JPH05184370A - Flavonoid hydroxylase gene - Google Patents

Abstract

PURPOSE:To provide a genetic manipulation technique capable of introducing hydroxyl group even to the 5'-site of a flavonoid of an ornamental garden flower such as rose and chrysanthemum and preparing a new variety having unique color which is impossible to produce by combination breeding. CONSTITUTION:A DNA chain coding a protein having a flavonoid 3',5'- hydroxylase activity or arbitrary fragment of the DNA chain. A recombinant vector containing the DNA chain or its arbitrary fragment. Introduction of the DNA chain into a plant free from delphinidin such as rose, chrysanthemum, fringed pink and tulip by conventional introduction method is remarkably effective for increasing the variety of the color such as flower color of the plant, especially for creating a blue-colored flower.

Description

Detailed Description of the Invention

[Background of the Invention]

TECHNICAL FIELD The present invention relates to a DNA encoding a flavonoid 3 ′, 5′-hydroxylase, which is one of the enzymes that modifies a flavonoid pigment, which is a major pigment present in the petals of plants. It is about chains.

[0002]

2. Description of the Related Art The color of plants such as flowers and stems is brought about by pigments produced by plants. As is well known, new flowers
There is a great demand for plants with stem and leaf colors. Until now, there has been no choice but to use cross breeding to produce plants such as flowers having a new color. However, such a classical plant breeding method has good compatibility with various plant species used for cross breeding or a combination depending on the combination of strains, and introduction of undesired traits other than the target trait, etc. There were many problems inherent. It is mainly the flavonoids, carotenoids and betalains that give various colors to the plant, but the most important and widely distributed are the flavonoids (Fig. 1). Among flavonoid compounds, various anthocyanin compounds play a central role in the development of color tone of plants, and these compounds can give a wide range of color tone from orange to yellow, red, purple and blue. Anthocyanins are glycosides to which sugars are bound, and the part (aglycone) excluding the sugars is called anthocyanidin. Ten or more kinds of anthocyanidins are known, but the main ones are pelargonidin, cyanidin, and delphinidin. The color tone of the anthocyanin pigment depends on the structure of the anthocyanidin, but the hydroxylation of the flavonoid skeleton is particularly important.
Of the three major anthocyanidins mentioned above, part 3
Delphinidin, which is all hydroxylated at the ', 4', 5'positions (on the B ring, see FIG. 1), is a purple to blue dye, while pelargonidin, which is hydroxylated only at the 4'position, is pink to orange. ,
Cyanidine hydroxylated at the 3'and 4'positions is a red dye. Some plants, such as Rosa , Rosa , Chrysanthemum , Dianthus , and Tulipa , have pelargonidin and cyanidin but cannot synthesize delphinidin. Therefore, these plant species do not have purple to blue flowers. Of the hydroxyl groups on the B ring of anthocyanidin, the 4'-position hydroxyl group is derived from 4-coumaric acid, which is a precursor of flavonoid compounds, but the 3'- and 5'-position hydroxyl groups are introduced after the flavonoid skeleton is formed. (Fig. 2). Therefore, it is understood that the important (key) enzymatic reaction in delphinidin synthesis is the hydroxylation reaction at the 5'position. That is, roses and chrysanthemums can hydroxylate the 3'position of flavonoids, but they cannot hydroxylate the 5'position. The lack of the 5'-hydroxylation ability of flavonoids is due to the fact that the enzyme that catalyzes this reaction, flavonoid 3 ', 5'-hydroxylase (F3', 5'H) (Fig. 2), is present in the plant species. This is because they are genetically lacking. For example, it is considered extremely difficult to produce blue roses by conventional breeding methods because there are no varieties with an enzyme that introduces a hydroxyl group into the 5'of flavonoids [Forkmann, G. , Plant Br
eeding, 106, 1-26, (1991)]. One solution to this is to make a large number of mutant strains by mutagenesis and screen the desired individual from among them. However, it is clear that performing such an operation on a crop of wood such as roses requires a great physical or time burden, and in general, there is a possibility that mutation occurs in a site other than the target trait. Is also high. On the other hand, due to the recent development of plant genetic engineering, it is becoming possible to give various useful traits to plants by using gene recombination techniques. Since the gene recombination technique does not depend on mating, it has an advantage that a gene can be introduced from a plant that cannot be crossed with a target plant, and further from microorganisms, animals, and the like. For example, since petunia ( Petunia hybrida ) dihydroflavonol-4-reductase (DFR) cannot use dihydrokaempferol as a substrate, petunia with pelargonidin cannot be obtained in cross breeding (Fig. 2).
reference). So Meyer et al. [Nature, 330, 6
77-678 (1987); Japanese Patent Application Laid-Open No. 2-2305] discloses that a DFR gene derived from maize ( Zea mays ) having a wide substrate selectivity is introduced into a petunia mutant in which dihydrokaempferol accumulates by gene recombination and expressed. Created a new petunia with a brick-colored flower color. By using such a method, an enzyme capable of hydroxylating the 5'position of flavonoids, that is, a gene of F3 ', 5'H is introduced into roses and chrysanthemums to be expressed, and roses having a delphinidin blue pigment. There is a possibility that you can create a chrysanthemum. However, until now, the F3 ', 5' H gene, namely F3 ', 5'
Little was known about the DNA strand encoding H. There is a very recent report [Nikkei Biotech, No. 238, published on August 26, 1991] that a gene encoding this enzyme was obtained from Petunia, but in this report, the nucleotide sequence of the obtained DNA chain and Not to mention the physical properties, there is no description about the details of the acquisition method.
The hydroxylases that catalyze the hydroxylation reactions at the 5'and 3'positions of the flavonoids described above are biochemically both cytochrome P-450 type monooxygenases (hereinafter P-450).
Type enzyme) [Forkmann, G., Plant Breedin
g, 106, 1-26 (1991)]. However, plant P-4
Regarding type 50 enzyme, there are few examples of structural studies of genes for hydroxylases of flavonoids as well as hydroxylases for other substances, and only a small amount of the natural enzyme protein encoded in avocado fruit is found. Only one gene with unknown substrate and function has been cloned [Bozak, KR et al., Proc. Na.
tl. Acad. Sci., 87, 3904-3908 (1990)]. As described above, it is not possible with conventional techniques to manipulate flavonoid 3 ', 5'-hydroxylase genes to produce varieties with various new colors in flowers such as chrysanthemums and roses. It was

[0003]

DISCLOSURE OF THE INVENTION The present invention provides the ability to introduce a hydroxyl group at the 5′-position of flavonoids into ornamental plants such as roses and chrysanthemums by using a genetic engineering technique, whereby hybrid breeding is performed. The aim is to create new varieties of these plants with impossible new colors.

[0004]

[Means for Solving the Problems]

SUMMARY OF THE INVENTION The present invention provides a DNA chain encoding a protein having flavonoid 3 ′, 5′-hydroxylase activity or any fragment of this DNA chain. This D
By introducing the NA chain into the target plant, a variety having a new color can be produced. The present invention also relates to a recombinant vector containing the above DNA chain or any fragment of this DNA chain.

