Genes encoding proteins regulating the pH of vacuoles Number:6,803,500 from the United States Patent and Trademark Office (PTO) owispatent There is provided a gene encoding a protein that has an activity of regulating the pH of vacuoles, for example a gene derived from morning glory encoding a protein that has the amino acid sequence as set forth in SEQ ID NO: 2. By introducing this gene into a plant, the flower color can be regulated via the control of the pH of vacuoles.<H regulating, vacuoles, Genes, encoding, , 6803500.html, Patent, pto, 6803500

Title: Genes encoding proteins regulating the pH of vacuoles

Abstract: There is provided a gene encoding a protein that has an activity of regulating the pH of vacuoles, for example a gene derived from morning glory encoding a protein that has the amino acid sequence as set forth in SEQ ID NO: 2. By introducing this gene into a plant, the flower color can be regulated via the control of the pH of vacuoles.

Patent Number: 6,803,500 Issued on 10/12/2004 to Iida, et al.

Inventors: Iida; Shigeru (Okazaki, JP); Tanaka; Sachiko (Okazaki, JP); Inagaki; Yoshishige (Okazaki, JP)
Assignee: Suntory Limited (Osaka, JP)

Appl. No.: 09/830,123

Filed: April 24, 2001

PCT Filed: August 24, 1999

PCT No.: PCT/JP00/05722

PCT Pub. No.: WO01/14560

PCT Pub. Date: March 01, 2001

Foreign Application Priority Data


Aug 24, 1999 [JP]			11-236800

Current U.S. Class: 800/282 ; 435/252.3; 435/320.1; 435/419; 435/468; 536/23.6; 800/298; 800/323

Field of Search: 424/93.2 435/320.1,419,468,252.3 536/23.6 800/282,298,323

References Cited [Referenced By]

U.S. Patent Documents


5910627	June 1999	Chuck et al.

Foreign Patent Documents


94/23561	Oct., 1994	WO

Other References

Rhoads et al. Regulation of the cyanide-resistant alternative oxidase of plant mitochondria. J. Biol. Chem., Nov. 1998, vol. 273, No. 46, pp. 30750-30756.* .
A. Fukuda et al, "Molecular cloning and expression of the Na +/H + exchanger gene in Oryza sativa" Biochim. Biophys. Acta, vol. 1446, pp. 149-155, Jul. 1999. .
R. Gaxiola et al, "The Arabidopsis thaliana proton transporters, AtNhxL and Avpl, can function in cation detoxification in yeast", Proc. Natl. Acad. Sci. USA, vol. 96, pp. 1480-1485, Feb. 1999. .
Y. Lu et al, "AtMRPI gene of Arabidopsis encodes a glutathione S-conjugate pump: Isolation and functional definition of a plant ATP-binding cassette transporter gene", Proc. Natl. Acad. Sci. USA, vol. 94, pp. 8243-8248, Jul. 1997. .
K. Marrs et al, "A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2", Nature, vol. 375, No. 6530, pp. 397-400, Jun. 1995. .
S. Tanaka et al, "Colour-enhancing protein in blue petals", Nature, vol. 407, p. 581, Oct. 2000..

Primary Examiner: Fox; David T.
Assistant Examiner: Collins; Cynthia
Attorney, Agent or Firm: Burns, Doane, Swecker & Mathis, L.L.P.

Claims

What is claimed is:

1. An isolated nucleic acid encoding a protein that has the amino acid sequence as set forth in SEQ ID NO: 2 and that has an activity of regulating the pH of vacuoles in plant cells.

2. A vector comprising the nucleic acid sequence according to claim 1.

3. A host cell transformed with the vector according to claim 2.

4. A transgenic plant in which the nucleic acid sequence according to claim 1 has been introduced or a progeny of said plant in which said nucleic acid sequence has been introduced in said progeny and in which the pH of vacuoles in the plant cells are regulated and flower color is altered, or a tissue thereof.

5. A cut flower of the plant according to claim 4 or a progeny of said plant in which said nucleic acid sequence has been introduced in said progeny and in which the pH of vacuoles in the plant cells are regulated and flower color is altered.

6. A method of regulating the pH of vacuoles comprising introducing the nucleic acid sequence according to claim 1 into a plant or plant cells and then allowing said nucleic acid sequence to be expressed in said plant or plant cells.

7. A method of controlling the flower color of a plant comprising introducing the nucleic acid sequence according to claim 1 into a plant or plant cells and then allowing said nucleic acid sequence to be expressed in said plant or plant cells.

Description

TECHNICAL FIELD

The present invention relates to genes encoding proteins that regulate the pH of vacuoles, and the uses thereof.

BACKGROUND

In the flower industry, the development of novel or varied cultivars of flowering plants is important, and flower color is one of the most important traits of flowers. Although cultivars of various colors have been bred using conventional breeding by crossing, it is rare that a single plant species has cultivars of all colors. Thus, there is a need for the development of cultivars having a variety of colors.

The main components of flower color are a group of flavonoid compounds termed anthocyanins. It is known that a variety of anthocyanins occur in plants, and the structure of many of them have already been determined. The color of anthocyanins depends partly on their structures. Progress has been made in the study on the enzymes and genes involved in the biosynthesis of anthocyahins, and in some studies molecular biological techniques and gene introductions into plants were used to change the structure of anthocyanins, leading to changes in the color of flowers (Holton and Cornish, Plant Cell, 7:1071 (1995); Tanaka et al., Plant Cell Physiol. 39:1119 (1998)). The color of anthocyanins also depends on the pH of the aqueous solution, and the same anthocyanin may appear blue when the pH of the aqueous solution is neutral to weakly alkaline (Saito and Honda, Genda Kadaku (Chemistry Today), May 1998, pp. 25).

It is also known that since anthocyanins are present in the vacuole of the cell, the pH of vacuoles has a great impact on the color of flowers (Holton and Cornish, Plant Cell, 7 (1995); Mol et al., Trends Plant Sci. 3:212 (1998)). For example, in morning glory (Ipomea tricolor), it is known that the reason why red-purple buds bloom into blue flowers is that the pH of vacuoles in petal epithelium rises from 6.6 to 7.7 (Yoshida et al., Nature 373:291 (1995)).

It is thought that the vacuole of plant cells is regulated by vacuolar proton-transporting ATPase and vacuolar proton-transporting pyrophosphatase (Leigh et al., The Plant vacuole (1997), Academic Press), but the mechanism of how these proton pumps are involved in the color of flowers has not been elucidated. It was also known that a sodium ion-proton antiporter (hereinafter referred to as Na.sup.+ --H.sup.+ antiporter) exits in plant vacuoles and that the Na.sup.+ --H.sup.+ antiporter transports sodium ions into vacuoles, depending on the proton concentration gradient between the outside and the inside of vacuoles, whereupon protons are transported outside of vacuoles resulting a reduced proton concentration gradient.

Furthermore, the Na.sup.+ --H.sup.+ antiporter is thought to be a protein with a molecular weight of about 170,000. However, there are many unknown factors involved in the regulation of pH of vacuoles, and the mechanism of regulating the pH of vacuoles, in particular the petal vacuoles, is uncertain (Leigh et al., The Plant Vacuole (1997), Academic Press). The pH of plant vacuoles has never been artificially raised, nor have any industrially useful traits been obtained, and its association with flower color is unknown.

It is known that the Na.sup.+ --H.sup.+ antiporter gene, with a molecular weight of about 70,000, has been cloned from Arabidopsis, and a yeast into which this gene was introduced has acquired salt tolerance (Gaxiola et al., Proc. Natl. Acad. Sci. USA 96:1480-1485 (1999)), but it is not known how this antiporter regulates the pH of vacuoles in plant cells or how it is associated with flower color.

On the other hand, in petunias, seven loci are known to be involved in the pH regulation of petal vacuoles, and it has been proposed that the pH of petal vacuoles increases when one of them turns homozygously recessive (van Houwelingen et al., Plant J. 13:39 (1998); Mol et al., Trends Plant Sci. 3:212 (1998)). One of them, Ph6, has already been cloned and was found to be a kind of transcription regulating factor (Chuck et al., Plant Cell 5:371 (1993)), but the actual biochemical mechanism involved in the pH regulation of vacuoles is unknown.

