Peptides or their salts or solvates and their uses

Abstract

To provide a peptide that promotes the expression of sucrose transporters, which is useful for plant protection and increasing the sugar content of plants, and its applications. [Solution] Any of the following peptides (A1) to (A4), or a salt thereof, or a solvate thereof. (A1) A peptide consisting of the amino acid sequence shown in Sequence ID No. 1 (A2) A peptide consisting of an amino acid sequence in which 1 to 2 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, and which has an effect of promoting the expression of sucrose transporters. (A3) A peptide consisting of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, and which has the effect of promoting the expression of sucrose transporters. (A4)(A1)~(A3) peptides in which the N-terminus is acetylated and / or the C-terminus is amidated.

Description

Technical Field

[0001] The present invention relates to a peptide having an action of promoting the expression of sucrose transporter, or a salt thereof, or a solvate thereof. The present invention also relates to a composition containing the above peptide, or a salt thereof, or a solvate thereof, a method for cultivating plants, and the like.

Background Art

[0002] In global agricultural production, environmental problems such as salt damage and climate change have become issues. For example, global warming also causes an increase in pests and plant diseases, leading to a decrease in crop yields. Therefore, there is a need for a technology that can enhance the tolerance of plants to environmental stress and pests and diseases.

[0003] In recent years, functional peptides have attracted attention and are being used in fields such as food and medicine. In plant research, peptides with physiological activity have also been identified. Plant elicitor peptide (PEP) is an endogenous peptide that causes a plant defense response. AtPEP3, a PEP derived from Arabidopsis thaliana, has been reported to play a role in plant salt tolerance (Non-Patent Document 1). Regarding PEP (SlPep) derived from tomato, it has been reported that it can reduce the severity of infection in tomato seedlings exposed to bacteria (Non-Patent Document 2). Also, in tomato, it has been reported that salt stress promotes the accumulation of sugar in fruits (Non-Patent Document 3).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0005] Non-patent document 3 describes that one mechanism by which salt stress promotes the accumulation of sugar content in fruits is that salt stress causes an increase in the expression of sucrose transporters (SUTs) in leaves. When the sucrose concentration in the plant increases due to the promotion of sucrose synthesis induced by external environmental stress, the osmotic pressure in the plant increases, improving tolerance to environmental stresses such as salt tolerance, cold tolerance, and drought tolerance. Sucrose transporters are present throughout the plant and transport synthesized sucrose to the phloem tubes, where it is translocated to various organs of the plant. Therefore, substances that promote the expression of sucrose transporters in plants are useful, for example, for plant protection (e.g., improving the plant's tolerance to environmental stress) and increasing the sugar content of plants.

[0006] The present invention aims to provide a peptide that promotes the expression of sucrose transporters, which is useful for plant protection and increasing the sugar content of plants, and for applications thereof. [Means for solving the problem]

[0007] As a result of diligent research to solve the above problems, the inventors of the present invention have found that a peptide consisting of the amino acid sequence shown in Sequence ID No. 1 promotes the expression of sucrose transporters in plants.

[0008] The present invention relates to any of the peptides (A1) to (A4) below, or their salts or solvates; any of the polynucleotides (a1) to (a3) ​​below; a composition comprising the peptide or its salt or its solvate; a method for cultivating plants by applying the polypeptide or its salt or its solvate to plants; and the use of the peptide or its salt or its solvate for increasing the sugar content of plants or for plant protection. (A1) A peptide consisting of the amino acid sequence shown in Sequence ID No. 1 (A2) A peptide consisting of an amino acid sequence in which 1 to 2 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, and which has an effect of promoting the expression of sucrose transporters. (A3) A peptide consisting of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, and which has the effect of promoting the expression of sucrose transporters. (A4)(A1)~(A3) peptides in which the N-terminus is acetylated and / or the C-terminus is amidated. (a1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 2 (a2) A polynucleotide encoding a peptide having a sucrose transporter expression-promoting effect, consisting of a nucleotide sequence in which 1 to 7 bases are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 2. (a3) A polynucleotide encoding a peptide that has a sequence identity of 90% or more with respect to the sequence of Sequence ID No. 2 and has an effect of promoting the expression of sucrose transporters. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a peptide that has the effect of promoting the expression of sucrose transporters, which is useful for plant protection and increasing the sugar content of plants, as well as its uses. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the reverse-phase HPLC chromatogram of the synthesized peptide (BvPEP). [Figure 2] Figure 2 shows the results of mass spectrometry of the synthesized peptide (BvPEP). [Figure 3] Figure 3 is a graph showing the results of examining the expression of sucrose transporter 1 (SUT1) in sugar beets after providing them with water containing sodium chloride (NaCl). [Figure 4]Figure 4 is a graph showing the results of examining SUT1 expression in sugar beets treated with BvPEP (2 μM or 10 μM). [Figure 5] Figure 5 shows photographs of sugar beet seedlings two days after application of BvPEP (2 μM or 10 μM) and of sugar beet seedlings treated with water. [Figure 6] Figure 6 is a graph showing the results of examining SUT1 expression in tomatoes treated with BvPEP (10 μM). [Figure 7] Figure 7 shows photographs of tomato seedlings 30 days after application of BvPEP (10 μM), and tomato seedlings treated with water or 0.15 M NaCl. [Modes for carrying out the invention]

[0011] The peptide of the present invention is one of the following peptides (A1) to (A4). (A1) A peptide consisting of the amino acid sequence shown in Sequence ID No. 1 (A2) A peptide consisting of an amino acid sequence in which 1 to 2 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, and which has an effect of promoting the expression of sucrose transporters. (A3) A peptide consisting of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, and which has the effect of promoting the expression of sucrose transporters. (A4)(A1)~(A3) peptides in which the N-terminus is acetylated and / or the C-terminus is amidated. The peptide of the present invention is a polypeptide, and can also be described as a protein.

