CN1624136A

CN1624136A - Genes coding for flavone synthases

Info

Publication number: CN1624136A
Application number: CNA2004101000768A
Authority: CN
Inventors: 水谷正子; 田中良和; 久住高章; 绫部真一; 明石智义
Original assignee: Suntory Flower Industry Co Ltd; Suntory Ltd
Current assignee: Suntory Ltd
Priority date: 1999-07-19
Filing date: 2000-06-30
Publication date: 2005-06-08
Also published as: AU5572100A; IL138718A0; WO2001005981A1; EP1114162A1; NZ507150A; CN1310762A; CA2324228A1; KR20010071128A

Abstract

A DNA encoding an enzyme capable of converting flavanone directly into flavone which is obtained from, for example, antirrhinum and torenia, and utilization thereof. The amino acid sequences of this DNA and the enzyme encoded thereby are represented by, for example, SEQ ID NO: 1 and 2. Transfer of this gene into a plant makes it possible to, for example, change the flower color of the plant.

Description

The gene of coding for flavone synthases

The application is to be the dividing an application of Chinese patent application 00800102.2 on June 30th, 2000 applying date.

Technical field

The present invention relates to by genetic engineering technique control and utilize the biosynthesizing of flavones, flavones is to the color of flower in the plant, and the provide protection of antiultraviolet is influential with the symbiosis of microorganism etc.More specifically, the present invention relates to the gene and the application thereof of coded protein, described protein has the activity by the synthetic flavones of flavanone.

The background of invention technology

Multicoloured different pattern is a pleasant thing in the life, and it can enrich people's thoughts and inner world.By quickening plant-growth with microorganisms symbiosis, thereby or make by the number that increases the pulse family nitrogen-fixing bacteria that the increase of nitrogen content improves productivity of plant in the soil, hope can increase grain yield to cater to the demand that population in the future increases.Seek out the agricultural of compliance with environmental protection requirements more, need to eliminate or reduce the use of agricultural chemicals, this just need improve the soil by above-mentioned biological method, and needs plant that infected by microbes is had higher resistance.Another wants that the target that reaches is to obtain the stronger plant of antiultraviolet protecting function to avoid the destroyed influence of ozonosphere with protective plant.

" flavonoid " is the common name with one group of compound of C6-C3-C6 carbon skeleton, and they are distributed widely in the vegetable cell.The known class flavones has the function that attracts insect and other pollinator, but protective plant is avoided ultraviolet invasion and attack, and the interaction of participation and soil microorganisms (BioEssays, 16 (1994), Koes etc., p123; Trends in Plant Science, 1 (1997), Shirley, B.W., p.377).

In flavonoid, flavones plays an important role in the interaction of plant and microorganism, and is especially all the more so in leguminous plants, wherein they have participated in the initial step (vegetable cell with the pulse family bacteria paragenesis, 7 (1995), Dixon and Paiva, p.1085; Plant pathology academic year comments, and 33 (1995), Spaink, p.345).Flavones in the petal aspect insect identification, work and can be used as accessory pigment and with cyanin form mixture (Gendai Kagaku, (May, 1998), Honda and Saito, p.25; Prog.Chem.Org.Natl.Prod., 52 (1987), Goto, T., p.114).Known when flavones and cyanin formation mixture, the absorbancy maximum value of cyanin promptly is offset toward blue direction toward long wavelength shift.

Broad research the biosynthetic pathway of flavonoid (vegetable cell, 7 (1995), p.1071) Holton and Cornish, have isolated the gene that participates in anthocyanidin 3-glucoside and biosynthetic all enzymes of flavonol.Yet, do not isolate as yet and participate in the biosynthetic gene of flavones.The enzyme of synthetic flavones comprises enzyme (flavone synthase I) that belongs to dioxygenase family and depend on the 2-oxoglutarate and the monooxygenase (flavone synthase II) that belongs to Cytochrome P450 family.These enzymes are the diverse enzymes that do not have structural homology.

It is reported, in parsley, the dioxygenase catalysis that depends on the 2-oxoglutarate by naringenin (a kind of flavanone) produce apigenin (a kind of flavones) reaction (Z.Naturforsch., 36C (1981), Britsch etc., p.742; Arch.Biochem.Biophys., 282 (1990), Britsch, p.152).The flavone synthase II of known other type be present in Common Snapdragon (Z.Naturforsch., 36C (1981), Stotz and Forkmann, p.737) and soybean (Z.Naturforsch., 42C (1987), Kochs and Grisebach, p.343; Planta, 171 (1987), Kochs etc., p.519) in.Recently, the someone reported locus in the Herba Leibnitziae petal and the dependency between the flavone synthase II activity (vegetable chemistry, 49 (1998), Martens and Forkmann, p.1953).Yet unmanned report has been isolated gene or the highly purified flavone synthase II of flavone synthase I and II.