In morning glory (Ipomea nil), the analysis of mutants revealed that a number of loci are associated with the color and shape of leaves and flowers and that 19 of them are highly mutable (Iida et al., Shokubutsu Saibo Kogaku Series (Plant Cell Engineering Series) 5 (1996) pp. 132, Shujunsha; Iida et al., Annal. New York Acad. Sci. (1999) pp. 870). Among them, the one locus defined by the recessive mutation that results in purple flowers instead of blue flowers is termed the Purple locus (T. Hagiwara, The genetics of flower colours in Phrarbitis nil. J. Coll. Agr. Imp. Univ. Tokyo 51:241-262 (1931); Y. Imai, Analysis of flower colour in Pharbitis nil. J. Genet. 24:203-224 (1931)), and one allele of mutable mutation that results in flowers that produce blue sectors in purple petals was termed purple-mutable (pr-m) (Imai, J. Coll. Agric. Imp. Univ. Tokyo 12:479 (1934)). The gene derived from the Purple locus is termed Purple gene.

The blue portion is believed to be derived from somatic reverse mutation from the recessive purple, and germ cell revertants can also be separated. An allele produced from the reverse mutation of these revertants are termed herein Purple-revertant (Pr-r). Such a classical method of genetic analysis had been performed on this Purple gene, but the identity of the Purple gene and its association etc. with the pH regulation of petal vacuoles were totally unknown.

It is believed that if the pH of vacuoles could be modified, for example if the pH of vacuoles could be raised, flower color could be turned blue. Representative plant species that lack blue colors include roses, chrysanthemums, carnations, gerberas and the like, which are very important cut flowers. Though the importance of modifying pH of vacuoles has been recognized, the identities of proteins that regulate the pH of petal vacuoles are unknown and therefore the isolation of genes encoding them has been in great demand.

DISCLOSURE OF THE INVENTION

The present invention provides a gene of a protein that regulates the pH of vacuoles in plant cells, preferably a gene of a protein that transports protons in vacuoles, more preferably a Na.sup.+ --H.sup.+ antiporter gene. By introducing the gene of the present invention into a plant and allowing it to be expressed, flower color can be controlled and, preferably, can be turned blue.

Thus, the present invention provides a gene encoding a protein that regulates the pH of vacuoles. This gene is, preferably, a gene encoding a Na.sup.+ --H.sup.+ antiporter, for example a gene encoding a protein that has the amino acid sequence as set forth in SEQ ID NO: 2, or a gene encoding a protein that has an amino acid sequence modified by the addition or deletion of one or a plurality of amino acids and/or substitution with other amino acids in the amino acid sequence as set forth in SEQ ID NO: 2 and that, has an activity of regulating the pH of vacuoles; a gene encoding a protein that has an amino acid sequence having a identity of 20% or more with the amino acid sequence as set forth in SEQ ID NO: 2 and that has an activity of regulating the pH of vacuoles; or, a gene that hybridizes to part or all of a nucleic acid having a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 2 under a stringent condition, and that encodes a protein having an activity of regulating the pH of vacuoles.

The present invention also provides a vector comprising the above gene.

The present invention also provides a host cell transformed with the above vector.

The present invention also provides a protein encoded by the above gene.

The present invention further provides a method of producing a protein that has an activity of regulating the pH of vacuoles, said method comprising culturing or growing the above host cell and then harvesting said protein from said host cell.

The present invention also provides a plant in which said gene or said vector has been introduced or a progeny thereof having the same property as said plant, or a tissue thereof.

The present invention also provides a cut flower of the above plant or a progeny thereof.

The present invention further provides a method of regulating the pH of vacuoles comprising introducing the above gene or the above vector into a plant or plant cells and then allowing it to be expressed.

The present invention further provides a method of controlling the flower color of plants comprising introducing the above gene or the above vector into a plant or plant cells and then allowing said gene to be expressed.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a drawing showing the structure of plasmid pSPB607.

FIG. 2 is a drawing showing the structure of plasmid pSPB608.

FIG. 3 is a drawing showing the structure of plasmid pINA145.

FIG. 4 is a drawing showing the structure of plasmid pINA147.

BEST MODE FOR CARRYING OUT THE INVENTION

The color of the petal of morning glory is blue when the locus Purple is dominant, and the blue petal turns purple when it is homozygously recessive. It is clear that the locus is associated with flower color but the mechanism thereof is unknown.

First, the chemical analysis of the pigments in the petal of the pr-m mutant and a revertant thereof detected no difference in the composition of the pigments. The change in flower color of the blue-colored morning glory from the reddish purple buds to the blue flowers accompanied by flowering is believced, as mentioned above, to be caused by pH changes in the vacuole of petal cells.

In the pr-m mutant, flowering is not associated with a color change to blue, and the pH of vacuoles of petal cells of flowers that bloomed was lower in the pr-m mutant than in Pr-r. Thus, the Purple gene is considered to be a gene that regulates the pH of vacuoles of petal cells dduring flowering thereby controls flower color. Accordingly, using a pr-m mutant, and a revertant thereof, by the transposon display method, fragments of genomic DNA containing the Purple gene sequence specifically present in pr-m were identified and then the Purple gene was identified. Surprisingly, the Purple gene thus obtained had a homology with the Na.sup.+ --H.sup.+ antiporter from Arabidopsis etc., and, in the pr-m mutation, a transposon had been inserted in the 5'-untranslated region the Purple gene.

As the gene of the present invention, there can be mentioned, for example, one that encodes the amino acid sequence as set forth in SEQ ID NO: 2. It is known, however, that proteins having an amino acid sequence modified by the addition or deletion of one or a plurality of amino acids and/or substitution with other amino acids also retain an activity equal to that of the original protein. Thus in accordance with the present invention, a protein that has an amino acid sequence modified by the addition or deletion of one or a plurality of amino acids and/or substitution with other amino acids in the amino acid sequence as set forth in SEQ ID NO: 2, and a gene encoding said protein, are encompassed in the present invention as long as the protein is a protein that has an activity of regulating the pH of vacuoles.

The present invention also relates to a gene that hybridizes to the nucleotide sequence as set forth in SEQ ID NO: 1, a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 2, or a nucleotide sequence encoding part of these nucleotide sequences at a stringent condition, for example at 5.times.SSC and 50.degree. C., and that encodes a protein having an activity of regulating the pH of vacuoles. As used herein, a suitable hybridization temperature varies with the nucleotide sequence and the length of the nucleotide sequence, and when, for example, a DNA fragment comprising 18 bases encoding 6 amino acids is used as a probe, a temperature of 50.degree. C. or lower is preferred.

Genes selected, based on such hybridization, include those obtained from nature, for example from plants such as petunia and torenia, but a gene derived from sources other than plants may be used. Genes selected based on hybridization may be cDNA or genomic DNA.

The Na.sup.+ --H.sup.+ antiporter genes form a superfamily (Debrov et al., FEBS Lett. 424:1 (1998)), and have an amino acid homology of 20% or more (Orlowski et al., J. Biol. Chem. 272:22373 (1997)).

Thus, the present invention relates to a gene encoding a protein that has an amino acid sequence with a homology of about 20% or more, preferably 50% or more, for example 60% or 70% or more, and that has an activity of regulating the pH of vacuoles.

A gene having an intact nucleotide sequence is obtained, as specifically illustrated in Examples, by, for example, screening CDNA libraries. DNA encoding a protein having a modified amino acid sequence can be synthesized by commonly used site-directed mutagenesis or the PCR method based on DNA having an intact nucleotide sequence. For example, a DNA fragment that is to be modified may-be obtained by restriction enzyme treatment of the intact cDNA or genomic DNA, which is used as a template in the site-directed mutagenesis, or by the PCR method using primers in which desired mutation has been introduced to obtain a DNA fragment in which the desired modification has been introduced. Thereafter, the mutated DNA fragment may be ligated to a DNA fragment encoding another portion of the enzyme of interest.

Alternatively, in order to obtain DNA encoding a protein comprising a shortened amino acid sequence, an amino acid sequence longer than the amino acid sequence of interest, for example, DNA encoding the full-length amino acid sequence, may be cleaved with a desired restriction enzyme, and when the resultant DNA fragment was found not to encode the entire amino acid sequence of interest, a DNA fragment comprising the sequence of the lacking portion may be synthesized and ligated thereto.

The present invention is not limited to a gene encoding a protein that has an activity of regulating the pH of vacuoles derived from morning glory, but the sources may be plants, animals, or microorganisms, and all they need is to have a topology that pumps protons out of the vacuole.

