WO2001092536A1 - Nouveaux genes de l'acyltransferase aliphatique - Google Patents
Nouveaux genes de l'acyltransferase aliphatique Download PDFInfo
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- WO2001092536A1 WO2001092536A1 PCT/JP2001/004677 JP0104677W WO0192536A1 WO 2001092536 A1 WO2001092536 A1 WO 2001092536A1 JP 0104677 W JP0104677 W JP 0104677W WO 0192536 A1 WO0192536 A1 WO 0192536A1
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- C—CHEMISTRY; METALLURGY
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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
- 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
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
Definitions
- the present invention relates to a gene encoding a protein having an activity of transferring an aliphatic acyl group to a sugar at the 5-position of a flavonoid, and a method of using the same.
- anthocyanins The major component of flower color is a group of compounds in Flavonoids, collectively called anthocyanins. It is known that a variety of anthocyanins exist in plants, and many of their structures have already been determined. The color of anthocyanin mainly depends on its structure (Harborne (1986) The Flavonoids, p565).
- Enzymes involved in the biosynthesis of anthocyanin and its genes are also being studied, and examples of the use of molecular biology techniques and gene transfer into plants to change the structure of anthocyanin and change the color of flowers (Plant Cell, 7 (1995) Holton and Cornish, p. 1071, Plant Cell Physiol. 39 (1998) Tanaka et al. Plll9;).
- the biosynthetic pathway of anthocyanin is common to most flowering plants up to anthocyanidin 3 -glucoside (Holton e t al. (1995) Plant Cell, 7, pl071). Thereafter, anthocyanidin 3-dalcoside undergoes various species- and variety-specific modifications. This diversity contributes to the variety of flower colors.
- Anthocyanin is an unstable compound in neutral solution, but its stability is improved by modification with sugar or acyl group (Forkmann
- anthocyanin becomes slightly red by glycosylation, and becomes blue by the addition of an aromatic acyl group (Forkmann (1991) Plant Breeding, 106, pi). Roughly speaking, there are an aromatic group (for example, caffeoyl group and coumaroyl group) and an aliphatic group (for example, malonyl group and acetyl group). Is not known other than increasing the solubility of anthocyanin.
- Flavonol 3MaT Enzyme that catalyzes the transfer of a malonyl group to the sugar at position 3 of flavonol
- Flavone / Flavonol 7MaT the reaction that transfers the malonyl group to the sugar at position 7 of flavone and flavonol
- Archives of Biochemistry and Biophysics, 1983, 224, 26 to 271 Measurement of activity in organs of Flameol 3MaT and Flavone / Flavonol 7MaT of parsley)
- Archives of Biochemistry and Biophysics, 1983, 226, 206-217 Purification of Flavonol 3MaT and Flavone / Flavonol 7MaT in
- the effect of malonylation on the flower color in the acylation of anthocyanin by an aliphatic acyltransferase is clarified, and a gene encoding a protein having an aliphatic acyltransferase activity, preferably an anthocyanin,
- a gene encoding a protein having an aliphatic acyltransferase activity preferably an anthocyanin
- An object was to obtain a gene encoding a protein having an aliphatic acyl transfer activity.
- the flower color can be changed by introducing and expressing the gene encoding the protein having an aliphatic acyl transfer activity obtained in the present invention into a plant.
- anthocyanin machinylation As described above, there is no report on the effect of anthocyanin machinylation on flower color. To elucidate its effects, three types of anthocyanins, namely delphinidin 3,5-diglucosid, apadin (delphinidin 3- (coumaroyl) darcoside-5-glucoside), malonyradivanin Comparison of the color of the solution of (delphinidin 3- (coumaroyl) darcoside-5- (malonyl) darcoside) showed that the color of malonylapapanin was the bluest, indicating that malonylation contributed to the bluening of anthocyanin. Was done.
- the present invention relates to a protein having the amino acid sequence of SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, and having an activity of transferring an aliphatic acyl group to a sugar at position 5 of flavonoid.
