Protein capable of forming dimer

Abstract

The present invention pertains to a protein capable of forming a dimer in the presence of a chemically inducible dimerization (CID) inducer, said protein comprising: a protein comprising an amino acid sequence represented by any of SEQ ID NOs: 1-2 and 139-140, or a variant thereof; a protein comprising an amino acid sequence represented by any of SEQ ID NOs: 3-4 and 167, or a variant thereof; a protein comprising an amino acid sequence represented by any of SEQ ID NOs: 5-6 and 89, or a variant thereof; a protein comprising an amino acid sequence represented by any of SEQ ID NOs: 7-8, or a variant thereof; or a protein which comprises an amino acid sequence represented by SEQ ID NO: 201, or a variant thereof, and in which the amino acid residue at the position corresponding to position 291 in the amino acid sequence represented by SEQ ID NO: 201 is a threonine residue.

Description

Proteins that have the ability to form dimers

[0001] This invention relates to a protein having the ability to form dimers.

[0002] Chemically induced dimerization (CID) is a phenomenon in which, for example, a specific chemical substance (CID inducer) binds to two different proteins, bringing them closer together and forming heterologous protein dimers. It is also a phenomenon in which a specific chemical substance (CID inducer) binds to a specific protein, causing a change in the protein's three-dimensional structure, which in turn causes it to bind to other proteins and form heterologous protein dimers. Using this phenomenon, CID methods have been developed to control biological processes temporally and spatially. Specifically, CID methods are used to regulate gene expression in cells and organisms, alter protein localization, and manipulate signaling molecules.

[0003] Representative proteins used in the CID method include FK506-binding protein (FKBP) and FKB-rapamycin-binding protein (FRB), which dimerize in the presence of rapamycin, a CID inducer (Non-Patent Literature 1). In addition, a portion of the Arabidopsis thaliana abscisic acid receptor protein (PYL1) and a portion of the Arabidopsis thaliana abscisic acid-dependent receptor-binding protein (ABI), which dimerize in the presence of abscisic acid, a plant hormone, as CID inducers, are also known to be usable in the CID method (Non-Patent Literature 1, 2, and 4). Furthermore, a portion of the Arabidopsis thaliana gibberellin receptor protein (GID1) and a portion of the Arabidopsis thaliana gibberellin-dependent receptor-binding protein (GAI), which dimerize in the presence of gibberellin, a plant hormone, as CID inducers, are also known to be usable in the CID method (Non-Patent Literature 1, 3, and 4).

[0004] DeRose R, et al., “Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology.”, Pflugers Arch. 2013 Mar;465(3):409-17.Liang FS, et al., “Engineering the ABA plant stress pathway for regulation of induced proximity.”, Sci Signal. 2011 Mar 15;4(164):rs2.Miyamoto T, et al., “Rapid and orthogonal logic gating with a gibberellin-induced dimerization system.”, Nat Chem Biol. 2012 Mar25;8(5):465-70.Gao Y, et al., “Complex transcriptional modulation with orthogonal and inducible dCas9 regulators.”, Nat Methods. 2016 Dec;13(12):1043-1049.

[0005] On the other hand, CID methods using a portion of the Arabidopsis thaliana PYL1 protein and a portion of the Arabidopsis thaliana ABI protein, or a portion of the Arabidopsis thaliana GID1 protein and a portion of the Arabidopsis thaliana GAI protein, as described in Non-Patent Documents 1 to 4, only showed CID activity under conditions where abscisic acid or gibberellin was present at high concentrations on the order of μM (e.g., 10 μM or more) (see also the results of Experimental Examples 3 and 5). When CID-inducing substances such as plant hormones are present at high concentrations, there are problems such as causing cell death, stressing cells, and reducing the reproducibility of experiments.

[0006] Therefore, the present invention aims to provide a protein that exhibits CID activity when used in combination with a specific protein, even when the concentration of the CID-inducing substance in chemically induced dimerization (CID) is less than the order of μM, i.e., a protein that has the ability to form dimers. The present invention also aims to provide a method for forming protein dimers that can be carried out by using such proteins in combination.

[0007] The inventors have found that the GID1 protein and a portion of the GAI protein from corn (Zea mays), and the GID1 protein and a portion of the GAI protein from rice (Oryza sativa), exhibit CID activity even in the presence of gibberellin at concentrations of 100 nM or less. Furthermore, the inventors have found that a portion of the PYL1 protein and a portion of the ABI protein from corn, and a portion of the PYL1 protein and a portion of the ABI protein from rice, exhibit CID activity in the presence of abscisic acid at concentrations of 100 nM or less. Based on these novel findings, the present invention was completed.

[0008] In other words, the present invention provides, for example, the following invention: [1] A protein having dimerization ability in the presence of a chemically induced dimerization (CID) inducer, wherein the protein is a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 7-8, or a protein or variant thereof consisting of an amino acid sequence shown in SEQ ID NOs: 201, wherein the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NOs: 201 is a threonine residue. [2] A protein or variant thereof consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 1-2 and 139-140 has a sequence identity of 90% or more with the amino acid sequence shown in any of the above SEQ ID NOs: 1-2 and 139-140; A protein or variant thereof consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 3-4 and 167 has a sequence identity of 90% or more with the amino acid sequence shown in any of the above SEQ ID NOs: 3-4 and 167; A protein or variant thereof consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 5-6 and 89 has a sequence identity of 90% or more with the amino acid sequence shown in any of the above SEQ ID NOs: 5-6 and 89; A protein or variant thereof consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 7-8 has a sequence identity of 90% or more with the amino acid sequence shown in any of the above SEQ ID NOs: 7-8; The protein according to [1], wherein the protein comprising the amino acid sequence shown in Sequence ID No. 201 or a variant thereof has 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 201, and the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in Sequence ID No. 201 is a threonine residue.[3] The protein according to [1] or [2], wherein in the presence of a CID inducer, a protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, or a variant thereof, and a protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, or a variant thereof, form a dimer. [4] The protein according to [1] or [2], wherein in the presence of a CID inducer, a protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, or a variant thereof, and a protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8, or a variant thereof, form a dimer. [5] The protein according to [3], wherein the CID inducer comprises gibberellin. [6] The protein according to [5], wherein the gibberellin comprises at least one selected from the group consisting of gibberellin A3, gibberellin A1, gibberellin A4, and gibberellin A7. [7] The protein according to [4], wherein the CID inducer comprises at least one selected from the group consisting of abscisic acid, mandipropamide, and azinephosethyl. [8] A protein according to any one of [1] to [7], further comprising a functional protein. [9] A nucleic acid comprising a nucleotide sequence encoding a protein according to any one of [1] to [8].

[10] An expression vector comprising the nucleotide sequence of the nucleic acid according to [9].

[11] A cell into which the nucleic acid according to [9] or the expression vector according to

[10] has been introduced.

[12] A method for forming a dimer of a first protein and a second protein, comprising the step of contacting the first protein, the second protein, and a chemically induced dimerization (CID) inducing substance, wherein the first protein comprises a first dimerizing protein, the second protein comprises a second dimerizing protein, the first dimerizing protein and the second dimerizing protein are proteins that have dimerizing ability in the presence of a chemically induced dimerization (CID) inducing substance, and the first dimerizing protein is a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89 A method for obtaining a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7 to 8, or a protein or a variant thereof consisting of the amino acid sequence shown in SEQ ID NOs: 201, wherein the amino acid residue at position 291 in the amino acid sequence shown in SEQ ID NOs: 201 is a threonine residue.

[13] A protein or variant thereof consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 1-2 and 139-140 has a sequence identity of 90% or more with the amino acid sequence shown in any of the above SEQ ID NOs: 1-2 and 139-140; A protein or variant thereof consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 3-4 and 167 has a sequence identity of 90% or more with the amino acid sequence shown in any of the above SEQ ID NOs: 3-4 and 167; A protein or variant thereof consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 5-6 and 89 has a sequence identity of 90% or more with the amino acid sequence shown in any of the above SEQ ID NOs: 5-6 and 89; A protein or variant thereof consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 7-8 has a sequence identity of 90% or more with the amino acid sequence shown in any of the above SEQ ID NOs: 7-8; The method according to

[12] , wherein the protein or variant thereof consisting of the amino acid sequence shown in SEQ ID NO: 201 has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 201, and the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NO: 201 is a threonine residue.

[14] The method according to

[12] or

[13] , wherein one of the first and second dimer-forming proteins is a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, and the other is a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167.

[15] The method according to

[12] or

[13] , wherein one of the first and second dimer-forming proteins is a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, and the other is a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8.

[16] The method according to

[14] , wherein the CID inducer contains gibberellin.

[17] The method according to

[16] , wherein the gibberellin is selected from the group consisting of gibberellin A3, gibberellin A1, gibberellin A4, and gibberellin A7.

[18] The method according to

[15] wherein the CID inducer comprises at least one selected from the group consisting of abscisic acid, mandipropamide, and azinephosethyl.

[19] The method according to any one of

[12] to

[18] wherein the first protein further comprises a first functional protein, and the second protein further comprises a second functional protein.

[20] A kit comprising nucleic acids including a protein encoding a protein, wherein the protein has the ability to form dimers in the presence of a chemically induced dimerization (CID) inducer, and the protein is a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8, or a protein or a variant thereof consisting of the amino acid sequence shown in SEQ ID NOs: 201, wherein the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NOs: 201 is a threonine residue.

[21] The kit according to

[20] , comprising a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of the above SEQ ID NOs: 1-2 and 139-140, and a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of the above SEQ ID NOs: 3-4 and 167, or a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of the above SEQ ID NOs: 5-6 and 89, and a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of the above SEQ ID NOs: 7-8, or a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in the above SEQ ID NO: 201.

[22] The kit according to

[20] or

[21] , further comprising one or more selected from the group consisting of nucleic acids having a base sequence encoding a functional protein, a CID inducer, and cells.

[0009] According to the present invention, it is possible to provide a protein that exhibits CID activity even when the concentration of the CID inducer is less than the order of μM, by being used in combination with a specific protein. Furthermore, according to the present invention, it is possible to provide a method for forming a protein dimer by using such a protein in combination.

[0010] This is a schematic diagram showing an example of chemically induced dimerization (CID). This is a schematic diagram showing the mechanism when the CID method is used in various applications. This is a schematic diagram of the four types of expression vectors prepared in the experimental examples. This is a graph showing the results of luciferase activity analysis in cells prepared in Experimental Example 1. This is a graph showing the results of luciferase activity analysis in cells prepared in Experimental Example 2. This is a graph showing the results of luciferase activity analysis in cells prepared in Experimental Example 3. This is a graph showing the results of luciferase activity analysis in cells prepared in Experimental Example 4. This is a graph showing the results of luciferase activity analysis in cells prepared in Experimental Example 5. This is a graph showing the results of luciferase activity analysis in cells prepared in Experimental Example 6. This is a graph showing the results of luciferase activity analysis in cells prepared in Experimental Example 7. This is a graph showing the results of luciferase activity analysis in cells prepared in Experimental Example 8. This is a graph showing the results of luciferase activity analysis in cells prepared in Experimental Example 9. This figure shows the results of principal component analysis of transcriptome analysis in Experimental Example 11. This figure shows the results of structural comparison of the GID1 protein in Experimental Example 12. This figure shows the results of multiple alignment of the GID1 protein in Experimental Example 12. This graph shows the results of CID activity evaluation of the AtGID1 mutant in Experimental Example 12. This graph shows the results of CID activity evaluation of the ZmGID1 mutant in Experimental Example 12. This schematic diagram shows the system overview of Switch-ON CAR and the vector configuration used in Experimental Example 13. This graph shows the results of CAR activity evaluation (CD69-positive cell rate) in Experimental Example 13.

[0011] The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

[0012] [Proteins with Dimer-Forming Ability] The proteins with dimer-forming ability according to this embodiment (hereinafter also referred to as "proteins according to this embodiment") include specific proteins that have the activity to form protein dimers in the presence of chemically induced dimerization (CID) inducers. Here, the specific proteins may be proteins or variants thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, proteins or variants thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, proteins or variants thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, proteins or variants thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8, or proteins or variants thereof consisting of the amino acid sequence shown in SEQ ID NOs: 201, wherein the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NOs: 201 is a threonine residue. In other words, the protein according to this embodiment includes an amino acid sequence of a specific protein having the activity to form a protein dimer in the presence of a chemically induced dimerization (CID) inducer, and may be a fusion protein in which such amino acid sequence is linked to another amino acid sequence (such as the amino acid sequence of a functional protein).

[0013] The dimer-forming protein according to this embodiment may be the protein or a variant thereof listed in Tables 4 and 5. Preferably, the protein or variant thereof listed in Tables 4 and 5 is a protein or variant thereof consisting of the amino acid sequence shown in SEQ ID NOs: 139-140, 131-132, 137, 147-148, 151, 163, 166-167, 170-171, and 173.

[0014] In this specification, "a protein having the activity to form a dimer in the presence of a CID inducer" means a protein that induces CID only in the presence of a CID inducer and can be used as one of two different proteins in CID. CID will be described later.

[0015] Whether or not a protein is "a protein that has the activity to form a protein dimer in the presence of a CID inducer" can be analyzed by known methods. For example, one such method is to apply the CID method to the applications described later and perform the analysis. For example, to analyze whether the protein under analysis forms a dimer with a predetermined protein, a fusion protein of the protein under analysis and its DNA-binding domain, and a fusion protein of the predetermined protein and a transcription activator are expressed in a predetermined cell, and then a CID inducer is added. If a reporter gene is expressed by the transcription activator, or if the activity of its gene product is detected, then it can be determined that the protein under analysis is a protein that has the ability to form a dimer with the predetermined protein (exhibits CID activity). In other words, the level of CID activity can be evaluated and compared using the activity of a reporter gene product, such as luciferase. When a protein under evaluation has "high CID activity" compared to a comparison protein, it means, for example, that when the activity of the reporter gene products of both the protein under evaluation and the comparison protein is evaluated using the same reporter assay system, the activity of the reporter gene product of the protein under evaluation is higher than the activity of the reporter gene product of the comparison protein. Furthermore, for a protein under evaluation to be "highly sensitive to CID activity" compared to a comparison protein, this means, for example, using the same reporter assay system, gradually changing the concentration of the CID inducer (for example, changing it 10 times in increments from 1 nM to 10 μM), evaluating the activity of the reporter gene products of both the protein under evaluation and the comparison protein, deriving the minimum concentration of the CID inducer for both the protein under evaluation and the comparison protein at which the activity of the reporter gene product can be measured, and finding that the minimum concentration of the CID inducer in the protein under evaluation is lower than the minimum concentration of the CID inducer in the comparison protein. Also, if the value obtained by dividing the minimum concentration of the CID inducer in the comparison protein by the minimum concentration of the CID inducer in the protein under evaluation is "X", then it can be said that the protein under evaluation has CID activity that is X times more sensitive than the comparison protein.

