JP4879314B2 - In vivo proteolysis system using ubiquitin-dependent proteolysis system, and protein function elucidation method using the system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of analyzing a proteinic function by selectively decomposing the protein by using an ubiquitin-dependent proteolysis system. <P>SOLUTION: The method of analyzing the proteinic function includes: expressing a protein A-RFD in a living body; forming a composite of a protein A and a protein X; transferring the ubiquitin bonded to an ubiquitin bonding enzyme interacting with an RFD to the protein X to decompose the protein X; and inactivating the function of the protein X or protein A-X composite. The RFD is a protein having an amino acid sequence in which the 134 to 181 amino acid sequence or 1 to 4 amino acid residual groups are replaced, defected, added or inserted in a specific amino acid sequence; and the protein A-RFD is made by bonding the protein A and the RFD with a linker having the amino acid sequence described in another specific sequence or a linker having the amino acid sequence in which 1 to 4 amino acid residual groups are replaced, defected, added or inserted from the amino acid residual group described in the specific sequence. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a protein function analysis method, a function suppression method, and the like. More specifically, the present invention uses a ubiquitin-dependent proteolytic system, which is a universal function in eukaryotic cells, to analyze and suppress the function of a protein by selectively degrading a specific protein. And a method for producing knockout organisms using the same.

  Many proteins do not function alone but form a complex to exert their functions. Therefore, it is important to investigate what kind of protein the protein of interest interacts with in vivo and what function it has. Therefore, it is expected to establish a method for determining the binding partner of a specific protein and a method for elucidating the function of the complex.

  On the other hand, in higher animal and plant cells, proteins that have undergone ubiquitination are selectively degraded, and this is considered to play an important role in the maintenance of living organisms. The ubiquitin-dependent proteolytic system exists universally in the living body, and various studies have been carried out (literature name: Pickart, C.M. (2001) Annu. Rev. Biochem. 70, 503-533). For example, the partial structure and activity relationship of an enzyme called EL5, one of the enzymes (ubiquitin ligase: E3) that selectively ubiquitinates the target protein in the rice ubiquitin-dependent proteolytic system, was elucidated, and the ring finger domain was It is known to be essential for the interaction with the ubiquitin-conjugating enzyme (E2) (Non-patent Document 1 below (Katoh et al. (2003), J. Biol. Chem. 278 (17), 15341-15348) reference.). However, there is no report that elucidates or suppresses the function of a protein using a ubiquitin-dependent proteolytic system.

Katoh et al. (2003), J. Biol. Chem. 278 (17), 15341-15348.

  An object of the present invention is to provide a method for analyzing a protein function by selectively degrading the protein using a ubiquitin-dependent proteolytic system. Another object of the present invention is to provide a new functional analysis method capable of elucidating the function of a protein whose function cannot be analyzed by RNAi or the like.

  Another object of the present invention is to provide a method for producing a non-human knockout animal or knockout plant in which the function of the protein is suppressed by degrading the protein using a ubiquitin-dependent proteolytic system.

  The present invention basically applies the function that a predetermined protein is decomposed by ubiquitin modification. That is, a protein (protein A-RFD) complex in which a target protein (protein A) and a protein (RFD) capable of interacting with a ubiquitin-binding enzyme are fused is expressed. And it decomposes | disassembles by carrying out ubiquitin modification of the protein X couple | bonded with the protein A. As a result, the functions of protein X and the like are elucidated.

The first aspect of the present invention is:
Using a protein (protein A-RFD) in which a target protein (protein A) and a protein (RFD) capable of interacting with a ubiquitin-binding enzyme are fused,
The function of the protein (protein X) that forms a complex with the target protein and is decomposed by being modified with ubiquitin, or the complex of the protein A and the protein X (protein AX complex) A method of analyzing the function,
Expressing the protein A-RFD in vivo,
Forming a complex of the protein A and the protein X;
Transferring the ubiquitin bound to the ubiquitin-conjugating enzyme that interacts with the RFD to the protein X, thereby decomposing the protein X and deactivating the function of the protein X or protein AX complex;
Analyzing the function of protein X or protein AX complex,
Here, the “protein capable of interacting with ubiquitin-binding enzyme (RFD)”
A protein consisting of amino acid sequences 134-181 in the amino acid sequence set forth in SEQ ID NO: 1,
Or a protein consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence of 134-181 in the amino acid sequence described in SEQ ID NO: 1.
A protein functional analysis method,
In the protein A-RFD, protein A and RFD are bound by a linker,
The linker is a linker comprising the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14,
Or a linker consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence described in SEQ ID NO: 13 or SEQ ID NO: 14.
The present invention relates to a protein functional analysis method.

The second aspect of the present invention uses a protein (protein A-RFD) in which a target protein (protein A) and a protein capable of interacting with a ubiquitin-binding enzyme (RFD) are used to form a complex with the target protein. A non-human knockout animal that disrupts the function of the protein (protein X) that is degraded by being modified with ubiquitin, or the function of the complex protein (protein AX) of the protein A and the protein X, or A method for producing a knockout plant, comprising:
Expressing the protein A-RFD in vivo,
Forming a complex of the protein A and the protein X;
By transferring ubiquitin bound to the ubiquitin-conjugating enzyme that interacts with the RFD to the protein X, the function of the protein X or the function of the protein AX is deactivated,
Producing non-human knockout animals or knockout plants;
Here, the “protein capable of interacting with ubiquitin-binding enzyme (RFD)”
A protein consisting of amino acid sequences 134-181 in the amino acid sequence set forth in SEQ ID NO: 1,
Or a non-human knockout animal or knockout plant, which is a protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence of 134-181 in the amino acid sequence described in SEQ ID NO: 1 It relates to the manufacturing method.

In a preferred embodiment of the second aspect of the present invention, the protein A-RFD is obtained by binding the protein A and RFD with a linker,
The linker is a linker comprising the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14,
Or a linker consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence described in SEQ ID NO: 13 or SEQ ID NO: 14.
The present invention relates to a method for producing the non-human knockout animal or knockout plant described above.

In a preferred embodiment of the second aspect of the present invention, the protein A is
A protein comprising the amino acid sequence set forth in SEQ ID NO: 5,
A protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence set forth in SEQ ID NO: 5;
A domain protein having amino acids 117-211 in the amino acid sequence shown in SEQ ID NO: 5,
Or a protein consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence 117-211 of the amino acid sequence shown in SEQ ID NO: 5,

The protein X is
The protein described in SEQ ID NO: 6 (SLR1),
Or a protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added, or inserted from the amino acid sequence set forth in SEQ ID NO: 6 as to the method for producing a non-human knockout animal or knockout plant as described above .

In a preferred embodiment of the second aspect of the present invention, the protein A is
The protein described in SEQ ID NO: 7 (MAPKP),
A domain protein having amino acids 362-467 of the amino acid sequence set forth in SEQ ID NO: 7,
A protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence set forth in SEQ ID NO: 7;
Or a protein consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the 362-467th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 7,

The protein X is
The protein described in SEQ ID NO: 8 (CaM1),
A protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence set forth in SEQ ID NO: 8;
The protein described in SEQ ID NO: 9 (CaM3),
And the non-human knockout described above, wherein one to four amino acid residues from the amino acid sequence set forth in SEQ ID NO: 9 are substituted, deleted, added or inserted, or a protein comprising the amino acid sequence. The present invention relates to a method for producing an animal or a knockout plant.

In a preferred embodiment of the second aspect of the present invention, the protein A is
The protein described in SEQ ID NO: 10 (Gγ),

Or a protein consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence set forth in SEQ ID NO: 10,
The protein X is
The protein described in SEQ ID NO: 11 (Gβ),
Or a protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added, or inserted from the amino acid sequence set forth in SEQ ID NO: 11, and relates to the method for producing a non-human knockout animal or knockout plant as described above .

In a preferred embodiment of the second aspect of the present invention, the protein A is
The protein described in SEQ ID NO: 12 (CIGR2),
Or a protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added, or inserted from the amino acid sequence set forth in SEQ ID NO: 12. The present invention relates to the method for producing a non-human knockout animal or knockout plant as described above. .

  ADVANTAGE OF THE INVENTION According to this invention, the method of analyzing the function of protein can be provided by selectively decomposing | disassembling protein using a ubiquitin-dependent proteolytic system.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the non-human knockout animal or knockout plant which suppressed the function of the protein can be provided by degrading protein using a ubiquitin-dependent proteolytic system.

