WO2015011604A1

WO2015011604A1 - Ascorbate transporter

Info

Publication number: WO2015011604A1
Application number: PCT/IB2014/063104
Authority: WO
Inventors: Yoshinori Moriyama; Takaaki Miyaji; Hiroshi Omote; Takashi Kuromori; Kazuo Shinozaki
Original assignee: National University Corporation Okayama University
Priority date: 2013-07-26
Filing date: 2014-07-15
Publication date: 2015-01-29
Also published as: JP6412127B2; JP2015023831A; JP2016526897A

Abstract

A novel method of imparting stress tolerance, particularly light stress tolerance and oxidation stress tolerance, to a plant is provided. A transporter that transports an ascorbate to a chloroplast was identified and role of the transporter in imparting tolerance to a plant was clarified. The present invention utilizes an ascorbate transporter and a gene encoding the ascorbate transporter, thereby providing a composition and a method that impart stress tolerance, particularly light stress tolerance and oxidation stress tolerance, to a plant.

Description

[DESCRIPTION]

[Title of Invention] ASCORBATE TRANSPORTER

[Technical Field]

[0001]

The present invention relates to an ascorbate transporter protein and a gene encoding the ascorbate transporter. The present invention further relates to a composition and a method for imparting stress tolerance (in particular, tolerance for light stress and/or oxidation stress) to a plant.

[Background Art]

[0002]

A plant performs biosynthesis of an ascorbate and transports the ascorbate to a destination location (chloroplast) to exert various physiological actions. In particular, typical examples thereof are roles as a coenzyme in an enzymatic reaction in a chloroplast and an antioxidation action (removal of active oxygen) .

[0003]

As an enzyme and/or a mutant enzyme utilizing an ascorbate, mutant ascorbate peroxidase (Patent Literature 1), oxidation-resistant ascorbate peroxidase (Patent Literature

2) and violaxanthin de-epoxidase (Patent Literature 3) are known. An attempt has been made to impart stress tolerance to a plant using these enzymes and/or mutant enzymes. However, the effect thereof is not sufficient, and many aspects of the mechanism of stress tolerance of a plant are unknown.

[Citation List]

[Patent Literature]

[0004]

[PTL 1]

Japanese Laid-Open Publication No. 2006-296209

[PTL 2]

Japanese Laid-Open Publication No. 2008-253248

[PTL 3]

Japanese National Phase PCT Laid-open Publication No. 2000-500016

[Summary of Invention]

[Technical Problem]

[0005]

The ob ective of the present invention is to provide a novel method for imparting stress tolerance, particularly light stress tolerance and oxidation stress tolerance, to a plant. [Solution to Problem]

[0006]

The above-described objective has been achieved by identifying a transporter which transports an ascorbate to a chloroplast, and clarifying the role of the transporter in imparting tolerance to a plant.

[0007]

For example, the present invention provides the following.

(Item 1)

A composition for imparting stress tolerance to a plant, wherein the composition comprises a nucleic acid selected from the group consisting of:

(a) a nucleic acid that hybridizes under highly stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, and encodes a polypeptide having ascorbate transport activity;

(b) a nucleic acid that comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence set forth in SEQ ID NO: 1, and encodes a polypeptide having ascorbate transport activity;

(c) a nucleic acid that consists of the nucleic acid sequence set forth in SEQ ID NO: 1;

(d) a nucleic acid that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and

(e) a nucleic acid that encodes a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 2, and having ascorbate transport activity.

(Item 2)

The composition of item 1, wherein the composition comprises a nucleic acid encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

(Item 3)

The composition of item 1, wherein the stress tolerance is tolerance for stress selected from the group consisting of high temperature, low temperature, low pH, low oxygen, oxidation, salt, osmotic pressure, dryness, water, flooding, cadmium, copper, ozone, air pollution, ultraviolet rays, light, pathogens, pathogenic germs, harmful insects, herbicides and aging .

(Item 4)

The composition of item 1, wherein the stress tolerance is selected from the group consisting of tolerance for light stress and tolerance for oxidation stress.

(Item 5)

A method for imparting stress tolerance to a plant, comprising a step of introducing into the plant a nucleic acid selected from the group consisting of:

(b) a nucleic acid that comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence set forth in SEQ ID NO : 1, and encodes a polypeptide having ascorbate transport activity;

(d) a nucleic acid that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; and (e) a nucleic acid that encodes a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 2, and having ascorbate transport activity.

(Item 6)

The method of item 5, wherein the nucleic acid introduced into the plant is a nucleic acid encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

(Item 7)

The method of item 5, wherein the stress tolerance is tolerance for stress selected from the group consisting of high temperature, low temperature, low pH, low oxygen, oxidation, salt, osmotic pressure, dryness, water, flooding, cadmium, copper, ozone, air pollution, ultraviolet rays, light, pathogens, pathogenic germs, harmful insects, herbicides and aging .

(Item 8)

The method of item 5, wherein the stress tolerance is selected from the group consisting of tolerance for light stress and tolerance for oxidation stress.

(Item 9)

A method for imparting stress tolerance to a plant body, comprising the steps of:

(1) introducing into a plant cell a nucleic acid selected from the group consisting of:

(b) a nucleic acid that comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence set forth in SEQ ID NO: 1, and encodes a polypeptide having ascorbate transport activity; (c) a nucleic acid that consists of the nucleic acid sequence set forth in SEQ ID NO: 1 ;

(e) a nucleic acid that encodes a polypeptide having an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 2, and having ascorbate transport activity; and

(2) regenerating the plant body from a plant cell obtained in step ( 1 ) .

(Item 10)

The method of item 9, wherein the nucleic acid introduced into the plant cell is a nucleic acid encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

(Item 11)

The method of item 9, wherein the stress tolerance is tolerance for stress selected from the group consisting of high temperature, low temperature, low pH, low oxygen, oxidation, salt, osmotic pressure, dryness, water, flooding, cadmium, copper, ozone, air pollution, ultraviolet rays, light, pathogens, pathogenic germs, harmful insects, herbicides and aging .

(Item 12)

The method of item 9, wherein the stress tolerance is selected from the group consisting of tolerance for light stress and tolerance for oxidation stress.

(Item 13)

An expression vector comprising:

(1) a nucleic acid selected from the group consisting of:

(2) a transcription/translation regulatory sequence operably linked to the nucleic acid of (1) .

(Item 14)

The expression vector of item 13, wherein the nucleic acid of (1) is a nucleic acid encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

(Item 15)

A plant cell, wherein the plant cell is:

a plant cell transformed by using the composition of any one of items 1 to 4;

a plant cell prepared by the method of any one of items 5 to 8; or

a plant cell comprising the expression vector of item 13 or 14.

(Item 16)

A plant body, wherein the plant body is:

a plant body transformed by using the composition of any one of items 1 to 4;

a plant body prepared by the method of any one of items 9 to 12; or

a plant body comprising the expression vector of item 13 or 14.

(Item 17)

A plant body regenerated from the plant cell of item 15. (Item 18)

A progeny or hybrid strain of the plant body of item 16 or 17.

(Item 19)

A plant cell, wherein the plant cell is:

a plant cell obtained from the plant body of item 16 or 17; or

a plant cell obtained from the progeny or hybrid strain of item 18.

[Advantageous Effects of Invention]

[0008]

Introduction of the gene of the present invention into a plant has enabled impartation of stress tolerance, particularly light stress tolerance and oxidation stress tolerance, to the plant . The present invention provides a composition and a method for imparting stress tolerance to a plant . The present invention also provides a plant cell and a plant body in which stress tolerance is imparted.

[Brief Description of Drawings]

[0009]

[Fig. 1A] Fig. 1A is a graph showing the results of measuring ascorbate transport activity. Triangles (Δ) are results obtained by using a liposome in which an ascorbate transporter is not reconstituted. White circles (o) are results obtained by using a reconstituted proteoliposome in which an ascorbate transporter is reconstituted, without the addition of valinomycin. Black circles (·) are results obtained by using a reconstituted proteoliposome in which an ascorbate transporter is reconstituted, with the addition of valinomycin. [Fig. IB] Fig. IB is a graph showing the results of measuring ascorbate transport activity. White circles (o) are results obtained by using a reconstituted proteoliposome in which an ascorbate transporter is reconstituted, without the addition of valinomycin. Black circles (·) are results obtained by a reconstituted proteoliposome in which an ascorbate transporter is reconstituted, with the addition of valinomycin. Chloride ions at various concentrations shown in the figure were used. [Fig. 1C] Fig. 1C is a graph showing the results of measuring ascorbate transport activity. The transportation of a [¹⁴C] -ascorbate by the transporter of the present invention (left) was competitively inhibited by a cold ascorbate (center), but the transportation was not inhibited by a cold dehydroascorbate (right) .

