JP2004173617A - Cyclamen anthocyanin synthase gene - Google Patents

Abstract

The present invention provides a DNA encoding an enzyme involved in flower color synthesis of cyclamen, particularly a flavonoid 3 ', 5'-hydroxylase.
A cDNA is synthesized from mRNA prepared from cyclamen, a DNA encoding a part of flavonoid 3 ', 5'-hydroxylase is amplified by reverse transcription PCR, and the obtained DNA fragment is used as a probe to obtain a cyclamen. A DNA having a specific nucleotide sequence is isolated from the cDNA library.
[Selection diagram] None

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel DNA encoding a cyclamen flower color synthase protein. More specifically, the present invention relates to a novel protein having cyclamen flavonoid 3 ', 5'-hydroxylase activity, and a novel DNA encoding the same.
[0002]
[Prior art]
Anthocyanins, a type of flavonoid, play an important role as pigments in flowers, and anthocyanins are compounds that produce a wide range of flower colors from yellow to red, purple, and blue. Anthocyanidins, which are pigment bodies as aglycones obtained by removing glycoside moieties from anthocyanin molecules, are classified into three types: pelargonidin, cyanidin and delphinidin according to the number and position of hydroxyl groups bonded to the B ring of the flavonoid basic skeleton. being classified.
[0003]
It is known that delphinidin, which is a compound in which all of the 3 ′, 4 ′ and 5 ′ positions of the B ring are hydroxylated, exhibits a bluish purple color. In particular, flavonoid 3 ′, 5′-hydroxylase (hereinafter abbreviated as “F3 ′, 5′H”) in the anthocyanin synthesis pathway is an enzyme that hydroxylates the 3 ′ and 5 ′ positions of the flavonoid B ring, It is considered a key enzyme for the biosynthesis of delphinidin, a blue pigment. F3 ′, 5′H has an important role in the field of research on the improvement of the flower color of flowering plants, which involves the use of recent genetic engineering techniques.
[0004]
Heretofore, the following are known as the nucleotide sequences of genes encoding F3 'and 5'H of flower plants.
[0005]
(1) Petunia F3 ', 5'H gene (see Patent Document 1)
(2) F3 ′, 5′H gene of eggplant (see Patent Document 2)
(3) F3 ', 5'H gene of Cinellaria (see Patent Document 3)
(4) Torenia F3 ', 5'H gene (see Non-Patent Document 1)
(5) F3 ′, 5′H gene of Catharanthus roseus (see Non-Patent Document 2)
(6) F3 ', 5'H gene of ginger (see Non-Patent Document 3)
However, the genes encoding F3 ', 5'H from cyclamen plants are not known.
[0006]
[Patent Document 1] JP-A-2000-23686
[Patent Document 2] JP-A-5-184370
[Patent Document 3] Japanese Patent Application No. 2001-362459
[Non-Patent Document 1] Mol. Breed. 6,239-246 2000
[Non-Patent Document 2] Plant J. 19 (2), 183-193 1999
[Non-Patent Document 3] Plant Cell Physiol. 37 (5), 711-716 1996
[0007]
[Problems to be solved by the invention]
An object of the present invention is to obtain DNA encoding a novel cyclamen F3 ′, 5′H from cyclamen.
[0008]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above problems. Then, a 1745 bp DNA fragment described in SEQ ID NO: 1 in the sequence listing was successfully obtained from the cyclamen cDNA library by the genetic engineering technique described below.
[0009]
Furthermore, a DNA sequence having a size of 1524 bp having a base sequence of a region from base number 90a at the 5 'side to base number 1613 t in the 1745 bp size DNA fragment is obtained by combining cyclamen F3' and 5'H. It was found to be a DNA sequence that encodes. As a result, the present invention has been completed.
That is, the gist of the present invention is as follows.
[0010]
(1) A DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and having F3 ′, 5′H activity.
(2) The DNA of (1) having a base sequence consisting of base numbers 90 to 1613 of SEQ ID NO: 1 in the sequence listing.
(3) a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, wherein the protein is a flavonoid 3 ′, 5′-hydroxylase DNA encoding a protein having activity.
(4) a DNA having a nucleotide sequence partially different from the nucleotide sequence consisting of nucleotide numbers 90 to 1613 of SEQ ID NO: 1 in the sequence listing, but showing homology to the nucleotide sequence described in SEQ ID NO: 1, In addition, it is a DNA that can hybridize to a DNA having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing under stringent conditions and encodes a protein having F3 ′, 5′H activity. DNA of (3).
