JP2006042730A - Method for producing male sterile body of monocotyledon, plant obtained by using the method and use of the plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology producing the male sterile body of a plant by suppressing transcription of a gene contributing to flower organ formation. <P>SOLUTION: A chimeric protein obtained by fusing a transcription factor with a functional peptide is produced in plant cells by introducing a gene encoding the transcription factor promoting transcription of the gene contributing to flower organ formation and a polynucleotide encoding a functional peptide converting the transcription factor into a transcription suppressing factor. The chimeric protein suppresses the expression of the gene contributing to flower organ formation, and thereby producing a male sterile body of the plant incapable of forming a normal pollen. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a technique for producing a male sterile body of a plant, and in particular, a production method for producing a male sterile body of a monocotyledonous plant by suppressing transcription of a gene involved in flower organ formation and using the same. The present invention relates to a plant obtained and its use.

  When crossed between different varieties and made a hybrid, a trait superior to the parents appears in the child. This is called a hybrid strength. It is now common to produce excellent varieties by hybridizing agricultural products by crossing using this hybrid strength. For example, most of the major varieties of major vegetables and cereals have been improved through such crossings.

  It is necessary to cross between different varieties in order to receive the benefits of hybrid strength. For this reason, it is necessary to prevent self-pollination from being performed in the varieties to be crossed. Here, in plants such as corn where male and female flowers are separated, self-pollination can be avoided by artificially cutting male flowers. However, this work requires a great deal of human labor and is very troublesome. In addition, in self-propagating plants typified by rice, a large number of flowers gather, and a pistil and a stamen envelop the petals. Therefore, it is difficult to artificially cut male flowers, and it is very difficult to avoid self-pollination.

  Therefore, it is desirable to use so-called male sterile bodies that do not allow normal pollen formation in order to perform crosses using hybrid stress. In fact, so far, male sterile bodies have been obtained and utilized for breeding in many plants such as tomatoes and cucumbers.

  However, on the other hand, it is also true that there are many plant varieties for which male sterile bodies have not been established. In these plants, when a new male sterile body is newly obtained by mutation, it is necessary to leave the result to chance, so long-term mating is required. Therefore, much labor and cost are required, and it is practically difficult to tackle in the current agriculture.

Thus, several attempts have been reported to artificially establish male sterile bodies using genetic recombination techniques. Non-patent document 1 artificially causes male sterility due to nuclear genes. Technology is disclosed. In this technique, a chalcone synthase antisense gene is introduced into the penitature to suppress the activity of chalcone synthase. This suppresses the biosynthesis of chalcone, the biosynthetic precursor of flavonoids, and the transformed penitula becomes a male sterile body.

  Non-Patent Document 2 discloses a technique for producing a male sterile body by eliminating tobacco cocoons with a toxic substance. In this technology, the argE gene, which uses N-acetyl-L-ornithine deacetylase as a transcription / translation product, is fused with a DNA sequence similar to the TA29 promoter that functions specifically in tapet tissue. Introduce. The tobacco is then administered non-toxic N-acetyl-L-phosphinothricin. Then, N-acetyl-L-phosphinothricin is deacetylated by N-acetyl-L-ornithine deacetylase expressed in sputum and becomes toxic L-phosphinothricin. This toxic substance causes necrosis and disappears so that transformed tobacco becomes a male sterile body in which no pollen is formed.

  Non-Patent Document 3 discloses a technique for artificially causing cytoplasmic male sterility. In this technique, a mitochondrial atp9 gene derived from wheat and not subjected to RNA editing is introduced into tobacco cells. Thereby, an inactive ATP9 protein is expressed and moves to mitochondria. As a result, the mitochondria function is inhibited, and the transformed tobacco becomes a male sterile body.

  Furthermore, in Non-Patent Document 4, another transformed tobacco into which an antisense gene of a mitochondrial atp9 gene not subjected to RNA editing was introduced was prepared, and a mitochondrial atp9 gene not subjected to RNA editing was introduced. Techniques have been disclosed for producing transformed tobacco that recovers fertility in the next generation by crossing with transformed tobacco.

  By the way, it is known that the Arabidopsis APETALA3 gene which is a MADS-box gene is a homeotic gene involved in flower organ formation. In this mutant lacking the APETALA3 gene, the petals are converted into sepals, and the stamens are converted into the female pods that can be formed inside.

  Similarly, it has been reported that the SPDS gene of rice, which is a MADS-box gene, is a homolog (ortholog) of the APRTALA3 gene (see, for example, Non-Patent Document 5). Furthermore, it has been reported that the function of the APRTALA3 gene as a homolog was analyzed by suppressing the expression of this rice SPW1 gene with RNAi (for example, Non-Patent Document 6).

