EP1114162A1 - Genes codant pour les synthetases de flavones - Google Patents

Genes codant pour les synthetases de flavones

Info

Publication number
EP1114162A1
EP1114162A1 EP00940914A EP00940914A EP1114162A1 EP 1114162 A1 EP1114162 A1 EP 1114162A1 EP 00940914 A EP00940914 A EP 00940914A EP 00940914 A EP00940914 A EP 00940914A EP 1114162 A1 EP1114162 A1 EP 1114162A1
Authority
EP
European Patent Office
Prior art keywords
gene
gene according
flavones
plant
flavanones
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00940914A
Other languages
German (de)
English (en)
Inventor
Masako Mizutani
Yoshikazu Tanaka
Takaaki Kusumi
Shin-Ichi Ayabe
Tomoyoshi Akashi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suntory Ltd
Original Assignee
Suntory Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP20522999A external-priority patent/JP4368005B2/ja
Application filed by Suntory Ltd filed Critical Suntory Ltd
Publication of EP1114162A1 publication Critical patent/EP1114162A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis

Definitions

  • 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 relates to genes encoding proteins with activity of synthesizing flavones from flavanones, and to their utilization.
  • the abundance of different flower colors is one of 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- 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 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 plants from the destruction of the ozone layer.
  • Flavonoids 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 and other pollinators, protecting plant from ultraviolet rays, and participating in interaction with soil microorganisms (BioEssays, 16 (1994), Koes at al., p.123; Trends in Plant Science, 1 (1997), Shirley, B.W., p.377).
  • flavone plays an important role in interaction of plants with microorganisms, especially in legumes, where they participate in the initial steps of the symbiosis with leguminous bacteria (Plant Cell, 7
  • Flavones in petals play a role in recognition by insects and act as copigments which form complexes with anthocyanins .
  • 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 P450 family (flavone synthase II). These groups of enzymes are completely different enzymes with no structural homology.
  • 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).
  • 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.
  • CYPGe-3 cytochrome P450 No.CYP ⁇ lEl
  • CYPGe-5 cytochrome P450 NO.CYP93B1, hereinafter indicated as CYP93B1
  • CYP93B1 cytochrome P450 NO.CYP93B1
  • 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 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 enzyme, so that it was concluded that a total of two enzymes were necessary.
  • flavone synthase genes preferably flavone synthase II genes, and more preferably genes for flavone synthases 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 .
  • the present invention therefore provides genes 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 genes encoding P450 proteins having the amino acid sequences listed as SEQ.ID. No.
  • the invention further provides a gene encoding proteins having amino acid sequences with at least 55% identity with the amino acid sequences listed as SEQ.ID. No. 2 of the Sequence Listing and possessing activity of synthesizing flavones from flavanones.
  • the invention still further provides genes encoding proteins possessing activity of synthesizing flavones from flavanones, and hybridizing with all or a part of the nucleotide sequences listed as SEQ.ID. No. 1 of the Sequence List under the conditions of 5 x SSC, 50°C.
  • the invention still further provides a vector, particularly an expression vector, containing any one of the aforementioned genes.
  • the invention still further provides a host transformed with the aforementioned vector.
  • the invention still further provides a protein encoded by any of the aforementioned genes.
  • the invention still further provides a process for producing the aforementioned protein which is 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 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 amounts and compositions of flavonoid using the aforementioned genes; a method of altering amounts of flavones using the aforementioned genes; a method of altering flower colors using the aforementioned genes; a method of bluing the color of flowers using the 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 aforementioned genes.
  • Flavanone 2-hydroxylase encoded by the Glycyrrhiza CYP93B1 gene produces 2-hydroxyflavanones from flavanones as the substrates, and the products are converted to 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 containing a large amount of flavones, to thus obtain cDNA encoding proteins with activity of synthesizing flavones directly from flavanones as substrates.
  • a cDNA library of perilla which contains a large amount of flavones is screened using the Glycyrrhiza-derived cDNA, CYP93B1 as a probe, to obtain cDNA encoding a novel cytochrome P450 ( see Example 1 ) .
  • the perilla-derived cDNA was expressed in yeast and reacted with naringenin, a flavanone, as a substrate which resulted in production not of 2-hydroxynaringenin but rather of the flavone apigenin, without acid treatment (see Example 2).
  • 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 example, one encoding the amino acid sequence listed as SEQ.ID. No. 2 of the Sequence Listing.
  • the present invention also relates to genes that have the nucleotide sequence listed as 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 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, 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 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 DNA.
  • the invention also relates to genes encoding proteins that have amino acid sequences with identity of at least 55%, preferably at least 70%, such as 80% or greater and even 90% or greater, with the amino acid sequence listed as SEQ.ID. No. 2 of the Sequence Listing, 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 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.
  • a DNA fragment into 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 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.
  • DNA encoding an enzyme consisting of a shortened amino acid sequence for 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 acid sequence, it may be linked with synthesized DNA comprising the rest of the sequence.
