WO1997035984A1

WO1997035984A1 - Plant phosphate transporter gene and method of regulating plant growth with said gene

Info

Publication number: WO1997035984A1
Application number: PCT/JP1997/000975
Authority: WO
Inventors: Norihiro Mitsukawa; Satoru Okumura; Yumiko Shirano; Daisuke Shibata
Original assignee: Mitsui Plant Biotechnology Research Institute
Priority date: 1996-03-25
Filing date: 1997-03-24
Publication date: 1997-10-02
Also published as: AU1945097A; JP3474882B2

Abstract

A gene encoding a phosphate transporter protein has been isolated from Arabidopsis thaliana and its structure has been elucidated. By integrating the gene into a highly active expression vector to effect transformation into cultured tobacco cells, it has become possible to remarkably enhance the phosphate absorption by the transgenic cultured tobacco cells and to increase the growth rate of the cells. This technique is expected to be widely applicable to plants.

Description

Description Plant phosphate transporter gene and use of the gene

How to control plant growth

The present invention relates to the field of genetic engineering. In particular, it relates to the production of useful transgenic plants using genetic engineering.

Plant growth, such as germination, flower bud formation, and flowering, can be said to be controlled by various environmental factors such as daylight hours, hormonal conditions, temperature, and nutritional conditions. Therefore, it is possible to regulate the growth of plants by regulating these environmental factors, and various techniques have been considered based on this idea.

For example, to control the daylight hours, short-day plants such as chrysanthemums and kalanchoe are illuminated after sunset or midnight to suppress flowering. Techniques for blocking and promoting flowering are being implemented in flower cultivation. In order to control the temperature conditions, the early flowering time has been applied to lilies and tulips by low-temperature treatment. Furthermore, as a means of regulating hormonal conditions, the promotion of flowering by gibberellin treatment for long-day plants has been put to practical use. (The above is the cultivation and management of flowers, Yokendo, Seitaro Yokogi, Hiroshi Watanabe, Hiroshi Watanabe, )), It is known that administration of a gibberellin biosynthesis inhibitor is effective in suppressing growth, and at the same time, increases the photosynthetic rate of plants (Hitoshi Sekimoto, Journal of Soil Fertilizer Science, 66 (1995) 681-681). 682)). However, there were drawbacks such as limitations on applicable plant species and the need for labor and materials for implementation. In addition, since the technology is for flowers and horticultural crops, it is virtually impossible to apply it to large-scale farmland and forests. It is generally known that nutrient conditions are regulated by excessively applying nitrogen fertilizer to suppress flowering or to promote flowering by administering nitrogen when nitrogen is deficient ( Walter R., Flowering Physiology and Cultivation of Horticultural Plants, Seibundo Shinkosha (1978)) In Arabidopsis, flowering is delayed by reducing the total nutrient dose (Myerscough PJ "et al., 72). (1973) 595-617), and it is considered that general rules do not hold for nutrients and flowering time.

In addition, with the recent development of genetic engineering technology, it has been reported, to a small extent, that plant growth is controlled by introducing genes into plants and transforming plant traits.

For example, transformation of tobacco with horseradish peroxidase has been used to increase the growth rate of tobacco and, consequently, to accelerate the flowering time of tobacco (Yoshida K. et al. Abstracts 6th International Meeting on Arabidopsis Research, (1995) p.337). However, when transformed with Arabidopsis thaliana beloxylase, similar results cannot be obtained, and its application range is narrow.

On the other hand, the genes for the inorganic phosphate transport protein localized in the plasma membrane are expressed in yeast (Bun-ya M. et al. Mol. Cell. Biol. 11, 3229-3238 (1991)) and erawaw. WK Gene 153, 135-139 (1995)) and VA mycorrhizal fungi (Harrison MJ et al. Nature, 378, 626-629 (1995)) confirmed the function of the gene product. Furthermore, a partial cDNA showing homology with the amino acid sequence of yeast inorganic phosphate transposase was isolated in rice (GenBank D25132 and D25087) and Arabidopsis (GenBank Z33763), and cDNA was isolated from potato. (Leggewie G. et al. Abstracts of 10th International Workshop on Plant Membrane Biology (1995) p.R32).

However, the structure of the phosphate transport protein gene in plants has not been clarified, and the function of the protein encoded by the isolated cDNA has not been confirmed. <<Disclosure of the Invention

Phosphate is an important nutrient for plants and is closely related to plant growth, so isolating the gene and elucidating its structure is important in establishing technologies to control plant growth. It will be a great step. Furthermore, if the gene can be introduced into a plant and expressed in the plant, it will be an important technique for controlling plant growth.

Therefore, an object of the present invention is to isolate a novel phosphate transport protein gene and elucidate its structure. Another object of the present invention is to introduce the gene into a plant to improve the ability of the plant to absorb phosphate, thereby increasing the growth of the plant. As a result of intensive research, we succeeded in isolating the gene for the phosphate transport protein from Arabidopsis, and succeeded in elucidating the structure.

Furthermore, the present inventors have remarkably enhanced the phosphate absorption capacity of the transformed tobacco cultured cells by incorporating the gene into a high expression vector and transforming the tobacco cultured cells, and Was successfully increased.

That is, the present invention

(1) Using a plant-derived protein that has the function of transporting phosphate from outside to inside a cell by utilizing the actively created hydrogen ion concentration gradient inside and outside the cell

D N A,

(2) the protein of any one of SEQ ID NOs: 9 to 13, or a protein in which a part of the amino acid sequence of the protein has been mutated by deletion, substitution, or insertion of another amino acid Therefore, a protein that has the function of transporting phosphate from outside the cell to the inside of the cell by utilizing the actively created hydrogen ion concentration gradient inside and outside the cell DNA,

(3) the DNA of (1) or (2), which encodes a protein derived from Arabidopsis thaliana;

(4) A vector containing the DNA according to any one of (1) to (3),

(5) a plant cell into which the DNA according to any one of (1) to (3) has been introduced,

(6) a protein described in any of SEQ ID NOs: 9 to 13 or a protein in which a part of the amino acid sequence of the protein has been mutated by deletion, substitution, or insertion of another amino acid; A protein that has the function of transporting phosphate from outside the cell to the inside of the cell by utilizing the active hydrogen ion concentration gradient inside and outside the cell.

(7) A plant containing a cell into which the DNA according to any one of (1) to (3) has been introduced.

(8) A method for producing a transformed plant, comprising the steps of: introducing the DNA according to any one of (1) to (3) into a cell; and producing a plant from the transformed cell.

(9) A method for promoting phosphate uptake in a plant or a plant cell, comprising introducing the DNA according to any one of (1) to (3) into a plant cell.

The amino acid sequence of IPT1 is shown in SEQ ID NO: 9, the amino acid sequence of IPT4 is shown in SEQ ID NO: 10, the amino acid sequence of IPT3 is shown in SEQ ID NO: 11, and the amino acid sequence of IPT2 is shown in SEQ ID NO: 12. SEQ ID NO: 13 describes the amino acid sequence of IPT5.

The methods required for general gene recombination such as DNA cleavage and ligation, E. coli transformation, gene sequencing, hybridization, etc. described below are commercially available reagents and machinery used for each operation. And the like, and the description in an experimental manual (for example, see “Molecular Cloning (Maniatis T. et al., Cold Spring Harbor Laboratory Press)”).

1) Isolation and identification of phosphate transporter gene The DNA (eg, cDNA and genomic DNA) of the present invention may be, for example, a nucleotide sequence encoding a phosphate transporter or a deduced amino acid sequence of a phosphate transporter (eg, SEQ ID NOs: 1 to 6). A PCR method using a pair of oligonucleotides as primers, or a method based on the sequence or amino acid sequence described above. It can be isolated from plant cells, for example, plant tissues of Arabidopsis thaliana, by the hybridization method using a lignonucleotide as a probe. It can also be prepared by the following method. For example, mRNA was extracted from Arabidopsis thaliana plants two weeks after germination without excessive administration of phosphate, cDNA was synthesized using reverse transcriptase, and double-stranded by polymerase reaction. Is inserted into a commercially available vector to transform Escherichia coli, thereby preparing a cDNA library. Probes used for screening include partial-length cDNAs of transgenic plants such as Arabidopsis registered in sequence databases such as GenBank and DDBJ, and Arabidopsis Biological Resource Centerj (in the US, Ohio State University). Or using a pair of oligonucleotide primers based on the partial length cDNA sequence to amplify the DNA fragment by the PCR method using Arabidopsis chromosome DNA etc. .

Then, by colony hybridization or black hybridization using the obtained probe, a clone positive for hybridization is selected from the prepared cDNA library to prepare the DNA of the present invention. It is possible to manufacture. In addition, the base sequence can be appropriately determined by a conventional method such as the Maxam-Gilbert method or the Sanger method.

The nucleotide sequences of the DNA of the present invention thus obtained are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The gene shown in SEQ ID NO: 1 was named IPT1, and the gene shown in SEQ ID NO: 2 was named # 4. Translation product of the gene of SEQ ID NO: 1 (in SEQ ID NO: 9 Described proteins) include yeast (Bun-ya M. et al. Mol. Cell. Biol. 11, 3229-3238 (1991)), Neurospora crassa (Versaw WK Gene 153, 135-139 (1995)), and VA bacteria. Shows 34%, 34%, and 43 homology with the amino acid sequence of the phosphate transporter gene isolated from the root fungus (Harrison MJ et al. Nature, 378, 626-629 (1995)), respectively. Was.

The translation product of the gene of SEQ ID NO: 2 (the protein described in SEQ ID NO: 10) is 32%, 30%, and 40%, respectively, of the amino acid sequence of the yeast transport protein of yeast, Akapankabi, and VA mycorrhizal fungi. Showed homology.

Furthermore, once the cDNA is obtained, the hybridisation method using these cDNA sequences is used to obtain DNA from the chromosomal DN DN library prepared using the P1 phage vector, λ phage vector, etc. The phosphotransport gene can be isolated from the chromosomal DNA by PCR using chromosomal DNA and the like with a pair of oligonucleotide primers based on the PCR.

The sequences of the thus obtained DNA fragment containing the gene for the Arabidopsis phosphate transporter are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. Further, Escherichia coli (92C10 XLl-blue) carrying the P1 genomic clone (92C10) containing the DNA fragments of SEQ ID NO: 3 and SEQ ID NO: 4 obtained in the below-described Examples was isolated on March 14, 1996. 〒 305, Tsukuba City, Ibaraki Prefecture, Japan 1-3-1, Higashi Tsumo International deposit with the Institute of Biotechnology and Industrial Technology, Ministry of Industry and Industry (Accession No. FEHM BP-5474).

Note that a region that controls the expression of the gene of the present invention is included upstream of the coding region of the gene of the present invention. These regions include at least a part of the sequence represented by base numbers 1 to 1944 of SEQ ID NO: 3, and at least a part of the sequence represented by base numbers 1 to 2560 of SEQ ID NO: 4. Region, a region containing at least a part of the sequence represented by base numbers 4249 to 9110 of SEQ ID NO: 4, base numbers 1 to 18 of SEQ ID NO: 5 Replacement sheet (Rule 26) 6/1

A region containing at least a part of the sequence represented by 75, base numbers 1 to 19 of SEQ ID NO: 6 Replacement paper (Rule 26) P97 / 00975

A region including at least a part of the sequence represented by 80 is exemplified.

<2> Preparation of phosphate transporter protein

The protein of the present invention can be prepared as a recombinant protein by, for example, inserting the DNA of the present invention into an appropriate vector, introducing the vector into a host, and purifying from the host.

Examples of the host used for producing the protein of the present invention include Escherichia coli and yeast.

The vector used for producing the protein of the present invention is not particularly limited. For example, if the host is rspodoptera frugiperdaj, “pBacPAK8” (manufactured by Clontech) may be used, and if the host is a strain derived from Escherichia coli BL21. For example, “pET vector series” (Stratagene) and pESP-1 (Stratagene) when the host is yeast SP-Q01.

Purification of the recombinant protein of the present invention from the transformant thus obtained is performed, for example, when the protein of the present invention is expressed as a fusion protein with a label such as 6X histidine- ゃ -glu-thione S-transferase. The labeling can be performed by affinity chromatography for the label.

It is common practice for those skilled in the art to obtain a functional equivalent of the protein of the present invention by appropriately substituting amino acids in the protein of the present invention described in SEQ ID NOs: 9 to 13. Therefore, a part of the amino acid sequence of the protein described in SEQ ID NOs: 9 to 13 has an amino acid sequence mutated by deletion, substitution, insertion of another amino acid, etc. A protein having a function of transporting phosphate from the outside of a cell to the inside of a cell by utilizing the hydrogen ion concentration gradient is also included in the scope of the present invention. Conventional methods include, for example, site-directed mutagenesis (for example, Zoller, M.J. and Smith, M. (1983) Methods in Enzymology 100, p468).