- the amino acid sequence thereof which has been modified by the addition, deletion and / or substitution of another amino acid to one or more amino acid sequences thereof, and the presence of a flavonoid.
- a gene encoding a protein having an activity of transferring an aliphatic acyl group to the sugar at position 5.
- the present invention also provides an amino acid sequence (J having 50% or more homology to the amino acid sequence described in SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29). And a gene encoding a protein having an activity of transferring an aliphatic acyl group to a sugar at the 5-position of a flavonoid.
- the present invention also relates to 5 ⁇ SSC: 50 ° to part or all of the nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29.
- a gene encoding a protein that hybridizes under the conditions of C and has an activity of transferring an aliphatic acyl group to the 5-position sugar of flavonoids.
- the present invention further provides a setter comprising the above gene.
- the present invention also provides a host transformed with the vector.
- the present invention also provides a protein encoded by any one of the above genes. '
- the present invention also relates to a method of culturing or growing said host,
- the present invention provides a method for producing a protein having an activity of transferring an aliphatic acyl group to a saccharide at the 5-position of a flavonoid from the above.
- the present invention also provides a plant into which the above-described gene has been introduced, or progeny of the plant or a tissue thereof having the same properties.
- the present invention further provides a cut flower of the above plant or a progeny of the plant having the same properties.
- the present invention further provides a method for changing the color of a flower using the above gene.
- the present invention further provides a method for making a flower blue using the above gene.
- Examples of the gene of the present invention include those encoding the amino acid sequence described in SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29.
- a protein having an amino acid sequence modified by addition or deletion of a plurality of amino acids and / or substitution with another amino acid also has the same enzymatic activity as the original protein. It has been known. Accordingly, the present invention relates to the following: SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29 as long as the protein has an activity to transfer an aliphatic acyl group to the sugar at position 5 of the flaponoid.
- a protein having an amino acid sequence modified by addition or deletion of one or more amino acid sequences to the amino acid sequence and / or substitution with another amino acid, and the protein The gene encoding the gene also belongs to the present invention.
- the present invention also relates to a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, or a part of the nucleotide sequence, preferably 6 amino acids
- a nucleotide sequence encoding the above for example, a nucleotide sequence encoding six or more amino acid sequences in the consensus region
- a gene that hybridizes to a 5xSSC at 50 ° C and encodes a protein having an activity to transfer an aliphatic acyl group to the sugar at position 5 of the flavonoid. is there.
- the appropriate hybridization temperature depends on the base sequence and the length of the base sequence.For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, Temperatures below 50 ° C are preferred.
- Genes selected by such hybridization include those derived from natural sources, for example, those derived from plants, for example, genes derived from petunia trennia, and those derived from non-plants. Good.
- the gene selected by the hybridization may be cDNA or genomic DNA.
- the present invention further relates to an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, of about 50% or more, preferably 60% or 70% or more, or further 80%. Or a gene encoding a protein having an amino acid sequence having a homology of 90% or more and having an activity of transferring an aliphatic acyl group to the sugar at position 5 of the flaponoid. It is about usage.
- the gene having the native nucleotide sequence can be obtained, for example, by screening a cDNA library, as specifically shown in Examples.
- DNA encoding a protein having a modified amino acid sequence can be synthesized using conventional site-specific mutagenesis or PCR based on DNA having a native nucleotide sequence.
- a DNA fragment to be modified is obtained by treating a native cDNA or genomic DNA with a restriction enzyme, converting the fragment into type III, and performing site-directed mutagenesis or PCR using a primer into which a desired mutation has been introduced. Then, a DNA fragment into which the desired modification has been introduced is obtained.
- the DNA fragment into which this mutation has been introduced may be ligated to a DNA fragment encoding another part of the target protein.
