WO2000049155A1 - Genes codant des proteines ayant une activite de transfert de sucre sur l'aurone - Google Patents
Genes codant des proteines ayant une activite de transfert de sucre sur l'aurone Download PDFInfo
- Publication number
- WO2000049155A1 WO2000049155A1 PCT/JP2000/000876 JP0000876W WO0049155A1 WO 2000049155 A1 WO2000049155 A1 WO 2000049155A1 JP 0000876 W JP0000876 W JP 0000876W WO 0049155 A1 WO0049155 A1 WO 0049155A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- protein
- activity
- plant
- auron
- Prior art date
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
- C12N15/63—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
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
-
- 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)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
Definitions
- the present invention relates to a gene encoding a protein having an activity of transferring sugar to auron.
- the present invention relates to a gene encoding a protein having an activity of transferring sugar to auron, a protein, and a method of using the same.
- Flower color is mainly based on three types of colorants: flavonoids, carotenoids and betalains.
- the yellow color is mainly derived from carotenoids and betalains, but the yellow color of some plants is derived from flavonoids.
- the main pigments that are thought to be involved in the development of yellow flowers can be divided into three groups: chalcones, aurons, and yellow flavonols (Saito, Bioholty 1, p49 -57, 1 990) o
- Auron is a substance in which two phenyl groups are bonded via three carbon atoms of dihydrofuran, and as auron, 4, 6, 4'-trih Droxylone, auredin, sunolefretin, black teatin and the like are known.
- auron 4, 6, 4'-trih Droxylone, auredin, sunolefretin, black teatin and the like are known.
- goldfish plants have aureidin and black teatin
- statices have aureticin
- morning glory have aureticin
- dalian have sulfretin
- wheat wheat has black teatin.
- Jerusalem artichoke contains sulfretin.
- flavonoids are modified by acylation, glycosylation, methylation, etc., and carotenoids and betalains are often glycosylated. Glycosylation among various modifications is due to the following: 1) Dye stability and solubility 2) Presence of acylation, which has a significant effect on color tone, 3) Glossation of flavonoids, such as the co-pigmentation effect of flower colors. Plays an important role.
- one of the flavonoids is glycosylated at position 6 corresponding to position 7 in flavonoids. Glycosylation occurs in plants containing other aurons, because aurons exist as glycosides, and glycosylation occurs because of the stable presence of aurons. Deemed necessary.
- UDP-glucose flavonoid 3
- darcosyltransferase (3GT) which transfers sugars to position 3 of flavonoids
- UDP-glucose flavonoid 5 — glucosyltransferase (5GT), which transfers sugars to the 5th position of flavonoids, are perilla, trennia and verpena. (International Publication Number; W099 / 05287).
- PS.b UFGTl a glycosyltransferase gene that has the activity to transfer glucose to position 7 of baicalein, one of the flavones, was isolated from hairy roots of the Labiatae plant Scutellaria.
- this gene product can also transfer sugars to position 7 of anthocyanidins and flavonols (presented at the 15th Annual Meeting of the Japanese Society for Plant Cell and Molecular Biology).
- This gene product can also transfer sugars to position 7 of anthocyanidins and flavonols (presented at the 15th Annual Meeting of the Japanese Society for Plant Cell and Molecular Biology). Has not been reported.
- glycosyltransferases using flavonoids as substrates have large differences in substrate specificity between flavonoids.
- the flavonoid 3 -glycosyltransferase gene derived from gentian was cloned and expressed in Escherichia coli, and the activity was measured. The activity is 61% for, and 38% for pelargonidin, showing good activity for anthocyanidins. On the other hand, it shows only 7.0%, 6.5% and 4.4% activity against the flavonols, camprol, quercetin and myricetin, respectively. Furthermore, it does not transfer sugar at all to dihydroflavonol (Tanaka et al. Plant Cell Physiol.
- glycosyltransferases can recognize the type of flavonoids, and that the glycosyltransferase activity of one flavonoid cannot be easily inferred from the glycosyltransferase activity of another flavonoid. ing.
- glycosyltransferases that use flavonoids as substrates have large differences in substrate specificity, and predict the activity of glycosyltransferases from known glycosyltransferases to specific flavonoids. It was difficult.
- the inventors of the present invention completed the present invention with an object of obtaining a gene encoding a protein having an activity of transferring sugar to aurone among flavonoid dyes.
