WO2006059433A1 - Nouveau gene de l’anthocyanidine glucosyltransferase - Google Patents

Nouveau gene de l’anthocyanidine glucosyltransferase Download PDF

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Publication number
WO2006059433A1
WO2006059433A1 PCT/JP2005/019294 JP2005019294W WO2006059433A1 WO 2006059433 A1 WO2006059433 A1 WO 2006059433A1 JP 2005019294 W JP2005019294 W JP 2005019294W WO 2006059433 A1 WO2006059433 A1 WO 2006059433A1
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gene
plant
protein
group
anthocyanin
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PCT/JP2005/019294
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English (en)
Japanese (ja)
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Jun Ogata
Yoshiaki Kanno
Hidehito Tsugawa
Masahiko Suzuki
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Aomori Prefecture
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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
    • 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
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)

Definitions

  • the present invention encodes a protein having an activity of transferring a darcosyl group to the hydroxyl group at the 5-position of anthocyanin and an activity of transferring Z or darcosyl group to the hydroxyl group at the 3-position of anthocyanin 5-darcoside. It relates to genes and their use.
  • Anthocyanins are the most common among plant pigments, and anthocyanins exhibit many colors from light blue to deep red. Industrially, plant color is one of the most important traits, and as seen in the diversification of flower color, good color development of fruits, and stabilization of coloration, it is uniform. It is a big economic factor in vegetables. Color development by anthocyanins, a directly identifiable trait, has been the subject of many genetic, biochemical and molecular biological studies, and today there are genes involved in flavonoid biosynthesis systems, including flavonoids containing anthocyanins. Flowers such as petunia, goldfish, arabidopsis, fruits such as apples and grapes, and vegetables such as eggplant and perilla are also crawling. In addition, the mechanism of anthocyanin expression is being elucidated by natural product chemistry and physiological analysis.
  • anthocyanidin is an aglycone that forms the skeleton of anthocyanin, but it does not exist in the plant body as it is, but it always exists in a glycosylated form (anthocyanin).
  • anthocyanins become non-toxic anthocyanins and become stable, and they become soluble in water and become soluble in the vacuole of cells.
  • Anthocyanins form complex pigment macromolecules by forming intramolecular associations, intermolecular associations, and coordination bonds with auxiliaries such as flavones / flavonols and metal ions in the vacuole, and exhibit unique colors of each plant. . Therefore, anthocyanin glycosides are important as the first reaction to form anthocyanins and are essential chemical reactions for the subsequent color development.
  • the sugar residue that binds to the hydroxyl group of anthocyanin is the 3-position of the C ring, the 5-position, 7-position of the A-ring, and the 3 ' Forces found at the 5 'position, etc.
  • the order in which these sugar residues bind is considered to be a common reaction pathway across plants, as reported by various plant species. It is known in many plants that the glycoside of the hydroxyl group of anthocyanin begins the 3-position force of the C ring, and this is generally recognized as the first reaction process of the glycoside. The It is thought that sugar residues bind to the hydroxyl groups at the 3rd and 5th positions, and glycosides are generated in the order of 3rd to 5th positions. Glycosyltransferases are still found both enzymatically and molecularly.
  • glycosyltransferase glycoside enzyme
  • Patent Document 1 International Publication No. 99/05287 Pamphlet
  • Patent Document 2 International Publication No. 01/92509 Pamphlet
  • Patent Document 3 JP-A-10-113184
  • the present invention relates to a gene encoding a protein having an activity of transferring a darcosyl group, preferably an activity of transferring a darcosyl group to the hydroxyl groups at the 5-position and 3-position of anthocyanin in an order different from the known order. It was an object to obtain a gene encoding a protein having a protein.
  • a gene encoding a protein having a dalcosyl group transfer activity obtained in the present invention or a similar gene is introduced into a plant and expressed to modify the type of flavonoid compound to be accumulated, so that flower color, fruit, etc. It is possible to change the color of the plant body. In rose, gene expression control by RNAi method, gene disruption method, etc.
