019] The problem to be solved by the present invention is to produce a transformed plant having uniform and stable blue flower color (RHS chromatogram standard 5th edition: Violet-Blue group / Blue group and / or hue angle: 339.7° to 270.0°) by identifying the cause of redness in flower color.

[0020] The inventors of the present application have conducted in-depth research to solve the above-mentioned problems and have repeatedly conducted experiments. As a result, they have found that the cause of redness is flavonoid di-C-glycosides, and that flavonoid mono-C-glycosides are significantly higher than flavonoid di-C-glycosides in terms of the co-pigment effect with delphinidin-type anthocyanins. Furthermore, the inventors of the present application have successfully accumulated only flavonoid mono-C-glycosides significantly in the petals of plants by introducing a CGT gene from buckwheat among various CGT genes. Based on such insights, the present invention has been completed.

[0021] The present invention is as follows.

[0022] [1] A CGT gene or a homologous gene from buckwheat, which is a C-glycosidase from buckwheat, i.e., a CGT gene or a homologous gene thereof, characterized in that the CGT gene or a homologous gene from buckwheat is selected from:

[0023] (1-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 11;

[0024] (1-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 11 and has the same activity as the polynucleotide described in (1-a);

[0025] (1-c) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO:12.

[0026] [2] The CGT gene from buckwheat or its homologous gene according to 1, characterized in that it is attached with the untranslated region (5'-UTR) from the ADH gene from Arabidopsis thaliana, i.e., sequence number 15, or the untranslated region (5'-UTR) from the HSPRO gene from Arabidopsis thaliana, i.e., sequence number 13.

[0027] [3] A vector comprising a CGT gene or a homologous gene derived from buckwheat, wherein the CGT gene or a homologous gene derived from buckwheat is selected from:

[0028] (1-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 11;

[0029] (1-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 11 and has the same activity as the polynucleotide described in (1-a);

[0030] (1-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 12;

[0031] (1-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 12, and having the same activity as the protein encoded by the polynucleotide described in (1-c);

[0032] (1-e) A polynucleotide encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 12 and having the same activity as the protein encoded by the polynucleotide described in (1-c).

[0033] [4] The vector according to 3, characterized in that the untranslated region (5'-UTR) from the ADH gene from Arabidopsis thaliana, i.e., sequence number 15, or the untranslated region (5'-UTR) from the HSPRO gene from Arabidopsis thaliana, i.e., sequence number 13, is attached to the CGT gene from buckwheat or its homologous gene.

[0034] [5] The vector according to 3 or 4, characterized in that it further contains flavanone 2-hydroxylase, i.e., F2H gene or its homologous gene, and dehydratase, i.e., FDH gene or its homologous gene.

[0035] [6] The vector according to 5, characterized in that the F2H gene or its homologous gene is selected from:

[0036] (2-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 5;

[0037] (2-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 5 and has the same activity as the polynucleotide described in (2-a);

[0038] (2-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6;

[0039] (2-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 6, and having the same activity as the protein encoded by the polynucleotide described in (2-c);

[0040] (2-e) a polynucleotide encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 6 and having the same activity as the protein encoded by the polynucleotide described in (2-c),

[0041] The FDH gene or its homologous gene is selected from:

[0042] (3-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9;

[0043] (3-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 9 and has the same activity as the polynucleotide described in (3-a);

[0044] (3-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10;

[0045] (3-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO:10, and having the same activity as the protein encoded by the polynucleotide described in (3-c);

[0046] (3-e) A polynucleotide encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 10 and having the same activity as the protein encoded by the polynucleotide described in (3-c).

[0047] [7] The vector according to any one of 3 to 6, further comprising a flavonoid F3'5' hydroxylase, ie, F3'5'H gene or a homologous gene thereof, and a methyltransferase, ie, MT gene or a homologous gene thereof.

[0048] [8] The vector according to item 7, characterized in that the F3'5'H gene or its homologous gene is selected from:

[0049] (4-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 1;

[0050] (4-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1 and has the same activity as the polynucleotide described in (4-a);

[0051] (4-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;

[0052] (4-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 2, and having the same activity as the protein encoded by the polynucleotide described in (4-c);

[0053] (4-e) a polynucleotide encoding a protein having an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2 and having the same activity as the protein encoded by the polynucleotide described in (4-c), and,

[0054] The MT gene or its homologous gene is selected from:

[0055] (5-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 3;

[0056] (5-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 3 and has the same activity as the polynucleotide described in (5-a);

[0057] (5-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4;

[0058] (5-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 4, and having the same activity as the protein encoded by the polynucleotide described in (5-c);

[0059] (5-e) A polynucleotide encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 4 and having the same activity as the protein encoded by the polynucleotide described in (5-c).

[0060] [9] A transformed plant or its self-propagated or hybrid progeny, characterized by containing the CGT gene or a homologous gene derived from buckwheat described in 1 or 2, or the vector described in any one of 3 to 8.

[0061]

[10] The transformed plant or its self-propagated or hybridized progeny according to 9, characterized in that the plant is selected from rose, chrysanthemum, carnation or lily.

[0062]

[11] The transformed plant or its self-propagated or hybridized progeny according to item 10, characterized in that the plant is rose.

[0063]

[12] A vegetative propagation product, a part of a plant, a tissue or a cell, characterized in that it is a vegetative propagation product, a part of a plant, a tissue or a cell of the transformed plant according to any one of 9 to 11 or its self-propagated or hybrid progeny.

[0064]

[13] A cut flower, or a processed product made from the cut flower, characterized in that it is a cut flower of the transformed plant according to any one of 9 to 11 or its self-propagated or hybrid progeny, or a processed product made from the cut flower.

[0065]

[14] A method for producing a transformed plant having a blue flower color, characterized in that it comprises a step of introducing a C-glycosidase from buckwheat, i.e., a CGT gene or a homologous gene thereof, into a host plant so that delphinidin-type anthocyanins and flavonoid mono-C-glycosides coexist in cells of the plant.

[0066]

[15] The method according to item 14 is characterized in that the flavonoid mono-C-glycoside is apigenin 6-C-glycoside, luteolin 6-C-glycoside, triticum flavonoid 6-C-glycoside, apigenin 8-C-glycoside, luteolin 8-C-glycoside, triticum flavonoid 8-C-glycoside or their derivatives.

[0067]

[16] The method according to 14 or 15, characterized in that the delphinidin-type anthocyanin is selected from delphinidin, delphinidin, petunidin and a combination thereof.

[0068]

[17] The method according to any one of 14 to 16, characterized in that it comprises the step of introducing the CGT gene or a homologous gene derived from buckwheat according to 1 or 2, or the vector according to any one of 3 to 8, into cells of a host plant.

[0069]

[18] The method according to any one of 14 to 17, characterized in that the plant is selected from rose, chrysanthemum, carnation or lily.

[0070]

[19] The method according to 18, characterized in that the plant is rose.

[0071] According to the present invention, a plant variety having a uniform and stable blue flower color (RHS chromatogram standard 5th edition: Violet-Blue group / Blue group and / or hue angle: 339.7° to 270.0°) can be produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Represents the biosynthetic pathway of flavonoid mono-C-glycosides in plants.

[0073] Figure 2 The structure of pSPB6486 is shown.

[0074] Figure 3 The structure of pSPB7473 is shown.

[0075] Figure 4 The detailed structure of pSPB7473 is shown.

[0076] Figure 5 The detailed structure of pSPB7472 is shown.

[0077] Figure 6 The detailed structure of pSPB7808 is shown.

[0078] Figure 7 The detailed structure of pSPB7809 is shown. DETAILED DESCRIPTION

[0079] Anthocyanins are a group of pigments widely found in plants, showing red, blue and purple flower colors. According to the number of hydroxyl groups on the B ring of anthocyanidins, which are glycosides, they are classified into three systems: pelargonidin, cyanidin and delphinidin. The chromophore is the glycoside part, pelargonidin-type anthocyanins show orange, cyanidin-type anthocyanins show red, and delphinidin-type anthocyanins show purple to blue. In the specification of this application, for example, "delphinidin-type anthocyanins" can include derivatives thereof with delphinidin, cyanidin and petunidin as the skeleton, preferably cyanidin.

[0080] By making delphinidin-type anthocyanins coexist with substances such as flavonoids, flavonols, organic acid esters, tannins, etc., they interact with each other intermolecularly, thereby sometimes expressing a bluish color. This phenomenon is called a co-pigmentation, and the substance that causes this phenomenon is called a co-pigment (co-pigment). The co-pigmentation not only has a dark effect that causes blue expression, but also has a dark color effect and an effect of improving color stability. The inventors confirmed that the petals of roses express blue through the co-pigmentation of delphinidin-type anthocyanins and flavonoid C-glycosides (patent document 2).

[0081] Flavonoids are a type of organic compound, a cyclic ketone of a flavanoid derivative, which exists mainly as a glycoside in plants. Flavonoids in a narrow sense refer to 15 H 10 O 2, a compound with a molecular weight of 222.24, 2,3-didehydroflavan-4-one, flavonoids in a broad sense are one of the classifications of flavonoids. Among flavonoids, flavonoids with a flavonoid structure as the basic skeleton and no hydroxyl group at the 3-position are classified as "flavonoids". In the present application specification, "flavonoid C-glycoside" refers to flavonoids in a broad sense, that is, a glycoside in which aglycone is directly bonded to the terminal carbon of aldose in a glycoside belonging to a derivative of flavonoids. Examples of flavonoid C-glycosides include luteolin C-glycoside, triticum flavonoid C-glycoside, apigenin C-glycoside, and acacetin C-glycoside, but are not limited thereto. Flavonoid C-glycosides also include glycosides of apigenin, luteolin, triticum flavonoids, and acacetin derivatives. In plants, as one of the biosynthetic pathways of flavonoid C-glycosides, it is known that Figure 1 In this pathway, flavonoid C-glycosides are produced through the action of F2H, CGT and FDH.

[0082] In such a synthesis route, in addition to flavonoid mono-C-glycosides, flavonoid di-C-glycosides such as Vicenin-2 (apigenin 6,8-di-C-glycoside) are also synthesized, and the inventors and others surprisingly found that the cause of redness is flavonoid di-C-glycosides. In terms of the co-pigment effect with delphinidin-type anthocyanins, flavonoid mono-C-glycosides are higher than flavonoid di-C-glycosides. In order to produce a transformed plant with uniform and stable blue flower color, it is necessary to minimize the accumulation of flavonoid di-C-glycosides and only make flavonoid mono-C-glycosides significantly accumulate in the petals. Flavonoid mono-C-glycosides are typically flavonoid 6-C-glycoside or flavonoid 8-C-glycoside, preferably flavonoid 6-C-glycoside. Examples of such flavonoid mono-C-glycosides include apigenin 6-C-glycoside (isovitexin), luteolin 6-C-glycoside (isoorientin), triticum flavonoid 6-C-glycoside, apigenin 8-C-glycoside (vitexin), luteolin 8-C-glycoside (orientin), triticum flavonoid 8-C-glycoside or their derivatives.