Here, the present inventor has found various peptides that convert a transcription factor into a transcriptional repression factor (see, for example, Patent Documents 1 to 7 and Non-Patent Documents 7 and 8). This peptide is excised from a Class II ERF (Ethylene Responsive Element Binding Factor) protein or a plant zinc finger protein (Zinc Finger Protein, such as Arabidopsis SUPERMAN protein) and has a very simple structure.
JP 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) Ingrid M. van der Meer, Maike E. Stam, Arjen J. van Tunen, Joseph NM Mol, and Antoine R. Stuitje. The Plant Cell, Vol 4, pp 253-262, March, 1992 G. Kriete, K. Niehaus, AM Perlick, A. Puehler and I. Broer, The Plant Journal, Vol 9, pp 809-818, 1996 Michel Hernould, Sony Suharsono, Simon Litvak, Alejandro Araya, and Armand Mouras., Proc. Natl. Acad. Sci. USA, Vol 90, pp. 2370-2374, March, 1993 Eduardo Zabaleta, Armand Mouras, Michel Hernould, Suharsono, and Alejandro Araya., Proc. Natl. Acad. Scl. USA, Vol 93, pp 11259-11263, October, 1996 Nagasawa, N., Miyoahi, M., Sano, Y., Satoh, H., Hirano, H., Sakai, H., Nagato, Y., Development 130, 705-718, 2003 Xiao, H., Wang, Y., Liu, D., Wang, W., Li, X., Zhao, X., Xu, J., Zhai, W., Zhu, L., Plant Mol Biology 52, 957-966. 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

  However, in the method of suppressing the expression of SPW1 by RNAi reported in Non-Patent Document 6 above, among the 185 flowers examined in one transformed individual, six stamens possessed by the wild-type plant body Among them, the number of flowers in which one stamen changed into a percutaneous skin was 70, 2 flowers in which a stamen changed into a percutaneous skin, and only 4 flowers in which three stamens changed into a percutaneous skin. 81 flowers have been reported to be normal. Moreover, the flower which four or more stamens changed into the heart skin was not seen. Thus, the method of suppressing the expression of SPW1 by RNAi cannot obtain a complete male sterile individual.

  In addition, by producing a chimeric protein consisting of a transcription factor that promotes transcription of genes involved in flower organ formation and a functional peptide that converts the transcription factor into a transcriptional repressor, The technology to produce is not known.

  The present invention efficiently produces a male sterile body by producing in a plant a chimeric protein of a transcription factor that promotes transcription of a gene involved in flower organ formation and a functional peptide that converts the transcription factor into a transcription repressor. It aims at providing the production method of the male sterile body of a plant to produce.

  As a result of diligent studies to solve the above-mentioned problems, the present inventor has converted almost all SPW1 protein, which is one of transcription factors that promote transcription of genes involved in flower organ formation, into transcription repressors. The present inventors have found that a male sterile plant in which the formation of stamens is inhibited by the flowers of the plant can be completed.

That is, the method for producing a male sterile body of a plant according to the present invention is a transcription factor that promotes the transcription of a gene involved in flower organ formation in order to solve the above-mentioned problems, and the following (a) or (b)
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 134,
(B) The amino acid sequence shown in SEQ ID NO: 134 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and promotes transcription of genes involved in flower organ formation. Functional protein,
A chimera protein produced by fusing a transcription factor consisting of the protein described in 1. and a functional peptide that converts a transcription factor into a transcriptional repressor is produced in a plant, and transcription of genes involved in flower organ formation is suppressed. It is said. In the above production method, it is preferable that the male sterile body of the plant is one in which at least formation of the male stamen is inhibited.

  Thereby, the chimeric protein can effectively suppress the transcription of the gene targeted by the transcription factor. Therefore, there is an effect that the plant body from which the chimeric protein is produced becomes a male sterile body.

  The production method further includes a transformation step of introducing a recombinant expression vector containing a chimeric gene comprising a gene encoding the transcription factor and a polynucleotide encoding the functional peptide into a plant cell. 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 the plant body becomes a male sterile body.

  Moreover, it is preferable to use the gene described in the following (c), (d) or (e) as a gene encoding the transcription factor. (C) A gene consisting of the base sequence represented by SEQ ID NO: 135. (D) A gene having, as an open reading frame region, a base sequence consisting of positions 216 to 890 of the base sequence shown in SEQ ID NO: 135. (E) encodes a transcription factor that hybridizes with a gene comprising a base sequence complementary to either (c) or (d) above under stringent conditions and promotes transcription of a gene involved in flower organ formation. gene.

