WO2019009257A1 - マロニル基転移酵素遺伝子の使用 - Google Patents
マロニル基転移酵素遺伝子の使用 Download PDFInfo
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- WO2019009257A1 WO2019009257A1 PCT/JP2018/025101 JP2018025101W WO2019009257A1 WO 2019009257 A1 WO2019009257 A1 WO 2019009257A1 JP 2018025101 W JP2018025101 W JP 2018025101W WO 2019009257 A1 WO2019009257 A1 WO 2019009257A1
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- flavone
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- amino acid
- glucoside
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/02—Flowers
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
Definitions
- the present invention uses a polynucleotide encoding a protein having an activity of transferring a malonyl group to the 6th position of glucose in the 7th position of the flavone 7-glucoside, and copigmenting a flavone wherein a malonyl group is added to the 7th position of glucose.
- the present invention relates to a method of producing a plant variety having a modified flower color, preferably a flower color that is more blue than existing varieties, and a method of producing a flavone in which a malonyl group is added to glucose at position 7.
- flavonoids exhibit various colors such as yellow, red, purple and blue.
- flavonoids one group exhibiting red, purple and blue is generically called anthocyanin, and can be classified into three groups of pelargonidin, cyanidin and delphinidin according to the structure of aglycone.
- anthocyanins are modified by one or more aromatic acyl groups, (ii) anthocyanins coexist with copigments such as flavone and flavonol (Iii) the coexistence of iron ion and aluminum ion with anthocyanin, (iv) the pH of vacuoles in which anthocyanin is localized increases from neutral to slightly alkaline, or (v) anthocyanins, copigments, metals It is believed that either of the ions forming a complex (such anthocyanins are called metalloanthocyanins) is required (Non-patent Document 1).
- Non-patent Document 2 The biosynthetic pathways of flavonoids and anthocyanins are well studied, and relevant biosynthetic enzymes and genes encoding them have been identified (Non-patent Document 2).
- flavone which is one of the flavonoids, has the effect of making the flower color darker when it coexists with anthocyanin, and genes encoding flavone biosynthetic enzymes have been identified from many plants.
- Enzyme genes that modify anthocyanins and flavones are also obtained from many plants, such as glycosyltransferase gene, acyl transferase gene, methyl transferase gene and the like.
- Anthocyanins and flavones undergo various species- and cultivar-specific modifications by these enzymes, and this diversity contributes to the diversity of flower color.
- a gene encoding a protein having an activity of transferring a malonyl group to position 6 of glucose at position 3 of anthocyanin 3-glucoside has been isolated from chrysanthemum, dahlia, cineraria (Non-patent Document 3, Patent Document 1) ).
- a gene encoding a protein having an activity of transferring a malonyl group to the 6th position of glucose at the 5th position of anthocyanin 5-glucoside has been isolated from Salvia and Arabidopsis thaliana (Non-patent Document 4, Patent Document 1).
- a gene encoding a protein having an activity of transferring a malonyl group to position 6 of glucose at position 7 of isoflavone 7-glucoside has been isolated from soybean, medicac, etc. (Non-Patent Documents 5 and 6).
- a gene encoding a protein having an activity of transferring a malonyl group to position 6 of glucose at position 7 of flavonol 7-glucoside has been isolated from tobacco (Non-patent Document 7).
- Patent Document 2 a blue chrysanthemum that produces a blue pigment delphinidin derivative has been produced.
- the petal bluening factor is considered to be due to the copigment effect of luteolin 7-malonyl glucoside, which is one of the coexisting flavone.
- a gene derived from chrysanthemum which encodes a protein having an activity of transferring a malonyl group to position 6 of glucose at position 7 of luteolin 7-glucoside has not been identified.
- the problem to be solved by the present invention is to identify a polynucleotide encoding a protein having an activity of specifically transferring a malonyl group to position 6 of glucose at position 7 of flavone 7-glucoside in chrysanthemum It is to provide a method for producing flavone in which a malonyl group is added to glucose at position 7 using this. Furthermore, such a polynucleotide is used to form a flavone in which a malonyl group is added to glucose at position 7, and the copigment effect of the flavone causes a modified flower color, preferably a flower color that is bluer than existing varieties. It is to create the plant variety which it has.
- the inventor of the present invention has conducted intensive studies and repeated experiments. As a result, it is derived from chrysanthemum as a protein having an activity of specifically transferring a malonyl group to the 6th position of 7th glucose of flavone 7-glucoside.
- Anthocyanin malonyl group transfer enzyme homolog (Dm3MaT3) was identified, and the present invention has been completed. That is, the present invention is as follows.
- a flavone in which a malonyl group is added to glucose at position 7, thereby producing a plant variety having a modified flower color, preferably a blue flower color than existing varieties. it can.
- a protein solution crudely extracted from E. coli expressing Dm3 MaT3, a protein solution crudely extracted from E. coli expressing Dm3 MaT1, and a protein solution crude extracted from E. coli expressing Dm3 MaT2 were enzymatically reacted with luteolin 7-glucoside It is a high performance liquid chromatogram of the reaction liquid. Cyanidin 3-glucoside was enzymatically reacted with each of a protein solution roughly extracted from E. coli in which Dm3MaT3 was expressed, a protein solution roughly extracted from E. coli in which Dm3MaT1 was expressed, and a protein solution roughly extracted from E.
- coli in which Dm3MaT2 was expressed It is a high performance liquid chromatogram of the reaction liquid. It is a high performance liquid chromatogram of the reaction liquid which carried out the enzyme reaction of Dm3MaT3 protein solution and luteolin 7-glucoside. It is a high performance liquid chromatogram of the reaction liquid which carried out the enzyme reaction of Dm3MaT3 protein solution and luteolin 4'-glucoside. It is the figure which put together the reactivity with respect to the various flavonoid board
- the polynucleotide (SEQ ID NO: 1) used in the present invention encodes Dm3MaT3.
- the term "polynucleotide” means DNA or RNA.
- the polynucleotide used in the present invention is not limited to one consisting of a polynucleotide encoding a protein consisting of the base sequence of SEQ ID NO: 1 or the corresponding amino acid sequence (SEQ ID NO: 2), and the base sequence or a complementary sequence thereof Or a specific sequence homology, preferably sequence identity, with the amino acid sequence, and the activity of specifically transferring the malonyl group to the 6th position of the 7th glucose of flavone 7-glucoside It contains a polynucleotide encoding a protein that it has.