DETAILED DESCRIPTION OF THE INVENTION (1) Overview (i) Flavonoid Hydroxylase Gene The flavonoid hydroxylase gene according to the present invention comprises flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5 ′ H). ) It has been described above that it is a DNA chain encoding a protein having activity, and in a preferred embodiment, the above-mentioned protein is a protein derived from a plant belonging to the genus Solanum, such as eggplant, or the like. D
Any fragment of the NA chain. The “arbitrary fragment” in the present invention is a portion of the DNA chain which is the F3 ′, 5 ′ H gene of the present invention, as described in detail in the section (6) below. By a protein or polypeptide encoded by, alone or in the form of a fusion with a fragment or portion of another related enzyme, is meant a DNA strand having F3 ', 5'H activity. The DNA strand of the present invention includes degenerate isomers of each codon for the same amino acid as described below. A specific preferred example of the gene of the present invention is a DNA chain in which the amino acid sequence of a protein having F3 ′, 5 ′ H activity is substantially that shown in SEQ ID NO: 1 or any fragment of this DNA chain. As used herein, "the amino acid sequence substantially shown in SEQ ID NO: 1" means that the encoded protein or polypeptide has F3 ', 5'H activity, as described in detail in the section (6) below. It means that some of the amino acids may have substitutions, deletions, additions and the like as long as they have the amino acid, and therefore the present invention D
The NA chain also includes the base sequence corresponding to this amino acid change. In order to express this DNA chain and produce the protein or polypeptide encoded by it, D corresponding to the amino acid sequence of the polypeptide is required.
In addition to the NA sequence (coding region), an expression control sequence (upstream promoter, etc.) is required. Therefore, the DNA chain of the present invention is a DNA containing this expression control sequence.
It also includes sequences. (ii) Preparation of flavonoid hydroxylase gene The flavonoid hydroxylase gene according to the present invention as described above is used to irradiate long-wavelength light (preferably red light) to seeds of plants having delphinidin synthesizing ability (eg, eggplant). Flavonoids 3 ', 5' after being cultivated in, then cultivated under irradiation with light containing short wavelength (preferably white light and ultraviolet light)
-Selectively inducing the expression of a gene (mRNA) encoding a protein having hydroxylase activity, and then isolating the mRNA gene from this plant, and complementing it with cD
It can be obtained by preparing NA. Also, according to the method of the present invention, an enzyme gene involved in flavonoid synthesis can be prepared more easily than before (details will be described later). Hereinafter, the present invention will be described in more detail with reference to specific examples.

(2) Method for inducing flavonoid pigment synthesis Flavonoid 3 ', 5'-hydroxylase (F3', 5 'H) is a membrane-bound protein, but it is inactive when solubilized in addition to its small content. Therefore, it has not been refined at all [Forkma
nn, G., Plant Breeding, 106, 1-26 (1991)]. As a matter of course, no antibody against F3 ', 5'H has been obtained, and there is no information about the amino acid sequence of this enzyme. Therefore, in order to obtain the F3 ', 5'H gene, it is essential to develop a system capable of cloning the gene without purifying the protein. Among such cloning methods, one of the most reliable methods applicable to higher plants is to use a material that induces delphinidin synthesis under certain conditions as a gene source, and to induce expression in parallel with the induction of delphinidin synthesis. It is a method of selecting a gene. This method is commonly referred to as differential hybridization, and several successful cases have been reported [Sambrook et al., "Molecular Cloning, second edit
ion ", pp. 10.38-10.43, Cold Spring Harbor Laborato
ry Press, New York (1989)]. Differential
In order to achieve successful hybridization, the target gene is not expressed in the tissue before induction, or the expression level is extremely lower than that after induction, in other words, the expression induction of the target gene is clearly controlled. It is important that you As a system for inducing flavonoid pigment synthesis including delphinidin, it is performed to compare young buds with mature buds under natural conditions. However, although the expression of flavonoid synthesis system genes certainly differs depending on the developmental stage of the bud, the difference ("level" of induction) is not so large, and screening by differential hybridization was considered difficult (Petunia). An example in the case of a flower is shown in FIG. 7). It is also conceivable to perform a differential screening between a tissue containing delphinidin and a tissue not containing delphinidin (eg petals and leaves), or a variety containing delphinidin (eg petunia) and a variety not containing delphinidin. It was virtually impossible to detect the target gene because the difference in gene expression patterns due to the difference or the difference in varieties was too large. Therefore, the inventors first searched for a system in which delphinidin synthesis was clearly induced. As a result, the seeds of eggplant ( Solanum melongena ) were cultivated under red light for about 2 weeks, and then white light and ultraviolet light were mixed. It was found that the flavonoids containing delphinidin can be induced very clearly by cultivating a plant (hereinafter referred to as white light) for 2 days or more. The change in the content of anthocyan (flavonoid pigment) for 5 days after white light irradiation was observed by the method of the inventors.
Is. It can be seen that the irradiation of white light increases the flavonoid content about 20 times. Induction of synthesis occurs very quickly, reaching half maximum at about 48 hours post irradiation.
Examination of changes in mRNA of a known gene during this period revealed that chalcone synthase (CH) located before and after the flavonoid hydroxylation reaction at the 3'and 5'positions in the flavonoid pigment synthesis system.
It was found that S) and DFR (see FIG. 2) mRNAs could not be detected before induction, and gradually increased from 7 hours after induction (FIG. 4). On the other hand, phenylalanine ammonia lyase (PAL) and 4-coumaric acid: CoA ligase (4-CL), which act further upstream of the flavonoid synthesis system and are included in the more general metabolic system phenylpropanoid synthesis system (Fig. The mRNA (see 2) was present before induction, although the amount increased by induction. This is a reaction in which the phenylpropanoid synthesis system is common not only to flavonoids but also to the synthesis systems of substances more essential for plant growth such as phytoalexin and lignin [Ishikura, "Plant Metabolism Physiology, pp. 162-168, Morikita Shuppan (1987)], so it is considered that they are expressed to some extent even under red light. Therefore, regarding the cinnamic acid 4-hydroxylase (C4H) gene, which exists between PAL and 4-CL and is enzymatically P-450 type hydroxylase, the PAL and 4-CL genes Similarly, clear induction is not required, and it is presumed that the expression level increases only to some extent. Genes that do not induce such strong induction are unlikely to be selected by the method of the present invention. In the process of transferring from the dark to the white light, which is commonly used in biochemical studies of plants, most of the induced genes are related to photosynthesis, and flavonoids are among them. Choosing something about is extremely inefficient. In the case of the method of the present invention, ribulose-1,5-2 which is a typical enzyme of the photosynthetic system
The amount of the phosphoric acid carboxylase / oxygenase small subunit mRNA was almost unchanged before and after the induction with white light (FIG. 4). That is, in the system using the eggplant seedlings of the present invention, a clone corresponding to the mRNA of the photosynthetic gene, which is present in a much larger amount than the flavonoid system, can be efficiently removed. Induction of flavonoid synthesis according to the present invention is not limited to eggplants, plants that accumulate flavonoid compounds in seedlings when cultivated under white light,
For example, it is also possible to use petunia or corn. Therefore, the induction system of the present invention can be utilized to efficiently clone various flavonoid synthesis system genes from these plants. In the case of eggplant, CHS and DFR gene mRNAs are specifically expressed in the hypocotyl in seedlings (FIG. 6), so it is desirable to extract and use only the hypocotyl. Selected material is F3
Whether or not it contains ', 5' H can be confirmed by measuring the enzyme activity of F3 ', 5' H. For example, the following method can be used. A dihydroquercetin (DHQ) sold by Serva is used to request a labeling service with tritium from New England Nucleer to obtain ³ H-DHQ. This is reacted with the membrane fraction of the extract of plant material, the product is extracted with ethyl acetate and concentrated under reduced pressure. This is developed by cellulose-based thin layer chromatography (developing solvent; chloroform: acetic acid: water = 10: 9: 1), and then a sensitizer is sprayed and analyzed by fluorography. The inventors have found that F3 ′, 5 ′ H activity, that is, the activity of converting DHQ into dihydromyrcetine, is detected in the buds of blue petunia and eggplant seedlings after white light irradiation by such a method. It was confirmed that this activity was not detected in eggplant seedlings before light induction.

