WO2011030793A1 - Utilisation d'un gène impliqué dans l'absorption d'aluminium par les plantes - Google Patents

Utilisation d'un gène impliqué dans l'absorption d'aluminium par les plantes Download PDF

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WO2011030793A1
WO2011030793A1 PCT/JP2010/065426 JP2010065426W WO2011030793A1 WO 2011030793 A1 WO2011030793 A1 WO 2011030793A1 JP 2010065426 W JP2010065426 W JP 2010065426W WO 2011030793 A1 WO2011030793 A1 WO 2011030793A1
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aluminum
osnrat1
gene
uptake
plant
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PCT/JP2010/065426
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Japanese (ja)
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建鋒 馬
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国立大学法人岡山大学
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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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8259—Phytoremediation

Definitions

  • the present invention relates to utilization of a gene involved in aluminum uptake in plants.
  • Aluminum ions have a strong toxicity to plants. Aluminum contained in the soil elutes as aluminum ions under acidic conditions (about pH 5 or less). Even at low concentrations (several ⁇ M), it quickly causes root elongation inhibition, and nourishing water from the roots. Inhibits absorption.
  • Non-patent Document 2 Aluminum is also involved in the determination of flower color.
  • the present inventor has reported that in Hydrangea, the color of a flower changes from red to blue by binding delphinidin, which is a pigment, and aluminum in the plant body (Non-patent Document 2).
  • the present invention has been made in view of the above problems, and an object thereof is to identify a gene involved in aluminum uptake in a plant and provide a method for using the gene.
  • the present inventor has intensively studied to solve the above problems. As a result, it was discovered for the first time that the OsNrat1 gene isolated from rice by the inventor was involved in aluminum transport in plants, and the present invention was completed. That is, the present invention includes the following inventions.
  • the method for producing a transformed plant according to the present invention is a method for producing a transformed plant in which uptake of aluminum is promoted, and introduces the following polynucleotide (a) or (b) into a plant so as to allow expression: It is characterized by: (A) a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having an activity of promoting aluminum uptake Polynucleotide.
  • the transformed plant according to the present invention is characterized by being produced by the method for producing a transformed plant according to the present invention.
  • the kit according to the present invention is a kit for producing a transformed plant in which uptake of aluminum is promoted, and is characterized by comprising the following polynucleotide (a) or (b): (A) a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having an activity of promoting aluminum uptake Polynucleotide.
  • the method for producing a transformed plant according to the present invention there is an excellent effect that a transformed plant in which the uptake of aluminum is promoted can be produced. Specifically, it is possible to produce a transformed plant that promotes aluminum uptake compared to a wild-type plant.
  • the petal color can be changed to blue by, for example, cultivation in soil containing aluminum. It can also be used to create aluminum-resistant plants.
  • kit according to the present invention a transformed plant in which uptake of aluminum is promoted can be easily produced.
  • FIG. 1 shows the evolutionary relationship between OsNrat1 (Oryza sativa Nramp Aluminum transporter 1) and other genes.
  • the length of the thick solid line in FIG. 1 represents a length with an evolutionary distance of 0.1 in the phylogenetic tree.
  • FIG. 2 shows the insertion positions of retrotransposon Tos-17 in the OsNrat1 gene of NE7009 and NF7046, respectively. It is a figure showing the result of RT-PCR.
  • FIG. 10 is a graph showing the percent elongation of roots of wild-type rice, NE7009, and NF7046 after 24 hours exposure to a 0.5 mM CaCl 2 solution containing 30 ⁇ M aluminum.
  • FIG. 7 represents the change over time in the aluminum concentration present in the root cell symplastic solution
  • (b) represents the change over time in the aluminum content in the cell wall
  • (c) Represents a pH-dependent change in the aluminum concentration in root cells.
  • It is a figure showing the result of tissue RT-PCR.
  • It is a graph showing the result of RT-PCR according to root region.
  • FIG. 2 is a graph showing the results of RT-PCR, (a) shows the expression of the OsNrat1 gene relative to other metals, (b) in the roots of rice when the wild type strain was treated with a solution containing 20 ⁇ M aluminum.
  • the time course of the expression of the OsNrat1 gene is shown, and (c) shows the expression of the OsNrat1 gene in the art1 mutant. It is a figure showing the result of the immunohistochemical dyeing
  • FIG. 13 is a figure showing the growth of yeast
  • (b) is a graph showing the uptake of aluminum in yeast
  • (c) is a graph showing the change over time of the uptake of aluminum in yeast. is there.
  • 2 is a graph showing the uptake of aluminum, aluminum-citrate complex, and aluminum-oxalate complex in yeast. It is a figure showing the result of the functional analysis of OsNrat1 protein using yeast.
  • (A) of FIG. 15 is a figure showing the growth of the yeast in the medium which restricted iron
  • (b) is a figure showing the growth of the yeast in the medium which restricted manganese
  • (c) is a figure showing the cadmium of yeast. It is a graph showing uptake.
  • FIG. 2 is a graph showing the results of RT-PCR of Nrat1 overexpressing body.
  • “NE7009” represents an OsNrat1 gene disruption strain
  • “5-5” and “5-7” represent OsNrat1 gene overexpression strains.