- the polynucleotide used in the present invention Is not limited to the gene derived from chrysanthemum, and it is possible to use a plant or animal as a source of a gene encoding a protein having an activity to specifically transfer a malonyl group to the 6th position of the 7th position of glucose in flavone 7-glucoside It may be any microorganism, and as long as it has an activity to specifically transfer the malonyl group to position 6 of glucose at position 7 of flavone 7-glucoside, it can be used to change the flower color in plants regardless of its origin It is.
- the polypeptide used in the present invention is a polynucleotide which hybridizes under polynucleotide stringent conditions consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, which comprises glucose at position 7 of flavone 7-glucoside. Also included are polynucleotides encoding proteins having an activity to specifically transfer the malonyl group at position 6.
- stringent conditions is conditions that allow selective and detectable specific binding of a polynucleotide or oligonucleotide to genomic DNA. Stringent conditions are defined by a suitable combination of salt concentration, organic solvent (eg, formamide), temperature, and other known conditions.
- the stringency is increased by reducing the salt concentration, increasing the organic solvent concentration, or raising the hybridization temperature.
- post-hybridization wash conditions also affect stringency.
- the wash conditions are also defined by salt concentration and temperature, with decreasing salt concentration and increasing temperature increasing the stringency of the wash. Therefore, the term "stringent conditions” means that the degree of "identity” or "homology" between each base sequence is, for example, about 80% or more on average, preferably about 90% or more, more preferably about the whole. It means conditions that allow specific hybridization only between base sequences having high homology, such as about 95% or more, more preferably 97% or more, and most preferably 98% or more.
- stringent conditions include conditions where the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the pH is 6 to 8 at a temperature of 60 ° C. to 68 ° C. Specific examples thereof include 5 Hybridization is performed under the conditions of ⁇ SSC (750 mM NaCl, 75 mM trisodium citrate), 1% SDS, 5 ⁇ Denhardt's solution 50% formaldehyde, and 42 ° C., 0.1 ⁇ SSC (15 mM NaCl, 1.5 mM trisodium citrate) And 0.1% SDS at 55 ° C.).
- Hybridization may be performed by any method known in the art, such as, for example, the method described in Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). It can carry out according to the method according to it. In addition, when using a commercially available library, it can be performed according to the method described in the attached instruction manual.
- the genes selected by such hybridization may be those of natural origin, such as those of plants or those other than plants.
- the gene selected by hybridization may be cDNA, and may be genomic DNA or chemically synthesized DNA.
- the DNA according to the present invention can be synthesized chemically by methods known to those skilled in the art, for example, the phosphoramidite method etc., using a plant nucleic acid sample as a template and using primers designed based on the nucleotide sequence of the target gene. It can be obtained by a nucleic acid amplification method or the like.
- the polypeptide used in the present invention consists of the amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, and of flavone 7-glucoside Also included is a polynucleotide encoding a protein having an activity of specifically transferring a malonyl group to position 6 of glucose at position 7.
- amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added is, for example, any of 1 to 20, preferably 1 to 5, more preferably 1 to 3 Means an amino acid sequence in which a number of amino acids are deleted, substituted, inserted and / or added.
- Site-directed mutagenesis which is one of the genetic engineering techniques, is useful because it is a technique that can introduce a specific mutation at a specific position, and is useful in Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory. It can carry out according to the method as described in Press, Cold Spring Harbor, NY, 1989 grade
- This mutant DNA By expressing this mutant DNA using an appropriate expression system, it is possible to obtain a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added.
- the polypeptide used in the present invention has an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and a malonyl group at position 6 of glucose at position 7 of flavone 7-glucoside
- a polynucleotide encoding a protein having an activity of specifically transferring
- Such a polypeptide encodes a protein having an amino acid sequence having preferably at least about 95%, more preferably at least 97%, most preferably at least 98% identity to the amino acid sequence of SEQ ID NO: 2.
- identity refers to any combination of amino acid residues that make up the chain between two chains in a polypeptide sequence (or amino acid sequence) or polynucleotide sequence (or base sequence).
- the numerical value of "identity" described herein may be a numerical value calculated using a homology search program known to those skilled in the art, but preferably MacVector Values calculated using the ClustalW program of the application (version 14.5.2 (24), Oxford Molecular Ltd., Oxford, England).
- polynucleotides used in the present invention are those which "code” the protein of interest.
- encoding means that the protein of interest is expressed with its activity.
- encoding includes the meaning of both encoding the protein of interest as a continuous structural sequence (exon) or encoding via an intervening sequence (intron).
- a gene having a native base sequence can be obtained, for example, by analysis with a DNA sequencer as described in the following examples.
- DNA encoding an enzyme having a modified amino acid sequence can be synthesized using routine site-directed mutagenesis or PCR based on DNA having a natural nucleotide sequence.
- a DNA fragment to be modified is obtained by restriction enzyme treatment of native cDNA or genomic DNA, which is used as a template, site-directed mutagenesis or PCR is carried out using primers into which a desired mutation has been introduced, and the desired A modified DNA fragment is obtained. Thereafter, the DNA fragment into which this mutation has been introduced may be ligated with the DNA fragment encoding the other part of the target enzyme.
- a DNA encoding an enzyme consisting of a truncated amino acid sequence for example, an amino acid sequence longer than the target amino acid sequence
- a DNA encoding a full length amino acid sequence is cleaved with a desired restriction enzyme.
- a DNA fragment consisting of the missing sequence may be synthesized and ligated.
- the obtained polynucleotide is expressed using a gene expression system in E. coli and yeast, and the enzyme activity is measured, whereby the obtained polynucleotide is malonyl at position 6 of glucose at position 7 of flavone 7-glucoside. It can be confirmed to encode a protein having an activity of specifically transferring a group. Furthermore, by expressing the polynucleotide, it is possible to obtain a protein having an activity of specifically transferring a malonyl group to position 6 of glucose at position 7 of the polynucleotide product flavone 7-glucoside.
- an antibody against a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 a protein having the activity of specifically transferring a malonyl group to the 6th position of glucose at the 7th position of flavone 7-glucoside can be obtained.
- Such an antibody can also be used to clone a polynucleotide encoding a protein having an activity of specifically transferring a malonyl group to position 6 of glucose at position 7 of flavone 7-glucoside derived from other organisms.