[0016] Figure 1 is a schematic diagram showing an example of chemically induced dimerization (CID). As shown in Figure 1(A), in the absence of CID inducer 3, the first dimer-forming protein 1 and the second dimer-forming protein 2 cannot bind to each other and do not form a dimer. On the other hand, as shown in Figure 1(B), when CID inducer 3 is present, the binding of CID inducer 3 to the first dimer-forming protein 1 changes the conformation of the first dimer-forming protein. This change in conformation allows the first dimer-forming protein 1 and the second dimer-forming protein 2 to bind and form a dimer. Thus, CID is a phenomenon in which the addition of a specific chemical substance (CID inducer) acts as a switch, and depending on its presence or absence, for example, two different proteins form a dimer or dissociate. The protein according to this embodiment includes, for example, the first dimer-forming protein or the second dimer-forming protein shown in Figure 1.

[0017] Figure 2 is a schematic diagram illustrating an example of the control of a biological process using the CID method. In this specification, "CID method" means a method that utilizes the CID phenomenon to control various biological processes. In this specification, "biological process" refers to chemical, physical, or genetic reactions and dynamics that occur within living organisms. The control of a biological process may be, for example, the control of intracellular processes, or the control of the activity and localization of proteins within cells, and more specifically, it may be the control of gene expression induction, the control of cell activation, and the control of apoptosis induction in cells.

[0018] When controlling biological processes using the CID method, each protein (the first dimerizing protein and the second dimerizing protein) can be genetically fused with any protein or peptide (collectively called a "functional protein") that can induce a biological process through the occurrence of CID. For example, a first fusion protein 111, which is a fusion protein of the first dimerizing protein 1 and the first functional protein 11, and a second fusion protein 221, which is a fusion protein of the second dimerizing protein 2 and the second functional protein 21, are expressed in cells. Subsequently, by adding a CID inducer 3 to induce CID, the first fusion protein 111 and the second fusion protein 221 dimerize and form a complex. This makes it possible for the first functional protein 11 and the second functional protein 21 to be in close proximity, or for the first functional protein 11 or the second functional protein 21 to be located in a desired region in the three-dimensional structure of the complex, and the desired function is exerted through the interaction of the functional proteins. In the CID method, CID can be induced at any desired time using a CID-inducing substance, allowing cells to exert desired functions or to turn off (inhibit) desired functions.

[0019] When the CID method is used to control cell activation as a control of intracellular processes, the cells may be, for example, chimeric antigen receptor (CAR) T cells (CAR-T cells). When the CID method is used to control the activation of CAR-T cells, for example, scFV (single-chain antibody)-hinge-Tm (transmembrane domain) may be used as the first functional protein, and Tm-CD3zeta (T cell activation domain) may be used as the second functional protein. By inducing CID, a complex containing scFV and CD3zeta is formed, and CD3zeta is activated, thereby activating CAR-T cells. The fusion protein of the first dimerizing protein and scFV-hinge-Tm may be, for example, a fusion protein consisting of the amino acid sequence shown in any of SEQ ID NOs. 216, 218, 220, and 222, and is encoded by the amino acid sequence shown in any of SEQ ID NOs. 217, 219, 221, and 223. The fusion protein of the second dimerizing protein and Tm-CD3zeta may be, for example, a fusion protein consisting of the amino acid sequence shown in SEQ ID NOs. 212 or 214, and is encoded by the nucleotide sequence shown in SEQ ID NOs. 213 or 215. Furthermore, when the CID method is used to control apoptosis induction in cells as a control of intracellular processes, the cells may be, for example, CAR-T cells. When the CID method is used to induce apoptosis in CAR-T cells, for example, Caspase9 gene products may be used as the first and second functional proteins. By inducing CID, it is possible to induce Caspase9 homodimerization at the necessary time, which in turn can induce apoptosis (Stavrou M, et al., Mol Ther. 2018 May 2;26(5):1266-1276). When the CID method is used in CAR-T therapy, it is possible to induce cell death in CAR-T cells that have experienced cytokine storms, etc., thereby reducing side effects.

[0020] When using the CID method for gene expression induction, for example, a DNA-binding protein (e.g., dCas9 protein) may be used as the first functional protein, and a transcription activator (e.g., VPR, VP16) may be used as the second functional protein. By inducing CID, the presence of a transcription activator near the region to which the DNA-binding domain binds activates the nearby promoter, thereby inducing downstream gene expression, i.e., transcription. The dCas9 protein is a nuclease that cleaves a target DNA sequence depending on the presence or absence of guide RNA (sgRNA), and is a mutant of Cas9 in which amino acid mutations are added to the active site to eliminate its enzymatic activity.

[0021] The protein according to this embodiment has the ability to form dimers in the presence of a CID-inducing substance, and preferably exhibits CID activity even under conditions where the concentration of the CID-inducing substance in CID is less than the order of μM. Examples of less than the order of μM include greater than 0 pM and less than 1 μM, 1 pM or more and less than 1 μM, 10 pM or more and less than 1 μM, 50 pM or more and less than 1 μM, 100 pM or more and less than 1 μM, 1 nM or more and less than 1 μM, 10 nM or more and 100 nM or less, and 50 nM or more and 100 nM or less.

[0022] The protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is the wild-type GID1 protein derived from maize; the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is the wild-type GID1 protein derived from rice with three amino acids deleted from the C-terminus (SEQ ID NO: 10) (hereinafter referred to as the rice-derived GID1 protein); the protein consisting of the amino acid sequence shown in SEQ ID NO: 139 is the wild-type GID1b protein derived from cassava; and the protein consisting of the amino acid sequence shown in SEQ ID NO: 140 is the wild-type GID1a protein derived from cassava. Hereinafter, the protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140 will also be referred to as the "GID1 protein".

[0023] The GID1 protein is the Gibberellin Insensitive Dwarf 1 protein. It is a gibberellin receptor activated by gibberellin binding and plays a central role in the gibberellin signaling pathway. When gibberellin binds to GID1, it is known to bind to other proteins that mediate the gibberellin response, particularly the Della protein.

[0024] The wild-type GID1 protein derived from maize is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and is encoded by the nucleotide sequence shown in, for example, SEQ ID NO: 9 or 108. The GID1 protein derived from rice is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and is encoded by the nucleotide sequence shown in, for example, SEQ ID NO: 109. The wild-type GID1 protein derived from rice (SEQ ID NO: 10) is encoded by the nucleotide sequence shown in, for example, SEQ ID NO: 107. The wild-type GID1a protein derived from cassava is a protein consisting of the amino acid sequence shown in SEQ ID NO: 140, and is encoded by the nucleotide sequence shown in, for example, SEQ ID NO: 119. The wild-type GID1b protein derived from cassava is a protein consisting of the amino acid sequence shown in SEQ ID NO: 139, and is encoded by the nucleotide sequence shown in, for example, SEQ ID NO: 118. The GID1a and GID1b proteins derived from cassava are orthologs of the GID1 protein in cassava. The wild-type GID1 protein derived from Arabidopsis thaliana (SEQ ID NO: 35) is encoded by the nucleotide sequence shown in SEQ ID NO: 106.

[0025] The GID1 protein or its variant according to this embodiment has CID activity and can be used in the CID method. The variant of the GID1 protein according to this embodiment is a protein in which at least one amino acid residue or amino acid residue region is substituted, deleted, or added to the amino acid sequence (SEQ ID NOs: 1-2 and 139-140) of the GID1 protein according to this embodiment, and is a protein that has the ability to form dimers. Substitution, deletion, or addition may occur simultaneously.

[0026] A variant of the GID1 protein according to this embodiment may have an amino acid sequence in which, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, or added to the amino acid sequence of the GID1 protein according to this embodiment (SEQ ID NOs: 1-2 and 139-140). An example of a variant having 1 to 100 amino acid residue deletions in the amino acid sequence of the GID1 protein according to this embodiment is one in which some (preferably 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more) or all of the amino acid residues corresponding to positions 200-300 in SEQ ID NOs: 1 or 2 are deleted.

[0027] The variant of the GID1 protein according to this embodiment may be a protein or a variant thereof consisting of the amino acid sequence shown in SEQ ID NO: 201, wherein the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NO: 201 is a threonine residue.

[0028] The amino acid sequence shown in SEQ ID NO: 201 is the same as that of the wild-type GID1 protein (SEQ ID NO: 35) derived from Arabidopsis thaliana, but with the 291st amino acid residue replaced by a threonine residue instead of an isoleucine residue. The protein consisting of the amino acid sequence shown in SEQ ID NO: 201 is encoded by, for example, the nucleotide sequence shown in SEQ ID NO: 202.

[0029] Here, for example, "the amino acid residue at the 291st position" refers to the amino acid residue in the amino acid sequence of the target protein that corresponds to the position of the 291st amino acid residue in SEQ ID NO: 201 when the amino acid sequence of the target protein is aligned with the amino acid sequence shown in SEQ ID NO: 201.

[0030] The presence of a threonine residue at position 291 is thought to contribute to the formation of new hydrogen bonds around the ligand-binding pocket, thereby improving the stability of the complex of CID-inducing substances such as gibberellins and dimer-forming proteins. This, in turn, is thought to improve CID activity and its sensitivity. However, the mechanism by which the presence of a threonine residue at this position improves CID activity and sensitivity is not limited to this.

[0031] The GID1 protein or its variant according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. This sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and may even be 100%. In this case, from the viewpoint of high CID activity and high sensitivity of CID activity, the amino acid residue at the position corresponding to position 297 in the amino acid sequence shown in SEQ ID NO: 1 may be a threonine residue.

[0032] The GID1 protein or its variant according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 2. This sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. In this case, from the viewpoint of high CID activity and high sensitivity of CID activity, the amino acid residue at position 298 in the amino acid sequence shown in Sequence ID No. 2 may be a threonine residue.

[0033] The GID1 protein or its variant according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 139. This sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or even 100%. In this case, from the viewpoint of high CID activity and high sensitivity of CID activity, the amino acid residue at position 290 in the amino acid sequence shown in SEQ ID NO: 139 may be a threonine residue.

[0034] The GID1 protein or its variant according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 140. This sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or even 100%. In this case, from the viewpoint of high CID activity and high sensitivity of CID activity, the amino acid residue at position 290 in the amino acid sequence shown in SEQ ID NO: 140 may be a threonine residue.

[0035] The GID1 protein or its variant according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 201, and the amino acid residue at the position corresponding to position 291 in the amino acid sequence shown in SEQ ID NO: 201 may be a threonine residue. This sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0036] In this specification, sequence identity refers to the percentage (%) of matching bases or residues when the base sequences or amino acid sequences being compared are multiple-aligned. Multiple alignment refers to aligning base sequences or amino acid sequences by inserting appropriate gaps so that corresponding base sequences or amino acid sequence portions are aligned, in order to make the base sequences or amino acid sequences comparable to each other. For multiple alignment, a known multiple alignment program can be used. For example, Crystal W, Crystal X, and the BLAST program can be suitably used.

[0037] The GID1 protein or its variant according to this embodiment may form a dimer with any protein in the presence of a CID inducer, may form a dimer with a variant of the GAI protein, or may form a dimer with a variant of the GAI protein of any plant origin. Examples of plants from which GAI proteins are derived include corn, rice, Arabidopsis thaliana, quinoa, tomato (Solanum lycopersicum), sesame (Sesamum indicum), beet (Beta vulgaris), soybean (Glycine max), cassava (Manihot esculenta), cotton (Gossypium hirsutum), eucalyptus (Eucalyptus grandis), lotus (Nelumbo nucifera), wheat (Triticum aestivum), and Selaginella. Other suitable options include moellendorffii, cowpea (Vigna unguiculata), amborella (Amborella trichopoda), loblolly pine (Pinus taeda), bracken fern (Pteridium aquilinum), adzuki bean (Vigna angularis), monkey flower (Mimulus guttatus), etc. If the above-mentioned GID1 protein or its variant is derived from maize, it is preferable that it forms a dimer with a maize-derived GAI protein variant in the presence of a CID inducer. If the GID1 protein or its variant is derived from rice, it is preferable that it forms a dimer with a rice-derived GAI protein variant in the presence of a CID inducer. If the GID1 protein or its variant is derived from cassava, it is preferable that it forms a dimer with a cassava-derived GAI protein variant in the presence of a CID inducer. If it is derived from Arabidopsis thaliana, it is preferable that it forms a dimer with an Arabidopsis-derived GAI protein variant in the presence of a CID inducer.The GAI protein may be a truncated form of the GAI protein according to the present embodiment described later or a variant thereof. More preferably, in the presence of a CID inducer, a protein consisting of the amino acid sequence represented by any one of SEQ ID NOs: 1-2 and 139-140 or a variant thereof preferably forms a dimer with a protein consisting of the amino acid sequence represented by any one of SEQ ID NOs: 3-4 and 167 or a variant thereof. A protein consisting of the amino acid sequence represented by SEQ ID NO: 201 or a variant thereof preferably forms a dimer with a protein consisting of the amino acid sequence represented by SEQ ID NO: 36 or a variant thereof. However, suitable GAI protein variants can be selected from other combinations. The CID inducer will be described later.

[0038] The GID1 protein or a variant thereof according to the present embodiment can form a dimer in the presence of a CID inducer when it is a variant of a gibberellin-dependent receptor-binding protein belonging to the DELLA protein family other than a GAI protein variant. The GID1 protein or a variant thereof according to the present embodiment may have, for example, activity to form a dimer with a variant of RGA protein which is a DELLA protein variant, a variant of RGL1 protein, a variant of RGL2 protein, or a variant of RGL3 protein. The variant of RGA protein, variant of RGL1 protein, variant of RGL2 protein, and variant of RGL3 protein may, for example, have 60% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4.

[0039] Examples of amino acid sequences having 60% or more sequence identity with the amino acid sequence shown in Sequence ID No. 4 include the amino acid sequence shown in Sequence ID No. 102 (the amino acid sequence at positions 1 to 111 of the Arabidopsis thaliana-derived RGA protein (TAIR accession number: AT2G01570)), the amino acid sequence shown in Sequence ID No. 103 (the amino acid sequence at positions 1 to 111 of the Arabidopsis thaliana-derived RGL1 protein (TAIR accession number: AT1G66350)), and the amino acid sequence shown in Sequence ID No. 104 The amino acid sequence may be the amino acid sequence at positions 1-111 of the RGL2 protein (TAIR accession number: AT3G03450) derived from Arabidopsis thaliana, or the amino acid sequence shown in Sequence ID No. 105 (the amino acid sequence at positions 1-111 of the RGL3 protein (TAIR accession number: AT5G17490) derived from Arabidopsis thaliana). TAIR is a gene database for Arabidopsis thaliana (https: / / www.arabidopsis.org / ).

[0040] The protein consisting of the amino acid sequence shown in SEQ ID NO: 3 is a part (truncated form) of the wild-type GAI protein derived from maize, the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is a part (truncated form) of the wild-type GAI protein derived from rice, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 167 is a part (truncated form) of the wild-type GAI protein derived from cassava. Hereafter, the protein consisting of the amino acid sequence shown in SEQ ID NOs: 3-4 and 167 will also be referred to as the "truncated form of GAI protein."

[0041] GAI (Gibberellic Acid Insulator) proteins are proteins involved in the gibberellin signaling pathway and are known to be gibberellin-dependent receptor-binding proteins belonging to the DELLA protein family (e.g., Murase K, et al., Nature. 2008 Nov 27;456(7221):459-63).