[Fig. 2A] Fig. 2A is a photograph comparing the growth of wild-type plants and knockout-type (knockout mutant) plants. A substantial difference was not recognized between the both types of plants.

[Fig. 2B] Fig. 2B is a result showing that an ascorbate transporter is not expressed in the knockout-type (knockout mutant) plants.

[Fig. 2C] Fig. 2C is a graph showing results of measuring photosynthesis by chlorophyll fluorescence and absorption in wild-type plants and knockout-type (knockout mutant) plants under high light stress.

[Fig. 2D] Fig. 2D shows the results of analyzing ascorbate transporter knockout plants. Fig. 2D shows that the production of zeaxanthin significantly decreased in ascorbate transporter knockout plants under light irradiation.

[Fig. 2E] Fig. 2E shows the results of measuring the production of antheraxanthin, violaxanthin, lutein and neoxanthin in addition to zeaxanthin in an experiment that is the same as the experiment for which the results are shown in Fig. 2D. 1, 2, 3, 4, and 5, corresponding to zeaxanthin, antheraxanthin, violaxanthin, lutein, and neoxanthin, respectively.

[Fig. 3] Fig. 3 is a schematic diagram showing the physiological significance of an ascorbate transporter that is conceivable from the results of Fig. 1 and Fig. 2.

[Fig. 4] Fig. 4 is a schematic diagram that describes the schematic of Fig. 3 in further detail and extensiveness . [Fig. 5] Fig. 5 is a result obtained by measuring substrate specificity of an ascorbate transporter.

[Description of Embodiments]

[0010]

Hereinafter, the present invention will be described. Throughout the present specification, it should be understood that unless particularly stated otherwise, an expression in its singular form also includes the conception of plurality. It should be also understood that unless particularly stated otherwise, the terms used in the present specification have the meanings that are conventionally used in the art. Therefore, unless defined otherwise, all technical and scientific terms used in the present specification have the same meanings as commonly understood by those having ordinary skill in the art to which the present invention pertains . In the case of conflict, the present specification, including the definitions, will control .

[0011]

(Definition of terms)

Hereinafter, the definitions of the terms that are used particularly in the present specification will be listed.

[0012]

The term "stress" as used herein refers to any stress from the environment. Examples of the stress include, but are not limited to, high temperature, low temperature, low pH, low oxygen, oxidation, salt, osmotic pressure, dryness, water, flooding, cadmium, copper, ozone, air pollution, ultraviolet rays, light (high light and/or low light) , pathogens, pathogenic germs, harmful insects, herbicides and aging.

[0013]

The term "imparting stress tolerance" as used herein means that healthy growth impaired under the presence of stress (for example, decrease in growth rate and/or decrease in degree of growth) comes closer to growth in the absence of stress. [0014]

The term "transporter" as used herein means a substance which transports a substance that cannot permeate through a lipid bilayer membrane (for example, an organic acid such as an ascorbate) across the lipid bilayer membrane. Typically, a transporter is a membrane protein present in the lipid bilayer membrane. The transporter, which is a protein, is used interchangeably with "transport protein" in the present specification .

[0015]

The term "transport activity" as used herein means an activity of transporting a substance that cannot permeate through a lipid bilayer membrane (for example, an organic acid such as an ascorbate), across a lipid bilayer membrane. The transport activity of an ascorbate will be referred to in the present specification as "ascorbate transport activity".

[0016]

The term "artificial membrane" as used herein is a membrane artificially prepared by using lipids as raw materials, and is preferably a lipid bilayer membrane, but is not limited thereto . An example of the "artificial membrane" may be a liposome, but is not limited thereto.

[0017]

The term "activity regulator of an ascorbate transport protein" as used herein means a substance that exerts influence on the transport activity of an ascorbate transport protein. The "activity regulator of an ascorbate transport protein" may be a substance that promotes the transport activity, or may be a substance that inhibits the transport activity.

[0018]

Unless particularly stated otherwise, the "plant" as used herein means any of a plant body, a plant organ, a plant tissue, a plant cell and a seed. The plant of the present invention may or may not be a transgenic plant, but preferably is a transgenic plant. Examples of the plant organ include a root, a leaf, a stem, a flower and the like. Examples of the plant cell include a callus and a suspension cell culture. In certain embodiments, a plant may refer to a plant body. Examples of the plant species that may be used in the present invention are not limited to Arabidopsis thaliana but include a plant having an ascorbate transporter consisting of an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 2. Examples thereof include poplar, corn, rice, soybean, castor bean, Medicago, grape, false brome grass and Sorghum, but are not limited thereto.

[0019]

As used herein, the "progeny" of the present invention such as a progeny of a transgenic plant is either: produced from a plant or a transgenic plant; generated by a plant or a transgenic plant; or derived from a plant or a transgenic plant.

Accordingly, a "progeny" plant, that is, an "Fl" generation plant is posterity (offspring and/or descendant) of a transgenic plant that is prepared by the method of the invention The progeny of a transgenic plant may comprise a desired polynucleotide that is incorporated into a cell of a parent transgenic plant by the method described in the present specification, in at least one, or part or all of the cellular genome thereof. Accordingly, a desired polynucleotide is "transmitted" or "passed down" to a progeny plant. The term "progeny" as used herein may also be regarded as posterity (offspring and/or descendant) of a group of plants.

[0020]

As used herein, a "hybrid" refers to a progeny that is obtained by "hybridization" of plants. A method for hybridization of plants is well known. For example, hybridization can be performed by carrying out the following steps: (a) planting seeds of first ( starting lineage ) and second (donor plant lineage including a desired mutation, a transgene and/or an allelic gene) parent plants; (b) growing the seeds of the first and second parent plants into blooming plants; (c) pollinating a flower from the first parent plant with pollen from the second parent plant; and (d) harvesting seeds produced in the parent plant of the blooming pollinated flower.

[0021]

As used herein, "regeneration" of a plant means that the entirety of an individual is restored from a part of the individual. In a plant body, a part of an individual includes a cell, as well as a piece of tissue such as callus, a leaf, a leaf stem and a root. Amethod for regeneration is a well-known technique in the art, although optimum conditions differ depending on plant species.

[0022]

A "polynucleotide", "nucleic acid" or "nucleic acid molecule" may refer to a ribonucleotide (adenosine, guanosine, uridine or cytidine; "RNA molecule " ) or a deoxyribonucleotide (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine ( "DNA molecule" ) in the form of a phosphoric acid ester polymer, which is in a single strand form, a double strand form or some other form, or any phosphoester analogues thereof (for example, phosphorothioate and thioester) .

[0023]

A "polynucleotide sequence", "nucleic acid sequence" or "nucleotide sequence" is a series of nucleotide bases (also referred to as "nucleosides") present in a nucleic acid (for example, DNA or RNA) , and means any strand of two or more nucleotides or a complementary strand thereof. Preferable nucleic acids of the present invention include the nucleic acid shown in SEQ ID NO: 1, and a complementary strand, a variant and a fragment thereof. Examples of the variant include a nucleic acid that comprises a nucleic acid sequence having 70% or more identity, 75% or more identity, 80% or more identity, 85% or more identity, 90% or more identity, 95% or more identity, or 98% or more identity to the nucleic acid sequence shown in SEQ ID NO: 1, and encodes a protein having ascorbate transport activity, but are not limited thereto. Alternatively, examples of the variant include a nucleic acid that consists of a nucleic acid sequence having 70% or more identity, 75% or more identity, 80% or more identity, 85% or more identity, 90% or more identity, 95% or more identity, or 98% or more identity to the nucleic acid sequence shown in SEQ ID NO: 1, and encodes a protein having ascorbate transport activity, but are not limited thereto. Alternatively, examples of the variant include a nucleic acid that comprises a nucleic acid sequence having 70% or more homology, 75% or more homology, 80% or more homology, 85% or more homology, 90% or more homology, 95% or more homology, or 98% or more homology to the nucleic acid sequence shown in SEQ ID NO: 1, and encodes a protein having ascorbate transport activity, but are not limited thereto. Alternatively, examples of the variant include a nucleic acid that hybridizes under highly stringent conditions with a nucleic acid consisting of the nucleic acid sequence shown in SEQ ID NO: 1, and encodes a protein having ascorbate transport activity, or a nucleic acid that hybridizes under moderately stringent conditions with a nucleic acid consisting of the nucleic acid sequence shown in SEQ ID NO: 1, and encodes a protein having ascorbate transport activity, but are not limited thereto.

