NZ507150A

NZ507150A - Genes coding for flavone synthases

Info

Publication number: NZ507150A
Application number: NZ507150A
Authority: NZ
Inventors: Masako Mizutani; Yoshikazu Tanaka; Takaaki Kusumi; Shin-Ichi Yabe; Tomoyoshi Akashi
Original assignee: Suntory Ltd; Suntory Flowers Ltd
Priority date: 1999-07-19
Filing date: 2000-02-28
Publication date: 2003-11-28
Also published as: AU5572100A; WO2001005981A1; CN1310762A; IL138718A0; CN1624136A; CA2324228A1; EP1114162A1; KR20010071128A

Abstract

An isolated gene encodes a protein having the amino acid sequence of SEQ ID NO:2 and a protein with 70% homology to SEQ ID NO:2 and which shows activity of synthesizing flavones from flavanones. A method for altering the flavonoid composition and/or its amount in a plant uses the gene. Methods for altering the colour of flowers, bluing or reddening the colour of flowers, altering the photosensitivity of a plant, promoting a symbiotic interaction between a plant and microorganism and increasing plant resistance to microorganisms using the gene are described.

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number 507150 507150 - 1 - DESCRIPTION GENES CODING FOR FLAVONE SYNTHASES Technical Field 5 The present invention relates to the control and utilization of biosynthesis of flavones, which have effects on flower color, protection from ultraviolet ray, symbiosis with microorganisms, etc. in plants, by a genetic engineering technique. More specifically, it 10 relates to genes encoding proteins with activity of synthesizing flavones from flavanones, and to their utilization. Background Art The abundance of different flower colors is one of 15 the pleasant aspects of life that enriches human minds and hearts. It is expected to increase food production to meet future population increase by the means of accelerating the growth of plants through symbiosis with microorganisms, or by increasing the number of nitrogen-20 fixing leguminous bacteria, thus improving the plant productivity as a result of increasing the content of nitrogen in the soil. Elimination or reduction of the use of agricultural chemicals is also desirable to achieve more environmentally friendly agriculture, and 25 this requires improvement of the soil by the above- mentioned biological means, as well as higher resistance of plants against microbial infection. Another desired goal is to obtain plants with high protective functions against ultraviolet rays as a means of protecting the 30 plants from the destruction of the ozone layer. "Flavonoid" is a general term for a group of compounds with a C6-C3-C6 carbon skeleton, and they are widely distributed throughout plant cells. Flavonoids are known to have such functions as attracting insects 35 and other pollinators, protecting plant from ultraviolet rays, and participating in interaction with soil microorganisms (BioEssays, 16 (1994), Koes at al., p.123; - 2 - Trends in Plant Science, 1 (1997), Shirley, B.W., p.377). Of flavonoids, flavone plays an important role in interaction of plants with microorganisms, especially in legumes, where they participate in the initial steps of 5 the symbiosis with leguminous bacteria (Plant Cell, 7 (1995), Dixon and Paiva, p.1085; Annu. Rev. Phytopathol., 33 (1995), Spaink, p.345). Flavones in petals play a role in recognition by insects and act as copigments which form complexes with anthocyanins. (Gendai Kagaku, 10 (May, 1998), Honda and Saito, p.25; Prog. Chem. Org. Natl. Prod., 52 (1987), Goto, T., p.114). It is known that when flavone forms a complex with anthocyanin, the absorption maximum of the anthocyanin shifts toward the longer wavelength, i.e. toward blue. 15 The biosynthesis pathways for flavonoids have been widely studied (Plant Cell, 7 (1995), Holton and Cornish, p.1071), and the genes for all of the enzymes involved in the biosynthesis of anthocyanidin 3-glucoside and flavonol, for example, have been isolated. However, the 20 genes involved in the biosynthesis of flavones have not yet been isolated. The enzymes that synthesize flavones include those belonging to the dioxygenase family (flavone synthase I) that depends on 2-oxoglutaric acid and monooxygenase enzymes belonging to the cytochrome 25 P450 family (flavone synthase II). These groups of enzymes are completely different enzymes with no structural homology. It has been reported that in parsley, 2-oxoglutaric acid-dependent dioxygenase catalyzes a reaction which 30 produces apigenin, a flavone, from naringenin, a flavanone (Z. Naturforsch., 36c (1981), Britsch et al., p.742; Arch. Biochem. Biophys., 282 (1990), Britsch, p.152). The other type, flavone synthase II, is known to exist in snapdragon (Z. Naturforsch., 36c (1981), Stotz 35 and Forkmann, p.737) and soybean (Z. Naturforsch., 42c (1987), Kochs and Grisebach, p.343; Planta, 171 (1987), Kochs et al., p.519). A correlation has been recently reported between a gene locus and flavone synthase II activity in the petals of gerbera (Phytochemistry, 49 (1998), Martens and Forkmann, p.1953). However, there are no reports that the genes for these flavone synthases I and II were isolated or that flavone synthase II was highly purified. The properties of a cytochrome P450 protein, which had licodione-synthesizing activity that was induced when cultured cells of licorice (Glycyrrhiza echinata) were treated with an elicitor, were