WO2007119381A1

WO2007119381A1 - Gene participating in aluminum tolerance and utilization of the same

Info

Publication number: WO2007119381A1
Application number: PCT/JP2007/055284
Authority: WO
Inventors: Jian Feng Ma
Original assignee: National University Corporation Okayama University
Priority date: 2006-03-16
Filing date: 2007-03-15
Publication date: 2007-10-25
Also published as: JPWO2007119381A1

Abstract

A gene participating in aluminum tolerance (Als 1 gene) is identified and isolated as a novel gene by map base cloning with the use of individuals belonging to the F2 generation obtained by crossing an aluminum-sensitive mutant (als 1 mutant) with Kasalath. Thus, it becomes possible to identify a gene participating in aluminum tolerance, which has never been identified so far, and provide a method of using the gene.

Description

Specification

Genes involved in aluminum resistance and use thereof

Technical field

[0001] The present invention relates to a novel gene involved in aluminum resistance and use thereof.

Background art

[0002] Acidic soil accounts for about 40% of the world's arable land area. Acidic soil is a soil in which inhibiting plant growth is a problem. Plant growth inhibition is caused by aluminum toxicity. Aluminum ions quickly cause root elongation inhibition even at low concentrations (several M) and inhibit the absorption of nutrients from the roots. As a result, plants become vulnerable to various stresses. For this reason, the productivity of plants in acidic soil is very low.

[0003] Growth inhibition by aluminum varies depending on the type of plant. In other words, the aluminum tolerance varies greatly depending on the type of plant. Gramineae (Aceae) are considered to be highly resistant to aluminum. In particular, the aluminum tolerance of rice is the strongest among grasses. The aluminum tolerance of Gramineae plants is in the order of rice, rye> wheat> barley.

[0004] Aluminum tolerance varies greatly among plant varieties. For example, Japanese rice varieties are more resistant to aluminum than Indian rice varieties.

[0005] However, regions with high rice production are often acidic soils. For example, it is often an acid sulfate soil paddy rice cultivation area and an acid land cultivation area. In regions where rice production is high, rice varieties with weak aluminum tolerance are cultivated. For this reason, rice productivity is very low. For this reason, in order to improve plant productivity in acidic soil, it is required to produce rice with strong aluminum tolerance.

[0006] Thus, aluminum ions are the largest factor that inhibits plant growth.

[0007] The inventor of the present application is energetically researching aluminum tolerance of plants (see, for example, Patent Documents 1 to 3, Non-Patent Documents 1 to 4).

Patent Document 1: Japanese Patent Publication “JP 2004-105164 Publication (April 8, 2004)” Patent Document 2: Japanese Published Patent Publication “Japanese Patent Laid-Open No. 2004-344024 (published on December 9, 2004)”

Patent Document 3: Japanese Patent Publication “JP 2005-058022 Publication (published on March 10, 2005)”

Non-patent literature 1: Ma, J. F. 2005. Plant root responses to three aoundant soil mineral: s ilicon, aluminum and iron. Crit. Rev. Plant ¾ci. 24, 2b / — 281.

Non-Patent Document 2: Ma, J. F., Nagao, S., Huang, C. F., Nishimura, M. 2005. Isolation and characterization of a rice mutant hypersensitive to Al. Plant Cell Physiol. 46, 1054—1061.

Non-Patent Document 3: Ma, JF, Shen, R "Zhao, Z., Wissuwa, M., Takeuchi, Y., Ebitani, T. and Yano, M .: Response of rice to Al stress and identification of quantitative trait 1 oci for Al tolerance. Plant Cell Physiol. 43: 652—659 (2002).

Non-Patent Document 4: Delhaize E, Ryan PR, Hebb DM, Yamamoto Y, Sasaki T and Matsumo to H 2004: Engineering high-level aluminum tolerance in barley with the ALMTl ge ne.Proc. Natl. Acad. Sci. USA, 101 : 15249-15254

Disclosure of the invention

[0008] However, a gene involved in aluminum tolerance has not yet been identified, and the mechanism of aluminum tolerance in plants should be elucidated.

[0009] An object of the present invention is to identify a gene involved in aluminum resistance and provide a method for using the gene.

[0010] The present inventor has intensively studied a gene involved in strong aluminum resistance that has not been obtained so far. As a result, we succeeded in identifying the gene by map-based cloning using an F2 population obtained by mating with an aluminum-sensitive mutant (alsl mutant) and Kasalath. Thus, the present invention has been completed.