[0083] Accumulation of flavonoid C-glycosides in plant cells can be achieved by transforming host plants with a vector containing the genes necessary for the above-mentioned synthesis pathway (i.e., F2H gene, CGT gene, and FDH gene) or their homologous genes. Furthermore, by using a CGT gene from buckwheat or a homologous gene thereof as a CGT gene, particularly a CGT gene from buckwheat or a homologous gene thereof to which the untranslated region (5'-UTR) (SEQ ID NO: 15) from the ADH gene of Arabidopsis thaliana or the untranslated region (5'-UTR) (SEQ ID NO: 13) from the HSPRO gene of Arabidopsis thaliana is attached as a translation enhancing sequence, flavonoid mono-C-glycosides can be significantly accumulated in petals compared to flavonoid di-C-glycosides.

[0084] The CGT gene from buckwheat or its homologous gene is selected from the following polynucleotides:

[0085] (1-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 11;

[0086] (1-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 11 and has the same activity as the polynucleotide described in (1-a);

[0087] (1-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 12;

[0088] (1-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 12, and having the same activity as the protein encoded by the polynucleotide described in (1-c);

[0089] (1-e) A polynucleotide encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 12 and having the same activity as the protein encoded by the polynucleotide described in (1-c).

[0090] The F2H gene or its homologous gene, as long as it has the desired function, is not particularly limited in terms of its origin, and is preferably a F2H gene or its homologous gene from licorice, selected from the following polynucleotides:

[0091] (2-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 5;

[0092] (2-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 5 and has the same activity as the polynucleotide described in (2-a);

[0093] (2-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6;

[0094] (2-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 6, and having the same activity as the protein encoded by the polynucleotide described in (2-c);

[0095] (2-e) A polynucleotide encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 6 and having the same activity as the protein encoded by the polynucleotide described in (2-c).

[0096] The FDH gene or its homologous gene, as long as it has the desired function, has no particular limitation on its origin, and is preferably an FDH gene or its homologous gene from Lotus japonicus, selected from the following polynucleotides:

[0097] (3-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9;

[0098] (3-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 9 and has the same activity as the polynucleotide described in (3-a);

[0099] (3-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10;

[0100] (3-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO:10, and having the same activity as the protein encoded by the polynucleotide described in (3-c);

[0101] (3-e) A polynucleotide encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 10 and having the same activity as the protein encoded by the polynucleotide described in (3-c).

[0102] The accumulation of delphinidin-type anthocyanins in plant cells can be achieved by introducing the flavonoid F3'5'hydroxylase (F3'5'H) gene or its homologous gene and the methyltransferase (MT) gene or its homologous gene into the host plant (Patent Document 3). Thus, by transforming the host plant with a vector further containing the F3'5'H gene or its homologous gene and the MT gene or its homologous gene in addition to the essential genes in the synthesis pathway of the above-mentioned flavonoid mono-C-glycoside or their homologous genes, delphinidin-type anthocyanins and flavonoid mono-C-glycosides can coexist in the cells of the host plant.

[0103] The F3'5'H gene or its homologous gene is not particularly limited in its origin as long as it has the desired function. It is preferably a F3'5'H gene or its homologous gene from Campanula, selected from the following polynucleotides:

[0104] (4-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 1;

[0105] (4-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1 and has the same activity as the polynucleotide described in (4-a);

[0106] (4-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;

[0107] (4-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 2, and having the same activity as the protein encoded by the polynucleotide described in (4-c);

[0108] (4-e) A polynucleotide encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 2 and having the same activity as the protein encoded by the polynucleotide described in (4-c).

[0109] The MT gene or its homologous gene is not particularly limited in its origin as long as it has the desired function. It is preferably a MT gene or its homologous gene from torenia, selected from the following polynucleotides:

[0110] (5-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 3;

[0111] (5-b) a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 3 and has the same activity as the polynucleotide described in (5-a);

[0112] (5-c) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4;

[0113] (5-d) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of SEQ ID NO: 4, and having the same activity as the protein encoded by the polynucleotide described in (5-c);

[0114] (5-e) A polynucleotide encoding a protein having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 4 and having the same activity as the protein encoded by the polynucleotide described in (5-c).

[0115] The MT gene or its homologous gene may be supplemented with the untranslated region (5'-UTR) from the Arabidopsis thaliana ADH gene (SEQ ID NO: 15) or the untranslated region (5'-UTR) from the Arabidopsis thaliana HSPRO gene (SEQ ID NO: 13) as a translation enhancing sequence.

[0116] In this specification, the term "polynucleotide" refers to DNA or RNA.

[0117] In this specification, the term "stringent conditions" refers to conditions that enable polynucleotides or oligonucleotides to selectively and detectably bind to genomic DNA. Stringent conditions are defined by appropriate combinations of salt concentration, organic solvent (e.g., formamide), temperature and other known conditions. That is, stringency is increased by reducing salt concentration, increasing organic solvent concentration, or increasing hybridization temperature. The washing conditions after further hybridization also affect stringency. The washing conditions can also be defined according to salt concentration and temperature, and the stringency of washing is increased by the reduction of salt concentration and the rise of temperature. Thus, the term "stringent conditions" refers to the "consistency" degree only between each base sequence, for example, the overall average is about 80% or more, preferably about 90% or more, more preferably about 95% or more, further preferably 97% or more, and most preferably 98% or more base sequences with high consistency, the conditions for specific hybridization. As "stringent conditions", for example, a temperature of 60°C to 68°C, a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, and a pH of 6 to 8 can be listed. As a specific example, hybridization can be carried out under the conditions of 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 1% SDS, 5×Denhard solution 50% formaldehyde and 42°C, and washing can be carried out under the conditions of 0.1×SSC (15 mM NaCl, 1.5 mM trisodium citrate), 0.1% SDS and 55°C.

[0118] Hybridization can be implemented according to methods known to the industry such as the method recorded in the molecular biology modern laboratory guide (Current protocols inmolecular biology (edited by Frederick M.Ausubel et al., 1987)) or the method based on it. In addition, when using commercially available libraries, it can be implemented according to the method recorded in the additional instruction manual. As the gene selected by such hybridization, it can be a natural gene, for example, a gene from a plant, or a gene from outside the plant. In addition, the gene selected by hybridization can be cDNA, genomic DNA, or chemically synthesized DNA.

[0119] In this specification, the so-called "amino acid sequence obtained by deletion, substitution, insertion and / or addition of one or more amino acids" refers to, for example, an amino acid sequence obtained by deletion, substitution, insertion and / or addition of any number of amino acids from 1 to 20, preferably from 1 to 5, and more preferably from 1 to 3. The site-specific induced mutagenesis method, one of the methods of genetic engineering, is a method for introducing a specific mutation at a specific position, and is therefore useful, and can be implemented according to the method described in Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, etc. By using an appropriate expression system to express the mutant DNA, a protein consisting of an amino acid sequence obtained by deletion, substitution, insertion and / or addition of one or more amino acids can be obtained.

[0120] In addition, polynucleotides can be obtained by methods known to those skilled in the art, such as chemical synthesis by the phosphoramidite method, nucleic acid amplification using primers designed based on the nucleotide sequence of the target gene using a plant nucleic acid sample as a template, etc.

[0121] In this specification, the term "consistency" refers to the amount (number) of substances that can be determined to be consistent in the suitable relationship between each amino acid residue or each base constituting the chain between two chains in a polypeptide sequence (or amino acid sequence) or a polynucleotide sequence (or base sequence). It is a term that expresses the degree of sequence relatedness between two polypeptide sequences or two polynucleotide sequences, and "consistency" can be simply calculated. Many methods for determining the identity between two polynucleotide sequences or polypeptide sequences are known, and the term "identity" is well known to those skilled in the art (for example, see Lesk, AM (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin, AM & Grifin, HG (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.).

[0122] In addition, the numerical value of "identity" recorded in this specification, unless otherwise specified, can be a numerical value calculated using a consistency search program known to those skilled in the art, preferably a numerical value calculated using the ClustalW program of MacVector application (version 9.5 Oxford Molecular Ltd., Oxford, England). In the present invention, the degree of "identity" between each polynucleotide sequence or 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.

[0123] The polynucleotide (nucleic acid, gene) of the present invention is a substance that "encodes" the protein of interest. Here, "encoding" means expressing the protein of interest in a state in which it has its activity. In addition, "encoding" has two meanings: encoding the protein of interest as a continuous structural sequence (exon) or encoding it through an intervening sequence (intron).

[0124] Genes with natural base sequences can be obtained, for example, by being analyzed by a DNA sequencer. In addition, DNA encoding an enzyme with a modified amino acid sequence can be synthesized using a DNA with a natural base sequence as a basis using a conventional site-specific mutagenesis method and a PCR method. For example, a DNA fragment to be modified is obtained by treating a restriction enzyme of natural cDNA or genomic DNA, and using the primers introduced with the desired mutation as a template to implement site-specific mutagenesis method and a PCR method, thereby obtaining the desired modified DNA fragment. Thereafter, the DNA fragment introduced with the mutation is connected to the DNA fragment encoding the other parts of the target enzyme.

[0125] Alternatively, in order to obtain a DNA encoding an enzyme consisting of a shortened amino acid sequence, for example, a DNA encoding a full-length amino acid sequence is cut by, for example, a desired restriction enzyme to encode an amino acid sequence longer than the target amino acid sequence, and as a result, the resulting DNA fragment cannot encode the entire target amino acid sequence, it is sufficient to synthesize a DNA fragment consisting of the insufficient portion of the sequence and ligate them.

[0126] Furthermore, by expressing the obtained polynucleotide using a gene expression system in Escherichia coli or yeast and measuring the enzyme activity, it can be confirmed that the obtained polynucleotide encodes a protein having the desired activity.

[0127] The present invention also relates to a (transgenic) vector, in particular an expression vector, containing the above polynucleotide, and further relates to a plant transformed by the vector.

[0128] In addition, the vectors of the present invention contain expression control regions, such as promoters, terminators, and replication origins, depending on the type of host plant into which they are introduced. Examples of promoters that constitutively express a polynucleotide in plant cells include the 35S promoter of cauliflower mosaic virus, the E1 promoter that connects the enhancing region of two 35S promoters, and the promoter of the cauliflower mosaic virus. 2 35S promoter, rd29A gene promoter, rbcS promoter, mac-1 promoter, etc. In addition, for tissue-specific gene expression, a promoter of a gene that is specifically expressed in the tissue can be used.

