Fusion peptides, the polynucleotides encoding them, and their applications

Abstract

To provide a fusion peptide that promotes the expression of sucrose transporters and is suitable for production by microorganisms, a polynucleotide encoding the same, and applications thereof. [Solution] A fusion peptide or a salt thereof, or a solvate thereof, comprising a tag peptide and one of the peptides (A1) to (A3) below, from the amino terminus to the carboxyl terminus. (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.

Description

Technical Field

[0001] The present invention relates to a fusion peptide or a salt thereof or a solvate thereof, and a polynucleotide encoding the fusion peptide. The present invention also relates to a composition containing the fusion peptide or a salt thereof or a solvate thereof, a method for cultivating plants, and the like.

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

[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 induces a plant's defense response. AtPEP3, a PEP derived from Arabidopsis thaliana, has been reported to play a role in the salt tolerance of plants (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 sugars 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] One technique for industrially producing functional peptides involves introducing polynucleotides encoding the target peptide into microorganisms and allowing the microorganisms to produce the peptide. However, depending on the peptide to be expressed, microbial production can be difficult.

[0007] The present invention aims to provide a fusion peptide that promotes the expression of sucrose transporters and is suitable for production by microorganisms, a polynucleotide encoding the same, and applications thereof. [Means for solving the problem]

[0008] The present inventors have diligently studied to solve the above problems and have arrived at the present invention. Specifically, the present invention comprises a fusion peptide or a salt thereof, or a solvate thereof, comprising a tag peptide and one of the peptides (A1) to (A3) below, from the amino terminus to the carboxyl terminus; a polynucleotide comprising a polynucleotide encoding the tag peptide and one of the polynucleotides (a1) to (a3) ​​below, from the 5' terminus to the 3' terminus; an expression vector comprising the above polynucleotide; a transformant, which is a microorganism into which the above polynucleotide or the above expression vector has been introduced; a method for producing the fusion peptide, comprising the step of culturing the above transformant; a composition comprising the above fusion peptide or a salt thereof, or a solvate thereof; a method for cultivating plants by applying the above fusion peptide or a salt thereof, or a solvate thereof, to plants; and the use of the above fusion peptide or a salt thereof, or a solvate thereof, 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. (a1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 4 (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. 4. (a3) A polynucleotide encoding a peptide that has a sequence identity of 90% or more with respect to the sequence of Sequence ID No. 4 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 fusion peptide that has the effect of promoting the expression of sucrose transporters and is suitable for production by microorganisms, a polynucleotide encoding the same, and applications thereof. [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 3A shows the results of confirming the expression of the fusion peptide (Fh8-BvPEP) using SDS-PAGE. Figure 3B shows the results of confirming the expression of the fusion peptide (P17-BvPEP) using SDS-PAGE. [Figure 4] Figure 4 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 5] Figure 5 is a graph showing the results of examining SUT1 expression in sugar beets treated with the fusion peptide (Fh8-BvPEP), the fusion peptide (P17-BvPEP), or BvPEP. [Figure 6] Figure 6 is a graph showing the results of examining SUT1 expression in tomatoes treated with the fusion peptide (Fh8-BvPEP), the fusion peptide (P17-BvPEP), or BvPEP. [Figure 7] Figures 7A, 7B, 7C, 7D, and 7E are photographs of tomato seedlings treated with water, BvPEP, fusion peptide (P17-BvPEP), fusion peptide (Fh8-BvPEP), or 0.15M NaCl (Figure 7A: water, Figure 7B: BvPEP, Figure 7C: fusion peptide (P17-BvPEP), Figure 7D: fusion peptide (Fh8-BvPEP), Figure 7E: 0.15M NaCl). [Modes for carrying out the invention]

[0011] The fusion peptide of the present invention contains a tag peptide and a peptide of any one of the following (A1) to (A3) from the amino terminus towards the carboxy terminus. (A1) A peptide consisting of the amino acid sequence shown in SEQ 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 SEQ ID NO: 1 and having an action of promoting the expression of sucrose transporter (A3) A peptide consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having an action of promoting the expression of sucrose transporter The fusion peptide of the present invention is a polypeptide and can also be described as a fusion protein.

[0012] In this specification, unless otherwise specified, the amino acid sequence of a peptide is represented by 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 amino terminus (N-terminus) and the right end is the carboxy terminus (C-terminus). In the present invention, when an amino acid can have an optical isomer, the L-form amino acid is shown unless otherwise specified.

[0014] In this specification, the addition of an amino acid also includes the meaning of insertion into a sequence. In the present invention, when one or more amino acids are deleted, substituted or added in an amino acid sequence, it means that there is a deletion, substitution or addition of one or more amino acids at an arbitrary position in one or more amino acid sequences in the same sequence, and two or more of deletion, substitution and addition may occur simultaneously. For example, when one or two amino acids are deleted, substituted or added in an amino acid sequence, it means that there is a deletion, substitution or addition of one or two amino acids at an arbitrary position in one or two amino acid sequences in the same sequence, and two or more of deletion, substitution and addition may occur simultaneously.

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

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

[0017] The identity of an amino acid sequence or a base sequence can be calculated by using analysis software such as BLAST with default parameters.

[0018] The peptides (A1) to (A3) described above are peptides that promote the expression of sucrose transporters. The peptides (A1) to (A3) are polypeptides and can also be described as proteins. 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. When the peptides (A1) to (A3) described above are used in plants, they can promote the expression of sucrose transporters in those plants. Among the peptides (A1) to (A3) described above, peptide (A1) is preferred.

[0019] 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, 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 expressed as an increase in the expression level of sucrose transporter or an increase in sucrose transporter expression. 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.

[0020] The fusion peptide of the present invention contains a tag peptide at the N-terminus of any of the peptides (A1) to (A3) above. The tag peptide can also be described as a tag protein. The tag peptide used in the fusion peptide of the present invention is a tag peptide that can be used in the production of recombinant peptides by microorganisms such as E. coli, and when placed at the N-terminus of the peptides (A1) to (A3) above, the fusion protein containing the tag peptide and any of the peptides (A1) to (A3) above exhibits a sucrose transporter expression promoting effect. In the present invention, a tag peptide consisting of 20 to 100 amino acids (polypeptide tag) is preferred as the tag peptide, and a tag peptide consisting of 30 to 80 amino acids is more preferred. In the present invention, a soluble tag peptide is preferred as the tag peptide. A soluble tag peptide is a peptide that has the effect of improving the solubility of the fusion peptide containing the tag peptide. A fusion peptide containing a soluble tag peptide at the N-terminus of the peptides (A1) to (A3) above is easily expressed in a soluble form (soluble peptide) in microorganisms. Preferred soluble tag peptides are those that improve the in-host expression stability of the fusion peptide containing the peptide in question. Examples of soluble tag peptides include peptides encoded by the Fh8 gene of the liver fluke (Fasciola hepatica) (Fh8 tag peptide), P17 tag (P17 tag peptide), Thioredoxin (Trx tag peptide), Maltose binding protein (MBP tag peptide), Small ubiquitin related modifier (SUMO tag peptide), Glutathione S-transferase (GST tag peptide), Nutrition substance A (Nus tag peptide), etc.