  • the method for producing a transformed plant according to the present invention is a method for producing a transformed plant in which uptake of aluminum is promoted, and introduces the following polynucleotide (a) or (b) into a plant so as to allow expression: Includes steps: (A) a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having an activity of promoting aluminum uptake Polynucleotide.
  • the present inventor newly identified a gene (OsNrat1 gene) involved in aluminum transport into cells from rice.
  • the polypeptide shown in SEQ ID NO: 1 is a translation product of the OsNrat1 gene. That is, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is a protein newly identified from rice by the present inventors and involved in the transport of aluminum into cells. That is, it can be said that the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 has an activity of promoting plant aluminum uptake.
  • polynucleotide (a) or (b) examples include polynucleotides corresponding to the 56th A to 1693rd G of SEQ ID NO: 2 showing the entire base sequence of the OsNrat1 gene. .
  • the phrase “promoted aluminum uptake” means that in a transformed plant, compared to a wild-type plant into which the polynucleotide (a) or (b) is not introduced. It is intended to promote the uptake of aluminum ions (Al 3+ ).
  • the amount may be confirmed by comparing the amounts, or may be confirmed by comparing the amount of aluminum accumulated in the tissue expressing the polynucleotide (a) or (b) in the transformed plant.
  • the polynucleotide (a) or (b) is expressed in the root tissue, it is confirmed by comparing the amount of aluminum accumulated in the root of the wild type plant and the root of the transformed plant. be able to.
  • the amount of aluminum accumulated in the plant can be measured, for example, by flameless atomic absorption shown in the examples described later.
  • polypeptide can also be referred to as “peptide” or “protein”.
  • the above “one or several amino acids are substituted, deleted, and / or added” means that substitution, deletion, or addition is performed by a known mutant polypeptide production method such as site-directed mutagenesis.
  • amino acids as possible (for example, 20 or less, preferably 10 or less, more preferably 7 or less, even more preferably 5 or less, particularly preferably 3 or less) amino acids are substituted, deleted, and / or added.
  • Such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by a known mutant polypeptide production method, but is a product obtained by isolating and purifying a similar naturally occurring mutant polypeptide. It may be.
  • polynucleotide can also be referred to as “nucleic acid” or “nucleic acid molecule”, and is intended to be a polymer of nucleotides.
  • the “base sequence” can also be referred to as “nucleic acid sequence” or “nucleotide sequence”, and is shown as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T).
  • the “polynucleotide consisting of the base sequence shown in SEQ ID NO: 1” refers to a polynucleotide consisting of the sequence shown by each deoxynucleotide A, G, C and / or T of SEQ ID NO: 1.
  • the method for obtaining the polynucleotide is not particularly limited, but can be obtained by a known technique. For example, it can be obtained by a method using amplification means such as PCR. Specifically, primers corresponding to the 5 ′ side and 3 ′ side sequences (or their complementary sequences) are prepared for the polynucleotide (a) or (b), and genomic DNA ( Alternatively, a large amount of DNA fragments containing the above-described polynucleotide can be obtained by performing PCR or the like using a cDNA) as a template and amplifying a DNA region sandwiched between both primers.
  • amplification means such as PCR.
  • a primer capable of amplifying the OsNrat1 gene region is designed, and using that primer, the genomic DNA (or cDNA) or RT-PCR product is used as a template, and the OsNrat1 gene region is designed.
  • the polynucleotide can also be obtained by amplifying.
  • a polynucleotide can be obtained by a method of isolating and cloning a DNA fragment containing an oligonucleotide containing the polynucleotide or a partial sequence thereof.
  • a probe that specifically hybridizes with a part of the nucleotide sequence of the polynucleotide (a) or (b) above may be prepared, and a genomic DNA library or cDNA library may be screened.
  • those having any sequence and / or length can be used as long as they specifically hybridize with at least a part of the base sequence of the polynucleotide or its complementary sequence.
  • polypeptide which consists of an amino acid sequence shown to sequence number 1 originates in the rice cultivar Nipponbare, a rice plant (rice, It is preferable to use corn or the like.
  • RNA for example, mRNA
  • DNA for example, cDNA or genome
  • the “plant” into which the polynucleotide is introduced is not particularly limited and can be appropriately selected according to the purpose.
  • a grass family plant, a solanaceous plant, a legume plant, a hydrangea plant, a rose plant, etc. can be mentioned.
  • Examples of the above “Poaceae plants” include rice, barley, wheat, corn, rye, sorghum and the like.
  • Examples of the above “Solanaceae plants” include eggplants and the like.
  • Examples of the “leguminous plant” include soybean.
  • Examples of the “hydrangea plant” include hydrangea and the like.
  • Examples of the “Rosaceae plant” include roses.
  • the polynucleotide (a) or (b) above or a recombinant expression vector containing the polynucleotide is introduced, and There is no particular limitation as long as it expresses a polypeptide having the activity of promoting uptake.
  • the above-mentioned “activity for promoting aluminum uptake” refers to “in a transformed plant as compared with a wild-type plant into which the polynucleotide (a) or (b) is not introduced”. It is intended to have an activity that promotes aluminum uptake.
  • the type of the “recombinant expression vector” is not particularly limited as long as it includes the polynucleotide (a) or (b).
