WO2005085467A1 - Méthode de détection d’un complexe protéique et trousse de détection d’un complexe protéique - Google Patents

Méthode de détection d’un complexe protéique et trousse de détection d’un complexe protéique Download PDF

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WO2005085467A1
WO2005085467A1 PCT/JP2005/003797 JP2005003797W WO2005085467A1 WO 2005085467 A1 WO2005085467 A1 WO 2005085467A1 JP 2005003797 W JP2005003797 W JP 2005003797W WO 2005085467 A1 WO2005085467 A1 WO 2005085467A1
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protein
chimeric
complex
leu
gene
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PCT/JP2005/003797
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Japanese (ja)
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Masaru Takagi
Keiichiro Hiratsu
Kyoko Matsui
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Japan Science And Technology Agency
National Institute Of Advanced Industrial Science And Technology
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Priority to JP2006510754A priority Critical patent/JP4631003B2/ja
Publication of WO2005085467A1 publication Critical patent/WO2005085467A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a technique for detecting the formation of a complex by two proteins. More specifically, the present invention relates to a technique for suppressing trans-transcriptional suppression, which suppresses transcription of a reporter gene whose transcription is activated by a transcription factor. As an index, the present invention relates to a protein complex detection method for detecting complex formation, and a protein complex detection kit.
  • GAL4 binds to DNA, a DNA-binding domain (abbreviated as DBD) 112, Domain (abbreviated as AD) 122.
  • DBD DNA-binding domain
  • AD Domain
  • a chimeric protein 110 in which X protein 111 is linked to DBD112 of GAL4 is designed.
  • a chimeric protein 120a is also designed, in which the Y protein 121 is linked to the AD122 of GAL4.
  • genes encoding the chimeric proteins 110 and 120a, respectively, are ligated to plasmids to construct two types of plasmids.
  • these plasmids have a CIS element (abbreviated to CIS) 132 to which DBD112 can bind, so that GAL4-dependent transcriptional activity of reporter gene 131 (denoted as Reporter in the figure) occurs. It has been modified.
  • these plasmids are introduced into yeast having the reporter gene 131.
  • chimeric proteins 110 and 120a are synthesized from the plasmid introduced into yeast by gene expression.
  • X protein 111 and Y protein 121 interact, they form a complex. If formed, the chimeric protein 110 and the chimeric protein 120a form a complex 140 through this complex formation, as shown in FIG. 6 (b). The DBD 112 and AD 122 then form a complex and functionally reconstitute the transcription factor GAL4.
  • the reconstituted GAL4 promotes (activates) the transcription of the reporter gene 131.
  • the X protein 111 and the Y protein 121 have formed a complex in the conventional method.
  • a chimeric protein 120b in which a Z protein 123 which does not form a complex with the X protein 111 and an AD122 are linked is used instead of the chimeric protein 120a.
  • no complex 140 is formed. Therefore, functional reconstitution of GAL4 by DBD112 and AD122 does not occur, nor does transcription of reporter gene 131 be promoted.
  • the transcriptional activation of the reporter gene 131 cannot be detected, it can be determined that the X protein 111 and the Z protein 123 do not form a complex.
  • Non-Patent Document 1 A two-hybrid system having the above features is disclosed in Non-Patent Document 1, for example.
  • JP-A 2001-269177 (Published date: October 2, 2001)
  • JP-A 2001-269178 (Published date: October 2, 2001 (2001))
  • JP 2001-292776 Gazette (Published date: 2001 (2001) 10 May 2)
  • JP-A 2001-292777 (Published date: October 23, 2001)
  • JP-A 2001-269176 (Published date: October 2, 2001)
  • JP-A 2001-269179 (Published date: October 23, 2001)
  • JP-A 2001-269176 (Published date: October 2, 2001)
  • the two-hybrid system has a problem that complex formation cannot be detected even though a complex is actually formed in some cases.
  • the cause of this problem is that the transcriptional activity of reporter gene 131 by the reconstituted transcription factor (GAL4) is used as an index when detecting the complex formation of two different proteins using this system. It is in.
  • the transcription activation of the reporter gene 131 is indicated.
  • the formation of the complex is confirmed. Therefore, for example, when the X protein 111 or the Y protein 121 shown in FIGS. 6 (a) and 6 (b) has a property capable of promoting the transcription of the reporter gene 131 by itself, these proteins Transcription of reporter gene 131 is promoted even before body formation. On the other hand, even when the X protein 111 and the Y protein 121 form a complex, the reconstituted GAL4 promotes the transcription of the reporter gene 131.
  • a protein having a function of activating gene transcription (a number of transcription factors) is selected as a protein to be detected for complex formation.
  • this conventional method has a problem that complex formation between a transcription factor and another protein cannot be detected.
  • the present invention has been made in view of the above problems, and an object of the present invention is to detect, in addition to a general protein, a complex formed by a transcription factor and a protein other than the transcription factor. Another object of the present invention is to provide a protein complex detection method and a protein complex detection kit.
  • the inventors of the present application have conducted intensive studies to solve the above-mentioned problems. As a result, when a protein to which a functional peptide that converts an arbitrary transcription factor into a transcription repressor is bound, binds to the transcription factor, The inventors have found that a transcription factor is converted into a transcription repressor into trans, and have completed the present invention.
  • the protein complex detection method provides a method for detecting whether or not a first protein and a second protein form a complex,
  • a protein synthesis system capable of producing a protein by expression
  • a first chimeric gene encoding a first chimeric protein containing a first protein
  • a second chimeric gene encoding a second chimeric protein containing a second protein
  • chimeric gene expression And a complex confirmation step of detecting the formation of a complex with the first protein and the second protein, wherein the first chimeric protein encoded by the first chimeric gene comprises a promoter sequence of a reporter gene.
  • the second chimeric protein encoded by the second chimera gene may convert any transcription factor into a transcription repressing factor.
  • a functional peptide that is converted into a protein, and the above-mentioned second protein is bound, and the first protein part of the first chimeric protein and the second protein part of the second chimeric protein are bound to form a complex.
  • the transcription of the reporter gene is suppressed, and in the complex confirmation step, the reporter gene is By transfer of over gene to check the power whether it is suppressed, it is characterized in that is adapted to detect the complex formation.
  • Preferred examples of the protein complex detection method according to the present invention include the following methods.
  • the first protein and the second protein containing the first protein are used.
  • the second chimeric protein encoded by the second chimeric gene is used.
  • transcription of the reporter gene is suppressed, and in the complex confirmation step, transcription of the reporter gene is suppressed.
  • a method for detecting a protein complex comprising detecting complex formation by detecting whether or not the protein complex has a force.
  • the reporter gene be incorporated into an expression vector and introduced into a plant cell.
  • the first protein containing the first protein is used.
  • the first chimeric protein encoded by the first chimeric gene comprises a DNA-binding peptide that specifically recognizes a promoter sequence of a reporter gene and the first protein.
  • the second chimeric protein encoded by the second chimeric gene is an arbitrary one.
  • the transcription of the reporter gene is suppressed, and in the complex confirmation step, whether or not the transcription of the reporter gene is suppressed should be confirmed.
  • the functional peptide may be represented by the following formula (1)-(4)
  • XI represents 0-10 amino acid residues
  • X2 represents Asn or Glu
  • X3 represents at least 6 amino acid residues.
  • Z1 represents Leu, Asp-Leu or Leu-Asp-Leu
  • Z2 represents Glu, Gin or Asp
  • Z3 represents 0-10 amino acid residues.
  • Z4 represents Glu, Gin or Asp.
  • it has an amino acid sequence represented by any one of the above.
  • the functional peptide is represented by the following formula (5):
  • oc 1 represents Asp, Asn, Glu, Gln, Thr or Ser
  • ⁇ 1 represents Asp, Gln, Asn, Arg ⁇ Glu, Thr, Ser or His
  • ⁇ 1 represents Arg , Gln, Asn, Thr, Ser ⁇ His, or Lys.
  • Preferable examples of the protein complex detection method according to the present invention include the following methods.
  • a first chimeric protein containing the first protein is encoded in a protein synthesis system capable of producing a protein by gene expression.
