Flavanone 2-hydroxylase gene and application thereof

Abstract

The present invention provides a transgenic plant having enhanced stress resistance and / or with a change in flower color, preferably with a change in flower color to blue, or a self-fertilized or filial generation thereof, or a part of a plant body, a tissue or a cell thereof, and provides a method capable of producing 2-hydroxyflavanone in a simple and efficient manner. The present invention uses a polynucleotide encoding an enzyme belonging to the 2-ketoglutaric acid dependent dioxygenase (ODD) family and having an activity of hydroxylating position 2 of a flavanone.

Description

Technical Field

[0001] The present invention relates to a novel polynucleotide and its application, and the polynucleotide encodes an enzyme belonging to the 2-oxoglutarate-dependent dioxygenase (ODD) family and having the activity of hydroxylating the 2-position of flavanone. Background Art

[0002] Flavonoid compounds, as representative plant secondary metabolites, start from phenylalanine and can be classified into more than a dozen categories such as flavanone, flavone, isoflavone, flavonol, and anthocyanin according to the C-ring structure. The enzymes related to their biosynthesis or the genes encoding the enzymes have been studied in depth. Isoflavone and flavone are both biosynthesized from flavanone. For isoflavone, isoflavone synthase catalyzes the hydroxylation of the 2-position of flavanone and the migration of the B-ring (Non-Patent Document 1), and further becomes isoflavone through the action of 2-hydroxyisoflavone dehydratase (Non-Patent Document 2). Isoflavone synthase belongs to the cytochrome P450 family, and 2-hydroxyisoflavone dehydratase belongs to the carboxylesterase family. Flavones mostly exist in plants in the form of aglycone, C-glycoside, and O-glycoside (Non-Patent Document 3). For example, carnation (Dianthus caryophyllus) or Dianthus sp. of the Caryophyllaceae family produces flavone O-glycoside and flavone C-glycoside (Non-Patent Documents 4, 5, and 6). In addition, gentian, wasabi, buckwheat, or plants of the Gramineae family also produce flavone C-glycoside. The functions of flavonoids in plants include ultraviolet protection, interaction with microorganisms, biological resistance protection as phytoalexin, and bluing of anthocyanin based on the copigmentation effect. For example, flavone C-glycosides such as isovitexin function as copigments in iris or genetically modified carnations to achieve bluing of flower color (Non-Patent Documents 7 and 8).

[0003] Flavonoids are synthesized from flavanones via two pathways (Non-Patent Document 3). Flavonoid synthase catalyzes the reaction of the conversion of flavanones to flavonoids. It is known that flavonoid synthase also includes flavonoid synthase I belonging to the dioxygenase family (Non-Patent Document 9) and flavonoid synthase II belonging to the cytochrome P450 family (Non-Patent Document 10), and the genes encoding them. The generated flavonoids are metabolized to flavonoid O-glycosides or flavonoid C-glycosides by UDP-glucose-dependent glycosyltransferase (UGT). For example, the flavonoid O-glycosyltransferase gene of Petunia hybrida has been obtained (Non-Patent Document 11), and the flavonoid C-glycosyltransferase genes of Gentiana triflora or Wasabia japonica (Non-Patent Documents 12 and 13). Another synthetic pathway uses 2-hydroxyflavanone as an intermediate, and first generates 2-hydroxyflavanone through the catalytic action of flavanone 2-hydroxylase (Non-Patent Document 14). Then, glycosyltransferase acts on the hydroxyl group at the 2-position thereof to generate 2-hydroxyflavanone C-glycoside. The gene of this glycosyltransferase has been reported (Non-Patent Document 15). Under in vitro conditions, 2-hydroxyflavanone C-glycoside is converted into a mixture of flavone 6-C-glycoside and flavone 8-C-glycoside (typically isovitexin, vitexin) by a spontaneous dehydration reaction (Non-Patent Documents 15 and 16). The enzymes involved in flavonoid biosynthesis vary in molecular species depending on the plant species, and are considered to be related to enzymes belonging to the P450 family or enzymes belonging to the soluble 2-oxoglutarate-dependent dioxygenase (ODD) family. However, to date, it has not been determined that the enzymes belonging to the ODD family have the activity of specifically hydroxylating the 2-position of flavanones.

[0004] Non-Patent Document [Non-Patent Document 1]Plant Physiol.1999 121:821 - 828 [Non-Patent Document 2]Plant Physiol 2005 137:882 - 91 [Non-Patent Document 3]Phytochemistry 2005 66:2399 - 2407 [Non-Patent Document 4]Z.Naturforsch.63c,161D168(2008) [Non-Patent Document 5]Chem.Pharm.Bull.59(9)1141 - 1148(2011) [Non-Patent Document 6]Phytochemistry.2003May;63(1):15 - 23 [Non-Patent Document 7]Plant Physiol Biochem.2013 72:116 - 24 [Non-Patent Document 8]Phytochemistry.2003;63:15 - 23 [Non - Patent Document 9] Phytochemistry 2001 58:43 - 6 [Non - Patent Document 10] Plant Cell Physiol. 1999 40:1182 - 6 [Non - Patent Document 11] Plant Cell Physiol. 2018 59:2075 - 2085 [Non - Patent Document 12] FEBS Lett. 2015 589:182 - 7 [Non - Patent Document 13] Plant Cell Physiol. 2019 Dec 1; 60(12):2733 - 2743 [Non - Patent Document 14] FEBS Lett. 1998 Jul 17; 431(2):287 - 90 [Non - Patent Document 15] J Biol Chem. 2009 3; 284:17926 - 34 [Non - Patent Document 16] Metabolic Engineering 2013 16:11 - 20 Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a transgenic plant or its self - fertilized or hybrid offspring or their vegetative propagules, a part of the plant body, a tissue or a cell thereof with enhanced stress resistance and / or accompanied by a change in flower color, preferably changed to a blue - based flower color, and to provide a simple and efficient method for producing 2 - hydroxyflavanone.

[0006] Generally, in the secondary metabolic system of plants, even if the plants are of different species, the enzymes that carry out the same reaction show a high degree of conservation in their base and amino acid sequences. That is, they are genes derived from a common ancestral gene and are the same genes formed along with species differentiation, and are considered to have an orthologous relationship. However, the biosynthetic mechanism of flavonoids and related enzymes vary depending on the plant species, and it is speculated that they have independently acquired metabolic systems in each plant. The present inventors have conducted in - depth research based on the publicly available RNA sequencing data and biochemical tests, and as a result, have made the amazing discovery that the enzyme belonging to the ODD family from Caryophyllaceae plants has the activity of specifically hydroxylating the 2 - position of flavanone, thus completing the present invention.