[0042] A wild-type GAI protein derived from corn is a protein consisting of the amino acid sequence represented by SEQ ID NO: 11, and is encoded by, for example, the nucleotide sequence represented by SEQ ID NO: 12. A wild-type GAI protein derived from rice is a protein consisting of the amino acid sequence represented by SEQ ID NO: 13, and is encoded by the nucleotide sequence represented by SEQ ID NO: 14. A wild-type GAI protein derived from cassava is a protein consisting of the amino acid sequence represented by SEQ ID NO: 167, and is encoded by, for example, the nucleotide sequence represented by SEQ ID NO: 156.

[0043] A truncated GAI protein or a variant thereof according to the present embodiment has CID activity and can be used in the CID method. The variant of the truncated GAI protein according to the present embodiment is a protein in which at least one amino acid residue or an amino acid residue region is substituted, deleted, or added to the amino acid sequence (SEQ ID NOs: 3 to 4 and 167) of the truncated GAI protein according to the present embodiment. Substitution, deletion, or addition may occur simultaneously.

[0044] The variant of the truncated GAI protein according to the present embodiment may have an amino acid sequence in which, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, or added in the amino acid sequence (SEQ ID NOs: 3 to 4 and 167) of the truncated GAI protein according to the present embodiment.

[0045] The truncated GAI protein or a variant thereof according to the present embodiment may, for example, have 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, and the sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or may be 100%. The protein consisting of the amino acid sequence represented by SEQ ID NO: 3 is encoded by, for example, the nucleotide sequence represented by SEQ ID NO: 15. The amino acid sequence represented by SEQ ID NO: 3 is one in which the amino acid residues corresponding to positions 112 to 626 in the amino acid sequence represented by SEQ ID NO: 11 are deleted.

[0046] A variant of the GAI protein according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4, and this sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is encoded by, for example, the nucleotide sequence shown in SEQ ID NO: 16. The amino acid sequence shown in SEQ ID NO: 4 is characterized by the deletion of amino acid residues corresponding to positions 112 to 625 in the amino acid sequence shown in SEQ ID NO: 13.

[0047] A variant of the GAI protein according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 167, and this sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The protein consisting of the amino acid sequence shown in SEQ ID NO: 167 is encoded by, for example, the nucleotide sequence shown in SEQ ID NO: 156. The amino acid sequence shown in SEQ ID NO: 167 is characterized by the deletion of amino acid residues corresponding to positions 121 to 634 in the amino acid sequence shown in SEQ ID NO: 175.

[0048] The truncated form of the GAI protein or its variant according to this embodiment may form a dimer with any protein in the presence of a CID inducer, or with the GID1 protein or its variant, or with the GID1 protein or its variant derived from any plant. The plants from which they are derived are as described above. If the truncated form of the GAI protein or its variant according to this embodiment is derived from maize, it is preferable that it forms a dimer with the maize-derived GID1 protein or its variant in the presence of a CID inducer. If the truncated form of the GAI protein or its variant according to this embodiment is derived from rice, it is preferable that it forms a dimer with the rice-derived GID1 protein or its variant in the presence of a CID inducer, and if the truncated form of the GAI protein or its variant according to this embodiment is derived from cassava, it is preferable that it forms a dimer with the cassava-derived GID1 protein variant in the presence of a CID inducer. The GID1 protein may be the GID1 protein according to this embodiment as described above. In the presence of a CID inducer, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167 preferably forms a dimer with a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140. However, a suitable GID1 protein or variant thereof can be selected from other combinations.

[0049] The protein consisting of the amino acid sequence shown in SEQ ID NO: 5 is a part (truncated form) of the PYL1 protein derived from corn, the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is a part (truncated form) of the PYL1 protein derived from rice, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 89 is a variant of a part (truncated form) of the PYL1 protein derived from rice. Hereinafter, the protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89 will also be referred to as the "truncated form of the PYL1 protein."

[0050] The PYL1 protein belongs to the PYR (pyrabactin resistance) / PYL (PYR1 like) / RCAR (regulatory components of ABA receptor) family and is known to be an abscisic receptor that is activated upon abscisic acid (ABA) binding and modulates ABA-dependent signaling pathways via binding to protein phosphatase 2C (PP2C). The wild-type PYL1 protein derived from maize is a protein consisting of the amino acid sequence shown in SEQ ID NO: 17, and is encoded by the nucleotide sequence shown in, for example, SEQ ID NO: 18. The wild-type PYL1 protein derived from rice is a protein consisting of the amino acid sequence shown in SEQ ID NO: 19, and is encoded by the nucleotide sequence shown in, for example, SEQ ID NO: 20.

[0051] The truncated form of the PYL1 protein or its variant according to this embodiment has CID activity and can be used in the CID method. The variant of the truncated form of the PYL1 protein is a protein in which at least one amino acid residue or amino acid residue region is substituted, deleted, or added to the amino acid sequence (SEQ ID NOs. 5-6 and 89) of the truncated form of the PYL1 protein. The substitution, deletion, or addition may occur simultaneously.

[0052] The truncated variant of the PYL1 protein according to this embodiment may have an amino acid sequence in which, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, or added in the amino acid sequence of the truncated PYL1 protein according to this embodiment (SEQ ID NOs: 5-6 and 89).

[0053] The truncated form or variant thereof of the PYL1 protein according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and this sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The protein consisting of the amino acid sequence shown in SEQ ID NO: 5 is encoded by, for example, the nucleotide sequence shown in SEQ ID NO: 21. The amino acid sequence shown in SEQ ID NO: 5 is characterized by the deletion of amino acid residues corresponding to positions 196 to 212 in the amino acid sequence shown in SEQ ID NO: 17 (17 amino acid residues from the C-terminus in SEQ ID NO: 17).

[0054] The truncated form or variant thereof of the PYL1 protein according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, and this sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is encoded by, for example, the nucleotide sequence shown in SEQ ID NO: 22. The amino acid sequence shown in SEQ ID NO: 6 is characterized by the deletion of amino acid residues corresponding to positions 195 to 212 in the amino acid sequence shown in SEQ ID NO: 19 (18 amino acid residues from the C-terminus in SEQ ID NO: 19).

[0055] The truncated form or variant thereof of the PYL1 protein according to this embodiment may, for example, have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 89, and this sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The protein consisting of the amino acid sequence shown in SEQ ID NO: 89 is encoded by, for example, the nucleotide sequence shown in SEQ ID NO: 90. The amino acid sequence shown in SEQ ID NO: 89 has a deletion of amino acid residues corresponding to positions 195-212 in the amino acid sequence shown in SEQ ID NO: 19, and has mutations such that lysine at position 70 becomes arginine, phenylalanine at position 126 becomes alanine, valine at position 99 becomes isoleucine, and phenylalanine at position 174 becomes leucine.

[0056] The truncated form of the PYL1 protein or its variant according to this embodiment may form a dimer with any protein in the presence of a CID inducer, or with an ABI protein, or with a variant of an ABI protein derived from any plant. The plants from which they are derived are as described above. If the truncated form of the PYL1 protein or its variant according to this embodiment is derived from maize, it is preferable that it forms a dimer with a variant of an ABI protein derived from maize. If the truncated form of the PYL1 protein or its variant according to this embodiment is derived from rice, it is preferable that it forms a dimer with a variant of an ABI protein derived from rice. The ABI protein may be the ABI protein according to this embodiment described later. In the presence of a CID inducer, it is preferable that a protein or its variant consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 5-6 and 89 forms a dimer with a protein or its variant consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 7-8. However, a suitable variant of the ABI protein can be selected from other combinations.

[0057] The protein consisting of the amino acid sequence shown in Sequence ID No. 7 is a part (truncate) of the ABI protein derived from corn, and the protein consisting of the amino acid sequence shown in Sequence ID No. 8 is a part (truncate) of the ABI protein derived from rice. Hereafter, the protein consisting of the amino acid sequence shown in any of Sequence ID Nos. 7 to 8 will also be referred to as the "truncate of the ABI protein."

[0058] The ABI protein is a member of protein phosphatase 2C (PP2C) and a negative regulator in the ABA signaling pathway. The Arabidopsis thaliana ABI protein is also known to be an abscisic acid-dependent receptor-binding protein. The wild-type ABI protein from maize consists of the amino acid sequence shown in SEQ ID NO: 23 and is encoded by the nucleotide sequence shown in, for example, SEQ ID NO: 24. The wild-type ABI protein from rice consists of the amino acid sequence shown in SEQ ID NO: 25 and is encoded by the nucleotide sequence shown in SEQ ID NO: 26.

[0059] The truncated variant of the ABI protein according to this embodiment has CID activity and can be used in the CID method. The truncated variant of the ABI protein according to this embodiment is a protein in which at least one amino acid residue or a continuous amino acid residue region is substituted, deleted, or added to the amino acid sequence (SEQ ID NOs: 7-8) of the ABI protein according to this embodiment. Substitution, deletion, or addition may occur simultaneously.

[0060] A truncated variant of the maize-derived ABI protein having the amino acid sequence shown in Sequence ID No. 7 according to this embodiment may have an amino acid sequence in which, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence (Sequence ID No. 8) of the truncated variant of the maize-derived ABI protein.

[0061] As a truncated variant of the ABI protein according to this embodiment, for example, it may have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and this sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The protein consisting of the amino acid sequence shown in SEQ ID NO: 7 is encoded by the nucleotide sequence shown in SEQ ID NO: 27, for example. The amino acid sequence shown in SEQ ID NO: 7 is characterized by the deletion of amino acid residues corresponding to positions 1-140 and 476-484 in the amino acid sequence shown in SEQ ID NO: 23.

[0062] A mutant of the rice-derived ABI protein having the amino acid sequence shown in Sequence ID No. 8 according to this embodiment may have an amino acid sequence in which, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues are deleted, substituted, or added in the amino acid sequence (Sequence ID No. 8) of the mutant of the rice-derived ABI protein having the amino acid sequence (Sequence ID No. 8).

[0063] As a truncated variant of the ABI protein according to this embodiment, for example, it may have 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, and this sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The protein consisting of the amino acid sequence shown in SEQ ID NO: 8 is encoded by the nucleotide sequence shown in SEQ ID NO: 28, for example. The amino acid sequence shown in SEQ ID NO: 8 is characterized by the deletion of amino acid residues corresponding to positions 1-67 and 390-396 in the amino acid sequence shown in SEQ ID NO: 25.

[0064] The truncated form of the ABI protein or its variant according to this embodiment may form a dimer with any protein in the presence of a CID inducer, or with the PYL1 protein or its variant, or with the PYL1 protein or its variant derived from any plant. The plants from which they are derived are as described above. If the truncated form of the ABI protein or its variant according to this embodiment is derived from maize, it is preferable that it forms a dimer with the maize-derived PYL1 protein or its variant. If the truncated form of the ABI protein or its variant according to this embodiment is derived from rice, it is preferable that it forms a dimer with the rice-derived PYL1 protein or its variant. The PYL1 protein may be the PYL1 protein according to this embodiment as described above. In the presence of a CID inducer, it is preferable that the protein or its variant consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 7 to 8 forms a dimer with the protein or its variant consisting of the amino acid sequence shown in any of the above SEQ ID NOs: 5 to 6 and 89. However, a suitable PYL1 protein or its variant can be selected from other combinations.

[0065] As explained with reference to Figure 2, the protein according to this embodiment may be a fusion protein such as a first fusion protein 111 and a second fusion protein 221, and may further contain functional proteins in addition to the above protein. That is, the protein according to this embodiment may be a protein fused with a functional protein, and more specifically, the amino acid sequence of the functional protein may be linked to the amino acid sequence of the above protein. The functional protein may be any protein that can induce a biological process by generating CID, and the protein according to this embodiment may be a protein containing multiple functional proteins. Examples of functional proteins include those mentioned above.

[0066] If the protein according to this embodiment further includes a functional protein, the variant of the protein and the functional protein may be fused via a non-peptide linker and / or a peptide linker, or directly. That is, the protein according to this embodiment may be a protein consisting of a variant of the protein and a functional protein, or a protein consisting of a variant of the protein, a linker, and a functional protein.

[0067] Peptide linkers are not particularly limited, but examples include linkers consisting of 1 to 50 amino acids, preferably 1 to 20 amino acids, or linkers consisting of 15 to 20 amino acids, such as peptide linkers consisting of 1 to 5 or 1 to 6 naturally occurring L-amino acids or repeating sequences thereof. Specifically, examples include linkers consisting of the amino acid sequence shown in SEQ ID NO: 101, or linkers consisting of repeating sequences thereof. In addition, linkers generally used to link protein domains, such as GSSG linkers, can be used. Non-peptide linkers (linkers in chemical linking) are also not particularly limited, but examples include ε-aminocaproic acid, β-aminoalanine, γ-aminobutyric acid, 7-aminoheptanoic acid, 12-aminolauric acid, glutamic acid, p-aminobenzoic acid, etc.

[0068] In the protein according to this embodiment, the protein or its variant may be fused to either the N-terminus or the C-terminus, or both, of the functional protein. Furthermore, in the protein according to this embodiment, multiple variants of the protein may be fused to a single functional protein. That is, identical or different variants of the protein may be fused to both the N-terminus and the C-terminus of the functional protein, and the variant of the protein fused to the functional protein may be further fused with identical or different variants of the protein.

[0069] [CID Inducing Substances] CID inducers can be any compound capable of inducing dimerization of two different proteins, and are not particularly limited, as they can be appropriately selected by those skilled in the art according to the desired proteins. For example, when the two different proteins are the GID1 protein or a variant thereof and a variant of the GAI protein, gibberellin can be mentioned.