investigated. The protein is believed to catalyze the hydroxylation of 2-position of liquiritigenin which is a 5-deoxyflavanone, followed by non-enzymatic hemiacetal ring opening to produce licodione (Plant Physiol., 105 (1994), Otani et al., p.1427). For cloning of licodione synthase, a cDNA library was prepared from elicitor-treated Glycyrrhiza cultured cells, and 8 gene fragments encoding cytochrome P450 were cloned (Plant Science, 126 (1997), Akashi et al., p.39). From these fragments there were obtained two different full-length cDNA sequences, each encoding a cytochrome P450, which had been unknown until that time. Specifically, they were CYPGe-3 (cytochrome P450 NO.CYP81E1) and CYPGe-5 (cytochrome P450 NO.CYP93B1, hereinafter indicated as CYP93B1) (Plant Physiol., 115 (1997), Akashi et al., p.1288). By further expressing the CYP93B1 cDNA in a system using cultured insect cells, the protein derived from the gene was shown to catalyze the reaction synthesizing licodione from liquiritigenin, a flavanone, and 2-hydroxynaringenin from naringenin, also a flavanone. 2-Hydroxynaringenin was converted to apigenin, a flavone, by acid treatment with 10% hydrochloric acid (room temperature, 2 hours). Also, eriodictyol was converted to luteolin, a flavone, by reacting eriodictyol with microsomes of CYP93Bl-expressing yeast followed by acid treatment. It was therefore demonstrated that the - 4 - cytochrome P450 gene encodes the function of flavanone 2-hydroxylase activity (FEBS Lett., 431 (1998), Akashi et al., p.287). Here, production of apigenin from naringenin required. CYP93B1 as well as another unknown 5 enzyme, so that it was concluded that a total of two enzymes were necessary. However, no genes have yet been identified for enzymes with activity of synthesizing flavones (such as apigenin) directly from flavanones (such as naringenin) 10 without acid treatment. Thus, despite the fact that flavones have numerous functions in plants, no techniques have yet been reported for controlling their biosynthesis in plants, and improving the biofunctions in which flavones are involved, such as flower color. The 15 discovery of an enzyme which by itself can accomplish synthesis of flavones from flavanones and acquisition of its gene, and introduction of such a gene into plants, would be more practical and industrially applicable than the introduction into a plant of genes for two enzymes 20 involved in the synthesis of flavones from flavanones. Disclosure of the Invention It is an aim of the present invention to provide flavone synthase genes, preferably flavone synthase II genes, and more preferably genes for flavone synthases 25 with activity of synthesizing flavones directly from flavanones. The obtained flavone synthase genes may be introduced into plants and over-expressed to alter flower colors. Moreover, in the petals of flowers that naturally 30 contain large amounts of flavones, it is expected that controlling expression of the flavone synthase genes by an antisense method or a cosuppression method can also alter flower colors. Also, expression of the flavone synthase genes in the appropriate organs, in light of the 35 antibacterial activity of flavones and their interaction with soil microorganisms, will result in an increase in the antibacterial properties of plants and improvement in - 5 - the nitrogen fixing ability of legumes due to promoted symbiosis with rhizosphere microorganisms, as well as a protective effect against ultraviolet rays and light. The present invention therefore provides genes 5 encoding proteins that can synthesize flavones directly from flavanones. The genes are, specifically, genes encoding flavone synthase II that can synthesize flavones from flavanones by a single-enzyme reaction (hereinafter referred to as "flavone synthase II"). More specifically, the present invention provides an isolated gene encoding a protein having the amino acid sequence of SEQ.ID. No. 2 and showing activity of synthesizing flavones from flavanones, or a gene encoding a protein having one of these amino acid sequences wherein the amino acid sequence has been modified within at least 70% identity with the amino acid sequence of SEQ ID No. 2, and possessing activity of synthesizing flavones from flavanones. Described is a gene encoding \ 20 proteins having amino acid sequences with at least 55% identity with the amino acid sequence of SEQ.ID. No. 2 and possessing activity of synthesizing flavones from flavanones. The invention still further provides isolated genes encoding 25 proteins possessing activity of synthesizing flavones from flavanones, and hybridizing with all or a part of the nucleotide sequence of SEQ.ID. No. 1 under the conditions of 5 x SSC, 50°C. The invention still further provides a vector, 30 particularly an expression vector, containing any one of the aforementioned genes. The invention still further provides a non-human host transformed with the aforementioned vector. The invention still further provides a protein 35 encoded by any of the aforementioned genes. Described is a process for producing the aforementioned protein which is intellectual office of n.z. 23 SEP 2003 - 6 - characterized by culturing or growing the aforementioned host, and collecting the protein with flavone-synthesizing activity from the host. The invention still further provides a plant into 5 