JPWO2011030793A1 - Utilization of genes involved in aluminum uptake in plants - Google Patents

Abstract

A gene involved in aluminum uptake in plants is identified, and a method for using the gene is provided. The present invention relates to a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or one or several amino acids substituted, deleted, and / or added in the amino acid sequence shown in SEQ ID NO: 1. A polynucleotide encoding a polypeptide consisting of an amino acid sequence and having an activity of promoting aluminum uptake is introduced into a plant so that it can be expressed.

Description

  The present invention relates to the use of genes involved in aluminum uptake in plants.

  Aluminum ions are highly toxic to plants. Aluminum contained in the soil elutes as aluminum ions under acidic conditions (about pH 5 or less). Even at low concentrations (several μM), it quickly causes root elongation inhibition, and nourishing water from the roots. Inhibits absorption.

  Thus, aluminum ions are the largest factor that inhibits plant growth in acidic soil, but it has been found that aluminum uptake in plants is involved in the mechanism of plant aluminum tolerance. The present inventor makes plants with high aluminum tolerance such as buckwheat, tea, hydrangea, etc. absorb aluminum in the soil and form a complex of aluminum and an organic acid in the vacuole of cells such as leaves to detoxify it. It is reported that the aluminum resistance is acquired by this (nonpatent literature 1).

  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).

Ma, J. F., Zheng, S. J., Hiradate, S. and Matsumoto, H. 1997. Detoxifying aluminum with buckwheat. Nature 390: 569-570. Ma, J. F., Hiradate, S., Nomoto, K., Iwashita, T. and Matsumoto, H. 1997. Internal detoxification mechanism of Al in hydrangea. Identification of Al form in the leaves.Plant Physiol. 113: 1033-1039.

  Elucidating the mechanism of aluminum uptake into cells in plants is considered to be a useful clue for elucidating the mechanism of aluminum tolerance in plants and artificially changing the color of flowers.

  However, it was unclear what gene is involved in aluminum uptake (transport) in plants.

  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.

  Other objects, features, and advantages of the present invention will be fully understood from the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.

  According to the method for producing a transformed plant according to the present invention, there is an excellent effect that a transformed plant in which 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.

  Since the uptake of aluminum is promoted in the transformed plant according to the present invention, it is considered that 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.

  Moreover, if the kit which concerns on this invention is used, the transformed plant which promoted the uptake | capture of aluminum can be produced easily.

It 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. FIG. 2 is a diagram showing 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 μM aluminum. It is a graph showing the elongation percentage (%) of roots 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. And NE7009, and NE7046, wild-type and rice, aluminum, cadmium (Cd), lanthanum after exposure for 24 hours in 0.5 mM CaCl ₂ solution containing either (Lt), wild-type rice, NE7009 and NE7046 It is a graph showing the elongation rate (%) of the root of the root. It is a graph showing the result of having measured the aluminum density | concentration which exists in a cell and a cell wall about NE7009 and wild type rice. FIG. 7 (a) shows the change over time in the aluminum concentration present in the root cell symplastic solution, (b) shows 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 RT-PCR classified by tissue. It is a graph showing the result of RT-PCR classified by root part. It is a graph showing the result of RT-PCR, (a) shows the expression of OsNrat1 gene with respect to other metals, (b) is in the root of rice when a wild type strain was treated with a solution containing 20 μM 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 | staining in the base located in 15 mm from the front-end | tip of the root of a GFP transformed plant, or the front-end | tip of a root. It is a figure showing the localization of OsNrat1-GFP fusion protein in an onion epidermal cell. It is a figure showing the result of the 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, and (c) is a graph showing the change over time of the uptake of aluminum in yeast. is there. It is a graph showing the uptake | capture of aluminum, an aluminum-citrate complex, and an aluminum-oxalic acid 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. It is a graph showing the result of RT-PCR of the overexpressed body of Nrat1. The root elongation rate (%) of wild type rice and OsNrat1 gene overexpression strain after exposure of OsNrat1 gene overexpression strain and wild type rice to 0.5 mM CaCl ₂ solution containing 30 μM aluminum for 24 hours. It is a graph. It is a graph showing the amount of aluminum taken in the root cell about OsNrat1 gene overexpression strain and wild type rice. It is a figure showing accumulation | storage of aluminum in the cell of a root. In FIG. 19, “NE7009” represents an OsNrat1 gene disruption strain, and “5-5” and “5-7” represent OsNrat1 gene overexpression strains. It is a graph which shows the influence of cadmium and manganese with respect to the uptake | capture of aluminum in yeast. It is a figure which shows the result of an immuno-staining, (a) shows the result in the root of the wild-type rice which is not aluminum-treated, (b) is the result in the root of the OsNrat1 gene disruption strain which is not treated with aluminum (C) shows the results for the roots of wild-type rice treated with 30 μM aluminum for 12 hours, and (d) shows the results for the roots of the OsNrat1 gene-disrupted strain treated with 30 μM aluminum for 12 hours. It is a figure which shows the result of a complementation test. It is a figure which shows the influence of the temperature in the uptake | capture of aluminum of a root. (A) shows the aluminum concentration in the root cell symplast solution, and (b) shows the aluminum concentration in the root cell wall.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” indicating a numerical range indicates “A or more and B or less”.