(5) A protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, the protein having F3 ′, 5′H activity.
(6) a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, wherein the protein is a flavonoid 3 ', 5'-hydroxylase A protein having activity.
[0011]
In the present invention, the “F3 ′, 5′H activity” means an activity of converting the 3 ′ and 5 ′ positions of the B ring of the flavonoid into a hydroxyl group to catalyze a reaction for generating delphinidin.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the method for obtaining DNA according to the present invention from cyclamen will be described in detail.
[0013]
A first form of the DNA of the present invention is a protein having the amino acid sequence shown in SEQ ID NO: 2, which encodes a protein having F3 ', 5'H activity. An example of the DNA of the present invention encoding this amino acid sequence is shown in SEQ ID NO: 1. The DNA of the present invention was obtained from a cyclamen cDNA library as described later when completing the present invention. Since the nucleotide sequence of the DNA of the present invention has been elucidated, it can also be obtained by chemical synthesis based on the amino acid sequence shown in SEQ ID NO: 2 or the nucleotide sequence shown in SEQ ID NO: 1. When a synthetic oligonucleotide probe or oligonucleotide primer devised and prepared based on the base sequence of SEQ ID NO: 1 is used, hybridization or polymerase chain reaction (PCR) can be performed from a cyclamen cDNA library by a known method. ) Can also be obtained.
[0014]
Hereinafter, a DNA fragment that forms a partial region of the DNA of the present invention is obtained from the total RNA of cyclamen by RT (reverse transcription) -PCR method, and further obtained by a plaque hybridization method using the DNA fragment as a probe. The method for obtaining the DNA of the present invention will be exemplified.
[0015]
(1) Preparation of cyclamen mRNA and construction of cyclamen cDNA library
(I) After extracting total RNA from petals of cyclamen (Cyclamen persicum) by a conventional method, proteins, polysaccharides, and other contaminants are removed from the extract of the total RNA. Furthermore, when total RNA containing no impurities is treated using a column filled with oligo dT cellulose, Poly (A) ⁺ RNA can be purified, and thereby mRNA can be obtained.
[0016]
(Ii) Next, cDNA is synthesized from the above mRNA using a reverse transcriptase. The synthesized cDNA is incorporated into a phage vector, such as λZAPII, to obtain a recombinant phage. The obtained recombinant phage is infected to a host Escherichia coli (Escherichia coli), and a large number of plaques of the recombinant phage can be obtained. The phage of this plaque is used as a cyclamen cDNA library. Since a cDNA cloning kit is commercially available for these series of operations, this kit may be used.
[0017]
(2) Preparation of cloning probe DNA and screening of required recombinant phage from cDNA library
(I) Next, from the cyclamen cDNA library obtained as described above, an operation of isolating a recombinant phage containing the target F3 ′, 5′H cDNA of cyclamen is performed. For this purpose, an oligonucleotide designed based on a known base sequence conserved in a DNA encoding a known F3 ′, 5′H such as a petunia plant is used as a primer and described in the above (1) (i). By the RT-PCR method using the extracted cyclamen RNA as a template, a cDNA fragment encoding a partial region of the DNA encoding F3 ′, 5′H of cyclamen is obtained as a probe DNA.
[0018]
(Ii) Next, the DNA probe is hybridized with the plaque of the recombinant phage prepared in the above (1) (ii) to obtain the desired cyclamen cDNA library (recombinant phage). Recombinant phage containing DNA encoding F3 ', 5'H therein can be selected and isolated as a positive clone.
[0019]
(3) Recovery of plasmid (phagemid) DNA from positive clones
The phage is collected from the plaques selected and isolated by the above hybridization. Further, the collected phage (positive clone) is infected with E. coli as a host together with a helper phage. As a result, phagemid DNA into which cDNA which seems to encode cyclamen F3 ', 5'H is inserted from phage DNA can be cut out in E. coli (by in vivo excision system). Further, this phagemid DNA is introduced into another E. coli for transformation, and the phagemid DNA is recovered from the culture of this E. coli.
[0020]
(4) Determination of base sequence by sequencing
The collected phagemid DNA is treated with a restriction enzyme so that the inserted DNA fragment is cut, and the digested solution is subjected to electrophoresis on an agarose gel, and the obtained DNA fragment is examined. Based on the results, the base sequence of the inserted DNA fragment that is considered to retain the full length of F3 ', 5'H in the phagemid DNA can be determined by the dideoxy method.