[0012] In this specification, unless otherwise specified, the amino acid sequence of a peptide is represented in the conventional one-letter notation as follows. A: alanine residue, R: arginine residue, N: asparagine residue, D: aspartic acid residue, C: cysteine residue, Q: glutamine residue, E: glutamic acid residue, G: glycine residue, H: histidine residue, I: isoleucine residue, L: leucine residue, K: lysine residue, M: methionine residue, F: phenylalanine residue, P: proline residue, S: serine residue, T: threonine residue, W: tryptophan residue, Y: tyrosine residue, V: valine residue

[0013] In this specification, according to the convention of peptide notation, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxy terminus). In the present invention, when an amino acid can have an optical isomer, unless otherwise specified, the L-form amino acid is indicated.

[0014] In the above (A2), the addition of an amino acid also includes the meaning of insertion into the sequence. In the present invention, in an amino acid sequence, when 1 to 2 amino acids are deleted, substituted or added, it means that there is a deletion, substitution or addition of 1 or 2 amino acids at an arbitrary position in 1 or 2 amino acid sequences in the same sequence, and two or more of deletion, substitution and addition may occur simultaneously.

[0015] The peptide in the above (A2) preferably consists of an amino acid sequence in which 1 amino acid is deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and has an action of promoting the expression of a sucrose transporter.

[0016] The sequence identity of the peptide in the above (A3) is 90% or more, preferably 91% or more or 92% or more, more preferably 93% or more or 94% or more, still more preferably 95% or more, 96% or more or 97% or more, still more preferably 98% or more, particularly preferably 99% or more.

[0017] The peptide (A4) described above is a peptide in which the N-terminus and / or C-terminus of any of the peptides (A1) to (A3) are modified. By amidation of the N-terminus and / or acetylation of the C-terminus of the peptide, degradation can be prevented and stability improved while maintaining the peptide's function.

[0018] The identity of amino acid sequences and base sequences can be calculated using analysis software such as BLAST with default parameters.

[0019] The peptides of the present invention may also be in the form of salts. The salts of the peptides in the present invention are not particularly limited and may be either acidic salts or basic salts. Examples of acidic salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, and phosphate; organic acid salts such as formate, acetate, citrate, maleate, malate, oxalate, lactate, succinate, fumarate, and propionate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; ammonium salts; and salts with organic bases such as triethylamine, triethanolamine, and pyridine. These salts are usable in plants and can be used as salts of peptides in the present invention. Among these, hydrochloride, formate, acetate, phosphate, citrate, lactate, aspartate, glutamate, sodium salt, and potassium salt are preferred as salts of peptides, and hydrochloride, formate, and acetate are more preferred.

[0020] The peptide or salt thereof of the present invention may be in the form of a solvate. The solvent that forms the solvate is not particularly limited and includes, for example, water, ethanol, methanol, glycerol, etc., and is preferably water.

[0021] The method for producing the peptide of the present invention, its salt, or its solvate is not particularly limited. The peptide of the present invention can be produced, for example, by known peptide synthesis methods. When the peptide of the present invention is obtained by peptide synthesis, it can be synthesized by either a solid-phase or liquid-phase method. The peptide obtained by synthesis can be purified by conventional purification methods such as reverse-phase high-performance liquid chromatography or affinity chromatography. The peptide of the present invention can also be biosynthesized using microorganisms. When using microorganisms, an expression vector into which the gene encoding the peptide of the present invention is introduced is prepared, and the expression vector is introduced into a host microorganism to produce a transformant. The transformant can be cultured, and the peptide of the present invention can be purified from the culture. The host microorganism is not particularly limited, and for example, Escherichia coli, yeast, etc. can be used. Separation and purification from the culture can be carried out by conventional purification methods. Acetylation of the N-terminus and amidation of the C-terminus of the peptide can be carried out by methods known in the art. The salts and solvates of the peptide can be readily prepared by those skilled in the art by any method known in the art.

[0022] Polynucleotides encoding the peptides of the present invention are also included in the present invention. In this specification, polynucleotide means DNA or RNA, preferably DNA. The base sequence of a polynucleotide encoding the peptide of the present invention can be designed, for example, by replacing the corresponding codons based on the amino acid sequence of the peptide.

[0023] As the polynucleotide encoding the peptide of the present invention, any of the following polynucleotides (a1) to (a3) ​​are preferred. (a1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 2 (a2) A polynucleotide encoding a peptide having a sucrose transporter expression-promoting effect, consisting of a nucleotide sequence in which 1 to 7 bases are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 2. (a3) A polynucleotide encoding a peptide that has a sequence identity of 90% or more with respect to the sequence of Sequence ID No. 2 and has an effect of promoting the expression of sucrose transporters.

