JP2006006248A - Method for producing plant body of which leaf morphogenesis is regulated, plant body obtained by using the same and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a plant body of which ratio of the length of leaf blade in a side directional axial direction to that in a base part to tip end part axial direction is changed. <P>SOLUTION: This modified plant body so that the ratio of the length of the leaf blade in the side directional axial direction to that in the base part to tip end part axial direction becomes larger is provided by introducing a chimera gene of a gene encoding a transcription factor associated with the regulation of extending growth of the leaf body in the base part to tip end part axial direction with a polynucleotide encoding a functional peptide for converting an optional transcription factor to a transcription inhibitory factor into plant cells to produce a chimera protein obtained by fusing the transcription factor with the peptide in the plant cells. By excessively expressing the gene encoding the transcription factor in the plant body, the modified plant body of which the ratio of the length of the leaf blade in the side directional axial direction to that in the base part to tip end part axial direction becomes smaller, is produced. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a plant body in which leaf morphogenesis is controlled, a plant body obtained by using the plant body, and use thereof, and particularly to a lateral direction with respect to the axial length of the base end of the leaf blade. The present invention relates to a method for producing a plant body in which the ratio of lengths in the axial direction is modified, a plant body obtained using the plant body, and use thereof.

  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 shape (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 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 an arbitrary transcription factor into a transcriptional repression 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 ratio of the length in the lateral axial direction to the length in the axial direction at the distal end of the base of the leaf blade is modified has advantages such as high horticultural value and space saving in plant cultivation. Have Until now, in order to modify a trait related to the form of a leaf of a plant, 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 the traits of plant leaf morphology 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 not been known a technique for modifying the ratio of the length in the lateral axis direction to the length in the axial direction of the proximal end of the leaf blade by a genetic engineering method using a gene encoding a transcription factor.

  An object of the present invention is to use a genetic engineering technique using a gene encoding a transcription factor, and to easily and in a short period, the ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end of the leaf blade. Is to provide a method for producing a modified plant.

  As a result of intensive studies to solve the above problems, the present inventor has converted a transcription factor that is a TCP family protein having a TCP domain and whose function has not been clarified, and an arbitrary transcription factor into a transcription repressor. Modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the proximal end of the leaf blade is increased by producing a chimeric protein fused with the functional peptide We found for the first time that a body was obtained. Further, when the same transcription factor is excessively produced in the plant body, a plant body modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade is reduced is obtained. I also found out. Further, from these findings, it has been found for the first time that such a TCP transcription factor is a transcription factor involved in the control of the elongation growth in the axial direction of the distal end of the base of the leaf blade, and the present invention has been completed.

  That is, the method for producing a plant according to the present invention fuses a transcription factor involved in the control of elongation growth in the axial direction of the proximal tip of the leaf blade and a functional peptide that converts any transcription factor into a transcription repressor. By producing the chimeric protein thus produced in the plant body, the transcription of the gene targeted by the transcription factor is suppressed, and the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade is It is characterized by being modified to be larger.

  Thereby, the chimeric protein can effectively suppress the transcription of the gene targeted by the transcription factor. Therefore, there is an effect that modification is made so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade is increased.

  The plant production method includes a transformation step of introducing a recombinant expression vector containing a chimeric gene comprising a gene encoding a transcription factor and a polynucleotide encoding the functional peptide into a plant cell. Is preferred. 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 of being modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base part of the leaf blade is increased.

  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: 137, 139 or 141; (B) In the amino acid sequence shown in SEQ ID NO: 137, 139 or 141, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and the tip of the base of the leaf blade A protein involved in the control of elongation in the axial direction.

  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: 138, 140 or 142 as an open reading frame region. (D) The length in the axial direction of the base end of the leaf blade, which hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 138, 140 or 142 under stringent conditions A gene that encodes a protein involved in leaf morphogenesis with respect to the ratio of the length in the lateral axis to the.

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.

  Moreover, it is preferable that the said functional peptide is a peptide which has 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 having 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 transcription of any transcription factor is repressed. A peptide having a conversion function as a factor.

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 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. A peptide having the amino acid sequence described above may be used.

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

  The functional peptide is a peptide having an amino acid sequence represented by any of the above formulas or any of the peptides represented by the above SEQ ID NOs, 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, there is an effect that the expression of the target gene can be effectively suppressed.

  In addition, the plant body production method according to the present invention allows the plant body to produce excessively a transcription factor involved in the control of elongation growth in the axial direction of the distal end of the base of the leaf blade, whereby the axial direction of the proximal end of the leaf blade It may be modified so that the ratio of the length in the lateral axis direction to the length of the length becomes smaller.

  Thereby, the transcription factor produced in excess can promote the transcription of the gene targeted by the transcription factor. Therefore, there is an effect that the length of the leaf blade is modified so that the ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end portion of the leaf blade is reduced.

  Preferably, the plant body production method includes a transformation step of introducing a recombinant expression vector containing the gene encoding the transcription factor into a plant cell so as to express the gene encoding the transcription factor. . The production method may further include an expression vector construction step for constructing the recombinant expression vector.

