JP2006042729A - Method for producing double-petaled plant, plant obtained by using the method and use of the plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily and surely producing a double-petaled plant in a short term, by converting a transcription factor contributing to pistil formation into a transcription suppressing factor and suppressing the transcription of a target gene of the transcription factor. <P>SOLUTION: A chimeric protein obtained by fusing the transcription factor with a functional peptide is produced in plant cells by introducing into the plant cells a chimeric gene of a polynucleotide encoding the transcription factor contributing to pistil formation and a polynucleotide encoding a functional peptide converting the optional transcription factor into the transcription suppressing factor. The chimeric protein suppresses expression of gene targeting the transcription factor to produce double-petaled plant. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing double-flowered plants, a plant obtained using the same, and use thereof, and in particular, a method for producing double-flowered plants by suppressing gene transcription and obtained using the same. The present invention relates to a plant body and its use.

  Generally, it consists of four organs: flower postcards, petals, stamens and pistils. When the flower bud meristem is divided into four concentric regions, that is, from one to four, the four ones in whol1 and the two in one Four petals, six stamens for whol3, and two pistils with two heart skins fused to whol4. It is known that the traits of these organs are determined by floral homeotic genes (Non-patent Document 1). Flower homeotic genes are classified into three classes, A, B, and C, and encode transcription factors. Therefore, the combination of these gene groups determines the type of flower organ formed in the flower bud meristem. That is, only class A works, but petals are formed when classes A and B cooperate, stamens form when classes B and C cooperate, and pistils form when only class C works. Therefore, when the function of a homeotic gene is lost due to mutation or the like, one organ is transformed into a trait of another organ. Such a change is called homeotic conversion, and in plants, homeotic genes involved in determination of floral organ traits have been clarified in Arabidopsis (Non-patent Document 1).

  The agamous gene (hereinafter referred to as “AG gene”) is the above-mentioned class C gene. In the AG mutant in which the function of the AG gene is lost, the function of class A extends to the entire region of the flower and the stamens to the petal. It is known that in the wild type, a new flower is formed in a region that is a pistil, and the form of the flower is double bloom (Non-patent Document 1).

  Until now, when the shape of a flower is changed for the purpose of producing a double blooming plant body, it is common to carry out cross breeding by crossing varieties of plants having the target character.

  In recent years, RNA interference (RNAi) has been widely used as a method for suppressing the action of a specific gene by putting double-stranded RNA into a cell and degrading cellular mRNA that is homologous to the sequence. It is coming.

  By the way, the present inventor has found various peptides that convert an arbitrary transcription factor into a transcriptional repression factor (see, for example, Patent Documents 1 to 7, Non-Patent Documents 2 and 3). This peptide is excised from Class II ERF (Ethyrene Responsive Element Binding Factor) protein and plant zinc finger protein (Zinc Finger Protein, such as Arabidopsis SUPERMAN protein, etc.) and has a very simple structure.

Furthermore, the present inventor has attempted to introduce a gene encoding a fusion protein (chimeric protein) in which various transcription factors are fused with the above peptide into a plant body. As a result, the transcription factor is converted into a transcription repressing factor, and the transcription factor has succeeded in producing a plant in which the expression of a target gene that promotes transcription is suppressed.
JP 2001-269177 A (released on October 2, 2001) JP 2001-269178 A (published on October 2, 2001) JP 2001-292776 A (published on October 2, 2001) JP 2001-292777 A (released on October 23, 2001) JP 2001-269176 A (released on October 2, 2001) JP 2001-269179 A (published October 2, 2001) International Publication No. WO03 / 055903 pamphlet (published July 10, 2003) Sakai, “Molecular mechanisms that determine plant shape”, Shujunsha, pages 150-155 Ohta, M .; , Matsui, K .; , Hiratsu, K .; , Shinshi, H .; and Ohme-Takagi, M .; , The Plant Cell, Vol. 13, 1959-1968, August, 2001. Hiratsu, K .; Ohta, M .; , Matsui, K .; Ohme-Takagi, M .; , FEBS Letters 514 (2002) 351-354.

  However, in the conventional method of cross breeding, in order to produce a plant having the target character, a long period of time and experience of an expert are required. Therefore, it is very difficult to obtain a double blooming plant easily and reliably in a short period of time.

  In addition, it is conceivable to suppress the function of the AG gene by the above RNAi method to obtain a double blooming plant body. However, the RNAi method is difficult to determine a target site for suppressing gene expression. There are various problems such as the need for mistakes, the difficulty in constructing constructs, the low transfection efficiency of some cells, and the limited effects of RNA interference. Therefore, it is very difficult to obtain a double-flowered plant body easily and reliably in a short period of time by the RNAi method.

  The present invention has been made in view of the above-mentioned problems, and its purpose is to convert a transcription factor involved in pistil formation into a transcription repressor and to suppress transcription of a target gene of the transcription factor. Another object of the present invention is to provide a method for producing a double bloom plant in a short period of time with ease and reliability.

  As a result of diligent studies to solve the above problems, the present inventor converted a transcription factor involved in pistil formation into a transcriptional repressing factor, and repressed transcription of the target gene of the transcription factor, thereby producing a double blooming plant. It was revealed for the first time that the body can be produced easily and reliably in a short period of time, and the present invention has been completed.

  That is, the method for producing a double bloom plant according to the present invention produces a chimeric protein in which a transcription factor involved in pistil formation and a functional peptide that converts any transcription factor into a transcription repressor is fused in the plant. It is characterized by making the flower form into double bloom.

  According to the said structure, the said transcription factor can be converted into a transcriptional repression factor, and transcription | transfer of the target gene of the said transcription factor is suppressed in a plant body. In addition, since the trait that suppresses transcription of the target gene of the transcription factor is dominant, it is not necessary to isolate an inferior homo individual as in the AG mutant prepared by the conventional technique. Therefore, a double-flowered plant can be produced easily and reliably in a short period of time.

  Moreover, according to the said structure, the double bloom plant body obtained turns into a sterile plant body in which a seed is not formed. Therefore, the target plant can be made into a sterile plant very easily without using a complicated gene recombination technique.

  In addition, the method for producing a double-flowered plant according to the present invention comprises a trait for introducing a recombinant expression vector containing a chimeric gene comprising a gene encoding the transcription factor and a polynucleotide encoding the functional peptide into a plant cell. It preferably includes a conversion step.

  According to the above configuration, the chimeric protein can be expressed in a plant cell simply by incorporating the transcription factor gene into a cassette vector to which the functional peptide is added and introducing the gene into the plant cell. Thus, transcription of a transcription factor target gene can be easily suppressed. Therefore, a double bloom plant can be produced easily and reliably in a short time.

  Moreover, it is preferable that the production method of the double bloom plant according to the present invention further includes an expression vector construction step of constructing the above recombinant expression vector.

  The transcription factor is preferably a protein described in the following (a) or (b). (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 134; (B) A protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 134.

Moreover, it is preferable to use the gene of the following (c) or (d) as a gene which codes the said transcription factor.
(C) A gene having the base sequence represented by SEQ ID NO: 135 as an open reading frame region.
(D) A gene that hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 135 under stringent conditions and encodes a protein involved in pistil formation.

  According to the said structure, the said transcription factor is converted into a transcriptional repression factor by the said functional peptide, and transcription | transfer of the target gene of the said transcription factor is repressed. Therefore, a double bloom plant can be produced easily and reliably in a short time.

In addition, the functional peptide has the following formulas (1) to (4).
(1) X1-Leu-Asp-Leu-X2-Leu-X3
(In the formula, X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Wherein, Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)
(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Wherein, Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)
(4) Asp-Leu-Z4-Leu-Arg-Leu
(However, in the formula, Z4 represents Glu, Gln or Asp.)
It is preferable that it has the amino acid sequence represented by either.

  Moreover, it is preferable that the said functional peptide is a peptide which has an amino acid sequence shown in either of sequence number 1-17.

  The functional peptide may be a peptide described in the following (e) or (f). (E) A peptide having the amino acid sequence shown in SEQ ID NO: 18 or 19. (F) A peptide having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 18 or 19.

