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WO1997035984A1 - Gene transporteur de phosphate vegetal et procede de regulation de la croissance d'une plante au moyen de ce gene - Google Patents

Gene transporteur de phosphate vegetal et procede de regulation de la croissance d'une plante au moyen de ce gene Download PDF

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Publication number
WO1997035984A1
WO1997035984A1 PCT/JP1997/000975 JP9700975W WO9735984A1 WO 1997035984 A1 WO1997035984 A1 WO 1997035984A1 JP 9700975 W JP9700975 W JP 9700975W WO 9735984 A1 WO9735984 A1 WO 9735984A1
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PCT/JP1997/000975
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Japanese (ja)
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Norihiro Mitsukawa
Satoru Okumura
Yumiko Shirano
Daisuke Shibata
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Mitsui Plant Biotechnology Research Institute
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Priority to AU19450/97A priority Critical patent/AU1945097A/en
Priority to JP53424197A priority patent/JP3474882B2/ja
Publication of WO1997035984A1 publication Critical patent/WO1997035984A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • 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.
  • 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.
  • the early flowering time has been applied to lilies and tulips by low-temperature treatment.
  • the promotion of flowering by gibberellin treatment for long-day plants has been put to practical use.
  • 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).
  • 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.
  • 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.
  • 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.
  • (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 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.
  • 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
  • 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 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).
  • 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. .
  • 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 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.
  • the hybridisation method using these cDNA sequences is used to obtain DNA from the chromosomal DN DN library prepared using the P1 phage vector, ⁇ 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.
  • 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.
  • a region including at least a part of the sequence represented by 80 is exemplified.
  • 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.
  • the host is rspodoptera frugiperdaj
  • pBacPAK8 manufactured by Clontech
  • the host is a strain derived from Escherichia coli BL21.
  • pET vector series (Stratagene)
  • 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- ⁇ -glu-thione S-transferase.
  • the labeling can be performed by affinity chromatography for the label.
  • 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.
  • 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.
  • a promoter that can be expressed in plants
  • CaMV cauliflower mosaic virus
  • 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.
  • a vector used for plant transformation is a vector such as PBI121.
  • 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.
  • herbaceous dicotyledonous plants such as Arabidopsis tobacco
  • monocotyledonous plants such as rice and corn
  • 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.
  • 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 ( ⁇ ).
  • IPT1 phosphate transport gene
  • 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
  • ⁇ indicates neomycin phosphotransferase (kanamycin resistance gene)
  • 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.
  • PCR Polymerase Chain Reaction
  • pIPT-PCil pBluescript II SK + (Stratagene)
  • 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"
  • IPT Arabidopsis inorganic phosphate transporter
  • 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
  • pIPTC19 11 cDNA clones were obtained (hereinafter, this clone is referred to as “pIPTC19”) (SEQ ID NO: 1).
  • Arabidopsis Coldup stage
  • P1 genomic library Liu Y.-G. et al. Plant J. 7, 351-358 (1995)
  • Arabidopsis thaliana Colombia strain
  • ZapII Genome library-1 (Stratagene) was used.
  • 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.
  • 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).
  • Figure 2 shows the restriction map of the whole genome clone.
  • the entire nucleotide sequences of PIPTGZ33 and pIPTGZ37 were determined.
  • SEQ ID NO: 3 shows the determined nucleotide sequence of the IPT1 gene.
  • 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.
  • 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.
  • 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 983 ⁇ 4.
  • chromosome mapping was performed to determine the distribution of the five isolated IPT genes on the chromosome.
  • the three IPT genes (IPT1, ⁇ 2, ⁇ 3) are located on the lower arm of chromosome 5, and the ⁇ 4 and ⁇ 5 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 ⁇ gene that is surely expressed in plants.
  • 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.
  • PIPTC19 which is the cDNA of the IPT1 gene
  • hybridization was performed, and washing was performed under conditions of 0.1X SSC, 0.13 ⁇ 4SDS, and 65 ° C. Transcripts were detected.
  • the IPT1 gene was specifically expressed in the root (FIG. 4).
  • the construct for overexpression 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) ⁇
  • 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.
  • CaMV35S cauliflower mosaic virus 35S
  • 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, 33 ⁇ 4 sucrose, pg / mithiamine-HC1, 0.2 ⁇ g / 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.
  • a modified MS medium 2.5 mM pho sphate, 0.1 mg / il myo-inositol, 33 ⁇ 4 sucrose, pg / mithiamine-HC1, 0.2 ⁇ g / g Rotational culture at 25 ° C and 100 pm in ml 2,4-D and 2 ⁇ g / ml glycine, 50
  • 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.
  • 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.
  • UOC40HV25 0.45 im centrifugal filtration tube
  • 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).
  • 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.
  • a cell strainer Cell Strainer
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Sequence type nucleic acid
  • Organism name Arabidopsis thaliana
  • Sequence type nucleic acid
  • Organism name Arabidopsis thai i ana
  • 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
  • Sequence type nucleic acid
  • Organism name Arabidopsis thal iana
  • Trp Arg lie He Val Met Phe Gly Ala Leu Pro Ala Ala Leu Thr
  • 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
  • Sequence type nucleic acid
  • IPT2 Characteristic symbol CDS 40 Location: 9662.. 10832
  • IPT2 Characteristic symbol intron
  • GCTCGAGAGA TTAGTCACTA TTTCGACCTA GATTATGGTT ACTTAAGATA CTGATATCTA 3071 GACGATTATA TATAG G TTT TGG CTC GGG TTT GGC ATT GGA GGT GAC TAC 3120
  • Trp Arg lie Val Met Phe Gly Ala Leu Pro
  • 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
  • TTTAATTCGG TTCTTTAGTC AAACCGTTTA ATTATACCAG ATATCATAAA TTCTAAACTT 12572
  • Sequence type nucleic acid
  • Organism name Arabidopsis thai i ana
  • GAATTCCTAC AATGTTGAAT AAACGTAGGT AGTGGCTACT TAATTTCTTC GATTTCTTAA 60 GTGCTTAGTA CTTTTCAACA TTAAAAATGT TGTTACCAAG TCTAAATTTT CTTCACAACT 120 TGTAACTAAA CTTTTCATTA TGTGTAATCG TAAAGGATTA GCGCTACAAA TAGATGGTGA 180
  • AAGAA ACTAATTT AATAGGA CTTCTCT CTTCA TTTCTAACCTTACCCG- AAGTCA TCAC TTCCATT TTTTTTAC ATAGCCTCCCTC CCCACAT TTTCCCC
  • AGT AGA ACG GTC CCA ATA GTT TAGGTGATAT AATACGCCTT TTGTAATAAT 4635 Ser Arg Thr Val Pro lie Val
  • TTTCGTTTTT TCTTTCTCCT TGTCTCTAGC AACTCAAGTT GTTCTTTGTG TAATCCATTG 4695 ATACCTAATT AATGCTAGAG AAATCAAAAT TTTCATGAGT CGATTTTAAA TCAGCTCCTA 4755 AATTGAAGAT TTATTAGACT TGACGAAGCC GCGCAAGTTG TGCATGACGATCATACGATCATGACCATGCATCATGACT
  • Sequence type nucleic acid
  • Organism name Arabidopsis thal iana
  • TCTTTCTCTA GATATGCCTT AAGCAATAAA AGGGGGAAAA ACATTTTTTG ATGACAAATT 480
  • TTAATTGAT TCTAACGTT AAGCG '' AAAGG ATTAGATT TTTCGG
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • J3 ⁇ 4 ⁇ is ⁇ o ng BV ⁇ ⁇ ⁇ ⁇ usv ⁇ 3 ⁇ 4 ⁇ B IV ⁇ TV 3 ⁇ 4IV ⁇ ⁇ ⁇ SIH OJ ⁇ OJJ
  • Gly Arg Phe Lys lie Gin Leu Asn Gly Phe Phe Met Met Thr Val Phe 370 375 380 385
  • Lys Asn Ser Leu lie Met Leu Gly Val Leu Asn Phe lie Gly Met Leu
  • 205 210 215 lie lie Leu Met Ala Gly Ala lie Pro Ala Ala Met Thr Tyr Tyr Ser