which any one of the aforementioned genes has been introduced, or progenies of the plant or a tissue thereof, such as cut flowers, which exhibit the same properties. The invention still further provides a method of altering the flavonoid composition and/or its amount in a plant using the aforementioned genes; a method of altering the amount of flavone in a plant using the aforementioned genes; a method of altering flower colors using the aforementioned genes; a method of bluing the color of flowers using the 15 . aforementioned genes; a method of reddening the color of flowers using the aforementioned genes; a method of modifying the photosensitivity .of plants using the aforementioned genes; and a method of controlling the interaction between plants and microbes using the 20 aforementioned genes. Embodiments for Carrying out the Invention Flavanone 2-hydroxylase encoded by the Glycyrrhiza CYP93B1 gene produces 2-hydroxyflavanones from flavanones as the substrates, and the products are converted to 25 flavones by acid treatment. The present inventors viewed that it would be possible to obtain a gene encoding a flavone synthase II, which was an object of the invention, by using the Glycyrrhiza-derived cDNA, CYP93B1 for screening of a cDNA library of, for example, a flower 30 containing a large amount of flavones, to thus obtain cDNA encoding proteins with activity of synthesizing flavones directly from flavanones as substrates. According to the invention, a cDNA library of perilla which contains a large amount of flavones is 35 screened using the Glycyrrhiza-derived cDNA, CYP93B1 as a probe, to obtain cDNA encoding a novel cytochrome P450 (see Example 1). intellectual property office of n.z. 23 SEP 2003 RECEIVE!* ;- 7 - ;The perilla-derived cDNA was expressed in yeast and reacted with naringenin, a flavanonfe, as a substrate which resulted in production not of 2-hydroxynaringenin but rather of the flavone apigenin, without acid 5 treatment (see Example 2). In other words, this enzyme directly produced flavones from flavanones without acid treatment, and its gene was confirmed to be a flavone synthase II which had never been cloned. ;The genes of the present invention may be, for 10 example, one encoding the amino acid sequence of ;SEQ.ID. No. 2. However, it is known that proteins whose amino acid sequences are modified by additions or deletions of multiple amino acids and/or substitutions with different amino acids can 15 . maintain the same enzyme activity as the original protein. Consequently, proteins having the amino acid sequence of SEQ.ID. No. 2 ;wherein the amino acid sequence is modified within at least 70% ;identity with the amino acid sequence of SEQ ID No. 2, and genes ;* encoding those proteins, are also encompassed by the present invention so long as they maintain the activity of producing flavones directly from flavanones. The present invention also relates to genes that 25 have the nucleotide sequence of SEQ.ID. No. 1 and nucleotide sequence encoding the amino acid sequences listed therein, or that hybridize with portions of the nucleotide sequence under conditions of 5 x SSC, 50°C, for example, providing they encode proteins possessing 30 activity of producing flavones from flavanones. The suitable hybridization temperature will differ depending on nucleotide sequences and the length of nucleotide sequences, and for example, when the probe used is a DNA fragment comprising 18 bases coding for 6 amino acids, 35 the temperature is preferably not higher than 50°C. A gene selected by such hybridization may be a naturally derived one, such as a plant-derived gene, for intellectual property office of n.z. 23 SEP 2003 B F I? P I V F T> - 8 - example, a gene derived from snapdragon, torenia or perilla; it may also be a gene from another plant, such as gentian, verbena, chrysanthemum, iris, or the like. A gene selected by hybridization may be cDNA or genomic 5 DNA. The invention also relates to genes encoding proteins that have amino acid sequences with identity of at least 70%, such as 80% or greater and even 90% or greater, with the amino acid 10 sequence of SEQ ID.No. 2, and that possess activity of synthesizing flavones from flavanones. A gene with the natural nucleotide sequence can be obtained by screening of a cDNA library, for example, as 15 demonstrated in detail in the examples. DNA encoding enzymes with modified amino acid sequences can be synthesized using common site-directed mutagenesis or a PCR method, using DNA with a natural nucleotide sequence as a starting material. For example, a DNA fragment into 20 which a modification is to be introduced may be obtained by restriction enzyme treatments of natural cDNA or genomic DNA and then used as a template for site-directed mutagenesis or PCR using a primer having the desired mutation introduced therein, to obtain a DNA fragment 25 having the desired modification introduced therein. Mutation-introduced DNA fragments may then be linked to a DNA fragment encoding another portion of a target enzyme. Alternatively, in order to obtain DNA encoding an enzyme consisting of a shortened amino acid sequence, for 30 example, DNA encoding an amino acid sequence which is longer than the aimed amino acid sequence, such as the full length amino acid sequence, may be cut with desired restriction endonucleases, and if the DNA fragment obtained thereby does not encode the entire target amino 35 acid sequence, it may be linked with synthesized DNA comprising the rest of the sequence. Thus obtained genes may be expressed in an intellectual property office of n.z. 23 SEP 2003 - 9 - expression system using E. coli or yeast and its enzyme activity measured to confirm that the obtained gene encodes flavone synthase. By expressing the gene, it is also possible to obtain the flavone synthase protein as 5 the gene product. Alternatively, it is also possible to obtain a flavone synthase protein even using antibodies for a full or a partial amino acid sequence listed as SEQ.ID. No. 2, and such antibodies may be used for cloning of a flavone synthase gene in another organism. 