  • the present inventor has found an enzyme that catalyzes the reaction of transferring the darcosyl group to the hydroxyl group at the 3-position after transferring the darcosyl group to the hydroxyl group at the 5-position of anthocyanin.
  • the gene was clawed.
  • RT-PCR was performed using degenerate primers synthesized based on the nucleotide sequence that is common to glycosyltransferases.
  • the cDNA fragment was amplified and its nucleotide sequence was determined.
  • the protein coding region of the gene was cloned by RT-PCR.
  • the enzyme activity was analyzed using a protein obtained by utilizing a recombinant protein synthesis system of Escherichia coli.
  • the present invention has been completed based on the above findings.
  • the present invention provides the following [1] to [16].
  • the first aspect of the present invention is the activity of transferring a darcosyl group to the hydroxyl group at the 5-position of anthocyanin and the activity of transferring Z or darcosyl group to the hydroxyl group at the 3-position of anthocyanin 5-darcoside. It is a gene that encodes a protein it has.
  • a second aspect of the present invention is the gene according to [1], which encodes the following proteins (a) to (:
  • the third aspect of the present invention is that stringent conditions are applied to a part or all of the nucleic acid represented by the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 2.
  • the fourth aspect of the present invention is that both the activity of transferring a darcosyl group to the hydroxyl group at the 5-position of anthocyanin and the activity of transferring the darcosyl group to the hydroxyl group at the 3-position of anthocyanin 5-darcoside It is a gene according to any one of [1] to [3] that encodes a protein having [5]
  • a fifth aspect of the present invention is a vector comprising the gene according to any one of [1] to [3].
  • a sixth aspect of the present invention is a vector containing the gene according to [4].
  • a seventh aspect of the present invention is a host cell transformed with the vector according to [5] or [6].
  • An eighth aspect of the present invention is a protein encoded by the gene according to any one of [1] to [4].
  • a ninth aspect of the present invention is that the host cell according to [7] is cultured or grown, and then the host cell force darcosyl group is transferred to the hydroxyl group at the 5-position of anthocyanin.
  • a method for producing a protein comprising collecting a protein having an activity and an activity of transferring Z or a dalcosyl group to a hydroxyl group at the 3-position of anthocyanin 5-darcoside.
  • a tenth aspect of the present invention is a method for producing the protein by a cell-free protein synthesis system using the gene according to any one of [1] to [4].
  • An eleventh aspect of the present invention is a plant transformed by introducing the gene according to any one of [1] to [4] or the vector according to [5] or [6] It is.
  • a twelfth aspect of the present invention is a progeny of the plant having the same properties as the plant according to [11].
  • the thirteenth aspect of the present invention is the plant according to [11] or the progeny tissue of the plant according to [12].
  • a fourteenth aspect of the present invention is a cut flower of the plant according to [11] or the progeny of the plant according to [12].
  • the fifteenth aspect of the present invention is an anthocyanin by introducing the gene according to [4] or the vector according to [6] into a plant or plant cell and expressing the gene. This is a method in which the darcosyl group is transferred to the hydroxyl group at the 5-position of gin and then transferred to the hydroxyl group at the 3-position.
  • the gene according to any one of [1] to [4] or the vector according to [5] or [6] is introduced into a plant or plant cell, By expressing the gene, It is a method of adjusting the flower color of a plant body.
  • the flower color of the plant body is regulated by suppressing the expression of the gene. It is a method to do.
  • the gene of the present invention encodes a protein having an activity of transferring a darcosyl group to the hydroxyl group at the 5-position of anthocyanin and an activity of transferring Z or darcosyl group to the hydroxyl group at the 3-position of anthocyanin 5-darcoside. is there.
  • Examples of the gene of the present invention include the following genes (A) to (D).
  • “having the amino acid sequence described in SEQ ID NO: 2” means that a plurality of amino acids are added to the N-terminal or C-terminal side of such a protein that includes only the protein having only the amino acid sequence described in SEQ ID NO: 2.
  • the purpose is to include the purified protein.