  • a recombinant expression vector in which a cDNA corresponding to the 56th A to the 1693rd G of SEQ ID NO: 2 is inserted can be mentioned.
  • a plasmid, a cosmid or the like can be used, but the present invention is not limited to these.
  • the “recombinant expression vector” is not particularly limited as long as it can express the inserted gene in plant cells (hereinafter also referred to as “host cells”).
  • host cells plant cells
  • a recombinant expression vector is introduced into a plant by a method using Agrobacterium (Agrobacterium infection method)
  • a binary vector such as pBI as the recombinant expression vector.
  • binary vectors include pBIG, pBIN19, pBI101, pBI121, and pBI221.
  • the “recombinant expression vector” is preferably a vector having a promoter capable of expressing a gene in cells of a plant to be introduced (plant to be introduced).
  • a known promoter can be preferably used as the promoter. Examples thereof include cauliflower mosaic virus 35S promoter (CaMV35S), ubiquitin promoter, actin promoter, and the like.
  • the cauliflower mosaic virus 35S promoter it is preferable to use the cauliflower mosaic virus 35S promoter.
  • a transformed plant in which aluminum uptake is further promoted can be produced.
  • the OsNrat1 gene may be expressed in the whole plant or specifically in a specific site of the plant.
  • the OsNrat1 gene is specifically expressed in the petal of a plant (for example, hydrangea) having a pigment (for example, delphinidin) that changes color by binding to aluminum. By accumulating aluminum in the petals, the petal color can be changed to blue.
  • OsNrat1 protein is expressed in the roots of plants. Therefore, from the viewpoint of suitably functioning the OsNrat1 protein in the transformed plant, it is preferable to introduce the polynucleotide (a) or (b) so that it can be expressed specifically in the root of the plant.
  • the polynucleotide (a) or (b) described above under the control of a promoter known to control a gene specifically expressed in roots, the above (a) or (b ) Can be specifically expressed in the roots of the plant.
  • a promoter for example, the OsNrat1 promoter can be used.
  • the method for introducing the polynucleotide or the recombinant expression vector into a plant is not particularly limited.
  • the polynucleotide or the recombinant expression vector may be integrated into a chromosome, or the polynucleotide may be integrated into a specific site of the chromosome by homologous recombination.
  • the polynucleotide or the recombinant expression vector may be transiently expressed in a plant.
  • a conventionally known genetic engineering technique (gene manipulation technique) can be used.
  • conventionally known methods such as Agrobacterium infection method, electroporation method (electroporation method), calcium phosphate method, protoplast method, lithium acetate method, and particle gun method can be suitably used.
  • Agrobacterium infection method the method described in Plant, J. 6: 271-282 (1994) can be used.
  • the polynucleotide or the recombinant expression vector is introduced into the host cell, and whether or not it is reliably expressed in the host cell can be confirmed using various markers.
  • a drug resistance gene that gives resistance to antibiotics such as hygromycin is used as a marker, and a plasmid containing this marker and the polynucleotide (a) or (b) above is introduced into a host cell as an expression vector.
  • a method can be mentioned. By using this method, it can be confirmed by drug selection whether or not the introduced gene is reliably expressed in the host cell.
  • the transformed plant produced by the method for producing a transformed plant according to the present invention is introduced with the polynucleotide (a) or (b) above or the above recombinant expression vector, and promotes aluminum uptake.
  • a polypeptide having the activity to be expressed is, the transformed plant produced by the method for producing a transformed plant according to the present invention is compared with a wild-type plant into which the polynucleotide (OsNrat1 gene) described above (a) or (b) is not introduced, Aluminum uptake is promoted.
  • the polynucleotide (a) or (b) corresponds to the OsNrat1 gene derived from rice (Nipponbare). For this reason, in the transformed plant into which the polynucleotide (a) or (b) is introduced, the uptake of aluminum in the plant is promoted as compared to the wild type plant into which these polynucleotides are not introduced. it is conceivable that.
  • a transformed plant that promotes aluminum uptake can be easily produced.
  • the transformed plant according to the present invention is characterized by being produced by the method for producing a transformed plant according to the present invention.
  • the “method for producing a transformed plant according to the present invention” is the same as that described in the section “1. Method for producing a transformed plant” above, and is therefore omitted here.
  • the category of transformed plants according to the present invention includes not only plant bodies but also various forms of plant cells such as suspension culture cells, protoplasts, leaf sections, and callus.
  • a transformed plant in which the above-mentioned polynucleotide (a) or (b) is incorporated into a plant chromosome is obtained by the method for producing a transformed plant according to the present invention, seeds obtained from the plant are also obtained.
  • the above polynucleotide has been introduced. Accordingly, the present invention also includes seeds obtained from transformed plants.
  • the transformed plant according to the present invention can be used for various purposes because aluminum uptake is promoted. For example, it is thought that it can be set as the plant which changed the color of the flower into blue.
  • the kit according to the present invention is a kit for producing a transformed plant in which uptake of aluminum is promoted, and is characterized by comprising the following polynucleotide (a) or (b): (A) a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having an activity of promoting aluminum uptake Polynucleotide.
  • polynucleotide is the same as that described in the section “1. Method for producing transformed plant”, and is omitted here.
