Fusion peptide, polynucleotide encoding same, and use therefor

Abstract

The present invention relates to a fusion peptide, a salt thereof, or a solvate thereof, the fusion peptide containing, from the amino terminus toward the carboxy terminus, a tag peptide and any peptide among (A1)-(A3). (A1) A peptide that comprises the amino acid sequence represented by SEQ ID NO: 1; (A2) a peptide that comprises an amino acid sequence obtained by deleting, substituting, or adding 1-4 amino acids in the amino acid sequence represented by SEQ ID NO: 1 and that has a pore density increasing effect; and (A3) a peptide that comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and that has a pore density increasing effect.

Description

Fusion peptides, the polynucleotides encoding them, and their applications

[0001] This invention relates to fusion peptides or salts thereof or solvates thereof, and polynucleotides encoding said fusion peptides. The invention also relates to compositions containing fusion peptides or salts thereof or solvates thereof, methods for cultivating plants, and the like.

[0002] Environmental problems such as salinization and climate change are posing challenges to global agricultural production. For example, global warming is causing an increase in pests, diseases, and plant blights, leading to reduced crop yields. Therefore, there is a need for technologies that can enhance plants' resistance 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. Stomagent is an endogenous peptide that has the effect of increasing stomatal density in plants. As one of the mechanisms that regulates stomatal differentiation, when epidermal pattern-forming factors (EPF1, EPF2) are bound as ligands to the cell surface receptors TOO MUCH MOUTH (TMM) and ERECTA family receptor-like kinases (ER, ERL1, ERL2), they act as negative regulators of stomatal density, while when Stomagent is bound, it acts as a positive regulator of stomatal density. It has been reported that Stomagent, EPF1, and EPF2 compete with each other for the receptor TMM and have antagonistic effects (Non-Patent Literature 1).

[0004] “Stomagen positively regulates stomatai density in Arabidopsis”, Nature, 463, 2010, 241-244 (2010)

[0005] In plants, an increase in stomatal density leads to increased carbon dioxide uptake, promoting photosynthesis, and is expected to result in increased crop yield (number of harvested crops) and higher sugar content (sucrose concentration). Therefore, there is a need to industrially produce Stomagen, which positively regulates stomatal density.

[0006] Generally, a technique for industrially producing functional peptides and other peptides involves introducing a polynucleotide encoding the target peptide into a microorganism and allowing the microorganism to produce the peptide. However, depending on the peptide to be expressed, microbial production may be difficult, or even if production is possible, recovering the produced peptide in an industrially usable form may be challenging.

[0007] Non-patent document 1 describes how, in order to express Stomagen in E. coli, a DNA fragment encoding Stomagen was amplified by PCR and cloned into a vector. However, when Stomagen itself is expressed alone in microorganisms such as E. coli, the produced Stomagen is not easily secreted outside the cell, making Stomagen purification difficult.

[0008] The present invention aims to provide a fusion peptide that has a porosity-increasing effect and can be secreted extracellularly when produced by microorganisms, a polynucleotide encoding the same, and applications thereof.

[0009] The present inventors have diligently studied to solve the above problems and have arrived at the present invention. Specifically, the present invention relates to 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, protecting plants, or increasing plant yield. (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 4 amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, and which has a porosity-increasing effect. (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 a porosity-increasing effect. (a1) A polynucleotide consisting of the base sequence shown in Sequence ID No. 7. (a2) A polynucleotide encoding a peptide consisting of a base sequence in which 1 to 14 bases are deleted, substituted, or added in the base sequence shown in Sequence ID No. 7, and which has a porosity-increasing effect. (a3) ​​A polynucleotide encoding a peptide consisting of a base sequence having 90% or more sequence identity with the base sequence shown in Sequence ID No. 7, and which has a porosity-increasing effect.

[0010] According to the present invention, it is possible to provide a fusion peptide that has the effect of increasing porosity density and can be secreted extracellularly when produced by microorganisms, a polynucleotide encoding the same, and applications thereof.

[0011] Figure 1A shows the results of confirming the expression of the fusion peptide (PelB-His-Fh8-Stomagen) using SDS-PAGE. Figure 1B shows the results of confirming the expression of the fusion peptide (Fh8-Stomagen) using SDS-PAGE. Figure 1C shows the results of confirming the expression of the fusion peptide (PelB-His-ZZ-Stomagen) using SDS-PAGE. Figure 1D shows the results of confirming the expression of the fusion peptide (PelB-His-Z-Stomagen) using SDS-PAGE. Figure 1E shows the results of confirming the expression of the fusion peptide (ZZ-Stomagen) using SDS-PAGE. Figure 2 is a graph showing the results of examining the expression of TMM in cucumbers treated with the fusion peptide (PelB-His-Fh8-Stomagen). Figure 3 is a graph showing the results of TMM expression in cucumbers treated with the fusion peptide (PelB-His-Fh8-Stomagen). Figure 4A is a graph showing the results of TMM expression after 7 hours in tomatoes treated with the fusion peptide (Fh8-Stomagen) or water. Figure 4B is a graph showing the results of TMM expression after 24 hours in tomatoes treated with the fusion peptide (Fh8-Stomagen) or water. Figure 4C is a graph showing the results of TMM expression after 48 hours in tomatoes treated with the fusion peptide (Fh8-Stomagen) or water. Figure 5A is a graph showing the results of TMM expression after 7 hours in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen), water, or PelB-His-Fh8 peptide. Figure 5B is a graph showing the results of TMM expression 24 hours after treatment with the fusion peptide (PelB-His-Fh8-Stomagen), water, or PelB-His-Fh8 peptide. Figure 5C is a graph showing the results of TMM expression 48 hours after treatment with the fusion peptide (PelB-His-Fh8-Stomagen), water, or PelB-His-Fh8 peptide. Figure 6A is a graph showing the results of TMM expression 41 hours after treatment with the fusion peptide (PelB-His-Fh8-Stomagen) or water.Figure 6B is a graph showing the results of TMM expression 48 hours after application of the fusion peptide (PelB-His-Fh8-Stomagen) or water to tomatoes. Figure 6C is a graph showing the results of TMM expression 65 hours after application of the fusion peptide (PelB-His-Fh8-Stomagen) or water to tomatoes. Figure 6D is a graph showing the results of TMM expression 72 hours after application of the fusion peptide (PelB-His-Fh8-Stomagen) or water to tomatoes. Figure 7A is a graph showing the results of TMM expression 2 days after application of the fusion peptide (Fh8-Stomagen) or water to sweet potatoes. Figure 7B is a graph showing the results of TMM expression 2 days after application of the fusion peptide (Fh8-Stomagen) or water to sweet potatoes. Figure 7C is a graph showing the results of examining TMM expression two days after application of the fusion peptide (Fh8-Stomagen) or water three times. Figure 8A is a graph showing the results of summarizing the number of cucumbers harvested (number of cucumbers harvested per plant) over six months after application of the fusion peptide (PelB-His-Fh8-Stomagen) or water. Figure 8B is a graph showing the results of summarizing the number of cucumbers harvested (number of cucumbers harvested per plant) by month after application of the fusion peptide (PelB-His-Fh8-Stomagen) or water. Figure 9 is a graph showing the results of determining the Brix value of sweet potatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen) or water. Figure 10 is a graph showing the results of examining SUT1 expression in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen), water, or Pelb-His-Fh8 peptide.

[0012] The fusion peptide of the present invention comprises a tag peptide and one of the following peptides (A1) to (A3), 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 consisting of an amino acid sequence in which 1 to 4 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, and which has a porosity-increasing effect (A3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and which has a porosity-increasing effect The fusion peptide of the present invention is a polypeptide and can also be described as a fusion protein.

[0013] In this specification, unless otherwise specified, the amino acid sequence of a peptide is indicated by the following conventional single-letter abbreviations: 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

[0014] In this specification, the amino acid sequence of a peptide follows the convention of peptide notation, with the leftmost end being the amino terminus (N-terminus) and the rightmost end being the carboxyl terminus (C-terminus). In this invention, if an amino acid may have optical isomers, the L-isomer is referred to unless otherwise specified.

[0015] In this specification, the addition of amino acids includes the meaning of insertion into a sequence. In the present invention, the deletion, substitution, or addition of one or more amino acids in an amino acid sequence means that there is a deletion, substitution, or addition of one or more amino acids at any position in one or more amino acid sequences within the same sequence, and two or more types of deletion, substitution, and addition may occur simultaneously. For example, the deletion, substitution, or addition of one to four amino acids in an amino acid sequence means that there is a deletion, substitution, or addition of one to four amino acids at any position in one to four amino acid sequences within the same sequence, and two or more types of deletion, substitution, and addition may occur simultaneously.

[0016] The peptide (A2) described above is preferably 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. 1, and is a peptide that has a porosity-increasing effect; more preferably 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 is a peptide that has a porosity-increasing effect; and even more preferably an amino acid sequence in which 1 amino acid is deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, and is a peptide that has a porosity-increasing effect.

[0017] The sequence identity of the peptide (A3) 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.

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

[0019] The peptide (A1) described above is a peptide that has a stomatal density-increasing effect. The peptides (A2) to (A3) can be any peptide that has a stomatal density-increasing effect. The peptides (A1) to (A3) are polypeptides and can also be described as proteins. In the present invention, the stomatal density-increasing effect can be confirmed, for example, by increasing the expression level of TOO MUCH MOUTH (TMM) in plants. TOO MUCH MOUTH (TMM) is a transmembrane protein present in the cell membrane of plants and has an extracellular receptor site. When TMM expression is promoted, transcription factors involved in stomatal formation are activated by intracellular signal transduction via ERECTA family receptor-like kinases, and stomatal density increases. The peptides (A1) to (A3) described above can promote TMM expression in plants when used in those plants. In the present invention, if TMM expression is promoted to some extent (expression level increases) in plants to which a certain substance has been applied compared to plants to which the substance has not been applied, then the substance can be said to have a stomatal density increasing effect. The peptides (A1) to (A3) described above preferably have a stomatal differentiation promoting effect or a photosynthesis promoting effect.

[0020] In the present invention, TMM expression promotion includes the promotion of TMM mRNA expression and the promotion of TMM protein expression. In the present invention, if, when a certain substance is used on a plant, TMM expression 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 TMM expression promoting effect (exhibit an expression promoting effect). TMM expression promotion can also be expressed as an increase in the expression level of TMM or an increase in TMM expression. The plant may be the whole plant or a part of the plant. Parts of the plant include leaves, stems, flowers, fruits, trunks, branches, seeds, roots, buds, etc.

[0021] In one embodiment, the peptides (A1) to (A3) described above are preferably peptides that have an effect of promoting the expression of sucrose transporters. Sucrose transporters are proteins that transport sucrose and are present in the cell membranes of plants. Sucrose transporter 1 (SUT1) is an example of a sucrose transporter. When the peptides (A1) to (A3) described above are used in plants, it is preferable that they promote the expression of sucrose transporters in the plants. Among the peptides (A1) to (A3) described above, peptide (A1) is preferred.

[0022] 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 the plant include leaves, stems, flowers, fruits, trunks, branches, seeds, roots, buds, etc.

[0023] 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 positioned 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) exhibits a porosity-increasing effect. Alternatively, as the tag peptide, a peptide can be used that causes a microorganism to secrete the fusion peptide containing the tag peptide into the extracellular space of the host microorganism when the fusion peptide containing the tag peptide is produced by the microorganism. Secretion into the extracellular space of the microorganism includes secretion into the periplasm. In the present invention, a tag peptide consisting of 20 to 130 amino acids (polypeptide tag) is preferred, and a tag peptide consisting of 30 to 120 amino acids is more preferred. In the present invention, a soluble tag peptide is also preferred. A soluble tag peptide is a peptide that has the effect of improving the solubility of the fusion peptide containing the tag peptide. Fusion peptides containing a soluble tag peptide at the N-terminus of the peptides (A1) to (A3) described above are easily expressed in a soluble form (soluble peptide) in microorganisms. The soluble tag peptide is preferably one that improves the expression stability of the fusion peptide containing the tag peptide within the host.Examples of tag peptides usable in the present invention include PelB tag (PelB tag peptide), peptide encoded by the Fh8 gene of the liver fluke (Fasciola hepatica) (Fh8 tag), ZZ tag (ZZ tag peptide), Z tag (Z tag peptide), His tag (His 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), Nutilication substrate A (Nus tag peptide), and N-terminal extension. Examples include sequence (NEXT tag peptide). In the present invention, one of these tag peptides can be used alone or in combination with others as a tag peptide.

[0024] In the present invention, the tag peptide is preferably a peptide containing one or two of the following (B1) to (B3), (C1) to (C3), (D1) to (D3), and (E1) to (E3). (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 2 amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ 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 SEQ 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 6 amino acids are deleted, substituted, or added to 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. (D1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 4. (D2) A peptide consisting of an amino acid sequence in which 1 to 11 amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 4. (D3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. (E1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 5. (E2) A peptide consisting of an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 5. (E3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 5.

[0025] The peptide (B1) above is a Pelb-tagged peptide. The peptide (C1) above is an Fh8-tagged peptide. The peptide (D1) above is a ZZ-tagged peptide. The peptide (E1) above is a Z-tagged peptide. The fusion protein preferably contains at least one selected from the group consisting of (B1), (C1), (D1), (E1), (B2), (C2), (D2), and (E2) as a tag peptide, and (B1) and (C1), (B1) and (D1), (B1) and (E1), (C1) and (D1), (C1) and (E1), (D1) and (E1), (B2) and (C1), (B2) and (D1), (B2) and ( It is more preferable that the tag peptide contains E1), (C2) and (B1), (C2) and (D1), (C2) and (E1), (D2) and (B1), (D2) and (C1), (D2) and (E1), (E2) and (B1), (E2) and (C1), (E2) and (D1), (B2) and (C2), (B2) and (D2), (B2) and (E2), (C2) and (D2), (C2) and (E2), or (D2) and (E2). When the tag peptide contains the above peptides, when the fusion peptide containing the tag peptide is expressed in microorganisms such as E. coli, the extracellular secretion of the fusion peptide is further promoted. This makes purification easier when producing the fusion peptide by microorganisms. It is also preferable that the fusion protein contains the above tag peptides because it increases the solubility of the fusion protein. Furthermore, it is also preferable that the fusion protein contains the above tag peptides because it improves the expression stability in E. coli.