In addition, "Phosphoric acid is converted using the active hydrogen ion concentration gradient inside and outside the cell. The function of transporting from outside the cell to the inside of the cell "can be detected, for example, by the method described in the literature (Berhe, A., et al. Eur. J. Biochem. 227, 566-572 (1995)). is there.

<3> Preparation of transgenic plant into which the gene of the present invention has been introduced

All or a part of the DNA of the present invention is ligated so as to be transcribed in the forward direction with a promoter that can be expressed in plants, such as the CaMV (cauliflower mosaic virus) 35S promoter. By transforming a plant with recombinant DNA containing the protein, it is possible to produce a plant that expresses the phosphotransport protein, and it is also possible to produce so-called co-repression (a gene encoding a protein is transcribed in the forward direction). (A method in which the expression of the endogenous protein is suppressed by introducing a recombinant DNA) to produce a plant in which the expression level of the protein is suppressed. One example of a vector used for plant transformation is a vector such as PBI121.

Further, all or a part of the DNA of the present invention is bromo-ligated so as to be transcribed in the reverse direction, and a recombinant DNA containing the DNA is transformed into a plant to express so-called antisense RNA, Plants in which the amount of phosphate transport protein expression is suppressed can be produced.

The gene of the present invention can be introduced into, for example, herbaceous dicotyledonous plants such as Arabidopsis tobacco, monocotyledonous plants such as rice and corn, and woody plants such as lucifera poplar.

Transformation of plant cells can be carried out by conventional methods such as particle gun method, electoral poration method (electroporation method), and agropacterium infection method such as vacuum infiltration. This can be done by introducing DNA into the body.

Transformed plants or transformed plant cells incorporate a drug resistance gene for kanamycin or hygromycin into the recombinant DNA used for transformation, and are cultivated or cultured in a solid agar medium or liquid medium containing these drugs. To do It is possible to maintain more stable. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the analysis of the copy number of the Arabidopsis thaliana phosphate transporter overnight gene. The IPT1 gene cDNA (pIPTC19) was used as a probe and washed under conditions that hybridize with the homolog gene.

FIG. 2 shows a restriction enzyme map of an Arabidopsis thaliana phosphate transporter gene genomic clone. From the top, the restriction maps of the IPT1, IPT3, and IPT2, IPT4, and IPT5 genes are shown. The box indicates the position of the code area.

Figure 3 shows the location of the phosphate transporter gene on the Arabidopsis chromosome. Each gene sits at the position of the arrow. The numbers at the top indicate chromosome numbers, and the horizontal lines indicate the positions of known RFLP markers.

FIG. 4 shows the expression analysis of the phosphate transport gene (IPT1) in each organ of Arabidopsis thaliana. The tissue from which RNA was extracted is shown at the top, and the position of the transcript is indicated by an arrow (→).

FIG. 5 shows a transformation construct containing the phosphate transbo overnight gene (IPT1 gene). The upper panel shows the restriction enzyme map and gene structure of the IPT1 gene. The black portions indicate the untranslated transcription regions, and the white boxes indicate the coding regions. The lower part shows the structure of the T-DNA region of P35S-IPT1. pnos indicates nopaline synthase gene promoter, ΝΡΤΠ indicates neomycin phosphotransferase (kanamycin resistance gene), and tnos indicates nopaline synthase gene terminator.

FIG. 6 shows the relationship between the expression level of the phosphate transgenic protein and the phosphate absorption rate. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to Examples. It is not limited to 10.

[Example 1] Isolation of Phosphotransport Yuichi cDNA clone

A part of the Arabidopsis thaliana cDNA (GenBank Z33763) showing homology to the yeast inorganic phosphate transporter gene (PH084) (Bun-ya M. et al. Mol. Cell. Biol. 11, 3229-3238 (1991)). Based on the base sequence, an oligonucleotide primer was synthesized based on the base sequence (synthesis was outsourced to Glyna Japan). Hind III was added to the primer on the fifth side, and a Pst I recognition site was added to the primer on the third side. The array of Brimers —

2854F1: 5, -AAAGCTTCAAAACTATGGCTTGTTCTC -3, (5 'side primer)

2854R1: 5,-ACTGCAGAACTGTGAACCAGTACCCTG-3, (3, side primer) (sequence numbers: 7 and 8, respectively). As a template, "AmpliTaq DM" was used using genomic DNA prepared from the roots of Arabidopsis thaliana (Columbia strain) by the urea-phenol method described in the literature (Cell, 35 (1983)).

PCR (Polymerase Chain Reaction) was performed using (Perkin-Elmer) according to the protocol attached to the enzyme. After the amplified PCR product is cut with Hind III and Pst I, it is cloned into Hind III and Pst I of pBluescript II SK + (Stratagene) (hereinafter, this cloned product is called “pIPT-PCil”). The nucleotide sequence was determined from both ends using "ABI 373A DM sequencer ^ (Perkin-Elmer)" according to the attached instructions. As a result, a 251 bp PCil product and a partial cDNA of Arabidopsis thaliana were determined. It was confirmed that 248 bp of the nucleotide sequence was matched.

Arabidopsis inorganic phosphate transporter (hereinafter simply referred to as "IPT") cDNA clones of genes uses the "Arabidopsis cDNA Library in the Uni-ZIP ™ XR V ectorj (Stratagene , Inc.), 4.0 × 10 ⁵ pieces of recombinants The hybridization was performed using the insert (251 bp) of pIPT-PCR as a probe.The hybridization was performed according to the method of Liu et al. (Liu Y.-G. et al. Theor. Apple. Genet. 84, 535-543 (1992)). The washing conditions were as follows: 0.1X SSC, 0.1, SDS, 62 ° C. / 00975

11 cDNA clones were obtained (hereinafter, this clone is referred to as “pIPTC19”) (SEQ ID NO: 1).

In addition, screening was performed using the EcoRI-XhoI fragment of PIPTGZ7, a genomic clone of the IPT4 gene described below, as a probe. According to the method of Liu et al., The hybridization was washed under the conditions of 0.1X SSC, 0.1 ° SDS, and 65 ° C, and positive clones were selected to obtain 5 positive clones. Among these clones, there was no full-length cDNA clone, but a clone on the 5 'side (hereinafter, this clone is referred to as “pIPTC402”) and a clone on the third side (hereinafter, this clone is referred to as “pIPTC403”). As a result, the entire open reading frame (hereinafter, referred to as “0RF”) could be covered (SEQ ID NO: 2).

[Example 2] Isolation of phosphate transporter genomic clone

For isolation of genomic clones, Arabidopsis (Columbia strain) P1 genomic library (Liu Y.-G. et al. Plant J. 7, 351-358 (1995)) and Arabidopsis thaliana (Colombia strain) ZapII Genome library-1 (Stratagene) was used.

For screening of the P1 genomic library, DNA of 10,000 strains of Escherichia coli holding the P1 clone was immobilized on a nylon membrane to prepare a filter. The Insato of PIPT-PCR was labeled with ³² P with respect to the fill evening one using a probe, Liu et al. Method (Liu Y.-G. et al. Theor. Apple. Genet. 84, 535-543 ( 1992)). The washing conditions were 0.1X SSC, 0.1¾ SDS, 62 ° C, and the signal was detected with “Bio Image Analyzer I BAS2000” (manufactured by Fuji Photo Film Co., Ltd.). As a result, one positive clone (hereinafter, this clone is referred to as “92C10”) was obtained.

For screening of the ZapII genomic library, 400,000 entered phages were infected with the E. coli host XL1-Blue (Stratagene), thereby plaque-forming, and transferred to a nylon membrane. Probes use pIPT-PCR inserts W

12 Screening conditions were hybridization at 55 ° C, 0.2X SS 0.1¾ SDS, 42. Washing was performed under the conditions of C. As a result, four positive clones (hereinafter, these clones are referred to as “pIPTGZl”, “pIPTGZ2j”, “pIPTGZ4”, and “pIPTGZ7”) were obtained. Determination of the nucleotide sequence of the ends of the clones and analysis of the restriction enzyme fragments revealed that these clones were genomic clones of different genes that did not overlap with each other.

However, since these clones did not contain the entire gene, screening was performed again using the PIPTC19 insert as a probe. The screening conditions were hybridization at 55 ° C, and washing was performed under conditions of 0.2X SSC, 0.1% SDS and 42 ° C to select positive clones. Screening about 200,000 plaques resulted in 10 positive clones.

From the nucleotide sequence determination of the clone ends and the analysis of restriction enzyme fragments, two clones (GZ33 and GZ37) showed one inorganic phosphate transporter gene (hereinafter simply referred to as “IPT1 gene”) and one clone (GZ36) Of the inorganic phosphate transporter gene (hereinafter simply referred to as “ΙΡΤ2 gene j”), 6 clones (GZ34, GZ35, GZ38, GZ39, GZ40, GZ41) One clone (GZ32) was a genomic clone of another inorganic phosphate transporter gene (hereinafter, simply referred to as ΠΡΤ5 gene). Also, P1 clone 92C10 contained three of the four IPT genes described above (IPT1, ΙΡΤ2, ΙΡΤ3).

Since a full-length clone of PIPTGZ7 could not be obtained, screening was again performed on about 160,000 plaques of the human ZapI I genomic library using the EcoRI-XhoI fragment of PIPTGZ7 as a probe. Hybridization was performed at 55 ° C, washing was performed under the conditions of 0.1X SSC, 0.1 0SDS, and 65 ° C, and positive clones were selected. As a result, one positive clone (PIPTGZ20) was isolated.

In addition, genomic Southern printing was performed using IPT1 as a probe (Fig. 1). 13 Genome Southern blotting was performed on Arabidopsis thaliana Columbia strains (lane 1, 3, 5, 7, 9) and Landsberg erecta strain (lane 2, 4, 6, 8, 10). DNA prepared from BamHI (lanes 1, 2), Dral (lanes 3, 4), EcoRI (lanes 5, 6), EcoRV (lanes 7, 8), and Hindi II (lanes 9, 10) went. Both ends in the figure are size markers. As a result, it was confirmed that Arabidopsis thaliana IPT has a large number of homologous genes (Fig. 1).

This suggests that the IPT gene forms a multigene family composed of at least five or more types of genes. Figure 2 shows the restriction map of the whole genome clone.

[Example 3] Analysis of phosphate transport Yuichi genomic clone

For the IPT1 gene, the entire nucleotide sequences of PIPTGZ33 and pIPTGZ37 were determined. SEQ ID NO: 3 shows the determined nucleotide sequence of the IPT1 gene. Comparison with the nucleotide sequence of pIPTC19, which is the IPT1 gene cDNA clone described in SEQ ID NO: 1, shows that the 5 'untranslated region and the intron reading frame (hereinafter referred to as "0RF") have two introns. There was.

For the IPT2 gene and the IPT3 gene, the entire nucleotide sequences of pIPTGZ34, pIPTGZ36, pIPTGZ38 and pIPTGP2 were determined. Note that pIPTGP2 is a subclone obtained by inserting a fragment of about 7.5 kb obtained by cutting the insert of 92C10 with Pstl into pBluescript. The determined nucleotide sequences of the IPT2 gene and the IPT3 gene are shown in SEQ ID NO: 4. As expected, each had at least one intron at each location. The 0RF of IPT2 was located in the same direction downstream of the 0RF of IPT3, and the distance between the ORFs of both genes was about 5 kb.

For the IPT4 gene, the base sequence of pIPTGZ20 was determined for the portion including the upstream of the coding region and the entire 0RF. The nucleotide sequence of the determined IPT4 gene is shown in SEQ ID NO: 5. No intron was found in the translated region, but an approximately lkbp intron was present in the untranslated region. The upstream of the coding region was about 2 kbp.

For IPT5, the entire nucleotide sequence of pIPTGZ32 was determined. The base of the determined IPT5 gene The 14 sequence is shown in SEQ ID NO: 6. No cDNA has been isolated for the IPT5 gene, but at least two introns may be present in 0RF. The upstream of the coding region of the PIPTGZ32 clone was about 2 kb.

The amino acid sequence of each gene deduced from the analysis of cDNA and genomic clones was compared with the amino acid sequence of the phosphoric acid transport gene of yeast, A. niger and VA mycorrhizal fungi. The homology of the amino acid sequences of the Arabidopsis thaliana genes to the yeast and A. niger genes was around 30%. The amino acid sequences of the Arabidopsis thaliana showed homology of about 75% or more. In particular, the homology between IPT1 and IPT2 was high, at 98¾.