  • the kit according to the present invention may contain components other than the polynucleotide described above.
  • a plasmid for preparing a recombinant expression vector containing the above polynucleotide a reagent necessary for preparing the recombinant expression vector, a buffer, a reagent necessary for transforming a plant, etc. Also good.
  • a transformed plant that promotes aluminum uptake can be easily produced.
  • the inventor identified a large number of rice genes induced by aluminum by microarray analysis, and performed functional analysis of OsNrat1 therein.
  • FIG. 1 is a phylogenetic tree showing the evolutionary relationship between OsNrat1 (Oryza sativa Nramp Aluminum transporter 1) and other genes.
  • the length of the thick solid line in FIG. 1 represents a length with an evolutionary distance of 0.1 in the phylogenetic tree.
  • the phylogenetic tree in FIG. 1 was created using ClustalW (DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/)).
  • ClustalW DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/)
  • OsNrat1 belongs to the Nramp family. However, as shown in the examples described below, unlike other members of the Nramp family, which are known to transport divalent metal ions, they are found to be transporters that transport trivalent aluminum ions. It was. OsNrat1 showed low homology with other members (36% -59% at the amino acid level).
  • FIG. 2 shows the insertion positions of retrotransposon Tos-17 in the OsNrat1 gene of NE7009 and NF7046, respectively.
  • PCR reaction conditions are as follows. Composition of the reaction solution: 1 ⁇ L of buffer, 0.2 mM dNTP, one set of 0.5 ⁇ M primer each, 10 ⁇ L of reaction solution, 1 ⁇ L of reverse transcription product diluted 20 times PCR conditions: 95 ° C. for 30 seconds, (95 ° C. for 15 seconds, 58 ° C. for 15 seconds, 72 ° C. for 30 seconds) ⁇ 30 cycles.
  • FIG. 3 shows the results of RT-PCR.
  • the amount of Histone H3 was confirmed as an internal standard.
  • the expression of the OsNrat1 gene was not observed in NE7009 and NF7046. From this result, in NE7009 and NF7046, it was confirmed that the OsNrat1 gene was surely destroyed.
  • FIG. 4 is a graph showing the percent elongation of roots of wild-type rice, NE7009, and NF7046 after 24 hours exposure to a 0.5 mM CaCl 2 solution containing 30 ⁇ M aluminum.
  • the vertical axis of the graph represents the above-mentioned wild-type rice, NE7009, and NF7046 root elongation rates when the root elongation in the control is 100%.
  • FIG. 5 is a graph showing root elongation percentage (%) of wild type rice and NE7009 after exposure of NE7009 and wild type rice to 0.5 mM CaCl 2 solution containing different concentrations of aluminum for 24 hours. is there.
  • the vertical axis of the graph represents the above-described wild-type rice and NE7009 root elongation rates when the root elongation in the control is 100%.
  • the OsNrat1 gene-disrupted strain when treated with 0.5 mM CaCl 2 solution containing any concentration of aluminum, the OsNrat1 gene-disrupted strain was more inhibited in root elongation than wild-type rice. That is, in the OsNrat1 gene-disrupted strain, aluminum resistance was lower than that of wild-type rice.
  • FIG. 6 shows that NE7009, NE7046, and wild-type rice were exposed to 0.5 mM CaCl 2 solution containing either aluminum, cadmium (Cd), or lanthanum (Lt) for 24 hours. , NE7009 and NE7046 root elongation percentage (%).
  • the vertical axis of the graph represents the rate of root elongation of the wild-type rice, NE7009 and NE7046 when the root elongation in the control is 100%.
  • NE7009 and NE7046 intracellular aluminum concentrations were compared for wild-type rice and OsNrat1 gene disruption lines. Specifically, NE7009, NE7046 and wild type rice were treated for 0-10 hours in a 0.5 mM CaCl 2 solution containing 30 ⁇ M aluminum. Every 2 hours after treatment, an area of 0-1 cm was collected from the tip of the root. After removing the extracellular solution by centrifuging the collected roots, the roots were frozen at ⁇ 80 ° C. Thereafter, the roots were returned to room temperature, and cell symplasts were obtained by centrifugation.
  • the residue after centrifugation was washed with 80% ethanol three times, and 2N hydrochloric acid was added to elute aluminum bound to the cell wall.
  • the amount of aluminum contained in the cell symplast solution and the cell wall extract was measured by a conventionally known flameless atomic absorption method.
  • NE7009 in 0.5 mM CaCl 2 solution containing 30 ⁇ M aluminum buffered in the pH range of 4.0 to 6.9 with 10 mM homopipes.
  • NE7046 and wild type rice were treated for 8 hours.
  • FIG. 7 is a graph showing the results of measuring the concentration of aluminum present in cells and in the cell wall for NE7009 and wild-type rice.
  • FIG. 7 (a) shows the change over time in the aluminum concentration present in the cell symplastic solution, (b) shows the change over time in the aluminum content in the cell wall, and (c) shows the cell simp. It represents a pH-dependent change in the concentration of aluminum in the last solution. Data are shown as mean ⁇ standard deviation.
  • NE7009 has the tendency for the amount of aluminum couple
  • NE7009, NE7046 and wild type rice were treated in 0.5 mM CaCl 2 solution (pH 4.2) containing 30 ⁇ M aluminum for 8 hours at 4 ° C. or 25 ° C. did.