(3) Preparation of cDNA library enriched with clones encoding genes involved in flavonoid synthesis Induction of RNA from induced eggplant hypocotyl and enrichment of mRNA in it should be carried out by a conventional method. You can
The synthesis of double-stranded cDNA is a well-established technique, for example
Stratagene, Pharmacia, Amersham, Invitrogen
A kit sold by a company may be used. For example, in the case of using Stratagene's kit, double-stranded cDNA is synthesized according to the kit's usage, attached with EcoRI adapter, and then cut with restriction enzyme XhoI. From the above, a 1 to 4 kB DNA fragment is separated by a known means such as agarose gel electrophoresis. Then, for example, λZA sold by Stratagene
After inserting into a lambda phage vector in which EcoRI / XhoI sites such as P II are easily cloned, in vitro packaging (for example, a kit such as Gigapack II Gold sold by Stratagene can be used) to prepare a cDNA library. make. The host E. coli used to infect the packaged phage can be any cell capable of propagating the phage. PLK for λZAP II
F'is preferred. The above-described method for preparing a library is an example, and known cDNA synthesis kits, adapters, vectors and the like can be appropriately selected. After preparing a cDNA library, phage clones were allowed to form plaques on host E. coli, and the DNA in the plaques was formed.
Are transferred to 2 or 3 nylon membranes according to a known method, hybridized with respective probes and compared with each other to select a target clone induced by white light. For example, mRNA is extracted from each eggplant before and after the induction with white light, the corresponding cDNA is synthesized and labeled with ³² P to obtain a probe. And it hybridizes strongly with the eggplant-derived cDNA before induction,
By selecting a plaque group that does not react with the eggplant-derived cDNA after induction, a clone corresponding to a gene induced by white light can be selected (differential screening). For more efficient screening, a cDNA probe derived from eggplant after induction is reacted with mRNA present in eggplant before induction, and hybridizes with mRNA in eggplant before induction to form a double-stranded chain. Previously removed the probe (subtract probe). The double-stranded probe is selectively removed by separation of single-stranded cDNA and cDNA-mRNA double-stranded by phosphate gradient hydroxy apatite column chromatography (for example, Bio-Rad),
Alternatively, pre-induction mRNA derived from eggplant was biotinylated in advance, avidin was further bound to biotin on the mRNA after hybridization, and the nucleic acid to which avidin was bound was extracted into the phenol layer, whereby unhybridized cDNA. This can be carried out by a method such as collecting only the aqueous layer (for example, a kit from Invitrogen can be used). Screening with the subtractive probe thus obtained and differential screening were performed on the same plate.
By performing in parallel to the copy nylon film,
Induced clones can be obtained with high accuracy (Fig. 5). When the clones obtained as described above are repeatedly screened twice or more using the same probe, the accuracy is dramatically improved, and a library subset rich in white light-inducible clones is obtained. When this subset was screened with known genes CHS and DFR, clones of both genes were
The frequency increased more than 10 times. That is, although 10,000 clones before enrichment were screened by CHS and DFR, only 2 and 0 clones could be selected respectively, whereas the enriched library resulted in 4 and 1 clones out of 1,000 clones, respectively. was gotten.

(4) Acquisition of clone encoding P-450 type enzyme In order to subclone the phage clone obtained as described above into a plasmid, generally, after purifying the phage DNA by a conventional method, CDNA inserted with the same restriction enzyme used for cloning double-stranded cDNA
A is cut out and subcloned into a plasmid (for example Bluescript) cut with the same enzymes. In the case of λZAP II, it is also possible to directly obtain the subcloned plasmid according to the instruction manual of the manufacturer. In addition, in order to easily obtain the inserted cDNA for the purpose of using it as a probe or for confirming the size, a primer corresponding to the vector DNA outside the cloning site, for example, a universal and reverse primer is used in combination with a polymerase.・ Chain reaction (PCR) may be performed. In this case, the PCR reaction conditions and the like may be in accordance with the operating instructions of the supplier (Cetus). The interrelationship of the obtained cDNAs is examined by cross-hybridization assay between clones or comparison of cleavage patterns with appropriate restriction enzymes, and cDNA clones considered to correspond to different mRNAs are selected. In order to investigate how the DNA fragments presumed to be derived from different genes are expressed in the plant, the hypocotyls of eggplants before and after induction or other plants such as petunia flowers can be used. Northern analysis may be performed using the mRNAs obtained from the above-mentioned cDNAs and these cDNAs as probes according to a conventional method.
The nucleotide sequences of the clones selected as described above are analyzed according to a conventional method, and the obtained nucleotide sequences or the amino acid sequences encoded thereby are compared with the sequences of P-450 type enzymes known in database analysis software such as GENETYX. As a result of comparison, the amino acid sequence deduced to be encoded by one of the obtained clones, pC152, has the above-mentioned P-450 type enzyme of avocado fruit [Bozak, KR et al., Proc. Na.
tl. Acad. Sci., 87, 3904-3908 (1990)] and chicken steroids 17 α-monooxygenase.
It showed significant homology with the 450 type enzyme (Fig. 8). However, by comparison with the amino acid sequences of those genes, pC15
Since it was found that 2 was missing the 5'side, cDNA
PCR was performed using the phage DNA of the library as a template and a part of the internal sequence of pC152 and the sequence on the vector as primers at both ends to obtain a DNA of about 400 bp. This D
The 3'-end sequence of NA and the 5'-end sequence of pC152 are completely identical,
An open reading frame consisting of 1,542 bases encoding a polypeptide of 513 amino acid residues was found across both. Therefore, it is considered that the plasmid pC152N obtained by ligating the both contains the entire structural gene of the protein considered to be the P-450 type enzyme. Such a full-length clone can also be obtained by rescreening the library itself using pC152 as a probe. The entire nucleotide sequence of pC152N is shown in SEQ ID NO: 2 in the Sequence Listing, and the amino acid sequence predicted therefrom is shown in SEQ ID NO: 1.