[0070] Gibberellins are a general term for plant hormones known to be involved in promoting cell elongation, seed germination and dormancy breaking, and senescence. Examples of gibberellins include gibberellin A1 ((1R,2R,5S,8S,9S,10R,12S)-5,12-dihydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01.02,8]heptadecane-9-carboxylic acid), gibberellin A3 ((1R,2R,5S,8S,9S,10R,12S)-5,12-dihydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02, 8)Heptadeca-13-ene-9-carboxylic acid), Gibberellin A20 ((1R,2R,5S,8S,9S,10R)-5-hydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadeca-9-carboxylic acid), Gibberellin A8 ((1R,2R,5S,8S,9S,10R,12R,13S)-5,12,13-trihydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2. [15,8.01,10.02,8]heptadecane-9-carboxylic acid), gibberellin A53 ((1S,2S,3S,4R,8S,9S,12S)-12-hydroxy-4,8-dimethyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4-dicarboxylic acid), gibberellin A44 diacid ((1S,2S,3S,4R,8R,9R,12S)-12-hydroxy-8-(hydroxymethyl)-4-methyl-13-methylidenetetracyclo[10.2.1.01,9.03, 8] Pentadecane-2,4-dicarboxylic acid), Gibberellin A14 aldehyde ((1R,2S,3S,4S,5S,8S,9S,12R)-2-formyl-5-hydroxy-4,8-dimethyl-13-methylidenetetracyclo[10.2.1.0.1,9.03,8]pentadecane-4-carboxylic acid), Gibberellin A51 catabolite ((1R,2S,3S,4R,9R,12R)-4-methyl-13-methylidene-6-oxotetracyclo[10.2.1.01,9.03,8]pentadeca-7-ene-2,Gibberellin A29 catabolite ((1S,2S,3S,4R,9R,12S)-12-hydroxy-4-methyl-13-methylidene-6-oxotetracyclo[10.2.1.01,9.03,8]pentadeca-7-ene-2,4-dicarboxylic acid), Gibberellin A6 ((1R,2R,5S,8S,9S,10R,11S,12R,14S)-5-hydroxy-11-methyl-6-methylidene-17-oxo-13,16-dioxahexacyclo[9.4.2.15,8.01,10.02,8.012,14]octadecane-9-cal Gibberellin A12 ((1R,2S,3S,4R,8S,9S,12R)-4,8-dimethyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4-dicarboxylic acid), Gibberellin A14 ((1R,2S,3S,4S,5S,8S,9S,12R)-5-hydroxy-4,8-dimethyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4-dicarboxylic acid), Gibberellin A37 ((1R,2S,3S,4S,5S,8R,9R,12R)-5-hydroxy-8-(H (Droxymethyl)-4-methyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4-dicarboxylic acid, Gibberellin A15 ((1R,2S,3S,4R,8R,9R,12R)-8-(hydroxymethyl)-4-methyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4-dicarboxylic acid), Gibberellin A24 ((1R,2S,3S,4R,8R,9R,12R)-8-formyl-4-methyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4-dicarboxylic acid), Gibberellin A24 ((1R,2S,3S,4R,8R,9R,12R)-8-formyl-4-methyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4-dicarboxylic acid) Gibberellin A36 ((1R,2S,3S,4S,5S,8R,9R,12R)-8-formyl-5-hydroxy-4-methyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4-dicarboxylic acid), Gibberellin A9 ((1R,2R,5R,8R,9S,10R)-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid), Gibberellin A4 ((1R,2R,5R,8R,9S,10R,12S)-12-hydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid), Gibberellin A51 ((1R,2R,5R,8R,9S,10R,13R)-13-hydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid), 2,3-didehydrogibberellin A9 ((1R,2R,5R,8R,9S,10 R)-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadec-12-ene-9-carboxylic acid), Gibberellin A7 ((1R,2R,5R,8R,9S,10R,12S)-12-hydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadec-13-ene-9-carboxylic acid), Gibberellin A34 ((1R,2R,5R,8R,9S,10R,12R,13S)-1213 -Dihydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid), Gibberellin A34 catabolite ((1R,2S,3S,4S,5R,9R,12R)-5-hydroxy-4-methyl-13-methylidene-6-oxotetracyclo[10.2.1.01,9.03,8]pentadec-7-ene-2,4-dicarboxylic acid), Gibberellin A8 catabolite ((1S,2S,3S,4S,5R,9R,12S)-5,12-dihydro Roxy-4-methyl-13-methylidene-6-oxotetracyclo[10.2.1.01,9.03,8]pentadec-7-ene-2,4-dicarboxylic acid), Gibberellin A5 ((1R,2R,5S,8S,9S,10R)-5-hydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadec-12-ene-9-carboxylic acid), Gibberellin A53 aldehyde ((1S,2S,3S,4R,8S,9S,12S)-2-formyl-12-hydroxy-4,8-dimethyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-4-carboxylic acid), gibberellin A44 ((1R,2R,5S,8S,9S,10S,11S)-5-hydroxy-11-methyl-6-methylidene-12-oxo-13-oxapentacyclo[9.3.3.15,8.01,10.02,8]octadecane-9-carboxylic acid), gibberellin A3 O-β-D-glucose ((1R,2R,5S,8S,9S,10R,12S)-5-hydroxy-11-methyl-6-methylidene-16-oxo-12-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadeca-13-ene-9-carboxylic acid), gibberellin A17 ((1S,2S,3R,4R,8R,9R,12S)-12-hydroxy-4-methyl-13-methylidenetetracyclo[10.2.1.01,9.0 Gibberellin A28 ((1S,2S,3S,4S,5S,8R,9R,12S)-5,12-dihydroxy-4-methyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4,8-tricarboxylic acid), Gibberellin A29 ((1R,2R,5S,8S,9S,10R,13R)-5,13-dihydroxy-11-methyl-6-methylidene-16-oxo-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid), Gibberellin A2 O-β-D-glucose ((1R,2R,5R,6R,8R,9S,10R,12S)-6-hydroxy-6,11-dimethyl-16-oxo-12-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxane-2-yl]oxy-15-oxapentacyclo[9.3.2.15,8.01,10.02,8]heptadecane-9-carboxylic acid), gibberellin A19 ((1S,2S,3S,4R,8R,9R,12S)-8-formyl-12-hydroxy-4-methyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-2,4-dicarboxylic acid), gibberellin A12 aldehyde ((1R,This includes (2S, 3S, 4R, 8S, 9S, 12R)-2-formyl-4,8-dimethyl-13-methylidenetetracyclo[10.2.1.01,9.03,8]pentadecane-4-carboxylic acid), etc.

[0071] The gibberellin may include at least one selected from the group consisting of gibberellin A3, gibberellin A1, gibberellin A4, and gibberellin A7, and is preferably, for example, gibberellin A4.

[0072] The CID inducer can be any compound capable of inducing dimerization of two different proteins, and is not particularly limited, as it can be appropriately selected by those skilled in the art to suit the desired proteins. For example, if the two different proteins are the PYL1 protein or a variant thereof and a variant of the ABI protein, it may include at least one selected from the group consisting of abscisic acid, mandipropamide, and azinephosethyl.

[0073] Abscisic acid (ABA) is (2Z,4E)-5-[(1S)-1-hydroxy-2,6,6-trimethyl-4-oxocyclohexa-2-en-1-yl]-3-methylpenta-2,4-dienoic acid and is a type of plant hormone.

[0074] Mandipropamide (CAS: 374726-62-2) is 2-(4-chlorophenyl)-N-[3-methoxy-4-(propa-2-inyloxy)phenethyl]-2-(propa-2-inyloxy)acetamide, a bactericide having a mandelic acid amide structure.

[0075] Adinphosethyl (CAS: 2642-71-9) is O,O-diethyl{[(4-oxo-3,4-dihydro-1,2,3-benzotriazin-3-yl)methyl]sulfanyl}phosphonothioate, a broad-spectrum phosphate ester insecticide.

[0076] The protein according to this embodiment may be produced by protein purification or by genetic engineering techniques. The production method is not particularly limited, but for example, it can be obtained by using the cDNA of the wild-type protein as a template, designing primers to introduce the desired mutation, obtaining nucleic acid amplified by PCR, functionally linking it to an expression promoter, optionally linking a tag as well, introducing it into an appropriate expression vector, and expressing it in various cells. The mutant protein produced may be purified as needed. The purification method is not particularly limited, but examples include purification by affinity chromatography column or the like.

[0077] The localization of the protein according to this embodiment within the cell may be in the cytoplasm, on the cell membrane, or in an intracellular organelle. It may be a transmembrane protein or a cell membrane-bound protein, and it may or may not be integrated into the cell's genome.

[0078] [Nucleic Acids] The nucleic acids in this embodiment include a base sequence encoding the protein according to the present invention. The nucleic acids in this embodiment are not particularly limited as long as they include a base sequence encoding the protein according to the present invention. In particular, the base sequence may be optimized for the codons of the host organism. More specific examples of nucleic acids in this embodiment are as described above.

[0079] As described above, the nucleic acids according to this embodiment can be obtained, for example, by PCR using the cDNA of a wild-type protein as a template and designing primers to introduce the desired mutation, or by artificial gene synthesis.

[0080] [Expression Vector] The expression vector according to this embodiment is not particularly limited as long as it contains the nucleic acid according to the present invention and is capable of expressing the protein of the present invention. For example, it may be a viral vector, a plasmid vector (sometimes simply called a plasmid), a cosmid vector, an artificial chromosome vector, etc., or a fragment thereof. It may be an episomal plasmid vector, a constitutive expression vector, an inducible expression vector, or a transient expression vector. Furthermore, after being introduced into cells, part or all of the introduced plasmid vector may be inserted into the chromosome. It can be appropriately selected depending on the type of cells to be introduced and the purpose.

[0081] The expression vector according to this embodiment may be capable of autonomous replication within the host cell, or it may be incorporated into the host chromosome and replicated along with chromosome replication.

[0082] The expression vector according to this embodiment has, in addition to the nucleic acid according to the present invention, one or more regulatory sequences operably linked to the base sequence of the nucleic acid. The regulatory sequences are sequences that control protein expression in the host (e.g., promoters, enhancers, ribosome-binding sequences, transcription termination sequences, etc.) and can be appropriately selected depending on the type of host.

[0083] The expression vector according to this embodiment may include the base sequence of a selection marker gene for selecting a transformed strain. Examples of selection marker genes include genes that confer antibiotic resistance and genes that complement nutritional requirements.

[0084] The expression vector according to this embodiment is not particularly limited as long as it contains the base sequence of the nucleic acid according to the present invention and can express the protein of the present invention, but for example it may contain a base sequence having 90% or more sequence identity with the base sequences shown in SEQ ID NOs. 29-32, 184-185, and 198-199. Furthermore, as a promoter for constitutive expression, in addition to the PGK promoter and CAG promoter described in SEQ ID NOs. 29-32, 184-185, and 198-199, generally known promoters such as the CVM promoter, EF1α promoter, SSFV promoter, and CBh promoter can be used. Depending on the purpose, tissue / cell type specific promoters can also be used. To express two genes for GAI and GID1, or PYL1 and ABI / CID, in addition to the method of expression using independent expression cassettes as in SEQ ID NOs. 29-34, 184-185, and 198-199, generally known 2A peptides and IRES sequences can be utilized. The vector backbone can be any of the above-mentioned vectors, such as pUC, pPB, pHR, etc. The expression vector according to this embodiment may contain a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NOs. 176-177, 182, 192-193, and 196. The above sequence identity may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or it may be 100%.

[0085] [Cells] The cells according to this embodiment are introduced with the nucleic acid or expression vector according to the present invention. The cells according to this embodiment are not particularly limited as long as they are host cells into which the nucleic acid or expression vector according to the present invention is introduced, and which are capable of expressing the protein according to the present invention.

[0086] The method for incorporating a specific base sequence into the cell genome according to this embodiment is not particularly limited and can be carried out by methods commonly used in the art. For example, it can be carried out by methods such as random recombination, homologous recombination, or site-directed recombination.

[0087] The method for introducing a specific base sequence into cells according to this embodiment is not particularly limited and can be carried out by methods commonly used in the art. If the cells are prokaryotic cells, for example, competent cells can be prepared and the process can be carried out by a transformation method such as the heat shock method. If the cells are eukaryotic cells, for example, the process can be carried out by a lipofection method using a transfection reagent, a calcium phosphate method, an electroporation method, etc.

[0088] The cells according to this embodiment are not particularly limited as long as they can be used in the CID method, and any cells from prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used, with animal cells being preferred and mammalian cells being more preferred. Examples of mammalian cells include cells derived from humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, etc., with human-derived cells being preferred. Examples of human-derived cells include HEK293FT cells.

[0089] The cells according to this embodiment may be cells used in a method for forming dimers, as described later.

[0090] [Method for forming a dimer] The method for forming a dimer according to this embodiment (hereinafter also referred to as "the method according to this embodiment") is a method for forming a dimer of a first protein and a second protein, and includes a step of contacting a specific first protein, a specific second protein, and a chemically induced dimerization (CID) inducing substance (contact step).

[0091] The first protein contains the first dimer-forming protein. The second protein also contains the second dimer-forming protein. That is, the first protein contains the amino acid sequence of the first dimer-forming protein. The second protein also contains the amino acid sequence of the second dimer-forming protein. Here, the first dimer-forming protein may be a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8, or a protein or variant thereof consisting of the amino acid sequence shown in SEQ ID NO: 201, wherein the amino acid residue at position 291 in the amino acid sequence shown in SEQ ID NO: 201 is a threonine residue, and may also be a protein or variant thereof listed in Tables 4 and 5. The proteins or variants thereof listed in Tables 4 and 5 are as described above. These proteins or their variants and CID inducers are as described above.

[0092] In the method according to this embodiment, one of the first and second dimer-forming proteins may be a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, and the other may be a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167. Alternatively, in the method according to this embodiment, one of the first and second dimer-forming proteins may be a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, and the other may be a variant of a protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8.

[0093] The first protein according to one embodiment may further contain the first functional protein, and the second protein according to one embodiment may further contain the second functional protein. That is, the first protein may further contain the amino acid sequence of the first functional protein, and the second protein may further contain the amino acid sequence of the second functional protein. The first functional protein and the second functional protein may be the same or different. The first protein and the second protein are as described in the protein according to this embodiment.

[0094] In the contact step of the method according to this embodiment, the first protein and the second protein induce CID, and they form a protein dimer. When the first protein contains a first functional protein and the second protein contains a second functional protein, the first protein and the second protein induce CID, causing the first functional protein and the second functional protein to perform predetermined functions, thereby enabling the control of a predetermined biological process (the expression or inhibition of a desired biological process).

[0095] In the method according to this embodiment, the first functional protein may be, for example, scFV-hinge-Tm, in which case the second functional protein may be, for example, Tm-CD3zeta. The first protein may include the amino acid sequence of the first dimerizing protein and the amino acid sequence of scFV (single-chain antibody)-hinge-Tm, and may be a protein consisting of the amino acid sequence shown in any of SEQ ID NOs. 216, 218, 220, and 222. The second protein may include the amino acid sequence of the second dimerizing protein and the amino acid sequence of Tm-CD3zeta, and may be a protein consisting of the amino acid sequence shown in SEQ ID NOs. 212 or 214. In this case, the method according to this embodiment can be used, for example, to regulate the activity of T cells expressing chimeric antigen receptors (CARs) (CAR-T cells).

[0096] The method for carrying out the contact step according to this embodiment is not particularly limited. For example, it can be carried out by mixing the first protein, the second protein, and the CID inducer, or, if the first protein and the second protein are expressed in cells, by bringing the cells into contact with the CID inducer. That is, the method according to this embodiment may include a step of preparing cells that express the first protein and the second protein (preparation step). The preparation step can be carried out by producing cells that express the first protein and the second protein by the method described above. If the method according to this embodiment includes a preparation step, the contact step may be a step of bringing the cells obtained in the preparation step into contact with the CID inducer.

[0097] If the method according to this embodiment includes a preparation step, the contact step can be carried out, for example, by administering the cells obtained in the preparation step and the CID-inducing substance to the target, and by culturing the cells obtained in the preparation step in a culture medium containing the CID-inducing substance.

[0098] When the contact step is carried out by administering the cells obtained in the preparation step and the CID inducer, the method of administration may be, for example, oral or parenteral administration. Parenteral administration can be further divided into systemic administration and local administration (e.g., subcutaneous administration, transdermal administration, transmucosal administration, or transrectal administration).

