WO2003072774A1

WO2003072774A1 - K+ transporter and use thereof

Info

Publication number: WO2003072774A1
Application number: PCT/JP2003/002085
Authority: WO
Inventors: Nobuyuki Uozumi; Teruo Ogawa; Nobuyuki Matsuda
Original assignee: Proteinexpress Co., Ltd.
Priority date: 2002-02-26
Filing date: 2003-02-25
Publication date: 2003-09-04
Also published as: AU2003211710A1; JPWO2003072774A1

Abstract

It is intended to provide a novel protein having a K+ transporter activity and a plant capable of growing even under salt stress or osmotic stress. The K+ transporter protein has an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO:1.

Description

Description K + transporters and their uses Technical field

The present invention relates to the use of a new K + transporter. More specifically, the present invention relates to a protein having a novel K + transporter activity, and a plant capable of growing under salt stress. Background art

Cali- dium ion (K +) is a major intracellular cation maintained at about 100 mM in the cells of living organisms (microorganisms, animals and plants). By maintaining the intracellular K + concentration at a high level, a constant biomembrane potential is formed. By utilizing this electrochemical potential, the transport and transport of various substances is achieved. Energy conversion is taking place.

Each organism has various transport systems to maintain high levels of intracellular K + .For example, animal cells consume ATP via Na + / K + exchange transporters. It excretes Na + and transports K + into cells. On the other hand, in plant cells, K + channels and K + transporters are present in the cells, maintain the K + concentration in the cells, and prevent Na + from entering the cells. Ion K + is preventing. By using such transporters, organisms respond to changes in the external environment and transmit information within cells.

In addition, the genome of cyanobacteria (Synechocystis sp.) Contains Na ⁺ / K ⁺ transgenic plant, which is one of the major families of K ⁺ transposers. It is known that there is a gene that shows high homology with the K + transporter (KtrB) of the porter (HKT) and the marine bacterium Vibrio alginoly ticus. However, the structure of the gene, etc., was not clear.

On the other hand, due to the recent heavy use of chemical fertilizers and long-term continuous cropping, salts have been accumulating in soil at high concentrations. Soil near coasts and estuaries is rich in salt. Furthermore, desertification is progressing mainly in arid regions around the world, and it is said that a vast area of about 600,000 hectares is desertified every year. Since the total cultivated area of Japan is about 540,000 hectares, the area of desertification in one year exceeds the cultivated area of Japan in the whole world. Such desertified soils are rich in salt. As described above, soil containing a large amount of salt is increasing year by year, and in such soil, plants are exposed to salt stress, and it is difficult for plants to grow. The problem exists. No radical solution to this problem has yet been found, and reduced cultivated area may make it difficult to provide a stable supply of food.

As described above, plants cultivated in soils containing high amounts of salt are exposed to salt stress, which is a major factor inhibiting the growth of many higher plants. Exposure to such salt stress has caused a problem in that the production of agricultural products has decreased, making it difficult to provide a stable supply of food.

Therefore, in order to enable a stable supply of food, it is desired to produce agricultural crops that can grow in harsh lands where they are exposed to environmental stress such as salt stress.

Therefore, an object of the present invention is to provide a protein having a novel K + transporter activity and a plant that can grow under salt stress. Disclosure of the invention

The present inventors have conducted intensive studies, and as a result, the gene product derived from the cyanobacterium (Synechocystis sp. PCC6803) was transformed into K + from the extracellular medium (culture solution) together with the gene product derived from the cyanobacterium of unknown function. K + transporter to transport has been found to have one activity, and the present invention has been completed.

That is, the present invention relates to a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or the amino acid sequence represented by SEQ ID NO: 1. A protein consisting of an amino acid sequence in which some amino acids have been deleted, replaced or added, or a partial peptide thereof, or its amide or its ester or its salt (hereinafter referred to as the present (Also referred to as "K tr B protein" in the specification).

The present invention also relates to (a) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or the amino acid represented by SEQ ID NO: 1 A protein consisting of an amino acid sequence in which some amino acids have been deleted, substituted or added in the sequence, a partial peptide thereof, or its amide or its ester or its salt ;

(b) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a part of the amino acid sequence represented by SEQ ID NO: 2; A protein consisting of an amino acid sequence in which amino acid has been deleted, substituted or added, or its partial peptide, its amide or its ester or its salt (hereinafter referred to as “the present specification”). Also known as "K tr A protein";

(c) a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 3, or represented by SEQ ID NO: 3 In the amino acid sequence obtained, a protein consisting of an amino acid sequence in which some amino acids have been deleted, substituted or added, or a partial peptide thereof, or the amino acid or its amino acid sequence Provided is a protein composition having K + transporter activity, comprising an ester thereof or a salt thereof (hereinafter, also referred to as “K tr C protein” in the present specification).

The present invention also provides a gene having a DNA having a nucleotide sequence encoding a KtrB protein, a recombinant vector containing the DNA, and a transformant transformed with the recombinant vector. It is.

The present invention also provides a transgenic plant containing the recombinant vector. Plants used for the production of transgenic plants include plants, plant organs, plant tissues or plant culture cells. In addition, the type of plant may be one belonging to any family selected from the group consisting of Brassicaceae, Solanaceae, Poaceae, and Legume.

The present invention also provides an environmental stress (particularly salt stress, high osmotic pressure and low osmotic pressure) -tolerant plant obtained by culturing or cultivating a transgenic plant into which the recombinant vector has been introduced. Things.

Further, the present invention provides a DNA having a nucleotide sequence encoding a KtrB protein, a DNA having a nucleotide sequence encoding a KtrA protein, and a DNA having a nucleotide sequence encoding a KtrC protein. And a recombinant vector containing the DNA, and a transformant transformed with the vector.

Further, the present invention provides a transgenic plant containing the above-mentioned recombinant vector. Plants used for producing transgenic plants are the same as those described above.

Further, the present invention provides an environmental stress (especially salt stress, high osmotic pressure) obtained by culturing or cultivating a transgenic plant into which the recombinant vector has been introduced. And low osmotic pressure).

Further, the present invention provides a method for producing a KtrB protein (or a KtrB protein, a KtrA protein and a KtrC protein) by culturing the transformant and accumulating the KtrB protein. The present invention provides a method for producing a KtrB protein (or a KtrB protein, a KtrA protein, and a KtrC protein), characterized in that a KtrB protein is collected.

The present invention also provides an antibody against the KtrB protein, the KtrA protein, and the KtrC protein. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a photograph of a plate medium that has grown cyanobacteria.

Figure 2 is a photograph of a plate medium on which E. coli was grown.

FIG. 3 is a graph showing a growth curve of E. coli.

FIG. 4 is a graph showing the amount of K + uptake.

FIG. 5 is a graph showing the K + uptake amount.

FIG. 6 is a graph showing the amount of K + uptake.