- An exogenous polynucleotide encoding a protein having an activity of specifically transferring a malonyl group to position 6 of glucose at position 7 of flavone 7-glucoside thus obtained for example, a (recombinant) vector
- a (recombinant) vector for example, by introducing it into a plant using an expression vector and containing it in the cells of the plant, it contains a flavone in which a malonyl group is added to glucose at position 7 and thereby a modified flower color is obtained.
- transgenic plants or their progeny or parts or tissues thereof (including cells) having a flower color that is more blue than existing varieties can be produced.
- the form of the portion or tissue can be cut flowers.
- plants that can be transformed include rose, carnation, chrysanthemum, snapdragon, cyclamen, orchid, free spinach, freesia, gerbera, gladiolus, gypsophila, kalanchoe, lily, pelargonium, geranium, petunia, torenia, thurip, anceria, moth orchid Rice, barley, wheat, rapeseed, potato, tomato, poplar, banana, eucalyptus, eucalyptus, sweet potato, soybean, alfalsa, rubin, corn, cauliflower, dahlia and the like, without being limited thereto.
- a processed product (processed cut flower product) using cut flowers of the genetically modified plant obtained according to the above or the progeny thereof.
- the cut flower processed products include, but are not limited to, pressed flowers using the cut flowers, prizzard flowers, dried flowers, resin-sealed products, and the like.
- a polynucleotide encoding a protein having an activity of specifically transferring a malonyl group to position 6 of glucose at position 7 of flavone 7-glucoside, a malonyl group donor such as flavone 7-glucoside and / or malonyl CoA The malonyl is introduced into the 7-position glucose by introducing it into the non-human host containing it, culturing or growing the non-human host, and recovering the flavone in which the malonyl group is added to the 7-position glucose from the non-human host
- the flavone to which a group is added can be easily produced.
- the target protein can be obtained by recovery and purification.
- the flavone in which a malonyl group is added to glucose at position 7 produced by the production method of the present invention can be used for applications such as production of food, medicine, cosmetics and the like.
- Prokaryotic or eukaryotic organisms can be used as non-human hosts.
- bacteria for example, bacteria belonging to the genus Escherichia, for example, Escherichia coli, bacteria of the genus Bacillus, for example, commonly used hosts such as Bacillus subtilis can be used.
- eukaryotes lower eukaryotes, for example, eukaryotic microbes such as yeast which is a fungus or filamentous fungi can be used.
- yeast examples include, for example, microorganisms of the genus Saccharomyces (eg, Saccharomyces cerevisiae), and examples of filamentous fungi include microorganisms of the genus Aspergillus (eg, Aspergillus oryzae). Aspergillus niger (Aspergillus niger), Penicillium (Penicillium) genus microorganisms are mentioned.
- animal cells or plant cells can be used as hosts, and cell lines such as mice, hamsters, monkeys and humans are used as animal cells, and insect cells such as silkworm cells and silkworm adults themselves are also used. Used as a host. In the method of the present invention, preferably plant cells are used.
- nonrecombinant vectors containing the above-mentioned polynucleotides in particular the expression vector is used to introduce the polynucleotide into the non-human host, and the technology to constitutively or tissue-specifically express the polynucleotide is used can do.
- the introduction of a polynucleotide into a non-human host can be carried out by methods known to those skilled in the art, for example, the Agrobacterium method, binary vector method, electroporation method, PEG method, particle gun method and the like.
- the expression vector used in the present invention contains an expression control region such as a promoter, a terminator, an origin of replication and the like depending on the type of non-human host into which they are introduced.
- an expression control region such as a promoter, a terminator, an origin of replication and the like depending on the type of non-human host into which they are introduced.
- promoters for bacterial expression vectors commonly used promoters such as trc promoter, tac promoter and lac promoter are used, and as yeast promoters, for example, glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter and the like are used
- yeast promoters for example, glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter and the like are used
- a promoter for filamentous fungi for example, amylase promoter, trpC promoter and the like are used.
- promoters for animal cell hosts viral promoters such as SV40 early promoter, SV40 late promoter and the like are used.
- promoters that constitutively express polynucleotides in plant cells include the 35S RNA promoter of cauliflower mosaic virus, rd29A gene promoter, rbcS promoter, mac-1 promoter and the like.
- tissue specific gene expression a promoter of a gene specifically expressed in the tissue can be used.
- the production of the expression vector can be performed according to a conventional method using a restriction enzyme, a ligase or the like.
- Example 1 Experiment for measuring enzyme activity of a protein candidate having an activity of transferring a malonyl group to position 6 of glucose at position 7 of flavone 7-glucoside (when using a protein solution crudely extracted from E. coli)] ⁇ Preparation of expression vector for E. coli expression>
- the unfunctionalized Dm3MaT3 described in Non-Patent Document 8 is a protein candidate having the activity of transferring a malonyl group to the 6th position of the glucose at the 7th position of flavone 7-glucoside, and is manufactured using pET32a (Novagen). According to the protocol recommended in the above, an E.
- coli expression vector (pET32a-Dm3MaT3) containing the full-length Dm3MaT3 was prepared. ⁇ Expression of malonyl transferase in E. coli> pET32a-Dm3MaT3 was introduced into E. coli strain BL21 using One Shot BL21 (DE3) (invitorgen) according to the protocol recommended by the manufacturer to obtain transformed E. coli. The E. coli was cultured using Overnight Express Autoinduction System 1 (Novagen) according to the protocol recommended by the manufacturer. In 2 ml of the prepared culture, transformed E. coli was cultured at 37 ° C. until the OD600 value became 0.5 (about 4 hours). This E.
- coli solution was added to 50 ml of culture solution as a preculture solution, and main culture was carried out overnight at 25 ° C. The overnight main culture E. coli solution was centrifuged (3000 rpm, 4 ° C., 15 minutes), and the collected cells were collected in 5 ml sonic buffer (composition; TrisHCl (pH 7.0): 2.5 mM, dithiothreitol (DTT) ): Suspended in 1 mM, amidinophanylmethanesulfonyl fluoride hydrochloride (APMSF): 10 ⁇ M, and after crushing E.
- composition TrisHCl (pH 7.0): 2.5 mM, dithiothreitol (DTT)
- stop buffer (90% aqueous acetonitrile containing 0.1% TFA) was added to stop the reaction, and the reaction solution was analyzed by high performance liquid chromatography (Prominence (Shimadzu Corp.)).