[0099] Suitable dosage forms for oral administration include, for example, tablets, pills, granules, powders, capsules, drops, sublingual preparations, lozenges, and liquids. Suitable dosage forms for parenteral administration include, for example, liquids (including suspensions), emulsions (creams), gels, ointments (including pastes), plasters, powders, or suppositories.

[0100] For the contact process, the daily dose of cells administered to a target is, for example, 0.01 × 10⁻⁶ when administered to an adult male (weighing 60 kg). ８ cells / kg body weight ~10×10 ８ Cells / kg body weight, or 0.1 × 10⁻⁶ ８ cells / kg body weight ~2.5×10 ８The dose may be cells / kg body weight. As for the dose of the CID inducer to the target in the contact process, when administered to an adult human male (60 kg body weight), the daily dose of the CID inducer may be, for example, 1 mg / kg body weight to 50 mg / kg body weight, or 5 mg / kg body weight to 10 mg / kg body weight.

[0101] The administration interval of cells and CID-inducing substances in the contact process can be set as appropriate depending on the purpose. For example, the administration interval may be once a day, once every two days, once every three days, or once every seven days (one week).

[0102] Examples of target organisms for administration include animals such as mammals, birds, reptiles, amphibians, and fish, as well as plants, with mammals being preferred. Specific examples of mammals include mice, rats, rabbits, dogs, monkeys, and humans. Furthermore, the target organisms may also be those other than humans.

[0103] If the contact step is carried out by culturing the cells obtained in the preparation step in a medium containing a CID inducer, the cell culture can be performed under general culture conditions used by those skilled in the art. The cell culture can be performed under suitable culture conditions depending on the type of cells used. The culture temperature may be 20°C to 40°C or 30°C to 37°C. The pH of the medium may be 6 to 8 or 7.2 to 7.4. The culture time may be 1 to 2 days or 3 to 5 days. The culture medium used is not particularly limited and can be any medium capable of culturing the cells obtained in the preparation step, and can be appropriately selected depending on the cell type, etc. Examples of culture media used in the culture step include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), etc. The medium may be a serum-added medium or a serum-free medium. The medium may be a mixed medium obtained by mixing two types of mediums.

[0104] The concentration of CID-inducing substances in the culture medium during the culture process is not particularly limited, but may be, for example, less than the order of μM. Specific concentrations are as described above.

[0105] [Kit] The kit according to this embodiment includes a protein having dimerization ability in the presence of a chemically induced dimerization (CID) inducing substance, wherein the protein is a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8, or a protein or variant thereof consisting of the amino acid sequence shown in SEQ ID NOs: 201, wherein the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NOs: 201 is a threonine residue, and includes a nucleic acid containing a protein-coding base sequence, including a protein or variant thereof. The protein may be a protein or variant thereof listed in Tables 4 and 5. The proteins or variants thereof listed in Tables 4 and 5 are as described above. The variants of the protein and the CID inducing substance are as described above.

[0106] The kit according to this embodiment may include a nucleic acid comprising a nucleotide sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, and a nucleic acid comprising a nucleotide sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, or a nucleic acid comprising a nucleotide sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, and a nucleic acid comprising a nucleotide sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 7-8. The kit according to this embodiment may further include one or more selected from the group consisting of nucleic acids having a nucleotide sequence encoding a functional protein, a CID inducer, and cells. The functional protein and cells are as described above.

[0107] The kit according to this embodiment can be used to produce an expression vector or the like containing a base sequence encoding the fusion protein according to the present invention, and the produced expression vector or the like can be used to produce cells according to the present invention. The kit according to this embodiment may also be a kit for use in a method for forming a dimer according to the present invention.

[0108] The kit according to this embodiment may further contain reagents necessary for preparing the cells used in the method of the present invention and for carrying out the method of the present invention, in addition to the nucleic acid, CID inducer, and cells mentioned above. Examples of such reagents include PCR reagents (e.g., DNA polymerase, dNTPs, etc.), nucleic acid purification reagents, transfection reagents, and buffer solutions.

[0109] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0110] <1. Search for Orthologous Genes> In rice (Oryza sativa), maize (Zea mays), or quinoa (Chenopodium quinova), we searched for orthologs of the abscisic acid receptor PYL gene and its corresponding transcription factor ABI gene, and the gibberellin receptor GID1 gene and its corresponding transcription factor GAI gene. The following databases were used as targets. The amino acid sequences of the proteins expressed by the PYL1 gene, ABI gene, GID1 gene, and GAI gene of Arabidopsis thaliana (SEQ ID NOs. 33-36, respectively) were used as query sequences and the search was performed using the BLAST program. - Rice: RAP db (IRGSP-1.0, The Rice Annotation Project Database, https: / / rapdb.dna.affrc.go.jp / download / irgsp1.html) - Maize: Phytozome13 (RefGen_V4, The Joint Genome Institute, USD Department of Energy, https: / / phytozome-next.jgi.doe.gov / info / Zmays_RefGen_V4) - Quinoa: Phytozome13 (Chenopodium quinoa v1.0, The Joint Genome Institute, US Department of Energy, https: / / phytozome-next.jgi.doe.gov / info / Cquinoa_v1_0)

[0111] Next, using the CrystalW program, multiple sequence alignment of amino acid sequences was performed on the amino acid sequences of the proteins presumed to be encoded by the ortholog candidate genes of the extracted rice, maize, or quinoa genes, and the amino acid sequences of the proteins encoded by the respective Arabidopsis thaliana genes, and genes with high similarity were extracted. As a result, if the first two letters of the rice-derived gene are written as Os, the first two letters of the quinoa-derived gene are written as Cq, and the first two letters of the maize-derived gene are written as Zm, then Os10g0573400 was found as the OsPYL1 gene. Os01g0583100 was found as the OsABI gene. Os05g0407500 was found as the OsGID1 gene. Os03g0707600 was found as the OsGAI gene. Os06g0562200 was found as the OsPYL2 gene. The following genes were identified: Os06g0527800 as the OsPYL8 gene; AUR62007289 as the CqPYL1 gene; C. quinoa v1.0|AUR62029021 as the CqABI gene; AUR62000900 as the CqGID1 gene; AUR62014191 as the CqGAI gene; Zm00001eb024490 as the ZmPYL1 gene; Zm00001eb160470 as the ZmABI gene; Zm00001eb287800 as the ZmGID1 gene; and ZmLH145.01G43330 as the ZmGAI gene. Each gene ID will conform to the description method used in the databases mentioned above.

[0112] <2. Evaluation of CID Activity - Overview of Plasmid Vectors> In this experimental example, four different types of gene expression vectors were created: effector vector #1, effector vector #2, reporter vector, and reference vector. Schematic diagrams of these four types of expression vectors are shown in Figure 3. Effector vector #1 uses the pHR plasmid as its backbone. Downstream of the PGK promoter, a CDS (Coding Sequence) of the GAI gene or a part thereof - dCas9 gene - ABI CDS or a part thereof sequence cassette is inserted. Downstream of the CAG promoter, a CDS of GID1 or a part thereof - VPR CDS is inserted. The proteins expressed from each CDS or gene sequence are designed to fuse via a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 101. Effector vector #2 has a pPB plasmid as its backbone, with a single-guide RNA (sgRNA) gene targeting the TRE3G promoter inserted downstream of the U6 promoter, and a CDS of PYL1 or a portion thereof - VPR inserted downstream of the CMV promoter. The reporter vector has a CDS of firefly luciferase (FLUC) inserted downstream of the TRE3G promoter. The reference vector has a CDS of Renilla luciferase (RLUC) inserted downstream of the SV40 promoter. In Figure 3, GAI, ABI, GID1, and PYL1, enclosed in solid lines, represent Arabidopsis thaliana and, as described in 1. above, respectively. The orthologs were replaced with those derived from rice, maize, and quinoa, which were identified in the study.

[0113] <3. Principle of CID Activity Evaluation Method> When the above-mentioned effector vector #1, effector vector #2, reporter vector, and reference vector are simultaneously introduced into HEK293FT cultured cells, CID activity can be evaluated by the following mechanism. Effector vector #1 expresses the GID1-VPR fusion protein and the GAI-tagBFP-SpdCas9 (dCas9 derived from S. pyogenes)-ABI fusion protein. Effector vector #2 expresses the sgRNA (SpsgTRE3G) that recognizes the sequence near the TRE3G promoter that expresses the downstream reporter gene firefly luciferase (Firefly LUC) in the reporter vector, and the PYL1-VPR fusion protein. SpsgTRE3G and SpdCas9 form a complex, and the GAI-tagBFP-SpdCas9-ABI fusion protein binds to the nucleotide sequence near the TRE3G promoter via SpdCas9. As a result, SpdCas9 represses the expression of firefly luciferase from the reporter vector. If the ortholog candidates of GID1, GAI, PYL1, and ABI, which are components of the CID system, have the ability to form dimers, the transcriptional repression by SpdCas9-SpsgTRE3G is released via the transcription activator VPR in the GID1-VPR fusion protein or the PYL1-VPR fusion protein, and firefly luciferase is expressed from the activated TRE3G promoter. Since sea urticaria luciferase is constitutively expressed in the reference vector, the activity of the firefly luciferase can be normalized by comparing it with the activity of sea urticaria luciferase. This normalized firefly luciferase activity is thought to reflect the dimerization ability of the candidate ortholog, i.e., the CID activity.

[0114] <4. Preparation of Plasmid Vectors> The components of the plasmid vectors used in Experimental Examples 1 to 9 in this disclosure are shown in Tables 1 and 2 below.

[0115]

[0116]

[0117] The following describes in detail the method for preparing the plasmid vectors used to evaluate CID activity in the experimental examples in this disclosure. Individual plasmid vector names are shown in parentheses. (pOSA0001) pOSA0001 is effector vector #1 in which the sequences of Arabidopsis thaliana GAI and ABI, as well as the sequence of GID1 (hereinafter referred to as AttGAI (SEQ ID NO: 37), AttABI (SEQ ID NO: 38), and AtGID1 (SEQ ID NO: 39), respectively) are inserted. pOSA0001 was prepared using pSLQ2821 (Addgene #84257) used in Gao et al., Nature Methods 13, 1043-1049 2016.

[0118] (pOSA0002) pOSA0002 is effector vector #2 with a portion of the sequence of PYL1 from Arabidopsis thaliana (AttPYL1, sequence number 40) inserted. pOSA0002 was created using pSLQ2804 (Addgene #84258) used in Gao et al., Nature Methods 13, 1043-1049 2016.

[0119] (Reporter Vector) The reporter vector (pTetON-LUC) used was VB_0152_pGAL4-TATA-LUC, which was used in Gao et al., Nature Methods 13, 1043-1049 2016, and Mitsuda et al., Plant and Cell Physiology, Volume 51, Issue 12, December 2010, Pages 2145-2151, https: / / doi.org / 10.1093 / pcp / pcq161.

[0120] (Reference Vector) The reference vector used was pMLS-SV40-hRLUC, which is a combination of pMLS-SV40-EGGFP (Addgene #46919) and VB0150_pRLHSP_GWB5, as used in Yoshida et al., Front.Plant Sci., Volume 4 - 2013 | https: / / doi.org / 10.3389 / fpls.2013.00383.

[0121] (pJR0027) pJR0027 is a plasmid in which the vector type is effector vector #1, and in which AttGAI is replaced with the CDS of rice GAI (OsGAI, the wild-type OsGAI gene with intron deletion and codon optimization to human codons, SEQ ID NO: 14) in pOSA0001, AtGID1 is replaced with the CDS of rice GID1 (OsGID1, SEQ ID NO: 10), and AttABI is replaced with the CDS of rice ABI (OsABI, SEQ ID NO: 26).

[0122] First, an OsGAI DNA fragment with a linker sequence (Twist Bioscience) for Gibson assembly was artificially synthesized (SEQ ID NO: 41). Next, using pOSA0001 as a template, a PCR product of a linear plasmid that did not contain the AttGAI base sequence to be substituted was obtained by PCR using primer A (SEQ ID NO: 42) and primer B (SEQ ID NO: 43). Subsequently, in order to substitute AttGAI for OsGAI in pOSA0001, the above PCR product and the synthesized OsGAI DNA fragment were mixed, and a Gibson assembly reaction was performed using NEBuilder HiFi (NEB). Subsequently, E. coli DH5α was transformed using this reaction product to obtain plasmid 1 in which AttGAI was substituted for OsGAI. Similarly to the above, a DNA fragment of OsABI (SEQ ID NO: 44) with a linker sequence for Gibson assembly was artificially synthesized. Then, using plasmid 1 as a template, a PCR product (linear plasmid) without the AttABI base sequence was obtained by PCR using primer C (SEQ ID NO: 45) and primer D (SEQ ID NO: 46) in the same manner as described above. Subsequently, in order to replace AttABI with OsABI in plasmid 1, the artificially synthesized gene and the PCR product were mixed and a Gibson assembly reaction was performed in the same manner as above. Then, E. coli DH5α was transformed using this reaction product to obtain plasmid 2 in which AttABI was replaced with OsABI. Furthermore, similar to the above, a DNA fragment of OsGID1 (SEQ ID NO: 47) with a linker sequence for Gibson assembly was artificially synthesized. Then, using plasmid 2 as a template, a PCR product (linear plasmid) without the AtGID1 base sequence was obtained by PCR using primer E (SEQ ID NO: 48) and primer F (SEQ ID NO: 49). Subsequently, a Gibson assembly reaction was performed in plasmid 2 in the same manner as described above to replace AtGID1 with OsGID1. Then, E. coli DH5α was transformed using the reaction product to obtain pJR0027 (SEQ ID NO: 50) in which AtGID1 was replaced with OsGID1.

[0123] (pJR0039) pJR0039 is an effector vector #1 plasmid in which AtGID1 is replaced with a portion of the rice GAI sequence (OstGID1, SEQ ID NO: 109) in pOSA0001, AttGAI is replaced with a portion of the rice GAI sequence (OstGAI, SEQ ID NO: 16), and AttABI is replaced with a portion of the rice ABI sequence (OstABI, SEQ ID NO: 28). It was prepared using pJR0027 as a template.

[0124] First, a DNA fragment of OstGAI with a linker sequence for Gibson assembly added was prepared (SEQ ID NO: 51), similar to the preparation of pJR0027. Next, a PCR product of a linear plasmid that did not contain the GAI base sequence to be substituted was obtained, similar to the preparation of pJR0027. Subsequently, in order to replace OsGAI with OstGAI in pJR0027, the Gibson assembly reaction was performed, similar to the preparation of pJR0027. Then, E. coli DH5α was transformed using this reaction product to obtain plasmid 1 in which OsGAI was replaced with OstGAI. Similarly, a DNA fragment of OstABI with a linker sequence for Gibson assembly added (SEQ ID NO: 52) was prepared. Subsequently, using plasmid 1 as a template and primer C (SEQ ID NO: 45) and primer D (SEQ ID NO: 46), a PCR product (linear plasmid) that did not contain the ABI base sequence was obtained by PCR. Subsequently, a Gibson assembly reaction was performed in plasmid 1 in the same manner as described above to replace OsABI with OstABI. Then, E. coli DH5α was transformed using the reaction product to obtain pJR0039 (SEQ ID NO: 53) in which OsABI was replaced with OstABI.