That is, the polynucleotide according to the present invention is a polynucleotide involved in aluminum resistance,

The following polynucleotide (a) or (b): (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1;

(b) Polynucleids that are hybridized under stringent conditions with either (i) or (ii) below:

(i) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; or

(ii) It is characterized by having a polynucleotide power that is complementary to the base sequence shown in SEQ ID NO: 1! /

[0012] Here, in the present invention, "aluminum tolerance" refers to the ability of a plant to grow normally even in the presence of aluminum. In other words, “aluminum tolerance” refers to resistance to growth inhibition by aluminum. “Aluminum” may be ionized or may form a salt. “Aluminum” indicates aluminum and a compound containing aluminum. “Involved in aluminum resistance” means having (providing) aluminum resistance.

[0013] According to the above polynucleotide, a polypeptide involved in aluminum resistance can be obtained as a translation product.

[0014] The polypeptide according to the present invention is a polypeptide involved in aluminum resistance, and the following polypeptide (a) or (b):

(a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2;

(b) The amino acid sequence shown in SEQ ID NO: 2 is characterized by being a polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted or added.

[0015] According to the above polypeptide, aluminum resistance can be imparted.

[0016] Such a polypeptide involved in aluminum resistance exists, for example, in a region containing an amino acid encoded by a gene seated on the sixth chromosome in rice. Rice is particularly resistant to aluminum. That is, rice has a polypeptide (polynucleotide) that contributes to aluminum resistance. However, polypeptides lacking some of the amino acids encoded in this region have little aluminum resistance. For example, in the amino acid sequence shown in SEQ ID NO: 2, a polypeptide in which amino acids 123 to 127 are deleted Etc. have little aluminum resistance (aluminum sensitivity). The polypeptide represented by SEQ ID NO: 2 is an amino acid sequence of a polypeptide involved in rice aluminum tolerance.

[0017] The polypeptide according to the present invention may prevent root elongation inhibition by aluminum. As a result, root force aluminum can be eliminated and growth inhibition by aluminum can be prevented.

[0018] In addition, the polynucleotide according to the present invention may encode any of the above polypeptides. According to the above polynucleotide, a polypeptide involved in aluminum resistance can be obtained as a translation product. Examples of the polynucleotide include the polynucleotide (a) or (b) described above.

[0019] A marker gene for selecting a transformant that is useful in the present invention is one having the above-described polynucleotide power.

[0020] The polynucleotide useful in the present invention is capable of imparting aluminum resistance to cells (especially plant cells) in which it is expressed.

[0021] These marker genes can also be used for 用 IJ to select varieties with high aluminum tolerance.

[0022] A recombinant expression vector useful in the present invention includes any of the above-described polynucleotides. The above-described recombinant expression vector can be used as a recombinant expression vector for introducing a polynucleotide useful for the present invention into a cell. It can also be used as a recombinant expression vector for introducing these genes into cells.

[0023] A transformant that is effective in the present invention is one in which the above-described polynucleotide or the above-described thread-and-replaceable expression vector is introduced, and a polypeptide involved in aluminum resistance is expressed. . Here, since the polynucleotide is a polynucleotide involved in aluminum tolerance, the transformant is preferably a plant (transformed product).

[0024] This transformant is introduced together with a promoter that promotes the expression of the above-mentioned polynucleotide or a polypeptide involved in recombinant expression vector ability aluminum resistance. Yes. For this reason, the aluminum tolerance of this transformant can be increased by expressing a polypeptide involved in aluminum tolerance.

[0025] The transformant with enhanced aluminum tolerance has reduced growth inhibition by aluminum, and thus can increase productivity in acidic soil.

[0026] A transformation kit according to the present invention comprises at least one of the above-described polynucleotides or the above-described recombinant expression vector. By using the above transformation kit, a transformant expressing the polypeptide of the present invention can be obtained simply and efficiently.

[0027] According to the polynucleotide of the present invention, aluminum resistance can be imparted by producing a polypeptide involved in aluminum resistance. Since the transformant of the present invention in which the polynucleotide of the present invention or the thread-replaceable expression vector containing the polynucleotide is introduced together with a promoter that promotes the expression of the polypeptide is imparted with aluminum resistance, Growth inhibition by aluminum can be reduced.

[0028] Still other objects, features, and advantages of the present invention will be sufficiently enhanced by the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.

Brief Description of Drawings

[0029] FIG. 1 is a schematic diagram showing the amino acid sequences of the Alsl gene and a polypeptide encoded by the gene.

FIG. 2 is a schematic diagram showing Alsl gene and the insertion site of T-DNA insertion strain and Tosl7 disruption strain.

FIG. 3 is a diagram showing the results of analyzing the expression level of the Alsl gene in Example 2 by quantitative RT-PCR.

FIG. 4 is a schematic diagram mapping the Alsl gene.

[FIG. 5 (a)] Comparison of growth state between wild-type rice and alsl mutant.

[FIG. 5 (b)] Comparison of growth state between wild-type rice and alsl mutant.

FIG. 6 is a graph comparing the relative elongation of roots in Example 3.

[Fig.7 (a)] Alsl gene with and without aluminum in roots and shoots of wild-type rice It is the graph which compared the expression level of.

