JP4437936B2 - Method for producing plant body in which cleavage of cocoon is suppressed, plant body obtained by using the same, and use thereof - Google Patents

Description

  The present invention relates to a method for producing a plant in which cleavage of cocoons is suppressed, a plant obtained using the same, and use thereof, and in particular, to suppress transcription of genes involved in cocoon cleavage. The present invention relates to a method for producing a plant in which the dehiscence of cocoons is suppressed, a plant obtained using the plant, and the use thereof.

  Conventionally, the NAC family is known as a plant-specific transcription factor family in plants. In Arabidopsis, more than 100 genes belonging to the NAC family have been reported so far. The NAC family isolated so far has been reported as a transcription factor necessary for the formation and maintenance of shoot apical meristem, flower organ formation, lateral root formation, etc., and it is becoming clear that it has various functions. . However, the cis sequence to which the NAC family specifically binds is not known, and analysis of its function is awaited (for example, see Non-Patent Document 1).

  By the way, it is now common practice to produce excellent varieties by crossing plants between different varieties and hybridizing agricultural products. This utilizes the hybrid stress that when a plant is crossed between different varieties to create a hybrid, a trait superior to the parents appears in the child. It is necessary to cross between different varieties in order to receive the benefits of hybrid strength. For this reason, it is necessary to prevent self-pollination from being performed in the varieties to be crossed. Such methods include methods of artificially removing male organs, methods of artificial mating, methods of using chemical substances that inhibit pollen maturation, etc., but these are limited to plants that can use the method. In addition, it is a laborious work requiring a great deal of human labor. For this reason, a method using a male sterile body that has lost fertility due to the inability to form seeds due to incomplete formation of male gametes (pollen) is widely used. So far, male sterile bodies obtained by mutation have been used for breeding in various plants. In addition, there are many plant varieties for which male sterile bodies have not been established, and attempts to artificially establish male sterile bodies using genetic recombination techniques have been reported (for example, see Patent Documents 1 and 2). ). Patent Document 1 discloses a method for producing a male sterile plant by controlling the expression of the gene DAD1, which controls the dehiscence of cocoons and the maturation of pollen.

  On the other hand, as an attempt to suppress the cleavage of cocoon, cytotoxic barnase was artificially killed by binding the TA56 promoter that expresses the moth-specific gene and introducing it into tobacco. It has been reported that in the case of cigarette buds, no cocoon dehiscence occurs (see, for example, Non-Patent Document 2). Further, Non-Patent Document 2 reports that in order for cleavage to occur, oral cells need to be sufficiently functional until cell death occurs. However, little knowledge has been obtained at the molecular level in the cleavage of wrinkles. A relatively large number of mutants such as delayed-dehiscence 1 to delayed-dehiscence 5, non-dehiscence 1, msH and the like are known as mutants with abnormal cleavage of the anther (see, for example, Non-Patent Document 3). . However, only the above-mentioned DAD1 etc. have been clarified so far as the causal genes that control the cleavage of the wrinkles.

Here, the present inventors have found various peptides that convert an arbitrary transcription factor into a transcription repressing factor (see, for example, Patent Documents 3 to 9, Non-Patent Documents 4 and 5). This peptide is excised from a Class II ERF (Ethylene Responsive Element Binding Factor) protein or a plant zinc finger protein (Zinc Finger Protein, such as Arabidopsis SUPERMAN protein) and has a very simple structure.
JP 2000-3000273 (released on October 31, 2000) JP 2004-24108 A (published January 29, 2004) 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) Xie, Q., Frugis, G., Colgan, D., Chua, NH., Genes Dev. 14,3024-3036,2000 Beals, TP, Goldberg, RB, The Plant Cell, Vol.9,1527-1545, September, 1997 Edited by Yasuyoshi Hinata, "Flower-Molecular biology of sex and reproduction", Japan Society for the Science of Publishing, p116-p117 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

  In the said patent document 1, it does not occur that the dehiscence of a cocoon occurs and that a pollen does not have fertility. However, self-pollination can be avoided in crosses using hybrid stress if pod dehiscence does not occur regardless of the presence or absence of pollen fertility.

  Moreover, since the method using the male sterile body conventionally used turns into a hybrid automatically when it bears, it is an effective method for creating the first hybrid. However, since it does not bear fruit in the next generation, fertility must be restored in the next generation. On the other hand, the dehiscence of cocoons is suppressed, but if the pollen has fertility, it is possible to create a plant in which self-pollination does not occur while leaving the fertility in the pollen itself. Very useful in breeding. There is no known technique for suppressing such cleavage of cocoons by suppressing transcription of genes involved in cocoon cleavage.

  An object of the present invention is to provide a method for producing a plant in which cleavage of a cocoon is suppressed by suppressing transcription of a gene involved in the cleavage of the cocoon.

  As a result of intensive studies to solve the above problems, the present inventor fused a peptide that converts an arbitrary transcription factor to a transcription repressor with a protein encoded at the At2g46770 locus, which is one of NAC family proteins. Then, when expressed in plants, it was found that the cleavage of the cocoon does not occur completely or only occurs incompletely. This also allows this NAC family protein (protein encoded at the At2g46770 locus, hereinafter referred to as “NACAD1” (NAC involving to Anther Development) as appropriate) to promote transcription of genes involved in cleaving of sputum. It was revealed for the first time that it is a transcription factor, and the present invention was completed.