Studied the characteristic of cytochrome p450 protein, this protein has glycyrdione-composite reactive, can induce this activity when Radix Glycyrrhizae (Glycyrrhiza echinata) cell handled with inducer through cultivating.But it is believed that the hydroxylation of the 2nd of this protein catalysis liquiritigenin (it is 5-deoxidation flavanone), follow non-enzymatic and open the hemiacetal ring, and the generation glycyrdione (plant physiology, 105 (1994), Otani etc., p.1427).In order to clone the glycyrdione synthase, prepare the cDNA library by the Radix Glycyrrhizae culturing cell of handling through inductor, clone 8 Codocyte cytochrome p 450s gene segment (plant science, 126 (1997), Akashi etc., p.39).

Obtain 2 different full length cDNA sequences by these segments, each Codocyte cytochrome p 450 is until they are still unknown at that time.Specifically, they be CYPGe-3 (Cytochrome P450 No.CYP81E1) and CYPGe-5 (Cytochrome P450 No.CYP93B1 hereinafter is referred to as CYP93B1) (plant physiology, 115 (1997), Akashi etc., p.1288).By in the system that uses the insect cell of cultivating, further expressing CYP93B1 cDNA, but the protein catalysis that confirms this genes encoding is by the reaction of the synthetic glycyrdione of liquiritigenin (a kind of flavanone) and the reaction of being produced 2-hydroxyl naringenin by naringenin (a kind of flavanone).

Make 2-hydroxyl naringenin change apigenin (a kind of flavones) into by carry out acid treatment (room temperature, 2 hours) with 10% hydrochloric acid.By making eriodictyol and the zymic microsome reaction of expressing CYP93B1, then carry out acid treatment and eriodictyol can be changed into luteolin (a kind of flavones).Illustrate thus cytochrome P450 gene coding flavanone 2-hydroxylase activity function (FEBS Lett., 431 (1998), Akashi etc., p.287).Here, producing apigenin by naringenin needs CYP93B1 and another unknown enzyme, and the conclusion that draws thus is to need two kinds of enzymes altogether.

Yet, do not identify as yet at present and have without acid treatment directly by the active enzyme of the synthetic flavones (as apigenin) of flavanone (as naringenin).Therefore, although flavones has multiple function in plant, the flavones biosynthesizing in the controlling plant and improve the biological function that flavones participates in does not appear in the newspapers as yet as the technology of the color of flower.Compare with the gene transfered plant cell that will participate in by two kinds of enzymes of the synthetic flavones of flavanone, but catalysis own is by the discovery of the enzyme of the synthetic flavones of flavanone, the acquisition of this enzyme gene, and will have stronger operability and industrial applicibility in this gene transfered plant.

Disclosure of the Invention

The purpose of this invention is to provide the flavone synthase gene, be preferably flavone synthase II gene, more preferably have the gene that directly synthesizes the active flavone synthase of flavones by flavanone.Can with in the gained flavone synthase gene transfered plant and overexpression to change colored color.

In addition, in the natural petal that contains a large amount of flavones, expection also can change colored color by antisense method or the control of inhibition method altogether flavone synthase expression of gene.In addition; express in suitable organ according to the antibacterial activity of flavones and with the interaction of soil microorganisms that the flavone synthase gene can increase the plant antibacterial activity and because of having promoted to improve fabaceous nitrogen fixing capacity, and can obtain the provide protection of antiultraviolet and illumination with the symbiotic relationship of rhizospheric microorganism.

Therefore, the invention provides coding can directly be synthesized flavones by flavanone proteinic gene, this gene is specially the gene of coding for flavone synthases II, and flavone synthase II can be by the reaction (hereinafter be referred to as " flavone synthase II ") of single enzyme reaction catalysis by the synthetic flavones of flavanone.

More specifically, the invention provides the proteic gene of coding P450, described protein has the aminoacid sequence shown in the SEQ ID NO:2 in the sequence table, and has a activity by the synthetic flavones of flavanone, also providing coding to have a modified aminoacid sequence but still have active proteinic gene by the synthetic flavones of flavanone, wherein said modification is finished by adding in above-mentioned aminoacid sequence or lacking one or more amino acid and/or replace with different aminoacids.

The present invention also provide coding have with sequence table in aminoacid sequence shown in the SEQ ID NO:2 have the proteinic gene of the aminoacid sequence of 55% identity at least, described protein has the activity by the synthetic flavones of flavanone.

The present invention also provides coding to have active proteinic gene by the synthetic flavones of flavanone, and this gene is at 5 * SSC, under 50 ℃ the condition can with all or part of hybridization of nucleotide sequence shown in the SEQ ID NO:1 in the sequence table.

The present invention also provides carrier, expression vector especially, and it contains above-mentioned any gene.

The present invention also provides by above-mentioned carrier host transformed.

The present invention also provides the protein by above-mentioned any genes encoding.

The present invention also provides and has produced above-mentioned method of protein, and described method is characterised in that cultivates above-mentioned host and collect the protein with flavones composite reactive from the host.

The present invention also provides and has wherein imported the plant of above-mentioned any gene, or shows plant offspring or its tissue of identical characteristics, as cut-flower.