[1. (Transformed plant production method)
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.

  Examples of the polynucleotide (a) or (b) include polynucleotides corresponding to the 56th A to 1693rd G of SEQ ID NO: 2 showing the entire base sequence of the OsNrat1 gene. .

Here, in the present specification, 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 fact that the uptake of aluminum ions (Al ³⁺ ) promoted the cultivation of the wild-type plant and the transformed plant under the same conditions, and the accumulation of aluminum in the whole plant of the wild-type plant and the transformed plant after cultivation. 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. For example, when 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.

  The “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. As many 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. Means that. 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.

  The “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.

  In addition, based on known Nipponbare sequence information, 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 The polynucleotide can also be obtained by amplifying.

  The polynucleotide can also be obtained by a method of isolating and cloning a DNA fragment containing an oligonucleotide containing the polynucleotide or a partial sequence thereof. For example, 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. As such a probe, 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.

  Although it does not specifically limit as a supply source for acquiring the said polynucleotide, Since the polypeptide (OsNrat1 protein) 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.

  The form of the “polynucleotide” introduced into the plant is not particularly limited as long as it can be introduced into the plant so that it can be expressed. The form of RNA (for example, mRNA) or the form of DNA (for example, cDNA or genome) DNA).

  The “plant” into which the polynucleotide is introduced is not particularly limited and can be appropriately selected depending on the purpose. For example, 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.

  In the method for producing a transformed plant in which uptake of aluminum according to the present invention is promoted, 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. In the present specification, 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). For example, 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. For the production of a recombinant expression vector, 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”). For example, when a recombinant expression vector is introduced into a plant by a method using Agrobacterium (Agrobacterium infection method), it is preferable to use a binary vector such as pBI as the recombinant expression vector. Examples of 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 a cell 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. By using these promoter sequences and the above-mentioned polynucleotide (a) or (b) as a recombinant expression vector incorporated into a plasmid or the like, the introduced polynucleotide can be suitably expressed in plant cells. Among these, from the viewpoint of highly expressing the OsNrat1 protein, it is preferable to use the cauliflower mosaic virus 35S promoter. For example, by highly expressing the OsNrat1 protein, a transformed plant in which aluminum uptake is further promoted can be produced.

  Moreover, the OsNrat1 gene may be expressed in the whole plant or may be specifically expressed in a specific site of the plant. For example, it is conceivable that 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.

  It was revealed by the inventors that OsNrat1 protein is expressed in plant roots. 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.

  For example, by introducing 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. As such a promoter, for example, the OsNrat1 promoter can be used.

  A method for introducing the polynucleotide or the recombinant expression vector into a plant, that is, a transformation method is not particularly limited. For example, 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. Further, the polynucleotide or the recombinant expression vector may be transiently expressed in a plant.

  As the transformation method, conventionally known genetic engineering techniques (gene manipulation techniques) can be used. For example, 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. For example, as the “Agrobacterium infection method”, the method described in Plant, J. 6: 271-282 (1994) can be used.

  Further, whether or not 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. For example, 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. That 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.