  That is, in order to solve the above-mentioned problem, a method for producing a plant body in which cleavage of cocoons according to the present invention is suppressed comprises a transcription factor that promotes transcription of a gene involved in cocoon cleavage and an arbitrary transcription factor. A chimera protein fused with a functional peptide that is converted into a transcriptional repressor is produced in a plant body, thereby suppressing the cleavage of cocoons.

  In the plant body, the female organ has fertility. Furthermore, it is preferable that the pollen itself has fertility.

  The production method preferably includes a transformation step of 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. .

  The production method may further include an expression vector construction step for constructing the 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) In the amino acid sequence shown in SEQ ID NO: 134, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and transcription of a gene involved in cleavage of a fold A protein that has a function to promote.

  The transcription factor is a protein having a homology of 70% or more with respect to the amino acid sequence shown in SEQ ID NO: 134 and a function of promoting transcription of a gene involved in cleavage of the cocoon. Also good.

  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 transcription factor that hybridizes under stringent conditions with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 135, and promotes transcription of the gene involved in cleavage of the fold A gene encoding

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.)
It is preferable that it has the amino acid sequence represented by either.

  Further, the functional peptide may be a peptide having an amino acid sequence represented by any of SEQ ID NOs: 3 to 19.

  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: 20 or 21. (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: 20 or 21.

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
(Wherein 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.

The functional peptide has an amino acid sequence represented by SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 133. It may be a peptide.

  The functional peptide may be a peptide having the amino acid sequence shown in SEQ ID NO: 38 or 39.

  Moreover, the plant body concerning this invention is produced by the said production method, It is characterized by the dehiscence of a cocoon being suppressed. 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 plant cocoon dehiscence suppression kit according to the present invention is a kit for carrying out the above production method, comprising: a gene encoding a transcription factor that promotes transcription of a gene involved in cocoon dehiscence; And a recombinant expression vector containing at least a polynucleotide encoding a functional peptide that converts any transcription factor into a transcription repressor and a promoter. The kite dehiscence suppression kit may further contain a reagent group for introducing the recombinant expression vector into plant cells.

Moreover, the production method of a plant body in which cleavage of the cocoon according to the present invention is controlled includes a gene encoding the protein described in the following (a) or (b),
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) In the amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, A protein having a function of promoting,
Or the gene described in the following (c) or (d),
(C) a gene having the base sequence shown in SEQ ID NO: 2 as an open reading frame region,
(D) a transcription factor which hybridizes under stringent conditions with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2 and which promotes transcription of the gene involved in the cleavage of the anther It uses the gene which codes.

  As described above, the production method of a plant body in which cleavage of the cocoon according to the present invention is suppressed includes a transcription factor that promotes transcription of a gene involved in cleavage of the cocoon and an arbitrary transcription factor as a transcriptional repression factor. Since the plant has a structure in which a chimeric protein fused with a functional peptide to be converted is produced in a plant body, the expression of genes involved in the cleavage of the cocoon is suppressed, and the plant body in which the cleavage of the cocoon is suppressed Can be produced. Therefore, if a target plant is transformed with a chimeric gene encoding the chimeric protein, a plant in which the cleavage of the cocoon is suppressed can be produced, and it is very simple without using a complicated gene recombination technique. There is an effect that the dehiscence of the target plant can be suppressed.

  In addition, since the chimeric protein used in the present invention acts dominantly on the endogenous gene, the chimeric protein according to the present invention is a diploid or double diploid plant, Alternatively, even if there is a duplicated gene in the plant, it is possible to uniformly suppress the expression of genes involved in cocoon dehiscence, making it easy to convert any plant into which a gene can be introduced into a plant body in which cocoon dehiscence is suppressed There is an effect that can be transformed into.

  In addition, since the amino acid sequence of a transcription factor used in the present invention that promotes transcription of a gene involved in the cleavage of cocoons is considered to be highly conserved among many plants of different species, a specific model plant By introducing the chimeric protein constructed in (1) into other plants, it is possible to produce plants in which various types of plants can easily suppress the dehiscence of wrinkles.

  An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.

  The present invention relates to a technique for suppressing cocoon dehiscence in plants, a transcription factor that promotes transcription of genes involved in cocoon dehiscence, and a functional peptide that converts any transcription factor into a transcription repressor Is produced in a plant body. In the plant obtained by this, transcription of a gene involved in cocoon cleavage is suppressed, and a plant in which cocoon cleavage is suppressed can be produced.

  Here, the cleavage of the wrinkles is suppressed as follows. That is, the transcription factor-derived DNA binding domain in the chimeric protein binds to a target gene that is presumed to be involved in the cleavage of sputum. The transcription factor is converted into a transcription repressing factor, and transcription of the target gene is repressed. This reduces the production of proteins presumed to be involved in cocoon dehiscence, and as a result, it is possible to suppress cocoon dehiscence in the resulting plant.

  Plants in which the cleavage of the cocoon produced by the production method according to the present invention is suppressed include a plant in which the cleavage of the cocoon does not occur completely and a plant in which the cleavage of the cocoon occurs only incompletely. It is. Here, the cocoon is a part of stamen and is a bag-like part that makes pollen. In those that undergo fertilization (eg, legumes), pollen germinates in the cocoon and extends the pollen tube through a soft cocoon wall that does not develop a fibrous cell layer. Release pollen out of the cage. The dehiscence of the fold occurs when the oral cells cause cell death, where the fold wall is cut. In addition, when the limbal cells are cut, the cocoon does not open its mouth, and in order for pollen to be released, the cocoon wall needs to contract and warp. The plant body in which cleavage of the cocoon in the present invention is suppressed may be a plant body in which the limbal cells are cut, and the limbal cell is cut and the heel wall is warped to open the mouth. It may be a plant body. The oral cells are tissues composed of smaller cells that correspond to the part where the fold opens the mouth.