  • FIG. 23A shows the concentration of aluminum in the root cell symplast
  • FIG. 23B shows the concentration of aluminum in the root cell wall.
  • FIGS. 23 (a) and 23 (b) at 4 ° C., there was no difference in the uptake of OsNrat1 gene-disrupted strain and wild-type root cell symplast solution and cell wall aluminum.
  • the concentration of aluminum in the root cell symplast was higher in the wild type strain than in the OsNrat1 gene disrupted strain.
  • the wild-type strain had a lower aluminum concentration in the root cell wall than the OsNrat1 gene disrupted strain. This result suggests that aluminum uptake by OsNrat1 protein is an active process.
  • the uptake of other cations in the OsNrat1 gene disrupted strain and the wild type strain was compared. Specifically, the wild type strain and NE7009 were cultured for 1 month using 1/2 Kimura B solution. The concentration of the cation was measured by a conventionally known flameless atomic absorption method after digestion with HNO 3 .
  • 1/2 Kimura B solution is macronutrient (mM): MgSO 4 (0.28), (NH 4 ) 2 SO 4 (0.18), Ca (NO 3 ) 2 (0.18), KNO 3 (0.09), KH 2 PO 4 (0.09), and micronutrients (mM): Fe (II) SO 4 (10), H 3 BO 3 (3), MnCl 2 (0.5), A culture medium containing CuSO 4 (0.2), ZnSO 4 (0.4), (NH 4 ) 6 Mo 7 O 24 (1).
  • the 1/2 Kimura B solution was adjusted to pH 5.4 with 1N NaOH.
  • the obtained transformed strain, wild type strain, NE7009 was treated with a solution containing 30 ⁇ M aluminum for 8 hours. Thereafter, the root cell symplast solution was extracted by the freeze-thaw method described above, and the amount of aluminum was measured by a conventionally known flameless atomic absorption method.
  • the OsNrat1 gene was introduced into the OsNrat1 gene disrupted strain, the aluminum concentration in the root cell symplast increased to the same level as in the wild type rice. From this result, it was further confirmed that the OsNrat1 gene is involved in the phenotype in the OsNrat1 gene disrupted strain.
  • the above-mentioned “terrestrial part” intends a plant body (for example, a leaf, a stem, etc.) excluding roots.
  • RT-PCR was performed using the obtained RNA, and the expression level of the OsNrat1 gene was examined. The reverse transcription reaction was performed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen).
  • PCR reaction conditions are as follows. Composition of the reaction solution: 1 ⁇ L of buffer, 0.2 mM dNTP, one set of 0.5 ⁇ M primer each, 10 ⁇ L of reaction solution, 1 ⁇ L of reverse transcription product diluted 20 times PCR conditions: 95 ° C. for 30 seconds, (95 ° C. for 15 seconds, 58 ° C. for 15 seconds, 72 ° C. for 30 seconds) ⁇ 30 cycles.
  • FIG. 8 is a diagram showing the results of tissue-specific RT-PCR.
  • the amount of Histone H3 was confirmed as an internal standard.
  • the OsNrat1 gene was mainly expressed in the roots, and the expression of the OsNrat1 gene was not observed in the above-ground part. Moreover, it became clear that the expression of OsNrat1 gene in roots is increased by aluminum.
  • the effect of aluminum on the expression of the OsNrat1 gene in wild-type rice roots was examined.
  • the root was divided into a region of 0 to 1 cm from the tip and a region of 1 to 2 cm from the tip, and RNA was extracted from each root region.
  • RT-PCR was performed using the obtained RNA, and the expression level of the OsNrat1 gene was examined.
  • the reverse transcription reaction was performed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). PCR reaction conditions are as follows.
  • Composition of the reaction solution 20 ⁇ L of reaction solution, 1 ⁇ SYBR Premix Ex Taq (manufactured by Takara Bio Inc.), one set of 0.2 ⁇ M each primer, 2 ⁇ L of reverse transcription product diluted 20 times PCR conditions: 95 ° C. for 30 seconds, (95 ° C. for 15 seconds, 58 ° C. for 15 seconds, 72 ° C. for 30 seconds) ⁇ 40 cycles PCR was performed using Mastercycler ep realplex (Eppendorf).
  • FIG. 9 is a graph showing the results of RT-PCR for each root region. As shown in FIG. 9, similar to the RT-PCR results in FIG. 8, the expression of the OsNrat1 gene in roots was induced by aluminum. It was also revealed that the OsNrat1 gene expression level was higher at the base of the root (region of 1 to 2 cm from the tip) than at the tip of the root (region of 0 to 1 cm from the tip). The vertical axis of the graph in FIG. 9 represents the relative expression level when the expression level of the OsNrat1 gene is 1 in the root of the region 0 to 1 cm from the tip in the absence of aluminum.
  • the reverse transcription reaction was performed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen).