  • the number of amino acids to be added is not particularly limited as long as the above darcosyl group transfer activity is not lost, but it is usually within 400, preferably within 50.
  • one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2 have an amino acid sequence added, deleted, and substituted with Z or other amino acids, and the two enzyme activities
  • a gene encoding a protein having either one or both of the above proteins having an amino acid sequence that has been added, deleted, or substituted may be natural or artificial.
  • the number of amino acids to be added, deleted or substituted is not particularly limited as long as the above darcosyl group transfer activity is not lost, but is usually within 20 and preferably within 5.
  • (C) encodes a protein having an amino acid sequence exhibiting a certain homology to the amino acid sequence of SEQ ID NO: 2 and having either one or both of the two enzyme activities Gene
  • a certain degree of homology usually means a homology of 20% or more, preferably 50% or more, more preferably 60% or more, most preferably 70% or more. Intention Taste.
  • part of the nucleic acid means, for example, a portion encoding 6 or more amino acid sequences in the consensus sequence region.
  • the "stringent condition” refers to a condition in which only specific hybridization occurs and non-specific hybridization does not occur.
  • the temperature is 50 °. C
  • salt concentration is 5 X SSC (or equivalent salt concentration).
  • the appropriate hybridization temperature depends on the base sequence of the nucleic acid and the length of the nucleic acid.For example, when a DNA fragment of 18 bases encoding 6 amino acids is used as a probe, the temperature is 50 ° C. The following temperatures are preferred.
  • Examples of the gene selected by such hybridization include natural genes, for example, plant-derived genes, particularly rose-derived genes. Further, the gene selected by hybridization may be cDNA or genomic DNA.
  • the vector of the present invention can be produced by inserting the gene (1) into a known expression vector.
  • the known expression vector used here is not particularly limited as long as it contains an appropriate promoter, terminator, replication origin and the like.
  • the trc promoter, tac promoter, lac promoter, etc. can be used if the promoter is expressed in bacteria, and if expressed in yeast, the dalyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, etc. can be used.
  • Can be used and expressed in filamentous fungi If present, the amylase promoter, trpC promoter, etc. can be used, and if expressed in animal cells, the SV40 early promoter, SV40 rate promoter, etc. can be used.
  • the transformed host cell in the present invention is a host cell transformed with the vector (2).
  • the host cell may be either prokaryotic or eukaryotic.
  • prokaryotes for example, Escherichia coli, Bacillus subtilis, etc. can be used.
  • eukaryotes include yeast and filamentous fungi, as well as animal and plant cultured cells.
  • yeasts include Saccharomvces cerevisk (Saccharomvces cerevi Sk ⁇ ) and the like.
  • filamentous fungi include Aspergillus orvzae and Aspergillus nkgi.
  • Animal cells include: Examples thereof include cultured cells such as mouse, mouse, muster, monkey, human and silkworm.
  • the method of transforming with the vector of (2) is not particularly limited, and can be performed according to a conventional method.
  • Proteins encoded by the gene (1) are also included in the present invention. This protein can be produced, for example, by the method (5) described later.
  • the method for producing a protein of the present invention comprises culturing or growing the host cell of (3), and then transferring the host cell force darcosyl group to the hydroxyl group at the 5-position of anthocyanin and Z or dalcosyl. It is characterized by collecting a protein having an activity of transferring a group to the hydroxyl group at the 3-position of anthocyanin 5-darcoside, or by producing a protein using a cell-free protein synthesis system or the like.
  • the culture or growth of the host cell can be performed according to a method according to the type of the host cell.
  • protein production by a cell-free protein synthesis system can be performed according to a conventional method.
  • the protein can be collected according to a conventional method.
  • the protein can be filtered and removed from the culture.
  • the target protein can be obtained by collecting and purifying by heart separation, cell disruption, gel filtration, ion exchange chromatography and the like.
  • the plant of the present invention is transformed by introducing the gene (1) or the vector (2).