  In addition, in the plant body in which the dehiscence of the cocoon of the present invention is suppressed, the female organ (pistil) has fertility. Thereby, the pollen of another kind can be pollinated by the plant body in which the dehiscence of the cocoon of the present invention is suppressed. Therefore, a hybrid first generation (F1) variety can be obtained by crossing using a hybrid strength without the trouble of artificially removing male organs or crossing them artificially.

  Moreover, in the plant body in which the dehiscence of the cocoon of the present invention is suppressed, it is sufficient that the dehiscence of the cocoon is suppressed, and the pollen itself may or may not have fertility. However, it is more preferable that the pollen itself has fertility. Thereby, it becomes possible to produce a plant body that does not cause self-pollination while leaving fertility in the pollen itself, which is useful for breeding and the like. That is, in a male sterile body in which the pollen itself does not have fertility, its own pollen cannot be used, so that a homozygous individual by selfing cannot be created and maintained. Although homozygous individuals can be produced by self-pollination of heterozygous individuals, only 1/4 of homozygous individuals can be obtained. On the other hand, by leaving fertility in the pollen itself, it becomes possible to create and maintain homozygous individuals, which can be applied to breeding.

  In the following description, the chimeric protein used in the method for producing a plant body in which cleavage of the cocoon according to the present invention is suppressed, an example of the method for producing the plant body according to the present invention, the plant body obtained thereby and its usefulness , And their use will be described.

(I) Chimeric protein used in the present invention As described above, the chimeric protein used in the present invention uses a transcription factor that promotes transcription of genes involved in cleaving of wrinkles and an arbitrary transcription factor as a transcription repressor. It is a fusion of a functional peptide that converts.

  In addition, since the chimeric protein used in the present invention acts dominantly on the endogenous gene, the chimeric protein according to the present invention is a diploid or double diploid plant, Alternatively, even if a functionally duplicated gene exists in a plant, the expression of a gene involved in cocoon dehiscence can be uniformly suppressed. Therefore, it becomes possible to easily transform any plant into which a gene can be introduced into a plant body in which cleavage of the cocoon is suppressed.

  Hereinafter, each of the transcription factor and the functional peptide will be described.

(I-1) Transcription factor that promotes transcription of genes involved in cleavage of sputum The transcription factor used in the present invention is not particularly limited as long as it is a transcription factor that promotes transcription of genes involved in cleaving of sputum. It is not something. In general, the cocoon breaks and releases pollen out of the cocoon. Therefore, transcription factors that promote transcription of genes involved in cocoon dehiscence are conserved in many plants. Therefore, transcription factors used in the present invention include transcription factors having similar functions that are conserved in various plants.

  As a representative example of the transcription factor used in the present invention, for example, NACAD1 protein can be mentioned. The NACAD1 protein is a protein having the amino acid sequence shown in SEQ ID NO: 134, and is one of the NAC family proteins of Arabidopsis as described above. In the present invention, for example, NACAD1 protein, which is a transcription factor, is converted into a transcriptional repressing factor by fusing a functional peptide described later to this NACAD1 protein.

  The transcription factor used in the present invention is not limited to the NACAD1 protein having the amino acid sequence shown in SEQ ID NO: 134, and may be any transcription factor that promotes transcription of a gene involved in cleaving of the anther. Specifically, in the amino acid sequence shown in SEQ ID NO: 134, even a protein consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added has the above function. If so, it can be used in the present invention. The range of “one or several” in the above “amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids are substituted, deleted, inserted and / or added” is Although not particularly limited, for example, it means 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and particularly preferably 1 to 3.

  The transcription factor is a protein having a homology of 20% or more, preferably 50% or more, more preferably 60% or 70% or more with respect to the amino acid sequence shown in SEQ ID NO: 134, and A protein having a function of promoting transcription of a gene involved in cleavage of the cocoon is also included. Here, “homology” is the ratio of the same sequence in the amino acid sequence, and the higher this value, the closer the two are.

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

  The gene encoding the transcription factor is not particularly limited as long as it corresponds to the amino acid sequence of the transcription factor based on the genetic code. As a specific example, for example, when NACAD1 protein is used as a transcription factor, a gene encoding this NACAD1 protein (referred to as NACAD1 gene for convenience of description) can be mentioned. As a specific example of the NADAD1 gene, for example, a polynucleotide containing the base sequence represented by SEQ ID NO: 135 as an open reading frame (ORF) can be mentioned.

  Of course, the NADAD1 gene or the gene encoding the transcription factor used in the present invention is not limited to the above example, and may be a gene having homology with the base sequence shown in SEQ ID NO: 135. Good. Specifically, for example, a gene that hybridizes under stringent conditions with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 135 and encodes the above transcription factor, etc. Can do. Here, hybridizing under stringent conditions means binding at 60 ° C. under 2 × SSC washing conditions.

  The hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989). Generally, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize).