[0024]

A "complementary strand" means a strand of nucleotides that is likely to form base pairs with a certain nucleic acid sequence . For example, the respective strands of a double-stranded DNA have base sequences that are complementary to each other, and from the viewpoint of one strand, the other strand is a complementary strand.

[0025]

A "coding sequence", or a sequence that "encodes" an expression product (for example, RNA, polypeptide, protein or enzyme) , is a nucleotide sequence leading to the generation of the product when expressed.

[0026]

A "protein", "peptide" or "polypeptide" contains a consecutive string of two or more amino acids. Preferable peptides of the present invention include the peptide shown in SEQ ID NO: 2, and a variant and a fragment thereof. Examples of the variant include a protein that comprises an amino acid sequence having 70% or more identity, 75% or more identity, 80% or more identity, 85% or more identity, 90% or more identity, 95% or more identity, or 98% or more identity to the amino acid sequence shown in SEQ ID NO: 2, and has ascorbate transport activity, but are not limited thereto. Alternatively, examples of the variant include a protein that consists of an amino acid sequence having 70% or more identity, 75% or more identity, 80% or more identity, 85% or more identity, 90% or more identity, 95% or more identity, or 98% or more identity to the amino acid sequence shown in SEQ ID NO: 2, and has ascorbate transport activity, but are not limited thereto. Alternatively, examples of the variant include a protein that comprises an amino acid sequence having 70% or more homology, 75% or more homology, 80% or more homology, 85% or more homology, 90% or more homology, 95% or more homology, or 98% or more homology to the amino acid sequence shown in SEQ ID NO: 2, and has ascorbate transport activity, but are not limited thereto. Alternatively, examples of the variant include a protein which is encoded by a nucleic acid that hybridizes under highly stringent conditions with a nucleic acid consisting of the nucleic acid sequence shown in SEQ ID NO: 1 and encodes a protein having ascorbate transport activity, or a protein which is encoded by a nucleic acid that hybridizes under moderately stringent conditions with a nucleic acid consisting of the nucleic acid sequence shown in SEQ ID NO: 1 and encodes a protein having ascorbate transport activity, but are not limited thereto.

[0027]

A "protein sequence", "peptide sequence" or "polypeptide sequence" or "amino acid sequence" refers to a series of two or more amino acids present in a protein, a peptide or a polypeptide. [0028]

The "homology" of genes (for example, nucleic acid sequence, amino acid sequence, or the like) as used herein refers to the degree of identity with each other between two or more gene sequences. Furthermore, the identity of sequences (nucleic acid sequences, amino acid sequences, and the like) as used herein refers to the degree of sequences (individual nucleic acids, amino acids, and the like) being identical with each other, between two or more comparable sequences. Therefore, as the homology of two certain genes is higher, the identity or similarity of those sequences is high. Whether two kinds of genes are homologous may be investigated by directly comparing the sequences, or in the case of nucleic acids, according to a method of hybridization under stringent conditions. In the case of directly comparing two gene sequences, if the DNA sequence between those gene sequences is representatively at least 50% identical, preferably if at least 70% identical, and more preferably if at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical, those genes are homologous. In the present specification, the "similarity" of genes (for example, nucleic acid sequences, amino acid sequences, or the like) refers, with respect to the homology, to the degree of identity with each other of two or more gene sequences when conservative substitution is regarded as positive (identical) . Therefore, if there is conservative substitution, homology and identity differ from each other owing to the presence of the conservative substitution. Furthermore, if there is no conservative substitution, homology and identity represent the same value.

[0029]

In the present specification, comparison of the similarity, identity and homology of amino acid sequences and base sequences is calculated using FASTA, which is a tool for sequence analysis, and using default parameters.

[0030]

In the present specification, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of

I to n-1, with respect to the full length polypeptide or polynucleotide (having a length n) . The length of the fragment may be appropriately modified according to the purpose, and for example, the lower limit of the length in the case of a polypeptide may be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, while those lengths represented by integers that are not specifically listed herein (for example,

II or the like) may also be appropriate as the lower limit. Also, in the case of a polynucleotide, the lower limit may be 5, 6,

7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides , and those lengths represented by integers that are not specifically listed herein (for example, 11 or the like) may also be appropriate as the lower limit. In the present specification, the lengths of a polypeptide and a polynucleotide can be represented by the number of amino acids and the number of nucleic acids, respectively, as described above; however, such a referred number is not to be regarded as absolute, and as long as the same functions are exhibited, the referred number as the upper limit or lower limit is intended to include those having several units above or below (or, for example, 10% above or below) the referred number. In order to express such intention, in the present specification, the expression "about" may come before the number. However, in the present specification, it should be understood that the presence or absence of the expression "about" is not intended to affect the interpretation of the value. A useful length of a fragment for use in the present specification can be determined on the basis of whether at least one function among the functions of the full length protein, which serves as the reference of the fragment, is maintained.

[0031]

An "isolated" biological factor (for example, nucleic acid, protein or the like) as used herein refers to a product that has been substantially separated or purified from other naturally occurring biological factors in the cells of an organism (for example, in the case of a nucleic acid, factors other than nucleic acids, and nucleic acids having nucleic acid sequences other than that of a nucleic acid of interest; in the case of a protein, factors other than proteins, and proteins having amino acid sequences other than that of a protein of interest; and the like) . "Isolated" nucleic acids and proteins include nucleic acids and proteins that have been purified according to standard purification methods. Therefore, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

[0032]

A "purified" biological factor (for example, nucleic acid, protein or the like) as used herein refers to a product obtained by removing at least a part of factors that are naturally accompanying the biological factor. Therefore, the purity of a biological factor in regard to the biological factor in its purified state is usually higher than the purity of the biological factor in its usual state (that is, the factor is concentrated) .

[0033]

The terms "purified" and "isolated" as used herein imply that a biological factor of a same type is present at a proportion of preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight.

[0034]

In the present specification, the phrase "polynucleotide that hybridizes under stringent conditions" implies the well-known conditions that are conventionally used in the art.

Such a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like, while using a polynucleotide selected from the polynucleotides of the present invention as a probe . Specifically, such a polynucleotide means a polynucleotide that can be identified by carrying out hybridization at 65°C in the presence of 0.7 to 1.0 M NaCl, using a filter having a colony- or plaque-derived DNA immobilized thereon, and then washing the filter under the conditions of 65°C using an SSC (saline-sodium citrate) solution at a 0.1- to 2-fold concentration (the composition of an SSC solution at a one-fold concentration includes 150 mM sodium chloride and 15 mM sodium citrate) . Hybridization can be carried out according to methods described in experiment manuals such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, and DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995) . Here, from the sequence that hybridizes under stringent conditions, preferably, a sequence which includes a sequence of A only or a sequence of T only is excluded. A "hybridizable polynucleotide" refers to a polynucleotide which can hybridize to another polynucleotide under the above-described hybridization conditions. Specific examples of the hybridizable polynucleotide include a polynucleotide having at least 60% or more homology with the base sequence of a DNA that encodes a polypeptide having the amino acid sequence specifically shown in the present invention, preferably such a polynucleotide having at least 80% or more homology, and more preferably such a polynucleotide having at least 95% or more homology.