[Example 4] Chromosome mapping of phosphate transporter gene

Although Arabidopsis has five chromosomes, chromosome mapping was performed to determine the distribution of the five isolated IPT genes on the chromosome.

In order to perform chromosome mapping, it is necessary to detect RFLP (restriction fragment length polymorphism), and its probes include three genes (IPT1, ΙΡΤ2, ΙΡΤ3) contained in P1 clone 92C10. ), The insert of pIPT-PCR was used. For IPT4 gene and IPT5, P1 clone was isolated and used again. The isolated P1 clones were named 83A1 (IPT4 gene) and 88C10 (IPT5 gene). Using these probes, genomic DNA prepared from Columbia and Landsberg etrecta strains was digested with restriction enzymes, electrophoresed, and a filter was used to transfer the DNA to a nylon membrane. Restriction enzyme fragments showing polymorphism between both strains were searched. A 100-line RI line (F8 generation crossing a Colombian strain and a Ransberg erecta strain) cut with a restriction enzyme showing RFLP (Lister et al., Plant J, 4 (1993) 745-750) filter was used. Genotype of each RI line was determined. Using the separated data, `` Mapmaker Macint osh V2.0 '' (Lander et al. Genomics, 1 (1987) 174-181), LOD score 6.0, rKosambi mapping function j (osambi D, D, Ann, Eugen, 12 ( 1944) 172-175) (This was analyzed. 15 As a result, the three IPT genes (IPT1, ΙΡΤ2, ΙΡΤ3) are located on the lower arm of chromosome 5, and the ΙΡΤ4 and ΙΡΤ5 genes are located on the lower arm of chromosome 2. Then t. The distance between the two genes, IPT4 and IPT5, was 5.4 cM (Figure 3). This locus does not match the locus of pho2 (Delhaize E. et al., Plant Physiol, 107 (1995) 207-213), which has been reported as an absorption mutant that over-absorbs phosphate. It was close.

[Example 5] Expression analysis of phosphate transporter gene in plant tissue Among the genes of the present invention, cDNA was isolated, and Northern blot analysis was also performed on the IPT1 遗 gene that is surely expressed in plants. Was done. RNA was prepared from the roots, rosette leaves, stems, cowline leaves, and flowers of Arabidopsis thaliana grown for 6 weeks, electrophoresed, and transferred to a nylon membrane to prepare a fill. For this filter, using the insert of PIPTC19, which is the cDNA of the IPT1 gene, as a probe, hybridization was performed, and washing was performed under conditions of 0.1X SSC, 0.1¾SDS, and 65 ° C. Transcripts were detected. As a result, the IPT1 gene was specifically expressed in the root (FIG. 4).

[Example 6] Relationship between expression level of phosphate transporter gene and phosphate absorption rate

Ά

(1) Production of transformed cultured cells

The construct for overexpression (P35SIPT1) was obtained by inserting the Sstl l-EcoRI fragment of pKI121 (pBI121 (Clonetech)) into the Sstl and EcoRI sites of pGA643 (Pharmacia), and the intermediate product The GUS gene of the Brassmid constructed by inserting the Sail fragment of pACYC177 (accession no. The blunted Sac I-Hind 111 fragment (including exon 2, intron 2, and exon 3) of the genomic clone was ligated (Fig. 5) ο

To analyze the function of the phosphate transport gene, the coding region of the IPT1 gene containing the second intron was placed downstream of the cauliflower mosaic virus 35S (CaMV35S) promoter. 16 and transferred to cultured tobacco BY-2 cells. Gene transfer was performed using a particle gun, and the vector PKI121 was also introduced into cultured cells as a control. Transformants were selected on a medium containing kanamycin to obtain a large number of transgenic strains. In order to select a cultured cell line in which the introduced gene was expressed, 16 P35SIPT1-introduced lines were cultivated in turbidity and Northern analysis was performed using IPTlcDNA as a probe. Of the 16 lines analyzed, 10 lines (P2, P3, P10, Pll, P20, P27, P29, P33, P34, P38) showed mRNA expression. The PKI121-introduced cultured cells were stained for GUS activity. Of the 30 lines analyzed, the GUS gene was functional in 12 calli, and 40% of the kanamycin-resistant calli were found to have introduced the adjacent GUS gene so that the adjacent GUS gene was also expressed normally. Of these, pKI121-introduced cultured cells of 6 lines (C3, C8, Cll, C16, C18, C23) were subjected to turbid culture.

(2) Maintenance of tobacco cultured cells and measurement of phosphate absorption rate

The transformed tobacco BY-2 cultured cells were cultured in suspension, and 100 ml of a modified MS medium (2.5 mM pho sphate, 0.1 mg / il myo-inositol, 3¾ sucrose, pg / mithiamine-HC1, 0.2 μg / g Rotational culture at 25 ° C and 100 pm in ml 2,4-D and 2 ^ g / ml glycine, 50 ig / ml kanamycine, pH 5.7), and subculture 3 ml of a saturated culture solution every 7 days did. Cultured cells used for the measurement of phosphate absorption were obtained by transferring the cells 7 to 9 days after the passage into the above medium without kanamycin to a new MS liquid medium and having passed 23 to 27 hours. Cell culture and more attracted to the natural sedimentation, free phosphoric acid medium containing squeaking de cyclohexane of 100〃M (5mM MES-Tris (pH6.0) , 20.6mM NH, N0., 18.8mM 3.0mM CaCb · H 2 0 , 1.5 mM MgSO. · H! 0, 100 iM cycroheximide, 3% sucrose) 4 times. Imagine the above medium to a final concentration of about 0.1 lg / ml, 9.5 ml cells! 2 hours Nigoeki in 5 Οιη 1 brass tick tube, 25 ⁰ C, and pre-cultured at 200 rpm. The reaction was started by adding 0.5 ml of 2 mM phosphoric acid containing 50 zCi "P-phosphate (500 mCi / mol final concentraton, manufactured by Amersham) to a final concentration of 100 µM. Immediately, remove cells with a 0.22〃m centrifugal filtration tube Ultrafree C3GV (UFC30GVOO, manufactured by Millipore) and filter the filtrate. W 984 JP97 / 00975

The amount of Pi in the medium was measured by liquid scintillation counting (Beckman), and the amount of Pi absorbed by the cultured cells was calculated. The cell concentration (g fwt / ml) of the cell suspension was measured using a 0.45 im centrifugal filtration tube (UFC40HV25, manufactured by Millipore) from 2 ml of the cell suspension after the absorption measurement, and the wet weight was measured. Was calculated. In addition, to analyze the relationship between the gene expression level and the phosphate absorption rate, the phosphate absorption rate was measured, and the expression of the introduced phosphate transpo-yuichi gene was performed by Northern analysis (Fig. 6). The phosphate absorption rate of the cultured cells was measured at the initial phosphate concentration of 100 M in pre-culture in a high phosphate medium and in the presence of a protein synthesis inhibitor. As a result, the phosphate absorption rate of the overexpressing cells was 193 to 596 nmol Pi / h / g fwt, and the absorption activity was correlated with the expression level of IPTlmRNA. The phosphate absorption rates of the control cells ranged from 101 to 203 nniol Pi / h / g fwt (Fig. 6).

[Example 7] Awakening of Pfl harmful agent against increase in phosphate absorption activity due to expression of IPT1 gene

The endogenous phosphate-absorbing activity of cultured tobacco cells and the activity of the Arabidopsis thaliana phosphate transporter—an inhibitor of phosphate-absorbing activity increased by overexpression of the Yuichi gene IPT1—were analyzed (Table 1). In addition, among the following substrates, except for “phosphoric acid (100 // Μ)” and “phosphoric acid (2 mM)”, the phosphoric acid concentration was 100 zM.

18

Relative absorption of phosphoric acid)

PKI121 P35SIPT1 None 100 100

2, 4-D 0 17 at 100 ί / Μ

CCCP 15 12

100 zM Getyl Still Vestrol 24 18

Phosphoric acid (100〃M) 39 39

Phosphoric acid (2mM) 10 9

100〃M arsenate 63 61

2 mM arsenate 21 20

As a result, both 2,4-dinitrophenol (2,4-DNP) and cyanide m-clomouth phenylhydrazone (CCCP), which are protonophores, strongly inhibited phosphate absorption. Stilditrol, an inhibitor of plasma membrane H + -ATPase activity, also showed strong inhibition. Phosphoric acid and its analog, arsenate, inhibited the absorption of phosphate to a similar extent in all cells. These results indicate that the phosphate absorption mechanism of the Arabidopsis thaliana phosphate transporter gene IPT1 gene product is cotransportation of proton and phosphate, and that the activity of the IPT1 gene product is dependent on the endogenous phosphorylation of cultured tobacco cells. It was shown that the acid absorption activity and the response to the inhibitor were similar.

[Example 8] Effect of IPT1 gene transfer on growth of cultured tobacco cells

One week after passage, the cultured cells of the stationary leaf aids are filtered with a cell strainer (Cell Strainer), and 0.5 g of the cells are weighed into a 60-thigh disposable dish. This was subcultured in a modified MS medium (-Km, 100 ml). After 24 hours, the culture was filtered through a cell filter, washed with 10 ml of a phosphate-free MS medium (phosphate-starved medium), weighed and filtered into a phosphate-starved medium (-Km , 100ml). Subculture was performed by flowing 15 ml of the medium from the opposite side of the cell strainer into the flask. Thereafter, the weight of the cell strainer was measured again to calculate the amount of cells that had been transferred. After culturing in a phosphate-starved medium for 4 days, the cell weight was measured by the following method. Set “ADVANTEC” (made by T0Y0) ^ .3 filter paper (55 thighs) in the filter holder (glass filter base), moisten the filter paper with 10 ml of fresh phosphoric acid starvation medium, and aspirate and then moisten the filter paper. The weight of the filter paper was measured. The filter paper was set again, and the culture solution cultured for 4 days was subjected to suction filtration, and the weight of the filter paper was measured. From this value, the cell growth amount (wet weight) was calculated. Further, the weight of the filter paper after drying overnight in a desiccator was measured, and the weight of the dried filter paper was subtracted to obtain the dry weight. In addition, the measurement was performed on the growth amount of the cells under the phosphate starvation state for two lines (Pll, P38) and one control line (C3) of the cells into which the phosphate transfer gene was introduced. As a result, there was no significant difference between the wet weight after the first day of treatment with a normal medium rich in phosphate and that of the phosphate transfer gene-transfected cells ′ control cells. However, on day 4 after the passage of the phosphate-deficient medium, the phosphate transfer gene-transferred strain was about 40% higher in both wet weight and dry weight compared to the control strain (Table 2).

20 Table 2 Initial weight of cell line (a> 4) (b) Ratio (b / a)

(g) (g) (g)

Pll 0.70 ± 0.05 1.52 ± 0.01 2.19 0.15 ± 0.00 P38 0.80 ± 0.08 1.65 ± 0.05 2.07 0.16 ± 0.00

C3 0.73 ± 0.04 1.09 ± 0.01 1.51 0.10 ± 0.01

It is considered that the amount of phosphoric acid absorbed before the phosphate starvation treatment affected the cell growth amount after the phosphate starvation treatment. From studies on conventional cultured cells, it is known that the increase in the weight of cultured cells and the amount of nitrogen and carbon absorbed are in parallel. It is also known that phosphate absorption rapidly absorbs phosphoric acid in the culture medium at the initial stage of growth and does not absorb phosphate during the latter half of growth. In the transformant produced in this example, the phosphate absorption rate was remarkably increased, so that when the transformant was cultured for 24 hours in a medium containing a normal amount of phosphate, more phosphate was obtained than in the control cells. It was considered that this absorbed and accumulated, and this reflected the growth amount after transplantation to the phosphate-starved medium. According to this example, it was found that it is possible to increase the growth amount of a plant by manipulating one gene.