[0007] The present invention is as follows. [1] A polynucleotide which is a polynucleotide encoding an enzyme belonging to the 2 - ketoglutarate - dependent dioxygenase (ODD) family and having the activity of hydroxylating the 2 - position of flavanone, characterized in that the polynucleotide is selected from the following (A) to (E): (A) A polynucleotide consisting of the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (B) A polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (C) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12; (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 appended in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12; and, (E) A polynucleotide encoding a protein having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12. [2] The polynucleotide according to 1, which is characterized in that it is a polynucleotide consisting of the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11. [3] The polynucleotide according to 1, which is characterized in that it is a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12. [4] A protein, which is characterized in that it is encoded by the polynucleotide according to any one of 1 to 3. [5] A vector, which is characterized in that it contains the polynucleotide according to any one of 1 to 3. [6] A transgenic plant or its self-fertilized or hybrid offspring, which is characterized in that it contains the polynucleotide according to any one of 1 to 3. [7] A vegetative propagule, a part of a plant, a tissue or a cell, which is characterized in that it is a vegetative propagule, a part of a plant, a tissue or a cell of the transgenic plant according to 6 or its self-fertilized or hybrid offspring. [8] A cut flower or a processed product made from the cut flower, which is characterized in that it is a cut flower of the transgenic plant according to 6 or its self-fertilized or hybrid offspring or a processed product made from the cut flower. [9] A method, which is a method for producing a transgenic plant, and is characterized in that it includes a step of introducing a polynucleotide encoding an enzyme belonging to the 2-oxoglutarate-dependent dioxygenase (ODD) family and having the activity of hydroxylating the 2-position of flavanone into a plant cell.

[10] The method according to 9, which is characterized in that the polynucleotide is selected from the following (A) to (E): (A) A polynucleotide consisting of the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (B) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11; (C) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12; (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 appended in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12; and, (E) A polynucleotide encoding a protein having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12.

[11] A non-human host, characterized by comprising the polynucleotide according to any one of 1 to 3.

[12] A method for producing an enzyme having the activity of hydroxylating the 2-position of flavanone, characterized by comprising the step of culturing the non-human host according to 11, and, The step of extracting from the non-human host an enzyme having the activity of hydroxylating the 2-position of flavanone.

[13] A method for producing 2-hydroxyflavanone, characterized by comprising contacting the enzyme produced by the method according to 12 with flavanone.

[0008] By the present invention, there is provided a transgenic plant or its self-fertilized or hybrid offspring or their vegetative propagules, a part of the plant body, a tissue, or a cell having enhanced stress resistance and / or accompanied by a change in flower color, preferably changed to a blue flower color system. In addition, by the present invention, there is provided a non-human host (such as yeast or bacteria, etc.) expressing an enzyme having the activity of hydroxylating the 2-position of flavanone. By using this host, 2-hydroxyflavanone can be produced simply and efficiently. Description of the Drawings

[0009] Figure 1 Shown is the biosynthetic pathway for generating flavone C-glycosides in plants via 2-hydroxyflavanone as an intermediate. Figure 2 It is a high-performance liquid chromatography chart of an enzyme reaction solution after an enzyme reaction of a protein solution crudely extracted from Escherichia coli expressing SvF2H-1 or SvF2H-2 with naringenin. Figure 3 It is a high-performance liquid chromatography chart of an enzyme reaction solution after an enzyme reaction of a protein solution crudely extracted from Escherichia coli expressing AgF2H or DsF2H-1 with naringenin. Figure 4It is a high performance liquid chromatography map of the enzyme reaction solution after the enzyme reaction of naringenin with a protein solution crudely extracted from yeast expressing D_DN4746 or Dca16994.1. Detailed implementation mode

[0010] Flavonoids are contained as pigments in the flowers or leaves of plants. Flavonoids are a kind of organic compounds, which are cyclic ketones of flavan derivatives and mainly exist as glycosides in plants. Narrowly defined, flavonoids refer to the compound 2,3-dehydroflavan-4-one with the chemical formula C 15 H 10 O2 and a molecular weight of 222.24. Broadly defined, flavonoids (flavonoid) are one of the classifications of flavonoids. Among flavonoids, flavonoids with a flavonoid structure as the basic skeleton and without a hydroxyl group at the 3-position are classified as "flavonoids".

[0011] It is known that in plants, flavonoids are distributed in the form of glycosides in addition to the free form, mainly producing flavone O-glycosides and flavone C-glycosides. In particular, due to the coexistence of flavone C-glycosides and anthocyanins, which are a group of pigments widely present in plants, especially delphinidin-type anthocyanins such as delphinidin, malvidin, and petunidin, they interact intermolecularly with them, thus strongly showing the co-pigment effect of expressing a blue color. The co-pigment effect not only has a darkening effect of causing the expression of blue, but also has a thickening effect or an effect of improving the color stability.

[0012] In the specification of this application, "flavone C-glycoside" refers to flavonoids in a broad sense, that is, among the glycosides of derivatives belonging to flavonoids, the glycoside in which the aglycone is directly bonded to the anomeric carbon of an aldose. Examples of flavone C-glycosides include luteolin C-glycoside, tricin C-glycoside, apigenin C-glycoside, and acacetin C-glycoside, but are not limited thereto. Flavone C-glycosides also include glycosides of derivatives of apigenin, luteolin, tricin, and acacetin. In plants, there are two known biosynthetic pathways for flavone C-glycosides. Among them, in the pathway via 2-hydroxyflavanone as an intermediate, flavone 6-C-glycoside and flavone 8-C-glycoside are produced through the action of flavanone 2-hydroxylase (F2H), flavone C-glycosyltransferase (2HCGT), and dehydratase (2HDH) ( Figure 1 ). Therefore, it is speculated that by introducing the flavanone 2-hydroxylase (F2H) gene, one of the essential genes in this pathway, into the host plant, flavone C-glycosides can be accumulated in plant cells, thereby changing the flower color of the plant.

[0013] The inventors have successfully obtained a novel polynucleotide which encodes an enzyme belonging to the 2-oxoglutarate-dependent dioxygenase (ODD) family and having the activity of hydroxylating the 2-position of flavanone. The present invention relates to a polynucleotide encoding an enzyme belonging to the 2-oxoglutarate-dependent dioxygenase (ODD) family and having the activity of hydroxylating the 2-position of flavanone.

[0014] 2-oxoglutarate-dependent dioxygenase (ODD) is a water-soluble dioxygenase containing divalent iron, and can catalyze various oxidation reactions such as hydroxylation or demethylation for various biomolecules from small molecule compounds to proteins or DNA. ODD is widely present in bacteria, plants and animals. There are about 60 in humans, and 2OGD genes accounting for 0.5% are present in the genomes of various plant species, but a classification nomenclature based on phylogenetic analysis has not been established yet.

[0015] The enzyme belonging to the ODD family used in the present invention is typically from plants of the Caryophyllaceae family. Examples of plants of the Caryophyllaceae family include Silene vulgaris, Dianthus superbus, Agrostemma githago, Dianthus caryophyllus, Dianthus hybrida, etc., but are not limited to these.

[0016] The polynucleotide related to the present invention is preferably selected from the following (A) to (E): (A) A polynucleotide consisting of the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (B) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (C) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12; (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 in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12; and, (E) A polynucleotide encoding a protein having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12.