[0026] If the tag peptide contains two of the above (B1) to (B3), (C1) to (C3), (D1) to (D3), and (E1) to (E3), it is preferable that the tag peptide contains (B1) or (B2) and (C1), (C2), (D1), (D2), (E1) or (E2), and more preferably (B1) or (B2) and (C1), (D2) or (E1). In this case, it is preferable that the tag peptide contains (B1) or (B2) closer to the N-terminus than (C1), (C2), (D1), (D2), (E1) or (E2). (B1) or (B2) and (C1), (C2), (D1), (D2), (E1) or (E2) may be linked via the His tag (SEQ ID NO: 6). In one embodiment, the tag peptide is preferably a peptide comprising (B1) or (B2) above, a His tag (SEQ ID NO: 6), and (C1), (D2), or (E1). In one embodiment, the peptide (C2) or (E2) above is preferred as the tag peptide.

[0027] The peptides (B2), (B3), (C2), (C3), (D2), (D3), (E2), and (E3) above may include peptides that promote extracellular secretion of the fusion peptide when the fusion peptide containing the peptide is expressed, for example, in E. coli. These tag peptides preferably have a solubility-enhancing effect. The peptides (B2), (B3), (C2), (C3), (D2), (D3), (E2), and (E3) above preferably have an effect of improving the expression stability of the fusion peptide containing it in microorganisms (preferably E. coli).

[0028] In the peptide described in (B2) above, the number of deleted, substituted, or added amino acids is preferably one. As the peptide described in (B2) above, a peptide consisting of an amino acid sequence in which one to two amino acids (preferably glutamic acid or aspartic acid) are added to the C-terminus of the amino acid sequence shown in SEQ ID NO: 2 is preferred. In the peptide described in (C2) above, the number of deleted, substituted, or added amino acids is preferably one to five, more preferably one to four, even more preferably one to three, particularly preferably one to two, and most preferably one. As the peptide described in (C2) above, a peptide consisting of an amino acid sequence in which one amino acid (preferably methionine) is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 3 is preferred. In the peptide described in (D2) above, the number of deleted, substituted, or added amino acids is preferably one to ten or one to nine, more preferably one to eight or one to seven, even more preferably one to six, even more preferably one to five, even more preferably one to four, even more preferably one to three, particularly preferably one to two, and most preferably one. In one embodiment, the peptide (D2) above is preferably a peptide consisting of an amino acid sequence in which 1 to 3 amino acids are added to the C-terminus of the amino acid sequence shown in SEQ ID NO: 4. In another embodiment, the peptide (D2) above is preferably a peptide consisting of an amino acid sequence in which 1 amino acid (preferably methionine) is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 4, and 1 to 3 amino acids are added to the C-terminus. In the peptide (E2) above, the number of deleted, substituted, or added amino acids is preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, and particularly preferably 1.

[0029] The sequence identity of the peptides (B3), (C3), (D3), and (E3) 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.

[0030] In the present invention, the peptides described in (B1) (PelB tag peptide), (C1) (Fh8 tag), (D1) (ZZ tag peptide), and (E1) (Z tag peptide) are more preferred as tag peptides, and the peptides described in (B1) and (C1) are even more preferred. The peptides described in (B1), (C1), (D1), and (E1) are also preferred from the viewpoint of not affecting the porosity-increasing effect, TMM expression-promoting effect, and sucrose transporter expression-promoting effect of the peptides described in (A1) to (A3).

[0031] 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 a TMM expression promoting effect by containing any of the peptides (A1) to (A3). The fusion peptide of the present invention has a porosity increasing effect. From the viewpoint of obtaining the porosity increasing 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.

[0032] 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 two to twenty amino acids (peptide linker). Known peptide linkers can be used. A peptide consisting of three to fifteen amino acids is preferred as the peptide linker, and a peptide consisting of five to ten amino acids is more preferred. The fusion peptide of the present invention may have an acetylated N-terminus and 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.

[0033] 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.

[0034] 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: 12, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 13, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 14, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 15, and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 16. The amino acid sequence of SEQ ID NO: 12 is the amino acid sequence of a fusion peptide consisting of the peptide (B1) above, a His-tagged peptide, a peptide (C1), and a peptide (A1). The amino acid sequence of SEQ ID NO: 13 is the amino acid sequence of a fusion peptide consisting of a peptide having an amino acid sequence in which one amino acid is added to the peptide (C1) above, and a peptide (A1). The amino acid sequence of SEQ ID NO: 14 is the amino acid sequence of a fusion peptide consisting of a peptide having an amino acid sequence in which two amino acids are added to the peptide (B1) above, a His-tagged peptide, a peptide having an amino acid sequence in which three amino acids are added to the peptide (D1), and a peptide (A1). The amino acid sequence of Sequence ID No. 15 is the amino acid sequence of a fusion peptide consisting of a peptide with an amino acid sequence in which two amino acids are added to the peptide (B1) above, a His-tagged peptide, the peptide (E1), and the peptide (A1). The amino acid sequence of Sequence ID No. 16 is the amino acid sequence of a fusion peptide consisting of a peptide with an amino acid sequence in which three amino acids are added to the peptide (D1) above, and the peptide (A1).

[0035] The fusion peptide of the present invention may be in the form of a salt. The salts of the peptide in the present invention are not particularly limited and may be either acidic salts or basic salts. Examples of acidic salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, etc.; organic acid salts such as formate, acetate, citrate, maleate, malate, oxalate, lactate, succinate, fumarate, propionate, etc.; and amino acid salts such as aspartate, glutamate, etc. Examples of basic salts include alkali metal salts such as sodium salt, potassium salt, etc.; alkaline earth metal salts such as calcium salt, magnesium salt, etc.; ammonium salt; and salts with organic bases such as triethylamine, triethanolamine, pyridine, etc. These salts are salts that can be used in plants and can be used as salts of the fusion peptide in the present invention. Among them, as salts of the fusion peptide, hydrochloride, formate, acetate, phosphate, citrate, lactate, aspartate, glutamate, sodium salt, and potassium salt are preferred, and hydrochloride, formate, and acetate are more preferred.

[0036] The fusion peptide of the present invention or a salt thereof may be in the form of a solvate. The solvent forming the solvate is not particularly limited, and examples thereof include water, ethanol, methanol, glycerol, etc., and water is preferred.

[0037] 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.

[0038] Polynucleotides encoding the fusion peptide 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 fusion peptide of the present invention can be designed, for example, by replacing the corresponding codons based on the amino acid sequence of the fusion peptide.

[0039] 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 one of the above-mentioned peptides (A1) to (A3). As the polynucleotide encoding the fusion peptide of the present invention, a polynucleotide comprising, from the 5'-end to the 3'-end, a polynucleotide encoding the tag peptide and a polynucleotide encoding any one of the following (a1) to (a3) is preferred. (a1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7. (a2) A polynucleotide consisting of a nucleotide sequence in which 1 to 14 bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 7, and encoding a peptide having an action of increasing stomatal density. (a3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 7, and encoding a peptide having an action of increasing stomatal density.

[0040] A polynucleotide comprising, in this order, a polynucleotide encoding the tag peptide and a polynucleotide encoding any one of the above-mentioned (a1) to (a3) from the 5'-end to the 3'-end is preferred as the polynucleotide encoding the fusion peptide of the present invention described above. Among the polynucleotides of the above-mentioned (a1) to (a3), the polynucleotide of the above-mentioned (a1) is preferred.

[0041] As a polynucleotide encoding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 7 (ATTGGTAGCAACGGGCTCCGACCACTATAACGAGTGCCAGAGGGCTCGCTCTACAAGTGGCGCGCCAGAGCCAGGTTTCCGGTGAAGGAATCCGAGTTAACGAGCGCGTATACATTATCGTGTGCTCTCCATCGT), and the base sequence shown in SEQ ID NO: 22 (ATTGGCCAACGCGCGCCAACGTGCCAACGTGTGCATATAACGAGTGCGCGCGGTTGCGCTCTACAAAATGCGCGC A polynucleotide consisting of AGAGCAGGTTCCGGTTGAAGGTATGAACCCGATTATACAGGCCTATCATTCGTTGTGTCTCATCGT is preferred, and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 23 (ATTGGTAGCAACCGCACGCACCTGTGTATATGAATGTTCCGTTGTTCGTATATAAATGTTCCGGTCAGAACAGGTTTCCGGTTGAAGGTATATGAATCCCGATTAATAGGCCATATCATCATTCGTTGTGTGTGCCAACCGT is more preferred.

[0042] 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 14 bases means that there is a deletion, substitution, or addition of 1 to 14 bases at any position in one to 14 base sequences within the same sequence, and two or more types of deletion, substitution, and addition may occur simultaneously. In (a2) above, the deleted, substituted, or added bases are preferably 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 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.

[0043] 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.

[0044] As the polynucleotide encoding the tag peptide, a polynucleotide encoding any of the peptides listed above (B1) to (B3), (C1) to (C3), (D1) to (D3), and (E1) to (E3) is preferred. In one embodiment, as the polynucleotide encoding the tag peptide, a polynucleotide containing one or two of the following (b1) to (b3), (c1) to (c3), (d1) to (d3), and (e1) to (e3) is preferred. (b1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 (b2) A polynucleotide consisting of a nucleotide sequence in which 1 to 6 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 8 (b3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 8 (c1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 (c2) A polynucleotide consisting of a nucleotide sequence in which 1 to 18 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 9 (c3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 9 (d1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 (d2) A polynucleotide consisting of a nucleotide sequence in which 1 to 33 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 10 (d3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 10 (e1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 11 (e2) A polynucleotide consisting of a nucleotide sequence in which 1 to 15 bases are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 11. (e3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 11.

[0045] A polynucleotide consisting of the base sequence shown in SEQ ID NO: 8 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: 9 is preferred as a polynucleotide encoding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3. A polynucleotide consisting of the base sequence shown in SEQ ID NO: 10 is preferred as a polynucleotide encoding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4. A polynucleotide consisting of the base sequence shown in SEQ ID NO: 11 is preferred as a polynucleotide encoding a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5.

[0046] The polynucleotides (b2), (b3), (c2), (c3), (d2), (d3), (e2) and (e3) described above are preferably polynucleotides encoding peptides that improve the extracellular secretion of the fusion peptide containing the tag peptide, and more preferably polynucleotides encoding peptides that improve the extracellular secretion and solubility of the fusion peptide. The polynucleotides (b2), (b3), (c2), (c3), (d2), (d3), (e2) and (e3) are also preferably polynucleotides encoding peptides that improve the expression stability of the fusion peptide containing the tag peptide in microorganisms (preferably Escherichia coli). In (b2) described above, the number of deleted, substituted or added bases 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 (c2) above, the number of deleted, substituted, or added bases is preferably 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 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. In (d2) above, the number of deleted, substituted, or added bases is preferably 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 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. In (e2) above, the number of deleted, substituted, or added bases is preferably 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 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.

[0047] The sequence identity in (b3), (c3), (d3), and (e3) 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.

[0048] In one embodiment, the polynucleotide of the present invention is preferably a polynucleotide comprising the polynucleotide of (b1), a polynucleotide encoding a His-tagged peptide, the polynucleotide of (c1), and the polynucleotide of (a1) from the 5' end to the 3' end.

[0049] 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, more preferably a polynucleotide comprising the base sequence shown in SEQ ID NO: 17, a polynucleotide comprising the base sequence shown in SEQ ID NO: 18, a polynucleotide comprising the base sequence shown in SEQ ID NO: 19, a polynucleotide comprising the base sequence shown in SEQ ID NO: 20, and a polynucleotide comprising the base sequence shown in SEQ ID NO: 21, and even more preferably a polynucleotide comprising the base sequence shown in SEQ ID NO: 17. The polynucleotide comprising the base sequence shown in SEQ ID NO: 17 is a polynucleotide comprising the polynucleotide of (b1), a polynucleotide encoding the His tag peptide, a polynucleotide of (c1), and a polynucleotide of (a1). The polynucleotide comprising the base sequence shown in SEQ ID NO: 18 is a polynucleotide comprising a base sequence in which three bases are added and 29 bases are substituted in the base sequence shown in SEQ ID NO: 9, and a polynucleotide comprising a base sequence in which 15 bases are substituted in the base sequence shown in SEQ ID NO: 7 (SEQ ID NO: 22). The polynucleotide consisting of the base sequence shown in Sequence ID No. 19 is a polynucleotide consisting of a base sequence in which 12 bases are substituted and 6 bases are added in the base sequence shown in Sequence ID No. 8, a polynucleotide encoding a His-tag peptide, a polynucleotide consisting of a base sequence in which 9 bases are added in the base sequence shown in Sequence ID No. 10, and a polynucleotide (Sequence ID No. 23) consisting of a base sequence in which 23 bases are substituted in the base sequence shown in Sequence ID No. 7. The polynucleotide consisting of the base sequence shown in Sequence ID No. 20 is a polynucleotide consisting of a base sequence in which 12 bases are substituted and 6 bases are added in the base sequence shown in Sequence ID No. 8, a polynucleotide encoding a His-tag peptide, the polynucleotide of (e1), and a polynucleotide (Sequence ID No. 23) consisting of a base sequence in which 23 bases are substituted in the base sequence shown in Sequence ID No. 7.The polynucleotide consisting of the base sequence shown in Sequence ID No. 21 is a polynucleotide consisting of a base sequence in which nine bases have been added to the base sequence shown in Sequence ID No. 10, and a polynucleotide (Sequence ID No. 23) consisting of a base sequence in which 23 bases have been substituted to the base sequence shown in Sequence ID No. 7.

[0050] The polynucleotides of the present invention can be obtained by known genetic engineering or synthetic methods.

[0051] 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.

[0052] 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 polynucleotide encoding the tag peptide, and preferred embodiments thereof are as described above. As the polynucleotide encoding the tag peptide, a polynucleotide containing one or two of the above (b1) to (b3), (c1) to (c3), (d1) to (d3), and (e1) to (e3) is preferred.

[0053] 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; and fungi such as yeasts of the Saccharomyces, Schizosaccharomyces, and Pichia genus, molds, and filamentous fungi. Among these, bacteria or fungi are preferred as host microorganisms, bacteria are more preferred, and Escherichia coli is even more preferred.

[0054] 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 a pET vector, pCold ＴＭ Examples of vectors include Takara Bio Inc., pQE vector (QIAGEN Inc.), pMW vector (Nippon Gene Inc.), and pACYC vector (Nippon Gene Inc.). When transforming fungi such as yeast, an example of a vector would be pYE22m.

[0055] 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 sequences (ori). For example, as promoters for bacterial expression vectors, the 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. can be used. 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] As described above, the transformant of the present invention can express a fusion peptide having the above-mentioned porosity-increasing effect or TMM expression-promoting effect by expressing the polynucleotide of the present invention. For this reason, the transformant of the present invention can be used to produce a fusion peptide having the porosity-increasing effect or TMM expression-promoting effect.

[0066] 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.

[0067] 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 the present 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 a component composition adjusted to promote the expression of the target fusion peptide.