[0021]
By examining the positions of the translation initiation codon and stop codon in the determined nucleotide sequence of the inserted fragment, the coding region of the protein (ie, open reading frame) can be determined. Then, by examining the amino acid sequence to be defined, the amino acid sequence encoded by the cyclamen cDNA retained by the inserted DNA fragment can be determined. Further, when this base sequence and amino acid sequence are compared with the base sequence and amino acid sequence of F3 ', 5H cDNA of known petunia and the like to confirm homology, the inserted DNA fragment encodes the desired cyclamen F3', 5'H. Can be confirmed.
[0022]
On the other hand, in general, even when one or more amino acids are added, deleted or substituted in an amino acid sequence encoding a protein having a specific function or biological activity, the function or biological activity is maintained. It is widely recognized by those skilled in the art that this is the case. The DNA of the present invention may have a DNA sequence encoding a protein having such a modification and having F3 ', 5'H activity. That is, the second form of the DNA of the present invention is a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. And a modified DNA encoding a protein having F3 ', 5'H activity. Such a modified DNA can be obtained, for example, by modifying the base sequence of the DNA of the present invention so that amino acids at a specific site are deleted, substituted or added by site-directed mutagenesis.
[0023]
The “several” is preferably 2 to 100, more preferably 2 to 50, and particularly preferably 2 to 20. Alternatively, the “several” is a value such that the homology with the amino acid sequence of SEQ ID NO: 2 is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more.
[0024]
In addition, the first form of the DNA of the present invention or a cell containing the same is subjected to a mutation treatment, and then the mutated DNA or a cell containing the same is subjected to, for example, the base described in SEQ ID NO: 1 in the sequence listing A fragment of the modified DNA of the present invention can also be obtained by selectively separating DNA that hybridizes under stringent conditions from DNA having the nucleotide sequence of Nos. 90 to 1613.
[0025]
The term "stringent conditions" as used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify these stringent conditions clearly, one example is that two nucleic acids having high homology, such as 70% or more, preferably 80%, and more preferably 95% or more homology The condition is that two DNAs having similarity hybridize with each other, but on the other hand, two nucleic acids with lower homology do not hybridize with each other. More specifically, for example, conditions in which nucleic acids hybridize with each other in a hybridization solution (500 mM NaPi buffer (pH 7.2), 7% SDS, 1 mM EDTA) at 68 ° C. or higher can be mentioned.
[0026]
Furthermore, when the DNA of the present invention or a DNA fragment of a partial region thereof is used as a probe DNA from the cyclamen chromosome itself, a DNA fragment encoding F3 ′, 5′H can be obtained by a conventional method. However, the DNA fragment encoding F3 ', 5'H derived from the cyclamen chromosome is expected to contain introns. Such a DNA fragment separated by an intron is also included in the DNA of the present invention as long as it is a DNA encoding F3 ′, 5′H.
[0027]
The cyclamen F3 ', 5'H cDNA fragment cloned into the recombinant lambda phage DNA obtained in the following Examples was ligated to a Bluescript plasmid vector, and the resulting recombinant plasmid was introduced. The Escherichia coli thus obtained was named Escherichia coli DH5α (CpFH-1), and deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary on November 8, 2002 as Accession No. FERM P-19093.
[0028]
The protein F3 ', 5'H can be produced by expressing the DNA of the present invention encoding F3', 5'H in an appropriate host, for example, Escherichia coli. Examples of the host include bacteria such as Escherichia coli and Bacillus subtilis, yeast, cultured insect cells, cultured animal cells, cultured plant cells, and the like. The host cell is transformed by introducing the DNA of the present invention encoding F3 ′, 5′H operably linked downstream of an expression control sequence such as a promoter that functions in the host cell into the host cell. Such transformation can be performed by ligating the DNA of the present invention to a plasmid to construct a recombinant plasmid, and introducing the obtained recombinant plasmid into a host. Alternatively, the host can be transformed by integrating the DNA of the present invention into chromosomal DNA in the host by homologous recombination or the like. By culturing the obtained transformed cell under conditions where the promoter expression regulatory sequence functions, proteins F3 'and 5'H are produced.