The functional peptide is represented by the following formula (5):
(5) α1-Leu-β1-Leu-γ1-Leu
(Wherein α1 represents Asp, Asn, Glu, Gln, Thr or Ser, β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, and γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys, or Asp are shown.)
It may have an amino acid sequence represented by

Moreover, the functional peptide is represented by the following formulas (6) to (8).
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(In the formula, α1 represents Asp, Asn, Glu, Gln, Thr or Ser, α2 represents Asn, Glu, Gln, Thr or Ser, and β1 represents Asp, Gln, Asn, Arg, Glu. , Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, and γ2 represents Gln, Asn, Thr, Ser, His, Lys or Asp.)
It may have an amino acid sequence represented by any of the above.

  In addition, the functional peptide is SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, or 40. A peptide having the amino acid sequence shown in FIG.

  The functional peptide may be a peptide having the amino acid sequence shown in SEQ ID NO: 36 or 37.

  The functional peptide is a peptide having an amino acid sequence represented by any of the above formulas or any of the peptides represented by the above SEQ ID NOs, and most of them are extremely short peptides, so that synthesis is easy, Transcriptional repression of the target gene of the transcription factor can be performed efficiently. The functional peptide has a function of suppressing the expression of a target gene in preference to the activity of other transcription factors that are functionally redundant (redundant). Therefore, there is an effect that the expression of the target gene can be effectively suppressed.

  Moreover, the plant body concerning this invention is the double bloom plant body produced by the said production method, It is characterized by the above-mentioned. The plant body preferably contains at least one of a grown plant individual, a plant cell, a plant tissue, a callus, and a seed.

  A kit for producing double-flowered plants according to the present invention is a kit for performing the production method described above, and has a functionality for converting a polynucleotide encoding the transcription factor and an arbitrary transcription factor into a transcriptional repression factor. It is characterized by comprising at least a recombinant expression vector comprising a polynucleotide encoding a peptide and a promoter. The production kit for the double bloom plant may further include a reagent group for introducing the recombinant expression vector into a plant cell.

  Further, the method for producing a sterile plant according to the present invention comprises a chimera in which a transcription factor involved in the formation of a sterile plant is fused with a functional peptide that converts any transcription factor into a transcriptional repressor. It is characterized in that the plant body is sterilized by producing the protein in the plant body.

  According to the said structure, the obtained plant body becomes a sterile plant body in which a seed is not formed. Therefore, the target plant can be made into a sterile plant very easily without using a complicated gene recombination technique.

  As described above, the production method of the double-flowered plant according to the present invention includes a chimeric protein obtained by fusing a transcription factor involved in pistil formation and a functional peptide that converts an arbitrary transcription factor into a transcription repressor. It is produced in the body, and has the structure of producing the double-bloomed plant by suppressing the transcription of the target gene of the transcription factor, so that the double-bloomed plant can be produced easily and reliably in a short period of time. There is an effect.

Hereinafter, the production method of the double blooming plant according to the present invention, the plant obtained using the same, and the use thereof will be described in detail.
(1) Production method of double blooming plant The present invention is a technique for producing double blooming plant, comprising a transcription factor involved in pistil formation and a functional peptide that converts any transcription factor into a transcriptional repressor. By producing the fused chimeric protein in a plant body, a double bloom plant body is produced.

  In the method for producing the chimeric protein, the transcription factor is converted into a transcription repressor by the functional peptide. Therefore, when the DNA binding domain derived from the transcription factor in the chimeric protein binds to the target gene of the transcription factor, transcription of the target gene of the transcription factor is suppressed. As a result, the functions of genes involved in pistil formation are lost, and the above-mentioned class A functions extend to the entire flower area, so that the stamens change into petals, and in the wild type, new flowers are created in the areas that are pistil. As a result, a double blooming plant is formed. That is, as shown in FIG. 4A, flowers are repeatedly formed in the order of petals and petals from the outside.

  Moreover, the trait that the chimeric protein suppresses transcription of the target gene of the transcription factor is dominant. That is, even when a normal gene involved in pistil formation exists, a mutant gene in which transcription of a target gene of a transcription factor is suppressed is predominantly expressed. Therefore, it is not necessary to isolate inferior homozygous individuals, for example, as in the case of a mutant prepared by conventional techniques such as mating or RNAi method.

  Therefore, it is possible to produce a double bloom plant body easily and reliably in a short period of time.

  In addition, the resulting double blooming plant body is a sterile plant body in which seeds are not formed. In addition to the completely sterile plants in which no stamens and pistils are formed, incomplete stamen-like organs and / or pistil-like organs are formed in the above-mentioned sterile plants, but the formation of seeds is suppressed Sterile plants are also included.

  No seeds are formed in the sterile plant. Moreover, in the said completely sterile plant body, the dispersion | distribution of pollen does not arise. Therefore, it is possible to prevent the genetically modified plant from spreading into the environment.

(I) Chimeric protein used in the present invention As described above, the chimeric protein used in the present invention comprises a transcription factor involved in pistil formation and a functional peptide that converts any transcription factor into a transcription repressor. It is a fusion. Therefore, each of the transcription factor and the functional peptide will be described.

(I-1) Transcription factor The transcription factor used in the present invention is not particularly limited as long as it is a transcription factor involved in pistil formation. In one embodiment, the transcription factor used in the present invention is a protein consisting of the amino acid sequence shown in SEQ ID NO: 134, or a variant of a protein consisting of the amino acid sequence shown in SEQ ID NO: 134. A protein consisting of the amino acid sequence shown in SEQ ID NO: 134 is a transcription factor encoded by the AG gene, and is involved in the formation of pistils.

  Variants include deletions, insertions, inversions, repeats, and type substitutions (eg, replacement of a hydrophilic residue with another residue, but usually a strongly hydrophilic residue to a strongly hydrophobic residue Are not substituted). In particular, “neutral” amino acid substitutions in a protein generally have little effect on the activity of the protein.

  It is well known in the art that some amino acids in the amino acid sequence of a protein can be easily modified without significantly affecting the structure or function of the protein. Furthermore, it is also well known that there are variants in natural proteins that do not significantly alter the structure or function of the protein, not only artificially modified.

  One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a protein using well-known techniques. For example, according to a known point mutation introduction method, any base of a polynucleotide encoding a protein can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a protein. Furthermore, if the method described in this specification is used, it can be easily determined whether the produced mutant has a desired activity.

  Preferred variants have conservative or non-conservative amino acid substitutions, deletions, or additions. Silent substitution, addition, and deletion are preferred, and conservative substitution is particularly preferred. These do not change the protein activity according to the invention.

  Representative conservative substitutions are substitutions of one amino acid for another in the aliphatic amino acids Ala, Val, Leu, and Ile; exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

  As detailed above, further guidance on which amino acid changes are likely to be phenotypically silent (ie likely not to have a significantly detrimental effect on function) can be found in Bowie, J. . U. “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substations”, Science 247: 1306-1310 (1990), which is incorporated herein by reference.

The transcription factor according to this embodiment is preferably a protein described in the following (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 134;
(B) A protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 134.

  In the amino acid sequence shown in the above-mentioned “amino acid sequence described in (i), wherein one or several amino acids are substituted, deleted, inserted, and / or added” and “SEQ ID NO: 134, The range of “one or several” in the “amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added” is not particularly limited, but for example, 1 to 20, preferably 1 To 10, more preferably 1 to 7, even more preferably 1 to 5, and particularly preferably 1 to 3. As described above, such a mutant protein is not limited to a protein having a mutation artificially introduced by a known mutant protein production method, but is a protein obtained by isolating and purifying a naturally occurring protein. May be.

  The protein according to the present invention is not limited to this as long as it is a protein in which amino acids are peptide-bonded, and may be a complex protein including a structure other than a protein. As used herein, examples of the “structure other than protein” include sugar chains and isoprenoid groups, but are not particularly limited.

  The protein according to the present invention may include an additional protein. Examples of the additional protein include epitope-tagged proteins such as His, Myc, and Flag.