[0021] In the present invention, the following peptides (B1) to (B3) and (C1) to (C3) are preferred as tag peptides. (B1) A peptide consisting of the amino acid sequence shown in Sequence ID No. 2 (B2) A peptide consisting of an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 2. (B3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 2. (C1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (C2) A peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 3. (C3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3.

[0022] The peptide (B1) described above is an Fh8-tagged peptide. The peptide (C1) described above is a P17-tagged peptide. It is preferable for the fusion protein to contain (B1) or (C1) because it increases the solubility of the fusion protein. Furthermore, it is preferable for the fusion peptide containing (B1) or (C1) to exhibit good expression stability in E. coli. In addition, when the fusion protein contains the peptide (B1) or (C1), the extracellular secretion of the fusion peptide is promoted when the fusion protein is expressed in microorganisms such as E. coli. This also has the advantage of making purification easier when producing the fusion protein using microorganisms.

[0023] The peptides (B2), (B3), (C2), and (C3) described above may include peptides that improve the solubility of the fusion peptide containing the peptide. The peptides (B2), (B3), (C2), and (C3) are preferably those that improve the expression stability of the fusion peptide containing the peptide in microorganisms (preferably Escherichia coli). The peptides (B2), (B3), (C2), and (C3) are preferably those that promote the extracellular secretion of the fusion peptide when the fusion peptide containing the peptide is expressed, for example, in Escherichia coli.

[0024] In the peptide (B2) described above, the number of deleted, substituted, or added amino acids is preferably 1 to 5, more preferably 1 to 4, even more preferably 1 to 3, particularly preferably 1 to 2, and most preferably 1. In the peptide (C2) described above, the number of deleted, substituted, or added amino acids is preferably 1 to 2, more preferably 1.

[0025] The sequence identity of the peptides (B3) and (C3) described 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.

[0026] In the present invention, the peptide (B1) (Fh8 tag peptide) and the peptide (C1) (P17 tag) are more preferred as tag peptides. The peptides (B1) and (C1) are also preferred from the viewpoint that they do not interfere with the sucrose transporter expression promoting effect of the peptides (A1) to (A3).

[0027] In the fusion peptide of the present invention, the tag peptide and any of the peptides (A1) to (A3) above are arranged in this order from the N-terminus to the C-terminus. The fusion peptide of the present invention has the effect of promoting the expression of sucrose transporters by containing any of the peptides (A1) to (A3). From the viewpoint of obtaining the sucrose transporter expression promoting effect by the peptides (A1) to (A3) above more sufficiently, it is preferable that the peptides (A1) to (A3) above be located at the C-terminus of the fusion peptide. It is preferable that the fusion peptide of the present invention has any of the peptides (A1) to (A3) above at its C-terminus. Furthermore, it is preferable that the fusion peptide of the present invention has the tag peptide at its N-terminus.

[0028] In the fusion peptide of the present invention, the tag peptide and any of the peptides (A1) to (A3) above may be directly linked by an amide bond. Alternatively, the tag peptide and any of the peptides (A1) to (A3) above may be linked via a peptide consisting of one amino acid or 2 to 20 amino acids (peptide linker). Known peptide linkers can be used. A peptide consisting of 3 to 15 amino acids is preferred as the peptide linker, and a peptide consisting of 5 to 10 amino acids is preferred. The fusion peptide of the present invention may have an acetylated N-terminus or an amidated C-terminus. In one embodiment, the fusion peptide of the present invention is preferably a peptide in which the C-terminal amino acid of the tag peptide and the N-terminal amino acid of any of the peptides (A1) to (A3) above are (directly) linked by an amide bond.

[0029] In one embodiment, the fusion peptide of the present invention is preferably composed of a tag peptide and any of the peptides (A1) to (A3) above. The fusion peptide composed of a tag peptide and any of the peptides (A1) to (A3) above has the tag peptide at its N-terminus and any of the peptides (A1) to (A3) above at its C-terminus.

[0030] In one embodiment, the fusion peptide of the present invention is preferably a peptide consisting of the amino acid sequence shown in SEQ ID NO: 7 and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence of SEQ ID NO: 7 is the amino acid sequence of a fusion peptide consisting of the peptide (B1) and the peptide (A1). The amino acid sequence of SEQ ID NO: 8 is the amino acid sequence of a fusion peptide consisting of the peptide (C1) and the peptide (A1).

[0031] The fusion peptide of the present invention may also be in the form of a salt. The salt of the peptide in the present invention is not particularly limited and may be either an acidic salt or a basic salt. 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 salt; and salts with organic bases such as triethylamine, triethanolamine, and pyridine. These salts are usable in plants and can be used as salts of the fusion peptide in the present invention. Among these, hydrochloride, formate, acetate, phosphate, citrate, lactate, aspartate, glutamate, sodium salt, and potassium salt are preferred as salts of the fusion peptide, and hydrochloride, formate, and acetate are more preferred.

[0032] The fusion peptide of the present invention or a salt thereof 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.

[0033] The fusion peptide of the present invention can be biosynthesized using microorganisms. When using microorganisms, an expression vector into which the gene encoding the fusion peptide of the present invention, as described later, 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 fusion peptide of the present invention can be purified from the culture. Alternatively, the fusion peptide of the present invention can also be produced by known peptide synthesis methods, for example. When obtaining the fusion peptide of the present invention by peptide synthesis, it can be synthesized by either a solid-phase or liquid-phase method. The peptide can be purified by conventional purification methods such as reverse-phase high-performance liquid chromatography or affinity chromatography. Salts and solvates of the fusion peptide can be readily prepared by those skilled in the art by any method known in the art. The fusion peptide of the present invention, its salts, and their solvates are preferably used in plants, as described later.

[0034] Polynucleotides encoding the fusion 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.

[0035] The polynucleotide encoding the fusion peptide of the present invention comprises, from the 5' end to the 3' end, a polynucleotide encoding the tag peptide and a polynucleotide encoding any of the peptides (A1) to (A3) above. Preferably, the polynucleotide encoding the fusion peptide of the present invention comprises, from the 5' end to the 3' end, a polynucleotide encoding the tag peptide and a polynucleotide of any of the following (a1) to (a3). (a1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 4 (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. 4. (a3) A polynucleotide encoding a peptide that has a sequence identity of 90% or more with respect to the sequence of Sequence ID No. 4 and has an effect of promoting the expression of sucrose transporters.

[0036] A polynucleotide comprising, from the 5' end to the 3' end, a polynucleotide encoding the tag peptide and one of the polynucleotides (a1) to (a3) ​​above in this order is preferred as the polynucleotide encoding the fusion peptide of the present invention described above. Among the polynucleotides (a1) to (a3) ​​above, polynucleotide (a1) is preferred.

[0037] The polynucleotide consisting of the base sequence shown in Sequence ID No. 4 (GCTACCACGGCGGCAATTAAGAAACCGCCGCGTCCGCCAATCAGCACCGGCCGTGGTGGTCAGATTCAC) is preferred as a polynucleotide that encodes a peptide consisting of the amino acid sequence shown in Sequence ID No. 1.