- the present inventors have clarified that the pS.b UFGT1 gene product derived from Scutellaria has a transglycosylation activity to auron, and further used this gene as a probe.
- a gene encoding a protein having an activity to transfer sugar to gold was obtained from goldfish (Antirrrhinum majus).
- the present invention provides a gene encoding a protein having an activity to transfer sugar to auron. Further, the present invention provides a gene encoding a protein having an activity of transferring a sugar to an aurone having the amino acid sequence of SEQ ID NO: 2, 8, or 10. The present invention further relates to the addition or deletion of one or more amino acids to the amino acid sequence described in SEQ ID NO: 2, 8, or 10, and Z or other amino acids. It is intended to provide a gene encoding a protein having an amino acid sequence which has been modified by the substitution of a sugar chain and having an activity of transferring sugar to sugar.
- the present invention further relates to a nucleic acid having a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 2, 8 and 10, or a nucleic acid having a portion thereof, under stringent conditions. It is intended to provide a gene encoding a protein which hybridizes and has an activity to transfer sugars to itself.
- the present invention also provides a vector comprising the above gene.
- the present invention further provides a host transformed with the above vector. This host may be a cell such as a microorganism, a plant cell, or an animal cell, or may be a plant.
- the present invention can also provide a method for producing a protein having an activity of transferring sugar to auron by culturing, cultivating, or breeding the host.
- the present invention also relates to a plant having auron, wherein the above gene is introduced into a plant, the gene is expressed, and sugar is transferred to auron in the plant by the produced protein, whereby the plant is transformed into a plant.
- FIG. 1 is a diagram showing a method for producing plasmid pESBGT-1.
- FIG. 2 shows a method for preparing plasmid pETAmGTl.
- cDNA library for the yellow goldfish petals.
- the obtained cDNA library is screened using pS.b UFGTl, a flavonoid 7-glycosyltransferase gene derived from Scutellaria, to obtain clones. Then, the plasmid obtained from this clone is separated and its nucleotide sequence is determined.
- a protein having an enzymatic activity has a region essential for the enzyme activity and a region not essential for the enzyme activity, and the non-essential region has one or more amino acid deletions, additions and / or deletions. It is known that the enzyme activity is maintained even when modified by substitution with another amino acid. Therefore, the present invention provides not only the protein having the amino acid sequence shown in SEQ ID NO: 2, 8 or 10, but also the amino acid sequence shown in SEQ ID NO: 2, 8 or 10, Has an amino acid sequence modified by deletion, addition, and / or substitution with another amino acid sequence of the amino acid sequence, and has an activity of transferring a sugar to aurone.
- the present invention also includes a protein and a gene encoding the protein.
- the number of amino acids to be modified is, for example, 50 or less, preferably 30 or less, for example, 20 or 10 or less.
- the gene encoding the protein having the amino acid sequence shown in SEQ ID NO: 2, 8 or 10 of the present invention can be obtained from goldfish or petunia as cDNA or genomic DNA. Methods for cloning cDNA are specifically described in Examples 2-3 and 6. Genomic DNA can be obtained by preparing a genomic library from goldfish or petunia according to a conventional method and screening it with the cDNA or a fragment thereof according to a conventional method.
- the gene encoding a protein having an amino acid sequence modified with respect to the amino acid sequence shown in SEQ ID NO: 2, 8 or 10 of the present invention is a system number: 2, 8 or 10
- DNA encoding a protein having an amino acid sequence shown in (1) for example, a cDNA by modifying a nucleotide sequence according to a conventional method for manipulating a gene, such as site-directed mutagenesis or PCR. Can be.
- a nucleic acid that hybridizes to the gene or a portion thereof encodes a similar enzymatic activity and is similar to the original protein. It often encodes the acid sequence. Therefore, the present invention hybridizes under stringent conditions to a nucleic acid having a nucleotide sequence encoding the amino acid sequence described in any of SEQ ID NOs: 2, 8 or 10, or a nucleic acid having a portion thereof, And a gene encoding a protein having an activity to transfer sugar to aurone.
- washing conditions are preferably 5X SSC, 0.1% SDS, 50 ° C, and more preferably 2X SSC, 0.1% SDS. , 50 ° C, more preferably 0.1 x SSC, 0.1% SDS, 50 ° C.
- nucleic acid having a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 2, 8, or 10 is used in the above hybridization
- the length of the nucleic acid It has a length of at least 17 base pairs and preferably has a length of at least 100 base pairs.