- a DNA encoding a protein consisting of a shortened amino acid sequence for example, a DNA encoding an amino acid sequence longer than the desired amino acid sequence, for example, a DNA encoding the full-length amino acid sequence, is desired. If the DNA fragment obtained by cutting with a restriction enzyme does not encode the entire amino acid sequence of interest, a DNA fragment consisting of the missing sequence may be synthesized and ligated.
- the obtained gene encodes a protein having an aliphatic acyl transfer activity. Can be confirmed. Further, by expressing the gene, it is possible to obtain a gene product, a protein having an activity of transferring an aliphatic acyl group. Alternatively, using an antibody against the amino acid sequence of SEQ ID NO: 2, 4, 6, 23, 25, 27 or 29, a protein having an activity of transferring an aliphatic acyl group can be obtained. It can also be used to clone a gene encoding a protein having an aliphatic acyl transfer activity from another organism.
- the present invention also relates to a recombinant vector containing the above-mentioned gene, particularly an expression vector, and a host transformed with the vector.
- Prokaryote or eukaryote can be used as a host.
- a prokaryote a common host such as a bacterium, for example, a bacterium belonging to the genus Escherichia, for example, a microorganism belonging to the genus Escherichia coli or Bacillus, for example, a bacterium of Pasillus subtilis, for example, Bacillus subtilis, should be used.
- eukaryotic hosts lower eukaryotes, for example, eukaryotic microorganisms, for example, fungi such as yeast or filamentous fungi can be used.
- yeast examples include microorganisms belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae. Aspergillus (Aspergillus), genus microorganisms such as Aspergillus oryzae, Aspergillus niger, Aspergillus niger, Examples include microorganisms of the genus Penicillium.
- animal cells or plant cells can be used, and as animal cells, cell lines such as mouse, hamster, sal, and human are used. Insect cells, such as silkworm cells, or adult silkworms themselves, are also used as hosts.
- the expression vector of the present invention contains an expression control region, for example, a promoter and a terminator, an origin of replication, and the like, depending on the type of host into which they are to be introduced.
- Usable promoters for bacterial expression vectors include conventional promoters such as trc promoter, tac promoter, lac promoter and the like, and yeast promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter.
- an amylase promoter, a trpC promoter, or the like is used as a filamentous fungal promoter.
- a promoter for an animal cell host a viral promoter, for example, an SV40 early promoter, an SV40 rate promoter or the like is used.
- the production of the expression vector can be carried out using a restriction enzyme, a ligase and the like according to a usual manner.
- transformation of the host with the expression vector can be performed according to a conventional method.
- the present invention is not limited to only the gene encoding a protein having an activity of transferring an aliphatic acyl group from Salvia.
- the present invention relates to the use of a gene encoding a protein having a transposition activity.
- the source of the protein having an aliphatic transacylation activity may be a plant, an animal, or a microorganism, and the protein having the aliphatic transacylation activity can be similarly used for flower color conversion.
- the present invention relates to a plant or a progeny thereof whose color is regulated by introducing a gene encoding a protein having an aliphatic acyl transfer activity, or a tissue thereof.
- the form may be a cut flower.
- transformable plants include roses, chrysanthemums, carnations, goldfish, cyclamen, orchids, lisianthus, freesia, gerberas, gladioli, kasumi, kalanchoe, lilies, peranoregonids, geranids.
- Example 1 Color change of various anthocyanins depending on pH Delphinidin 3,5-didacoside, apapanin (denolefinidin 3- (coumarinoid) gnolecoside-5-gnorecoside), malonylazine (delphinidin) 3- (Kumaroinore) Darukoshido -. 5- (Ma b two Honoré) Darukoshido) each 0 ⁇ 1 ⁇ , 0.3 mM, Mack Rubei down buffer at a concentration of 0.5 mM (pH5.3, pH5.6, P H6.