[0017] SEQ ID NO: 1 is the nucleotide sequence of a gene encoding a protein (SvF2H-1) having the activity of hydroxylating the 2-position of flavanone from Silene vulgaris, SEQ ID NO: 3 is the nucleotide sequence of a gene encoding a protein (SvF2H-2) having the activity of hydroxylating the 2-position of flavanone from Silene vulgaris, SEQ ID NO: 5 is the nucleotide sequence of a gene encoding a protein (DsF2H-1) having the activity of hydroxylating the 2-position of flavanone from Dianthus superbus, SEQ ID NO: 7 is the nucleotide sequence of a gene encoding a protein (AgF2H) having the activity of hydroxylating the 2-position of flavanone from Agrostemma githago, SEQ ID NO: 9 is the nucleotide sequence of a gene encoding a protein (D_DN4746) having the activity of hydroxylating the 2-position of flavanone from Dianthus caryophyllus, and SEQ ID NO: 11 is the nucleotide sequence of a gene encoding a protein (Dca16994.1) having the activity of hydroxylating the 2-position of flavanone from Dianthus caryophyllus.

[0018] In the present specification, the term "polynucleotide" refers to DNA or RNA. In the present specification, the term "stringent conditions" refers to conditions that allow a polynucleotide or oligonucleotide to selectively and detectably specifically bind to genomic DNA. Stringent conditions are defined by an appropriate combination of salt concentration, organic solvents (e.g., formamide), temperature, and other known conditions. That is, the stringency is increased by decreasing the salt concentration or increasing the organic solvent concentration, or by increasing the hybridization temperature. Further, the washing conditions after hybridization also affect the stringency. The washing conditions can also be defined by salt concentration and temperature, and the stringency of washing is increased by decreasing the salt concentration and increasing the temperature. Thus, the term "stringent conditions" refers to conditions that allow specific hybridization only between nucleotide sequences having a high degree of identity, for example, with an overall average identity of 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. As "stringent conditions", for example, conditions of 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 mentioned. As a specific example, hybridization is carried out 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 washing is carried out under the conditions of 0.1×SSC (15 mM NaCl, 1.5 mM trisodium citrate), 0.1% SDS, and 55°C.

[0019] Hybridization can be carried out according to methods well-known in the art, such as those described in Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987), or methods based thereon. In addition, when using a commercially available library, it can be carried out according to the methods described in the attached instruction manual. The gene selected by such hybridization can be a gene from nature, such as 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.

[0020] SEQ ID NO: 2 is the amino acid sequence of a protein (SvF2H-1) from Silene vulgaris with the activity of hydroxylating the 2-position of flavanone, SEQ ID NO: 4 is the amino acid sequence of a protein (SvF2H-2) from Silene vulgaris with the activity of hydroxylating the 2-position of flavanone, SEQ ID NO: 6 is the amino acid sequence of a protein (DsF2H-1) from Dianthus superbus with the activity of hydroxylating the 2-position of flavanone, SEQ ID NO: 8 is the amino acid sequence of a protein (AgF2H) from Agrostemma githago with the activity of hydroxylating the 2-position of flavanone, SEQ ID NO: 10 is the amino acid sequence of a protein (D_DN4746) from Dianthus caryophyllus with the activity of hydroxylating the 2-position of flavanone, and SEQ ID NO: 12 is the amino acid sequence of a protein (Dca16994.1) from Dianthus caryophyllus with the activity of hydroxylating the 2-position of flavanone.

[0021] 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 1 to 20 amino acids, preferably 1 to 5 amino acids, more preferably 1 to 3 amino acids. Site-directed mutagenesis, one of the methods of genetic engineering, is a method of introducing a specific mutation at a specific position, and thus is useful 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 the mutant DNA using an appropriate expression system, a protein composed of an amino acid sequence obtained by deletion, substitution, insertion and / or addition of one or more amino acids can be obtained. In addition, polynucleotides can be obtained by methods well-known to those skilled in the art, such as chemical synthesis methods such as the phosphoramidite method, nucleic acid amplification methods using primers designed based on the nucleotide sequence of the target gene with a nucleic acid sample of a plant as a template.

[0022] In this specification, the term "identity" refers to the amount (number) of substances that can be determined to be identical in the fitness relationship between each amino acid residue or each base constituting two strands in a polypeptide sequence (or amino acid sequence) or a polynucleotide sequence (or base sequence), and is a term representing the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. "Identity" 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, refer to 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.).

[0023] In addition, the value of "identity" described in this specification, unless otherwise specified, can be a value calculated using an identity search program well-known to those skilled in the art, preferably a 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, still more preferably about 98% or more, and most preferably 99% or more.

[0024] The polynucleotide (nucleic acid, gene) of the present invention is a substance that "encodes" the protein of interest. Here, the so-called "encoding" means expressing the protein of interest in a state with its activity. In addition, the so-called "encoding" includes two meanings: encoding the protein of interest as a continuous structural sequence (exon) or encoding it through intervening sequences (introns).

[0025] Genes with natural base sequences can be obtained, for example, by analysis using a DNA sequencer. In addition, DNA encoding an enzyme with a modified amino acid sequence can be synthesized based on DNA with a natural base sequence using common site-specific mutagenesis methods or PCR methods. For example, a DNA fragment to be modified is obtained by restriction enzyme treatment of natural cDNA or genomic DNA, and the site-specific mutagenesis method or PCR method is carried out using a primer into which the desired mutation has been introduced with this as a template, thereby obtaining the desired modified DNA fragment. Then, as long as the DNA fragment into which the mutation has been introduced is ligated to the DNA fragment encoding the other part of the target enzyme. Alternatively, in order to obtain DNA encoding an enzyme composed of a shortened amino acid sequence, for example, when DNA encoding an amino acid sequence longer than the target amino acid sequence (e.g., the full-length amino acid sequence) is cut by a desired restriction enzyme and the resulting DNA fragment cannot encode the entire target amino acid sequence, a DNA fragment composed of the lacking part of the sequence can be synthesized and ligated.

[0026] In addition, by expressing the obtained polynucleotide using the gene expression systems in Escherichia coli and yeast and measuring the enzyme activity, it can be confirmed that the obtained polynucleotide encodes a protein with the desired activity.

[0027] In addition, the present invention also relates to (transgenic) vectors containing the above polynucleotide, especially expression vectors. 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 constitutive expression of polynucleotides in plant cells include the 35S promoter of cauliflower mosaic virus, the El235S promoter connecting the enhancer regions of two 35S promoters, the rd29A gene promoter, the rbcS promoter, the mac-1 promoter, etc. In addition, for tissue-specific gene expression, the promoter of a gene specifically expressed in that tissue can be used.

[0028] The production of the vector can be carried out according to common methods using restriction enzymes, ligases, etc. In addition, the transgene of the host plant based on the expression vector can also be carried out according to common methods.