- Nucleic acids to be subjected to high-purification are, for example, nucleic acids prepared from Scutellaria, goldfish, petunia, statice, morning glory, dahlias, barley chrysanthemum, jerusalem artichoke, and preferably genomic.
- a DNA library or cDNA library is used.
- the present invention also relates to the above protein having glycosyltransferase activity to aurone.
- a manufacturing method is provided. This method comprises introducing a vector comprising the DNA encoding the protein into a host, culturing or growing the host, and collecting the protein as desired. And features.
- the host may be a host cell or an organism such as a plant.
- prokaryotic cells especially bacterial cells, such as Escherichia coli and Bacillus bacteria, such as Bacillus subtilis ⁇ Bacillus brevis, are used.
- Lower eukaryotes for example, fungi, for example, yeasts, for example, yeasts of the genus Saccharomyces, for example, Saccharomyces' Saccharomyces cerev is iae, or thread;
- filamentous fungi belonging to the genus Aspergillus such as Aspergillus oryzae, Aspergillus oryzae, and Aspergilulus nicer, Aspergi 1 lus niger, can be mentioned.
- higher eukaryotic cell hosts include insect cells, such as silkworm cells, animal cells, such as CH0 cells, human cultured cells, such as HeLa cells, and the like.
- the gene of the present invention can also be expressed in organisms such as plants.
- a vector comprising the DNA of the present invention, particularly an expression vector contains an expression control region, and the expression control region depends on the host cell.
- trc promoter, tac promoter, lac promoter, T7 promoter, etc. can be used as the promoter of the bacterial expression vector.
- the promoter of the yeast expression vector include, for example, A glycolytic enzyme gene promoter, for example, glyceroaldehyde 3-phosphate dehydrogenase-promoter, galactokinase promoter, etc. can be used, and an animal cell expression vector promoter can be used.
- the virus promoter can be used as the heater.
- isolation and purification of the protein such as liquid chromatography, affinity chromatography, etc. The common means used in the above can be used.
- this cDNA can also be linked under the control of a constitutive or inductive micromotor to produce a system using an aggressive vehicle or a particle gun, an electroporator.
- Plants such as petunias, roses, and powers — such as petunia, chrysanthemum, trennia, verbena, gerbera, tobacco, strawberry, eustoma, gentian, gladiolus, and chew.
- a gene encoding an auron synthase gene is introduced and expressed, and at the same time, a gene encoding a protein having the activity of transferring sugar to auron of the present invention is also introduced. By expressing it, it is possible to stably express auron and provide a new plant having a yellow color tone. Examples of the plant that does not have auron include petunia, trennia, and tobacco. Example
- Example 1 Translocation of p S .b UFGT1 gene product from Aspergillus oryzae to orange Measurement of glycosyl transfer activity
- the transglycosylation activity of the pS.b UFGTl gene derived from Aspergillus oryzae was measured using the expression vector pE SiGT-1 in Escherichia coli prepared by the method described below.
- the pS.b UFGTl gene was subjected to a PCR reaction using the following two types of primers, and Ndel and BamHI sites were introduced.
- the PCR reaction solution consists of 300 ng of pSBGT-1, lx Native Pfu DNA polymerase reaction buffer (Stratagene), 0.2 mM dNTPs, 4 pg / ⁇ 1 each of primers, and 2.5 U of Native Pfu DNA polymerase. The volume was adjusted to 50 ⁇ 1. The reaction was carried out at 95 ° C for 3 minutes, then 95 ° C for 1 minute, 50 ° C for 2 minutes, and 72 ° C for 2 minutes as one cycle, and then 30 cycles, and finally Treated at 72 ° C for 7 minutes.
- the obtained PCR product was digested with Ndel and BamHI, and ligated with a pET-3a vector (Stratagene) also digested with Ndel and BamHI to construct pESBGT-1 (FIG. 1).
- pESBGT-1 and pET-3a vectors were used to transform Epicurian Coli (Epicurian Coli) BL2KDE3) (Stratagene), respectively.
- the transformant was cultured with shaking at 37 ° C. overnight in 3 ml of LB medium containing 50 g / ml of ampicillin.
- IPTG isopropyl-/ 3 thiogalacto virano
- the pellet was suspended in 5 ml of a buffer (lOmM sodium phosphate, pH 6.5, ImM / 3-mercaptoethanol (2-ME)), and E. coli was disrupted with an ultrasonic disrupter. After that, centrifugation (15,000 rpm, 5 minutes, 4 ° C) The supernatant was used as a crude enzyme solution and used in the following enzyme reaction.