[Example 9] Production of overexpressing transformed plant

As the transformation method of Arabidopsis thaliana, p35S-IPTl and pKI121 were introduced using "improved vacuum infiltration method" (Shirano, unpublised, Bechtold et al., 1993). Transformants were grown for 2 weeks on 0.8 Paga plate containing 1/2 B5, 1/1000 HYP0Nex (pH 5.7) containing 75 μg / ml kanamycin, and resistant individuals were selected. Plants are grown at 20 ° C or 23 ° C, 16L / 8D or `` continuous light j with 1/1000 HYPONex. 21 Industrial Applicability

According to the present invention, a phosphate transport protein gene of Arabidopsis thaliana was isolated and its structure was elucidated. Further, according to the present invention, it has become possible to increase the phosphate absorption capacity of tobacco cultured cells by introducing the Arabidopsis phosphate transport protein gene into tobacco cultured cells. Therefore, according to the present invention, for example, it is possible to improve the growth of a plant in a poorly phosphate-neutral soil such as an acidic soil with a small amount of phosphoric acid in a form usable by the plant. In the natural environment where the environment is rapidly changing, the amount of phosphoric acid in the soil is constantly changing.However, plants into which the gene of the present invention has been introduced rapidly absorb and accumulate phosphate in the presence of phosphoric acid. Therefore, according to the present invention, it is possible to produce a plant that is resistant to environmental changes. In the present invention, it has been found that the Arabidopsis phosphate transfer protein gene functions in cultured tobacco cells, and thus the present invention is expected to be widely applied to plants.

22 Sequence Listing SEQ ID NO: 1

Array length: 1755

Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: cDNA to iRNA

Origin

Organism name: Arabidopsis thaliana

Stock Name: Colombia

Array features

Characteristic symbol: CDS

Location: 55.. 1626 sequence

CTCTCCAGAG AAGTTCTTAA TTTCTCCTGC CAAGCTGATT AAGAGCTCTA GGAA ATG 57

Met

1

GCC GAA CAA CAA CTA GGA GTG CTA AAG GCA CTC GAT GTT GCG AAG ACG 105 Ala Glu Gin Gin Leu Gly Val Leu Lys Ala Leu Asp Val Ala Lys Thr

5 10 15

CAA CTT TAT CAT TTC ACG GCG ATT GTC ATC GCC GGT ATG GGT TTC TTT 153 Gin Leu Tyr His Phe Thr Ala lie Val lie Ala Gly Met Gly Phe Phe

20 25 30

ACC GAT GCC TAC GAT CTT TTT TGC GTG TCC TTG GTG ACG AAA CTC CTT 201 991

MI m ^ ΐο ^ 19 W BIV BIV BIV 3Π 8Md

S8S DXX 31V 103 010 loo oiv αοο IDO VOO OXV 3X1 IDO 039

091 991 091

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sXo Π9ΐ ¾iv usy FA STH ojj ojj

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S9 09 99 OS ojd sXq ¾iv J9S nio ojj usy sqd 丄 911 SJV ^ ίθ

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SL6O0 / L6d £ / lDd t ^ 86S £ / .6 OAV twenty four

GCT GGA GGT TTC GTG GCA CTC GCA GTA TCT TCT ATA TTC GAC AAA AAG 633 Ala Gly Gly Phe Val Ala Leu Ala Val Ser Ser He Phe Asp Lys Lys

180 185 190

TTC CCA GCT CCA ACA TAT GCA GTA AAC AGG GCC CTC TCA ACG CCT CCT 681 Phe Pro Ala Pro Thr Tyr Ala Val Asn Arg Ala Leu Ser Thr Pro Pro

195 200 205

CAA GTT GAC TAC ATT TGG CGA ATC ATC GTC ATG TTT GGT GCT TTA CCC 729 Gin Val Asp Tyr lie Trp Arg lie lie Val Met Phe Gly Ala Leu Pro

210 215 220 225

GCA GCT TTG ACT TAC TAC TGG CGT ATG AAG ATG CCT GAA ACT GCC CGT 777 Ala Ala Leu Thr Tyr Tyr Trp Arg Met Lys Met Pro Glu Thr Ala Arg

230 235 240

TAC ACC GCT TTG GTT GCC AAG AAC ATC AAA CAA GCC ACA GCC GAC ATG 825 Tyr Thr Ala Leu Val Ala Lys Asn lie Lys Gin Ala Thr Ala Asp Met

245 250 255

TCC AAG GTC TTA CAA ACA GAT ATC GAG CTT GAG GAA AGG GTG GAG GAT 873 Ser Lys Val Leu Gin Thr Asp lie Glu Leu Glu Glu Arg Val Glu Asp

260 265 270

GAC GTC AAA GAC CCC AAA CAA AAC TAT GGC TTG TTC TCC AAG GAA TTC 921 Asp Val Lys Asp Pro Lys Gin Asn Tyr Gly Leu Phe Ser Lys Glu Phe

275 280 285

CTT AGA CGC CAT 6GG CTT CAT CTC CTT GGA ACT ACC TCC ACA TGG TTT 969 Leu Arg Arg His Gly Leu His Leu Leu Gly Thr Thr Ser Thr Trp Phe

290 295 300 305

TTG CTT GAC ATT GCC TTC TAC AGC CAA AAC TTG TTC CAG AAG GAT ATT 1017 Leu Leu Asplie Ala Phe Tyr Ser Gin Asn Leu Phe Gin Lys Asp He 6 ^ ΐ VDX m X39 339 IVX VXD Oil 93911103D VOO XID IIV 33D V99 丄 :) 3

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Ala Gly Ala lie Val Gly Ala Phe Gly Phe Leu Tyr Ala Ala Gin Ser

450 455 460 465

CAA GAC AAG GCC AAG GTA GAC GCA GGA TAC CCA CCA GGC ATC GGA GTT 1497

Gin Asp Lys Ala Lys Val Asp Ala Gly Tyr Pro Pro Gly lie Gly Val

470 475 480

AAG AAC TCA TTG ATC ATG CTT GGT GTT CTT AAC TTT ATC GGT ATG CTC 1545 Lys Asn Ser Leu lie Met Leu Gly Val Leu Asn Phe lie Gly Met Leu

485 490 495

TTC ACC TTC CTT GTC CCA GAG CCC AAA GGC AAG TCC CTT GAA GAA CTC 1593 Phe Thr Phe Leu Val Pro Glu Pro Lys Gly Lys Ser Leu Glu Glu Leu

500 505 510

TCT GGT GAG GCT GAG GTT AGC CAT GAC GAG AAA TAATTATGTA TGTTTATTTT 1646 Ser Gly Glu Ala Glu Val Ser His Asp Glu Lys

515 520

GTTATTTGGA GTGCGATGTT TGGTTTTGTT TTCATTTTAT TGGCTCGTTG ACCTTAAGTT 1706 ATGATGTTAT AAGAATATTT ATGATAAAAA AAAAAAAAAA AAAAAAAAA 1755 SEQ ID NO: 2

Sequence length: 1766

Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: cDNA to mRNA

Origin

Organism name: Arabidopsis thai i ana

Stock Name: Colombia 001 36 06 jqi ^ 3W ^ 19 丄 ΐ¾ sl SJV ^ T9 naq s ^ i dsy 9 na cUx

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TTG ATG GTC ATG GTC CTT TGT TCA ATA GCC TCT GGT CTC TCT TTC GGA 390

Leu Met Val Met Val Leu Cys Ser lie Ala Ser Gly Leu Ser Phe Gly

105 110 115 120

CAT GAG CCA AAA GCT GTG ATG GCC ACG CTC TGT TTT TTT CGG TTT TGG 438

His Glu Pro Lys Ala Val Met Ala Thr Leu Cys Phe Phe Arg Phe Trp

125 130 135

CTT GGA TTT GGC ATC GGT GGT GAC TAC CCT TTA TCC GCA ACC ATC ATG 486 Leu Gly Phe Gly He Gly Gly Asp Tyr Pro Leu Ser Ala Thr He Met

140 145 150

TCT GAA TAT GCG AAC AAG AAG ACT CGC GGA GCC TTT GTC TCT GCG GTT 534 Ser Glu Tyr Ala Asn Lys Lys Thr Arg Gly Ala Phe Val Ser Ala Val

155 160 165

TTT GCT ATG C GGG TTC GGA ATC ATG GCT GGT GGT ATT TTC GCT ATT 582 Phe Ala Met Gin Gly Phe Gly lie Met Ala Gly Gly lie Phe Ala lie

170 175 180

ATA ATT TCC TCT GCT TTT GAA GCT AAG TTT CCA TCC CCG GCC TAT GCG 630 l ie l ie Ser Ser Ala Phe Glu Ala Lys Phe Pro Ser Pro Ala Tyr Ala

185 190 195 200

GAT GAT GCC TTG GGA TCC ACG ATT CCT C GCT GAT TTG GTA TGG CGG 678 Asp Asp Ala Leu Gly Ser Thr He Pro Gin Ala Asp Leu Val Trp Arg

205 210 215

ATA ATC CTG ATG GCT GGT GCT ATC CCT GCG GCT ATG ACG TAT TAC TCA 726 lie lie Leu Met Ala Gly Ala lie Pro Ala Ala Met Thr Tyr Tyr Ser

220 225 230

AGG TCG AAG ATG CCT GAG ACC GCA AGG TAC ACG GCT TTG GTT GCT AAG 774 Arg Ser Lys Met Pro Glu Thr Ala Arg Tyr Thr Ala Leu Val Ala Lys 29

235 240 245

GAC GCA AAG CAG GCA GCT TCG GAC ATG TCT AAG GTT CTG CAA GTG GAG 822 Asp Ala Lys Gin Ala Ala Ser Asp Met Ser Lys Val Leu Gin Val Glu

250 255 260

ATA GAG CCA GAA CAA CAG AAA TTG GAA GAG ATC TCA AAG GAG AAG TCC 870 lie Glu Pro Glu Gin Gin Lys Leu Glu Glu lie Ser Lys Glu Lys Ser

265 270 275 280

AAG GCC TTT GGA TTG TTC TCA AAG GAA TTC ATG AGT CGC CAT GGG CTT 918 Lys Ala Phe Gly Leu Phe Ser Lys Glu Phe Met Ser Arg His Gly Leu

285 290 295

CAT TTG CTA GGC ACT ACA TCG ACA TGG TTC CTT CTC GAC ATT GCT TTC 966 His Leu Leu Gly Thr Thr Ser Thr Trp Phe Leu Leu Asp He Ala Phe

300 305 310

TAC AGT CAA AAC CTT TTC CAA AAG GAT ATT TTC AGC GCG ATC GGA TGG 1014 Tyr Ser Gin Asn Leu Phe Gin Lys Asp lie Phe Ser Ala lie Gly Trp

315 320 325

ATT CCT CCC GCG CAA AGC ATG AAC GCA ATT CAA GAG GTT TTC AAG ATT 1062 l ie Pro Ala Gin Ser Met Asn Ala lie Gin Glu Val Phe Lys l ie

330 335 340

GCC CGT GCG CAA ACG CTA ATC GCC TTG TGT AGC ACG GTA CCT GGT TAC 1110 Ala Arg Ala Gin Thr Leu He Ala Leu Cys Ser Thr Val Pro Gly Tyr

345 350 355 360

TGG TTC ACA GTT GCG TTC ATC GAC GTC ATT GGA AGA TTT GCG ATT CAG 1158 Trp Phe Thr Val Ala Phe lie Asp Val lie Gly Arg Phe Ala lie Gin

365 370 375

ATG ATG GGT TTC TTT TTC ATG ACG GTC TTT ATG TTT GCT CTG GCT ATT 1206 02S 9Γ9 019 90S dsy nig usy nio s W nig ng jas sA ^ A \ Q s J9s nig

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AAT GAG AAT AGC AAC AAT GAT AGT AGA ACG GTC CCA ATA GTT 1632 Asn Glu Asn Ser Asn Asn Asp Ser Arg Thr Val Pro lie Val

525 530

TAGGTGATAT AATACGCCTT TTGTAATAAT TTTCGTTTTT TCTTTCTCCT TGTCTCTAGC 1692

AACTCAAGTT GTTCTTTGTG TAATCCATTG ATACCTAATT AATGCTAGAG AAATCAAAAA 1752

AAAAAAAAAA AAAA 1766 SEQ ID NO: 3

Array length: 5810

Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: Genomic DNA

Origin

Organism name: Arabidopsis thal iana

Array features

Characteristic symbol: CDS

Location: 2226 · · 2626 Character code: CDS

Location: 2778 3948 Characteristic code: exon

Location: 1945.. 1978 Characteristic symbol: intron 32 Location: 1979.. 2214 Characteristic code: exon

Location: 2215.. 2626 Characteristic symbol: intron

Location: 2627.. 2777 Characteristic symbol: exon

Location: 2778 ... 4054 sequence

GAATTCCCTG ATAAGTTTTT TTTCTTTACT TCTTAATCATCAT TGTTTCTTCA TAAAAACAAC 60 ATATGAAGGA TATGATAGAA ATTTGACTAA AAAAAACTTG ATGGATGAGT CTACGTACAT 120 ATATAAATGC AAGGAATATC ATATATAAAA ATTGCATTGA TCTTTGATGC TTTAATACTT 180