[0038] In the present invention, the addition of a base also includes the meaning of insertion into a sequence. In a polynucleotide, the deletion, substitution, or addition of one or more bases means that there is a deletion, substitution, or addition of one or more bases at any position in one or more base sequences within the same sequence, and two or more types of deletion, substitution, and addition may occur simultaneously. For example, in a polynucleotide, 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 one to 7 base sequences within the same sequence, 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.

[0039] 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. Examples of the polynucleotides in (a2) or (a3) ​​above include the polynucleotide consisting of the base sequence shown in SEQ ID NO: 14 (GCTACCACCGCGGCAATTAAAAAACCGCCACGTCCGCCGATCAGCACCGGTCGTGGTGGCCAAATCCAC). The polynucleotide of the base sequence in SEQ ID NO: 14 is the polynucleotide that encodes the peptide shown in SEQ ID NO: 1.

[0040] As the polynucleotide encoding the tag peptide, a polynucleotide encoding any of the peptides (B1) to (B3) and (C1) to (C3) above is preferred. In one embodiment, as the polynucleotide encoding the tag peptide, any of the polynucleotides (b1) to (b3) and (c1) to (c3) below is preferred. (b1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 5 (b2) A polynucleotide consisting of a nucleotide sequence in which 1 to 20 bases are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 5. (b3) Polynucleotides consisting of a base sequence having 90% or more sequence identity with the base sequence of Sequence ID No. 5 (c1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 6 (c2) A polynucleotide consisting of a nucleotide sequence in which 1 to 9 bases are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 6. (c3) A polynucleotide consisting of a nucleotide sequence that has more than 90% sequence identity with the nucleotide sequence of Sequence ID No. 6.

[0041] A polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 is preferred as a polynucleotide encoding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2. A polynucleotide consisting of the base sequence shown in SEQ ID NO: 6 is preferred as a polynucleotide encoding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3.

[0042] The polynucleotides (b2), (b3), (c2), and (c3) described above are preferably polynucleotides encoding a soluble tag peptide, and more preferably polynucleotides encoding a peptide that improves the solubility and extracellular secretion of the fusion peptide containing it. The polynucleotides (b2), (b3), (c2), and (c3) are also preferably polynucleotides encoding a peptide that improves the expression stability of the fusion peptide containing it in microorganisms (preferably Escherichia coli). In (b2) above, the number of deleted, substituted, or added bases is preferably 1 to 15, more preferably 1 to 10, 1 to 9, 1 to 8, or 1 to 7, even more preferably 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, particularly preferably 1 to 2, and most preferably 1. In (c2) above, the number of deleted, substituted, or added bases is preferably 1 to 8, more preferably 1 to 7, even more preferably 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, particularly preferably 1 to 2, and most preferably 1.

[0043] The sequence identity in (b3) and (C3) 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.

[0044] In one embodiment, the polynucleotide of the present invention is preferably a polynucleotide containing the polynucleotide of (b1) and the polynucleotide of (a1) from the 5' end to the 3' end, and a polynucleotide containing the polynucleotide of (c1) and the polynucleotide of (a1) from the 5' end to the 3' end.

[0045] In one embodiment, the polynucleotide of the present invention is preferably a polynucleotide comprising a polynucleotide encoding a tag peptide and any of the polynucleotides (a1) to (a3) ​​above, and more preferably a polynucleotide comprising the base sequence shown in SEQ ID NO: 12 and a polynucleotide comprising the base sequence shown in SEQ ID NO: 13. The polynucleotide comprising the base sequence shown in SEQ ID NO: 12 is a polynucleotide comprising the polynucleotide of (b1) and the polynucleotide of (a1). The polynucleotide shown in SEQ ID NO: 13 is a polynucleotide comprising the polynucleotide of (c1) and the polynucleotide comprising the base sequence shown in SEQ ID NO: 14.

[0046] The polynucleotides of the present invention can be obtained by known genetic engineering or synthetic methods.

[0047] The polynucleotides of the present invention can be used, for example, in the production of fusion peptides encoded by the polynucleotides of the present invention, in the production of expression vectors that express the peptides, and in the production of transformants that express the peptides. Preferably, the polynucleotides of the present invention are introduced into a host in an appropriate expression vector. For example, it is preferable to introduce the polynucleotides into the host using an expression vector of the present invention as described later.

[0048] The present invention also includes expression vectors containing the polynucleotides of the present invention described above. In the expression vector of the present invention, one of the polynucleotides (a1) to (a3) ​​above is positioned at the 3' end (downstream) of the polynucleotide encoding the tag peptide. The polynucleotides (a1) to (a3) ​​above, the polynucleotides encoding tag peptides, and preferred embodiments thereof are as described above. The polynucleotides (b1) to (b3) and (c1) to (c3) above are preferred as polynucleotides encoding tag peptides.

[0049] Using the expression vector of the present invention, for example, the polynucleotide of the present invention can be easily introduced into a host. Furthermore, the expression of the polynucleotide of the present invention in the host can be easily controlled. The expression vector of the present invention only needs to contain the polynucleotide so that the fusion 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, but microorganisms are preferred. Examples of microorganisms include bacteria of the Escherichia genus such as Escherichia coli; yeasts of the Saccharomyces, Schizosaccharomyces, Pichia genus, molds, filamentous fungi, etc. Among these, bacteria or fungi are preferred as host microorganisms, bacteria are more preferred, and Escherichia coli is even more preferred.

[0050] The expression vector of the present invention can be prepared, for example, by inserting the polynucleotide of the present invention into a skeletal vector (hereinafter also referred to as the "basic vector"). The type of vector is not particularly limited and can be appropriately selected, for example, depending on the type of host to be introduced. When performing transformation in bacteria such as E. coli, for example, the basic vector can be the pET vector, pCold TM Examples of vectors include Takara Bio Inc., pQE vector (QIAGEN), pMW vector (Nippon Gene Inc.), and pACYC vector (Nippon Gene Inc.). When performing transformation on fungi such as yeast, examples of vectors include pYE22m.

[0051] The expression vector of the present invention preferably has a regulatory sequence that regulates the expression of the polynucleotide described above. Examples of regulatory sequences include promoters, terminators, enhancers, polyadenylation signal sequences, and origin of replication (ori) sequences. For example, as a promoter for bacterial expression vectors, the following can be used: araBAD promoter, lac promoter, lacUV5 promoter, phoA promoter, pL promoter, pR promoter, rhaBAD promoter, Sp6 promoter, T3 promoter, T5 promoter, T7 promoter, T7lac promoter, tac promoter, tet promoter, trc promoter, trp promoter, etc. Among these, the T7 promoter is preferred. Examples of yeast promoters include the GAL promoter, CTR promoter, CUP1 promoter, CYC1 promoter, MET25 promoter, and GPD promoter. Examples of filamentous fungal promoters include the trpC promoter.

[0052] In the expression vector of the present invention, the arrangement of the regulatory sequence is not particularly limited. The regulatory sequence only needs to be arranged so as to functionally regulate the expression of the polynucleotide of the present invention, and can be arranged according to known methods. Furthermore, the regulatory sequence may be appropriately selected depending on the type of host to be introduced. The regulatory sequence may be, for example, a sequence already present in the basic vector, or an regulatory sequence may be inserted into the basic vector. The regulatory sequence present in the basic vector may also be replaced with another regulatory sequence.