[0129] The vector can be prepared by conventional methods using restriction enzymes, ligases, etc. In addition, the transformation of host plants using the expression vector can also be carried out by conventional methods.

[0130] At the current state of the art, techniques for introducing polynucleotides into plants and expressing the polynucleotides constitutively or tissue-specifically can be used. 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.

[0131] In the present invention, the plant that can be used as the host is not particularly limited, and the plants of the genus Rosa of the Rosaceae family, the genus Chrysanthemum of the Asteraceae family, the genus Dianthus of the Caryophyllaceae family (carnation, etc.), and the genus Lilium of the Liliaceae family can be used, and the cultivated rose (scientific name Rosa hybrida) of the genus Rosa of the Rosaceae family is particularly preferred. In addition, the term "rose plant" used in this specification refers to the cultivated rose (scientific name Rosa hybrida) of the genus Rosa of the Rosaceae family in taxonomic positioning. Roses are mainly divided into hybrid tea roses (Hybrid tea), floribunda, and polyantha according to the tree shape and the size of the flower. The main pigments (anthocyanins) contained in the petals of any system are only cyanidin and pelargonium. In the present invention, the type of rose plant that can be used as the host is not particularly limited, and these varieties or systems can be appropriately used. For example, rose varieties that can be used as hosts include Ocean song, Noblesse, Rita·Perfumera, Cool water, Fame, Topless, and Peach avalanche.

[0132] According to the present invention, a transformed plant having a uniform and stable blue flower color can be prepared, preferably a plant of the genus Rosa of the Rosaceae family, the genus Chrysanthemum of the Asteraceae family, or the genus Dianthus (carnation, etc.) of the Caryophyllaceae family, and particularly preferably a rose plant. The obtained transformed plant exhibits a flower color of the Blue group or the Violet-Blue group under the RHS chromatographic standard, and / or a hue angle of 339.7° to 270.0°, preferably 315° or less, of the CIEL*a*b* color system.

[0133] Furthermore, the present invention also relates to a cut flower of the transformed plant obtained by the above method or its self-propagated or hybridized progeny, their vegetative propagation bodies, plant parts, tissues or cells, or a processed product made from the cut flower (especially a cut flower processed product). Here, the cut flower processed product includes pressed flowers, preserved flowers, dried flowers, resin-sealed products, etc. using the cut flower, but is not limited thereto.

[0134] The present invention is specifically described below by way of examples.

[0135] Example

[0136] [Example 1: Simulation of the co-pigment effect of flavonoid C-glycoside on anthocyanin (dimethicone)]

[0137] In order to simulate the copigment effect of flavonoid C-glycoside on anthocyanin (delphinidin), delphinidin and flavonoid C-glycoside were adjusted. Delphinidin (delphinidin 3,5-bioside) and flavonoid C-glycosides (vitexin (apigenin 8-C-glycoside), isovitexin (apigenin 6-C-glycoside), orientin (luteolin 8-C-glycoside), isoorientin (luteolin 6-C-glycoside), vicinin-2 (apigenin 6,8-di-C-glycoside)) used in this experiment were purchased from Nacalai Tesque Co., Ltd.

[0138] To the thus obtained dandrin, each flavonoid C-glycoside (vitexin, isovitexin, orientin, isoorientin, and vecinin-2) was added at a molar concentration ratio of 5 equivalents in a buffer solution at pH 5.0, and the absorption spectrum was measured. The concentration of dandrin was set to 0.5 mM.

[0139] [Table 1]

[0140] Absorption extreme value (λmax) and hue angle (°) of the aqueous solution of damifloxacin when flavonoid C-glycoside was added

[0141]

[0142] By adding flavonoid C-glycoside, the absorbance of the aqueous solution of dimethoate increased. When any flavonoid C-glycoside was added, the absorption extreme value (λmax) shifted to the long wavelength side compared with the case of dimethoate alone. In addition, the effect was confirmed to shift the absorption extreme value to the long wavelength side in the order of isovitexin>isoorientin>orientin>vitexin>vecin-2. It was further confirmed that the hue angle also became smaller in the order of isovitexin>isoorientin>orientin>vitexin>vecin-2. From the above, it is clear that the copigment effect of flavonoid mono-C-glycoside is higher than that of flavonoid di-C-glycoside. It is further clarified that among flavonoid mono-C-glycosides, the copigment effect of flavonoid 6-C-glycoside is particularly high.

[0143] [Example 2: Introduction of the F3'5'H gene from Campanula, the MT gene from Torenia, the F2H gene from Licorice, the CGT gene with altered codon usage from Rice, and the FDH gene from Lotus japonicus into the rose variety "Ocean Song"]

[0144] pSPB6486 is based on pBINPLUS and contains the following five expression cassettes.

[0145] (1) El 2 35S promoter and F3'5'H full-length cDNA from Campanula (SEQ ID NO. 1) and D8 terminator

[0146] (2) El 2 35S promoter, MT full-length cDNA from Torenia odoratus (SEQ ID NO: 3), and NOS terminator

[0147] (3) 35S promoter, F2H full-length cDNA from licorice (SEQ ID NO: 5), and AT terminator from Perilla frutescens

[0148] (4) 35S promoter and CGT full-length cDNA with altered codon usage from rice (SEQ ID NO: 7) and HSP terminator from Arabidopsis thaliana

[0149] (5) 35S promoter and FDH full-length cDNA from Lotus japonicus (SEQ ID NO: 9) and HSP terminator from Arabidopsis thaliana

[0150] In plants, this plasmid constitutively expresses the F3'5'H gene of Campanula, the MT gene of Torenia, the F2H gene of Licorice, the CGT gene with altered codon usage of Rice, and the FDH gene of Lotus japonicus.

[0151] The pSPB6486 prepared in this way was introduced into the blue rose variety "Ocean Song", and a total of 27 transformants were obtained. As a result of the pigment analysis of these, the accumulation of delphinidin was confirmed in 26 individuals, and the delphinidin content was the highest at 74.5% (average 57.0%). Furthermore, as flavonoid C-glycosides, isovitexin (apigenin 6-C-glycoside), vitexin (apigenin 8-C-glycoside), isoorientin (luteolin 6-C-glycoside), orientin (luteolin 8-C-glycoside), and vecinin-2 (apigenin 6,8-C-diglycoside) were identified and quantified. Flavonoid C-glycosides were detected in all individuals in which delphinidin was detected, and vecinin-2, which is a flavonoid di-C-glycoside, was detected as the main component. The order of production was vitexin-2>isovitexin>vitexin>isoorientin>orientin, with the highest total amount being 1.563 mg per 1 g of fresh petal. In addition, the total amount of flavonoid C-glycosides was about 10 times greater than that of delphinidin.

[0152] The analytical values ​​of representative transformants are shown in Table 2 below.

[0153] [Table 2]

[0154]

[0155] Host: Song of the Sea

[0156] Del: delphinidin, Cya: cyanidin, Pet: petunidin, Pel: pelargonidin, Mal: ​​cyanidin

[0157] M: Myricetin, Q: Quercetin, K: Kaempferol

[0158] Tri: quercetin, Lut: luteolin, Api: apigenin, Vic2: venetin-2, VX: vitexin, IVX: isovitexin, Ori: orientin, Iori: isoorientin

[0159] Mal(%): The ratio of malachite in total anthocyanins

[0160] [Example 3: Introduction of the F3'5'H gene from Campanula, the MT gene from Torenia, the F2H gene from Licorice, the CGT gene with altered codon usage from Fagopyrum serrata, and the FDH gene from Lotus japonicus into the rose variety "Ocean Song"]

[0161] pSPB7473 is based on pBINPLUS and contains the following five expression cassettes.

[0162] (1) El 2 35S promoter and F3'5'H full-length cDNA from Campanula (SEQ ID NO. 1) and D8 terminator

[0163] (2) El 2 35S promoter and MT full-length cDNA from Torenia thaliana (SEQ ID NO: 3) (5'-UTR (SEQ ID NO: 13) from Arabidopsis thaliana HSPRO gene attached to the 5' side) and HSP terminator from Arabidopsis thaliana

[0164] (3)El 2 35S promoter and F2H full-length cDNA from Licorice (SEQ ID NO: 5) and AT terminator from Perilla

[0165] (4)El 2 35S promoter and CGT full-length cDNA (SEQ ID NO. 11) with altered codon usage from buckwheat (5'-UTR (SEQ ID NO. 13) from Arabidopsis thaliana HSPRO gene added to the 5' side) and HSP terminator from Arabidopsis thaliana

[0166] (5)El 2 35S promoter and FDH full-length cDNA from Lotus japonicus (SEQ ID NO: 9) and HSP terminator from Arabidopsis thaliana

[0167] In plants, this plasmid constitutively expresses the F3'5'H gene of Campanula, the MT gene of Torenia, the F2H gene of Licorice, the CGT gene with altered codon usage of Fagopyrum truncatum, and the FDH gene of Lotus japonicus.

[0168] The pSPB7473 prepared in this way was introduced into the blue rose variety "Ocean Song", and a total of 35 transformants were obtained. As a result of the pigment analysis of these, the accumulation of delphinidin was confirmed in 21 individuals, and the delphinidin content was the highest at 20.8% (average 9.1%). Furthermore, as flavonoid C-glycosides, isovitexin (apigenin 6-C-glycoside), vitexin (apigenin 8-C-glycoside), isoorientin (luteolin 6-C-glycoside), orientin (luteolin 8-C-glycoside), and vicinin-2 (apigenin 6,8-C-diglycoside) were identified and quantified. Flavonoid C-glycosides were detected in all individuals in which delphinidin was detected, and isovitexin, which is a flavonoid 6-C-glycoside, was detected as the main component. The order of production was isovitexin>ovitexin>vecin-2>isoorientin>orientin, with the highest total amount being 7.418 mg per 1 g of fresh petal weight, which was a very high content. Compared with the OS / 6486 system shown in Example 2, almost all individuals showed a high content of flavonoid C-glycosides of more than 3 mg per 1 g of fresh petal weight, which was about 100 times more than the production amount of delphinidin.

[0169] The analytical values ​​of representative transformants are shown in Table 3 below.