[0024] The polynucleotide consisting of the base sequence shown in Sequence ID No. 2 (GCTACCACGGCGGCAATTAAGAAACCGCCGCGTCCGCCAATCAGCACCGGCCGTGGTGGTCAGATTCAC) is preferred as a polynucleotide that encodes a peptide consisting of the amino acid sequence shown in Sequence ID No. 1.

[0025] In (a2) above, the addition of a base also includes insertion into a sequence. In the polynucleotide in (a2) above, the deletion, substitution, or addition of 1 to 7 bases means that there is a deletion, substitution, or addition of 1 to 7 bases at any position in the sequence of 1 to 7 bases, and two or more types of deletion, substitution, and addition may occur simultaneously. In (a2) above, the number of deleted, substituted, or added bases is preferably 1 to 6, more preferably 1 to 5, even more preferably 1 to 4, even more preferably 1 to 3, particularly preferably 1 to 2, and most preferably 1.

[0026] The sequence identity in (a3) ​​above is 90% or more, preferably 91% or more or 92% or more, more preferably 93% or more or 94% or more, even more preferably 95% or more, 96% or more or 97% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0027] The polynucleotides of the present invention can be obtained by known genetic engineering or synthetic methods.

[0028] The polynucleotide of the present invention can be used, for example, in the production of peptides encoded by the polynucleotide of the present invention, in the production of expression vectors that express the peptide, in the production of transformants that express the peptide, and so on.

[0029] The method of using the polynucleotide of the present invention is not particularly limited, but for example, it is preferable to introduce it into a host. By introducing the polynucleotide of the present invention into a host, it is possible to produce, for example, a peptide that promotes the expression of sucrose transporters, or a transformant that expresses the peptide. The host is not particularly limited and can be a microorganism, etc. Preferably, the polynucleotide of the present invention is introduced into the host in an inserted state in an appropriate expression vector. The expression vector only needs to contain the polynucleotide so that the peptide encoded by the polynucleotide of the present invention can be expressed in the host into which it is introduced, and other components are not particularly limited. The host is not particularly limited and may be appropriately selected depending on the purpose of use of the expression vector. The method of introducing the expression vector into the host is not particularly limited and can be carried out by known methods, which can be appropriately selected depending on the type of host, the type of expression vector, etc.

[0030] The peptides of the present invention (peptides (A1) to (A4) above) are polypeptides that promote the expression of sucrose transporters. Sucrose transporters are proteins that transport sucrose and are present in the cell membranes of plants. Sucrose transporter 1 (SUT1) is an example of a sucrose transporter. The peptides of the present invention, their salts, or their solvates can be used to promote the expression of sucrose transporters in plants. The plant may be the whole plant or a part of the plant. Parts of a plant include leaves, stems, flowers, fruits, trunks, branches, seeds, roots, buds, etc.

[0031] In the present invention, the promotion of sucrose transporter expression includes the promotion of sucrose transporter mRNA expression and the promotion of protein expression. In the present invention, the promotion of sucrose transporter expression may be the promotion of expression in a part of a plant, and it is preferable that the promotion of sucrose transporter expression be in the plant body. In the present invention, if, when a certain substance is used on a plant, the expression of sucrose transporter is promoted to some extent (expression level increases) compared to when the substance is not used, then the substance can be said to have a sucrose transporter expression promoting effect (exhibit an expression promoting effect). The promotion of sucrose transporter expression can also be described as an increase in the expression level of sucrose transporter or an increase in sucrose transporter expression.

[0032] The peptides, salts thereof, or solvates thereof of the present invention can be preferably used in plants. When used in plants, the peptides, salts thereof, or solvates thereof of the present invention can promote the expression of sucrose transporters in the plants. The plants are not particularly limited, but at least one plant selected from the group consisting of Amaranthaceae, Euphorbia, Brassicaceae, Solanaceae, and Vitaceae plants is preferred. Examples of these plants are listed below. When used in any of these plants, it is preferable to promote the expression of sucrose transporters in the plants. Among these, Amaranthaceae and Solanaceae plants are more preferred, and sugar beet (Beta vulgaris ssp. vulgaris) of the Amaranthaceae family and tomato (Solanum lycopersicum) of the Solanaceae family are even more preferred. In one embodiment, the peptide of the present invention is preferably a peptide that promotes the expression of sucrose transporters in plants of the Amaranthaceae family, more preferably sugar beet (Beta vulgaris ssp. vulgaris) of the Amaranthaceae family. In another embodiment, the peptide of the present invention is preferably a peptide that promotes the expression of sucrose transporters in plants of the Solanaceae family, more preferably tomato (Solanum lycopersicum) of the Solanaceae family.

[0033] When the expression of sucrose transporters is promoted in plants, the translocation of sucrose to various organs of the plant is enhanced, and the uptake of sucrose into cells increases. As a result, the sucrose content in cells increases, leading to an increase in sugar content. Furthermore, the increase in sucrose content in cells confers, improves, or induces tolerance to environmental stresses, such as tolerance to osmotic stress (e.g., salt tolerance), drought tolerance, high temperature tolerance, and low temperature tolerance. Therefore, promoting the expression of sucrose transporters in plants can provide a plant protection effect against environmental stresses.