[0053] The expression vector of the present invention may further have, for example, one or more coding sequences for selection markers. Examples of selection markers include drug resistance markers, fluorescent protein markers, enzyme markers, and the like.

[0054] The method for introducing the expression vector of the present invention into a host is not particularly limited and can be carried out by known methods. The method for introducing the expression vector into a host can be appropriately determined depending on, for example, the type of host, the type of expression vector, etc.

[0055] The transformant of the present invention is a microorganism into which the polynucleotide of the present invention or the expression vector of the present invention has been introduced. Hereinafter, "introduction of the polynucleotide of the present invention" also means "introduction of the expression vector of the present invention" unless otherwise specified.

[0056] The transformant of the present invention may be any microorganism into which the polynucleotide of the present invention has been introduced in an expressible manner, and its other components are not particularly limited. "Polynucleotide expressible" means that the fusion peptide encoded by the polynucleotide is expressible. The above microorganism is preferably a bacterium or fungus as described above, more preferably a bacterium, and even more preferably Escherichia coli.

[0057] The method for introducing the above-mentioned polynucleotide or expression vector into a host microorganism is not particularly limited, and known transformation methods can be used. Examples of introduction methods include the calcium phosphate method, the heat shock method using chemical compicellus, the polyethylene glycol method, the lipofection method, the electroporation method, the ultrasonic nucleic acid introduction method, the DEAE-dextran method, and methods using introduction aids. The introduction method should be appropriately selected depending on the type of host, etc.

[0058] Transformants obtained by introducing the polynucleotide or expression vector of the present invention into a microorganism may be cultured. The transformants of the present invention also include microorganisms obtained by further culturing them after introducing the polynucleotide or expression vector of the present invention into them.

[0059] Whether or not the polynucleotide of the present invention has been introduced into a host can be confirmed by PCR, Southern hybridization, Northern hybridization, etc. For example, an expression vector into which the polynucleotide of the present invention has been introduced can be extracted from a transformant, and a primer specific to the polynucleotide of the present invention can be designed and PCR can be performed. The amplification product obtained by PCR can be subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, stained with a nucleic acid staining reagent such as ethidium bromide, and the amplification product can be detected as a single band to confirm that transformation has occurred. Alternatively, the amplification product can be detected by performing PCR using primers that have been previously labeled with a fluorescent dye or the like.

[0060] In the transformant of the present invention, it is preferable that the fusion peptide described above is expressed by the introduced polynucleotide of the present invention. Furthermore, in the transformant of the present invention, the fusion peptide encoded by the introduced polynucleotide of the present invention may be expressed by further culturing, for example.

[0061] As described above, the transformant of the present invention can express a fusion peptide having the sucrose transporter expression-promoting effect described above by the expression of the polynucleotide of the present invention. For this reason, the transformant of the present invention can be used in the production of a fusion peptide having the sucrose transporter expression-promoting effect.

[0062] A method for producing a fusion peptide, which includes the step of culturing the transformant described above, is also part of the present invention. By culturing the transformant described above in a culture medium, the polynucleotide of the present invention is expressed and the fusion peptide of the present invention is produced. Liquid culture medium is usually used as the culture medium.

[0063] The culture medium used should be selected according to the type of microorganism, but typically a medium containing a carbon source and a nitrogen source is used. Examples of commercially available media that can be used in this invention include, but are not limited to, Luria bethani (LB) broth, Sabouraud dextrose (SD) broth, and yeast medium (YM) broth. Synthetic media with a clearly defined composition can also be used, or they can be used with components adjusted to promote the expression of the target fusion peptide.

[0064] The carbon source used in the culture medium is not particularly limited, as long as it is a carbon source that the transformants can utilize. Examples of carbon sources include monosaccharides (glucose, fructose, galactose, etc.), oligosaccharides (sucrose, lactose, etc.), and polysaccharides (starch, cellulose, etc.). One carbon source may be used, or a combination of two or more may be used.

[0065] The nitrogen source is not particularly limited and can be any nitrogen source that microorganisms can utilize. Examples of nitrogen sources include inorganic nitrogen sources (urea, nitrates, ammonium salts, etc.) and organic nitrogen sources (peanuts, fish meal, peptone, etc.). One nitrogen source may be used, or a combination of two or more may be used.

[0066] The carbon source in the culture medium is preferably at a concentration of 0.02 to 5% by weight at the start of cultivation, and more preferably at 0.1 to 2% by weight. The nitrogen source in the culture medium is preferably at a concentration of 0.01 to 5% by weight at the start of cultivation, and more preferably at 0.1 to 2% by weight.

[0067] The liquid culture medium may contain components other than the carbon and nitrogen sources mentioned above. For example, to promote the expression of fusion peptides, it is preferable that the medium contains appropriate minerals, vitamins, salts, cofactors, and buffers (e.g., dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium citrate). Antibiotics known to modulate catabolite inhibition (e.g., kanamycin, ampicillin, carbenicillin, chloramphenicol) may also be incorporated into the medium.

[0068] The pH of the culture medium is preferably 5.0 to 9.0, and more preferably 6.0 to 9.0. In this specification, pH refers to the pH at 25°C. Known acids or alkalis can be used to adjust the pH. Examples include hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, butyric acid, lactic acid, succinic acid, maleic acid, malic acid, oxalic acid, citric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, and aqueous ammonia.

[0069] Culturing can be carried out under aerobic or anaerobic conditions, but aerobic conditions are preferred. Culturing under aerobic conditions can be carried out by, for example, shaking culture or stirring culture of the culture medium containing the transformants. Dissolved oxygen can be maintained by increasing the stirring or by introducing air into the bioreactor.

[0070] The culture temperature should be set to a temperature suitable for the type of microorganism. For example, for E. coli, 20-50°C is preferred, and 25-40°C is more preferred.

[0071] In culturing transformants, the expression of the target fusion peptide may be induced once the transformants have grown to a certain extent. For example, in the case of E. coli transformants, isopropyl-β-thiogalactopyranoside (IPTG) may be added to promote the production of the fusion peptide. The cell density, IPTG concentration, pH, temperature, and airflow rate during expression induction can be adjusted to obtain favorable production conditions for the target peptide.

[0072] The culture time is not particularly limited and can be set as appropriate, but for example, aerobic culture is preferably performed for 20 to 48 hours, and more preferably for 20 to 28 hours. During the culture, the fusion peptide accumulates in the culture material.

[0073] In the method for producing fusion peptides of the present invention, a step of purifying the fusion peptide from a culture in which the fusion peptide has accumulated may be performed. By purifying the fusion peptide from a culture in which the transformant has been cultured, the desired fusion peptide can be obtained. The purification of the fusion peptide can be carried out according to the usual methods for purifying peptides or proteins. The degree of purification of the fusion peptide is not particularly limited as long as it has the effect of promoting the expression of sucrose transporters. The above-mentioned culture or crude purified fusion peptide can also be used as long as the effects of the present invention are achieved. The term "culture" refers to any of the following: culture medium, cultured cells, or lysates of cultured cells.