[0170] [Table 3]

[0171]

[0172] Host: Song of the Sea

[0173] Del: delphinidin, Cya: cyanidin, Pet: petunidin, Pel: pelargonidin, Mal: ​​cyanidin

[0174] M: Myricetin, Q: Quercetin, K: Kaempferol

[0175] Tri: quercetin, Lut: luteolin, Api: apigenin, Vic2: venetin-2, VX: vitexin, IVX: isovitexin, Ori: orientin, Iori: isoorientin

[0176] Mal(%): The ratio of malachite in total anthocyanins

[0177] [Example 4: Introduction of the F3'5'H gene from Campanula, the MT gene from Torenia, the F2H gene from Licorice, the CGT gene from Fagopyrum serrata, and the FDH gene from Lotus japonicus into the rose variety "Ocean Song"]

[0178] pSPB7472 is based on pBINPLUS and contains the following five expression cassettes.

[0179] (1) El 2 35S promoter and F3'5'H full-length cDNA from Campanula (SEQ ID NO. 1) and D8 terminator

[0180] (2) El 2 35S promoter and MT full-length cDNA from Torenia thaliana (SEQ ID NO: 3) (5'-UTR (SEQ ID NO: 13) from Arabidopsis thaliana HSPRO gene attached to the 5' side) and HSP terminator from Arabidopsis thaliana

[0181] (3)El 2 35S promoter and F2H full-length cDNA from Licorice (SEQ ID NO: 5) and AT terminator from Perilla

[0182] (4)El 2 35S promoter and full-length CGT cDNA from buckwheat (SEQ ID NO: 14) (5'-UTR (SEQ ID NO: 13) from Arabidopsis HSPRO gene attached to the 5' side) and HSP terminator from Arabidopsis

[0183] (5)El 2 35S promoter and FDH full-length cDNA from Lotus japonicus (SEQ ID NO: 9) and HSP terminator from Arabidopsis thaliana

[0184] In plants, this plasmid constitutively expresses the F3'5'H gene of Campanula, the MT gene of Torenia, the F2H gene of Licorice, the CGT gene of Fagopyrum, and the FDH gene of Lotus japonicus.

[0185] The pSPB7472 prepared in this way was introduced into the blue rose variety "Ocean Song", and a total of 33 transformants were obtained. Next, as flavonoid C-glycosides, identification and quantification of isovitexin (apigenin 6-C-glycoside), vitexin (apigenin 8-C-glycoside), isoorientin (luteolin 6-C-glycoside), orientin (luteolin 8-C-glycoside), and vecinin-2 (apigenin 6,8-C-bioside) were carried out, and as anthocyanins, identification and quantification of delphinidin, cyanidin, petunidin, pelargonidin, and delphinidin were carried out. As a result, the accumulation of flavonoid C-glycosides and delphinidin was confirmed in 11 individuals. The average content of flavonoid C-glycosides was 3.75 mg per 1 g of fresh weight of petals, and isovitexin as a flavonoid 6-C-glycoside was detected as the main component. Furthermore, the content of delphinidin was the highest at 15.6% (average 8.8%).

[0186] As shown above, the average content of flavonoid C-glycoside per 1 g of fresh petal weight was as high as 4.19 mg in the OS / 7473 system shown in Example 3. That is, it was shown that the CGT gene with the modified codon can produce flavonoid C-glycoside more efficiently than the original CGT gene derived from buckwheat.

[0187] The analytical values ​​of representative transformants are shown in Table 4 below.

[0188] [Table 4]

[0189]

[0190] Host: Song of the Sea

[0191] Del: delphinidin, Cya: cyanidin, Pet: petunidin, Pel: pelargonidin, Mal: ​​cyanidin

[0192] M: Myricetin, Q: Quercetin, K: Kaempferol

[0193] Tri: quercetin, Lut: luteolin, Api: apigenin, Vic2: venetin-2, VX: vitexin, IVX: isovitexin, Ori: orientin, Iori: isoorientin

[0194] Mal(%): The ratio of malachite in total anthocyanins

[0195] [Example 5: Introduction of the F3'5'H gene from Campanula, the MT gene from Torenia, the F2H gene from Licorice, the CGT gene from Fagopyrum serrata, and the FDH gene from Lotus japonicus into the rose variety "Ocean Song"]

[0196] pSPB7808 is based on pBINPLUS and contains the following five expression cassettes.

[0197] (1) El 2 35S promoter and F3'5'H full-length cDNA from Campanula (SEQ ID NO. 1) and D8 terminator

[0198] (2) El 2 35S promoter and MT full-length cDNA from Torenia odoratus (SEQ ID NO. 3) and HSP terminator from Arabidopsis thaliana

[0199] (3)El 2 35S promoter and F2H full-length cDNA from Licorice (SEQ ID NO: 5) and AT terminator from Perilla

[0200] (4)El 235S promoter and full-length CGT cDNA from buckwheat (SEQ ID NO: 14) (5'-UTR (SEQ ID NO: 15) from the alcohol dehydrogenase (ADH) gene of Arabidopsis thaliana added to the 5' side) and HSP terminator from Arabidopsis thaliana

[0201] (5)El 2 35S promoter and FDH full-length cDNA from Lotus japonicus (SEQ ID NO: 9) and HSP terminator from Arabidopsis thaliana

[0202] In plants, this plasmid constitutively expresses the F3'5'H gene of Campanula, the MT gene of Torenia, the F2H gene of Licorice, the CGT gene of Fagopyrum, and the FDH gene of Lotus japonicus.

[0203] The pSPB7808 prepared in this way was introduced into the blue rose variety "Ocean Song", and a total of 65 transformants were obtained. Next, as flavonoid C-glycosides, identification and quantification of isovitexin (apigenin 6-C-glycoside), vitexin (apigenin 8-C-glycoside), isoorientin (luteolin 6-C-glycoside), orientin (luteolin 8-C-glycoside), and vecinin-2 (apigenin 6,8-C-bioside) were carried out, and as anthocyanins, identification and quantification of delphinidin, cyanidin, petunidin, pelargonidin, and delphinidin were carried out. As a result, the accumulation of flavonoid C-glycosides and delphinidin was confirmed in 32 individuals. The average content of flavonoid C-glycosides was 3.00 mg per 1 g of fresh weight of petals, and isovitexin as a flavonoid 6-C-glycoside was detected as the main component. In addition, the content of delphinidin was the highest at 69.2% (average 43.9%).

[0204] The analytical values ​​of representative transformants are shown in Table 5 below.

[0205] [Table 5]

[0206]

[0207] Host: Song of the Sea

[0208] Del: delphinidin, Cya: cyanidin, Pet: petunidin, Pel: pelargonidin, Mal: ​​cyanidin

[0209] M: Myricetin, Q: Quercetin, K: Kaempferol

[0210] Tri: quercetin, Lut: luteolin, Api: apigenin, Vic2: venetin-2, VX: vitexin, IVX: isovitexin, Ori: orientin, Iori: isoorientin

[0211] Mal(%): The ratio of malachite in total anthocyanins

[0212] [Example 6: Introduction of the F3'5'H gene from Campanula, the MT gene from Torenia, the F2H gene from Licorice, the CGT gene with altered codon usage from Fagopyrum serrata, and the FDH gene from Lotus japonicus into the rose variety "Ocean Song"]

[0213] pSPB7809 is based on pBINPLUS and contains the following five expression cassettes.

[0214] (1) El 2 35S promoter and F3'5'H full-length cDNA from Campanula (SEQ ID NO. 1) and D8 terminator

[0215] (2) El 2 35S promoter and MT full-length cDNA from Torenia odoratus (SEQ ID NO. 3) and HSP terminator from Arabidopsis thaliana

[0216] (3)El 2 35S promoter and F2H full-length cDNA from Licorice (SEQ ID NO: 5) and AT terminator from Perilla

[0217] (4)El 2 35S promoter and CGT full-length cDNA (SEQ ID NO. 11) with altered codon usage from buckwheat (5'-UTR (SEQ ID NO. 15) from the alcohol dehydrogenase (ADH) gene of Arabidopsis thaliana added to the 5' side) and HSP terminator from Arabidopsis thaliana

[0218] (5)El 2 35S promoter and FDH full-length cDNA from Lotus japonicus (SEQ ID NO: 9) and HSP terminator from Arabidopsis thaliana

[0219] In plants, this plasmid constitutively expresses the F3'5'H gene of Campanula, the MT gene of Torenia, the F2H gene of Licorice, the CGT gene with altered codon usage of Fagopyrum truncatum, and the FDH gene of Lotus japonicus.

[0220] The pSPB7809 prepared in this way was introduced into the blue rose variety "Ocean Song", and a total of 143 transformants were obtained. Next, as flavonoid C-glycosides, identification and quantification of isovitexin (apigenin 6-C-glycoside), vitexin (apigenin 8-C-glycoside), isoorientin (luteolin 6-C-glycoside), orientin (luteolin 8-C-glycoside), and vecinin-2 (apigenin 6,8-C-bioside) were implemented, and as anthocyanins, identification and quantification of delphinidin, cyanidin, petunidin, pelargonidin, and dimethoate were implemented. As a result, the accumulation of flavonoid C-glycosides was confirmed in 58 individuals. The average content of flavonoid C-glycosides was 3.24 mg per 1 g of fresh weight of petals, and isovitexin as a flavonoid 6-C-glycoside was detected as the main component. Furthermore, the content of delphinidin was the highest at 80.3% (average 46.6%).

[0221] As shown above, the average content of flavonoid C-glycoside per 1 g of fresh petal weight was higher in the OS / 78093 system shown in this example. Thus, similar to the results obtained in Examples 3 and 4, the CGT gene with the codon modified can produce flavonoid C-glycoside more efficiently than the original CGT gene derived from buckwheat.

[0222] The analytical values ​​of representative transformants are shown in Table 6 below.

[0223] [Table 6-1]

[0224]

[0225] [Table 6-2]

[0226]

[0227] Host: Song of the Sea

[0228] Del: delphinidin, Cya: cyanidin, Pet: petunidin, Pel: pelargonidin, Mal: ​​cyanidin

[0229] M: Myricetin, Q: Quercetin, K: Kaempferol

[0230] Tri: quercetin, Lut: luteolin, Api: apigenin, Vic2: venetin-2, VX: vitexin, IVX: isovitexin, Ori: orientin, Iori: isoorientin

[0231] Mal(%): The ratio of malachite in total anthocyanins

[0232] [Example 7: Evaluation of the Flower Color of Roses Containing Flavonoid C-Glycoside]

[0233] For the transformants prepared in Examples 2 and 3 (using the rose variety "Ocean Song" as the host), the color of the petals was measured using a spectrophotometer CM-2022 (Minolta Co., Ltd.) at a 10-degree field of view and D65 light source, and analyzed using the color management software SpectraMagicTM (Minolta Co., Ltd.).