Figure 7 is a photograph of the liquid medium in which the cyanobacteria grew. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

In the protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the present invention, or in the amino acid sequence represented by SEQ ID NO: 1 K + transporter containing a protein consisting of an amino acid sequence in which a part of the amino acid has been deleted, substituted or added, or a partial peptide thereof, or its amide or its ester or a salt thereof A protein having one activity (K tr B protein) will be described. K tr B data down Nono ⁰ click proteins are cyanobacteria (Synechocystis sp. PCC6803) Ri gene products der from, the K tr B proteins, K tr A proteins and K tr C protein and jointly extracellular (culture Liquid), which transports K + into cells, and has a so-called K + transporter activity. In addition, the KtrB protein has an activity to excrete Na + from the cell in cooperation with the KtrA protein and the KtrC protein.

As described above, the KtrB protein is a gene product derived from a cyanobacterium (Synechocystis sp, PCC6803). In the present invention, the KtrB protein is produced from the DNA by using a DNA cloned from a cyanobacterium and cloned. The used protein is used. In addition, the KtrB protein may be synthesized by a conventionally known protein synthesis method.

Next, in the protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 of the present invention, or the amino acid sequence represented by SEQ ID NO: 2, A protein consisting of an amino acid sequence in which some amino acids have been deleted, substituted or added, or a partial peptide or its amide or its ester or its salt (K tr A protein) ), And a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 of the present invention, or the amino acid sequence represented by SEQ ID NO: 3 Some amino acids have been deleted or substituted, and some of the amino acids have been deleted. Substituted amino acids have been added to proteins or partial peptides or their amides or their esters or their salts (K tr C protein ) Will be described.

Like the KtrB protein, the KtrA protein and the KtrC protein are gene products derived from the cyanobacterium (Synechocystis sp.PCC6803), and the KtrA protein and the KtrC protein cooperate with each other. Is a protein that exerts K + transporter activity on the KtrB protein. You. The KtrA protein and the KtrC protein may be synthesized proteins as in the case of the KtrB protein, but in the present invention, the KtrA protein and the KtrC protein are cloned to form a protein. Using a DNA that has been roung, a protein produced from the DNA is used.

Cloning of genes for the KtrB protein, the KtrA protein and the KtrC protein was performed by creating PCR primers (oligonucleotides) based on the nucleotide sequences of the gene database at the Kazusa DNA Research Institute. The genes of the KtrB protein, the KtrA protein and the KtrC protein are multiplied, and cloning is performed by a conventionally known E. coli introduction method such as a calcium ion method or an electrophoresis method.

As used herein, “substantially the same” means that the activities of the proteins, ie, the K + transporter activities, are substantially the same. If some amino acids are deleted, substituted or added, and the activity is the same, the deleted, substituted or added protein is substantially the same as the protein without deletion, substitution or addition. Are identical.

Generally, the degree of homology with the entire amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 is about 80% or more, preferably 90% or more of the whole. If the protein has the amino acid sequence of, it is interpreted that they are substantially the same. Therefore, a part (preferably about 1 to 20, more preferably about 1 to 10 倜, and most preferably several) of the amino acids in the amino acid sequence represented by SEQ ID NO: 1 Proteins consisting of deleted, substituted or added amino acid sequences are also substantially identical.

The K tr B protein is a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or represented by SEQ ID NO: 1. Some amino acids are missing in the amino acid sequence Those produced using a transformant transformed with a DNA-containing gene encoding a protein comprising a lost, substituted or added amino acid sequence can be used.

As the trA protein, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or the amino acid represented by SEQ ID NO: 2 Manufactured using a transformant transformed with a DNA-containing gene encoding a protein comprising an amino acid sequence in which some amino acids have been deleted, substituted or added in the sequence Can be used.

As the KtrC protein, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 or ί the amino acid represented by SEQ ID NO: 3 Manufactured using a transformant transformed with a DNA-containing gene encoding a protein comprising an amino acid sequence in which some amino acids have been deleted, substituted or added in the sequence Anything can be used.

The K tr B protein, K tr A protein and K tr C protein of the present invention usually have a carboxyl group (—COOH) or carboxylate (—COO—) at the C-terminus, but have an amino acid at the C-terminus. It may be de (one CO NH ₂ ) or ester (one COOR). Here, R in the ester is, for example, an alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, cycloalkyl, cycloalkyl, and the like. Cycloalkyl groups having 3 to 8 carbon atoms, such as rohexyl; for example, aryl groups having 6 to 12 carbon atoms, such as phenyl and α-naphthyl; and bivaloyloxymethyl, which is widely used as an oral ester Esters.

In addition, the above protein has a carboxyl group (or In the case of (xylate), those having a carboxyl group amidated or esterified can also be used. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Furthermore, in the protein of the present invention, the amino group of the N-terminal methionine residue is protected by a protective grave (for example, an acyl group having 1 to 6 carbon atoms such as a formyl group and an acetyl group). N-glutamic acid residue generated by cleavage in vivo, pyroglutamated N-terminal glutamate residue, on the side chain of amino acid in the molecule, such as OH, COOH, NH ₂ , SH, etc. are protected with an appropriate protecting group (for example, a formyl group, an acetyl group, etc., having 1 to 6 carbon atoms), or a complex protein such as a so-called glycoprotein to which a sugar chain is bound. Quality is also included.

The salt of the protein or its partial peptide is particularly preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, For example, salts with fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, lingic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid are used.

A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a part of the amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 As a DNA encoding a protein consisting of a lost, substituted or added amino acid sequence, it is preferable to use a DNA having a base sequence represented by SEQ ID NO: 4. In addition, the DNA hybridized with the DNA represented by SEQ ID NO: 4 under stringent conditions, and the same or substantially the same amino acid as the amino acid sequence represented by SEQ ID NO: 1 In the protein having an acid sequence or the amino acid sequence represented by SEQ ID NO: 1, some amino acids are deleted, substituted or added. The protein may be a DNA substantially identical to the protein consisting of the amino acid sequence.

A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a part of the amino acid sequence in the amino acid sequence represented by SEQ ID NO: 2; It is preferable to use a DNA having the base sequence represented by SEQ ID NO: 5 | as a DNA encoding a protein comprising a deleted, substituted or added amino acid sequence. In addition, it hybridizes with the DNA represented by SEQ ID NO: 5 under stringent conditions, and is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2. A protein having a sequence or an amino acid sequence represented by SEQ ID NO: 2 which is substantially identical to a protein comprising an amino acid sequence in which some amino acids have been deleted, substituted or added; The coding DNA may be used.

A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 or a part of the amino acid sequence in the amino acid sequence represented by SEQ ID NO: 3 As a DNA encoding a protein comprising a lost, substituted or added amino acid sequence, it is preferable to use a DNA having a base sequence represented by SEQ ID NO: 6. In addition, it hybridizes with the DNA represented by SEQ ID NO: 6 under stringent conditions, and produces an amino acid identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 3. A protein having an acid sequence or substantially the same as a protein consisting of an amino acid sequence in which a part of the amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 3 It may be a DNA that encodes a protein.