  By the way, floral homeotic genes have been isolated from Arabidopsis thaliana, snapdragons, etc., and are thought to function similarly in monocotyledons as well as dicotyledons. Therefore, the amino acid sequences of transcription factors involved in pistil formation, such as transcription factors encoded by the AG gene, are considered to be highly conserved among many plants of different species. Therefore, it is not always necessary to isolate a transcription factor and its gene involved in pistil formation in individual plants that form double bloom plants. That is, by introducing a chimeric protein constructed in Arabidopsis thaliana, which will be described later in Examples, into other plants, double-flowered plants can be produced in various types of plants.

  When producing the chimeric protein used in the present invention, a known gene recombination technique can be suitably used as described later. Therefore, the gene (polynucleotide) encoding the transcription factor can also be suitably used in the plant production method according to the present invention.

  The gene encoding the transcription factor can be present in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). The DNA may be double stranded or single stranded. Single-stranded DNA or RNA may be the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand).

  The gene encoding the transcription factor may further be a mutant of the gene encoding the transcription factor involved in pistil formation. Variants can occur naturally, such as a natural allelic variant. By “allelic variant” is intended one of several interchangeable forms of a gene occupying a given locus on the chromosome of an organism. Non-naturally occurring variants can be generated, for example, using mutagenesis techniques well known in the art.

  Examples of such mutants include mutants in which one or several bases have been deleted, substituted, or added in the nucleotide sequence of the polynucleotide encoding the transcription factor. Variants can be mutated in coding or non-coding regions, or both. Mutations in the coding region can generate conservative or non-conservative amino acid deletions, substitutions, or additions.

  The gene encoding the transcription factor includes a gene encoding the transcription factor or a polynucleotide that hybridizes to the gene under stringent hybridization conditions.

In one embodiment, the gene encoding the transcription factor is preferably a polynucleotide described in the following (c) or (d).
(C) A gene having the base sequence represented by SEQ ID NO: 135 as an open reading frame region.
(D) A gene that hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 135 under stringent conditions and encodes a protein involved in pistil formation.

  The above “stringent conditions” means that hybridization occurs only when at least 90% identity, preferably at least 95% identity, most preferably 97% identity exists between sequences. Means that.

  The hybridization is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. As hybridization conditions, conventionally known conditions can be preferably used, and are not particularly limited. For example, 42 ° C., 6 × SSPE, 50% formamide, 1% SDS, 100 μg / ml salmon sperm DNA, 5 × Denhardt's solution (however, 1 × SSPE; 0.18M sodium chloride, 10 mM sodium phosphate, pH 7.7, 1 mM EDTA. 5 × Denhardt's solution; 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone).

  The method for obtaining the gene encoding the transcription factor is not particularly limited, and a method for isolating and cloning a DNA fragment containing a polynucleotide encoding the transcription factor by a known technique can be used. For example, a probe that specifically hybridizes with a part of the base sequence of the gene encoding the transcription factor may be prepared, and a genomic DNA library or cDNA library may be screened. As such a probe, any probe of any sequence and / or length can be used as long as it specifically hybridizes to at least part of the base sequence of the gene encoding the transcription factor or its complementary sequence. Also good.

  Alternatively, as a method for obtaining a gene encoding the above transcription factor, a method using amplification means such as PCR can be mentioned. For example, among the cDNAs of polynucleotides encoding the transcription factors, primers are prepared from 5 ′ and 3 ′ sequences (or their complementary sequences), and genomic DNA (or cDNA) is prepared using these primers. PCR is performed using the above as a template, and a DNA region sandwiched between both primers is amplified to obtain a large amount of DNA fragments containing the polynucleotide encoding the transcription factor.

(I-2) Functional peptide that converts an arbitrary transcription factor into a transcriptional repressing factor The functional peptide used in the present invention that converts an arbitrary transcription factor into a transcriptional repressing factor (referred to as a transcriptional repressing conversion peptide for convenience of explanation) ) Is not particularly limited as long as it is a peptide that can suppress transcription of a target gene controlled by the transcription factor by forming a chimeric protein fused with the transcription factor. Specifically, for example, transcription-repressing conversion peptides found by the present inventors (see Patent Documents 1 to 7, Non-Patent Documents 2, 3, etc.) can be mentioned.

  The present inventor has found that a protein obtained by binding an AtERF3 protein, an AtERF4 protein, an AtERF7 protein, or an AtERF8 protein derived from Arabidopsis thaliana, which is one of the Class II ERF gene groups, remarkably suppresses gene transcription. Obtained. Thus, an effector plasmid containing a gene encoding each of the above proteins and DNA excised therefrom was constructed, and this was introduced into plant cells, thereby succeeding in actually suppressing the transcription of the gene (for example, Patent Documents 1 to 3). 4). Moreover, it is one of the genes encoding tobacco ERF3 protein (see, for example, Patent Document 5), rice OsERF3 protein (see, for example, Patent Document 6) which is one of the Class II ERF genes, and zinc finger protein. A similar test as described above was performed for a gene encoding a certain Arabidopsis thaliana ZAT10 and ZAT11, and it was found that transcription of the gene was suppressed. Furthermore, the present inventor has revealed that these proteins have a common motif including aspartic acid-leucine-asparagine (DLN) in the carboxyl group terminal region. As a result of examining the proteins having this common motif, the protein that suppresses gene transcription may be a peptide having a very simple structure, and the peptide having this simple structure can be used as a transcriptional repressor. It has been found that it has a function of converting to.

  Further, the present inventor has found that the Arabidopsis SUPERMAN protein has a motif that does not match the above-mentioned common motif, but has a function of converting an arbitrary transcription factor into a transcriptional repressor, and a gene encoding the SUPERMAN protein. It has also been found that a chimeric gene bound to a DNA-binding domain of a transcription factor or a gene encoding a transcription factor produces a protein having a strong transcription repressing ability.

  Therefore, as an example of the transcriptional repression converting peptide used in the present invention, in the present embodiment, the AtERF3 protein, the AtERF4 protein, the AtERF7 protein, the AtERF8 protein, the tobacco ERF3 protein, the rice ERF3 protein, the rice, which are Class II ERF proteins. Examples include OsERF3 protein, one of zinc finger proteins, Arabidopsis thaliana ZAT10 protein, ZAT11 protein and other proteins, SUPERMAN protein, peptides excised from these, synthetic peptides having the above functions, and the like.

A specific structure of an example of the transcription repressing conversion peptide is an amino acid sequence represented by any of the following formulas (1) to (4).
(1) X1-Leu-Asp-Leu-X2-Leu-X3
(In the formula, X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Wherein, Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)
(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Wherein, Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)
(4) Asp-Leu-Z4-Leu-Arg-Leu
(However, in the formula, Z4 represents Glu, Gln or Asp.)
(I-2-1) Transcriptional Repression Conversion Peptide of Formula (1) In the transcriptional repression conversion peptide of the above formula (1), the number of amino acid residues represented by X1 should be in the range of 0-10. That's fine. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by X1 is not specifically limited, What kind of thing may be sufficient. In other words, in the transcription repressor converting peptide of the above formula (1), an oligomer consisting of one arbitrary amino acid or 2 to 10 arbitrary amino acid residues may be added to the N-terminal side. However, no amino acid may be added.

  The amino acid residue represented by X1 is preferably as short as possible in view of the ease of synthesizing the transcriptional repression converting peptide of formula (1). Specifically, it is preferably 10 or less, and more preferably 5 or less.

  Similarly, in the transcription repressor converting peptide of the above formula (1), the number of amino acid residues represented by X3 may be at least 6. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by X3 is not specifically limited, What kind of thing may be sufficient. In other words, in the transcription repressor converting peptide of the above formula (1), an oligomer composed of 6 or more arbitrary amino acid residues may be added to the C-terminal side. If the amino acid residue represented by X3 is at least 6, it can exhibit the above function.