  More specifically, 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.

  According to the method for producing a transformed plant according to the present invention, a transformed plant in which uptake of aluminum is promoted can be easily produced.

[2. Transformed plant)
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”, and is omitted here.

  The category of the transformed plant according to the present invention includes not only plant bodies but also various forms of plant cells such as suspension cultured cells, protoplasts, leaf sections, and callus. In addition, once 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 applications 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.

[3. kit〕
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 “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. For example, 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.

  If the kit which concerns on this invention is used, the transformed plant which promoted the uptake | capture of aluminum can be produced easily.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to this.

  The inventor identified many rice genes whose expression is 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/)). As shown in FIG. 1, 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).

(1. Functional analysis of OsNrat1 gene using OsNrat1 gene disruption strain)
In order to analyze the function of OsNrat1, 2 lines (NE7009 and NF7046) of OsNrat1 gene disruption strain (Tos-17 insertion strain) were obtained. These Tos-17 insertion strains were ordered from the Rice Genome Resource Center, National Institute of Agrobiological Sciences. NE7009 and NF7046 are rice (Nipponbare) in which the OsNrat1 gene was disrupted by insertion of the retrotransposon Tos-17. In this example, “wild type rice” is intended to be wild type Nipponbare. FIG. 2 is a diagram showing the insertion positions of retrotransposon Tos-17 in the OsNrat1 gene of NE7009 and NF7046, respectively.

First, wild-type rice, NE7009 and NF7046 were hydroponically cultivated, RNA was extracted from the roots, RT-PCR was performed to examine the expression level of OsNrat1. 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 μL of buffer, 0.2 mM dNTP, one set of 0.5 μM primer each, 10 μL of reaction solution, 1 μL 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 is a diagram showing the results of RT-PCR. The amount of Histone H3 was confirmed as an internal standard. As shown in FIG. 3, 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.

Next, wild type rice (n = 10), NE7009 (n = 9), and NF7046 (n = 5) were exposed to 0.5 mM CaCl ₂ solution containing 30 μM aluminum for 24 hours to increase root elongation. It was measured. At this time, wild type rice, NE7009, and NF7046 exposed to a 0.5 mM CaCl ₂ solution containing no aluminum for 24 hours were used as controls.

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 μM 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%.

  As shown in FIG. 4, in the OsNrat1 gene disrupted strain, root elongation was further inhibited compared to wild-type rice. This indicates that the OsNrat1 gene-disrupted strain has lower aluminum resistance than wild-type rice. From this result, it was predicted that the OsNrat1 gene is a gene involved in aluminum tolerance in plants.

Next, NE7009 (each n = 10) and wild type rice (each n = 10) were exposed to 0.5 mM CaCl ₂ solution containing different concentrations of aluminum for 24 hours to measure root elongation. At this time, wild type rice and NE7009 exposed to a 0.5 mM CaCl ₂ solution containing no aluminum for 24 hours were used as controls.

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%.

As shown in FIG. 5, 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.

Therefore, NE7009 (each n = 10), NE7046 (each n = 10), and wild-type rice (each n = 10) are added to a 0.5 mM CaCl ₂ solution containing any one of aluminum, cadmium, and lanthanum. After 24 hours of exposure, root elongation was measured. At this time, the concentration of each metal was adjusted to be 30 μM aluminum, 10 μM cadmium, and 5 μM lanthanum. In addition, wild type rice, NE7009 and NE7046 exposed to a 0.5 mM CaCl ₂ solution containing no aluminum for 24 hours were used as controls.

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%.

As shown in FIG. 6, when treated with a 0.5 mM CaCl ₂ solution containing cadmium or lanthanum, OsNrat1 gene disruption strains (NE7009 and NE7046) and wild-type rice root elongation were inhibited to the same extent. That is, the resistance to cadmium and lanthanum was almost the same in the OsNrat1 gene-disrupted strain and wild-type rice. On the other hand, when treated with a 0.5 mM CaCl ₂ solution containing aluminum, root elongation of the OsNrat1 gene-disrupted strain was further inhibited compared to wild-type rice. From this result, it was considered that the OsNrat1 gene is a gene specifically involved in aluminum tolerance of plants.