  Thereby, the transcription factor can be overexpressed in the transformed plant cell. Therefore, the transcription of the gene targeted by the transcription factor is promoted, and the effect is that the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade is modified to be small.

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: 137, 139 or 141;
(B) In the amino acid sequence shown in SEQ ID NO: 137, 139 or 141, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and the tip of the base of the leaf blade A protein involved in the control of elongation in the axial direction.

  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: 138, 140 or 142 as an open reading frame region. (D) The length in the axial direction of the base end of the leaf blade, which hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 138, 140 or 142 under stringent conditions A gene that encodes a protein involved in leaf morphogenesis with respect to the ratio of the length in the lateral axis to the.

  Moreover, the plant body concerning this invention is produced by the said production method, The ratio of the length of the side axial direction with respect to the length of the base part front-end | tip part axial direction of a leaf blade is changed, 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 leaf shape modification kit according to the present invention is a kit for performing the production method described above, and a function of converting a gene encoding the transcription factor and an arbitrary transcription factor into a transcriptional repression factor. It comprises at least a recombinant expression vector comprising a polynucleotide encoding a sex peptide and a promoter.

  A plant leaf shape modification kit according to the present invention is a kit for performing the production method described above, and includes at least a recombinant expression vector comprising a gene encoding the transcription factor and a promoter. It may be.

  The plant leaf shape modification kit may further contain a reagent group for introducing the recombinant expression vector into plant cells.

Moreover, the production method of the plant body in which the ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end of the leaf blade according to the present invention is modified by the protein described in the following (a) or (b) The gene encoding,
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 137, 139 or 141,
(B) In the amino acid sequence shown in SEQ ID NO: 137, 139 or 141, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and the tip of the base of the leaf blade Proteins involved in the control of triaxial elongation growth,
Or the gene described in the following (c) or (d),
(C) a gene having the base sequence represented by SEQ ID NO: 138, 140 or 142 as an open reading frame region,
(D) An extension growth in the axial direction of the proximal end of the leaf blade, which hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 138, 140 or 142 under stringent conditions. A gene encoding a protein involved in the regulation of
It includes a transformation step of introducing a recombinant expression vector containing the above into a plant cell.

  That is, the plant production method according to the present invention, as described above, is a function that converts a transcription factor involved in the control of elongation growth in the axial direction of the proximal end of the leaf blade and an arbitrary transcription factor into a transcriptional repressor. By producing a chimeric protein fused with a sex peptide in the plant body, the transcription of the gene targeted by the transcription factor is suppressed, and the axial length in the lateral direction relative to the axial length of the base of the leaf blade is suppressed. It is characterized in that the length ratio is modified so as to increase.

  Thereby, the chimeric protein can effectively suppress the transcription of the gene targeted by the transcription factor. Therefore, there is an effect that modification is made so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade is increased.

  In addition, as described above, the method for producing a plant according to the present invention causes an excessive production of a transcription factor involved in the control of elongation growth in the axial direction of the distal end of the base of the leaf blade. A modification is made such that the ratio of the length in the lateral axial direction to the length in the axial direction of the base distal end portion is reduced.

  Thereby, the transcription factor produced in excess can promote the transcription of the gene targeted by the transcription factor. Therefore, there is an effect that the length of the leaf blade is modified so that the ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end portion of the leaf blade is reduced.

  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 body in which the ratio of the length in the lateral axial direction to the length in the axial direction of the base end of the leaf blade is modified, one of which is the axial direction of the base distal end portion of the leaf blade A chimeric protein is produced in a plant by fusing a transcription factor that is involved in the control of elongation growth of the protein and a functional peptide that converts an arbitrary transcription factor into a transcription repressor. One of them is that the above transcription factor is excessively produced in a plant body.

  In the plant obtained by producing the chimeric protein in the plant, since the transcription of the target gene of the transcription factor is suppressed, the length in the lateral axial direction with respect to the length in the axial direction of the proximal end of the leaf blade Plants modified so as to increase the ratio of can be produced.

  In the method for producing the chimeric protein, the modification is performed as follows so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade is increased. That is, the DNA binding domain derived from the transcription factor 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 plant body obtained can be modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the distal end portion of the base of the plant body is increased.

  In addition, in the plant obtained by excessive production of the transcription factor in the plant, transcription of the target gene of the transcription factor is promoted, so that the lateral axis relative to the axial length of the base part of the leaf blade Plants modified so that the ratio of the lengths in the direction is reduced can be produced.