- Adipanin is obtained by removing the 5-malonyl moiety of malonyl apanin (Tetrahedron Lett, 24, 4863-4866, 1983). Also, maloninoleazinonin was obtained by extracting from a plant body by the method described in Tetrahedron Lett, 24, 4863-4866, 1983. The larger the number in the color chart, the bluer the color. In the same figure, A is deeper. The results are summarized in Table 1. At any concentration and pH, malonyl apapanin had a blue color, indicating that the malonyl group contributed to the blue coloration of anthocyanin.
- the activity of the malonyltransferase was measured using 100 ⁇ l of the reaction mixture (10 mM shisonine dissolved in 0.01% trifluoroacetic acid (TFA) in 20 mM potassium phosphate, pH 7.0). ⁇ g, malonyl-CoA 10 ⁇ g, and the enzyme preparation to be measured) at 30 ° C for 20 minutes, then stop by adding ice-cold 0.05% TFA aqueous solution 200 1 did.
- the quantitative determination of shisonine and malonylshisonin was performed by reversed-phase high-performance liquid chromatography (DYNAMAX HPLC system) using a Shodex Asahipak ODP-50 4E column, using a 0.5% TFA aqueous solution as solution A.
- Example 3 Purification of Salvia Malonyltransferase Protein Purification of malolyltransferase was carried out using 2,664 g of red flowers of Salvia splendens. Salvia flowers collected the entire flower, including the calyx, immediately before flowering, and were stored at -80 ° C until used in experiments.
- ammonium sulfate fractionation was performed, and ammonium sulfate was collected as a precipitate of a 20-50% saturated fraction, and buffer A (100 mM potassium phosphate (pH 7.0), 30 mM 2-menolecaptoethanol) was dissolved in 1 mM EDTA, 0.1 mM PMSF) (2920 ml).
- buffer A 100 mM potassium phosphate (pH 7.0), 30 mM 2-menolecaptoethanol
- 1 mM EDTA, 0.1 mM PMSF 2920 ml
- 280 ml of Octyl Sepharose Fast Flow was added, and with gentle stirring, ammonium sulfate was gradually added to a final concentration of 30% saturation. After sufficiently stirring, the mixture was allowed to stand still.
- the gel in the serum was collected by suction filtration (filter paper, Whatmanll4).
- the gel was sufficiently washed with buffer B (20 mM potassium phosphate (pH 7.0), 30 mM 2-mercaptoethanol, 20% saturated ammonium sulfate) while suction-filtering.
- Buffer C (20 mM potassium phosphate ( ⁇ 70), 15 mM 2-mercaptoethanol, 50% ethylene glycol, 0.1% 3-[(3-colamidopropyl) dimethyl ammonium O] -1-Propanesulfonic acid (CHAPS) ⁇ 280 ml was added, and the mixture was gently stirred for 30 minutes, and then a malonyltransferase active fraction was collected by suction filtration. Perform elution with buffer C for 10 This was repeated twice to collect an active fraction.
- CHAPS 3-[(3-colamidopropyl) dimethyl ammonium O] -1-Propanesulfonic acid
- MIMETIC Yellow 2 (Nacalai Tesque, Inc.) was filled into Econoram (approx. 1.0 cm ⁇ 120 cm, Nippon Pyo. Rad Laboratories Co., Ltd.) and equilibrated with buffer D. After abundant enzyme solution 1 buffer, buffer solution! ), And elute the protein bound to the column with buffer E (20 mM calcium phosphate (pH 7.0), 30 mM 2_mercaptoethanol, 0.05% CHAPS) did. The active fraction was concentrated by Pellicon and ultrafiltration (YM-10, MILLIPORE CORPORATION) to obtain 32 ml of enzyme solution 2.
- MIMETIC Red 3 (Nacalai Tester Co., Ltd.) is filled in an Econo column ( ⁇ 1.5 cm x 30 cm, Nippon Biotechnology, Inc., Lad Laboratories Co., Ltd.), and buffer solution! 7 (5 mM calcium phosphate ( ⁇ 7.0 ) And 15 mM 2-mercaptoethanol, 0.03% Triton X-100). After applying the entire amount of Enzyme Solution 2, wash with Buffer F, then Buffer G (5 mM potassium phosphate (pH 7.0), 30 mM 2-mercaptoethanol, 0.03% Triton X-100, 0.1 The protein bound to the column was eluted with mM acetyl CoA).