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

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

  The functional peptide may be a peptide described in the following (f) or (g). (F) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 20 or 21. (G) The amino acid sequence shown in SEQ ID NO: 20 or 21 comprises an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and the transcription factor is used as a transcription repressor. A peptide having a function to convert.

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

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

  The functional peptide is 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 may be a peptide consisting of an amino acid sequence.

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

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

  The plant according to the present invention is a male sterile plant produced by the above production method. The plant body preferably contains at least one of a grown plant individual, a plant cell, a plant tissue, a callus, and a seed.

  The plant male sterile body production kit according to the present invention is a kit for performing the production method described above, and is a transcription factor that promotes transcription of a gene involved in flower organ formation. Or (b) at least a recombinant expression vector comprising a gene encoding a transcription factor comprising the protein described above, a polynucleotide encoding a functional peptide that converts the transcription factor into a transcription repressor, and a promoter It is said. The plant male sterile body production kit may further include a reagent group for introducing the recombinant expression vector into plant cells.

  In the method for producing a male sterile body of a plant according to the present invention, as described above, it is a transcription factor that promotes transcription of a gene involved in flower organ formation, and comprises the protein described in (a) or (b) above. A male sterile body of a plant by producing a chimeric protein fused with a transcription factor and a functional peptide that converts the transcription factor into a transcriptional repressor in the plant and suppressing the expression of genes involved in flower organ formation. To produce. Therefore, if a target plant is transformed with a chimeric gene encoding the above chimeric protein, a male sterile plant can be produced, and the target plant can be transformed into a male without any complicated genetic recombination techniques. There is an effect that it can be sterilized.

  Hereinafter, an embodiment of the present invention will be described. In addition, this invention is not limited to the following embodiment.

  The present invention is a technique for producing a male sterile body of a plant, which is a transcription factor that promotes transcription of a gene involved in flower organ formation, and comprises the protein described in (a) or (b) above And a chimeric protein in which a transcription factor is fused with a functional peptide that converts a transcription factor into a transcriptional repressor. Since the plant body obtained by this cannot form normal pollen, a male sterile plant body can be produced by the present invention.

  Here, the failure to form normal pollen occurs as follows. That is, the transcription factor-derived DNA binding domain in the chimeric protein binds to a target gene presumed to be involved in flower organ formation. The transcription factor is converted into a transcription repressing factor, and transcription of the target gene is repressed. As a result, a male sterile plant that cannot form normal pollen can be obtained, for example, by inhibiting the formation of stamens.

  The male sterile bodies of plants (the male sterile bodies) produced by the production method of the present invention are those that cannot normally form pollen. That is, examples of this male sterile body include those in which the formation of stamens is inhibited and pollen is not formed at all, stamens are formed, but pods are not formed, and pollen is not formed, and stamens are also included. Although wrinkles are also formed, there are those in which the amount of pollen formed is small and does not lead to cleavage of wrinkles, and in which the formed pollen becomes enlarged and sticks to each other and does not scatter at all.

  In this male sterile body, the female moth has fertility. For this reason, pollen of other species can be pollinated to the male sterile body. Therefore, a first-generation hybrid can be obtained by crossing using the hybrid strength.

  In addition, in this male sterile body, in addition to the inability to form normal pollen, the formation of other tissues may not be performed normally. For example, the male sterile body may be one in which petals, sepals, scales, inner wings, outer wings, etc. are formed in a different shape from the usual one, or one that is not formed at all. For example, if no petals, sepals, scales, inner pods, outer pods, etc. are formed, the female pods will be exposed, so the trouble of pollinating other types of pollen (petals, gargles, scales, inner pods, outer Etc.) can be simplified.

  In the following description, the chimeric protein used in the method for producing a male sterile plant according to the present invention, an example of the method for producing the plant according to the present invention, the plant obtained thereby, its usefulness, and its use Each will be explained.

(I) Chimeric protein used in the present invention As described above, the chimeric protein used in the present invention is a transcription factor that promotes transcription of genes involved in flower organ formation, and is described in (a) or (b) above. These are fusions of a transcription factor comprising the above protein and a functional peptide that converts the transcription factor into a transcriptional repressor.

  The chimeric protein used in the present invention acts dominantly on endogenous genes. That is, the chimeric protein according to the present invention is involved in flower organ formation, which is controlled by the relevant transcription factor even when the plant is diploid or double diploid, or even when a functional duplication gene exists in the plant. Gene expression can be uniformly suppressed. Therefore, any plant capable of gene transfer can be easily transformed into a male sterile body.