[0029]
The DNA of the present invention encoding F3 ′, 5′H can be introduced into flowers or other plants, alone or in combination, to transform the plants and thereby to synthesize pigment in the plants. Can be regulated. In order to introduce the DNA of the present invention into a plant, (a) Agrobacterium method (edited by Draper, J et al., "Plant Genetic Transformation and Gene Expression A Laboratory Manual", Blackwell Scientific Pub. (B) Whisker introduction method (US Pat. No. 5,302,523, US Pat. No. 7,472,538, Japanese Patent No. 3,328,867, Kaepper, HF, et al., 1992, Theoretical and Applied Genetics, 84, 560-566). Pp. Asano, Y. et al., 1991, Plant Science, 79, 247-252); (Jorsbo, M. et al., 1990, Plant Cell Reports, Vol. 9, pp. 207-210, Joersbo, M. et al., 1992). Physiological Plantarum, Vol. 85, pp. 230-234); (d) Particle gun method (Sanford, JC, U.S. Pat. No. 5,100,792).
[0030]
A first form of the protein of the present invention is a protein having F3 ', 5'H activity of cyclamen and having an amino acid sequence consisting of 508 amino acid residues shown in SEQ ID NO: 2 in the sequence listing.
[0031]
Further, the protein of the present invention is a protein to which one or more amino acids have been added, deleted or substituted so long as the F3 ′, 5′H activity is not substantially impaired. Is also good. That is, the second aspect of the present invention is a modified protein comprising an amino acid sequence shown in SEQ ID NO: 2 in the sequence listing in which one or several amino acids have been deleted, substituted, inserted, or added. Thus, it is a modified protein having cyclamen F3 ', 5'H activity.
[0032]
The protein of the present invention can be produced by expressing the DNA of the present invention in a suitable host by the method described above.
[0033]
【Example】
Hereinafter, the present invention will be specifically described by way of examples of the present invention, but the scope of the present invention is not limited thereto.
[0034]
Unless otherwise stated, the following experimental procedures were performed according to the method described in “Molecular Cloning 2nd Edition” (J. Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).
[0035]
Embodiment 1
(1) Preparation of cyclamen mRNA
4 g of petals of cyclamen (cultivar: fragrance mini) during flowering were frozen in the presence of liquid nitrogen, and the frozen petals were ground using a pestle mortar. 40 ml of 2 × CTAB solution (2% cetyltrimethylammonium bromide, 0.1 M Tris-HCl (pH 9.5), 20 mM EDTA, 1.4 M NaCl and 4% β-mercaptoethanol) was added to the pulverized material. After stirring the resulting mixture, it was incubated at 65 ° C. for 10 minutes. The incubated mixture was then subjected to chloroform extraction twice. After isopropanol was added to the extracted aqueous phase and mixed by inversion, the mixture was allowed to stand at room temperature for 10 minutes to precipitate nucleic acids. The nucleic acid precipitate was centrifuged at 8000 g at 4 ° C. for 15 minutes to recover the nucleic acid. The nucleic acid was dissolved in 4 ml of TE (10 mM Tris (pH 8.0), 1 mM EDTA), and 1 ml of a 10 M lithium chloride solution was added to the resulting solution. The obtained mixture was placed on ice for 2 hours, and then centrifuged at 19000 g and 4 ° C. for 10 minutes, whereby RNA was precipitated. The RNA was dissolved in distilled water, and the RNA solution was purified by phenol extraction and ethanol precipitation. Thus, 0.4 mg of purified total RNA was obtained. Further, mRNA was isolated from the purified product of total RNA thus obtained using an mRNA purification kit (Oligotex-Mag mRNA purification Kit, manufactured by Takara Bio Inc.) to obtain about 6 μg of cyclamen mRNA.
[0036]
(2) Preparation of cyclamen cDNA library
A double-stranded cyclamen cDNA was obtained from the cyclamen mRNA obtained in (1) above using a cDNA synthesis kit (manufactured by Amersham Life Sciences, cDNA synthesis module). An adapter EcoRI-NotI-BamHI Adapter (manufactured by Takara Bio Inc.) having an EcoRI site as a sticky end was ligated to the double-stranded cyclamen cDNA, and the adapter was phosphorylated. The thus-phosphorylated adapter portion was ligated, and the retained cyclamen double-stranded cDNA was ligated to the EcoRI site of a λZAPII phage vector (manufactured by STRATAGENE, Lambda ZAP Predigested EcoRI / CIAP-Treated Vector Kit). Using the obtained ligation vector, a recombinant lambda phage containing cyclamen cDNA was reconstructed using an in vitro packaging kit (manufactured by STRATAGENE, Gigapack III Gold Packaging Kit) (packaging). The thus obtained recombinant lambda phage was infected to Escherichia coli XL1-Blue MRF '(manufactured by STRATAGENE) and allowed to proliferate to obtain a large number of the above-mentioned recombinant lambda phage. This recombinant lambda phage was used below as a cyclamen cDNA library.