  • PCR reaction conditions are as follows. Composition of the reaction solution: 20 ⁇ L of reaction solution, 1 ⁇ SYBR Premix Ex Taq (manufactured by Takara Bio Inc.), one set of 0.2 ⁇ M each primer, 2 ⁇ L of reverse transcription product diluted 20 times PCR conditions: 95 ° C. for 30 seconds, (95 ° C. for 15 seconds, 58 ° C. for 15 seconds, 72 ° C. for 30 seconds) ⁇ 40 cycles PCR was performed using Mastercycler ep realplex (Eppendorf).
  • FIG. 10 (a) is a graph showing the expression of the OsNrat1 gene relative to other metals.
  • FIG. 10 shows the expression of the OsNrat1 gene in the art1 mutant.
  • OsNrat1 As shown in FIG. 10 (c), it was confirmed that the expression of the OsNrat1 gene was not induced by aluminum in the art1 mutant. This result indicates that the expression of OsNrat1 gene is regulated by ART1 (Yamaji, N., Huang, CF, Nagao, S., Yano, M., Sato, Y., Nagamura, Y., and Ma, JF (2009).
  • ART1 Yamaji, N., Huang, CF, Nagao, S., Yano, M., Sato, Y., Nagamura, Y., and Ma, JF (2009).
  • a Zn-finger transcription factor ART1 regulates multiple genes implicated in aluminum tolerance in rice. Plant Cell 21: 3339-3349).
  • a recombinant expression vector containing a fusion gene incorporating a gene encoding a fluorescent protein GFP (green fluorescent protein) under the control of the promoter of the OsNrat1 gene ( ProOsNrat1-GFP vector) was prepared, and the recombinant expression vector was transformed into wild-type rice. Transformation was performed by the Agrobacterium method.
  • the expression site of the OsNrat1 protein in the root tissue can be examined. Therefore, immunohistochemical staining was performed on the roots of GFP-transformed plants using an anti-GFP antibody (A11122, manufactured by Molecular Probes) as a primary antibody, and the localization of GPF in the roots was examined.
  • an anti-GFP antibody A11122, manufactured by Molecular Probes
  • FIG. 11 is a diagram showing the result of immunohistochemical staining at the root tip of a GFP-transformed plant or at the base located 15 mm from the root tip. As shown in FIG. 11, GFP expression was observed in all cells at the root tip. In addition, at the base of the root, expression of GFP was observed in all cells except the epidermis. That is, it was revealed that the OsNrat1 protein is expressed in all cells at the tip of the root and in all cells in the base other than the base epidermis.
  • a recombinant gene (35S: :) was prepared by preparing a fusion gene of the OsNrat1 gene and the GFP gene and incorporating the fusion gene under the control of the cauliflower mosaic virus 35S promoter.
  • OsNrat1-GFP vector was prepared.
  • a recombinant expression vector (35S :: GFP vector) incorporating the GFP gene under the control of the cauliflower mosaic virus 35S promoter was prepared.
  • Each of these recombinant expression vectors was introduced into onion epidermal cells, and the intracellular localization of GFP was observed. Transformation was performed with a particle gun.
  • the GFP gene was fused in-frame to the C-terminal side of the OsNrat1 gene.
  • FIG. 12 shows the localization of the OsNrat1-GFP fusion protein in onion epidermal cells. As shown in FIG. 12, it was revealed that the OsNrat1 protein was localized in the cell membrane.
  • the antibody was obtained by immunizing a rabbit with a synthetic peptide corresponding to the amino acid sequence from position 1 to position 18 of the amino acid sequence of the OsNrat1 protein (SEQ ID NO: 1).
  • Immunostaining was performed using wild-type rice treated with 30 ⁇ M aluminum for 12 hours and the roots of the OsNrat1 gene-disrupted strain.
  • the immunostaining procedure was performed according to a conventionally known method (see Yamaji, N., and Ma, JF (2007). Spatial distribution and temporal variation of the rice silicon transporter Lsi1. Plant Physiol. 143: 1306-1313) .
  • the fluorescence was observed using a laser scanning confocal microscope (LSM700; Carl Carl Zeiss).
  • FIG. 21 is a diagram showing the results of immunostaining, (a) shows the results of OsNrat1 protein immunostaining in the roots of wild-type rice not treated with aluminum, and (b) shows no aluminum treatment. (C) shows the results of OsNrat1 protein immunostaining in the roots of wild-type rice treated with 30 ⁇ M aluminum for 12 hours, (d) shows the results of 30 ⁇ M aluminum.
  • stock processed for 12 hours is shown.
  • the scale bar in the figure corresponds to 100 ⁇ m.
  • OsNrat1 protein As shown in (a) and (c) of FIG. 21, in wild-type rice, OsNrat1 protein was expressed in all root cells except epidermis cells, and the expression level was increased by aluminum.
  • the OsNrat1 gene and the AtNramp4 gene (negative control) were expressed in yeast, and the aluminum transport activity of the OsNrat1 protein was analyzed. Specifically, each gene was introduced into yeast (BY4741 strain (MATa his2 ⁇ 0 met15 ⁇ 0 ura3 ⁇ 0)) using the expression vector pYES2 (manufactured by Invitrogen) by a conventionally known lithium acetate method. Furthermore, only the pYES2 vector was introduced as a negative control.
  • the obtained transformed yeast was inoculated on an agar medium containing 0 ⁇ M, 100 ⁇ M or 200 ⁇ M aluminum and cultured at 30 ° C. for 2 days or 3 days.