  The plant body in which the ratio of the length in the lateral axial direction to the length in the axial direction of the base distal portion of the leaf blade produced by the production method of the present invention is modified. Modified so that the ratio of the length in the lateral axial direction to the length in the axial direction of the base of the base part of the plant body and the leaf blade was modified so that the ratio of the length in the lateral axis direction relative to It is a concept including a plant body. The plant body in which the ratio of the length in the lateral axial direction to the length in the axial direction at the base distal end of the leaf blade is at least the length in the lateral axial direction relative to the length in the axial direction at the proximal distal end of the leaf blade Any plant that has been modified to have a large or small ratio may be used. Therefore, the plant body in which the ratio of the length in the lateral axial direction to the length in the axial direction at the base distal end of the leaf blade is modified is the length in the lateral axial direction with respect to the length in the axial direction at the proximal distal end of the leaf blade. It may be a plant whose shape of other leaves is not modified, or the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade Plants in which other leaf forms are also modified at the same time as is modified. Among these, plants with modified leaf morphology are modified only in the ratio of the length in the lateral axial direction to the length in the axial direction of the base of the base of the leaf blade, and other leaf shapes are modified. More preferably, the plant body is not. Further, the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade may be modified, and at the same time, the shape of other leaves may be slightly modified. Examples of such a plant include a plant whose peripheral surface of the leaf blade is curved at the same time as the ratio of the length in the lateral axial direction to the length in the axial direction of the base tip of the leaf blade is modified. Can do. Further, the leaf blade is changed from, for example, an ellipse to a shape close to a circle by modifying the leaf blade so that the ratio of the length in the lateral axial direction to the length in the axial direction of the distal end of the base portion of the leaf blade is increased. Similarly, the leaf blade changes from, for example, an ellipse to a more vertically elongated ellipse by changing the ratio of the length in the lateral axial direction to the length in the axial direction of the base end of the leaf blade. To do. Therefore, the plant body in which the ratio of the length in the lateral axis direction to the length in the axial direction of the base portion of the leaf blade is modified can be regarded as a plant body in which the shape of the leaf blade has changed. The ratio of the length in the lateral axial direction to the length in the axial direction of the base end of the leaf blade is modified so that the length in the lateral axial direction relative to the length in the axial direction of the base distal end of the leaf blade The ratio is not particularly limited as long as the ratio is increased as compared with that before modification. Further, it is modified so that the ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end of the leaf blade is reduced. There is no particular limitation as long as the ratio of the lengths decreases as compared with that before modification. The axial direction of the base end of the leaf blade refers to the direction along the axis connecting the base and the distal end of the leaf blade, and the side axial direction refers to the axial direction of the base distal end on the surface of the leaf blade. The vertical direction.

  In the following description, a method for producing a plant body modified so that the ratio of the length in the lateral axial direction to the length in the axial direction of the base distal end portion of the leaf blade according to the present invention is increased, and the leaf blade according to the present invention Plant production methods modified so that the ratio of the length in the lateral axial direction to the length in the axial direction at the base tip of the plant is reduced, the plants obtained by these production methods, their usefulness, and their use Each will be described.

(I) Method for producing plant body modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade is increased (I-1) Chimeric protein used in the present invention As described above, the chimeric protein used in the present invention is a fusion of a transcription factor involved in the control of elongation growth in the axial direction of the proximal tip of the leaf blade and a functional peptide that converts any 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 controls the elongation growth in the axial direction of the proximal end of the leaf blade, even if the plant is diploid or diploid or the plant has a function duplication gene. Can uniformly suppress the expression of genes involved in. Therefore, the plant body into which the chimeric protein has been introduced is effectively converted into a plant body that has been modified so that the ratio of the length in the lateral axial direction to the length in the axial direction of the base end of the leaf blade is increased. Can be transformed.

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

(I-1-1) Transcription factor involved in the control of elongation growth in the axial direction of the proximal tip of the leaf blade The transcription factor used in the present invention is involved in the control of elongation growth in the axial direction of the proximal tip of the leaf blade The transcription factor is not particularly limited. 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 TCP5, TCP13, TCP17, and the like, which are transcription factors including a TCP domain, but the transcription factor is not limited thereto.

  Representative examples of transcription factors used in the present invention include Arabidopsis TCP5 protein, TCP13 protein, and TCP17 protein.

  The TCP5 protein is a protein having the amino acid sequence shown in SEQ ID NO: 137, the TCP13 protein is a protein having the amino acid sequence shown in SEQ ID NO: 139, and the TCP17 protein has the amino acid sequence shown in SEQ ID NO: 141. It is a protein. In the present invention, for example, the TCP5 protein, TCP13 protein, or TCP17 protein, which is a transcription factor, is converted into a transcriptional repressing factor by fusing a functional peptide described later to the TCP5 protein, TCP13 protein, or TCP17 protein.

  The transcription factor used in the present invention is not limited to the TCP5 protein, TCP13 protein, or TCP17 protein having the amino acid sequences shown in SEQ ID NOs: 137, 139, or 141, respectively. Any transcription factor may be used as long as it is involved in the control of axial growth. Specifically, in the amino acid sequence shown in SEQ ID NO: 137, 139 or 141, even a protein consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, If it has the said function, it can be used by this invention. In addition, “one or several amino acids in the amino acid sequence represented by SEQ ID NO: 137, 139 or 141 in which one or several amino acids are substituted, deleted, inserted, and / or added” ”Is not particularly limited, but means, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 3 To do.