[0074] In the production method of the present invention, it is preferable that the fusion peptide accumulates in the culture medium. For example, if the fusion peptide includes, as a tag peptide, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, the fusion peptide is usually accumulated in the culture medium. After the completion of cultivation, the culture supernatant containing the target fusion peptide can be obtained by separating the transformed bacterial cells and the culture supernatant by a conventional method (e.g., centrifugation, filtration, etc.). The production of the fusion peptide can be confirmed by a method such as SDS-PAGE. In addition to the culture supernatant, the fusion peptide may also be contained within the cultured bacterial cells. If the fusion peptide accumulates within the cultured bacterial cells, after cultivation, the bacterial cells can be disrupted by a known method (e.g., ultrasound, lysozyme, freeze-thaw cycle, etc.), and then a bacterial cell extract containing the fusion peptide can be obtained by centrifugation, filtration, etc. For separation or purification, methods such as dialysis, ultrafiltration, ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and reverse-phase high-performance liquid chromatography can be used individually or in appropriate combinations. Salts and solvates of the fusion peptide can be readily prepared by those skilled in the art by any method known in the art.

[0075] The fusion peptide or salt thereof, or its solvate, of the present invention can be preferably used in plants. When used in plants, the fusion peptide or salt thereof, or its solvate, of the present invention can promote the expression of sucrose transporters in those 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 those 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 fusion 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 fusion 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.

[0076] 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.

[0077] The fusion peptide or its salt or solvate of the present invention may be used individually or in combination of two or more. The fusion peptide or its salt or solvate of the present invention can be used, for example, for plant cultivation. Furthermore, the fusion peptide or its salt or solvate of the present invention can be used to increase the sugar content of plants or for plant protection. The fusion peptide or its salt or solvate 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 fusion peptide or its salt or solvate of the present invention to plants are also included in the present invention. The fusion peptide or its salt or solvate of the present invention can be used alone or in the form of a composition by mixing the peptide or its salt or solvate with other components. In plant cultivation methods, etc., the fusion peptide or its salt or solvate of the present invention may be applied directly to plants, or a composition containing the peptide or its salt or solvate may be applied to plants. The fusion peptide or its salt, or its solvate, 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 plant can be cultivated using known methods appropriate to the plant, except for the application of the fusion peptide or its salt, or its solvate, of the present invention.

[0078] Compositions comprising the fusion 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-described fusion 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-described fusion peptide of the present invention, a salt thereof, or a solvate thereof as an active ingredient.

[0079] 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.

[0080] Plant protection includes improving plant defense mechanisms and enhancing plant adaptive responses. The fusion 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 stresses. Plant protection also includes improving tolerance to abiotic stresses and improving tolerance to biological stresses. Examples of tolerance to abiotic stresses include tolerance to environmental stresses, such as tolerance to osmotic stress (e.g., salt tolerance), drought tolerance, high temperature tolerance, and low temperature tolerance. Examples of biological stresses include pathogens and pests. In one embodiment, the fusion peptides or salts thereof, or their solvates, and compositions of the present invention can be preferably used to improve tolerance to abiotic stresses such as environmental stresses. In one embodiment, the plant protection composition is preferably a biostimulant. Also in one embodiment, the fusion 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.

[0081] Other components in the composition besides the fusion 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 fusion peptide of the present invention or its salt or solvate in the composition is not particularly limited; for example, the total content of the fusion 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.

[0082] 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.

[0083] When the composition of the present invention is a liquid preparation, the total content of the fusion peptide or its salt or its solvate is preferably 0.001 to 20% by weight, and more preferably 0.002 to 2.5% by weight, as the content of the fusion peptide in the composition. When the composition of the present invention is a solid preparation, the total content of the fusion peptide or its salt or its solvate is preferably 0.0001 to 1% by weight, and more preferably 0.001 to 0.1% by weight, as the content of the fusion peptide in the composition.

[0084] 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, humectants, disintegrants, lubricants, diluents, excipients, amino acids, peptides (peptides different from the fusion peptide of the present invention), fertilizer elements, and components derived from natural products. Examples of liquid carriers include media capable of dissolving or dispersing the fusion peptide of the present invention or its salt, or its solvate, as described above. Examples include water; alcohols such as 1-propanol and butanol; polyhydric alcohols such as ethylene glycol and propylene glycol; and hydrocarbons such as xylene.

[0085] 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.

[0086] The method of applying the fusion peptide or its salt, 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 the hydroponic solution of the plant, or mixing into the soil. Among these, it is preferable to apply the fusion peptide or its salt, or its solvate, or the composition of the present invention to plants by foliar spraying or irrigation. A suitable formulation for foliar spraying 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 fusion peptide or its salt, or its solvate, or the composition of the present invention, mixed with water, should be irrigated onto the plant. The total content of the fusion peptide or its salt, or its solvate, in the composition used for foliar spraying (preferably a liquid formulation) is preferably 0.001 to 20% by weight, and more preferably 0.002 to 2.5% by weight, as the content of the fusion peptide. When applied to plants by irrigation, the total content of the fusion peptide, its salt, or its solvate in the irrigation solution is preferably 0.001 to 20% by weight, and more preferably 0.002 to 2.5% by weight, as the content of the fusion peptide.

[0087] The timing of application of the fusion peptide or its salt, 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 fusion peptide or its salt, 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 fusion peptide or its salt, or its solvate, can be appropriately set according to the plant and is not particularly limited. In one embodiment, the application amount of the fusion peptide or its salt, or its solvate, is preferably 0.01 to 20 mg per plant, and more preferably 0.05 to 10 mg per application.

[0088] The plants to which the fusion peptide or salt thereof, or solvate thereof, or composition containing the same 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, and Amaranthaceae or Solanaceae plants are more preferred.

[0089] 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.

[0090] 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.

[0091] 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.).

[0092] In one embodiment, the fusion 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 fusion 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.

[0093] This specification discloses the following: (1) of this disclosure is a fusion peptide or a salt thereof, or a solvate thereof, comprising a tag peptide and any of the peptides (A1) to (A3) below, from the amino terminus to the carboxyl terminus. (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.

[0094] Disclosure (2) is a fusion peptide or a salt thereof, or a solvate thereof, as described in Disclosure (1), wherein the tag peptide is one of the peptides (B1) to (B3) and (C1) to (C3) below. (B1) A peptide consisting of the amino acid sequence shown in Sequence ID No. 2 (B2) A peptide consisting of an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 2. (B3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 2. (C1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (C2) A peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 3. (C3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3.

[0095] (3) of this disclosure is a polynucleotide comprising a polynucleotide encoding a tag peptide from the 5' end to the 3' end, and one of the polynucleotides (a1) to (a3) ​​below. (a1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 4 (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. 4. (a3) A polynucleotide encoding a peptide that has a sequence identity of 90% or more with respect to the sequence of Sequence ID No. 4 and has an effect of promoting the expression of sucrose transporters.