Examples of the DNA that hybridizes with the DNA represented by SEQ ID NO: 4, 5, or 6 under stringent conditions include, for example, SEQ ID NO: 4, 5, Is a D having a nucleotide sequence having a homology of about 90% or more, preferably about 95% or more, more preferably about 98% or more with the nucleotide sequence represented by 6. Hybridization can be carried out by a method known in the art or a method analogous thereto, as described in Molecular Cloning, ^2ηα , J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989. In the case of using a commercially available library, the method can be performed according to the method described in the attached instruction manual. The “stringent conditions” refer to, for example, when a probe is labeled with DIG DNA Labeling (Roche Diagnostics), at 32 ° C. in a DIG Easy Hyb solution (Roche's The conditions for washing the membrane in a 0 IxSSC solution (including 0.1 [w / v] SDS) at 40 ° C (IxSSC is 0.15M NaCl, 0.015M sodium citrate ) Refers to a Southern blot Tohaiburi Dizesho down in the present invention DNA probe enough to high pre soybean conditions in.

It hybridizes with the DNA represented by SEQ ID NO: 4, 5 or 6, or the DNA represented by SEQ ID NO: 4, 5 or 6 under stringent conditions, and obtains K tr B protein and K tr As a means for cloning a DNA encoding a protein substantially identical to the A protein or the KtrC protein, the KtrB protein, the KtrA protein or the KtrC protein may be substantially cloned. Amplification by PCR using a synthetic DNA primer having an appropriate nucleotide sequence such as the same protein part as described above, or Ktr B protein, Ktr A protein or Ktr It can be selected by hybridization with a DNA fragment coding for a part or the entire region of the C protein or labeled with synthetic DNA. Hydration The method can be carried out, for example, by the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. The conversion of the DNA sequence can be performed by using a known kit, for example, Superscript II reverse transcriptase kit (Invitrogen), or other known methods such as the Gapped duplex method and the Kunkel method. It can be done according to the same method. The DNA encoding the cloned polypeptide can be used as it is depending on the purpose, or can be digested with a restriction enzyme or added with a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end, and may have TAA, TGA or TAG as a translation termination codon at the 3' end. . These translation start codons and translation stop codons can be added using an appropriate synthetic DNA adapter.

-Once the nucleotide sequence of the gene of the KtrB protein, KtrA protein or KtrC protein of the present invention has been determined, then the cDNA synthesized by chemical synthesis or cloned is designated as type II. The gene of KtrB protein, KtrA protein or KtrC protein can be obtained by PCR or by hybridizing with a DNA fragment having the nucleotide sequence as a probe. Furthermore, it is also possible to synthesize a mutant form of the gene of the KtrB protein, the KtrC protein or the KtrA protein of the present invention by site-specific mutagenesis or the like.

As a method for introducing a mutation into a gene, for example, a known method such as the Kunkel method or the gapped duplex method or a method similar thereto can be adopted. For example, for mutagenesis using site-directed mutagenesis Mutants can be introduced using a kit (Mutant-K (TAKARA) or Mutant-G (TAKARA)).

A recombinant vector containing DNA encoding the KtrB protein and a recombinant vector containing the KtrB protein, the KtrA protein and the KtrC protein can be obtained by methods known in the art. It can be created according to For example, it can be obtained by ligating (introducing) the gene of the KtrB protein of the present invention or the genes of the KtrB protein, the KtrA protein and the KtrC protein to an appropriate vector. Can be. The vector is not particularly limited as long as it can be replicated in the host, and examples thereof include plasmid DNA and phage DNA.

A KtrB protein gene of the present invention or a DNA fragment containing DNA encoding the KtrB protein, KtrA protein and KtrC protein is cut out, and the DNA fragment is expressed in an appropriate expression vector. It is implemented by connecting to the downstream of the middle promoter. Examples of the vector include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC18, pUC19, pUC118, pB1uescript, etc.) ), Plasmid derived from Bacillus subtilis (eg, pUBllO, pTP5 or pC194), plasmid derived from yeast (eg, pSH19, pSH15, YEpl3 or YCP50, etc.), pacteriophage such as λ phage, and animal viruses such as retrovirus, vaccinia virus or baculovirus can be used. The promoter used in the present invention may be any promoter suitable for the host used for gene expression. For example, when the host is Escherichia coli, the trp promoter, lac promoter, rec A promoter, PL promoter, lpp promoter, T7 promoter, T3 promoter, araB AD promoter, etc. If it is a bacterium, use SPOl promoter, penP promoter When the host is Bacillus subtilis, SPOL promoter, SP02 promoter, pen P promoter, etc., and when the host is yeast, PHQ5, XYL promoter, HWP promoter, CWP promoter, etc. Micromotors, promoters, GAP promoters, ADH promoters, etc. are preferred. When an animal cell is used as a host, the promoter includes the SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like. When used as a host for insect cells, a polyhydrin promoter, an OplE2 promoter and the like are preferable.

In addition to the above, expression vectors include, if desired, an enhancer, a splicing signal, a poly-A addition signal, a selection primer, a SV40 replication origin (hereinafter referred to as an SV), which are known in the art. 40 ori). Further, if necessary, the protein encoded by the DNA of the present invention can be expressed as a fusion protein with another protein (for example, glutathione S transferase and protein A). . Such a fusion protein can be cleaved using a site-specific protease and separated into the respective proteins.

As host cells, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, animal cells, and the like are used. Specific examples of the genus Escherichia include Escherichia coli K12 · DH1 (Proc. Natl. Acad. Sci. USA, 60, 16 (19) 6 8)), JM 103 (Nucleic Acids Research, Vol. 9, 309 (1991)), JA221 (Journal of Molecular Biology, Vol. 120, Vol. 51 (197) 8)), HB101 (Journal of Molecular Biology, vol. 41, 559 (19669)), DH5α and JM109 are used. Examples of Bacillus bacteria include, for example, Bacillus subtilis Mill 4 (Gene, 24, 25 5 (1993)), 20 7 — 21 [Journal Biochemistry, 95, 8 7 (1984)] and Bacillus brevis etc. are used. . Examples of yeast include, for example, Saccharomyces cerevisiae.

(Saccaromyces cerevisiae) A H 2 2, A H 2 2 R-, Ν A 8 7 — 11 A, D K D — 5 D, 20 Β — 12

(Schizosaccaromyces pombe) NCYC193, NCYC236, Pichia pastoris, Hansenula polymorpha and the like are used. Examples of animal cells include monkey cells COS—7, Vero, Chinese hamster cells CHO

(Hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster one-cell CHO (hereinafter abbreviated as CH◦ (dhfr-) cells), mouse L cells, mouse AtT_20, mouse myeloma cells, GH3, human FL cells, HEK293 cells and the like are used.