  In the transcription repressor converting peptide of the above formula (1), the specific sequences of pentamers (5mers) consisting of 5 amino acid residues excluding X1 and X3 are shown in SEQ ID NOs: 41 and 42. The amino acid sequence when X2 is Asn is the amino acid sequence shown in SEQ ID NO: 41, and the amino acid sequence when X2 is Glu is the amino acid sequence shown in SEQ ID NO: 42.

(I-2-2) Transcriptional Repression Conversion Peptide of Formula (2) The transcriptional repression conversion peptide of Formula (2) is represented by Y1 as described above for X1 of the transcriptional repression conversion peptide of Formula (1). The number of amino acid residues should just be in the range of 0-10. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by Y1 is not specifically limited, What kind of thing may be sufficient. In other words, in the transcriptional repression converting peptide of the above formula (2), as in the transcriptional repression converting peptide of the above formula (1), at the N-terminal side, one arbitrary amino acid or 2-10 arbitrary An oligomer composed of amino acid residues may be added, or no amino acid may be added.

  The amino acid residue represented by Y1 should be as short as possible in view of the ease of synthesizing the transcriptional repression converting peptide of formula (2). Specifically, it is preferably 10 or less, and more preferably 5 or less.

  Similarly, in the transcription repressor converting peptide of the above formula (2), the number of amino acid residues represented by Y3 may be at least 6 as in the case of X3 of the transcription repressing converting peptide of the above formula (1). . Moreover, the specific kind of amino acid which comprises the amino acid residue represented by Y3 is not specifically limited, What kind of thing may be sufficient. In other words, in the transcription repressor converting peptide of the above formula (2), as in the transcription repressing convertible peptide of the above formula (1), an oligomer composed of 6 or more arbitrary amino acid residues is added to the C-terminal side. It only has to be done. If the amino acid residue represented by Y3 is at least 6, the above function can be exhibited.

  In the transcription repressor converting peptide of the above formula (2), the specific sequences of pentamers (5mers) consisting of 5 amino acid residues excluding Y1 and Y3 are shown in SEQ ID NOs: 43 and 44. The amino acid sequence when Y2 is Phe is the amino acid sequence shown in SEQ ID NO: 43, and the amino acid sequence when Y2 is Ile is the amino acid sequence shown in SEQ ID NO: 44. A specific sequence of a tetramer (4mer) consisting of 4 amino acid residues excluding Y2 is shown in SEQ ID NO: 45.

(I-2-3) Transcriptional Repression Conversion Peptide of Formula (3) In the transcriptional repression conversion peptide of the above formula (3), the amino acid residue represented by Z1 has Leu within a range of 1 to 3. It is included. The case of 1 amino acid is Leu, the case of 2 amino acids is Asp-Leu, and the case of 3 amino acids is Leu-Asp-Leu.

  On the other hand, in the transcriptional repression converting peptide of the above formula (3), the number of amino acid residues represented by the above Z3 is in the range of 0 to 10 like X1 of the transcriptional repression converting peptide of the above formula (1). If it is. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by Z3 is not specifically limited, What kind of thing may be sufficient. In other words, in the transcription repressor converting peptide of the above formula (3), an oligomer consisting of one arbitrary amino acid or 2 to 10 arbitrary amino acid residues may be added to the C-terminal side. However, no amino acid may be added.

  The amino acid residue represented by Z3 should be as short as possible from the viewpoint of ease of synthesis of the transcriptional repressor converting peptide of formula (3). Specifically, it is preferably 10 or less, and 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 and the like. It is not limited.

  In addition, the number of amino acid residues in the entire transcriptional repressor conversion peptide represented by the formula (3) is not particularly limited, but may be 20 amino acids or less from the viewpoint of ease of synthesis. preferable.

  In the transcriptional repression converting peptide of the above formula (3), the specific sequence of the oligomer consisting of 7 to 10 amino acid residues excluding Z3 is shown in SEQ ID NOs: 46 to 54. The amino acid sequence when Z1 is Leu and Z2 is Glu, Gln or Asp is the amino acid sequence shown in SEQ ID NO: 46, 47 or 48, respectively, and Z1 is Asp-Leu and Z2 is Glu, Gln or Asp. Is the amino acid sequence shown in SEQ ID NO: 49, 50 or 51, respectively, and the amino acid sequence when Z1 is Leu-Asp-Leu and Z2 is Glu, Gln or Asp is SEQ ID NO: 52, The amino acid sequence shown in 53 or 54.

(I-2-4) Transcriptional Repression Conversion Peptide of Formula (4) The transcriptional repression conversion peptide of the above formula (4) is a hexamer (6mer) consisting of 6 amino acid residues, and its specific sequence is: SEQ ID NOs: 5, 14, and 55 are shown. 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 Gln. The sequence is the amino acid sequence shown in SEQ ID NO: 55.

  In particular, the transcriptional repression converting peptide used in the present invention may be a peptide having a minimum sequence such as a hexamer represented by the above formula (4). For example, the amino acid sequence shown in SEQ ID NO: 5 corresponds to the 196th to 201st amino acid sequence of Arabidopsis SUPERMAN protein (SUP protein), and as described above, the present inventor newly found as the above transcription repressor conversion peptide. It is.

(I-2-5) More specific example of transcriptional repression converting peptide As a more specific example of the transcriptional repressing converting peptide represented by each formula described above, for example, any one of SEQ ID NOs: 1 to 17 is shown. And a peptide having an amino acid sequence. These oligopeptides have been found by the present inventor to be the above-described transcriptional repression converting peptide (see, for example, Patent Document 7).

Furthermore, the other oligopeptide of the following (e) or (f) description can be mentioned as another specific example of the said transcription repression conversion peptide.
(E) A peptide consisting of any amino acid sequence shown in SEQ ID NO: 18 or 19.
(F) A peptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in any one of the amino acid sequences shown in SEQ ID NO: 18 or 19.

  The peptide consisting of the amino acid sequence shown in SEQ ID NO: 18 is a SUP protein. In addition, “one or a number in the amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in any amino acid sequence shown in SEQ ID NO: 18 or 19” above. The range of “pieces” is not particularly limited, but for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3 means.

  Deletion, substitution or addition of the amino acid can be performed by modifying the base sequence encoding the peptide by a technique known in the art. Mutation can be introduced into a base sequence by a known method such as Kunkel method or Gapped duplex method or a method similar thereto, for example, a mutation introduction kit using site-directed mutagenesis (for example, Mutant- Using K, Mutant-G (both trade names, manufactured by TAKARA)) or the like, or LA PCR in vitro Mutagenesis series kit (trade name, manufactured by TAKARA) is introduced.

  The functional peptide is not limited to the peptide having the full-length sequence of the amino acid sequence shown in SEQ ID NO: 18, and may be a peptide having a partial sequence thereof.

  Examples of the peptide having the partial sequence include a peptide having the amino acid sequence shown in SEQ ID NO: 19 (the 175th to 204th amino acid sequence of the SUP protein). Examples of the peptide having the partial sequence include (3 ).

(I-3) Other Examples of Transcriptional Repression Conversion Peptides The present inventors have further studied the structure of the motif, and as a result, have newly found a motif composed of six amino acids. Specifically, this motif is a peptide having an amino acid sequence represented by the following general formula (5). These peptides are also included in the above-mentioned transcription repressor conversion peptide.
(5) α1-Leu-β1-Leu-γ1-Leu
However, in the formula (5), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, and γ1 represents Arg. , Gln, Asn, Thr, Ser, His, Lys or Asp.

For convenience, the peptide represented by the general formula (5) is changed to a peptide having the amino acid sequence represented by the following general formula (6), (7), (8) or (9). Classify.
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(9) Asp-Leu-β3-Leu-Arg-Leu
In the above formulas, α1 represents Asp, Asn, Glu, Gln, Thr or Ser, and α2 represents Asn, Glu, Gln, Thr or Ser. Β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, and β3 represents Glu, Asp or Gln. Further, γ2 represents Gln, Asn, Thr, Ser, His, Lys, or Asp.