- the detector detected flavone at 330 nm using Shimadzu PDA SPD-M20A.
- the column used Shim-Pack ODS 150 mm * 4.6 mm (Shimadzu Corporation).
- solution A (0.1% TFA aqueous solution
- solution B 90% methanol aqueous solution containing 0.1% TFA
- Dm3MaT2 a vector for E.
- Dm3MaT3 is different from Dm3MaT1 and 2, and a gene encoding a protein having an activity of transferring a malonyl group to the 6th position of glucose in the 3rd position of anthocyanin 3-glucoside, the 6th position of glucose in the 3rd position is malonyl Not a gene encoding a protein having an activity to transfer the malonyl group to the 3-position of glucose in position 3 of the anthocyanin, but it has an activity to transfer the malonyl group to the 6-position of glucose at the 7-position of flavone 7-glucoside It has been shown that it may be a gene encoding a protein.
- Example 2 Experiment for measuring the enzyme activity of a protein having an activity of transferring a malonyl group to position 6 of glucose at position 7 of flavone 7-glucoside (when using a protein solution obtained by purifying a protein to which His-Tag has been added from E. coli )] ⁇ Expression of malonyl transferase in E. coli and protein purification> E. coli strain BL21 into which pET32a-Dm3MaT3 was introduced described in Example 1 was cultured using Overnight Express Autoinduction System 1 (Novagen) according to the protocol recommended by the manufacturer. In 8 ml of the prepared culture, transformed E. coli was cultured at 37 ° C.
- E. coli solution was added to 200 ml of culture solution as a pre-culture solution, and main culture was performed at 25 ° C. overnight. The overnight main culture E. coli solution is centrifuged (1000 ⁇ g, 4 ° C., 10 minutes), and the collected cells are extracted with 20 ml of an extract (composition; buffer (KCl: 300 mM, KH 2 PO 4 : 50 mM, Imidazole: 5 mM) (pH 8.0), amidinophanylmethanesulfonyl fluoride hydrochloric acid (APMSF): 10 ⁇ M), and after E.
- composition composition; buffer (KCl: 300 mM, KH 2 PO 4 : 50 mM, Imidazole: 5 mM) (pH 8.0), amidinophanylmethanesulfonyl fluoride hydrochloric acid (APMSF): 10 ⁇ M
- APIMSF amidinophanylmethanesulfonyl flu
- coli was disrupted by sonication, centrifuged (1400 ⁇ g, 4 ° C., 20 minutes) And the supernatant was collected. The supernatant was passed through a 0.45 ⁇ m filter and His-Tag purified using Profinia (Bio-Rad) according to the protocol recommended by the manufacturer. The resulting purified protein solution is centrifuged (7500 ⁇ g, 4 ° C., 15 minutes) using centrifugal filters (Ultracel-10K) (Amicon Ultra), and the concentrated protein solution is “Dm3MaT3 protein solution ". Avanti HP-26XP (rotor: JA-2) was used for centrifugation (BECKMAN COULTER).
- stop buffer (90% aqueous acetonitrile containing 0.1% TFA) was added to stop the reaction, and the reaction solution was analyzed by high performance liquid chromatography (Prominence (Shimadzu Corp.)).
- the detector detected flavone at 330 nm using Shimadzu PDA SPD-M20A.
- the column used Shim-Pack ODS 150 mm * 4.6 mm (Shimadzu Corporation).
- solution A (0.1% TFA aqueous solution
- solution B 90% methanol aqueous solution containing 0.1% TFA
- a linear gradient of 16 minutes from the mixture of both 8: 2 to the mixture of 0:10 was followed by elution with a mixture of 0:10 for 6 minutes.
- the flow rate was 0.6 ml / min.
- luteolin 7-malonyl glucoside was synthesized in the enzyme reaction solution.
- the reaction rate (the rate at which the substrate was converted) was 81.13% (see FIGS. 3 and 5).
- Apigenin 7-malonyl glucoside was synthesized when the enzyme reaction was carried out under the same reaction conditions with the substrate as 500 ⁇ g / ml apigenin 7-glucoside (dissolved in 50% aqueous acetonitrile containing 0.1% TFA) .
- the reaction rate was 85.80% (FIG. 5).
- Dm3MaT3 protein is similar to apigenin 7-glucoside and luteolin 7-glucoside, when examined for reactivity with cyanidin 3-glucoside, cyanidin 3,5-diglucoside, delphinidin, delphinidin 3-glucoside, delphinidin 3,5-diglucoside).
- Glucose is selectively malonylated at position 7 of the flavone 7-glucoside and is known to be a malonyl transferase with high substrate specificity. It has become one (see Figure 5).
- Dm3MaT3 and a known glycosyltransferase were analyzed.
- Dm3MaT3 and the same malonyl transferase derived from chrysanthemum were compared, the identity of the amino acid sequences between Dm3MaT3 and Dm3MaT1 and between Dm3MaT3 and Dm3MaT2 was 55% and 53%, respectively (see FIG. 6).
- the amino acid sequence with the highest identity to Dm3MaT3 is Dm3MaT1 (see FIG. 7).
- Dm3MaT1 and Dm3MaT2 do not have the activity to transfer the malonyl group to position 6 of glucose at position 7 of flavone 7-glucoside
- Dm3MaT3 of the present application is a malonyl transferase having different functions between Dm3MaT1 and Dm3MaT2 It is.
- Example 3 Expression in Rose of Dm3MaT3 Gene
- pSPB7136 was constructed (see FIG. 8) and introduced into rose (ocean song).
- pB7136 pBINPLUS (Van Engel et al., Transgenic Research 4, 288) is used as a basic skeleton, and the El235S promoter (Mitsuhara et al., (1996) Plant Cell Physiol.
- RNA isolation was obtained using the Plant RNAeasy Kit (QIAGEN) according to the protocol recommended by the manufacturer, and cDNA synthesis using the GeneRacer Kit (invitrogen) according to the protocol recommended by the manufacturer went.