The present invention also provides and has used said gene to change the amount of flavonoid and the method for composition; Use said gene to change the method for flavones amount; Use said gene to change the method for the color of flower; Use said gene to make colored color become blue method; Use said gene to make the redder method of colored color; Use the method for the photosensitivity of said gene modified plant; Use the interactional method between said gene controlling plant and the microorganism.

Implement concrete scheme of the present invention

Flavanone 2-hydroxylase by Radix Glycyrrhizae CYP93B1 genes encoding has produced 2-hydroxyl flavanone by the substrate flavanone, changes product into flavones by acid treatment.The inventor thinks that the cDNA CYP93B1 screening that derives from Radix Glycyrrhizae by use for example contains the cDNA library of the flower of a large amount of flavones, have the active proteinic cDNA that directly synthesizes flavones thereby obtain coding, can obtain the gene (this is one of purpose of the present invention) of coding for flavone synthases II by the substrate flavanone.

According to the present invention, use the cDNA CYP93B1 that derives from Radix Glycyrrhizae to be that the probe screening contains the cDNA library of the purple perilla of a large amount of flavones, the cDNA (seeing embodiment 1) of the new Cytochrome P450 that can obtain encoding.

Expression derives from the cDNA of purple perilla and reacts with substrate naringenin (a kind of flavanone) in yeast, and the result need not can produce flavones apigenin rather than 2-hydroxyl naringenin (seeing embodiment 2) through acid treatment.In other words, this enzyme can directly produce flavones by flavanone, and need not through acid treatment, and is verified, the flavone synthase II gene of its gene for never cloning in the past.

Gene of the present invention can be the gene of aminoacid sequence shown in the SEQ ID NO:2 in for example code sequence tabulation.Yet known its aminoacid sequence is through adding or lacking a plurality of amino acid and/or replaced by different aminoacids and adorned protein can keep the enzymic activity identical with urporotein.Therefore, has aminoacid sequence shown in the SEQ ID NO:2 in the sequence table, but aminoacid sequence wherein is through adding or lacking one or more amino acid and/or replaced and adorned protein by different aminoacids, and the gene of code for said proteins is also contained among the present invention, as long as their keep directly being produced by flavanone the activity of flavones.

The invention still further relates to nucleotide sequence with nucleotide sequence shown in the SEQ ID NO:1 and coding aminoacid sequence described herein, or at for example 5 * SSC, under 50 ℃ the condition can with the gene of the part of described nucleotide sequence hybridization, condition is that they can be encoded and have the active protein of being produced flavones by flavanone.Suitable hybridization temperature is different and different with nucleotide sequence and nucleotide sequence length, for example, and when used probe is that preferred temperature is no more than 50 ℃ when containing the dna segment of 6 amino acid whose 18 bases length of coding.

The gene that described hybridization is selected can be natural, for example Common Snapdragon freely, the plant gene of HUDIECAO or purple perilla; Can be the gene of another kind of plant also, as rough gentian, vervain, chrysanthemum, the gene of White flag etc.Hybridizing selected gene can be cDNA or genomic dna.

The invention still further relates to coding have with sequence table in aminoacid sequence shown in the SEQ ID NO:2 have 55% at least, preferably at least 70%, as 80% or above even 90% or the proteinic gene of the aminoacid sequence of above identity, described protein has the activity by the synthetic flavones of flavanone.

Can obtain having the gene of natural nucleus glycoside acid sequence by screening cDNA library as described in embodiment.Can use DNA as initial substance, the DNA that site-directed mutagenesis or the PCR method composite coding by routine has the enzyme of modified aminoacid sequence with natural nucleus glycoside acid sequence.For example, by handle natural cDNA or genomic dna with Restriction Enzyme,, can wherein be imported the dna segment of required modification then as the template of site-directed mutagenesis or PCR (having imported required sudden change in the primer of use).The dna segment that imports sudden change can be connected with the dna segment of coding target enzyme another part then.

Perhaps, the DNA of the enzyme of forming by the aminoacid sequence that shortens in order to obtain encoding, for example, available required restriction endonuclease cutting coding is longer than the DNA of the aminoacid sequence (as full length amino acid sequence) of purpose aminoacid sequence, the complete target amino acid sequence if the gained dna segment is not encoded can be connected this segment with the synthetic DNA that contains all the other sequences.

Can in using intestinal bacteria or zymic expression system, express the gene of gained, detect its enzymic activity to confirm gained genes encoding flavone synthase.By expressing gene, also can obtain flavone synthase albumen as gene product.Perhaps, even can use the antibody of all or part of aminoacid sequence shown in the SEQ ID NO:2 to obtain flavone synthase albumen, can use the flavone synthase gene in the another kind of biology of this antibody cloning.

Therefore, the invention still further relates to the recombinant vectors that contains said gene, expression vector especially, and relate to by these carrier host transformed.Used host can be protokaryon or eukaryote.The procaryotic example that is commonly used for the host comprises the bacterium of Escherichia, as the microorganism of intestinal bacteria and bacillus, as subtilis.