10 Consequently, the invention also relates to recombinant vectors, and especially expression vectors, containing the aforementioned genes, and to hosts transformed by these vectors. The hosts used may be prokaryotic or eukaryotic organisms. Examples of 15 prokaryotic organisms that may commonly be used as hosts include bacteria belonging to the genus Escherichia, such as Escherichia coli, and microorganisms belonging to the genus Bacillus, such as Bacillus subtilis. Examples of eukaryotic hosts that may be used 20 include lower eukaryotic organisms, for example, eukaryotic microorganisms, for example, Eumycota such as yeast and filamentous fungi. As yeast there may be mentioned microorganisms belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae, and as 25 filamentous fungi there may be mentioned microorganisms belonging to the genus Aspergillus, such as Aspergillus oryzae and Aspergillus niger and microorganisms belonging to the genus Penicillium. Animal cells and plant cells may also be used, the animal cells being cell lines from 30 mice, hamsters, monkeys or humans. Insect cells, such as silkworm cells, or the adult silkworms themselves, may also be used. The expression vectors of the invention will include expression regulating regions such as a promoter and a 35 terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. Examples of promoters for bacterial expression vectors - 10 - which may be used include conventional promoters such as trc promoter, tac promoter, lac promoter, etc., examples of yeast promoters that may be used include glyceraldehyde-3-phosphate dehydrogenase promoter, PH05 5 promoter, etc., and examples of filamentous fungi promoters that may be used include amylase promoter, trpC, etc. Examples of animal cell host promoters that may be used include viral promoters such as SV40 early promoter, SV40 late promoter, etc. 10 The expression vector may be prepared according to a conventional method using restriction endonucleases, ligases and the like. The transformation of a host with an expression vector may also be carried out according to conventional methods. 15 The hosts transformed by the expression vector may be cultured, cultivated or raised, and the target protein may be recovered and purified from the cultured product, etc. according to conventional methods such as filtration, centrifugal separation, cell crushing, gel 20 filtration chromatography, ion-exchange chromatography and the like. The present specification throughout discusses flavone synthase II derived from perilla that is capable of synthesizing flavones directly from flavanones, and it 25 is also known that the cytochrome P450 genes constitute a superfamily (DNA and Cell Biology, 12 (1993), Nelson et al., p.l) and that cytochrome P450 proteins within the same family have 40% or greater identity in their amino acid sequences while cytochrome P450 proteins within a 30 subfamily have 55% or greater identity in their amino acid sequences, and their genes hybridize to each other (Pharmacogenetics, 6 (1996), Nelson et al., p.l). For example, a gene for flavonoid 3',5'-hydroxylase, which was a type of cytochrome P450 and participated in 35 the pathway of flavonoid synthesis, was first isolated from petunia (Nature, 366 (1993), Holton et al., p.276), and the petunia flavonoid 3',5'-hydroxylase gene was used -lias a probe to easily isolate a flavonoid 3',5'-hydroxylase gene from gentian (Plant Cell Physiol., 37 (1996), Tanaka et al., p.711), prairie-gentian, bellflower (W093/18155 (1993), Kikuchi et al.), lavender, 5 torenia and verbena (Shokubutsu no Kagaku Chosetsu, 33 (1998), Tanaka et al., p.55). Thus, a part or all of the flavone synthase II gene of the invention derived from perilla, which is capable of synthesizing flavones directly from flavanones, can be 10 used as a probe, in order to obtain flavone synthase II genes capable of synthesizing flavones directly from flavanones, from different species of plants. Furthermore, by purifying the perilla-derived flavone synthase II enzymes described in this specification which 15 can synthesize flavones directly from flavanones, and obtaining antibodies against the enzymes by conventional methods, it is possible to obtain different flavone synthase II proteins that react with the antibodies, and obtain genes coding for those proteins. 