The above-described transformation of the host cell can be performed according to a method known in the art. For example, the following literature describes a method for transforming a host cell. Natl. Acad. Sci. USA, 69 Vol. 2 11 0 (1972); Gene, 17 Vol., 107 (1992); Molecular & General Genetics, 1668 Vol. 1 1 1 (1977); Methods in Enzymology, 194, 18 2 — 18 7 (1991); Proc. Natl. Acad. Sci. USA, 75, 192 9 (1978); Cell Engineering Separate Volume 8 New Cell Engineering Experiment Protocol. 26 3 — 26 7 (1995) (published by Shujunsha); and Χβ Virology, 52 volumes, 45 6 (1 9 7 3).

When a transgenic plant is obtained by transforming a gene encoding the KtrB protein of the present invention or a gene encoding the KtrB protein, the KtrA protein and the KtrC protein into a plant. For example, ^ electroporation method (electroporation method), agglomerated battery method, Genes can be introduced into plants by the Ticklegun method, the PEG method, or the like. For example, in the case of using the electoral poration method, the electroporation device equipped with a pulse controller is used for processing at a voltage of 500 to 600 V, 100 tF, and 20 msec. And introduce the gene into the host.

When the Agrobacterium method is used, the constructed plant expression vector is introduced into an appropriate Agrobacterium tumefaciens, such as Agrobacterium tumefaciens, and the strain is transformed into a plasmid. The sterilization of the host is performed according to the method described in the Muffin Refinery Method (Bechtold et al. (1993) C. R. Acad. Sci. Ser. Ill Sci. Vie, 316, 1194-1199). A transgenic plant can be obtained by infecting cultured leaf pieces.

When using the particle gun method, the plant, plant organs (eg, leaves, petals, stems, roots, seeds, etc.) and plant tissues (eg, epidermis, phloem, soft tissue, xylem, vascular bundle, etc.) themselves are used as they are. Use after preparing mosquito slices, or prepare and use protoplasts. Use a sample introduction device (eg, BIOLISTIC P0S 1000 / He; BioRad, etc.) to remove the sample prepared in this way! /, To process. The processing conditions vary depending on the plant or sample, but are usually performed at a pressure of about 1000 to 1100 psi and a distance of about 5 to: L0 cm.

Plants used for transformation include, for example, Arabidopsis thaliana of the Brassicaceae, Nicotiana tabacum of the Solanaceae, corn (Zeamays) of the Poaceae, and rice. (Oryza sativa), legume soybean (Clycine max) and the like, but are not limited thereto, and are useful for other agricultural crops and trees. The plant may be any of conifers, hardwoods, dicots, monocots, and the like. Once in the genome, the K tr B protein and K tr A protein of the present invention If a transformed plant into which DNA encoding the KtrC protein has been introduced can be obtained, progeny can be obtained from the plant by sexual or asexual reproduction. Propagation materials (for example, seeds, fruits, cuttings, tubers, tubers, strains, calli, protoplasts, etc.) are obtained from the plant, its progeny or clone, and the plant is mass-produced therefrom. It is also possible. According to the present invention, a plant cell into which DNA encoding the KtrB protein, the KtrA protein and the KtrC protein of the present invention has been introduced, a plant containing the cell, progeny of the plant, and a clone And the propagation material of the plant, its progeny and the clone.

The callus, tumor tissue, hairy root, and the like obtained by the above-described transformation method can be used for cell culture, tissue culture, or organ culture as it is. Also, by adding plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylen, brassinolide, etc.) at an appropriate concentration by a conventionally known plant tissue culture method. It can be regenerated into plants.

The method of introducing the recombinant vector into a bacterium such as Escherichia coli is not particularly limited as long as it can introduce DNA into the bacterium, and for example, using a potention ion. Method (Cohen, SN et al .: Proc. Natl. Acad. Sci., USA, 69: 2110 (1972)), and the electoral port-shion method.

When a yeast is used as a host, the method of introducing the recombinant vector into the yeast is not particularly limited as long as it can introduce DNA into the yeast. For example, the electroporation method, Sueproplus And the lithium acetate method.

When an animal cell is used as a host, the method of introducing the recombinant vector into the animal cell is not particularly limited as long as it can introduce DNA into the animal cell. However, for example, there are an election port method, a calcium phosphate method, a reboaction method, and the like.

When an insect cell is used as a host, the method of introducing the recombinant vector into the insect cell is not particularly limited as long as it can introduce DNA into the insect cell. For example, the calcium phosphate method, the lipofection And the electro-rotation method.

As a method for confirming whether or not the gene has been integrated into the host, for example, a PCR method, a Southern hybridization method, a Northern hybridization method, or the like can be used. For example, DNA is prepared from a transformant, DNA-specific primers are designed, and PCR is performed. The PCR is performed under the same conditions as those used for preparing the above-mentioned plasmid. Next, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, and stained with bromide TC or SYBR Green solution. As a result, the transformation can be confirmed. PCR may be performed using a primer that has been labeled with a fluorescent dye or the like in advance, and the amplification product may be detected. Further, a method of binding the amplification product to a solid phase such as a microplate, and then confirming the amplification product using fluorescence or an enzymatic reaction can also be used.

The KtrB protein, the KtrA protein and the KtrC protein of the present invention are obtained by culturing the transformant, and transforming the KtrB protein or the KtrB protein, the KtrA protein and the KtrC protein. It can be produced by producing, storing, and collecting the protein. The fact that the protein is accumulated after culturing means not only the culture supernatant but also any of cultured cells or cultured cells or cells or crushed cells. The method of culturing the transformant in the present invention is not particularly limited, In the usual manner used in culturing a host.

For example, when the host is a microorganism such as Escherichia coli or yeast, the culture medium for culturing the transformant contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be used by the microorganism, to efficiently culture the transformant. Either a natural medium or a synthetic medium can be used as long as it can be performed. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol. Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other inorganic or organic acid ammonium salts, and other nitrogen-containing compounds, as well as peptone and meat kiss. And corn stays. Inorganic substances include potassium phosphate (II), potassium phosphate (II), magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, Calcium carbonate and the like. The cultivation is usually performed under aerobic conditions such as infiltration culture or aeration and stirring culture. When the host is Escherichia coli, the culture is performed at a temperature of about 15 to 43 ° C for about 12 to 48 hours. When the host is Bacillus, the reaction is performed at a temperature of about 30 to 40 ° C for about 12 to 100 hours. When the host is a yeast, the reaction is carried out at a temperature of about 20 to 35 ° C for about 24 to 100 hours. In addition, ventilation and stirring can be added as needed. When the pH needs to be adjusted, use an inorganic or organic acid, an alkaline solution, or the like.