[0034] The peptides, salts thereof, or solvates of the present invention may be used individually or in combination of two or more. The peptides, salts thereof, or solvates of the present invention can be used, for example, for plant cultivation. Furthermore, the peptides, salts thereof, or solvates of the present invention can be used to increase the sugar content of plants or for plant protection. The peptides, salts thereof, or solvates of the present invention can be applied to plants for plant cultivation, to increase sugar content, or for plant protection. Methods for plant cultivation, methods for increasing plant sugar content, or methods for plant protection that involve applying the peptides, salts thereof, or solvates of the present invention to plants are also included in the present invention. The peptides, salts thereof, or solvates of the present invention can be used alone or in the form of compositions by mixing the peptides, salts thereof, or solvates with other components. In plant cultivation methods, the peptides, salts thereof, or solvates of the present invention may be applied directly to plants, or a composition containing the peptides, salts thereof, or solvates may be applied to plants. The peptides or salts thereof, or their solvates, of the present invention may be used in combination with other agricultural materials such as fertilizers, soil conditioners, and horticultural potting soils. The plant cultivation method, plant sugar content increase method, or plant protection method of the present invention can be applied to plants such as vegetables, fruit trees, flowers, and trees. In the plant cultivation method, plant sugar content increase method, and plant protection method, the plants can be cultivated using known methods appropriate to the plant, except for the application of the peptides or salts thereof, or their solvates, of the present invention.

[0035] Compositions comprising the peptide of the present invention, a salt thereof, or a solvate thereof are also included in the present invention. A composition of the present invention comprises any of the above-mentioned peptide of the present invention, a salt thereof, or a solvate thereof, or a mixture of two or more thereof. A composition of the present invention may contain the above-mentioned peptide of the present invention, a salt thereof, or a solvate thereof as an active ingredient.

[0036] The compositions of the present invention are preferably compositions for use in plants (plant compositions). For example, the compositions of the present invention can be used to promote the expression of sucrose transporters in plants. For example, the compositions of the present invention can be preferably used to increase the sugar content of plants or to protect plants. In one embodiment, the compositions of the present invention are preferably used as compositions for increasing the sugar content of plants or as compositions for protecting plants. Compositions for increasing the sugar content of plants can also be called sugar content increasing agents for plants. Compositions for protecting plants can also be called plant protective agents.

[0037] Plant protection includes improving plant defense mechanisms and enhancing plant adaptive responses. The peptides or salts thereof, or their solvates, and compositions of the present invention can be used, for example, to improve plant defense mechanisms or plant adaptive responses. Improving plant defense mechanisms includes improving resistance to diseases and pests. Improving plant adaptive responses includes improving adaptive responses to environmental stress. Plant protection also includes improving tolerance to abiotic stress and improving tolerance to biological stress. Examples of tolerance to abiotic stress include tolerance to environmental stress, such as tolerance to osmotic stress (e.g., salt tolerance), drought tolerance, high temperature tolerance, and low temperature tolerance. Examples of biological stress include pathogens and pests. In one embodiment, the peptides or salts thereof, or their solvates, and compositions of the present invention can be preferably used to improve tolerance to abiotic stress such as environmental stress. In one embodiment, the plant protection composition is preferably a biostimulant. Also in one embodiment, the peptides or salts thereof, or their solvates, and compositions of the present invention can be preferably used to increase the sugar content of plant parts, such as fruits or roots.

[0038] Other components in the composition besides the peptide of the present invention or its salt or solvate can be appropriately selected depending on the use and form of the composition. When the composition of the present invention is used on plants, components that can be used on plants can be included. The content of the peptide of the present invention or its salt or solvate in the composition is not particularly limited; for example, the total content of the peptide or its salt or solvate can be 0.0001 to 20% by weight, preferably 0.001 to 20% by weight, and more preferably 0.002 to 2.5% by weight.

[0039] The composition of the present invention can take various forms depending on the method of application to plants. The dosage form of the composition of the present invention is not limited, but it can be a solid (e.g., tablet, granule, powder) or a liquid. When applying to plants by spraying, a liquid or a dosage form that can be made liquid at the time of application is preferred. The composition of the present invention can also be used, for example, as a tablet, granule, powder, or concentrated liquid during distribution and storage, and then dissolved or suspended in water at the time of use to obtain an appropriate concentration.

[0040] When the composition of the present invention is a liquid preparation, the total content of the peptide or its salt or solvate is preferably 0.001 to 20% by weight, and more preferably 0.002 to 2.5% by weight, as the peptide content in the composition. When the composition of the present invention is a solid preparation, the total content of the peptide or its salt or solvate is preferably 0.0001 to 1% by weight, and more preferably 0.001 to 0.1% by weight, as the peptide content in the composition.

[0041] The composition of the present invention may contain other optional components, depending on the dosage form and shape, as long as they do not impair the effects of the present invention. Examples of other optional components include liquid carriers, spreading agents, emulsifiers, dispersants, fillers, bulking agents, binders, humicants, disintegrants, lubricants, diluents, excipients, amino acids, peptides (peptides different from those of the present invention), fertilizer elements, and components derived from natural products. Examples of liquid carriers include media capable of dissolving or dispersing the peptides or salts thereof of the present invention described above, or their solvates, such as water; alcohols such as 1-propanol and butanol; polyhydric alcohols such as ethylene glycol and propylene glycol; and hydrocarbons such as xylene.

[0042] The composition of the present invention may contain other active ingredients, as long as they do not impair the effects of the present invention. For example, it may contain known agents for plant diseases. The composition of the present invention may be used alone on plants, or it may be used in combination with other agricultural materials such as fertilizers, soil conditioners, and horticultural potting soils.