[0125] (pJR0042) pJR0042 is an effector vector #2, a plasmid in which AttPYL1 in pOSA0002 is replaced with a portion of the sequence of rice PYL1 (OstPYL1, SEQ ID NO: 22).

[0126] Similar to pJR0027, an OstPYL1 DNA fragment (SEQ ID NO: 54) with a linker sequence for Gibson assembly was artificially synthesized. Next, using pOSA0002 as a template and primers G (SEQ ID NO: 55) and H (SEQ ID NO: 56), a PCR product (linear plasmid) that did not contain the AttPYL1 base sequence to be substituted was obtained by PCR. Subsequently, in pOSA0002, a Gibson assembly reaction was performed in the same manner as with pJR0027 to replace AttPYL1 with OstPYL1. Then, E. coli DH5α was transformed using this reaction product to obtain pJR0042 (SEQ ID NO: 57) in which AttPYL1 was replaced with OstPYL1.

[0127] (pJR0028) pJR0028 is an effector vector #2, a plasmid in which AttPYL1 in pOSA0002 is replaced with the CDS of rice PYL1 (OsPYL1, SEQ ID NO: 20).

[0128] Similar to pJR0027, an OsPYL1 DNA fragment (SEQ ID NO: 58) with a linker sequence for Gibson assembly was artificially synthesized. Next, a PCR product (linear plasmid) without the AttPYL1 base sequence to be substituted was obtained in the same manner as pJR0042. Subsequently, in pOSA0002, a Gibson assembly reaction was performed in the same manner as pJR0027 to substitute AttPYL1 with OsPYL1. Then, E. coli DH5α was transformed using this reaction product to obtain pJR0028 (SEQ ID NO: 59) in which AttPYL1 was substituted with OsPYL1.

[0129] (pJR0029) pJR0029 is an effector vector #2, a plasmid in which AttPYL1 in pOSA0002 is replaced with the CDS of rice PYL2 (OsPYL2, SEQ ID NO: 60).

[0130] Similarly to the above, pJR0029 (SEQ ID NO: 62) was obtained in the same manner as pJR0028, except that an OsPYL2 DNA fragment (SEQ ID NO: 61) with a linker sequence for Gibson assembly was artificially synthesized.

[0131] (pJR0030) pJR0030 is an effector vector #2, a plasmid in which AttPYL1 in pOSA0002 is replaced with the CDS of rice PYL8 (OsPYL8, SEQ ID NO: 63).

[0132] Similarly to the above, pJR0030 (SEQ ID NO: 65) was obtained in the same manner as pJR0028, except that a DNA fragment of OsPYL8 (SEQ ID NO: 64) with a linker sequence for Gibson assembly was artificially synthesized.

[0133] (pJR0033) pJR0033 is a plasmid of the effector vector #1, in which AttABI in pOSA0001 is replaced with the rice ABI CDS (OsABI, SEQ ID NO: 26).

[0134] Similarly to the above, pJR0033 (SEQ ID NO: 67) was obtained in the same manner as pJR0027, except that an OsABI DNA fragment (SEQ ID NO: 66) with a linker sequence for Gibson assembly was artificially synthesized.

[0135] (pJR0043) pJR0043 is an effector vector #2, a plasmid in which AttPYL1 in pOSA0002 is replaced with the domain of rice PYL2 (OstPYL2, Sequence ID No. 68).

[0136] Similarly to the above, pJR0043 (SEQ ID NO: 70) was obtained in the same manner as pJR0030, except that a DNA fragment of OstPYL2 with a linker sequence for Gibson assembly (SEQ ID NO: 69) was artificially synthesized.

[0137] (pJR0044) pJR0044 is an effector vector #1 plasmid in which AttABI in pOSA0001 is replaced with a portion of the rice ABI sequence (OstABI, sequence number 28).

[0138] Similarly to the above, pJR0044 (SEQ ID NO: 72) was obtained in the same manner as pJR0027, except that an OstABI DNA fragment (SEQ ID NO: 71) with a linker sequence for Gibson assembly was artificially synthesized.

[0139] (pJR0078(Zm#1)) pJR0078(Zm#1) is an effector vector #1 plasmid in which AttGAI is replaced with a portion of the sequence of maize GAI (ZmtGAI, SEQ ID NO: 15) in pOSA0001, AttABI is replaced with a portion of the sequence of maize ABI (ZmtABI, SEQ ID NO: 27), and AtGID1 is replaced with maize GID1 (ZmGID1, SEQ ID NO: 9).

[0140] Similarly to the above, pJR0078 (Zm#1) (SEQ ID NO: 76) was obtained in the same manner as pJR0039, except that a DNA fragment of ZmtGAI (SEQ ID NO: 73), a DNA fragment of ZmtABI (SEQ ID NO: 74), and ZmGID1 (SEQ ID NO: 75) with linker sequences for Gibson assembly were artificially synthesized.

[0141] (pJR0076(Zm#2)) pJR0076(Zm#2) is a plasmid in which the vector type is effector vector #2, and AttPYL1 in pOSA0002 is replaced with a portion of the sequence of maize PYL1 (ZmtPYL1, sequence number 21).

[0142] Similarly to the above, pJR0076 (Zm#2) (SEQ ID NO: 78) was obtained in the same manner as pJR0028, except that a DNA fragment of ZmtPYL1 with a linker sequence for Gibson assembly (SEQ ID NO: 77) was artificially synthesized.

[0143] (pJR0077(Cq#1)) pJR0077(Cq#1) is an effector vector #1 plasmid in which AttGAI is replaced with a portion of the sequence of quinoa GAI (CqtGAI1, SEQ ID NO: 79) in pOSA0001, AttABI is replaced with a portion of the sequence of quinoa ABI (CqtABI, SEQ ID NO: 80), and AtGID1 is replaced with a portion of the sequence of quinoa GID1 (CqGID1, SEQ ID NO: 81).

[0144] Similarly to the above, pJR0077 (Cq#1) (SEQ ID NO: 85) was obtained in the same manner as pJR0039, except that DNA fragments of CqtGAI (SEQ ID NO: 82), CqtABI (SEQ ID NO: 83), and CqtGID1 (SEQ ID NO: 84) with linker sequences for Gibson assembly were artificially synthesized.

[0145] (pJR0087(Cq#2)) pJR0087(Cq#2) is an effector vector #2 plasmid in which AttPYL1 in pOSA0002 is replaced with a portion of the sequence of quinoa PYL1 (CqtPYL1, sequence number 86).

[0146] Similarly to the above, pJR0087 (Cq#2) (Sequence ID 88) was obtained in the same manner as pJR0028, except that a DNA fragment of CqtPYL1 with a linker sequence for Gibson assembly (Sequence ID 87) was artificially synthesized.

[0147] Table 3 shows the vector type for each plasmid and the combinations of the GID1 gene, GAI gene, PYL1 gene, and ABI gene inserted into effector vector #1 or effector vector #2.

[0148]

[0149] <5. Introduction of Plasmid Vector into Cells> HEK293FT cells (ATCC) were cultured in Dulbecco's Modified Eagle Medium (WAKO) supplemented with 10% FBS (Biosera) at 37°C and 5% CO2. ２The plasmids were maintained in a carbon dioxide incubator. For pJR0031, a plasmid solution was prepared at a concentration of 77 ng / μl, and for the other plasmids, a plasmid solution was prepared at a concentration of 300 ng / μl. Transfection of cells was performed using ViaFect® transfection reagent (Promega) with a 4:1 reagent-to-plasmid solution ratio to prepare the transfection complex, following the manufacturer's instructions. The plasmids used in the transfection complex were effector vector #1, effector vector #2, reporter plasmid, and reference vector. Effector vector #1 and effector vector #2 were replaced with appropriate plasmids as needed for each experiment. After incubating the transfection complex at room temperature for 10 minutes, the final concentration was 1 × 10⁻⁶. ４ Cells were added to a 96-well plate to a cell / mL concentration, and cells into which each plasmid had been transfected were obtained. Transfection and induction efficiency were evaluated by monitoring mCherry (expressed from effector vector #2) under a fluorescence microscope.

[0150] <6. Luciferase Assay> 24 hours after transfection, 500-fold concentrated abscisic acid (50 mM), or GA3, GA3-AM, or GA4 (5 mM) dissolved in DMSO was added to each well to the specified concentration to induce luciferase expression in the cells. After incubating the cells for 24 hours, the medium was removed, the cells were washed with PBS, treated with trypsin for cell detachment, and the cells were collected. Subsequently, a luciferase assay was performed using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Firefly and sea urchin luciferase activity was measured using a luminometer (TECAN, Infinite Pro200 M PLEX).

[0151] [Experimental Example 1: Comparison of Full-Length Protein and Protein Domain (1)] HEK293FT cells were prepared using the procedure described above, with pOSA0001 (AttGAI and AtGID1) and pOSA0002 introduced, pJR0027 (OsGAI and OsGID1) and pJR0042 introduced, and pJR0039 (OstGAI and OstGID1) and pJR0042 introduced. GA4 was added to these cells to a final concentration of 10 μM, and the CID activity was evaluated by performing a luciferase assay as described above. The same experiment was also performed without GA. The results are shown in Figure 4.

[0152] As reported in the non-patent literature (Gao et al., Nature Methods 13, 1043-1049 2016), in cells into which AttGAI and AtGID1 were introduced, the addition of 10 μM GA resulted in induced luciferase activity, confirming the presence of CID activity (Figure 4, "Arabidopsis thaliana," first and second bar graphs from the left). Furthermore, in cells into which full-length OsGAI and OsGID1 were introduced, the firefly luciferase activity remained at the same level as in the untreated cells, even with the addition of 10 μM GA (Figure 4, "Rice, full length," third and fourth bar graphs from the left). From these results, it was confirmed that full-length OsGAI and OsGID1 do not exhibit CID activity. On the other hand, in cells into which OstGAI and OstGID1 were introduced, the addition of 10 μM GA significantly increased the activity of luciferase expression, confirming that they possessed CID activity (Figure 4, "Rice-derived, Truncate," bar graphs 5th and 6th from the left). Furthermore, since the luciferase activity of cells into which OstGAI and OstGID1 were introduced was approximately 1.6 times higher than that of cells into which conventional AttGAI and AtGID1 were introduced, it was suggested that rice GAI and GID1 orthologs exhibit higher CID activity than those of Arabidopsis thaliana.

[0153] [Experimental Example 2: Comparison of full-length protein and protein domain (2)] HEK293FT cells introduced with pOSA0002 (AttABI) and pOSA0001 (AttPYL1), HEK293FT cells introduced with pJR0033 (OsABI), pJR0028 (OsPYL1) and pJR0044 (OstABI), HEK293FT cells introduced with pJR0033 (OsABI), pJR0029 (OsPYL2) and pJR0044 (OstABI), JR0033 (OsABI) and pJR003 HEK293FT cells were prepared using the procedure described above, with the following cell types: HEK293FT cells introduced with 0 (OsPYL8) and pJR0044 (OstABI), HEK293FT cells introduced with JR0044 (OstABI) and pJR0042 (OstPYL1), HEK293FT cells introduced with JR0044 (OstABI) and pJR0043 (OstPYL2), and HEK293FT cells introduced with JR0044 (OstABI) and pJR0030 (OsPYL8). Abscisic acid (ABA) was added to these cells to a final concentration of 100 μM, and CID activity was evaluated by luciferase assay as in Experimental Example 1. As a control experiment, luciferase assay was also performed in the case without ABA addition. The results are shown in Figure 5.

[0154] As described in the non-patent literature (Gao et al., Nature Methods 13, 1043-1049 2016), in cells into which AttABI and AttPYL1 were introduced, the addition of 100 μM ABA resulted in induced luciferase activity, confirming the presence of CID activity (Figure 5, "Arabidopsis thaliana," bar graphs 1st to 2nd from the left). Furthermore, in cells into which full-length OsPYL1, OsPYL2, OsPYL8, and OsABI were introduced, luciferase activity remained at the same level as in the untreated cell, even with the addition of 100 μM ABA (Figure 5, "Rice, full length," bar graphs 3rd to 8th from the left). These results confirmed that full-length OsPYL1, OsPYL2, OsPYL8, and OsABI did not exhibit CID activity. On the other hand, cells into which OstPYL1 and OstABI, and OstPYL2 and OstABI were introduced all showed luciferase activity upon addition of 100 μM ABA, thus confirming that they possessed CID activity (Figure 5, "Rice-derived, Truncate," bar graphs 9th to 12th from the left). Furthermore, cells into which OstPYL2 and OstABI were introduced had approximately 2.3 times higher luciferase activity than cells into which conventional AttABI and AttPYL1 were introduced, and cells into which OstPYL1 and OstABI were introduced had approximately twice as high luciferase activity as cells into which OstPYL2 and OstABI were introduced. These results suggest that rice PYL1 and ABI orthologs exhibit higher CID activity than those of Arabidopsis thaliana.

[0155] [Experimental Example 3: Evaluation of Concentration Dependence of CID Activity (1)] HEK293FT cells introduced with pOSA0001 (AttGAI and AtGID1) and pOSA0002, and HEK293FT cells introduced with pJR0039 (OstGAI and OstGID1) and pJR0042 were prepared using the procedure described above. GA4 was added to these cells to a final concentration of 1 nM, 10 nM, 100 nM, 1 μM, or 10 μM, and the GA4 concentration dependence of CID activity was evaluated by performing a luciferase assay as described above. The same experiment was also performed when GA4 was not added. The results are shown in Figure 6.

[0156] In all cells, both those introduced with AttGAI and AtGID1 ("AtGID1-AttGAI" in Figure 6) and those introduced with OstGAI and OstGID1 ("OstGID1-OstGAI" in Figure 6), GA4 concentration-dependent luciferase activity was observed. Furthermore, the luciferase activity induced by 100 nM GA4 in cells introduced with OstGAI and OstGID1 was at the same level as the luciferase activity induced by 10 μM GA4 in cells introduced with AttGAI and AtGID1. From these results, it was confirmed that rice GAI and GID1 orthologs exhibited approximately 100 times more sensitive CID activity than those of Arabidopsis thaliana when GA4 was used as a CID inducer.

[0157] [Experimental Example 4: Evaluation of Concentration Dependence of CID Activity (2)] The concentration dependence of CID activity on GA3-AM was evaluated in the same manner as in Experimental Example 3, except that GA3-AM was used instead of GA4. The results are shown in Figure 7.

[0158] In cells introduced with AttGAI and AtGID1 (labeled "AtGID1-AttGAI" in Figure 7) and cells introduced with OstGAI and OstGID1 (labeled "OstGID1-OstGAI" in Figure 7), GA3-AM concentration-dependent luciferase activity was observed. Furthermore, in cells introduced with OstGAI and OstGID1, high luciferase activity was observed even at a low concentration of GA3-AM of 1 nM. Therefore, extrapolating the results shown in Figure 7, it is expected that luciferase activity will be observed even at a concentration of GA3-AM of 100 pM. From the above, it was confirmed that rice GAI and GID1 orthologs exhibited CID activity that was approximately 300 times more sensitive than that of Arabidopsis thaliana when GA3-AM was used as a CID inducer. The results from Experimental Examples 3 and 4 showed that when GA3-AM was used as a CID inducer, luciferase activity was higher and CID activity was also higher than when GA4 was used.