- a buffer LOmM sodium phosphate, pH 6.5, ImM / 3-mercaptoethanol (2-ME)
- Enzyme activity was measured using naringenin or luteolin as a substrate in addition to aureungin.
- enzymatic activity was measured as follows. 0.1M Tris-HCl, pH 8.0, 0.05% 2-ME 150 ⁇ 1 was added to the crude enzyme solution and incubated at 30 ° C for 10 minutes. Then, 4.66 mM auredin 5-1 and 5 mM UDP-glucose 50-1 were added and reacted at 30 ° C. for 1 hour. The reaction was stopped by adding 200% acetonitrile 200 ⁇ 1 containing 5% TFA (trifluoroacetic acid), followed by centrifugation at 15,000 rpm for 3 minutes at 4 ° C. The supernatant was filtered to remove insolubles using a filter (pore size: 0.45 ex1, 4 mm Mi11ex-LH, Millipore). This was analyzed by liquid high-speed chromatography.
- TFA trifluoroacetic acid
- Crude enzyme solution 201 0.1 M citrate-phosphate buffer, pH 6.5, 25 ⁇ 1 5 ⁇ M of each substrate 5 ⁇ 1, containing 5 mM UDP-glucose 25 ⁇ 1 in a total volume of 250 // 1
- the reaction was performed at 30 ° C for 30 minutes. After stopping the reaction by adding 90% acetonitrile 200 1 containing 5% TFA, the mixture was centrifuged at 15,000 rpm for 3 minutes at 4 ° C, and the resulting supernatant was filtered. (Pore size 0.45 ⁇ m, 4 mm Millex-LH, Millipore) to remove insolubles. This was analyzed by high performance liquid chromatography.
- the analysis conditions for naringenin are as follows. Column using YMC J 'sp here ODS-M80 (4.6mm0 xl50mm, YM C) and the mobile phase was a H 2 0, B solution containing TFA 0.1% in the A dissolved solution TFA0.13 ⁇ 4 the B80% after B80% of Riniagura Gen bets 10 minutes B20% with 90% CH 3 CN containing and held for 5 minutes. The flow rate was 0.6 ml / min. Detection was performed using A290nm and Shimadzu PDA detector SPD-M6A to measure the absorption spectrum at 250 to 400nm.
- the analysis conditions for luteolin are as follows. YMC J 'sphere 0DS-M 80 using (4. G ⁇ xl50mm), 90 the mobile phase containing TFA 0.1% in the H 2 0, B solution containing TFA 0.1% in the solution A A linear gradient from B20% to B80% was used for 10 minutes using% CH 3 CN, and then B80% was held for 5 minutes. The flow rate was 0.6 ml / min. Detection was performed using A330nm and Shimadzu PDA detector SPD-M6A to measure the absorption spectrum at 250-400nm.
- baicalein can be glycosylated at the 7-position.
- baicalein almost 100% was detected as 7-glycoside after the reaction, but almost no reaction for naringenin, and S. serrata pS.b UFG Tl gene expression The product was also found to have high substrate specificity.
- the petal cDNA library was prepared by the following method. 5 g of fresh petals of yellow goldfish (Ero Butterfly) from guanidine thiocyanoate / cesium chloride as detailed in R. McGookin et al., Method in Molecular Biology vol. 2 (Humana Press Inc. 1984) RNA was obtained in the same manner as described above, and polyA + RNA was purified using Origotex dT30 (Nippon Roche). From this polyA + RNA, a cDNA library was constructed using a cDNA synthesis kit, an XR vector kit (Stratagene). Library one obtained had summer 1.6 10 5 plaque forming Interview two Tsu preparative (pfu).
- the goldfish grass cDNA library obtained in Example 2 was Screening was performed using the full length of pS.b UFGT1, a flavonoid 7-glycosyltransferase gene derived from hairy roots. Screening of the library was performed using a non-radiosystem DNA detection kit (Boehringer). Neubridization is 37. C. The final letter was washed with 5 ⁇ SSC, 0.1% SDS at 50 ° C. for 30 minutes. About 200,000 plaques were screened and finally two clones were obtained. METHODS: The Molecular Cloning was by (Sambrook e t. Al. Cold Spring Harbour L aboratory Press, 1989) c
- the nucleotide sequence was determined by synthesizing an oligonucleotide primer and using a DNA Sequencer model 310 (Applied Biosystems). The nucleotide sequence and the deduced amino acid sequence are shown in Sequence Listing and SEQ ID NO: 1.