FIG. 1 is a conceptual diagram showing an outline of the method of the present invention. FIG. 1A is a conceptual diagram of a fusion protein of protein A and RFD (described later). In the example shown in FIG. 1A, protein A and RFD are connected by a linker. FIG. 1B is a conceptual diagram showing how ubiquitin and protein X physically approach each other and protein X is modified with ubiquitin. FIG. 1C is a conceptual diagram showing a state in which protein X is continuously modified with polyubiquitin and a polyubiquitin tag is formed. FIG. 1D is a conceptual diagram showing that polyubiquitinated protein X is decomposed. FIG. 1E is a conceptual diagram showing that the function of the complex of protein A and protein X has disappeared because protein E has been decomposed. In FIG. 1E, protein X is represented by a dotted line to indicate that protein X has been decomposed. FIG. 2 is a model diagram of the SLR1 decomposition system. FIG. 2 (A) is a conceptual diagram showing an outline of a protein degradation system. FIG. 2B is a diagram showing how rice grows up. FIG. 3 is a model diagram of the CaM1 and CaM3 decomposition system. FIG. 4 is a diagram showing an ubiquitination experiment of S-protein by S-tag-RFD fusion protein. . 4A is a conceptual diagram of an experimental system in Example 1. FIG. FIG. 4 (B) is a gel electrophoresis photograph in place of a drawing showing that a given protein has been ubiquitinated. FIG. 5 is a gel electrophoresis photographic image instead of a drawing showing the experimental results of verifying the ubiquitination efficiency by the length of the linker. FIG. 6 is a gel electrophoresis photographic diagram instead of a drawing showing the results of verification of control of ubiquitination efficiency by RFD mutants. FIG. 7 is a diagram showing the structure of a plasmid. FIG. 7 (A) is a cleavage map of plasmid “pBI-EN4-GID2”. FIG. 7B is a cleavage map of the plasmid “pBI-EN4-GID2RING”.

(1. Overview of protein functional analysis method or non-human knockout animal or knockout plant production method)
Hereinafter, the “method for analyzing / suppressing the function of a protein” of the present invention (hereinafter also referred to as “method for analyzing the function of a protein”) of the present invention will be described with reference to the drawings. FIG. 1 is a diagram for explaining a system used in the method of the present invention. FIG. 1A is a conceptual diagram of a fusion protein of protein A and RFD (described later). In the example shown in FIG. 1A, protein A and RFD are connected by a linker. FIG. 1B is a conceptual diagram showing how ubiquitin and protein X physically approach each other and protein X is modified with ubiquitin. FIG. 1C is a conceptual diagram showing a state in which protein X is continuously modified with polyubiquitin and a polyubiquitin tag is formed. FIG. 1D is a conceptual diagram showing that polyubiquitinated protein X is decomposed. FIG. 1E is a conceptual diagram showing that protein X is decomposed and the function of the complex of protein A and protein X disappears. In FIG. 1E, protein X is represented by a dotted line to indicate that protein X has been decomposed.

  As shown in FIG. 1, the “method for analyzing protein function” of the present invention is as follows. A composite protein (A-RFD) is formed by fusing a target protein (protein A) and a protein capable of interacting with a ubiquitin-conjugating enzyme (RFD) (FIG. 1 (A)). Then, the target protein (protein A) of the protein A-RFD is allowed to interact with protein X that is degraded by ubiquitin modification to form a complex (protein A-X complex) (FIG. 1 (B)). . Then, ubiquitin (FIG. 1 (C)) linked to the ubiquitin-conjugating enzyme (E2) interacting with RFD is transferred to protein X, and protein X is decomposed by being ubiquitin-modified (FIG. 1 (D )). In this way, the function of protein X or a complex protein of protein X and protein A can be analyzed. In addition, creatures that have destroyed their functions can be created. Hereinafter, terms used in this specification will be described. In the present specification, the term “protein” includes a polypeptide.

(1.1.
Protein X)
“Proteins that are degraded by ubiquitin modification” (hereinafter also referred to as “protein X”) are those that are degraded by ubiquitin modification. Protein X is particularly limited as long as it forms a complex by interacting with protein A described later, and as a result, protein X or a complex protein of protein A and protein X exhibits a function. Not. Preferably, the function of protein X is elucidated by the present invention, and the function is suppressed (destroyed).

  Protein X is “ubiquitin modified” when the ε (epsilon) -amino group of the lysine residue of protein X is condensed with the carboxyl group of the C-terminal glycine of ubiquitin to form an isopeptide bond. It means that the ε-amino group of the lysine residue of ubiquitin repeats a condensation reaction with the carboxyl group of the C-terminal glycine of another ubiquitin to form a polyubiquitin tag (or ubiquitin tag). As a result, in the present invention, protein X is decomposed and its function is destroyed. Formation of such a polyubiquitin tag (ubiquitin modification) is caused by the physical proximity of protein X to a ubiquitin-binding enzyme in which ubiquitin is thioesterified. In general, “ubiquitin modification” means a post-translational modification reaction of a protein with ubiquitin, and is a reaction in which a carboxyl group of ubiquitin and an ε-amino group of a lysine residue of a protein are bound. Of the amino acid residues of the first ubiquitin bound to the protein, the second ubiquitin binds to the ε-amino group of lysine, and this reaction is repeated to bind many ubiquitin molecules to specific lysine residues of the protein. (Polyubiquitination). The polyubiquitinated protein is degraded by a proteolytic enzyme such as 26S proteasome.

  Specifically, as protein X, a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (SLR1), a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (CaM1), and an amino acid sequence shown in SEQ ID NO: 9 A protein consisting of (CaM3), a protein consisting of the amino acid sequence set forth in SEQ ID NO: 11 (Gβ), and 1 to several (2,3,4,5) amino acids from these amino acid sequences are substituted or deleted. Examples include added or inserted proteins.

(1.2.
Protein A)
A target protein (hereinafter also referred to as “protein A”) is a protein that can be linked to a protein (RFD) that can interact with a ubiquitin-binding enzyme by a linker or the like. Protein A forms a complex with protein X in vivo in a state where a fusion protein is formed with RFD. Protein A and protein X may express their functions by forming a complex protein (protein A-X complex).

  Specific protein A is a protein (GID2) consisting of the amino acid sequence described in SEQ ID NO: 5 (NCBI accession (Accession) No. BAC81428), and the 117-211th amino acid sequence described in SEQ ID NO: 5 is a domain protein. , A protein having an amino acid described in SEQ ID NO: 7 (NCBI accession No. BAD00043) (MAPKP), a domain protein having amino acids 362 to 467 of the amino acid sequence described in SEQ ID NO: 7, SEQ ID NO: 10 (NCBI accession No. BAA07405) protein consisting of the amino acid sequence described in (Gγ), SEQ ID NO: 12 (NCBI accession No. AAL62810) protein consisting of the amino acid sequence (CIGR2), and one to several (2) , 3, 4, 5) amino acids are substituted, deleted, added or inserted.

The above protein A forms a complex with the above protein X.
“Gomi et al., (2004) Plant J. 37, 626-34.”; “Yamakawa et al., (2004) J Biol Chem. 279, 928-36.”; And “kato et al., (2004 ) Plant J. 2004 Apr; 38 (2): 320-31.

(1.3.
RFD)
“Protein capable of interacting with ubiquitin-binding enzyme” (referred to as “RFD” after the ring finger domain, which is a typical example in this specification), is a protein that can interact with ubiquitin-binding enzyme, There is no particular limitation as long as it can form a fusion protein (protein A-RFD).

As a specific RFD, a protein consisting of amino acid sequences 134-181 of the amino acid sequence described in SEQ ID NO: 1 (NCBI accession No. BAD16550 (EL5)) (Takai et al.,
(2002) Plant J. 30 (4), 447-55.), SEQ ID NO: 2 (NCBI accession No. NP 201297 (CIP8)), a protein comprising the amino acid sequence of positions 257-297 (Hardtke et al., (2002 ) Plant J. 30 (4), 385-94.), Protein consisting of amino acid sequence 48-101 of SEQ ID NO: 3 (NCBI accession No. NP 974506 (Rma1)) (Matsuda et al.,
(2001) J. Cell Sci. 114, 1949-1957.), SEQ ID NO: 4 (NCBI accession No. NP AAL26903 (Hrd1)) protein consisting of amino acid sequences 272-343 (Marjolein et al., (2004) J. Biol. Chem. 279 3525-3534.) And proteins in which one to several (2, 3, 4, or 5) amino acids are substituted, deleted, added or inserted from these amino acid sequences. It is done.

  The gene of the protein A-RFD fusion protein to be introduced is preferably selected according to the host. For example, in plants, the RFD region is a gene encoding a protein consisting of amino acid sequences 134-181 of the amino acid sequence described in SEQ ID NO: 1 (NCBI accession No. BAD16550 (EL5)), SEQ ID NO: 2 (NCBI accession No. NP 201297 (CIP8)) gene coding for a protein consisting of amino acids 257-297, coding for a protein consisting of amino acids 48-101 of SEQ ID NO: 3 (NCBI accession No. NP 974506 (Rma1)) Genes are preferred. On the other hand, for an animal (human or non-human animal), a gene encoding a protein comprising the amino acid sequence at positions 272 to 343 of SEQ ID NO: 4 (NCBI accession No. NP AAL26903 (Hrd1)) is preferable.

(1.4.
E2)
The ubiquitin-binding enzyme (hereinafter also referred to as “E2”) is not particularly limited as long as it is an enzyme that can transfer ubiquitin to another protein. However, E2 interacts with the introduced RFD has specificity. For example, OsUbc5b, Ubc4, and UbcH5 can be mentioned as E2 corresponding to RFD consisting of amino acid sequence 134-181 of the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 1 (Takai et al. (2002). ) Plant J.
30 (4) 1-10.). UbcH5a is exemplified as E2 corresponding to the RFD consisting of the amino acid sequences of 257 to 297 in SEQ ID NO: 2 or SEQ ID NO: 2 (Matsuda et al., (2001) J. Cell Sci. 114 (10) 1949- 1957.) Moreover, AtUBC7 is mentioned as E2 corresponding to RFD which consists of the 48th to 101st amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 3 (Hardtke et al., (2002) Plant J.30 (4) 385- 94.). Ubc4p, UbcH5b and Ubc7 are mentioned as E2 corresponding to the RFD consisting of the amino acid sequence of SEQ ID NO: 4 or 272-343 of SEQ ID NO: 4 (Kikkert et al., (2004)
279 3525-3534.). However, the selectivity of E2 is conserved across species.