  • genes may be expressed in an expression system using E. coli or yeast and its enzyme activity measured to confirm that the obtained gene encodes flavone synthase.
  • the gene By expressing the gene, it is also possible to obtain the flavone synthase protein as 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. 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.
  • 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 .
  • eukaryotic hosts examples include lower eukaryotic organisms, for example, eukaryotic microorganisms, for example, Eumycota such as yeast and filamentous fungi.
  • yeast there may be mentioned microorganisms belonging to the genus Saccharomyces , such as Saccharomyces cerevisiae
  • 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 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 terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced.
  • promoters for bacterial expression vectors which may be used include conventional promoters such as trc promoter, tac promoter, lac promoter, etc.
  • yeast promoters examples include glyceraldehyde-3-phosphate dehydrogenase promoter, PH05 promoter, etc.
  • filamentous fungi promoters examples include amylase promoter, trpC, etc.
  • animal cell host promoters examples include viral promoters such as SV40 early promoter, SV40 late promoter, etc.
  • 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.
  • 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 filtration chromatography, ion-exchange chromatography and the like.
  • flavone synthase II derived from perilla that is capable of synthesizing flavones directly from flavanones
  • 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 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).
  • a gene for flavonoid 3 ', 5 ' -hydroxylase which was a type of cytochrome P450 and participated in 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 - li ⁇
  • flavone synthase II gene of the invention derived from perilla which is capable of synthesizing flavones directly from flavanones
  • flavone synthase II genes capable of synthesizing flavones directly from flavanones, from different species of plants.
  • purifying the perilla-derived flavone synthase II enzymes described in this specification which 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.
  • 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 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 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 or their tissues, which may also be in the form of cut flowers.
  • flavone synthases II or their genes which have been cloned according to the invention 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 the amount of flavones in the flower petals, thus allowing the colors of the flowers to be modified toward the blue, for example.
  • transformable plants there may be 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, sweet potato, soybean, alfalfa, lupin, corn, etc., but there is no limitation to these.
  • flavones have various physiological activities as explained above, they can impart new physiological activity or economic value to plants. For 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, animals or insects. Examples
  • RNA was extracted from leaves of red perilla (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 ⁇ gt 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 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. 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 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.
  • 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 ⁇
  • the phage clone #3 obtained in Example 1 was used as 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 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.
  • 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 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 ⁇ g/ml leucine, 20 ⁇ g/ml tryptophan and 5 mg/ml casamino acid), at 30°C for 24 hours.
  • selective medium 6.7 mg/ml amino acid-free yeast nitrogen base (Difco)
  • 20 mg/ml glucose 20 mg/ml glucose
  • 30 ⁇ g/ml leucine 20 ⁇ g/ml tryptophan
  • 5 mg/ml casamino acid 5 mg/ml casamino acid
  • the harvested yeast cells were cultured at 30 °C for 48 hours in an expressing medium (10 mg/ml yeast extract, 10 mg/ml peptone, 2 ⁇ g/ml hemin, 20 mg/ml galactose). After collecting the yeast cells, they were washed by suspending in water and collecting them. Glass 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 fraction.
  • an expressing medium (10 mg/ml yeast extract, 10 mg/ml peptone, 2 ⁇ g/ml hemin, 20 mg/ml galactose). After collecting the yeast cells, they were washed by suspending in water and collecting them. Glass 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
  • the acid treatment involved dissolution of the evaporator- dried sample in 150 ⁇ l of ethanol containing 10% hydrochloric acid, and stirring for 30 minutes. This was diluted with 1.3 ml of water, 800 ⁇ l 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 ⁇ l of methanol, and analyzed by HPLC. The yeast expressing pYFS3 yielded apigenin from naringenin without acid treatment of the reaction mixture.
  • perilla pFS3 cDNA encodes a protein with flavone synthase II activity.
  • Industrial Applicability 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.
  • flavone synthase genes not only in petals but also in entire plants or their appropriate organs, it is possible to increase the resistance agasint microorganisms of plants or to improve the nitrogen 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Nutrition Science (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne des ADN obtenus, par exemple, à partir de perilla et codant une enzyme capable de transformer directement les flavanones en flavones, ainsi que l'utilisation de ceux-ci; le listage des séquences d'acides aminés et de l'ADN destinés à l'enzyme codée se présente comme SEQ.ID. No. 1 & 2. L'insertion de ce gène dans les plantes peut, par exemple, modifier la couleur des fleurs d'une plante.
EP00940914A 1999-07-19 2000-06-30 Genes codant pour les synthetases de flavones Withdrawn EP1114162A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP20522999A JP4368005B2 (ja) 1999-01-29 1999-07-19 フラボン合成酵素をコードする遺伝子
JP20522999 1999-07-19
PCT/JP2000/004379 WO2001005981A1 (fr) 1999-07-19 2000-06-30 Genes codant pour les synthetases de flavones

