JP2006020607A - Method for producing plant body having modified leaf shape, plant body obtained by using the method, and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a plant body having a stalk in a modified shape. <P>SOLUTION: A kimera protein having a functional peptide fused with a transcription factor is produced in a plant cell by introducing a kimera gene of a gene encoding the transcription factor concerned with the control of a leaf shape, and a polynucleotide encoding the functional peptide for converting the transcription factor to a transcription repressor. The plant body having the stalk in the modified shape is produced thereby because the kimera protein inhibits the expression of a target gene targeted by the transcription factor. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a plant having a modified leaf form, a plant obtained by using the same, and a use thereof, and more particularly to a method for producing a plant having a modified petiole shape and the method. The present invention relates to a plant obtained by use and its use.

  Conventionally, mutants lacking the corn TEOSINTE BRANCHED1 (TB1) gene and the snapdragon CINCINNATA (CIN) gene have been reported to show abnormalities in branching and leaf morphology (for example, Non-Patent Document 1). 2). That is, the maize mutant lacking the TB1 gene does not have the top bud dominance seen in the wild type and grows without suppressing the side buds. In addition, the snapdragon mutant lacking the CIN gene has excessive growth at the edge of the leaf, and the leaf becomes wavy.

  In the above Non-Patent Documents 1 and 2, it is reported by analysis using transposon tagging that both the TB1 gene and the CIN gene have a base sequence encoding a homologous domain shared by TCP family proteins. Has been. The TCP domain, which is a common domain of this TCP family protein, has been identified as a region that is commonly encoded by the maize TB1 gene, the snapdragon CYCLOIDEA (CYC) gene, and the rice PCF1 and PCF2 genes. In addition, it is suggested that this TCP family protein is a transcription factor family because PCF1 and PCF2 proteins function as DNA-binding proteins.

  Furthermore, the TB1 gene and the CYC gene are involved in the growth control of axillary and flower bud meristems, respectively, the CIN gene is presumed to be involved in the control of site-specific cell division in leaves, and the PCF1 and PCF2 genes are required for cell division Since these are transcription factors that bind to the promoter region of various genes and control their transcription, these proteins are presumed to be involved in cell division control among the TCP family proteins. However, the functions of most transcription factors belonging to the TCP family are still unknown.

  By the way, the present inventor has found various peptides that convert a transcription factor into a transcription repressing factor (see, for example, Patent Documents 1 to 7, Non-Patent Documents 3 and 4). 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.

  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.

In addition, the plant in which the form of the petiole is modified has advantages such as space saving of plant cultivation, in addition to the advantage of high horticultural value. So far, in order to modify a trait such as the shape of a leaf of a plant, a cross breeding in which varieties of plants having the target trait are crossed is generally used. However, conventional cross breeding requires a long time and experience of a skilled person in order to produce a plant having the desired trait. In recent years, attempts have been made to modify plant traits using genetic recombination techniques. As such an attempt, for example, by overexpressing the KNAT1 and KNAT2 genes of Arabidopsis belonging to the knox transcription factor family, various expression types such as split leaves and ectopic leaves are reported. (For example, see Non-Patent Document 5).
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) Doebley, J., Stec, A., Hubbard, L., Nature, 386, 485-488 (1997) Nath, U., Crauford, BCW, Carpenter, R., Coen, C., Science, 299, 1404-1407 (2003) 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 Lincoln, C., Long, J., Yamaguchi, J., Serikawa, K., Hake, S., The Plant Cell, Vol. 6, 1859-1876, December, 1994

  However, conventionally, there has been no known technique for altering the petiole morphology by suppressing gene transcription.

  An object of the present invention is to provide a method for producing a plant having a modified petiole morphology easily in a short period of time by suppressing gene transcription.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has converted a transcription factor that has a TCP domain and whose function has not been clarified and an arbitrary transcription factor into a transcriptional repressor. It was revealed for the first time that a plant body having a modified petiole shape can be produced by producing a chimeric protein fused with a functional peptide to be produced in the plant body, and the present invention has been completed.