[0029] Furthermore, the present invention also relates to transgenic plants containing the polynucleotide or vector, or the self-fertilized or hybrid offspring thereof.

[0030] When producing transgenic plants, at the current technical level, techniques for introducing polynucleotides into plants and enabling constitutive or tissue-specific expression of the polynucleotides can be utilized. The introduction of DNA into plants can be carried out by methods well-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, and the like.

[0031] In the present invention, the plants that can be used as hosts are not particularly limited, and 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, and the genus Lilium in the Liliaceae family can be used. Particularly preferred is the cultivated rose (scientific name: Rosa hybrida) of the genus Rosa in the Rosaceae family. In addition, the term "rose plant" used in this specification refers to the cultivated rose (scientific name: Rosa hybrida) taxonomically classified as the genus Rosa in the Rosaceae family. Roses are mainly classified into Hybrid tea, Floribunda, Polyantha, etc. according to the tree form and flower size. In any system, the main pigments (anthocyanins) contained in the petals are only two types, namely the cyanidin type and the pelargonidin type. In the present invention, the species of rose plants that can be used as hosts are 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, Peach avalanche, etc.

[0032] By introducing the polynucleotide according to the present invention into plant cells, flavone C-glycosides can be accumulated in plant cells, and by functioning as phytoalexins, the stress resistance can be enhanced. Moreover, by coexisting with delphinidin-type anthocyanins, a co-pigmentation effect can be exerted, and thus transgenic plants with preferably improved flower colors to the blue series or their self-fertilized or hybrid offspring can be obtained.

[0033] Furthermore, the present invention also relates to cut flowers of the transgenic plants obtained by the above method or their self-fertilized or hybrid offspring, their vegetative propagules, parts of the plant body, tissues or cells, or processed products made from cut flowers (especially cut flower processed products). 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.

[0034] The present invention also relates to non-human hosts comprising the polynucleotide or vector. As non-human hosts, prokaryotes or eukaryotes can be used. As prokaryotes, bacteria can be used, such as bacteria belonging to the genus Escherichia, such as Escherichia coli, and microorganisms belonging to the genus Bacillus, such as Bacillus subtilis, etc., which are commonly used hosts. As eukaryotes, lower eukaryotes can be used, such as eukaryotic microorganisms, such as yeast or filamentous fungi as fungi. As yeast, for example, microorganisms belonging to the genus Saccharomyces can be cited, such as Saccharomyces cerevisiae, etc. In addition, as filamentous fungi, microorganisms belonging to the genus Aspergillus can be cited, such as Aspergillus oryzae, Aspergillus niger, and microorganisms belonging to the genus Penicillium. As non-human hosts, animal cells or plant cells can further be used. As animal cells, cell lines of mice, hamsters, monkeys, humans, etc. can be used, and further insect cells, such as silkworm cells, and the adult silkworms themselves can also be used as hosts.

[0035] Furthermore, the present invention relates to a method for producing an enzyme having an activity of hydroxylating the 2-position of flavanone, which is characterized by comprising the steps of introducing the polynucleotide according to the present invention into a non-human host, culturing the non-human host, and extracting from the non-human host an enzyme having an activity of hydroxylating the 2-position of flavanone, and a method for producing 2-hydroxyflavanone, which is characterized by comprising the step of bringing the enzyme produced by the above method into contact with flavanone. By these methods, 2-hydroxyflavanone can be produced simply and efficiently.

[0036] The present invention will be specifically described below by way of examples. Examples

[0037] [Example 1: Cloning of the full-length cDNA of a candidate gene encoding a protein having an activity of hydroxylating the 2-position of flavanone from Silene vulgaris and functional analysis thereof] After obtaining the transcriptome data of Silene vulgaris (SRA037583) from the SRA database of DDBJ, contigs were constructed by de novo assembly using Trinity. With respect to the constructed contigs, the amino acid sequence of Arabidopsis F3H (AT3G51240) was used as a query, and TBLASTN alignment was performed to screen out two candidate genes (SvF2H-1 and SvF2H-2) showing about 60% identity.

[0038] The nucleotide sequence of the SvF2H-1 gene is shown below.

[0039] The amino acid sequence of SvF2H-1 is shown below. MTKEESITVPEWEGEKSVKQTYVRDEDDRPKVAYNEFSDDIPVISLSGIDSADERAHIRSQITEACEKYGIFQVVNHGIDTDLISTMARLCSEFFNLPGHEKQRFGASEGKPGSFFVSSPFKGELVKNWRETVTQCTFPIKSGDYTLWPDKPDGWKAVTKEYSEKMMDLSHQLLGLLSEAMGLETEALSKACVTMDQRVMINYYPKCPQPDLTLGLPRHTDPGTITLLYQDLVGGLQATRDDGKTWITVPPIAGALVVNLGDHAYYVSNGRFTSAEHRAVVNSNQSRLSIATFQYPTAEATVFPLAIRQGEKPVMEEAITFAEMYRRKMSSDIERPKLKKLDGKMDKSKLESRPIGEIFV*(SEQ ID NO: 2)

[0040] The base sequence of the SvF2H-2 gene is shown below.

[0041] The amino acid sequence of SvF2H-2 is shown below. MTKEKATTLTELQDQNCINQSFVRDEDDRPKVAYNDFTDVIPVISLSGIDAHDERAEIRRKITEACETWGLFQVIDHGVELEIISEMARLSTEFFHLPASEKQQFAASDGKPGSFFVSSPFKRELVQNWRETVTQCTYPIKSGDYTLWPDKPEGWRKITKEYSDKMMELSHKLLGILSEAMGLETDALSKACVEMDQRVMINYYPKCPQPDLTLGLLRHTDPGTITLLYQDXVGGLXATCDDGVTWXTVPPIPGALVVNLGDHSHFVSNGRFVSAEHRAVVNSNTGRLSMVTFQYPTAEATVYPLTLREGETPIMEEAITFGEMYRRKMSKDLELPRNKKLDTKMEKSELESRPIDAIFI* (SEQ ID NO: 4)

[0042] The seeds of Silene vulgaris (Campion Bladder; Mitsuke Horticulture) were placed on filter paper moistened with sterilized water and germinated for 6 days under light (16 hours) / dark (8 hours) conditions. The young plant bodies were recovered, and after separating total RNA using the SV Total RNA Isolation System (Promega), cDNA was synthesized using SuperScriptIII Reverse Transcriptase (Invitrogen). Using this cDNA as a template, primers (SvF2H: 5′-ATGACAAAAGAGGAATCAATAACCG-3′ (SEQ ID NO: 13) and 5′-TCAAACAAAGATCTCGCCGATGG-3′ (SEQ ID NO: 14); SvF2H2: 5′-ATGACAAAAGAAAAGGCAACAACG-3′ (SEQ ID NO: 15) and 5′-TTAAATAAAGATGGCGTCAATGGG-3′ (SEQ ID NO: 16)) and PrimeSTAR MAX DNA Polymelase (Takara) were used to amplify the CDS. After adding dA to the amplification product using TaKaRa Ex Taq (Takara), it was introduced into the pCR8 / GW / TOPO Vector (Invitrogen). Further, an LR reaction was carried out using Gateway LR clonase II enzyme Mix (Invitrogen) and introduced into the pET-53-DEST vector (Novagen), and the Escherichia coli Rosetta2(DE3) strain was transformed. Additionally, as a control vector, a pET-53-DEST vector into which the GUS gene was introduced was used.