(5) Flavonoid 3 ', 5'-hydroxylase c
Identification of DNA The expression pattern of pC152N was examined as follows.
First, for mRNA obtained from eggplant seedlings, pC152N
Northern analysis was carried out by using as a probe (Fig. 6). pC15
Approximately 1,800 bases of mRNA that hybridizes with 2 was not detected in eggplant seedlings grown under red light but was detected for the first time after irradiation with white light, and in seedlings, it was localized in hypocotyls rather than cotyledons. Was shown to do. The white light inducibility and the hypocotyl localization are features common to the expression patterns of CHS and DFR, as described above in (1) (FIG. 4, FIG.
6). Next, when mRNA was obtained from buds or leaves of three stages of petunia blueflower varieties with different developmental stages and Northern analysis was performed, a band of approximately 2,000 bases hybridizing with pC152 was detected from the bud mRNA (FIG. 7). This indicates that there is a homologous gene in pC152 clone derived from eggplant also in petunia. However, the mRN of the gene homologous to pC152 present in this petunia
A was different in size from the eggplant pC152 mRNA, and it was estimated from the hybridization strength that the homology with A was moderate. The bud was the largest in the middle stage of the developmental stage, and its increase / decrease pattern was in good agreement with other flavonoid synthesis genes, CHS, DFR, and chalcone flavanone isomerase (CFI). No mRNA hybridizing with pC152N could be detected in petunia leaves, indicating that this mRNA is also localized in pigment-producing tissues in petunia. The above expression pattern strongly suggests that the protein encoded by this pC152N clone is involved in anthocyanidin synthesis. By the way, petunia has long been regarded as a genetic research material, and many mutant strains are known [Sink, KC, "Petunia", pp. 34-7.
6, Springer-Verlag, BerlinHeidelberg (1984)], in which many mutants of genes controlling flower color are collected. Therefore, mRNA was extracted from flower buds from a mutant strain lacking flavonoid hydroxylation ability and a wild strain having a blue flower, and Northern analysis was performed using pC152N as a probe. In the wild strain, a band of about 2,000 bases was detected as described above, but in the mutant strain lacking F3 ', 5'H, mRNA hybridizing to pC152 could not be detected. Among the enzymes involved in the anthocyanidin synthesis system, biochemically P-450 type enzymes are cinnamic acid 4-hydroxylase (C4H) and flavonoid 3'-.
Hydroxylase (F3'H) and F3 ', 5'H are three [Forkmann, G., Plant Breeding, 106, 1-26 (199).
1)]. Among them, C4H is an enzyme of the phenylpropanoid synthesis system at the upstream side. As described in (2), the gene group of the phenylpropanoid synthesis system is not cloned in the gene acquisition method of the present invention because the white light induction system using eggplant hypocotyls according to the present invention does not induce a clear expression. Unlikely to occur. Furthermore, the F3 ′, 5 ′ H-deficient mutant strain used in the Northern analysis experiment of the above-mentioned petunia mutant strain was C
Since 4H is a normal strain, the P-450 type enzyme encoded by pC152N is not C4H. In addition, this petunia mutant strain is known to be normal for F3'H. Therefore, from the above results, pC152N is considered to be a cDNA clone encoding F3 ', 5'H.

(6) Conclusion The above (2) to (5) are summarized as follows. The mRNA corresponding to pC152N is strongly induced in the eggplant hypocotyl by white light, but does not exist before induction. The pattern of this expression induction is CH of anthocyanidin synthesis system.
Same as S, CFI and DFR, but different from PAL and 4-CL in phenylpropanoid synthesis system. In the petunia bud, mRNA corresponding to pC152N and having a slightly higher molecular weight than that of eggplant was also detected. The expression of this mRNA was maximized in the middle stage of the bud development stage, similar to the expression pattern of anthocyanidin synthesis system genes. As described above, the expression pattern of the gene encoded by pC152N is more similar to the genes of the anthocyanidin synthesis system such as CHS and DFR than the genes of the phenylpropanoid synthesis system such as PAL and 4-CL. C4H
It is strongly suggested that it is a gene of anthocyanidin synthesis system. pC152N has a base sequence as shown in SEQ ID NO: 2, and the amino acid sequence deduced therefrom (SEQ ID NO: 1) is judged to be a typical P-450 type hydroxylase sequence. The activity of flavonoid F3 ', 5'H is not detected in eggplant hypocotyls grown under red light, but is detected only after white light induction. It is not known whether F3'H is present in the eggplant hypocotyl. C4H and F3'H are normal, but F3 ', 5'H is deficient in the bud mRNA of the petunia mutant strain.
No mRNA corresponding to pC152N was detected. this is
It strongly suggests that the gene encoded by pC152N is F3 ', 5'H. From the above, it is concluded that pC152N encodes the flavonoid 3 ′, 5′-hydroxylase, which is a key enzyme for synthesizing delphinidin.