The example of operable eucaryon host comprises lower eukaryotes, as eukaryotic microorganisms (as yeast in the Mycophyta and filamentous fungus).As for yeast, what can mention is the microorganism that belongs to saccharomyces, and as yeast saccharomyces cerevisiae, as for filamentous fungus, what can mention is the microorganism that belongs to Aspergillus, as the microorganism of aspergillus oryzae and aspergillus niger and Penicillium.Also can use zooblast and vegetable cell, zooblast can be a mouse, hamster, monkey or people's clone.Also can use insect cell, as bombyx mori cell or adult silkworm itself.

Expression vector of the present invention can comprise the expression regulatory region, as promotor and terminator, and replication orgin etc., this depends on the host type that their desires import.The example of operable bacterial expression vector promotor comprises conventional promotor, as the trc promotor, and tac promotor, lac promotor etc.The example of operable Yeast promoter comprises the glyceraldehyde-3-phosphate dehydrogenase promotor, PH05 promotor etc.The example of operable filamentous fungus promotor comprises amylase promotor, trpC etc.The example of operable zooblast host promotor comprises viral promotors, as the SV40 early promoter, and SV40 late promoter etc.

According to conventional methods, use restriction endonuclease, ligase enzyme etc. can prepare expression vector.Transforming the host with expression vector also can carry out according to conventional methods.

Can cultivate, cultivation or fermentation can be according to conventional methods by the expression vector host transformed, as filtering, and centrifugation, cytoclasis, gel permeation chromatography, ion exchange chromatography etc. reclaim from cultured products and the purifying target protein.

This specification has been discussed the flavone synthase II that derives from purple perilla, this enzyme can be directly by the synthetic flavones of flavanone, known cytochrome P450 gene has constituted superfamily (DNA and cytobiology, 12 (1993), Nelson etc., p.1), the aminoacid sequence of the cytochrome p450 protein in the identical family has 40% or higher identity, and the aminoacid sequence of the cytochrome p450 protein in the subfamily has 55% or higher identity, their gene can be hybridized (Pharmacogenetics mutually, 6 (1996), Nelson etc., p.1).

For example, from Petunia, isolate the gene of flavonoid 3 ', 5 '-hydroxylase first, described enzyme is a kind of of Cytochrome P450 and participates in flavonoid route of synthesis (nature, 366 (1993), Holton etc., p.276), use petunia flavonoid 3 ', 5 '-'-hydroxylase gene is easy to from rough gentian (plant cell physiology, 37 (1996), Tanaka etc. as probe, p.711), prairie gentian, bellflower (WO93/18155 (1993), Kikuchi etc.), lavandula angustifolia, HUDIECAO and vervain (Shokubutsu no Kagaku Chosetsu, 33 (1998), Tanaka etc., p.55) isolate flavonoid 3 ', 5 '-'-hydroxylase gene in.

Therefore, of the present invention can be directly by the perilla flavone synthase II gene of the synthetic flavones of flavanone all or part of can be used as probe with obtain in the kindred plant never can be directly by the perilla flavone synthase II gene of the synthetic flavones of flavanone.In addition, by the perilla flavone synthase II that can directly synthesize flavones described in this specification sheets of purifying by flavanone, obtain the antibody of anti-this enzyme by ordinary method, can obtain the different flavone synthase II albumen with antibody response, and these proteinic genes that obtain encoding.

Therefore, the present invention is not limited in and can also relates to the flavone synthase II that can directly be synthesized flavones by flavanone that derives from multiple other plant directly by the perilla flavone synthase II gene of the synthetic flavones of flavanone.Except purple perilla as herein described, the source of described flavone synthase II gene can also be a rough gentian, vervain, and chrysanthemum, White flag, Herba Commelinae, Minor centaury, Salvia japonica Thunb., happiness woods grass etc., but scope of the present invention is not limited to these plants.

The invention still further relates to by importing and one or morely can be directly make the adorned plant of color by the flavone synthase II gene of the synthetic flavones of flavanone, and the offspring of plant or its tissue, described organizing also can be the form of cut-flower.The flavone synthase II of the application of the invention or its be by cloned genes, can only produce flavones seldom do not produce the plant species of flavones or mutation in produce flavones.By in petal, expressing one or more flavone synthase II genes, can increase the amount of flavones in the petal, thereby make colored color past for example, blue direction changes.

On the contrary, synthetic in petal by suppressing flavones, can make colored color past for example, red direction changes.Yet flavones is very big to the effect of pattern, so the change of pattern is not limited to the above-mentioned type.With regard to present state of the art, can still, also can suppress target gene expression with gene transfered plant and with composing type or tissue-specific mode expressing gene by antisense method or inhibition method altogether.

The example of transformable plant comprises rose, chrysanthemum, carnation, Common Snapdragon, Cyclamen persicum, Herba Orchidis Latifoliae, prairie gentian, xiangxuelan, African daisy, gladiolus, Stem and leaf of Hongkong Pavetta, Laciniate Kalanchoe Herb, lily, Flos Pelargonii, heroubill, green winter eggplant, HUDIECAO, turmeric, paddy rice, barley, wheat, coleseed, potato, tomato, white poplar, banana, eucalyptus, sweet potato, soybean, clover, lupine, corn etc., but be not limited to these plants.