  “Ubiquitin-conjugating enzyme (“ E2 ”) interacts with RFD” mainly means that they bind, but it is bound by hydrophobic interaction, electrical interaction, hydrogen bond, etc. It includes that a state is formed.

(1.5.
Ub)
Ubiquitin (hereinafter also referred to as “Ub”) is a protein composed of 76 amino acids. Ubiquitin is universally present in eukaryotes. Ubiquitin (GenBank M26880) is linked to the cysteine residue of E1 (ubiquitin activating enzyme) by a glycine residue at the C-terminal, and then transferred from E1 to E2 (ubiquitin-binding enzyme). Then, isopeptide bonds to the ε (epsilon) -amino group of the lysine side chain of the protein recognized by E3 (ubiquitin ligase). The C-terminus of ubiquitin repeats isopeptide bonds to the 48th lysine residue of ubiquitin bound to the lysine residue of the protein. Thereby, a polyubiquitin chain is formed. The polyubiquitin chain added by the ubiquitin system functions as a recognition signal by the regulatory subunit of the 26S proteasome, and the ubiquitinated protein is degraded by the proteasome. Ubiquitin is known to be involved in various biological responses such as regulation of gene expression, stress response, ribosome biosynthesis, DNA repair, and cell cycle control, in addition to functioning as a marker for proteolysis.

(1.6.
Complex of protein A and protein X)
The “complex of protein A and protein X” is a complex protein containing protein X and protein A.

(1.7.
Fusion protein)
A “fusion protein” is a protein in which protein A and RFD are linked. In the fusion protein, these proteins may be linked in this order. The order of protein A and RFD may be reversed. Moreover, some amino acid residues (linker) may be inserted between each. In particular, it is preferable that a linker part consisting of a predetermined amino acid residue is provided between protein A and RFD. Since it has such a linker part, protein X and ubiquitin are likely to interact in vivo. Therefore, from this point of view, the number of amino acid residues in the linker portion between protein A and RFD is 1 to 100, 5 to 40, and 5 to 20, preferably 30 to 60. More preferably, it is 40-50. For example, in the S-tag system, protein A is preferably designed so that the number of amino acid residues in this linker part is 30-40. It is desirable that the amino acid sequence used for the linker does not form a specific secondary structure but forms a random coil. Examples of such a linker include a linker consisting of the amino acid sequences described in SEQ ID NO: 13 and SEQ ID NO: 14, a linker consisting of these partial sequences, and 1 to 4 from the amino acid sequences described in SEQ ID NO: 13 and SEQ ID NO: 14. Examples include linkers composed of amino acid sequences in which individual amino acid residues are substituted, deleted, added, or inserted.

(2. Action)
Protein X is ubiquitinated by the transfer reaction of ubiquitin from ubiquitinated E2 bound to RFD, and is degraded by the proteasome. When the protein X is decomposed, the function of the complex protein AX or the function of the protein X is destroyed. If this action is utilized, the function of the target protein, complex protein AX, or the function of protein X can be analyzed and suppressed. Note that protein X is not necessarily one type, and there may be a plurality of types (X1, X2,...). Protein X includes those constituting a plurality of domains. Therefore, in the following, the method for analyzing the function of the protein of the present invention will be described more specifically by dividing protein X into cases.

(2.1)
A system in which protein X interacts with protein A will be described below for a system in which protein X interacts with protein A. In this system, first, protein A is linked to RFD to produce a fusion protein of RFD and protein A (protein A-RFD). Thereafter, protein A and protein X are allowed to interact with each other, and protein X is decomposed by ubiquitin modification via E2.

FIG. 2 is a conceptual diagram showing an example in which rice protein is decomposed using the “method for analyzing protein function” of the present invention. FIG. 2 (A) is a conceptual diagram showing an outline of a protein degradation system. FIG. 2B is a diagram showing how rice grows up. As shown in FIG. 2, SLR1 (corresponding to protein X) consisting of the amino acid sequence set forth in SEQ ID NO: 6, which is a negative regulator of gibberellin signal in rice, is GID2 ( Interacts with protein A) (Gomi literature)
et al., (2004) Plant J. 37, 626-34.). Therefore, a fusion protein of GID2 (or a domain protein consisting of the 117th to 211st amino acid sequences of the amino acid sequence described in SEQ ID NO: 5) and RFD is prepared, and ubiquitin is converted into SLR1 by the action of E2 bound to RFD. To metastasize. SLR1 is recognized and degraded by the proteasome by this ubiquitin tag. Since SLR1 has a function of suppressing the plant height of rice, the rice after SLR1 is degraded by the proteasome is prone.

Hereinafter, an example of another system in which protein X is one type will be described. It is known that plant trimeric G protein involved in signal transduction has an important role in plant morphogenesis. Among these, it is known that the interaction between subunit Gβ (SEQ ID NO: 11) and Gγ is particularly strong (references kato et al.,
(2004) Plant J. 2004 Apr; 38 (2): 320-31.). Focusing on this function, rice that destroys the function of the G protein is created. That is, rice with Gγ-RFD introduced is produced for the purpose of decomposing rice trimeric G protein Gβ subunit. First, a fusion protein of G protein Gγ and RFD having the amino acid sequence set forth in SEQ ID NO: 10 is prepared by a known method. Then, ubiquitin is transferred to G protein Gβ by the action of E2 bound to RFD. The G protein Gβ is recognized and degraded by the proteasome by this ubiquitin tag. Rice is dwarfed by the decomposition of the Gβ subunit and becomes a stem-opening type. In this way, an organism that knocks out the function of a given protein can be created.

(2.2)
In the following, a system in which protein X is a plurality of types will be described. Among proteins, there are proteins that have similar functions and form a family. For example, calmodulin (CaM) is a very important calcium-binding protein involved in intracellular signal transduction. In animal cells, it is reported that only one or two types of CaM exist in one kind of organism, but in plants, it is reported that multiple CaM molecular species exist from the same plant (literature name). : Ishida et al., (2001) J. Biol. Chem. 129, 745-753.). Since these CaM molecular species are well conserved among plants, each molecular species is functionally shared and is thought to be evolutionarily conserved. Among CaM molecular species, CaM1 and CaM3 are MAP kinase phosphatase (MAPKP: SEQ ID NO: 7) or MAPKP domain protein (domain protein consisting of amino acid sequences 362 to 467 of the amino acid sequence described in SEQ ID NO: 7). It is known that protein functions are similar, such as binding and being involved in disease resistance. In such a case, it is possible to confirm the function and phenotype because the functions complement each other even if either CaM1 (SEQ ID NO: 8) or CaM3 (SEQ ID NO: 9) is knocked out using a technique such as RNAi. Have difficulty.

  Even in such a system, if the “method for producing a non-human knockout animal or knockout plant” of the present invention is used, CaM1 and CaM3 can be decomposed simultaneously, so that the functions of CaM1 and CaM3 can be completely suppressed. FIG. 3 is a diagram for explaining a function elucidation / suppression method of CaM1 and CaM3 using the method of the present invention. That is, a partial region of MAPKP that binds to CaM1 and CaM3 (for example, a domain consisting of amino acid sequences 362 to 467 of the amino acid sequence shown in SEQ ID NO: 7) and an RFD fusion protein are designed, and CaM1 and CaM3 are designated as At the same time, it may be modified by ubiquitin modification. That is, the present invention can be used particularly effectively for a system in which protein X is a plurality of types.

  In addition, MAPKP (SEQ ID NO: 7) and MAPKP domain protein (domain protein having amino acids 362-467 of the amino acid sequence described in SEQ ID NO: 7), CaM1 (SEQ ID NO: 8) and CaM3 (SEQ ID NO: 9) Is described in “Yamakawa et al., (2004) J Biol Chem. 279, 928-36.”.

  Although not specifically described in detail, even in a system in which a plurality of types of protein X are used, an organism in which the function of a predetermined protein is knocked out can be created in the same manner as in a system in which protein X is one type.

(2.3)
There are many known genes whose expression is changed by environmental environmental stimuli for which protein X is unknown. However, their functions are hardly elucidated. In addition, many proteins are unclear how they interact with other proteins and what functions they express. If the method of this invention is used, the protein which expresses a function by interacting with a certain protein can be identified. That is, a gene encoding a fusion protein between a certain protein and a protein capable of interacting with a ubiquitin-binding enzyme is constructed. This constructed gene is introduced into a host such as a plant body. Protein is extracted from the obtained host and proteome analysis is performed. For example, a protein extracted from a plant body is subjected to two-dimensional electrophoresis analysis and mass spectrometry to identify a protein whose amount is reduced by ubiquitin degradation.