Next, intracellular aluminum concentrations were compared for wild-type rice and OsNrat1 gene disruption lines (NE7009 and NE7046). Specifically, NE7009, NE7046 and wild type rice were treated for 0-10 hours in a 0.5 mM CaCl ₂ solution containing 30 μM 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.

In addition, in order to investigate the effect of pH on aluminum uptake, NE7009, in 0.5 mM CaCl ₂ solution containing 30 μM aluminum buffered in the pH 4.0-6.9 range using 10 mM homopipes. NE7046 and wild type rice (each n = 3) were treated for 8 hours.

  After the treatment, an area of 0 to 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. Further, the residue after centrifugation was washed three times with 70% ethanol, 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.

  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.

  As shown in FIG. 7 (a), in wild-type rice, the concentration of aluminum present in the cell symplast solution at the tip of the root (region of 0 to 1 cm from the tip) according to the treatment time with the aluminum solution. Tended to increase over time. On the other hand, in NE7009, regardless of the treatment time in the aluminum solution, the concentration of aluminum present in the cell symplast solution at the tip of the root (region of 0 to 1 cm from the tip) hardly increased. Further, as shown in FIG. 7 (b), in wild-type rice and NE7009, the amount of aluminum in the cell wall of the tip of the root (region of 0 to 1 cm from the tip) depends on the treatment time with the aluminum solution. It became clear that it increased with time. And it became clear that NE7009 has the tendency for the amount of aluminum couple | bonded with the cell wall to increase rather than wild-type rice when the processing time in an aluminum solution becomes long. From this result, it was considered that the OsNrat1 gene has an activity of transporting aluminum into cells.

  As shown in FIG. 7 (c), when the pH was lower than 5.0, the wild-type strain had a significantly higher aluminum concentration than the OsNrat1 gene disrupted strain. However, above pH 5.5, there was no difference in the aluminum concentration of the OsNrat1 gene disrupted strain and the wild type strain.

Further, to investigate the effect of temperature on aluminum uptake, NE7009, NE7046 and wild type rice were treated in 0.5 mM CaCl ₂ solution (pH 4.2) containing 30 μM aluminum for 8 hours at 4 ° C. or 25 ° C. did.

  After the treatment, an area of 0 to 1 cm was collected from the tip of the root, and a cell symplast solution and an extract from the cell wall were obtained by the freeze-thaw method described above. 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.

  The results are shown in FIG. FIG. 23A shows the concentration of aluminum in the root cell symplast, and FIG. 23B shows the concentration of aluminum in the root cell wall. As shown in 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. In contrast, at 25 ° C., the concentration of aluminum in the root cell symplast was higher in the wild type strain than in the OsNrat1 gene disrupted strain. At 25 ° C., 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.

Furthermore, 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 . In addition, 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 results are shown in Table 1. Data are shown as mean ± standard deviation (n = 3). “-” In the table indicates that no detection was possible.

As shown in Table 1, in the root and aerial parts of the OsNrat1 gene disrupted strain and wild type strain, the concentration of macronutrients containing potassium, calcium and magnesium and the concentration of micronutrients containing iron, copper, zinc and manganese are different. There was no.

  A complementation assay was then performed. To perform a complementation assay, a 6.915 kb promoter containing the promoter region of the OsNrat1 gene (2.1 kb before the start codon (ATG)), ORF and 3′-URT (1 kb after the stop codon (TGA)) The DNA fragment was amplified from Nipponbare genomic DNA by the PCR method. The DNA fragment was inserted into the pPZP2H-lac vector and transformed into NE7009 strain, which is an OsNrat1 gene disruption strain, by the Agrobacterium method.

  The obtained transformed strain, wild type strain, NE7009 was treated with a solution containing 30 μM 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 results are shown in FIG. FIG. 22 is a diagram showing the results of complementarity test, and the data are shown as mean ± standard deviation (n = 3). As shown in FIG. 22, in the transformed strain in which 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.