  As more specific examples of the transcriptional repression converting peptide having the amino acid sequence represented by the above formulas (5) to (9), SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, Examples thereof include peptides having an amino acid sequence represented by 29, 30, 31, 32, 33, 34, 35, 38, 39, or 40. Among these, the peptide of SEQ ID NO: 27, 28, 30, 32, 38, 39, or 40 corresponds to the peptide represented by the general formula (6), and the peptide of SEQ ID NO: 20, 23, 33, 34, or 35 is The peptide of SEQ ID NO: 24, 25, 26, 29, or 31 corresponds to the peptide of general formula (8), and the peptide of SEQ ID NO: 21 or 22 Corresponds to the peptide represented by the general formula (9).

  In addition to the peptides represented by the general formulas (5) to (9), a transcription repressing conversion peptide having the amino acid sequence represented by SEQ ID NO: 36 or 37 can also be used.

(I-4) Chimeric protein production method The various transcription repressor converting peptides described in (I-2) and (I-3) above are fused with the transcription factor described in (I-1) above to form a chimeric protein. By doing so, the transcription factor can be used as a transcriptional repression factor. Therefore, in the present invention, a chimeric protein can be produced by obtaining a chimeric gene with a polynucleotide encoding a transcription factor using the polynucleotide encoding the transcription repressing conversion peptide.

  Specifically, a chimeric gene is constructed by linking a polynucleotide encoding the transcription repressing conversion peptide (referred to as a transcription repressing conversion polynucleotide for convenience of explanation) and a polynucleotide encoding the transcription factor, Introduce into plant cells. Thereby, a chimeric protein can be produced. A specific method for introducing a chimeric gene into a plant cell will be described in detail in the section (II) described later.

  The specific base sequence of the transcriptional repression conversion polynucleotide is not particularly limited, and may include a base sequence corresponding to the amino acid sequence of the transcriptional repression conversion peptide based on the genetic code. In addition, if necessary, the transcription repressing / converting polynucleotide may include a base sequence serving as a linking site for linking with a polynucleotide encoding a transcription factor. Furthermore, when the amino acid reading frame of the transcription repressor conversion polynucleotide does not match the reading frame of the polynucleotide encoding the transcription factor, an additional base sequence for matching them may be included.

  Specific examples of the transcription repressing conversion polynucleotide include, for example, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 92, A polynucleotide having the base sequence shown in 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132 Can be mentioned. Also, SEQ ID NOs: 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, or 133 is a polynucleotide that is complementary to the polynucleotide exemplified above, respectively. It is a nucleotide. In addition, other specific examples of the transcription repression-converting polynucleotide include the polynucleotides shown in SEQ ID NOs: 90 and 91, for example. These polynucleotides correspond to the amino acid sequences shown in SEQ ID NOs: 1 to 40 as shown in Table 1 below.

  The chimeric protein used in the present invention can be obtained from the above chimeric gene in which a gene encoding a transcription factor and a transcriptional repression conversion polynucleotide are linked. Accordingly, the chimeric protein is not particularly limited as long as it includes the transcription factor site and the transcription repressing conversion peptide site. For example, various additional agents such as a polypeptide having a linker function for connecting between a transcription factor and a transcription repressor conversion peptide, and a polypeptide for epitope-labeling a chimeric protein such as His, Myc, Flag, etc. A polypeptide may be included. Furthermore, the chimeric protein may contain a structure other than a polypeptide, such as a sugar chain or an isoprenoid group, as necessary.

(II) Example of production method of double bloom plant according to the present invention The plant production method according to the present invention includes a process of introducing the chimeric protein described in (I) above into a plant and producing an double bloom plant. Although it will not specifically limit if it contains, if the production method of the plant body concerning this invention is shown in a specific process, for example, production including processes, such as an expression vector construction process, a transformation process, and a selection process It can be mentioned as a method. Among these, in the present invention, at least the transformation step may be included. Hereinafter, each step will be specifically described.

(II-1) Expression vector construction step The expression vector construction step carried out in the present invention comprises the polynucleotide encoding the transcription factor described in (I-1) above and the transcription repression conversion described in (I-4) above. It is not particularly limited as long as it is a step of constructing a recombinant expression vector containing a polynucleotide and a promoter.

  Various vectors known in the art can be used as the parent vector of the recombinant expression vector. For example, a plasmid, phage, cosmid or the like can be used, and can be appropriately selected according to the plant cell to be introduced and the introduction method. Specific examples include pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, pBI vectors, and the like. In particular, when the method for introducing a vector into a plant is a method using Agrobacterium, it is preferable to use a pBI binary vector. Specific examples of the pBI binary vector include pBIG, pBIN19, pBI101, pBI121, pBI221, and the like.

  The promoter is not particularly limited as long as it is a promoter capable of expressing a gene in a plant body, and a known promoter can be suitably used. Examples of such promoters include cauliflower mosaic virus 35S promoter (CaMV35S), actin promoter, nopaline synthase promoter, tobacco PR1a gene promoter, tomato ribulose 1,5-diphosphate carboxylase oxidase small subunit promoter and the like. Can be mentioned. Among these, cauliflower mosaic virus 35S promoter or actin promoter can be used more preferably. When each of the above promoters is used, the resulting recombinant expression vector can strongly express any gene when introduced into a plant cell.

  The promoter is not limited as long as it is linked so as to express a chimeric gene in which a gene encoding a transcription factor and a transcriptional repression-converting polynucleotide are linked and introduced into a vector. The structure is not particularly limited.

  The recombinant expression vector may further contain other DNA segments in addition to the promoter and the chimeric gene. The other DNA segment is not particularly limited, and examples thereof include a terminator, a selection marker, an enhancer, and a base sequence for improving translation efficiency. The recombinant expression vector may further have a T-DNA region. The T-DNA region can increase the efficiency of gene transfer particularly when Agrobacterium is used to introduce the recombinant expression vector into a plant body.

  The terminator is not particularly limited as long as it has a function as a transcription termination site, and may be a known one. For example, specifically, the transcription termination region (Nos terminator) of the nopaline synthase gene, the transcription termination region of the cauliflower mosaic virus 35S (CaMV35S terminator) and the like can be preferably used. Of these, the Nos terminator can be more preferably used.

  In the above transformation vector, by placing the terminator at an appropriate position, after introduction into a plant cell, an unnecessarily long transcript is synthesized, or a strong promoter reduces the copy number of the plasmid. Such a phenomenon can be prevented from occurring.

  As the selection marker, for example, a drug resistance gene can be used. Specific examples of such drug resistance genes include drug resistance genes for hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol and the like. Thereby, the transformed plant body can be easily selected by selecting the plant body growing in the medium containing the antibiotic.

  Examples of the base sequence for improving the translation efficiency include omega sequences derived from tobacco mosaic virus. By placing this omega sequence in the untranslated region (5′UTR) of the promoter, the translation efficiency of the chimeric gene can be increased. Thus, the transformation vector can contain various DNA segments depending on the purpose.

  The method for constructing the recombinant expression vector is not particularly limited, and the promoter, the gene encoding the transcription factor, the transcription repressor conversion polynucleotide, and, if necessary, the mother vector appropriately selected. What is necessary is just to introduce | transduce said other DNA segment so that it may become a predetermined order. For example, a chimeric gene is constructed by linking a polynucleotide encoding a transcription factor and a transcriptional repression-converting polynucleotide, and then linking this chimeric gene and a promoter (such as a terminator if necessary) to express the expression cassette. May be constructed and introduced into a vector.

  In the construction of the chimeric gene and the expression cassette, for example, it is possible to define the order of the DNA segments by setting the cleavage sites of each DNA segment as complementary protruding ends and reacting with ligation enzymes. . When the terminator is included in the expression cassette, the promoter, the chimeric gene, and the terminator may be in order from the upstream. Further, the types of reagents for constructing the recombinant expression vector, that is, the types of restriction enzymes and ligation enzymes are not particularly limited, and commercially available ones may be appropriately selected and used.

  Moreover, the propagation method (production method) of the recombinant expression vector is not particularly limited, and a conventionally known method can be used. In general, Escherichia coli may be used as a host and propagated in the E. coli. At this time, a preferred E. coli type may be selected according to the type of vector.