In the present invention, nucleoproteins whose expression is early induced by chitin oligosaccharide elicitor and gibberellin can be analyzed. As the nucleoprotein, for example, CIGR2 (NCBI accession No.
AAL62810). CIGR2 is thought to be involved in the induction of PR protein gene expression and the suppression of gibberellin-repressed protein expression, and CIGR2 does not have a motif that directly binds to DNA, so it interacts with other proteins. It is thought that the function is expressed. Therefore, it is possible to identify a protein that interacts with CIGR2.

(2.4. Examples of protein combinations)
Preferred combinations of the above-mentioned protein X, protein A and RFD include the following.
(1) Protein X: a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 (SLR1),
Protein A: a protein consisting of the amino acid sequence described in SEQ ID NO: 5 (GID2), and / or a protein consisting of the 117th to 211st amino acid sequence of the amino acid sequence described in SEQ ID NO: 5,
RFD: protein consisting of amino acid sequence 134-181 of the amino acid sequence described in SEQ ID NO: 1 (EL5) (Gomi et al.,
(2004) Plant J. 37, 626-34.);
(2) Protein X: a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (CaM1) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 9 (CaM3),
Protein A: a protein consisting of the amino acid sequence described in SEQ ID NO: 7 (MAPKP) and / or a domain protein having amino acids 362 to 467 of the amino acid sequence described in SEQ ID NO: 7,
RFD: protein consisting of amino acid sequence 134-181 of the amino acid sequence described in SEQ ID NO: 1 (EL5) (Yamakawa et al.,
(2004) J Biol Chem. 279, 928-36.); And (3) Protein X: a protein (Gβ) comprising the amino acid sequence set forth in SEQ ID NO: 11,
Protein A: a protein (Gγ) comprising the amino acid sequence set forth in SEQ ID NO: 10,
RFD: a protein comprising the amino acid sequence 134-181 of the amino acid sequence described in SEQ ID NO: 1 (EL5) (kato et al.,
(2004) Plant J. 2004 Apr; 38 (2): 320-31.).

  Although not described in detail in detail, even in a system in which protein X is unknown, it is possible to create an organism in which protein X is knocked out in the same manner as a single type of system.

(3. Functional analysis / suppression method)
Hereinafter, an example of the “method for analyzing and suppressing the function of a protein” according to the present invention will be described. The method of the present invention can be basically achieved by using a known method in the field of biotechnology, without needing to be particularly described.

(3.1.
Fusion protein production process)
In order to produce a fusion protein (protein A-RFD), a gene encoding a fusion protein containing protein A and RFD (hereinafter referred to as “fusion gene (A -RFD) "), and the protein may be generated. In designing the gene, preferably, as described above, the gene serving as a linker portion is designed so that ubiquitin can easily interact with protein X.

  A gene encoding a fusion protein containing protein A and RFD can be obtained by a cloning technique commonly used by those skilled in the art. Specifically, a method of preparing and screening a cDNA library from mRNA can be mentioned. Preparation of mRNA, RT-PCR method and the like can be easily performed by using a commercially available kit. For example, ISOGEN (manufactured by Nippon Gene) or Oligotex series kit (Takara Bio) can be used for mRNA preparation, and “SuperScriptTM One-StepTM RT-PCR System” or the like can be used for RT-PCR. it can. The base sequence of each gene may be obtained from a commercially available library and chemically synthesized.

  A recombinant vector is prepared by incorporating the DNA thus obtained into an appropriate cloning vector such as a plasmid. In addition, cloning vectors other than plasmids such as λ phage can be used.

When the cDNA base sequence is partially known, a so-called RACE (Rapid Amplification cDNA ends) method may be employed. RACE methods are also well known in the art and are commercially available kits such as GENE
It can be carried out using RACER (Invitrogen), “SMART RACE cDNA Amplification kit” (Clontech) or the like.

  The fusion gene (A-RFD) is obtained by cleaving one DNA with an appropriate restriction enzyme at a predetermined restriction enzyme site in a vector, and adding the other DNA cut with the same restriction enzyme to the DNA. The amino acid sequence of the protein encoded by can be linked and amplified so as not to deviate from the reading frame. At this time, the direction of the gene is arbitrary, and it may be the same direction or the direction facing each other.

  As described above, in the present invention, an artificial nucleotide sequence (linker) is provided in a plurality of gene binding regions used for fusing genes so that each gene translation product has a proper functioning conformation. It is also preferable to insert. For example, in FIG. 1, a sequence serving as a linker is inserted between the protein A gene and the RFD gene. The length of the linker can be appropriately set in consideration of the three-dimensional structure of protein A, experimental results, etc. For example, when the linker is short (0 to 20 bases), ubiquitination is inhibited or reduced. Preferably it is 30-50 bases. The linker can be obtained by ordinary chemical synthesis.

  In order to confirm whether the fusion gene is the target, the base sequence of the obtained clone or DNA fragment may be determined. The base sequence may be determined by a known method. For example, the base sequence may be determined using an automatic base sequencer.

  A gene encoding a fusion protein containing protein A and RFD can also be used by preparing a mutant by site-directed mutagenesis known in the art depending on the purpose of functional analysis. Mutagenesis can be performed using a commercially available site-directed mutagenesis kit (for example, “TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km”, etc.).

A transformant can be obtained by introducing the fusion gene into a host so that the target gene can be expressed. Transformation can be appropriately selected depending on the host, etc., for example, methods using calcium ions, electroporation method, spheroplast method, lithium acetate method, calcium phosphate method, lipofection method, Agrobacterium method, etc. Specifically, “Sambrook J and Russel D. Molecular Cloning, A Laboratory Manual, 3rd
edition, CSHL Press, 2001 ”.

  The host for the transformation is not particularly limited. Examples of the host include bacteria such as Escherichia and Bacillus, yeasts such as Saccharomyces and Schizosaccharomyces, plant bodies (including callus, plant culture cells, and plant organisms), animal cells, and insect cells.

(3.2.
(Proteolytic process)
In the present invention, protein X undergoes ubiquitin transfer from a ubiquitin-binding enzyme that interacts with RFD, and protein X is ubiquitinated by ubiquitin modification, and as a result, protein X is degraded. As a result, it is possible to analyze how the function of the target protein changes. Similarly, the function of the target protein can be suppressed. By creating an organism that suppresses the function of the target protein, an organism that knocks out the function of a given protein can be created.

(4. Function check)
The function of the protein can be confirmed by a known method. Below, the specific example which confirms that the function was suppressed and the evaluation method are demonstrated.

  As an assay for confirming the function, there is a method in which the fusion gene is expressed in a living body such as a plant body, and a reaction product obtained is extracted and analyzed. As the analysis method, a known method such as two-dimensional electrophoresis or Western analysis can be adopted.

  In addition to ubiquitin and the above fusion genes, ubiquitination may include the presence of ubiquitin-activating enzyme, ubiquitin-binding enzyme, and small molecules necessary for ubiquitination, so the amount of these substances may be measured as an aid. .

Specifically, the ubiquitination assay may be basically performed according to the method of Matsuda et al. (Matsuda te al., (2001) J. Cell. Sci., 114, 1949-1957.). Below, the example of the specific method is demonstrated. Total volume of 75 μl reaction solution (40 mM
Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM ATP, 2 mM DTT, 300 ng / μl bovine ubiquitin (Sigma), 50 ng mouse E1, 100 ng OsUBC5b: E2), 300 ng A-RFD Add fusion protein and react at 30 ° C for 2 hours. After completion of the reaction, add an equal volume of SDS-PAGE sample buffer, heat at 100 ° C for 5 minutes, and perform SDS-PAGE on 7.5% polyacrylamide gel. After electrophoresis, the gel was subjected to PVDF membrane (Immobilon-P;
Millipore) and PBS-Tween20 (8.1 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 2.68 mM) containing 5% (w / v) skim milk
Block by shaking in KCl, 0.1% Tween 20, pH 7.4) for 30 minutes at room temperature. The membrane is reacted overnight at 4 ° C. in PBS-Tween 20 containing 1/5000 volume of anti-MBP antibody (New England Biolabs, Beverly, Mass.). After washing with PBS-Tween20 for 5 minutes x 3 times, 1/4000 volume of peroxidase-labeled anti-rabbit IgG antibody (Jacskon Immuno
Reaction with PBS-Tween20 containing (Research) for 1 hour at room temperature. Color is developed using DAB after washing with PBS-Tween20 for 5 minutes x 3 times and with PBS for 5 minutes.

(5. Identification of protein X)
By using the above method, protein X that forms a complex with protein A can be identified.
That is, protein X uses a protein (protein A-RFD) in which a target protein (protein A) and a protein (RFD) capable of interacting with a ubiquitin-binding enzyme are used to form a complex with the target protein and interact with ubiquitin. A method for identifying a protein (protein X) that is degraded by acting, wherein the complex protein A-RFD is expressed in vivo according to the method described above, and the protein A and the protein X are combined. Ubiquitin bound to the ubiquitin-binding enzyme that interacts with the RFD is transferred to the protein X, the protein X is destroyed, the protein is extracted from the living body in which the protein X is destroyed, By identifying protein X by analyzing the protein Protein X is a protein that binds to the protein can be identified.