When culturing a transformant transformed by an expression vector using an inducible promoter as a promoter, add an inducer to the medium as needed to perform the culturing. For example, in the case of an expression vector using a T7 promoter, culture may be performed by adding IPTG or the like to a medium. In the case of an expression vector using a trp promoter inducible with indoleacetic acid (IAA), IAA or the like may be added to the medium. When culturing a transformant obtained using animal cells as a host, a commonly used RPMI164 medium, DMEM medium, or a fetal calf A medium to which serum or the like is added can be used. The cultivation is usually performed in the presence of about 5% carbon dioxide at a temperature of about 37 ° C for 1 to 30 days.

After culturing, if the KtrB protein, KtrA protein or KtrC protein is produced in the cells or cells, the cells or cells are collected by a known method and collected. After suspending the cells in a buffer, disrupting the cells or cells by sonication, lysozyme, or thawing or freeze-thawing, a method of obtaining a crude protein extract by centrifugation or filtration may be used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (registered trademark). When the KtrB protein, the KtrA protein or the KtrC protein is secreted into the culture, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected. The protein contained in the culture supernatant or extract obtained in this way can be purified by appropriately combining known separation and purification methods. That is, the target protein can be produced by using, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, or the like, alone or in an appropriate combination.

The protein of the present invention thus obtained can be converted into a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained with a salt, a known method or a method analogous thereto. , Free form or other salts can be converted. Furthermore, the protein produced by the recombinant is fragmented arbitrarily before or after purification by the action of an appropriate proteinase such as trypsin or chymotrypsin. You can. Also, Modification can also be made arbitrarily by the action of a protein-modifying enzyme such as a kinase. The presence of the KtrB protein, the KtrA protein and the KtrC protein or a salt thereof of the present invention can be measured by various binding assays and enzyme immunoassays using specific antibodies.

When a recombinant vector incorporating the KtrB protein, the KtrA protein and the KtrC protein of the present invention is introduced into a microorganism or a plant, the activity of incorporating K + into cells is enhanced, The activity of excreting Na + to the outside of the cell is enhanced, and the microorganism or plant has improved salt tolerance, and is a microorganism or plant resistant to environmental stress, particularly salt stress, high osmotic pressure and low osmotic pressure. Become a plant. Such plants are capable of growing in, for example, desertified soil, arid regions, cold regions and soil near the coast.

Such plants can also be used as experimental plants for studying salt stress. In addition, salt stress refers to a state in which salt is contained in a high concentration in soil and ordinary plants cannot grow.

On the other hand, plants with reduced salt stress tolerance cannot grow on desertified lands as described above, and therefore, by reducing the salt stress tolerance of weeds and the like. Can be excluded from the environment. Therefore, compounds that inhibit the activity of KtrB protein, KtrA protein and KtrC protein (especially K + transporter activity) of the present invention are screened, or KtrB protein, KtrB An antibody against the A protein and the K tr protein is prepared, and the compound or the antibody is used to spray the soil on a soil with a high salt stress, thereby reducing the resistance to salt stress such as weeds, and then growing the crop. You can also.

The above-described protein having the K + transporter activity of the present invention Certain K tr B, K tr A and K tr C proteins can be used to produce antibodies as described below. Hereinafter, antibodies against the KtrB protein, the KtrA protein and the KtrC protein of the present invention will be described.

The antibodies against the KtrB protein, the KtrA protein and the KtrC protein of the present invention may be poly-antibodies as long as they can recognize the KtrB protein, the KtrA protein and the KtrC protein. Either a clonal antibody or a monoclonal antibody may be used. The antibody can be produced using a KtrB protein, a KtrA protein and a KtrC protein as antigens according to a conventionally known method for producing an antibody or antiserum. The method for producing a monoclonal antibody and a polyclonal antibody is described below.

[Preparation of Monoclonal Antibody]

(a) Preparation of monoclonal antibody-producing cells

The protein of the present invention is administered to a warm-blooded animal at a site capable of producing an antibody by administration, itself or together with a carrier and a diluent. In order to enhance the antibody-producing ability upon administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. The administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. The warm-blooded animals used include, for example, monkeys, rabbits, dogs, guinea pigs, mice, rats, sheds, sheep, goats, and nits, but mice and rats Is preferably used. When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with the antigen, for example, a mouse with an antibody titer was selected from the mouse, and the spleen or lymph node was collected 2 to 5 days after the final immunization. Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody-producing cells contained therein with myeloma cells of the same or different species. In antiserum The antibody titer can be measured, for example, by reacting a labeled protein described below with an antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed by a known method, for example, the method of Koehler and Millstein [Nature (Nature) 256, 495 (1975) 3]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.

The myeloma cells include, for example, myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. . The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG100 to PEG600) is used. Is added at a concentration of about 10 to 80% and incubated at 20 to 40 ^, preferably at 30 to 37 ° C for 1 to 10 minutes, to increase the efficiency of cells. Fusion can be performed. Various methods can be used to screen the monoclonal antibody-producing hybridomas. For example, protein antigens can be directly or directly adsorbed on a solid phase (eg, microplate). An anti-immunoglobulin antibody labeled with a hybridoma culture supernatant and then labeled with a radioactive substance or an enzyme (if the cell used for cell fusion is a mass, an anti-mouse immunoglobulin antibody is used) Alternatively, add protein A to detect monoclonal antibodies bound to the solid phase, add the hybridoma culture supernatant to the solid phase to which the anti-immunoglobulin antibody or protein A has been adsorbed, and add the radioactive substance. And a method in which a protein labeled with an enzyme or the like is added to detect a monoclonal antibody bound to a solid phase. The selection of hybridomas can be carried out according to a method known per se or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a breeding medium, any medium can be used as long as it can grow a hybridoma. For example, RPMI 164 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd. Co., Ltd.) or serum medium for hybridoma culturing (SFM-101, Nissui Pharmaceutical Co., Ltd.). The culture temperature is usually from 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The cultivation can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(b) Monoclonal antibody purification

Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, , DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase or active adsorbent such as protein A or protein Q to collect only the antibody and dissociate the bond to obtain the antibody. Specific purification method].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced by a method known per se or a method analogous thereto. For example, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. The antibody can be produced by collecting an antibody-containing substance against the protein of the present invention and separating and purifying the antibody. With respect to the complex of the immunizing antigen used to immunize warm-blooded animals and the carrier protein, the mixing ratio of the carrier protein and the carrier to the hapten depends on the carrier. If antibodies can be efficiently produced against haptens immunized by cross-linking, what is cross-linked at what ratio However, for example, serum serum albumin, psiloglobulin, hemocyanin, etc. may be used in a weight ratio of about 0.1 to 20 and preferably about 1 to 5 with respect to hapten 1. A coupling method is used. In addition, various condensing agents can be used for capping of hapten and carrier, but it contains dartal aldehyde carbodiimide, maleimide active ester, thiol group and dithioviridyl group. An active ester reagent or the like is used. The condensation product is administered to a warm-blooded animal at a site where antibody production is possible, or together with a carrier and a diluent. In order to enhance the antibody-producing ability upon administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. The administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 times. The polyclonal antibody can be collected from the blood of a warm-blooded animal immunized by the method described above. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the above-described measurement of the antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as in the above-mentioned separation and purification of the monoclonal antibody.