(II-2) Transformation Step In the transformation step performed in the present invention, the recombinant expression vector described in (II-1) above is introduced into plant cells to produce the chimeric protein described in (I) above. It only has to come to let you.

  The method (transformation method) for introducing the recombinant expression vector into a plant cell is not particularly limited, and any conventionally known method suitable for the plant cell can be used. Specifically, for example, a method using Agrobacterium or a method of directly introducing into plant cells can be used. As a method using Agrobacterium, for example, Transformation of Arabidopsis thaliana by vacuum information (http://www.bch.msu.edu/pammgreen/protocol.htm) can be used.

  As a method for directly introducing a recombinant expression vector into a plant cell, for example, a microinjection method, an electroporation method (electroporation method), a polyethylene glycol method, a particle gun method, a protoplast fusion method, a calcium phosphate method, or the like may be used. it can.

  Examples of plant cells into which the recombinant expression vector is introduced include cells of each tissue, callus, suspension culture cells, etc. in plant organs such as flowers, leaves and roots.

  Here, in the method for producing a plant according to the present invention, the recombinant expression vector may be appropriately constructed according to the type of plant to be produced. An expression vector may be constructed in advance and introduced into plant cells. That is, in the method for producing a plant according to the present invention, the recombinant expression vector construction step described in (II-1) above may or may not be included.

(II-3) Other steps and other methods In the production method of the double blooming plant according to the present invention, it is sufficient that the transformation step is included, and further the recombinant expression vector construction step is included. However, other steps may be included. Specifically, the selection process etc. which select an appropriate transformant from the plant body after transformation can be mentioned.

  The selection method is not particularly limited. For example, the selection may be performed on the basis of drug resistance such as hygromycin resistance, and after growing the transformant, the plant itself is selected from the form of the flower. Also good. For example, as an example of selecting from the form of a flower, there can be mentioned a method of comparing the form of a flower of a transformant with the form of a flower of a plant that has not been transformed (see Examples below). In particular, the form of the flower can be selected simply by comparing, and the effect itself of the present invention of producing a double bloom plant body can be confirmed.

  In the production method of the double bloom plant according to the present invention, since the chimeric gene is introduced into the plant, it is possible to obtain offspring from which the flower form is double bloom by sexual reproduction or asexual reproduction. Become. It is also possible to obtain propagation materials such as plant cells, seeds, fruits, strains, calluses, tubers, cut ears, lumps, etc. from the plants and their progeny, and mass-produce the plants based on these. . Therefore, in the production method of the plant body concerning this invention, the reproduction process (mass production process) which reproduces the plant body after selection may be included.

  The plant body in the present invention includes at least one of a grown plant individual, plant cell, plant tissue, callus, and seed. That is, in this invention, if it is a state which can be made to grow finally to a plant individual, all will be considered as a plant body. The plant cells include various forms of plant cells. Such plant cells include, for example, suspension culture cells, protoplasts, leaf sections and the like. Plants can be obtained by growing and differentiating these plant cells. In addition, the reproduction | regeneration of the plant body from a plant cell can be performed using a conventionally well-known method according to the kind of plant cell. Therefore, in the method for producing a plant according to the present invention, a regeneration step for regenerating the plant from the plant cell may be included.

(III) Method for producing sterile plant according to the present invention The method for producing a sterile plant according to the present invention comprises introducing the gene expressing the chimeric protein described in (I) above into a plant, If the process of producing a fertile plant is included, it will not specifically limit.

  In the present invention, the “sterile plant” is a completely sterile plant in which stamens and pistils are not formed, and incompatible plants, that is, stamens and pistils are formed but seeds cannot be formed. Plants are also included. Also included are plants that have incomplete stamen-like and / or pistil-like organs but are unable to form seeds. In addition, for example, sterile plants, in which stamen formation is inhibited and no pollen is formed, stamens are formed, but sterile plants, stamens and pods in which no pollen is formed because no stamens are formed. However, the amount of pollen formed is small, sterile plants that do not lead to cleavage of the cocoons, and so-called sterile plants that do not scatter at all because the pollen formed is enlarged and stuck together. Male sterile plants are also included.

  If a target plant is transformed with a chimeric gene encoding the chimeric protein, a sterile plant can be obtained very simply without using a complicated gene recombination technique.

  The above sterile plant cannot form seeds. Also, pollen dispersal does not occur in completely sterile plants and male sterile plants. Therefore, it is possible to prevent the genetically modified plant from spreading into the environment.

  The male sterile plants cannot be self-pollinated, but can be crossed between different varieties. Therefore, it can be suitably used for crossing utilizing hybrid strength, and breeding of a hybrid first generation having excellent traits can be performed efficiently.

  Moreover, since male sterility is a hybrid, it is not necessary to remove stamens for mating, and a great deal of labor is reduced. Therefore, it is suitable for improving the efficiency of the mating experiment.

(2) Plant obtained by the present invention, its usefulness, and its utilization The production method of the double bloom plant according to the present invention is based on expressing the chimeric protein in the plant. A DNA binding domain derived from a transcription factor in the chimeric protein binds to a target gene. The transcription factor is converted to a transcription repressor, and transcription of the target gene is repressed. As a result, the shape of the leaves of the obtained plant can be modified. Therefore, the present invention includes a plant obtained by the above-described method for producing a plant.

(2-1) Specific examples of plants according to the present invention The specific types of the double-flowered plants according to the present invention are not particularly limited. Plants that increase can be mentioned. Such a plant may be an angiosperm or a gymnosperm. Further, the angiosperm may be a monocotyledonous plant or a dicotyledonous plant, but a dicotyledonous plant is more preferable. The dicotyledonous plant may be a petal flower subclass or a joint flower subclass. As the joint flower subclass, for example, gentian eyes, eggplant eyes, perilla eyes, armored eyes, plantain eyes, asteraceae eyes, omanohasa eyes, red-eyed eyes, antaceae eyes, and chrysanthemum eyes can be mentioned. In addition, examples of the petal flower subclass include bivalamidae, camellia, blue mushrooms, crayfish eyes, diptera, violets, willows, scallops, azaleas, crabs, oysters, primulas, etc. be able to.

(2-2) Usefulness of the Present Invention According to the present invention, a double blooming plant can be produced, but the usefulness of the present invention is not particularly limited, and is more effective in producing a double blooming plant. Any field is acceptable. Examples of such fields include application to the creation of new horticultural varieties.

  First, an application example to the creation of a new horticultural variety will be described. By using the plant production method according to the present invention, a transcription factor gene is incorporated into a cassette vector to which the functional peptide has been added, thereby producing plant cells. Can be expressed in plant cells, and transcription of the transcription factor target gene can be easily suppressed. In addition, since the trait that suppresses transcription of the target gene of the transcription factor is dominant, it is not necessary to isolate the inferior homozygous individual as in the mutants produced by conventional techniques such as mating and RNAi method. Therefore, it is possible to produce a double blooming plant easily and reliably in a short period of time, which is very useful for horticulture.

  In addition, seed formation is not performed in double-flowered plants or sterile plants produced according to the present invention, and furthermore, no pollen disperses in completely sterile plants and male sterile plants. It can prevent the spread of recombinant plants to the environment and is very safe.

  Furthermore, the double-flowered plant or the sterile plant produced according to the present invention can be suitably used for crossing utilizing hybrid stress, and efficiently breeding a hybrid first generation having an excellent trait This is very useful for agriculture.

(2-3) Example of Use of the Present Invention The field of use and method of use of the present invention are not particularly limited, but as an example, a kit for carrying out the method for producing a plant according to the present invention, that is, an double bloom plant The body production kit can be mentioned.

  As a specific example of this double blooming plant production kit, it is sufficient to include at least a recombinant expression vector containing a chimeric gene comprising a polynucleotide encoding the transcription factor and the transcription repressor conversion polynucleotide. More preferably, it contains a group of reagents for introducing the vector into plant cells. Examples of the reagent group include enzymes, buffers, and the like corresponding to the type of transformation. In addition, if necessary, experimental materials such as microcentrifuge tubes may be attached.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and FIG. 1 thru | or FIG. 7, this invention is not limited to a following example.