[0234] In the comparison of the average values ​​of hue angle, no difference was observed in the hue angle of the petals between the roses whose CGT gene was derived from buckwheat (Example 3) and the roses whose CGT gene was derived from rice (Example 2). However, when looking at the individual aspects, the roses into which the CGT gene from buckwheat was introduced had many individuals with hue angles below 315°, and among them, an individual with a hue angle of 294.5° was obtained, which was the bluest individual so far. From the above results, it was confirmed that the production of flavonoid C-glycosides, especially mono-C-glycosides, can be significantly increased by using the CGT gene from buckwheat, and the coexistence of these with anthocyanins can make the color of the petals blue.

[0235] The results are shown in Table 7.

[0236] [Table 7]

[0237] Sequence Listing <110> Suntory Holdings Co., Ltd. <120> CGT gene from buckwheat and its application <130> P200445WO <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 1927 <212> DNA <213> Bluebells <400> 1 accaaatgag ctttgtaatt tgagattaat cataattgca tgctcaacta acattctgta 60 ttcatatatc catatgtatt ttgacctata gatattacat tacaccttga ggcctttata 120 tatagagagt gtatctactt cccttaatat caccttttca ttcaacaagt gaagccacca 180 tgtctataga catatccacc ctcttctatg aacttgttgc agcaatttca ctctacttag 240 ctacctactc tttcattcgt ttcctcttca aaccctctca ccaccaccac ctccctcccg 300 gcccaaccgg atggccgatc atcggagccc ttccactctt aggcaccatg ccacatgttt 360 ccttagccga catggccgtt aaatacggtc ctataatgta cctaaaactt ggttcaaagg 420 gcaccgtcgt ggcctcaaat ccaaaagccg cccgagcctt cttgaaaacc catgatgcca 480 atttttctaa ccgtccgatt gatgggggcc ctacctacct cgcgtataat gcacaagaca 540 tggtttttgc agaatatggc ccaaaatgga agcttttgcg aaagctatgt agcttgcaca 600 tgttaggccc gaaggcactc gaggattggg ctcatgtcaa agtttcagag gtcggtcata 660 tgctcaaaga aatgtacgag caatcgagta agtcagtgcc agtgccagtg gtggtgccag 720 agatgttaac ttatgccatg gctaatatga ttggacgaat catactcagc cgacgccctt 780 ttgttatcac gagcaaatta gactcgtctg cttctgcttc tgcttctgtt agtgaattcc 840 aatatatggt tatggagctc atgaggatgg cagggttgtt caatattggt gatttcatac 900 catatattgc atggatggat ttgcaggca ttcaacgtga tatgaaggtt atacagaaaa 960 agtttgatgt cttgttgaac aaatgatca aggacatac agaatccgct catgatcgca 1020 aagataatcc tgatttctt gatttctt tggcggctac ccagaaaac acggagga 1080 ttcagcttaa tcttgtaat gttaaggcac ttctttgga tttatcacg gcggggcacgg 1140 atacatcatc aagtgtgatc gatgggcac tagccgaat gttgaaccat cgacagatcc 1200 taaaccggggc ccacgagaa atggaccaag tcattggcag aaacagaga ctagaacat 1260 ctgacatacc aaacttgcca tattccaag ccatatgcaa agaacattc cgaaaacacc 1320 cttccacgcc cttaaacctc ccaagaatct cAcagaagc atgtgaagtg gacggattc 1380 acataccaaaaacactaga ctaatagtga acatagggc atagggagg gacctaag 1440 tgtgggaaaa tccattagat tttactccgg aacgttctt gagtgaaaaa cacgcgaaaa 1500 ttgatccgcg aggtaatcat ttgagttaa tcccattgg ggctggacga aggatatgtg 1560 caggggctag aatgggagcg gcctcggtcg agtacatatt aggtacattg gtgcactcat 1620 ttgattggaa attgcctgat ggagttgtgg aagttaatat ggaagagagc tttgggatcg 1680 cattgcaaaa aaaagtgcct ctttctgcta ttgttactcc aagattgcct ccaagttctt 1740 acactgtcta ggcaaatgct tatatatatg aataattgat tgagttgttt agttgtatga 1800 aagatttgag aaaataaatt attaggtttt gcaccattat gttgagatgg ttgttgttag 1860 tgttaaggaa gtcgattgta gtaataataa ttttattttt ttcgaaaaaa aaaaaaaaaa 1920 aaaaaaa 1927 <210> 2 <211> 523 <212> PRT <213> Campanula <400> 2 Met Ser Ile Asp Ile Ser Thr Leu Phe Tyr Glu Leu Val Ala Ala Ile 1 5 10 15 Ser Leu Tyr Leu Ala Thr Tyr Ser Phe Ile Arg Phe Leu Phe Lys Pro 20 25 30 Ser His His His His Leu Pro Pro Gly Pro Thr Gly Trp Pro Ile Ile 35 40 45 Gly Ala Leu Pro Leu Leu Gly Thr Met Pro His Val Ser Leu Ala Asp 50 55 60 Met Ala Val Lys Tyr Gly Pro Ile Met Tyr Leu Lys Leu Gly Ser Lys 65 70 75 80 Gly Thr Val Val Ala Ser Asn Pro Lys Ala Ala Arg Ala Phe Leu Lys 85 90 95 Thr His Asp Ala Asn Phe Ser Asn Arg Pro Ile Asp Gly Gly Pro Thr 100 105 110 Tyr Leu Ala Tyr Asn Ala Gln Asp Met Val Phe Ala Glu Tyr Gly Pro 115 120 125 Lys Trp Lys Leu Leu Arg Lys Leu Cys Ser Leu His Met Leu Gly Pro 130 135 140 Lys Ala Leu Glu Asp Trp Ala His Val Lys Val Ser Glu Val Gly His 145 150 155 160 Met Leu Lys Glu Met Tyr Glu Gln Ser Ser Lys Ser Val Pro Val Pro 165 170 175 Val Val Val Pro Glu Met Leu Thr Tyr Ala Met Ala Asn Met Ile Gly 180 185 190 Arg Ile Ile Leu Ser Arg Arg Pro Phe Val Ile Thr Ser Lys Leu Asp 195 200 205 Ser Ser Ala Ser Ala Ser Ala Ser Val Ser Glu Phe Gln Tyr Met Val 210 215 220 Met Glu Leu Met Arg Met Ala Gly Leu Phe Asn Ile Gly Asp Phe Ile 225 230 235 240 Pro Tyr Ile Ala Trp Met Asp Leu Gln Gly Ile Gln Arg Asp Met Lys 245 250 255 Val Ile Gln Lys Lys Phe Asp Val Leu Leu Asn Lys Met Ile Lys Glu 260 265 270 His Thr Glu Ser Ala His Asp Arg Lys Asp Asn Pro Asp Phe Leu Asp 275 280 285 Ile Leu Met Ala Ala Thr Gln Glu Asn Thr Glu Gly Ile Gln Leu Asn 290 295 300 Leu Val Asn Val Lys Ala Leu Leu Leu Asp Leu Phe Thr Ala Gly Thr 305 310 315 320 Asp Thr Ser Ser Ser Val Ile Glu Trp Ala Leu Ala Glu Met Leu Asn 325 330 335 His Arg Gln Ile Leu Asn Arg Ala His Glu Glu Met Asp Gln Val Ile 340 345 350 Gly Arg Asn Arg Arg Leu Glu Gln Ser Asp Ile Pro Asn Leu Pro Tyr 355 360 365 Phe Gln Ala Ile Cys Lys Glu Thr Phe Arg Lys His Pro Ser Thr Pro 370 375 380 Leu Asn Leu Pro Arg Ile Ser Thr Glu Ala Cys Glu Val Asp Gly Phe 385 390 395 400 His Ile Pro Lys Asn Thr Arg Leu Ile Val Asn Ile Trp Ala Ile Gly 405 410 415 Arg Asp Pro Lys Val Trp Glu Asn Pro Leu Asp Phe Thr Pro Glu Arg 420 425 430 Phe Leu Ser Glu Lys His Ala Lys Ile Asp Pro Arg Gly Asn His Phe 435 440 445 Glu Leu Ile Pro Phe Gly Ala Gly Arg Arg Ile Cys Ala Gly Ala Arg 450 455 460 Met Gly Ala Ala Ser Val Glu Tyr Ile Leu Gly Thr Leu Val His Ser 465 470 475 480 Phe Asp Trp Lys Leu Pro Asp Gly Val Val Glu Val Asn Met Glu Glu 485 490 495 Ser Phe Gly Ile Ala Leu Gln Lys Lys Val Pro Leu Ser Ala Ile Val 500 505 510 Thr Pro Arg Leu Pro Pro Ser Ser Tyr Thr Val 515 520 <210> 3 <211> 720 <212> DNA <213> Blue pig ear hybrid variety <400> 3 atgaaagata agttctatgg caccattttg cagagcgaag ccctcgcaaa gtatctgtta 60 gagacaagtg cctatccacg agaacatccg cagctcaaag aactaaggag cgcaactgtg 120 gacaagtatc aatattggag cttgatgaat gttccagctg atgaggggca gttcatttca 180 atgttactga aaattatgaa cgcaaaaaag acaattgaag ttggagtttt cacaggctac 240 tcactcctat caactgctct ggctctacct gatgatggca aaatcgttgc cattgatcct 300 gatagagaag cttatgagac tggtttgcca tttatcaaga aagcaaacgt ggctcataaa 360 atccaataca tacaatctga tgccatgaaa gtcatgaatg acctcattgc tgccaaggga 420 gaagaagaag aggggagctt tgactttggg ttcgtggatg cagacaaaga aaactacata 480 aactaccacg agaaactgtt gaagctggtt aaggttggag ggatcatagg atacgacaac 540 actctgtggt ctggaacagt tgctgcatct gaagacgatg agaataatat gcaagactac 600 ttaagaggtt gcagagggca tatcctcaaa ctaaactcct ttctcgcaaa cgatgatcgg 660 attgaattgg ctcacctctc tattggagat ggactcacct tgtgcaaacg tctcaaataa 720 <210> 4 <211> 239 <212> PRT <213> Torenia hybrid <400> 4 Met Lys Asp Lys Phe Tyr Gly Thr Ile Leu Gln Ser Glu Ala Leu Ala 1 5 10 15 Lys Tyr Leu Leu Glu Thr Ser Ala Tyr Pro Arg Glu His Pro Gln Leu 20 25 30 Lys Glu Leu Arg Ser Ala Thr Val Asp Lys Tyr Gln Tyr Trp Ser Leu 35 40 45 Met Asn Val Pro Ala Asp Glu Gly Gln Phe Ile Ser Met Leu Leu Lys 50 55 60 Ile Met Asn Ala Lys Lys Thr Ile Glu Val Gly Val Phe Thr Gly Tyr 65 70 75 80 Ser Leu Leu Ser Thr Ala Leu Ala Leu Pro Asp Asp Gly Lys Ile Val 85 90 95 Ala Ile Asp Pro Asp Arg Glu Ala Tyr Glu Thr Gly Leu Pro Phe Ile 100 105 110 Lys Lys Ala Asn Val Ala His Lys Ile Gln Tyr Ile Gln Ser Asp Ala 115 120 125 Met Lys Val Met Asn Asp Leu Ile Ala Ala Lys Gly Glu Glu Glu Glu 130 135 140 Gly Ser Phe Asp Phe Gly Phe Val Asp Ala Asp Lys Glu Asn Tyr Ile 145 150 155 160 Asn Tyr His Glu Lys Leu Leu Lys Leu Val Lys Val Gly Gly Ile Ile 165 170 175 Gly Tyr Asp Asn Thr Leu Trp Ser Gly Thr Val Ala Ala Ser Glu Asp 180 185 190 Asp Glu Asn Asn Met Gln Asp Tyr Leu Arg Gly Cys Arg Gly His Ile 195 200 205 Leu Lys Leu Asn Ser Phe Leu Ala Asn Asp Asp Arg Ile Glu Leu Ala 210 215 220 His Leu Ser Ile Gly Asp Gly Leu Thr Leu Cys Lys Arg Leu Lys 225 230 235 <210> 5 <211> 1920 <212> DNA <213> Glycyrrhiza <400> 5 ccccaatttc ctctatcata agccattccg ttgattgagc ttcctttccg tgaaaaaaat 60 aactaagcga tatggaacct caactcgtag cagtgtctgt gttggtttca gcacttatct 120 gctacttctt tttccggcca tatttccacc gttacggaaa aaaccttcca ccatctcctt 180 ttttccggct tccaataatt ggccacatgc acatgttagg tccccttctt caccaatcct 240 tccacaacct ctcccaccgt tacggtcctc tgttttcact taactttggc tctgttctct 300 gtgtcgttgc ttcaacccct cactttgcca aacaactcct tcaaaccaac gaactcgcct 360 ttaactgtcg cattgaatca accgccgtta aaaaactcac ttacgagtct tccttggcct 420 