- the reverse transcription PCR reaction was performed in 20 ⁇ l using cDNA as a template and AmpliTaq Gold DNA Polymerase (Thermo Fisher Scientific) according to the protocol recommended by the manufacturer (hold at 94 ° C for 5 minutes, 94 ° C After repeating the cycle of holding for 30 seconds at 55 ° C. for 30 seconds and holding at 72 ° C. for 1 minute and 30 seconds for 30 cycles, the temperature was kept at 4 ° C. for 7 minutes at 72 ° C.). At that time, a primer (forward primer: ATGGCTTCTCTTCCCATCTTG, reverse primer: TTAAAGGTATGCTTTTAGTCC) was designed and used such that the full-length cDNA of Dm3MaT3 is specifically amplified. The reaction product was analyzed by agarose gel electrophoresis, and a band of 1365 bp corresponding to full-length cDNA was detected, which confirmed that the Dm3MaT3 gene was transcribed in rose.
- Example 4 Functional analysis of Dm3MaT3 in rose
- a crude enzyme solution was prepared from the petals of the rose strain from which the full-length cDNA Dm3MaT3 transcript was synthesized, and the presence or absence of the activity to transfer the malonyl group to the 6-position of the 7-position glucose of flavone 7-glucoside was evaluated.
- the obtained protein solution was centrifuged (10000 rpm, 4 ° C., 10 minutes), and ammonium sulfate was added to the collected supernatant to a saturation concentration of 35%. After stirring at 4 ° C.
- the supernatant was recovered by centrifugation (10000 rpm, 4 ° C., 10 minutes). Ammonium sulfate was added to the obtained supernatant to a saturation concentration of 70%, stirred at 4 ° C. for 3 hours, and centrifuged (10000 rpm, 4 ° C., 10 minutes) to obtain a precipitate.
- the detector detected flavone at 330 nm using Shimadzu PDA SPD-M20A.
- the column used Shim-Pack ODS 150 mm * 4.6 mm (Shimadzu Corporation).
- solution A (0.1% TFA aqueous solution
- solution B 90% acetonitrile aqueous solution containing 0.1% TFA
- a 5-minute linear concentration gradient from the mixture to the 0:10 mixture was followed by elution with the 0:10 mixture for 1 minute.
- the flow rate was 0.6 ml / min.
- a crude enzyme solution was prepared from the petals in the same manner for non-genetically modified rose, and an enzyme activity measurement experiment was performed.
- apigenin compounds 71.24% of apigenin and 28.76% of apigenin 7-glucoside in the enzyme reaction solution using non-genetically modified rose petal crude enzyme solution, apigenin 7-malonylglucoside was not detected.
- the peak of apigenin 7-malonyl glucoside accounts for 2.04%, and the remaining 97.96% corresponds to the non-control.
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Abstract
Description
アントシアニンやフラボンを修飾する酵素遺伝子も多くの植物から得られており、例えば糖転移酵素遺伝子、アシル基転移酵素遺伝子、メチル基転移酵素遺伝子などがある。アントシアニンやフラボンは、これらの酵素によって、種および品種特異的に多様な修飾を受け、この多様性が花色の多様さの一因となっている。例えば、アントシアニン 3-グルコシドの3位のグルコースの6位にマロニル基を転移する活性を有するタンパク質をコードする遺伝子は、キク、ダリア、シネラリアから単離されている(非特許文献3、特許文献1)。アントシアニン 5-グルコシドの5位のグルコースの6位にマロニル基を転移する活性を有するタンパク質をコードする遺伝子は、サルビア、シロイヌナズナから単離されている(非特許文献4、特許文献1)。イソフラボン 7-グルコシドの7位のグルコースの6位にマロニル基を転移する活性を有するタンパク質をコードする遺伝子は、ダイズ、メディカゴなどから単離されている(非特許文献5、6)。フラボノール 7-グルコシドの7位のグルコースの6位にマロニル基を転移する活性を有するタンパク質をコードする遺伝子は、タバコから単離されている(非特許文献7)。
かかる状況の下、本発明が解決しようとする課題は、キクにおいてフラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチドを同定し、これを用いて、7位のグルコースにマロニル基が付加されたフラボンの製造方法を提供することである。さらに、このようなポリヌクレオチドを用いて7位のグルコースにマロニル基が付加されたフラボンを生成し、当該フラボンのコピグメント効果により、改変された花色、好ましくは、既存の品種よりも青い花色を有する植物品種を作出することである。
すなわち、本発明は以下の通りのものである。
[1] 以下の(a)~(e):
(a)配列番号1の塩基配列からなるポリヌクレオチド;
(b)配列番号1の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリジェント条件下でハイブリダイズするポリヌクレオチドであって、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