[Example 1]
In Example 1 below, a 12 amino acid peptide LDLDLELLRLGFA (SRDX) (SEQ ID NO: 17), which is one of transcription repressor conversion peptides, is encoded between the cauliflower mosaic virus 35S promoter and the transcription termination region of the nopaline synthase gene. A recombinant expression vector incorporating a polynucleotide in which a polynucleotide to be bound to the downstream of the AG gene was constructed, and this was introduced into Arabidopsis thaliana using the Agrobacterium method, thereby transforming Arabidopsis thaliana.

<Construction of vector for construction of transformation vector>
As shown in FIG. 1, p35SG, a vector for constructing a transformation vector, was constructed as shown in the following steps (1) to (4).

  (1) The respective regions of attL1 and attL2 on the pENTR vector manufactured by Invitrogen were used as primers attL1-F (SEQ ID NO: 136), attL1-R (SEQ ID NO: 137), attL2-F (SEQ ID NO: 138), attL2-R ( Amplified by PCR using SEQ ID NO: 139). The obtained attL1 fragment was digested with restriction enzymes HindIII and attL2 fragment with EcoRI and purified. The PCR reaction conditions were 25 cycles, with a denaturation reaction at 94 ° C. for 1 minute, an annealing reaction at 47 ° C. for 2 minutes, and an extension reaction at 74 ° C. for 1 minute. All subsequent PCR reactions were carried out under the same conditions.

  (2) After cutting the plasmid pBI221 made by Clontech (Clontech, USA) with restriction enzymes XbaI and SacI, the GUS gene was removed by agarose gel electrophoresis, and the cauliflower mosaic virus 35S promoter (in the following description, for convenience, A 35S-Nos plasmid fragment DNA containing the transcription termination region of the nopaline synthase gene (referred to as Nos-ter in the following description for convenience) was obtained.

(3) A DNA fragment having the sequences of SEQ ID NOS: 140 and 141 below was synthesized, heated at 90 ° C. for 2 minutes, then heated at 60 ° C. for 1 hour, and then allowed to stand at room temperature (25 ° C.) for 2 hours. Annealing was performed to form a double strand. This was ligated to the XbaI-SacI region of the 35S-Nos plasmid fragment DNA to complete the p35S-Nos plasmid. The DNA fragments having the sequences of SEQ ID NOs: 140 and 141 include a BamHI restriction enzyme site at the 5 ′ end, an omega sequence derived from tobacco mosaic virus that enhances translation efficiency, and restriction enzyme sites SmaI, SalI, and SstI in this order.
5'-ctagaggatccacaataccacaacaacaacaacaaacaacaaacaacatatacaatattacagatccccgggggtaccgtcgacgagctc-3 '(SEQ ID NO: 140)
5'-cgtcgacggtacccccggatctgtataattgtatagtgtgtttgtttgtttgtttgtttgtttgtgtattgtgtgattcct-3 '(SEQ ID NO: 141)
(4) The p35S-Nos plasmid was digested with the restriction enzyme HindIII, and the attL1 fragment was inserted. This was further digested with EcoRI and the attL2 fragment was inserted to complete the vector p35SG.

<Construction of a construction vector incorporating a polynucleotide encoding a transcriptional repressor conversion peptide>
As shown in FIG. 2, p35SSRDG, which is a construction vector incorporating a polynucleotide encoding a transcription repressing conversion peptide, was constructed as shown in the following steps (1) to (2).

(1) DNAs encoding the 12 amino acid transcription repressor conversion peptide LDLDLELLRLGFA (SRDX) and designed to have a stop codon TAA at the 3 ′ end, each having the following sequence were synthesized and heated at 70 ° C. for 10 minutes. Thereafter, it was annealed by natural cooling to obtain double-stranded DNA.
5′-ggggcttgattctgatctagaactccgttttgggtttcgcttaag-3 ′ (SEQ ID NO: 142)
5′-tcgacttaagcgaaacccaacgggagtttagataccagatacagcccc-3 ′ (SEQ ID NO: 143)
(2) p35SG was digested with restriction enzymes SmaI and SalI, and double-stranded DNA encoding the above SRDX was inserted into this region to construct p35SSRDXG.

<Construction of transformation vector>
As shown in FIG. 3, pBIGCKH, which is a plant transformation vector having two att sites for recombination with a DNA fragment sandwiched between att sites of the construction vector, is transformed into the following steps (1) to (3 ).

  (1) pBIG (Becker, D. Nucleic Acids Res. 18: 203, 1990) transferred from Michigan State University, USA was digested with restriction enzymes HindIII and EcoRI, and the GUS and Nos regions were removed by electrophoresis.

(2) Fragment A of Gateway (registered trademark) vector conversion system purchased from Invitrogen was inserted into the EcoRV site of plasmid pBluescript. This was digested with HindIII-EcoRI and the Fragment A fragment was recovered.

  (3) The recovered Fragment A fragment was ligated with the above pBIG plasmid fragment to construct pBIGCKH. These can grow only in E. coli DB3.1 (Invitrogen) and are resistant to chloramphenicol and kanamycin.

<Incorporation of AG coding region into construction vector>
A DNA sequence or gene encoding a transcription factor AG protein derived from Arabidopsis was incorporated into the construction vector p35SSRDXG as shown in the following steps (1) to (3).

(1) A DNA fragment containing only the coding region of the AG polynucleotide (At4g18960) excluding the stop codon was amplified by PCR using Arabidopsis thaliana full-length cDNA pda01673 as a template and the following primers.
Primer 1 5′-atgaccgcgtaccatacgggagtagtaggg-3 ′ (SEQ ID NO: 144)
Primer 2 5′-catactactggaggcgggtttgggtctggcg-3 ′ (SEQ ID NO: 145)
The amino acid sequence encoded by the AG polynucleotide and the cDNA of the AG polynucleotide are shown in SEQ ID NOs: 134 and 135, respectively.

  (2) As shown in FIG. 2, the obtained DNA fragment of the AG coding region was ligated to the SmaI site of the construction vector p35SSRDXG previously digested with the restriction enzyme SmaI.

  (3) Escherichia coli was transformed with this plasmid, the plasmid was prepared, the nucleotide sequence was determined, and the clone inserted in the forward direction was isolated to obtain a chimeric gene with SRDX.

<Construction of recombinant expression vector>
An expression vector using a plant as a host was constructed by recombining a DNA fragment containing the CaMV35S promoter, chimeric gene, Nos-ter, etc. on the above construction vector into the plant transformation vector pBIGCKH. The recombination reaction was performed as described in the following steps (1) to (3) using Gateway (registered trademark) LR clone (registered trademark) manufactured by Invitrogen.

  (1) First, LR buffer 4.0 μL and TE buffer (10 mM TrisCl pH 7.0, 1 mM EDTA) 5.5 μL diluted 5-fold into p35SSRDXG 1.5 μL (about 300 ng) and pBIGCKH 4.0 μL (about 600 ng) were added. .

  (2) LR clone 4.0 μL was added to this solution and incubated at 25 ° C. for 60 minutes. Subsequently, 2 μL of proteinase K was added and incubated at 37 ° C. for 10 minutes.

  (3) Thereafter, 1-2 μL of this solution was transformed into E. coli (DH5a, etc.) and selected with kanamycin.

<Production of plant body transformed with recombinant expression vector>
Next, as shown in the following steps (1) to (3), Arabidopsis thaliana is transformed with pBIG-AGSRDX which is a plasmid in which the DNA fragment containing the chimeric gene is incorporated into pBIGCKH. Produced. Transformation of Arabidopsis plants was in accordance with Transformation of Arabidopsis thaliana by vacuum information (http://www.bch.msu.edu/pammgreen/protocol.htm). However, I did not use vacuum to infect it, but just dipped it.