Because flavones has aforesaid multiple physiologically active, they can give new physiologically active of plant or economic worth.For example,, can promote the microbial growth favourable, thereby promote plant-growth this plant by in root, expressing the gene that produces flavones.Also can synthesize the people, show the flavones of physiologically active in animal or the insect.

The following example will be explained the present invention in more detail.Unless stated otherwise, can carry out molecular biology method according to molecular cloning (Sambrook etc., 1989).

Embodiment 1. clone's perilla flavone synthase II genes

From purple perilla (Perilla frutescens) leaf, extract RNA, obtain polyA+RNA by Oligotex.According to Gong etc. (molecular biology of plants, 35 (1997), Gong etc., method p.915) prepares cDNA library to use λ gt10 (Stratagene) as carrier as template with polyA+RNA.Use total length CYP93B1 cDNA to be probe screening cDNA library.According to the method that manufacturer is recommended, under low stringent condition, use DIG-DNA-mark and detection kit (Boehringer) to carry out the screening and the detection of positive colony.

Specifically; under 42 ℃; use hybridization buffer (5 * SSC, 30% methane amide, 50mM sodium phosphate buffer (pH7.0); 1%SDS; 2% blocking-up reagent (Boehringer), 0.1% lauroyl sarcosine, 80 micrograms/ml salmon sperm DNA) prehybridization 2 hours; the probe that adds the DIG mark then spends the night the mixture placement.Under 65 ℃, film was embathed 1.5 hours with the 5 * SSC washing soln that contains 1%SDS.Obtain a positive colony, be referred to as phage clone #3.By 5 ' of mensuration #3 cDNA terminal nucleotide sequence, the flavanone 2-hydroxylase that expection #3 cDNA coding and Radix Glycyrrhizae CYP93B1 encode has the sequence of high identity, infers that its encoding function is similar to the P450 of flavanone 2-hydroxylase.

Gained #3 cDNA encoded protein matter has 52% identity with the coded flavanone 2-hydroxylase of CYP93B1 on amino acid levels.The nucleotides sequence of purple perilla clone #3 cDNA is shown in SEQ ID NO.1, is shown in SEQ ID NO.2 by its aminoacid sequence of inferring.

Embodiment 2. expresses perilla flavone synthase II gene in yeast

Carry out following experiment to detect the enzymic activity of embodiment 1 gained purple perilla cDNA#3 encoded protein matter.The phage clone #3 of embodiment 1 gained is used as PCR (using λ Arm primer (Stratagene)) template.The PCR condition be 98 ℃ 1 minute; 98 ℃ 15 seconds, 55 ℃ 10 seconds, 74 ℃ of totally 20 circulations in 30 seconds; Then 74 ℃ 10 minutes.With the dna segment subclone of amplification to the EcoRV site of pBluescript KS (-).Be chosen in the clone that the SalI site of pBluescript KS (-) has purple perilla #3 cDNA initiator codon, be referred to as pFS3.Measure the nucleotide sequence of pFS3 cDNA, carry out PCR to confirm not exist mistake.

Dna segment that will be by about 1.8kb of obtaining with SalI and XbaI digestion pFS3 be connected generation plasmid pYFS3 with the predigested pYES2 of XhoI and XbaI.Then the gained plasmid is imported BJ2168 yeast (Nihon Gene).By (FEBS Lett., 431 (1998), Akashi etc., p.287) described method mensuration enzymic activitys such as Akashi.30 ℃, select in the substratum (6.7mg/ml does not have amino acid whose yeast nitrogenous base (Difco), 20mg/ml glucose, 30 micrograms/ml leucine, 20 micrograms/ml tryptophane and 5mg/ml casamino acids) transformed yeast cells to be cultivated 24 hours at 20ml.

After the centrifugal collection yeast cell, 30 ℃, in expression substratum (10mg/ml yeast extract, 10mg/ml peptone, 2 micrograms/ml teichmann's crystals, 20mg/ml semi-lactosi), the yeast cell of collecting was cultivated 48 hours.Collect after the yeast cell, by be suspended in the water and collecting cell to wash.With granulated glass sphere smudge cells 10 minutes, then with the rotating speed eccentric cell of 8000g 10 minutes, with 15, the further centrifuged supernatant of the rotating speed of 000g 10 minutes was to obtain thick enzyme fraction.

Under 30 ℃, (R, S)-naringenin (being dissolved in the 30 microlitre 2-methoxyethanols), (the total reaction mixture volume: 1.05ml) reaction is 2 hours for the mixture of thick enzyme solution of 1ml and 1mM NADPH with 15 micrograms.By adding after the 30 microlitre acetic acid termination reactions, add the 1ml ethyl acetate and mix.After centrifugal, use the evaporator dry ethyl acetate layer, residuals is dissolved in 100 microliter methanol and with HPLC analyzes.According to Akashi etc. (FEBS Lett., 431 (1998), Akashi etc., p.287) described method is analyzed.Acid treatment comprises that the sample dissolution with evaporator dry contains in the ethanol of 10% hydrochloric acid in 150 microlitres, stirs 30 minutes.With the dilution of 1.3ml water, add 800 microlitre ethyl acetate, mix, centrifugal, reclaim ethyl acetate layer, dry then, be dissolved in 200 microliter methanol and and analyze with HPLC.