- Example 3 To the protein recovered as a single peptide in Example 3, 2 pmol of trypsin (Promega Co., Ltd.) was added, digested at 37 ° C for 30 hours, and an attempt was made to determine the structure of each peptide fragment. Each peptide fragment was separated from the trypsin digest by reverse phase HPLC (RPC C2 / C18, Amersham Pharmacia Biotech). The separation conditions were as follows: 0.1% trifluoroacetic acid, linear concentration gradient of 8% to 80% acetonitrile in 60 minutes, absorption at 215 nm at a flow rate of 0.1 ml / min. Only the peak fraction was collected.
- RPC C2 / C18 reverse phase HPLC
- MTT21-1 Leu-Leu-Phe-Tyr-His-His-Pro-Ser-Ser-Lys (SEQ ID NO: 8)
- MTT21-2 Ser-Gly-Asp-Lys-Ser-Asp-Glu-Asn-Ala-Pro-Glu-Leu-Phe-Ile-Ile-Pro-Ala-Asp-Ala (SEQ ID NO: 9)
- MTT22-1 Met-Ala-Ala-Phe-Glu-Glu-Val-Phe (SEQ ID NO: 10)
- MTT23 Trp-Leu-His-Tyr-His-Pro-Val (Disposition 'J number: 11)
- MTT26 Gly-Ala-Glu-Asn-Trp-Met-Ser-Asp-Ile-Phe-Lys (SEQ ID NO: 12)
- MTT27-2 Leu-Ala-Ala-Glu-Xaa-Gly-Phe- Ala-Vato Ala-Ala- Ala-A1 a-Ile-Gly_Gly-Gly-lie-lie-Gly (SEQ ID NO: 13)
- MTT28 Ser-Phe-Ile-Asn-Asp-Pro-Asn-Lys-Ile-Asp-Ala-Ile-Phe ( SEQ ID NO: 14)
- MTT141 Thr-Ala-Ser-Phe-Pro-Leu-Pro-Thr-Asn-Arg (SEQ ID NO: 15)
- MTT291 Asp-Ala-Asp-Gln-Phe-Tyr-Asp-Leu-Leu-Pro-Pro-Ile-Pro-
- Example 5 Amplification of Salvia Malonyltransferase Gene Fragment The following primers were prepared based on the partial amino acid sequences MTT20 and MT142 obtained in Example 4.
- MTT20-1 5 TA (T / C) GCI GCI GGI GA (T / C) TCI GTI CCI GT -3 '
- MTT20-3 5 '-GTI CCI GTI ACI AT (A / T / C) GCI GC-3' (Distribution 'J number
- ATCRr2 5'- (T / C) TT ICC CCA ICC (A / G) AA (A / G) TC IGC one 3 '(Toriki Column No .: 21)
- PCR was performed using the following reaction mixture composition, using cDNA prepared from Salvia flowers as type II in a total amount of 100 ⁇ l: IxTAKARA PCR buffer, 200 mM dNTPs, Sanorebi, ⁇ cDNA 100 ng, MTT20-1 primer lpmol / 1 , ATC Rr2 Primer lpmol / ⁇ 1, TAKARA rTaq 2.5 units.
- the reaction was carried out at 96 ° C for 1 minute, followed by 30 cycles of 96 ° C for 1 minute, 42 ° C for 2 minutes, 72 ° C for 3 minutes, and further at 72 ° C for 7 minutes. Allowed to react for minutes.