TTAGTGCTTT TTTTAAACAT AACCAATTTT ATATTTTGTT TGATCACACT TTTTTATACT 240

ATGAACATGT GTAAAGAGGC TTTTTTTATA TTATGAACAT GTGTAAAATA TCCTCTCTTT 300

TTTTTTTACT ATAAGCATGT GTTAAGTGGC CTTTTCTTTT TCTTTGCTTG TAGCCTTGTA 360

AATAATAGGA ATGCCCAAGG GCAAGTCCCT TGAAGAGCTC TCCGGTGAGG CTGAGGTTGA 420

GAAATGATTA CGCCATAATA TTTTCAAATT CTTAATTGGT TCGATGAATT ACATCTTCTA 480

TTACGATTTA CTTTATTTTT GCGATGCGTA ATGTGATATT TCATGTTCGT AATGGTGGAT 540

CTCTACATTT ATTTTTTTGT GTGACAAAAT GTAATCAATC GATATTTAGT GGTCATTTAT 600

GATATTTATT TGACTGCTTA TCTCTACTAC ACAGCTACTA TCAAATTCCT AATCTTTGTT 660

GTTATTATGG TTATATTTAA TTTATGCATG TAATACCTCT CGTATAACAA CAAAACATTC 720

AAACCATTTA AAACATTAAC GTGTACATAT CGAAGTCCAG AAAACCCGTC ATTTAAAACA 780

AATACAATCC TCACATTTAT ATATATATAT GGTCTGAGCT GGATTGGTTG ATATTAAATC 840 Three

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S m 600 / "df / 丄 0 J fr86SC / i6 OAV 35

G G TTT TGG TTG GGA TTT GGT ATT GGA GGT GAC TAC CCA CTT TCT GCC 2823 Phe Trp Leu Gly Phe Gly lie Gly Gly Asp Tyr Pro Leu Ser Ala

135 140 145

ACC ATC ATG TCT GAA TAC GCA AAC AAG AAG ACC CGT GGG GCT TTC ATC 2871 Thr He Met Ser Glu Tyr Ala Asn Lys Lys Thr Arg Gly Ala Phe lie

150 155 160 165

GCA GCT GTC TTC GCC ATG CAA GGT GTC GGT ATC TTG GCT GGA GGT TTC 2919 Ala Ala Val Phe Ala Met Gin Gly Val Gly lie Leu Ala Gly Gly Phe

170 175 180

GTG GCA CTC GCA GTA TCT TCT ATA TTC GAC AAA AAG TTC CCA GCT CCA 2967 Val Ala Leu Ala Val Ser Ser lie Phe Asp Lys Lys Phe Pro Ala Pro

185 190 195

ACA TAT GCA GTA AAC AGG GCC CTC TCA ACG CCT CCT CAA GTT GAC TAC 3015 Thr Tyr Ala Val Asn Arg Ala Leu Ser Thr Pro Pro Gin Val Asp Tyr

200 205 210

ATT TGG CGA ATC ATC GTC ATG TTT GGT GCT TTA CCC GCA GCT TTG ACT 3063 lie Trp Arg lie He Val Met Phe Gly Ala Leu Pro Ala Ala Leu Thr

215 220 225

TAC TAC TGG CGT ATG AAG ATG CCT GAA ACT GCC CGT TAC ACC GCT TTG 3111 Tyr Tyr Trp Arg Met Lys Met Pro Glu Thr Ala Arg Tyr Thr Ala Leu

230 235 240 245

GTT GCC AAG AAC ATC AAA CAA GCC ACA GCC GAC ATG TCC AAG GTC TTA 3159 Val Ala Lys Asn l ie Lys Gin Ala Thr Ala Asp Met Ser Lys Val Leu

250 255 260

CAA ACA GAT ATC GAG CTT GAG GAA AGG GTG GAG GAT GAC GTC AAA GAC 3207 Gin Thr Asp He Glu Leu Glu Glu Arg Val Glu Asp Asp Val Lys Asp 36

265 270 275

CCC AAA CAA AAC TAT GGC TTG TTC TCC AAG GAA TTC CTT AGA CGC CAT 3255 Pro Lys Gin Asn Tyr Gly Leu Phe Ser Lys Glu Phe Leu Arg Arg His

280 285 290

GGG CTT CAT CTC CTT GGA ACT ACC TCC ACA TGG TTT TTG CTT GAC ATT 3303 Gly Leu His Leu Leu Gly Thr Thr Ser Thr Trp Phe Leu Leu Asplie

295 300 305

GCC TTC TAC AGC CAA AAC TTG TTC CAG AAG GAT ATT TTC TCG GCC ATC 3351 Ala Phe Tyr Ser Gin Asn Leu Phe Gin Lys Asp lie Phe Ser Ala lie

310 315 320 325

GGA TGG ATC CCA AAG GCA GCC ACC ATG AAC GCC ACC CAT GAG GTT TTC 3399 Gly Trp lie Pro Lys Ala Ala Thr Met Asn Ala Thr His Glu Val Phe

330 335 340

AGG ATT GCT AGG GCT CAG ACT CTT ATC GCC CTT TGC AGT ACA GTC CCA 3447 Arg He Ala Arg Ala Gin Thr Leu lie Ala Leu Cys Ser Thr Val Pro

345 350 355

GGC TAC TGG TTC ACA GTT GCG TTT ATT GAT ACC ATT GGA AGG TTT AAG 3495 Gly Tyr Trp Phe Thr Val Ala Phe lie Asp Thr lie Gly Arg Phe Lys

360 365 370

ATC CAA CTA AAT GGA TTT TTC ATG ATG ACC GTG TTT ATG TTT GCC ATT 3543 He Gin Leu Asn Gly Phe Phe Met Met Thr Val Phe Met Phe Ala He

375 380 385

GCC TTC CCT TAC AAC CAC TGG ATC AAA CCA GAA AAC CGT ATC GGA TTT 3591 Ala Phe Pro Tyr Asn His Trp lie Lys Pro Glu Asn Arg lie Gly Phe

390 395 400 405

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AACATATATT AACTAATTGA CGAAAAATGT GTGGGTTCAA CATCCCGCAA CGAATAAAAT 5778 CTTTTGACAA TGATTCATAA ACATAGGAAT TC 5810 SEQ ID NO: 4

Sequence length: 12987

Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: Genomic DNA

Origin

Animal name: Arabidopsis thaliana

Stock Name: Colombia

Array features

Characteristic symbol: CDS

Location: 2561.. 2961

Other information: IPT3 Characteristic symbol: CDS

Location: 3087 .. 4248

Other information: IPT3 Characteristic symbol: CDS

Location: 9111.. 9511

Other information: IPT2 Characteristic symbol: CDS 40 Location: 9662.. 10832

Other information: IPT2 Characteristic symbol: intron

Location: 2962.. 3086 Characteristic symbol: intron

Location: 4249.. 9110 sequence

GCTTTTAAAA AATTTACAAA GATTTTTAAA AGCATTTTTT TGGCTGGGAA AAAAATTTTA 60 CGAGAAAACA TTTTTGGCGG GAAATTTTTT TTTTGGCGGG AAAAAAAGTT TGGCGGAAAT 120 TTTTTGTTTG GCGGGAAAAA TAAATTTTGG TACATGTTAT TATTAAATGA GGGATT

GTCATTCTGT TCAATAGAAG GGATATTTTT AAAAATAAAC AATATAAAAT GGTATTGTTA 240

CAAAAGGGTA GTAAAAAAAG GGTAGTTTTG CAAATCTCCC TCGAGAATTT GCAATTTCTC 300

TCTCATAGCA TAACATGTCT TTTTTTTAGT AAATTTCAAG ATGACAAAAA GAAAAAAAAA 360

ACATAAAGAA ACAAATTGAA AGCAACATCT ATTTTTCTGA CTTGTAAAAA TGTGGTGTTA 420

CTAGCAAATA TCAAATTTTT GTATCTATAA ACTAGATTTT AACCCGCGGT ATACTAGGAG 480

AACGATTTAT TTTTTAAAGT TAATATATAT ACAAGTTTAC AAATTGAATA TATTTATAAA 540

AAAAATAAAT TTTTTAGTTT ACAATTATTA TTAGGTAACA TCCCGCCAAA TCTGTTCCAT 600

CAAAAAGCTT ATTAATTTTA TTAATGTTAA CCTTAGTTAT GATATGATTC TAAATCATTG 660

TCTAGATATT TTAGCCAATG TTAGGACGTT GAACCCACAT AGTTTTCGTC AATTGAATAA 720

TATATGTTCG TTTTATAAAT TTCGAATCAC AATTTATGCA GAAAATGTTT TGAAATTTTT 780

CTTAACHTA CATATTATAC AATAAAAAAT CAAACAAAGA TGATAGGAAA TTCATAGTTT 840

TAAATCTTAA TCAAATCTTC TAAATATCAA TATTGTTAAA TATAGAAGTA ATGAGTATAA 900

GAGTTGGGTT TTATTTGAAC CAACCCAACT AAGATTTAAT GAACATCATA AACTAATGGG 960 M & A 6 S

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120 125 130

AG GTACAATTTA TTTAGCCACA AACCTAATAT CACATACGTC ACAGATACAA 3011 Arg

GCTCGAGAGA TTAGTCACTA TTTCGACCTA GATTATGGTT ACTTAAGATA CTGATATCTA 3071 GACGATTATA TATAG G TTT TGG CTC GGG TTT GGC ATT GGA GGT GAC TAC 3120

Phe Trp Leu Gly Phe Gly lie Gly Gly Asp Tyr

135 140 145

CCT CTA TCT GCC ACC ATC ATG TCT GAA TAC GCT AAC AAG AAG ACT CGT 3168

Pro Leu Ser Ala Thr lie Met Ser Glu Tyr Ala Asn Lys Lys Thr Arg

150 155 160

GGG GCT TTC ATC GCG GCA GTG TTC GCC ATG CAA GGT GTA GGT ATC TTG 3216

Gly Ala Phe He Ala Ala Val Phe Ala Met Gin Gly Val Gly He Leu

165 170 175

GCG GGA GGT TTT GTG GCA CTT GCA GTT TCT TCC ATA TTT GAC AAA AAG 3264

Ala Gly Gly Phe Val Ala Leu Ala Val Ser Ser lie Phe Asp Lys Lys

180 185 190

TTC CCA TCG CCG ACG TAT GAG CAA GAC AGG TTT CTA TCA ACG CCT CCT 3312

Phe Pro Ser Pro Thr Tyr Glu Gin Asp Arg Phe Leu Ser Thr Pro Pro

195 200 205

CAA GCT GAT TAC ATT TGG CGA ATC ATC GTC ATG TTT GGT GCT TTA CCC 3360

Gin Ala Asp Tyr lie Trp Arg lie lie Val Met Phe Gly Ala Leu Pro

210 215 '220 225

GCA GCT TTG ACT TAC TAT TGG CGT ATG AAG ATG CCT GAA ACA GCC CGT 3408

Ala Ala Leu Thr Tyr Tyr Trp Arg Met Lys Met Pro Glu Thr Ala Arg

230 235 240

TAC ACC GCT TTA GTT GCC AAG AAC ATC AAA CAA GCC ACA GCA GAC ATG 3456 Three

44

Tyr Thr Ala Leu Val Ala Lys Asn He Lys Gin Ala Thr Ala Asp Met

245 250 255

TCC AAG GTC TTA CAA ACA GAT CTC GAG CTT GAG GAA AGG GTG GAG GAT 3504 Ser Lys Val Leu Gin Thr Asp Leu Glu Leu Glu Glu Arg Val Glu Asp

260 265 270

GAC GTC AAG GAC CCC AAA AAA AAC TAT GGC TTG TTC TCC AAG GAA TTC 3552 Asp Val Lys Asp Pro Lys Lys Asn Tyr Gly Leu Phe Ser Lys Glu Phe

275 280 285

CTT AGA CGC CAT GGG CTT CAT CTC CTT GGG ACT ACC TCC ACT TGG TTT 3600 Leu Arg Arg His Gly Leu His Leu Leu Gly Thr Thr Ser Thr Trp Phe

290 295 300 305

TTG CTT GAC ATC GCC TTC TAC AGC CAA AAC TTG TTC CAA AAG GAT ATT 3648 Leu Leu Asp lie Ala Phe Tyr Ser Gin Asn Leu Phe Gin Lys Asp lie

310 315 320

TTC TCG GCC ATT GGA TGG ATC CCA AAG GCA GCC ACT ATG AAC GCC ATC 3696 Phe Ser Ala lie Gly Trp He Pro Lys Ala Ala Thr Met Asn Ala He