[0035]

The term "highly stringent conditions" as used herein refers to the conditions designed to allow hybridization of a DNA strand having high complementarity to a nucleic acid sequence, but to exclude hybridization of DNA with significant mismatch. The stringency of hybridization is determined mainly by the conditions of temperature, ion strength, and a denaturant such as formamide. Examples of such "highly stringent conditions" in terms of hybridization and washing may be 0.0015 M sodium chloride and 0.0015 M sodium citrate at 65 to 68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate and 50% formamide at 42 °C. In regard to such highly stringent conditions, see Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y., 1989) ; and Anderson et al . , Nucleic Acid Hybridization: a Practical Approach, IV, IRL Press Limited (Oxford, England) . Limited, Oxford, England. If necessary, more stringent conditions (for example, higher temperature, lower ion strength, higher formamide, or another denaturant ) may also be used . Other agents may be included in the hybridization buffer solution and washing buffer solution, for the purpose of reducing non-specific hybridization and/or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodS0₄ or SDS), Ficoll, Denhardt solution, ultrasonication-treated salmon sperm DNA (or other non-complementary DNA) , and dextran sulfate, but other appropriate agents may also be used. The concentration and form of these additives may be modified without substantially affecting the stringency of the hybridization conditions.

Hybridization experiments are usually performed at pH 6.8 to 7.4, but under representative ion strength conditions, the rate of hybridization is almost pH-independent . See Anderson et al . , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England) .

[0036]

As factors affecting the stability of double-stranded DNA, there may be mentioned the composition of bases, length, and the degree of base pair mismatch. The conditions of hybridization may be adjusted by a person of ordinary skill in the art, and by applying these variables , it can be made possible that DNA with different sequence relatedness form hybrids . The melting temperature of a completely matched double-stranded DNA can be estimated by the following formula:

T_m(°C)=81.5+16.6(log[Na⁺] )+0.41 (%G+C) -600/N-O .72 (%formamide )

Here, N is the length of the double strand being formed; [Na⁺] is the molar concentration of sodium ions in the hybridization solution or washing solution; and % G+C is the percentage of ( guanine+cytosine ) bases in the hybrid. In regard to an incompletely matched hybrid, the melting temperature is decreased by about 1°C for every 1% mismatch.

[0037]

The term "moderately stringent conditions" as used herein refers to the conditions in which a double-stranded DNA having a higher degree of base pair mismatch than that may occur under "highly stringent conditions", may be formed. Representative examples of the "moderately stringent conditions" maybe 0.015 M sodium chloride, 0.0015 M sodium citrate at 50 to 65°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate and 20% formamide at 37 to 50°C. For instance, "moderately stringent" conditions of 0.015 M sodium ions at 50°C allow about 21% mismatch .

[0038]

For example, with 0.015 M sodium ions (no formamide), the melting temperature of a completely matched, long DNA is about 71°C. Under washing at 65°C (the same ion strength), this condition allows about 6% mismatch. In order to capture more distantly related sequences, a person having ordinary skill in the art may simply lower the temperature, or may increase the ion strength.

[0039]

In regard to an oligonucleotide probe having up to about 20 nucleotides, an appropriate estimation of the melting temperature at 1 M NaCl is provided by the expression: Tm=(2°C for a single A-T base)+(4°C for a single G-C base pair) . In addition, the sodium ion concentration in 6<citric acid sodium salt (SSC) is 1 M ( see Suggs , et al ., Developmental Biology Using Purified Genes, page 683, Brown and Fox (Ed) (1981)) .

[0040] A natural nucleic acid that encodes a protein such as a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a variant or fragment thereof, is easily separated from, for example, a cDNA library having a PCR primer containing a part of the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof, and a hybridization probe. The nucleic acid that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a variant or fragment thereof or the like, may be hybridized with the sequences shown in SEQ ID NO: 1, or a part thereof, under less stringent conditions that are defined by a hybridization buffer solution essentially containing 1% bovine serum albumin (BSA) ; 500 mM sodium phosphate (NaP0₄) ; 1 mM EDTA; and 7% SDS at a temperature of 42 °C, and a washing buffer solution essentially containing 2xSSC (600 mM NaCl; 60 mM sodium citrate); and 0.1% SDS at 50°C, more preferably under less stringent conditions that are defined by a hybridization buffer solution essentially containing 1% bovine serum albumin (BSA) ; 500 mM sodium phosphate (NaP0₄) ; 15% formamide; 1 mM EDTA; and 7% SDS at a temperature of 50°C, and a washing buffer solution essentially containing lxSSC (300 mM NaCl; 30 mM sodium citrate); and 1% SDS at 50°C, and most preferably under less stringent conditions that are defined by a hybridization buffer solution essentially containing 1% bovine serum albumin (BSA) ; 200 mM sodium phosphate (NaP0₄) ; 15% formamide; 1 mM EDTA; and 7% SDS at a temperature of 50°C, and a washing buffer solution essentially containing 0.5<SSC (150 mM NaCl; 15 mM sodium citrate); and 0.1% SDS at 65°C.

[0041]

The percentages of "identity", "homology" and "similarity" of sequences (amino acid, nucleic acid, or the like) in the present specification are determined by comparing two sequences that are optimally aligned over a comparison window. Here, the portion of the polynucleotide sequence or polypeptide sequence within the comparison window may include additions or deletions (that is, gaps), as compared with the reference sequence for the optimal alignment of the two sequences (if the other sequence includes additions, gaps may occur, but the reference sequence as used herein is defined to have neither additions nor deletions) . The number of matched positions is determined by determining the number of positions where the same nucleic acid bases or amino acid residues are recognized in both of the sequences, and the number of matched positions is divided by the total number of positions in the comparison window, followed by multiplication of the obtained result by 100, thereby calculating the percentage of identity. In the case of use in the search, the homology is evaluated using appropriate ones among those various sequence comparing algorithms and programs that are well known in the art. Examples of these algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW ( Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85(8) : 2444-2448; Altschul et al . , 1990, J. Mol. Biol., 215(3) : 403-410; Thompson et al . , 1994, Nucleic Acids Res., 22(2) : 4673-4680; Higgins et al . , 1996, Methods Enzymol., 266: 383-402; Altschul et al . , 1990, J. Mol. Biol., 215(3) : 403-410; and Altschul et al . , 1993, Nature Genetics, 3: 266-272), but these examples are not intended to be limiting. In a particularly preferred exemplary embodiment, the homology of protein and nucleic acid sequences is evaluated using Basic local Alignment Search Tool (BLAST) that is conventionally well known in the art (see, for example, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA, 87: 2267-2268; Altschul et al . , 1990, J. Mol. Biol., 215: 403-410; Altschul et al . , 1993, Nature Genetics , 3: 266-272; and Altschul et al., 1997, Nuc . Acids Res . , 25: 3389-3402) . Particularly, comparison or search may be achieved by performing the following operations using five customized BLAST programs.

[0042]

(1) Compare an amino acid query sequence against a protein sequence database using BLASTP and BLAST3;

(2) compare a nucleotide query sequence against a nucleotide sequence database using BLASTN;

(3) compare a conceptual translation product obtained by converting a nucleotide query sequence (both strands) into six reading frames, against a protein sequence database using BLASTX;

(4) compare a protein query sequence against a nucleotide sequence database converted in all six reading frames (both strands), using TBLASTN; and

(5) compare a six-reading frame conversion product of a nucleotide query sequence, against a nucleotide sequence database converted in six reading frames, using TBLASTX.

[0043]

BLAST programs are designed to identify homologous sequences by specifying similar segments called "high-score segment pairs" between an amino acid query sequence or a nucleic acid query sequence, and preferably a subj ect sequence obtained from a protein sequence database or a nucleic acid sequence database . It will be preferable if many high-score segment pairs are identified (that is, aligned) by a scoring matrix that is well known in the art. Preferably, BLOSUM62 matrix (Gonnet et al., 1992, Science, 256: 1443-1445; and Henikoff and Henikoff, 1993, Proteins, 17: 49-61) is used as the scoring matrix. Although it is not preferable as this matrix, PAM matrix or PAM250 matrix may also be used (see, for example, Schwartz and Dayhoff, eds . , 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation) . The BLAST programs evaluate the statistical significance of all identified high-score segment pairs, and select a segment which satisfies the threshold level of the significance that a user uniquely sets up, preferably such as the homology ratio unique to the user. It is preferable to evaluate the statistical significance of high-score segment pairs using Karlin' s formula, which determines statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA, 87: 2267-2268) . [0044]

The term "primer" as used herein refers to a substance required for the initiation of a reaction of a high molecular weight compound to be synthesized, in an enzymatic reaction for high molecular weight compound synthesis. In a reaction for synthesizing a nucleic acid molecule, a nucleic acid molecule (for example, DNA, RNA or the like) which is complementary to the sequence of a part of the high molecular weight compound to be synthesized may be used.