By expressing the obtained gene using a gene expression system in Escherichia coli or yeast and determining the activity, it can be confirmed that the gene obtained encodes a protein that has an activity of regulating the pH of vacuoles. Furthermore, by expressing said gene, a protein, the gene product, having an activity of regulating the pH of vacuoles can be obtained. Alternatively, a protein can also be obtained that has an activity of regulating the pH of vacuoles using an antibody against the amino acid sequence as set forth in SEQ ID NO: 2, and a protein that has an activity of regulating the pH of vacuoles derived from other organisms can be cloned using an antibody.

Thus, the present invention also relates to a recombinant vector comprising the above-mentioned gene, specifically an expression vector, and a host cell transformed with said vector. As a host, there can be used a prokaryotic or eukaryotic organism. As a prokaryotic organism, for example, there can be used such a common host as a bacterium belonging to the genus Escherichia such as Escherichia coli, a bacterium belonging to the genus Bacillus such as Bacillus subtilis, and the like. As a eukaryotic host, there can be used a lower eukaryotic organism, for example an eukaryotic microorganism such as a fungus, a yeast or a mold.

As yeast, there can be mentioned a microorganism belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, and as a mold, there can be mentioned a microorganism belonging to the genus Aspergillus such as Aspergillus oryzae and Aspergillus niger, and a microorganism belonging to the genus Penicillium. Furthermore, animal cells or plant cells can be used: as animal cells, there can be used cell lines derived from mouse, hamster, monkey, human and the like. Insect cells such as silkworm cells or adult silkworms per se can also be used as hosts.

The vectors of the present invention may contain expression regulatory regions such as a promoter, a terminator, an origin of replication, and the like, depending on the type of the host into which said vector is to be introduced. As promoters for bacterial expression vectors, there can be used commonly used promoters such as trc promoter, tac promoter, lac promoter, and the like; as promoters for yeasts, there can be used the glyceraldehyde-3-phosphate dehydrogenase promoter, PHO5 promoter, and the like; and as mold promoters, there can be used amylase promoter, trpC promoter, and the like.

As promoters for animal cell hosts, there can be used viral promoters such as SV40 early promoter, SV40 late promoter, and the like. The construction of expression vectors may be performed according to conventional methods using restriction enzymes, ligase, etc. The transformation of host cells can also be performed according to conventional methods.

Host cells transformed with the above expression vectors may be cultured, cultivated or bred, and from the culture the desired protein can be recovered and purified according to conventional methods such as filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, and the like.

The present invention also relates to a plant or its progenies or tissues thereof of which hue of color has been controlled by introducing a gene encoding a protein that has an activity of regulating the pH of the vacuoles, specifically a Na.sup.+ --H.sup.+ antiporter gene. They may be cut flowers in shape. Using a gene encoding a protein that has an activity of regulating the pH of vacuoles obtained by the present invention, the pumping of proton into the cytoplasm from the vacuole and the pumping of sodium ion into the vacuole can be performed, so that anthocyanins accumulated in the vacuole can be turned blue and, as a result, the flower color can be turned blue.

It is also possible to lower the pH of vacuoles by suppressing the expression of the gene of the present invention. With the state-of-the-art technology, it is possible to introduce a gene into plants, and allow the gene to be expressed in a constitutive or tissue-specific manner, and also to suppress the expression of the gene of interest by the antisense method or the co-suppression method.

Examples of plants that can be transformed include, but not limited to, roses, chrysanthemums, carnations, snapdragons, cyclamens, orchids, lisianthus, freesias, gerberas, gladioluses, gypsophilas, kalanchoes, lilies, pelargoniumas, geraniums, petunias, torenias, tulips, rice, barley, whieat, rapeseeds, potatoes, tomatoes, poplars, bananas, eucalyptuses, sweet potatoes, soy beans, alfalfas, lupins, corns, and the like.

EXAMPLES

The present invention will now be explained in further details with reference to the following Examples. Molecular biological techniques used were performed according to Molecular Cloning (Sambrook et al., 1989), unless otherwise specified.

Example 1

Obtaining a Germ Cell Revertant

Obtaining a germ cell revertant has already been reported (Iida et al., Shokubutsu Saibo Kogaku Series (Plant Cell Engineering Series) 5 (1996) pp. 132, Shujunsha; Iida et al., Annal. New York Acad. Sci. (1999) pp. 870; Inagaki et al., Plant Cell, 6:375 (1994); Inagaki et al., Theor. Appl. Genet. 92:499 (1996)).

Morning glory having the genotype (Pr-r/pr-m) (Iida et al., pp. 870; Inagaki et al., Plant Cell, 6:375 (1994); Inagaki et al., Theor. Appl. Genet. 92:499 (1996)) was subjected to self-fertilization and the seeds of the progeny were planted. The flowers of the self-fertilized progeny were observed to select individuals that bloom with blue flowers by back mutation. Furthermore, in this self-fertilized progeny of the germ cell revertant, it was proved whether it is homozygous or heterozygous based on whether or not isolates that bloom with purple flowers can be obtained. Those having the genotype (Pr-r/Pr-r) and (pr-m/pr-m) were selected.

Example 2

Anthocyanins in the Petals of Revertants

Anthocyanins contained in morning glory are mainly heavenly blue anthocyanin and several other anthocyanins (Lu et al., Phytochemistry 31:659 (1992)). When the open petals of the Pr-r/Pr-r strain and the pr-m/pr-m strain obtained in Example 1 were similarly analyzed, the anthocyanins contained in both of them were almost identical.

A cellophane tape was stuck to the front side of a petal and then peeled off to recover one layer of epithelium, from which the cell liquid was scraped with a scalpel etc., which was then centrifuged to obtain juice. The pH of the juice was measured using the Horiba B212 pH meter (Horiba Seisakusho). pH of the petal epithelium of the Pr-r/Pr-r strain was about 7.1 whereas that of the pr-m/pr-m strain was about 6.5. This result indicates that the change in flower color by mutation of purple was not due to the structure of anthocyanins but to the change of vacuolar pH.

Example 3

Isolation of a Aenome Fragment Specifically Present in pr-m

For the isolation of a gene, the transposon display method (Frey et al., Plant J. 13:717 (1998); Van den Broeck et al., Plant J. 13:121 (1998)) or a similar method (Dosho et al., Shokubutsu Saibo Kogaku Series (Plant Cell Engineering Series) 7 (1997) pp. 144, Shujunsha) was used to search for DNA bands that were present in the pr-m/pr-m strain and the Pr-w/pr-m strain but not in the Pr-r/Pr-r strain or in the wild strain. Since Tpn1-related transposon is thought to be mainly associated with mutability in morning glory, special note was given to the Tpn1-related transposon.

Specifically, chromosomal DNA was extracted from the pr-m/pr-m strain, and 125 ng of it was digested with MseI in 20 .mu.l. To the digested DNA was added 80 pmole of MseI adaptor (obtained by annealing 5'-GACGATGAGTCCTGAG-3' (SEQ ID NO: 3) and 5'-TACTCAGGACTCAT-3' (SEQ ID NO: 4)) in 25 .mu.l at 20.degree. C. for 2 hours. After keeping it at 75.degree. C. for 10 minutes, it was stored at -20.degree. C. After diluting this ten-fold, 2 .mu.l was used as a template, which was PCR-amplified using 4.8 pmole of TIR primer (5'-TGTGCATTTTTCTTGTAGTG-3' (SEQ ID NO: 5), this includes the inverted terminal repeat of the transposon Tpnl) and 4.8 pmole of MseI primer (5'-GATGAGTCCTGAGTAA-3') (SEQ ID NO: 6) in 20 .mu.l.

PCR was performed with Taq polymerase (Takara Shuzo) for 20 cycles with one cycle comprising 94.degree. C. for 0.5 minute, 56.degree. C. for 1 minute, and 72.degree. C. for 1 minute, and the volume was diluted ten-fold. Two .mu.l of it was used as a template in a PCR using 4.8 pmole of TIR+N primer (5'-TGTGCATTTTTCTTGTAGN-3' (SEQ ID NO: 7) N=A, C, G or T. Four different species were synthesized instead of a mixture) and 4.8 pmole of MseI+N primer (5'-GATGAGTCCTGAGTAAN-3' (SEQ ID NO: 8) N=A, C, G or T. Four different species were synthesized instead of a mixture. The 5'-end was labeled with fluorescein (using Amersham Pharmacia Biotek, Vistra fluorescence 5'-oligo labeling kit)) in 20 .mu.l.