[0159] [Experimental Example 5: Evaluation of Concentration Dependence of CID Activity (3)] The ABA concentration dependence of CID activity was evaluated in the same manner as in Experimental Example 3, except that ABA was used instead of GA4 as the CID inducer. In Experimental Example 5, a luciferase assay was performed comparing HEK293FT cells introduced with pOSA0001 (AttABI) and pOSA0002 (AttPYL1) with HEK293FT cells introduced with pJR0044 (OstABI) and pJR0042 (OstPYL1). The results are shown in Figure 8.

[0160] In cells introduced with AttABI and AttPYL1 (in Figure 8, "AttPYL1-AttABI") and cells introduced with OstABI and OstPYL1 (in Figure 8, "OstPYL1-OstABI"), ABA concentration-dependent luciferase activity was observed. Furthermore, in cells introduced with OstABI and OstPYL1, high luciferase activity was observed even at a low concentration of ABA of 100 nM. From these results, it was confirmed that rice ABI and PYL1 orthologs exhibited CID activity approximately 300 times more sensitive than those of Arabidopsis thaliana when ABA was used as a CID inducer.

[0161] [Experimental Example 6: Comparison of CID Activity Among Derived Plant Species (1)] The differences in CID activity and sensitivity among the derivated plants of GAI and GID1 orthologs were investigated as follows. The GA4 concentration dependence of CID activity was evaluated in the same manner as in Experimental Example 3, except that HEK293FT cells were created by introducing pJR0078 (Zm#1) (maize-derived ZmtGAI and ZmGID1) and pJR0076 (Zm#2), and HEK293FT cells were created by introducing pJR0077 (Cq#1) (quinoa-derived CqtGAI and CqGID1) and pJR0087 (Cq#2). The results are shown in Figure 9.

[0162] As shown in Figure 9, in cells into which quinoa-derived CqtGAI and CqGID1 were introduced (in Figure 9, "CqGID1-CqtGAI"), the luciferase activity was at the same level as in cells without CID inducers, indicating that they did not possess CID activity. Furthermore, the luciferase activity of cells into which maize-derived ZmtGAI and ZmGID1 were introduced (in Figure 9, "ZmGID1-ZmtGAI") showed a higher degree of identification or higher CID activity compared to cells into which OstGAI and OstGID1 were introduced (in Figure 9, "OstGID1-OstGAI") as shown in Experimental Example 3 (Figure 6).

[0163] [Experimental Example 7: Comparison of CID activity among different plant species (2)] The GA3-AM concentration dependence of CID activity was evaluated in the same manner as in Experimental Example 6, except that GA3-AM was used instead of GA4. The results are shown in Figure 10.

[0164] The same trend was observed when GA3-AM was used as when GA4 was used. Specifically, as shown in Figure 10, cells into which CqtGAI and CqGID1 were introduced (in Figure 10, "CqGID1-CqtGAI") showed almost no luciferase activity and were found to lack CID activity. Furthermore, as shown in Experimental Example 4, in cells into which OstGAI and OstGID1 were introduced (in Figure 10, "OstGID1-OstGAI"), the CID activity showed a significantly higher dependence on GA3-AM concentration compared to cells into which AttGAI and AtGID1 were introduced (in Figure 10, "AtGID1-AttGAI"). Furthermore, in cells into which ZmtGAI and ZmGID1 were introduced (in Figure 10, labeled "ZmGID1-ZmtGAI"), CID activity showed a significantly higher dependence on GA3-AM concentration compared to cells into which AttGAI and AtGID1 were introduced. When GA3-AM was used, high luciferase activity was observed even at 1 nM in cells into which OstGAI and OsGID1 were introduced, and in cells into which ZmtGAI and ZmGID11 were introduced, indicating very high sensitivity of CID activity.

[0165] [Experimental Example 8: Comparison of CID activity among different plant species (3)] The ABA concentration dependence of CID activity was evaluated in the same manner as in Experimental Example 6, except that ABA was used instead of GA4. The results are shown in Figure 11.

[0166] In cells into which CqtABI and CqtPYL1 were introduced (in Figure 11, labeled "CqtPYL1-CqtABI"), CID activity was similar to or slightly lower than that of cells into which AttABI and AttPYL1 were introduced (in Figure 11, labeled "AttPYL1-AttABI"). Furthermore, as shown in Experimental Example 5, in cells into which OstABI and OstPYL1 were introduced (in Figure 11, labeled "OstPYL1-OstABI"), CID activity showed a significantly higher ABA concentration dependence compared to cells into which AttABI and AttPYL1 were introduced. Furthermore, in cells into which ZmtABI and ZmtPYL1 were introduced (in Figure 11, labeled "ZmtPYL1-ZmtABI"), CID activity showed a significantly higher ABA concentration dependence compared to cells into which AttABI and AttPYL1 were introduced.

[0167] [Experimental Example 9: Evaluation of Concentration Dependence of CID Activity (4)] In Arabidopsis thaliana, it is known that when a modified AttPYL1 is created with increased mandipropamide binding affinity and decreased ABA binding affinity, the sensitivity of CID activity increases (Ziegler, M. J et al., Nat Chem Biol 18, 64-69 (2022)). Therefore, we analyzed whether CID activity would be further increased by creating a similar modified PYL1 in rice.

[0168] In OstPYL1, a mutation was introduced such that, when described in the full length of wild-type OsPYL1, the lysine at position 70 becomes arginine, the phenylalanine at position 126 becomes alanine, the valine at position 99 becomes isoleucine, and the phenylalanine at position 174 becomes leucine. This resulted in a modified OstPYL1 (OstPYL1mandi, amino acid sequence: SEQ ID NO: 89, nucleotide sequence: SEQ ID NO: 90) with increased mandipropamide binding and decreased ABA binding. For the designed OstPYL1mandi, pJR0069 (SEQ ID NO: 92) was obtained in the same manner as pJR0042, except that a DNA fragment of OstPYL1mandi (SEQ ID NO: 91) with a linker sequence for Gibson assembly added was artificially synthesized, similar to 2.

[0169] In AtPYL1, a mutation was introduced such that, when described in the full length of wild-type AtPYL1, the lysine at position 86 becomes arginine, the phenylalanine at position 135 becomes alanine, the valine at position 108 becomes isoleucine, and the phenylalanine at position 189 becomes leucine. This resulted in a modified AtPYL1 (AtPYL1mandi, amino acid sequence: SEQ ID NO: 93, nucleotide sequence: SEQ ID NO: 94) with increased mandipropamide binding and decreased ABA binding. For the designed AtPYL1mandi, pJR0072 (SEQ ID NO: 96) was obtained in the same manner as pJR0042, except that a DNA fragment of AtPYL1mandi (SEQ ID NO: 95) with a linker sequence for Gibson assembly added was artificially synthesized, similar to 2.

[0170] To introduce a mutation that would result in V276R when described in full length as wild-type OsABI, pJR0039 (SEQ ID NO: 53) was used as a template, and linear DNA fragments were amplified by inverse PCR using primers SEQ ID NO: 97 and SEQ ID NO: 98. These fragments were then circularized by self-ligation to produce the plasmid vector pJR0056, which expresses the mutant OstABI.

[0171] To introduce a mutation that would result in V308R when described in the full length of wild-type AtABI, a plasmid vector pJR0063 expressing the mutant AtABI was constructed by amplifying a linear DNA fragment using pSLQ2821 as a template and primers SEQ ID NO: 99 and SEQ ID NO: 100 via inverse PCR, and then circularizing it by self-ligation.

[0172] HEK293FT cells introduced with pJR0063 (AttABI_V308R) and pJR0072 (AttPYL1mandi) (SEQ ID NO: 96), and HEK293FT cells introduced with pJR0056 (OstABI_V276R) and pJR0069 (OstPYL1mandi) were prepared using the procedure described above. Mandipropamide was added to these cells to a final concentration of 1 nM, 10 nM, 100 nM, or 1 μM, and the mandipropamide concentration dependence of CID activity was evaluated by performing a luciferase assay as described above. The same experiment was also performed when GA4 was not added. The results are shown in Figure 12.

[0173] As shown in Figure 12, in cells introduced with AttABI_V308R and AttPYL1mandi (labeled "AttPYL1mandi-AttABI" in Figure 12), luciferase activity was almost absent, and CID activity was almost absent under conditions of mandipropamide concentrations of 1 nM, 10 nM, 100 nM, or 1 μM. This was consistent with previous findings (Ziegler, MJ et al., Nat Chem Biol 18, 64-69 (2022)). On the other hand, in cells introduced with OstABI_V276R and OstPYL1mandi (labeled "OstPYL1mandi-OstABI" in Figure 12), luciferase activity was observed and CID activity was confirmed under conditions of mandipropamide concentration of approximately 50 nM. Notably, in cells into which OstABI_V276R and OstPYL1mandi were introduced, luciferase activity was significantly reduced under untreated conditions. Therefore, it was suggested that using rice-derived ABI and PYL1mandi can reduce the nonspecific binding, or "leakage," of highly active CID proteins.

[0174] The results of the above experimental examples 1 to 9 suggest that a portion of GID1-GAI derived from rice or maize, or a portion of PYL1-ABI derived from rice or maize, exhibits high CID activity in the presence of low concentrations of CID inducers of about 10 to 100 nM.

[0175] [Example 10: Comparison of CID activity among derived plant species (4)] In addition to the three GID1 gene orthologs and GAI gene orthologs (truncate type; hereinafter referred to as "tGAI") examined above, AtGID1 and AttGAI from Arabidopsis thaliana, OsGID1 and OstGAI from Oryza sativa, and ZmGID1 and ZmtGAI from corn (Zea mays), 11 more plant species were examined: tomato (Solanum lycopersicum), sesame (Sesamum indicum), beet (Beta vulgaris), soybean (Glycine max), and cassava (Manihot). Esculenta), cotton (Gossypium hirsutum), eucalyptus (Eucalyptus), lotus (Nelumbo nucifera), wheat (Triticum aestivum), dog cypress (Selaginella moellendorffii), adzuki bean (Vigna angularis), cowpea (Vigna Orthologs of the GID1 gene and the GAI gene (tGAI) derived from Arabidopsis thaliana were cloned after optimizing the human codons. On effector vector #1, the base sequence of the Arabidopsis thaliana-derived GID1 gene ortholog was replaced with that of the other GID1 gene orthologs, and further, the Arabidopsis thaliana-derived tGAI gene was replaced with that of the tGAI from each plant to construct a vector for evaluating CID activity. Due to genome duplication, multiple orthologs may exist for the same gene in the above plant species. Therefore, the genes shown in Tables 4 and 5 are assigned branch numbers, and the reference accession numbers are indicated to clarify the base sequence of each wild-type gene.

[0176]

[0177]

[0178] The GA4 concentration dependence of CID activity was evaluated in the same manner as in Experimental Examples 3 and 6. The results of evaluating CID activity in response to 10 μM GA4 treatment are shown in Table 6.

[0179]

[0180] The results in Table 6 show that the luciferase activity (LUC activity) after treatment with 10 μM GA4 was equivalent when using cassava-derived GID1 gene orthologs and tGAI, and when using maize-derived GID1 gene orthologs and tGAI. In particular, it was suggested that highly sensitive CID activity (even in the presence of low concentrations of CID inducers of about 10-100 nM) was generated when using cassava-derived GID1 gene orthologs and tGAI.

[0181] [Example 11: Evaluation of the effects of CID-inducing substances on cells by transcriptome analysis] To investigate the transcriptome response of cells to CID-inducing drugs, RNA was extracted from Jurkat cells treated with various plant hormones or compounds, and a library for sequencing was prepared. Specifically, the following procedure was followed.

[0182] Jurkat cells were cultured in a culture medium containing RPMI-1640 (L-glutamine, phenol red) (Fujifilm Wako Pure Chemical Corporation, catalog number 189-02025) with 2% FBS (Biosera, catalog number 515-99055) added, resulting in 2 × 10⁶ cells. ６ Cells / mL were seeded in 100 μL portions into 96-well plates, and 5% CO2 was added. ２Cells were cultured at 37°C for 4 hours in a humidified atmosphere containing the compounds. Subsequently, cells were treated with each compound to the following concentrations: 10 μM GA3, 10 μM GA3-AM, 10 μM GA4, 100 μM ABA, 100 μM mandipropamide, 100 nM rapamycin, and 100 nM limitusid (AP1903). Cells treated with DMSO were used as a negative control. After culturing under the same conditions for 24 hours, cells were collected by centrifugation at 200 × g for 5 minutes at 4°C. The supernatant was removed, and the cells were washed with 50 μL of phosphate-buffered saline (PBS) to remove residual medium. Centrifugation was performed again under the same conditions, and the resulting pellet was transferred to an RNA-free tube, rapidly frozen in liquid nitrogen, and stored at -80°C until RNA extraction. RNA extraction was performed using the RNeasy Plant Mini Kit (QIAGEN, catalog number 74106), and the purified RNA was used to prepare a library for RNA-seq using the Kapa mRNA HyperPrep Kit (KapaBiossystems, catalog number KK8580).

[0183] Sequencing was performed using Nextseq 2000 (Illumina), PE100, and it was confirmed that a minimum of 20 million reads were obtained. The obtained reads were quality trimmed using fastp, mapped to the GRCh38 genome using STAR to distinguish splicing variants, and converted into a count table for each gene ID using featureCounts. DESeq2 was used for normalization, and principal component analysis was performed on each sample. The results are shown in Figure 13.

[0184] As shown in Figure 13, in principal component analysis, PC1 and PC2 had explanatory powers (percentages explaining the overall variability) of 24% and 18%, respectively. Furthermore, when mapping the rapamycin-treated sample group using these two principal components, it was confirmed that the samples were plotted at significantly different positions compared to the DMSO and GA4-treated conditions.

[0185] [Example 12: Structural Comparison of GID1 Proteins and Analysis of One-Amino Acid Substitution Mutants] (1) Structural Comparison The three-dimensional structures of Arabidopsis thaliana GID1 protein (AtGID1), rice GID1 protein (OsGID1), and maize GID1 protein (ZmGID1) were compared. Specifically, for AtGID1, Protein Data Bank (PDB) ID: 2ZSI was used as the three-dimensional structure; for OsGID1, PDB ID: 3EBL was used; and for ZmGID1, the predicted structure by AlphaFold2 (ID A0A1D6M449) was used. The results of the comparative structural modeling are shown in Figures 14(A) to (C).

[0186] Comparative structural modeling revealed that a single amino acid residue, distinct from the gibberellin (GA) binding pocket, may play a crucial role in ligand sensitivity. Isoleucine (labeled I291), the 291st amino acid residue in AtGID1, corresponds to threonine (T297), the 297th amino acid in ZmGID1. T297 in ZmGID1 may form three hydrogen bonds not present in AtGID1, supported by a comparison of the X-ray crystal structures of OsGID1 (a relative of ZmGID1) and AtGID1 (hydrogen bonds are present in Figures 14(A) and 14(C) (indicated by dotted lines), but absent in Figure 14(B)).