  • a method for detecting a protein complex comprising detecting whether or not reporter gene transcription has been suppressed, thereby detecting complex formation.
  • the protein complex detection method according to the present invention it is preferable to use a plant cell as the protein synthesis system.
  • both the first chimeric gene and the second chimeric gene are incorporated into an expression vector and introduced into a plant cell.
  • the reporter gene be incorporated into an expression vector and introduced into a plant cell.
  • a cell-free protein synthesis system is used.
  • the protein complex detection method it is preferable to use a transcription factor or a DNA-binding domain contained in the transcription factor as the DNA-binding peptide.
  • the reporter gene may be any substance that influences the expression of a protein complex according to the present invention.
  • the first chimeric gene, the second chimeric gene, and the reporter gene are each constructed as an expression vector and then introduced into a protein synthesis system.
  • a protein synthesis system Preferably.
  • the protein complex detection kit according to the present invention is characterized by being a kit for performing the above-described protein complex detection method in order to solve the above-mentioned problems.
  • another protein complex detection kit provides a first chimera in which a DNA-binding peptide that specifically recognizes a promoter sequence of a reporter gene and a first protein are combined.
  • a first chimeric gene expression vector capable of producing a protein, a functional peptide that converts any transcription factor into a transcription repressor, and a second protein And a second chimeric gene expression vector capable of producing a second chimeric protein bound to a protein.
  • the first chimeric gene expression vector further comprises at least (a) a DBD which is a polynucleotide encoding the DNA binding domain (DBD).
  • a segment comprising (b) a first RE recognition segment which is a polynucleotide having a nucleotide sequence which is adjacent to the DBD segment and has a nucleotide sequence recognized by at least one type of restriction enzyme (RE);
  • the gene expression vector comprises at least (c) a functional peptide segment which is a polynucleotide encoding the above functional peptide, and (d) at least one type of restriction enzyme (RE) adjacent to the functional peptide segment.
  • a second RE recognition segment which is a polynucleotide having a recognized nucleotide sequence, and the first chimeric gene expression vector
  • the first chimeric protein By incorporating the gene encoding the first protein into the (b) first RE recognition segment, the first chimeric protein can be produced, and the second chimera gene expression vector contains the (d) second RE recognized gene. It is preferable that the second chimeric protein can be produced by incorporating a gene encoding the second protein into the recognition segment.
  • the protein complex detection kit according to the present invention preferably further contains a reporter gene expression vector that expresses a reporter gene.
  • the protein complex detection kit at least one of Sarakoko, the first chimeric gene expression vector, the second chimeric gene expression vector, and the reporter gene expression vector is contained in the kit.
  • the promoter the cauliflower mosaic virus 35S promoter is included! /, Preferably! / ,.
  • the protein complex detection method of the present invention is a method for detecting protein interaction by suppressing the transcriptional activity that rises! It is easier to detect if the reporter has some activity. Therefore, the reporter gene expression vector preferably contains an enhancer such as the cauliflower mosaic virus 35S promoter.
  • the first chimeric gene expression vector, the second chimeric gene expression vector, and the reporter gene expression vector It is preferable that at least one of the terminators contains a terminator of the nopaline synthase gene as a terminator!
  • a transcription factor or a DNA binding domain contained in the transcription factor is further used as the DNA binding peptide.
  • the DNA binding domain of the yeast GAL4 protein is used as the DNA binding peptide.
  • a gene encoding a protein capable of relatively confirming its increase or decrease in appearance be used as the reporter gene. ,.
  • a luciferase gene is further used as the reporter gene!
  • XI represents 0-10 amino acid residues
  • X2 represents Asn or Glu
  • X3 represents at least 6 amino acid residues.
  • Z1 represents Leu, Asp-Leu or Leu-Asp-Leu
  • Z2 represents Glu, Gin or Asp
  • Z3 represents 0-10 amino acid residues.
  • Z4 represents Glu, Gin or Asp.
  • it has an amino acid sequence represented by any one of the above.
  • the functional peptide is further expressed by the following formula (5)
  • the functional peptide is further expressed by the following formulas (6)-(8)
  • oc 1 represents Asp, Asn, Glu, Gln, Thr or Ser
  • a2 represents Asn, Glu, Gln, Thr or Ser
  • j81 represents Asp, Gln, Asn , Arg, Glu, Thr, Ser or His
  • j82 represents Asn, Arg, Thr, Ser or His
  • ⁇ 2 represents Gln, Asn, Thr, Ser, His or Lys.
  • it has an amino acid sequence represented by any one of the above.
  • the functional peptide is preferably a peptide having an amino acid sequence represented by any one of SEQ ID NOS: 138.
  • the protein complex detection kit further comprises (a) a group of reagents for introducing an expression vector into a plant cell, (b) a first chimeric gene expression vector or a second chimeric gene expression. At least one of a reagent group for incorporating the gene encoding the first protein or the gene encoding the second protein into the vector, and (c) a reagent group for confirming a change in the expression level of the reporter gene. It is preferable to include a group of reagents.
  • FIG. 1 (a) A diagram schematically showing the mechanism of the protein complex detection method according to the present invention.
  • FIG. 3 is a diagram showing a state before forming a complex.
  • FIG. 1 (b) A drawing schematically showing the mechanism of the protein complex detection method according to the present invention, showing a state in which a complex is formed.
  • FIG. 2 is a diagram schematically illustrating a state where two different proteins do not form a complex in the protein complex detection method according to the present invention.
  • FIG. 3 is a view showing an experimental result of detecting the formation of a complex between a JUN protein and a FOS protein in one example of the present invention.
  • FIG. 4 is a view showing an experimental result of detecting a complex formation between a JUN protein and a FOS protein in another example of the present invention.
  • FIG. 5 is a view showing an experimental result of detecting a complex formation between a PI protein and an AP3 protein in another example of the present invention.
  • FIG. 6 (a) is a view schematically showing a conventional mechanism for detecting a protein complex using a two-hybrid system, and showing a state before a complex is formed.
  • FIG. 6 (b) is a view schematically showing a conventional mechanism of a protein complex detection method using a two-hybrid system, and is a view showing a state in which a complex is formed.
  • FIG. 3 is a diagram schematically showing a state where two different proteins do not form a complex.
  • FIG. 8 (a) is a view showing seeds of a wild-type Arabidopsis thaliana.
  • FIG. 8 (b) shows Arabidopsis seeds transformed with a gene obtained by adding SRDX to the TTG1 gene.
  • FIG. 8 (c) is a diagram showing Arabidopsis seeds transformed with the TTG1 gene to which SRDXm, a domain in which the repression domain (SRDX) is mutated, is attached.
  • FIG. 9 (a) is a view showing leaves of a wild-type Arabidopsis thaliana in which a trichome is formed.
  • FIG. 9 (b) is a view showing that trichome formation is suppressed in the leaves of Arabidopsis thaliana transformed with a gene obtained by adding SRDX to the TTG1 gene.
  • FIG. 10 (a) is a view showing a wild type Arabidopsis thaliana plant in which anthocyanin is accumulated.
  • FIG. 10 (b) is a view showing that the accumulation of anthocyanin is suppressed in Arabidopsis thaliana plants transformed with a gene obtained by adding SRDX to the TTG1 gene.
  • FIG. 10 (c) is a diagram showing that anthocyanins are accumulated in a plant of Arabidopsis thaliana transformed with a TTG1 gene to which SRDXm has been added in the same manner as in the wild type.
  • FIG. 11 (a) shows the roots of wild-type Arabidopsis thaliana.
  • FIG. Ll (c) shows that Arabidopsis roots transformed with the TTG1 gene to which SRDXm has been added show the same phenotype as the wild type.
  • FIG. 12 (a) is a view showing that cormela is formed on the seed surface of wild-type Arabidopsis thaliana.
  • FIG. 12 (b) is a view showing that the formation of cormella is suppressed on the seed surface of Arabidopsis thaliana transformed with a gene obtained by adding SRDX to the TTG1 gene.
  • FIG. 12 (c) is a view showing that a colmela is formed on the seed surface of Arabidopsis thaliana transformed with the TTG1 gene to which SRDXm has been added, similarly to the wild type.
  • FIG. 13 is a process chart showing a method for constructing a construction vector for constructing a recombinant expression vector used in Examples.