[0096] Disclosure (4) is the polynucleotide described in Disclosure (3), wherein the polynucleotide encoding the tag peptide is one of the polynucleotides (b1) to (b3) and (c1) to (c3) below. (b1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 5 (b2) A polynucleotide consisting of a nucleotide sequence in which 1 to 20 bases are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 5. (b3) Polynucleotides consisting of a base sequence having 90% or more sequence identity with the base sequence of Sequence ID No. 5 (c1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 6 (c2) A polynucleotide consisting of a nucleotide sequence in which 1 to 9 bases are deleted, substituted, or added in the nucleotide sequence of Sequence ID No. 6. (c3) A polynucleotide consisting of a nucleotide sequence that has more than 90% sequence identity with the nucleotide sequence of Sequence ID No. 6.

[0097] Disclosure (5) is an expression vector comprising the polynucleotide described in Disclosure (3) or (4).

[0098] Disclosure (6) is the expression vector described in Disclosure (5), wherein the polynucleotide encoding the tag peptide is one of the polynucleotides described in (b1) to (b3) and (c1) to (c3) above.

[0099] Disclosure (7) is a transformant, which is a microorganism into which the polynucleotide described in Disclosure (3) or (4) or the expression vector described in Disclosure (5) or (6) has been introduced.

[0100] This disclosure (8) is a transformant as described in this disclosure (7), wherein the microorganism is a bacterium or a fungus.

[0101] The present disclosure (9) is a method for producing a fusion peptide, comprising the step of culturing the transformant described in the present disclosure (7) or (8).

[0102] Disclosure (10) is a composition comprising a fusion peptide or a salt thereof, or a solvate thereof, as described in Disclosure (1) or (2).

[0103] Disclosure (11) is the composition described in Disclosure (10), which is a composition for increasing the sugar content of plants or a composition for protecting plants.

[0104] This disclosure (12) is a composition according to this disclosure (11), wherein plant protection is the enhancement of the plant's defense mechanism or adaptive response.

[0105] (13) of this disclosure is a composition according to (11) or (12) of this disclosure, wherein the plant protection composition is a biostimulant.

[0106] The present disclosure (14) is a method for cultivating plants, comprising applying a fusion peptide or a salt thereof, or a solvate thereof, as described in the present disclosure (1) or (2) to the plants.

[0107] Disclosure (15) relates to the use of a fusion peptide or salt thereof, or a solvate thereof, as described in Disclosure (1) or (2), for increasing the sugar content of plants or for plant protection.

[0108] 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]

[0109] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0110] <Test 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: 9, 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).

[0111] 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 (SEQ ID NO: 10), 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: 11)

[0112] 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 11: 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 10), the homology was 71%. The peptide consisting of the amino acid sequence of sequence number 1 may be referred to as BvPEP below.

[0113] <Test 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

[0114] 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.

[0115] <Example 1> Cloning, expression, and purification of Tag+BvPEP A polynucleotide encoding a fusion peptide in which a tag peptide was attached to the N-terminus of BvPEP was constructed and expressed in E. coli. The Fh8 tag peptide and the P17 tag were used as the tag peptides. The amino acid sequences of the Fh8 tag peptide and the P17 tag are described below. Fh8 tag peptide: MPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSILSS (SEQ ID NO: 2) P17 tag:MKNESSTNATNTKQWRDETKGFRDEAKRFKNTAG (Sequence ID 3)

[0116] As a polynucleotide encoding a fusion peptide containing an Fh8-tagged peptide (SEQ ID NO: 2) and BvPEP (SEQ ID NO: 1), we constructed a polynucleotide (Fh8-BvPEP) with the base sequence shown in SEQ ID NO: 12. Fh8-BvPEP:ATGCCCTCAGTTCAAGAAGTAGAGAAACTACTCCATGTCCTGGACCGCAATGGTGATGGCAAGGTGTCGGCGGAAGAATTGAAGGCGTTCGCCGATGATAGCAAATGCCCGCTGGACTCCAACAAAATCAAGGCGTT TATCAAGGAGCACGATAAAAACAAGGACGGCAAACTGGACCTGAAAGAGTTGGTTAGCATTCTGTCTAGCGCTACCACGGGCGCAATTAAGAAACCGCCGCGTCCGCCAATCAGCACCGGCCGTGGTGGTCAGATTCAC (SEQ ID NO: 12) The amino acid sequence of the fusion peptide encoded by the above polynucleotide (Fh8-BvPEP) is shown below. The underlined portion is BvPEP. MPSVQEVEKLLHVLDRNDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSILSS ATTAAIKKPPRPPISTGRGGQIH (Sequence ID 7)

[0117] As a polynucleotide encoding a fusion peptide containing the P17 tag (SEQ ID NO: 3) and BvPEP (SEQ ID NO: 1), we constructed a polynucleotide (P17-BvPEP) with the base sequence shown in SEQ ID NO: 13. P17-BvPEP: ATGAAAAACGAATCAAGTACAAATGCTACTAACACGAAGCAGTGGCGTGATGAAACCAAGGGCTTCCGCGACGAGGCCAAGCGCTTTAAAAATACTGCGGGTGCTACCACCGCGGCAATTAAAAAACCGCCACGTCCGCCGATCAGCACCGGTCGTGGTGGCCAAATCCAC (SEQ ID NO: 13) The amino acid sequence of the fusion peptide encoded by the above polynucleotide (P17-BvPEP) is shown below. MKNESSTNATNTKQWRDETKGFRDEAKRFKNTAG ATTAAIKKPPRPPISTGRGGQIH (Sequence 8)

[0118] Vector cloning We commissioned Eurofins Genomics to synthesize plasmids (Plasmid names: pEX-A2J2-Fh8-BvPEP, pEX-A2J2-P17-BvPEP) containing nucleotide sequences encoding a fusion peptide including BvPEP (Fh8-BvPEP (SEQ ID NO: 12) or P17-BvPEP (SEQ ID NO: 13)). pEX-A2J2-Fh8-BvPEP is a plasmid in which Fh8-BvPEP (SEQ ID NO: 12) is incorporated into the pEX-A2J2 vector. pEX-A2J2-P17-BvPEP is a plasmid in which P17-BvPEP (SEQ ID NO: 13) is incorporated into the pEX-A2J2 vector.

[0119] pEX-A2J2-Fh8-BvPEP, pEX-A2J2-P17-BvPEP, and pET22b were digested with NdeI and HindIII restriction enzymes, and agarose gel electrophoresis was performed. The Fh8-BvPEP fragment (288 bp), P17-BvPEP fragment (183 bp), and pET22b vector fragment (5400 bp) were recovered from the gel.

[0120] DNA fragments were extracted from the gel, and the Fh8-BvPEP fragment was ligated with the pET22b vector fragment to form pET22b(Fh8-BvPEP). The resulting pET22b(Fh8-BvPEP) was introduced into E. coli DH5a strain for transformation, and colonies were sorted on ampicillin-containing LB agar plates. In addition, the P17-BvPEP fragment was ligated with the pET22b vector fragment to form pET22b(P17-BvPEP). The resulting pET22b(P17-BvPEP) was introduced into E. coli DH5a strain for transformation, and colonies were sorted on ampicillin-containing LB agar plates.