  • the presence or absence of aluminum transport activity was determined using yeast growth as an index. That is, it was considered that the transformed yeast introduced with a gene encoding a protein having an aluminum transport activity takes up aluminum into the cell, and thus its growth is suppressed by the toxicity of aluminum. Also, the uptake of aluminum into yeast for a short time (2 h) was measured.
  • the obtained transformed yeast was treated with a 50 ⁇ M AlCl 3 solution (pH 4.2), and the change over time in the uptake of aluminum was measured.
  • FIG. 13 is a diagram showing the results of functional analysis of OsNrat1 protein using yeast.
  • A of FIG. 13 is a figure showing the growth of yeast
  • (b) is a graph showing the uptake of aluminum in yeast
  • (c) is a graph showing the change over time of the uptake of aluminum in yeast. is there.
  • the yeast in which the OsNrat1 protein was expressed was inhibited from growing in a medium containing aluminum as compared with the yeast into which the pYES2 vector was introduced as a negative control. From this result, it was revealed that the OsNrat1 protein has aluminum transport activity. On the other hand, the growth of the yeast expressing the AtNramp4 protein belonging to the same Nramp family as the OsNrat1 protein was the same as that of the yeast introduced with the pYES2 vector as a negative control.
  • net uptake indicates the difference in aluminum uptake between the transformed yeast and the negative control. As shown in FIG. 13 (c), it was revealed that aluminum uptake increases linearly with time in the transformed yeast.
  • the transformed yeast was treated with a solution containing an equal concentration of cadmium or manganese for 2 hours in the presence of 50 ⁇ M aluminum.
  • Aluminum uptake in yeast was measured by flameless atomic absorption.
  • yeast expressing OsNrat1 protein contains only aluminum, aluminum-citrate complex, or aluminum-oxalate complex
  • the cells were seeded in a liquid medium and cultured at 4 ° C. or 30 ° C. for 2 hours.
  • the aluminum-citric acid complex used was a mixture of aluminum and citric acid at a ratio of 50: 500 ( ⁇ M).
  • As the aluminum-oxalic acid complex a mixture of aluminum and oxalic acid at a ratio of 50: 500 ( ⁇ M) was used.
  • capture of aluminum in yeast was measured by the method similar to (b) of FIG.
  • FIG. 14 is a graph showing the uptake of aluminum, aluminum-citrate complex, and aluminum-oxalic acid complex in yeast.
  • yeast expressing OsNrat1 protein showed improved aluminum ion transport activity, but did not show transport activity for aluminum-citrate complexes or aluminum-oxalate complexes.
  • the transport activity of aluminum ions by OsNrat1 protein is suppressed under 4 ° C. culture conditions. It is believed that plants secrete organic acids such as citric acid to form a complex with aluminum to detoxify aluminum extracellularly. From the results shown in FIG. 14, it was considered that the OsNrat1 protein took in free aluminum ions that could not form a complex into the cells.
  • OsNrat1 protein against divalent metals was examined. Specifically, yeast expressing OsNrat1 protein, yeast expressing AtNramp4 protein as positive control, and yeast introduced with pYES2 vector as negative control were used. As the yeast strain, fet3fet4 mutant lacking iron absorption ability was used for iron, smf1 mutant lacking manganese absorption ability was used for manganese, and BY4741 strain was used for cadmium.
  • yeasts were inoculated on an agar medium not containing iron or manganese (including traces) and cultured at 30 ° C. for 3 days.
  • bathophenanthroline disulfonic acid BPDS
  • BPDS bathophenanthroline disulfonic acid
  • EGTA was added to the medium at a concentration of 0 mM or 2 mM.
  • cadmium uptake in yeast was measured by flameless atomic absorption spectrometry after culturing at 30 ° C. for 2 hours in a liquid medium containing 0 ⁇ M, 2 ⁇ M, 5 ⁇ M, 10 ⁇ M or 20 ⁇ M cadmium.
  • FIG. 15 is a diagram showing the results of functional analysis of the OsNrat1 protein using yeast.
  • A of FIG. 15 is a figure showing the growth of the yeast in the medium which restricted iron
  • (b) is a figure showing the growth of the yeast in the medium which restricted manganese
  • (c) is a figure showing the cadmium of yeast. It is a graph showing uptake.
  • the yeast expressing the AtNramp4 protein grew in a medium restricted with iron or manganese, but the yeast expressing the OsNrat1 protein was introduced with the pYES2 vector. As in the case of yeast, growth was suppressed in a medium restricted with iron or manganese. This indicates that AtNramp4 protein has the activity of transporting iron and manganese, whereas OsNrat1 protein has no activity of transporting iron and manganese.
  • cadmium uptake was promoted in the yeast in which the AtNramp4 protein was expressed, compared to the yeast in which the OsNrat1 protein was expressed and the yeast into which the pYES2 vector was introduced.
  • the uptake of cadmium by the yeast expressing the OsNrat1 protein was similar to that of the yeast introduced with the pYES2 vector.
  • RT-PCR was performed on the whole root of the obtained OsNrat1 gene overexpression strain to examine the expression of the OsNrat1 gene.
  • RNA extraction was performed using RNeasy plant extraction mini kit (Qiagen).