The antibody produced as described above may be, for example, a compound that inhibits the activity of the protein or its partial peptide or its amide or its ester or its salt, as described above. In this case, when a monoclonal antibody is used, the inhibitory activity of the protein or its partial peptide or its amide or its ester or its motif is simple. Because it is one, there is an advantage that it is easy to use.

In addition, the antibody prepared as described above can be used for, for example, examining the amount of KtrB protein, KtrA protein, and KtrC protein expressed. .

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be noted that the present invention It goes without saying that the box is not limited to such an embodiment.

Example 1

KtrB in the blue-green algae (Synechocystis sp. PCC6803). An experiment was performed to confirm that the protein exhibited K + transporter activity. The experiment was performed as follows.

Cyanobacteria (Synechocystis sp. Created a blue-green algae. Hereinafter, the cyanobacterium in which the KtrB gene has been destroyed is referred to as ΔKtrB.

Based on the nucleotide sequence of the gene database of Kazusa DNA Research Institute, PCR primers (original nucleotides) represented by SEQ ID NOS: 9 and 10 were prepared. The gene for the expressed protein (K tr B protein) was propagated and incorporated into a PBR322-derived wide vector (manufactured by Takara Shuzo Co., Ltd.) which was replicable in Escherichia coli to obtain a recombinant vector. Escherichia coli (JM109) was transformed with the recombinant betta by the calcium decion method to prepare a transformant. The transformant was cultured to obtain a protein (KtrB protein) gene represented by SEQ ID NO: 1. The KtrB protein gene was introduced into the chromosome by gene transfer by homologous recombination. Hereinafter, the cyanobacterium into which the KtrB protein gene has been introduced is referred to as + KtrB.

The AKtrB, + KtrB, and wild-type (WT) cyanobacterium (Synechocystis sp. PCC6803) obtained as described above were transformed into a BG11 medium (liquid medium) containing no sodium salt. ), Pre-cultured, and then placed on BG11 medium without sodium chloride (plate medium) and BG11 medium containing 0.4 M sodium chloride (plate medium). Were transplanted and observed for growth at a temperature of 30 in the presence of 0.3% CO ₂ . Grow for 5-7 days, The plate medium was observed. The results are shown in Figure 1. Figure 1 is a photograph of a plate medium on which cyanobacteria have grown.

The upper part of FIG. 1 shows the growth state of BG11 medium without sodium chloride, and the lower part of FIG.1 shows the growth state of BG11 medium containing 0.4 M sodium chloride. It is a figure showing a growing state. Both upper and lower rows show the growth status of ΔKtrB, ΔKtrB, WT, and + ΚtrB from the left.

As shown in FIG. 1, all were able to grow in the BG11 medium containing no sodium chloride. In contrast, in the medium containing 0.4% sodium chloride, W and + K tr B could grow, but ΔK tr B could not grow. .

From the above results, it is clear that the KtrB gene encodes a protein having a K + transporter activity involved in salt tolerance of cyanobacteria, Example 2.

Based on the nucleotide sequence in the gene database of Kazusa DNA Research Institute, a DNA primer for PCR (oligonucleotide) represented by SEQ ID NO: 7 and 8 was prepared, and the DNA represented by SEQ ID NO: 2 was prepared. The gene for the protein (K trA protein) was propagated and incorporated into a pTWV228 amplification vector (Takara Shuzo Co., Ltd.) which can be replicated in E. coli to obtain a recombinant vector. Next, the obtained recombinant vector was introduced into E. coli LB203 strain by the potassium ion method together with the recombinant vector obtained in Example 1, cloned, and the KtrB protein and KtrB tr A protein-producing Escherichia coli (hereinafter referred to as “K tr AB”) was obtained.

The LB203 strain is a strain deficient in Kdp, Trk and Kup, and has no K + uptake function. In other words, since the LB203 strain does not have the function of taking up K +, it cannot grow in the presence of low concentrations of potassium, but grows only in the presence of high concentrations of potassium. thing Can be.

Similarly, DNA primers for PCR (polynucleotides) represented by SEQ ID NOS: 11 and 12 were prepared based on the nucleotide sequence of the gene database of Kazusa DNA Research Institute, and SEQ ID NO: The gene of the protein (KtrB protein) represented by 1 and the gene of the protein (KtrC protein) represented by SEQ ID NO: 3 are amplified, and the pBR322-derived amplification vector (Takara Shuzo) capable of replicating in E. coli And the recombinant vector was obtained. Next, the obtained recombinant vector was introduced into E. coli LB203 strain by the calcium ion method, cloned, and used to produce KtrB protein and KtrC protein-producing Escherichia coli (hereinafter, referred to as “K tr BC”).

'Furthermore, a recombinant vector of the gene of the protein represented by SEQ ID NO: 2 (K tr A protein), the protein represented by SEQ ID NO: 1 (K tr B protein) and the protein represented by SEQ ID NO: 3 (K tr trC protein) was introduced into the E. coli LB203 strain by the calcium ion method, cloned, and the KtrA protein and KtrB protein were cloned. And K tr C protein-producing Escherichia coli (hereinafter referred to as “K tr ABC”).

Next, Ktr AB, 1 ^ 1: 8 (and 1 r ABC) obtained as described above were first pre-cultured in a minimal medium containing 12 OmM potassium chloride. The cells were transplanted to a minimal medium containing 1 O mM or 3 O mM sodium chloride, and the growth was observed at a temperature of 30 ° C. The LB203 strain (empty vectors) was used. The results are shown in Fig. 2. The results are shown in Fig. 2. Fig. 2 is a photograph of the plate medium on which E. coli was grown.

The upper part of Fig. 2 shows the growth state in a medium containing 10 mM chloride room. The lower part of FIG. 2 is a view showing the growth state in a medium containing 30 mM chloride rim. The upper row and the lower row show the growth state of empty vectors, K tr AB, K tr BC and K tr ABC from the left.

As shown in FIG. 2, all Escherichia coli were able to grow in the medium containing 30 mM potassium chloride. On the other hand, in the medium containing 1 O mM potassium chloride, empty vectors, K tr AB and K tr BC could not grow, and only K tr ABC could grow. . From these results, it was found that K tr AB and K tr BC could not impart the function of taking up potassium to E. coli, that is, K tr A and K tr B or K tr B and K tr B When C is expressed in Escherichia coli, the function of taking up calcium is not added, and KtrA, 18 and 1:13: <When all are expressed in Escherichia coli, the function of taking up potassium is exhibited. You can see this.