325 330 335

CAT GAG GTT TTC AAG ATT GCT AGG GCT CAG ACT CTC ATT GCC CTC TGC 3744 His Glu Val Phe Lys He Ala Arg Ala Gin Thr Leu lie Ala Leu Cys

340 345 350

ACT ACA GTC CCA GGT TAC TGG TTC ACA GTA GCC TTT ATT GAT ATC ATT 3792 Ser Thr Val Pro Gly Tyr Trp Phe Thr Val Ala Phe lie Asp lie lie

355 360 365

GGA AGG TTT GCG ATC CAA CTA ATG GGA TTT TTC ATG ATG ACC GTT TTT 3840 Gly Arg Phe Ala He Gin Leu Met Gly Phe Phe Met Met Thr Val Phe

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M3TTTTiAA TA GCGMMTA¾ GAAAGGCC AA ATTTGCAG_

MAA GCGTC CTTTAAA TTAA 44GTGGT.A CTA AACTA58GC T2TATGGTC ATACTAGTTGTCI

ATGCGAGT AAT GGTTATATT GM ATAT 43TG GTTATTCTTT AATGTGGT98CG ACCTG CTTTTTG_- GAAT TTTAAT TATCTTT 43ATA TTAT38TTACTCTT.ΊΤ TCG iG-

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ATAGTTGTTT ATATCAATAG AAACTTTTGT TTCTAAATTG TAATCGCTTA AATTATTTGA 8958 TTTATGTATA GGTTTAATTA TAAATGTTAT TCTGTTGGTT GTATTGGTAC TAAACTAAAA 9018 TAATTTAGTA GCATCTTTCC AATATTATTA TAAAATTATA GTTAAGATAT GATCGA AGGA GATCATA GAGA GATCATA GAGA GAGA TGA

Met Ala Glu Gin Gin Leu Gly

1 5

GTG CTA AAG GCA CTC GAT GTT GCG AAG ACG CAA CTT TAT CAT TTC ACG 9179 Val Leu Lys Ala Leu Asp Val Ala Lys Thr Gin Leu Tyr His Phe Thr

10 15 20

GCG ATT GTC ATC GCC GGT ATG GGT TTC TTT ACC GAT GCG TAC GAT CTT 9227 Ala lie Val lie Ala Gly Met Gly Phe Phe Thr Asp Ala Tyr Asp Leu

25 30 35

TTT TGC GTG TCC TTG GTA ACA AAG CTC CTT GGC CGC ATC TAC TAC TTC 9275 Phe Cys Val Ser Leu Val Thr Lys Leu Leu Gly Arg lie Tyr Tyr Phe

40 45 50 55

AAT CCG GAG TCA GCG AAG CCT GGC TCA CTT CCC CCT CAT GTT GCG GCC 9323 Asn Pro Glu Ser Ala Lys Pro Gly Ser Leu Pro Pro His Val Ala Ala

60 65 70

GCT GTC AAT GGT GTG GCC CTT TGT GGA ACC CTT TCT GGT CAA CTC TTC 9371 Ala Val Asn Gly Val Ala Leu Cys Gly Thr Leu Ser Gly Gin Leu Phe

75 80 85

TTC GGT TGG CTC GGT GAC AAA CTC GGA CGG AAA AAA GTG TAC GGT CTT 9419 Phe Gly Trp Leu Gly Asp Lys Leu Gly Arg Lys Lys Val Tyr Gly Leu

90 95 100

ACT TTG ATC ATG ATG ATC TTA TGC TCT GTC GCT TCT GGC CTC TCT TTT 9467 Thr Leu lie Met Met He Leu Cys Ser Val Ala Ser Gly Leu Ser Phe OJd% 3 s SJV dJX

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225 230 235

GAA ACT GCC CGT TAC ACC GCT TTG GTT GCC AAG AAC ATC AAA CAA GCC 10019 Glu Thr Ala Arg Tyr Thr Ala Leu Val Ala Lys Asn He Lys Gin Ala

240 245 250

ACA GCC GAC ATG TCC AAG GTC TTA CAA ACA GAT ATC GAG CTT GAG GAA 10067 Thr Ala Asp Met Ser Lys Val Leu Gin Thr Asp lie Glu Leu Glu Glu

255 260 265

AGG GTG GAG GAT GAC GTC AAA GAC CCC AGA CAA AAC TAT GGC TTG TTC 10115 Arg Val Glu Asp Asp Val Lys Asp Pro Arg Gin Asn Tyr Gly Leu Phe

270 275 280 285

TCC AAG GAA TTC CTT AGA CGC CAT GGA CTT CAT CTC CTT GGA ACT ACC 10163 Ser Lys Glu Phe Leu Arg Arg His Gly Leu His Leu Leu Gly Thr Thr

290 295 300

TCC ACT TGG TTT TTG CTT GAC ATT GCC TTC TAC AGC CAA AAC TTG TTC 10211 Ser Thr Trp Phe Leu Leu Asp lie Ala Phe Tyr Ser Gin Asn Leu Phe

305 310 315

CAG AAG GAT ATT TTC TCG GCC ATC GGA TGG ATC CCA AAG GCA GCC ACC 10259 Gin Lys Asp lie Phe Ser Ala He Gly Trp lie Pro Lys Ala Ala Thr

320 325 330

ATG AAC GCC ACC CAT GAG GTT TTC AGG ATT GCT AGG GCT CAG ACT CTT 10307 Met Asn Ala Thr His Glu Val Phe Arg He Ala Arg Ala Gin Thr Leu

335 340 345

ATC GCC CTT TGC AGT ACA GTC CCA GGC TAC TGG TTC ACA GTT GCG TTT 10355 He Ala Leu Cys Ser Thr Val Pro Gly Tyr Trp Phe Thr Val Ala Phe

350 355 360 365

ATT GAT ACC ATT GGA AGG TTT AAG ATC CAA CTA AAT GGA TTT TTC ATG 10403 S09 009 96 ^

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GTTAACATTT AAAATTATGA TTTTTCTATC ATTTTTTTTA TTTTTTAAAC TACTTTGAAT 12272

CATCATATAA TACATAACAC TAATTAAAAT CTAATTTTTT CTGGATATAT TGTGATTCGG 12332

ATGTTTGAAA ACAGCCATAT ATTACTCACA TAATGAAATA AAATTATTTG TGTTCAAAGT 12392

CTCACATCAG CTAAAACTTT GTGTCCAAAA TCTCATAAAT AAGACCAAAT TGAACCCAAA 12452

GAAATATGAG TGAGTAGGAA TCCTAACTTA ACAAACATCC AAATTTAAAT ATTTGTTCAG 12512

TTTAATTCGG TTCTTTAGTC AAACCGTTTA ATTATACCAG ATATCATAAA TTCTAAACTT 12572

TTTAAAAAAT TAAAATATTA TAATTGATCT ATTTAAAGTG TATAAAAACT TCAAAATATT 12632

ATCAACGTTT AACTTATAAA AAGTTTAAAA TATCACGGTA TGGGTTATAT GGTTTGACGA 12692

GTTTGAATCT TCATTAATAT AAATTTAAAT ATCATTAACT CGGTGGTACA ACATGGGTAA 12752

GTCATCATTT TAATATTTTG TTAACACTAG CAATATTATA ATGATATGTC AAGTTAAATT 12812

GATTCAATTA TGATTATGTA TTAAAACAAG ATATATAAGT ATTAGTTTTC TAGGTATCTG 12872

CAATATTAGA AACCAGATAT CCAAAAGTCA CGGATCTGGA TTGTTAAAGC ACAGTTCTGA 12932

ATACTTTTCA AATTCTAGGA TATCCGGATC CATTCTACCA ATTACAATCC TGCAG 12987 SEQ ID NO: 5

Array length: 4864

Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: Genomic DNA

Origin

Organism name: Arabidopsis thai i ana

Stock Name: Colombia

Array features

Characteristic symbol: CDS

Location: 3004 · · 4605 55

Characteristic symbol: exon

Location: 1876 .. 1893 Characteristic symbol: intron

Location: 1894 .. 2991 Characteristic symbol: exon

Location: 2992 .. 4724 Sequence

GAATTCCTAC AATGTTGAAT AAACGTAGGT AGTGGCTACT TAATTTCTTC GATTTCTTAA 60 GTGCTTAGTA CTTTTCAACA TTAAAAATGT TGTTACCAAG TCTAAATTTT CTTCACAACT 120 TGTAACTAAA CTTTTCATTA TGTGTAATCG TAAAGGATTA GCGCTACAAA TAGATGGTGA 180

TTCCCTTCTA ATGGACGAGT TGACATTGAC GCATTATGTC TCTGGTTAGC TAGTCCGACG 240

TTTGAACAAG TACTCTTACC GCTCTCGAAA CAAAATTAAA ACCAAAATTT TATAGATCTA 300

TTAGTAAAAT CTACTATTGT TAATTTTATC ACATAGTCCA TGTGTGTGTT AATATTAAGG 360

ATGAAGTCAA TGTATATATA TATATCAAAT CTCTATTCCT ACTAGATATG GGAATCACCT 420

ACTTGTATAA ATGGCAAACT CATTCAACGA GCTACACACG ACTTTTCCAA CTTATTTCAG 480

TGTTTGAGAT CATTTTAATG CAACAACTAT ATGTTAAAGG GAAATTGGTC TAGAAGCGGC 540

TATTTCTTGG TCTTGAAATC ATATTGTTCT TCTATAGTGT AGTGACATTT CCTATAATTA 600

ATTTGAAAAA AGGAAGAAAT TGTGTTGGCA ATGAAAACAT CATATGTATG GTGTGAAGTA 660

TATACAAAAA AAAAATCCCA TTCGTGAATG AAAACTACGG TGTATATATG TGAAAGACAT 720

ATATGGAGCC TTCACTATAC GGTGTAGTTC ATTTACATAA GAATGGTTGG AAATGGAGAT 780

GCCATATTTT TTTTATTTTT TTTTTCCACA ATGGAGATGC CATATCTATA AAAAAAGAAA 840

AGAGGTTGAA CTAGTTGGGT CGGCGCGACG AAAAGAGAAA ATACAACTTG CTGGGCTAAA 900 TCAGTGTCTTCTAC CAAG AT

GTAATGTGTT TGCAAC AATG

T 234AGTAA0T ACTCTCATG TCGC

—3

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GCA AGG TAC ACG GCT TTG GTT GCT AAG GAC GCA AAG CAG GCA GCT TCG 3768 Ala Arg Tyr Thr Ala Leu Val Ala Lys Asp Ala Lys Gin Ala Ala Ser

240 245 250 255

GAC ATG TCT AAG GTT CTG CAA GTG GAG ATA GAG CCA GAA CAA CAG AAA 3816 Asp Met Ser Lys Val Leu Gin Val Glu lie Glu Pro Glu Gin Gin Lys

260 265 270

TTG GAA GAG ATC TCA AAG GAG AAG TCC AAG GCC TTT GGA TTG TTC TCA 3864 Leu Glu Glu lie Ser Lys Glu Lys Ser Lys Ala Phe Gly Leu Phe Ser

275 280 285

AAG GAA TTC ATG AGT CGC CAT GGG CTT CAT TTG CTA GGC ACT ACA TCG 3912 Lys Glu Phe Met Ser Arg His Gly Leu His Leu Leu Gly Thr Thr Ser

290 295 300

ACA TGG TTC CTT CTC GAC ATT GCT TTC TAC AGT CAA AAC CTT TTC CAA 3960 Thr Trp Phe Leu Leu Asp lie Ala Phe Tyr Ser Gin Asn Leu Phe Gin

305 310 315

AAG GAT ATT TTC AGC GCG ATC GGA TGG ATT CCT CCC GCG CAA AGC ATG 4008 Lys Asp lie Phe Ser Ala lie Gly Trp lie Pro Pro Ala Gin Ser Met

320 325 330 335

AAC GCA ATT CAA GAG GTT TTC AAG ATT GCC CGT GCG CAA ACG CTA ATC 4056 Asn Ala He Gin Glu Val Phe Lys He Ala Arg Ala Gin Thr Leu He

340 345 350

GCC TTG TGT AGC ACG GTA CCT GGT TAC TGG TTC ACA GTT GCG TTC ATC 4104 Ala Leu Cys Ser Thr Val Pro Gly Tyr Trp Phe Thr Val Ala Phe He

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GAG GAA ATG TCC GGT GAA AAT GAA GAC AAT GAG AAT AGC AAC AAT GAT 4584 Glu Glu Met Ser Gly Glu Asn Glu Asp Asn Glu Asn Ser Asn Asn Asp