Reactions were performed for combinations of primers to a total of 16 reactions. PCR was performed for 13 cycles with one cycle comprising 94.degree. C. for 0.5 minute, 65.degree. C. (with a decrement of 0.7.degree. C. for each cycle) for 1 minute, and 72.degree. C. for 1 minute, and further for 13 cycles with one cycle comprising 94.degree. C. for 0.5 minute, 56.degree. C. for 1 minute, and 72.degree. C. for 1 minute. A similar procedure was performed for chromosomal DNA obtained from the Pr-r/Pr-r strain, subjected to electrophoresis using a sequence gel of the DNA Sequencer 377 (PE Biosystems Japan), and the bands were detected using FMBIOII (Takara Shuzo).

When bands derived from the Pr-r/Pr-r strain and the pr-m/pr-m strain were compared, an about 130 bp DNA fragment was specifically expressed in the strain having pr-m. The 130 bp DNA fragment was recovered, and amplified by PCR (for 30 cycles with one cycle comprising 94.degree. C. for 0.5 minute, 56.degree. C. for 1 minute, and 72.degree. C. for 1 minute) using 20 pmole TIR primer and 20 pmole MseI primer, which was then subcloned into the pGEM-T vector (Promega Corporation), and then the nucleotide sequence was determined. The sequence was 5'-TGAGCATTTTTCTTGTAGTG CTGAGATTTTCCTCCATTTGTCTGAAGCTCTTCATCCTTCAACAC TACCCCCACATCTCACCTTTCAAG GTCCAATCTTTATCATTCATCT TTACTCAGGACTCATCGTC-3' (SEQ ID NO: 9) (the single-underlined portion corresponds to a used primer, the double-underlined portion corresponds to an exon, and the rest corresponds to an intron). After the sequence as set forth in SEQ ID NO: 9 was used as a probe in Northern analysis, a transcription product of about 2.3 kb was found in the bud of morning glory having Pr-r, but a corresponding transcription product was not found in the pr-m/pr-m strain. Thus, it can be seen that this 2.3 kb transcription product corresponds to the Purple gene.

Example 4

Isolation of cDNA

About 6 million clones of a CDNA library (Inagaki et al., Plant Cell 6:375 (1994)) derived from the wild strain morning glory (Pr-w/Pr-w) were screened using the 130 bp DNA fragment as a probe, with a result that two positive clones were obtained. One of these clones had a 2237 bp CDNA, among which a 1626 bp-long open reading frame was observed (SEQ ID NO: 1). The predicted amino acid sequence had an identity of 29.3% and 73.4% with the Na.sup.+ --H.sup.+ antiporter of yeast and Arabidopsis, respectively (Nhx1 and AtNhx1 , respectively, Gaxiola et al., Proc. Natl. Acad. Sci. USA 96:1480-1485 (1999)).

The result revealed that the Purple gene of morning glory encodes a Na.sup.+ --H.sup.+ antiporter. Incidentally, although the Na.sup.+ --H.sup.+ antiporter obtained from Arabidopsis is attracting attention as a protein that gives salt resistance to yeast, this is the first time that an association of the Na.sup.+ --H.sup.+ antiporter with flower color was observed.

Example 5

Complementation Experiment of Yeast Na.sup.+ --H.sup.+ Antiporter

The predicted amino acid sequence encoded by the Purple gene of morning glory has a homology with those of the Na.sup.+ --H.sup.+ antiporters of yeast and Arabidopsis. Thus, in order to confirm whether the Purple gene product of morning glory can function as a Na.sup.+ --H.sup.+ antiporter protein, a complementation experiment was performed using a yeast Na.sup.+ --H.sup.+ antiporter mutant.

First, the following two DNA fragments were synthesized:

CBSC1-Linker (22 mer) 5'-CGA TAG ATC TGG GGG TCG ACA T-3' (SEQ ID NO: 12)

CSBD2-Linker (22 mer) 5'-CGA TGT CGA CCC CCA GAT CTA T-3' (SEQ ID NO: 13)

From these two fragments, a linker having restriction enzyme sites ClaI-BglII-SalI-ClaI is formed. A plasmid pINA145 (FIG. 3) was constructed by inserting the above linker according to a standard method into the ClaI site of the pYES2 vector (Invitrogen Corporation) so that the BglII site is located at the URA3 gene side. A plasmid pINA147 (FIG. 4) was constructed by ligating a 2 kb DNA fragment obtained by digesting plasmid pJJ250 (Jones and Prakash, Yeast 6:363-366 (1990)) with BamHI and SalI to plasmid pINA145 digested with BglII and SalI. Plasmid pIAN151 was constructed by ligating Purple cDNA thereto under the control of the GAL 1 promoter of plasmid pINA147. pINA147 and pIAN151 were transformed respectively to the yeast R101 strain which is a mutant strain of the Na.sup.+ --H.sup.+ antiporter. Due to the mutation of the Na.sup.+ --H.sup.+ antiporter, the yeast R101 strain cannot grow on a 400 mM NaCl-added APG medium (Nass et al., J. Biol. Chem. 272:26145 (1997); Gaxiola et al., 96:1480-1485 (1999)). The pINA147-transformed R101 strain could not grow either, and only the pIAN151-transformed R101 strain could grow on the 400 mM NaCl-added APG medium. The result has shown that the gene product of the morning glory Purple gene has the Na.sup.+ --H.sup.+ antiporter function.

Example 6

Construction of an Expression Vector in Plants

With 10 ng of morning glory Purple cDNA as template, PCR was performed using synthetic primers PR-5 (5'-GGGATCCAACAAAAATGGCTGTCGGG-3') (SEQ ID NO: 10) and PR-3 (5'-GGGTCGACTAAGCATCAAAACATAGAGCC-3') (SEQ ID NO: 11). The polymerase used was Taq polymerase (Toyoboseki), and the reaction was performed, after reaction at 95.degree. C. for 45 seconds, for 25 cycles with one cycle comprising 95.degree. C. for 45 seconds, 50.degree. C. for 45 seconds, and 72.degree. C. for 45 seconds, and then further reacted at 72.degree. C. for 10 minutes. An about 1.6 kb DNA fragment obtained was ligated to pCR2.1-Topo (Clontech) to make pCR-purple. It was confirmed that there were no errors due to PCR in the nucleotide sequence of Purple cDNA on this plasmid.

pBE2113-GUS (Mitsuhara et al., Plant Cell Physiol. 37:49 (1996)) was digested with SacI and blunt-ended. Then a XhoI linker (Toyoboseki) was inserted thereto, and the plasmid obtained was termed pBE2113-GUSx. This was digested with EcoRI and HindIII to obtain an about 2.7 kb DNA fragment, which was ligated to the HindIII and EcoRI digest of pBinPLUS, and the plasmid obtained was termed pBEXP.

On the other hand, an about 1.2 kb DNA fragment obtained by digesting pCGP484 (Kohyo (National Publication of Translated Version) No. 8-511683) with HindIII and XbaI, an about 1.6 kb DNA fragment obtained by digesting pCR-purple with XbaI and SalI, and an about 13 kb DNA fragment obtained by digesting pBEXP with HindIII and XhoI were ligated to obtain pSPB607 (FIG. 1). This plasmid is a binary vector for use in the Agrobacterium-mediated transformation of plants, and on this plasmid Purple cDNA is under the control of a chalcone synthase promoter derived from snapdragon and a nopaline synthase terminator derived from Agrobacterium.

An about 0.8 kb DNA fragment obtained by digesting pCGP669 (Kohyo (National Publication of Translated version) No. 8-511683) with HindIII and BamHI, an about 1.6 kb DNA fragment obtained by digesting pCR-purple with BamHI and SalI, and an about 13 kb DNA fragment obtained by digesting pBEXP with HindIII and XhoI were ligated to obtain pSPB608 (FIG. 2). This plasmid is a binary vector for use in the Agrobacterium-mediated transformation of plants, and on this plasmid Purple CDNA is under the control of a chalcone synthase promoter derived from petunia and a nopaline synthase terminator derived from Agrobacterium.

By transforming plants using the expression vectors thus obtained, the pH of vacuoles can be regulated and thereby flower color can be controlled.