- a cDNA library from Salvia guaranitica flowers was constructed using Stratagene; I ZAP II directional cDNA synthesis kit according to the manufacturer's recommendations. About 200,000 clones from this library were screened under the washing conditions (5x SS 0.1% SDS, 37 ° C) using the 889 bp DNA fragment obtained in Example 3 as a probe, 0 positive clones were obtained. They could be divided into three groups and the longest clones in each group were named SgMaTl, SgMaTl 'and SgMaT2. Screening of the library was performed by a known method (for example, Fujiwara et al, 1998, PI ant. J.16, 421).
- the deduced amino acid sequence of SgMaTl there was a portion in which the partial amino acid sequence of the purified maxillary transferase clarified in Example 4 was partially different, but all were confirmed. Some differences are likely due to differences in the species of salvia used.
- SgMaTl and SgMaTl 'genes encode an enzyme that transfers a malonyl group to the 5-position sugar of anthocyanin.
- SgMaT2 had a 1530 bp cDNA, which contained an open reading frame of 1260 bp, encoding the full length.
- SgMaTl is 52% identical to SgMaT2 showed that.
- both genes are compatible with the transferases of other plants.
- SgMaTl SgMaTl 'and SgMaT2 are shown in SEQ ID NOs: 1, 3 and 5, respectively, and the amino acid sequences deduced from these nucleotide sequences are shown in SEQ ID NOs: 2, 4 and 6, respectively.
- Plasmid pSgMaTl (EcoRI of pBluescriptSr (Stratagene), containing the SgMaTl gene at the Xhol site, and addition of isopropyl beta thiogalactoside (IPTG) to LB medium containing ampicillin at a final concentration of 50 mg / L.
- IPTG isopropyl beta thiogalactoside
- a single colony of Escherichia coli transfected with the gene was inoculated, and precultured at 37 ° C. under vacuum. The culture (2 ml) was inoculated into 100 ml of an LB medium containing ampicillin, and main-cultured at 30 ° C until the absorbance at 600 nm became 0.5.
- IPTG was added to the culture solution to a final concentration of ImM, and the cells were cultured at 30 ° C. for 9 hours after the addition of IPTG to collect the cells.
- the collected cells are suspended in a buffer (0.1 M KPB, pH 7.0, 30 mM 2-mercaptoethanol, ImM EDTA, 0.1 mM PMSF, 0.1% TritonX-100), and the cells are ultrasonically disrupted while cooling forever. did.
- the enzyme activity was measured using the supernatant (soluble fraction) obtained by centrifugation.
- E. coli containing only pBluescriptSIT as a control was treated in the same manner.
- the activity was measured using shisonin as a substrate.
- Example 2 was used.
- Example 8 Isolation of Salvia-derived malonyltransferase cDNA (2) ZAP-cDNA Synthesis Kit (Stratagene) using a petal cDNA library of Sanolevia (Salvia splendens) using ⁇ ZAP II (Stratagene
- SsMaTl showed 92% identity to SgMaTl and 52% identity to SgMaT2 at the amino acid level.
- SsMaT2 The nucleotide sequence is shown in SEQ ID NO: 24, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 25.
- SsMaT2 was 53% identical to SgMaTl, 96% identical to SgMaT2, and 52% identical to SsMaTl.
- a young red leaf was collected from perilla (Perlla frutescens), and a cDNA library was prepared on the basis thereof using the ZAP-cDNA Synthesis Kit (Stratagene) using I ⁇ (Stratagene) as a vector. It was constructed using the recommended method. This library is used in the same manner as in Example 8. Screening was performed using SgMaTl as a probe, and the clone having the longest cDNA among the obtained clones was designated as PfMaTl.
- the nucleotide sequence of PfMaTl is shown in SEQ ID NO: 26, and the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 27.
- PfMaTl showed 67% identity to SgMaTl, 57% to SgMaT2, 65% to SsMaTl, and 57% to SsMaT2 at the amino acid level.