  (1) First, the obtained plasmid, pBIG-AGSRDX, was introduced into a soil bacterium strain ((Agrobacterium tumefaciens strain GV3101 (C58C1Rifr) pMP90 (Gmr) (koncz and Sahell 1986)) by an electroporation method. Culturing was carried out in 1 liter of YEP medium containing antibiotics (kanamycin (Km) 50 μg / ml, gentamicin (Gm) 25 μg / ml, rifampicillin (Rif) 50 μg / ml) until the OD600 reached 1. The bacterial cells were collected and suspended in 1 liter of an infection medium (Table 2 below).

  (2) Arabidopsis thaliana cultivated for 14 days was immersed in this solution for 1 minute to infect, and then bred and seeded again. The collected seeds were sterilized with 25% bleach and 0.02% Triton X-100 solution for 7 minutes, rinsed three times with sterilized water, and seeded on a sterilized hygromycin selective medium (Table 3 below).

  (3) On average, 50 transformed plants that were resistant to hygromycin were obtained from about 5000 seeds that were cultivated. Total RNA was prepared from these plants, and it was confirmed that the AGSRDX gene was introduced using RT-PCR.

  Next, the plant body transformed with pBIG-AGSRDX will be described with reference to FIGS. FIG. 4 (a) shows the Arabidopsis flowers transformed with pBIG-AGSRDX, and the complete Arabidopsis flowers are in bloom, and FIG. 4 (b) is a diagram showing the whole Arabidopsis flowers in which the flower forms are in bloom. . As shown in FIG. 4 (a), Arabidopsis flowers transformed with pBIG-AGSRDX formed complete double-flowered plants in 16 of the 28 individuals tested. FIG. 5 (a) shows wild-type Arabidopsis flowers, and FIG. 5 (b) is a diagram showing AG mutant Arabidopsis flowers. The wild-type Arabidopsis thaliana has four struts, four petals, six stamens, and one pistil, whereas Arabidopsis transformed by the method of the present invention changes to stahl4 where the stamens become petals and become pistil. A new flower was formed. The AG mutant also has the same configuration, but the Arabidopsis transformed by the method according to the present invention formed a beautiful double-flowered plant with a narrower spacing between petals and an evenness compared to the AG mutant. FIG. 6 shows 10 Arabidopsis flowers out of 28 transformed with pBIG-AGSRDX. In the above 10 individuals, double-bloomed plant bodies were formed although incomplete. FIG. 7 shows two Arabidopsis flowers out of 28 transformed with pBIG-AGSRDX. In the above two individuals, a flower close to the wild type was formed.

  Further, the 16 individuals became completely sterile plants in which neither stamens or pistils were formed. In the above 10 individuals, incomplete stamen-like and pistil-like organs were formed, but seeds were not formed. That is, a sterile plant was formed. In the two individuals, stamens and pistils were formed, but the number of seeds formed was very small. Thus, all the plants transformed with pBIG-AGSRDX were sterile plants.

  Thus, in the present invention, a double-flowered plant body or a sterile plant body can be obtained by suppressing the transcription of the gene targeted by the AG transcription factor. Therefore, the present invention can be used for various agriculture, horticulture, landscaping, agribusiness, and the like, and is considered to be very useful.

It is process drawing which shows the construction method of the vector for construction for constructing the recombinant expression vector used in an Example. FIG. 3 is a process diagram for incorporating a gene encoding a transcription repressor conversion peptide SRDX and an AG gene into a construction vector p35SG used in Examples. It is process drawing which shows the construction method of vector pBIGCKH for transformation. FIG. 4 (a) shows the Arabidopsis flowers transformed with pBIG-AGSRDX, and the complete Arabidopsis flowers are in bloom, and FIG. 4 (b) is a diagram showing the whole Arabidopsis flowers in which the flower forms are in bloom. . FIG. 5 (a) shows wild-type Arabidopsis flowers, and FIG. 5 (b) is a diagram showing AG mutant Arabidopsis flowers. It is a figure which shows the Arabidopsis flower which was transformed by the recombinant expression vector pBIG-AGSRDX, and became the incomplete double bloom. It is a figure which shows the flower of the Arabidopsis thaliana transformed by the recombinant expression vector pBIG-AGSRDX, and became the form close | similar to a wild type.

Claims (17)

  It is characterized by producing a double-bloomed flower by producing a chimeric protein in the plant that fuses a transcription factor involved in pistil formation and a functional peptide that converts any transcription factor into a transcriptional repressor. The production method of the double blooming plant.   2. A transformation step of introducing into a plant cell a recombinant expression vector comprising a chimeric gene comprising a gene encoding the transcription factor and a polynucleotide encoding the functional peptide. The production method of the double blooming plant body of description.   Furthermore, the production method of the double bloom plant body of Claim 2 characterized by including the expression vector construction process which constructs | assembles the said recombinant expression vector. The method for producing double-flowered plants according to any one of claims 1 to 3, wherein the transcription factor is a protein described in (a) or (b) below.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 134;
(B) A protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 134. The method for producing a double bloom plant according to any one of claims 1 to 3, wherein the gene described in (c) or (d) below is used as the gene encoding the transcription factor.
(C) A gene having the base sequence represented by SEQ ID NO: 135 as an open reading frame region.
(D) A gene that hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 135 under stringent conditions and encodes a protein involved in pistil formation. The functional peptide is represented by the following formulas (1) to (4):
(1) X1-Leu-Asp-Leu-X2-Leu-X3
(In the formula, X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Wherein, Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)
(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Wherein, Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)
(4) Asp-Leu-Z4-Leu-Arg-Leu
(However, in the formula, Z4 represents Glu, Gln or Asp.)
The method for producing a double bloom plant according to any one of claims 1 to 5, wherein the plant has an amino acid sequence represented by any one of the above.   The said functional peptide is a peptide which has an amino acid sequence shown in either of sequence number 1-17, The production method of the double bloom plant body of any one of Claim 1 to 5 characterized by the above-mentioned. The said functional peptide is the peptide of the following (e) or (f) description, The production method of the double bloom plant body of any one of Claim 1 to 5 characterized by the above-mentioned.
(E) A peptide having the amino acid sequence shown in SEQ ID NO: 18 or 19.
(F) A peptide having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 18 or 19. The functional peptide is represented by the following formula (5)
(5) α1-Leu-β1-Leu-γ1-Leu
(Wherein α1 represents Asp, Asn, Glu, Gln, Thr or Ser, β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, and γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys, or Asp are shown.)
It has an amino acid sequence represented by these, The production method of the double bloom plant body of any one of Claim 1 to 5 characterized by the above-mentioned. The functional peptide is represented by the following formulas (6) to (8):
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(In the formula, α1 represents Asp, Asn, Glu, Gln, Thr or Ser, α2 represents Asn, Glu, Gln, Thr or Ser, and β1 represents Asp, Gln, Asn, Arg, Glu. , Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, and γ2 represents Gln, Asn, Thr, Ser, His, Lys or Asp.)
The method for producing a double bloom plant according to any one of claims 1 to 5, wherein the plant has an amino acid sequence represented by any one of the above.   The functional peptide is an amino acid represented by SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, or 40 It is a peptide which has a sequence, The production method of the double bloom plant body of any one of Claim 1 to 5 characterized by the above-mentioned.   The method for producing a double bloom plant according to any one of claims 1 to 5, wherein the functional peptide is a peptide having an amino acid sequence represented by SEQ ID NO: 36 or 37.   A double-flowered plant produced by the production method according to any one of claims 1 to 12.   The double bloom plant according to claim 13, wherein the double bloom plant includes at least one of a grown plant individual, a plant cell, a plant tissue, a callus, and a seed. A kit for performing the production method according to any one of claims 1 to 12,
A double bloom plant production comprising at least a recombinant expression vector comprising a gene encoding the above transcription factor, a polynucleotide encoding a functional peptide that converts any transcription factor into a transcription repressor, and a promoter. kit.   Furthermore, the double bloom plant body production kit of Claim 15 characterized by including the reagent group for introduce | transducing the said recombinant expression vector into a plant cell.   Plant sterility by producing a chimeric protein that fuses a transcription factor involved in the formation of stamens or pistil with a functional peptide that converts any transcription factor into a transcriptional repressor. A method for producing sterile plants characterized by the above.

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