Publications (1)

Publication Number Publication Date
EP1114162A1 true EP1114162A1 (fr) 2001-07-11

Family

ID=16503554

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00940914A Withdrawn EP1114162A1 (fr) 1999-07-19 2000-06-30 Genes codant pour les synthetases de flavones

Country Status (8)

Country Link
EP (1) EP1114162A1 (fr)
KR (1) KR20010071128A (fr)
CN (2) CN1310762A (fr)
AU (1) AU5572100A (fr)
CA (1) CA2324228A1 (fr)
IL (1) IL138718A0 (fr)
NZ (1) NZ507150A (fr)
WO (1) WO2001005981A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002086110A2 (fr) * 2001-04-20 2002-10-31 Technische Universität München Enzymes de flavone synthase i et leur utilisation
CN101514344B (zh) * 2009-02-19 2010-12-08 上海交通大学 黄酮合成酶基因及其编码的多肽
CN106754989B (zh) * 2016-12-21 2020-04-21 广东药科大学 布渣叶黄烷酮-2-羟基化酶及其编码基因与应用
CN114940982B (zh) * 2022-06-08 2023-01-03 百事基材料（青岛）股份有限公司 一种由基因工程菌制备的芹菜素及其在涤纶纤维制造中应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2130800C (fr) * 1992-03-02 2002-05-14 Yasuhiro Kikuchi Genes vegetaux codant pour le flavonoide-3',5'-hydroxylase
JPH09224671A (ja) * 1996-02-19 1997-09-02 Mitsui Gyosai Shokubutsu Bio Kenkyusho:Kk 新規なチトクロームｐ４５０をコードするｄｎａ
AUPN838696A0 (en) * 1996-03-01 1996-03-21 Florigene Limited Genetic sequences encoding flavonoid pathway enzymes and uses therefor
JP4368005B2 (ja) * 1999-01-29 2009-11-18 インターナショナル フラワー ディベロプメンツ プロプライアタリー リミティド フラボン合成酵素をコードする遺伝子

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
None *
See also references of WO0105981A1 *

Also Published As

Publication number Publication date
WO2001005981A1 (fr) 2001-01-25
NZ507150A (en) 2003-11-28
CN1310762A (zh) 2001-08-29
CN1624136A (zh) 2005-06-08
KR20010071128A (ko) 2001-07-28
IL138718A0 (en) 2001-10-31
AU5572100A (en) 2001-02-05
CA2324228A1 (fr) 2001-01-19

Similar Documents

Publication Publication Date Title
US7465569B2 (en) Flavone synthases, methods of using flavone synthases, and plants expressing flavone synthases
JPH07505772A (ja) フラボノイド経路の酵素をコードする遺伝子配列およびその用途
US20060242735A1 (en) Nucleic acid fragments encoding isoflavone synthase
EP1114162A1 (fr) Genes codant pour les synthetases de flavones
CA2590286C (fr) Genes codant pour les synthetases de flavones
JP4259886B2 (ja) 新規糖転移活性を有する蛋白質をコードする遺伝子
AU2005201464B2 (en) Gene encoding flavone synthase
AU2005246960A1 (en) Genes coding for flavone synthases
JPH05184370A (ja) フラボノイド水酸化酵素遺伝子

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17P Request for examination filed

Effective date: 20010620

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: SUNTORY FLOWERS LIMITED

Owner name: SUNTORY LIMITED

17Q First examination report despatched

Effective date: 20030422

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: SUNTORY LIMITED

17Q First examination report despatched

Effective date: 20030422

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20080815