  That is, in the method for producing a plant having a modified petiole morphology according to the present invention, a transcription factor involved in leaf morphology control and a functional peptide that converts an arbitrary transcription factor into a transcription repressor are fused. It is characterized in that transcription of a target gene targeted by the transcription factor is suppressed by producing a chimeric protein in a plant body.

  Thereby, the said chimeric protein can suppress effectively transcription | transfer of the target gene of the said transcription factor. Therefore, there is an effect that the form of the petiole is modified.

  It is preferable that the plant body in which the form of the petiole is modified is a plant body modified so that at least the length of the petiole is shortened.

  This makes it possible to increase the horticultural value of the plant body and to save space for plant cultivation.

  The plant production method 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. It is preferable. The production method may further include an expression vector construction step for constructing the recombinant expression vector.

  Thereby, the chimeric protein can be expressed in the transformed plant cell. Therefore, the chimeric protein suppresses the transcription of the target gene and has an effect that the shape of the petiole is modified.

  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 represented by SEQ ID NO: 134, and is involved in leaf morphology control;

  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 leaf morphology control.

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 consists of the amino acid sequence represented by either.

  Moreover, it is preferable that the said functional peptide is a peptide which consists of an amino acid sequence shown in either of sequence number 3-19.

  The functional peptide may be a peptide described in the following (e) or (f). (E) A peptide consisting of the 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 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 consist of 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 consist of an amino acid sequence represented by any of the above.

  The functional peptide is shown in SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 133, 59, or 60. It may be a peptide consisting of an amino acid sequence.

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

  The functional peptide is a peptide consisting of the amino acid sequence represented by any one of the above formulas or any one of the peptides shown in the above SEQ ID NOs, most of which are extremely short peptides, and therefore are easily synthesized. Transcriptional repression of the target gene can be performed efficiently. Further, the functional peptide has a function of suppressing the transcription (expression) of the target gene in preference to the activity of other transcription factors that are functionally redundant (redundant). Therefore, it has the effect that the expression of the target gene can be effectively suppressed.

  Moreover, the plant body concerning this invention is produced by the said production method, The shape of a petiole is modified, 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 plant petiole shape modification kit according to the present invention is a kit for performing the production method described above, and includes a gene encoding the transcription factor and a functional peptide that converts the transcription factor into a transcriptional repression factor. It includes at least a recombinant expression vector containing a polynucleotide that encodes and a promoter. The petiole shape modification kit may further include a reagent group for introducing the recombinant expression vector into a plant cell.

  That is, as described above, the method for producing a plant having a modified petiole shape according to the present invention includes a transcription factor involved in leaf shape control and a functional peptide that converts any transcription factor into a transcription repressor. Is characterized in that the transcription factor suppresses the transcription of a target gene that promotes transcription by producing a chimeric protein in which the transcription factor is fused in a plant body.

  Thereby, in the obtained plant body, transcription of a gene involved in leaf shape control is suppressed, and it becomes possible to produce a plant body in which the shape of the petiole is modified.

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

  The present invention is a technique for producing a plant having a modified petiole shape, and is a chimeric protein in which a transcription factor relating to leaf shape control is fused with a functional peptide that converts an arbitrary transcription factor into a transcriptional repressor Is produced in a plant body. In a plant obtained by producing the chimeric protein in a plant, transcription of a target gene targeted by the transcription factor can be suppressed, and a plant with a modified petiole morphology can be produced.

  Here, alteration of the shape of the petiole occurs as follows. That is, the transcription factor-derived DNA binding domain in the chimeric protein binds to the 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 petiole of the obtained plant can be modified.

  The plant body with a modified petiole form produced by the production method of the present invention is not particularly limited as long as it is a plant body with a modified petiole form. Here, examples of the form of the petiole include, but are not limited to, the length of the petiole, the thickness of the petiole, the shape of the cross section of the petiole, the state of the surface of the petiole, and the like. In addition, the plant body in which the form of the petiole is modified may be a plant body in which the form of the petiole is modified and the form of the other leaves is not modified, or the form of the petiole is modified. At the same time, other leaf forms may be modified.