[0043] Escherichia coli was inoculated into 10 mL of LB liquid medium (50 mg / L kanamycin) and cultured overnight at 37°C. Then, 10 mL of the preculture solution was added to 240 mL of the same LB liquid medium and cultured with shaking at 37°C until the OD600 reached 0.4 - 0.6. Then, IPTG with a final concentration of 0.5 mM was added and cultured at 28°C for 4 hours. After recovering the cells by centrifugation (9,000 x g, 2 minutes), they were disrupted using glass beads in 50 mM Tris-HCl buffer (pH 8.0). The supernatant was obtained by centrifugation (4°C, 9,000 x g, 10 minutes) for enzyme assay. In the presence of 50 mM Tris-HCl (pH 8.0), 1 mM ketoglutaric acid, 0.4 mM FeSO4, and 4 mM sodium ascorbate, the substrate S-naringenin (10 μg) and the crude enzyme solution were added and incubated at 30°C for 10 minutes. After partitioning the reaction solution with ethyl acetate, the ethyl acetate layer was analyzed by HPLC.

[0044] In the reaction based on SvF2H-1 and SvF2H-2, it can be seen that 2-hydroxy naringenin is generated from S-naringenin ( Figure 2 ).

[0045] [Example 2: Obtaining a candidate gene encoding a protein with the activity of hydroxylating the 2-position of flavanone from Agrostemma githago and Dianthus superbus and analyzing its function] <Screening of candidate genes> The search for F2H was carried out using Agrostemma githago and Dianthus superbus as objects. Based on the publicly available RNA sequencing data, de novo assembly was performed to generate contigs. Then, sequences with a sequence identity of 75% or more with SvF2H and the putative amino acid were retrieved, and three candidate genes (DsF2H-1, DsF2H-2, AgF2H) were screened out.

[0046] The nucleotide sequence of the DsF2H-1 gene is shown below.

[0047] The amino acid sequence of DsF2H-1 is shown below. MTKEDKKNVTTLTELVDQKCINQSFVRDEDERPKVAYNEFSDEIPVISLLGIDDDSERTHIRRKITEACESWGIFQVVDHGVDLLLISEMARLSTEFFNLPAHEKQRFDTSGGKPGSFFVSSPFKGELVQNWRETVTQCTYPIKSGDFNLWPDKPEGWRKVTKDYGDKMMGLSHKILGLLSEAMGLETEALSKACVEMDQRVMINYYPKCPQPDLTLGLLRHTDPGTITLLYQDQVGGLQATRDDGVTWITVTPVPGALVVNLGDHAYYASNGRFTSAEHRAVVNSNQGRLSMATFQYPTAEAMVYPLTVSEGEKPVMEEAITFAEMYRRKMSKDLERPHLKKLDNTMDKSKLEGRAINEIFI*(SEQ ID NO: 6)

[0048] The base sequence of the AgF2H gene is shown below.

[0049] The amino acid sequence of AgF2H is shown below. MPQEESIITVSEMQEDKSVEQKFVRDEDDRPKVAYNEFSDDIPVISLSGIDSPDDRDHIRRQVTEACEEYGIFQVVDHGIDSDIISTMARQSSQFFNLPAPEKQRFAAAEGKPGSFFVSSPFKGELVQNWRETVTQCTFPIKSGDYTLWPDKPEGWRSVTKEYSEKMMDLSHKLLGVLSEAMGLETEALSNACVTMDQRVMINYYPKCPQPDLTLGLPRHTDPGTITLLYQDLVGGLQATRDDGKTWITVPPIPGALVVNLGDHAFYASNGRFTSAEHRAVVNSNQSRLSIATFQYPTAEAMVYPLAIREGETAVMEEAITFAEMYRRKMSSDIERPKHKKVDGKMDKSKLESRPMEEIFV*(SEQ ID NO: 8)

[0050] <cDNA Synthesis> Total RNA was extracted and purified from Silene gallica (about 40 mg) and Dianthus superbus (40 mg) within 7 days of germination using ISOSPIN Plant RNA (NIPPON GENE). The concentration of the purified total RNA was measured using Nano drop, and the purity was confirmed by electrophoresis on a 1% agarose gel. Then, reverse transcription reactions were performed on each purified total RNA and Oligo dT primer using SuperScript IV Reverse Transcriptase (Invitrogen) to synthesize cDNA.

[0051] <Cloning and Transformation of Candidate Genes> Using each of the above-synthesized cDNAs, the candidate gene sequences were amplified by PCR together with PrimeSTAR MAX (TAKARA BIO), a heat-resistant polymerase. The reaction system, primers used, and PCR conditions are as follows. (Reaction System) (Primers Used) DsF2H-1 Forward: ATGACAAAAGAAGATAAAAAAAATGTTAC (SEQ ID NO: 17) Reverse: TTAGATAAAGATCTCATTAATGGCC (SEQ ID NO: 18) DsF2H-2 Forward: ATGACAAAGGAAACACCCTCGACCG (SEQ ID NO: 19) Reverse: CTACACGAAGATTTCTCCAATAGC (SEQ ID NO: 20) AgF2H Forward: ATGCCTCAAGAAGAGTCAATAATAAC (SEQ ID NO: 21) Reverse: TCAAACAAAGATCTCTTCCATGGGC (SEQ ID NO: 22) (PCR conditions) 98°C for 1 minute → (98°C for 5 seconds → 53°C for 15 seconds → 72°C for 1 minute) × 35 cycles → 72°C for 1 minute → 12°C

[0052] <Purification of target DNA> For each amplified candidate gene, the target DNA was purified using the Wizard SV Gel and PCR Clean-Up System (Promega), and the concentration was measured using a Nano drop.

[0053] <Adding A to the 3' side of the target DNA> 10 μl of the target DNA was mixed with 10 μl of 2× SapphireAmp Fast PCR Master Mix (TAKARA BIO), and A was added to the 3' side of the target DNA at 60°C for 15 minutes using a thermal cycler.

[0054] <pCR8 / GW / TOPO and annealing> To introduce the product with added A into the pCR8 / GW / TOPO vector (Invitrogen) (T vector) as the introduction vector, the following reaction system was reacted at room temperature for 15 minutes. (Reaction system)

[0055] <Transformation> 3 μl of the T vector with the introduced gene was added to 100 μl of Escherichia coli DH5α melted on ice, slowly mixed, and then refrigerated in the refrigerator for 5 minutes. Then, 100 μl of S.O.C. Medium (Invitrogen), the basic medium for Escherichia coli, was added, and it was incubated at 37°C for 75 min without stirring. -1Oscillate and culture for 30 minutes under the following conditions. Then, add all the bacterial solution to LB agar medium containing spectinomycin (Spe) [LB Medium (Miller), granulated (Kanto Chemical), 25 g / L; AGAR, 15 g / L; 0.1 mM screening antibiotic], spread the bacterial solution using a spreader, and culture at 37 °C for 16 - 20 hours.