20 Consequently, the present invention is not limited merely to perilla-derived genes for flavone synthases II capable of synthesizing flavones directly from flavanones, but further relates to flavone synthases II derived from numerous other plants, which are capable of 25 synthesizing flavones directly from flavanones. The sources for such flavone synthase II genes may be, in addition to perilla described here, also gentian, verbena, chrysanthemum, iris, commelina, centaurea, salvia, nemophila and the like, although the scope of the 30 invention is not limited to these plants. The invention still further relates to plants whose colors are modified by introducing a gene or genes for flavone synthases II that can synthesize flavones directly from flavanones, and to progenies of the plants 35 or their tissues, which may also be in the form of cut flowers. By using the flavone synthases II or their genes which have been cloned according to the invention, - 12 - it is possible to produce flavones in plant species or varieties that otherwise produce little or absolutely no flavones. By expressing the flavone synthase II gene or the genes in flower petals, it is possible to increase 5 the amount of flavones in the flower petals, thus allowing the colors of the flowers to be modified toward the blue, for example. Conversely, by repressing synthesis of flavones in flower petals, it is possible to modify the colors of the 10 flowers toward the red, for example. However, flavones have myriad effects on flower colors, and the changes in flower colors are therefore not limited to those mentioned here. With the current level of technology, it is possible to introduce a gene into a plant and express 15 the gene in a constitutive or tissue-specific manner, while it is also possible to repress the expression of a target gene by an antisense method or a cosuppression method. As examples of transformable plants there may be 20 mentioned rose, chrysanthemum, carnation, snapdragon, cyclamen, orchid, prairie-gentian, freesia, gerbera, gladiolus, baby's breath, kalanchoe, lily, pelargonium, geranium, petunia, torenia, tulip, rice, barley, wheat, rapeseed, potato, tomato, poplar, banana, eucalyptus, 25 sweet potato, soybean, alfalfa, lupin, corn, etc., but there is no limitation to these. Because flavones have various physiological activities as explained above, they can impart new physiological activity or economic value to plants. For 30 example, by expressing the gene to produce flavones in roots, it is possible to promote growth of microorganisms that are beneficial for the plant, and thus promote growth of the plant. It is also possible to synthesize flavones that exhibit physiological activity in humans, 35 animals or insects. - 13 - Examples The invention will now be explained in further detail by way of the following examples. Unless otherwise specified, the molecular biological methods 5 were carried out according to Molecular Cloning (Sambrook et al., 1989). Example 1. Cloning of perilla flavone synthase II gene RNA was extracted from leaves of red perilla 10 (Perilla frutescens), and polyA+ RNA was obtained by an Oligotex. This polyA+ RNA was used as a template to prepare a cDNA library using a Xgt 10 (Stratagene) as the vector according to the method of Gong et al. (Plant Mol. Biol., 35 (1997), Gong et al., p. 915). The cDNA library 15 was screened using the full length CYP93B1 cDNA as the probe. The screening and detection of positive clones were carried out using a DIG-DNA-labeling and detection kit (Boehringer) based on the method recommended by the same company, under a low stringent condition. 20 Specifically, a hybridization buffer (5 x SSC, 30% formamide, 50 mM sodium phosphate buffer (pH 7.0), 1% SDS, 2% blocking reagent (Boehringer), 0.1% lauroylsarcosine, 80 ^g/ml salmon sperm DNA) was used for prehybridization at 42°C for 2 hours, after which the 25 DIG-labeled probe was added and the mixture was kept overnight. The membrane was rinsed in 5 x SSC washing solution containing 1% SDS at 65°C for 1.5 hours. One positive clone was obtained, and it was designated as a phase clone #3. Upon determining the nucleotide sequence 30 at the 5' end of #3 cDNA it was expected that #3 cDNA encodes a sequence with high identity with the flavanone 2-hydroxylase encoded by licorice CYP93B1, and it was assumed that it encoded a P450 with a function similar to that of flavanone 2-hydroxylase. 35 The protein encoded by #3 cDNA obtained here exhibited 52% identity on the amino acid level with flavanone 2-hydroxylase encoded by CYP93B1. The - 14 - nucleotide sequence of perilla clone #3 cDNA is listed as SEQ.ID. No.1, and the amino acid sequence deduced therefrom is listed as SEQ.ID. No.2. Example 2. Expression of perilla flavone synthase II 5 aene in veast The following experiment was conducted in order to detect the enzyme activity of the protein encoded by the perilla cDNA #3 obtained in Example 1. The phage clone #3 obtained in Example 1 was used as 10 a template for PCR using Lambda Arm primer (Stratagene). The PCR conditions were 98°C for one minute, 20 cycles of (98°C for 15 seconds, 55°C for 10 seconds, 74°C for 30 seconds), followed by 74°C for 10 minutes. The amplified DNA fragment was subcloned at the EcoRV site of 15 pBluescript KS(-). A clone with the initiation codon of the perilla #3 cDNA on the Sail side of pBluescript KS (-) was selected, and was designated as pFS3. The nucleotide sequence of the pFS3 cDNA was determined and the PCR was conducted to confirm the absence of errors. 