[0045]

A nucleic acid molecule that is usually used as a primer may be one such molecule having a nucleic acid sequence having a length of at least 8 contiguous nucleotides, which is complementary to the nucleic acid sequence of a gene of interest Such a nucleic acid sequence may be a nucleic acid sequence having preferably a length of at least 9 contiguous nucleotides, more preferably a length of 10 contiguous nucleotides , even more preferably a length of 11 contiguous nucleotides, a length of 12 contiguous nucleotides, a length of 13 contiguous nucleotides, a length of 14 contiguous nucleotides, a length of 15 contiguous nucleotides, a length of 16 contiguous nucleotides, a length of 17 contiguous nucleotides, a length of 18 contiguous nucleotides, a length of 19 contiguous nucleotides, a length of 20 contiguous nucleotides, a length of 25 contiguous nucleotides, a length of 30 contiguous nucleotides, a length of 40 contiguous nucleotides, or a length of 50 contiguous nucleotides. The nucleic acid sequence used as a primer includes a nucleic acid sequence that is at least 70% homologous, more preferably at least 80% homologous, even more preferably 90% homologous, and most preferably 95% homologous, to the sequence described above. A sequence appropriate as a primer may vary depending on the nature of the sequence intended to be synthesized (amplified) , but a person having ordinary skill in the art can appropriately design a primer in accordance with the intended sequence. Design of such a primer is well known in the art, and may be carried out manually or using a computer program (for example, LASERGENE, PrimerSelect , or DNAStar) .

[0046]

In the present specification, the term "substitution, addition or deletion" of a polypeptide or a polynucleotide means that an amino acid or a substitute thereof, or a nucleotide or a substitute thereof is respectively substituted, added or removed with respect to the original polypeptide or polynucleotide. Such technologies of substitution, addition or deletion are well known in the art, and examples of such technologies include site-specific mutagenesis technologies and the like. The substitution, addition or deletion may occur at any number as long as it is one or greater, and such a number can be made large, as long as the function of interest (for example, signal transduction functions of hormone and cytokines, and the like) is maintained in a variant having the substitution, addition or deletion. For example, that number could be 1 or several, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, and preferably within 20% or within 10% of the full length, or 100 or smaller, 50 or smaller, 25 or smaller, or the like.

[0047]

The molecular biological techniques, biochemical techniques and microbiological techniques as used herein are well known and conventionally used in the art, and are described in, for example, Sambrook J. et al . , (1989) . Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001) ; Ausubel, F. M. (1987) . Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M.

(1989) . Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M. A. (1990) . PCR Protocols: A Guide to Methods and Applications , Academic Press ; Ausubel, F. M. (1992) . Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. M. (1995) . Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995) . PCR Strategies, Academic Press; Ausubel, F. M. (1999) . Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates ; Sninsky, J. J. et al . (1999) PCR Applications : Protocols for Functional Genomics, Academic Press; Supplement Experimental Medicine "Experimental Methods in Transfection & Expression Analysis, " Yodosha, 1997; and the like, the disclosure of which is incorporated herein by reference, in part (maybe in their entirety) as related to the present specification.

[0048]

The DNA synthesis technologies and nucleic acid chemistry for the production of artificially synthesized genes are described in, for example, Gait, M. J. (1985) . Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990) . Oligonucleotide Synthesis: A Practical Approach, IRL Press;

Eckstein, F. (1991) . Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al . (1992) . The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al . (1994) . Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al . (1996) . Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (1996) . Bioconjugate Techniques, Academic Press; and the like, the disclosure of which is incorporated herein by reference, in part as related to the present specification.

[0049]

To confirm the presence of nucleic acid in the present specification, evaluation may be made by any appropriate methods, including molecular biological measurement methods such as a radiation method, a fluorescence method, a Northern blotting method, a dot blotting method, and a PCR method. [0050]

A gene of the present invention can have its expression knocked down (suppressed) using a siRNA. The methods for producing a siRNA from a predetermined gene are well known, and for example, annealed synthetic double-stranded siRNAs are available from siRNA suppliers that are known in the art (for example, Nippon EGT Co., Ltd., Toyama, Japan) . Such a synthetic siRNA is dissolved in an RNAse-free solution, and the solution is adjusted to a final concentration of 20μΜ, and then is introduced into cells. In the case of producing a siRNA, for example, conditions such as that: (1) there are no 4 or more contiguous G or C bases, (2) there are no 4 or more contiguous A or T bases, or (3) there are no 9 or more G or C bases, may also be added. The siRNA of the present invention is 19 bases long, 20 bases long, 21 bases long, 22 bases long, 23 bases long, 24 bases long, 25 bases long, 26 bases long, 27 bases long, 28 bases long, 29 bases long or 30 bases long. The siRNA of the present invention preferably has a length of 19 bases . The siRNA of the present invention also preferably has a length of 20 bases The siRNA of the present invention also preferably has a length of 21 bases. The siRNA of the present invention also preferably has a length of 22 bases. The siRNA of the present invention also preferably has a length of 23 bases . The siRNA of the present invention also preferably has a length of 24 bases.

[0051]

The terms "expresses" and "expression" mean enabling or causing the information in a gene, an RNA sequence or a DNA sequence to be made known (for example, producing a protein by activating cellular functions that participate in the transcription and translation of the corresponding gene) . A DNA sequence is expressed so as to form an "expression product" (for example, an RNA (for example, mRNA) or a protein) within a cell or under the action of a cell. The expression product itself may be said to be "expressed" by the cell.

[0052] The term "transformation" means introducing a nucleic acid into a cell . The gene or sequence to be introduced may be called a "clone". The host cell receiving the introduced DNA or RNA is "transformed", and this is a " transformant " or a "clone". The DNA or RNA that is introduced into a host cell may be derived from any source, and may be derived from a cell of the same genus or species as the host cell, or derived from a cell of a different genus or species.

[0053]

The term "vector" includes a medium (for example, a plasmid) which allows a DNA sequence or an RNA sequence to be introduced into a host cell, so that the medium transforms the host, and if necessary, promotes expression and/or replication of the introduced sequence.

[0054]

Examples of the vector that may be used in the present invention include plasmids, viruses, bacteriophages, integratable DNA fragments, and other vehicles which can promote the integration of nucleic acid into the genome of the host. Plasmids are vectors of the most generally used form and all of which that provide equivalent functions or are known or are becoming known in the art, are appropriate for use in the present specification. See, for example, Pouwels, et al . , Cloning Vectors: A Laboratory Manual, 1985, and Supplements, Elsevier, N.Y., and Rodriguez, et al . (Ed), Vectors: A Survey of Molecular Cloning Vectors and Their Uses , 1988, Buttersworth, Boston, MA.

[0055]

The term "expression system" means a host cell and a compatible vector, which can express a protein or nucleic acid that is carried by the vector and introduced into the host cell under appropriate conditions. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. [0056]

Expression of a nucleic acid that encodes the polypeptide set forth in SEQ ID NO: 2 of the present invention may be carried out, preferably in eukaryotic cells, according to a conventional method. As for host cells that are appropriate for the expression of nucleic acid, higher eukaryotes may be mentioned, and preferably are plant cells.

[0057]

Higher eukaryotic tissue culture cells may also be used for the recombinant production of the polypeptide set forth in SEQ ID NO: 2 of the present invention. Any higher eukaryotic tissue culture cell lines (insect baculovirus expression systems may be mentioned) may be used, but plant cells are preferable. An expression vector for such cell lines usually contains an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used) , a polyadenylation site, and a transcription termination site. Such a vector also usually contains a selection gene or an amplification gene. A suitable expression vector may be a plasmid, virus or retrovirus that carries a promoter derived from a source such as, for example, adenovirus, SV40, parvovirus, vaccinia virus or cytomegalovirus. Examples of the expression vector include pCR (registered trademark) 3.1, pCDNAl, pCD (Okayama, et al . , Mol. Cell. Biol. 5: 1136), pMCl neo Poly-A (Thomas, et al., (1987) Cell 51: 503), pREP8, pSVSPORT, derivatives thereof, and baculovirus vectors (for example, pAC373 or pAC610) .