(c)配列番号2のアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(d)配列番号2のアミノ酸配列において、1又は数個のアミノ酸が欠失、置換、挿入、及び/又は付加されたアミノ酸配列からなり、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;及び
(e)配列番号2のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を有し、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
からなる群から選ばれるポリヌクレオチドを宿主植物に導入することを含む、7位のグルコースにマロニル基が付与されたフラボンを生成する遺伝子組換え植物又はその子孫を生産する方法。
[2] 前記フラボンがルテオリン又はアピゲニンである、1に記載の方法。
[3] 前記遺伝子組換え植物が改変された花色を有する、1又は2に記載の方法。
[4] 以下の(a)~(e):
(a)配列番号1の塩基配列からなるポリヌクレオチド;
(b)配列番号1の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリジェント条件下でハイブリダイズするポリヌクレオチドであって、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
(c)配列番号2のアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(d)配列番号2のアミノ酸配列において、1又は数個のアミノ酸が欠失、置換、挿入、及び/又は付加されたアミノ酸配列からなり、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;及び
(e)配列番号2のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を有し、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
からなる群から選ばれるポリヌクレオチドを含むことを特徴とする、7位のグルコースにマロニル基が付与されたフラボンを生成する遺伝子組換え植物若しくはその子孫又はそれらの部分若しくは組織。
[5] 前記フラボンがルテオリン又はアピゲニンである、4に記載の遺伝子組換え植物若しくはその子孫又はそれらの部分若しくは組織。
[6] 前記遺伝子組換え植物が改変された花色を有する、4又は5に記載の植物若しくはその子孫又はそれらの部分若しくは組織。
[7] 切花である、4~6のいずれかに記載の植物の部分。
[8] 7に記載の切花を用いた切花加工品。
[9] 以下の(a)~(e):
(a)配列番号1の塩基配列からなるポリヌクレオチド;
(b)配列番号1の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリジェント条件下でハイブリダイズするポリヌクレオチドであって、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
(c)配列番号2のアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(d)配列番号2のアミノ酸配列において、1又は数個のアミノ酸が欠失、置換、挿入、及び/又は付加されたアミノ酸配列からなり、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;及び
(e)配列番号2のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を有し、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
からなる群から選ばれるポリヌクレオチドを非ヒト宿主に導入し、
該非ヒト宿主を培養し又は生育させること、
を含む、7位のグルコースにマロニル基が付加されたフラボンの製造方法。
[10] 前記非ヒト宿主が植物細胞である、9に記載の方法。
[11] 前記フラボンがルテオリン又はアピゲニンである、9又は10に記載の方法。
本明細書では、キク由来のフラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードする遺伝子について述べているが、本発明で用いられるポリヌクレオチドは、キク由来の遺伝子に限定されるものではなく、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードする遺伝子の起源としては植物でも動物でも微生物であってもよく、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有している限り、起源を問わず、植物における花色の変更に利用可能である。
本発明に係るDNAは、当業者に公知の方法、例えば、ホスホアミダイド法等により化学的に合成する方法、植物の核酸試料を鋳型とし、目的とする遺伝子のヌクレオチド配列に基づいて設計したプライマーを用いる核酸増幅法などによって得ることができる。
あるいは短縮されたアミノ酸配列からなる酵素をコードするDNAを得るには、例えば、目的とするアミノ酸配列より長いアミノ酸配列、例えば、全長アミノ酸配列をコードするDNAを所望の制限酵素により切断し、その結果得られたDNA断片が目的とするアミノ酸配列の全体をコードしていない場合は、不足部分の配列からなるDNA断片を合成し、連結すればよい。
植物細胞内でポリヌクレオチドを構成的に発現させるプロモーターの例としては、カリフラワーモザイクウィルスの35S RNAプロモーター、rd29A遺伝子プロモーター、rbcSプロモーター、mac-1プロモーター等が挙げられる。また、組織特異的な遺伝子発現のためには、その組織で特異的に発現する遺伝子のプロモーターを用いることができる。
発現ベクターの作製は、制限酵素、リガーゼなどを用いて常法に従って行うことができる。
[実施例1:フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を転移する活性を有するタンパク質候補の酵素活性測定実験(大腸菌から粗抽出したタンパク質溶液を用いる場合)]
<大腸菌発現用ベクターの作製>
非特許文献8に記載された機能未同定のDm3MaT3をフラボン 7-グルコシドの7位のグルコースの6位にマロニル基を転移する活性を有するタンパク質候補とし、pET32a(Novagen社)を用いて、製造者に推奨されているプロトコールに従って、Dm3MaT3の完全長を含む大腸菌発現用ベクター(pET32a-Dm3MaT3)を作製した。
<マロニル転移酵素の大腸菌での発現>
pET32a-Dm3MaT3を、One Shot BL21(DE3)(invitorgen)を用いて、製造者に推奨されているプロトコールに従い、大腸菌株BL21へ導入し、形質転換大腸菌を取得した。この大腸菌をOvernight Express Autoinduction System1(Novagen社)を用いて、製造者に推奨されているプロトコールに従い、培養した。調製した培養液2mlで、形質転換大腸菌をOD600値が0.5になるまで37℃で培養した(約4時間)。この大腸菌液を前培養液として、50mlの培養液に加え、25℃で一晩本培養した。
一晩本培養した大腸菌液を遠心分離(3000rpm、4℃、15分間)し、集菌した菌体を5mlのソニックバッファー(組成;TrisHCl(pH7.0):2.5mM、ジチオスレイトール(DTT):1mM、アミジノファニルメタンスルフォニルフルオライド塩酸(APMSF):10μM)に懸濁し、超音波処理により大腸菌を粉砕した後、遠心分離(15000rpm、4℃、10分間)して、上清を回収した。その上清を、Dm3MaT3を発現させた大腸菌から粗抽出したタンパク質溶液とした。遠心分離には、Avanti HP-26XP(ローター:JA-2)を使用した(BECKMAN COULTER社)。