  Among these, the plant body in which the form of the petiole of the present invention is modified is preferably a plant body modified so that at least the length of the petiole is shortened. Therefore, the plant body in which the form of the petiole is modified may be a plant body in which the length of the petiole is shortened and the form of other leaves is not modified, or the length of the petiole is short. At the same time, the form of other leaves may be modified. Among these, the plant body in which the form of the petiole is modified is more preferably a plant body in which the length of the petiole is shortened and the form of other leaves is not modified. Further, the length of the petiole may be shortened, and at the same time, the shape of other leaves may be slightly modified. The fact that the length of the petiole is shortened is not particularly limited as long as the length of the petiole is shorter than that before modification, and includes those in which the petiole is extremely short and becomes no-foliate. The length of the petiole means the length in the axial direction connecting the base of the petiole and the blade and the border of the petiole. “Leaf” includes leaf blades, petiole and bamboo leaves. Here, “leaf blade” refers to the main part of the leaf composed of epidermis, mesophyll, and veins, and “petiole” refers to the portion of the leaf that supports the leaf blade and adheres to the stem. The term “bamboo leaf” refers to a leafy organ that develops on the petiole or on the stem near the base of the petiole.

  In the following description, a chimeric protein used in a method for producing a plant with a modified petiole according to the present invention, an example of a method for producing a plant according to the present invention, a plant obtained thereby and its usefulness, Each of these will be described.

(I) Chimeric protein used in the present invention As described above, the chimeric protein used in the present invention fuses a transcription factor involved in leaf morphology control and a functional peptide that converts the transcription factor into a transcription repressor. It has been made.

  Moreover, the chimeric protein used in the present invention acts dominantly on the endogenous gene. That is, the chimeric protein according to the present invention promotes transcription by a transcription factor involved in leaf morphology control even if the plant is diploid or diploid, or a functionally duplicated gene exists in the plant. Can uniformly suppress the target gene expression. Therefore, the plant into which the chimeric protein is introduced can be effectively transformed into a plant having a modified petiole shape.

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

(I-1) Transcription factor involved in leaf morphology control The transcription factor used in the present invention is not particularly limited as long as it is a transcription factor involved in leaf morphology control. Such transcription factors are widely conserved in the plant kingdom. Therefore, transcription factors used in the present invention include transcription factors having similar functions that are conserved in various plants.

  Examples of such transcription factors include TCP1 protein, which is a transcription factor containing a TCP domain, but the transcription factor is not limited thereto.

  As a typical example of the transcription factor used in the present invention, for example, Arabidopsis TCP1 protein can be mentioned. The TCP1 protein is a protein consisting of the amino acid sequence shown in SEQ ID NO: 134. In the present invention, for example, the TCP1 protein, which is a transcription factor, is converted into a transcription repressing factor by fusing the functional peptide described later to the TCP1 protein.

  The transcription factor used in the present invention is not limited to the TCP1 protein consisting of the amino acid sequence shown in SEQ ID NO: 134, and may be any transcription factor involved in leaf morphology control. 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. In addition, the range of “one or several” in the above “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” 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 leaves It may be a transcription factor involved in morphological control. Here, “homology” is the ratio of the same sequence in the amino acid sequence, and the higher this value, the closer the two are.

  Further, the amino acid sequences of transcription factors involved in leaf morphology control used in the present invention are considered to be highly conserved among many plants of different species. Therefore, it is not always necessary to isolate a unique transcription factor and its gene involved in leaf shape control in individual plants that want to modify the petiole shape. That is, by introducing the chimeric protein constructed in Arabidopsis thaliana, which will be described later in Examples, into other plants, it is possible to easily produce plants with modified petiole morphology 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, 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. For example, when the TCP1 protein is used as the transcription factor, the gene encoding the TCP1 protein (for convenience of explanation, (Referred to as TCP1 gene). As a specific example of the TCP1 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 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. 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 the base sequence of a gene encoding a known transcription factor 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 a transcription factor into a transcriptional repressing factor As used in the present invention, a functional peptide that converts a transcription factor into a transcriptional repressing factor (referred to as a transcription repressing conversion peptide for convenience of explanation) The peptide is not particularly limited as long as it is a peptide capable of suppressing 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 conversion peptides found by the present inventors (see Patent Documents 1 to 7, Non-Patent Documents 1 and 2, 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 a plant cell, thereby succeeding in actually suppressing the transcription of the gene (for example, Patent Documents 1 to 3). 4). In addition, a gene encoding tobacco ERF3 protein (see, for example, Patent Document 5), a rice OsERF3 protein (see, for example, Patent Document 6), which is one of the Class II ERF gene groups, and one of the zinc finger protein gene groups. 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 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 converts a transcription factor into a transcriptional repressor. It has been found that it has a function to