[0058]

The present invention also includes a fusion product containing the polypeptide set forth in SEQ ID NO: 2 and the polynucleotide set forth in SEQ ID NO: 1, and a second polypeptide moiety or a second polynucleotide moiety (may be referred to as a "tag") . A fusion polypeptide of the present invention may be conveniently constructed by, for example, inserting the polynucleotide of the present invention or a fragment thereof into an expression vector. The fusion product of the present invention may contain a tag that facilitates purification or detection. Examples of such a tag include glutathione-S-transferase (GST), hexahistidine (His6) tags, maltose binding protein (MBP) tags, hemagglutinin (HA) tags, cellulose binding protein (CBP) tags, and myc tags . Detectable tags (for example, ³²P, ³⁵S, ³H, ^99mTc, ¹²³I, ⁱ In, ⁶⁸Ga, ¹⁸F, ¹²⁵I, ¹³¹I, ^113mIn, ⁷⁶Br, ⁶⁷Ga, ^99mTc, ¹²³I, ^ι Ιη and ⁶⁸Ga) may also be used to label the polypeptide and the polynucleotide of the present invention. Methods for constructing and using such fusion products are well known in the art.

[0059]

The term "operably linked" as used herein implies that a sequence of interest is located such that expression (operation) thereof is under the control of a certain transcription/translation regulatory sequence (for example, a promoter, an enhancer or the like) or translation regulatory sequence. In order for a promoter to be operably linked to a gene, the promoter is usually located immediately upstream to the gene, but is not necessarily located adjacently.

[0060]

Any technology may be used herein for the introduction of a nucleic acid molecule into cells, and examples include transformation, transduction, transfection and the like. Such technologies for the introduction of nucleic acid molecules are well known and commonly used in the art, and are described in, for example, edited by Ausubel F. A. et al . , (1998), Current Protocols in Molecular Biology, Wiley, New York, N.Y.; Sambrook J, et al . (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. and 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Supplement Experimental Medicine "Experimental Methods in Transfection & Expression Analysis " , Yodosha, 1997; and the like. Introduction of genes can be confirmed by using the methods described in the present specification, such as Northern blot analysis and Western blot analysis, or other well-known, commonly used technologies. [0061]

As for the method of introducing a vector, any of the above-described methods for introducing DNA into cells can be used, and for example, Agrobacterium-mediated transformation transfection, transduction, transformation, and the like (for example, a calcium phosphate method, a liposome method, a DEAE dextran method, an electroporation method, a method using a particle gun (gene gun) , and the like) may be used.

[0062]

(Method for the Screening of Activity Regulator of Ascorbate

Transporter)

It is possible to perform screening of an activity regulator of an ascorbate transporter, according to various methods using the protein of the present invention. For example, it is possible to perform screening of an activity regulator of an ascorbate transport protein by:

(a) reconstituting the membrane protein of the present invention into liposomes, and further adding valinomycin;

(b) adding (1) radiolabeled ascorbate only, or (2) radiolabeled ascorbate and a candidate agent, respectively, to the liposomes, and incubating the liposomes;

(c) precipitating the liposomes through centrifugation, and comparing the amounts of radiolabeled ascorbate taken up into the liposomes in the instances of (1) and (2); and

(d) determining whether the candidate agent has affected the ascorbate transport activity.

[0063]

Alternatively, it is also possible to perform screening of an activity regulator of an ascorbate transport protein by: (a) preparing cells that express the membrane protein of the present invention (for example, transforming cells using the gene of the present invention) ;

(b) adding (1) radiolabeled ascorbate only, or (2) radiolabeled ascorbate and a candidate agent, respectively, to the cells, and incubating the cells; (c) disrupting the cells, preparing membrane fractions, and comparing the amounts of radiolabeled ascorbate present in the membrane fraction in the instances of (1) and (2); and

[0064]

Hereinafter, the present invention will be described by way of examples, but the following examples are provided only for the purpose of illustration. The scope of the invention is intended to be limited to neither the detailed description of the invention nor the following examples, and is defined by the claims .

[Examples ]

[0065]

(Example 1: Cloning of ascorbate transporter)

A viral vector for cloning and expression of an ascorbate transporter cDNA was prepared based on the nucleic acid sequence of accession number NM_116261.4 by using Baculovirus Expression System with Gateway technology (Invitrogen No. 11827-011) . 6xhistidine tags were added to the amino-terminal domain of the ascorbate transporter.

[0066]

(Example 2: Purification of ascorbate transporter)

A High 5 cell (insect cell) was infected with a recombinant baculovirus (modified such that transporters are abundantly expressed) at MOI=l, to abundantly express transporters. The transporters were ultrasonically fragmented (OUTPUT 4, 30 seconds, 8 times) (TOMY UD200 tip sonicator) and ultra-centrifuged (16, 000xg, one hour, 4°C) . The precipitate (membrane fraction) was solubilized in 2% octyl glucoside (OG: surfactant) buffer solution and ultra-centrifuged (26,000xg, 30 minutes, 4°C) . The supernatant was mixed with Ni-NTA affinity resin lmL and incubated for 4 hours at 4°C. This resin was washed in a washing solution 20mL and 60mM imidazole was added to obtain an ascorbate transporter. [0067]

The 2% octyl glucoside buffer solution includes 2% octyl glucoside, 2 OmM MOPS-Tris (pH7.0) and 10% glycerol . The washing solution includes 1% octyl glucoside, 20mM MOPS-Tris (pH7.0) and 20% glycerol. The eluate includes 2% octyl glucoside, 20mM MOPS-Tris (pH7.0), 20% glycerol and 60mM imidazole.

[0068]

(Example 3: Reconstitution of ascorbate transporter into artificial membrane vesicle)

20\iq of purified ascorbate transporters and 500 g of artificial membrane vesicles prepared from

L-a-phosphatidylcholine Typell-S ( Sigma No . P-5638 ) were mixed The mixture was frozen for 15 minutes at -80°C, and promptly dissolved. Then the mixture was diluted 30-fold with a reconstituted buffer comprising a surfactant, and ultra-centrifuged (200, 000xg, one hour, 4°C) . The precipitate was suspended in the reconstituted buffer to prepare a proteoliposome .

[0069]

The reconstituted buffer includes 20mM MOPS-Tris (pH7.0),

150mM sodium acetate and 5mM magnesium acetate.

[0070]

(Example 4: Measurement of ascorbate transport activity)

2μΜ valinomycin and ΙΟΟμΜ [¹⁴C] -ascorbate were added to a reaction solution, and the mixture was incubated at 27°C. The reconstituted proteoliposome was added to this mixture. The mixture was added to Sephadex G-50 column (GE Healthcare) after 1, 2 and 5 minutes, and centrifuged (700xg, 2 minutes, 4°C) . Radioactivity measurement was performed on the eluate with a liquid scintillation counter (manufactured by Perkin Elmer) to quantify the transported ascorbate. The principle of the driving force is that when valinomycin, which is potassium ionophore, is added, the potassium flows from the outside to the inside, and the inside takes on + charges compared to the outside . The reaction solution includes 2 OmM MOPS/Tris (pH7.0), 150mM potassium acetate, 5mM magnesium acetate and 4mM KC1.

[0071]

The results are shown in Fig. 1. Triangles (Δ) are results obtained by using a liposome in which an ascorbate transporter is not reconstituted. White circles (o) are results obtained by using a reconstituted proteoliposome in which an ascorbate transporter is reconstituted, without the addition of valinomycin. Black circles (·) are results obtained by using a reconstituted proteoliposome in which an ascorbate transporter is reconstituted, with the addition of valinomycin.