Example 3

The growth of Escherichia coli transfected with empty vectors, KtrAB, KtrBC and KtrABC used in Example 2 in a liquid medium was examined.

First, each cell was pre-cultured in a minimal medium containing 12 O mM chloride medium, and the cells were washed twice with a minimal medium containing no potassium chloride, and then washed with 30 mM chloride medium. The cells were transplanted to a minimal medium containing a fermentation medium, and cultured at a temperature of 30 ^ with 100 reciprocal shakings per minute. The absorbance at 0 n x n was measured to create a growth curve, and the results are shown in Fig. 3. The Q0610 at the start of the culture in the liquid medium was 0.05.

In FIG. 3, the horizontal axis represents time, and the vertical axis represents absorbance at 61 nm.

As shown in Figure 3, empty vectors, K tr AB and K tr BC It was found that K tr ABC showed growth over time, while growth hardly occurred in somatic medium for up to 8 hours. This result indicates that the strain that does not produce the KtrB protein lacks the K + uptake function, and therefore has a low growth rate even under high concentrations of potassium. Also, due to the high growth rate of K tr ABC, the K tr B protein does not exert its own function of taking up potassium but cooperates with the K tr A protein and the K tr C protein. It can be seen that the above functions are exhibited.

Example 4

The K ⁺ uptake activity of empty vectors, K tr AB, K tr BC and K tr ABC was measured using E. coli as a protein expression host. First, each cell was grown on a minimal medium containing 3 OmM potassium chloride, and then washed twice with Na +-Hepes buffer (pH 7.5). Were suspended in 200 mM Hepes buffer (pH 7.5). 10 mM glucose was added 10 minutes before the start of measurement, and 0.5 M potassium chloride was added at the start of measurement. The measurement was performed at 25 ° C. and a pH of 7.5, and K ′ ⁺ incorporation was measured over time by measuring the amount of ions by atomic absorption spectrophotometry. Figure 4 shows the results. As shown in FIG. 4, empty vectors, K tr AB, have an activity to take up a small amount of K +, while K tr BC hardly takes up K +, whereas K tr ABC The K ⁺ uptake activity was high.

Example 5

K tr ABC was examined for Na + -dependent K + uptake activity. In the activity measurement in Example 4, 11 minutes after the start of the measurement, 5 mM sodium chloride was added, and the same experiment was performed. Figure 5 shows the results. As shown in FIG. 5, it was found that the K + uptake activity of K tr ABC was increased by the addition of Na ⁺ . That is, the K + uptake activity of K tr ABC is Na ⁺ Dependency.

Example 6

Using + KtrB, AKtrB obtained in Example 1, and a wild strain (WT) cyanobacterium (Synechocystis sp. PCC6803), the contribution of osmotic pressure regulation was examined. First, each cell was suspended in BG11 liquid medium, and culture was started. 30 seconds after the start of the culture, potassium chloride was added so that the concentration of potassium chloride became 5 mM. Next, 10 minutes after the start of the culture, sodium oxide or sorbitol was added so that the respective concentrations became 100 mM, s, or 200 mM. The K + concentration in the medium was determined over time. The measurement was performed under the conditions of 25 ° C and a pH of 7.5, and was performed for atomic absorption analysis. The osmotic pressure is the same for 10OmM sodium chloride and 20OmM sorbitol.

Figure 6 shows the results. As shown in FIG. 6, it was found that K + uptake activity increased in the presence of 10 O mM sodium chloride. In contrast, K ⁺ uptake activity was slightly increased in the presence of 200 mM sorbitol. From this result, it is clear that K + transport of K tr B is promoted by Na ^+, and the contribution of osmotic pressure is negligible.

Example 7

The + K tr B, AK tr B and wild strain (WT) blue-green algae (Synechocystis sp. PCC6803) obtained in Example 1 do not contain sodium salt! / ヽ BG11 medium, 0.15 M sodium chloride, 0.3 M sodium chloride, 0.15 M sorbitol and BG11 medium containing 0.3 M sorbitol ( ^ Somatic medium). The culture was carried out at a temperature of 30 3C in the presence of 0.3% CO ₂ for 7 days, and the liquid medium was observed. Figure 7 shows the results. Figure 7 is a photograph of the liquid medium in which the cyanobacteria grew. In the results shown in Fig. 7, from the top, BG11 medium without sodium chloride BG11 medium containing sodium chloride, 0.3 M sodium chloride, 0.15 M sorbitol and 0.3 M sorbitol, + s KtrΒ, Δs from left 2 shows the growth state of K tr B and WT.

As shown in FIG. 7, all of them were able to grow on the BG11 medium containing no sodium chloride. On the other hand, the growth was poor in the medium containing 0.3% sodium chloride and the medium containing 0.3% sorbitol. As shown in Fig. 7, AKtrB was able to grow in a medium containing 0.15 M sodium chloride, while the osmotic pressure was the same as that of 0.15 M sodium chloride. In the medium containing 0.3 M sorbitol, the growth of Δΐ B rB was poor. On the other hand, + KtrB also proliferated in the medium containing 0.3 M sorbitol. This result indicates that KtrB, ie, the K + transporter, is resistant to osmotic pressure (anti-osmotic activity) and is involved in the osmoregulation of bacteria.

As described in detail above, the activity of the K + transreporter of the present invention is promoted by Na +. Thus, proteins having a K + trans- porter activity of the present invention all SANYO imparting host salt tolerance when transformed into a host, a protein having a K ⁺ preparative lance transporter activity of the present invention The DNA to be coded can be used for imparting salt tolerance to plants and microorganisms, and can provide plants that can grow under high osmotic pressure and high salt stress, for example. . In addition, the protein having the Κ + transporter activity of the present invention is a protein involved in the regulation of osmotic pressure in bacteria.

Claims

The scope of the claims

1. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a part of the amino acid sequence represented by SEQ ID NO: 1 K + transporters including proteins consisting of amino acid sequences in which amino acids have been deleted, substituted or added, or partial peptides thereof, or their amides or their esters or salts thereof. A protein having one activity.

2. (a) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or the amino acid sequence represented by SEQ ID NO: 1 A protein comprising an amino acid sequence in which some amino acids have been deleted, substituted or added, or a partial peptide thereof, or an amide or an ester or a salt thereof;

(b) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a part of the amino acid sequence represented by SEQ ID NO: 2; A protein consisting of an amino acid sequence in which an amino acid has been deleted, substituted or added, or a partial peptide thereof, or its amide or its ester or a salt thereof; and

(c) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 or a part of the amino acid sequence represented by SEQ ID NO: 3; A K + transporter containing a protein consisting of an amino acid sequence in which an amino acid has been deleted, substituted or added, or a partial peptide thereof, or an amide or an ester or a salt thereof. An active protein composition.

3. A gene encoding the protein according to claim 1 or a partial peptide thereof; a gene having DNA having a base sequence.