[0068] The carbon source used in the culture medium is not particularly limited, as long as it is a carbon source that microorganisms can utilize. Examples of carbon sources include glycerol, sugar alcohols (such as mannitol), monosaccharides (such as glucose, fructose, and galactose), disaccharides (such as maltose), oligosaccharides (such as sucrose and lactose), and polysaccharides (such as starch and cellulose). One type of carbon source may be used, or a combination of two or more types may be used. When a carbon source is used in the culture medium, the amount of carbon source used (amount added) is preferably such that the concentration of the carbon source at the start of cultivation is 0.02 to 5% by weight, and more preferably 0.1 to 2% by weight.

[0069] 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 (e.g., soybean peptone), yeast autodegradation products, etc.). One type of nitrogen source may be used, or a combination of two or more types may be used. When a nitrogen source is used in the culture medium, the amount of nitrogen source used (amount added) is preferably such that the concentration of the nitrogen source at the start of cultivation is 0.01 to 5% by weight, and more preferably 0.1 to 2% by weight.

[0070] The culture medium preferably further contains a phosphorus source. The phosphorus source is not particularly limited as long as it is a phosphorus source that can be assimilated by microorganisms, for example, phosphorus pentoxide (P ２ O ５Examples include dipotassium hydrogen phosphate, dihydrogen dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate. One phosphorus source may be used, or a combination of two or more may be used. When a phosphorus source is used in the culture medium, the amount of phosphorus source used (amount added) is preferably such that the phosphorus source concentration at the start of culture is 0.3 to 2.0% by weight.

[0071] The liquid culture medium may contain components other than the carbon, nitrogen, and phosphorus sources mentioned above. For example, it is preferable that the medium contains appropriate minerals, vitamins, salts, cofactors, buffers (e.g., sodium citrate) to promote the expression of the fusion peptide. Antibiotics known to modulate catabolite inhibition (such as kanamycin, ampicillin, carbenicillin, and chloramphenicol) may also be incorporated into the medium.

[0072] The pH of the culture medium is preferably 5.0 to 9.5, and more preferably 6.0 to 9.0. For example, if the microorganism is Escherichia coli, the pH of the culture medium is preferably 5.0 to 9.5, 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.

[0073] 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.

[0074] The culture temperature should be set to a temperature suitable for the type of microorganism. For example, for E. coli, 20 to 50°C is preferred, and 25 to 40°C is more preferred.

[0075] 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 fusion peptide.

[0076] 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.

[0077] 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 a porosity-increasing effect or a TMM expression-promoting effect. The above-mentioned culture or crude purified fusion peptide can also be used as long as it achieves the effects of the present invention. The culture refers to any of the following: culture medium, cultured cells, or lysates of cultured cells.

[0078] 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, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5, the fusion peptide is usually accumulated in the culture medium. After the culturing is complete, 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 methods such as SDS-PAGE. In addition, the fusion peptide may be contained within the cultured bacterial cells as well as in the culture supernatant. If the fusion peptide accumulates within the cultured bacterial cells, after culturing, 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.

[0079] The fusion peptide of the present invention, its salt, or its solvate has the effect of increasing TMM expression. When the fusion peptide of the present invention, its salt, or its solvate is used in plants, TMM expression can be increased. As described above, when TMM expression is increased in plants, stomatal density increases. The fusion peptide of the present invention, its salt, or its solvate can be preferably used in plants. When the fusion peptide of the present invention, its salt, or its solvate is used in plants, it can increase stomatal density in those plants. The plants are not particularly limited, but it is preferable that they be at least one selected from the group consisting of Cucurbitaceae, Solanaceae, Convolvulaceae, Salicaceae, Rutaceae, Fabaceae, Rosaceae, Asteraceae, Amaranthaceae, Musaceae, Poaceae, and Amaryllidaceae. Examples of these plants are listed below. The fusion peptide of the present invention, its salt, or its solvate is preferably used in any of these plants to increase stomatal density or promote TMM expression in the plant. Among these, plants of the Cucurbitaceae, Solanaceae, and Convolvulaceae families are more preferred, with cucumber (Cucumis sativus) of the Cucurbitaceae family, tomato (Solanum lycopersicum) of the Solanaceae family, and sweet potato (Ipomoea batatas) of the Convolvulaceae family being even more preferred. In one embodiment, the fusion peptide of the present invention is preferably a peptide that increases stomatal density in Cucurbitaceae plants, more preferably cucumber (Cucumis sativus) of the Cucurbitaceae family. In one embodiment, the fusion peptide of the present invention is preferably a peptide that increases stomatal density or promotes TMM expression in Solanum plants, more preferably Solanum lycopersicum, of the Solanaceae genus.Furthermore, in one embodiment, the fusion peptide of the present invention is preferably a peptide that increases stomatal density or promotes TMM expression in plants of the Convolvulaceae family, more preferably sweet potato (Ipomoea batatas) of the Convolvulaceae family.

[0080] In plants, promoting TMM expression increases stomatal density, leading to increased carbon dioxide uptake and enhanced photosynthesis. Therefore, increased stomatal density in plants can result in increased yield (number of harvested plants) and higher sugar content (sucrose concentration). Furthermore, increased intracellular sugar content confers, improves, or induces tolerance to environmental stresses, such as osmotic stress (e.g., salt tolerance), drought tolerance, high temperature tolerance, and low temperature tolerance. Thus, increasing stomatal density in plants also provides a plant protection effect against environmental stresses.

[0081] The fusion peptide of the present invention, its salt, or its solvate is preferably used to promote the expression of sucrose transporters in plants. When the expression of sucrose transporters is promoted in plants, the translocation of sucrose to various organs of the plant is promoted, and the uptake of sucrose into cells increases. As a result, the sucrose content in cells increases, leading to an increase in sugar content. When the sucrose concentration in the plant increases, the osmotic pressure in the plant increases, which in turn 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 (cold tolerance). Sucrose transporters are present throughout the plant and transport synthesized sucrose to the phloem and translocate it to various organs of the plant. Therefore, promoting the expression of sucrose transporters in plants can result in an increase in sugar content and a plant protection effect against environmental stress. Thus, substances that promote the expression of sucrose transporters in plants are useful, for example, for increasing the sugar content of plants and for plant protection (e.g., improving the plant's tolerance to environmental stress).

[0082] The fusion peptides, salts thereof, or solvates thereof of the present invention may be used individually or in combination of two or more. The fusion peptides, salts thereof, or solvates thereof of the present invention can be used, for example, for plant cultivation. Furthermore, the fusion peptides, salts thereof, or solvates thereof of the present invention can be used to increase the sugar content of plants, for plant protection, or for increasing plant yield. The fusion peptides, salts thereof, or solvates thereof of the present invention can be applied to plants for plant cultivation, to increase sugar content, for plant protection, or for increasing plant yield. Methods for plant cultivation, methods for increasing plant sugar content, methods for plant protection, or methods for increasing plant yield by applying the fusion peptides, salts thereof, or solvates thereof to plants are also included in the present invention. The fusion peptides, salts thereof, or solvates thereof of the present invention can be used individually or in the form of compositions by mixing the fusion peptides, salts thereof, or solvates thereof with other components. In plant cultivation methods, the fusion peptide of the present invention, its salt, or its solvate may be applied directly to the plant, or a composition containing the fusion peptide, its salt, or its solvate may be applied to the plant. The fusion peptide, its salt, or its solvate may be used in combination with other agricultural materials such as fertilizers, soil conditioners, and horticultural potting soils. The plant cultivation method, method for increasing the sugar content of plants, method for protecting plants, or method for increasing plant yield of the present invention can be applied to plants such as vegetables, fruit trees, flowers, and trees. In plant cultivation methods, methods for increasing the sugar content of plants, methods for protecting plants, or methods for increasing plant yield of the present invention, plants can be cultivated using known methods appropriate to the plant, except for the application of the fusion peptide, its salt, or its solvate of the present invention.

[0083] 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-mentioned 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-mentioned fusion peptide of the present invention, a salt thereof, or a solvate thereof as an active ingredient.

[0084] The compositions of the present invention are preferably compositions used for plants (plant compositions). For example, the compositions of the present invention can be used to increase stomatal density in plants. In one embodiment, the compositions of the present invention can be used to promote the expression of TMM in plants. The compositions of the present invention can be preferably used, for example, to increase the sugar content of plants, protect plants, or increase plant yield. In one embodiment, the compositions of the present invention are preferably used as compositions for increasing the sugar content of plants, compositions for protecting plants, or compositions for increasing plant yield. 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. Compositions for increasing plant yield can also be called crop yield increasing agents.

[0085] Plant protection includes improving plant defense mechanisms and improving plant adaptive responses. Preferably, plant protection involves improving plant defense mechanisms or adaptive responses. The fusion peptides or salts thereof, or their solvates, and the compositions of the present invention can be used, for example, to improve plant defense mechanisms or adaptive responses. Improving plant defense mechanisms includes improving resistance to diseases and pests. Improving plant adaptive responses includes improving adaptive responses to environmental stress. Furthermore, plant protection includes improving tolerance to abiotic stress and improving tolerance to biological stress. Examples of tolerance to abiotic stress include tolerance to environmental stress, such as tolerance to osmotic stress (e.g., salt tolerance), drought tolerance, high temperature tolerance, and low temperature tolerance. Examples of biological stress include pathogens and pests. In one embodiment, the fusion peptides or salts thereof, or their solvates, and the compositions of the present invention can be preferably used to improve tolerance to abiotic stress such as environmental stress. In one embodiment, the plant protection composition is preferably a biostimulant. In one embodiment, the fusion peptide or salt thereof, or its solvate, and the compositions of the present invention can be preferably used to increase stomatal density in parts of plants, such as leaves. In another embodiment, the fusion peptide or salt thereof, or its solvate, and the compositions of the present invention can be preferably used to increase sugar content in parts of plants, such as fruits or roots (tubers).

[0086] 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 0.0005 to 10% by weight, and more preferably 0.001 to 2.5% by weight.

[0087] 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 dissolved or suspended in water at the time of use to obtain an appropriate concentration.

[0088] 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 0.0005 to 20% by weight, and more preferably 0.001 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.

[0089] 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 salts, or its solvates, such as water; alcohols such as 1-propanol and butanol; polyhydric alcohols such as ethylene glycol and propylene glycol; and hydrocarbons such as xylene.

[0090] 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.

[0091] 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 application or irrigation. A suitable formulation for foliar application is a liquid formulation. If the target plant is cultivated in soil, it is also preferable to irrigate the soil. When applying by irrigation, the fusion peptide or its salt, or its solvate, or the composition of the present invention, mixed with water, can be used to irrigate the plants. The total content of the fusion peptide or its salt, or its solvate, in the composition used for foliar application (preferably a liquid formulation) is preferably 0.0005 to 20% by weight, and more preferably 0.001 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 0.0005 to 20% by weight, and more preferably 0.001 to 2.5% by weight, as the content of the fusion peptide.

[0092] The timing of application of the fusion peptide or its salt or 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. The frequency of application of the fusion peptide or its salt or solvate, or the composition of the present invention, to plants is not particularly limited, but is preferably 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 solvate, or the composition of the present invention, 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 solvate, or the composition of the present invention, is preferably 0.01 to 20 mg per plant per application, and more preferably 0.05 to 10 mg.

[0093] 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 Cucurbitaceae family, Solanaceae family, Convolvulaceae family, Salicaceae family, Rutaceae family, Fabaceae family, Rosaceae family, Asteraceae family, Amaranthaceae family, Musaceae family, Poaceae family, Amaryllidaceae family, and the like. In the present invention, at least one plant selected from the group consisting of Cucurbitaceae, Solanaceae, Convolvulaceae, Salicaceae, Rutaceae, Fabaceae, Rosaceae, Asteraceae, Amaranthaceae, Musaceae, Poaceae, and Amaryllidaceae is preferred as the plant, with Cucurbitaceae, Solanaceae, or Convolvulaceae being more preferred.

[0094] Examples of plants in the Cucurbitaceae family include cucumbers (Cucumis sativus) of the genus Cucumis. Examples of plants in the Solanaceae family include tomatoes (Solanum lycopersicum) and potatoes (Solanum tuberosum) of the genus Solanum, with tomatoes being preferred. Examples of plants in the Convolvulaceae family include sweet potatoes (Ipomoea batatas) of the genus Ipomoea. Examples of plants in the Salicaceae family include poplars (Populus) of the genus Populus or Populus. Examples of plants in the Rutaceae family include the Satsuma mandarin (Citrus unshiu) of the Citrus genus. Examples of plants in the Fabaceae family include the pea (Pisum sativum) of the Pisum genus. Examples of plants in the Rosaceae family include the strawberry (Fragaria) of the Fragaria genus. Examples of plants in the Asteraceae family include the burdock (Arctium lappa) of the Arctium genus. Examples of plants in the Amaranthaceae family include the sugar beet (Beta vulgaris ssp. vulgaris) of the Beta vulgaris genus. Examples of plants in the Musaceae family include bananas (Musa spp.) of the genus Musa. Examples of plants in the Poaceae family include rice (Oryza sativa) of the genus Oryza; maize (Zea mays subsp. mays) of the genus Zea; and wheat (Triticum) of the genus Triticum. Examples of plants in the Amaryllidaceae family include onions (Allium cepa) of the genus Allium.

[0095] 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 promote TMM expression, increase stomatal density, or increase yield (number of harvested fruits) in cucumber (Cucumis sativus) of the Cucurbitaceae family. In another embodiment, the fusion peptide or salt thereof, or its solvate, or the composition of the present invention can be used, for example, to promote TMM expression, increase stomatal density, increase sugar content (preferably, increase sugar content of the fruit), or improve salt tolerance in tomato (Solanum lycopersicum) of the Solanaceae family. In yet another embodiment, the fusion peptide or salt thereof, or its solvate, or the composition of the present invention can be used, for example, to promote TMM expression, increase stomatal density, increase sugar content (preferably, increase sugar content of the root (tuber)), or improve salt tolerance in sweet potato (Ipomoea batatas) of the Convolvulaceae family.

[0096] Furthermore, as shown in the examples described later, the inventors' studies have revealed that the expression of various genes is promoted in plants to which the fusion peptide of the present invention or its salt, or its solvate (or a composition of the present invention containing the same) has been applied. Genes whose expression is promoted by application of the fusion peptide of the present invention or its salt, or its solvate (or a composition of the present invention containing the same) include amino acid biosynthesis, metabolism, and transport-related genes, autophagy-related genes, calcium signaling-related genes, carbohydrate metabolism, glycolysis, and sucrose synthesis-related genes, cell cycle-related genes, defense response and stress response-related genes, energy metabolism, ATP synthesis, and electron transport-related genes, hormone metabolism and signal transduction-related genes, lipid metabolism-related genes, membrane function-related genes, mitochondrial function-related genes, redox regulation and oxidative stress-related genes, nitrogen metabolism-related genes, transcription-related genes, transport and ion homeostasis-related genes, and water transport-related genes.