[0043] The method of applying the peptide or salt thereof, or its solvate, or the composition of the present invention to plants is not particularly limited and can be applied by general methods. Examples include spraying, coating, irrigation, adding to hydroponic solutions, and soil mixing. Among these, it is preferable to apply the peptide or salt thereof, or its solvate, or the composition of the present invention to plants by foliar application or irrigation. A suitable formulation for foliar application is a liquid formulation. If the target plant is cultivated in soil, it is also preferable to irrigate the soil. When applying by irrigation, the solution obtained by mixing the peptide or salt thereof, or its solvate, or the composition of the present invention with water can be irrigated onto the plants. The total content of the peptide or salt thereof, or its solvate, in the composition used for foliar application (preferably a liquid formulation) is preferably 0.001 to 20% by weight, and more preferably 0.002 to 2.5% by weight, as the peptide content. When applied to plants by irrigation, the total content of peptides, their salts, or their solvates in the irrigation solution is preferably 0.001 to 20% by weight, and more preferably 0.002 to 2.5% by weight, as the peptide content.

[0044] The timing of application of the peptide or salt thereof, or its solvate, or the composition of the present invention to plants is not particularly limited, but application from the seedling stage to before harvest is preferred. Furthermore, the frequency of application of the peptide or salt thereof, or its solvate, to plants is not particularly limited, but it is preferably applied once every 1 to 30 days, more preferably once every 7 to 14 days. The application amount of the peptide or salt thereof, or its solvate, can be appropriately set according to the plant and is not particularly limited. In one embodiment, the application amount of the peptide or salt thereof, or its solvate, is preferably 0.01 to 20 mg per plant, and more preferably 0.05 to 10 mg per application.

[0045] The plants to which the peptide or salt thereof, or solvate thereof, or composition containing the present invention is applied are not particularly limited, but examples include plants of the Amaranthaceae family, Euphorbia family, Brassicaceae family, Solanaceae family, and Vitaceae family. In the present invention, at least one plant selected from the group consisting of Amaranthaceae, Euphorbia, Brassicaceae, Solanaceae, and Vitaceae families is preferred as the plant, and Amaranthaceae or Solanaceae plants are more preferred.

[0046] Examples of plants in the Amaranthaceae family include sugar beet (Beta vulgaris ssp. vulgaris), Swiss chard (Beta vulgaris L.) of the genus Beta, and spinach (Spinacia oleracea L.) of the genus Spinach, with sugar beet being preferred. Examples of plants in the Euphorbiaceae family include poinsettia (Euphorbia pulcherrim), leopard plant (Euphorbia cyathophora), and snow-on-the-mountain (Euphorbia marginata) of the genus Euphorbia; cassava (Manihot esculenta) of the genus Manihot, rubber tree (Hevea brasiliensis) of the genus Hevea, castor bean (Ricinus communis) of the genus Ricinus, and Chinese tung tree (Aleurites cordata) of the genus Aleurites.

[0047] Examples of plants belonging to the Brassicaceae family include radishes (Raphanus sativus var. hortensis) of the genus Raphanus, cabbage (Brassica oleracea var. capitata), mustard greens (Brassica juncea), broccoli (Brassica oleracea var. italica), bok choy (Brassica rapa var. chinensis), mizuna (Brassica rapa var. nipposinica), and turnip (Brassica rapa var. rapa); Arabidopsis thaliana of the genus Arabidopsis, shepherd's purse (Capsella bursa-pastoris) of the genus Capsella, and wasabi (Eutrema japonicum) of the genus Eutrema.

[0048] Examples of plants in the Solanaceae family include eggplant (Solanum melongena), potato (Solanum tuberosum), and tomato (Solanum lycopersicum) of the Solanum genus; and bell pepper (Capsicum annuum var. grossum) and chili pepper (Capsicum annuum) of the Capsicum genus, with tomato (Solanum lycopersicum) being preferred. Examples of plants in the Vitaceae family include grapes (Vitis spp.).

[0049] In one embodiment, the peptide or salt thereof, or its solvate, or the composition of the present invention can be preferably used, for example, to improve the salt tolerance or increase the sugar content (preferably the sugar content of the roots) of sugar beet (Beta vulgaris ssp. vulgaris.) of the Amaranthaceae family. Also in one embodiment, the peptide or salt thereof, or its solvate, or the composition of the present invention can be used, for example, to improve the salt tolerance or increase the sugar content (preferably the sugar content of the fruit) of tomatoes.

[0050] This specification discloses the following: The disclosure (1) is any of the peptides (A1) to (A4) below, a salt thereof, or a solvate thereof. (A1) A peptide consisting of the amino acid sequence shown in Sequence ID No. 1 (A2) A peptide consisting of an amino acid sequence in which 1 to 2 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, and which has an effect of promoting the expression of sucrose transporters. (A3) A peptide consisting of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, and which has the effect of promoting the expression of sucrose transporters. (A4)(A1)~(A3) peptides in which the N-terminus is acetylated and / or the C-terminus is amidated.

[0051] The present disclosure (2) is a polynucleotide of any of the following (a1) to (a3). (a1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 2 (a2) A polynucleotide encoding a peptide having a sucrose transporter expression-promoting effect, consisting of a nucleotide sequence in which 1 to 7 bases are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 2. (a3) A polynucleotide encoding a peptide that has a sequence identity of 90% or more with respect to the sequence of Sequence ID No. 2 and has an effect of promoting the expression of sucrose transporters.