[0056] <Recovery of the target plasmid and next-generation sequencing> Add the selected colonies to 2 ml of LB liquid medium containing Spe [LB Medium, granulated, 25 g / L; 0.1 mM screening antibiotic], and culture overnight at 37 °C using a rotary shaker. The plasmid after culture was recovered using the Wizard Plus SV Minipreps DNA purification System (Promega), and the concentration was measured with a Nano drop. To confirm whether the plasmid has mutated, next-generation sequencing was used. PCR and ethanol precipitation were performed as preparations for sequencing. The reaction system and conditions for PCR are shown below. (Reaction system) Big Dye 1 μl ABI buffer 3.5 μl GW1 or GW2 Primer 2 μl (Diluted with 50 μM primer: water = 2:48) Plasmid X μl (300 - 500 ng, corresponding to the plasmid size) Water (DNAse RNAse Free) 13.5 - X μl (PCR conditions) 96 °C for 1 minute → (96 °C for 10 seconds → 50 °C for 5 seconds → 60 °C for 4 minutes) × 30 cycles → 4 °C

[0057] For ethanol precipitation, transfer all the sequencing reaction products to a 1.5 ml centrifuge tube, add 5 μl of 0.125 M EDTA and 60 μl of 99.5% ethanol, invert and stir several times, and then leave at room temperature for 15 minutes. Subsequently, centrifuge at 4 °C, 12,000 rpm for 15 minutes, aspirate the solution with a PIPETMAN without damaging the precipitate, add 100 μl of 70% ethanol, and centrifuge at 4 °C, 12,000 rpm for 10 minutes. Aspirate the solution again with a PIPETMAN without damaging the precipitate, and dry with a suction device for 3 - 5 minutes, and submit it to the Institute of Integrated Biosciences, Faculty of Bioresource Sciences, The University of Tokyo for DNA sequencing analysis.

[0058] <Introduction of the expression vector for the target plasmid> Based on the data obtained from the sequencing results, target colonies were screened and introduced into the pET-53-DEST vector. The reaction system was as follows, and a thermal cycler was used at 25 °C for 30 minutes. (Reaction system)

[0059] Then, 1 μl of Proteinase K (Invitrogen) was added to the reaction system, and a thermal cycler was used at 37 °C for 10 minutes. After the reaction, 3 μl of the reaction solution was added to 100 μl of DH5α, and they were slowly mixed and then placed in the refrigerator for 5 minutes. It was spread on a pre-incubated LB agar medium containing carbenicillin (Car) and cultured overnight at 37 °C.

[0060] <Recovery of target plasmid and next-generation sequencing> The propagated colonies were added to 2 ml of LB liquid medium containing Car and cultured overnight at 37 °C using a rotary shaker. The plasmid after culture was recovered using the Wizard Plus SV Minipreps DNA purification System, and the concentration was measured with a Nano drop. Next-generation sequencing was used to confirm whether the plasmid had mutated. PCR and ethanol precipitation were performed as preparations for sequencing. The reaction system and conditions for PCR are shown below. (Reaction system) Big Dye 1 μl ABI buffer 3.5 μl pET Upstream Primer 2 μl (Diluted with 50 μM primer: water = 2:48) Plasmid X μl (300 - 500 ng, corresponding to plasmid size) Water (DNAse RNAse Free) 13.5 - X μl (PCR conditions) 96 °C for 1 minute → (96 °C for 10 seconds → 50 °C for 5 seconds → 60 °C for 4 minutes) × 30 cycles → 4 °C

[0061] For ethanol precipitation, transfer all the sequencing reaction products to a 1.5-ml centrifuge tube, add 5 μl of 0.125 M EDTA and 60 μl of 99.5% ethanol. After inverting and stirring several times, let it stand at room temperature for 15 minutes. Then centrifuge at 4°C, 12,000 rpm for 15 minutes. Use a PIPETMAN to aspirate the solution without damaging the precipitate, add 100 μl of 70% ethanol, and centrifuge at 4°C, 12,000 rpm for 10 minutes. Again, use a PIPETMAN to aspirate the solution without damaging the precipitate, and dry it with a suction device for 3 - 5 minutes. Then commission for DNA sequencing analysis.

[0062] <Transformation of Escherichia coli for expression> For F2H, thaw 50 μl of Escherichia coli Rosetta2(DE3) on ice, add 2 μl of the plasmid of the colony confirmed to be introduced in the sequencing result, and spread it on the Car-containing LB agar medium pre-incubated at 37°C, and perform overnight culture at 37°C.

[0063] <Pre-culture, main culture and expression induction of F2H> Add the transformed Rosetta2(DE3) of one colony to 10 ml of Car-containing LB liquid medium, and perform overnight culture at 37°C and 180 rpm with an orbital shaker. The next day, add all the pre-culture broth to 240 ml of Car-containing LB liquid medium, and perform shaking culture at 37°C and 180 rpm until the OD600 reaches 0.4 - 0.6. Then add IPTG with a final concentration of 0.5 mM and perform shaking culture. After the shaking culture is completed, repeat the conditions of 4°C, 9000 x g for 2 minutes to collect only the cells.

[0064] <Preparation of the lysate of Escherichia coli in F2H> Place the collected cells on ice, add pre-cooled glass beads to the 5-ml mark in the tube, and add Binding Buffer to the 10-ml mark. Perform vortex stirring for 30 seconds, cool on ice for 30 seconds, and repeat this operation 3 times. Then centrifuge at 4°C, 9000 x g for 10 minutes, and collect only the supernatant. Add Binding Buffer again until it reaches the 10-ml mark, and repeat this operation 3 times. Filter the collected supernatant through a 0.45-μm filter. The composition of Binding Buffer is as follows.

[0065] <Purification of the enzyme protein> Pass 10 ml of distilled water through a His-Trap HP (GE Healthcare) column to remove the ethanol inside, and pass 10 ml of Binding Buffer to equilibrate the column. Pass the above-filtered supernatant through the column for adsorption, and then pass 10 ml of Binding Buffer through the column again to wash it. After washing, pass 5 ml of Elution Buffer through the column and fractionate it into 10 centrifuge tubes at 500 μl per tube. Recover 5 μl from each fraction, mix it with 100 μl of Protein Assay Bradford Reagent (Wako) by inverting and stirring, collect the blue-colored fractions, and transfer them to ice.