[0097] Genes related to amino acid biosynthesis, metabolism, and transport include glnA (GLUL), trpE, and cysK. glnA is a gene that codes for a subunit of glutamine synthase (GS), which plays an important role in nitrogen metabolism. trpE is a gene that codes for anthranilate synthase component I, a subunit that makes up anthranilate synthase, which is key to tryptophan biosynthesis. cysK is a gene that codes for cysteine ​​synthase, which is essential for sulfur metabolism.

[0098] Autophagy-related genes include ATG3, ATG8, and ULK2. ATG3 is a gene that encodes a ubiquitin-like enzyme (ubiquitin-conjugating enzyme ATG3) that is essential for autophagy membrane formation. ATG8 is a gene that encodes a ubiquitin-like protein (GABA(A) receptor-associated protein, LC3) that is used as a marker protein for autophagy. ULK2 is a gene that encodes a type of serine / threonine kinase (serine / threonine-protein kinase ULK2, ATG1) that is involved in autophagy.

[0099] Examples of calcium signaling-related genes include CALM, CPK, and CAMTA. CALM is the gene that codes for calmodulin, a representative calcium-binding protein. CPK is the gene that codes for creatine phosphokinase, a type of calcium-dependent protein kinase. CAMTA is the gene that codes for calmodulin-binding transcription activator.

[0100] Genes related to carbohydrate metabolism, glycolysis, and sucrose synthesis include pfkA, AMY, and TPS. pfkA is the gene that codes for PFK (6-phosphofructokinase 1), the rate-limiting enzyme in glycolysis. AMY is the gene that codes for α-amylase, known for its starch-breaking action. TPS is the gene that codes for trehalose 6-phosphate synthase (trehalose 6-phosphate synthase / phosphatease), which is involved in trehalose synthesis.

[0101] Cell cycle-related genes include CDK12, CDK13, CCNT, and TRIP13. CDK12 and CDK13 are genes that encode cyclin-dependent kinase 12 and cyclin-dependent kinase 13, respectively, which are involved in the regulation of the cell cycle. CCNT is a gene that encodes cyclin T, a type of transcription-related cyclin. TRIP13 is a gene that encodes thyroid receptor-interacting protein 13, which plays an important role in the chromosome segregation checkpoint.

[0102] Examples of genes related to defense and stress responses include NPR1, FLS2, and PR1. NPR1 is a major regulator of disease resistance and is the gene that codes for NPR1 (regulatory protein NPR1), a salicylate-dependent protein. FLS2 is the gene that codes for FLS2 (flagellin-sensitive 2), a receptor that recognizes flagellin, a type of protein found in bacteria. PR1 is the gene that codes for an infection-specific protein (Pathogenesis-related protein 1, PR1) and is a representative marker gene for disease response.

[0103] Genes related to energy metabolism, ATP synthesis, and electron transport include COX6B, CYC, and ATP5B (ATPeF1B). COX6B is the gene that codes for the cytochrome c oxidase subunit 6b, which is important in the respiratory chain. CYC is the gene that codes for cytochrome c, the central molecule of the electron transport chain. ATP5B is the gene that codes for the β subunit of ATP synthase.

[0104] Genes related to hormone metabolism and signal transduction include GH3, ERF1, and NCED. GH3 is an auxin-responsive gene that encodes an amidase that condenses amino acids with auxin. ERF1 is a gene that encodes ethylene-responsive transcription factor 1, which is involved in the stress response. NCED is a gene that encodes 9-cis-epoxycarotenoid dioxygenase (NCED), which is the rate-limiting enzyme in abscisic acid biosynthesis and is involved in the drought stress response.

[0105] The lipid metabolism-related genes include DGAT1, PLD1, PLD2, and ACOX1. DGAT1 is a key enzyme in triacylglycerol synthesis and encodes diacylglycerol O-acyltransferase 1, which is important for lipid accumulation. PLD1 and PLD2 are genes that encode Phospholipase D1 and Phospholipase D2, respectively, which are involved in phospholipid metabolism. ACOX1 is a gene that encodes Acyl-CoA Oxidase 1, the rate-limiting enzyme in fatty acid β-oxidation.

[0106] Examples of membrane function-related genes include TMEM33, STOML2 (SLP2), and TMEM222. TMEM33 is a gene that encodes transmembrane protein 33, which is involved in maintaining membrane structure. STOML2 is a gene that encodes stomatain-like protein 2, a mitochondrial membrane protein that plays an important role in the stress response. TMEM222 is a gene that encodes transmembrane protein 222.

[0107] Genes related to mitochondrial function include VDAC2, COX17, and PHB1. VDAC2 is a gene that encodes voltage-dependent anion channel protein 2 (VDAC2), a type of mitochondrial outer membrane channel that plays an important role in metabolism and apoptosis. COX17 is a gene that encodes a subunit of cytochrome c oxidase (COX), which is important in the respiratory chain (cytochrome c oxidase assembled protein subunit 17). PHB1 is a gene that encodes prohibitin 1, which is involved in maintaining the structure of the mitochondrial membrane.

[0108] Genes related to redox regulation and oxidative stress include SOD1, TXN (trxA), and GSR (gor). SOD1 is a gene that encodes superoxide dismutase, a central enzyme in the removal of reactive oxygen species (ROS) and an indicator of oxidative stress tolerance. TXN is a gene that encodes thioredoxin, which is important for maintaining the redox balance in cells and plays a central role in the antioxidant system. GSR is a gene that encodes glutathione reductase (NADPH), which reduces oxidized glutathione to produce reduced glutathione.

[0109] Genes related to nitrogen metabolism include nirA, cynS, and uaZ. nirA is the gene that encodes ferredoxin-nitrite reductase, which plays an important role in nitrogen assimilation. cynS is the gene that encodes cyanate lyase, which is involved in nitrogen metabolism. uaZ is the gene that encodes urate oxidase, which is involved in nitrogen metabolism.

[0110] Transcription-related genes include MYC2, WRKY22, and HD-ZIP. MYC2 is the gene that encodes the major transcription factor (transscription factor MYC2) of the jasmonic acid signaling pathway, which is important in the stress response. WRKY22 is the gene that encodes the transcription factor (WRKY transcription factor 22) involved in disease and stress responses. HD-ZIP is the gene that encodes the representative transcription factor (homeobox-leucine zipper protein) involved in plant development and environmental responses.

[0111] Genes related to transport and ion homeostasis include NRT2, SLAC1, and AMT. NRT2 is a gene that encodes a high-affinity nitrate transporter, which plays an important role in nitrogen uptake. SLAC1 is a gene that encodes an anion channel (S-type anion channel) involved in stomatal opening and closing. AMT is a gene that encodes an ammonium transporter, which plays an important role in nitrogen metabolism.

[0112] Examples of water transport-related genes include PIP, TIP, and NIP. PIP is a gene that encodes plasma membrane aquaporin (Plasma membrane intrinsic protein). TIP is a gene that encodes vacuolar membrane aquaporin (Tonoplast intrinsic protein). NIP is a gene that encodes aquaporin (Nodulin 26-like intrinsic protein) involved in the transport of silicon, boron, and other materials.

[0113] In the examples described later, the enhancement of the expression of each of the above-mentioned genes was confirmed by the application of the fusion peptide of the present invention or its salt, or its solvate (or a composition of the present invention containing the same), further supporting the fact that the fusion peptide of the present invention or its salt, or its solvate (or a composition of the present invention containing the same) can be preferably used to increase the sugar content of plants, protect plants, or increase plant yields.

[0114] This specification discloses the following: (1) This disclosure is 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 consisting of an amino acid sequence in which 1 to 4 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, and which has a porosity-increasing effect (A3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and which has a porosity-increasing effect

[0115] Disclosure (2) is a fusion peptide or a salt thereof, or a solvate thereof, as described in Disclosure (1), wherein the tag peptide is a peptide comprising one or two of the following (B1) to (B3), (C1) to (C3), (D1) to (D3) and (E1) to (E3). (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 2 amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ 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 SEQ 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 6 amino acids are deleted, substituted, or added to 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. (D1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 4. (D2) A peptide consisting of an amino acid sequence in which 1 to 11 amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 4. (D3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. (E1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 5. (E2) A peptide consisting of an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 5. (E3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 5.

[0116] (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 following polynucleotides (a1) to (a3): (a1) A polynucleotide consisting of the base sequence shown in Sequence ID No. 7; (a2) A polynucleotide consisting of a base sequence in which 1 to 14 bases are deleted, substituted, or added in the base sequence shown in Sequence ID No. 7, and which encodes a peptide that increases porosity density; (a3) ​​A polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence shown in Sequence ID No. 7, and which encodes a peptide that increases porosity density.

[0117] Disclosure (4) is the polynucleotide according to Disclosure (3), wherein the polynucleotide encoding the tag peptide is a polynucleotide comprising one or two of the following (b1) to (b3), (c1) to (c3), (d1) to (d3) and (e1) to (e3). (b1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 (b2) A polynucleotide consisting of a nucleotide sequence in which 1 to 6 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 8 (b3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 8 (c1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 (c2) A polynucleotide consisting of a nucleotide sequence in which 1 to 18 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 9 (c3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 9 (d1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 (d2) A polynucleotide consisting of a nucleotide sequence in which 1 to 33 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 10 (d3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 10 (e1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 11 (e2) A polynucleotide consisting of a nucleotide sequence in which 1 to 15 bases are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 11. (e3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 11.

[0118] Disclosure (5) is an expression vector comprising the polynucleotide described in Disclosure (3) or (4).

[0119] Disclosure (6) is the expression vector described in Disclosure (5), wherein the polynucleotide encoding the tag peptide is a polynucleotide comprising one or two of the following (b1) to (b3), (c1) to (c3), (d1) to (d3) and (e1) to (e3). (b1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 (b2) A polynucleotide consisting of a nucleotide sequence in which 1 to 6 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 8 (b3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 8 (c1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 (c2) A polynucleotide consisting of a nucleotide sequence in which 1 to 18 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 9 (c3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 9 (d1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 (d2) A polynucleotide consisting of a nucleotide sequence in which 1 to 33 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 10 (d3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 10 (e1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 11 (e2) A polynucleotide consisting of a nucleotide sequence in which 1 to 15 bases are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 11. (e3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 11.

[0120] Disclosure (7) is a transformant which is a microorganism into which a polynucleotide described in Disclosure (3) or (4) or an expression vector described in Disclosure (5) or (6) has been introduced.

[0121] Disclosure (8) is a transformant of Disclosure (7) wherein the microorganism is a bacterium or a fungus.

[0122] 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).

[0123] Disclosure (10) is a composition comprising a fusion peptide or a salt thereof, or a solvate thereof, as described in Disclosure (1) or (2).

[0124] The present disclosure (11) is the composition described in the present disclosure (10), which is a composition for increasing the sugar content of plants, a composition for protecting plants, or a composition for increasing the yield of plants.

[0125] The present disclosure (12) is a composition according to the present disclosure (11), wherein plant protection is an improvement of the plant's defense mechanism or adaptive response.

[0126] The present disclosure (13) is a composition according to the present disclosure (11) or (12), wherein the plant protection composition is a biostimulant.

[0127] Disclosure (14) is a composition according to any one of Disclosures (10) to (13) that is applied to plants by foliar spraying or irrigation.

[0128] The present disclosure (15) 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.

[0129] The present disclosure (16) is a method for cultivating the plant described in the present disclosure (15), wherein the plant is at least one selected from the group consisting of Cucurbitaceae plants, Solanaceae plants, Convolvulaceae plants, Salicaceae plants, Rutaceae plants, Fabaceae plants, Rosaceae plants, Asteraceae plants, Amaranthaceae plants, Musaceae plants, Poaceae plants, and Amaryllidaceae plants.

[0130] The present disclosure (17) is the use of a fusion peptide or salt thereof, or a solvate thereof, as described in the present disclosure (1) or (2), for increasing the sugar content of plants, protecting plants, or increasing plant yields.

[0131] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0132] <Example 1> Cloning, Expression, and Purification of Tag+Stomagen A polynucleotide encoding a fusion peptide in which a tag peptide was bound to the N-terminus of Stomagen was prepared and expressed in E. coli. Pelb tag peptide, Fh8 tag, ZZ tag peptide, Z tag peptide, and His tag peptide were used as tag peptides. The amino acid sequences of Pelb tag peptide, Fh8 tag, ZZ tag peptide, Z tag peptide, and His tag peptide are described below. Pelb-tagged peptide: MKKKKPTAAAGLLLLLAAQPAMA (SEQ ID NO: 2) Fh8-tagged peptide: PSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSILSS (SEQ ID NO: 3) ZZ-tagged peptide: DNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPKV (SEQ ID NO: 4) Z-tag peptide: DNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPKV (SEQ ID NO: 5) His-tag peptide: HHHHHH (SEQ ID NO: 6)

[0133] Preparation of PelB-His-Fh8-Stomagene A polynucleotide (PelB-His-Fh8-Stomagene) was prepared as a polynucleotide encoding a fusion peptide containing a PelB-tagged peptide (SEQ ID NO: 2), a His-tagged peptide (SEQ ID NO: 6), an Fh8 tag (SEQ ID NO: 3), and a Stomagene (SEQ ID NO: 1), with the base sequence shown in SEQ ID NO: 17. PelB-His-Fh8-Stomagen:ATGAAAAAAGAAAAAAACCCACAGCAGCTGCGGGCCTGTTGTTGTTGGCGGCACAGCCGGCTATGGCGCATCACCACCACCACCACCCAAGCG TTCAAGAAGTGGAGAAAACTGCTGCATGTGCTGGACCGTAATGGTGATGGTAAAGTTTCCGCGGAAGAACTGAAAGCCTTCGCCGATGACTCCAAATGCCCGCTGGACAGCAAT AAGATCAAGGCGTTTATCAAAAGAGCCAACGATAAAAAGGAGCCAAGTCCGACCTTTAAGGAGCGTGTTCCGATCCCTGTGTTAGCCATTTGGTAGCCAGCGCTCCGACCCTGTATACGAGTGCCAGAAGGCGCTCGCGCTAACAAGTGCGCGCCAGAAGCAGGTTTCCCCGGTGAAGTAACGATAGCGCGTATACCAGCGCGTATCCGTGCTCTCCATTCGT (Sequence ID 17) The amino acid sequence of the fusion peptide encoded by the above polynucleotide (PelB-His-Fh8-Stomagent) is shown below. The underlined portion is the amino acid sequence of Stomagen (Sequence ID 1). MKKKKPTAAAGLLLLLAAQPAMAHHHHHHPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSILSSIGSTAPTTCTYNEECRGCRYKCRAEEQVPVEGNDPINSAYHYRCVCHR (Sequence ID 12)