tcgcacctta cggtgattac tggaggttca ttaagaagct gagcatgaac gagcttttgg 480 gctctcgtag cataaacaac ttccaacacc tacgagcaca agagacccat caattgttaa 540 ggcttttgtc caacagggca agagcgtttg aggccgtgaa tatcaccgag gagcttctta 600 agttgaccaa caacgttatt tctataatga tggttgggga ggcagaggag gcaagggatg 660 tggtgcgtga tgtgacggag atatttggag agtttaatgt ttcggatttt atttggttgt 720 ttaagaagat ggacttgcag gggtttggga agaggattga ggatttgttt cagaggtttg 780 atacgttggt ggaaaggatt attagcaagc gggagcagac gaggaaagac agaaggagga 840 atgggaagaa gggtgagcag gggagtggtg atgggatcag agactttctt gatatcttgc 900 ttgactgtac tgaggatgag aattccgaga ttaaaatcca aagggttcac attaaggcct 960 tgattatgga tttcttcact gcagggacgg ataccacagc gatttcaaca gagtgggcat 1020 tagtggagct cgtcaagaaa ccctccgtgc tacaaaaagt tcgtgaagag atagacaatg 1080 tcgtaggaaa agacagactt gttgaggaat ctgattgtcc taatctccca tatctccaag 1140 ccattcttaa agaaacattc cgtttgcacc caccggttcc tatggttaca agaagatgcg 1200 tggcagagtg cacggtagag aattacgtca tcccagaaga ctcacttctc tttgtgaatg 1260 tttggtccat cggaagaaac ccaaagtttt gggacaaccc attggagttt cgccccgaac 1320 gattcttaaa actagaagga gattccagtg gagttgttga tgtgagggga agccattttc 1380 agcttctgcc atttgggtct ggaaggagga tgtgccctgg tgtgtccttg gctatgcaag 1440 aggtgccagc actacttggt gctataatcc agtgctttga tttccacgtt gtgggtccca 1500 aaggtgagat tttgaagggt gatgacatag tcattaatgt ggatgaaagg ccaggattga 1560 cggctccaag ggcacataac cttgtgtgtg ttcccgttga tagaacaagt ggcggtggac 1620 ccctcaaaat cattgaatgt tgattattcg tctcttgaat ttggatctgt gtgtggcttt 1680 gaataacatg tatggtgtat gtatgtatgt gttcttttct ttctttctaa ttctgatcag 1740 tagcgtacac taggcactag ccttcgttag tggcaataac ttcggcaaat taacgaacat 1800 atgctgttca gagatatttt ttgccatgta tcgtcattct attctaggtt gtttttccgt 1860 tttccttatt acattctatg ataaatataa taaattgagt aatattatag tctcttaatt 1920 <210> 6 <211> 523 <212> PRT <213> Glycyrrhiza <400> 6 Met Glu Pro Gln Leu Val Ala Val Ser Val Leu Val Ser Ala Leu Ile 1 5 10 15 Cys Tyr Phe Phe Phe Arg Pro Tyr Phe His Arg Tyr Gly Lys Asn Leu 20 25 30 Pro Pro Ser Pro Phe Phe Arg Leu Pro Ile Ile Gly His Met His Met 35 40 45 Leu Gly Pro Leu Leu His Gln Ser Phe His Asn Leu Ser His Arg Tyr 50 55 60 Gly Pro Leu Phe Ser Leu Asn Phe Gly Ser Val Leu Cys Val Val Ala 65 70 75 80 Ser Thr Pro His Phe Ala Lys Gln Leu Leu Gln Thr Asn Glu Leu Ala 85 90 95 Phe Asn Cys Arg Ile Glu Ser Thr Ala Val Lys Lys Leu Thr Tyr Glu 100 105 110 Ser Ser Leu Ala Phe Ala Pro Tyr Gly Asp Tyr Trp Arg Phe Ile Lys 115 120 125 Lys Leu Ser Met Asn Glu Leu Leu Gly Ser Arg Ser Ile Asn Asn Phe 130 135 140 Gln His Leu Arg Ala Gln Glu Thr His Gln Leu Leu Arg Leu Leu Ser 145 150 155 160 Asn Arg Ala Arg Ala Phe Glu Ala Val Asn Ile Thr Glu Glu Leu Leu 165 170 175 Lys Leu Thr Asn Asn Val Ile Ser Ile Met Met Val Gly Glu Ala Glu 180 185 190 Glu Ala Arg Asp Val Val Arg Asp Val Thr Glu Ile Phe Gly Glu Phe 195 200 205 Asn Val Ser Asp Phe Ile Trp Leu Phe Lys Lys Met Asp Leu Gln Gly 210 215 220 Phe Gly Lys Arg Ile Glu Asp Leu Phe Gln Arg Phe Asp Thr Leu Val 225 230 235 240 Glu Arg Ile Ile Ser Lys Arg Glu Gln Thr Arg Lys Asp Arg Arg Arg 245 250 255 Asn Gly Lys Lys Gly Glu Gln Gly Ser Gly Asp Gly Ile Arg Asp Phe 260 265 270 Leu Asp Ile Leu Leu Asp Cys Thr Glu Asp Glu Asn Ser Glu Ile Lys 275 280 285 Ile Gln Arg Val His Ile Lys Ala Leu Ile Met Asp Phe Phe Thr Ala 290 295 300 Gly Thr Asp Thr Thr Ala Ile Ser Thr Glu Trp Ala Leu Val Glu Leu 305 310 315 320 Val Lys Lys Pro Ser Val Leu Gln Lys Val Arg Glu Glu Ile Asp Asn 325 330 335 Val Val Gly Lys Asp Arg Leu Val Glu Glu Ser Asp Cys Pro Asn Leu 340 345 350 Pro Tyr Leu Gln Ala Ile Leu Lys Glu Thr Phe Arg Leu His Pro Pro 355 360 365 Val Pro Met Val Thr Arg Arg Cys Val Ala Glu Cys Thr Val Glu Asn 370 375 380 Tyr Val Ile Pro Glu Asp Ser Leu Leu Phe Val Asn Val Trp Ser Ile 385 390 395 400 Gly Arg Asn Pro Lys Phe Trp Asp Asn Pro Leu Glu Phe Arg Pro Glu 405 410 415 Arg Phe Leu Lys Leu Glu Gly Asp Ser Ser Gly Val Val Asp Val Arg 420 425 430 Gly Ser His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg Arg Met Cys 435 440 445 Pro Gly Val Ser Leu Ala Met Gln Glu Val Pro Ala Leu Leu Gly Ala 450 455 460 Ile Ile Gln Cys Phe Asp Phe His Val Val Gly Pro Lys Gly Glu Ile 465 470 475 480 Leu Lys Gly Asp Asp Ile Val Ile Asn Val Asp Glu Arg Pro Gly Leu 485 490 495 Thr Ala Pro Arg Ala His Asn Leu Val Cys Val Pro Val Asp Arg Thr 500 505 510 Ser Gly Gly Gly Pro Leu Lys Ile Ile Glu Cys 515 520 <210> 7 <211> 1416 <212> DNA <213> Japonica rice group <400> 7 atgccgagct ctggcgacgc ggcgggcagg cggccgcatg tggtgctcat cccgagcgcc 60 ggcatgggcc acctcgtccc cttcggccgc ctcgccgtgg cgctctcctc cggccacggc 120 tgcgacgtct ccctcgtcac ggtgctcccc acggtgtcca ccgcggagtc gaagcacctc 180 gacgcgctgt tcgacgcgtt cccggcggtg cggcggctcg acttcgagct cgcgccgttc 240 gacgcgtcgg agttccccag cgccgacccg ttcttcctcc ggttcgaggc catgcggcgg 300 tcggcgccgc tgctcggccc gctcctcacc ggcgccggcg catcggcgct cgccacggac 360 atcgcgctga catccgtcgt catacccgtg gcgaaggagc agggcctccc gtgccacatc 420 ctcttcaccg cctccgccgc gatgctctcc ctctgcgcct acttccccac atacctcgac 480 gccaacgctg gcgacggcgg cggcgtcggc gacgtcgaca tccccggcgt gtaccgcatc 540 cccaaggcct ccatcccgca ggcgctgcac gatcccaacc acctcttcac ccgccagttc 600 gtcgccaacg gccggagcct cacgagcgcc gccggcatcc tcgtcaacac gttcgacgcc 660 ttggagccgg aggccgtcgc ggccctgcag cagggcaagg tcgcctccgg cttcccgccg 720 gtgttcgccg tggggccact tctcccggcg agcaaccagg caaaagatcc gcaggcaaac 780 tacatggagt ggctcgacgc gcagccggcg cggtcggtgg tgtacgtgag cttcggcagc 840 cgcaaggcca tctcagggga gcagctcagg gagctcgccg ccgggctgga gaccagcggc 900 cacaggttcc tgtgggtggt gaagagcacc gtcgtggaca gggacgacgc cgccgagctc 960 ggcgagctgc tcggcgaggg gttcttgaag cgggtggaga agcgaggcct cgtcaccaag 1020 gcatgggtgg atcaggaaga ggtcctgaag cacgagtccg tggcgctgtt cgtgagccac 1080 tgcggctgga actcggtgac ggaggcggcg gcgagcggcg tcccggtgct ggcgctgccg 1140 aggttcggcg accagcgggt gaactccggc gtggtggcgc gcgccgggct cggcgtgtgg 1200 gcggacacct ggagctggga gggggaagcc ggggtgatcg gcgcagagga gatatcggag 1260 aaggtgaagg cggcgatggc ggacgaggcg ttgcgtagga aggcggcgag cctcgccaag 1320 gccgccgcga aggccgtcgc cggcggtgga tcgagccacc gttgtctggt cgagttcgcg 1380 cggctgtgcc aagggggaac atgtcgcact aattga 1416 <210> 8 <211> 471 <212> PRT <213> Oryza sativa japonica group <400> 8 Met Pro Ser Ser Gly Asp Ala Ala Gly Arg Arg Pro His Val Val Leu 1 5 10 15 Ile Pro Ser Ala Gly Met Gly His Leu Val Pro Phe Gly Arg Leu Ala 20 25 30 Val Ala Leu Ser Ser Gly His Gly Cys Asp Val Ser Leu Val Thr Val 35 40 45 Leu Pro Thr Val Ser Thr Ala Glu Ser Lys His Leu Asp Ala Leu Phe 50 55 60 Asp Ala Phe Pro Ala Val Arg Arg Leu Asp Phe Glu Leu Ala Pro Phe 65 70 75 80 Asp Ala Ser Glu Phe Pro Ser Ala Asp Pro Phe Phe Leu Arg Phe Glu 85 90 95 Ala Met Arg Arg Ser Ala Pro Leu Leu Gly Pro Leu Leu Thr Gly Ala 100 105 110 Gly Ala Ser Ala Leu Ala Thr Asp Ile Ala Leu Thr Ser Val Val Ile 115 120 125 Pro Val Ala Lys Glu Gln Gly Leu Pro Cys His Ile Leu Phe Thr Ala 130 135 140 Ser Ala Ala Met Leu Ser Leu Cys Ala Tyr Phe Pro Thr Tyr Leu Asp 145 150 155 160 Ala Asn Ala Gly Asp Gly Gly Gly Val Gly Asp Val Asp Ile Pro Gly 165 170 175 Val Tyr Arg Ile Pro Lys Ala Ser Ile Pro Gln Ala Leu His Asp Pro 180 185 190 Asn His Leu Phe Thr Arg Gln Phe Val Ala Asn Gly Arg Ser Leu Thr 195 200 205 Ser Ala Ala Gly Ile Leu Val Asn Thr Phe Asp Ala Leu Glu Pro Glu 210 215 220 Ala Val Ala Ala Leu Gln Gln Gly Lys Val Ala Ser Gly Phe Pro Pro 225 230 235 240 Val Phe Ala Val Gly Pro Leu Leu Pro Ala Ser Asn Gln Ala Lys Asp 245 250 255 Pro Gln Ala Asn Tyr Met Glu Trp Leu Asp Ala Gln Pro Ala Arg Ser 260 265 270 Val Val Tyr Val Ser Phe Gly Ser Arg Lys Ala Ile Ser Gly Glu Gln 275 280 285 Leu Arg Glu Leu Ala Ala Gly Leu Glu Thr Ser Gly His Arg Phe Leu 290 295 300 Trp Val Val Lys Ser Thr Val Val Asp Arg Asp Asp Ala Ala Glu Leu 305 310 315 320 Gly Glu Leu Leu Gly Glu Gly Phe Leu Lys Arg Val Glu Lys Arg Gly 325 330 335 Leu Val Thr Lys Ala Trp Val Asp Gln Glu Glu Val Leu Lys His Glu 340 345 350 Ser Val Ala Leu Phe Val Ser His Cys Gly Trp Asn Ser Val Thr Glu 355 360 365 Ala Ala Ala Ser Gly Val Pro Val Leu Ala Leu Pro Arg Phe Gly Asp 370 375 380 Gln Arg Val Asn Ser Gly Val Val Ala Arg Ala Gly Leu Gly Val Trp 385 390 395 400 Ala Asp Thr Trp Ser Trp Glu Gly Glu Ala Gly Val Ile Gly Ala Glu 405 410 415 Glu Ile Ser Glu Lys Val Lys Ala Ala Met Ala Asp Glu Ala Leu Arg 420 425 430 Arg Lys Ala Ala Ser Leu Ala Lys Ala Ala Ala Lys Ala Val Ala Gly 435 440 445 Gly Gly Ser Ser His Arg Cys Leu Val Glu Phe Ala Arg Leu Cys Gln 450 455 460 Gly Gly Thr Cys Arg Thr Asn 465 470 <210> 9 <211> 990 <212> DNA <213> Lotus japonicus <400> 9 atggcttctg caacaaccac cccaaccaaa gagatagaca gagacctccc tcctcttctc 60 cgagtctaca aagatggaac