(2. Analysis of expression pattern of OsNrat1 gene)
Next, the expression pattern of the OsNrat1 gene was examined. Specifically, separate seedlings of the wild type rice, 0.5 mM CaCl ₂ solution containing no aluminum or 6 hours in 0.5 mM CaCl ₂ solution containing 30μM aluminum, after which the roots and the aerial parts And RNA was extracted from each tissue. In the present embodiment, 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 μL of buffer, 0.2 mM dNTP, one set of 0.5 μM primer each, 10 μL of reaction solution, 1 μL 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. As shown in FIG. 8, 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.

Next, the effect of aluminum on the expression of the OsNrat1 gene in wild-type rice roots was examined. Specifically, the wild-type rice (each n = 3), and 6 hours at 0.5 mM CaCl ₂ solution containing 0.5 mM CaCl ₂ solution or 50μM aluminum, which does not contain aluminum. Thereafter, the root was cut 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 μL of reaction solution, 1 × SYBR Premix Ex Taq (manufactured by Takara Bio Inc.), one set of 0.2 μM each primer, 2 μL 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 of FIG. 8, the expression of the OsNrat1 gene in roots was induced by aluminum. It was also revealed that the expression level of the OsNrat1 gene 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 of 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.

Furthermore, it was examined whether the expression of the OsNrat1 gene was induced by the presence of a metal other than aluminum or a change in pH. Specifically, wild type rice (each n = 3) was treated for 6 hours in a 0.5 mM CaCl ₂ solution (pH 4.5) containing either 50 μM aluminum, 30 μM cadmium, or 10 μM lanthanum. Wild-type rice was also treated for 6 hours in a 0.5 mM CaCl ₂ solution containing no aluminum, adjusted to pH 4.5 or pH 5.6. Thereafter, RNA was extracted from the whole root. 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 μL of reaction solution, 1 × SYBR Premix Ex Taq (manufactured by Takara Bio Inc.), one set of 0.2 μM each primer, 2 μL 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).

  The results are shown in FIG. FIG. 10 (a) is a graph showing the expression of the OsNrat1 gene relative to other metals. In addition, the vertical axis | shaft of the graph of Fig.10 (a) represents the relative expression level when the expression level of the OsNrat1 gene is set to 1 in the condition of pH4.5 in absence of aluminum. Data are shown as mean ± standard deviation (n = 3).

  As shown in FIG. 10 (a), similar to the RT-PCR results of FIGS. 8 and 9, the expression of the OsNrat1 gene in roots was induced by aluminum. On the other hand, the expression of the OsNrat1 gene in roots was not induced by changes in pH or metals other than aluminum.

  In addition, the wild type strain was treated with a solution containing 20 μM aluminum, and then RNA was extracted from the whole root. RT-PCR was performed using the obtained RNA, and the expression level of the OsNrat1 gene was examined. The results are shown in FIG. FIG. 10 (b) shows the time course of OsNrat1 gene expression in rice roots when the wild-type strain was treated with a solution containing 20 μM aluminum. Data are shown as mean ± standard deviation (n = 3).

  As shown in FIG. 10 (b), it was revealed that the expression level of the OsNrat1 gene was rapidly increased by aluminum and reached the maximum 3 hours after the aluminum treatment.

  Furthermore, the expression of the OsNrat1 gene in rice mutants lacking the C2H2-type zinc finger transcription factor (art1 gene) was examined. Wild type strains and artl mutants were treated with 20 μM aluminum solution for 4 hours. The expression of OsNrat1 gene in roots was measured by quantitative real-time PCR. The results are shown in FIG. FIG. 10 (c) shows the expression of the OsNrat1 gene in the art1 mutant. In (c) of FIG. 10, the relative expression level with respect to histone H3 (internal standard) is shown. Data are shown as mean ± standard deviation (n = 3).

  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). A Zn-finger transcription factor ART1 regulates multiple genes implicated in aluminum tolerance in rice. Plant Cell 21: 3339-3349).

  Next, in order to investigate the localization of the OsNrat1 gene in the cell, a recombinant expression vector containing a fusion gene incorporating a gene encoding a fluorescent protein GFP (green fluorescent protein) under the control of the OsNrat1 gene promoter ( ProOsNrat1-GFP vector) was prepared, and the recombinant expression vector was transformed into wild-type rice. Transformation was performed by the Agrobacterium method.