  The method for obtaining the gene encoding the transcription factor is not particularly limited, and it can be isolated from many plants by a conventionally known method. For example, a primer pair prepared based on a known transcription factor base sequence can be used. Using this primer pair, the gene can be obtained by PCR using plant cDNA or genomic DNA as a template. The gene encoding the transcription factor can also be obtained by chemical synthesis by a conventionally known method.

(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 repressor converting peptides found by the present inventors (see Patent Documents 3 to 9, Non-Patent Documents 4, 5, 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 group, significantly suppresses gene transcription. Obtained. Thus, an effector plasmid containing a gene encoding each of the above proteins and DNA excised therefrom was constructed and introduced into plant cells, thereby succeeding in actually suppressing gene transcription (for example, Patent Documents 3 to 3). 6). In addition, a gene encoding tobacco ERF3 protein (see, for example, Patent Document 7), a rice OsERF3 protein (see, for example, Patent Document 8), which is one of the Class II ERF genes, and one of the zinc finger protein genes. 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 the transcription factor produces a protein having a strong transcription repressing ability.

  Therefore, as an example of the transcriptional repressor converting peptide used in the present invention, in this embodiment, an AtERF3 protein, an AtERF4 protein, an AtERF7 protein, an AtERF7 protein, a tobacco ERF3 protein, a rice ERF3 protein, a 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 transcriptional repressor converting peptide of the above formula (1), specific sequences of pentamers (5mers) consisting of 5 amino acid residues excluding X1 and X3 are shown in SEQ ID NOs: 40 and 41. The amino acid sequence when X2 is Asn is the amino acid sequence shown in SEQ ID NO: 40, and the amino acid sequence when X2 is Glu is the amino acid sequence shown in SEQ ID NO: 41.

(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 comprising 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), specific sequences of pentamers (5mers) consisting of 5 amino acid residues excluding Y1 and Y3 are shown in SEQ ID NOs: 42 and 43. The amino acid sequence when Y2 is Phe is the amino acid sequence shown in SEQ ID NO: 42, and the amino acid sequence when Y2 is Ile is the amino acid sequence shown in SEQ ID NO: 43. A specific sequence of a tetramer (4mer) consisting of 4 amino acid residues excluding Y2 is shown in SEQ ID NO: 44.

(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: 45 to 53. 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: 45, 46 or 47, respectively, and Z1 is Asp-Leu and Z2 is Glu, Gln or Asp. Is the amino acid sequence shown in SEQ ID NO: 48, 49 or 50, respectively, and the amino acid sequence when Z1 is Leu-Asp-Leu and Z2 is Glu, Gln or Asp is SEQ ID NO: 51, respectively. The amino acid sequence shown in 52 or 53.

(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: 7, 16, and 54 are shown. The amino acid sequence when Z4 is Glu is the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence when Z4 is Asp is the amino acid sequence shown in SEQ ID NO: 16, and the amino acid sequence when Z4 is Gln. The sequence is the amino acid sequence shown in SEQ ID NO: 54.

  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: 7 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: 3 to 19 is shown. And a peptide having an amino acid sequence. These oligopeptides have been found by the present inventor to be the above-mentioned transcription repressor conversion peptide (see, for example, Patent Document 9).

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: 20 or 21.
(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: 20 or 21.

  The peptide consisting of the amino acid sequence shown in SEQ ID NO: 20 is a SUP protein. In addition, “one or several in the 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: 20 or 21”. 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 based thereon, for example, a mutation introduction kit using site-directed mutagenesis (for example, Mutant- Anomalies are introduced 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).

  The functional peptide is not limited to the peptide having the full-length sequence of the amino acid sequence shown in SEQ ID NO: 20, 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: 21 (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
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: 22, 23, 24, 25, 26, 27, 28, 29, 30, Mention may be made of peptides having the amino acid sequence represented by 31, 32, 33, 34, 35, 36, 37 or 133 . Among these, the peptide of SEQ ID NO: 29, 30, 32, 34 or 133 corresponds to the peptide represented by the general formula (6), and the peptide of SEQ ID NO: 22, 25, 35, 36 or 37 is represented by the general formula (7 The peptide represented by SEQ ID NO: 26, 27, 28, 31, or 33 corresponds to the peptide represented by the general formula (8), and the peptide represented by SEQ ID NO: 23 or 24 is represented by the general formula (8). It corresponds to the peptide shown in 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: 38 or 39 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 gene 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 gene encoding the transcription factor, Introduce into cells. Thereby, a chimeric protein can be produced. A specific method for introducing the chimeric gene into plant cells will be described in detail in the section (2) 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 transcriptional repression conversion polynucleotide may include a base sequence serving as a linking site for linking with a transcription factor gene. Further, when the amino acid reading frame of the transcription repressor conversion polynucleotide does not match the reading frame of the transcription factor gene, an additional base sequence for matching them may be included.