[0134] To produce Fh8-Stomagen, a polynucleotide with the base sequence shown in Sequence ID No. 18 (Fh8-Stomagen) was produced as a polynucleotide encoding a fusion peptide containing a peptide consisting of the amino acid sequence shown in Sequence ID No. 3 (Fh8 tag) with one amino acid added (a peptide in which methionine is bound to the N-terminus of the peptide consisting of the amino acid sequence shown in Sequence ID No. 3 (Fh8 tag)) and Stomagen (Sequence ID No. 1). Fh8-Stomagen: ATGCCCTCAGTTCAAGAAGTAGAGAAAACTATTACACGTGCTGGACCGTAATGGTGATGGCAAGGTGAGCGCGGAGGA ACTGAAGGCGTTCGCCGATGATTCCAAATGCCCGCTGGACTCCAACAAGATCAAGGCGTTTATCAAAGAGCACGATAAAAAAAGGGACGGC AAGCTGGACCTGAAAAGAATTTGGTTTCGAATCTGTCTAGCATTTGGGCACGCGCCCACACGTGCACACTATACGAGTGGCCGCGGTTGGCCGTTAACAAATGGCCGCGCAGAGCAGGTTTCCCGGTTGAAGGTTAATGAACCGCGAATTAACAGGCTTATACCATTTATCGTTGTGTCTTCCATCGT (Sequence ID 18) In the above sequence ID 18, the ununderlined portion (numbers 1 to 207) is a polynucleotide consisting of a sequence in which 3 bases are added and 29 bases are substituted in the sequence shown in sequence ID 9. The underlined portion is a polynucleotide (Sequence ID 22) consisting of a sequence in which 15 bases are substituted in the sequence shown in sequence ID 7. The amino acid sequence (SEQ ID NO: 13) of the fusion peptide encoded by the above polynucleotide (Fh8-Stomagegen) is shown below. The underlined portion is the amino acid sequence of Stomagegen (SEQ ID NO: 1). MPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSILSSIGSTAPTTCTYNECRGCRYKCRAEEQVPVEGNDPINSAYHYRCVCHR (SEQ ID NO: 13)

[0135] To create PelB-His-ZZ-Stomagegen, a polynucleotide with the base sequence shown in SEQ ID NO: 19 (PelB-His-ZZ-Stomagegen) was created as a polynucleotide encoding a fusion peptide containing a peptide with an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 2 (PelB-tagged peptide), a peptide with an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 4 (ZZ-tagged peptide), a peptide with an amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 4, a peptide with an amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of SEQ ID NO: 4, a peptide with an amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of the amino acid sequence consisting of SEQ ID NO: 2, a His-tagged peptide (SEQ ID NO: 6), and Stomagegen (SEQ ID NO: 1). PelB-His-ZZ-Stomagen:ATGAAAAAAGAAAAAAACCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAAC CTGCCATGGCCGAAGAACATCATCATCACCATCACGATAACAAAATTTAACAAAGAACAGCAGAACGCCTTCTACGA AATTCTGCATCTGCCGAATCTGAATGAAGAACAGCGTAATGCATTTATCCAGAGCCTGAAAGATGATCCGAGCCAG AGCGCAATCTGCTGGCCGAAGCAAAAAAACTGAATGATGCACAGGCACCGAAAGTCGACAATAAGTTCAATAAAGA GCAACAGAACGCGTTTTATGAGATCCTGCATTTACCGAACTTAAACGAGGAACAGCGCAACGCGTTTATTCAGTCA CTGAAAAGACGACCCGTCACAGTCAGCCAACCTGCTGGCGGAGGCCAAAAGTTAAACGATGCACAAGCCCCTAAGGT CGACGGATCCATTTGGTAGCGCGCACCGACCTGTTAACCTAATGAATGTTCGTGGTTGTTCGTTTATAAATGTTCGTGTGCCAGAACAGGTTCCCGGTTGAAGGTTAATGATCCGATTAATAGGCCATATCATCATTCGTTTGTGTGTGCCAACCGT (Sequence ID 19) In the above sequence ID 19, the underlined portion at the 5' end (1st to 72nd) is a polynucleotide consisting of a sequence in which 12 bases are substituted and 6 bases are added in the sequence shown in sequence ID 8.In the base sequence of Sequence ID No. 19, the region from positions 91 to 447 is a polynucleotide consisting of a base sequence in which nine bases have been added to the base sequence shown in Sequence ID No. 10. In the base sequence of Sequence ID No. 19, the underlined portion at the 3' end (positions 448 to 582) is a polynucleotide (Sequence ID No. 23) consisting of a base sequence in which 23 bases have been substituted to the base sequence shown in Sequence ID No. 7. The amino acid sequence (Sequence ID No. 14) of the fusion peptide encoded by the above polynucleotide (PelB-His-ZZ-Stomagent) is shown below. The underlined portion is the amino acid sequence of Stomagent (Sequence ID No. 1). MKKKKPTAAAGLLLLLAAQPAMAEEHHHHHHDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPKVDGSIGSTAPTTCTYNECRGCRYKCRAEQVPVEGNDPINSAYHYRCVCHR (Sequence ID 14).

[0136] To create PelB-His-Z-Stomagegen, a polynucleotide with the base sequence shown in Sequence ID No. 20 (PelB-His-Z-Stomagegen) was created as a polynucleotide encoding a fusion peptide containing a peptide with an amino acid sequence obtained by adding two amino acids to a peptide consisting of the amino acid sequence shown in Sequence ID No. 2 (PelB-tagged peptide), a His-tagged peptide (Sequence ID No. 6), a Z-tagged peptide (Sequence ID No. 5), and Stomagegen (Sequence ID No. 1). PelB-His-Z-Stomagen:ATGAAAAAAGAAAAAAACCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAACCTGCCATGGCCGAAGAACATCATCATCACCA TCACGATAACAAATTTAACAAAAGAACAGCAGAACGCCTTCTACGAAATTCTGCATCTGCCGAATCTGAATGAAGAACAGCGTAATGCATTTATCCAGAGCCTGAAAG ATGATCCGAGCCCAGAGCGCCAAAAATCTGCCTGGCCGAAGCAAAAAAAACTGAATGAATGCCAACAGGCACGCGAAAAAGTCCATTTGGTAGCCAACCGCACGCGTGTACCTAATGAATGTTCCGGTTTGTTCCGTTATATAAATGTTCCGGTGCCAGAACAGGTTCCCCGGTGAAGGTTAATGAATCCCGATATATAGGCCATATATCATGTGTGTGTGTGCCAACCGT (Sequence ID 20) In the above sequence ID 20, the underlined portion at the 5' end (1st to 72nd) is a polynucleotide consisting of a sequence in which 12 bases are substituted and 6 bases are added in the sequence shown in sequence ID 8. In the nucleotide sequence of Sequence ID No. 20 shown above, the underlined portion at the 3' end (positions 265-399) is a polynucleotide (Sequence ID No. 23) consisting of a nucleotide sequence in which 23 bases have been substituted in the nucleotide sequence shown in Sequence ID No. 7. The amino acid sequence (Sequence ID No. 15) of the fusion peptide encoded by the above polynucleotide (PelB-His-Z-Stomagent) is shown below. The underlined portion is the amino acid sequence of Stomagent (Sequence ID No. 1).MKKKKPTAAAGLLLLLAAQPAMAEEHHHHHHDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPKVIGSTAAPTCTYNEECRGCRYKCRAEQVPVEGNDPINSAYHYRCVCHR (Sequence ID 15).

[0137] To create ZZ-Stomagen, a polynucleotide with the base sequence shown in Sequence ID No. 21 (ZZ-Stomagen) was created as a polynucleotide encoding a peptide consisting of an amino acid sequence in which three amino acids were added to a peptide consisting of the amino acid sequence shown in Sequence ID No. 4 (ZZ-tag peptide), and a fusion peptide containing Stomagen (Sequence ID No. 1). ZZ-Stomagen: ATGGATAACAAATTTAACAAAGAACAGCAGAACGCCTTCTACGAAATTCTGC ATCTGCCGAATCTGAATGAAGAACAGCGTAATGCATTTATCCAGAGCCTGAAAGATGATCCGAG CCAGAGCGCAAATCTGCTGGCCGAAGCAAAAAAACTGAATGATGCACAGGCACCGAAAGTCGAC AATAAGTTCAATAAAGAGCAACAGAACGCGTTTTATGAGATCCTGCATTTACCGAACTTAAACG AGGAACAGCGCAACGCGTTTATTCAGTCACTGAAAGACGACCCGTCACAGTCAGCCAACCTGCT GGCGGAGGCCAAAAGTTAAACGATGCACAAGCCCCTAAGGTCGGATCCATTGGTAGCACCGCA CCGACCTGTACCTAATAATGAATGTCGTGGTTGTCGTTATAAAATGTCGTGCAGAACAGGTTCCGG TTGAAGGTAATGATCCGATTAATAGCGCATATCATTATCGTTGTGTGCCACCGT (SEQ ID NO: 21) In the nucleotide sequence of Sequence ID No. 21 above, the ununderlined portion (positions 1 to 357) is a polynucleotide consisting of a nucleotide sequence in which 9 bases have been added to the nucleotide sequence shown in Sequence ID No. 10. In the nucleotide sequence of Sequence ID No. 21 above, the underlined portion is a polynucleotide (Sequence ID No. 23) consisting of a nucleotide sequence in which 23 bases have been substituted to the nucleotide sequence shown in Sequence ID No. 7. The amino acid sequence (Sequence ID No. 16) of the fusion peptide encoded by the above polynucleotide (ZZ-Stomagent) is shown below. The underlined portion is the amino acid sequence of Stomagent (Sequence ID No. 1).MDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPKVGSIGSSTAAPTCTYNECRGCRYKCRAEQVPVEGNDPINSAYHYRCVCHR (Sequence ID 16).

[0138] Vector cloning Plasmid having a nucleotide sequence encoding a fusion peptide containing Stomagen (PelB-His-Fh8-Stomagen (SEQ ID NO: 17), Fh8-Stomagen (SEQ ID NO: 18), PelB-His-ZZ-Stomagen (SEQ ID NO: 19), PelB-His-Z-Stomagen (SEQ ID NO: 20), or ZZ-Stomagen (SEQ ID NO: 21)) We performed contract synthesis of the following products (name: pEX-A2J2-PelB-His-Fh8-Stomagen, pEX-A2J2-Fh8-Stomagen, pEX-A2J2-PelB-His-ZZ-Stomagen, pEX-A2J2-PelB-His-Z-Stomagen, pEX-A2J2-ZZ-Stomagen) (Eurofins Genomics Co., Ltd.). pEX-A2J2-PelB-His-Fh8-Stomagen is a plasmid in which PelB-His-Fh8-Stomagen (SEQ ID NO: 17) is incorporated into the pEX-A2J2 vector. pEX-A2J2-Fh8-Stomagent is a plasmid in which Fh8-Stomagent (SEQ ID NO: 18) is incorporated into the pEX-A2J2 vector. pEX-A2J2-PelB-His-ZZ-Stomagent is a plasmid in which PelB-His-ZZ-Stomagent (SEQ ID NO: 19) is incorporated into the pEX-A2J2 vector. pEX-A2J2-PelB-His-Z-Stomagent is a plasmid in which PelB-His-Z-Stomagent (SEQ ID NO: 20) is incorporated into the pEX-A2J2 vector. pEX-A2J2-ZZ-Stomagent is a plasmid in which ZZ-Stomagent (SEQ ID NO: 21) is incorporated into the pEX-A2J2 vector.

[0139] pEX-A2J2-PelB-His-Fh8-Stomagent, pEX-A2J2-Fh8-Stomagent, pEX-A2J2-PelB-His-ZZ-Stomagent, pEX-A2J2-PelB-His-Z-Stomagent, pEX-A2J2-ZZ-Stomagent, and pET22b were digested with restriction enzymes NdeI and HindIII, and then subjected to agarose gel electrophoresis. The following fragments were recovered from the gel: PelB-His-Fh8-Stomagen (423 bp), Fh8-Stomagen (342 bp), PelB-His-ZZ-Stomagen (582 bp), PelB-His-Z-Stomagen (399 bp), ZZ-Stomagen (492 bp), and pET22b vector fragment (5400 bp).

[0140] Each DNA fragment was extracted from the gel, and the PelB-His-Fh8-Stomagen fragment was ligated with the pET22b vector fragment to obtain pET22b(PelB-His-Fh8-Stomagen). The Fh8-Stomagen fragment was also ligated with the pET22b vector fragment to obtain pET22b(Fh8-Stomagen). Furthermore, the PelB-His-ZZ-Stomagen fragment was ligated with the pET22b vector fragment to obtain pET22b(PelB-His-ZZ-Stomagen). Furthermore, the PelB-His-Z-Stormagen fragment and the pET22b vector fragment were ligated to obtain pET22b(PelB-His-Z-Stormagen). Also, the ZZ-Stormagen fragment and the pET22b vector fragment were ligated to obtain pET22b(ZZ-Stormagen). The obtained pET22b(PelB-His-Fh8-Stomagent), pET22b(Fh8-Stomagent), pET22b(PelB-His-ZZ-Stomagent), pET22b(PelB-His-Z-Stomagent), and pET22b(ZZ-Stomagent) were introduced into Escherichia coli DH5a strains for transformation, and the colonies were sorted using ampicillin-containing LB agar plates.

[0141] Colony PCR was performed using T7pro (5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO: 24)) and T7term (5'-ATGCTAGTTTATTGCTCAGCGG-3' (SEQ ID NO: 25)) primers to confirm the introduction of PelB-His-Fh8-Stomagent, Fh8-Stomagent, PelB-His-ZZ-Stomagent, PelB-His-Z-Stomagent, and ZZ-Stomagent, respectively. Furthermore, colonies in which the introduction of PelB-His-Fh8-Stomagen, Fh8-Stomagen, PelB-His-ZZ-Stomagen, PelB-His-Z-Stomagen, or ZZ-Stomagen was confirmed were grown, and pET22b(PelB-His-Fh8-Stomagen), pET22b(Fh8-Stomagen), pET22b(PelB-His-ZZ-Stomagen), pET22b(PelB-His-Z-Stomagen), and pET22b(ZZ-Stomagen) were recovered from the bacterial cells, and it was confirmed that the target base sequence had been introduced. The base sequences of each pET22b (PelB-His-Fh8-Stomagen), pET22b (Fh8-Stomagen), pET22b (PelB-His-ZZ-Stomagen), pET22b (PelB-His-Z-Stomagen), and pET22b (ZZ-Stomagen) were confirmed in E. coli Lemo21 (DE3) strain (New England Biolabs Japan). By introducing and transforming the cells (purchased from Inc.), we obtained Lemo21(DE3)_pET22b(PelB-His-Fh8-Stomagent), Lemo21(DE3)_pET22b(Fh8-Stomagent), Lemo21(DE3)_pET22b(PelB-His-ZZ-Stomagent), Lemo21(DE3)_pET22b(PelB-His-Z-Stomagent), and Lemo21(DE3)_pET22b(ZZ-Stomagent). The transformed Lemo21(DE3)_pET22b(PelB-His-Fh8-Stomagent) was pre-cultured in LB medium (2-5 mL) at 30°C with shaking (150 rpm) for 18-24 hours. The pre-culture solution was then inoculated into the main medium (70-90 mL).LB medium was used for this culture medium. The pH of this medium was adjusted to 6.5-7.0 by adding lactic acid or ammonia. The transformants Lemo21(DE3)_pET22b(Fh8-Stomagent), Lemo21(DE3)_pET22b(PelB-His-ZZ-Stomagent), Lemo21(DE3)_pET22b(PelB-His-Z-Stomagent), and Lemo21(DE3)_pET22b(ZZ-Stomagent) were also cultured with shaking under the same conditions as Lemo21(DE3)_pET22b(PelB-His-Fh8-Stomagent).