  By examining the localization of GFP in the obtained GFP-transformed plant, the expression site in the root tissue of the OsNrat1 protein 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.

  FIG. 11 is a diagram showing the results 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.

  In order to examine the intracellular localization of the OsNrat1 protein in more detail, 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. As a positive control, 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 is a diagram showing 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.

  Furthermore, immunostaining was performed to examine the cellular and intracellular localization of the OsNrat1 protein. 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 μM 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 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 μM aluminum for 12 hours, (d) shows the results of 30 μM aluminum. The result of the OsNrat1 protein immunostaining in the root of the OsNrat1 gene disruption strain | stump | stock processed for 12 hours is shown. The scale bar in the figure corresponds to 100 μm.

  As shown in FIGS. 21 (a) and 21 (c), in wild-type rice, OsNrat1 protein was expressed in all root cells except epidermal cells, and the expression level was increased by aluminum.

  As shown in FIGS. 21 (b) and (d), no signal was observed in the OsNrat1 gene disrupted strain, confirming the specificity of the antibody used for immunostaining.

  Further, in order to examine intracellular localization, anti-OsNrat1 antibody and DAPI were double-stained. As a result, it was revealed that the OsNrat1 protein was localized in the cell membrane (not shown).

(3. Analysis of aluminum transport activity of OsNrat1 protein)
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 his2Δ0 met15Δ0 ura3Δ0)) 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 μM, 100 μM, or 200 μM 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.

Furthermore, the obtained transformed yeast was treated with a 50 μM 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, and (c) is a graph showing the change over time of the uptake of aluminum in yeast. is there. Aluminum uptake in yeast was measured by flameless atomic absorption. Data are shown as mean ± standard deviation (n = 3).

  As shown in FIG. 13 (a), the yeast in which the OsNrat1 protein was expressed was inhibited from growing in a medium containing aluminum as compared to 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 the yeast introduced with the pYES2 vector as a negative control.

  In addition, as shown in FIG. 13 (b), it was revealed that the uptake of aluminum into yeast is increased by the expression of OsNrat1.

  In FIG. 13 (c), 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.

  Furthermore, in order to investigate the influence of cadmium and manganese on the uptake of aluminum, transformed yeast was treated with a solution containing an equal concentration of cadmium or manganese for 2 hours in the presence of 50 μM aluminum. Aluminum uptake in yeast was measured by flameless atomic absorption.

  The results are shown in FIG. FIG. 20 is a graph showing the effect of cadmium and manganese on aluminum uptake in yeast. Data are shown as mean ± standard deviation (n = 3). As shown in FIG. 20, it was confirmed that the incorporation of aluminum was not affected by the presence of equimolar concentrations of divalent ions (cadmium and manganese).

  Further analysis was performed on the aluminum transport activity of the OsNrat1 protein. Specifically, yeast that expressed OsNrat1 protein, yeast that expressed AtNramp4 protein as a negative control, and yeast introduced with pYES2 vector contain 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. In addition, the aluminum-citric acid complex used what mixed aluminum and a citric acid in the ratio of 50: 500 (micromol). As the aluminum-oxalic acid complex, a mixture of aluminum and oxalic acid at a ratio of 50: 500 (μM) was used. Moreover, the uptake | 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-oxalate complex in yeast. As shown in FIG. 14, yeast expressing OsNrat1 protein showed improved aluminum ion transport activity, but did not show transport activity for aluminum-citrate complexes or aluminum-oxalate complexes. Moreover, it became clear that 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.

  Furthermore, the transport activity of the OsNrat1 protein against divalent metals (Fe, Mn, Cd) 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.

  These yeasts were inoculated on an agar medium containing no iron or manganese (including traces) and cultured at 30 ° C. for 3 days. In order to further reduce the effectiveness of iron and manganese contained in traces, bathophenanthroline disulfonic acid (BPDS) was added to the medium at a concentration of 0 μM, 8 μM or 10 μM for iron as a chelate compound. In some cases, EGTA was added to the medium at a concentration of 0 mM or 2 mM. Further, 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 μM, 2 μM, 5 μM, 10 μM or 20 μM cadmium.

  FIG. 15 is a diagram showing the results of 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.