- Example 10 Isolation of lavender-derived malonyltransferase cDNA
- a primer that introduces a BamHI site into the 5'-end of the SsMaTl gene obtained in Example 8 (Primer # l: 5'-GGA TCC ATC GAG GGA CGC ATG ACA ACA ACA ACA AC-3, (SEQ ID NO: 30))), 3-Primer for introducing a BamHI site at the-terminus of the SsMaTl gene (Primer # 2: 5, -GGA TCC TTA CAA TGG TTC GAC GAG CGC CGG AGA-3 '(Distribution' J number: 3 1)) and a primer that deletes the BamHI site in the SsMaTl gene (Primer # 3: 5'-G GAC CCG CCG ATA CCG GAA AAT TAC TTC-3 '(SEQ ID NO: 32)) was synthesized. .
- Primer # l is the SsMaTl start code
- a Factor Xa cleavage site (Ile-Glu-Gly-
- PCR reaction solution composition: pBK-CMV- SsMaTl 100 ng, l Xpfu buffer, 200 ⁇ ⁇ dNTPs, ⁇ ⁇ ⁇ Primer # 1, 100 ng of first PCR product, 2.5 U pfu DNA polymerase (Stratagene); Reaction condition: 96.C for 7 minutes, (96 ° C for 2 minutes, 70 ° C for 1 minute, 72 ° C for 7 minutes) Min) X30 cycle, 72 ° C, 10 min).
- the second PCR product was treated with calo with A-tail (100 ng of the second PCR product, 1X ExTaq buffer, 2 mM dATP, TaKaRa ExTaq; 70 ° C, 30 minutes), and then pCR2.1- Cloned into T0P0 vector (Clonetech, Inc.).
- pCR2.1-SsMaTl plasmid into which full-length SsMaTl was inserted was obtained.
- a DNA sequencer it was confirmed that the DNA sequence of the SsMaTl gene did not contain erroneous nucleotide incorporation by PCR.
- pCR2.1-SsMaTl was completely digested with BamHI, and the generated DNA fragment of about 1400 bp was recovered. This DNA fragment was subcloned into the Escherichia coli expression vector pQE-30 (QIAGEN) at the BamHI site to give pQE_30Xa-SsMaTl.
- the expression of SsMaTl in E. coli containing pQE-30Xa-SsMaTl followed the method described in Example 7. The activity of the enzyme was measured according to the method described in Example 2.
- Example 7 using the plasmid prepared so that the PfMaTl gene obtained in Example 9 can be expressed as a fusion protein with LacZ protein, expressed in Escherichia coli as in Example 7, It was used for activity measurement. The activity of the enzyme was measured according to the method described in Example 2.
- an aliphatic acyltransferase is involved in the control of flower color.
- this protein By expressing this protein in petals and modifying anthocyanin, the color of the flower can be changed.
- Aliphatic acyltransferases include the monkeys mentioned here. Not only genes from beer, perilla and lavender but also genes with similar enzymatic activities from other organisms can be used.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01934510A EP1291426B1 (en) | 2000-06-02 | 2001-06-01 | Novel aliphatic acyltransferase genes |
AU60700/01A AU785496B2 (en) | 2000-06-02 | 2001-06-01 | Novel aliphatic acyltransferase genes |
US10/048,897 US7186886B2 (en) | 2000-06-02 | 2001-06-01 | Aliphatic acyl transferase genes |
DE60123787T DE60123787T2 (de) | 2000-06-02 | 2001-06-01 | Neuartige, aliphatische acyltransferase-gene |
JP2002500728A JP4827043B2 (ja) | 2000-06-02 | 2001-06-01 | 新規脂肪族アシル基転移酵素遺伝子 |
Applications Claiming Priority (4)
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JP2000-170435 | 2000-06-02 | ||
JP2000170435 | 2000-06-02 | ||
JP2001-34467 | 2001-02-09 | ||
JP2001034467 | 2001-02-09 |
Publications (1)