- pAmGTl contained the 1751 bp gene AmGTl which encodes a protein consisting of 481 amino acids and having a molecular weight of 53.9 kDa.
- AmGTl gene expression was performed using the pET System (Stratagene).
- PETAmGT5 ' 5'-ATA ACT ACA TAT GGG AAA ACT TCA C_3' (Sequence number: 5)
- pETAmGT3 ' 5'-GAA CAG GAT CCA CAC ACT AGA AGT CA-3' (SEQ ID NO: 6)
- the PCR reaction solution was pAmGTl lOOng, lx cloned Pfu DNA polymerase reaction buffer (Stratagene), 0.2 mM dNTPs, and the primer—0.5 pmo each.
- the total volume was adjusted to 100 ⁇ 1 comprising M 1 and cloned Pfu DNA polymerase 5.0.
- the reaction is performed at 95 ° C for 45 seconds, followed by 25 cycles of 95 ° C for 45 seconds, 50 ° C for 45 seconds and 72 ° C for 2 minutes, and finally for 10 minutes at 72 ° C. Processed.
- the obtained PCR product was subcloned into pCR2.1 T0P0 vector (IN VITR0GEN).
- Example 4 The transformant obtained in Example 4 was cultured and extracted as in Example 1, and the enzyme activity was measured.
- the retention time of the aleicidin 6-glycoside for the substance evolving at 10.98 minutes was the same as the retention time of the aleicidin 4-glycoside for the substance evolving at 11.85 minutes. From the above results, it was revealed that AmGTl can glycosylate the 6th and 4th positions of auredin. The absorption spectrum of the substance detected at 11.27 minutes is presumed to be aurethidine glycoside.
- Example 6 Acquisition of auronic glycosyltransferase gene derived from petunia
- a cDNA library derived from petals of a petunia cultivar Alldgrilli blue was obtained in Example 3 using cDNA library (Nature366, 276-279, 1993). Screening was performed using the full length of the gene AmGTl. Screening of the library was performed using a non-radiosystem DNA detection kit (Belinger). Hybridization was performed at 37 ° C., and filter washing was performed using 5 ⁇ SSC, 0.1% SDS at 50 ° C. for 30 minutes. Approximately 200,000 plaques were screened, and finally two types of clones were obtained. The method was based on Molecular Cloning (Sambrook et. Al. Cold Spring Harbor Laboratory Press, 1989).
- the two clones were named pPh7GTa and pPh7GTb, respectively, and their base sequences were determined.
- the nucleotide sequence was determined by synthesizing the oligonucleotide primer and using DNA Sequencer model 310 (Applied Biosystems).
- P The nucleotide sequence and predicted amino acid sequence at the insertion site in Ph7GTa are shown in SEQ ID NOs: 7 and 8, respectively, and the nucleotide sequence and predicted amino acid sequence at the insertion site in pPh7GTb are sequenced, respectively. No .: Shown in 9 and 10.
- pPh7GTa contained a 1750 bp gene Ph7GTa encoding a protein consisting of 488 amino acids
- pPh7GTb contained a 1669 bp gene Ph7GTb encoding a protein consisting of 476 amino acids.
- AmGTl derived from goldfish and pS.b UFGT1 derived from Scutellaria obtained in Example 3 were compared with PhGTGT, and AmGTl and pS.b UFGT1 were identified as Ph7GTa. Indicate 50% and 51% homology, respectively. Was.
- Ph7GTa gene expression was performed using the pET System (Stratagene). To introduce the Ndel and BamHI sites first, the following two primers were used: pETPh7GTa5 '[5'-ATA ACT ACA TAT GGC TAT TCC CAC A-3' (SEQ ID NO: 11)] and pETPh7GTa3 '[ 5'-GAA CAG GAT CCT AAA AGG ACC T-3 '(SEQ ID NO: 12)]
- the PCR reaction solution was pAmGTl lOOng, lx cloned Pfu DNA polymerase reaction buffer (Stratagene), 0.2 mM dNTPs, each primer 0.5 pmo 1/1, cloned Pfu DNA polymerase. 5. Adjusted to a total volume of 100 1 consisting of OUn it. The reaction is performed at 95 ° C for 45 seconds, followed by 25 cycles of 95 ° C for 45 seconds, 50 ° C for 45 seconds, and 72 ° C for 2 minutes, and finally for 10 minutes at 72 ° C. Processed. The obtained PCR product was subcloned into pCR2.1 T0P0 vector (INVI TR0GEN).