In nature, a large number of Ps corresponding to various substrates
-450 type enzyme is present [Nebert et al., Ann.
Rev. Biochem., 56, 945-993 (1987)], or using these fragments or combining different P-450 type enzyme fragments to form a fusion protein, there is a possibility that the enzyme activity may be significantly changed. Has been done. For example, yeast P-
N-terminal side 2/3 of P-450 P1 which is a 450 type enzyme and P-450 LM4
It was shown that the fusion with the C-terminal 1/3 of P. elegans was much more active than either of them [Pompon et al., G
ene, 83, 15-24 (1989)]. In the oxidation reaction catalyzed by the P-450 type enzyme, electrons are often supplied from the P-450 reductase. In such a case, the P-450 type enzyme and the corresponding P-450 reductase are supplied. When fused, P-4
It is also known that the activity of type 50 enzyme is more than doubled [Shibata, et al., DNA Cell Biol., 9, 27-36 (199).
0)]. Therefore, D encoding F3 ', 5'H of the present invention
It will be apparent that any fragment of the NA chain can be used alone or as part of the DNA chain encoding the fusion protein. In addition, commonly found DNA
When a polypeptide that has a certain amino acid sequence is encoded, there are multiple genetic codes (codons) corresponding to one amino acid, so there are multiple DNA chains corresponding to one amino acid sequence (degenerate isomerism). body). It goes without saying that any genetic code can be used in the DNA chain encoding F3 ', 5'H of the present invention as long as the amino acid sequence of the polypeptide encoded by it does not change. Furthermore, even if a variant polypeptide in which various amino acid substitutions, deletions, additions, or insertions have been added to F3 ′, 5 ′ H or any fragment thereof, F3 ′, 5 ′ H
As long as the activity is retained, it can be used for the purpose of the present invention. In a preferred embodiment of the present invention, the term "substantially" when the encoded "the amino acid sequence of a protein having flavonoid 3 ', 5'-hydroxylase activity is substantially as shown in SEQ ID NO: 1" The present invention DN
It is meant that the A chain includes not only a DNA chain encoding a protein having F3 ′, 5 ′ H activity that occurs in nature but also a DNA chain encoding such a mutant hydroxylase polypeptide. In addition, in order to express a DNA chain or a fragment thereof to produce a protein or polypeptide encoded by the DNA chain, an expression control sequence is required in addition to the DNA sequence (coding region) corresponding to the amino acid sequence. Therefore, the DNA chain of the present invention also includes a DNA sequence containing such an expression control sequence. Of particular importance among these expression control sequences are the promoter sequence upstream of the coding region and the poly A addition signal sequence downstream. Promoter and poly A
The additional signal is known to function in plants, for example, cauliflower mosaic virus 35S promoter, nopaline synthase promoter, ribulose diphosphate carboxylase / oxygenase small subunit promoter, and nopaline synthase promoter. A poly A addition signal, a poly A addition signal of octopine synthase, and the like can be appropriately combined and used. When the obtained DNA sequence is cDNA, in order to express F3 ', 5'H in the host, the cDNA
It is necessary to ligate the above-mentioned expression control sequence upstream and downstream of. When the obtained DNA sequence is a genomic gene, if the DNA sequence contains an expression control sequence, it can be used as it is. As mentioned above, the present invention also relates to a recombinant vector containing the above-mentioned DNA chain or a fragment thereof. This recombinant vector is obtained by ligating the above-mentioned DNA chain or a fragment thereof to a vector, and known vectors such as plasmid, phage or cosmid can be used. By introducing this recombinant vector DNA into a host such as an appropriate plant (for example, rose, chrysanthemum, dianthus, tulip, etc.) or a microorganism (for example, yeast) cell, etc., F3 ′, 5 ′ H can be expressed in the host. Produced.

[0012]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Induction of flavonoid pigments Eggplant seeds (variety “Sinsahara eggplant” produced by Tohoku Seedling Co., Ltd.) were sown in vermiculite and passed through a red acrylic plate (Mayvan, thickness 1 mm) to obtain 300 lux red light. It was continuously irradiated and cultivated at 25 ° C. for 2 weeks. Next, 8000 lux white fluorescent lamp (Toshiba, 6W / m ² at seedling position) and ultraviolet light (Toshiba, FL-20SE, FL-20SBLB; 0.5W / m ²⁾
) [This condition is called white light in the present invention] was irradiated for 5 days. The distance from the plant to the light source was 15 to 25 cm. After irradiation with white light, 0.2 g of seedlings were periodically extracted with 1% methanolic hydrochloric acid to measure the anthocyan content, and the absorbance at 540 nm was measured. When the dye amount was maximized on the 5th day, it had already reached half of the maximum value on the 2nd day (Fig. 3). In parallel, the method of Chargwin [Chirgwin, J.
M. et al., Biochemistry, 18, 5294-5299 (1979)], mRNA was extracted from each part of the seedling, and after agarose electrophoresis under denaturation with formaldehyde, a nylon membrane (Hybond N; Amersham) was used. Transcription according to the instructions of Petunia petal chalcone synthase (CH
S) and dihydroflavonol-4-reductase (D
FR), and the entire structural gene of ribulose-1,5-2 dicarboxyl carboxylase / oxygenase small subunit of tobacco leaves, each of which has a known nucleotide sequence [Koes et.
al., Gene, 81, 245-257 (1989); Beld et al., Plant
Mol. Biol., 13, 491-502 (1989) ； Mazur et al., Nu
cl. Acids Res., 13, 2373-2386 (1985)]
It was cloned by the CR method. PCR conditions are Cetus
I followed the instructions in the company's instructions. Using each of the obtained genes as a probe, hybridization was performed on Northern blots of eggplant mRNA. Hybridization is 5 x SSPE [1 x SSPE is 10 mM phosphate buffer (pH
7.0), 1 mM EDTA, 0.15M NaCl], 0.2% SDS, 100 μ
It was carried out at 60 ° C for 16 hours in a liquid consisting of g / ml herring sperm DNA and 100 µg / ml yeast RNA, and then the membrane was transferred to 2 x SSC [1 x
SSC was washed in a 0.15 M NaCl, 15 mM sodium citrate solution at room temperature for 15 minutes x 2 times, and then in 0.1 x SSC solution at 60 ° C for 15 minutes x 2 times. The result of autoradiography is shown in FIG.

Example 2 Preparation of cDNA Library Concentrated with Genes Related to Flavonoid Synthesis According to Example 1, eggplant seedlings which had been cultivated under red light for 2 weeks and then induced with white light for 72 hours were used as materials. Used as. MRNA was prepared in the same manner as in Example 1, and Stra
Double-stranded c using a cDNA synthesis kit (λZAP II cloning kit) from tagene, according to the manufacturer's instructions.
DNA was synthesized and cloned into the EcoRI / XhoI site of λZAP II to prepare a cDNA library. On the other hand, mRNA was extracted from eggplant seedlings before and after white light induction, 100 ng of each mRNA was annealed with 60 times amount of oligo (dT) primer, and then 20 μM dCTP, 300 μCi ³² P-dC.
TP (3000 Ci / mmol; Amersham), 200 μM each dGTP / d
ATP / dTTP, 20 mM DTT, 50 mM Tris-HCl (pH 7.5), 75 mM K
Cl, 3 mM MgCl _{2 in} the presence of 1 μl of MMLV reverse transcriptase (10
0 U / μl; BRL) at 37 ° C. for 1 hour to prepare a ³² P-labeled cDNA probe for each mRNA, which was used for differential screening. In addition, the cD after white light induction created in this way
A subtracted probe was prepared from NA as follows. Using a kit from Invitrogen, mRNA from eggplant seedlings before white light induction was biotinylated according to the instruction manual. ³² P-labeled post-induction cDNA and 20 volumes of biotinylated pre-induction mRNA were added to 20 μl of 0.2% SD.
S, 50 mM Hepes (pH 7.6), 0.5 M NaCl, 2 mM EDTA, 50
Solution containing% (V / V) formamide [Crowell, DN et a
L., Proc. Natl. Acad. Sci., 87, 8815-8819 (1990)]
Hybridization was carried out at 40 ° C. for 40 hours. After the reaction was completed, add 80 μl of 50 mM Hepes (pH7.6), 0.5M NaCl, 2 mM to this solution.
A mixed solution of EDTA and 10 μg streptavidin (Vector) was added, reacted at room temperature for 10 minutes, extracted with 100 μl of a phenol / chloroform mixed solution, and the upper layer was recovered and used as a subtract probe. In accordance with the standard method, 3 Phage libraries plated on an agar medium were used to form 3 Hybond
Transferred to N nylon membrane. These membranes were screened with the above-mentioned cDNA probe or subtract probe before or after white light induction, and positive clones were selected. Hybridization is 5 x SSPE, 0.2%
SDS, 100 μg / ml herring sperm DNA, 100 μg / ml yeast RNA in a liquid at 65 ° C for 16 hours, then membrane
It was washed in the × SSC solution at room temperature for 15 minutes × 2 times, and then in the 0.1 × SSC solution at 65 ° C for 15 minutes × 2 times. An example of the result of this experiment is shown in FIG. It can be seen that the ratio of signal noise is much higher by using the subtract probe as compared with the case of using the cDNA probe before induction or directly after induction (differential screening).