  • FIG. 14 is a process chart in which a gene encoding a transcription repressor converting peptide SRDX and a TTG1 gene are incorporated into a construction vector p35SG used in Examples.
  • FIG. 15 is a process diagram showing a method for constructing a transformation vector pBIGCKH.
  • the protein complex detection method according to the present invention is a method including at least a chimeric gene expression step and a complex confirmation step. ⁇ Outline of this method>
  • An object of the present invention is to detect whether a first protein and a second protein form a complex. Therefore, as shown in FIG. 1A, first, the first protein is referred to as X protein 11, and the second protein is referred to as Y protein 21.
  • the chimeric protein 10 encoded by the first chimeric gene is obtained by binding a DNA binding peptide (DBD) 12 and an X protein 11.
  • DBD12 is a peptide that specifically recognizes the CIS element (CIS) 32 contained in the reporter gene 31, as described below.
  • CIS32 In the upstream region of CIS32, there is a non-enzyme containing a cauliflower mosaic virus 35S promoter.
  • the chimeric protein 20a encoded by the second chimeric gene is obtained by binding a functional peptide 22 for converting an arbitrary transcription factor to a transcription repressor and a Y protein 21.
  • the reporter gene 31 has its transcription activated by the gene-enhancing enzyme 33 such as the 35S promoter, irrespective of the transcriptional activity of the DNA-binding peptide (DBD) 12.
  • complex formation with X protein 11 and Y protein 21 is detected. Specifically, as shown in FIG. 1 (b), when the X protein 11 portion of the first chimeric protein 10 and the Y protein 21 portion of the second chimeric protein 20a bind to form a complex 40. In addition, since the transcription of the reporter gene 31 is suppressed, the formation of the complex is detected by confirming whether or not the transcription of the reporter gene 31 is suppressed.
  • the complex 40 contains the functional peptide 22.
  • the DBD12 suppresses the transcription of the reporter gene 31 to trans (indirectly).
  • the X protein 11 and the Y protein 21 form a complex by confirming the transcription suppression in the complex confirmation step.
  • the Z protein cannot form a complex 40 that cannot be formed by the Y protein 21 that forms the complex 40 with the second protein X protein 11.
  • the second chimeric protein 20b produced in the protein synthesis system is obtained by binding the functional peptide 22 to the Z protein 23.
  • the X protein 11 portion of the first chimeric protein 10 and the Z protein 23 portion of the second chimeric protein 20b cannot bind to each other, and the complex 40 is not formed. Therefore, transcription of the reporter gene 31 is not suppressed while being activated by the function of the DBD12 portion of the chimeric protein 10 or by its own enzyme 133. Therefore, by confirming that this transcriptional repression does not occur, it is possible to detect that X protein 11 and Z protein 23 do not form a complex.
  • the first chimeric gene encoding chimeric protein 10 and the second chimeric gene encoding chimeric protein 20a were combined with a protein containing reporter gene 31. To be expressed.
  • X protein 11 and DBD12 contained in chimeric protein 10 are referred to as
  • the Y protein 21 and the functional peptide 22 contained in the chimeric protein 20a will be described in detail. Further, the reporter gene 31 and the protein synthesis system are also described in detail.
  • the X protein 11 is a protein for which it is desired to determine whether it forms a complex by interacting with the Y protein 21 described below, and can be any protein in principle. However, the X protein 11 does not include a protein that alone suppresses the transcription of the reporter gene 31 described below. Such proteins are used in the present invention.
  • the X protein 11 may be any protein of any length. Furthermore, unlike the conventional protein complex detection method using the one-hybrid system, in this method, the X protein 11 may be an arbitrary transcription factor or a protein having the property of binding to a transcription factor. it can. This is because the transcriptional suppression of the reporter gene 31 by the chimeric protein complex 40 described later is predominantly performed in an environment where the transcription factor X protein 11 is present.
  • DBD12 used in the present method has the ability to activate transcription of reporter gene 31 by binding to CIS32 (cis element) contained in reporter gene 31 described below, or has no transcriptional activity. It is a DNA binding peptide.
  • This DBD12 may be a transcription factor itself or a DNA binding domain within the transcription factor.
  • DBD12 is a transcription factor such as GAL4, PAP1, EIN3, CUC1, CUC2, AtMYB23, or a DNA binding domain contained therein, but is not limited thereto.
  • DBD12 does not necessarily need to be powerful in all of the amino acid sequences constituting the transcription factor or the DNA binding domain contained in the transcription factor. That is, DBD12 may be a part of the entire amino acid sequence of the transcription factor and its DNA binding domain.
  • the Y protein 21 is a protein whose ability to form a complex by interacting with the X protein 11 described above, and can be any protein in principle. However, the Y protein 21 does not include a protein that alone suppresses the transcription of the reporter gene 31 described below. Such proteins are used in the present invention. This is because it is not possible to determine whether or not to form a complex.
  • Y protein 21 can independently suppress the transcription of reporter gene 31, it suppresses the transcription of reporter gene 31 even before forming a complex with X protein 11. On the other hand, even when the Y protein 21 and the X protein 11 form a complex, the transcription of the reporter gene 31 is also suppressed. Therefore, in any case, since transcription repression is confirmed in the complex confirmation step, it cannot be distinguished whether the Y protein 21 and the X protein 11 have formed a complex.
  • the Y protein 21 may also be a polypeptide of any length. Furthermore, unlike the conventional protein complex detection method using a one-hybrid system, in this method, the Y protein 21 may be an arbitrary transcription factor or a protein having a property of binding to a transcription factor. it can. This is because the transcriptional suppression of the reporter gene 31 by the chimeric protein complex 40 described later is predominantly performed in an environment where the transcription factor Y protein 21 is present.
  • the functional peptide 22 used in the present invention which converts an arbitrary transcription factor into a transcription repressor, is controlled by the transcription factor by forming a chimeric protein fused with the transcription factor, which is not particularly limited. Any peptide can be used as long as it can suppress the transcription of the target gene (see Patent Documents 17 to 17, Non-Patent Documents 2 and 3).
  • This peptide has a very simple structure, which is also derived from Class II ERF (Ethylene Responsive Element Binding Factor) protein and plant zinc finger protein (Zinc Finger Protein, such as Arabidopsis SUPER MAN protein). RU
  • a specific structure of an example of the functional peptide 22 is an amino acid sequence represented by any of the following formulas (1) and (4).
  • XI represents 0-10 amino acid residues
  • X2 represents Asn or Glu
  • X3 represents at least 6 amino acid residues.
  • Z1 represents Leu, Asp-Leu or Leu-Asp-Leu
  • Z2 represents Glu, Gin or Asp
  • Z3 represents 0-10 amino acid residues.
  • Z4 represents Glu, Gin or Asp
  • the number of amino acid residues represented by the above XI may be in the range of 0 to 10. Further, the type of the specific amino acid constituting the amino acid residue represented by XI is not particularly limited, and may be any type. In other words, in the functional peptide 22 of the above formula (1), at the N-terminal side, one arbitrary amino acid or an oligomer having 2 to 10 arbitrary amino acid residue powers is added, and V, Yes, and with or without any amino acids.
  • the amino acid residue represented by XI is preferably as short as possible. Specifically, the number is preferably 10 or less, more preferably 5 or less.
  • the number of amino acid residues represented by the above X3 may be at least six.
  • the type of the specific amino acid constituting the amino acid residue represented by X3 is not particularly limited, and may be any type.
  • an oligomer having an arbitrary amino acid residue power of 6 or more is added to the C-terminal side. The function described above can be exhibited if there are at least six amino acid residues represented by X3.
  • a specific sequence of a pentamer (5mer) consisting of 5 amino acid residues excluding XI and X3 is shown in SEQ ID NOS: 39 and 40.
  • the amino acid sequence when X2 is Asn is the amino acid sequence shown in SEQ ID NO: 39
  • the amino acid sequence when X2 is Glu is the amino acid sequence shown in SEQ ID NO: 40.