  Specific examples of the transcription repression conversion polynucleotide include, for example, SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 137, 139, 141 or 143 Mention may be made of polynucleotides. Also, SEQ ID NOs: 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 138, 140, 142, 144 are complementary to the polynucleotides exemplified above, respectively. It is a polynucleotide. In addition, other specific examples of the above transcription repressor conversion polynucleotide include the polynucleotides shown in SEQ ID NOs: 95 and 96, for example. These polynucleotides correspond to the amino acid sequences shown in SEQ ID NOs: 3-39 and 133-136 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. Therefore, the chimeric protein is not particularly limited as long as it contains the site of the transcription factor and the site of the transcription repressor conversion peptide. 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) An example of a method for producing a plant according to the present invention The method for producing a plant according to the present invention comprises the step of producing the chimeric protein described in (I) above in a plant and suppressing the dehiscence of wrinkles. 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 gene encoding the transcription factor described in (I-1) above and the transcriptional repression conversion poly described in (I-4) above. There is no particular limitation as long as it is a step of constructing a recombinant expression vector containing a nucleotide 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, and pBI vectors. 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 these promoters are used, the resulting recombinant expression vector can strongly express an arbitrary gene when introduced into a plant cell. The promoter is more preferably a promoter capable of expressing a gene in a specific manner. Examples of such promoters include TA56 promoter, AtMYB26 promoter, DAD1 promoter and the like. By using such a promoter, it becomes possible to express the gene encoding the chimeric protein only in the cocoon and suppress the cleavage of the cocoon without affecting other tissues. The promoter is particularly preferably a promoter of a gene encoding a similar transcription factor conserved in NACAD1 and various plants. By using such a promoter, it becomes possible to express the gene specifically in the time of expression and the tissue of the gene, and the cleavage of the wrinkles can be more effectively suppressed.

  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 gene 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 form an expression cassette. What is necessary is just to construct | assemble and introduce this 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 infiltration (http://www.bch.msu.edu/pamgreen/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 (I-1) above may or may not be included.

(II-3) Other Steps and Other Methods In the method for producing a plant according to the present invention, it is sufficient if 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, or may be selected from the state of dehiscence of the cocoon after growing the transformant. Good. For example, as an example of selecting from the state of cleavage of the cocoon, a method of observing the shape of the cocoon using an electron microscope, a stereomicroscope, or the like can be cited (see examples described later).

  In the method for producing a plant according to the present invention, since the chimeric gene is introduced into the plant, it is possible to obtain offspring from which the dehiscence of moths is suppressed 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, a plant cell, a plant tissue, a callus, and a 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.

  Moreover, the production method of the plant body concerning this invention is not limited to the method of transforming with a recombinant expression vector, You may use another method. Specifically, for example, the chimeric protein itself may be administered to a plant body. In this case, the chimeric protein may be administered to the young plant so that the dehiscence of the cocoon can be suppressed at the site of the plant finally used. Also, the administration method of the chimeric protein is not particularly limited, and various known methods may be used.

(III) Plant obtained by the present invention, its usefulness, and use thereof The method for producing a plant according to the present invention is based on expressing a gene encoding the chimeric protein in the plant. A transcription factor-derived DNA binding domain in the chimeric protein binds to a target gene presumed to be involved in wrinkle cleavage. The transcription factor is converted to a transcription repressor, and transcription of the target gene is repressed. Thereby, tearing of a wrinkle can be suppressed. Therefore, the present invention includes a plant obtained by the above-described method for producing a plant.

(III-1) Specific Examples of Plants According to the Present Invention Here, the specific types of plant bodies in which the cleavage of the cocoon according to the present invention is suppressed are not particularly limited. Plants whose usefulness is enhanced by inhibition can be mentioned. Such a plant may be an angiosperm or a gymnosperm. As a gymnosperm, for example, a cedar family, a pine family, a cypress family or a asteraceae plant can be mentioned. Further, the angiosperm may be a monocotyledonous plant or a dicotyledonous plant. Examples of dicotyledonous plants include plants such as Brassicaceae such as Arabidopsis thaliana and camellia. In addition, examples of monocotyledonous plants include plants such as rice, corn, wheat, etc.

  Moreover, the plant body in which the cleavage of the cocoon according to the present invention is suppressed may be a plant using fruits and seeds as a product, or a foliage plant (floral plant) using products such as flowers and plants themselves. Therefore, specific examples of the plant body in which cleavage of the cocoon according to the present invention is restricted include rapeseed, potato, spinach, soybean, cabbage, lettuce, tomato, cauliflower, soy bean, turnip, mekabu, radish, broccoli, There are various edible plants such as melon, orange, watermelon, leek and burdock, or ornamental plants such as roses, chrysanthemum, hydrangea and carnations.

(III-2) Usefulness of the Present Invention The present invention has utility in a field having a certain effect by suppressing the dehiscence of cocoons in plants. Some specific examples are given below, but the usefulness of the present invention is not limited thereto.

  First, by the technique of the present invention, a plant body in which the dehiscence of cocoons is suppressed can be produced, and can be used for breed improvement by crossing using hybrid strength. In the plant body in which the dehiscence of the cocoon of the present invention is suppressed, the pollen is not released outside the cocoon, and therefore self-pollination is not performed even for self-propagating plants such as rice. Therefore, crossing between species can be performed simply by pollinating pollen of other species. Thereby, the search for the primary hybrid of the excellent variety using the hybrid strength can be easily and efficiently performed.

  The technique of the present invention can also be applied to other bred plants such as corn. In other breeding plants, self-pollination is avoided by manual reaping of stamens (male removal work), and varieties are improved by pollinating pollen of other varieties. On the other hand, if the plant body in which the dehiscence of the cocoon is suppressed is produced by the technique of the present invention, such labor is not required, so it is necessary for time and cost required for breed improvement or cultivation of excellent varieties. Can be significantly reduced compared to the current situation.