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Abstract

On a isolé d'Arabidopsis thaliana un gène codant une protéine transportant un phosphate et on a déterminé sa structure. En intégrant le gène dans un vecteur d'expression extrêmement actif pour procéder à la transformation en cellules du tabac mises en culture, il a été possible de considérablement améliorer l'absorption du phosphate par les cellules transgéniques du tabac mises en culture et d'accroître leur rythme de croissance. Cette technique peut être appliquée à grande échelle sur les plantes.
PCT/JP1997/000975 1996-03-25 1997-03-24 Gene transporteur de phosphate vegetal et procede de regulation de la croissance d'une plante au moyen de ce gene WO1997035984A1 (fr)

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AU19450/97A AU1945097A (en) 1996-03-25 1997-03-24 Plant phosphate transporter gene and method of regulating plant growth with said gene
JP53424197A JP3474882B2 (ja) 1996-03-25 1997-03-24 植物のリン酸トランスポーター遺伝子及び該遺伝子を用いた植物の成長を制御する方法

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JP9479096 1996-03-25
JP8/94790 1996-03-25

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998005760A3 (fr) * 1996-07-31 1998-10-08 Univ Kingston Proteines pouvant etre induites en cas de privation de phosphate
WO1999058657A3 (fr) * 1998-05-13 2000-07-27 Pioneer Hi Bred Int Genes transporteurs de phosphate de zea mays
KR100401007B1 (ko) * 2000-08-11 2003-10-08 윤성중 담배의 인산수송자 유전자

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998005760A3 (fr) * 1996-07-31 1998-10-08 Univ Kingston Proteines pouvant etre induites en cas de privation de phosphate
WO1999058657A3 (fr) * 1998-05-13 2000-07-27 Pioneer Hi Bred Int Genes transporteurs de phosphate de zea mays
KR100401007B1 (ko) * 2000-08-11 2003-10-08 윤성중 담배의 인산수송자 유전자

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AU1945097A (en) 1997-10-17
JP3474882B2 (ja) 2003-12-08

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