- pETPhGTa was used to transform Epicurian Co1i BL21 (DE3) (Stratagene).
- pETPh7GTb5 ′ [5′-ATA ACT ACA TAT GGG TCA GCT CCA-3 ′ (SEQ ID NO: 13)] and pETPh7GTb3
- plasmid pETPhGTb was similarly obtained.
- Example 8 The transformant obtained in Example 8 was cultured and extracted as in Example 1, and the enzyme activity was measured. Enzyme activity was measured using auredin as a substrate. The enzyme activity was measured in the same manner as described in Example 1.
- Ph7GTa and Ph7GTb peaks having the same retention time and spectrum as those of auredin 6-glycoside were detected as reaction products.
- Ph7GTa one kind of peak expected to be an aurone glycoside from the absorption spectrum was obtained
- Ph7GTb two kinds of peaks were obtained.
- Ph7GTa and Ph7GTb encode an enzyme having an activity of glycosylating aurethidine.
- aurone could be glycosided. This enabled stable expression of olone in plant cells.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Nutrition Science (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002325385A CA2325385A1 (en) | 1999-02-16 | 2000-02-16 | Genes encoding proteins having activity of transferring sugar onto aurone |
US09/673,300 US6770747B1 (en) | 1999-02-16 | 2000-02-16 | Genes encoding protein having activity of transferring sugar onto aurone |
EP00903988A EP1072684A4 (en) | 1999-02-16 | 2000-02-16 | GENES ENCODING PROTEINS HAVING AURONE-SUGAR TRANSFER ACTIVITY |
AU25722/00A AU2572200A (en) | 1999-02-16 | 2000-02-16 | Genes encoding proteins having activity of transferring sugar onto aurone |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3680199 | 1999-02-16 | ||
JP11/36801 | 1999-02-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000049155A1 true WO2000049155A1 (fr) | 2000-08-24 |
Family
ID=12479900
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2000/000876 WO2000049155A1 (fr) | 1999-02-16 | 2000-02-16 | Genes codant des proteines ayant une activite de transfert de sucre sur l'aurone |
Country Status (6)
Country | Link |
---|---|
US (1) | US6770747B1 (ja) |
EP (1) | EP1072684A4 (ja) |
AU (1) | AU2572200A (ja) |
CA (1) | CA2325385A1 (ja) |
NZ (1) | NZ507563A (ja) |
WO (1) | WO2000049155A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005059141A1 (ja) * | 2003-12-17 | 2005-06-30 | Suntory Limited | フラボノイド合成系の制御による黄色の花の作製方法 |
JP2009213413A (ja) * | 2008-03-11 | 2009-09-24 | Iwate Prefecture | 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 |
US10899727B2 (en) | 2016-04-11 | 2021-01-26 | Middle Tennessee State University | Therapeutic aurones |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE60329505D1 (de) | 2002-08-20 | 2009-11-12 | Suntory Holdings Ltd | Neue glycosyltransferase-gene |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999005287A1 (fr) * | 1997-07-25 | 1999-02-04 | Suntory Limited | Genes codant pour des proteines a activite de transglycolsylation |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9611420D0 (en) * | 1996-05-31 | 1996-08-07 | Univ York | Use of salicyclic acid-glucosyl transferase |
US6011145A (en) * | 1997-08-13 | 2000-01-04 | Cornell Research Foundation, Inc. | Chain length specific UDP-GLC: fatty acid glucosyltransferases |
FR2780415A1 (fr) * | 1998-06-26 | 1999-12-31 | Rhone Poulenc Agrochimie | Procede enzymatique de glucosylation de derives aromatiques |
-
2000
- 2000-02-16 EP EP00903988A patent/EP1072684A4/en not_active Withdrawn
- 2000-02-16 US US09/673,300 patent/US6770747B1/en not_active Expired - Fee Related
- 2000-02-16 NZ NZ507563A patent/NZ507563A/xx unknown
- 2000-02-16 AU AU25722/00A patent/AU2572200A/en not_active Abandoned
- 2000-02-16 CA CA002325385A patent/CA2325385A1/en not_active Abandoned
- 2000-02-16 WO PCT/JP2000/000876 patent/WO2000049155A1/ja not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999005287A1 (fr) * | 1997-07-25 | 1999-02-04 | Suntory Limited | Genes codant pour des proteines a activite de transglycolsylation |