20 An approximately 1.8 kb DNA fragment obtained by digesting pFS3 with Sail and Xbal was ligated with pYES2 predigested with Xhol and Xbal to produce a plasmid designated as pYFS3. The resultant plasmid was then introduced into BJ2168 yeast (Nihon Gene). The enzyme 25 activity was measured by the method described by Akashi et al. (FEBS Lett., 431 (1998), Akashi et al., p.287). The transformed yeast cells were cultured in 20 ml of selective medium (6.7 mg/ml amino acid-free yeast nitrogen base (Difco), 20 mg/ml glucose, 30 yg/ml 30 leucine, 20 pg/ml tryptophan and 5 mg/ml casamino acid), at 30°C for 24 hours. After harvesting the yeast cells with centrifugation, the harvested yeast cells were cultured at 30°C for 48 hours in an expressing medium (10 mg/ml 35 yeast extract, 10 mg/ml peptone, 2 jjg/ml hemin, 20 mg/ml galactose). After collecting the yeast cells, they were washed by suspending in water and collecting them. Glass - 15 - beads were used for 10 minutes of disrupting the cells, after which the cells were centrifuged at 8000 x g for 10 minutes. The supernatant was further centrifuged at 15,000 x g for 10 minutes to obtain a crude enzyme 5 fraction. A mixture of 15 yg of (R,S)-naringenin (dissolved in 30 fj 1 of 2-methoxyethanol), 1 ml of crude enzyme solution and 1 mM NADPH (total reaction mixture volume: 1.05 ml) was reacted at 30°C for 2 hours. After terminating the 10 reaction by addition of 30 jjI of acetic acid, 1 ml of ethyl acetate was added and mixed therewith. After centrifugation, the ethyl acetate layer was dried with an evaporator. The residue was dissolved in 100 of methanol and analyzed by HPLC. The analysis was carried 15 out according to the method described by Akashi et al. (FEBS Lett., 431 (1998), Akashi et al., p. 287). The acid treatment involved dissolution of the evaporator-dried sample in 150 /j1 of ethanol containing 10% hydrochloric acid, and stirring for 30 minutes. This was 20 diluted with 1.3 ml of water, 800 /j1 of ethyl acetate was further added and mixed therewith, and after centrifugation, the ethyl acetate layer was recovered. This was then dried, dissolved in 200 pi of methanol, and analyzed by HPLC. 25 The yeast expressing pYFS3 yielded apigenin from naringenin without acid treatment of the reaction mixture. This demonstrated that perilla pFS3 cDNA encodes a protein with flavone synthase II activity. Industrial Applicability 30 It is possible to alter flower colors by linking cDNA of the invention to an appropriate plant expression vector and introducing it into plants to express or inhibit expression of flavone synthases. Furthermore, by expressing the flavone synthase genes not only in petals 35 but also in entire plants or their appropriate organs, it is possible to increase the resistance against microorganisms of plants or to improve the nitrogen intellectual property office of n.z. 23 SEP 2003 - 16 - fixing ability of legumes by promoting association with rhizosphere microorganisms, as well as to improve the protective effects of plants against ultraviolet rays and light. it 50 7 1^0 SEQUENCE LISTING W V / I v/ V <110> SUNTORY LIMITED <120> Gene coding for flavone synthesizing enzyme <130> <160> 2 <210> 1 <211> 1770 <212> DNA <213> Perilla frutescens <220> <223> Nucleotide sequence encoding a protein having an activity to directly convert flavanone to flavone <400> 1 tgtcgacgga gcaagtggaa atg gca ctg tac gcc gcc ctc ttc ctc ctg tcc 53 Met Ala Lau Tyr Ala Ala Leu Phe Lou Leu Ser 15 10 gcc gcc gtg gtc cgc tcc gtt ctg gat cga aaa cgc ggg egg ccg ccc 101 Ala Ala Val Val Arg Ser Val Lau Asp Arg Lys Arg Gly Arg Pro Pro 15 20 25 tac cct ccc ggg ccg ttc cct ctt ccc ate ate ggc cac tta cac ctc 149 Tyr Pro Pro Gly Pro Phe Pro Leu Pro lie lie Gly His Leu His Leu 30 35 40 ctc ggg ccg aga ctc cac caa acc ttc cac gat ctg tcc caa egg tac 197 Leu Gly Pro Arg Leu His Gin Thr Phe His Asp Leu Ser Gin Arg Tyr 45 50 55 ggg ccc tta atg cag ctc cgc ctc ggg tcc ate cgc tgc gtc att get 245 Gly Pro Leu Met Gin Leu Arg Leu Gly Ser lie Arg Cys Val- lie Ala 60 65 70 75 507 150 18 gcc teg ccg gag ctc gcc aag gaa tgc ctc aag aca cac gag ctc gtc 293 Ala Ser Pro Glu Leu Ala Lys Glu Cys Leu Lys Thr His Glu Leu Val 80 85 90 ttc tcc tcc cgc aaa cac tcc acc gcc att gat ate gtc acc tac gat 341 Phe Ser Ser Arg Lys His Ser Thr Ala lie Asp lie Val Thr Tyr Asp 95 100 105 tea tcc ttc get ttc tct ccc tac ggg cct tac tgg aaa ttc ate aag 389 Ser Ser Phe Ala Phe Ser Pro Tyr Gly Pro Tyr Trp Lys Phe lie Lys 110 115 120 aaa tta tgc acc tac gag ctg ctc ggg gcc cga aat ctc gcc cac ttt 437 Lys Leu Cys Thr Tyr Glu Leu Leu Gly Ala Arg Asn Leu Ala His Phe 125 130 135 cag ccc ate agg act ctc gaa gtc aag tct ttc ctc caa att ctt atg 485 Gin Pro lie Arg Thr Leu Glu Val Lys Ser Phe Leu Gin lie Leu Met 140 145 150 155 cgc aag ggt gaa teg ggg gag age ttc aac gtg act gag gag ctc gtg 533 Arg Lys Gly Glu Ser Gly Glu Ser Phe Asn Val Thr Glu Glu Leu Val 160 165 170 aag ctg acg age aac gtc ata teg cat atg atg ctg age ata egg tgt 581 Lys Leu Thr Ser Asn Val lie Ser His Met Met Leu Ser lie Arg Cys 175 180 185 tea gag acg gag teg gag gcg gag gcg gcg agg acg gtg att egg gag 629 Ser Glu Thr Glu Ser Glu Ala Glu Ala Ala Acg Thr Val lie Arg Glu 190 195 _ 200 gtc acg cag ata ttt ggg gag ttc gac gtc tcc gac ate ata tgg ctt 677 Val Thr Gin lie Phe Gly Glu Phe Asp Val Ser Asp lie lie Trp Leu 205 210 215 tgt aag aac ttc gat ttc caa ggt ata agg aag egg tcc gag gat ate 725 Cys Lys Asn Phe Asp Phe Gin Gly lie