cgtggaacgt ctcctaggct ctcctgtcgt tccagcaatc 120 cctcatgacc cagaaacaga ggtctcatca aaagacatag tcatctcaca aaccccctta 180 atctctgctc gtatccacct cccaaaacag agcaaccccc aaaacccaaa ggttccaata 240 ttgatctact accatggtgg tgcgttttgc cttgaatcag ctttctcctt cctccaccaa 300 cgctacctca acatcgtggc ttcacgatca aacgttgtgg tggtttccgt cgagtacagg 360 ctcgcgccag agcatcctct gcctgcagca tatgaagatg gttgggaagc tctgaaatgg 420 gttacctctc attccaccga caacaaaccc atcaactctg agccatggtt gatcgaacat 480 ggtgatttca gcagattcta catcggaggt gacacttcag gtgccaacat cgcataccat 540 gtgggtctcc gtgtcggtgg tggagttgag aaattgccag gggatgtgaa aattgcaggg 600 gcgttacttg cttttcccct gttttggagt tcataccctg ttttggaaga acctgttgag 660 gggtttgaac agagtttgag caggaaggtt tggaactttg tgtacccaga tgcacctggt 720 gggatcgaca accctctgat caatcctttg gctgatgggg ctccaagctt gacaacgttt 780 ggaagcaaca agatgttgat ctttgttgca gggaatgatg aactgagaga cagaggaatc 840 tggttctatg aggctgtgaa gaagagtgag tgggaaggtg atgtggaact cattcgagtg 900 gatggagagg agcattgctt ccagatttac catcctgaat ctgagaattc taaagacatg 960 atgaagcgca tagcttcttt ccttgtttga 990 <210> 10 <211> 329 <212> PRT <213> Lotus japonicus <400> 10 Met Ala Ser Ala Thr Thr Thr Pro Thr Lys Glu Ile Asp Arg Asp Leu 1 5 10 15 Pro Pro Leu Leu Arg Val Tyr Lys Asp Gly Thr Val Glu Arg Leu Leu 20 25 30 Gly Ser Pro Val Val Pro Ala Ile Pro His Asp Pro Glu Thr Glu Val 35 40 45 Ser Ser Lys Asp Ile Val Ile Ser Gln Thr Pro Leu Ile Ser Ala Arg 50 55 60 Ile His Leu Pro Lys Gln Ser Asn Pro Gln Asn Pro Lys Val Pro Ile 65 70 75 80 Leu Ile Tyr Tyr His Gly Gly Ala Phe Cys Leu Glu Ser Ala Phe Ser 85 90 95 Phe Leu His Gln Arg Tyr Leu Asn Ile Val Ala Ser Arg Ser Asn Val 100 105 110 Val Val Val Ser Val Glu Tyr Arg Leu Ala Pro Glu His Pro Leu Pro 115 120 125 Ala Ala Tyr Glu Asp Gly Trp Glu Ala Leu Lys Trp Val Thr Ser His 130 135 140 Ser Thr Asp Asn Lys Pro Ile Asn Ser Glu Pro Trp Leu Ile Glu His 145 150 155 160 Gly Asp Phe Ser Arg Phe Tyr Ile Gly Gly Asp Thr Ser Gly Ala Asn 165 170 175 Ile Ala Tyr His Val Gly Leu Arg Val Gly Gly Gly Val Glu Lys Leu 180 185 190 Pro Gly Asp Val Lys Ile Ala Gly Ala Leu Leu Ala Phe Pro Leu Phe 195 200 205 Trp Ser Ser Tyr Pro Val Leu Glu Glu Pro Val Glu Gly Phe Glu Gln 210 215 220 Ser Leu Ser Arg Lys Val Trp Asn Phe Val Tyr Pro Asp Ala Pro Gly 225 230 235 240 Gly Ile Asp Asn Pro Leu Ile Asn Pro Leu Ala Asp Gly Ala Pro Ser 245 250 255 Leu Thr Thr Phe Gly Ser Asn Lys Met Leu Ile Phe Val Ala Gly Asn 260 265 270 Asp Glu Leu Arg Asp Arg Gly Ile Trp Phe Tyr Glu Ala Val Lys Lys 275 280 285 Ser Glu Trp Glu Gly Asp Val Glu Leu Ile Arg Val Asp Gly Glu Glu 290 295 300 His Cys Phe Gln Ile Tyr His Pro Glu Ser Glu Asn Ser Lys Asp Met 305 310 315 320 Met Lys Arg Ile Ala Ser Phe Leu Val 325 <210> 11 <211> 1374 <212> DNA <213> Buckwheat <400> 11 atgatgggcg acttaaccac atcttttcca gcaaccacac tcaccacgaa tgatcaacct 60 cacgtggtgg tttgctcagg agctggtatg gggcatttga caccgttcct taacttggcc 120 tctgctcttt catctgcgcc gtataactgc aaggttactc tgttaatcgt gattccgctt 180 atcacagatg ccgaaagtca ccacattagc agttctttct cttcacatcc cactatccac 240 aggctagact tccacgtgaa tttaccggct ccaaagccca atgtggatcc gttctttctc 300 cgttacaaga gcatctcaga ctctgcacat agacttcctg tccacttatc ggccttgagt 360 cccccgatat ctgccgtttt ctctgacttc ctcttcactc agggtctgaa cactactttg 420 cctcatcttc ccaactacac gttactacg acttctgctc ggtttttcac cttgatgtcc 480 tatgttcctc acttggccaa atcgagctca tcaagtcctg tagagattcc tggccttgag 540 ccgtttccaa ctgacaacat ccctccccct ttttcaacc cagagcacat ctttaccagt 600 ttcaccatat ccaatgccaa gtacttctcg ttgagcaaag ggattttggt gaataccttc 660 gactccttg agcccgagac actctctgca ttgaactcgg gagacacatt gtcagatctc 720 cctccggtca tccctattgg cccactgaac gaactcgagc ataacaaaca ggaggaactc 780 ctgccatggc tcgaccaaca acctgagaaa tccgtcctttt acgtctcttt tgggaatcga 840 acagccatga gtagcgatca gattctcgag ctaggaatgg gattagagcg tagtgattgt 900 cgctttattt gggtcgttaa gacgtccaag atagacaagg acgataagag tgagctacgt 960 aaactctttg gtgaagagct ctaccttaaa ctgtccgaga agggaaagct tgtgaaatgg 1020 gtgaaccaaa ccgagattct cgggcatact gctgtaggtg gtttcctgtc tcattgtggg 1080 tggaattcgg tcatggaagc agccagaaga ggtgttccga tattggcttg gccacaacac 1140 ggtgatcagc gggaaaatgc atgggttgtt gaaaaggctg gcttgggagt gtgggaaagg 1200 gagtgggcaa gcgggattca ggcagccatt gttgagaagg tcaagatgat catgggcaat 1260 aacgaccttc gcaaaagcgc tatgaaggtg ggtgaagagg caaagcgagc atgtgatgta 1320 ggtgggagtt ccgctaccgc cttgatgaat attatcgggt cattgaagag gtaa 1374 <210> 12 <211> 457 <212> PRT <213> Buckwheat <400> 12 Met Met Gly Asp Leu Thr Thr Ser Phe Pro Ala Thr Thr Leu Thr Thr 1 5 10 15 Asn Asp Gln Pro His Val Val Val Cys Ser Gly Ala Gly Met Gly His 20 25 30 Leu Thr Pro Phe Leu Asn Leu Ala Ser Ala Leu Ser Ser Ala Pro Tyr 35 40 45 Asn Cys Lys Val Thr Leu Leu Ile Val Ile Pro Leu Ile Thr Asp Ala 50 55 60 Glu Ser His His Ile Ser Ser Phe Phe Ser Ser His Pro Thr Ile His 65 70 75 80 Arg Leu Asp Phe His Val Asn Leu Pro Ala Pro Lys Pro Asn Val Asp 85 90 95 Pro Phe Phe Leu Arg Tyr Lys Ser Ile Ser Asp Ser Ala His Arg Leu 100 105 110 Pro Val His Leu Ser Ala Leu Ser Pro Pro Ile Ser Ala Val Phe Ser 115 120 125 Asp Phe Leu Phe Thr Gln Gly Leu Asn Thr Thr Leu Pro His Leu Pro 130 135 140 Asn Tyr Thr Phe Thr Thr Thr Ser Ala Arg Phe Phe Thr Leu Met Ser 145 150 155 160 Tyr Val Pro His Leu Ala Lys Ser Ser Ser Ser Ser Pro Val Glu Ile 165 170 175 Pro Gly Leu Glu Pro Phe Pro Thr Asp Asn Ile Pro Pro Pro Phe Phe 180 185 190 Asn Pro Glu His Ile Phe Thr Ser Phe Thr Ile Ser Asn Ala Lys Tyr 195 200 205 Phe Ser Leu Ser Lys Gly Ile Leu Val Asn Thr Phe Asp Ser Phe Glu 210 215 220 Pro Glu Thr Leu Ser Ala Leu Asn Ser Gly Asp Thr Leu Ser Asp Leu 225 230 235 240 Pro Pro Val Ile Pro Ile Gly Pro Leu Asn Glu Leu Glu His Asn Lys 245 250 255 Gln Glu Glu Leu Leu Pro Trp Leu Asp Gln Gln Pro Glu Lys Ser Val 260 265 270 Leu Tyr Val Ser Phe Gly Asn Arg Thr Ala Met Ser Ser Asp Gln Ile 275 280 285 Leu Glu Leu Gly Met Gly Leu Glu Arg Ser Asp Cys Arg Phe Ile Trp 290 295 300 Val Val Lys Thr Ser Lys Ile Asp Lys Asp Asp Lys Ser Glu Leu Arg 305 310 315 320 Lys Leu Phe Gly Glu Glu Leu Tyr Leu Lys Leu Ser Glu Lys Gly Lys 325 330 335 Leu Val Lys Trp Val Asn Gln Thr Glu Ile Leu Gly His Thr Ala Val 340 345 350 Gly Gly Phe Leu Ser His Cys Gly Trp Asn Ser Val Met Glu Ala Ala 355 360 365 Arg Arg Gly Val Pro Ile Leu Ala Trp Pro Gln His Gly Asp Gln Arg 370 375 380 Glu Asn Ala Trp Val Val Glu Lys Ala Gly Leu Gly Val Trp Glu Arg 385 390 395 400 Glu Trp Ala Ser Gly Ile Gln Ala Ala Ile Val Glu Lys Val Lys Met 405 410 415 Ile Met Gly Asn Asn Asp Leu Arg Lys Ser Ala Met Lys Val Gly Glu 420 425 430 Glu Ala Lys Arg Ala Cys Asp Val Gly Gly Ser Ser Ala Thr Ala Leu 435 440 445 Met Asn Ile Ile Gly Ser Leu Lys Arg 450 455 <210> 13 <211> 71 <212> DNA <213> Arabidopsis thaliana <400> 13 acaaattcaa aatttaacac acaaacacaa acacacacac caaaaaaaac acagacctta 60 aaaaaataaa a 71 <210> 14 <211> 1374 <212> DNA <213> Buckwheat <400> 14 atgatgggag atttaacaac ttcttttccg gcaaccacat taaccaccaa tgaccaaccc 60 catgttgtcg tttgttcggg tgcggggatg ggccacttaa ccccattcct caacctagct 120 tccgcccttt cctccgcacc ctacaactgt aaagtcaccc tactcattgt catccctctt 180 atcaccgatg ctgaatccca ccatatctcg tccttctttt cctctcaccc caccatccac 240 cgcctcgact tccacgtcaa cctccccgcc cccaaaccta acgtcgaccc tttcttctta 300 cgctacaaaa gcatctccga ctcggcccac cgcctccccg tccatctctc agccctctcg 360 ccacccatct ccgccgtgtt ttccgacttt ctattcacac aaggactcaa cactactctc 420 cctcacctcc ctaactacac cttcaccacc acctccgcaa ggttcttcac tctcatgtct 480 tacgttcctc acttggctaa atcgtcatcg tcctcgcccg tcgagattcc cggccttgaa 540 ccttttccga ccgacaacat ccctcctcct ttcttcaatc ccgaacacat cttcacttcc 600 ttcacaatct ccaacgctaa gtatttttct ctttccaaag ggattctcgt caacacattc 660 gactccttcg aaccggaac attatcggcg ctcaatccg gcgatactct ttccgatctc 720 cctccggtaa tccctatagg gcctcttaat gaacttgaac ataataaca agaggagtta 780 ctcccttggt tggatcaca accggaaa tccgtactgt acgtatcatt cgggaatagg 840 acggcgatgt cgagcgatca gatacttgag ctgggatgg gactggagag gagtgattgc 900 aggttcattt gggtggtga aaccagcaag attgacagg atgataatc ggagctacgg 960 aagctattcg gcgaggagtt gtacctgaag ctaagtgaga aagggaagtt agtgaatgg 1020 gtgaatcaa cggatttt agggcatacg gcggtcggag ggttttgag ccattgtggg 1080 tggaactccg tgatggaagc tgctcggcgc ggagttccga tcttagcg gccgcagcac 1140 ggggatcaga gagagaatgc ttggttggtg gagaaggcgg ggcttggagt tgggagaga 1200 gagtggcgt cggggattca ggcggcgatt gtggagaagg tgagatgat ttgggaat 1260 aatgatctga gaagaagtgc aatgaaggtt gggaggaag cgaagaggggc atgtgatgtt 1320 ggtggaagct ctgcaactgc attgatgaac atcatcggaa gtcttaaacg ttga 1374 <210> 15 <211> 58 <212> DNA <213> Arabidopsis thaliana <400> 15 tacatcacaa tcacacaaaa ctaacaaaag atcaaaagca agttcttcac tgttgata 58