[0142] In this culture, the pH was maintained at 6.5–7.0 for 9–12 hours after the start of culture. Then, the pH was increased to 8.0–9.5 and maintained until the end of culture. The cell density was adjusted so that 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. 22–26 hours after the addition of IPTG, the culture medium in the culture vessel was centrifuged (13000 rpm) to separate the supernatant fraction from the precipitate. The fusion peptide contained in the supernatant and precipitate was confirmed by SDS-PAGE.

[0143] Figure 1A shows the results of SDS-PAGE analysis of the expression of the fusion peptide (PelB-His-Fh8-Stomagen). Figure 1B shows the results of SDS-PAGE analysis of the expression of the fusion peptide (Fh8-Stomagen). Figure 1C shows the results of SDS-PAGE analysis of the expression of the fusion peptide (PelB-His-ZZ-Stomagen). Figure 1D shows the results of SDS-PAGE analysis of the expression of the fusion peptide (PelB-His-Z-Stomagen). Figure 1E shows the results of SDS-PAGE analysis of the expression of the fusion peptide (ZZ-Stomagen). In Figures 1A, 1B, 1C, 1D, and 1E, "sup" indicates the supernatant fraction and "pellet" indicates the precipitate. SDS-PAGE revealed bands for the fusion peptide (PelB-His-Fh8-Storomagen) (Molecular Weight: 15.0 kDa), fusion peptide (Fh8-Storomagen) (Molecular Weight: 12.8 kDa), fusion peptide (PelB-His-ZZ-Storomagen) (Molecular Weight: 23.0 kDa), fusion peptide (PelB-His-Z-Storomagen) (Molecular Weight: 15.0 kDa), and fusion peptide (ZZ-Storomagen) (Molecular Weight: 20.8 kDa). For each peptide, a band was present in the supernatant fraction (sup), confirming that it was secreted 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, and the concentrations were 1.3 g / L for the fusion peptide (PelB-His-Fh8-Stomagent), 1.1 g / L for the fusion peptide (Fh8-Stomagent), 1.1 g / L for the fusion peptide (PelB-His-ZZ-Stomagent), 1.1 g / L for the fusion peptide (PelB-His-Z-Stomagent), and 1.1 g / L for the fusion peptide (ZZ-Stomagent).

[0144] The purified supernatant fraction is then processed in a desalting and concentration apparatus (AKTA flux). ＴＭDesalting and concentration were performed using a UF membrane (MWCO 5000) with Cytiva (Global Life Science Technologies Japan Co., Ltd.). Electrical conductivity was 5 mS / cm. ２ Desalting was performed until the following was achieved. In this way, the fusion peptide (PelB-His-Fh8-Stomagen) (a fusion peptide in which the PelB tag peptide, His tag peptide, and Fh8 tag are attached to the N-terminus of Stomagen), fusion peptide (Fh8-Stomagen) (a fusion peptide in which the Fh8 tag is attached to the N-terminus of Stomagen), and fusion peptide (PelB-His-ZZ-Stomagen) (the N-terminus of Stomagen) We obtained a fusion peptide in which PelB-tagged peptide, His-tagged peptide, and ZZ-tagged peptide were bound, a fusion peptide (PelB-His-Z-Stomagen) (a fusion peptide in which PelB-tagged peptide, His-tagged peptide, and Z-tagged peptide were bound to the N-terminus of Stomagen), and a fusion peptide (ZZ-Stomagen) (a fusion peptide in which ZZ-tagged peptide was bound to the N-terminus of Stomagen).

[0145] Application of Fusion Peptides and Evaluation of Gene Expression In Examples 2 to 10 below, the fusion peptides obtained in Example 1 were applied to plants, and their effects on the gene expression of TOO MUCH MOUTH (TMM) were investigated. Peptide solutions were prepared by mixing the fusion peptide (PelB-His-Fh8-Stomagent) with deionized water, the fusion peptide (Fh8-Stomagent) with deionized water, the fusion peptide (PelB-His-ZZ-Stomagent) with deionized water, the fusion peptide (PelB-His-Z-Stomagent) with deionized water, and the fusion peptide (ZZ-Stomagent) with deionized water, and these solutions were applied to plants.

[0146] <Example 2> Preparation of cucumber seedlings and application of fusion peptide (1) Cucumber seeds were placed one by one into Jiffy-7 (manufactured by Sakata Seed Corporation, the same applies to the following examples) and watered by bottom watering. The sown cucumbers 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 to 8:00 AM; 20°C, 8:00 AM to 9:00 AM; 22.5°C, 9:00 AM to 7:00 PM; 25°C; humidity 60%), 12 hours of dark (7:00 PM to 7:00 AM; 16°C; humidity 60%)). Seedlings with approximately four true leaves were selected, and a 10 μM fusion peptide (PelB-His-Fh8-Stomagen) solution was foliar-sprayed at a rate of 2 mL per plant. Cucumber leaves were collected by punching them out at 0 hours (immediately after spraying), 2 hours, 6 hours, and 18 hours after spraying the peptide solution. The concentration of the fusion peptide (PelB-His-Fh8-Stomagen) in the peptide solution was 0.015% by weight.

[0147] <Example 3> Preparation of cucumber seedlings and application of fusion peptide (2) Cucumber seeds were placed one by one in Jiffy-7 pellets and watered by bottom watering. The sown cucumbers were cultivated in an artificial climate chamber (12 hours of light (7:00 AM to 8:00 AM; 20°C, 8:00 AM to 9:00 AM; 22.5°C, 9:00 AM to 7:00 PM; 25°C; humidity 60%), 12 hours of dark (7:00 PM to 7:00 AM; 16°C; humidity 60%)). Seedlings with about four true leaves were selected and foliar sprayed with water (peptide solution) containing 10 μM fusion peptide (PelB-His-Fh8-Stomagen) at a rate of 2 mL / plant. Cucumber leaves were collected by punching them out at 0 days (immediately after spraying), 1 day (20-24 hours later), 2 days (45-48 hours later), and 5 days (116-120 hours later) after spraying the peptide solution. The concentration of the fusion peptide (PelB-His-Fh8-Stomagent) in the peptide solution was 0.015% by weight.

[0148] <Example 4> Preparation of tomato (variety: Regina) seedlings and application of peptide (1) Paper wipes (Kimwipes, Nippon Paper Crecia Co., Ltd., the same applies to the following examples) were moistened with water and placed in a petri dish, and tomato seeds were sown on top of them. The sown tomatoes were cultivated in an artificial climate chamber (12 hours of light (7:00 AM to 8:00 AM; 20°C, 8:00 AM to 9:00 AM; 22.5°C, 9:00 AM to 7:00 PM; 25°C; humidity 60%), 12 hours of dark (7:00 PM to 7:00 AM; 16°C; humidity 60%)). Two weeks after the start of cultivation, the germinated seedlings were transplanted into Jiffy-7 pellets and continued to be cultivated in the artificial climate chamber under the above conditions. Tomato plants 40 days old after sowing were foliar-sprayed with water (peptide solution) containing 10 μM of the fusion peptide (Fh8-Stomagen prepared in Example 1) at a rate of 2 mL / plant. Tomato leaves were collected by punching them out 7, 24, and 48 hours after spraying the peptide solution. The concentration of the fusion peptide (Fh8-Stomagen) in the peptide solution was 0.013% by weight.

[0149] <Comparative Example 1> Except that ion-exchanged water (which does not contain the above-mentioned fusion peptide) was used instead of the peptide solution in Example 4, tomatoes were grown from seed in the same manner as in Example 4, and the tomato leaves were collected by punching them out.

[0150] <Example 5> Preparation of tomato (variety: Regina) seedlings and application of peptide (2) A paper cloth was moistened with water and placed in a petri dish, and tomato seeds were sown on it. The sown tomatoes were cultivated in an artificial climate chamber (12 hours of light (7:00 AM to 8:00 AM; 20°C, 8:00 AM to 9:00 AM; 22.5°C, 9:00 AM to 7:00 PM; 25°C; humidity 60%), 12 hours of dark (7:00 PM to 7:00 AM; 16°C; humidity 60%)). Two weeks after the start of cultivation, the germinated seedlings were transplanted into Jiffy-7 pellets and continued to be cultivated in the artificial climate chamber under the above conditions. Tomato plants 40 days old after sowing were foliar-sprayed with water containing 10 μM of the fusion peptide (PelB-His-Fh8-Stomagen) (peptide solution) at a rate of 2 mL / plant. Tomato leaves were collected by punching them out 7, 24, and 48 hours after spraying the peptide solution. The concentration of the fusion peptide (PelB-His-Fh8-Stomagen) in the peptide solution was 0.015% by weight.

[0151] <Comparative Example 2> Except that ion-exchanged water (which does not contain the above-mentioned fusion peptide) was used instead of the peptide solution in Example 5, tomatoes were grown from seed in the same manner as in Example 5, and the tomato leaves were collected by punching them out.

[0152] <Reference Example 1> In the preparation of PelB-His-Fh8-Stomagen in Example 1, the vector was cloned (culture of Lemo21(DE3)_pET22b (PelB-His-Fh8)) and the supernatant fraction was purified in the same manner as in Example 1, except that the polynucleotide encoding Stomagen (SEQ ID NO: 1) was deleted, to obtain PelB-His-Fh8 peptide (a fusion peptide to which PelB-tagged peptide, His-tagged peptide, and Fh8 tag are bound). In Example 5, tomatoes were grown from seed in the same manner as in Example 5, except that instead of a peptide solution, water containing 10 μM PelB-His-Fh8 peptide (a peptide solution that does not contain the Stomagen region) was used for cultivation (negative control), and the tomato leaves were punched out and collected.

[0153] <Example 6> Preparation of tomato (variety: Regina) seedlings and application of peptide (3) A paper cloth was moistened with water and placed in a petri dish, and tomato seeds were sown on it. The sown tomatoes were cultivated in an artificial climate chamber (12 hours of light (7:00 AM to 8:00 AM; 20°C, 8:00 AM to 9:00 AM; 22.5°C, 9:00 AM to 7:00 PM; 25°C; humidity 60%), 12 hours of dark (7:00 PM to 7:00 AM; 16°C; humidity 60%)). Two weeks after the start of cultivation, the germinated seedlings were transplanted into Jiffy-7 pellets and continued to be cultivated in an artificial climate chamber under the above conditions. Tomatoes 40 days old were treated with a fusion peptide (PelB-His-Fh8-Stomagent) by one of the following methods. (Method 1) Spray 2 mL / plant with water containing 10 μM fusion peptide (peptide solution) as a foliar spray. (Method 2) Irrigate the base of plants with 2 mL / plant with water containing 10 μM fusion peptide (peptide solution). (Method 3) Irrigate the base of plants with 2 mL / plant with water containing 100 μM fusion peptide (peptide solution). Tomato leaves were collected by punching them out 41, 48, 65, and 72 hours after spraying or irrigating with the peptide solution. The concentration of the fusion peptide (PelB-His-Fh8-Stomagen) in the peptide solution (10 μM) was 0.015% by weight, and the concentration of the fusion peptide (PelB-His-Fh8-Stomagen) in the peptide solution (100 μM) was 0.15% by weight.

[0154] <Comparative Example 3> Except that ion-exchanged water (which does not contain the above-mentioned fusion peptide) was used instead of the peptide solution, tomatoes were grown from seed in the same manner as in Example 6 (Method 1), and the tomato leaves were collected by punching them out.

[0155] <Example 7> Preparation of sweet potato seedlings and application of peptide Sweet potato seedlings with 7-8 true leaves and vines 20-30 cm long were planted at an angle. The sweet potato seedlings were cultivated in an artificial climate chamber (14 hours of light (7:00 AM - 9:00 PM); 28°C; 60% humidity, 10 hours of dark (9:00 PM - 7:00 AM); 25°C; 60% humidity). 21 days after planting, 2 mL / plant of water containing 10 μM fusion peptide (Fh8-Stomagent) (peptide solution) was foliar sprayed onto the sweet potatoes. The number of foliar sprays was 1, 2, or 3 times. When the number of foliar sprays was 2, the second spray was performed 1 week after the first spray. When foliar spraying was performed three times, the second spraying was performed one week after the first spraying, and the third spraying was performed one week after the second spraying. When foliar spraying was performed once, sweet potato leaves were punched out and collected two days (45-48 hours) after the first spraying. When foliar spraying was performed twice, the leaves were punched out and collected two days (45-48 hours) after the second spraying. When foliar spraying was performed three times, the leaves were punched out and collected two days (45-48 hours) after the third spraying. The concentration of the fusion peptide (Fh8-Stomagen) in the peptide solution was 0.013% by weight.

[0156] <Comparative Example 4> Sweet potato seedlings were cultivated in the same manner as in Example 7, except that tap water (which did not contain the above-mentioned fusion peptide) was used instead of the peptide solution. Sweet potato leaves were punched out and collected. The foliar spray was applied once.

[0157] <Gene Expression Evaluation> The effect of fusion peptide spraying on increasing stomatal density was evaluated by measuring the gene expression level of TOO MUCH MOUTH (TMM). Eppendorf tubes containing cucumber, tomato, or sweet potato leaves collected in Examples 2-7, Comparative Examples 1-4, and Reference Example 1 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. The RNA concentration was determined by absorbance (A260).

[0158] For evaluating TMM gene expression levels, RNA-direct® SYBR ＴＭ qRT-PCR was performed 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). As an internal standard, the 40S Ribosomal protein S8 (40SRPS8) gene was used for cucumbers, and the Actin gene was used for tomatoes and sweet potatoes. Gene expression levels were compared using the ΔΔCt method with the Ct values ​​obtained by qRT-PCR. The primer sequences used are as follows.