The yeast of expressing pYFS3 has produced apigenin by naringenin, and need not to use the acid treatment reaction mixture.This shows that purple perilla pFS3 cDNA coding has the active protein of flavone synthase II.

Industrial applicibility

By cDNA of the present invention is connected with suitable plant expression vector, and with its importing plant In to express or to suppress the expression of flavone synthase, can change colored color. In addition, by not Only in petal, also in whole plant or its suitable organ, express the flavone synthase gene, can increase Add anti-flora microbial resistance, perhaps by promoting to improve beans with the relation of rhizosphere microorganism The nitrogen fixing capacity of section plant, and the protective effect of improving plant antiultraviolet and illumination.

Sequence table

<110>SUNTORY?LIMITED

＜120〉gene of coding for flavone synthetic enzyme

<130>

<160>2

<210>1

<211>1770

<212>DNA

＜213〉purple perilla

<220>

＜223〉coding has the active proteinic nucleotide sequence that directly flavanone is changed into flavones

<400>1

tgtcgacgga?gcaagtggaa?atg?gca?ctg?tac?gcc?gcc?ctc?ttc?ctc?ctg?tcc 53

Met?Ala?Leu?Tyr?Ala?Ala?Leu?Phe?Leu?Leu?Ser

1 5 10

gcc?gcc?gtg?gtc?cgc?tcc?gtt?ctg?gat?cga?aaa?cgc?ggg?cgg?ccg?ccc 101

Ala?Ala?Val?Val?Arg?Ser?Val?Leu?Asp?Arg?Lys?Arg?Gly?Arg?Pro?Pro

15 20 25

tac?cct?ccc?ggg?ccg?ttc?cct?ctt?ccc?atc?atc?ggc?cac?tta?cac?ctc 149

Tyr?Pro?Pro?Gly?Pro?Phe?Pro?Leu?Pro?Ile?Ile?Gly?His?Leu?His?Leu

30 35 40

ctc?ggg?ccg?aga?ctc?cac?caa?acc?ttc?cac?gat?ctg?tcc?caa?cgg?tac 197

Leu?Gly?Pro?Arg?Leu?His?Gln?Thr?Phe?His?Asp?Leu?Ser?Gln?Arg?Tyr

45 50 55

ggg?ccc?tta?atg?cag?ctc?cgc?ctc?ggg?tcc?atc?cgc?tgc?gtc?att?gct 245

Gly?Pro?Leu?Met?Gln?Leu?Arg?Leu?Gly?Ser?Ile?Arg?Cys?Val?Ile?Ala

60 65 70 75

gcc?tcg?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?atc?gtc?acc?tac?gat 341

Phe?Ser?Ser?Arg?Lys?His?Ser?Thr?Ala?Ile?Asp?Ile?Val?Thr?Tyr?Asp

95 100 105

tca?tcc?ttc?gct?ttc?tct?ccc?tac?ggg?cct?tac?tgg?aaa?ttc?atc?aag 389

Ser?Ser?Phe?Ala?Phe?Ser?Pro?Tyr?Gly?Pro?Tyr?Trp?Lys?Phe?Ile?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?atc?agg?act?ctc?gaa?gtc?aag?tct?ttc?ctc?caa?att?ctt?atg 485

Gln?Pro?Ile?Arg?Thr?Leu?Glu?Val?Lys?Ser?Phe?Leu?Gln?Ile?Leu?Met

140 145 150 155

cgc?aag?ggt?gaa?tcg?ggg?gag?agc?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?agc?aac?gtc?ata?tcg?cat?atg?atg?ctg?agc?ata?cgg?tgt 581

Lys?Leu?Thr?Ser?Asn?Val?Ile?Ser?His?Met?Met?Leu?Ser?Ile?Arg?Cys

175 180 185

tca?gag?acg?gag?tcg?gag?gcg?gag?gcg?gcg?agg?acg?gtg?att?cgg?gag 629

Ser?Glu?Thr?Glu?Ser?Glu?Ala?Glu?Ala?Ala?Arg?Thr?Val?Ile?Arg?Glu

190 195 200

gtc?acg?cag?ata?ttt?ggg?gag?ttc?gac?gtc?tcc?gac?atc?ata?tgg?ctt 677

Val?Thr?Gln?Ile?Phe?Gly?Glu?Phe?Asp?Val?Ser?Asp?Ile?Ile?Trp?Leu

205 210 215

tgt?aag?aac?ttc?gat?ttc?caa?ggt?ata?agg?aag?cgg?tcc?gag?gat?atc 725

Cys?Lys?Asn?Phe?Asp?Phe?Gln?Gly?Ile?Arg?Lys?Arg?Ser?Glu?Asp?Ile

220 225 230 235

cag?agg?aga?tat?gat?gct?ctg?ctg?gag?aag?atc?atc?acc?gac?aga?gag 773

Gln?Arg?Arg?Tyr?Asp?Ala?Leu?Leu?Glu?Lys?Ile?Ile?Thr?Asp?Arg?Glu

240 245 250

aag?cag?agg?cgg?acc?cac?ggc?ggc?ggt?ggc?ggc?ggc?ggg?gaa?gcc?aag 821

Lys?Gln?Arg?Arg?Thr?His?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Glu?Ala?Lys