FIG. 7 (b) is a diagram showing the results of quantitative RT-PCR in which the expression of Alsl gene and lactin was confirmed for wild-type rice roots and shoots.

FIG. 8 (a) is a diagram showing the results of quantitative RT-PCR in which expression of Alsl gene and lactin was confirmed for different sites and shoots of wild-type rice roots.

[FIG. 8 (b)] A graph comparing the expression levels of the Alsl gene in different shoots and shoots of wild-type rice.

FIG. 8 (c) is a diagram showing the positions of the root tip and the root base in FIGS. 8 (a) and 8 (b).

FIG. 9 is a graph showing the results of analysis of aluminum resistance of transformants into which the alsl mutant Alsl gene was introduced.

FIG. 10 is a diagram showing the results of staining aluminum present in the roots of each plant in FIG. 9.

[Fig. 11] A diagram showing the expression site of Alsl protein in a transformant introduced with an Alsl-GFP fusion gene.

FIG. 12 shows the results of antibody staining using an antibody against Alsl protein.

FIG. 13 is a graph showing the relationship between the expression level of the Alsl gene and the elapsed time after aluminum treatment.

FIG. 14 is a graph showing the relationship between the expression level of the Alsl gene and the aluminum treatment concentration.

FIG. 15 shows intracellular aluminum in Koshihikari and alsl mutants.

BEST MODE FOR CARRYING OUT THE INVENTION

[0030] An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this. SEQ ID NO: 1 is the base sequence of the Alsl gene (cDNA). SEQ ID NO: 2 is the amino acid sequence of the Alsl protein. SEQ ID NOs: 3 and 4 are base sequences of complete cDNA (full cDNA) formed by binding an untranslated region (UTR) to the base sequence of SEQ ID NO: 1. SEQ ID NO: 5 is the base sequence of the genomic gene of wild-type rice (Koshihikari) containing the Als gene.

[0031] (1) Polynucleotide useful for the present invention

Polynucleotides that are useful in the present invention are polypeptides that are involved in aluminum resistance. It is to be

Here, 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 a “nucleic acid sequence” or a “nucleotide sequence”, and is indicated as a sequence of deoxyribonucleotides (abbreviated as A, G, C and T). The “polynucleotide having the base sequence ability shown in SEQ ID NO: 1” refers to a polynucleotide comprising the sequence shown by each of the dioxynucleotides A, G, C and Z or T of SEQ ID NO: 1. .

[0033] The polynucleotides of the invention may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). The DNA may be double stranded or single stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand)! /.

[0034] The polynucleotide useful in the present invention is a polynucleotide involved in aluminum resistance, and is the following polynucleotide (a) or (b).

[0035] (a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1;

(i) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; or

(ii) a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1.

[0036] The polynucleotide (a) or (b) is a polynucleotide involved in aluminum resistance (resistance).

[0037] The above "stringent conditions" refer to hybrids only when at least 90% identity, preferably at least 95% identity, most preferably at least 97% identity exists between sequences. This means that a sesion occurs, for example, binding at 60 ° C under 2 X SSC wash conditions. The above hybridization can be performed by a conventionally known method such as the method described in “Molecular Cloning (Third Edition)” (J-bambroo & DW Russell, old Spring Harbor Laboratory Press, 2001). Generally, the higher the temperature and the lower the salt concentration, the higher the stringency. [0038] Among the above polynucleotides, the polynucleotide having the nucleotide sequence shown by SEQ ID NO: 1 is a gene involved in aluminum tolerance, which was first identified in the plant kingdom.

[0039] The polynucleotide of SEQ ID NO: 1 is the base sequence (cDNA sequence) of the Alsl gene derived from wild type (Japanese type) rice. Rice is a typical plant with strong aluminum tolerance. In particular, Japanese rice varieties have strong aluminum tolerance, so even if they are cultivated in acidic soil, their growth is not easily inhibited. Thus, SEQ ID NO: 1 is a polynucleotide (wild-type Alsl gene) involved in aluminum resistance derived from wild-type rice having high aluminum resistance.

[0040] The polynucleotide useful in the present invention is a polynucleotide that encodes a polypeptide involved in aluminum resistance, and a polynucleotide that encodes the following polypeptide (a) or (b).

[0041] (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2;

(b) A polypeptide having an amino acid sequence ability in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 2.

[0042] The above "one or several amino acids have been deleted, substituted or added" means deletion, substitution or addition by a known mutant polypeptide production method such as site-directed mutagenesis. (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) of amino acids are substituted, deleted, or It means to be added. Such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by a known mutant polypeptide production method, but a similar naturally occurring mutant polypeptide is isolated and purified. It may be

[0043] SEQ ID NO: 2 is a polypeptide involved in aluminum resistance found by the present invention.

This polypeptide is encoded by, for example, the wild-type Alsl gene shown in SEQ ID NO: 1.