Example 7

Isolation of a Homoloas of the Purple Gene

cDNA libraries derived from the petals of petunia (Petunia hybrida cv. Old Glory Blue), Nierembergia (Nierembergia hybrida.cv. NB17), and Torenia (Torenia hybrida cv. Summerwave Blue) were each constructed using the cDNA synthesis kit (Stratagene, USA). The method of construction was as recommended by the manufacturer. About 200,000 clones each were screened according to a standard method. For washing the membrane, an aqueous solution of 5.times.SSC and 0.1% SDS was used and the incubation was performed three times at 50.degree. C. for 10 minutes. Among the positive clones obtained, the nucleotide sequence of the longest clone was determined for each clone. The nucleotide sequence of the clone of Petunia and the corresponding amino acid sequence are shown in SEQ ID NO: 14 and 15, the nucleotide sequence of the clone of Nierembergia and the corresponding amino acid sequence are shown in SEQ ID NO: 16 and 17, and the nucleotide sequence of the clone of Torenia and the corresponding amino acid sequence are shown in SEQ ID NO: 18 and 19. Homologs of the Purple gene of Petunia, Nierembergia, and Torenia had an identity on the amino acid level of 75%, 76%, and 71%, respectively, with the morning glory Purple gene.

Since the amino acid sequence of the Na.sup.+ --H.sup.+ antiporter encoded by the morning glory Purple gene and that of the Na.sup.+ --H.sup.+ antiporter encoded by Arabidopsis AtNhx 1 are about 73% identical, the homologs of the Purple gene of Petunia, Nierembergia, and Torenia obtained are judged to encode the Na.sup.+ --H.sup.+ antiporter.

Example 8

Isolation of the Clone of Mornina Glory Purple Chromosome

After chromosomal DNAs of a mutant morning glory (pr-m/pr-m) and a revertant morning glory (Pr-r/Pr-r) were cleaved with BglII, they were electrophoresed on a 0.8% agarose gel, and were subjected to genomic Southern analysis with cDNA of morning glory Purple as a probe. As a result, an about 7.5 kb band that was not present in the mutant morning glory was detected in the revertant morning glory.

After 50 .mu.g of chromosomal DNA of the wild type morning glory (Pr-w/Pr-w, the KKZSK2 strain) was digested with BglII, it was electrophoresed on a 0.8% agarose gel. An about 7-9 kb fragmently was recovered, from which DNA was extracted using the GENECLEAN III KIT (B10101). This DNA was ligated to the .lambda. Zap express vector (Stratagene, USA), which was screened with cDNA of morning glory Purple as a probe. The determination of nucleotide sequences of positive clones obtained revealed that, on this about 7.5 kb DNA fragment, there was a region from about 6.3 kb upstream of the Purple promoter to midway in exon 3. For this sequence, a sequence up to the initiation codon of the Purple gene is shown in SEQ ID NO: 20.

It has been demonstrated that the expression of the Purple gene is strongly induced only at about 24 hours before the flowering of morning glory, and that the expression of the Purple gene is suppressed by insertion of a transposon into the 5'-untranslated region. From this, it is clear that the promoter region of the Purple gene obtained contains a factor needed for the expression of the Purple gene in a developmental stage-specific and organ-specific manner in the petals of morning glory. By placing the gene of interest downstream of this promoter region, the expression of the gene of interest can be regulated in a developmental stage-specific and organ-specific manner.