  In addition, the amino acid sequence of a transcription factor used in the present invention, which is involved in the control of elongation growth in the axial direction of the proximal end of the leaf blade, is considered to be highly conserved among many plants of different species. Therefore, in individual plants that want to modify the ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end of the leaf blade, transcription involved in the control of elongation growth in the axial direction of the proximal portion of the leaf blade It is not necessary to isolate the factor or its gene. That is, by introducing the chimeric protein constructed in Arabidopsis thaliana shown in the examples to be described later into other plants, the lateral axial direction with respect to the length of the base end portion axial direction of the leaf blade is easily obtained in various types of plants. Plants modified so that the ratio of the lengths of the plants can be increased.

  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, but as a specific example, for example, when TCP5 protein is used as the transcription factor, the gene encoding the TCP5 protein (for convenience of explanation, (Referred to as TCP5 gene). As a specific example of the TCP5 gene, for example, a polynucleotide containing the base sequence represented by SEQ ID NO: 138 as an open reading frame (ORF) can be mentioned.

  In addition, for example, when TCP13 protein is used as a transcription factor, a gene encoding this TCP13 protein (referred to as TCP13 gene for convenience of description) can be exemplified. As a specific example of the TCP13 gene, for example, a polynucleotide containing the base sequence represented by SEQ ID NO: 140 as an open reading frame (ORF) can be mentioned.

  In addition, for example, when TCP17 protein is used as a transcription factor, a gene encoding this TCP17 protein (referred to as TCP17 gene for convenience of description) can be exemplified. As a specific example of the TCP17 gene, for example, a polynucleotide containing the base sequence represented by SEQ ID NO: 142 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: 138, 140 or 142. . 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: 138, 140 or 142, and which encodes the above transcription factor Etc. 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-1-2) Functional peptide that converts an arbitrary transcription factor into a transcriptional repressing factor The functional peptide used in the present invention that converts any transcription factor into a transcriptional repressing factor (for convenience of explanation, a transcriptional repressing conversion peptide 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 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 this was introduced into plant cells, 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 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 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-1-2-1) Transcriptional repression converting peptide of formula (1) In the transcriptional repression converting peptide of formula (1), the number of amino acid residues represented by X1 is in the range of 0 to 10 If it is. 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 repression 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-1-2-2) Transcriptional Repression Conversion Peptide of Formula (2) In the transcriptional repression conversion peptide of the above formula (2), Y1 is represented by the same as X1 of the transcriptional repression conversion peptide of the above formula (1). The number of amino acid residues to be used may be in the range of 0 to 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 to 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 transcriptional repression 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-1-2-3) Transcriptional Repression Conversion Peptide of Formula (3) In the transcriptional repression conversion peptide of Formula (3), the amino acid residue represented by Z1 is within the range of 1 to 3 Leu 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. It is the amino acid sequence shown in 52 or 53.

(I-1-2-4) Transcriptional Repression Conversion Peptide of Formula (4) The transcriptional repression conversion peptide of the above formula (4) is a hexamer consisting of 6 amino acid residues, and its specific sequence Are shown in SEQ ID NOs: 7, 16, 54. 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-1-2-5) More specific example of transcriptional repression conversion peptide As a more specific example of the transcriptional repression conversion peptide represented by each formula described above, for example, any one of SEQ ID NOS: 3 to 19 And peptides having the amino acid sequence shown in FIG. 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 any transcription factor A peptide having a function of converting a protein into a transcriptional repressor.

  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 the nucleotide sequence by a known method such as Kunkel method or Gapped duplex method or a method according thereto, 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-1-3) Other Examples of Transcriptional Repression-Converting Peptides The present inventors have further investigated the structure of the motif, and as a result, have newly found a motif consisting 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, 133, 59, or 60. 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 conversion peptide having the amino acid sequence represented by SEQ ID NO: 38 or 39 can also be used.

(I-1-4) Chimeric protein production method The various transcription repressor conversion peptides described in (I-1-2) and (I-1-3) above are the transcription described in (I-1-1) above. The transcription factor can be used as a transcription repressing factor by being fused with a factor to form a chimeric protein. 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 a chimeric gene into a plant cell will be described in detail in the section (I-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, having a 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.

(I-2) An example of the production method of the plant body concerning this invention The production method of the plant body concerning this invention makes the chimera protein demonstrated by said (I-1) produce in a plant body, and the front-end | tip part of a leaf blade The method for producing a plant according to the present invention is not particularly limited as long as it includes a process of suppressing transcription (expression) of a target gene by a transcription factor involved in the control of triaxial elongation. Can be mentioned as a production method including steps such as an expression vector construction step, a transformation step, and a selection step. Among these, in the present invention, at least the transformation step may be included. Hereinafter, each step will be specifically described.

(I-2-1) 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-1-1) above and the above (I-1-2). The step is not particularly limited as long as it is a step for constructing a recombinant expression vector containing the transcription-repression-converting polynucleotide described 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 repressing 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.

(I-2-2) Transformation Step In the transformation step performed in the present invention, the recombinant expression vector described in (I-2-1) above is introduced into a plant cell, and (I-1) above. What is necessary is just to be able to produce the described chimeric protein.

  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-2-1) may or may not be included.