  • the number of amino acid residues represented by the above Y1 may be in the range of 0-10. Just fine. Ma
  • the type of the specific amino acid constituting the amino acid residue represented by Yl is not particularly limited, and may be any type. In other words, in the functional peptide 22 of the above formula (2), like the functional peptide 22 of the above formula (1), one arbitrary amino acid or 2-10 An oligomer having any amino acid residue power may be added !, or no amino acid may be added or not!
  • the amino acid residue represented by Y1 is preferably as short as possible. Specifically, the number is preferably 10 or less, more preferably 5 or less.
  • the number of amino acid residues represented by the above Y3 is at least 6 as in X3 of the functional peptide 22 of the above formula (1). Any number is acceptable. Further, the type of the specific amino acid constituting the amino acid residue represented by Y3 is not particularly limited, and may be any type. In other words, in the functional peptide 22 of the above formula (2), similarly to the functional peptide 22 of the above formula (1), an oligomer having six or more arbitrary amino acid residue powers is added to the C-terminal side. Being done, If the number of amino acid residues represented by Y3 is at least 6, the above function can be exhibited.
  • a specific sequence of a pentamer (5mer) consisting of 5 amino acid residues excluding Y1 and Y3 is shown in SEQ ID NOS: 41 and 42.
  • the amino acid sequence when Y2 is Phe is the amino acid sequence shown in SEQ ID NO: 41
  • the amino acid sequence when Y2 is lie is the amino acid sequence shown in SEQ ID NO: 42.
  • a specific sequence of a tetramer (4mer) having four amino acid residues except for Y2 is shown in SEQ ID NO: 43.
  • the amino acid residue represented by Z1 contains Leu in a range of 113.
  • the case of one amino acid is Leu
  • the case of two amino acids is Asp-Leu
  • the case of three amino acids is Leu-Asp-Leu.
  • the number of amino acid residues represented by the above Z3 is 0 to 10 as in the case of the XI of the functional peptide 22 of the above formula (1). It may be within the range. Also, the specific type of amino acid constituting the amino acid residue represented by Z3 is particularly Any kind of thing may be used without limitation. In other words, in the functional peptide 22 of the above formula (3), one arbitrary amino acid or an oligomer having 2-10 arbitrary amino acid residue power is added to the C-terminal side. However, no amino acid may be added.
  • the amino acid residue represented by Z3 is preferably as short as possible. Specifically, the number is preferably 10 or less, more preferably 5 or less. Specific examples of the amino acid residue represented by Z3 include Gly, Gly-Phe-Phe, Gly-Phe-Ala, Gly-Tyr-Tyr, Ala-Ala-Ala, etc. It is not limited.
  • the number of amino acid residues in the entirety of the functional peptide 22 represented by the formula (3) is not particularly limited, but from the viewpoint of ease of synthesis, the number of amino acids is not more than 20 amino acids. It is preferable that
  • a specific sequence of an oligomer consisting of 7 to 10 amino acid residues excluding Z3 is shown in SEQ ID NOS: 44 to 52.
  • the amino acid sequence when Z1 is Leu and Z2 is Glu, Gin or Asp is the amino acid sequence shown in SEQ ID NO: 44, 45 or 46, respectively, wherein Z1 is Asp-Leu and Z2 is Glu, Gin or Asp.
  • the functional peptide 22 of the above formula (4) is a hexamer (6mer) having six amino acid residues, and its specific sequence is shown in SEQ ID NOS: 5, 14, and 53.
  • the amino acid sequence when Z4 is Glu is the amino acid sequence shown in SEQ ID NO: 5
  • the amino acid sequence when Z4 is Asp is the amino acid sequence shown in SEQ ID NO: 14, and the amino acid sequence when Z4 is Gin.
  • the amino acid sequence is the amino acid sequence shown in SEQ ID NO: 53.
  • the functional peptide 22 used in the present invention may be a peptide having a minimum sequence such as the hexamer represented by the above formula (4).
  • the amino acid sequence shown in SEQ ID NO: 5 corresponds to the 196th to 201th amino acid sequence of Arabidopsis thaliana SUPERMAN protein (SUP protein). It was found as a trans-repression converting peptide.
  • oligopeptides having an amino acid sequence represented by any one of SEQ ID NOS: 117.
  • These oligopeptides have been found by the present inventors to be the above-mentioned transcription repressor converting peptides (for example, see Patent Document 7).
  • the present inventor further studied the structure of the motif, and as a result, found six new motifs having amino acid power.
  • This motif is specifically represented by the following general formula (
  • ⁇ 1 represents Asp, Asn, Glu, Gln, Thr or Ser
  • ⁇ 1 represents Asp, Gln, Asn ⁇ Arg ⁇ Glu, Thr, Ser or His
  • ⁇ 1 Indicates Arg ⁇ Gln, Asn ⁇ Thr, Ser, His, or Lys.
  • the peptides having the amino acid sequence represented by (6), (7), (8) or (9) are classified as peptides.
  • 1 represents Asp, Asn, Glu, Gln, Thr or Ser
  • oc2 represents Asn, Glu, Gln, Thr or Ser
  • j81 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His
  • j82 represents Asn, Arg, Thr, Ser or His
  • j83 represents Glu, Asp or Gin. Show.
  • ⁇ 2 indicates Gln, Asn, Thr, Ser, His, or Lys.
  • More specific examples of the functional peptide 22 having the amino acid sequence represented by the above formulas (5) to (9) include a peptide having the amino acid sequence represented by SEQ ID NOS: 18 to 36.
  • the peptide of SEQ ID NO: 25, 26, 28 or 30 is represented by the general formula (6).
  • the peptide of SEQ ID NO: 18, 2521, 31, 32 or 33 corresponds to the peptide represented by the general formula (7), and the peptide of SEQ ID NO: 22, 23, 24, 27 or 29 is
  • the peptide corresponds to the peptide represented by the general formula (8), and the peptide of SEQ ID NO: 19 or 20 corresponds to the peptide represented by the general formula (9).
  • a functional peptide 22 having an amino acid sequence represented by SEQ ID NO: 37 or 38 can also be used.
  • the reporter gene 31 that can be used in the present invention is required to be a gene to which DBD12 can bind. It is not necessary to be a gene whose transcription is activated by DBD12. For example, if the reporter gene 31 has the enhancer 33 in the promoter region, the transcription need not necessarily be activated by DBD12. It is preferable that a promoter sequence and an effector region to which DBD12 binds are bound to this reporter gene 31!
  • the reporter gene 31 is a gene capable of confirming by some means whether or not the transcription of the gene is suppressed. Specifically, it is preferable that the reporter gene 31 is a gene encoding a protein capable of relatively confirming the increase or decrease in appearance.
  • genes corresponding to such conditions include a luciferase gene, a chloramue-coal acetyltransferase (CAT) gene, a 13-glucuroidase (GUS) gene, and a j8-galactosidase (lacZ) gene.
  • CAT chloramue-coal acetyltransferase
  • GUS 13-glucuroidase
  • lacZ j8-galactosidase
  • the reporter gene 31 can be prepared by cutting the reporter gene 31 with a restriction enzyme and then isolating the DNA by electrophoresis or the like.
  • the reporter gene 31 may be synthesized using a polynucleotide synthesizer.
  • the reporter gene 31 may be prepared in large amounts by using the base sequence of the reporter gene 31 as a type III and DNA amplification techniques such as PCR.
  • ⁇ Protein synthesis system> the above-described chimeric gene and reporter gene 31 are expressed in a protein synthesis system capable of producing a protein by gene expression.
  • a protein synthesis system capable of producing a protein by gene expression.
  • plant or animal cells it is preferable to use plant or animal cells as described later, but a cell-free protein synthesis system can also be used.
  • DNA-dependent RNA polymerase required to transcribe messenger RNA from gene DNA, nucleotides used as the material of messenger RNA, ribosomal RNA required to translate mRNA proteins, It contains a variety of amino acids that are used as materials for proteins that can be used to transcribe genomic messenger RNA and translate transcribed messenger RNA into proteins based on these enzymes, nucleotides, and amino acids.
  • An environment that can be performed can be mentioned as a cell-free protein synthesis system that can be used in the present method.
  • the above-described chimeric gene and reporter gene 31 are each incorporated into a recombinant expression vector and introduced into a protein synthesis system. Therefore, the following describes a recombinant expression vector that can be used in the present method.