  In addition, 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 a transcription factor into a transcriptional repressor, and a gene encoding the SUPERMAN protein is transcribed. It has also been found that a chimeric gene bound to a DNA encoding domain of a factor or a gene encoding a transcription factor produces a protein having a strong transcriptional repression ability.

  Therefore, as an example of the transcriptional repression converting peptide used in the present invention, in the present embodiment, the class II ERF protein, AtERF3 protein derived from Arabidopsis thaliana, the same AtERF4 protein, the same AtERF7 protein, the same AtERF8 protein, the tobacco ERF3 protein, the rice 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 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), 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 consisting of the 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: 20 or 21.
(F) any one of the amino acid sequences shown in SEQ ID NO: 20 or 21, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and transcription of transcription factors A peptide having a function of converting to a suppressor.

  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 according thereto, for example, a mutation introduction kit (for example, Mutant-) using site-directed mutagenesis. 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 a peptide consisting of 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 consisting of 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 consisting of 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 consisting of the amino acid sequence represented by the above formulas (5) to (9), SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, Peptides having an amino acid sequence represented by 31, 32, 33, 34, 35, 36, 37, 133, 59, or 60 can be mentioned. Among these, the peptide of SEQ ID NO: 29, 30, 32, 34, 133, 59, or 60 corresponds to the peptide represented by the general formula (6), and the peptide of SEQ ID NO: 22, 25, 35, 36, or 37 is The peptide of SEQ ID NO: 26, 27, 28, 31, or 33 corresponds to the peptide of general formula (8), and the peptide of SEQ ID NO: 23 or 24. 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 / converting peptide consisting of 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, Poly, consisting of the base sequence shown in 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 1, 55, or 57 Mention may be made of nucleotides. 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, 2, 56, or 58 are each a polynucleotide complementary to the polynucleotide exemplified above. It is a nucleotide. 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, 133, 59, 60 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 A method for producing a plant according to the present invention includes a step of introducing the chimeric protein described in (I) above into a plant and modifying the form of the petiole. However, if the production method of the plant body according to the present invention is shown by specific steps, for example, a production method including steps such as an expression vector construction step, a transformation step, and a selection step Can be mentioned. 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 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 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 after growing the transformant, the plant itself is selected from the petiole form. Also good. For example, as an example of selecting from the form of a petiole, a method of comparing the form of a petiole of a transformant with the form of a petiole of a plant body that has not been transformed can be mentioned (see Examples described later). In particular, the shape of the petiole can be selected simply by comparison, and the effect itself of the present invention of alteration of the form of the petiole can be confirmed.

  In the plant production method according to the present invention, since the chimeric gene is introduced into the plant body, it is possible to obtain offspring from which the petiole shape is simply modified 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) 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 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 petiole of the obtained plant can be modified. 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 form of the petiole according to the present invention is modified are not particularly limited. Plants whose usefulness is increased 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.

(III-2) Usefulness of the Present Invention In the present invention, the form of the petiole of the plant can be modified, but the usefulness of the present invention is not particularly limited, and the effect is obtained by modifying the form of the petiole. It may be in a certain field. Examples of such fields include application to creation of new horticultural varieties, application to space saving of plant cultivation, and the like.

  First, an application example for creation of a new horticultural variety will be described. For example, by using the plant production method according to the present invention, a new houseplant or flower plant having a large change in leaf morphology is produced. be able to.

  Moreover, when space saving of plant cultivation is demonstrated, for example, the plant body modified so that the petiole may be shortened can cultivate many plants in a small space. Therefore, it is expected to contribute to space saving in plant cultivation.

(III-3) Example of Utilization of the Present Invention The field of utilization and utilization method of the present invention are not particularly limited, but as an example, a kit for performing the plant production method according to the present invention, that is, a petiole Mention may be made of form modification kits.