[0037]
(3) Cloning probe preparation
(I) To prepare a probe for cloning F3 ', 5'H cDNA fragment using reverse transcription (RT) -PCR (Reverse Transcriptase-Polymerase Chain Reaction) method, it should be used for preparing probe DNA. Primers were designed first. That is, two types of oligonucleotides having the base sequences shown below were used as primer Nos. 1 and No. 1 2 was produced by chemical synthesis.
[0038]
(A) Primer No. 1 (shown in SEQ ID NO: 3)
5'-gckggwacrgayacwtcwtcwa-3 '
(B) Primer No. 2 (shown in SEQ ID NO: 4)
5'-ayccatatcaawgmwtgnaccaa-3 '
However, in the above two base sequences, k = g or t; w = a or t; r = g or a; y = t or c; n = inosine.
[0039]
The above primer No. 1 and No. 1 The oligonucleotide 2 was prepared by synthesizing an oligonucleotide using a DNA synthesizer (Model 391, manufactured by Applied Biosystems), and further purifying the synthesized product by ion exchange HPLC.
[0040]
(Ii) Next, for the production of cyclamen cDNA, using 1 μg of the total RNA obtained in the above (1) as a template, this total RNA was subjected to a reverse transcription reaction solution [10 mM Tris-HCl (pH 8.3), 50 mM KCl , 5 mM MgCl ₂ 1 mM dNTP (dATP, dGTP, dCTP, dTTP) mixture, 2.5 μM oligo (dT) primer (Oligo (dT) _12-18 primer (Invitrogen), RNase inhibitor (RNase Inhibitor (Takara Bio Inc.), 20 units) and reverse transcriptase (AMV Reverse Transcriptase XL (Takara Bio Inc., 5 units)). The volume of the solution was set to 20 μl, and a reverse transcription reaction was performed. The reverse transcription reaction was performed under the conditions of incubation at 30 ° C. for 10 minutes, 50 ° C. for 30 minutes, 99 ° C. for 5 minutes, and 4 ° C. for 5 minutes to obtain a reaction solution containing the generated cyclamen cDNA.
[0041]
(Iii) The primer No. 1 and No. 1 A PCR reaction solution [10 mM Tris-HCl (pH 8 .1) was used as a template with 0.3 μM of each of the two synthetic oligonucleotides constructed as 2 as primers and 1 μl of the reverse transcription reaction solution containing the cyclamen cDNA obtained above as a template. 3), 50 mM KCl, 1.5 mM MgCl ₂ , 0.2 mM dNTP (dATP, dGTP, dCTP, dTTP) mixture and Taq DNA Polymerase (TaKaRa Taq (manufactured by Takara Bio Inc.) 1 unit)] to make a total volume of 50 μl, and a DNA amplification reaction was performed. .
[0042]
The amplification reaction of cyclamen cDNA by the above-mentioned PCR method was performed using a PCR reaction device (manufactured by ASTEK, Program Temp Control System PC-700) for denaturation at 94 ° C for 30 seconds and annealing at 52 ° C for 1 minute. In addition, three reaction operations in which extension was performed at 72 ° C. for 1 minute were repeated 35 times.
[0043]
The amplification reaction product obtained above was ligated to a TA site of a TA vector pT7Blue Vector (manufactured by Navagen). By transforming Escherichia coli DH5α with the obtained ligated vector, transformed Escherichia coli having DNA as an amplification reaction product was obtained. From each of the thus transformed Escherichia coli cells, the cloned plasmid DNA was extracted using the alkaline SDS method.