50μlのDm3MaT3を発現させた大腸菌から粗抽出したタンパク質溶液、5μlの1mg/mlのマロニル-CoA、5μlの1M KPB(pH7.0)、5μlの500μg/mlのルテオリン 7-グルコシド(0.1%TFAを含む50%アセトニトリル水溶液に溶解)を混合し、水で100μlになるように氷上で調整した反応液を30℃で20分間保持した。その後、100μlの停止バッファー(0.1%TFAを含む90%アセトニトリル水溶液)を加えて反応を停止させ、反応液を高速液体クロマトグラフィー(Prominence(島津製作所))にて分析した。検出器は島津PDA SPD-M20Aを用い330nmでフラボンを検出した。カラムはShim-Pack ODS 150mm*4.6mm(島津製作所)を用いた。溶出には、A液(0.1%TFA水溶液)とB液(0.1%TFAを含む90%メタノール水溶液)を用いた。両者の8:2の混合液から0:10の混合液までの16分間の直線濃度勾配とそれにつづく6分間0:10の混合液による溶出を行なった。流速は0.6ml/分とした。コントロールとして、インサートを挿入しないpET32aベクターを導入した大腸菌から粗抽出したタンパク質溶液を用いて同様の実験を行った。
キクは、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を転移する活性以外に、アントシアニンの3位のグルコースの6位にマロニル基を転移する活性を有する(非特許文献8参照)。Dm3MaT3を、アントシアニンの3位のグルコースの6位にマロニル基を転移する活性を有するタンパク質をコードする遺伝子、及び3位のグルコースの6位がマロニル化されたアントシアニンの3位のグルコースの3位にマロニル基を転移する活性を有するタンパク質をコードする遺伝子としてすでに報告されているキク由来アントシアニジン3-O-グルコシド-6”-O-マロニル基転移酵素(Dm3MaT1)、及びキク由来アントシアニジン3-O-グルコシド-3”,6”-O-ジマロニル基転移酵素(Dm3MaT2)と差別化するため、Dm3MaT1、2についても同様に大腸菌発現用ベクターを作製し、大腸菌から粗抽出したタンパク質溶液を用いて酵素活性測定実験を行った。さらに、シアニジン 3-グルコシドを基質とした酵素反応も行い、Dm3MaT3の結果と比較した。その場合、反応液を高速液体クロマトグラフィー(Prominence(島津製作所))にて分析をするときには、検出器は島津PDA SPD-M20Aを用い520nmでアントシアニンを検出した。カラムはShodex RSpak DE-413L(Shodex)を用いた。溶出には、A液(0.1%TFA水溶液)とB液(0.1%TFAを含む90%アセトニトリル水溶液)を用いた。両者の8:2の混合液から0:10の混合液までの15分間の直線濃度勾配とそれにつづく5分間0:10の混合液による溶出を行なった。流速は0.6ml/分とした。
その結果、Dm3MaT3とルテオリン 7-グルコシドを酵素反応させると、基質として加えたルテオリン 7-グルコシド以外に、ルテオリン 7-マロニルグルコシドのピークが検出された。Dm3MaT1、2とルテオリン 7-グルコシドを酵素反応させた場合には、ルテオリン 7-マロニルグルコシドのピークは検出されなかった(図1参照)。
また、Dm3MaT3とシアニジン3-グルコシドを酵素反応させると、基質として加えたシアニジン 3-グルコシド以外に、シアニジン 3-マロニルグルコシドのピークが検出された。しかし、Dm3MaT1とシアニジン 3-グルコシドを反応させた場合と比較して、Dm3MaT3の場合では、シアニジン 3-グルコシドの消費量は明らかに少なかった(図2参照)。
これらの結果より、Dm3MaT3は、Dm3MaT1、2と異なり、アントシアニン3-グルコシドの3位のグルコースの6位にマロニル基を転移する活性を有するタンパク質をコードする遺伝子、3位のグルコースの6位がマロニル化されたアントシアニンの3位のグルコースの3位にマロニル基を転移する活性を有するタンパク質をコードする遺伝子ではなく、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を転移する活性を有するタンパク質をコードする遺伝子である可能性が示された。
<マロニル基転移酵素の大腸菌での発現とタンパク質精製>
実施例1で記載したpET32a-Dm3MaT3を導入した大腸菌株BL21をOvernight Express Autoinduction System1(Novagen社)を用いて、製造者に推奨されているプロトコールに従い、培養した。調製した培養液8mlで、形質転換大腸菌をOD600値が0.5になるまで37℃で培養した(約4時間)。この大腸菌液を前培養液として、200mlの培養液に加え、25℃で一晩本培養した。
一晩本培養した大腸菌液を遠心分離(1000×g、4℃、10分間)し、集菌した菌体を20mlの抽出液(組成;緩衝液(KCl:300mM、KH2PO4:50mM、イミダゾール:5mM)(pH8.0)、アミジノファニルメタンスルフォニルフルオライド塩酸(APMSF):10μM)に懸濁し、超音波処理により大腸菌を粉砕した後、遠心分離(1400×g、4℃、20分)して、上清を回収した。その上清を0.45μmフィルターに通し、Profinia(Bio-Rad)を用いて、製造者に推奨されているプロトコールに従って、His-Tag精製した。得られた精製タンパク質溶液を、centrifugal Filters(Ultracel-10K)(Amicon Ultra社)を用いて、遠心分離(7500×g、4℃、15分間)し、その濃縮されたタンパク質溶液を「Dm3MaT3タンパク質溶液」とした。遠心分離には、Avanti HP-26XP(ローター:JA-2)を使用した(BECKMAN COULTER社)。
30μlのDm3MaT3タンパク質溶液(10μg)、5μlの1mg/mlのマロニル-CoA、5μlの1M KPB(pH7.0)、5μlの500μg/mlのルテオリン 7-グルコシド(0.1%TFAを含む50%アセトニトリル水溶液に溶解)を混合し、水で100μlになるように氷上で調整した反応液を30℃で20分間保持した。その後、100μlの停止バッファー(0.1%TFAを含む90%アセトニトリル水溶液)を加えて反応を停止させ、反応液を高速液体クロマトグラフィー(Prominence(島津製作所))にて分析した。検出器は島津PDA SPD-M20Aを用い330nmでフラボンを検出した。カラムはShim-Pack ODS 150mm*4.6mm(島津製作所)を用いた。溶出には、A液(0.1%TFA水溶液)とB液(0.1%TFAを含む90%メタノール水溶液)を用いた。両者の8:2の混合液から0:10の混合液までの16分間の直線濃度勾配とそれにつづく6分間0:10の混合液による溶出を行なった。流速は0.6ml/分とした。
本発明のDm3MaT3遺伝子が、植物内でフラボン 7-グルコシドの7位のグルコースの6位にマロニル基を転移する活性を有するタンパク質をコードすることを確かめるため、Dm3MaT3を植物で発現させるためのバイナリーベクターpSPB7136を構築し(図8参照)、バラ(オーシャンソング)へ導入した。pSPB7136では、基本骨格としてpBINPLUS(Van Engel et al., Transgenic Research 4, 288)を、Dm3MaT3遺伝子を発現させるプロモーターとしてEl235Sプロモーター(Mitsuhara et al., (1996) Plant Cell Physiol. 37, p49)を、ターミネーターとしてHSPターミネーター(Plant Cell Physiol (2010) 51, 328-332)を使用した。
pSPB7136を導入した遺伝子組換えバラの若い葉を用いて、Dm3MaT3遺伝子の発現解析を行った。トータルRNA単離はPlant RNAeasy Kit(QIAGEN社)を用いて、製造者に推奨されているプロトコールに従って取得し、cDNA合成はGeneRacer Kit(invitrogen社)を用いて、製造者に推奨されているプロトコールに従って行った。逆転写PCR反応は、cDNAを鋳型として、AmpliTaq Gold DNA Polymerase(Thermo Fisher Scientific社)を用いて、製造者に推奨されているプロトコールに従い、20μlで行った(94℃で5分間保持し、94℃で30秒、55℃で30秒、72℃で1分30秒間保持のサイクルを30サイクル繰り返した後、72℃7分、4℃で保持した)。その際、Dm3MaT3の完全長cDNAが特異的に増幅するようなプライマー(フォワードプライマー:ATGGCTTCTCTTCCCATCTTG、リバースプライマー:TTAAAGGTATGCTTTTAGTCC)を設計し、利用した。反応産物をアガロースゲル電気泳動で解析したところ、完全長cDNAに相当する1365bpのバンドが検出されたことから、バラにおいてDm3MaT3遺伝子が転写されていることが確認された。