255 260 265

gat?ttt?ctt?gac?atg?ttc?ctc?gac?ata?atg?gag?agc?ggg?aaa?gcc?gaa 869

Asp?Phe?Leu?Asp?Met?Phe?Leu?Asp?Ile?Met?Glu?Ser?Gly?Lys?Ala?Glu

270 275 280

gtt?aaa?ttc?acg?agg?gag?cat?ctc?aaa?gct?ttg?att?ctg?gat?ttc?ttc 917

Val?Lys?Phe?Thr?Arg?Glu?His?Leu?Lys?Ala?Leu?Ile?Leu?Asp?Phe?Phe

285 290 295

acc?gcc?ggc?acc?gac?acg?acg?gcg?atc?gtg?tgt?gaa?tgg?gcg?ata?gca 965

Thr?Ala?Gly?Thr?Asp?Thr?Thr?Ala?Ile?Val?Cys?Glu?Trp?Ala?Ile?Ala

300 305 310 315

gaa?gtg?atc?aac?aat?cca?aat?gtg?ttg?aag?aaa?gct?caa?gaa?gag?att 1013

Glu?Val?Ile?Asn?Asn?Pro?Asn?Val?Leu?Lys?Lys?Ala?Gln?Glu?Glu?Ile

320 325 330

gcc?aac?atc?gtc?gga?ttc?gac?aga?att?ctg?caa?gaa?tcc?gac?gcc?cca 1061

Ala?Asn?Ile?Val?Gly?Phe?Asp?Arg?Ile?Leu?Gln?Glu?Ser?Asp?Ala?Pro

335 340 345

aat?ctg?ccc?tac?ctt?caa?gcc?ctc?atc?aaa?gaa?aca?ttc?cgg?ctc?cac 1109

Asn?Leu?Pro?Tyr?Leu?Gln?Ala?Leu?Ile?Lys?Glu?Thr?Phe?Arg?Leu?His

350 355 360

cct?cca?atc?cca?atg?ctg?gcg?agg?aaa?tcg?atc?tcc?gac?tgc?gtc?atc 1157

Pro?Pro?Ile?Pro?Met?Leu?Ala?Arg?Lys?Ser?Ile?Ser?Asp?Cys?Val?Ile

365 370 375

gac?ggc?tac?atg?att?ccg?gcc?aac?acg?ctg?ctc?ttc?gtc?aac?ctc?tgg 1205

Asp?Gly?Tyr?Met?Ile?Pro?Ala?Asn?Thr?Leu?Leu?Phe?Val?Asn?Leu?Trp

380 385 390 395

tcc?atg?ggg?cgg?aac?cct?aaa?atc?tgg?gac?tac?ccg?acg?gcg?ttc?cag 1253

Ser?Met?Gly?Arg?Asn?Pro?Lys?Ile?Trp?Asp?Tyr?Pro?Thr?Ala?Phe?Gln

400 405 410

ccg?gag?agg?ttt?ctg?gag?aag?gaa?aag?gcc?gcc?atc?gat?gtt?aaa?ggg 1301

Pro?Glu?Arg?Phe?Leu?Glu?Lys?Glu?Lys?Ala?Ala?Ile?Asp?Val?Lys?Gly

415 420 425

cag?cat?ttt?gag?ctg?cta?ccg?ttc?gga?acg?ggc?agg?aga?ggc?tgc?cca 1349

Gln?His?Phe?Glu?Leu?Leu?Pro?Phe?Gly?Thr?Gly?Arg?Arg?Gly?Cys?Pro

430 435 440

ggg?atg?ctt?tta?gcc?att?cag?gag?gtg?gtc?atc?ata?att?ggg?acg?atg 1397

Gly?Met?Leu?Leu?Ala?Ile?Gln?Glu?Val?Val?Ile?Ile?Ile?Gly?Thr?Met

445 450 455

att?caa?tgc?ttc?gat?tgg?aag?ctg?ccc?gac?ggc?tcc?ggc?cat?gtt?gat 1445

Ile?Gln?Cys?Phe?Asp?Trp?Lys?Leu?Pro?Asp?Gly?Ser?Gly?His?Val?Asp

460 465 470 475

atg?gca?gaa?cgg?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?Va1?Ser?Thr?Gln

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〉purple perilla

<220>

＜223〉has the active proteinic aminoacid sequence that directly flavanone is changed into flavones

<400>2

Met?Ala?Leu?Tyr?Ala?Ala?Leu?Phe?Leu?Leu?Ser?Ala?Ala?Val?Val?Arg

1 5 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?Ile?Ile?Gly?His?Leu?His?Leu?Leu?Gly?Pro?Arg?Leu