[0044]
1 μl of the obtained DNA was digested with 5 units of restriction enzyme EcoRI and 5 units of restriction enzyme PstI in 10 μl of H buffer. The digestion reaction solution was analyzed by agarose gel electrophoresis to confirm the DNA size of the amplification reaction product. Escherichia coli in which a DNA fragment as an amplification reaction product of the expected size was cloned was selected from primers designed from known petunia F3 ', 5'H, and the nucleotide sequence of the plasmid DNA carried by the Escherichia coli was analyzed. . Analysis of this base sequence was performed using a plasmid purification kit (QIAfilter PlasmidMidi Kit (manufactured by QIAGEN)) and a DNA sequencing service (Hokkaido System Science Co., Ltd.), and an automatic DNA sequencer (ABI PRISM 3100 Gentic Analyzer (Applied Biosystems)). Company))). As a result, the DNA fragment of the amplification reaction product was determined to be a part of cDNA consisting of 550 bases. By comparing the amino acid sequence predicted from the nucleotide sequence with the amino acid sequence of F3 ', 5'H of petunia which has been clarified so far, the DNA fragment which is the above amplification reaction product is obtained from the F3', 5 'of cyclamen. It was confirmed that the DNA fragment contained a partial region of the cDNA encoding H.
[0045]
A series of operations were performed as described above, and a DNA fragment containing a partial region of the cyclamen F3 ', 5'H cDNA was obtained as a probe. The probe DNA was used to screen a cDNA encoding the target cyclamen F3 ', 5'H from a cyclamen cDNA library (recombinant lambda phage shown in (ii) above).
[0046]
(4) Screening and cloning of cDNA encoding cyclamen F3 ', 5'H from cyclamen cDNA library
From the cyclamen cDNA library prepared in the above (2) (ie, the above-described recombinant lambda phage), plaque hybridization using the probe DNA prepared in the above (3) was carried out to obtain the cyclamen F3 ′, 5′H. An operation for screening a clone having a DNA sequence encoding was performed. This plaque hybridization method was performed as follows.
[0047]
(I) Label the probe DNA obtained in the above (3) and (iii) with digoxigenin (DIG) using a DNA labeling kit (Roche Diagnostics, DIG-ELISA DNA Labeling & Detection Kit). did. As a result, a labeled probe DNA was obtained.
[0048]
(Ii) Next, the recombinant lambda phage, which is the cyclamen cDNA library obtained in (2) above, was infected with Escherichia coli XL1-Blue MRF ′ as a host cultured on a 1.5% agar medium and allowed to grow. Thus, a plaque of the phage was formed on the medium.
[0049]
Plaques of the above-mentioned recombinant phage constituting the cyclamen cDNA library were transferred to a nylon membrane (Gene Screen (Perkin Elmer Life Science)). The nylon membrane onto which the phage plaque was transferred was washed with an alkaline denaturing solution (1.5 M NaCl, 0.5 M NaOH), a neutralizing solution (1 M Tris-HCl (pH 7.5)) and 2 × SSC (0.3 M NaCl and 30 mM sodium citrate) were successively treated for 10 minutes, air-dried, and further baked at 80 ° C. for 2 hours to remove the phage DNA contained in the plaque of the recombinant lambda phage into the nylon. Fixed on a membrane.
[0050]
(Iii) The nylon membrane on which the phage DNA was immobilized was immersed in a hybridization solution (500 mM NaPi buffer (pH 7.2), 7% SDS, 1 mM EDTA) and subjected to immersion treatment at 68 ° C. for 2 hours.
[0051]
Next, the DIG-labeled labeled probe DNA was incubated at 100 ° C. for 5 minutes (denature treatment). Then, this was added to the hybridization solution so that the probe DNA concentration in the hybridization solution was 10 ng / ml. The nylon membrane is immersed in a hybridization solution containing the labeled probe DNA, and further immersed at 68 ° C. for 15 hours, whereby the labeled probe DNA and the target phage DNA immobilized on the nylon membrane are separated. To form the required DNA-DNA hybrid.
[0052]
After the completion of the plaque hybridization reaction, the nylon membrane was washed three times for 20 minutes each with a washing solution (40 mM NaPi buffer (pH 7.2), 1% SDS) kept at 68 ° C. Thereafter, the required recombinant phage containing the DNA sequence encoding F3 ′, 5′H of cyclamen could be detected using the above-mentioned DIG-ELISA Labeling & Detection Kit.
[0053]
(Iv) As a result, out of 600,000 recombinant lambda phage plaques, a DNA sequence encoding cyclamen F3 ', 5'H was internally incorporated, and four phage plaques considered to be included therein were converted as positive clones. I was able to select.