The protein that is encoded by SEQ ID NO: 1 exhibited activity of specifically transporting an ascorbate (Fig. 1A) . In addition, this transport activity was activated by the presence of chloride ions (Fig. IB) . When an excess amount (lmM) of cold ascorbate was added to the reaction solution, transport of [¹⁴C] -ascorbate was inhibited by a competitive inhibition (Fig. 1C, center) . On the other hand, transport of [¹⁴C] -ascorbate was not inhibited when an excess amount (lmM) of dehydroascorbate was added (Fig. 1C, right) . This result demonstrates that the transporter of the present invention is an ascorbate specific transporter.

[0072]

(Example 5: Plant material and determination of genetic type) A plant body was potted and grown in a cycle of 16 hours of brightness under fluorescent light /8 hours of darkness at room temperature of 22°C, in a well-watered state. The pht4; 4-1 (ET4970) mutant and pht4 ; 4-2 (GT5039) mutant of Arabidopsis thaliana were Ds transposon inserts of the Landsberg ecotype and were purchased from Cold Spring Harbor Laboratory (New York, USA) .

[0073]

An automated nucleic acid extraction system (PI-50alpha, KURABO) was used for genome DNA extraction from Arabidopsis thaliana. Determination of the genetic type of the mutants was performed by a PCR method using a genome DNA as a template. In doing so, the following primers were used: PHT4_L2 (SEQ ID NO: 3: 5' -ATGGAGATGCGTTCTGTAGATT-3' ) ; PHT4_R (SEQ ID NO: 4: 5' -GGTTCCAACGAGTAGAAGATGA-3' ) ; Ds3-4 (SEQ ID NO: 5: 5' -CCGTCCCGCAAGTTAAATATG-3' ) ; and Ds5-3 (SEQ ID NO: 6: 5' -TACCTCGGGTTCGAAATCGAT-3' ) .

[0074]

The RNeasy Plant Mini Kit (Qiagen) was used for RNA extraction from Arabidopsis thaliana. RT-PCRwas performed with the PrimeScript One-Step RT-PCR kit (TAKARA BIO) by using the above-described primers PHT4_L2 and PHT4_R. Further, in order to use the Actin2 gene as control of a gene that is being expressed, the following primers were used: Actin2RT-F (SEQ ID NO: 7: 5' -GACCTGCCTCATCATACTCG-3 ' ) and Actin2RT-R (SEQ ID NO: 8: 5' -TTCCTCAATCTCATCTTCT TCC-3') .

[0075]

The results are shown in Fig. 2A and Fig. 2B. The pht4;4-l (ET4970) mutant (knockout strain 1 in Fig. 2B) and pht4;4-2 (GT5039) mutant (knockout strain 2 in Fig. 2B) both normally expressed the Actin2 gene, but an ascorbate transporter was not expressed. As shown in Fig. 2A, all of the knockout strains (knockout mutants) grew normally.

[0076]

(Example 6: Chlorophyll fluorescence measurement)

In order to investigate photosynthetic activity in a chloroplast, chlorophyll fluorescence measurement was performed by using FluorCam 700MF (Photon Systems Instruments, Drasov, Czech Republic) . A plant body, four weeks after seeding, was treated with high light (1, 700μπιο1 photons/m2 si) for 5 hours, and the rosette leaves were arranged on a 12-well multititer plate. After treatment with 15 minutes of darkness, a saturated light pulse was applied, and then actinic light (220μπιο1 photons/m2 si) was applied for 5 minutes. When Fm is the maximum PSII fluorescence after treatment in darkness and Fm' is the maximum PSII fluorescence after treatment with light, the value of NPQ is calculated as ( Fm-Fm' ) /Fm' . [0077]

The results of measuring photosynthesis by chlorophyll fluorescence and absorption under high light stress are shown in Fig.2C (p<0.01 ) . The light energy is used for photosynthesis, where there is a path in which the energy is emitted as fluorescence (photochemical quenching) and a path in which the energy is dissipated as heat (non-photochemical quenching) . As indicators of photochemical quenching, Fv/Fm, Fv'/Fm', (Fm'-Ft)/Fm' and qP were used. As indicators of non-photochemical quenching, qN and NPQ were used. Among these, only non-photochemical quenching was decreased in an ascorbate transporter gene disrupted plant. This amount of decrease was the same level as when major non-photochemical quenching paths were knocked out.

[0078]

The results show that a plant in which the ascorbate transporter of the present invention is knocked out has decreased non-photochemical quenching (that is, heat dissipation) of excess energy under high light stress. Excess energy under high light stress is a type of stress for a plant.

Therefore, tolerance for high light stress decreases in a plant having decreased non-photochemical quenching (that is, heat dissipation) of excess energy. Conversely, tolerance for high light stress is enhanced by increasing non-photochemical quenching (that is, heat dissipation) of excess energy.

Accordingly, the results show that tolerance for stress such as high light stress is enhanced in a plant that is genetically modified to abundantly express the ascorbate transporter of the present invention.

[0079]

(Example 7: Measurement of zeaxanthin production)

A leaf of a plant body, four to five weeks after seeding, was used as experimental material. After treatment with 15 minutes of darkness, a saturated light pulse was applied. Then actinic light (230μπιο1 photons/m² · s or 540μπιο1 photons/m² · s ) was applied for 2 minutes and the leaf was extracted in 80% (v/v) acetone. The extract was centrifuged for 5 minutes at 4°C, 900xg to collect a supernatant. HPLC measurement was taken thereon according to the method of Muller-Mole et al . (Plant Physiol. 128: 970-977, 2002) . In this light irradiation condition, qN and NPQ were also decreased.

[0080]

The measurement conditions are as follows:

column: Spherisorb S5 ODS14.6x250mm Cartridge Column (Waters, Milford, MA) ;

measuring wavelength: 445nm (reference 550nm) ;

mobile phase A: acetonitrile : methanol: 0.1M Tris-HCl (pH8.0)

= 84:2:14 (v/v) ;

mobile phase B: methanol: ethyl acetate = 68:32 (v/v)

(a gradient was linearly provided from 100% mobile phase A to 100% mobile phase B over 15 minutes); and

flow rate: 1.2mL/minute .

[0081]

The results are shown in Fig. 2D. When zeaxanthin, which is the end product of a xanthophyll cycle, was quantified by the HPLC method under light irradiation ("bright") , zeaxanthin production was significantly decreased in an ascorbate transporter knockout plant (p<0.05) .

[0082]

(Example 8: Measurement of production of substances other than zeaxanthin)

By using the same method as Example 7, the production of antheraxanthin, violaxanthin, lutein and neoxanthin in addition to zeaxanthin was measured. The values are relative amounts that are standardized with chlorophyl a (n=10~12; * denotes value for p<0.05 and ** denotes value for p<0.01) . The results are shown in Fig. 2E . The actual measurement values of the result of experiment in a bright place by using a leaf of a plant body that expresses a wild-type transporter were 13.7, 3.0, 26.4, 104.2 and 34.4 mmol/mol Chi a from the left. As is the case for zeaxanthin, the production of antheraxanthin was also reduced in a knockout-type plant.

[0083]

(Discussion)

From the knowledge so far, the ascorbate transporter that we have found is localized in the inner membrane of a chloroplast (PLANTA, 218: 406-416, 2004) . Accordingly, the ascorbate transporter raises the amount of ascorbate within the chloroplast and contributes to the heat dissipation mechanism of excess energy.

[0084]

An ascorbate functions as a coenzyme of a xanthophyll cycle, which is important for non-photochemical quenching (see Fig. 3) . Interestingly, an ascorbate was shown to be essential as a coenzyme of xanthophyll cycle reaction, which is one of the mechanisms that perform heat dissipation. While not wishing to be bound by theory, it is considered that light stress tolerance is imparted by utilizing the mechanism shown in Fig. 3.

[0085]

Further details thereof are as follows . Among violaxanthin, antheraxanthin and zeaxanthin, violaxanthin is a substance having high light condensing rate (that is, a substance with small amount of heat dissipation, which is non-photochemical quenching of excess energy) , while zeaxanthin is a substance having low light condensing rate (that is, a substance with large amount of heat dissipation, which is non-photochemical quenching of excess energy) . As described above, excess energy under high light stress is a type of stress for a plant, and a plant exhibits tolerance for such stress by non-photochemical quenching (that is, heat dissipation) of excess energy. That is to say, a plant exhibits tolerance for stress, that is, excess light energy, by synthesizing antheraxanthin from violaxanthin, and synthesizing zeaxanthin from antheraxanthin. Therefore, a gene that encodes the ascorbate transporter of the claimed invention imparts resistance to stress (in particular, excess light stress) to a plant.