[0159] (Cucumber) Cs40SRPS8_Fw:ACTCGACACTGGGAAAACTACTCG (Sequence number 26) Cs40SRPS8_Rv:CCTGAACACGGGCACTCTTT (Sequence number 27) CsTMM_Fw:TTGAACCGGTTCGATTCCTATC (Sequence number 28) CsTMM_Rv:GAACTCGGAAAGTGGGCTAGAG (Sequence number 29) (Tomato) SlTMM_Fw:CGGACTCCGGGTGCTCTAGTC (Sequence number 32) SlTMM_Rv:CGACCACGACAAAACATCAGG (Sequence number 33) SlActin_Fw: GGGAATGGAGAAAGTTTTGGGTGG (Sequence ID 34) SlActin_Rv: CTTCGAACCAAGGGGAATGGTGTTAGC (Sequence ID 35) (Sweet potato) IbβActin_Fw: GGTGTTAATGGTTGGGAATGGGGAC (Sequence ID 36) IbβActin_Rv: GGTAAAGAAGGAACAGGGGTGCTC (Sequence ID 37) IbTMM_Fw: ATTCCCCGACGTTTGTCGGG (Sequence ID 38) IbTMM_Rv: GTCGCACGTTCGGGAAAACG (Sequence ID 39)

[0160] <PCR reaction conditions> React at 95°C for 30 seconds, then at 61°C for 20 minutes, followed by 40 cycles of reaction at 95°C for 10 seconds, 63°C for 10 seconds, and 68°C for 30 seconds.

[0161] The results for Example 2 are shown in Figure 2, the results for Example 3 in Figure 3, the results for Example 4 and Comparative Example 1 in Figures 4A to 4C, the results for Example 5, Comparative Example 2 and Reference Example 1 in Figures 5A to 5C, the results for Example 6 and Comparative Example 3 in Figures 6A to 6D, and the results for Example 7 and Comparative Example 4 in Figures 7A to 7C. The vertical axis of the graphs shows the relative expression level of the TMM gene. In Figures 2, 3, 4A to 4C, 5A to 5C, 6A to 6D, and 7A to 7C, the results are shown as mean ± standard error (n=4). In Figures 7A to 7C, the analysis of differences between two independent groups was performed using the Student's t-test. Data were considered significant if the p-value was p < 0.05. To analyze differences between three or more independent groups, one-way ANOVA was performed followed by Dunnett's test for multiple comparisons. * indicates a statistically significant difference compared to Comparative Example 4 (water treatment) at p < 0.05, and ** indicates a statistically significant difference at p < 0.01.

[0162] Figure 2 is a graph showing the results of examining TMM expression in cucumbers treated with the fusion peptide (PelB-His-Fh8-Stomagen). Figure 3 is a graph showing the results of examining TMM expression in cucumbers treated with the fusion peptide (PelB-His-Fh8-Stomagen). The TMM expression levels in Figures 2 and 3 are shown as relative values, with the TMM expression level of cucumbers immediately after spraying with the peptide solution set to 1.

[0163] Figure 4A is a graph showing the results of TMM expression after 7 hours in tomatoes treated with the fusion peptide (Fh8-Stomagen) or water. Figure 4B is a graph showing the results of TMM expression after 24 hours in tomatoes treated with the fusion peptide (Fh8-Stomagen) or water. Figure 4C is a graph showing the results of TMM expression after 48 hours in tomatoes treated with the fusion peptide (Fh8-Stomagen) or water. The TMM expression levels in Figures 4A to 4C are shown as relative values ​​with the TMM expression level of Comparative Example 1 (water treatment) tomatoes set to 1.

[0164] Figure 5A is a graph showing the TMM expression levels after 7 hours in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen), water, or PelB-His-Fh8 peptide. Figure 5B is a graph showing the TMM expression levels after 24 hours in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen), water, or PelB-His-Fh8 peptide. Figure 5C is a graph showing the TMM expression levels after 48 hours in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen), water, or PelB-His-Fh8 peptide. The TMM expression levels in Figures 5A to 5C are shown as relative values, with the TMM expression level of the tomatoes in Comparative Example 2 (water treatment) set to 1.

[0165] Figure 6A is a graph showing the results of TMM expression after 41 hours in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen) or water. Figure 6B is a graph showing the results of TMM expression after 48 hours in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen) or water. Figure 6C is a graph showing the results of TMM expression after 65 hours in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen) or water. Figure 6D is a graph showing the results of TMM expression after 72 hours in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen) or water. The TMM expression levels in Figures 6A to 6D are shown as relative values ​​with the TMM expression level of the tomatoes in Comparative Example 3 (water treatment) set to 1.

[0166] Figure 7A is a graph showing the results of examining TMM expression two days after application to sweet potatoes treated with the fusion peptide (Fh8-Stomagen) or water once. Figure 7B is a graph showing the results of examining TMM expression two days after application to sweet potatoes treated with the fusion peptide (Fh8-Stomagen) or water twice. Figure 7C is a graph showing the results of examining TMM expression two days after application to sweet potatoes treated with the fusion peptide (Fh8-Stomagen) or water three times. The TMM expression levels in Figures 7A to 7C are shown as relative values, with the TMM expression level of sweet potatoes in Comparative Example 4 (water treatment) set to 1.

[0167] As shown in Figure 2, the expression level of TMM in cucumbers decreased significantly to about 10% of the level immediately after application two hours after spraying the peptide solution. However, it recovered to about 50% of the level immediately after application after six hours, and further increased to about twice the level immediately after application after eighteen hours. In addition, as shown in Figure 3, the expression level of TMM in cucumbers remained at more than 1.5 times the level immediately after application two days after spraying the peptide solution, and was maintained at about 1.3 times even after five days. This confirms that the TMM expression-promoting effect of the fusion peptide of the present invention is maintained for several days.

[0168] As shown in Figures 4A to 4C, when Fh8-Stomagen was used as the fusion peptide of the present invention, the TMM expression level in tomatoes was almost the same as that of water treatment (Comparative Example 1) 7 hours and 24 hours after spraying the peptide solution, but after 48 hours it improved significantly to about 1.6 times that of water treatment (Comparative Example 1). Furthermore, as shown in Figures 5A to 5C, when Pelb-His-Fh8-Stomagen was used as the fusion peptide of the present invention, the TMM expression level in tomatoes fell to about 85% of that of water treatment (Comparative Example 2) 7 hours after spraying the peptide solution, but after 24 hours it was about 1.3 times that of water treatment (Comparative Example 2), and after 48 hours it improved significantly to about twice that of water treatment (Comparative Example 2). Furthermore, when PelB-His-Fh8-Stomagent was used as the fusion peptide of the present invention, the expression level of TMM in tomatoes was higher than that of the negative control (Reference Example 1, treated with water containing PelB-His-Fh8 peptide without the Stomagent region) at 7, 24, and 48 hours. This proves that the effect of improving TMM expression is due to the Stomagent region in the fusion peptide of the present invention. Moreover, as shown in Figures 6A to 6D, the expression level of TMM in tomatoes increased significantly to approximately three times that of the water treatment case (Comparative Example 3) 48 hours after the start of irrigation with the peptide solution, not only when the peptide solution was applied by foliar spraying but also when it was applied by irrigation.

[0169] As shown in Figures 7A to 7C, the expression level of TMM in sweet potatoes increased significantly compared to the water treatment case (Comparative Example 4). Two days after the first application of the peptide solution, it increased to approximately 1.6 times; two days after the second application of the peptide solution, it increased to approximately 2.6 times; and two days after the third application of the peptide solution, it increased to approximately 4.3 times.

[0170] <Example 8> Field cultivation test of cucumbers treated with fusion peptide Young cucumber seedlings were uniformly transplanted into the field, and a solution containing 2 μM of the fusion peptide (PelB-His-Fh8-Stomagen) (peptide solution) was foliar sprayed once a week for 6 months at a rate of 10-50 mL / plant. The amount of cucumber harvested per plant over 6 months was tallied, and the average value was calculated (n=24). The concentration of the fusion peptide (PelB-His-Fh8-Stomagen) in the peptide solution (2 μM) was 0.003% by weight.

[0171] <Comparative Example 5> A field cultivation experiment of cucumbers was conducted in the same manner as in Example 8, except that tap water (which did not contain the above-mentioned fusion peptide) was used instead of the peptide solution, and the number of cucumbers harvested over a 6-month period was tallied (n=24).

[0172] The results for Example 8 and Comparative Example 5 are shown in Figures 8A and 8B. Figure 8A is a graph showing the total number of cucumbers harvested (number of cucumbers harvested per plant) over a six-month period when treated with the fusion peptide (PelB-His-Fh8-Stomagen) or water. Figure 8B is a graph showing the total number of cucumbers harvested (number of cucumbers harvested per plant) on a monthly basis when treated with the fusion peptide (PelB-His-Fh8-Stomagen) or water. The harvest numbers in Figure 8B are shown as relative values ​​with the monthly harvest of cucumbers in Comparative Example 5 (water treatment) set to 100.

[0173] Effect of increasing cucumber yield and reducing seedling fatigue As shown in Figure 8A, in Example 8, the number of cucumbers harvested (total over 6 months) increased by approximately 5% compared to Comparative Example 5 (water treatment). Furthermore, as shown in Figure 8B, when the number of cucumbers harvested in Example 8 is aggregated by month, the number of harvests in the first and second months (October and November) was slightly lower than that of Comparative Example 5 (water treatment), but from the third month (December) onward, the number of harvests increased compared to Comparative Example 5 (water treatment), and in particular, the number of harvests in the fifth and sixth months (February and March) increased by approximately 25-30% compared to Comparative Example 5 (water treatment). From these results, the effect of increasing yield (number of harvests) and reducing seedling fatigue by the fusion peptide of the present invention was confirmed. Note that the numbers on the horizontal axis of Figure 8B represent months.

[0174] <Example 9> Open-field cultivation test of sweet potatoes treated with fusion peptide Seedlings of sweet potato (variety: Beniharuka) were transplanted into a field in mid-June. From June 22 to October 4, a solution containing 10 μM of the fusion peptide (PelB-His-Fh8-Stomagen) (peptide solution) was foliar sprayed at a frequency of once every two weeks at a rate of 10-20 mL / plant (n=20). Sweet potatoes were harvested four months after transplanting. The concentration of the fusion peptide (PelB-His-Fh8-Stomagen) in the peptide solution (10 μM) was 0.015% by weight.

[0175] <Comparative Example 6> A field cultivation test of sweet potatoes was conducted in the same manner as in Example 9, except that tap water (which did not contain the above-mentioned fusion peptide) was used instead of the peptide solution. Sweet potatoes were harvested four months after planting (n=20).

[0176] <Measurement of Brix Value of Sweet Potatoes> The Brix value of sweet potatoes harvested in Example 9 and Comparative Example 6 was measured as an indicator of sugar content by the following method. Randomly selected sweet potatoes were steamed at 105°C for 10 minutes, allowed to cool at room temperature for one day, then the sweet potatoes were divided into approximately three equal parts lengthwise, and the middle section was further divided into approximately five equal parts. Approximately 10 g of sweet potato weighed from one of the five sections and approximately 20 g of deionized water were placed in a poly cup, and the sweet potato was ground until no solid matter was visible to prepare a sample solution. The sample solution was simply filtered with a paper cloth, and the Brix value of the filtrate was measured using a Brix meter (Pocket Refractometer PAL-1, manufactured by Atago Co., Ltd.). Using the obtained measurement values, a converted value considering the dilution ratio of the sample solution was calculated using the following formula. The converted value was calculated as follows: Conversion value = Measured Brix value of the filtrate × Dilution ratio ((Weight of sweet potato + Weight of water) / Weight of sweet potato). The converted value was calculated for all of the approximately five sections using the same method as above (n=5 per sweet potato), and the average of these values ​​was taken as the Brix value of one sweet potato. In each of Example 9 and Comparative Example 6, the Brix values ​​of five sweet potatoes were calculated (n=25), and the average of the Brix values ​​of the five sweet potatoes was taken as the Brix value for Example 9 and Comparative Example 6, respectively.

[0177] The results for Example 9 and Comparative Example 6 are shown in Figure 9. Figure 9 is a graph showing the Brix values ​​of sweet potatoes treated with the fusion peptide (PelB-His-Fh8-Stomagent) or water. In Figure 9, the results are shown as mean ± standard error (n=25). In Figure 9, the analysis of differences between two independent groups was performed using the Student's t-test. Data were considered significant if the p-value was p < 0.05. To analyze differences between three or more independent groups, one-way ANOVA was performed followed by Dunnett's test for multiple comparisons. * indicates a significant difference with p < 0.05 compared to Comparative Example 6 (water treatment).

[0178] As shown in Figure 9, the Brix value of sweet potatoes in Example 9 increased by approximately 1.17 times compared to Comparative Example 6 (water treatment). This result confirms the effect of the fusion peptide of the present invention on increasing the sugar content of sweet potatoes.

[0179] <Example 10> Preparation of tomato (variety: Regina) seedlings and application of peptide (4) A paper cloth was moistened with water and placed in a petri dish, and tomato seeds were sown on it. The sown tomatoes were cultivated in an artificial climate chamber (12 hours of light (7:00 AM to 8:00 AM; 20°C, 8:00 AM to 9:00 AM; 22.5°C, 9:00 AM to 7:00 PM; 25°C; humidity 60%), 12 hours of dark (7:00 PM to 7:00 AM; 16°C; humidity 60%)). Two weeks after the start of cultivation, the germinated seedlings were transplanted into Jiffy-7 pellets and continued to be cultivated in the artificial climate chamber under the above conditions. Tomato plants 40 days old were irrigated with water containing 5 μM of the fusion peptide (PelB-His-Fh8-Stomagen) at a rate of 5 mL / plant. Two days after irrigation with the peptide solution, tomato leaves were collected by punching them out. The concentration of the fusion peptide (PelB-His-Fh8-Stomagen) in the peptide solution was 0.0075% by weight.

[0180] <Comparative Example 7> Except that ion-exchanged water (which does not contain the above-mentioned fusion peptide) was used instead of the peptide solution in Example 10, tomatoes were grown from seed in the same manner as in Example 10, and the tomato leaves were collected by punching them out.

[0181] <Reference Example 2> Pelb-His-Fh8 peptide (a fusion peptide to which Pelb-tagged peptide, His-tagged peptide, and Fh8 tag are bound) was obtained by the same method as in Reference Example 1. Tomatoes were grown from seed in the same method as in Example 10, except that instead of the peptide solution in Example 10, water containing 5 μM Pelb-His-Fh8 peptide (a peptide solution that does not contain the Stomagen region) was used for cultivation (negative control), and the tomato leaves were collected by punching them out.