完全長cDNADm3MaT3の転写産物が合成されたバラ系統の花弁から粗酵素液を調製し、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を転移する活性の有無を評価した。2.5gの花弁サンプルを液体窒素で冷やしながら乳鉢ですりつぶし、30mlの抽出バッファー(組成;TrisHCl(pH7.5):100mM、ポリビニルピロリドン K-30:10mg/ml、1-チオグリセロール:1mg/ml、アミジノファニルメタンスルフォニルフルオライド塩酸(APMSF):10μM)に溶かした。得られたタンパク質溶液を遠心分離(10000rpm、4℃、10分間)し、回収した上清に35%の飽和濃度となるように硫酸アンモニウムを加えた。4℃で1時間撹拌した後、遠心分離(10000rpm、4℃、10分間)して上清を回収した。得られた上清に硫酸アンモニウムを飽和濃度70%となるように添加し、4℃で3時間撹拌した後、遠心分離(10000rpm、4℃、10分間)して沈澱を得た。この沈澱を1mlの溶出バッファー(組成;TrisHCl(pH7.5):20mM、DTT:1mM、アミジノファニルメタンスルフォニルフルオライド塩酸(APMSF):10μM)に溶かし、NAP-5Colums Sephadex G-25 DNA Grade(GE Healthcare社)を用いてカラム精製を行って、硫酸アンモニウムを取り除いた。この液を「花弁粗酵素液」とした。遠心分離には、Avanti HP-26XP(ローター:JA-2)を使用した(BECKMAN COULTER社)。
20μlの花弁粗酵素液、5μlの1mg/mlのマロニル-CoA、5μlの1M KPB(pH7.0)、2.5μlの1mMのアピゲニン(0.1%TFAを含む50%アセトニトリル水溶液に溶解)を混合し、水で100μlになるように氷上で調整した反応液を30℃で20分間保持した。その後、100μlの停止バッファー(0.1%TFAを含む90%アセトニトリル水溶液)を加えて反応を停止させ、反応液を高速液体クロマトグラフィー(Prominence(島津製作所))にて分析した。検出器は島津PDA SPD-M20Aを用い330nmでフラボンを検出した。カラムはShim-Pack ODS 150mm*4.6mm(島津製作所)を用いた。溶出には、A液(0.1%TFA水溶液)とB液(0.1%TFAを含む90%アセトニトリル水溶液)を用いた。両者の9:1の混合液から8:2の混合液までの20分間の直線濃度勾配、8:2の混合液から2:8の混合液までの15分間の直線濃度勾配、2:8の混合液から0:10の混合液までの5分間の直線濃度勾配とそれにつづく1分間0:10の混合液による溶出を行なった。流速は0.6ml/分とした。対照として、非遺伝子組換えバラについても同様にして、花弁から粗酵素液を調製し、酵素活性測定実験を行った。
非遺伝子組換えバラの花弁粗酵素液を用いた酵素反応液中においては、アピゲニン化合物のうちアピゲニンが71.24%、アピゲニン 7-グルコシドが28.76%を占めており、アピゲニン 7-マロニルグルコシドは検出されなかった。Dm3MaT3遺伝子を導入した遺伝子組換えバラの花弁粗酵素液を用いた酵素反応液中においては、アピゲニン 7-マロニルグルコシドのピークが2.04%を占め、残りの97.96%は、対照の非遺伝子組換えバラの花弁粗酵素液を用いた酵素反応液にも含まれるアピゲニン、アピゲニン 7-グルコシドであった。このことから、遺伝子組換えバラの花弁において、フラボン7-グルコシドの7位のグルコースにマロニル基を転移する活性を有するDm3MaT3が発現していることが確認された。
Claims (11)
- 以下の(a)~(e):
(a)配列番号1の塩基配列からなるポリヌクレオチド;
(b)配列番号1の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリジェント条件下でハイブリダイズするポリヌクレオチドであって、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
(c)配列番号2のアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(d)配列番号2のアミノ酸配列において、1又は数個のアミノ酸が欠失、置換、挿入、及び/又は付加されたアミノ酸配列からなり、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;及び
(e)配列番号2のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を有し、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
からなる群から選ばれるポリヌクレオチドを宿主植物に導入することを含む、7位のグルコースにマロニル基が付与されたフラボンを生成する遺伝子組換え植物又はその子孫を生産する方法。 - 前記フラボンがルテオリン又はアピゲニンである、請求項1に記載の方法。
- 前記遺伝子組換え植物が改変された花色を有する、請求項1又は2に記載の方法。
- 以下の(a)~(e):
(a)配列番号1の塩基配列からなるポリヌクレオチド;
(b)配列番号1の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリジェント条件下でハイブリダイズするポリヌクレオチドであって、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
(c)配列番号2のアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(d)配列番号2のアミノ酸配列において、1又は数個のアミノ酸が欠失、置換、挿入、及び/又は付加されたアミノ酸配列からなり、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;及び
(e)配列番号2のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を有し、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
からなる群から選ばれるポリヌクレオチドを含むことを特徴とする、7位のグルコースにマロニル基が付与されたフラボンを生成する遺伝子組換え植物若しくはその子孫又はそれらの部分若しくは組織。 - 前記フラボンがルテオリン又はアピゲニンである、請求項4に記載の遺伝子組換え植物若しくはその子孫又はそれらの部分若しくは組織。
- 前記遺伝子組換え植物が改変された花色を有する、請求項4又は5に記載の植物若しくはその子孫又はそれらの部分若しくは組織。
- 切花である、請求項4~6のいずれか1項に記載の植物の部分。
- 請求項7に記載の切花を用いた切花加工品。
- 以下の(a)~(e):
(a)配列番号1の塩基配列からなるポリヌクレオチド;
(b)配列番号1の塩基配列と相補的な塩基配列からなるポリヌクレオチドとストリジェント条件下でハイブリダイズするポリヌクレオチドであって、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
(c)配列番号2のアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(d)配列番号2のアミノ酸配列において、1又は数個のアミノ酸が欠失、置換、挿入、及び/又は付加されたアミノ酸配列からなり、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;及び
(e)配列番号2のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を有し、かつ、フラボン 7-グルコシドの7位のグルコースの6位にマロニル基を特異的に転移する活性を有するタンパク質をコードするポリヌクレオチド;
からなる群から選ばれるポリヌクレオチドを非ヒト宿主に導入し、
該非ヒト宿主を培養し又は生育させること、
を含む、7位のグルコースにマロニル基が付加されたフラボンの製造方法。 - 前記非ヒト宿主が植物細胞である、請求項9に記載の方法。
- 前記フラボンがルテオリン又はアピゲニンである、請求項9又は10に記載の方法。
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