(I-2-3) 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 the recombinant expression vector construction step is further 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, selection may be performed based on drug resistance such as hygromycin resistance, or after growing the transformant, selecting from the leaf form of the plant itself. Also good. For example, as an example of selecting from the form of leaves, there can be mentioned a method of comparing the form of leaves of a transformed body with the form of leaves of a plant body which has not been transformed (see Examples described later). In particular, the leaf morphology can be selected simply by comparison, and the effect itself of the present invention that the leaf morphology is modified can be confirmed.

  In the method for producing a plant according to the present invention, the chimera gene is introduced into the plant. Therefore, the plant body is simply lateral to the axial length of the base of the leaf blade by sexual reproduction or asexual reproduction. It is possible to obtain offspring modified so that the ratio of lengths in the axial direction is increased. 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.

(II) Method for producing plant body modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade is reduced (II-1) Transcription factor used in the present invention Since the transcription factor and the gene encoding the transcription factor used in the invention have been described in (I-1-1) above, description thereof is omitted here.

  In the present invention, if the gene encoding the transcription factor is used, the transcription factor can be excessively produced in the plant body. Specifically, a gene encoding the transcription factor is introduced into a plant so that the gene is expressed. Thereby, the transcription factor can be produced excessively.

(II-2) 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 is to produce the transcription factor described in the above (I-1-1) excessively in the plant, The plant according to the present invention is not particularly limited as long as it includes a process that promotes transcription (expression) of a target gene by a transcription factor involved in the control of elongation growth in the axial direction of the proximal end of the body. If the production method of a body is shown by a concrete process, it can mention as a production method including processes, such as an expression vector construction process, a transformation process, and a selection process, for example. Among these, in the present invention, at least the transformation step may be included. Hereinafter, each step will be specifically described.

(II-2-1) Expression vector construction step The expression vector construction step carried out in the present invention comprises a recombinant expression vector comprising a gene encoding the transcription factor described in (I-1-1) above and a promoter. If it is the process to construct | assemble, it will not specifically limit.

  The promoter may be linked so that a gene encoding a transcription factor can be expressed and introduced into the vector, and the specific structure as a recombinant expression vector is not particularly limited.

  The recombinant expression vector may further contain other DNA segments in addition to the promoter and the gene.

  The method for constructing the recombinant expression vector is not particularly limited, and the promoter, the gene encoding the transcription factor, and the other DNA segment as required are preliminarily selected on the appropriately selected parent vector. It may be introduced so that the order becomes. For example, an expression cassette may be constructed by linking a gene encoding a transcription factor and a promoter (such as a terminator if necessary), and then introducing this into a vector.

  In the construction of the expression cassette, for example, the order of the DNA segments can be defined by setting the cleavage sites of each DNA segment as complementary protruding ends and reacting with a ligation enzyme. When the terminator is included in the expression cassette, the promoter, the gene encoding the transcription factor, 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.

  Regarding the parent vector of the recombinant expression vector, the promoter, the other DNA segment, the terminator, the selection marker, the base sequence for increasing the translation efficiency, and the propagation method (production method) of the recombinant expression vector. Since this is the same as (I-2-1) above, description thereof is omitted here.

(II-2-2) Transformation step In the transformation step performed in the present invention, the recombinant expression vector described in (II-2-1) is introduced into a plant cell to produce the transcription factor. It only has to be.

  Here, in the method for producing a plant according to the present invention, the recombinant expression vector may be appropriately constructed according to the type of plant to be produced. An expression vector may be constructed in advance and introduced into plant cells. That is, in the method for producing a plant according to the present invention, the recombinant expression vector construction step described in (II-2-1) may or may not be included.

  The method for introducing the recombinant expression vector into a plant cell (transformation method) and the plant cell into which the recombinant expression vector is introduced are the same as in (I-2-2) above. Description is omitted.

(II-2-3) 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 the recombinant expression vector construction step is further 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.

  In the method for producing a plant according to the present invention, since the gene encoding the transcription factor is introduced into the plant, the length in the axial direction of the proximal end of the leaf blade is simply increased from the plant by sexual reproduction or asexual reproduction. It is possible to obtain a progeny modified so that the ratio of the length in the lateral axis direction to the height becomes small. It is also possible to obtain plant cells and propagation materials such as seeds, fruits, strains, callus, tubers, cut ears, lumps and the like from the plants and their descendants, 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 selection method and the plant in the present invention are the same as in (I-2-3) above, and thus the description thereof is omitted here.

(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 producing the above chimeric protein in a 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 leaf morphology can be modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base distal end portion of the leaf blade of the obtained plant body is increased. Moreover, the other production method of the plant body concerning this invention is based on making the said transcription factor produce excessively in a plant body. Thereby, transcription of the target gene targeted by the transcription factor is promoted. As a result, the leaf morphology can be modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base distal end portion of the leaf blade of the obtained plant body becomes small. Therefore, the present invention includes a plant obtained by the above-described method for producing a plant.