  • the recombinant expression vector containing the chimeric gene encoding chimeric protein 10 (the second chimeric gene expression vector) has a nucleotide sequence encoding the amino acid sequence of DBD12 and a nucleotide sequence encoding the amino acid sequence of X protein 11. Any configuration that includes the linked chimeric base sequence (first chimeric gene) is acceptable.
  • any method including a known technique described above as a method for obtaining the reporter gene 31 may be used.
  • polynucleotide corresponding to the above-mentioned chimeric nucleotide sequence When incorporated into the recombinant expression vector, the polynucleotide itself corresponding to the above-mentioned chimeric nucleotide sequence may be directly incorporated into the recombinant expression vector. Good. Alternatively, one of the polynucleotides corresponding to the nucleotide sequence encoding the amino acid sequence of DBD12 or X protein 11 is previously prepared. The other polynucleotide designed so that the reading frame matches the expression vector may be incorporated into the expression vector incorporated in! /.
  • the above-described polynucleotide may be incorporated into a plasmid, phage, or cosmid.
  • the recombinant expression vector containing the chimeric gene encoding the chimeric protein 20a (the second chimeric gene expression vector) has a nucleotide sequence encoding the amino acid sequence of the functional peptide 22 and a nucleotide sequence encoding the amino acid sequence of the Y protein 21 Conjugated, chimeric base sequence
  • any method including a known technique described above as a method for obtaining the reporter gene 31 may be used.
  • the polynucleotide when a polynucleotide corresponding to the above-described chimeric nucleotide sequence is incorporated into the recombinant expression vector, the polynucleotide itself may be directly incorporated into the recombinant expression vector.
  • the reading frame was designed to match the reading frame to an expression vector in which one of the polynucleotides corresponding to the amino acid sequence of the functional peptide 22 or the Y protein 21 was previously incorporated.
  • One polynucleotide may be incorporated.
  • nucleotide sequence encoding the amino acid sequence of the functional peptide 22 is based on the amino acid sequence represented by the general formula (1)-(9), SEQ ID NO: 16, or What is necessary is just to have the base sequence which codes the peptide of sequence number 17.
  • polynucleotide corresponding to the nucleotide sequence encoding the amino acid sequence of the functional peptide 22 SEQ ID NOs: 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 7 4, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 1 16, 118, 120, 122.
  • Polynucleotides having the nucleotide sequence shown by 124, 126 or 128 can be mentioned.
  • the positive nucleotides shown in 15, 117, 119, 121, 123, 125, 127, or 129 are polynucleotides complementary to the above-exemplified polynucleotides, respectively. These polynucleotides correspond to the amino acid sequence shown in SEQ ID NO: 138, as shown in Table 1 below.
  • nucleotide sequences may include a nucleotide sequence serving as a linking site for linking to the gene encoding Y protein 21, if necessary. Further, if the reading frame of these nucleotide sequences does not match the reading frame of the nucleotide sequence of the gene encoding the amino acid sequence of Y protein 21, an additional nucleotide sequence for matching them is inserted. May be included.
  • the above-described polynucleotide may be incorporated into a plasmid, phage, cosmid, or the like.
  • [0114] [Recombinant expression vector containing reporter gene 31]
  • the recombinant expression vector containing the reporter gene 31 may be a plasmid, phage, or cosmid into which a polynucleotide corresponding to the nucleotide sequence of the reporter gene 31 obtained as described above is incorporated. Just fine.
  • the above three types of recombinant expression vectors may contain various DNA segments.
  • at least one of these recombinant expression vectors contains an arbitrary promoter sequence for expressing a gene.
  • a rice actin promoter can also be used.
  • At least one of these recombinant expression vectors contains an arbitrary terminator region. At that time, it is preferable to use a terminator of the nopaline synthase gene as the terminator.
  • any of the above three types of recombinant expression vectors that can be used in the present invention can be produced (proliferated) using any known method.
  • Escherichia coli may be used as a host to grow in the cells of the bacteria.
  • a preferred type of Escherichia coli may be selected depending on the type of the vector.
  • the three types of recombinant expression vectors described above are introduced into arbitrary cells and transformed into transformants, thereby producing chimeric proteins 10 and 20a by gene expression in the transformants.
  • the three types of recombinant expression vectors described above are introduced into arbitrary cells and transformed into transformants, thereby producing chimeric proteins 10 and 20a by gene expression in the transformants.
  • the cells into which these recombinant expression vectors are introduced may be cells that can produce chimeric proteins 10 and 20a by gene expression.
  • Such cells include plant cells.
  • the plant cells referred to here include not only isolated plant cells but also cells contained in plants, cells contained in various tissues in plant organs such as flower 'leaf' roots, and the like. Cells in the callus and cells in suspension culture are also included.
  • the above-mentioned recombinant expression vector can be introduced into animal cells, human cells, or cultured cells derived from these cells, in addition to plant cells.
  • the recombinant expression vector may be introduced into cells other than the cell type in which X protein 11 and Y protein 21 are naturally present. That is, in the present invention, verification of complex formation by X protein 11 or Y protein 21 derived from various cell types can be performed in one type of cells of different types or the same type (eg, plant cells). In other words, in the present invention, in a cell into which a recombinant expression vector is introduced, formation of a protein complex that does not originally exist in the cell can also be verified.
  • a recombinant expression vector containing a gene encoding X protein 11 or Y protein 21 derived from yeast / human may be introduced into plant cells. Even in this case, the complex formation by the X protein 11 and the Y protein 21 can be verified.
  • the formation of a complex of these proteins can be performed under more natural conditions. Can be verified in an environment close to For example, if the X protein 11 or the Y protein 21 is a plant-derived protein, introducing the recombinant expression vector into a plant cell makes it possible to obtain a more natural in vivo environment than when introducing the animal cell. Protein complex formation can be verified under conditions close to.
  • the recombinant expression vector only needs to be able to be eventually introduced into cells. That is, the recombinant expression vector may be introduced into tissues, organs, or individuals (plants, animals, etc.) of various organisms, not only directly into isolated single cells. In this case, the recombinant expression vector may be introduced into a tissue, an organ or a cell of an individual.
  • the individual into which the recombinant expression vector is introduced may be a plant or an animal! / ⁇ . If the individual is a plant, the ability to be introduced into, for example, Arabidopsis thaliana, tobacco, rice, or ⁇ -thiure is not limited thereto. If it is an animal, the force that can be introduced into mice, rats, Drosophila, nematodes and the like is not limited to these.
  • any gene transfer method may be used. Such methods include a particle gun method, a protoplast Z-spheroidal plast method, an aglobatery method, an electoral poration method (electroporation method), and a phosphorus method. There are calcium acid method, ribosome, DEAE dextran method, etc., but these are not limited.
  • markers may be used to confirm whether the recombinant expression vector has been introduced into cells and that the gene has been reliably expressed in the cells into which the recombinant expression vector has been introduced, and whether or not the cells have been cultivated.
  • a gene deleted in the introduced cell is used as a marker, and a plasmid or the like containing the marker and the gene of the present invention is introduced into the cell as an expression vector. This makes it possible to confirm whether or not the gene of the present invention has been introduced, using the expression of the marker gene as an index.
  • a gene for visualizing and monitoring the expression site in the transformant can also be introduced into the recombinant expression vector.
  • One example of such a gene is the ⁇ -glucurodase (GUS) gene, which is not limited to this gene.
  • any method including a known method may be used. Specifically, the expression level of the protein encoded by the reporter gene 31 or the activity thereof may be measured.
  • the reporter gene 31 when the reporter gene 31 is a CAT gene, the enzyme activity of the CAT protein is measured.
  • the reporter gene 31 is a luciferase gene, the luminescence of luciferin caused by the luciferase protein is measured.
  • the transcript of the reporter gene 31 when the transcript of the reporter gene 31 has a property that causes a significant change in the morphology of a plant or an animal, the transcript may be used as a protein synthesis system in a plant or an animal (cells containing the same). Is used, the transcription activity of the reporter gene 31 can be measured using the degree of such morphological change as an index. (II) Protein complex detection kit
  • the protein complex detection kit (the kit) according to the present invention, first, a kit for performing the above-described protein complex detection method can be mentioned.