Claims

1. A vector comprising a CGT gene derived from buckwheat, characterized in that: The CGT gene from buckwheat is: (1-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 11; The CGT gene from buckwheat is attached with the untranslated region (5'-UTR) from the ADH gene from Arabidopsis thaliana, i.e., sequence number 15, or the untranslated region (5'-UTR) from the HSPRO gene from Arabidopsis thaliana, i.e., sequence number 13. The vector further contains the flavanone 2-hydroxylase, i.e., F2H gene, the dehydratase, i.e., FDH gene, the flavonoid F3'5'hydroxylase, i.e., F3'5'H gene, and the methyltransferase, i.e., MT gene, wherein: The F2H gene is selected from: (2-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 5, or (2-b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6; The FDH gene is selected from: (3-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 9, or (3-b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 10; The F3'5'H gene is selected from: (4-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 1, or (4-b) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2; and The MT gene is selected from: (5-a) a polynucleotide consisting of the base sequence of SEQ ID NO: 3, or (5-b) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO:

4.

2. A method for producing a transformed plant having a blue flower color or its self-propagated or hybridized progeny, or a vegetative propagation body, a plant tissue or a cell, characterized in that: The method comprises the step of introducing the vector according to claim 1 into a host plant so that delphinidin-type anthocyanin and flavonoid mono-C-glycoside coexist in cells of the plant.

3. The method according to claim 2, characterized in that The flavonoid mono-C-glycoside is apigenin 6-C-glycoside, luteolin 6-C-glycoside, triticum flavonoid 6-C-glycoside, apigenin 8-C-glycoside, luteolin 8-C-glycoside, or triticum flavonoid 8-C-glycoside.

4. The method according to claim 2 or 3, characterized in that: The delphinidin-type anthocyanin is selected from delphinidin, delphinidin, or petunidin or a combination thereof.

5. The method according to claim 2 or 3, characterized in that: The plant is selected from rose, chrysanthemum, carnation or lily.

6. The method according to claim 5, characterized in that The plant is rose.

Images