[0054]
(5) cDNA subcloning
From the four plaques isolated in the above (4) (iv), the phage were respectively subjected to SM buffer (50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 8 mM MgSO 4). ₄ , 0.01% gelatin). According to the above-mentioned Lambda ZAPII Predicted EcoRI / CIAP-Treated Vector Kit, these four phages are separately infected with a helper phage ExAssist helper phage (manufactured by Stratagene) into Escherichia coli XL1-BlueMRF ′ as a host. From the phage DNA, a pBluescript SK (-) phagemid DNA (manufactured by STRATAGENE) into which a cDNA believed to encode cyclamen F3 ', 5'H was inserted was cut out. By further culturing this E. coli, a phage having the phagemid DNA was obtained in the culture supernatant. The culture supernatant containing these phages was infected with E. coli SOLR (manufactured by STRATAGENE) to obtain transformed E. coli. The subcloned phagemid DNA was extracted from each of the four types of Escherichia coli cells thus transformed by the alkaline SDS method. 1 μl of the obtained DNA was digested with 5 units of restriction enzyme EcoRI in 10 μl of H buffer. As a result of analyzing the digestion reaction solution by agarose gel electrophoresis, it was confirmed that all four types of phagemid DNAs retained a DNA fragment of about 1.7 kbp.
[0055]
(6) Sequencing of cDNA encoding cyclamen F3 ', 5'H
Since all of the four cDNA fragments subcloned as phagemid DNAs in (5) were expected to contain the full length of cyclamen F3 ', 5'H cDNA, one of the phagemid DNAs having 1.7 Kbp cDNA was expected. (Named CpFH-1) was selected, and its nucleotide sequence was analyzed. Analysis of this nucleotide sequence was performed using a plasmid purification kit (QIAfilter Plasmid Midi Kit (manufactured by QIAGEN)) and a DNA sequence contract service (Hokkaido System Science Co., Ltd.) using an automatic DNA sequencer (ABI PRISM 3100 Genic Analyzer (Applied Bio). Systems Inc.)). As a result, the DNA fragment having a size of 1.7 Kbp was determined to be a cDNA consisting of 1745 bases. This 1745 bp size cDNA contains the DNA sequence of a single open reading frame (ORF) consisting of 1524 bases. The nucleotide sequence of the 1745 bp cDNA is shown in SEQ ID NO: 1 in the sequence listing. The ORF is sandwiched between a 5 'untranslated region consisting of 89 bases and a 3' untranslated region of a DNA sequence consisting of 127 bases.
[0056]
The amino acid sequence of SEQ ID NO: 2 is 76% homologous when compared with the petunia F3 ', 5'H previously disclosed, but in other regions it is apparently petunia F3', 5'H. And the sequence is unique to cyclamen.
[0057]
The homology of the nucleotide sequences of the DNA of the present invention encoding the cyclamen F3 ', 5'H and the petunia F3', 5'H gene was found to be 61%. The homology was calculated using Gene Works 2.5.1 software (Teijin System Technology Co., Ltd.), using the Protein Alignment files of the cyclamen F3 ′, 5′H of the present invention and the petunia F3 ′, 5′H. This was done by creating with the initial settings of the software.
[0058]
【The invention's effect】
The gene encoding F3 ′, 5′H of cyclamen provided by the present invention is involved in anthocyanin synthesis in a plant of the same or different species from cyclamen when introduced alone or in combination with other DNA. The enzymatic reaction can be controlled. The DNA of the present invention can be used for breeding and developing plants for measuring the diversity of flower colors of plants.
[0059]
[Sequence list]

Claims (6)

A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing, the protein having flavonoid 3 ', 5'-hydroxylase activity. The DNA according to claim 1, which has a base sequence consisting of base numbers 90 to 1613 of SEQ ID NO: 1 in the sequence listing. A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and has a flavonoid 3 ', 5'-hydroxylase activity DNA encoding a protein. A DNA having a nucleotide sequence partially different from the nucleotide sequence consisting of nucleotide numbers 90 to 1613 of SEQ ID NO: 1 in the sequence listing, but showing homology to the nucleotide sequence of SEQ ID NO: 1; It is a DNA that can hybridize under stringent conditions to the DNA having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing and encodes a protein having flavonoid 3 ', 5'-hydroxylase activity. The DNA according to claim 3. A protein having an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, the protein having flavonoid 3 ', 5'-hydroxylase activity. A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and has a flavonoid 3 ', 5'-hydroxylase activity protein.