  • Plants to which genes and the like are to be introduced are not particularly limited. Gelaum, petunia, tulip, rice, barley, wheat, rapeseed, potato, tomato, poplar, banana, eucalyptus, sweet potato, soybean, alfalfa, lupine, corn, cauliflower, lobelia, apple, bud, peach, oyster, Plums, citrus fruits, etc. can be mentioned.
  • the progeny of the plant of the above (6) is also included in the present invention.
  • Tissues of the plant of the above (6) or the progeny of the plant of (7) are also included in the present invention.
  • Cut flowers of the plant of the above (6) or the progeny of the plant of (7) are also included in the present invention.
  • the method for transferring a darcosyl group of the present invention comprises the activity of transferring a darcosyl group to the hydroxyl group at the 5-position of anthocyanin in the gene of (1) above and the hydroxyl group at the 3-position of anthocyanin 5-glucoside.
  • the vector (2) which encodes a protein having both the activity of transferring to aldose, or the activity of transferring the dalcosyl group to the hydroxyl group at the 5-position of anthocyanin and the darcosyl group of anthocyanin 5-darcoside
  • the darcosyl group is transferred to the hydroxyl group at the 3-position.
  • the method for regulating flower color of the present invention comprises introducing the gene (1) or the vector (2) into a plant or a plant cell and expressing the gene, or a plant having the gene (1).
  • the color of the flower or fruit of the plant body is regulated by suppressing the expression of the gene.
  • the plant targeted for gene transfer is not particularly limited, and for example, the plant exemplified in (6) above can be used. Further, the plant to be suppressed by the gene (1) is not particularly limited as long as it is a plant having the gene (1).
  • the introduction and expression of the gene of (1) can be performed according to a conventional method.
  • suppression of gene expression in (1) can also be carried out according to conventional methods (for example, the antisense method, the cosuppression method, and the RNAi method).
  • a darcosyl group can be transferred to the hydroxyl group at the 5-position of anthocyanin, and then the darcosyl group can be transferred to the hydroxyl group at the 3-position. This makes it possible to modify plant tissues that are colored by the accumulation of anthocyanins such as flowers and fruits.
  • FIG. 1 The results of the reactions of [A. Cy (Cyanidin), B. Cy5G (Cyanidin 5-Og lucoside), C. Cy3G (Cyanidin 3-Q-glucoside)] by the rose petal extract enzyme solution. Show.
  • Rose dried petals HPLC (described later as a result of 520 analysis, containing 95% or more of ciazine 3,5-diglucoside in the crude extract: raw weight 100 g) in 1 L of 0.05% hydrochloric acid aqueous solution-left at room temperature
  • fractionation was performed with petroleum ether, black mouth form, and ethyl acetate, a glass column (30 mm id X 600 mm) packed with Amberlite XAD-7 Organo Co., Ltd. After equilibrating with 0.01% aqueous hydrochloric acid, the aqueous layer fraction diluted twice was passed through the column to adsorb the dye. After washing the unadsorbed material, the dye was eluted with 80% aqueous methanol solution containing 0.01% hydrochloric acid. The eluate was dried and solidified.
  • the dried and solidified pigment powder was redissolved in 0.05% aqueous hydrochloric acid solution, and an equal amount of 12% aqueous hydrochloric acid solution was added thereto, followed by heating at 80 ° C for 20 minutes for acid hydrolysis. After heating, an equal amount of isoamyl alcohol was added to the solution, and the mixture was vigorously stirred and centrifuged to remove the isoamyl alcohol layer. After this was performed three times, an equal amount of ethyl acetate was added, stirred, centrifuged, and the aqueous layer was fractionated. Paper chromatography (Whatman 3MM) was performed twice from the aqueous layer obtained by the above-mentioned method. 5-Dalcoside was isolated.
  • Reversed column WakosiHI5C18AR column (4.6 mm id) for HPLC analysis and fractionation X 250 mm) at a column temperature of 35 ° C.
  • a flow rate of lml / min an aqueous solution containing 0.5% TFA and 5% acetonitrile (A) with a linear concentration gradient of 20% of acetonitrile solution (B) containing 0.5% TFA at 5% force for 20 minutes
  • the elution time was detected using PDA.