(6.
Control of protein X degradation rate)
The degradation rate of protein X can be controlled as follows. The efficiency of ubiquitination depends on the strength of binding to the ubiquitin-binding enzyme that interacts with RFD. The interaction between RFD and ubiquitin-binding enzyme is mainly controlled by hydrophobic interaction and partly electrostatic interaction. By substituting amino acid residues that are directly involved in the interaction, RFD and ubiquitin binding The strength of binding with the enzyme can be controlled. For example, when an RFD mutant in which leucine 138 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with alanine (or a protein consisting of amino acid sequences 134 to 181 of such a mutant), the efficiency of ubiquitination can be obtained. Decrease. Moreover, the efficiency of ubiquitination also decreases in a mutant in which leucine 174 in the amino acid sequence described in SEQ ID NO: 1 is substituted with alanine (or a protein comprising the 134th to 181st amino acid sequence of such a mutant). In other words, these mutants indicate that the efficiency of ubiquitination can be controlled. If the efficiency of ubiquitination decreases, the efficiency recognized by the proteasome system also decreases, and the degradation is suppressed. That is, since the efficiency of ubiquitination changes when the amino acid sequence of RFD is slightly changed, the degradation rate of protein X can be controlled by designing the RFD according to the desired degradation rate. Such a specific example, as explained above, uses a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 as RFD (or a protein comprising the 134th to 181st amino acid sequence of such a variant). Among the amino acid sequences described in SEQ ID NO: 1, those using a protein in which the 138th amino acid is substituted with alanine (or a protein consisting of the 134th to 181st amino acid sequence of such a mutant) are described in SEQ ID NO: 1. The degradation rate of protein X is controlled by selectively using a protein in which amino acid sequence 174 is substituted with alanine (or a protein consisting of amino acid sequence 134-181 of such a mutant). is there.

(7.Description of sequence number)
Hereinafter, the protein or nucleic acid represented by the SEQ ID NO used in this specification will be described.
[1] SEQ ID NO: 1 relates to the amino acid sequence of NCBI accession No. BAD16550 (EL5). In the present specification, a protein consisting of amino acid sequences 134-181 of the amino acid sequence shown in SEQ ID NO: 1 was used as RFD.
[2] SEQ ID NO: 2 relates to the amino acid sequence of NCBI accession No. NP 201297 (CIP8). In the present specification, a protein comprising the amino acid sequence of positions 257 to 297 in the amino acid sequence shown in SEQ ID NO: 2 was used as RFD.
[3] SEQ ID NO: 3 relates to the amino acid sequence of NCBI accession No. NP 974506 (Rma1). In the present specification, a protein consisting of the 48th to 101st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 3 was used as RFD.
[4] SEQ ID NO: 4 relates to the amino acid sequence of NCBI accession No. AAL26903 (Hrd1). In the present specification, a protein consisting of amino acid sequences 272 to 343 of the amino acid sequence shown in SEQ ID NO: 4 was used as RFD.
[5] SEQ ID NO: 5 relates to the amino acid sequence described in NCBI accession No. BAC81428 (GID2). In the present specification, a protein (GID2) consisting of the amino acid sequence shown in SEQ ID NO: 5 and a domain protein consisting of the 117th to 211st amino acid sequences of the amino acid sequence shown in SEQ ID NO: 5 were used as protein A.
[6] SEQ ID NO: 6 relates to the amino acid sequence of NCBI accession No. BAB97367 (SLR1). In the present specification, a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 was used as protein X.
[7] SEQ ID NO: 7 relates to the amino acid sequence of NCBI accession No. BAD00043 (MAPKP). In the present specification, a protein consisting of the amino acid sequence described in SEQ ID NO: 7 (MAPKP) and a domain protein having amino acids 362 to 467 in the amino acid sequence described in SEQ ID NO: 7 were used as protein A.
[8] SEQ ID NO: 8 relates to the amino acid sequence of NCBI accession No. BAB61907 (CaM1). In the present specification, a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 was used as protein X.
[9] SEQ ID NO: 9 relates to the amino acid sequence of NCBI accession No. BAB61908 (CaM3). In the present specification, a protein consisting of the amino acid sequence shown in SEQ ID NO: 9 was used as protein X.
[10] SEQ ID NO: 10 relates to the amino acid sequence of NCBI accession No. BAA07405 (Gγ). In the present specification, a protein (Gγ) consisting of the amino acid sequence shown in SEQ ID NO: 10 was used as protein A.
[11] SEQ ID NO: 11 relates to the amino acid sequence of NCBI accession No. BAD15277 (Gβ). In the present specification, a protein (Gβ) consisting of the amino acid sequence shown in SEQ ID NO: 11 was used as protein X.
[12] SEQ ID NO: 12 relates to the amino acid sequence of NCBI accession No. AAL62810 (CIGR2). In the present specification, a protein consisting of the amino acid sequence shown in SEQ ID NO: 12 was used as protein A.

[13] SEQ ID NO: 13 relates to the amino acid sequence of linker 1.
[14] SEQ ID NO: 14 relates to the amino acid sequence of linker2.
[15] SEQ ID NO: 15 relates to the amino acid sequence of the S-tag-RFD fusion protein.
[16] SEQ ID NO: 16 relates to the amino acid sequence of MBP- (13) -RFD.
[17] SEQ ID NO: 17 relates to the amino acid sequence of MBP- (46) -RFD.
[18] SEQ ID NO: 18 relates to the amino acid sequence of MBP- (82) -RFD.
[19] SEQ ID NO: 19 relates to the amino acid sequence of MBP-RFD (L174A).
[20] SEQ ID NO: 20 relates to the amino acid sequence of MBP-RFD (L138A).
[21] SEQ ID NO: 21 relates to the amino acid sequence of RFD-GID2.
[22] SEQ ID NO: 22 relates to the gene sequence of NCBI accession No. AB045120 (EL5).
[23] SEQ ID NO: 23 relates to a primer sequence at the 5 ′ end when amplifying the S-tag-RFD gene.
[24] SEQ ID NO: 24 relates to the primer sequence at the 3 ′ end when amplifying the S-tag-RFD gene.
[25] SEQ ID NO: 25 relates to a primer sequence at the 5 ′ end when a gene is amplified when preparing MBP- (13) -RFD.
[26] SEQ ID NO: 26 relates to a primer sequence on the 3 ′ end side for amplifying a gene used for preparing MBP- (13) -RFD.
[27] SEQ ID NO: 27 relates to a primer sequence on the 5 ′ end side for amplifying a gene used for preparing MBP- (46) -RFD.
[28] SEQ ID NO: 28 relates to a primer sequence on the 3 ′ end side for amplifying a gene used for preparing MBP- (46) -RFD.
[29] SEQ ID NO: 29 relates to a primer sequence on the 5 ′ end side for amplifying a gene used for preparing MBP- (82) -RFD.
[30] SEQ ID NO: 30 relates to a primer sequence on the 3 ′ end side for amplifying a gene used for preparing MBP- (82) -RFD.

[31] SEQ ID NO: 31 relates to the gene sequence of SEQ ID NO: 17.
[32] SEQ ID NO: 32 relates to the 5 ′ terminal primer sequence used in preparing the L138A mutant of SEQ ID NO: 19.
[33] SEQ ID NO: 33 relates to the 3 ′ terminal primer sequence used in preparing the L138A mutant of SEQ ID NO: 19.
[34] SEQ ID NO: 34 relates to the 5 ′ terminal primer sequence used in preparing the L174A mutant of SEQ ID NO: 20.
[35] SEQ ID NO: 35 relates to the 3 ′ terminal primer sequence used in preparing the L174A mutant of SEQ ID NO: 20.
[36] SEQ ID NO: 36 relates to the 5 ′ terminal primer sequence used in preparing the RFD gene fragment used in preparing GID2-RFD.
[37] SEQ ID NO: 37 relates to the 3 ′ terminal primer sequence used in preparing the RFD gene fragment used in preparing GID2-RFD.
[38] SEQ ID NO: 38 relates to the gene sequence described in NCBI accession No. AB100246 (GID2).
[39] SEQ ID NO: 39 relates to the 5 ′ terminal primer sequence used in preparing the GID2 gene fragment used in generating GID2-RFD.
[40] SEQ ID NO: 40 relates to the 3 ′ terminal primer sequence used in preparing the GID2 gene fragment used in generating GID2-RFD.
[41] SEQ ID NO: 41 relates to the amino acid sequence of the GID2-RFD gene sequence.

-Model experiment with S protein-Method for preparing S-tag-RFD fusion protein (SEQ ID NO: 15) FIG. 4 is a conceptual diagram for explaining Example 1. FIG. 4A is a conceptual diagram of an experimental system in Example 1. FIG. FIG. 4B is a gel electrophoresis diagram instead of a drawing showing that a predetermined protein has been ubiquitinated. Primer 5'-CATGC CATG GGGGT CGACC with the NcoI site added to the end of the gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (cDNA SEQ ID NO: 22) as a template
CGGAG GTG-3 '(SEQ ID NO: 23) and primer 5'-CGGGA TCCTT ACACG with a BamHI site added at the end of 3
PCR was performed using ACGAC GGTGA GGC-3 ′ (SEQ ID NO: 24) according to a known method. That is, after 2 minutes at 98 ° C., one cycle was 98 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute, and the nucleic acid was amplified by repeating this cycle 25 times.