[0052] Disclosure (3) is a composition comprising the peptide or a salt thereof described in Disclosure (1), or a solvate thereof.

[0053] Disclosure (4) is the composition described in Disclosure (3), which is a composition for increasing the sugar content of plants or a composition for protecting plants.

[0054] Disclosure (5) is the composition described in Disclosure (4), wherein plant protection is the enhancement of the plant's defense mechanism or adaptive response.

[0055] Disclosure (6) is the composition according to Disclosure (4) or (5), wherein the plant protection composition is a biostimulant.

[0056] Disclosure (7) is a composition according to any one of Disclosures (3) to (6) that is applied to plants by foliar spraying or irrigation.

[0057] Disclosure (8) relates to a method for cultivating plants, comprising applying a polypeptide or salt thereof, or a solvate thereof, as described in Disclosure (1) to the plants.

[0058] The present disclosure (9) is a method for cultivating the plant described in the present disclosure (8), wherein the plant is at least one selected from the group consisting of plants of the Amaranthaceae, Euphorbia, Brassicaceae, Solanaceae, and Vitaceae families.

[0059] Disclosure (10) relates to the use of the peptides or salts thereof described in Disclosure (1), or solvates thereof, for increasing the sugar content of plants or for plant protection.

[0060] In this specification, a numerical range expressed by a lower limit and an upper limit, i.e., "lower limit to upper limit," includes those lower and upper limits. For example, a range expressed as "1 to 2" means 1 or more and 2 or less, including 1 and 2. In this specification, the upper and lower limits may be any combination of ranges. [Examples]

[0061] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0062] <Example 1> Search for plant elicitor peptides (PEPs) derived from sugar beet (Beta vulgaris subsp. vulgaris) PEP3 (AtPEP3) derived from Arabidopsis thaliana is known to play a role in salt tolerance (PNAS, 2018, 115(22), 5810-5815: Non-patent document 1 above). The amino acid sequence of AtPROPEP3, a precursor peptide of AtPEP3 (SEQ ID NO: 3, NCBI Reference Sequence: NP_569002.1), was subjected to BLAST (Protein BLAST: search protein databases using a protein query (nih.gov)), but no highly homologous amino acid sequences were found in sugar beet (Beta vulgaris subsp. vulgaris).

[0063] Based on Fig. 1A in J. Exp. Bot. 2015, 66(17), 5315-5325 (Reference 1), we used the PROPEP sequences arranged in order of close relatives from Arabidopsis thaliana in the phylogenetic tree and ran BLAST to search for amino acid sequences with high homology in sugar beet. As a result, we found that RcPROPEP5 (MKTEVEGIISSSQNEVTAKEKEAAVTTVNSNSYNYNIDRCFLIEVLLRCLGIETRTHQSCSSSSSSSSSEQNINGEVEEYGKEPSSTTELDPSTDPPLITEDIG) of castor bean (Ricinus communis) of the Euphorbiaceae family was found to be highly homologous. RVPARIKPPPKPPVSSGSGPQIN Searching using the sequence (Sequence No. 4), we found the following sequence A in sugar beet, which has a motif-like (underlined) amino acid sequence near the C-terminus and more than 70 amino acid residues. Sequence A (uncharacterized protein LOC104905694 isoform X1 [Beta vulgaris subsp. vulgaris], NCBI Reference Sequence: XP_010692606.1) MEMEVNEEEDEQLNFTTSPNTNNIYYNMFQGSYDFIEDVVRVILRCLGFEEENSSNSSQACCTSAAPAPVTTTTDAAAIGTTTTTPDLGGSDGGDADDSAEIADSGGQESSPDIGGGGGGDDDAGTVADPPGADTPSVGAQGRI ATTAAIKKPPRPPISTGRGGQIH (167 amino acid residues) (SEQ ID NO: 5)

[0064] The 23 amino acid residues near the C-terminus of sequence A (consisting of amino acids 145-167 of the amino acid sequence of sequence number 5: ATTAAIKKPPRPPISTGRGGQIH (sequence number 1)), which are considered to be the motif (underlined) of sequence A, were identified as the PEP sequence of sugar beet. When this amino acid sequence was compared with the motif of RcPROPPEP5 (consisting of amino acids 105-127 of the amino acid sequence of sequence number 4), the homology was 71%. The peptide consisting of the amino acid sequence of sequence number 1 may be referred to as BvPEP below.

[0065] <Example 2> BvPEP synthesis and verification A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 was obtained by contract synthesis (BEX Co., Ltd). The synthesized peptide was confirmed to be the peptide (BvPEP) consisting of the amino acid sequence shown in SEQ ID NO: 1 by reverse-phase HPLC. The reverse-phase HPLC conditions are shown below. A Waters Acquity PREMIER (Waters) was used. Column: C18 (Waters, product name BEH C18 1.7μm 2.1×50mm), Detection: UV: 220nm, Mobile phase: Solvent A: 0.1% TFA (trifluoroacetic acid) / H2O, Solvent B: 0.1% TFA / ACN (acetonitrile), Gradient: 0%~50% solvent B (0~30min), Flow rate: 1.0mL / min

[0066] Figure 1 shows the reverse-phase HPLC chromatogram of the synthesized peptide (BvPEP). The fraction with a retention time of 15.742 min was collected and mass spectrometry was performed under the following conditions. (Mass spectrometry) Equipment: Waters SYNAPT XS (manufactured by Waters) Ionization mode: ESI Positive Measurement range: m / z 100-2000 Data Independent Scan Mode Ion source temperature: 120℃ Mass spectrometry results showed an Observed MS reading of 2355.50, which closely matched the Calculated MS reading (2353.33), thus identifying the synthesized peptide as BvPEP. The results of mass spectrometry of the synthesized peptide (BvPEP) are shown in Figure 2.