515 520 525

AGT AGA ACG GTC CCA ATA GTT TAGGTGATAT AATACGCCTT TTGTAATAAT 4635 Ser Arg Thr Val Pro lie Val

530

TTTCGTTTTT TCTTTCTCCT TGTCTCTAGC AACTCAAGTT GTTCTTTGTG TAATCCATTG 4695 ATACCTAATT AATGCTAGAG AAATCAAAAT TTTCATGAGT CGATTTTAAA TCAGCTCCTA 4755 AATTGAAGAT TTATTAGACT TGACGAAGCC GCGCAAGTTG TGCATGACGATCATACGATCATGACCATGCATCATGACT

Sequence length: 4249

Sequence type: nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: Genomic DNA

Origin

Organism name: Arabidopsis thal iana

Stock Name: Colombia

Array features

Characteristic symbol: CDS

Location: 1677.. 1980 Characteristic symbol: CDS 62 Location: 2158.. 2630 Characteristic code: CDS

Location: 2729.. 3577 Characteristic symbol: intron

Location: 1981.. 2157 Characteristic symbol: intron

Location: 2631.. 2728 array

GAATTCCGGC ATGTGGCTAT AAAAACCCCA ATTCATGTGT CAAACAACCA GTTAACCATT 60 ACCATAGAGG TTGTGAAAAA ATTACCAGGT GTGCTCGTGA TGCTGCTCGT TATACTGAAT 120 CCTTCAATGT CGATGACGAT GAAAGTCCCA TTATTAATCT CCATTGAAAT TTAATCTTTA 180

ATTTACTTTC GTTGATACTA CAAAGATTCA TGTATTTATA CGTCGTGTAG CTTGAATATA 240

TTCATATACC TAATTATCAG TCTATTAAAA TGTGAAAGAT TATATTTTAT TAATTTGTGT 300

TTTTATGTAA AGTCACTAAT TGTTGTCTTA AATTTTTATG TATTCTATGC TTTTCCTGAG 360

ATTATTTATT TATTTGTTTA TAATTTTTTT TTAATTAAAA AATAAATTAT GTGTGACTAC 420

TCTTTCTCTA GATATGCCTT AAGCAATAAA AGGGGGAAAA ACATTTTTTG ATGACAAATT 480

AGTTTGTCAT ATGAATGAAA TTGTGGTTTA GATGAATTTC ATCCAAGAAT TCTTAATAGA 540

ACCGAAAATC TCTTTAAGTT TTAACATAAT CAACAAAGAT AAATTCTATG AACATTCCTA 600

ATATTTCCTT TAAGATTACT CAATGAATTC TTGTCCGAGC GCTTGCATTT GTCTAATCAT 660

CAACATAGAC GAATCATACA TTTGCTTGCG GCTAACGTCA CCTTCCTTTC CTCCTTCCTC 720

TTCGAAGAGC CTACCATCCA ATATACTCAG CACATAGACA AAAATCTATA TGAGTTACAA 780

AAATTCATCC ATTGAGTTGT TACAATTTAA TTTGATTGAA AATTGTTCAT TAGACATTAG 840 C73 • -3> C¾

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TAAGT TATATTTTTGAATATAT TTTTTCTGGGCAAT T TTTT TTAGcCG

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AAT GCG ACG ACG TTT GTT GTT CCT GCT GAG ATA TTT CCG GCG AGG TTG 3265 Asn Ala Thr Thr Phe Val Val Pro Ala Glu lie Phe Pro Ala Arg Leu

425 430 435

AGA TCG ACT TGT CAT GGG ATA TCG GCG GCG AGT GGT AAG GCG GGA GCG 3313 Arg Ser Thr Cys His Gly lie Ser Ala Ala Ser Gly Lys Ala Gly Ala

440 445 450

ATT GTT GGA GCG TTT GGG TTT CTT TAC GCG GCG CAG AGT TCG GAT TCG 3361 l ie Val Gly Ala Phe Gly Phe Leu Tyr Ala Ala Gin Ser Ser Asp Ser

455 460 465 470

GAG AAG ACT GAT GCT GGT TAT CCT CCT GGG ATT GGT GTT AGG AAT TCG 3409 Glu Lys Thr Asp Ala Gly Tyr Pro Pro Gly lie Gly Val Arg Asn Ser

475 480 485

TTG TTG ATG CTT GCT TGT GTT AAT TTC TTG GGG ATT GTT TTT ACT CTG 3457 Leu Leu Met Leu Ala Cys Val Asn Phe Leu Gly lie Val Phe Thr Leu

490 495 500

TTG GTT CCG GAG TCT AAA GGG AAG TCT TTG GAA GAG ATT TCG AGA GAA 3505 Leu Val Pro Glu Ser Lys Gly Lys Ser Leu Glu Glu lie Ser Arg Glu

505 510 515

GAT GAG GAA C AGC GGC GGA GAT ACG GTG GTG GAG ATG ACT GTG GCA 3553 Asp Glu Glu Gin Ser Gly Gly Asp Thr Val Val Glu Met Thr Val Ala

520 525 530

AAT TCC GGT AGA AAA GTC CCG GTT TGATTATTTT GTGTTACTCA TTTTGTAAAT 3607 Asn Ser Gly Arg Lys Val Pro Val

535 540

TTACTCTATA TATGAAAGTC CCTTGCATAA AGTTTTTCGA TACTCAATAT ATCAGAATAA 3667 CAGTTTTGCT GGAACTTTGC AACCAAATTA CTCTGTGAAT TGGAAAGTAT TATCTTACTT 3727 68

CTTCCTATAA AGCACCAGTG ATCTTTTTGT AAAGAGATTC ATAAGTCTAT TGGTTTATGG 3787

AACAATGCCT TCACATGCAA GTCATATTAT TTGTCCAGAC GACGAAGAGG AAGAATTTGT 3847

TGAACTAGAA AGCAGTGGCG ATAGAATCGT TCATTAAGAT AAATGGTTCG TATAAAACCA 3907

TTAGGATAAA TTCTATGTTT TGTGTTTCAT CATTGAATAT CTACCAGACC AAATGATTTT 3967

ATTTGGTTTT AGTTTCTTTT GTAAATCCCA AAAAAGGTAA AGTTATATCA AAACCAAACT 4027

ACAAACCAGA TGTTAATCCC TATTGTATTA ACCACAAACA TGTTACCAGA ATATATCAAA 4087

AGTGTATCAG TTTCAGTTTG CTACTAAGTA AAGACTTTCA TCTTTGTGTT GCTTAGGCTT 4147

CAAGTTCGTT TTATCTTGTC TTCTCTGTTT TACTCTGCTT TATGATGATG ATAAATAACG 4207

ATGGAACACA AGAGAGAAGA GCTCCATTAT GCACAAGAAT TC 4249 SEQ ID NO: 7

Array length: 27

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Other nucleic acid Synthetic DNA

Array

AAAGCTTCAA AACTATGGCT TGTTCTC 27 SEQ ID NO: 8

Array length: 27

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Other nucleic acid Synthetic DNA

Array SZI QZ \ 9Π

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6: ½§½2Ι a 9X003VI DVD3W9I9I DWOVOOXOV

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Leu Cys Phe Phe Arg Phe Trp Leu Gly Phe Gly lie Gly Gly Asp Tyr 130 135 140 145