[0121] Colony PCR was performed using T7pro(5'-TAATACGACTCACTATAGGG-3'(SEQ ID NO: 15)) and T7term(5'-ATGCTAGTTATTGCT CAGCGG-3'(SEQ ID NO: 16)) primers to confirm the introduction of Fh8-BvPEP and P17-BvPEP into E. coli. Furthermore, colonies in which the introduction of Fh8-BvPEP or P17-BvPEP was confirmed were grown, and pET22b(Fh8-BvPEP) and pET22b(P17-BvPEP) were recovered from the bacterial cells to confirm that the target nucleotide sequence had been introduced.

[0122] The pET22b(Fh8-BvPEP) and pET22b(P17-BvPEP) strains, whose base sequences were confirmed, were introduced into E. coli Lemo21(DE3) strain (purchased from New England Biolabs Japan Inc.) and transformed. When pET22b(Fh8-BvPEP) was introduced into E. coli Lemo21(DE3) strain, the transformant Lemo21(DE3)_pET22b(Fh8-BvPEP) was obtained. When pET22b(P17-BvPEP) was introduced into E. coli Lemo21(DE3) strain, the transformant Lemo21(DE3)_pET22b(P17-BvPEP) was obtained.

[0123] The transformed Lemo21(DE3)_pET22b(Fh8-BvPEP) was pre-cultured in LB medium (2-5 mL) at 30°C with shaking (150 rpm) for 18-24 hours. After pre-culture, the culture medium was inoculated with the main medium. The pH of the main medium was adjusted to 6.5-7.0 by adding lactic acid or ammonia. The transformed Lemo21(DE3)_pET22b(P17-BvPEP) was also cultured with shaking under the same conditions as Lemo21(DE3)_pET22b(Fh8-BvPEP). The main culture was maintained at a pH of 6.5-7.0 for 9-12 hours from the start of culture. Then, the pH was increased to 8.0-9.5 and maintained until the end of culture. Cell density was maintained until the OD600 reached 15-20 6 hours after the start of culture. Three hours after the start of culture, isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to a concentration of 0.1–1 mM to promote the production of the target fusion peptide. Twenty-two to twenty-six hours after the addition of IPTG, the culture medium in the culture vessel was centrifuged (13,000 rpm) to separate the supernatant fraction from the precipitate. The proteins (peptides) contained in the supernatant and precipitate were identified by SDS-PAGE.

[0124] Figure 3A shows the results of SDS-PAGE analysis of the expression of the fusion peptide (Fh8-BvPEP). Figure 3B shows the results of SDS-PAGE analysis of the expression of the fusion peptide (P17-BvPEP). In Figures 3A and 3B, "sup" represents the supernatant fraction and "pellet" represents the precipitate. SDS-PAGE revealed bands for both the fusion peptide (Fh8-BvPEP) (Molecular Weight: 10.0 kDa) and the fusion peptide (P17-BvPEP) (Molecular Weight: 6.3 kDa). Bands were present in the supernatant fraction (sup) for both peptides, confirming their secretion into the culture medium. Furthermore, using bovine serum albumin (BSA) as a calibration curve, the concentrations were calculated from the band intensity of the supernatant fraction, resulting in concentrations of 1.3 g / L for the fusion peptide (Fh8-BvPEP) and 1.1 g / L for the fusion peptide (P17-BvPEP).

[0125] Refinement of the upper fraction The recovered supernatant fraction is processed using a desalination and concentration device (AKTA flux TM Desalting and concentration were performed using a UF membrane (MWCO 5000) with Cytiva (Global Life Science Technologies Japan Co., Ltd.). Electrical conductivity was 5 mS / cm². 2 The peptides were desalted until the desired result was obtained. Fusion peptides (Fh8-BvPEP) (a fusion peptide in which an Fh8 tag peptide is attached to the N-terminus of BvPEP) and fusion peptides (P17-BvPEP) (a fusion peptide in which a P17 tag is attached to the N-terminus of BvPEP) were obtained.

[0126] <Example 2> Application of fusion peptides and evaluation of gene expression The fusion peptides obtained in Example 1 were applied to plants, and their effects on the gene expression of sucrose transporter 1 (SUT1) were investigated. Peptide solutions were prepared by mixing the fusion peptide (Fh8-BvPEP) with deionized water, and peptide solutions were prepared by mixing the fusion peptide (P17-BvPEP) with deionized water, and these were applied to plants.

[0127] 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 approximately four true leaves were selected, and 5 mL of water containing 10 μM of fusion peptide (Fh8-BvPEP) or (P17-BvPEP) (peptide solution) was used to irrigate the base of each plant. Two days (45-48 hours) after irrigation with the peptide solution, the concentration of the fusion peptide (Fh8-BvPEP) in the peptide solution collected by punching out sugar beet leaves was 0.01% by weight. The concentration of the fusion peptide (P17-BvPEP) in the peptide solution was 0.0063% by weight.

[0128] <Comparative Example 1> In Example 2, instead of the peptide solution, ion-exchanged water (without the above-mentioned fusion peptide) was provided (water treatment). Otherwise, sugar beets were cultivated from seed in the same manner as in Example 2, and the sugar beet leaves were harvested by punching them out.

[0129] <Reference example 1> In Example 2, 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 2, and the sugar beet leaves were harvested by punching them out.

[0130] <Reference example 2> A solution (BvPEP concentration: 10 μM (0.023 wt%)) was prepared by mixing BvPEP (a peptide consisting of the amino acid sequence shown in Sequence ID No. 1) obtained in the synthesis of Test Example 2 with deionized water. Sugar beets were cultivated from seed in the same manner as in Example 2, except that this BvPEP solution was used for irrigation instead of the peptide solution containing the fusion peptide. The leaves of the sugar beets were then punched out and collected.

[0131] <Example 3> Preparation of tomato (variety: Regina) seedlings and application of fusion peptides Kimwipes moistened with water were placed in a petri dish, and Regina seeds were sown on 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 (peptide solution) containing 10 μM fusion peptide (Fh8-BvPEP) or (P17-BvPEP). 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.

[0132] <Comparative Example 2> In Example 3, instead of the peptide solution, deionized water (without the above-mentioned fusion peptide) was used (water treatment). Otherwise, tomatoes (Regina) were grown from seed using the same method as in Example 3, and the leaves were collected by punching them out. After collecting the leaves, cultivation was continued for another 30 days.

[0133] <Reference example 3> In Example 3, instead of the peptide solution, an aqueous NaCl solution with a concentration of 0.15 M was used for irrigation. Otherwise, tomatoes (Regina) were grown from seed using the same method as in Example 3, and the leaves were collected by punching them out. After collecting the leaves, cultivation was continued for another 30 days.

[0134] <Reference example 4> A solution (BvPEP concentration: 10 μM (0.023 wt%)) was prepared by mixing BvPEP (a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1), obtained in the synthesis of Test Example 2, with deionized water. In Example 3, the same method as in Example 3 was used for cultivation from tomatoes (Regina), except that this BvPEP solution was used for irrigation instead of the peptide solution containing the fusion peptide. Leaves were collected by punching them out. After collecting the leaves, cultivation was continued for another 30 days.