[0067] <Example 3> BvPEP application and gene expression evaluation In Examples 3 and 4, the peptide (BvPEP) described in Example 2 was applied to plants, and its effect on the gene expression of sucrose transporter 1 (SUT1) was investigated. A peptide solution was prepared by mixing the peptide (BvPEP) with deionized water, and this solution was applied to the plants.

[0068] Preparation of sugar beet seedlings and application of peptides The soil used was a mixture of vermiculite (1L), potting soil (1L), and magnesium lime (0.3g), with an appropriate amount of water added and adjusted to a pH of 6.2-6.3. The mixed soil was shaken well and divided into paper envelopes. Holes 5-8cm deep were made, one sugar beet seed was placed in each, and the seeds were lightly covered with soil. The seedlings were then gently watered with a watering can. The sown sugar beets were cultivated in an artificial climate chamber (manufactured by Nippon Ika Kikai Seisakusho Co., Ltd., product name BiOTRON; the same applies to the following examples) (12 hours of light (7:00 AM - 7:00 PM); 21℃; humidity 60%, 12 hours of dark (7:00 PM - 7:00 AM); 16℃; humidity 60%). Seedlings with about four true leaves were selected, and 5 mL of water containing a peptide (BvPEP) at a predetermined concentration (2 μM (0.0046 wt%) or 10 μM (0.023 wt%)) was used to irrigate the base of each plant. Two days (45-48 hours) after irrigating with the peptide solution, the sugar beet leaves were punched out and collected.

[0069] <Comparative Example 1> In Example 3, instead of the peptide solution, deionized water (which did not contain the peptide) was used (water treatment). Otherwise, sugar beets were grown from seed in the same manner as in Example 3, and the sugar beet leaves were harvested by punching them out.

[0070] <Reference example 1> In Example 3, instead of the peptide solution, an aqueous sodium chloride (NaCl) solution with a concentration of 0.1 M or 0.3 M was used for irrigation. Otherwise, sugar beets were cultivated from seed in the same manner as in Example 3, and the sugar beet leaves were harvested by punching them out.

[0071] <Example 4> Preparation of tomato (variety: Regina) seedlings and application of peptides Kimwipes moistened with water were placed in a petri dish, and Regina tomatoes were sown on top of them and germinated in an artificial climate chamber. The sown tomatoes were cultivated in an artificial climate chamber (temperature: 20°C from 7:00 AM to 8:00 AM, 22.5°C from 8:00 AM to 9:00 AM, 25°C from 9:00 AM to 7:00 PM, 12 hours of light (7:00 AM to 7:00 PM); humidity 60%, 12 hours of dark (7:00 PM to 7:00 AM); 16°C; humidity 60%). After two weeks, the germinated seedlings were transplanted into Jiffy-7 pellets (product name, manufactured by Sakata Seed Corporation) and continued to be cultivated in the artificial climate chamber. Tomatoes 40 days old were watered at the base with 5 mL / plant of water containing 10 μM peptide (BvPEP) (peptide solution). Two days (45-48 hours) after irrigating with the peptide solution, tomato leaves were removed using a punch and collected. After collecting the leaves, cultivation was continued for another 30 days. No further application of the peptide solution was performed during the cultivation period after leaf collection.

[0072] <Comparative Example 2> In Example 4, instead of the peptide solution, deionized water (which did not contain the peptide) was used (water treatment). Otherwise, tomatoes (Regina) were grown from seed using the same method as in Example 4, and the leaves were collected by punching them out. After collecting the leaves, cultivation was continued for another 30 days.

[0073] <Reference example 2> In Example 4, a 0.15 M NaCl aqueous solution was used for irrigation instead of the peptide solution. Otherwise, tomatoes (Regina) were grown from seed using the same method as in Example 4, and the leaves were collected by punching them out. After collecting the leaves, cultivation was continued for another 30 days.

[0074] <Gene Expression Evaluation> The ability of peptide application to confer salt tolerance and other resistances was evaluated by measuring the gene expression level of sucrose transporter 1 (SUT1). Eppendorf tubes containing sugar beet or tomato leaves collected in Examples 3-4, Comparative Examples 1-2, and Reference Examples 1-2 were frozen with liquid nitrogen and thoroughly crushed with a pestle until the leaves became powdery. 20-100 mg of the powdered leaves were transferred to a 1.5 mL centrifuge tube, and RNA was extracted using the Maxwell® RSC Plant RNA kit from Promega. RNA concentration was determined by absorbance (A260).

[0075] For evaluating the gene expression level of SUT1, RNA-direct(registered trademark) SYBR TM We performed qRT-PCR using Green Realtime PCR Master Mix (manufactured by Toyobo Co., Ltd.) and a PCR system (QuantStudio® 3 real-time PCR system manufactured by Thermo Fisher Scientific). The Actin gene was used as the internal standard. Using the Ct values ​​obtained by qRT-PCR, gene expression levels were compared using the ΔΔCt method. The primer sequences used are as follows.