[0086]

As shown in Fig. 3, synthesis of antheraxanthin from violaxanthin, and synthesis of zeaxanthin from antheraxanthin are performed by an enzyme de-epoxidase, and an ascorbate is required for the enzymatic reaction as a coenzyme. It is considered that the reason why the production of zeaxanthin and the production of antheraxanthin decrease in an ascorbate transporter knockout strain is because a coenzyme ascorbate is not transported to a stroma, and thus an enzyme de-epoxidase is unable to perform sufficient synthetic reaction.

[0087]

The relationships of the various substances measured in Fig. 2E are shown in Fig. 4. Xanthophylls are converted from lycopene to β-carotene, and then they become zeaxanthin, antheraxanthin and violaxanthin. These three substances are converted by enzymes (ZEPandVDE) in accordance with light , thereby changing light condensing rate or heat dissipating excess light energy, and this is called a xanthophyll cycle (surrounded by the dotted line in Fig. 4) . The reactions in the xanthophyll cycle include a reaction by de-epoxidase (VDE: violaxanthin de-epoxidase) that uses an ascorbate (Asc) as a coenzyme, and an enzymatic reaction that involves zeaxanthin epoxidase (ZEP), which is a reverse reaction (this reaction does not utilize an ascorbate as a coenzyme) . When light intensity is strong (high light) , the reaction of the xanthophyll cycle proceeds to synthesize zeaxanthin, which is a substance having low light condensing rate. In contrast, when light intensity is weak (low light), the reaction of the xanthophyll cycle proceeds to synthesize violaxanthin, which is a substance having high light condensing rate. Since lutein is a structural isomer of zeaxanthin and it is unrelated to the xanthophyll cycle, the numerical values do not change in Fig. 2E .

[0088]

(Example 9: Substrate specificity of ascorbate transporter) A leaf of a wild-type plant and a leaf of a gene knockout-type plant were treated with light as in Fig. 2D, and an ascorbate was extracted and the total amount of ascorbate, the amount of reduced ascorbate and the amount of oxidized ascorbate were determined according to Porra et al . (Biochim. Biophys . Acta 975, 384-394 (1989)) and Queval&Noctor (Anal. Biochem. 363, 58- 69 (2007) ) as follows :

Leaves of the same batch were batched off and were promptly frozen with liquid nitrogen. The frozen leaves were grinded into fine powders and were extracted for 30 minutes with 400 L of 0.2N HC1. The extract was neutralized with 0.2N NaOH and 0.2M NaH₂PC>4 (pH5.6), and was centri fuged with 16000 ^χ g for 10 minutes at 4°C, thereby pooling supernatants . The amount of reduced ascorbate was determined based on the difference in UV absorbance at 265nm before and after oxidizing all the ascorbates using an ascorbate oxidase. In order to obtain the amount of oxidized ascorbate and the total amount of ascorbate, 5mM of dithiothreitol was added and a sample was reduced. The total amount of ascorbate is the sum of the reduced form and the oxidized form. The value is a relative amount that is standardized with chlorophyl a (n=4~6; * denotes value for p<0.05) .

As is clear from the results shown in Fig. 5, when the ascorbate transporter was knocked out, only the amount of reduced ascorbate decreased and the amount of oxidized ascorbate did not decrease. This result is consistent with the result that when transport activity of the ascorbate transporter was measured, transport was not competitively inhibited in the oxidized ascorbate. It shows that the ascorbate transporter is specific to the reduced form and does not use the oxidized form as a substrate.

[Industrial Applicability]

[0089]

[0090]

Light stress has been considered as one of the major global problems in agriculture and environment (global warming and the like) . However, the plant in which the ascorbate transporter of the present invention is overexpressed is able to acquire tolerance for light and oxidation stress.

[Sequence Listing Free Text]

[0091]

SEQ ID NO: 1 is the nucleic acid sequence that encodes an ascorbate transporter of Arabidopsis thaliana.

[0092]

SEQ ID NO: 2 is the amino acid sequence of the ascorbate transporter of Arabidopsis thaliana.

[0093]

SEQ ID NO: 3 is the nucleic acid sequence of the primer

PHT4_L2 that was used for the determination of the genetic type of a mutant of Arabidopsis thaliana.

[0094]

SEQ ID NO : 4 is the nucleic acid sequence of the primer PHT4_R that was used for the determination of the genetic type of a mutant of Arabidopsis thaliana.

[0095]

SEQ ID NO: 5 is the nucleic acid sequence of the primer Ds3-4 that was used for the determination of the genetic type of a mutant of Arabidopsis thaliana.

[0096]

SEQ ID NO: 6 is the nucleic acid sequence of the primer Ds5-3 that was used for the determination of the genetic type of a mutant of Arabidopsis thaliana.

[0097] SEQ ID NO: 7 is the nucleic acid sequence of the control primer Actin2RT-F for amplifying the Actin2 gene.

[0098]

SEQ ID NO: 8 is the nucleic acid sequence of the control primer Actin2RT-R for amplifying the Actin2 gene.

Claims

[Claims ]

[Claim 1]

[Claim 2]

The composition of claim 1, wherein the composition comprises a nucleic acid encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[Claim 3]

The composition of claim 1, wherein the stress tolerance is tolerance for stress selected from the group consisting of high temperature, low temperature, low pH, low oxygen, oxidation, salt, osmotic pressure, dryness, water, flooding, cadmium, copper, ozone, air pollution, ultraviolet rays, light, pathogens, pathogenic germs, harmful insects, herbicides and aging.

[Claim 4] The composition of claim 1, wherein the stress tolerance is selected from the group consisting of tolerance for light stress and tolerance for oxidation stress.

[Claim 5]

[Claim 6]

The method of claim 5, wherein the nucleic acid introduced into the plant is a nucleic acid encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[Claim 7]

The method of claim 5, wherein the stress tolerance is tolerance for stress selected from the group consisting of high temperature, low temperature, low pH, low oxygen, oxidation, salt, osmotic pressure, dryness, water, flooding, cadmium, copper, ozone, air pollution, ultraviolet rays, light, pathogens, pathogenic germs, harmful insects, herbicides and aging .

[Claim 8]

The method of claim 5, wherein the stress tolerance is selected from the group consisting of tolerance for light stress and tolerance for oxidation stress.

[Claim 9]

(2) regenerating the plant body from a plant cell obtained in step ( 1 ) .

[Claim 10]

The method of claim 9, wherein the nucleic acid introduced into the plant cell is a nucleic acid encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[Claim 11] The method of claim 9, wherein the stress tolerance is tolerance for stress selected from the group consisting of high temperature, low temperature, low pH, low oxygen, oxidation, salt, osmotic pressure, dryness, water, flooding, cadmium, copper, ozone, air pollution, ultraviolet rays, light, pathogens, pathogenic germs, harmful insects, herbicides and aging .

[Claim 12]

The method of claim 9, wherein the stress tolerance is selected from the group consisting of tolerance for light stress and tolerance for oxidation stress.

[Claim 13]

An expression vector comprising:

(1) a nucleic acid selected from the group consisting of:

[Claim 14]

The expression vector of claim 13, wherein the nucleic acid of (1) is a nucleic acid encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[Claim 15]

A plant cell, wherein the plant cell is:

a plant cell transformed by using the composition of any one of claims 1 to 4 ;

a plant cell prepared by the method of any one of claims 5 to 8; or

a plant cell comprising the expression vector of claim 13 or 14.

[Claim 16]

A plant body, wherein the plant body is:

a plant body transformed by using the composition of any one of claims 1 to 4 ;

a plant body prepared by the method of any one of claims

9 to 12; or

a plant body comprising the expression vector of claim 13 or 14.

[Claim 17]

A plant body regenerated from the plant cell of claim 15.

[Claim 18]

A progeny or hybrid strain of the plant body of claim 16 or 17.

[Claim 19]

A plant cell, wherein the plant cell is:

a plant cell obtained from the plant body of claim 16 or 17; or

a plant cell obtained from the progeny or hybrid strain of claim 18.