Arg Lys Arg Ser Glu Asp lie 220 225 230 235 cag agg aga tat gat get ctg ctg gag aag ate ate acc gac aga gag 773 Gin Arg Arg Tyr Asp Ala Leu Leu Glu Lys lie lie Thr Asp Arg Glu 240 245 250 aag cag agg egg acc cac ggc ggc ggt ggc ggc ggc ggg gaa gcc aag 821 Lys Gin Arg Arg Thr His Gly Gly Gly Gly Gly Gly Gly Glu Ala Lys 255 260 265 gat ttt ctt gac Asp Phe Leu Asp 270 gtt aaa ttc acg Val Lys Phe Thr 285 acc gcc ggc acc Thr Ala Gly Thr 300 gaa gtg ate aac Glu Val lie Asn gcc aac ate gtc Ala Asn lie Val 335 aat ctg ccc tac Asn Leu Pro Tyr 350 cct cca ate cca Pro Pro lie Pro 365 gac ggc tac atg Asp Gly Tyr Met 380 tcc atg ggg egg Ser Met Gly Arg ccg gag agg ttt Pro Glu Arg Phe 415 cag cat ttt gag Gin His Phe Glu 430 ggg atg ctt tta Gly Met Leu Leu 445 atg ttc ctc gac Met Phe Leu Asp 275 agg gag cat ctc Arg Glu His Leu 290 gac acg acg gcg Asp Thr Thr Ala 305 aat cca aat gtg Asn Pro Asn Val 320 gga ttc gac aga Gly Phe Asp Arg ctt caa gcc ctc Leu Gin Ala Leu 355 atg ctg gcg agg Met Leu Ala Arg 370 att ccg gcc aac lie Pro Ala Asn 385 aac cct aaa ate Asn Pro Lys lie 400 ctg gag aag gaa Leu Glu Lys Glu ctg eta ccg ttc Leu Leu Pro Phe 435 gcc att cag gag Ala lie Gin Glu 450 ata atg gag age lie Met Glu Ser aaa get ttg att Lys Ala Leu lie 295 ate gtg tgt gaa lie Val Cys Glu 310 ttg aag aaa get Leu Lys Lys Ala 325 att ctg caa gaa lie Leu Gin Glu 340 ate aaa gaa aca lie Lys Glu Thr aaa teg ate tcc Lys Ser lie Ser 375 acg ctg ctc ttc Thr Leu Leu Phe 390 tgg gac tac ccg Trp Asp Tyr Pro 405 aag gcc gcc ate Lys Ala Ala lie 420 gga acg ggc agg Gly Thr Gly Arg gtg gtc ate ata Val Val lie lie 455 ggg aaa gcc gaa Gly Lys Ala Glu 280 ctg gat ttc ttc Leu Asp Phe Phe tgg gcg ata gca Trp Ala lie Ala 315 caa gaa gag att Gin Glu Glu lie 330 tcc gac gcc cca Ser Asp Ala Pro 345 ttc egg ctc cac Phe Arg Leu His 360 gac tgc gtc ate Asp Cys Val lie gtc aac ctc tgg Val Asn Leu Trp 395 acg gcg ttc cag Thr Ala Phe Gin 410 gat gtt aaa ggg Asp Val Lys Gly 425 aga ggc tgc cca Arg Gly Cys Pro 440 att ggg acg atg He Gly Thr Met 20 '50 att caa tgc ttc gat tgg aag ctg ccc gac ggc tcc ggc cat gtt gat 1445 lie Gin Cys Phe Asp Trp Lys Leu Pro Asp Gly Ser Gly His Val Asp 460 465 470 475 atg gca gaa egg cca ggg ctc acg gca ccg cga gag acc gat ttg ttt 1493 Met Ala Glu Arg Pro Gly Leu Thr Ala Pro Arg Glu Thr Asp Leu Phe 480 485 490 tgc cgt gtg gtg ccg cga gtt gat ccg ttg gtt gtt tcc acc cag 1538 Cys Arg Val Val Pro Arg Val Asp Pro Leu Val Val Ser Thr Gin 495 500 505 tgatcacccc ctttaaattt attaatgata tatttttatt ttgagaaaaa ataaaaatgc 1598 taattgtttt gtttcatgat gtaattgtta attagtttct attgtgcgct gtcgcgtgtc 1658 gcgtggctta agataagatt gtatcattgg tacctaggat gtattttcat tttcaataaa 1718 ttattttgtg ctgtgtatat taaaaaaaaa aaagaaaaaa aaaaaaaaaa aa 1770 <210> 2 <211> <212> PRT <213> Perilla frutescens <220> <223> Amino acid sequence of a'protein having an activity to directly convert flavanone to flavone <400> 2 Met Ala Leu Tyr Ala Ala Leu Phe Leu Leu Ser Ala Ala Val Val Arg 15 10 15 Ser Val Leu Asp Arg Lys Arg Gly Arg Pro Pro Tyr Pro Pro Gly Pro 20 25 30 Phe Pro Leu Pro lie lie Gly His Leu His Leu Leu Gly Pro Arg Leu 35 40 45 His Gin Thr Phe His Asp Leu Ser Gin Arg Tyr Gly Pro Leu Met Gin 50 55 60 Leu Arg Leu Gly Ser He Arg Cys Val lie Ala Ala Ser Pro Glu Leu 65 70 75 80 Ala Lys Glu Cys Leu Lys Thr His Glu Leu Val Phe Ser Ser Arg Lys 85 90 95 His Ser Thr Ala lie Asp lie Val Thr Tyr Asp Ser Ser Phe Ala Phe 100 105 110 21 t 3 Ser Pro Tyr Gly Pro Tyr Trp Lys Phe lie Lys Lys Leu Cys Thr Tyr 115 120 125 Glu Leu Leu Gly Ala Arg Asn Leu Ala His Phe Gin Pro lie Arg Thr 130 135 140 Leu Glu Val Lys Ser Phe Leu Gin lie Leu Met Arg Lys Gly Glu Ser 145 150 155 160 Gly Glu Ser Phe Asn Val Thr Glu Glu Leu Val Lys Leu Thr Ser Asn 165 170 175 Val lie Ser His Met Met Leu Ser lie Arg Cys Ser Glu Thr Glu Ser 180 185 190 Glu Ala Glu Ala Ala Arg Thr Val lie Arg Glu Val Thr Gin lie Phe 195 200 205 Gly Glu Phe Asp Val Ser Asp lie lie Trp Leu Cys Lys Asn Phe Asp 210 215 220 Phe Gin Gly lie Arg Lys Arg Ser Glu Asp lie Gin Arg Arg Tyr Asp 225 230 235 240 Ala Leu Leu Glu Lys lie lie Thr Asp Arg Glu Lys Gin Arg Arg Thr 245 250 255 His Gly Gly Gly Gly Gly Gly Gly Glu Ala Lys Asp Phe Leu Asp Met 260 265 270 Phe Leu Asp lie Met Glu Ser Gly Lys Ala Glu Val Lys Phe Thr Arg 275 280 285 Glu His Leu Lys Ala Leu lie Leu Asp Phe Phe Thr Ala Gly Thr Asp 290 295 300 Thr Thr Ala lie Val Cys Glu Trp Ala lie Ala Glu Val lie Asn Asn 305 310 315 320 Pro Asn Val Leu Lys Lys Ala Gin Glu Glu lie Ala Asn lie Val Gly 325 330 335 Phe Asp Arg lie Leu Gin Glu Ser Asp Ala Pro Asn Leu Pro Tyr Leu 340 345 350 Gin Ala Leu lie Lys Glu Thr Phe Arg Leu His Pro Pro lie Pro Met 355 360 365 Leu Ala Arg Lys Ser lie Ser Asp Cys Val lie Asp Gly Tyr Met lie 370 375 380 Pro Ala Asn Thr Leu Leu Phe Val Asn Leu Trp Ser Met Gly Arg Asn 385 390 395 400 Pro Lys lie Trp Asp Tyr Pro Thr Ala Phe Gin Pro Glu Arg Phe Leu 405 410 415 « </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 22 Glu Lys Glu Lys Ala Ala lie Asp Val Lys Gly Gin His Phe Glu Leu 420 425 430 Leu Pro Phe Gly Thr Gly Arg Arg Gly Cys Pro Gly Met Leu Leu Ala 435 440 445 Zle Gin Glu Val Val lie lie lie Gly Thr Met lie Gin Cys Phe Asp 450 455 460 Trp Lys Leu Pro Asp Gly Ser Gly His Val Asp Met Ala Glu Arg Pro 465 470 475 480 Gly Leu Thr Ala Pro Arg Glu Thr Asp Leu Phe Cys Arg Val Val Pro 485 490 495 Arg Val Asp Pro Leu Val Val Ser Thr Gin 500 505 23 Claims