Example 3 Obtainment of clones encoding P-450 type enzyme and structural analysis 30,000 clones were repeatedly screened by the method of Example 2 to obtain a library subset consisting of 70 white light-inducible clones. Obtained. From the selected phage clone, a cDNA fragment was subcloned into a plasmid Bluescript according to the instruction of λZAP II. The duplication between the obtained clones was checked by restriction enzyme mapping and dot hybridization, 10 clones were randomly extracted from the clones considered to be independent, and the nucleotide sequence was analyzed according to the Sanger method. The amino acid sequence encoded in the open reading frame found on the obtained nucleotide sequence was used as a known protein amino acid sequence database (NBRF-PDB, Rel. 27) using the GENETYX database analysis software (Software Development Co.). By comparison, one of the 10 clones, a polypeptide of 425 amino acids encoded in the open reading frame on pC152, showed homology to 20 P-450 type enzymes or fragments thereof. All known proteins showing a certain degree of homology with the polypeptide encoded by this clone were P-450 type enzymes or fragments thereof. One of them, the chicken steroid 17 α-monooxygenase, is compared with the polypeptide encoded by pC152 in FIG.
Shown in. Further, this polypeptide is a P-450 type enzyme of the avocado fruit described above [Bozak, KR et al., Proc. Nat.
l. Acad. Sci., 87, 3904-3908 (1990)] also showed significant homology. As is clear from FIG. 8, the open reading frame found on pC152 was presumed to be incomplete on the 5 ′ side. Therefore, a part of the base sequence of pC152 (base number of the base sequence shown in SEQ ID NO: 2) was found. 378 to 397
(20 bases of DNA) and a synthetic oligo DNA complementary to T3 primer (Promega) for Bluescript sequence.
A CR was done. This synthetic primer sequence is located at about 100 bp from the 5'end of the cDNA fragment on pC152 and contains a recognition sequence for the restriction enzyme NdeI therein. 0.2 μM each primer, 100 ng phage library DNA
, 10X buffer (included in the Cetus kit), 0.2 mM 4dNTPs, 2.5U Ampli Taq polymerase (C
etus) and 100 μl of mineral oil in a reaction solution at 95 ° C (1 minute), 55 ° C (1 minute) and 72 ° C.
The reaction cycle of (2 minutes) was performed 25 times. The product was extracted with an equal amount of chloroform, the aqueous layer was recovered, and ethanol precipitation was performed. The obtained DNA of about 400 bp was digested with the restriction enzyme BamHI.
After cleaving with (the recognition sequence is on the vector Bluescript) and NdeI, it was cloned between the BamHI site and the NdeI site of pC152, which was similarly cleaved. When the nucleotide sequence of the PCR fragment portion was analyzed according to a conventional method and the overlapping portion with the cDNA fragment on pC152 was compared, both were completely in agreement. In addition, in the sequence derived from the PCR fragment, a translation initiation codon (ATG) in which the open reading frame on pC152 was aligned with the reading frame was found. Therefore, it was concluded that the plasmid pC152N thus obtained contained the full-length open reading frame. pCN1
The entire nucleotide sequence of the 1696 bp cDNA fragment on 52N and the entire amino acid sequence of the 513 amino acid polypeptide encoded by it are shown in SEQ ID NOs: 2 and 1, respectively. The chicken steroid 17 α-monooxygenase consists of 503 amino acids, which is in good agreement with the size of the protein encoded by pC152N.

Example 4 Analysis of expression pattern of gene encoded by pC152N The expression pattern of the protein encoded by pC152N was examined as follows. Example 1 from eggplant seedling hypocotyls and avocado fruits before and after white light induction
MRNA was extracted in the same manner as above, and the 3'side fragment of pC152N (Ap
Northern hybridization was carried out using the entire 3'side of the aI site (base number 992 of SEQ ID NO: 2; hereinafter referred to as pC152-ApaI) as a probe. Hybridization is 5 × SSPE, 0.2% SDS, 100 μg / ml herring sperm D
NA, 100 μg / ml yeast RNA at 65 ℃, 16
The membrane for 2 hours at room temperature for 15 minutes x 2
Washed once and then twice in 0.1 × SSC solution at 65 ° C. for 15 minutes × 2 (Northern hybridization in the following examples was carried out under these conditions except that the hybridization and washing temperatures were appropriately changed) .. The results are shown in Figure 6a.
A strong signal of 1.8 kb was obtained only in the eggplant hypocotyls subjected to white light induction. Next, mRNA was similarly extracted from hypocotyls and cotyledons of eggplant seedlings after white light induction, and pC152-ApaI or the petunia-derived CHS and DF obtained in Example 1 were extracted.
Northern hybridization was performed using the R structural gene as a probe. The hybridization conditions were the same as described above when pC152-ApaI was used as the probe, and when the gene of petunia was used as the probe, the temperature was lowered to 60 ° C. The results are shown in Figure 6b. No matter which probe was used, a signal was obtained only in the hypocotyl, and the protein encoded by pC152N, CHS and DFR were obtained.
It was found that the expression pattern of the eggplants in eggplant seedlings was very similar. Furthermore, petunia varieties with blue flowers (“Blue Star”; Sakata Seed) were grown and buds were obtained over time. The development stage of buds is buds with a bud length of about 20% of the maximum length in the initial stage, 40% in the middle stage, and 80%
% Was the latter term. Each stage bud,
MRNA was extracted from the young leaves and young leaves in the same manner as in Example 1 to obtain pC152-ApaI or petunia petal-derived CH.
S, DFR (Example 1) and chalcone flavanone isomerase (CFI) structural gene (known nucleotide sequence in the same manner as in Example 1 [Tunen et al., EMBO J., 7, 1257-126.
3 (1988)] and cloned by PCR) as a probe. The hybridization temperature is pC152-Ap
The temperature was 60 ° C when aI was used as the probe, and 65 ° C when the gene of petunia was used as the probe. The results are shown in Fig. 7. When pC152-ApaI was used as a probe, a signal of about 2 kb was obtained in the buds of blue flower petunia. This 2kb
The expression level of mRNA varied depending on the developmental stage of the bud, with the highest expression level in the middle stage, but the pattern of this change was consistent with the changes in the expression levels of CHS, DFR, and CFI. Also, this 2 kb mRNA was not detected in petunia leaves, as in DFR. These results are
A gene homologous to the gene encoded by pC152N is present in blue flower petunia, and its expression pattern is CHS, DF.
It shows that it is very similar to the R and CFI genes. From the change in signal intensity when the hybridization temperature was changed, it was estimated that the homology between the gene hybridizing with pC152N of petunia and the gene encoded by pC152N was moderate. Finally, the wild strain V6 of Petunia and mutants R3 and W39 [These were given by Professor JNMMol of Vriji University in the Netherlands. ] And the variety "blue star" used in the above experiments (Sakata Seeds)
MRNA was extracted from the buds and the eggplant hypocotyls subjected to white light induction as a control in the same manner as in Example 1, and Northern hybridization was carried out at 60 ° C. using pC152-ApaI as a probe. Both R3 and W39 strains are mutant strains in which C4H and F3′H are normal but F3 ′, 5′H is deleted. As a result, a 1.8 kb or 2 kb signal was detected in V6 strains of eggplant hypocotyl and petunia and "blue star" (both producing delphinidin pigments) as in the above experiments. No signal hybridizing with pC152N was detected in the mutant strains R3 and W39 lacking 5,5'H. This result indicates that the gene encoded by pC152N is the flavonoid 3 ', 5'-hydroxylase gene.