Industrial Applicability

The gene obtained in the present invention was found, for the first time, to be involved in controlling the pH of vacuoles and flower color. By expressing the gene of the present invention on the flower petals, the pH of vacuoles can be increased and thereby the flower color can be turned blue. Furthermore, by suppressing the expression of the gene of the present invention, the pH of vacuoles can be lowered and thereby flower color can be turned red. As the gene encoding a protein that regulates the pH of vacuoles, there can be used not only those derived from morning glory obtained in the present invention but also similar genes derived from other organisms.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 20 <210> SEQ ID NO 1 <211> LENGTH: 2237 <212> TYPE: DNA <213> ORGANISM: Ipomoea nil <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(2237) <223> OTHER INFORMATION: Nucleotide sequence of DNA encoding for protein regulating the pH of vacuoles <400> SEQUENCE: 1 agaatgtagg ctacagaaat tttcagacag atagatacat aaatccgtat aatagagaca 60 gagaaacaga aaaagagaga gtcacgttaa tcctgagatt ttcctccatt tgtctgaagc 120 tcttcatcct tcaacactac ccccacatct cacctttcaa gtgatttgta tgttttcggg 180 agggattgga atgggcaacc cggatatgtg aacagaaacc acgacattgg gaaaagattt 240 attgcaaaaa ttgttttgat tgttttggat tttgtggtag aaaaagggga agaacaaaa 299 atg gcg ttc ggg ttg tct tct ttg ctc caa aat tcg gat ttg ttc acg 347 Met Ala Phe Gly Leu Ser Ser Leu Leu Gln Asn Ser Asp Leu Phe Thr 1 5 10 15 tct gat cat gct tcc gtt gtg tcg atg aac ctc ttt gtg gcg ttg ctt 395 Ser Asp His Ala Ser Val Val Ser Met Asn Leu Phe Val Ala Leu Leu 20 25 30 tgc gca tgc att gtt ctt ggc cat cta ctc gag gag aat cgc tgg gtg 443 Cys Ala Cys Ile Val Leu Gly His Leu Leu Glu Glu Asn Arg Trp Val 35 40 45 aac gaa tcc att act gcc ctt ata att ggt ttg tgc acc gga gtt gta 491 Asn Glu Ser Ile Thr Ala Leu Ile Ile Gly Leu Cys Thr Gly Val Val 50 55 60 att ttg ctc ctt agc gga gga aag agt tca cat ctt ctc gtc ttt agc 539 Ile Leu Leu Leu Ser Gly Gly Lys Ser Ser His Leu Leu Val Phe Ser 65 70 75 80 gaa gat ctt ttc ttt ata tat ctc ctg cca cct ata ata ttc aat gcg 587 Glu Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe Asn Ala 85 90 95 ggg ttt caa gtg aaa aag aag cag ttt ttc gtg aac ttc atg aca att 635 Gly Phe Gln Val Lys Lys Lys Gln Phe Phe Val Asn Phe Met Thr Ile 100 105 110 atg ctg ttt gga gct att ggc aca ctt att agc tgt tct att ata tca 683 Met Leu Phe Gly Ala Ile Gly Thr Leu Ile Ser Cys Ser Ile Ile Ser 115 120 125 ttt ggt gcg gtc aaa att ttc aag cac tta gac att gac ttt ctg gat 731 Phe Gly Ala Val Lys Ile Phe Lys His Leu Asp Ile Asp Phe Leu Asp 130 135 140 ttt gga gat tat tta gca att ggt gcg ata ttt gct gca acc gat tct 779 Phe Gly Asp Tyr Leu Ala Ile Gly Ala Ile Phe Ala Ala Thr Asp Ser 145 150 155 160 gtt tgc aca ttg cag gtg ctc agt cag gat gag acg ccc cta ctt tac 827 Val Cys Thr Leu Gln Val Leu Ser Gln Asp Glu Thr Pro Leu Leu Tyr 165 170 175 agt ctc gtg ttt gga gaa ggg gtc gtc aat gat gct aca tct gtg gtc 875 Ser Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser Val Val 180 185 190 ctt ttt aat gct att caa agt ttt gac atg act agt ttt gat cca aaa 923 Leu Phe Asn Ala Ile Gln Ser Phe Asp Met Thr Ser Phe Asp Pro Lys 195 200 205 att ggg ctt cat ttc att gga aac ttc ttg tat tta ttt ctc tcg agc 971 Ile Gly Leu His Phe Ile Gly Asn Phe Leu Tyr Leu Phe Leu Ser Ser 210 215 220 act ttt ttg ggc gtg gga att gga ctg ctt tgt gct tat att atc aaa 1019 Thr Phe Leu Gly Val Gly Ile Gly Leu Leu Cys Ala Tyr Ile Ile Lys 225 230 235 240 aag cta tac ttt ggc agg cac tca acc gat cgt gag gtt gcc ctt atg 1067 Lys Leu Tyr Phe Gly Arg His Ser Thr Asp Arg Glu Val Ala Leu Met 245 250 255 atg ctc atg tct tac ttg tct tat ata atg gcc gag tta ttc tat cta 1115 Met Leu Met Ser Tyr Leu Ser Tyr Ile Met Ala Glu Leu Phe Tyr Leu 260 265 270 agc ggc ata ctt act gta ttc ttc tgt gga att gtc atg tct cat tat 1163 Ser Gly Ile Leu Thr Val Phe Phe Cys Gly Ile Val Met Ser His Tyr 275 280 285 acc tgg cac aat gtt acc gag agc tca agg gtc act act agg cat tcc 1211 Thr Trp His Asn Val Thr Glu Ser Ser Arg Val Thr Thr Arg His Ser 290 295 300 ttt gca act ctg tca ttt gtc gca gag aca ttt atc ttc ctc tat gtt 1259 Phe Ala Thr Leu Ser Phe Val Ala Glu Thr Phe Ile Phe Leu Tyr Val 305 310 315 320 ggt atg gat gcc ttg gat atc gag aaa tgg aaa ttt gtg aaa aat agt 1307 Gly Met Asp Ala Leu Asp Ile Glu Lys Trp Lys Phe Val Lys Asn Ser 325 330 335 cag gga cta tca gtt gca gtg agc tca ata ttg gta ggc cta atc tta 1355 Gln Gly Leu Ser Val Ala Val Ser Ser Ile Leu Val Gly Leu Ile Leu 340 345 350 gta ggc aga gct gcg ttc gta ttc ccc ttg tcg ttt tta tcc aac tta 1403 Val Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser Asn Leu 355 360 365 gca aag aaa aac tct tcg gac aag ata tcc ttt agg caa caa ata ata 1451 Ala Lys Lys Asn Ser Ser Asp Lys Ile Ser Phe Arg Gln Gln Ile Ile 370 375 380 att tgg tgg gct ggc cta atg aga ggc gcc gtc tca ata gca ctt gcg 1499 Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Ile Ala Leu Ala 385 390 395 400 tat aat aag ttt aca acc tcg ggg cat acg tca ttg cac gag aac gca 1547 Tyr Asn Lys Phe Thr Thr Ser Gly His Thr Ser Leu His Glu Asn Ala 405 410 415 ata atg att aca agt act gtt acg gtt gtt ctg ttc agc aca gtt gta 1595 Ile Met Ile Thr Ser Thr Val Thr Val Val Leu Phe Ser Thr Val Val 420 425 430 ttc ggg ttg atg acg aag cct ctg ata aac ctt ctg cta ccc ccg cac 1643 Phe Gly Leu Met Thr Lys Pro Leu Ile Asn Leu Leu Leu Pro Pro His 435 440 445 aag cag atg cca agc ggt cat tcg tca atg aca aca tcc gaa ccc agt 1691 Lys Gln Met Pro Ser Gly His Ser Ser Met Thr Thr Ser Glu Pro Ser 450 455 460 agt ccg aag cac ttc acg gtg cca ctc ctg gac aac caa cct gac tca 1739 Ser Pro Lys His Phe Thr Val Pro Leu Leu Asp Asn Gln Pro Asp Ser 465 470 475 480 gaa agc gat atg ata acc gga cct gag gtt gct cga cca act gcc ttg 1787 Glu Ser Asp Met Ile Thr Gly Pro Glu Val Ala Arg Pro Thr Ala Leu 485 490 495 cgc atg ctg cta agg acg cca acc cac acc gtg cac cgc tac tgg cgt 1835 Arg Met Leu Leu Arg Thr Pro Thr His Thr Val His Arg Tyr Trp Arg 500 505 510 aag ttt gat gat tcg ttt atg cgt ccc gtg ttt ggc ggg cgg gga ttc 1883 Lys Phe Asp Asp Ser Phe Met Arg Pro Val Phe Gly Gly Arg Gly Phe 515 520 525 gtt ccg ttt gtc gcg ggc tca cca gtt gag cag agc cct aga tga 1928 Val Pro Phe Val Ala Gly Ser Pro Val Glu Gln Ser Pro Arg 530 535 540 ggtacaaagt acaaacaaga cactgttgct gggtgaaata gtgtaagttg tatcatagtt 1988 gattctggtt gcccctctta tgaaatgggc tgggtgaaag tcttctcact agctaggttg 2048 cattgcattg ctacttcata aatgttttat tttattttgt aaatgttggt gcattttagg 2108 tacttgtatt aacacctcat ttgtagcata ttatttggta cagagtattt tttttatgaa 2168 acaataatgg ctgaattatc aatttggctc tatgttttga tgcttagtaa aaaaaaaaaa 2228 aaaaaaaaa 2237 <210> SEQ ID NO 2 <211> LENGTH: 542 <212> TYPE: PRT <213> ORGANISM: Ipomea nil <220> FEATURE: <221> NAME/KEY: peptide <222> LOCATION: (1)..(542) <223> OTHER INFORMATION: Amino acid sequence of protein regulating the pH of vacuoles <400> SEQUENCE: 2 Met Ala Phe Gly Leu Ser Ser Leu Leu Gln Asn Ser Asp Leu Phe Thr 1 5 10 15 Ser Asp His Ala Ser Val Val Ser Met Asn Leu Phe Val Ala Leu Leu 20 25 30 Cys Ala Cys Ile Val Leu Gly His Leu Leu Glu Glu Asn Arg Trp Val 35 40 45 Asn Glu Ser Ile Thr Ala Leu Ile Ile Gly Leu Cys Thr Gly Val Val 50 55 60 Ile Leu Leu Leu Ser Gly Gly Lys Ser Ser His Leu Leu Val Phe Ser 65 70 75 80 Glu Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe Asn Ala 85 90 95 Gly Phe Gln Val Lys Lys Lys Gln Phe Phe Val Asn Phe Met Thr Ile 100 105 110 Met Leu Phe Gly Ala Ile Gly Thr Leu Ile Ser Cys Ser Ile Ile Ser 115 120 125 Phe Gly Ala Val Lys Ile Phe Lys His Leu Asp Ile Asp Phe Leu Asp 130 135 140 Phe Gly Asp Tyr Leu Ala Ile Gly Ala Ile Phe Ala Ala Thr Asp Ser 145 150 155 160 Val Cys Thr Leu Gln Val Leu Ser Gln Asp Glu Thr Pro Leu Leu Tyr 165 170 175 Ser Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser Val Val 180 185 190 Leu Phe Asn Ala Ile Gln Ser Phe Asp Met Thr Ser Phe Asp Pro Lys 195 200 205 Ile Gly Leu His Phe Ile Gly Asn Phe Leu Tyr Leu Phe Leu Ser Ser 210 215 220 Thr Phe Leu Gly Val Gly Ile Gly Leu Leu Cys Ala Tyr Ile Ile Lys 225 230 235 240 Lys Leu Tyr Phe Gly Arg His Ser Thr Asp Arg Glu Val Ala Leu Met 245 250 255 Met Leu Met Ser Tyr Leu Ser Tyr Ile Met Ala Glu Leu Phe Tyr Leu 260 265 270 Ser Gly Ile Leu Thr Val Phe Phe Cys Gly Ile Val Met Ser His Tyr 275 280 285 Thr Trp His Asn Val Thr Glu Ser Ser Arg Val Thr Thr Arg His Ser 290 295 300 Phe Ala Thr Leu Ser Phe Val Ala Glu Thr Phe Ile Phe Leu Tyr Val 305 310 315 320 Gly Met Asp Ala Leu Asp Ile Glu Lys Trp Lys Phe Val Lys Asn Ser 325 330 335 Gln Gly Leu Ser Val Ala Val Ser Ser Ile Leu Val Gly Leu Ile Leu 340 345 350 Val Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser Asn Leu 355 360 365 Ala Lys Lys Asn Ser Ser Asp Lys Ile Ser Phe Arg Gln Gln Ile Ile 370 375 380 Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Ile Ala Leu Ala 385 390 395 400 Tyr Asn Lys Phe Thr Thr Ser Gly His Thr Ser Leu His Glu Asn Ala 405 410 415 Ile Met Ile Thr Ser Thr Val Thr Val Val Leu Phe Ser Thr Val Val 420 425 430 Phe Gly Leu Met Thr Lys Pro Leu Ile Asn Leu Leu Leu Pro Pro His 435 440 445 Lys Gln Met Pro Ser Gly His Ser Ser Met Thr Thr Ser Glu Pro Ser 450 455 460 Ser Pro Lys His Phe Thr Val Pro Leu Leu Asp Asn Gln Pro Asp Ser 465 470 475 480 Glu Ser Asp Met Ile Thr Gly Pro Glu Val Ala Arg Pro Thr Ala Leu 485 490 495 Arg Met Leu Leu Arg Thr Pro Thr His Thr Val His Arg Tyr Trp Arg 500 505 510 Lys Phe Asp Asp Ser Phe Met Arg Pro Val Phe Gly Gly Arg Gly Phe 515 520 525 Val Pro Phe Val Ala Gly Ser Pro Val Glu Gln Ser Pro Arg 530 535 540 <210> SEQ ID NO 3 <211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: MseI adaptor <400> SEQUENCE: 3 gacgatgagt cctgag 16 <210> SEQ ID NO 4 <211> LENGTH: 14 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: MseI adaptor <400> SEQUENCE: 4 tactcaggac tcat 14 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: TIR primer <400> SEQUENCE: 5 tgtgcatttt tcttgtagtg 20 <210> SEQ ID NO 6 <211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: MseI primer <400> SEQUENCE: 6 gatgagtcct gagtaa 16 <210> SEQ ID NO 7 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: TIR+N primer <221> NAME/KEY: misc_feature <222> LOCATION: (19)..(19) <223> OTHER INFORMATION: Nucleotide 19 = "n" wherein "n" = any nucleotide <400> SEQUENCE: 7 tgtgcatttt tcttgtagn 19 <210> SEQ ID NO 8