1. An isolated gene encoding a protein having the amino acid sequence of SEQ.ID. No. 2 and showing activity of synthesizing flavones from flavanones, or a gene encoding a protein having one of these amino acid sequences wherein the amino acid sequence has been modified within at least 70% identity with the amino acid sequence of SEQ ID No. 2, and possessing activity of synthesizing flavones from flavanones.

2. A gene according to claim 1, which hybridizes with all or a part of SEQ.ID.No.1 under conditions of 5 x SSC, 50°C.

3. A vector comprising a gene according to claim 1 or 2.

4. A non-human host transformed with a vector according to claim 3.

5. A protein encoded by a gene according to claim 1 or 2.

6. A method of producing a protein with flavone-synthesizing activity, which is characterized by culturing or growing a non-human host according to claim 4 and recovering said protein from said host.

7. A plant into which a gene according to claim 1 or 2 has been introduced, or progenies of said plant or a tissue thereof, which exhibits the same properties.

8. A cut flower from a plant or a progeny thereof having the same properties, according to claim 7.

9. A method of altering the flavonoid composition and/or its amount in a plant using a gene according to claim 1 or 2.

10. A method of altering the amount of flavone in a plant using a gene according to claim 1 or 2.

11. A method of altering the color of a flower using a gene according to claim 1 or 2.

12. A method of bluing the color of a flower using a gene according to claim 1 or 2.

13. A method of reddening the color of a flower using a gene according to claim 1 or 2.

14. A method of altering the photosensitivity of a plant using a gene according to claim 1 or 2. 24

15. A method of promoting a symbiotic interaction between a plant and microorganisms using a gene according to claim 1 or 2.

16. A method of increasing plant resistance to microorganisms using a gene according to claim 1 or 2.

17. An isolated gene which encodes a protein as defined in claim 1 substantially as herein described with reference to any example thereof.

18. A vector as claimed in claim 3 substantially as herein described with reference to any example thereof.

19. A non-human host as claimed in claim 4 substantially as herein described with reference to any example thereof.

20. A protein as claimed in claim 5 substantially as herein described with reference to any example thereof.

21. A method of producing a protein with flavone-synthesizing activity as claimed in claim 6 substantially as herein described with reference to any example thereof.

22. A plant as claimed in claim 7 substantially as herein described with reference to any example thereof.

23. A cut flower as claimed in claim 8 substantially as herein described with reference to any example thereof.

24. A method as claimed in any one of claims 9 to 16 substantially as herein described with reference to any example thereof. intellectual property office of n.z. 23 SEP 2003 90386 1.DOC 1 T T) i </div>