  • the reverse transcription reaction was performed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen).
  • PCR reaction conditions are as follows. Composition of the reaction solution: 20 ⁇ L of reaction solution, 1 ⁇ SYBR Premix Ex Taq (manufactured by Takara Bio Inc.), one set of 0.2 ⁇ M each primer, 2 ⁇ L of reverse transcription product diluted 20 times PCR conditions: 95 ° C. for 30 seconds, (95 ° C. for 15 seconds, 58 ° C. for 15 seconds, 72 ° C. for 30 seconds) ⁇ 40 cycles PCR was performed using Mastercycler ep realplex (Eppendorf).
  • shaft of the graph of FIG. 16 represents the relative expression level when the expression level of the OsNrat1 gene is set to 1 in the absence of aluminum.
  • the expression of the OsNrat1 gene was about 80 times higher than that of wild-type rice.
  • the OsNrat1 gene overexpression strain and wild-type rice were exposed to a 0.5 mM CaCl 2 solution containing 30 ⁇ M aluminum for 24 hours to measure root elongation and aluminum uptake into cells.
  • wild type rice and an overexpression strain exposed to a 0.5 mM CaCl 2 solution containing no aluminum for 24 hours were used as controls.
  • FIG. 17 shows the growth rate of roots of wild-type rice and OsNrat1 overexpressing strains after exposing the OsNrat1 over-expressing strain and wild-type rice to 0.5 mM CaCl 2 solution containing 30 ⁇ M aluminum for 24 hours. %).
  • the vertical axis of the graph represents the root elongation rate of the wild-type rice and the OsNrat1 gene overexpression strain when the root elongation in the control is 100%.
  • FIG. 19 is a diagram showing aluminum accumulation in root cells.
  • “NE7009” represents an OsNrat1 gene disruption strain
  • “5-5” and “5-7” represent OsNrat1 gene overexpression strains.
  • the amount of accumulated aluminum was smaller in the OsNrat1 gene-disrupted strain compared to wild-type rice.
  • the OsNrat1 gene overexpression strain the amount of aluminum in the root cells was increased.
  • a transformed plant that promotes aluminum uptake can be produced. Since the uptake of aluminum is promoted in such a transformed plant, for example, it can be made into a plant having a modified flower color by cultivating it in a soil containing aluminum. It can also be used to produce aluminum-tolerant plants that can grow in acidic soil. Therefore, the present invention can be suitably used in agriculture.

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Abstract

Les buts de la présente invention sont d'identifier un gène impliqué dans l'absorption d'aluminium par les plantes et de fournir une méthode d'utilisation du gène. Spécifiquement, un polynucléotide codant un polypeptide qui comprend la séquence d'acides aminés représentée par SEQ ID NO : 1, ou un polynucléotide codant un polypeptide qui comprend une séquence d'acides aminés produite par la substitution, la délétion et/ou l'addition d'un ou plusieurs acides aminés dans la séquence d'acides aminés représentée par SEQ ID NO : 1 et possède une activité favorisant l'absorption d'aluminium est introduit dans une plante de telle sorte que le polynucléotide puisse être exprimé.
PCT/JP2010/065426 2009-09-10 2010-09-08 Utilisation d'un gène impliqué dans l'absorption d'aluminium par les plantes WO2011030793A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005185101A (ja) * 2002-05-30 2005-07-14 National Institute Of Agrobiological Sciences 植物の全長ｃＤＮＡおよびその利用
WO2009037279A1 (fr) * 2007-09-18 2009-03-26 Basf Plant Science Gmbh Plantes à rendement amélioré

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005185101A (ja) * 2002-05-30 2005-07-14 National Institute Of Agrobiological Sciences 植物の全長ｃＤＮＡおよびその利用
WO2009037279A1 (fr) * 2007-09-18 2009-03-26 Basf Plant Science Gmbh Plantes à rendement amélioré

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Title
DATABASE GENBANK [online] 14 February 2008 (2008-02-14), OHYANAGI H ET AL.: "Oryza sativa (japonica cultivar-group Os02g0131800 (Os02g0131800) mRNA, complete cds.", retrieved from http://www. ncbi.nlm.nih.gov/sviewer/viewer.fcgi?val= 115444028&sat=NCBI&satkey=20639534 Database accession no. NM 001052329 *
DATABASE GENBANK [online] 18 February 2008 (2008-02-18), SASAKI T ET AL.: "Oryza sativa Japonica Group genomic DNA, chromosome 2, BAC clone:OJ1007_ D04.", retrieved from http://www.ncbi.nlm. nih.gov/sviewer/viewer.fcgi?val=31621039&sat= DDBJ&satkey=7815453 Database accession no. AP004150 *
XIA J X ET AL.: "Identification of Al transporter in rice.", PLANT CELL PHYSIOL., vol. 51, 12 March 2010 (2010-03-12), pages 44, Retrieved from the Internet <URL:http://www.jspp.org> *
YAMAJI N ET AL.: "A zinc finger transcription factor ART1 regulates multiple genes implicated in aluminium tolerance in rice.", PLANT CELL, vol. 21, October 2009 (2009-10-01), pages 3339 - 3349, XP055140817, doi:10.1105/tpc.109.070771 *

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