[0044] Such amino acid mutations are caused by polynucleotide mutations (deletions, substitutions or additions) that are useful in the present invention. Sensitive to aluminum, as in the examples below. Mutants (alsl mutants) are less resistant to aluminum than wild-type rice. In the alsl mutant, the amino acid at positions 123 to 127 in the amino acid sequence shown in SEQ ID NO: 2 is deleted. This significantly reduces the aluminum resistance of the alsl mutant. For this reason, the alsl mutant is sensitive to aluminum and growth is inhibited by aluminum.

[0045] Therefore, the mutation that occurs in the amino acid represented by SEQ ID NO: 2 is preferably a mutation that retains the 123rd to 127th amino acids.

[0046] As shown in the examples described later, the present inventor is responsible for polynucleotide strength involved in the aluminum tolerance of rice, which is one of the polynucleotides of the present invention, to sit on chromosome 6 of rice. Revealed.

[0047] The polynucleotide useful in the present invention may be an oligonucleotide tide that is a fragment of the above-mentioned polynucleotide.

[0048] The polynucleotide or oligonucleotide useful for the present invention is not only a double-stranded DNA, but also a sense strand (coding strand) and an antisense strand (non-coding strand) t, each of which comprises single-stranded DNA. And RNA (eg, mRNA). In addition, the DNA includes, for example, cDNA and genomic DNA that can be obtained by cloning, chemical synthesis techniques, or a combination thereof. The base sequence shown in SEQ ID NO: 1, which is an example of a polynucleotide useful for the present invention, is the cDNA sequence of the polypeptide shown in SEQ ID NO: 2.

[0049] Furthermore, the polynucleotide or oligonucleotide according to the present invention may contain a sequence such as a sequence of an untranslated region (UTR) and a vector sequence (including an expression vector sequence). For example, the cDNA sequence shown in SEQ ID NO: 1 is the ORF (Open Reading Frame) of the polypeptide shown in SEQ ID NO: 2. The nucleotide sequences shown in SEQ ID NOs: 3 and 4 are full-length cDNA sequences including the untranslated region in the cDNA sequence shown in SEQ ID NO: 1. The base sequence shown in SEQ ID NO: 5 is a partial partition sequence of the genome sequence of wild-type Koshihikari chromosome 6. The base sequences shown in SEQ ID NOs: 3 and 4 are also cDNA sequences derived from the base sequence shown in SEQ ID NO: 5.

[0050] As a method for obtaining a polynucleotide or oligonucleotide useful for the present invention, a DN containing the polynucleotide or oligonucleotide useful for the present invention by a known technique. A method of isolating and crawling the A fragment can be mentioned. For example, prepare a probe that specifically hybridizes with a part of the nucleotide sequence of the polynucleotide of the present invention, and screen a genomic DNA library or cDNA library! As such a probe, if the probe specifically hybridizes to at least a part of the base sequence of the polynucleotide or the complementary sequence of the present invention, a misaligned sequence and Z or length can be used. A thing may be used. This probe is preferably one that specifically hybridizes to at least part of the sequence deleted in the alsl mutant in the Alsl gene shown in SEQ ID NO: 1. As a result, a gene involved in aluminum resistance can be obtained with certainty.

[0051] Further, examples of a method for obtaining a polynucleotide according to the present invention include a method using an amplification means such as PCR. For example, among the cDNA cDNAs of the present invention, primers are prepared from the 5 'side and 3, side sequences (or their complementary sequences), respectively, and genomic DNA (or cDNA) or the like is detected using these primers. A large amount of DNA fragments containing the polynucleotide according to the present invention can be obtained by performing PCR or the like in a mold and amplifying the DNA region sandwiched between both primers. In addition, for example, based on the known rice sequence information, a primer that can amplify the Alsl gene region is designed, and using that primer, genomic DNA (or cDNA) or RT-PCR product is made into a saddle shape, By amplifying the Alsl gene region, it is also possible to obtain a polynucleotide according to the present invention.

[0052] The source for obtaining the polynucleotide useful for the present invention is not particularly limited, but a grass plant is preferable. In the examples described later, the wild-type rice (Koshihikari) power is not limited to this, but the power to acquire one of the polynucleotides that are useful in the present invention.

[0053] It should be noted that the polynucleotide useful for the present invention can be used to elucidate the aluminum tolerance mechanism of plants, which has been clarified so far.

[0054] (2) Polypeptide according to the present invention

The polypeptide according to the present invention is a translation product of the polynucleotide described in (1) above, and is at least involved in aluminum resistance. Here, the “polypeptide” can also be referred to as “peptide” or “protein”. The “fragment” of a polypeptide indicates a partial fragment of the polypeptide.