(III-1) Specific Example of Plant According to the Present Invention Here, a specific example of a plant with a modified ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end of the leaf blade according to the present invention. The specific type is not particularly limited, and examples thereof include plants whose usefulness is enhanced by modification of the leaf form. 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 ratio of the length in the lateral axial direction to the length in the axial direction of the distal end of the base of the leaf of the plant body can be modified. The property is not particularly limited, and may be any field that is effective by such modification. 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 the creation of a new horticultural variety will be described. For example, by using the plant production method according to the present invention, the length in the lateral axial direction with respect to the length in the axial direction of the base end of the leaf blade It is possible to create new foliage plants and flowering plants that have a large change in the ratio.

  In addition, to explain the space-saving of plant cultivation, for example, the plant body modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base end of the leaf blade is increased due to less space. Many plants can be cultivated. Therefore, it is expected to contribute to space saving in plant cultivation.

(III-3) An example of the use of the present invention The field of use and the 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 leaf Mention may be made of form modification kits.

  Specific examples of the leaf shape modification kit include those containing at least a recombinant expression vector containing a chimeric gene comprising the gene encoding the transcription factor and the transcription repressor conversion polynucleotide. The leaf modification kit may contain at least a recombinant expression vector containing a gene encoding the transcription factor. Moreover, it is more preferable that the leaf shape modification kit according to the present invention includes a reagent group for introducing the recombinant expression vector 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. 8, 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 TCP5 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) Each region of attL1 and attL2 on the pENTR vector manufactured by Invitrogen Co., Ltd. Amplified by PCR using SEQ ID NO: 146). 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 having the following sequences of SEQ ID NOs: 147 and 148 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 having the sequences of SEQ ID NOs: 147 and 148 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: 147)
5'-cgtcgacggtacccccgggatctgtaattgtaatgttgtttgttgtttgttgttgttggtaattgtggatcct-3 '(SEQ ID NO: 148)
(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: 149)
5'-tcgacttaagcgaaacccaaacggagttctagatccagatcaagccc-3 '(SEQ ID NO: 150)
(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 TCP5 gene into construction vector>
A gene encoding a transcription factor TCP5 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 TCP5 gene excluding the stop codon was amplified by PCR using the following primers.
Primer 1 (TCP5-F) 5′-ATGAGATCAGGAGAATGTGATG-3 ′ (SEQ ID NO: 151)
Primer 2 (TCP5-R stopless) 5'-AGAATCTGATTCATTATCGCTAC-3 '(SEQ ID NO: 152)
The cDNA of TCP5 gene and the encoded amino acid sequence are shown in SEQ ID NOs: 138 and 137, respectively.

  (2) The obtained DNA fragment of the TCP5 coding region was ligated to the SmaI site of the construction vector p35SSRDXG previously digested with the restriction enzyme SmaI, as shown in FIG.

  (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, p35STCP5SRDXG in which a DNA fragment of the TCP5 coding region is inserted in a forward direction of 1.5 μL (about 300 ng), pBIGCKH 4.0 μL (about 600 ng) 5-fold diluted LR buffer 4.0 μL, 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-TCP5SRDX which is a plasmid in which the DNA fragment containing the chimeric gene is incorporated into pBIGCKH. 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-TCP5SRDX, 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 TCP5SRDX was introduced using RT-PCR.

  The plant body transformed with pBIG-TCP5SRDX and the wild type plant body are shown in FIG. In FIG. 4, the left end (indicated as WT in the figure) is a wild-type plant, and the center (indicated as # 1 in the figure) and the right end (indicated as # 2 in the figure) are pBIG-TCP5SRDX transformed plants. Show. As shown in FIG. 4, the wild-type leaf blades of Arabidopsis thaliana are elliptical, whereas the leaf blades of Arabidopsis transformed with pBIG-TCP5SRDX are In the leaves of transformed plants with a long lateral axis, a nearly circular shape, and a stronger phenotype, the peripheral surface of the leaf blade is curved, that is, the peripheral surface of the leaf blade is curled The shape was shown. Thus, the leaf blade of Arabidopsis transformed with pBIG-TCP5SRDX has been modified so that the ratio of the length in the lateral axis direction to the length in the axial direction of the base distal end portion is increased.

[Example 2]
A recombinant expression vector pBIG-TCP13SRDX was constructed in the same manner as in [Example 1] except that the Arabidopsis thaliana TCP13 gene was used as a gene encoding a transcription factor, and Arabidopsis thaliana was transformed.

The TCP13 gene is obtained by amplifying a DNA fragment containing only the coding region of the Arabidopsis TCP13 gene excluding the stop codon by PCR from a cDNA library prepared using mRNA prepared from Arabidopsis leaves. Obtained.
Primer 1 (TCP13-F) 5′-ATGAATATCGTCTCTTGGAAAGATG-3 ′ (SEQ ID NO: 153)
Primer 2 (TCP13-R stopless) 5'-CATTTGCCTCGGATCTAATGTG-3 '(SEQ ID NO: 134)
The cDNA of TCP13 gene and the encoded amino acid sequence are shown in SEQ ID NOs: 140 and 139, respectively.

  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 TCP13SRDX gene was introduced using RT-PCR.