Pro Leu Ser Ala Thr He Met Ser Glu Tyr Ala Asn Lys Lys Thr Arg

150 155 160

Gly Ala Phe He Ala Ala Val Phe Ala Met Gin Gly Val Gly lie Leu

165 170 175

Ala Gly Gly Phe Val Ala Leu Ala Val Ser Ser lie Phe Asp Lys Lys

180 185 190

Phe Pro Ala Pro Thr Tyr Ala Val Asn Arg Ala Leu Ser Thr Pro Pro

195 200 205

Gin Val Asp Tyr l ie Trp Arg lie He Val Met Phe Gly Ala Leu Pro 210 215 220 225

Ala Ala Leu Thr Tyr Tyr Trp Arg Met Lys Met Pro Glu Thr Ala Arg

230 235 240

Tyr Thr Ala Leu Val Ala Lys Asn lye Lys Gin Ala Thr Ala Asp Met

245 250 255

Ser Lys Val Leu Gin Thr Asp lie Glu Leu Glu Glu Arg Val Glu Asp

260 265 270

Asp Val Lys Asp Pro Lys Gin Asn Tyr Gly Leu Phe Ser Lys Glu Phe

275 280 285

Leu Arg Arg His Gly Leu His Leu Leu Gly Thr Thr Ser Thr Trp Phe 290 295 300 305

Leu Leu Asp lie Ala Phe Tyr Ser Gin Asn Leu Phe Gin Lys Asp He

310 315 320

Phe Ser Ala lie Gly Trp lie Pro Lys Ala Ala Thr Met Asn Ala Thr

325 330 335 71

His Glu Val Phe Arg lie Ala Arg Ala Gin Thr Leu lie Ala Leu Cys

340 345 350

Ser Thr Val Pro Gly Tyr Trp Phe Thr Val Ala Phe lie Asp Thr lie

355 360 365

Gly Arg Phe Lys lie Gin Leu Asn Gly Phe Phe Met Met Thr Val Phe 370 375 380 385

Met Phe Ala lie Ala Phe Pro Tyr Asn His Trp lie Lys Pro Glu Asn

390 395 400

Arg He Gly Phe Val Val Met Tyr Ser Leu Thr Phe Phe Phe Ala Asn

405 410 415

Phe Gly Pro Asn Ala Thr Thr Phe lie Val Pro Ala Glu lie Phe Pro

420 425 430

Ala Arg Leu Arg Ser Thr Cys His Gly lie Ser Ala Ala Ala Gly Lys

435 440 445

Ala Gly Ala lie Val Gly Ala Phe Gly Phe Leu Tyr Ala Ala Gin Ser 450 455 460 465

Gin Asp Lys Ala Lys Val Asp Ala Gly Tyr Pro Pro Gly lie Gly Val

470 475 480

Lys Asn Ser Leu lie Met Leu Gly Val Leu Asn Phe lie Gly Met Leu

485 490 495

Phe Thr Phe Leu Val Pro Glu Pro Lys Gly Lys Ser Leu Glu Glu Leu

500 505 510

Ser Gly Glu Ala Glu Val Ser His Asp Glu Lys

515 520 SEQ ID NO: 10 72 Sequence length: 534

Sequence type: amino acid

Topology: linear

Sequence type: Protein

Array

Met Ala Arg Glu Gin Leu Gin Val 1 5

Leu Asn Ala Leu Asp Val Ala Lys Thr Gin Trp Tyr His Phe Thr Ala

10 15 20

lie lie He Ala Gly Met Gly Phe Phe Thr Asp Ala Tyr Asp Leu Phe 25 30 35 40

Cys lie Ser Leu Val Thr Lys Leu Leu Gly Arg lie Tyr Tyr His Val

45 50 55

Glu Gly Ala Gin Lys Pro Gly Thr Leu Pro Pro Asn Val Ala Ala Ala

60 65 70

Val Asn Gly Val Ala Phe Cys Gly Thr Leu Ala Gly Gin Gin Leu Phe Phe

75 80 85

Gly Trp Leu Gly Asp Lys Leu Gly Arg Lys Lys Val Tyr Gly Met Thr

90 95 100

Leu Met Val Met Val Leu Cys Ser He Ala Ser Gly Leu Ser Phe Gly 105 110 115 120

His Glu Pro Lys Ala Val Met Ala Thr Leu Cys Phe Phe Arg Phe Trp

125 130 135

Leu Gly Phe Gly lie Gly Gly Asp Tyr Pro Leu Ser Ala Thr lie Met

140 145 150

Ser Glu Tyr Ala Asn Lys Lys Thr Arg Gly Ala Phe Val Ser Ala Val 73

155 160 165

Phe Ala Met Gin Gly Phe Gly lie Met Ala Gly Gly lie Phe Ala lie

170 175 180

lie lie Ser Ser Ala Phe Glu Ala Lys Phe Pro Ser Pro Ala Tyr Ala 185 190 195 200

Asp Asp Ala Leu Gly Ser Thr lie Pro Gin Ala Asp Leu Val Trp Arg

205 210 215 lie lie Leu Met Ala Gly Ala lie Pro Ala Ala Met Thr Tyr Tyr Ser

220 225 230

Arg Ser Lys Met Pro Glu Thr Ala Arg Tyr Thr Ala Leu Val Ala Lys

235 240 245

Asp Ala Lys Gin Ala Ala Ser Asp Met Ser Lys Val Leu Gin Val Glu

250 255 260

l ie Glu Pro Glu Gin Gin Lys Leu Glu Glu lie Ser Lys Glu Lys Ser 265 270 275 280

Lys Ala Phe Gly Leu Phe Ser Lys Glu Phe Met Ser Arg His Gly Leu

285 290 295

His Leu Leu Gly Thr Thr Ser Thr Trp Phe Leu Leu Asp lie Ala Phe

300 305 310

Tyr Ser Gin Asn Leu Phe Gin Lys Asp He Phe Ser Ala lie Gly Trp

315 320 325

lie Pro Pro Ala Gin Ser Met Asn Ala lie Gin Glu Val Phe Lys He

330 335 340

Ala Arg Ala Gin Thr Leu lie Ala Leu Cys Ser Thr Val Pro Gly Tyr 345 350 355 360

Trp Phe Thr Val Ala Phe lie Asp Val lie Gly Arg Phe Ala He Gin 74

365 370 375

Met Met Gly Phe Phe Phe Met Thr Val Phe Met Phe Ala Leu Ala lie

380 385 390

Pro Tyr Asn His Trp Thr His Lys Glu Asn Arg lie Gly Phe Val lie

395 400 405

Met Tyr Ser Leu Thr Phe Phe Phe Ala Asn Phe Gly Pro Asn Ala Thr

410 415 420

Thr Phe Val Val Pro Ala Glu lie Phe Pro Ala Arg Phe Arg Ser Thr 425 430 435 440

Cys His Gly lie Ser Ala Ala Ser Gly Lys Leu Gly Ala Met Val Gly

445 450 455

Ala Phe Gly Phe Leu Tyr Leu Ala Gin Asn Pro Asp Lys Asp Lys Thr

460 465 470

Asp Ala Gly Tyr Pro Pro Gly lie Gly Val Arg Asn Ser Leu lie Val

475 480 485

Leu Gly Val Val Asn Phe Leu Gly He Leu Phe Thr Phe Leu Val Pro

490 495 500

Glu Ser Lys Gly Lys Ser Leu Glu Glu Met Ser Gly Glu Asn Glu Asp 505 510 515 520

Asn Glu Asn Ser Asn Asn Asp Ser Arg Thr Val Pro lie Val

525 530 SEQ ID NO: 11

Sequence length: 521

Sequence type: amino acid

Topology: linear 75 Sequence type: Protein

Array

Met Ala Asp Gin Gin 1 5 Leu Gly Val Leu Lys Ala Leu Asp Val Ala Lys Thr Gin Leu Tyr His

10 15 20

Phe Thr Ala lie Val lie Ala Gly Met Gly Phe Phe Thr Asp Ala Tyr

25 30 35

Asp Leu Phe Cys Val Ser Leu Val Thr Lys Leu Leu Gly Arg Leu Tyr

40 45 50

Tyr Phe Asn Pro Thr Ser Ala Lys Pro Gly Ser Leu Pro Pro His Val

55 60 65

Ala Ala Ala Val Asn Gly Val Ala Leu Cys Gly Thr Leu Ala Gly Gin 70 75 80 85

Leu Phe Phe Gly Trp Leu Gly Asp Lys Leu Gly Arg Lys Lys Val Tyr

90 95 100

Gly lie Thr Leu lie Met Met lie Leu Cys Ser Val Ala Ser Gly Leu

105 110 115

Ser Leu Gly Asn Ser Ala Lys Gly Val Met Thr Thr Leu Cys Phe Phe

120 125 130

Arg Phe Trp Leu Gly Phe Gly lie Gly Gly Asp Tyr Pro Leu Ser Ala

135 140 145

Thr lie Met Ser Glu Tyr Ala Asn Lys Lys Thr Arg Gly Ala Phe He 150 155 160 165

Ala Ala Val Phe Ala Met Gin Gly Val Gly He Leu Ala Gly Gly Phe

170 175 180 76

Val Ala Leu Ala Val Ser Ser He Phe Asp Lys Lys Phe Pro Ser Pro

185 190 195

Thr Tyr Glu Gin Asp Arg Phe Leu Ser Thr Pro Pro Gin Ala Asp Tyr

200 205 210

He Trp Arg He lie Val Met Phe Gly Ala Leu Pro Ala Ala Leu Thr

215 220 225

Tyr Tyr Trp Arg Met Lys Met Pro Glu Thr Ala Arg Tyr Thr Ala Leu 230 235 240 245

Val Ala Lys Asn lie Lys Gin Ala Thr Ala Asp Met Ser Lys Val Leu

250 255 260

Gin Thr Asp Leu Glu Leu Glu Glu Arg Val Glu Asp Asp Val Lys Asp

265 270 275

Pro Lys Lys Asn Tyr Gly Leu Phe Ser Lys Glu Phe Leu Arg Arg His

280 285 290

Gly Leu His Leu Leu Gly Thr Thr Ser Thr Trp Phe Leu Leu Asp lie

295 300 305

Ala Phe Tyr Ser Gin Asn Leu Phe Gin Lys Asp lie Phe Ser Ala He 310 315 320 325

Gly Trp He Pro Lys Ala Ala Thr Met Asn Ala lie His Glu Val Phe

330 335 340

Lys lie Ala Arg Ala Gin Thr Leu lie Ala Leu Cys Ser Thr Val Pro

345 350 355

Gly Tyr Trp Phe Thr Val Ala Phe He Asp lie lie Gly Arg Phe Ala

360 365 370

l ie Gin Leu Met Gly Phe Phe Met Met Thr Val Phe Met Phe Ala l ie 375 380 385 77

Ala Phe Pro Tyr Asn His Trp lie Leu Pro Asp Asn Arg lie Gly Phe 390 395 400 405

Val Val Met Tyr Ser Leu Thr Phe Phe Phe Ala Asn Phe Gly Pro Asn

410 415 420

Ala Thr Thr Phe He Val Pro Ala Glu lie Phe Pro Ala Arg Leu Arg

425 430 435

Ser Thr Cys His Gly lie Ser Ala Ala Thr Gly Lys Ala Gly Ala lie

440 445 450

Val Gly Ala Phe Gly Phe Leu Tyr Ala Ala Gin Pro Gin Asp Lys Thr

455 460 465

Lys Thr Asp Ala Gly Tyr Pro Pro Gly lie Gly Val Lys Asn Ser Leu 470 475 480 485 lie Met Leu Gly Val lie Asn Phe Val Gly Met Leu Phe Thr Phe Leu

490 495 500

Val Pro Glu Pro Lys Gly Lys Ser Leu Glu Glu Leu Ser Gly Glu Ala

505 510 515

Glu Val Asp Lys

520 SEQ ID NO: 12

Sequence length: 524

Sequence type: amino acid

Topology: linear

Sequence type: Protein

Array

Met Ala Glu Gin Gin Leu Gly Val Leu Lys Ala Leu Asp 78

1 5 10

Val Ala Lys Thr Gin Leu Tyr His Phe Thr Ala lie Val lie Ala Gly

15 20 25

Met Gly Phe Phe Thr Asp Ala Tyr Asp Leu Phe Cys Val Ser Leu Val 30 35 40 45

Thr Lys Leu Leu Gly Arg lie Tyr Tyr Phe Asn Pro Glu Ser Ala Lys

50 55 60

Pro Gly Ser Leu Pro Pro His Val Ala Ala Ala Val Asn Gly Val Ala

65 70 75

Leu Cys Gly Thr Leu Ser Gly Gin Leu Phe Phe Gly Trp Leu Gly Asp

80 85 90

Lys Leu Gly Arg Lys Lys Val Tyr Gly Leu Thr Leu lie Met Met lie

95 100 105

Leu Cys Ser Val Ala Ser Gly Leu Ser Phe Gly Asn Glu Ala Lys Gly 110 115 120 125

Val Met Thr Thr Leu Cys Phe Phe Arg Phe Trp Leu Gly Phe Gly He

130 135 140

Gly Gly Asp Tyr Pro Leu Ser Ala Thr lie Met Ser Glu Tyr Ala Asn

145 150 155

Lys Lys Thr Arg Gly Ala Phe lie Ala Ala Val Phe Ala Met Gin Gly

160 165 170

Val Gly lie Leu Ala Gly Gly Phe Val Ala Leu Ala Val Ser Ser He

175 180 185

Phe Asp Lys Lys Phe Pro Ala Pro Thr Tyr Ala Val Asn Arg Ala Leu 190 195 200 205

Ser Thr Pro Pro Gin Val Asp Tyr l ie Trp Arg lie lie Val Met Phe 79

210 215 220

Gly Ala Leu Pro Ala Ala Leu Thr Tyr Tyr Trp Arg Met Lys Met Pro

225 230 235

Glu Thr Ala Arg Tyr Thr Ala Leu Val Ala Lys Asn lie Lys Gin Ala

240 245 250

Thr Ala Asp Met Ser Lys Val Leu Gin Thr Asp lie Glu Leu Glu Glu

255 260 265

Arg Val Glu Asp Asp Val Lys Asp Pro Arg Gin Asn Tyr Gly Leu Phe 270 275 280 285

Ser Lys Glu Phe Leu Arg Arg His Gly Leu His Leu Leu Gly Thr Thr

290 295 300

Ser Thr Trp Phe Leu Leu Asp He Ala Phe Tyr Ser Gin Asn Leu Phe

305 310 315

Gin Lys Asp lie Phe Ser Ala lie Gly Trp lie Pro Lys Ala Ala Thr

320 325 330

Met Asn Ala Thr His Glu Val Phe Arg lie Ala Arg Ala Gin Thr Leu

335 340 345

He Ala Leu Cys Ser Thr Val Pro Gly Tyr Trp Phe Thr Val Ala Phe 350 355 360 365 l ie Asp Thr He Gly Arg Phe Lys He Gin Leu Asn Gly Phe Phe Met

370 375 380

Met Thr Val Phe Met Phe Ala He Ala Phe Pro Tyr Asn His Trp He

385 390 395

Lys Pro Glu Asn Arg He Gly Phe Val Val Met Tyr Ser Leu Thr Phe

400 405 410

Phe Phe Ala Asn Phe Gly Pro Asn Ala Thr Thr Phe He Val Pro Ala 9C οε Z

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Ala Arg Asn Thr Lys Gin Ala Ala Ser Asp Met Ser Lys Val Leu Gin

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Val Asp Leu lie Ala Glu Glu Glu Ala Gin Ser Asn Ser Asn Ser Ser

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Asn Pro Asn Phe Thr Phe Gly Leu Phe Thr Arg Glu Phe Ala Arg Arg

280 285 290

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315 320 325 lie Gly Trp lie Pro Ala Ala Glu Thr Met Asn Ala lie His Glu Val

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Pro Gly Tyr Trp Phe Thr Val Ala Phe lie Asp lie Leu Gly Arg Phe

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Phe lie Gin Leu Met Gly Phe He Phe Met Thr He Phe Met Phe Ala 375 380 385 390

Leu Ala He Pro Tyr Asp His Trp Arg His Arg Glu Asn Arg lie Gly

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Phe Leu lie Met Tyr Ser Leu Thr Met Phe Phe Ala Asn Phe Gly Pro

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Asn Ala Thr Thr Phe Val Val Pro Ala Glu lie Phe Pro Ala Arg Leu

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Claims

84 Claims

1. A DNA derived from a plant that encodes a protein that has the function of transporting phosphate from the outside to the inside of a cell by using an actively created hydrogen ion concentration gradient inside and outside the cell.

2. A protein described in any of SEQ ID NOs: 9 to 13 or a protein in which a part of the amino acid sequence of the protein has been mutated by deletion, substitution, insertion of another amino acid, etc. DNA that encodes a protein that has the function of transporting phosphate from outside to inside the cell using the hydrogen ion concentration gradient created in the cell.

3. The DNA according to claim 1 or 2, which encodes a protein derived from Arabidopsis thaliana.

4. A vector comprising the DNA according to any one of claims 1 to 3.

5. The plant cell into which the DNA according to any one of claims 1 to 3 has been introduced.

6. A protein described in any of SEQ ID NOs: 9 to 13 or a protein in which a part of the amino acid sequence of the protein has been mutated by deletion, substitution, insertion of another amino acid, etc. A protein that has the function of transporting phosphate from outside to inside cells using the hydrogen ion concentration gradient created in the cell.

7. A plant comprising a cell into which the DNA according to any one of claims 1 to 3 has been introduced.

8. A method for producing a transformed plant, comprising the step of introducing the DNA according to any one of claims 1 to 3 into a cell, and producing a plant from the K-converted cell.

9. A method for promoting phosphate uptake in a plant or plant cell, comprising introducing the DNA according to any one of claims 1 to 3 into a plant cell.