  In particular, according to the technology of the present invention, the pollen has fertility, but can produce a plant body in which the dehiscence of the cocoon is suppressed. This is useful for breeding and the like. That is, it is possible to create and maintain homozygous individuals by leaving fertility in the pollen itself. By self-mating such a pure plant, it becomes possible to obtain a uniform seed breeding population, and it is possible to reduce the labor and time for performing the selection work.

  The technique of the present invention can also be applied to plants that use rhizomes such as onions and potatoes. In this type of plant, it is known that when pollination occurs, the growth of rhizomes is significantly inhibited and the commercial value is lowered. Therefore, at present, a male removal work is required to avoid pollination, and the labor and cost for that purpose are very large. According to the technique of the present invention, since a plant body in which the dehiscence of a plant with a rhizome as a product is suppressed is obtained, depilation is not required and pollination can be avoided. Therefore, the cost and time for producing a product by growing a plant body can be significantly reduced compared to the current situation.

  The technology of the present invention can also be suitably applied to plants that do not use fruits and flowers as commodities. One example is the prevention of hay fever. That is, in plants that disperse a large amount of pollen that causes pollinosis, for example, trees such as cedar, cypress, sawara, gramineous plants such as camodia, blue wagtail, nagahagusa, weeds such as ragweed, mugwort, canamgrass, If the plants in which the dehiscence of the cocoon is suppressed by the technique of the invention are produced, there is no fear of pollen scattering from these plants. Therefore, if the plant body in which the dehiscence of these anthers is suppressed is replaced with a wild-type plant body in the natural world, hay fever can be prevented because scattering of pollen causing hay fever is suppressed.

  The technology of the present invention can prevent undesirable diffusion of the pollen of the genetically modified plant into the natural world. For example, in Eucalyptus, the raw material of pulp, genetically engineered genetically modified plants with superior traits such as excellent salt tolerance and cold resistance and enormous tree growth have been created. Experiments to verify the introduced trait in the environment are being conducted. However, if such genetically modified plants are grown in an outdoor environment, the genetically modified plants may spread widely to nature through the diffusion of pollen using wind, insects, etc., and the natural environment may be altered. There is. Therefore, in order to deal with such a problem, it is necessary to conduct a verification experiment of a genetically modified plant in a special environment completely isolated from the outside world.

  However, if the genetically modified plant is transformed into a plant in which the dehiscence of buds is suppressed using the technique of the present invention, the genetically modified plant does not diffuse into the natural world due to pollen distribution. . Therefore, the verification test of the genetically modified plant can be performed under conditions closer to those in the actual outdoor environment as compared with the current situation. Thereby, the character introduced into the genetically modified plant can be verified in a more natural environment.

(III-3) An example of the use of the present invention The application field 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, a koji A dehiscence suppression kit can be mentioned.

  As a specific example of this kite dehiscence suppression kit, it suffices to contain at least a recombinant expression vector containing a chimeric gene comprising the gene encoding the transcription factor and the transcription repression-converting polynucleotide, and the recombinant expression described above. 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.

(IV) Method for producing plant body in which cleavage of cocoon is controlled The inventor of the present invention promotes transcription of genes involved in cleavage of cocoon by NACAD1 and similar transcription factors conserved in various plants. It was revealed for the first time that it is a transcription factor, and the present invention was completed. Therefore, the present invention also includes a method for producing a plant body in which cleavage of cocoons is controlled using a gene encoding such a transcription factor.

That is, according to the present invention, a method for producing a plant in which cleavage of a cocoon is restricted includes a gene encoding a protein described in the following (a) or (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) In the amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, A protein having a function of promoting,
Or the gene described in the following (c) or (d),
(C) a gene having the base sequence shown in SEQ ID NO: 2 as an open reading frame region,
(D) a transcription factor which hybridizes under stringent conditions with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2 and which promotes transcription of the gene involved in the cleavage of the anther The gene encoding,
Is used.

  This plant production method can be achieved by suppressing or over-expressing the gene encoding the transcription factor involved in cocoon dehiscence. Examples of the method for suppressing the expression of the gene include an antisense method, a gene targeting method, an RNAi method, a co-suppression method, and a gene disruption tagging method. Examples of a method for overexpressing the gene include a method of constructing a vector containing an appropriate promoter and the gene arranged downstream thereof and introducing the vector into a plant.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and FIG. 1 thru | or FIG. 6, this invention is not limited to a following example.

  In the present example, a polyenzyme encoding a 12 amino acid peptide LDLDLELRLGFA (SRDX) (SEQ ID NO: 19), which is one of transcription repressor conversion peptides, between the cauliflower mosaic virus 35S promoter and the transcription termination region of the nopaline synthase gene. A recombinant expression vector incorporating a polynucleotide having a nucleotide linked downstream of the NACAD1 gene was constructed and introduced into Arabidopsis thaliana using the Agrobacterium method to transform 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) Each region of attL1 and attL2 on the pENTR vector manufactured by Invitrogen Co., Ltd. is expressed as primers attL1-F (SEQ ID NO: 138), attL1-R (SEQ ID NO: 139), attL2-F (SEQ ID NO: 140), attL2-R ( Amplified by PCR using SEQ ID NO: 141). 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 cleaving the plasmid pBI221 of 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) DNA fragments having the sequences of SEQ ID NOS: 142 and 143 below were 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: 142 and 143 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'-ctagaggatccacaattaccaacaacaacaaacaacaaacaacattacaattacagatcccgggggtaccgtcgacgagctc-3 '(SEQ ID NO: 142)
5'-cgtcgacggtacccccgggatctgtaattgtaatgttgtttgttgtttgttgttgttggtaattgtggatcct-3 '(SEQ ID NO: 143)
(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 LDLDLELRLGFA (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'-gggcttgatctggatctagaactccgtttgggtttcgcttaag-3 '(SEQ ID NO: 144)
5'-tcgacttaagcgaaacccaaacggagttctagatccagatcaagccc-3 '(SEQ ID NO: 145)
(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) The Fragment A of Gateway (registered trademark) vector conversion system purchased from Invitrogen was inserted into the EcoRV site of the 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 NACAD1 gene into construction vector>
A gene encoding the Arabidopsis-derived transcription factor NACAD1 protein was incorporated into the construction vector p35SSRDXG as shown in the following steps (1) to (3).