  The amplified PCR product was subjected to electrophoresis and extracted from an agarose gel. The pUC19 vector (TAKARA) to be inserted was treated with HincII, and the target fragment was ligated using Ligaton high (TOYOBO), and then transformed into Escherichia coli to produce a clone.

Next, after this clone was cultured, plasmid extraction was performed from E. coli. The obtained plasmid was treated with NcoI and BamHI, which were sequences inserted at the time of primer design, and then subjected to electrophoresis to extract the target fragment from the agarose gel. In addition, pET32 vector (NOVAGEN) containing S-Tag
Similarly, after treating with NcoI and BamHI and ligating the fragment of interest using Ligaton high (TOYOBO), it was transformed into Escherichia coli to produce a clone of interest. Regarding the base sequence of the obtained plasmid, it was confirmed that the target base sequence was present by reading the sequence using a sequencer. Escherichia coli BL21 (DE3) was transformed with the obtained plasmid. Thus, mass expression of protein was performed using the obtained transformant (bacteria). The cells were pre-cultured in 2 × YT medium containing ampicillin at 37 ° C. for 4 hours, centrifuged at 10,000 × g for 5 minutes, and the collected cells were transferred again to the medium, and further cultured at 37 ° C. for 3 hours. Thereafter, IPTG at a final concentration of 1 mM was added to induce expression of the recombinant protein, and further cultured at 37 ° C. for 3 hours.

Thereafter, the cells were centrifuged at 5,000 × g for 15 minutes, recovered and stored at −80 ° C. After thawing frozen cells, 50 mM Tris-HCl, 500 mM
NaCl, 50 μM ZnSO4, 2.5 mM β-ME,
The cells were suspended at pH 7.4 and disrupted by sonication. The suspension was centrifuged at 27,000 × g for 30 minutes at 4 ° C., and the supernatant was collected. The obtained supernatant was subjected to Ni affinity chromatography. 50 mM Tris-HCl, 500
mM NaCl, 50 μM ZnSO4, 2.5 mM b-ME, pH 7.4 to 50 mM Tris-HCl, 500 mM NaCl, 50 μM ZnSO4, 500
Elution was performed with a linear gradient to mM imidazole, 2.5 mM β-ME, pH 7.4. The eluted fraction was subjected to Gel filtration chromatography. Elution was performed with 50 mM Tris-HCl, 100 mM NaCl, 20 mM ZnSO4, 2.5 mM β-ME, pH 7.4. Thrombin was added to this elution fraction and left at 25 ° C. for 3 hours for Q column chromatography. 50 mM Tris-HCl, 20 μM ZnSO4, 2.5 mM β-ME, pH 7.4 to 50 mM Tris-HCl, 600 mM NaCl, 20
Elution was performed with a linear gradient to mM ZnSO4, 2.5 mM b-ME, pH 7.4. Obtained elution fraction
It was subjected to affinity chromatography. The flow-through fraction was subjected to Gel filtration chromatography. Elution was performed with 50 mM Tris-HCl, 100 mM NaCl, 20 mM ZnSO4, 2.5 mM β-ME, pH 7.4. In this way, an S-tag-RFD fusion protein (SEQ ID NO: 15) was obtained.

Total volume of 75 μl reaction solution (40 mM
Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM ATP, 2 mM DTT, 300 ng / μl bovine ubiquitin (manufactured by Sigma),
50 ng mouse E1, 100 ng OsUBC5b: E2,
500 ng S-protein (manufactured by Sigma)) was added with 500 ng of S-tag-RFD fusion protein (SEQ ID NO: 15) and reacted at 30 ° C. for 16 hours. In this example, S-tag corresponds to protein A, and S protein corresponds to protein X.

After completion of the reaction, an equal volume of SDS-PAGE sample buffer was added, heated at 100 ° C for 5 minutes, and subjected to SDS-PAGE on 7.5% SDS polyacrylamide gel. After electrophoresis, the gel was transferred to a PVDF membrane (Immobilon-P; Millipore) and PBS-Tween20 containing 5% (w / v) skim milk (8.1 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 2.68 mM)
Blocking was performed by shaking in KCl, 0.1% Tween 20, pH 7.4) for 30 minutes at room temperature. This membrane was reacted in PBS-Tween20 containing 1/500 volume of anti-S-protein antibody for 1 hour at room temperature. After washing with PBS-Tween20 for 5 minutes × 3 times, the mixture was reacted overnight at 4 ° C. with PBS-Tween20 containing 1/4000 volume of peroxidase-labeled anti-rabbit IgG antibody (Jacskon Immuno Research). After washing with PBS-Tween20 for 5 minutes x 3 times and PBS for 5 minutes, the color was developed using DAB.

  The result is shown in FIG. In FIG. 4B, lane numbers are given from the left, the first, third and fifth lanes are after 0 hour, and the second, fourth and sixth lanes are after 12 hours. The first and second lanes indicate those using S-tag-RFD fusion protein. The third and fourth lanes show those using S protein. The fifth and sixth lanes show those using S-tag-RFD fusion protein and S protein. From FIG. 4 (B), ubiquitination of S-protein can be confirmed. This result indicates that ubiquitination of protein X interacting with protein A expressed by fusion with RFD is promoted, and that proteolysis can be artificially controlled by using the method of the present invention. It was done. Therefore, according to the method of the present invention, protein X (S protein) that forms a complex with protein A is expressed in vivo by expressing protein A-RFD in which protein A (S-tag) and RFD are fused. It was demonstrated that can be decomposed. It is clear that protein X other than S protein can be decomposed by using this method. Therefore, according to the present invention, it was demonstrated that the function of protein X or protein AX complex can be destroyed. It was also demonstrated that knockout organisms that destroyed such functions could be created. Furthermore, it was also demonstrated that the function of protein X or protein AX complex can be elucidated by analyzing the destroyed function.

-Verification of ubiquitination efficiency by linker length-
Method for Producing Maltose Linkage (MBP)-(Linker) -RFD Fusion Protein In the following, an experiment was conducted to examine the effect of the linker site in protein A-RFD on the ubiquitin modification of protein X. A linker containing 13, 46 and 82 amino acid residues was prepared. MBP- (13) -RFD, MBP- (46) -RFD, MBP- (82) -RFD (the length of the linker is shown in parentheses) is 5'-CCGA ATTCG GAGGA GGGGT CGACC
CGGAG-3 ': (SEQ ID NO: 25) and 5'-CCGGA
TCCTT ACACG ACGAC GGTGA GGCG-3 ': (SEQ ID NO: 26), 5'-CCGAA TTCGG AGGAG GGGTC GACCC GGAG-3': (SEQ ID NO: 27) and 5'-CCGGA TCCTT ACACG ACGAC GGTGA
GGCG-3 ': (SEQ ID NO: 28), 5'-CCGAA
TTCTG CTACT GCGAC GAGCG GCGC-3 ': (SEQ ID NO: 29) and 5'-CCGGA TCCTC AATCC GGACA TGCGC -3': (SEQ ID NO: 30) using both primers as a template for the full-length cDNA of EL5 (SEQ ID NO: 22) As a PCR. Specifically, after 2 minutes at 98 ° C., one cycle was 98 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 2 minutes, and the nucleic acid was amplified by repeating this cycle 25 times.

The resulting PCR product was digested with EcoRI and BamHI and ligated into pMAL-c2x previously digested with EcoRI and BamHI. The nucleotide sequence of the obtained plasmid was confirmed, and it was confirmed that no mutation was introduced. Escherichia coli BL21 (DE3) was transformed with the obtained plasmid. The obtained transformant (bacteria) was used for mass expression of protein. The cells were precultured in 2 × YT medium containing ampicillin at 37 ° C. for 4 hours, centrifuged at 10,000 × g for 5 minutes, and the collected cells were transferred again to the medium. Thereafter, the cells were further cultured at 37 ° C. for 3 hours. Thereafter, IPTG at a final concentration of 1 mM was added to induce expression of the recombinant protein. After further incubation at 37 ° C. for 3 hours, the cells were centrifuged at 5,000 × g for 15 minutes, recovered and stored at −80 ° C. After thawing the frozen cells, they were suspended in 50 mM Tris-HCl, 200 mM NaCl, 20 mM ZnSO4, 2.5 mM b-ME, pH 7.4, and the cells were disrupted by sonication. Centrifugation was performed at 27,000 × g for 30 minutes, and the supernatant was collected. The supernatant obtained was adsorbed on amylose resin and 50 mM
After washing with Tris-HCl, 200 mM NaCl, 20 mM ZnSO4, 2.5 mM b-ME, pH 7.4, 10 mM maltose, 50 mM Tris-HCl, 200 mM NaCl, 20 mM ZnSO4, 2.5 mM
Elutes with b-ME, pH 7.4. MBP is not protein A but a protein that is modified by ubiquitin. In this way, a maltose binding (MBP)-(linker) -RFD fusion protein (SEQ ID NOs: 16, 17, 18) was obtained.

Total volume of 75 μl reaction solution (40 mM
Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM ATP, 2 mM DTT, 300 ng / μl bovine ubiquitin (manufactured by Sigma),
50 ng mouse E1, 100 ng OsUBC5b: E2,
500 ng of maltose binding (MBP)-(linker) -RFD fusion protein (SEQ ID NOs: 16, 17, 18) was added and reacted at 30 ° C. for 1 hour. The length of the linker was examined with 13,46 and 82 amino acid residues.