<211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: MseI+N primer <221> NAME/KEY: misc_feature <222> LOCATION: (17)..(17) <223> OTHER INFORMATION: Nucleotide 17 = "n" wherein "n" = any nucleotide <400> SEQUENCE: 8 gatgagtcct gagtaan 17 <210> SEQ ID NO 9 <211> LENGTH: 130 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: MseI+N primer <400> SEQUENCE: 9 tgagcatttt tcttgtagtg ctgagatttt cctccatttg tctgaagctc ttcatccttc 60 aacactaccc ccacatctca cctttcaagg tccaatcttt atcattcatc tttactcagg 120 actcatcgtc 130 <210> SEQ ID NO 10 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PR-5 primer <400> SEQUENCE: 10 gggatccaac aaaaatggct gtcggg 26 <210> SEQ ID NO 11 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PR-3 primer <400> SEQUENCE: 11 gggtcgacta agcatcaaaa catagagcc 29 <210> SEQ ID NO 12 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CBSC1-linker <400> SEQUENCE: 12 cgatagatct gggggtcgac at 22 <210> SEQ ID NO 13 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: CBSC2-linker <400> SEQUENCE: 13 cgatgtcgac ccccagatct at 22 <210> SEQ ID NO 14 <211> LENGTH: 2423 <212> TYPE: DNA <213> ORGANISM: Petunia hybrida <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(2423) <223> OTHER INFORMATION: Nucleotide sequence of DNA encoding for protein regulating the pH of vacuoles <400> SEQUENCE: 14 attgcgcttc gtattttact gctgaatgaa atcgtgtttt tttattcagt tcgttgttat 60 taatttcaga gtttttttta ttaaaggtgt gtttggttga agaaattgta tttgctgaat 120 tttgcagaag tttttgagtt tttgctaaac tattgtgaga tctgattttg aatttttcca 180 gtggtgtttt aagctcaatt cgacgtcgtt tttactggaa ttctgatcag taaatagggc 240 tattttgatg taaggttgtg aaagtttaca gtttggaagt tgagttagtg aaaaagggga 300 aactttattg tgatattttc acaagtattt ggtgaattca ggttattgag a atg gct 357 Met Ala ttt gat ttt ggg acg ttg ttg gga aat gta gac agg tta tcg aca tct 405 Phe Asp Phe Gly Thr Leu Leu Gly Asn Val Asp Arg Leu Ser Thr Ser 5 10 15 gat cat caa tca gtt gtg tcg ata aac tta ttc gtt gct ctt att tgc 453 Asp His Gln Ser Val Val Ser Ile Asn Leu Phe Val Ala Leu Ile Cys 20 25 30 gcg tgt att gtg atc ggt cat ttg ttg gaa gaa aac aga tgg atg aat 501 Ala Cys Ile Val Ile Gly His Leu Leu Glu Glu Asn Arg Trp Met Asn 35 40 45 50 gag tcc ata act gcc tta gtg att ggt tct tgt act gga atc gtt att 549 Glu Ser Ile Thr Ala Leu Val Ile Gly Ser Cys Thr Gly Ile Val Ile 55 60 65 cta ctg ata agt gga gga aag aac tct cat att tta gtg ttc agt gaa 597 Leu Leu Ile Ser Gly Gly Lys Asn Ser His Ile Leu Val Phe Ser Glu 70 75 80 gat ctt ttc ttc att tac ctt ctt ccg cca atc att ttt aat gct ggg 645 Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe Asn Ala Gly 85 90 95 ttc cag gtg aaa aag aaa tcg ttc ttc cgc aat ttc agc act atc atg 693 Phe Gln Val Lys Lys Lys Ser Phe Phe Arg Asn Phe Ser Thr Ile Met 100 105 110 ctc ttt ggg gca ctt ggc acc ttg ata tca ttc att att ata tca tta 741 Leu Phe Gly Ala Leu Gly Thr Leu Ile Ser Phe Ile Ile Ile Ser Leu 115 120 125 130 ggt gcc att ggc att ttc aag aaa atg aat att gga agc ctt gaa att 789 Gly Ala Ile Gly Ile Phe Lys Lys Met Asn Ile Gly Ser Leu Glu Ile 135 140 145 gga gat tac ctt gca att ggg gca atc ttc tct gct aca gat tct gta 837 Gly Asp Tyr Leu Ala Ile Gly Ala Ile Phe Ser Ala Thr Asp Ser Val 150 155 160 tgc acc tta caa gtg ctt aat cag gat gaa aca ccc tta ttg tac agt 885 Cys Thr Leu Gln Val Leu Asn Gln Asp Glu Thr Pro Leu Leu Tyr Ser 165 170 175 cta gtt ttt ggg gaa ggt gtt gtg aat gat gcc aca tct gta gtt ctg 933 Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser Val Val Leu 180 185 190 ttc aat gct atc cag aac ttt gac tta tct cac atc gac acg ggc aaa 981 Phe Asn Ala Ile Gln Asn Phe Asp Leu Ser His Ile Asp Thr Gly Lys 195 200 205 210 gct atg gaa tta gtt gga aac ttt cta tac ttg ttt gcc tca agc act 1029 Ala Met Glu Leu Val Gly Asn Phe Leu Tyr Leu Phe Ala Ser Ser Thr 215 220 225 gcc cta gga gtt gct gct ggc cta ctg agc gcc tat att att aaa aaa 1077 Ala Leu Gly Val Ala Ala Gly Leu Leu Ser Ala Tyr Ile Ile Lys Lys 230 235 240 ctc tac ttt gga agg cac tca act gac cgt gag gtt gct ata atg ata 1125 Leu Tyr Phe Gly Arg His Ser Thr Asp Arg Glu Val Ala Ile Met Ile 245 250 255 ctc atg gct tac cta tct tac atg ctt gct gaa tta ttc tat tta agt 1173 Leu Met Ala Tyr Leu Ser Tyr Met Leu Ala Glu Leu Phe Tyr Leu Ser 260 265 270 gca atc ctc act gtg ttt ttc tct ggg atc gtg atg tct cac tac acc 1221 Ala Ile Leu Thr Val Phe Phe Ser Gly Ile Val Met Ser His Tyr Thr 275 280 285 290 tgg cat aat gtg act gag agc tcg aga gtc act acc aag cac act ttt 1269 Trp His Asn Val Thr Glu Ser Ser Arg Val Thr Thr Lys His Thr Phe 295 300 305 gct aca tta tca ttt att gct gaa ata ttc ata ttc ctt tat gtt ggt 1317 Ala Thr Leu Ser Phe Ile Ala Glu Ile Phe Ile Phe Leu Tyr Val Gly 310 315 320 atg gat gct ttg gac att gag aag tgg aag ttt gta agc gac agc cct 1365 Met Asp Ala Leu Asp Ile Glu Lys Trp Lys Phe Val Ser Asp Ser Pro 325 330 335 gga ata tca gtt cag gtt agc tca ata ttg ctg ggt ctt gtt ttg gtt 1413 Gly Ile Ser Val Gln Val Ser Ser Ile Leu Leu Gly Leu Val Leu Val 340 345 350 gga aga gca gca ttt gtt ttc cca ttg tca ttc ttg tcc aac ttg acc 1461 Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser Asn Leu Thr 355 360 365 370 aag aaa act cca gag gcg aaa att agt ttt aac cag cag gtt aca ata 1509 Lys Lys Thr Pro Glu Ala Lys Ile Ser Phe Asn Gln Gln Val Thr Ile 375 380 385 tgg tgg gct gga ctt atg aga ggt gcc gtt tct atg gcc ctt gct tat 1557 Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Met Ala Leu Ala Tyr 390 395 400 aat cag ttt acc agg gga ggt cat act cag tta cgc gca aat gca ata 1605 Asn Gln Phe Thr Arg Gly Gly His Thr Gln Leu Arg Ala Asn Ala Ile 405 410 415 atg atc aca agt act atc act gtt gtc ctt ttc agc aca gtc gtg ttt 1653 Met Ile Thr Ser Thr Ile Thr Val Val Leu Phe Ser Thr Val Val Phe 420 425 430 ggg ttg atg aca aaa cct ttg att aga ata ttg cta ccc tca cac aaa 1701 Gly Leu Met Thr Lys Pro Leu Ile Arg Ile Leu Leu Pro Ser His Lys 435 440 445 450 cac ttg agc aga atg atc tct tct gaa cca acg acc cca aaa tcc ttc 1749 His Leu Ser Arg Met Ile Ser Ser Glu Pro Thr Thr Pro Lys Ser Phe 455 460 465 att gtg cca ctt ctt gac agc aca caa gac tca gaa gct gat ctg gaa 1797 Ile Val Pro Leu Leu Asp Ser Thr Gln Asp Ser Glu Ala Asp Leu Glu 470 475 480 cgc cat gta ccc cgt ccc cac agt ttg cgg atg ctc ctt tca acc cca 1845 Arg His Val Pro Arg Pro His Ser Leu Arg Met Leu Leu Ser Thr Pro 485 490 495 tct cat aca gtg cat tat tac tgg aga aag ttt gac aat gca ttc atg 1893 Ser His Thr Val His Tyr Tyr Trp Arg Lys Phe Asp Asn Ala Phe Met 500 505 510 cgt cca gtt ttc ggt gga cga ggt ttt gta cct ttt gct cca gga tca 1941 Arg Pro Val Phe Gly Gly Arg Gly Phe Val Pro Phe Ala Pro Gly Ser 515 520 525 530 ccg aca gac cca gtt ggt gga aat ttg caa tgatggagat acagattgca 1991 Pro Thr Asp Pro Val Gly Gly Asn Leu Gln 535 540 aaaagtggtc ttggtgaggg aagagggcag ttttttggta atgaggttcc gttttcttta 2051 atgttaatag caagtgtggt taaaaagggg ttgtctagtt tataggtttt gcagatctca 2111 agtatattca tttgggtgat catgttttca gctcagttat tgcttttggt cattgctgac 2171 catcaatttc tgtggggaat tcctataggt tttctcccta acagttcttt tcttcatctt 2231 tttgcaattt atcgaaacac caaatgggtg tatattctgt aagcttgtgg catagctagc 2291 ttaattgtct tgtaaaattt cctacaggtt agagattggt tcttgatatg tagatttcat 2351 atgattgtaa cattcccatt tctcagaaaa gaaactataa tataaaattt ctggtggctg 2411 tcgcccgtgc tc 2423 <210> SEQ ID NO 15 <211> LENGTH: 540 <212> TYPE: PRT <213> ORGANISM: Petunia hybrida <220> FEATURE: <221> NAME/KEY: peptide <222> LOCATION: (1)..(540) <223> OTHER INFORMATION: Amino acid sequence of protein regulating the pH of vacuoles <400> SEQUENCE: 15 Met Ala Phe Asp Phe Gly Thr Leu Leu Gly Asn Val Asp Arg Leu Ser 5 10 15 Thr Ser Asp His Gln Ser Val Val Ser Ile Asn Leu Phe Val Ala Leu 20 25 30 Ile Cys Ala Cys Ile Val Ile Gly His Leu Leu Glu Glu Asn Arg Trp 35 40 45 Met Asn Glu Ser Ile Thr Ala Leu Val Ile Gly Ser Cys Thr Gly Ile 50 55 60 Val Ile Leu Leu Ile Ser Gly Gly Lys Asn Ser His Ile Leu Val Phe 65 70 75 80 Ser Glu Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe Asn 85 90 95 Ala Gly Phe Gln Val Lys Lys Lys Ser Phe Phe Arg Asn Phe Ser Thr 100 105 110 Ile Met Leu Phe Gly Ala Leu Gly Thr Leu Ile Ser Phe Ile Ile Ile 115 120 125 Ser Leu Gly Ala Ile Gly Ile Phe Lys Lys Met Asn Ile Gly Ser Leu 130 135 140 Glu Ile Gly Asp Tyr Leu Ala Ile Gly Ala Ile Phe Ser Ala Thr Asp 145 150 155 160 Ser Val Cys Thr Leu Gln Val Leu Asn Gln Asp Glu Thr Pro Leu Leu 165 170 175 Tyr Ser Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser Val 180 185 190 Val Leu Phe Asn Ala Ile Gln Asn Phe Asp Leu Ser His Ile Asp Thr 195 200 205 Gly Lys Ala Met Glu Leu Val Gly Asn Phe Leu Tyr Leu Phe Ala Ser 210 215 220 Ser Thr Ala Leu Gly Val Ala Ala Gly Leu Leu Ser Ala Tyr Ile Ile 225 230 235 240 Lys Lys Leu Tyr Phe Gly Arg His Ser Thr Asp Arg Glu Val Ala Ile 245 250 255 Met Ile Leu Met Ala Tyr Leu Ser Tyr Met Leu Ala Glu Leu Phe Tyr 260 265 270 Leu Ser Ala Ile Leu Thr Val Phe Phe Ser Gly Ile Val Met Ser His 275 280 285 Tyr Thr Trp His Asn Val Thr Glu Ser Ser Arg Val Thr Thr Lys His 290 295 300 Thr Phe Ala Thr Leu Ser Phe Ile Ala Glu Ile Phe Ile Phe Leu Tyr 305 310 315 320 Val Gly Met Asp Ala Leu Asp Ile Glu Lys Trp Lys Phe Val Ser Asp 325 330 335 Ser Pro Gly Ile Ser Val Gln Val Ser Ser Ile Leu Leu Gly Leu Val 340 345 350 Leu Val Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser Asn 355 360 365 Leu Thr Lys Lys Thr Pro Glu Ala Lys Ile Ser Phe Asn Gln Gln Val 370 375 380 Thr Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Met Ala Leu 385 390 395 400 Ala Tyr Asn Gln Phe Thr Arg Gly Gly His Thr Gln Leu Arg Ala Asn 405 410 415 Ala Ile Met Ile Thr Ser Thr Ile Thr Val Val Leu Phe Ser Thr Val 420 425 430 Val Phe Gly Leu Met Thr Lys Pro Leu Ile Arg Ile Leu Leu Pro Ser 435 440 445 His Lys His Leu Ser Arg Met Ile Ser Ser Glu Pro Thr Thr Pro Lys 450 455 460 Ser Phe Ile Val Pro Leu Leu Asp Ser Thr Gln Asp Ser Glu Ala Asp