35 40 45

His?Gln?Thr?Phe?His?Asp?Leu?Ser?Gln?Arg?Tyr?Gly?Pro?Leu?Met?Gln

50 55 60

Leu?Arg?Leu?Gly?Ser?Ile?Arg?Cys?Val?Ile?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?Ile?Asp?Ile?Val?Thr?Tyr?Asp?Ser?Ser?Phe?Ala?Phe

100 105 110

Ser?Pro?Tyr?Gly?Pro?Tyr?Trp?Lys?Phe?Ile?Lys?Lys?Leu?Cys?Thr?Tyr

115 120 125

Glu?Leu?Leu?Gly?Ala?Arg?Asn?Leu?Ala?His?Phe?Gln?Pro?Ile?Arg?Thr

130 135 140

Leu?Glu?Val?Lys?Ser?Phe?Leu?Gln?Ile?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?Ile?Ser?His?Met?Met?Leu?Ser?Ile?Arg?Cys?Ser?Glu?Thr?Glu?Ser

180 185 190

Glu?Ala?Glu?Ala?Ala?Arg?Thr?Val?Ile?Arg?Glu?Val?Thr?Gln?Ile?Phe

195 200 205

Gly?Glu?Phe?Asp?Val?Ser?Asp?Ile?Ile?Trp?Leu?Cys?Lys?Asn?Phe?Asp

210 215 220

Phe?Gln?Gly?Ile?Arg?Lys?Arg?Ser?Glu?Asp?Ile?Gln?Arg?Arg?Tyr?Asp

225 230 235 240

Ala?Leu?Leu?Glu?Lys?Ile?Ile?Thr?Asp?Arg?Glu?Lys?Gln?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?Ile?Met?Glu?Ser?Gly?Lys?Ala?Glu?Val?Lys?Phe?Thr?Arg

275 280 285

Glu?His?Leu?Lys?Ala?Leu?Ile?Leu?Asp?Phe?Phe?Thr?Ala?Gly?Thr?Asp

290 295 300

Thr?Thr?Ala?Ile?Val?Cys?Glu?Trp?Ala?Ile?Ala?Glu?Val?Ile?Asn?Asn

305 310 315 320

Pro?Asn?Val?Leu?Lys?Lys?Ala?Gln?Glu?Glu?Ile?Ala?Asn?Ile?Val?Gly

325 330 335

Phe?Asp?Arg?Ile?Leu?Gln?Glu?Ser?Asp?Ala?Pro?Asn?Leu?Pro?Tyr?Leu

340 345 350

Gln?Ala?Leu?Ile?Lys?Glu?Thr?Phe?Arg?Leu?His?Pro?Pro?Ile?Pro?Met

355 360 365

Leu?Ala?Arg?Lys?Ser?Ile?Ser?Asp?Cys?Val?Ile?Asp?Gly?Tyr?Met?Ile

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?Ile?Trp?Asp?Tyr?Pro?Thr?Ala?Phe?Gln?Pro?Glu?Arg?Phe?Leu

405 410 415

Glu?Lys?Glu?Lys?Ala?Ala?Ile?Asp?Val?Lys?Gly?Gln?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

Ile?Gln?Glu?Val?Val?Ile?Ile?Ile?Gly?Thr?Met?Ile?Gln?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?Gln

500 505

Claims

1. gene, its coding has aminoacid sequence shown in the SEQ ID NO:2 of sequence table and demonstrates active protein by the synthetic flavones of flavanone, or coding has a kind of of following aminoacid sequence and has active protein by the synthetic flavones of flavanone, and wherein these aminoacid sequences are by adding or lacking one or more amino acid and/or carry out one or more replacements and modified with different aminoacids.

2. the gene of claim 1, aminoacid sequence has 55% identity at least shown in the SEQ ID NO:2 of its amino acid sequence coded and sequence table, and has the activity by the synthetic flavones of flavanone.

3. claim 1 or 2 gene, it is at 5 * SSC, under 50 ℃, can with all or part of hybridization of nucleotide sequence shown in the SEQID NO:1 of sequence table, and coding has the active protein by the synthetic flavones of flavanone.

4. carrier, it contains each described gene in the claim 1 to 3.

5. by the protein of each described genes encoding in the claim 1 to 3.

6. produce method of protein, it is characterized in that cultivating or the carrier host transformed of the claim 4 of growing and from described host, reclaim described protein with flavones composite reactive.

7. the flavonoid that uses each described genes produce in the claim 1 to 3 to have change is formed and/or the method for the plant of flavonoid amount.

8. use each described genes produce in the claim 1 to 3 to have the method for plant of the flavones amount of change.

9. use each described genes produce in the claim 1 to 3 to have the method for plant of the colored color of change.

10. use the method for the plant of each described genes produce flower color change indigo plant in the claim 1 to 3.

11. use the method for the plant that each described genes produce flower color reddens in the claim 1 to 3.

12. use each described genes produce in the claim 1 to 3 to have the method for plant of the photosensitivity of change.

13. the interactional method of each described Gene Handling plant and microorganism in the use claim 1 to 3.