  • this kit produces chimeric protein 10 (first chimeric protein) in which DBD12 (DNA binding peptide) that specifically recognizes the promoter sequence of reporter gene 31 and X protein 11 (first protein) are linked.
  • a chimeric protein 20a (second chimeric protein) in which a first chimeric gene expression vector to be enabled is combined with a functional peptide 22 that converts an arbitrary transcription factor into a transcription repressor and a Y protein 21 (second protein)
  • a second chimeric gene expression vector capable of producing E. coli.
  • the first chimeric gene expression vector comprises at least (a) a DBD segment that is a polynucleotide encoding DBD21; and (b) at least one type of restriction vector adjacent to the DBD segment.
  • a first RE recognition segment which is a polynucleotide having a nucleotide sequence recognized by an enzyme (RE)
  • the second chimeric gene expression vector has at least (c) a functional peptide 22 A functional peptide segment which is a polynucleotide to be encoded; and (d) a second RE recognition segment which is a polynucleotide adjacent to the functional peptide segment and which has a nucleotide sequence recognized by at least one type of restriction enzyme (RE).
  • the first chimeric gene expression vector contains the gene encoding X protein 11 in the (b) first RE recognition segment.
  • the chimeric protein 10 can be produced, and the second chimeric gene expression vector is obtained by incorporating the gene encoding Y protein 21 into the (d) second RE recognition segment, thereby obtaining the chimeric protein 10. It is preferable to be able to produce 20a.
  • the present kit preferably contains a reporter gene expression vector that expresses reporter gene 31.
  • the present kit can be used for cells in which the reporter gene 31 is not present.
  • the kit includes (a) a group of reagents for introducing an expression vector into a plant cell, and (b) an X protein 11 in the first or second chimeric gene expression vector. Gene or gene encoding Y protein 21 It is preferable that at least one reagent group is included among the reagent group and (c) a reagent group for confirming a change in the expression level of the reporter gene 31.
  • the expression vector can be reliably introduced into plant cells, the gene can be reliably incorporated into the expression vector, or the change in the expression level of reporter gene 31 can be suppressed. It can be surely confirmed.
  • the “reagent group” mentioned here may include instruments and the like used for a series of operations in addition to general reagents such as enzymes and salts.
  • a functional peptide, a polynucleotide encoding a functional peptide, a protein synthesis system, a recombinant expression vector, a reporter gene, a transformant, a promoter, and a terminator that can be used in the present kit are described in the present invention. This is the same as the protein complex detection method according to the above.
  • PBI221 plasmid (Clontech) was digested with restriction enzymes Xhol and Sad. After the digest was subjected to blunt-end treatment with T4 polymerase, agarose gel electrophoresis was performed to remove the GUS gene from the digest.
  • CaMV35S cauliflower mosaic virus 35S
  • NOS terminator 1 the transcription termination region of the nopaline synthase gene
  • the PAS2-1 vector (Clontech) was digested with Hindlll restriction enzyme. From this digest, agarose gel electrophoresis was performed to isolate a 748 bp DNA fragment (hereinafter abbreviated as GAL4DB) encoding the DNA-binding region (111 amino acid residues) of the GAL4 protein.
  • GAL4DB 748 bp DNA fragment
  • the isolated GAL4DB was subjected to blunt-end treatment with T4 DNA polymerase.
  • the DNA fragment containing the GAL4DB coding region was inserted into the blunt-ended portion between CaMV35S and Noster in p35S-NOS. From the thus obtained vectors, those in which the open reading frames of the DNA binding region of the GAL4 protein were aligned in the forward direction with respect to CaMV35S were selected, and p35S-GAL4DBD was constructed.
  • the phosphorylated 5-terminal upper primer in the region corresponding to amino acid sequence 277-315 (SEQ ID NO: 134) of the protein encoded by the human JUN gene, designed to match the reading frame of GAL4DBD (SEQ ID NO: 135) and a three-terminal lower primer (SEQ ID NO: 136) having a site recognized by the restriction enzyme Sail were synthesized in a ligatory manner.
  • the amino acid sequence of the JUN protein corresponds to amino acids 277-315.
  • the coding region to be amplified was amplified by the PCR method to obtain a DNA fragment. At this time, denaturation reaction was performed at 95 ° C for 1 minute, annealing was performed at 58 ° C for 30 seconds, and extension reaction was performed at 74 ° C for 20 seconds. A series of these reactions was defined as one cycle, and a total of 30 cycles were performed to amplify DNA.
  • the DNA fragment thus obtained was digested with the restriction enzyme Sail.
  • the target DNA fragment was isolated from the obtained digest by agarose gel electrophoresis. This DNA fragment encoding JUN was digested with the restriction enzymes Smal and Sail in advance! And incorporated into p35S-GAL4DBD to construct an effector plasmid pGAL-JUN.
  • a chimeric protein containing the amino acid sequence 133-215 of the FOS protein interacting with JUN, and having the repression domain SRDX (functional peptide of the present invention, SEQ ID NO: 140) linked to the C-terminus The 5′-terminal upper primer (SEQ ID NO: 141) and the 3′-terminal lower primer (SEQ ID NO: 142) required to obtain DNA corresponding to the nucleotide sequence encoding the amino acid sequence of FIG.
  • a chimeric DNA corresponding to the nucleotide sequence encoding the amino acid sequence in which the above-mentioned SRDX was linked to the amino acid sequence 133 to 215 of the FOS protein was amplified by PCR, and the DNA fragment was amplified. Obtained. At this time, denaturation reaction was performed at 95 ° C for 1 minute, annealing was performed at 58 ° C for 30 seconds, and extension reaction was performed at 74 ° C for 30 seconds. One of these A series of reactions was defined as one cycle, and a total of 30 cycles were performed to amplify DNA fragments.
  • the DNA fragment thus obtained was digested with the restriction enzyme Sail, and then subjected to agarose gel electrophoresis to isolate the target DNA fragment from the obtained digest.
  • the target DNA fragment thus obtained was incorporated into p35S-NLS-NOS which had been digested with the restriction enzymes Smal and Sail in advance to construct an eflator plasmid pNLS-FOS-SRDX.
  • DNA corresponding to the nucleotide sequence encoding the amino acid sequence of the protein including amino acid sequence 133-215 of the FOS protein interacting with JUN
  • 5 'terminal upper primer (sequence) No. 141) and the lower primer at the 3 ′ end (SEQ ID NO: 143) were each chemically synthesized.
  • FOS-DNA was amplified by PCR to obtain a DNA fragment.
  • denaturation reaction was performed at 94 ° C for 1 minute
  • annealing was performed at 58 ° C for 30 seconds
  • extension reaction was performed at 74 ° C for 30 seconds.
  • a series of these reactions was defined as one cycle, and a total of 30 cycles were performed to amplify DNA fragments.
  • the DNA fragment thus obtained was digested with the restriction enzyme Sail, and then subjected to agarose gel electrophoresis to isolate the target DNA fragment from the digest.
  • the target DNA fragment thus obtained was digested with the restriction enzymes Smal and Sail, and then incorporated into p35S-NLS-NOS to construct an effector plasmid pNLS-FOS.
  • the pBI221 plasmid (Clontech) was digested with restriction enzymes EcoRI and Sstl, and a 270 bp DNA fragment containing Nos-ter was isolated from this digest by agarose gel electrophoresis. Then, the isolated DNA fragment was inserted into the EcoRI-Sstl site of the plasmid pUC18 that had been digested with the restriction enzymes EcoRI and Sstl.
  • DNA1 SEQ ID NO: 1434
  • DNA2 SEQ ID NO: 145) of complementary chains containing the TATA-Box of CaMV35S were synthesized.
  • the synthesized DNA was heated at 90 ° C for 2 minutes, and further heated at 60 ° C for 1 hour. Then, it was allowed to stand at room temperature (25 ° C.) for 2 hours for annealing to form a double strand.
  • the pUC18 plasmid having Nos-ter was digested with restriction enzymes Hindlll and BamHI. This The synthesized double-stranded DNA was inserted into Hindlll-BamHI site of pUC18 to construct a plasmid containing TATA-Box and Nos-ter. This plasmid was digested with the restriction enzyme Sstl, and blunt-ended with T4DNA polymerase.