Non-Patent Citations (4)
Title |
---|
DATABASE GENBANK 18 August 1999 (1999-08-18), HIROTANI M. ET AL.: "Scutellaria baicalensis ufgt mRNA for UDP-glucose: flavonoid 7-0-glucosyltransferase, complete cds" * |
HALBWIRTH H. ET AL.: "Enzymatic glucosylation of 4-deoxyaurones and 6'-deoxychalcones with enzyme extracts of Coreopsis grandiflora, Nutt. I.", PLANT SCI., vol. 122, no. 2, January 1997 (1997-01-01), pages 125 - 131, XP002924338 * |
HORVATH D.M. ET AL.: "Identification of an immediate-early salicylic acid-inducible tobacco gene and characterization of induction by other compounds", PLANT MOL. BIOL., vol. 31, no. 5, August 1996 (1996-08-01), pages 1061 - 1072, XP002924339 * |
See also references of EP1072684A4 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005059141A1 (ja) * | 2003-12-17 | 2005-06-30 | Suntory Limited | フラボノイド合成系の制御による黄色の花の作製方法 |
JPWO2005059141A1 (ja) * | 2003-12-17 | 2007-07-12 | 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 | フラボノイド合成系の制御による黄色の花の作製方法 |
US7750209B2 (en) | 2003-12-17 | 2010-07-06 | International Flower Developments Proprietary Limited | Method for producing yellow flower by controlling flavonoid synthetic pathway |
JP4641945B2 (ja) * | 2003-12-17 | 2011-03-02 | 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 | フラボノイド合成系の制御による黄色の花の作製方法 |
US8350125B2 (en) | 2003-12-17 | 2013-01-08 | Suntory Holdings Limited | Method for producing yellow flower by controlling flavonoid synthetic pathway |
JP2009213413A (ja) * | 2008-03-11 | 2009-09-24 | Iwate Prefecture | 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 |
US10899727B2 (en) | 2016-04-11 | 2021-01-26 | Middle Tennessee State University | Therapeutic aurones |
US11286245B2 (en) | 2016-04-11 | 2022-03-29 | Middle Tennessee State University | Therapeutic aurones |
Also Published As
Publication number | Publication date |
---|---|
EP1072684A4 (en) | 2004-11-17 |
US6770747B1 (en) | 2004-08-03 |
AU2572200A (en) | 2000-09-04 |
NZ507563A (en) | 2004-02-27 |
CA2325385A1 (en) | 2000-08-24 |
EP1072684A1 (en) | 2001-01-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7501271B2 (en) | Proteins having a flavonoid 5-O-glycosyltransferase activity (5GT) | |
Yoshihara et al. | cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Iris hollandica | |
CA2213082C (en) | Genes coding for proteins having acyl transferase activity | |
JP4927774B2 (ja) | Udp−グルクロン酸転移酵素およびそれをコードするポリヌクレオチド | |
IL195808A (en) | Genes encoding proteins synthesize Pleven | |
WO2000049155A1 (fr) | Genes codant des proteines ayant une activite de transfert de sucre sur l'aurone | |
JP4982782B2 (ja) | 新規糖転移酵素遺伝子 | |
JP4418865B2 (ja) | 新規グルコシル基転移酵素遺伝子 | |
JP4691720B2 (ja) | 新規アントシアニジングルコシル基転移酵素遺伝子 | |
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 | |
EP1114162A1 (en) | Genes coding for flavone synthases | |
EP1291426B1 (en) | Novel aliphatic acyltransferase genes | |
AU2005200905A1 (en) | Genes encoding proteins having activity of transferring sugar onto aurone | |
IL133469A (en) | Gene encoding a protein having aurone synthesis activity | |
CN110832080A (zh) | 丙二酰基转移酶基因的应用 | |
AU2005246960A1 (en) | Genes coding for flavone synthases |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP NZ US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
ENP | Entry into the national phase |
Ref document number: 2325385 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 25722/00 Country of ref document: AU Ref document number: 09673300 Country of ref document: US |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2000903988 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2000903988 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2000903988 Country of ref document: EP |