[0056] The polypeptide of the present invention may be isolated from a natural source or chemically synthesized. Here, an “isolated” polypeptide or protein refers to the polypeptide or protein from which its natural environmental forces have been removed. For example, recombinantly produced polypeptides and proteins expressed in host cells have been isolated, as are natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. Shall.

[0057] Polypeptides according to the present invention can be produced by natural purification products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insect cells, and mammals). (Including cells) Includes products produced by recombinant technology. Depending on the host used in the recombinant production procedure, the polypeptide of the present invention may be sugar chain-modified such as glycosyl potato. The polypeptide according to the present invention includes such a modified polypeptide.

[0058] The polypeptide according to the present invention is, for example, a polypeptide involved in at least aluminum resistance, and is the following polypeptide (a) or (b).

[0059] (a) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2;

[0060] The polypeptide having the amino acid sequence ability shown in SEQ ID NO: 2 is an Alsl protein having the 291 amino acid ability encoded by the Alsl gene. The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is, for example, a translation product of a polynucleotide consisting of the base sequence shown in any of SEQ ID NOs: 1 to 3. In the amino acid sequence shown in SEQ ID NO: 2, the 123rd to 127th amino acids are deleted in the alsl mutant. For this reason, the polypeptide of (b) above preferably retains the 123-127th amino acid in the amino acid sequence shown in SEQ ID NO: 2.

[0061] As shown in Fig. 1, the Alsl gene is encoded by the ATPase domain-like protein of the ABC transporter. [0062] The polypeptide is not limited to the polypeptide as long as it is a polypeptide in which amino acids are peptide-bonded, but may contain a structure other than the polypeptide. The structure other than the polypeptide here is not particularly limited as long as it can include sugar chains and isoprenoid groups.

[0063] (3) Marker gene for transformation

The polynucleotide according to the present invention can be used as a marker gene for transformation. That is, the marker gene for selecting a transformant according to the present invention may be any gene as long as it is composed of the polynucleotide useful for the present invention described in (1) above. The polynucleotides that contribute to the present invention confer aluminum resistance. For this reason, the cells into which the polynucleotide useful for the present invention has been introduced are not inhibited from growing even in the presence of aluminum. This is because aluminum accumulation is reduced as a result of eliminating aluminum. Therefore, the cells into which the polynucleotide has been introduced can be selected by measuring the growth state in the presence of aluminum or the amount of aluminum accumulated (amount of aluminum absorbed).

[0064] Specifically, in order to use the polynucleotide of the present invention as a marker gene for transformation, for example, an expression vector incorporating the polynucleotide is constructed, and the expression vector is introduced into a target cell. To do. When the expression vector is introduced, the amount of aluminum accumulated in cells expressing a polypeptide conferring aluminum resistance is reduced. Therefore, by culturing in the presence of aluminum and measuring the amount of aluminum accumulated before and after the introduction of the expression vector, cells in which the polynucleotide associated with aluminum is expressed can be selected. In addition, for example, it is possible to select varieties with strong aluminum resistance.

[0065] In the above-described example, the polynucleotide according to the present invention is used for both purposes of a gene that allows a transformed cell to express a polynucleotide and a marker gene. It is also possible to use it. In addition, for example, by using a transcription promoter specific to plant callus cells, it is possible to control the expression time as a selection marker of a polynucleotide that is useful in the present invention. In this case, an expression vector into which a gene encoding a protein to be expressed in the target cell is inserted may be constructed. And then transformed with the expression vector. It is also possible to introduce a polynucleotide useful for the present invention alone into a target cell without constructing an expression vector incorporating the polynucleotide according to the present invention.

[0066] In the polynucleotide shown in SEQ ID NO: 1, the 3182th to 3196th bases are deleted in the a lsl mutant. For this reason, a polynucleotide containing the 3182 to 3196th bases of the base sequence shown in SEQ ID NO: 1 can be used as a marker gene for the present invention.

[0067] For example, in the polynucleotide shown in SEQ ID NO: 1, the polynucleotide comprising the 3182th to 3196th base groups: a polynucleotide having LOO consecutive basic strengths is highly resistant to aluminum and is used to select cells. Can be used.

That is, such a polynucleotide can be used as a marker gene for selecting cells involved in aluminum resistance (strong aluminum resistance, cells).

[0069] (4) Recombinant expression vector and transformation kit for the present invention

The recombinant expression vector according to the present invention is not particularly limited as long as it contains the polynucleotide according to the present invention described in (1) above. For example, a recombinant expression vector into which the cDNAs shown in SEQ ID NOs: 1 to 3 are inserted can be mentioned. For the production of a recombinant expression vector, a plasmid, phage, cosmid or the like can be used, but it is not particularly limited. In addition, a manufacturing method may be performed using a known method.