  • PVPP polyvinylpolypyrrolidone
  • buffer A 100 mM potassium phosphate buffer (pHLO), 14 mM 2-mercaptoethanol, 1% polybutylpyrrolidone K-30 (PVP), lOOmM sodium chloride, 5 mM EDTA, 5 mM sodium ascorbate, 10% glycerol , 0.1% Triton X-100
  • the enzyme activity was measured using the above crude enzyme solution. HPLC analysis was performed according to the method described in Examples 1 and 2. As a result, when siadine was used as a substrate, the reaction products were siadine 5-darcoside, siadine 3,5-didalcoside, and trace amounts of siadine 3-darcoside. As a substrate, cyadin 3,5-diglucoside was not obtained. Furthermore, sheadin 3,5-didarcoside was obtained when sheadin 5-darcoside was used as a substrate. Figure 1 shows the reaction results for each substrate. In other words, it was thought that glycosylation at the 3rd position of anthocyanin, followed by glycosylation at the 5th position!
  • Total RNA was prepared from petals of rose cultivar, Krimson Erasmus by the modified CTAB method (Chang et al., 1993, Mukai 'Yamamoto Plant Cell Engineering Series 7 pp.57-62). using poly (A) + RNA Yoko total RNA (2 5 0 / zg) Power et Oligotex- dT30 super (Takara), was prepared in the manufacturer's recommended ways.
  • RNA was treated at 99 ° C for 2 minutes, cooled on ice, and then cDNA was synthesized using the first strand cDNA synthesis kit (Pharmacia) by the method indicated by the manufacturer.
  • Pharmacia the first strand cDNA synthesis kit
  • GTSPFd [WCICAYTGYGGIT GGAAYTC: SEQ ID NO: 3] designed based on the amino acid sequence and the sequence present in Not Id (T) used for cDNA synthesis.
  • PCR was performed using the designed NotlRl [AACTGGAAGAATTCGCGGC: SEQ ID NO: 4] as a primer.
  • the PCR reaction solution was 2 ⁇ 1 cDNA, lx Expand HF buffer (BOEHRINGE R MANNHEIM), 0.2 mM dNTPs ⁇ primer 1.6 pmol / ⁇ 1 each, Expand High Fidelity P CR system enzyme mix 1.75 unit total volume 50 ⁇ 1 Prepared.
  • the reaction was performed at 94 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 48 ° C for 30 seconds, 72 ° C and 30 seconds, and finally treated at 72 ° C for 1 minute. did.
  • PCR reaction was performed using CAGGAAT: SEQ ID NO: 5) as a primer.
  • the PCR reaction solution was 2 1 cD NA, lx Expand HF buffer (BOEHRINGER MANNHEIM), 0.2 mM dNTPs ⁇ Primer 1.6 pmol / ⁇ 1 each, Expand High Fidelity PCR system Enzyme mix 1.75 unit total volume 50 / zl Prepared.
  • the reaction was performed at 94 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 48 ° C for -30 seconds, 72 ° C for .30 seconds, and finally treated at 72 ° C for 1 minute. did.
  • the obtained reaction product was subjected to 0.8% agarose gel electrophoresis, and a DNA fragment of the expected size was recovered.
  • the recovered DNA product was cloned into pGEM Easy T vector (Promega). in this way The ABI TM BigDye TM Terminator cycle sequencing reaction ver.3.1 (Applied Biosystem) was used to determine the total nucleotide sequence using DNA sequencer model 3100.
  • a BLASTX search was performed, and a clone RhGT fra.42 that was found to be homologous to the glycosyltransferase gene was found.
  • CDNA for mRNA having 5 ′ end used for 5 ′ RACE was synthesized using GeneRacer kit (Invitrogen) according to the method indicated by the manufacturer. 1st-PCR using the GeneRacer 5 'primer (CGACTGGAGCACGAGGACACTGA: SEQ ID NO: 6) and primer 42R1 (TTATGCGGGCCCAACAT GCC: SEQ ID NO: 7) set to the internal sequence of RhGT Fra. went.