465 470 475 480 Leu Glu Arg His Val Pro Arg Pro His Ser Leu Arg Met Leu Leu Ser 485 490 495 Thr Pro Ser His Thr Val His Tyr Tyr Trp Arg Lys Phe Asp Asn Ala 500 505 510 Phe Met Arg Pro Val Phe Gly Gly Arg Gly Phe Val Pro Phe Ala Pro 515 520 525 Gly Ser Pro Thr Asp Pro Val Gly Gly Asn Leu Gln 530 535 540 <210> SEQ ID NO 16 <211> LENGTH: 2553 <212> TYPE: DNA <213> ORGANISM: Nierembergia hybrida <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)..(2553) <223> OTHER INFORMATION: Nucleotide sequence of DNA encoding for protein regulating the pH of vacuoles <400> SEQUENCE: 16 aattattatt atttctctcc aactctcatt tctcagtttg ttgtgacttt ttcagagctt 60 gaagttcagt taattcattt tccaatatat tgattgtttt catttgagcg cgagaggatt 120 tcgtcttctc aatctgcttt caaatccttt ttgtttgtga tattcgatat tattcactca 180 gtttacctta atatttcctc gcactttctg aattcgagtg ctttgaagtg tgttggattt 240 cgaaaagcgg aagaaaattc agcaaaaacg ctgttgctga atttgcagca gtttgagttt 300 ttgctaaata gctaagatct gattgaattt ttcactggtg cttataggga aattcgacgt 360 cgttttgact gcaatatttg tccgtgattc ggactttgtt gaaattttgc tatttgaaat 420 ttgaatgtaa ggttgtcata gctttgcca