After completion of the reaction, an equal volume of SDS-PAGE sample buffer was added, heated at 100 ° C for 5 minutes, and subjected to SDS-PAGE on 7.5% SDS polyacrylamide gel. After electrophoresis, the gel was transferred to a PVDF membrane (Immobilon-P; Millipore) and PBS-Tween20 containing 5% (w / v) skim milk (8.1 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 2.68 mM)
Blocking was performed by shaking in KCl, 0.1% Tween 20, pH 7.4) for 30 minutes at room temperature. This membrane was reacted overnight at 4 ° C. in PBS-Tween20 containing 1/500 volume of anti-S-protein antibody. After washing with PBS-Tween20 for 5 minutes × 3 times, the mixture was reacted with PBS-Tween20 containing 1/4000 volume of peroxidase-labeled anti-rabbit IgG antibody (Jacskon Immuno Research) for 1 hour at room temperature. After washing with PBS-Tween20 for 5 minutes x 3 times and PBS for 5 minutes, the color was developed using DAB.

  The result is shown in FIG. FIG. 5 is a gel electrophoresis photograph in place of a drawing showing the experimental results. The first and second lanes from the left indicate MBP. The third and fourth lanes from the left indicate MBP- (13) -RFD, the fifth through sixth lanes from the left indicate MBP- (46) -RFD, and the seventh through the left. The eighth lane shows MBP- (82) -RFD. Of the above lanes, odd-numbered lanes indicate after 0 hours, and even-numbered lanes indicate after 1 hour. In the figure, kDa indicates kilodaltons. From FIG. 5, it was confirmed that although the ubiquitin modification proceeds even when the length of the linker is changed, the progress rate can be controlled by the length of the linker. This also means that if a fusion protein of RFD and a protein that is degraded by ubiquitin is created, the protein can be degraded and the degradation rate can be controlled.

-Verification of control of ubiquitination efficiency by RFD mutants-
As described below, it was confirmed that the mutants in which leucine 138 or leucine 174 of SEQ ID NO: 1 was substituted with alanine each decreased in ubiquitination efficiency.

How to create a variant:
The L138A and L174A variants are Quickchange Site-Directed using the cDNA (SEQ ID NO: 31) encoding the amino acid sequence of SEQ ID NO: 17 as a template.
It was prepared by PCR using Mutagenesis Kit (stratagene). For PCR, L138A: 5'-GTGCG CGGTG TGCGC GGCGG
AGCTC G-3 '(SEQ ID NO: 32) and 5'-CGAGC
TCCGC CGCGC ACACC GCGCA C-3 '(SEQ ID NO: 33), L174A: 5'-CACTC CACCT GCCCG GCGTG CCGCC TCACC-3' (SEQ ID NO: 34) and 5'-GGTGA GGGGC ACGCC GGGCA GGTGG
AGTG-3 ′ (SEQ ID NO: 35) was used. PCR was performed at 95 ° C. for 30 seconds, 95 ° C. for 30 seconds, and 55 ° C. for 1 minute, and this cycle was repeated 12 times.

The base sequence of the obtained plasmid was confirmed, and it was confirmed that the mutation was introduced at the target site. Escherichia coli BL21 (DE3) was transformed with the obtained plasmid, and a large amount of protein was expressed using the obtained transformant (bacteria). The cells were pre-cultured in 2 × YT medium containing ampicillin at 37 ° C. for 4 hours, centrifuged at 10,000 × g for 5 minutes, and the collected cells were transferred again to the medium, and further cultured at 37 ° C. for 3 hours. Thereafter, IPTG at a final concentration of 1 mM was added to induce the expression of the recombinant protein. After further incubation at 37 ° C. for 3 hours, the cells were collected by centrifugation at 5,000 × g for 15 minutes and stored at −80 ° C. After thawing the frozen cells, suspend them in 50 mM Tris-HCl, 200 mM NaCl, 20 mM ZnSO4, 2.5 mM b-ME, pH 7.4, disrupt the cells by sonication, and suspend the suspension at 4 ° C. Centrifugation was performed at 27,000 × g for 30 minutes, and the supernatant was collected. The supernatant obtained was adsorbed on amylose resin and 50 mM
After washing with Tris-HCl, 200 mM NaCl, 20 mM ZnSO4, 2.5 mM b-ME, pH 7.4, 10 mM maltose, 50 mM Tris-HCl, 200 mM NaCl, 20 mM ZnSO4, 2.5 mM
Elutes with b-ME, pH 7.4. In this way, (MBP) -RFD mutant fusion protein (SEQ ID NOs: 19 and 20) was obtained.

Total volume of 75 μl reaction solution (40 mM
Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM ATP, 2 mM DTT, 300 ng / μl bovine ubiquitin (manufactured by Sigma),
50 ng mouse E1, 100 ng OsUBC5b: E2,
500 ng of (MBP) -RFD mutant fusion protein (SEQ ID NO: 19, 20) was added and reacted at 30 ° C. for 2 hours.

After completion of the reaction, an equal volume of SDS-PAGE sample buffer was added, heated at 100 ° C for 5 minutes, and subjected to SDS-PAGE on 7.5% SDS polyacrylamide gel. After electrophoresis, the gel was transferred to a PVDF membrane (Immobilon-P; Millipore) and PBS-Tween20 containing 5% (w / v) skim milk (8.1 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 2.68 mM)
Blocking was performed by shaking in KCl, 0.1% Tween 20, pH 7.4) for 30 minutes at room temperature. This membrane was reacted in PBS-Tween20 containing 1/500 volume of anti-S-protein antibody for 1 hour at room temperature. After washing with PBS-Tween20 for 5 minutes × 3 times, the mixture was reacted with PBS-Tween20 containing 1/4000 volume of peroxidase-labeled anti-rabbit IgG antibody (Jacskon Immuno Research) for 1 hour at room temperature. After washing with PBS-Tween20 for 5 minutes x 3 times and PBS for 5 minutes, the color was developed using DAB. The same applies to mutant R148A in which arginine 148 in SEQ ID NO: 1 is substituted with alanine, mutant V162A in which valine 162 in SEQ ID NO: 1 is replaced with alanine, and mutant D163A in which aspartic acid 163 in SEQ ID NO: 1 is replaced with alanine. Were manufactured and measured.

  The result is shown in FIG. In FIG. 6, from the left, the first and second lanes show MBP, the third and fourth lanes show no mutants, the fifth and sixth lanes show L138A mutants, and the seventh and eighth lanes The R148A mutant is shown, the 9th and 10th lanes indicate the V162A mutant, the 11th and 12th lanes indicate the D163A mutant, and the 13th and 14th lanes indicate the L174A mutant. In the fourth lane from the left of the unmutated mutant, bands derived from a plurality of polyubiquitinated proteins were observed. On the other hand, the mutant lane had fewer bands derived from polyubiquitination than the fourth lane. From this, it can be seen that the ubiquitination efficiency of protein X is inferior to that of the mutant without mutation. Moreover, from this result, it was confirmed that ubiquitin modification can be controlled by a mutant in which leucine 138 or leucine 174 is substituted with alanine, respectively.

-Verification of SLR1 proteolysis by GID2-RFD fusion protein-
Method for preparing GID2-RFD fusion protein Primer with 5% CACC sequence and cloning site (Xba I, Spe I, BamH I) added to template using gene (SEQ ID NO: 22) encoding SEQ ID NO: 13 as template: 5'-CACCT
Use CTAGA ACTAG TGGAT CCATG CCAGA TCTGG GTACC GACGA CG-3 '(SEQ ID NO: 36) and primer 5'-CCCGG GTCAC ACGAC GACGG TGAGG CGGCA-3' (SEQ ID NO: 37) with a stop codon and Sma I site added at the end of 3 PCR was performed. Specifically, after 2 minutes at 98 ° C., one cycle was 98 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 3 minutes, and the nucleic acid was amplified by repeating this cycle 25 times.

The amplified PCR product is electrophoresed and extracted from an agarose gel. TOPO cloning
Cloning was performed using a kit (Invitrogen) to prepare an RFD entry vector. Primer 5'-CACCT CTAGA ATGAA GTTCC GCTCT GATTC-3 'using the GID2 cDNA (SEQ ID NO: 38) as a template and adding the CACC sequence and Xba I site to the end of the gene of the protein represented by SEQ ID NO: 5 (SEQ ID NO: 39) and primer 5'- GGATC CCCCG CATTG with a stop codon deleted at the end of 3 and a BamHI site added
PCR was performed using GCCCC CTCCA TTCTT ATC-3 ′ (SEQ ID NO: 40). Specifically, after 2 minutes at 98 ° C., one cycle was 98 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 3 minutes, and the nucleic acid was amplified by repeating this cycle 25 times.
The amplified PCR product is electrophoresed and extracted from an agarose gel.
Cloning was performed using cloning kit (Invitrogen) to prepare a GID2 entry clone.

  Next, the GID2 entry plasmid was enzymatically treated with Xba I and BamH I, which were sequences inserted into primers, and subjected to electrophoresis to extract a GID2 fragment from an agarose gel. Furthermore, the RFD entry vector was similarly treated with Xba I and BamH I, and the GID2 fragment was ligated using Ligaton high (TOYOBO), and then transformed into E. coli to produce a GID2-RFD clone. Regarding the base sequence of the obtained plasmid, it was confirmed by sequencing that the target base sequence was present.