(1) From a cDNA library prepared using mRNA prepared from Arabidopsis leaves, a DNA fragment containing only the coding region of Arabidopsis NACAD1 gene excluding the stop codon was amplified by PCR using the following primers.
Primer 1 (NACAD1-F) 5′-GATGATGTCAAAATCTATGAGCATATC-3 ′ (SEQ ID NO: 136)
Primer 2 (NACAD1-R) 5′-TCCACTACCATTCGACACGTGAC-3 ′ (SEQ ID NO: 137)
The cDNA of NACAD1 gene and the encoded amino acid sequence are shown in SEQ ID NOs: 135 and 134, respectively.

  (2) As shown in FIG. 2, the obtained DNA fragment of the NACAD1 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 follows (1) to (3) using Gateway (registered trademark) LR clonase (registered trademark) of Invitrogen.

  (1) First, 4.0 μL of LR buffer diluted 5 times to 1.5 μL (about 300 ng) of p35SSRDXG and 4.0 μL (about 600 ng) of pBIGCKH and 5.5 μL of TE buffer (10 mM TrisCl pH7.0, 1 mM EDTA) were added. .

  (2) 4.0 μL of LR clonase 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-NACAD1SRDX which is a plasmid in which the DNA fragment containing the chimeric gene is incorporated into pBIGCKH, and a transformed plant is obtained. Produced. Transformation of Arabidopsis plants was according to Transformation of Arabidopsis thaliana by vacuum infiltration (http://www.bch.msu.edu/pamgreen/protocol.htm). However, I did not use vacuum to infect it, but just dipped it.

  (1) First, the obtained plasmid, pBIG-NACAD1SRDX, was introduced into a soil bacterium strain ((Agrobacterium tumefaciens strain GV3101 (C58C1Rifr) pMP90 (Gmr) (koncz and Sahell 1986)) by electroporation. 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 cells were collected and suspended in 1 liter of an infiltration 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. In the generation infected with Agrobacterium, the effect of the transformed gene is generally not exerted unless it prevents the survival of the ovule. For this reason, inhibition of sputum dehiscence did not occur, and the seeds were kneaded. 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 hygromycin-resistant plants were obtained from about 2000 seeds that were sown. Total RNA was prepared from these plants, and it was confirmed that the NACAD1SRDX gene was introduced using RT-PCR.

  For several plants transformed with pBIG-NACAD1SRDX, the shape of the cocoon was observed with a scanning electron microscope (JSM-6330F, manufactured by JEOL Data). The result is shown in FIG. FIG. 4B shows the result of observation of the shape of a wild-type Arabidopsis wing with a scanning electron microscope. As shown in FIG. 4 (a), no cleavage of the cocoon occurs in Arabidopsis transformed with pBIG-NACAD1SRDX. Thus, in Arabidopsis transformed with pBIG-NACAD1SRDX, it was confirmed that the cleavage of the wrinkles did not occur completely, or although not shown in the figure, it occurred only incompletely. As a result, as shown in the right side of FIG. 5, almost no fruits and seeds were formed in the Arabidopsis plant transformed with pBIG-NACAD1SRDX. In addition, the left side of FIG. 5 shows a wild strain of Arabidopsis thaliana.

  Moreover, about 37 plant bodies transformed with pBIG-NACAD1SRDX, the ratio of the mass of the seeds harvested by each individual to the dry weight of the above-ground parts other than the seeds is examined, and completely different Arabidopsis plants that normally bear fruit Compared to body groups. The result is shown in FIG. 6A and 6B, the vertical axis represents the number of individuals, and the horizontal axis represents the mass value of (mass of harvested seeds / dry weight of above-ground parts other than seeds) × 100. For example, 20 on the horizontal axis means that the calculated value of (mass of harvested seeds / dry weight of above-ground parts other than seeds) × 100 is greater than 10 and 20 or less. As shown in FIG. 6, in the plant group transformed with pBIG-NACAD1SRDX (FIG. 6 (a)), the individual group compared with the plant group (FIG. 6 (b)) that normally produces fruit. Many individuals were observed in which the ratio of the mass of the harvested seed to the dry weight of the above-ground parts other than the seed decreased. Here, the mass of seeds means the total mass of seeds harvested by one individual. This indicates that the plant group transformed with pBIG-NACAD1SRDX has almost no seeds since the cleavage of the cocoon is suppressed in the natural state. Moreover, the seeds obtained in the plant group transformed with pBIG-NACAD1SRDX were obtained by self-pollination of pollen released from incompletely split cocoons.