[0187] (2) Multiple Alignment In order to determine the polymorphism of the amino acid residue indicated by I291 of AtGID1 (the amino acid residue corresponding to T297 of ZmGID1), multiple sequence alignments were performed on the amino acid sequences of GID1 orthologs from various plants using the amino acid sequences indicated by SEQ ID NOs: 1, 2, 37, and 131-151, using the CrystalW program. For CrystalW, we used GenomeNet (https: / / www.genome.jp / tools-bin / clustalw). The results are shown in Figure 15.

[0188] As shown in Figure 15, when comparing the amino acid residue I291 in AtGID1 with that of other plants, GID1 from dicotyledonous plants often has leucine (L), serine (S), isoleucine (I), or valine (V) in this amino acid residue, while all monocotyledonous plant species investigated had threonine (T) in this amino acid residue.

[0189] (3) Production of mutants and evaluation of CID activity To verify the effect of the above amino acid mutations on CID, reciprocal mutants were generated. Specifically, one amino acid substitution mutants were created for T297 of ZmGID1 and I291 of AtGID1.

[0190] The AtGID1 mutants were created by substituting the nucleotide sequence encoding GID1 (AtGID1) of Arabidopsis thaliana pOSA0001 with either the nucleotide sequence encoding AtGID1_I291T (amino acid sequence: SEQ ID NO: 201) (SEQ ID NO: 202) or the nucleotide sequence encoding AtGID1_I291L (amino acid sequence: SEQ ID NO: 203) (SEQ ID NO: 204) using site-directed mutagenesis.

[0191] The ZmGID1 mutant was created by substituting the nucleotide sequence encoding ZmGID1 in pJR0078 with either the nucleotide sequence encoding ZmGID1_T297I (amino acid sequence: SEQ ID NO: 205) (SEQ ID NO: 206) or the nucleotide sequence encoding ZmGID1_T297L (amino acid sequence: SEQ ID NO: 207) (SEQ ID NO: 208) using site-directed mutagenesis.

[0192] For AtGID1 and its variants, the CID activity was evaluated using LUC luminescence with the prepared plasmid vectors, similar to Experimental Example 3. The experimental conditions for GAI and reporter were the same as in Experimental Example 3, and a LUC assay was performed comparing AtGID1, AtGID1_I291T, and AtGID1_I291L. The results are shown in Figure 16.

[0193] As a result, as shown in Figure 16, the threonine substitution of I291 in AtGID1 (AtGID1_I291T) significantly enhanced CID activity under 100 nM GA4 conditions compared to the unsubstituted form (AtGID1).

[0194] For ZmGID1 and its variants, the LUC assay was performed in the same manner as in Experimental Example 6, except that ZmGID1 was converted to ZmGID1_T297I or ZmGID1_T297L. The results are shown in Figure 17.

[0195] As shown in Figure 17, isoleucine or leucine substitution of T297 in ZmGID1 (ZmGID1_T297I or ZmGID1_T297L) resulted in a significantly reduced CID activity under 100 nM GA4 conditions compared to the unsubstituted ZmGID1.

[0196] [Example 13: Application to CAR-T Cells] The applicability of a CID system utilizing corn ZmGID1 and ZmGAI to Switch-ON CAR (CAR = Chimeric Antigen Receptor) was verified. In this example, CAR activity generally refers to the effect of making CD69 positive when introduced cells in T cells or T cell-derived cultured cells encounter target cells (cells with surface antigens that can be recognized by CAR). In this example, the percentage of CD69-positive Jurkat cells among the surviving Jurkat E6-1 cells 24 hours after mixing in a mixing experiment of target cells and Jurkat E6-1 cells was used as an indicator.

[0197] (1) The plasmid used for constructing the plasmid was pSLCAR-CD19-BBz (also known as pOSA0014 in this example; Sequence ID No. 209) described in the non-patent document (Bloemberg et al Mol Ther Methods Clin Dev. 2020 Jan 3209:238-254. doi: 10.1016 / j.omtm.2020.01.012). The DNA sequence (SEQ ID NO: 211) encoding the CAR expression portion (SEQ ID NO: 210) of pOSA0014 was replaced to express AtGID1 and AttGAI respectively, as described in non-patent literature (Wu et al., Science 2015, Vol 350, Issue 6258) (the two plasmids together are called AtSplitCAR), or the same was replaced to express ZmGID1 and ZmtGAI respectively (the two plasmids together are called ZmSplitCAR). Furthermore, plasmids containing the portion encoding GID1 were replaced with one-amino acid substitution mutants AtGID1_I291T or ZmGID1_T297I (combined with a vector expressing GAI, respectively, they are called AtSplitCAR_I291T and ZmSplitCAR_T297I). A schematic diagram of the expression vector used in Example 13 is shown in Figure 18.

[0198] The combinations of each SplitCAR system, the amino acid sequences of the CAR portion, and their coding sequences are as follows: • AtSplitCAR: pMI0158 (SEQ ID NOs. 221, 223) and pMI0011 (SEQ ID NOs. 216, 217) • ZmSplitCAR: pMI0159 (SEQ ID NOs. 214, 215) and pMI0012 (SEQ ID NOs. 218, 219) • AtSplitCAR_I291T: pMI0158 (SEQ ID NOs. 212, 213) and pMI0152 (SEQ ID NOs. 220, 221) • ZmSplitCAR_T297I: pMI0159 (SEQ ID NOs. 214, 215) and pMI0153 (SEQ ID NOs. 222, 223)

[0199] (2) Preparation of target cells As target cells, hCD19-expressing MDA-MB-231 cells were prepared. The preparation was performed in accordance with the non-patent literature (Lainscek et al. Mol Ther Oncolytics. 2023 Apr 26;29:77-90.). Specifically, for lentiviral introduction, human CD19 (DNA fragment; Twist) was cloned into the BamHI / EcoRI multiple cloning site of pLVX-Puro vector (Addgene, 141395). Furthermore, pVSV-G (Addgene, 138479) and psPAX2 (Addgene, 21560) were used as packaging plasmids. For preparation of hCD19-MDA-BR-231 cells, the cells were seeded at a density of 1×10 ５ cells / well in a 12-well plate. The next day, introduction was performed using a lentivirus in the presence of 8 μg / mL polybrene (Invivogen). After 7 days, selection with 0.5 mg / mL puromycin (Invitrogen) was performed for 3 weeks, and hCD19-positive cells were sorted. After selection, hCD19 expression was confirmed using flow cytometry.

[0200] (3) Evaluation of CAR activity For the prepared plasmids, transfection was performed on Jurkat E6-1 cells in the combinations described in Table 7 using the protocol for Jurkat cells of the Neon Transfection system, followed by incubation at 37°C under 5% CO ２ conditions.

[0201]

[0202] Specifically, the CAR activity evaluation assay was performed as follows. Transfected Jurkat E6-1 cells and target cells (MDA-MB-231 cells, or hCD19-expressing MDA-MB-231 cells) were placed in a V-bottom plate (Corning, Cat. #3894) at a ratio of 1:1 (2×10 ３ cells each). At this time, GA4 dissolved in DMSO was added to the cells at a final concentration of 10 nM or 10 μM, or only DMSO was added at a final concentration of 0.2%. The mixture was centrifuged at 500×g for 5 minutes, and incubated at 37°C under 5% CO ２The cells were incubated under the following conditions for 24 hours. After 24 hours of co-culture, the plates were centrifuged at 500 x g for 5 minutes, and the supernatant was removed. The cells were resuspended in 50 μL of buffer (PBS(-) (Fujifilm Wako Pure Chemical Corporation, catalog no. 166-23555) with 2% FBS (Biosera, catalog no. 515-99055), and then 0.2 μL of CD69-Brilliant Violet antibody (Becton) was added. Dickinson, Cat. #555533) was added and incubated at 37°C for 30 minutes. Subsequently, 100 μL of buffer was added and the cells were centrifuged. After removing the supernatant, the pellet was resuspended in 100 μL of buffer. The cells were analyzed using an SH-800 cell sorter (SONY), and Jurkat E6-1 cells were sorted based on GFP (confirmation of introduction of pMI0011, pMI0012, pMI0152, pMI0153, or pOSA0014), mCherry (confirmation of introduction of pMI0158, or pMI0159), and Brilliant Violet fluorescence (confirmation of CD69 expression), and the percentage of CD69-positive cells was determined. The results are shown in Figure 19.

[0203] The results of this experiment showed that in Jurkat E6-1 cells expressing AtSplitCAR, under conditions where the target cells expressed hCD19, the percentage of CD69-positive cells was approximately 2.25% under DMSO treatment conditions, while it gradually increased to approximately 5.23% when GA4 was added at 10 nM and approximately 12.38% at 10 μM. On the other hand, under conditions where the target cells did not express hCD19, the percentage of CD69-positive cells remained around 1.3% even when DMSO and GA4 were added at 10 μM, and no GA4-dependent induction of CD69 was observed. Furthermore, in Jurkat E6-1 cells expressing AtSplitCAR-I291T, under conditions where hCD19 was expressed in target cells, the response at low concentrations of GA4 (10 nM) was higher than that of AtSplitCAR: approximately 3.20% under DMSO treatment, approximately 7.17% under conditions with 10 nM GA4 added, and approximately 10.59% under treatment with 10 μM GA4.

[0204] Furthermore, in Jurkat E6-1 cells expressing ZmSplitCAR, under conditions where target cells expressed hCD19, the percentage of CD69-positive cells was approximately 1.96% under DMSO treatment conditions, compared to approximately 12.80% and 12.82% under conditions where GA4 was added at 10 nM and 10 μM, respectively, confirming higher CAR activity at lower concentrations than AtSplitCAR. On the other hand, under conditions where target cells did not express hCD19, CAR activity remained around 1.3%, regardless of whether GA4 was added or not. In Jurkat E6-1 cells expressing ZmSplitCAR-T297I, under conditions where hCD19 is expressed in target cells, the percentage of CD69-positive cells was approximately 1.62% under DMSO treatment and approximately 11.29% under conditions where GA4 was added at 10 μM. Although high CAR activity was maintained, the percentage was approximately 5.63% when GA4 was added at 10 nM, which was lower than the percentage of CD69-positive cells of approximately 12.8% under the same conditions for ZmSplitCAR.

[0205] In conventional_CAR, used as a control, the proportion of CD69-positive cells was high, approximately 20.92% under DMSO treatment conditions and approximately 12.75% under conditions with 10 μM of GA4 added, when hCD19 was expressed in the target cells. No GA4 response was observed, and the proportion remained low at around 1.3-1.4% when hCD19 was not expressed in the target cells.

[0206] 1...First dimer-forming protein, 2...Second dimer-forming protein, 3...CID inducer, 4,5...Dimers formed by CID, 11...First functional protein, 21...Second functional protein, 111...First fusion protein, 221...Second fusion protein.

Claims

1. A protein having dimerization ability in the presence of a chemically induced dimerization (CID) inducing substance, wherein the protein comprises a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 7-8, or a protein or variant thereof consisting of an amino acid sequence shown in SEQ ID NOs: 201, wherein the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NOs: 201 is a threonine residue.

2. A protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140 has 90% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140; A protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167 has 90% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167; A protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89 has 90% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89; A protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8 has 90% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 7-8; The protein according to claim 1, wherein the protein comprising the amino acid sequence shown in SEQ ID NO: 201 or a variant thereof has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 201, and the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NO: 201 is a threonine residue.

3. The protein according to claim 1, wherein, in the presence of a CID inducer, a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140 and a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167 form a dimer.

4. The protein according to claim 1, wherein, in the presence of a CID inducer, a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs. 5-6 and 89 forms a dimer with a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs. 7-8.

5. The protein according to claim 3, wherein the CID inducer comprises gibberellin.

6. The protein according to claim 5, wherein the gibberellin comprises at least one selected from the group consisting of gibberellin A3, gibberellin A1, gibberellin A4, and gibberellin A7.

7. The protein according to claim 4, wherein the CID inducer comprises at least one selected from the group consisting of abscisic acid, mandipropamide, and azinephosethyl.

8. The protein according to claim 1, further comprising a functional protein.

9. A nucleic acid comprising a base sequence encoding a protein according to any one of claims 1 to 8.

10. An expression vector comprising the base sequence of the nucleic acid described in claim 9.

11. A cell into which the nucleic acid described in claim 9 has been introduced.

12. A method for forming a dimer of a first protein and a second protein, comprising the step of contacting the first protein, the second protein, and a chemically induced dimerization (CID) inducing substance, wherein the first protein comprises a first dimerizing protein, the second protein comprises a second dimerizing protein, the first dimerizing protein and the second dimerizing protein are proteins that have dimerizing ability in the presence of a chemically induced dimerization (CID) inducing substance, and the first dimerizing protein is a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, or a protein or variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89. A method for obtaining a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7 to 8, or a protein or a variant thereof consisting of the amino acid sequence shown in SEQ ID NOs: 201, wherein the amino acid residue at position 291 in the amino acid sequence shown in SEQ ID NOs: 201 is a threonine residue.

13. A protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140 has 90% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140; A protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167 has 90% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167; A protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89 has 90% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89; A protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8 has 90% or more sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 7-8; The method according to claim 12, wherein the protein or mutant thereof consisting of the amino acid sequence shown in SEQ ID NO: 201 has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 201, and the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NO: 201 is a threonine residue.

14. The method according to claim 12, wherein one of the first and second dimer-forming proteins is a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, and the other is a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167.

15. The method according to claim 12, wherein one of the first and second dimer-forming proteins is a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs. 5-6 and 89, and the other is a protein or a variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs. 7-8.

16. The method according to claim 14, wherein the CID-inducing substance comprises gibberellin.

17. The method according to claim 16, wherein the gibberellin comprises at least one selected from the group consisting of gibberellin A3, gibberellin A1, gibberellin A4, and gibberellin A7.

18. The method according to claim 15, wherein the CID-inducing substance comprises at least one selected from the group consisting of abscisic acid, mandipropamide, and azinephosethyl.

19. The method according to any one of claims 12 to 18, wherein the first protein further comprises a first functional protein, and the second protein further comprises a second functional protein.

20. A kit comprising nucleic acids containing a protein-coding base sequence, wherein the protein has the ability to form dimers in the presence of a chemically induced dimerization (CID) inducing substance, and the protein is a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89, a protein or variant thereof consisting of the amino acid sequence shown in any of SEQ ID NOs: 7-8, or a protein or variant thereof consisting of the amino acid sequence shown in SEQ ID NOs: 201, wherein the amino acid residue at the position corresponding to the 291st position in the amino acid sequence shown in SEQ ID NOs: 201 is a threonine residue.

21. The kit according to claim 20, comprising: a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 1-2 and 139-140; a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 3-4 and 167; a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 5-6 and 89; a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in any of SEQ ID NOs: 7-8; or a nucleic acid comprising a base sequence encoding a protein or a variant thereof consisting of an amino acid sequence shown in SEQ ID NO:

201.

22. The kit according to claim 20, further comprising one or more selected from the group consisting of nucleic acids having a base sequence encoding a functional protein, CID inducers, and cells.

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