[0182] <Gene Expression Evaluation> The ability to confer salt tolerance and other resistances by spraying the fusion peptide was evaluated by measuring the gene expression level of sucrose transporter 1 (SUT1). Eppendorf tubes containing tomato leaves collected in Example 10, Comparative Example 7, and Reference Example 2 were frozen with liquid nitrogen and thoroughly crushed with a pestle until the leaves became powdery. 20-100 mg of the powdered leaves were transferred to a 1.5 mL centrifuge tube, and RNA was extracted using the Maxwell® RSC Plant RNA kit manufactured by Promega. The RNA concentration was determined by absorbance (A260).

[0183] For the evaluation of SUT1 gene expression levels, RNA-direct® SYBR ＴＭ qRT-PCR was performed using a PCR system (QuantStudio® 3 Real-Time PCR System, Thermo Fisher Scientific) with Green Realtime PCR Master Mix (manufactured by Toyobo Co., Ltd.). The Actin gene was used as the internal standard. Gene expression levels were compared using the ΔΔCt method with the Ct values ​​obtained by qRT-PCR. The primer sequences used are as follows.

[0184] (Tomato) SlSUT1_Fw: AACTCCCGGAGAAAAGAAG (Sequence ID 30) SlSUT1_Rv: TACAGTTTCGCCATCACCGAC (Sequence ID 31) SlActin_Fw: GGGATGGGAGAAGTTTTGGGTGG (Sequence ID 34) SlActin_Rv: CTTCGAACCAAGGGGAATGGGTTAGC (Sequence ID 35)

[0185] <PCR reaction conditions> React at 95°C for 30 seconds, then at 61°C for 20 minutes, followed by 40 cycles of reaction at 95°C for 10 seconds, 63°C for 10 seconds, and 68°C for 30 seconds.

[0186] The results for Example 10, Comparative Example 7, and Reference Example 2 are shown in Figure 10. In Figure 10, the results are shown as mean ± standard error (n=4). Figure 10 is a graph showing the results of examining the expression of SUT1 in tomatoes treated with the fusion peptide (PelB-His-Fh8-Stomagen), water, or PelB-His-Fh8 peptide. The expression levels of SUT1 in Figure 10 are shown as relative values ​​with the expression level of SUT1 in tomatoes from Comparative Example 7 (water treatment) set to 1.

[0187] As shown in Figure 10, the expression level of SUT1 in tomatoes increased significantly to approximately 1.6 times that of water treatment (Comparative Example 7) two days after irrigation with the peptide solution. Furthermore, in the case of the negative control (Reference Example 2, treated with water containing the PelB-His-Fh8 peptide which does not contain the Stomagen region), the expression level of SUT1 did not improve compared to water treatment (Comparative Example 7). This demonstrates that the effect of improving SUT1 expression is due to the Stomagen region in the fusion peptide of the present invention.

[0188] Furthermore, in Examples 1 to 10 described above, no plant damage caused by the fusion peptide of the present invention was observed in any of the cucumber, tomato, and sweet potato plants.

[0189] <Examples 11 to 29> By the same method as in Example 1, the transformant Lemo21(DE3)_pET22b(PelB-His-Fh8-Stomagen) was obtained. The transformant Lemo21(DE3)_pET22b(PelB-His-Fh8-Stomagen) was shaken (150 rpm) at 30 °C in LB medium (2 to 5 mL) for preculture for 18 to 24 hours. The preculture solution was inoculated into the main medium (70 to 90 mL). The main medium was prepared by adding the carbon source, nitrogen source, and phosphorus source (P ２ O ５ ) shown in Tables 1 and 2 to the LB medium and adjusting the pH. The carbon source, nitrogen source, and phosphorus source were added so that the concentrations of the carbon source, nitrogen source, and phosphorus source in the medium were the concentrations shown in Tables 1 and 2. The pH of the main medium was adjusted to 6.9 by adding lactic acid or ammonia.

[0190] In the main culture, from the start of the culture, the pH was maintained at 6.9 for the "time until pH change" shown in Tables 1 and 2 below. Then, when the "time until pH change" elapsed, the "substance to be added at the time of pH change" shown in Tables 1 and 2 was added, and the pH was raised to the "pH after change" shown in Tables 1 and 2 and maintained until the end of the culture. The cell density was adjusted so that OD600 reached 15 to 20 at 6 hours after the start of the culture. 3 hours after the start of the culture, isopropyl-β-thiogalactopyranoside (IPTG) was added to the medium to the "IPTG concentration" shown in Tables 1 and 2 to promote the production of the target fusion peptide. 22 to 26 hours after the addition of IPTG, the culture solution in the culture vessel was centrifuged (13000 rpm), and the supernatant fraction and the precipitate were separated. The fusion peptide contained in the supernatant was confirmed by SDS-PAGE.

[0191] SDS-PAGE analysis revealed a band for the fusion peptide (PelB-His-Fh8-Stomagent) (Molecular Weight: 15.0 kDa) in each of Examples 11-29. In all of Examples 11-29, the band was present in the supernatant fraction, confirming its secretion into the culture medium. Furthermore, using bovine serum albumin (BSA) as a calibration curve, the concentration of the fusion peptide (PelB-His-Fh8-Stomagent) was calculated from the band intensity in the supernatant fraction. The calculated concentrations (concentrations in the supernatant fraction) are shown in Tables 1 and 2.

[0192] The purified supernatant fraction is then processed in a desalting and concentration apparatus (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 / cm. ２ The mixture was desalted until it reached this state. In this way, a fusion peptide (PelB-His-Fh8-Stomagent) was obtained (a fusion peptide in which a PelB-tagged peptide, a His-tagged peptide, and an Fh8 tag are attached to the N-terminus of Stomagent).

[0193]

[0194]

[0195] Application of Fusion Peptide and Evaluation of Gene Expression In Examples 30 to 44 below, the fusion peptide (PelB-His-Fh8-Stomagen) obtained in Examples 11, 14, 17, 18, 20-22, 24-26, and 29 was applied to plants, and the effect on gene expression listed in Tables 3 and 4 below was investigated. A peptide solution was prepared by mixing Stomagen with ion-exchanged water, and a peptide solution was prepared by mixing the fusion peptide (PelB-His-Fh8-Stomagen) with ion-exchanged water, and these were applied to plants.

[0196] <Examples 30-44> Preparation of tomato seedlings (variety: Regina) and application of peptides (5) Paper rags were moistened with water and placed in a petri dish, and tomato seeds were sown on them. The sown tomatoes were cultivated in an artificial climate chamber (12 hours of light (7:00 AM - 8:00 AM; 20°C, 8:00 AM - 9:00 AM; 22.5°C, 9:00 AM - 7:00 PM; 25°C, humidity 60%), 12 hours of dark (7:00 PM - 7:00 AM; 16°C; humidity 60%)). Two weeks after the start of cultivation, the germinated seedlings were transplanted into Jiffy-7 pellets and continued to be cultivated in an artificial climate chamber under the above conditions. The fusion peptides listed in Table 3 were mixed with water to obtain the concentrations (unit: μM) listed in Table 3 to obtain peptide solutions. Tomato plants 40 days old were treated with the peptide solution obtained above by watering the base of each plant at a rate of 1 mL / plant. Subsequently, the peptide solution was applied once a week using the same method as described above. 48 hours after starting the peptide solution watering, tomato leaves were collected by punching them out.

[0197] <Comparative Example 8> Except that, in the same manner as in Example 30, ion-exchanged water (which does not contain the above-mentioned fusion peptide) was used instead of the peptide solution, tomatoes were grown from seed and the tomato leaves were collected by punching them out.

[0198] <Gene Expression Evaluation> Changes in the expression levels of each gene due to fusion peptide spraying were evaluated. Eppendorf tubes containing tomato leaves cultivated in Examples 30-44 and Comparative Example 8 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 total RNA was extracted using Maxwell® RSC Plant RNA kit from Promega. RNA concentration was determined by absorbance (A260). RNA sequencing (RNA-Seq) was performed on the total RNA extracted above using the following method, and genes whose expression levels in the tomato leaves cultivated in Examples 30-44 were significantly higher than those in the tomato leaves of Comparative Example 8 were identified. First, a library was prepared using Illumina Stranded mRNA Prep (Illumina Corporation). Poly-T magnetic beads (RNA Purification Beads included with the kit) were added to total RNA, and only mRNA with a Poly-A tail was captured, removing unwanted RNA such as rRNA. The magnetic beads were then washed, and the mRNA was fragmented into approximately 200-500 bp fragments using heat and divalent cations (magnesium, etc.). The fragmented mRNA was then mixed with the reaction solution included with the kit, and reverse transcription was performed at 25°C for 10 minutes, 42°C for 15 minutes, and 70°C for 15 minutes to synthesize cDNA. Next, a Second Strand cDNA synthesis reaction was performed. The 3' end was then reacted with the reagent included with the kit at 37°C for 30 minutes and 70°C for 5 minutes to add dATP. Finally, the adenylated cDNA was ligated with an adapter sequence by reacting at 30°C for 10 minutes. Finally, the reaction was amplified by PCR according to the <PCR reaction conditions> below, and excess primers and adapter dimers were removed by Bead Cleanup using the AMPure XP included with the kit to obtain the final library. The adapter sequences used are as follows: (Adapter sequences) F: ACACTCTTTCCCTACACGAACGCCTTCTCCGATCTNNNNNNNN (SEQ ID NO: 40) R: GTGACTGGAGTTTCAGACGTGTGCCTTCTCCGATCTNNNNNNNN (SEQ ID NO: 41)

[0199] The library prepared as described above was read using a next-generation sequencer (NGS) (MGI Tech, DNBSEC-G400) to obtain nucleotide sequence data. After that, mapping and quantification (evaluation) of the expression levels of each gene were performed.

[0200] <PCR reaction conditions> React at 95°C for 30 seconds, then at 61°C for 20 minutes, followed by 40 cycles of reaction at 95°C for 10 seconds, 63°C for 10 seconds, and 68°C for 30 seconds.

[0201] For Examples 30 to 44, the evaluation results of the expression levels of each gene were obtained by taking the base-2 logarithm (Log2 Fold Change) of the ratio of the gene expression levels in the Examples to the gene expression levels in Comparative Example 8. ２ Also written as FC. Represented as )) Log ２ FC is a value obtained by the following formula: Log ２ FC = Log ２ (Genetic expression level in the example / Genetic expression level in Comparative Example 8) For example, if the gene expression level in the example is the same as the gene expression level in Comparative Example 8, Log ２ The value of FC becomes 0, and if the gene expression level in the example is twice the gene expression level in Comparative Example 8, Log ２ The value of FC will be 1. Log ２ The FC value is preferably 0.6 or higher.

[0202] The results of the gene expression evaluation described above are shown in Tables 3 and 4. Table 3 also shows the concentration (in weight %) of the fusion peptide in the peptide solution used in each example. In Table 3 below, the "CDK12 / 13" column shows the base-2 logarithm of the ratio of the total expression levels of CDK12 and CDK13 in the example to the total expression levels of CDK12 and CDK13 in Comparative Example 8. In Table 4 below, the "PLD1 / 2" column shows the base-2 logarithm of the ratio of the total expression levels of PLD1 and PLD2 in the example to the total expression levels of PLD1 and PLD2 in Comparative Example 8.

[0203]

[0204]

[0205] Furthermore, no plant damage caused by the fusion peptide of the present invention was observed in the above Examples 30 to 44.

[0206] 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 listed below (A1) to (A3), 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 consisting of an amino acid sequence in which 1 to 4 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, and which has a pore density increasing effect. (A3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and which has a pore density increasing effect.

2. The fusion peptide or a salt thereof, or a solvate thereof, according to claim 1, wherein the tag peptide is a peptide comprising one or two of the following (B1) to (B3), (C1) to (C3), (D1) to (D3), and (E1) to (E3). (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 2 amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ 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 SEQ 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 6 amino acids are deleted, substituted, or added to 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. (D1) A peptide consisting of the amino acid sequence shown in SEQ ID NO:

4. (D2) A peptide consisting of an amino acid sequence in which 1 to 11 amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO:

4. (D3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

4. (E1) A peptide consisting of the amino acid sequence shown in SEQ ID NO:

5. (E2) A peptide consisting of an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO:

5. (E3) A peptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

5.

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) A polynucleotide consisting of the base sequence shown in Sequence ID No. 7; (a2) A polynucleotide consisting of a base sequence in which 1 to 14 bases are deleted, substituted, or added in the base sequence shown in Sequence ID No. 7, and which encodes a peptide that increases porosity density; (a3) ​​A polynucleotide consisting of a base sequence having 90% or more sequence identity with the base sequence shown in Sequence ID No. 7, and which encodes a peptide that increases porosity density.

4. The polynucleotide according to claim 3, wherein the polynucleotide encoding the tag peptide is a polynucleotide comprising one or two of the following (b1) to (b3), (c1) to (c3), (d1) to (d3), and (e1) to (e3). (b1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 (b2) A polynucleotide consisting of a nucleotide sequence in which 1 to 6 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 8 (b3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 8 (c1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 (c2) A polynucleotide consisting of a nucleotide sequence in which 1 to 18 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 9 (c3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 9 (d1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 (d2) A polynucleotide consisting of a nucleotide sequence in which 1 to 33 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 10 (d3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 10 (e1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 11 (e2) A polynucleotide consisting of a nucleotide sequence in which 1 to 15 bases are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No.

11. (e3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No.

11.

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 a polynucleotide comprising one or two of the following (b1) to (b3), (c1) to (c3), (d1) to (d3), and (e1) to (e3). (b1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 (b2) A polynucleotide consisting of a nucleotide sequence in which 1 to 6 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 8 (b3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 8 (c1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 (c2) A polynucleotide consisting of a nucleotide sequence in which 1 to 18 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 9 (c3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 9 (d1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 (d2) A polynucleotide consisting of a nucleotide sequence in which 1 to 33 bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 10 (d3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with respect to the nucleotide sequence shown in SEQ ID NO: 10 (e1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 11 (e2) A polynucleotide consisting of a nucleotide sequence in which 1 to 15 bases are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No.

11. (e3) A polynucleotide consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No.

11.

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, a composition for protecting plants, or a composition for increasing the yield of 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. The composition according to claim 10, which is applied to plants by foliar spraying or irrigation.

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

16. A method for cultivating a plant according to claim 15, wherein the plant is at least one selected from the group consisting of plants of the Cucurbitaceae family, Solanaceae family, Convolvulaceae family, Salicaceae family, Rutaceae family, Fabaceae family, Rosaceae family, Asteraceae family, Amaranthaceae family, Musaceae family, Poaceae family, and Amaryllidaceae family.

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

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