[0066] Pass 25 ml of 0.1 M potassium phosphate buffer (pH 7.5) through a Hi-Trap Desalting (GE Healthcare) column for washing and equilibration. Then pass the fraction solution transferred to ice through the column, pass 10 ml of 0.1 M potassium phosphate buffer (pH 7.5) through it, and fractionate it into 10 centrifuge tubes at 500 μl per tube. Recover 5 μl from each fraction, mix it with 100 μl of Protein Assay Bradford Reagent by inverting and stirring, recover the blue-colored fractions, measure the protein concentration by the Bradford method, and perform qualitative analysis by SDS-PAGE. The SDS-PAGE conditions are as follows: use XL-Ladder for the molecular weight marker and SuperSep for the gel plate. TM For the used His-Trap column, wash it with 10 ml of distilled water, rinse it with 10 ml of 20% ethanol, and store it refrigerated. Wash the Desalting column with 25 ml of 20% ethanol and store it refrigerated. The composition of the Elution Buffer is as follows.

[0067] <Enzyme assay in F2H> Dissolve the substrate S-type naringenin in methanol for use. To dissolve a specific amount of air-dried S-type naringenin, use 2-methoxyethanol, add Tris-HCl (pH 8.0), ketoglutaric acid, FeSO4, sodium ascorbate, distilled water, and then add the enzyme, and incubate it at 30 °C for 70 min. -1 Then add ethyl acetate and perform vortex stirring, and perform centrifugation for separation under the conditions of 4 °C, 9000 xg, and 2 minutes. Recover the ethyl acetate layer after centrifugation, dry it, dissolve it in 100 μl of methanol, and analyze it by HPLC. (Reaction system) [Table 1-1] Gene name Reaction time Substrate concentration range Enzyme amount SvF2H-1 15 minutes (30 minutes only at 5 μM) 5 μM to 100 μM 2.5 μg SvF2H-2 15 minutes 5 μM to 50 μM 1 μg AgF2H 15 minutes 2.5 μM to 50 μM 1 μg DsF2H-1 15 minutes 1 μM to 10 μM 0.5 μg [Table 1-2] Gene name Overall reaction system Amount of ethyl acetate added Amount of ethyl acetate recovered SvF2H-1 1ml 500 μl 300 μl SvF2H-2 3ml 1000 μl 500 μl AgF2H 1ml 500 μl 300 μl DsF2H-1 1ml 500 μl 300 μl Since the protein expression of DsF2H-2 was not confirmed, the test was not carried out.

[0068] <HPLC analysis> The test product obtained above was analyzed under the following HPLC analysis conditions. (Analysis conditions of HPLC) Column: TSK-Gel ODS-80TM Flow rate: 1.0 ml / minute Column temperature: 40 °C Gradient: Solvent A 40% → 100% (20 minutes) Solvent B 60% → 0% (20 minutes) Solvent A: Methanol (TFA 0.1%) Solvent B: Ultra-pure water (TFA 0.1%) Detection wavelength: 280 nm

[0069] <Results of functional analysis of AgF2H and DsF2H-1> The test of naringenin was carried out on the purified AgF2H and DsF2H-1, and the results were confirmed by HPLC. The results confirmed that 2-hydroxy naringenin was produced in the same way as the control SvF2H-1 ( Figure 3 ).

[0070] [Example 3: Obtaining a candidate gene encoding a protein having the activity of hydroxylating the 2-position of flavanone from Dianthus caryophyllus and Dianthus caryophyllus and its functional analysis] <Isolation of total RNA from Dianthus caryophyllus> Using the RNeasy Plant Mini Kit (QIAGEN), total RNA was isolated from the petals and leaves of the commercially available variety Miite Raspberry Rose (Suntory Flowers, Ltd.) of Dianthus caryophyllus according to the method recommended by the manufacturer.

[0071] <Analysis of cDNA expression level from Dianthus caryophyllus> Using the total RNA prepared above, a library for the next-generation sequencer NextSeq500 was prepared according to the procedures recommended by the manufacturer using the SureSelect Strand-Specific RNA Library Preparation Kit (Agilent Technologies). After determining the base sequence of the prepared library using NextSeq 500 (Illumina), the obtained reads were carefully examined. Subsequently, the reads of all samples were pooled and assembled using Trinity v2.12.0 to obtain contig sequences. Further, the paired-end reads of each sample were aligned to the obtained contig sequences using RSEM 1.3.0, and the FPKM values were calculated as the expression levels.

[0072] <Inference of gene function> For the contig sequences obtained above, DIAMOND searches against NCBI NR and BLAST searches against Araport11 (each with E-value ≤ 1.0) were performed, and functional annotation (inference of gene function) was carried out.

[0073] <Obtaining full-length cDNA of candidate genes from Dianthus and carnation> Using the amino acid sequence of SvF2H-1, a BLAST search was performed on the obtained contig sequences to screen for D_DN4746 (from Dianthus) with the highest identity. Further, similarly for Carnation DB (http: / / carnation.kazusa.or.jp / ), a BLAST search was performed using the amino acid sequence of SvF2H-1 to screen for Dca16994.1 (from carnation) with the highest identity. Based on the sequence of the assembled full-length cDNA, full-length cDNA clones were obtained by artificial gene synthesis. Additionally, the identity between D_DN4746 and Dca16994.1 at the amino acid level was 98.9%.

[0074] The base sequence of the D_DN4746 gene is shown below.

[0075] The amino acid sequence of D_DN4746 is shown below. MTKETPSTVTVWENETSVKQKFVRDEDDRPKVAYNEFSDEIPVISLNGIDTDNERAHIRSKITEACETWGIFQVVD HGVDPNLVSEMARLSTEFFNLPIHEKLRFGASDGKPGSFFVSSPFKGELVKNWRETVTQCTYPIKSGDYTLWPDSP DGWKNVTKEYSEKMMDLTHKLLGVLSEAMGLETEALSKACVDMDQRVMINYYPKCPQPDLTLGLPRHTDPGTITLL YQDQVGGLQATRDDGKSWITVPPVAGALVVNLGDHAYYVSNGRFTSAEHRAVVNSNQSRLSIATFQYPTAEATIYP LAIREGEEPVMEEAITFAEMYRRKMSSDIERPKLKKLDGTMDKSKLEARAIGEIFV*(SEQ ID NO: 10)

[0076] The nucleotide sequence of the Dca16994.1 gene is shown below.