  Furthermore, in the plant body transformed with pBIG-NACAD1SRDX and dehiscence of the cocoon was suppressed, it was examined whether or not the fruit was produced when pollen in the cocoon was taken out and pollinated. In FIG. 7, the case where the pollen is forcibly taken out by tweezers from the wrinkle that has not been cleaved and pollinated is indicated by an arrow, and the case where nothing is performed is indicated by an arrowhead. As shown by the arrows in FIG. 7, even when the dehiscence of the wrinkles is completely suppressed, the pollen itself has fertility because the pollen was taken out and pollinated. It was confirmed. From this result, it was found that the plant transformed with pBIG-NACAD1SRDX has fertility in the pollen itself, but cannot pollinate because the wrinkle does not cleave, resulting in no fruiting. In addition, when the buds were pollinated by a flower that did not dehiscence, the fruiting was confirmed, confirming that the female organ (stamen) has fertility.

  Thus, in this invention, the plant body by which the cleavage of the cocoon was suppressed by suppressing the transcription | transfer of the gene involved in the cleavage of the cocoon can be obtained. Therefore, the present invention can be used for various agriculture, forestry, agribusiness, industry for processing agricultural products, food industry, 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. 4 is a process diagram for incorporating a gene encoding the transcription repressor conversion peptide SRDX and the NACAD1 gene into the construction vector p35SG used in the Examples. It is process drawing which shows the construction method of vector pBIGCKH for transformation. (A) is a figure which shows the shape of the cocoon of Arabidopsis thaliana transformed by recombinant expression vector pBIG-NACAD1SRDX in the Example, (b) is a figure which shows the shape of the cocoon of wild-type Arabidopsis thaliana. It is a figure which shows the Arabidopsis thaliana (right side) and wild-type Arabidopsis thaliana (left side) transformed by recombinant expression vector pBIG-NACAD1SRDX in the Example. (A) plots the number of individuals against the class value of “mass of harvested seed × 100 / dry weight of above-ground parts other than seed” of Arabidopsis transformed with the recombinant expression vector pBIG-NACAD1SRDX in the Examples (B) is a graph in which the number of individuals is plotted against the class value of “mass of harvested seed × 100 / dry weight of above-ground parts other than seed” of wild-type Arabidopsis thaliana. In an Example, it is a figure which shows the result of having investigated whether it will produce when the pollen in a bud is taken out and pollinated in the plant body transformed by pBIG-NACAD1SRDX and the dehiscence of the bud was suppressed.

Claims (17)

By producing a chimeric protein in which a transcription factor, which is a protein described in the following (a) or (b), and a functional peptide that converts an arbitrary transcription factor into a transcriptional repression factor in a plant body, A method for producing a plant body in which cleavage of a cocoon is suppressed, characterized by suppressing cleavage of the cocoon.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 134;
(B) an amino acid sequence represented by SEQ ID NO: 134, comprising an amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted, and / or added; A protein having a function of promoting transcription.   The method for producing a plant according to claim 1, wherein the plant further has a fertile female organ.   The method for producing a plant according to claim 1 or 2, wherein the plant further has pollen itself. Claim, characterized in that it comprises a transformation step of a recombinant expression vector comprising a chimeric gene comprising a polynucleotide encoding the gene and the functional peptide encoding said transcription factor is introduced into plant cells 1-3 The method for producing a plant according to any one of the above . The method for producing a plant according to claim 4, wherein the gene described in (c) 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. The method for producing a plant according to claim 4 or 5, further comprising an expression vector construction step for constructing the recombinant expression vector. 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 plant according to any one of claims 1 to 6 , which has an amino acid sequence represented by any of the above. The method for producing a plant according to any one of claims 1 to 6 , wherein the functional peptide is a peptide having an amino acid sequence represented by any one of SEQ ID NOs: 3 to 19 . The method for producing a plant according to any one of claims 1 to 6 , wherein the functional peptide is a peptide described in the following (d), (e), or (f) .
(D) A peptide having the amino acid sequence shown in SEQ ID NO: 20 or 21.
(E) A peptide having an amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 20.
(F) A peptide having an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 21. 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.)
The plant production method according to any one of claims 1 to 6 , wherein the plant body has an amino acid sequence represented by formula (1). 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
(Wherein 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 plant production method according to any one of claims 1 to 6 , wherein the plant body has an amino acid sequence represented by any one of the above. The functional peptide is a peptide having the amino acid sequence shown in SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 36, 37, or 133. It exists, The production method of the plant body of any one of Claims 1-6 characterized by the above-mentioned. The method for producing a plant according to any one of claims 1 to 6, wherein the functional peptide is a peptide having the amino acid sequence represented by SEQ ID NO: 38 or 39. A plant produced by the production method according to any one of claims 1 to 13, in which cleavage of a cocoon is suppressed. The plant body according to claim 14, wherein the plant body 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 13,
A recombination comprising a gene encoding a transcription factor that is the protein described in (a) or (b) below, a polynucleotide encoding a functional peptide that converts any transcription factor into a transcription repressor, and a promoter A kit for inhibiting the dehiscence of plant wings, comprising at least an expression vector .
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 134;
(B) an amino acid sequence represented by SEQ ID NO: 134, consisting of an amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted and / or added, and A protein having a function of promoting transcription. Furthermore, the kit for inhibiting the cleavage of plant folds according to claim 16, further comprising a reagent group for introducing the recombinant expression vector into plant cells.

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