[0070] The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell may be appropriately selected. That is, if a promoter sequence is appropriately selected according to the type of host cell in order to reliably express the gene, and this and a polynucleotide useful for the present invention are incorporated into various plasmids or the like, it can be used as an expression vector.ヽ

[0071] The present recombinant expression vector can be used to express a polypeptide that is effective in the present invention! As a matter of course, the polynucleotide according to the present invention is used as a marker gene. And can be used as a recombinant expression vector for expressing a protein encoded by the other gene. [0072] Various markers may be used in order to confirm whether or not the polynucleotide of the present invention has been introduced into a host cell, and whether or not it is reliably expressed in the host cell. ! ヽFor example, a drug resistance gene that gives resistance to antibiotics such as hygromycin is used as the best force, and a plasmid or the like containing this marker and a polynucleotide useful for the present invention is introduced into a host cell as an expression vector. Thereby, the expression power of the marker gene can be confirmed.

[0073] The host cell is not particularly limited, and various conventionally known cells can be preferably used. Specifically, for example, the ability to raise rice, cucumber, rape, tomato, etc. is not particularly limited.

[0074] The method of introducing the above expression vector into a host cell, that is, the transformation method is not particularly limited. The agrobatterium infection method, electroporation method (elect mouth poration method), calcium phosphate method, protoplast Conventionally known methods such as a method, a lithium acetate method, and a particle gun method can be preferably used.

[0075] The transformation kit according to the present invention only needs to contain at least one of the polynucleotide according to the present invention described in (1) above or the recombinant expression vector according to the present invention. Other specific configurations are not particularly limited, and necessary kits may be selected by appropriately selecting necessary reagents and instruments. By using the transformation kit, transformed cells can be obtained simply and efficiently.

[0076] (5) Transformant that is effective in the present invention

The transformant according to the present invention is introduced with the polynucleotide according to the present invention described in (1) above or the recombinant expression vector described in (4) above, and is resistant to aluminum. The transformant is not particularly limited as long as the polypeptide involved in is expressed. Here, “transformant” means not only a cell / tissue organ but also an organism.

[0077] Here, "the polynucleotide has been introduced" means that it is introduced into a target cell (host cell) so that it can be expressed by a known genetic engineering technique (gene manipulation technique). However, in addition to this, the present invention includes the case where the polynucleotide of the present invention contained in the genome is expressed in vivo. [0078] The method for producing a transformant (production method) is not particularly limited, and examples thereof include a method for transformation by introducing the above-described recombinant expression vector into a host cell. In addition, the organisms to be transformed are not particularly limited, and examples thereof include plant cells exemplified as host cells in the above (4).

[0079] The transformant that is useful in the present invention is preferably a plant cell or a plant. Such transformed plants are endowed with aluminum tolerance. For this reason, the content (accumulation amount) of aluminum can be reduced in cells or plants. In the transformant introduced together with the promoter that promotes the expression of the polynucleotide or the recombinant expression vector force polypeptide, aluminum resistance is imparted, thereby eliminating the root strength aluminum, resulting in absorption of aluminum. The amount of accumulated aluminum can be reduced. This can reduce the growth hindrance caused by aluminum.

[0080] The transformed sickle of the present invention has aluminum resistance since the polynucleotide according to the present invention is introduced. For this reason, growth inhibition by aluminum can be reduced.

[0081] The recombinant expression vector used for transformation of the plant is not particularly limited as long as it can express the inserted gene in the plant cell. In particular, when the method for introducing a vector into a plant is a method using agrobacterium, it is preferable to use a binary vector such as pBI. Specific examples of the noinary vector include pBIG, pBIN19, pBI101, pBI121, and pBI221. Moreover, a vector having a promoter capable of expressing a gene in a plant is preferable. Known promoters can be preferably used as the promoter, and specific examples include cauliflower mosaic virus 35S promoter (CaMV35S) and ubiquitin promoter. The plant cells include various types of plant cells such as suspension culture cells, protoplasts, leaf sections, and callus.

[0082] Recombinant expression vector introduction into plant cells includes agrobacterium infection method, electroporation method (elect mouth position method), calcium phosphate method, protoplast method, lithium acetate Conventionally known methods such as a particle method and a particle gun method can be used. In addition, regeneration of a plant body from transformed cells can be performed by a known method depending on the type of plant cell.

[0083] Transformation sickle body power into which the polynucleotide according to the present invention has been introduced into the genome, and once it has been obtained, the polynucleotide has also been introduced into the seed obtained from the plant body power. The present invention also includes seeds obtained from transformed plants.

[0084] (6) Food according to the present invention

The food of the present invention contains the transformant according to the present invention. That is, this food contains a transformant imparted with aluminum resistance.

[0085] The food of the present invention includes feeds given to livestock as well as those taken by humans.

[0086] Rice is a high-consumption plant that is considered a staple food in various parts of the world, not only in Japan. Fruits and vegetables are also high in production and consumption. For this reason, safety is especially important for these crops.