[0016]

INDUSTRIAL APPLICABILITY The present invention provides a DNA chain encoding a flavonoid 3 ′, 5′-hydroxylase, which is a key enzyme in the biosynthesis of delphinidin, which is a major blue pigment of plant petals. Plants without delphinidin, such as roses, chrysanthemums, dianthus and tulips, can synthesize cyanidin and pelargonidin, but flavonoids 3 ', 5'.
-Definidine cannot be synthesized due to lack of hydroxylase. Therefore, when the DNA strand of the present invention is introduced into a target plant according to a known method, it is greatly useful for the variety of colors such as flower color, especially for creating a bluish color.

[0017]

[Sequence listing] SEQ ID NO: 1 Sequence length: 513 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin: Organism: Eggplant (Solanum melongena) Strain name: Shinsago Dorahara Met Val Ile Leu Pro Ser Glu Leu Ile Gly Ala Thr Ile Ile Tyr Ile 1 5 10 15 Ile Val Tyr Ile Ile Ile Gln Lys Leu Ile Ala Thr Gly Ser Trp Arg 20 25 30 Arg Arg Arg Leu Pro Pro Gly Pro Glu Gly Trp Pro Val Ile Gly Ala 35 40 45 Leu Pro Leu Leu Gly Gly Met Pro His Val Ala Leu Ala Lys Met Ala 50 55 60 Lys Lys Tyr Gly Pro Ile Met Tyr Leu Lys Val Gly Thr Cys Gly Met 65 70 75 80 Val Val Ala Ser Thr Pro Asn Ala Ala Lys Ala Phe Leu Lys Thr Leu 85 90 95 Asp Ile Asn Phe Ser Asn Arg Pro Pro Asn Ala Gly Ala Thr His Met 100 105 110 Ala Tyr Asn Ala Gln Asp Met Val Phe Ala Pro Tyr Gly Pro Arg Trp 115 120 125 Lys Leu Leu Arg Lys Leu Ser Asn Leu His Met Leu Gly Gly Lys Ala 130 135 140 Leu Glu Asn Trp Ala Asn Val Arg Ala Asn Glu Leu Gly His Met Leu 145 150 155 160 Lys Ser Met Phe Asp Ala Ser His Val Gly Glu Arg Ile Val Val Ala 165 170 175 Asp Met Leu Thr Phe Ala Met Ala Asn Met Ile Gly Gln Val Met Leu 180 185 190 Ser Lys Arg Val Phe Val Glu Lys Gly Lys Glu Val Asn Glu Phe Lys 195 200 205 Asn Met Val Val Glu Leu Met Thr Val Ala Gly Tyr Phe Asn Ile Gly 210 215 220 Asp Phe Ile Pro Gln Ile Ala Trp Met Asp Leu Gln Gly Ile Glu Lys 225 230 235 240 Gly Met Lys Lys Leu His Lys Lys Phe Asp Asp Leu Leu Thru Met 245 250 255 Phe Glu Glu His Glu Ala Thr Ser Asn Glu Arg Lys Gly Lys Pro Asp 260 265 270 Phe Leu Asp Phe Ile Met Ala Asn Arg Asp Asn Ser Glu Gly Glu Arg 275 280 285 Leu Ser Ile Thr Asn Ile Lys Ala Leu Leu Leu Asn Leu Phe Thr Ala 290 295 300 Gly Thr Asp Thr Ser Ser Ser Val Ile Glu Trp Ala Leu Thr Glu Met 305 310 315 320 Met Lys Asn Pro Thr Ile Phe Lys Lys Ala Gln Gln Glu Met Asp Gln 325 330 335 Ile Ile Gly Lys Asn Arg Arg Phe Ile Glu Ser Asp Ile Pro Asn Leu 340 345 350 Pro Tyr Leu Arg Ala Ile Cys Lys Glu Ala Phe Arg Lys His Pro Ser 355 360 365 Thr Pro Leu Asn Leu ProArg Val Ser Ser Asp Ala Cys Thr Ile Asp 370 375 380 Gly Tyr Tyr Ile Pro Lys Asn Thr Arg Leu Ser Val Asn Ile Trp Ala 385 390 395 400 Ile Gly Arg Asp Pro Asp Val Trp Glu Asn Pro Leu Glu Phe Ile Pro 405 410 415 Glu Arg Phe Leu Ser Glu Lys Asn Ala Lys Ile Glu His Arg Gly Asn 420 425 430 Asp Phe Glu Leu Ile Pro Phe Gly Ala Gly Arg Arg Ile Cys Ala Gly 435 440 445 Thr Arg Met Gly Ile Val Met Val Glu Tyr Ile Leu Gly Thr Leu Ile 450 455 460 His Ser Phe Asp Trp Lys Leu Pro Asn Asp Val Val Asp Ile Asn Met 465 470 475 480 Glu Glu Thr Phe Gly Leu Ala Leu Gln Lys Ala Val Pro Leu Glu Ala 485 490 495 Ile Val Thr Pro Arg Leu Ser Phe Asp Ile Tyr Gln Ser Ser Glu Pro 500 505 510 Phe