[0135] <Gene Expression Evaluation> The ability of fusion peptide spraying 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 2-3, Comparative Examples 1-2, and Reference Examples 1-4 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).

[0136] 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.

[0137] (Beet) BvSUT1_Fw : GGGATGCATTGTTTGGTGGG (Sequence ID 17) BvSUT1_Rv : ATTGACCCTCCGATTTTGGCT(Sequence ID 18) BvActin_Fw: TAAACCGAGATGGCTGATGC (SEQ ID NO: 19) BvActin_Rv: ATACTTGGGAAGACAGCCCT (SEQ ID NO: 10) (Tomato) SlSUT1_Fw: AACTCCCGGAGAAAGAAGAG (SEQ ID NO: 21) SlSUT1_Rv: TACAGTTTCGCATCACCGAC (SEQ ID NO: 22) SlActin_Fw: GGGATGGAGAAGTTTGGTGGTGG (SEQ ID NO: 23) SlActin_Rv: CTTCGACCAAGGGATGGTGTAGC (SEQ ID NO: 24)

[0138] <PCR reaction conditions> (Beet) Reacted at 95°C for 30 seconds and 61°C for 20 minutes, then performed reactions at 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 performed reactions at 95°C for 10 seconds, 63°C for 10 seconds, and 68°C for 30 seconds for a total of 40 cycles.

[0139] The results of Reference Example 1 are shown in Figure 4. Figure 4 is a graph showing the results of examining the expression of SUT1 by giving beets water containing NaCl. The results of Example 2, Comparative Example 1, and Reference Example 2 are shown in Figure 5. In Figures 4 and 5, the results are shown as mean ± standard error (n = 4). Figure 5 is a graph showing the results of examining the expression of SUT1 in beets treated with the fusion peptide (Fh8 - BvPEP), the fusion peptide (P17 - BvPEP), or synthetic BvPEP. The expression levels of SUT1 in Figures 4 and 5 are shown as relative values with the expression level of SUT1 in the beets of Comparative Example 1 (water treatment) set as 1.

[0140] 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, when synthetic BvPEP was applied (Reference Example 2), an increase of approximately 40% was observed compared to water treatment (Comparative Example 1) (Figure 5). When a fusion peptide (fusion protein) in which a tag peptide was bound to the N-terminus of BvPEP was applied, the expression of sucrose transporters also increased compared to water treatment. No plant damage was observed in sugar beets treated with either the fusion peptide or synthetic BvPEP.

[0141] The results for Example 3, Comparative Example 2, and Reference Examples 3-4 are shown in Figure 6. In Figure 6, the results are shown as mean ± standard error (n=4). Figure 6 is a graph showing the results of examining the expression of SUT1 in tomatoes treated with fusion peptide (Fh8-BvPEP), fusion peptide (P17-BvPEP), synthetic BvPEP, or 0.15M NaCl. The expression levels of SUT1 in Figure 6 are shown as relative values ​​with the expression level of SUT1 in tomatoes from Comparative Example 2 (water treatment) set to 1. In tomatoes as well, the expression of SUT1 increased with the application of fusion peptides. Figures 7A, 7B, 7C, 7D, and 7E are photographs of tomato seedlings 30 days after application of fusion peptide (Fh8-BvPEP), fusion peptide (P17-BvPEP), or BvPEP, and tomato seedlings treated with water or 0.15M NaCl (Figure 7A: water treatment, Figure 7B: BvPEP, Figure 7C: fusion peptide (P17-BvPEP), Figure 7D: fusion peptide (Fh8-BvPEP), Figure 7E: 0.15M NaCl treatment). No difference in growth was observed between tomatoes treated with fusion peptide or synthetic BvPEP and those treated with water, and no plant damage to tomatoes from fusion peptide or synthetic BvPEP was confirmed. [Industrial applicability]

[0142] This invention is useful in the fields of agriculture, horticulture, and the like.

Claims

1. A fusion peptide or a salt thereof, or a solvate thereof, comprising a tag peptide and one of the peptides (A1) to (A3) below, from the amino terminus to the carboxyl terminus. (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.

2. The fusion peptide or a salt thereof, or a solvate thereof, according to claim 1, wherein the tag peptide is one of the peptides (B1) to (B3) and (C1) to (C3) listed below. (B1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (B2) A peptide consisting of an amino acid sequence in which 1 to 6 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No.

2. (B3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

2. (C1) Peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (C2) A peptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO:

3. (C3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

3.

3. A polynucleotide comprising a polynucleotide encoding a tag peptide and one of the following polynucleotides (a1) to (a3), from the 5' end to the 3' end. (a1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 4 (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.

4. (a3) A polynucleotide encoding a peptide that has a sequence identity of 90% or more with respect to the sequence of Sequence ID No. 4 and has an effect of promoting the expression of sucrose transporters.

4. The polynucleotide according to claim 3, wherein the polynucleotide encoding the tag peptide is any of the following polynucleotides (b1) to (b3) and (c1) to (c3). (b1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 5 (b2) A polynucleotide consisting of a base sequence in which 1 to 20 bases are deleted, substituted, or added in the base sequence of Sequence ID No.

5. (b3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of Sequence ID No.

5. (c1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 6 (c2) A polynucleotide consisting of a base sequence in which 1 to 9 bases are deleted, substituted, or added in the base sequence of Sequence ID No.

6. (c3) A polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence of Sequence ID No.

6.

5. An expression vector comprising the polynucleotide described in claim 3.

6. The expression vector according to claim 5, wherein the polynucleotide encoding the tag peptide is any of the following polynucleotides (b1) to (b3) and (c1) to (c3). (b1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 5 (b2) A polynucleotide consisting of a base sequence in which 1 to 20 bases are deleted, substituted, or added in the base sequence of Sequence ID No.

5. (b3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of Sequence ID No.

5. (c1) Polynucleotide consisting of the base sequence shown in Sequence ID No. 6 (c2) A polynucleotide consisting of a base sequence in which 1 to 9 bases are deleted, substituted, or added in the base sequence of Sequence ID No.

6. (c3) A polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence of Sequence ID No.

6.

7. A transformant that is a microorganism into which the polynucleotide described in claim 3 or the expression vector described in claim 5 has been introduced.

8. The transformant according to claim 7, wherein the microorganism is a bacterium or a fungus.

9. A method for producing a fusion peptide, comprising the step of culturing the transformant described in claim 7.

10. A composition comprising the fusion peptide or a salt thereof, or a solvate thereof, according to claim 1 or 2.

11. The composition according to claim 10, which is a composition for increasing the sugar content of plants or a composition for protecting plants.

12. The composition according to claim 11, wherein plant protection is the improvement of the plant's defense mechanism or adaptive response.

13. The composition according to claim 11, wherein the plant protection composition is a biostimulant.

14. A method for cultivating plants, comprising applying the fusion peptide or a salt thereof, or a solvate thereof, according to claim 1 or 2, to the plants.

15. Use of the fusion peptide or salt thereof, or solvate thereof, according to claim 1 or 2, for increasing the sugar content of plants or for plant protection.

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