Therefore, the food containing the transformant imparted with high aluminum tolerance is preferably an agricultural product such as rice, vegetables, and fruits. This makes it possible to grow useful rice, vegetables, and fruits that are highly safe.

The present invention is not limited to the above-described embodiment, and various modifications can be made within the scope shown in the claims. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.

[0089] In the following examples, an aluminum resistance gene was successfully isolated by map-based cloning using an aluminum-sensitive mutant (alsl mutant). The al si mutant was previously obtained by the present inventor and was isolated from a Japanese rice cultivar (Koshihikari) with high aluminum tolerance. FIG. 5 (a) and FIG. 5 (b) are diagrams showing the growth state of wild-type rice (WT) and alsl mutants in the presence or absence of aluminum. As shown in Fig. 5 (a) and Fig. 5 (b), the alsl mutant is inhibited from growing in acidic soil (Fig. 5 (a)), and especially in the presence of aluminum, root elongation is inhibited. (Fig. 5 (b)). [Example 1]

First, rough mapping of the target gene, Alsl, was performed using the InDel marker, using the F2 population obtained by mating ability between the alsl mutant and Kasalath. As a result, it became clear that the Alsl gene sits on chromosome 6.

FIG. 4 is a schematic diagram of Alsl gene mapping. As shown in FIG. 4, the Alsl gene (Alsl in the figure) is present in the region of chromosome 6 between 113.4 and 115.6 cM. That is, the Alsl gene is present in the region between the marker MaOs0624 and the marker MaOs0617. Then, PAC clone AP003770 and PAC clone APOO 3771 containing this region were selected.

[0092] Next, using the selected clones, a new InDel marker and a CAPS marker were designed and fine mapping was performed. As a result, the candidate region of the Alsl gene could be narrowed down to 88 kb. A candidate region for the Alsl gene existed between marker MaOs0642 and marker MaOs0654.

[0093] From the prediction of TIGR, there were 14 genes (third row from the top in Fig. 4) in the candidate region of Alsl gene (region between marker MaOs0642 and marker Ma Os0654). Of these, 7 genes were retrotransposon-related genes, so the sequences of the remaining 7 gene translation regions were compared between the wild-type and the alsl mutant. As a result, a gene lacking 15 base pairs in the als 1 mutant was found, and the gene was assumed to be the Alsl gene.

[0094] Fig. 2 is a schematic diagram showing the Alsl gene and the insertion sites of the T-DNA insertion strain and Tosl7 disruption strain. The Alsl gene (genomic gene) has a total length of 3015 bp. The Alsl gene consists of 4 exons (876bP) and 3 introns. The four etasons in SEQ ID NO: 5 are the 354th to 894th bases, the 992st to 1179th bases, the 3126th to 3470th bases, and the 3567th to 4027th bases.

[Example 2]

Next, the cloned gene (presumed Alsl gene) in Example 1 was confirmed using a Tosl7-disrupted strain of the putative Alsl gene and a T-DNA insertion strain. For T-DNA inserts, the putative Alsl gene exon or intron is disrupted (3D-02176, 3A-02044) was used, and the Tosl7 disruption strain used was a system (NG0545) in which a foreign gene was inserted into Exon.

[0096] As a result, the aluminum resistance of the Tosl7-disrupted strain and the T-DNA insertion strain was significantly reduced.

FIG. 3 is a diagram showing the results of analyzing the expression level of the Alsl gene by quantitative RT-PCR. As shown in FIG. 3, Alsl gene expression confirmed in the wild type (WT) was not confirmed in the Tos 17 and T DNA systems. Actin was confirmed in all systems.

[Example 3]

Next, functional analysis was performed on wild-type rice, the alsl mutant, and the Tos-disrupted strain and T DNA-inserted strain of Example 2.

FIG. 6 is a graph comparing the amount of root elongation in each system. In this graph,% relative to the control (the amount of root elongation in the absence of aluminum) is shown. As shown in Fig. 6, the relative elongation of roots was significantly reduced in the lines other than wild-type rice.

FIG. 7 (a) is a graph comparing the expression levels of the Alsl gene in the presence and absence of aluminum in wild-type rice roots and shoots. Fig. 7 (b) shows quantitative RT-PCR in which the expression of the same Alsl gene and actin was confirmed. As shown in Figs. 7 (a) and (b), the Alsl gene was localized and expressed in the roots in the presence of aluminum.

[0101] Figure 8 (a) shows the quantification of wild-type rice that confirmed the expression of the Alsl gene and actin in the root tip (up to root force lcm), root base (root tip force further lcm), and shoot. FIG. 2 shows RT-PCR. FIG. 8 (b) is a diagram comparing the expression levels of the Als 1 gene in the same parts as in FIG. 8 (a). FIG. 8 (c) is a diagram showing the positions of the root tip and the root base in FIGS. 8 (a) and 8 (b). As shown in FIG. 8, the Alsl gene was expressed more in the root than in the shout (aboveground), and in particular, the expression at the tip of the root increased more with aluminum.