  As shown in FIGS. 15 (a) and 15 (b), 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.

  Further, as shown in FIG. 15 (c), the uptake of cadmium 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. These results revealed that the OsNrat1 protein is a specific aluminum transporter.

(4. Functional analysis of OsNrat1 gene using OsNrat1 gene overexpression rice)
In addition, a physiological analysis of the OsNrat1 protein was performed. Specifically, a recombinant expression vector in which the OsNrat1 gene is incorporated under the control of the ubiquitin promoter is prepared, the recombinant expression vector is introduced into rice (variety: Nipponbare) by the Agrobacterium method, and the OsNrat1 protein is overexpressed OsNrat1 gene overexpression strains (strain numbers: 5-5 and 5-7) were prepared.

First, RT-PCR was performed on the entire 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 μL of reaction solution, 1 × SYBR Premix Ex Taq (manufactured by Takara Bio Inc.), one set of 0.2 μM each primer, 2 μL 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. 16 is a graph showing the results of RT-PCR of Nrat1 overexpressors (each n = 3). In addition, the vertical axis | 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. In the OsNrat1 gene overexpression strain, it was revealed that the expression of the OsNrat1 gene was about 80 times higher than that of wild-type rice.

Therefore, the OsNrat1 gene overexpression strain and wild-type rice were exposed to a 0.5 mM CaCl ₂ solution containing 30 μM aluminum for 24 hours to measure root elongation and aluminum uptake into cells. At this time, 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 μM 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%. The number of data in each graph is 5-5 (n = 9), 5-7 (n = 8), and wild type rice (n = 13).

As shown in FIG. 17, when treated with a 0.5 mM CaCl ₂ solution containing 30 μM aluminum, the elongation of roots of the OsNrat1 gene overexpressing strain was significantly inhibited as compared with wild-type rice. That is, the aluminum tolerance of the OsNrat1 gene overexpression strain was reduced.

  FIG. 18 is a graph showing the amount of aluminum incorporated into root cells of OsNrat1 gene overexpression strain and wild-type rice. The number of data in each graph is 5-5 (n = 3), 5-7 (n = 3), and wild type rice (n = 3). As shown in FIG. 18, in the OsNrat1 gene overexpression strain, the aluminum uptake was promoted, and as a result, the aluminum concentration in the root cells was higher than that in the wild-type rice.

  Overexpression of the OsNrat1 gene results in excessive incorporation of aluminum into the root cells of the OsNrat1 gene overexpression strain. However, in the OsNrat1 gene overexpression strain, the mechanism of detoxifying aluminum incorporated into cells, for example, the activity of accumulating aluminum in vacuoles is the same as that of wild-type rice. It is considered that the growth of roots was inhibited by aluminum, and as a result, the aluminum tolerance of the OsNrat1 gene overexpressing strain was weaker than that of wild-type rice.

Subsequently, accumulation of aluminum in root cells was observed with an aluminum-specific fluorescent staining reagent Morin. Specifically, various rice plants were exposed to a 0.5 mM CaCl ₂ solution containing 50 μM aluminum for 24 hours, and sections were prepared for different root sites (2 mm region and 4 mm region from the tip of the root), Stained with Morin. Thereafter, the stained sections were observed under a microscope.

  FIG. 19 shows the accumulation of aluminum in root cells. In FIG. 19, “NE7009” represents an OsNrat1 gene disruption strain, and “5-5” and “5-7” represent OsNrat1 gene overexpression strains. As shown in FIG. 19, the amount of accumulated aluminum was smaller in the OsNrat1 gene disrupted strain than in the wild type rice. On the other hand, in the OsNrat1 gene overexpression strain, the amount of aluminum in the root cells was increased.

  From the above physiological analysis results, it was revealed that the OsNrat1 protein is involved in aluminum transport into plant cells.

  According to the present invention, 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.

Claims (3)

A method for producing a transformed plant that promotes the uptake of aluminum,
The following (a) or (b) polynucleotide is introduced into a plant so as to allow expression: a method for producing a transformed plant:
(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.   A transformed plant produced by the method for producing a transformed plant according to claim 1. A kit for producing a transformed plant in which uptake of aluminum is promoted,
A kit 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.