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WO2001092536A1 true WO2001092536A1 (fr) | 2001-12-06 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/JP2001/004677 WO2001092536A1 (fr) | 2000-06-02 | 2001-06-01 | Nouveaux genes de l'acyltransferase aliphatique |
Country Status (8)
Country | Link |
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US (1) | US7186886B2 (ja) |
EP (1) | EP1291426B1 (ja) |
JP (1) | JP4827043B2 (ja) |
AT (1) | ATE342370T1 (ja) |
AU (1) | AU785496B2 (ja) |
DE (1) | DE60123787T2 (ja) |
ES (1) | ES2274888T3 (ja) |
WO (1) | WO2001092536A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008120820A1 (ja) * | 2007-03-29 | 2008-10-09 | International Flower Developments Proprietary Limited | 表層キメラ形質転換植物の作出方法 |
Families Citing this family (1)
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JP2008161149A (ja) * | 2006-12-28 | 2008-07-17 | Kirin Holdings Co Ltd | 新規のアシル基転移酵素活性を有するタンパク質及びそれをコードする遺伝子 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996025500A1 (fr) * | 1995-02-17 | 1996-08-22 | Suntory Limited | Genes codant pour des proteines ayant une activite acyltransferase |
WO1999005287A1 (fr) * | 1997-07-25 | 1999-02-04 | Suntory Limited | Genes codant pour des proteines a activite de transglycolsylation |
-
2001
- 2001-06-01 ES ES01934510T patent/ES2274888T3/es not_active Expired - Lifetime
- 2001-06-01 US US10/048,897 patent/US7186886B2/en not_active Expired - Fee Related
- 2001-06-01 AT AT01934510T patent/ATE342370T1/de not_active IP Right Cessation
- 2001-06-01 AU AU60700/01A patent/AU785496B2/en not_active Ceased
- 2001-06-01 WO PCT/JP2001/004677 patent/WO2001092536A1/ja active IP Right Grant
- 2001-06-01 JP JP2002500728A patent/JP4827043B2/ja not_active Expired - Fee Related
- 2001-06-01 DE DE60123787T patent/DE60123787T2/de not_active Expired - Lifetime
- 2001-06-01 EP EP01934510A patent/EP1291426B1/en not_active Expired - Lifetime
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996025500A1 (fr) * | 1995-02-17 | 1996-08-22 | Suntory Limited | Genes codant pour des proteines ayant une activite acyltransferase |
WO1999005287A1 (fr) * | 1997-07-25 | 1999-02-04 | Suntory Limited | Genes codant pour des proteines a activite de transglycolsylation |
Non-Patent Citations (1)
Title |
---|
T. ISHIKAWA ET AL.: "An acetylated anthocy anin from the blue petals of salvia uliginosa", PHYTOCHEMISTRY, vol. 52, no. 3, 1999, pages 517 - 521, XP002944709 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008120820A1 (ja) * | 2007-03-29 | 2008-10-09 | International Flower Developments Proprietary Limited | 表層キメラ形質転換植物の作出方法 |
JPWO2008120820A1 (ja) * | 2007-03-29 | 2010-07-15 | 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 | 表層キメラ形質転換植物の作出方法 |
US8183434B2 (en) | 2007-03-29 | 2012-05-22 | Suntory Holdings Limited | Method for producing transgenic surface layer chimeric plant |
Also Published As
Publication number | Publication date |
---|---|
AU785496B2 (en) | 2008-06-19 |
DE60123787D1 (de) | 2006-11-23 |
DE60123787T2 (de) | 2007-10-11 |
AU6070001A (en) | 2001-12-11 |
EP1291426A4 (en) | 2004-08-04 |
US7186886B2 (en) | 2007-03-06 |
ATE342370T1 (de) | 2006-11-15 |
US20040132016A1 (en) | 2004-07-08 |
ES2274888T3 (es) | 2007-06-01 |
JP4827043B2 (ja) | 2011-11-30 |
EP1291426B1 (en) | 2006-10-11 |
EP1291426A1 (en) | 2003-03-12 |
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