  • a plasmid vector pGV-CS2 (trade name, manufactured by Toyo Ink Co., Ltd.) having a firefly luciferase gene (LUC) was digested with restriction enzymes Xbal and Ncol. This digest was subjected to blunt-end treatment with T4 DNA polymerase, and then a 1.65 kb DNA fragment containing the luciferase gene was isolated and purified from the digest by agarose gel electrophoresis. The purified DNA fragment was inserted into a plasmid containing the above TATA-Box and Nos-ter to construct a pTATA-LUC reporter gene.
  • plasmid pG5CAT (trade name, manufactured by Clontech) having 5 copies of the DNA binding sequence of yeast GAL4 protein was digested with restriction enzymes Smal and Xbal. The digest was subjected to blunt-end treatment with T4 DNA polymerase, and a DNA fragment containing 5 copies of the DNA binding sequence of GAL4 protein was isolated and purified from the digest by agarose gel electrophoresis. Furthermore, the pTATA-LUC vector was digested with the restriction enzyme Bglll, and the resulting digest was subjected to blunt-end treatment with T4 DNA polymerase. At this site, a DNA fragment containing the DNA binding sequence of 5 copies of the GAL4 protein with blunt ends was inserted. Among the thus obtained plasmids, those having the DNA binding sequence of the GAL4 protein oriented in the forward direction were selected, and a reporter gene pGAL4-LUC was constructed.
  • PCR was performed using plasmid PBI121 as a type III to obtain a DNA fragment containing the -80046th region of the CaMV35S nucleotide sequence.
  • the 5, terminal upper primer and 3 'terminal lower primer used in the PCR at this time are as shown in SEQ ID NOs: 146 and 147, respectively.
  • a reporter gene and an effector plasmid were introduced into Arabidopsis thaliana by a particle gun method, and the effect of the effector was examined by measuring the activity of the reporter gene.
  • PGAL4-LUC reporter gene 1.2 ⁇ g of total effector plasmid DNA and 0.4 ⁇ g of reference gene plasmid coated on 510 g of 1 ⁇ m diameter gold particles (Bio-Rad) did.
  • Arabidopsis thirty four leaves on the 21st day of growth were arranged in a 9 cm petri dish with filter paper moistened with water, and DNA was applied to the leaves using a Bio-Rad PDS-100 OZHe bombardment machine. .
  • the suspension was suspended in Passive Lysis Buffer 200 / zl attached to Dua LuciferaseTM Reporter Assay System (Promega), and then centrifuged to collect the supernatant.
  • This cell extract 201 is mixed with 100 ⁇ l of the measurement buffer attached to the Dua LuciferaseTM Reporter Assay System (Promega), and the luciferase activity is measured using a luminometer (TD20 / 20, Tunerer Design).
  • Firefly 'Luciferase' and 'Michitake' luciferase activities were measured by intensifying luminescence for 10 seconds in integration mode according to the instructions of the assay kit.
  • the activity value of the reference gene was divided by the activity value of the reporter gene, and the relative value, Relative lucifarase activity, was determined as a measured value.
  • Figure 3 shows the results of reporter gene activity for each type of effector gene introduced into Arabidopsis cells.
  • CONTOL shows the results when the effector plasmid was not introduced, and the others show the results when the effector genes shown in the figure were introduced.
  • FOS protein the polypeptide corresponding to the amino acid sequence 133-215 of the FOS protein
  • JUN protein the polypeptide corresponding to the amino acid sequence 277-315 of the JUN protein
  • protein Abbreviated as protein.
  • GAL4DB-JUN was introduced into Arabidopsis leaves simultaneously with p35S-GAL4-LUC containing the reporter gene, and a chimeric protein (GAL4-JUN) linked to GAL4 ⁇ JUN was expressed.
  • the luciferase activity of the reporter gene was 105 + -10, which was not much different from the control.
  • NLS-FOS-SRDX was introduced into Arabidopsis thaliana leaves, and a chimeric protein (F OS-) in which FOS and SRDX (functional peptide 22) were linked together
  • F OS- chimeric protein
  • SRDX functional peptide 22
  • the protein complex detection method of the present invention can be used to detect the formation of a complex between two proteins ⁇ JUN and FOS.
  • Example 2 the construction of a recombinant expression vector and the measurement of reporter gene activity
  • the same method as in Example 1 was used except that the protein linking GAL4 was FOS instead of JUN, and the protein linking SRDX was JUN instead of FOS.
  • Figure 4 shows the measurement results of the activity of the reporter gene for each type of effector gene introduced into Arabidopsis thaliana cells.
  • CONTOL shows the results when the plasmid was not introduced, and the other results show the results when the effector gene shown in the figure was introduced, respectively, as in FIG. 3 in Example 1. .
  • NLS-JUN-SRDX was introduced into Arabidopsis thaliana simultaneously with p35S-GAL4-LUC, and a chimeric protein (JUN) in which the JUN protein was linked to SRDX (functional peptide 22) was obtained.
  • JUN chimeric protein
  • SRDX functional peptide 22
  • PI and AP3 which are proteins derived from Arabidopsis thaliana and which are known to interact with each other, were used as X protein 11 and Y protein 21.
  • the entire amino acid sequence of PI is shown in SEQ ID NO: 153, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 154.
  • the entire amino acid sequence of AP3 is shown in SEQ ID NO: 155, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 156.
  • the same method as in Example 1 was used for construction of the recombinant expression vector and measurement of reporter gene activity.
  • 35S-GAL4-PI the 5-terminal upper primer shown in SEQ ID NO: 157 and the 3-terminal lower primer shown in SEQ ID NO: 158 were used.
  • NLS-AP3 a 5-terminal upper primer shown in SEQ ID NO: 159 and a 3-terminal lower primer shown in SEQ ID NO: 160 were used.
  • NLS-AP3-SRDX a 5-terminal upper primer shown in SEQ ID NO: 159 and a 3-terminal lower primer shown in SEQ ID NO: 161 were used.
  • Fig. 5 shows the measurement results of the activity of the reporter gene for each type of effector gene introduced into Arabidopsis thaliana cells.
  • CONTOL shows the results when the plasmid was not introduced, and the rest shows the results when the effector gene shown in the figure was introduced, respectively, as in FIG. 1 in Example 1. .
  • GAL4DB-PI was introduced into Arabidopsis thaliana leaves to express a chimeric protein (GAL4-P) in which GAL4 and PI were linked.
  • GAL4-P chimeric protein
  • NLS-AP3-SRDX was introduced into Arabidopsis thaliana at the same time as p35S-GAL4-LUC, and a chimeric protein (AP3-protein) in which the AP3 protein and SRDX (functional peptide 22) were linked together.
  • SRDX a chimeric protein
  • the luciferase activity of the reporter gene was 93.8 + —34.9, which was not much different from the control.
  • GAL4DB-PI and NLS-AP3 were introduced into Arabidopsis thaliana simultaneously with p35S-GAL4-LUC, GAL4-PI and AP3 protein were expressed simultaneously.
  • the luciferase activity was 89.3 + -25.1, which was not much different from the control.
  • the protein complex detection method of the present invention can be used to detect the formation of a complex between two proteins, PI and AP3.
  • Example 4 a 12 amino acid peptide LDLDLELRLGFA (SRDX) (SEQ ID NO: 140), which is one of the transcription repressor converting peptides, is located between the cauliflower mosaic virus 35S promoter and the transcription termination region of the nopaline synthase gene. And a polynucleotide encoding SRDXm, which is a mutant peptide obtained by converting leucine of SRDX, into a recombinant expression vector incorporating a polynucleotide linked downstream of the TTG1 gene. The Arabidopsis thaliana was transformed by introducing the DNA into the E. coli using the Agrobacterium terminus method.
  • SRDX 12 amino acid peptide LDLDLELRLGFA
  • the TTG1 protein is a protein classified into a group called WD40, and has been reported to bind to a transcription factor.
  • mutants of the TTG1 gene suppress trichome development, root hair hyperplasia (generation and differentiation of epidermal cells), control tannin synthesis, suppress anthocyanin biosynthesis, and inhibit the formation of cormela on the seed surface. It is reported to appear.