Gene transfer method
The GID2 gene (SEQ ID NO: 38) or GID2-RING gene (SEQ ID NO: 41) is linked between the promoter with 4 repeats of the enhancer region of the CaMV 35S promoter and the nopaline synthase gene-derived terminator, and the binary vector pBI121 (Clontech) ) Was introduced into plasmid pBI-HPT having a 35S promoter, hygromycin phosphotransferase (HPT) gene and nopaline synthase gene-derived terminator in the T-DNA region, and pBI-EN4-GID2 and pBI-EN4-GID2RING were introduced. It was constructed. The structures of these plasmids are shown in FIG. FIG. 7 (A) is a cleavage map of plasmid “pBI-EN4-GID2”. FIG. 7B is a cleavage map of the plasmid “pBI-EN4-GID2RING”. In the figure, 35S represents the CaMV 35S promoter, HPT represents the Escherichia coli hygromycin phosphotransferase gene (hygromycin resistance gene), CaMV3 ′ represents the CaMV terminator, and EN4 is upstream of the 35S promoter transcription start site. The artificial 35S promoter with 4 repeats of the -290 to -90 region is shown, NOS3 'is the terminator of the nopaline synthase gene, and RB is the T-DNA region right
Border sequence is shown, LB is T-DNA region left border sequence, and NPTIII is neomycin phosphotransferase gene (kanamycin resistance gene).

Transformed Agrobacterium can be obtained by the method of Nagel et al. (Nagel et al., (1990) Microbiol.
Lett., 67, 325.) After introducing the binary vectors pBI-EN4-GID2 and pBI-EN4-GID2RING into carcinoma (Agrobacterium tumefacience) by electroporation, 50 μg / ml kanamycin and It was obtained by culturing at 28 ° C. for 2 days on LB medium containing 50 μg / ml hygromycin.

Rice transformation was performed according to an ultra-rapid transformation method (Japanese Patent No. 3141084). That is, ripe rice seeds from which rice husks were removed were sterilized intact in a 2.5% sodium hypochlorite solution, washed thoroughly with sterilized water, and then 2,4-dichlorophenoxyacetic acid (2,4-D N6D medium (30 g / L sucrose, 0.3 g / L casamino acid, 2.8 g / L proline, 2 mg / L)
2,4-D, 4 g / L gellite, pH 5.8) and allowed to stand at 28 ° C. for 5 days.

After immersing the seeds previously cultured in the transformed Agrobacterium suspension, 2N6 · AS medium (30 g / L sucrose, 10 g / L glucose, 0.3 g / L casamino acid, 2 mg / L
2,4-D, 10 mg / L acetosyringone, 4 g / L gellite, pH 5.2), and co-cultured in the dark at 28 ° C. for 3 days.

  After thoroughly washing Agrobacterium from seeds using a washing solution in which 500 mg / L of carbenicillin, an antibiotic for Agrobacterium, was added to N6D medium excluding gellite, 50 mg of hygromycin was selected as a selective marker for carbenicillin 500 mg / L. The washed seeds were placed on N6D medium (selection medium) added with / L and cultured at 28 ° C. for 2 weeks.

  The hygromycin-resistant callus was subcultured four times in a new selection medium every two weeks to obtain a GID2 gene- or GID2-RING gene-introduced rice callus line.

Hygromycin-resistant callus is redifferentiated medium (500 mg / L carbenicillin, 30 mg / L hygromycin, 30 g / L sucrose, 30 g / L sorbitol, 2 g / L casamino acid, 2 mg / L kinetin, 0.002 mg / L
The cells were transferred to α-naphthylacetic acid (NAA), 4 g / L gellite, MS medium added to pH 5.8), and the culture was continued until redifferentiation at 28 ° C.

  The redifferentiated solid was placed on a rooting medium (hormone-free MS medium added to give hygromycin 30 mg / L) and assayed for hygromycin resistance. In the non-transformant, the new root did not grow and died in about one week, whereas the transformant considered to have the introduced gene showed resistance and showed the same growth as the wild type. When transformed plants grew, they were acclimated and transplanted into pots and grown in a greenhouse.

  The method for analyzing the function of the protein of the present invention can be used to analyze the function of the protein and the gene encoding the protein.

  In the method of analyzing the function of the protein of the present invention, since the function of the protein is analyzed by a method different from the method such as the gene disruption method or RNAi in particular, it is difficult to analyze the function of the multigene family. It can be used particularly effectively for analyzing.

  The method for suppressing the function of a protein of the present invention can be used for suppressing the function of a protein and a gene encoding the protein. It can also be used effectively to produce knockout animals that have been knocked out.

  Moreover, since the ubiquitin-dependent proteolytic system used in the present invention is universal for animals and plants, the method of the present invention has a wide range of applications.

Claims (7)

Using a protein (protein A-RFD) in which a target protein (protein A) and a protein (RFD) capable of interacting with a ubiquitin-binding enzyme are fused,
The function of the protein (protein X) that forms a complex with the target protein and is decomposed by being modified with ubiquitin, or the complex of the protein A and the protein X (protein AX complex) A method of analyzing the function,
Expressing the protein A-RFD in vivo,
Forming a complex of the protein A and the protein X;
Transferring the ubiquitin bound to the ubiquitin-conjugating enzyme that interacts with the RFD to the protein X, thereby decomposing the protein X and deactivating the function of the protein X or protein AX complex;
Analyzing the function of protein X or protein AX complex,
Here, the “protein capable of interacting with ubiquitin-binding enzyme (RFD)”
A protein consisting of amino acid sequences 134-181 in the amino acid sequence set forth in SEQ ID NO: 1,
Or a protein consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence of 134-181 in the amino acid sequence described in SEQ ID NO: 1,
In the protein A-RFD, protein A and RFD are bound by a linker,
The linker is a linker comprising the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14,
Or a linker consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence described in SEQ ID NO: 13 or SEQ ID NO: 14.
Protein function analysis method. By using a protein (protein A-RFD) fused with a target protein (protein A) and a protein (RFD) capable of interacting with a ubiquitin-conjugating enzyme, forming a complex with the target protein and being ubiquitin-modified A method for producing a knockout animal or knockout plant other than a human having disrupted the function of a protein to be degraded (protein X) or the function of a complex protein (protein AX) of the protein A and the protein X,
Expressing the protein A-RFD in vivo,
Forming a complex of the protein A and the protein X;
By transferring ubiquitin bound to the ubiquitin-conjugating enzyme that interacts with the RFD to the protein X, the function of the protein X or the function of the protein AX is deactivated,
Producing non-human knockout animals or knockout plants;
Here, the “protein capable of interacting with ubiquitin-binding enzyme (RFD)”
A protein consisting of amino acid sequences 134-181 in the amino acid sequence set forth in SEQ ID NO: 1,
Or a protein consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence of 134-181 in the amino acid sequence described in SEQ ID NO: 1.
A method for producing a non-human knockout animal or knockout plant. In the protein A-RFD, protein A and RFD are bound by a linker,
The linker is a linker comprising the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14,
Or a linker consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence described in SEQ ID NO: 13 or SEQ ID NO: 14.
The manufacturing method of the non-human knockout animal or knockout plant of Claim 2. Protein A is
A protein comprising the amino acid sequence set forth in SEQ ID NO: 5,
A protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence set forth in SEQ ID NO: 5;
A domain protein having amino acids 117-211 in the amino acid sequence shown in SEQ ID NO: 5,
Or a protein consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence 117-211 of the amino acid sequence shown in SEQ ID NO: 5,

The protein X is
The protein described in SEQ ID NO: 6 (SLR1),
Or a non-human knockout animal or knockout plant according to claim 2 or 3, which is a protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence described in SEQ ID NO: 6 Manufacturing method. Protein A is
The protein described in SEQ ID NO: 7 (MAPKP),
A domain protein having amino acids 362-467 of the amino acid sequence set forth in SEQ ID NO: 7,
A protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence set forth in SEQ ID NO: 7;
Or a protein consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the 362-467th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 7,

The protein X is
The protein described in SEQ ID NO: 8 (CaM1),
A protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence set forth in SEQ ID NO: 8;
The protein described in SEQ ID NO: 9 (CaM3),
The protein according to claim 2 or 3, which is any one or more of proteins consisting of amino acid sequences in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence shown in SEQ ID NO: 9. A method for producing a non-human knockout animal or knockout plant. Protein A is
The protein described in SEQ ID NO: 10 (Gγ),

Or a protein consisting of an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence set forth in SEQ ID NO: 10,
The protein X is
The protein described in SEQ ID NO: 11 (Gβ),
Or a non-human knockout animal or knockout plant according to claim 2 or 3, which is a protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence shown in SEQ ID NO: 11. Manufacturing method. Protein A is
The protein described in SEQ ID NO: 12 (CIGR2),
Or a non-human knockout animal or knockout plant according to claim 2 or 3, which is a protein comprising an amino acid sequence in which 1 to 4 amino acid residues are substituted, deleted, added or inserted from the amino acid sequence shown in SEQ ID NO: 12. Manufacturing method.

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