  • PCR reaction solution 1 ⁇ 1 cDNA, lx LA PCR Buffer II (Takara), 0.2 mM dNTPs ⁇ 3 ⁇ 1 GeneRacer 5 ′ primer (10 M), 2 1 42R1 (5 ⁇ M), 2 mM MgCl, Takara LA Taq The total volume of 2.5 units was adjusted to 50 ⁇ 1.
  • the reaction is
  • PCR reaction solution is 1 1 cDNA, lx LA PCR Buffer II (Takara), 0.2 mM dNTPs, 1 ⁇ 1 GeneRacer 5 'primer (10 M), 2 1 42R1 (5 ⁇ M), 2 mM MgCl, Takara LA Taq 2.5 The total volume of unit was adjusted to 50 ⁇ 1
  • the reaction was performed at 94 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 58 ° C for 30 seconds and 72 ° C for 2 minutes, and finally treated at 72 ° C for 2 minutes. did.
  • the reaction solution was subjected to 0.8% agarose electrophoresis, and the amplified product band was cut out.
  • the amplified product was recovered using the Qiagen Gel Extraction kit (Qiagen).
  • the obtained PCR product was cloned into pGEM Easy T vector (Promega).
  • the protein coding region predicted from the base sequence of the clone obtained in Example 5 was amplified by PCR and cloned into a pET vector for expression of E. coli.
  • CDNA for total RNA was synthesized using a first strand cDNA synthesis kit (Pharmacia) and reverse transcription reaction using Not I dT primer according to the method recommended by the manufacturer.
  • Primer RhGTFd (GACGACGACAAG ATGGGTGGTGATGCTATAGTTTG: SEQ ID NO: 9) and termination codon including the sequence for cloning into pET30 Ek / LIC (Novagen) for expression of E. coli and the start codon of the protein predicted from the base sequence obtained in Example 5
  • the PCR reaction solution is 2 ⁇ 1 cDNA, lx Expand HF Knoffer (BOEHRINGER MANNHEIM), 0.2 mM dNTPs, each primer 1.6 pmol / ⁇ 1, Expand High Fidelity PCR system Enzyme mix 1.75 units total volume 50 1 ⁇ did.
  • the reaction was performed at 94 ° C for 2 minutes, followed by 40 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C-2 minutes, and finally treated at 72 ° C for 2 minutes. did.
  • the obtained reaction product was subjected to 0.8% agarose gel electrophoresis, and the amplified product was purified from the gel using QIAquick Gel Extraction Kit (Qiagen). After ligating the obtained DNA to pET30Ek / LIC (Novagen) according to the method recommended by the manufacturer, it was transformed into E. coli JM109, and several clones were used with BigDye Terminator Ver. 3 using T7 promoter and T7 terminator primer. After performing Cycle Sequencing Reaction using 1., the base sequence was determined using ABI3100 Genetic Analyzer.
  • pETRhGTl consists of 1,422 base pairs (this nucleotide sequence is shown in SEQ ID NO: 1) and 473 amino acid residues (this amino acid sequence is shown in SEQ ID NO: 2).
  • Functional analysis was performed using this pETRh GT1.
  • pETRhGTl was transformed into E. coli BL21 (DE3) (Novagen).
  • the transformed strain was cultured at 37 ° C with shaking in 3 ml of LB medium containing 30 ⁇ g / ml kanamycin. 500 ⁇ l of this culture Add to LB medium containing 30 g / ml kanamycin and shake culture until the absorbance at 600 nm reaches 0.4, then add isopropyl- ⁇ -D-thiogalatatoside (IPTG) to a final concentration of 0.4 mM, 22 ° C After culturing for 12 hours, the cells were recovered by centrifuging (8000 rpm, 4 ° C, 20 minutes).
  • IPTG isopropyl- ⁇ -D-thiogalatatoside
  • the bacterial cell pellet was added to 1 ml of lysis buffer (pH 8.0) (100 mM Tris-HCl, 300 mM sodium chloride, 14 mM 2-mercaptoethanol 0.1% Protease and
  • Phosphatase Inhibitor Cocktails manufactured by SIGMA
  • SIGMA Stimulated GAA
  • the supernatant was used as a recombinant protein solution and used for enzyme activity measurement.