[0077] The amino acid sequence of Dca16994.1 is shown below. MTKEAPSTVTVWENETSVKQKFVRDEDDRPKVAYNEFSDEIPVISLNGIDTDNERAHIRSKITEACETWGIFQVVDHGFDPNLVSEMARLSTEFFNLPIHEKLRFGASDGKPGSFFVSSPFKGELVKNWRETVTQCTYPIKSGDYTLWPDSPDGWKNVTKEYSEKMMDLTHKLLGVLSEAMGLETEALSKACVDMDQRVMINYYPKCPQPDLTLGLPRHTDPGTITLLYQDQVGGLQATRDDGKSWITVPPVAGALVVNLGDHAYYVSNGRFTSAEHRAVVNSNQSRLSIATFQYPTAEATIYPLAIREGEEPVMEEAITFAEMYRRKMSSDIERPKQKLDGTMDKSKLEARAIGEIFV*(SEQ ID NO: 12)

[0078] The identities of D_DN4746 and the F2H genes (SvF2H-1, SvF2H-2, AgF2H, DsF2H-1) of Caryophyllaceae plants belonging to the ODD family were investigated at the amino acid level. The results showed that the identity with SvF2H-1 was 87.5%, with SvF2H-2 was 76.2%, with AgF2H was 81.5%, and with DsF2H-1 was 81.3%. On the other hand, the identities with the F2H genes of plants belonging to P450 (flavanone 2-hydroxylase (OsF2H) from rice (Oryza sativa), flavanone 2-hydroxylase (GeF2H) from Glycyrrhiza echinata L., flavanone 2-hydroxylase (MtF2H) from Medicago truncatula) were 7.7%, 7.8%, and 8.1% respectively. Further, the identities of Dca16994.1 and the F2H genes (SvF2H-1, SvF2H-2, AgF2H, DsF2H-1) of Caryophyllaceae plants belonging to the ODD family were investigated at the amino acid level. The results showed that the identity with SvF2H-1 was 87%, with SvF2H-2 was 75.6%, with AgF2H was 81.2%, and with DsF2H-1 was 80.5%. On the other hand, the identities with the F2H genes of plants belonging to P450 (flavanone 2-hydroxylase (OsF2H) from rice (Oryza sativa), flavanone 2-hydroxylase (GeF2H) from Glycyrrhiza echinata L., flavanone 2-hydroxylase (MtF2H) from Medicago truncatula) were 7.9%, 8.2%, and 7.3% respectively. Thus, D_DN4746 and Dca16994.1 can be clearly distinguished from the F2H genes belonging to P450. This indicates that the gene of the present invention is completely different from the enzyme genes of the P450 family.

[0079] <Production of yeast expression vector> Taking D_DN4746 and Dca16994.1 as protein candidates having the activity of hydroxylating the 2-position of flavanone, yeast expression vectors pSPB8380 and pSPB8379 containing the full length of D_DN4746 or Dca16994.1 were produced using pESC-TRP (Agilent Technologies) according to the method recommended by the manufacturer.

[0080] <Expression of flavanone 2-hydroxylase in yeast and enzyme reaction> pSPB8380 and pSPB8379 were introduced into the yeast strain YPH499 (Agilent Technologies) according to the method recommended by the manufacturer to obtain transgenic yeast. Using pESC Yeast Epitope Tagging Vectors (Agilent Technologies), these transgenic yeasts were made to express proteins according to the method recommended by the manufacturer, and 2 μg / mL of hemin and 100 μM of naringenin were added during cultivation to conduct in vivo enzymatic reactions. After centrifuging the obtained culture solution (3000 rpm, 4 °C, 10 minutes) to recover the supernatant, purification was carried out using a Sep-Pak C18 6cc Vac Cartridge (500 mg) (Waters Corporation) according to the method recommended by the manufacturer. After drying it with a SAVANT SpeedVac Concentrator SPD2010 (Thermo Fisher Scientific), it was dissolved in 200 μL of 50% aqueous acetonitrile solution and analyzed by high performance liquid chromatography (LC-2030C (Shimadzu Corporation)). The detector used was a Shimadzu PDA SPD-M20A, and detection was carried out at 280 nm. The column used was a Shim-Pack FC-ODS 150 mm × 4.6 mm (Shimadzu GL Sciences Inc.). The eluent used was solution A (0.1% aqueous formic acid solution) and solution B (100% methanol containing 0.1% formic acid). Elution was carried out based on the following process: a 12-minute linear concentration gradient from a 9:1 mixture of the two to a 4:6 mixture and a hold for 8 minutes, a 1-minute linear concentration gradient from a 4:6 mixture of the two to a 0:10 mixture and a hold for 1 minute, and a 0:10 mixture. The flow rate was 1.0 mL / min. As a control, the same experiment was carried out using transgenic yeast into which the pESC-TRP vector without an inserted fragment was introduced. As a result, reaction compounds were detected in the culture solution purified from the yeast expressing D_DN4746 and Dca16994.1. By comparison with the standard product, it was confirmed that this compound was 2-hydroxy naringenin in which the 2-position of the substrate naringenin was hydroxylated ( Figure 4 ).

[0081] From these results, it was clarified that D_DN4746 and Dca16994.1 exhibited activity for specifically hydroxylation at the 2-position of flavanone, indicating that D_DN4746 and Dca16994.1 were genes encoding proteins having activity for hydroxylation at the 2-position of flavanone. From the above results, these genes were identified as genes encoding proteins that hydroxylate the 2-position of flavanone.

Claims

1. A polynucleotide, which is a polynucleotide encoding an enzyme having an activity of hydroxylating the 2-position of flavanone, and is characterized in that, The polynucleotide is selected from the following (A) to (E): (A) A polynucleotide consisting of the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (B) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (C) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12; (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 in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12; and, (E) A polynucleotide encoding a protein having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12.

2. The polynucleotide according to claim 1, wherein Is a polynucleotide consisting of the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11.

3. The polynucleotide according to claim 1, wherein Is a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12.

4. A protein, characterized in that, Encoded by the polynucleotide according to any one of claims 1 to 3.

5. A carrier, characterized in that, Contains the polynucleotide according to any one of claims 1 to 3.

6. A transgenic plant or its self-fertilized or hybrid offspring, characterized in that, Contains the polynucleotide according to any one of claims 1 to 3.

7. A vegetative propagation body, a part of a plant body, a tissue or a cell, characterized in that: Is a vegetative propagule, a part of a plant body, a tissue or a cell of the transgenic plant according to claim 6 or its self-fertilized or hybrid offspring.

8. A cut flower or a processed product made from the cut flower, characterized in that, Is a cut flower of the transgenic plant according to claim 6 or its self-fertilized or hybrid offspring or a processed product made from the cut flower.

9. A method, which is a method for producing a transgenic plant, characterized in that, Contains a step of introducing a polynucleotide encoding an enzyme belonging to the 2-oxoglutarate-dependent dioxygenase (ODD) family and having the activity of hydroxylating the 2-position of flavanone into a plant cell.

10. The method according to claim 9, wherein The polynucleotide is selected from the following (A) to (E): (A) A polynucleotide consisting of the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (B) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (C) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12; (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 in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12; and, (E) A polynucleotide encoding a protein having an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12.

11. A non-human host, characterized in that, Contains the polynucleotide according to any one of claims 1 to 3.

12. A method, which is a method for producing an enzyme having an activity of hydroxylating the 2-position of flavanone, characterized in that, Contains a step of culturing the non-human host according to claim 11, and, A step of extracting an enzyme having the activity of hydroxylating the 2-position of flavanone from the non-human host.

13. A method, which is a method for manufacturing 2-hydroxyflavanone, characterized in that, Includes contacting the enzyme produced by the method according to claim 12 with flavanone.

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