  Specific examples of this petiole shape modification kit may include at least a recombinant expression vector containing a chimeric gene comprising a gene encoding the above transcription factor and the above transcription repressor conversion polynucleotide. It is more preferable if it contains a reagent group for introducing a bacterium into a plant cell. 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. 4, this invention is not limited to a following example.

[Example 1]
In Example 1 below, a 12 amino acid peptide LDLDLELRLGFA (SRDX) (SEQ ID NO: 19), 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 in which a polynucleotide in which a polynucleotide to be bound was downstream of the TCP1 gene was constructed was constructed and 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 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) A DNA fragment consisting of the following SEQ ID NOs: 140 and 141 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 fragment comprising the sequences of SEQ ID NOs: 140 and 141 contains 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: 140)
5'-cgtcgacggtacccccgggatctgtaattgtaatgttgtttgttgtttgttgttgttggtaattgtggatcct-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 consisting of the following sequences, which were designed to encode the 12 amino acid transcription repressor conversion peptide LDLDLELRLGFA (SRDX) and have a stop codon TAA at the 3 ′ end, were respectively 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: 142)
5'-tcgacttaagcgaaacccaaacggagttctagatccagatcaagccc-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) 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 TCP1 gene into construction vector>
A gene encoding a transcription factor TCP1 protein derived from Arabidopsis thaliana 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 TCP1 gene excluding the stop codon was amplified by PCR using the following primers.
Primer 1 (TCP1-F) 5′-ATGTCGTCTTCCACCAATGAC-3 ′ (SEQ ID NO: 144)
Primer 2 (TCP1-R stopless) 5′-GTTTACAAAAGAGTCTTGAATCC-3 ′ (SEQ ID NO: 145)
The cDNA of TCP1 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 TCP1 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, p35STCP1SRDXG in which a DNA fragment of the TCP1 coding region is inserted in the forward direction, LR buffer 4.0 μL diluted with 1.5 μL (about 300 ng) and pBIGCKH 4.0 μL (about 600 ng) and TE buffer (10 mM TrisCl) 5.5 μL of pH 7.0, 1 mM EDTA) was 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-TCP1SRDX, which is a plasmid in which the DNA fragment containing the chimeric gene is incorporated into pBIGCKH, and transformed plants are 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-TCP1SRDX, was introduced into a soil bacterium ((Agrobacterium tumefaciens strain GV3101 (C58C1Rifr) pMP90 (Gmr) (koncz and Sahell 1986)) strain by electroporation. Culturing was carried out in an YEP medium containing 1 liter of antibiotics (kanamycin (Km) 50 μg / ml, gentamicin (Gm) 25 μg / ml, rifampicillin (Rif) 50 μg / ml) until the OD600 was 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. 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 gene of TCP1SRDX was introduced using RT-PCR.

  Fig. 4 (b) shows a plant transformed with pBIG-TCP1SRDX, and Fig. 4 (a) shows a wild-type plant. As shown in FIGS. 4A and 4B, it was found that the Arabidopsis transformed with pBIG-TCP1SRDX had an extremely short petiole compared to the wild type. As a result, the plant transformed with BIG-TCP1SRDX showed a form in which the leaves overlapped.

[Reference example]
In the following reference examples, Arabidopsis thaliana-derived TCP1 gene is linked downstream of the CaMV35S promoter to construct a recombinant expression vector, which is introduced into Arabidopsis thaliana using the Agrobacterium method, thereby transforming Arabidopsis thaliana, Transformed plants overexpressing TCP1 were produced.

<Construction of vector for construction of transformation vector>
In the same manner as in Example 1, as shown in FIG. 1, p35SG, a vector for constructing a transformation vector, was constructed.

<Construction of transformation vector>
In the same manner as in Example 1, as shown in FIG. 3, a transformation vector pBIGCKH was constructed.