[0076] (Beet) BvSUT1_Fw: GGGATGCATTGTTTGGTGGG (SEQ ID NO: 6) BvSUT1_Rv: ATTGACCCTCCGATTTTGGCT (SEQ ID NO: 7) BvActin_Fw: TAAACCGAGATGGCTGATGC (SEQ ID NO: 8) BvActin_Rv: ATACTTGGGAAGACAGCCCT (SEQ ID NO: 9) (Tomato) SlSUT1_Fw: AACTCCCGGAGAAAGAAGAG (SEQ ID NO: 10) SlSUT1_Rv: TACAGTTTCGCATCACCGAC (SEQ ID NO: 11) SlActin_Fw: GGGATGGAGAAGTTTGGTGGTGG (SEQ ID NO: 12) SlActin_Rv: CTTCGACCAAGGGATGGTGTAGC (SEQ ID NO: 13)

[0077] <PCR reaction conditions> (Beet) Reacted at 95°C for 30 seconds and 61°C for 20 minutes, then carried out a reaction of 95°C for 10 seconds, 63°C for 10 seconds, and 68°C for 30 seconds for a total of 40 cycles. (Tomato) Reacted at 95°C for 30 seconds and 61°C for 20 minutes, then carried out a reaction of 95°C for 10 seconds, 63°C for 10 seconds, and 68°C for 30 seconds for a total of 40 cycles.

[0078] The results of Reference Example 1 are shown in Figure 3, and the results of Example 3 are shown in Figure 4. In Figures 3 and 4 and Figure 6 described below, the results are shown as mean ± standard error (n = 4). Figure 3 is a graph showing the results of examining the expression of SUT1 by giving sugar beet water containing NaCl. Figure 4 is a graph showing the results of examining the expression of SUT1 in sugar beet treated with BvPEP. The expression levels of SUT1 in Figures 3 and 4 are shown as relative values with the expression level of SUT1 in sugar beet of Comparative Example 1 (water treatment) set as 1.

[0079] As shown in Figure 3, when sugar beets were exposed to 0.3 M NaCl, the sucrose concentration in the plant increased to counteract osmotic stress, and the expression of sucrose transporters improved. Similarly, with BvPEP application, an increase of approximately 40% was observed with a 10 μM treatment compared to water treatment (Comparative Example 1) (Figure 4). Figure 5 shows photographs of sugar beet seedlings (Example 3) and water-treated sugar beet seedlings (Comparative Example 1) two days after application of BvPEP (2 μM or 10 μM). No difference in growth was observed between the sugar beets treated with BvPEP and those treated with water, and no plant damage to sugar beets by BvPEP was confirmed.

[0080] The results of Reference Example 2 and Example 4 are shown in Figure 6. Figure 6 is a graph showing the results of examining the expression of SUT1 in tomatoes treated with BvPEP (10 μM). The expression levels of SUT1 in Figure 6 are shown as relative values, with the expression level of SUT1 in tomatoes in Comparative Example 2 (water treatment) set to 1. In tomatoes as well, SUT1 expression increased after BvPEP application. Figure 7 is a photograph of tomato seedlings 30 days after application of BvPEP (10 μM) (Example 4) and tomato seedlings treated with water (Comparative Example 2) or 0.15 M NaCl (Reference Example 2). No difference in growth was observed between tomatoes treated with BvPEP and those treated with water, and no plant damage to tomatoes by BvPEP was confirmed. [Industrial applicability]

[0081] This invention is useful in the fields of agriculture, horticulture, and the like.

Claims

1. A peptide or salt thereof, or a solvate thereof, from any of the following (A1) to (A4). (A1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (A2) A peptide having an amino acid sequence in which one or two amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, and which has an effect of promoting the expression of sucrose transporters. (A3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1, and which has the effect of promoting the expression of sucrose transporters. (A4) A peptide in which any of (A1) to (A3) has an acetylated N-terminus and / or an amidated C-terminus.

2. One of the following polynucleotides (a1) to (a3). (a1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 2 (a2) A polynucleotide comprising a nucleotide sequence in which 1 to 7 bases are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 2, and which encodes a peptide that promotes the expression of sucrose transporters. (a3) A polynucleotide encoding a peptide that has a sequence identity of 90% or more with respect to the sequence of Sequence ID No. 2 and has an effect of promoting the expression of sucrose transporters.

3. A composition comprising the peptide or a salt thereof, or a solvate thereof, as described in claim 1.

4. The composition according to claim 3, which is a composition for increasing the sugar content of plants or a composition for protecting plants.

5. The composition according to claim 4, wherein plant protection is the improvement of the plant's defense mechanism or adaptive response.

6. The composition according to claim 4 or 5, wherein the plant protection composition is a biostimulant.

7. The composition according to any one of claims 3 to 5, which is applied to plants by foliar spraying or irrigation.

8. A method for cultivating plants, comprising applying the polypeptide described in claim 1, a salt thereof, or a solvate thereof to the plants.

9. The method for cultivating a plant according to claim 8, wherein the plant is at least one selected from the group consisting of plants of the Amaranthaceae family, Euphorbia family, Brassicaceae family, Solanaceae family, and Vitaceae family.

10. Use of the peptide or salt thereof, or solvate thereof, according to claim 1, for increasing the sugar content of plants or for plant protection.

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