  FIG. 5 shows a plant transformed with pBIG-TCP13SRDX. As shown in FIG. 5, the plant transformed with pBIG-TCP13SRDX was also modified so that the ratio of the length in the lateral axial direction to the length in the axial direction of the base distal end portion was increased.

Example 3
A recombinant expression vector pBIG-TCP17SRDX was constructed in the same manner as in [Example 1] except that the Arabidopsis thaliana TCP17 gene was used as a gene encoding a transcription factor, and Arabidopsis thaliana was transformed.

The TCP17 gene is obtained by amplifying a DNA fragment containing only the coding region of the Arabidopsis TCP17 gene excluding the stop codon by PCR from a cDNA library prepared using mRNA prepared from Arabidopsis leaves. Obtained.
Primer 1 (TCP17-F) 5′-ATGGGAATAAAAAAAGAAGATCAG-3 ′ (SEQ ID NO: 135)
Primer 2 (TCP17-R stopless) 5'-CTCGATATGGTCTGGTTGTGAG-3 '(SEQ ID NO: 136)
The cDNA of TCP17 gene and the encoded amino acid sequence are shown in SEQ ID NOs: 142 and 141, respectively.

  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 TCP17SRDX gene was introduced using RT-PCR.

  A plant transformed with pBIG-TCP17SRDX is shown in FIG. As shown in FIG. 6, the plant transformed with pBIG-TCP17SRDX was also modified so that the ratio of the length in the lateral axial direction to the length in the axial direction of the base distal end portion was increased.

Example 4
In Example 4 below, the Arabidopsis thaliana-derived TCP5 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. A transgenic plant overexpressing TCP5 was 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 TCP5 gene into construction vector>
The gene encoding the transcription factor TCP5 protein derived from Arabidopsis 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 TCP5 gene was amplified by PCR using the following primers.
Primer 1 (TCP5-F) 5′-ATGAGATCAGGAGAATGTGATG-3 ′ (SEQ ID NO: 154)
Primer 2 (TCP5-R) 5′-TCAAGAATCTGATTCATTATCGCTAC-3 ′ (SEQ ID NO: 155)
The cDNA of TCP5 gene and the encoded amino acid sequence are shown in SEQ ID NOs: 138 and 137, respectively.

  (2) The obtained DNA fragment of the TCP5 coding region was ligated to the SmaI site of the construction vector p35SG previously digested with the restriction enzyme SmaI, as shown in FIG.

  (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 the DNA fragment containing the CaMV35S promoter, TCP5 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-TCP5 which is a plasmid in which the DNA fragment containing the TCP5 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 gene of TCP5 was introduced using RT-PCR.

  FIG. 8 shows a transformed plant body and a wild-type plant body in which TCP5 is overexpressed using 35S-TCP5. In FIG. 8, the left end (indicated as WT in the figure) is a wild type plant, and the center (indicated as # 1 in the figure) and the right end (indicated as # 2 in the figure) are plants transformed with 35S-TCP5. Show. As shown in FIG. 8, the wild-type leaf blade of Arabidopsis thaliana is elliptical, whereas the leaf blade of Arabidopsis transformed with 35S-TCP5 has a length in the axial direction of the base tip. The length in the lateral axis direction has become extremely short. Thus, the leaf blade of the Arabidopsis transformant in which TCP5 was overexpressed with 35S-TCP5 had a small ratio of the length in the lateral axis direction to the length in the axial direction of the base tip. Thus, in the present Example and [Example 1], it was found that the transformed plant body showed an opposite phenotype. From this, it was confirmed that the TCP5 transcription factor is a transcription factor involved in the control of the elongation growth in the axial direction of the proximal end of the leaf blade.

  Thus, in the present invention, the length in the axial direction of the base distal end of the leaf blade is suppressed or suppressed by the transcription of the gene targeted by the transcription factor involved in the control of elongation growth in the axial direction of the proximal distal end of the leaf blade. A plant body in which the ratio of the length in the lateral axis direction to the length is modified 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. 3 is a process diagram for incorporating a gene encoding the transcription repressor conversion peptide SRDX and the TCP5 gene into the construction vector p35SG used in Example 1. It is process drawing which shows the construction method of vector pBIGCKH for transformation. It is a figure which shows the leaf of the wild type Arabidopsis thaliana, and the leaf of Arabidopsis thaliana transformed by recombinant expression vector pBIG-TCP5SRDX. It is a figure which shows the leaf of Arabidopsis thaliana transformed by recombinant expression vector pBIG-TCP13SRDX. It is a figure which shows the leaf of Arabidopsis thaliana transformed by recombinant expression vector pBIG-TCP17SRDX. FIG. 6 is a process diagram for incorporating the TCP5 gene into the construction vector p35SG used in Example 4. It is a figure which shows the leaf of the Arabidopsis thaliana transformed plant which overexpressed the TCP5 gene with the wild-type Arabidopsis thaliana leaf and the cauliflower mosaic virus 35S promoter.