<Incorporation of TCP1 gene into construction vector>
A gene encoding a transcription factor TCP1 protein derived from Arabidopsis thaliana was incorporated into the construction vector p35SG 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 containing the stop codon of Arabidopsis TCP1 gene was amplified by PCR using the following primers.
Primer 1 (TCP1-F) 5′-ATGTCGTCTTCCACCAATGAC-3 ′ (SEQ ID NO: 144)
Primer 2 (TCP1-R) 5′-TCAGTTTACAAAAGAGTCTTGAATCC -3 ′ (SEQ ID NO: 146)
The cDNA of TCP1 gene and the encoded amino acid sequence are shown in SEQ ID NOs: 135 and 134, respectively.

  (2) The obtained DNA fragment of the TCP1 coding region was ligated to the SmaI site of the construction vector p35SG 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.

<Construction of recombinant expression vector>
By recombining with the plant transformation vector pBIGCKH in the same manner as in [Example 1] except that a DNA fragment containing the CaMV35S promoter, TCP1 gene, Nos-ter, etc. on the above construction vector was used, An expression vector as a host was constructed.

<Production of plant body transformed with recombinant expression vector>
Next, except for using pBIG-TCP1, which is a plasmid in which the DNA fragment containing the TCP1 gene was incorporated into pBIGCKH, Arabidopsis thaliana was transformed in the same manner as in [Example 1] to produce a transformed plant body. .

  An average of 50 transformed plants that are hygromycin-resistant plants were obtained from about 5000 seeds that were sown. Total RNA was prepared from these plants, and it was confirmed that the TCP1 gene was introduced using RT-PCR.

  In the transformed plant body in which TCP1 was overexpressed using 35S-TCP1, the petiole length did not change compared to the wild type plant body. As a cause of this, since the TCP1 transcription factor is already sufficiently present in the plant body, it is considered that the change that shows the opposite phenotype did not occur even when overexpressed.

  As described above, in the present invention, a plant body having a modified petiole morphology can be obtained by suppressing transcription of a target gene targeted by a transcription factor involved in leaf morphology control. 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. 3 is a process diagram for incorporating a gene encoding a transcription repressor conversion peptide SRDX and a TCP1 gene into a construction vector p35SG used in Examples. It is process drawing which shows the construction method of vector pBIGCKH for transformation. (A) is a figure which shows the leaf of wild type Arabidopsis thaliana, (b) is a figure which shows the leaf of Arabidopsis thaliana transformed by recombinant expression vector pBIG-TCP1SRDX.

Claims (17)

  Targeting the transcription factor by producing in the plant a chimeric protein that fuses a transcription factor involved in leaf morphology control and a functional peptide that converts any transcription factor into a transcriptional repressor A method for producing a plant having a modified petiole shape, which comprises suppressing gene transcription.   The method for producing a plant according to claim 1, wherein the plant having a modified petiole shape is a plant modified so that at least the length of the petiole is shortened.   2. 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. Or the production method of the plant body of 2.   The plant production method according to claim 3, further comprising an expression vector construction step of constructing the recombinant expression vector. The method for producing a plant according to any one of claims 1 to 4, wherein the transcription factor is 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 represented by SEQ ID NO: 134, and is involved in leaf morphology control; The method for producing a plant according to claim 3 or 4, 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 leaf morphology control. 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 comprises an amino acid sequence represented by any one 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 comprising 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 (e) or (f) below.
(E) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 20 or 21.
(F) In the amino acid sequence shown in SEQ ID NO: 20 or 21, it consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and the transcription factor is a transcription repressor. A peptide having a function to convert. 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 method for producing a plant according to any one of claims 1 to 6, wherein the plant is composed of 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 method for producing a plant according to any one of claims 1 to 6, which comprises an amino acid sequence represented by any one of the above.   The functional peptide is an amino acid represented by SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 133, 59, or 60. It is a peptide consisting of a sequence, The plant production method according to any one of claims 1 to 6.   The method for producing a plant according to any one of claims 1 to 6, wherein the functional peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 38 or 39.   A plant having a modified petiole shape produced by the production method according to claim 1.   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 plant petiole pattern comprising at least a recombinant expression vector comprising a gene encoding the above transcription factor, a polynucleotide encoding a functional peptide that converts the transcription factor into a transcription repressor, and a promoter. Modification kit.   The plant petiole shape modification kit according to claim 16, further comprising a reagent group for introducing the recombinant expression vector into a plant cell.

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