  • Enzymatic reaction was performed using recombinant pETRhGTl protein.
  • the recombinant protein solution 30 1 was used in the same manner as in [Example 2].
  • a reverse layer column Wakosil-II5C18AR column (4.6 mm id ⁇ 250 mm) was used and the column temperature was 35 ° C.
  • Detection using PDA with an elution time of 20 minutes in a linear concentration gradient with a 20% 0.5% TFA solution containing 0.5% TFA and an aqueous solution containing 0.5% TFA and 5% acetonitrile at a flow rate of lml / min. did.
  • Figure 2 shows the reaction results for each substrate.
  • reaction products were identified as cyadin 3,5-didarcoside and cyadin 5-darcoside, respectively. Furthermore, when shia-zin 3-darcoside was used as a substrate, the reaction product was unable to be obtained.
  • the elution time of each compound under these conditions is as follows. Shea-jin 3,5-didarcoside: 6.31 minutes, Shea-jin 3-darcoside: 9.85 minutes, Shea-gin 5-darcoside: 10.68 minutes, Shea-jin: 16.34 minutes.
  • the recombinant enzyme of the pETRhGTl clone has both UDP-glucose: cyanidin 5-Q-glucosyltransferase and UDP-glucose: cyaniain 5-O-glucoside 3-Q-glucosyltransferase activities. It was. Therefore, the RhGTl gene encodes UDP—glucose: anthocyanidin 5,3—glucosyltransferase, which sequentially shifts the glycosyl group to the hydroxyl groups at the 5- and 3-positions of anthocyanin. ! /, Confirmed that.

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Abstract

L'invention concerne un gène obtenu à partir d’une rose et qui code pour une protéine ayant 473 résidus acides aminés. Le gène code pour une protéine dont l’activité consiste à transférer un groupe glucosyle au groupe hydroxy en position 5 de l’anthocyanidine, puis à transférer un groupe glucosyle au groupe hydroxy en position 3.
PCT/JP2005/019294 2004-11-30 2005-10-20 Nouveau gene de l’anthocyanidine glucosyltransferase WO2006059433A1 (fr)

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JP2004345322A JP4691720B2 (ja) 2004-11-30 2004-11-30 新規アントシアニジングルコシル基転移酵素遺伝子
JP2004-345322 2004-11-30

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WO2007094521A1 (fr) * 2006-02-17 2007-08-23 International Flower Developments Proprietary Limited Flavonoïde glycosyltransférase et son utilisation
CN114807160A (zh) * 2022-03-10 2022-07-29 上海师范大学 调控月季花瓣颜色的基因RcGT、蛋白、重组载体、重组转化体、应用和方法

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JP2008048612A (ja) * 2006-08-22 2008-03-06 Bio Taxol:Kk 糖転移酵素遺伝子
JP5279304B2 (ja) * 2008-03-11 2013-09-04 岩手県 ３−デオキシアントシアニジン配糖化酵素遺伝子とその利用
EP2664671A4 (fr) 2011-01-14 2014-09-10 Suntory Holdings Ltd Nouveau gène de glycosyltransférase et utilisation de celui-ci
RU2636463C2 (ru) 2012-01-17 2017-11-23 Сантори Холдингз Лимитед Новый ген гликозилтрансферазы и его применение

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007094521A1 (fr) * 2006-02-17 2007-08-23 International Flower Developments Proprietary Limited Flavonoïde glycosyltransférase et son utilisation
CN114807160A (zh) * 2022-03-10 2022-07-29 上海师范大学 调控月季花瓣颜色的基因RcGT、蛋白、重组载体、重组转化体、应用和方法
CN114807160B (zh) * 2022-03-10 2023-11-14 上海师范大学 调控月季花瓣颜色的基因RcGT、蛋白、重组载体、重组转化体、应用和方法

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