Claims (24)

  By producing in the plant a chimeric protein that fuses a transcription factor involved in the control of elongation growth in the axial direction of the proximal end of the leaf blade and a functional peptide that converts any transcription factor into a transcriptional repressor A plant body that suppresses transcription of a gene targeted by the transcription factor and is modified so that the ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end of the leaf blade is increased Production method.   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. A method for producing a plant according to claim 1.   Furthermore, the production method of the plant body of Claim 2 including the expression vector construction process which constructs | assembles the said recombinant expression vector. The method for producing a plant according to any one of claims 1 to 3, 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: 137, 139 or 141,
(B) In the amino acid sequence shown in SEQ ID NO: 137, 139 or 141, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and the tip of the base of the leaf blade A protein involved in the control of elongation in the axial direction. The method for producing a plant according to claim 2 or 3, wherein the gene described in (c) or (d) below is used as the gene encoding the transcription factor:
(C) a gene having the base sequence represented by SEQ ID NO: 138, 140 or 142 as an open reading frame region,
(D) An extension growth in the axial direction of the proximal end of the leaf blade, which hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 138, 140 or 142 under stringent conditions. A gene that encodes a protein involved in the regulation of the protein. 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 has the amino acid sequence represented by either of these, The production method of the plant body of any one of Claims 1-5 characterized by the above-mentioned.   The method for producing a plant according to any one of claims 1 to 5, 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 5, wherein the functional peptide is 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) 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 transcription of any transcription factor is repressed. A peptide having a function of converting to a factor. The functional peptide is represented by the following formula (5)
(5) α1-Leu-β1-Leu-γ1-Leu
(Wherein α1 represents Asp, Asn, Glu, Gln, Thr or Ser, β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, and γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys, or Asp are shown.)
It has an amino acid sequence represented by these, The production method of the plant body of any one of Claims 1-5 characterized by the above-mentioned. The functional peptide is represented by the following formulas (6) to (8):
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(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 has the amino acid sequence represented by either of these, The production method of the plant body of any one of Claims 1-5 characterized by the above-mentioned.   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 which has a sequence, The production method of the plant body of any one of Claims 1-5 characterized by the above-mentioned.   The method for producing a plant according to any one of claims 1 to 5, wherein the functional peptide is a peptide having an amino acid sequence represented by SEQ ID NO: 38 or 39.   The ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end of the leaf blade is caused by excessive production of transcription factors involved in the control of elongation growth in the axial direction of the distal tip of the leaf blade in the plant body. A method for producing a plant, wherein the plant body is modified so as to be small.   The method according to claim 13, further comprising a transformation step of introducing a recombinant expression vector containing the gene encoding the transcription factor into a plant cell so as to express the gene encoding the transcription factor. Plant production method.   The method for producing a plant according to claim 14, further comprising an expression vector construction step of constructing the recombinant expression vector. The said transcription factor is protein of the following (a) or (b) description, The production method of the plant body of any one of Claims 13-15 characterized by the above-mentioned.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 137, 139 or 141;
(B) In the amino acid sequence shown in SEQ ID NO: 137, 139 or 141, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and the tip of the base of the leaf blade A protein involved in the control of elongation in the axial direction. The method for producing a plant according to claim 14 or 15, 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: 138, 140 or 142 as an open reading frame region,
(D) An extension growth in the axial direction of the proximal end of the leaf blade, which hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 138, 140 or 142 under stringent conditions. A gene that encodes a protein involved in the regulation of the protein.   A plant produced by the production method according to any one of claims 1 to 12 and modified so that a ratio of a length in a lateral axial direction to a length in a axial direction of a proximal end portion of a leaf blade is increased. body.   A plant produced by the production method according to any one of claims 13 to 17 and modified so that a ratio of a length in a lateral axial direction to a length in a axial direction of a proximal end portion of a leaf blade is reduced. body.   The plant body according to claim 18 or 19, 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 12,
A plant leaf comprising at least a recombinant expression vector comprising a gene encoding the above transcription factor, a polynucleotide encoding a functional peptide that converts any transcription factor into a transcription repressor, and a promoter. The form modification kit. A kit for performing the production method according to any one of claims 13 to 17,
A plant leaf shape modification kit comprising at least a recombinant expression vector comprising a gene encoding the transcription factor and a promoter.   The plant leaf shape modification kit according to claim 21 or 22, further comprising a group of reagents for introducing the recombinant expression vector into plant cells. A gene encoding the protein described in (a) or (b) below,
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 137, 139 or 141,
(B) In the amino acid sequence shown in SEQ ID NO: 137, 139 or 141, consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and the tip of the base of the leaf blade Proteins involved in the control of triaxial elongation growth,
Or the gene described in the following (c) or (d),
(C) a gene having the base sequence represented by SEQ ID NO: 138, 140 or 142 as an open reading frame region,
(D) An extension growth in the axial direction of the proximal end of the leaf blade, which hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 138, 140 or 142 under stringent conditions. A gene encoding a protein involved in the regulation of
The ratio of the length in the lateral axial direction to the length in the axial direction of the proximal end of the leaf blade is modified, characterized by comprising a transformation step of introducing a recombinant expression vector containing Plant production method.

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