[Example 4]

Next, the isolated aluminum resistance gene (Alsl gene) was introduced into the alsl mutant and phased. Complementary experiments were performed. As a result, 4 lines of independent transformed plants (B79-1, 1, 1, 19-2, 35-1) were obtained. FIG. 9 is a graph showing the results of analyzing aluminum resistance of these transformants. For comparison, this graph also shows the results of root elongation for WT (wild-type rice), als mutants, and vector control lines. As shown in Fig. 9, aluminum resistance is conferred in the recombinant system (transformed sickle) into which the Als gene has been introduced. As a result, it was confirmed that the isolated Alsl gene was an aluminum resistance gene.

[0103] Fig. 10 shows each plant of Fig. 9 (alsl mutant (1), WT (1), transformed plant (TG)).

(2), vector control (VC) (2)) It is a figure which shows the result of having dye | stained the aluminum which exists in the root by Eriochrom Cyanine (Eriochrom Cyanine). As shown in FIG. 10, it was confirmed that the transformed plant (TG) into which the Als gene was introduced had less root aluminum deposition, as in the wild type (WT).

[Example 5]

Next, rice was transformed using a vector in which an Alsl promoter and a GFP-inducible gene were linked to the Alsl gene. FIG. 11 is a diagram showing the results of examining the expression site of Alsl protein for transformants. In Fig. 11, “+ Α1” indicates that the aluminum treatment is performed, “−Al” indicates that the aluminum treatment is not performed, and “2 mm and 20 mm” indicate the positions from the root tips, respectively. As shown in FIG. 11, it was confirmed that Alsl protein was expressed in all cells at the root tip and expressed in cells other than epidermal cells at the root base (lateral root). It was also confirmed that Alsl protein was localized in the cell membrane.

[0105] Fig. 12 is a diagram showing the results of antibody staining using an antibody against Alsl protein. As shown in Fig. 12, similar to the results of GFP in Fig. 11, Alsl protein is expressed in almost all cells at the root tip and localized in cells other than epidermal cells at the root base. It was done.

[Example 6]

Next, for Koshihikari and alsl mutants, the effects of aluminum treatment concentration and aluminum treatment elapsed time on the expression level of the Alsl gene were examined. Figure 13 FIG. 14 is a graph showing the relationship between the expression level of the Alsl gene and the elapsed time after aluminum treatment, and FIG. 14 is a graph showing the relationship between the expression level of the Alsl gene and the aluminum treatment concentration. As shown in these figures, it was confirmed that Alsl protein expression was induced about 2 hours after the start of aluminum treatment (Fig. 13) and induced by 5 M aluminum (Fig. 14). The vertical axis in FIG. 13 and FIG. 14 is the relative value of the expression level of the Alsl gene based on the expression level of actin.

[Example 7]

Next, for Koshihikari and alsl mutant, intracellular aluminum was observed by Morin. FIG. 15 shows the results. As shown in Fig. 15, aluminum signals were observed in the alsl mutant, whereas in the wild type (Koshihikari), the signal was not observed. As a result, it was confirmed that aluminum that had entered Alsl protein was released into the cell.

[0108] The specific embodiments or examples made in the detailed description section of the present invention are to clarify the technical contents of the present invention, and are limited to such specific examples. Therefore, the present invention should not be construed in a narrow sense, and can be carried out with modifications within the spirit of the present invention and the scope of the claims described below.

Industrial applicability

[0109] The polynucleotide of the present invention is a gene involved in aluminum tolerance, which was first identified in plants. By expressing this gene, inhibition of plant growth by aluminum can be reduced. Therefore, the present invention can be suitably used particularly for agriculture and the food industry.

Claims

The scope of the claims

[1] A polynucleotide involved in aluminum resistance,

The following polynucleotide (a) or (b):

(a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1;

(i) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; or

[2] A polypeptide involved in aluminum resistance,

The following polypeptide (a) or (b):

[3] The polypeptide according to claim 2, which prevents root elongation inhibition by aluminum.

[4] A polynucleotide encoding the polypeptide according to claim 2 or 3.

[5] A marker gene for selecting a transformant comprising the polynucleotide according to claim 1 or 4.

[6] A recombinant expression vector comprising the polynucleotide according to claim 1 or 4.

[7] A transformant into which the polynucleotide according to claim 1 or 4 or the recombinant expression vector according to claim 6 has been introduced and which expresses a polypeptide involved in aluminum resistance.

[8] A transformant into which a polynucleotide encoding the following polypeptide (a) or (b) has been introduced and which expresses a polypeptide involved in aluminum resistance.

[9] A food comprising the transformant according to claim 7 or 8. A transformation kit comprising the polynucleotide according to claim 1 or 4 or the recombinant expression vector according to claim 5.