SEQ ID NO: 2 Sequence length: 1696 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin: Organism name: Solanum melongena Strain name: Nakoto Sugihara Eggplant Sequence features: Characteristic symbol: CDS Location: 49..1590 Method of determining features: P sequence ATAAACTTTA TATTATAATT TATACATTTT TGATATTATT ATATTATCAT GGTTATACTT 60 CCTAGTGAGC TGATTGGAGC TGATTGGAGC ACATATTATA TATATCATAG TATATATTAT TATTCA TATATCATAG TATATATTAT TATTCA GTGATCGGAG CACTACCACT TTTAGGTGGC ATGCCACATG TTGCACTTGC AAAAATGGCC 240 AAAAAATATG GACCTATTAT GTATTTAAAA GTTGGCACTT GTGGGATGGT TGTTGCTTCA 300 ACTCCTAATG CTGCAAAAGC TTTCTTGAAA ACACTTGATA TTAATTTCTC CAATCGTCCA 360 CCTAATGCAG GTGCCACACA TATGGCCTAT AATGCCCAAG ACATGGTTTT TGCACCCTAT 420 GGACCTCGTT GGAAGTTGCT AAGGAAATTG AGCAACTTAC ACATGCTAGG TGGAAAAGCC 480 TTAGAAAATT GGGCCAATGT CCGGGCCAAT GAGCTAGGTC ACATGCTAAA ATCGATGTTC 540 GACGCGAGCC ATGTGGGT GA ACGCATAGTC GTGGCCGATA TGTTGACGTT CGCGATGGCA 600 AACATGATAG GTCAAGTGAT GTTAAGCAAG AGAGTGTTCG TCGAAAAAGG TAAAGAGGTC 660 AACGAATTCA AAAATATGGT CGTGGAACTG ATGACAGTTG CGGGGTACTT TAATATTGGC 720 GATTTTATTC CTCAAATAGC TTGGATGGAT TTACAAGGAA TTGAAAAGGG GATGAAAAAA 780 TTGCACAAAA AATTTGATGA TTTATTGACA AAAATGTTTG AGGAGCATGA GGCAACTAGC 840 AATGAAAGAA AAGGGAAACC TGATTTTCTT GATTTTATTA TGGCAAATAG GGATAATTCT 900 GAAGGAGAAA GGCTTAGTAT AACAAATATC AAAGCACTTT TACTGAATTT GTTCACAGCT 960 GGTACAGACA CCTCATCTAG CGTGATAGAA TGGGCCCTCA CAGAAATGAT GAAAAATCCC 1020 ACAATTTTCA AAAAAGCCCA ACAAGAAATG GACCAAATAA TTGGCAAAAA TAGACGTTTT 1080 ATTGAATCTG ATATCCCAAA TCTCCCTTAT TTACGTGCAA TTTGCAAAGA AGCATTTCGA 1140 AAACACCCTT CAACGCCACT AAATCTTCCT AGGGTGTCAA GTGATGCGTG CACGATCGAT 1200 GGTTACTATA TACCAAAAAA CACTAGGCTC AGCGTCAATA TATGGGCGAT TGGACGAGAC 1260 CCTGACGTGT GGGAGAATCC TCTCGAATTC ATTCCCGAGA GGTTTTTGAG TGAGAAAAAT 1320 GCAAAGATTG AGCATCGAGG AAATGATTTT GAATTGATTC CATTTGGTGC AGGACGAAGA 1380 ATTTGTGCCG GCACAAGGAT GGGAATAGT G ATGGTTGAGT ATATATTGGG AACTTTGATA 1440 CATTCATTTG ATTGGAAATT ACCAAATGAT GTTGTTGATA TAAATATGGA GGAAACTTTT 1500 GGATTAGCTT TGCAAAAAGC TGTCCCTCTT GAAGCTATAG TTACTCCAAG GCTATCTTTC 1560 GATATTTATC AGTCTAGCGA GCCTTTCTGA TCATCTACAT TGTAACGTAT TTGTTGAAGA 1620 CTTTAACAAA AGAAAACTCG CTTTATATAT GAAAAACTGA TAATGTCATA TCTCGGAGGA 1680 TTTTATTTTG TAAAAA 1696

[Brief description of drawings]

FIG. 1 is a view showing a skeleton of flavonoid.

FIG. 2 is a schematic diagram of reactions of a phenylpropanoid and flavonoid synthesis system.

FIG. 3 is an explanatory diagram showing changes over time in anthocyanin content in eggplant seedlings after irradiation with white light. It is expressed as a relative value with the maximum value set to 100.

FIG. 4 mRN of CHS, DFR, and ribulose-1,5-2 diphosphate carboxylase oxygenase small subunit (RuBisCO) in eggplant seedlings after white light irradiation.
It is an electrophoretic photograph which investigated the time-dependent change of the amount of A by Northern hybridization.

FIG. 5 is a photograph showing an example of screening for a clone encoding a gene whose expression is induced in eggplant hypocotyls by white light. MRNA from tissues before and after induction
Fig. 2 shows the results of differential screening using a cDNA probe for cDNA, and screening of a library using a probe in which cDNA for mRNA after induction was subtracted with a large excess of mRNA before induction. DNA was transferred from one plate to three filters and hybridized with each probe. The arrowhead indicates the same clone.

FIG. 6 (a) is an electrophoretic photograph showing the results of Northern analysis of mRNA derived from eggplant hypocotyls and avocado fruits before and after light induction using pC152N as a probe, and (b) and (c) of FIG. M taken by dividing the seedling into hypocotyl and cotyledon
4 is an electrophoretic photograph showing the results of Northern analysis of RNA using CHS, DFR, and pC152N as probes.

FIG. 7: mRNA obtained over time from buds of Aoba petunia cultivars using CHS, CFI, DFR and pC152N as probes, and for some probes mRNA obtained from induced eggplant hypocotyls and petunia leaves. 3 is an electrophoretic photograph showing the results of Northern analysis for

FIG. 8 is a diagram comparing the amino acid sequence of the polypeptide encoded by the open reading frame on pC152 with the amino acid sequence of chicken steroid 17 α-monooxygenase (SMO).

[Explanation of symbols]

 PAL: phenylalanine ammonia lyase C4H: cinnamic acid 4-hydroxylase 4-CL: 4-coumaric acid: CoA ligase CHS: chalcone synthase CFI: chalcone flavanone isomerase F3'H: flavonoid 3'-hydroxylase F3 ', 5'H: flavonoid 3 ', 5'-hydroxylase DFR: dihydroflavonol-4-reductase (these are related to FIG. 2) *: the matched amino acids :: similar amino acids (these are related to FIG. 8)

Claims (4)

[Claims] 1. A DNA chain encoding a protein having flavonoid 3 ′, 5′-hydroxylase activity or DN thereof.
Any fragment of the A chain. 2. The DNA chain or any fragment thereof according to claim 1, wherein the protein having flavonoid 3 ′, 5′-hydroxylase activity is a protein derived from a plant belonging to the genus Solanum. 3. The DNA chain or any fragment thereof according to claim 1, wherein the amino acid sequence of the protein having flavonoid 3 ′, 5′-hydroxylase activity is substantially as shown in SEQ ID NO: 1. 4. The DNA according to any one of claims 1 to 3.
A recombinant vector containing a chain or any fragment thereof.