4. The gene according to claim 3, which has a base sequence represented by SEQ ID NO: 4.

5. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a part of the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 1. A recombinant vector containing a DNA encoding a protein comprising an amino acid sequence in which amino acid has been deleted, substituted or added.

6. The DNA hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 4 or the DNA represented by SEQ ID NO: 4 under stringent conditions, and is represented by SEQ ID NO: 1. A protein having the same or substantially the same amino acid sequence as the amino acid sequence to be deleted or a portion of the amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 is deleted 6. The recombinant vector according to claim 5, which is a DNA encoding a protein substantially identical to a protein comprising a substituted or added amino acid sequence.

7. A transformant transformed with the recombinant vector according to claim 5.

8. A transgenic plant comprising the recombinant vector according to claim 5 or 6.

9. The transgenic plant according to claim 8, wherein the plant is a plant, a plant organ, a plant tissue or a plant culture cell.

10. An environmental stress-tolerant plant obtained by culturing or cultivating a transgenic plant into which the recombinant vector according to claim 5 or 6 has been introduced.

11. The environmental stress-tolerant plant according to claim 10, wherein the environmental stress is a salt stress.

12. The environmental stress-tolerant plant according to claim 10, wherein the environmental stress is a low osmotic stress or a high osmotic stress.

1 3. A DNA having a nucleotide sequence encoding the protein or its partial peptide (a) according to claim 2, a DNA having a nucleotide sequence encoding a protein or its partial peptide (b), and (4) A gene having a DNA having a base sequence encoding a protein or its partial peptide (c).

1 4. The nucleotide sequence encoding the protein or its partial peptide (a) is represented by SEQ ID NO: 4, and the nucleotide sequence encoding the protein or its partial peptide (b) is SEQ ID NO: 14. The DNA according to claim 13, wherein the DNA is represented by SEQ ID NO: 6, and the nucleotide sequence encoding the protein or its partial peptide (c) is represented by SEQ ID NO: 6.

15 (d) In the protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or in the amino acid sequence represented by SEQ ID NO: 1 A DNA encoding a protein consisting of an amino acid sequence in which some amino acids have been deleted, substituted or added;

(e) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a part of the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 2 DNA encoding a protein consisting of an amino acid sequence in which an acid has been deleted, substituted or added; and

(f) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3, or a part of the amino acid sequence represented by SEQ ID NO: 3 Recombinant vectors ¹ to containing DNA encoding a protein comprising an amino acid sequence in which the amino acid has been deleted, substituted or added.

16. The DNA (d) undergoes a stringent reaction with DNA having the base sequence represented by SEQ ID NO: 4 or DNA represented by SEQ ID NO: 4 under stringent conditions, and No .: a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by 1 or SEQ ID NO: Some amino acids are deleted in the amino acid sequence represented by 1. Coding of a protein substantially identical to the protein consisting of the substituted or added amino acid sequence To be DNA,

The DNA (e) hybridizes with the DNA having the base sequence represented by SEQ ID NO: 5 or the DNA represented by SEQ ID NO: 5 under stringent conditions, and SEQ ID NO: 2 A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by, or a part of amino acid in the amino acid sequence represented by SEQ ID NO: 2 A DNA encoding a protein substantially identical to a protein consisting of a lost, substituted or added amino acid sequence, and

The DNA (f) hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 6 or the DNA represented by SEQ ID NO: 6 under stringent conditions, and the sequence of SEQ ID NO: 3 is obtained. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by, or a part of the amino acid in the amino acid sequence represented by SEQ ID NO: 3 loss, a protein substantially identical to the protein consisting of substituted or added in the a Mi acid sequence is a code sul DNA, recombinant vector ₍₁ 7. claim of claim 1 5 A transformant transformed with the recombinant vector according to 15 or 16.

1 8. A transgenic plant comprising the recombinant vector according to claim 15 or 16.

19. The transgenic plant according to claim 18, wherein the plant is a plant, a plant organ, a plant tissue, or a plant cultured cell.

20. An environmental stress-tolerant plant obtained by culturing or cultivating a transgenic plant into which the recombinant vector according to claim 15 or 16 has been introduced.

21. The environment of claim 20, wherein the environmental stress is a salt stress. Stress-tolerant plant.

22. The environmental stress-tolerant plant according to claim 20, wherein the environmental stress is a low osmotic stress or a high osmotic stress.

23. The transformant according to claim 7 or claim 17 is cultured to have an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. In the amino acid sequence represented by the protein or SEQ ID NO: 1, a protein comprising an amino acid sequence in which a part of the amino acid has been deleted, substituted or added is generated and accumulated, and the protein is collected. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence represented by SEQ ID NO: 1; A method for producing a protein comprising an amino acid in which some amino acids have been deleted, substituted or added, or a partial peptide thereof, or its amide or its ester or its salt.

24. culturing the transformant according to claim 17,

A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a part of the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 1 A protein comprising an amino acid sequence in which no acid has been deleted, substituted or added,

A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a part of the amino acid sequence in the amino acid sequence represented by SEQ ID NO: 2 A protein comprising an amino acid sequence in which is deleted, substituted or added, and

A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 or a part of the amino acid sequence in the amino acid sequence represented by SEQ ID NO: 3; To produce and accumulate a protein consisting of an amino acid sequence in which amino acid has been deleted, substituted or added, and to collect the protein Characterized by

(a) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a part of the amino acid sequence represented by SEQ ID NO: 1 A protein consisting of an amino acid sequence in which the amino acid has been deleted, substituted or added, or its partial peptide, or its amide or its ester or its salt;

(b) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a part of the amino acid sequence represented by SEQ ID NO: 2; A protein consisting of an amino acid sequence in which an amino acid has been deleted, substituted or added, or a partial peptide thereof, or an amide or an ester or a salt thereof; and

(c) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 3; Of a protein comprising an amino acid sequence in which a part of the amino acid has been deleted, substituted or added, or a partial peptide thereof, or a amide thereof, or a protein mixture containing the ester or a salt thereof. Production method.

25. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a part of the amino acid sequence represented by SEQ ID NO: 1 An antibody against a protein consisting of an amino acid sequence in which an amino acid has been deleted, substituted or added, or a partial peptide thereof, or an amide or an ester or a salt thereof.

26. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a part of the amino acid sequence represented by SEQ ID NO: 2 A protein consisting of an amino acid sequence in which amino acids have been deleted, substituted or added, or a partial peptide or amino acid sequence thereof An antibody against the amide or its ester or its salt.

27. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 3 An antibody against a protein comprising an amino acid sequence in which a part of the amino acid has been deleted, substituted or added, or a partial peptide thereof, or an amide or an ester or a salt thereof.

28. The antibody according to claim 25, which is produced using the transformant according to claim 7 or claim 17.

29. The antibody according to claim 26 or 27, produced using the transformant according to claim 17.