  • P35SG a vector for transformation vector construction, was constructed according to the following steps (1)-(4) as shown in FIG.
  • AttLl and attL2 regions on the pENTR vector manufactured by Invitrogen were used as primers attLl-F (SEQ ID NO: 162), attLl-R (SEQ ID NO: 163), attL2-F (SEQ ID NO: 164), attL2 Amplified by PCR using —R (SEQ ID NO: 165).
  • the obtained attLl fragment was digested with the restriction enzyme HindIII and the attL2 fragment with EcoRI and purified.
  • the conditions of the PCR reaction were 25 cycles, with the denaturation reaction at 94 ° C for 1 minute, the anneal reaction at 47 ° C for 2 minutes, and the extension reaction at 74 ° C for 1 minute.
  • all PCR reactions were performed under the same conditions.
  • a DNA fragment having the following SEQ ID NOs: 166 and 167 was synthesized, heated at 90 ° C. for 2 minutes, heated at 60 ° C. for 1 hour, and then at room temperature (25 ° C.) The mixture was allowed to stand for 2 hours and allowed to elute to form a double strand. This was ligated to the Xbal-Sacl region of the 35S-Nos plasmid fragment DNA to complete the p35S-Nos plasmid.
  • the DNA fragment having the sequence of SEQ ID NOs: 166 and 167 contains a BamHI restriction enzyme site at the 5 'end, an omega sequence derived from tobacco mosaic virus for increasing translation efficiency, and restriction enzyme sites Smal, Sail, and Sstl in this order.
  • P35SSRDXG which is a construction vector incorporating a polynucleotide encoding a transcription repressor converting peptide, was constructed according to the following steps (1)-(2).
  • pBIGCKH a plant transformation vector having two att sites for recombination with a DNA fragment flanked by att sites in the construction vector, was subjected to the following steps (1) to (3). ).
  • Res. 18: 203, 1990 was digested with restriction enzymes 1-11 (1111, EcoRI), and the GUS and Nos regions were removed by electrophoresis.
  • PBIGCKH was constructed. They can grow only in Escherichia coli DB3.1 (Invitrogen) and are resistant to chloramphenol and kanamycin.
  • Arabidopsis-derived transcription factor TTG1 protein was added to the above-mentioned construction vector P35SSRDXG.
  • the polynucleotide encoding the protein was incorporated according to the following steps (1)-(3).
  • the obtained DNA fragment of the TTG1 coding region was ligated to the Smal site of the construction vector p35SSRDXG that had been digested with the restriction enzyme Smal in advance.
  • TTG1SRDXm which is a chimeric protein between TTG1 and SRDXm
  • TTGlSRDXm contains the coding region for TTG1.
  • the amino acid sequence encoded by the TTGlSRDXm polynucleotide is shown in SEQ ID NO: 175.
  • a DNA fragment containing the CaMV35S promoter, chimeric gene, Noster and the like on the above-mentioned construction vector was recombined into a plant transformation vector pBIGCKH to construct an expression vector using the plant as a host.
  • the recombination reaction was carried out using Gateway (registered trademark) LR clonase (registered trademark) manufactured by Invitrogen as described in the following steps (1)-(3).
  • TTG1 was incorporated into the above-mentioned construction vector or the above-mentioned TTG ISRDXm construct 1.
  • 4.0 ⁇ L of LR buffer diluted 5-fold in pBIGCKH4.L (about 600 ng) were prepared.
  • ⁇ Buffer solution (10 mM TrisCl pH 7.0, ImM EDTA) was added.
  • FIG. 8 (a) shows the seeds of wild-type Arabidopsis thaliana, and it was brown because tannin was synthesized.
  • Fig. 8 (b) shows Arabidopsis seeds expressing TTG1SRDX.Since the production of the chimeric gene caused a mutation in TTG1, tannin synthesis was suppressed and the seeds appeared pale yellow. did.
  • Figure 8 (c) shows that TTGlSRDXm is expressed. Of the Arabidopsis thaliana, and the seeds were dark brown like the wild type.
  • FIG. 9 (a) shows leaves of wild-type Arabidopsis thaliana, in which trichomes are formed.
  • FIG. 9 (b) shows leaves of Arabidopsis thaliana in which TTG1SRDX was expressed! /, And no trichome was formed.
  • FIG. 9 (c) shows Arabidopsis thaliana leaves expressing TTGlSRDXm, and trichomes were formed in the leaves as in the wild type.
  • FIG. 10 (a) shows a plant of a wild-type Arabidopsis thaliana, in which accumulation of anthocyanin was observed.
  • FIG. 10 (b) shows an Arabidopsis thaliana plant expressing TTG1SRDX, and the accumulation of anthocyanin was suppressed.
  • FIG. 10 (c) shows an Arabidopsis thaliana plant expressing TTG1SR DXm, and accumulation of anthocyanin was observed as in the wild type.
  • FIG. 11 (a) shows the roots of wild-type Arabidopsis.
  • FIG. 11 (b) shows the roots of Arabidopsis thaliana expressing TTG1SR DX, and excessive root hair formation was observed.
  • FIG. 11 (c) shows the roots of Arabidopsis thaliana expressing TTGlSRDXm, and the amount of root hair was similar to that of the wild type.
  • FIG. 12 (a) shows seeds of wild-type Arabidopsis thaliana, and a colmera was formed on the seed surface.
  • FIG. 12 (b) shows Arabidopsis seeds expressing TTG1SRDX, and no cormella formation was observed.
  • FIG. 12 (c) shows Arabidopsis seeds expressing TTGlSRDXm, and the formation of cormella was observed as in the wild type.
  • the present invention relates to a second chimeric protein encoding a second chimeric gene, wherein a functional peptide that converts an arbitrary transcription factor into a transcription repressor is bound to the second protein.
  • a functional peptide that converts an arbitrary transcription factor into a transcription repressor is bound to the second protein.
  • the present invention has an effect when it is possible to detect the formation of a complex of a wide range of proteins including, in addition to a general protein, a transcription factor or a protein capable of binding to the transcription factor.
  • the protein complex detection method and kit according to the present invention can be used not only for transcription factors or proteins having the property of binding to transcription factors, but also for complex formation in a wide range of proteins of various organisms including these. Because it can be detected, it can be used, for example, in the pharmaceutical industry.

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Abstract

Il est projeté de fournir une méthode de détection d’un complexe protéique, où la formation d’un complexe d’un facteur transcriptionnel ou d’une protéine capable de se lier à un facteur transcriptionnel avec une autre protéine peut être détecté, et une trousse pour détecter un complexe protéique. Dans un système de synthèse protéique où une protéine peut être produite par expression génétique, une protéine chimérique (10) comprenant un DBD (12) et une protéine X (11) qui lui est liée et une autre protéine chimérique (20a) comprenant un peptide fonctionnel (22), qui convertit un facteur transcriptionnel arbitraire en un répresseur transcriptionnel, liée à une protéine Y (21), sont produites. Dans le cas où la protéine X (11) et la protéine Y (21) forment ensemble un complexe, un complexe (40) est ainsi formé. Ce complexe (40) refoule la transcription d’un gène rapporteur (31) en raison de l’effet répressif transcriptionnel du peptide fonctionnel (22). En confirmant cette répression transcriptionnelle, la formation du complexe protéique par la protéine X (11) et la protéine Y (21) est détectée.
PCT/JP2005/003797 2004-03-05 2005-03-04 Méthode de détection d’un complexe protéique et trousse de détection d’un complexe protéique WO2005085467A1 (fr)

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US8847012B2 (en) 2007-12-05 2014-09-30 Toyota Jidosha Kabushiki Kaisha Genes that increase plant oil and method for using the same
US9045786B2 (en) 2008-03-04 2015-06-02 Toyota Jidosha Kabushiki Kaisha Gene that increases production of plant fat-and-oil and method for using the same
US9169488B2 (en) 2009-06-04 2015-10-27 Toyota Jidosha Kabushiki Kaisha Gene capable of improving material productivity in seed and method for use thereof
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US9169488B2 (en) 2009-06-04 2015-10-27 Toyota Jidosha Kabushiki Kaisha Gene capable of improving material productivity in seed and method for use thereof

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