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

(I-1) Transcription factor that promotes transcription of genes involved in flower organ formation The transcription factor used in the present invention is a transcription factor that promotes transcription of genes involved in flower organ formation, and the above (a) or ( If it is a transcription factor which consists of protein of b) description, it will not specifically limit. Transcription factors that promote transcription of genes involved in flower organ formation are conserved in many plants. Therefore, the transcription factors used in the present invention include proteins having similar functions that are conserved in various plants.

  Examples of such transcription factors include SPW1 protein, SI1 (SYLKY1) protein, and wheat WAP3 protein (Plant J, 29 (2), 169-181, Murai, K. et al.) Which are transcription factors including MADS-Box. 2002), barley HVAP3 protein (AY54165), and the like, but the transcription factor is not limited thereto.

  As a typical example of the transcription factor used in the present invention, for example, rice SPW1 protein can be mentioned. The rice SPW1 protein can include, for example, a protein consisting of the amino acid sequence represented by SEQ ID NO: 134, and as described above, it is known to be a transcription factor that promotes transcription of a gene involved in flower organ formation. In rice, it is known that the formation of scales and stamens is inhibited in a mutant spw1 of a gene encoding the SPW1 protein (referred to as SPW1 gene for convenience of explanation). In the present invention, for example, the rice SPW1 protein, which is a transcription factor, is converted into a transcription repressing factor by fusing a functional peptide described later to the rice SPW1 protein.

  As described above, it is known that the formation of stamens is inhibited when a mutation occurs in the rice SPW1 gene. However, for example, even if it is known that the SPW1 gene is a causative gene of the phenotype that the formation of stamens is inhibited, it is generally attempted to produce a plant having such a phenotype using genetic manipulation. In some cases, it is known that it is difficult to obtain the expected effect. The reason for this is that there are duplicate factors in the control factors to be genetically manipulated, and that they are supplemented by another route (the presence of a bypass).

  Therefore, for example, it is usually difficult to inhibit the formation of stamens only by suppressing transcription promoted by SPW1 protein. The method according to the present invention succeeded in overcoming such difficulties by fusing the functional protein with, for example, the SPW1 protein.

  In addition, as described above, the method of suppressing the action of the SPW1 gene by the RNAi method was not sufficient to inhibit the formation of stamens. On the other hand, in the method according to the present invention, the target plant is very efficiently introduced by introducing into the plant a chimeric gene in which the gene encoding the functional peptide is linked to the gene encoding the transcription factor. It becomes possible to inhibit the formation of stamens.

  The transcription factor used in the present invention is not limited to the SPW1 protein consisting of the amino acid sequence shown in SEQ ID NO: 134, and may be any transcription factor having a function of promoting transcription of a gene involved in flower organ formation. . Specifically, in the amino acid sequence shown in SEQ ID NO: 134, even a protein consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added has the above function. If so, it can be used in the present invention. The range of “one or several” in the above “amino acid sequence represented by SEQ ID NO: 1 in which one or several amino acids are substituted, deleted, inserted and / or added” is Although not particularly limited, for example, it means 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and particularly preferably 1 to 3.

  Further, even a protein having a homology of 50% or more, preferably 60% or more, more preferably 70% or 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 134 has the above function. Can be used in the present invention. Here, “homology” is the ratio of the same sequence in the amino acid sequence, and the higher this value, the closer the two are.

  In particular, it is known that there are a plurality of splicing variants resulting from variable splicing in higher organisms, and there are also a plurality of splicing variants in the SPW1 protein. Therefore, the SPW1 protein is not limited to the protein consisting of the amino acid sequence shown in SEQ ID NO: 134, and includes such splicing variants as long as it is a transcription factor having a function of promoting transcription of a gene involved in flower organ formation.

  In addition, the amino acid sequences of transcription factors used in the present invention that promote transcription of genes involved in flower organ formation are considered to be highly conserved among many plants of different species. Therefore, it is not always necessary to isolate a specific transcription factor or its gene that promotes transcription of a gene involved in flower organ formation in each individual plant body that wants to produce male sterile bodies. That is, it is considered that a male sterile body can be easily produced in various kinds of plants by introducing the chimeric protein constructed in rice shown in Examples described later into other plants.

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

  The gene encoding the transcription factor is not particularly limited, but a specific example includes, for example, the SPW1 gene when SPW1 protein is used as the transcription factor. The SPW1 gene is not particularly limited as long as it is a gene encoding the SPW1 gene. As a specific example, for example, a base consisting of positions 216 to 890 in the base sequence shown in SEQ ID NO: 135 is used. Mention may be made of genes having sequences as open reading frame regions. Of course, the SPW1 gene includes those that further contain an untranslated region (UTR) sequence. Examples of the SPW1 gene containing such UTR include a polynucleotide having the base sequence shown in SEQ ID NO: 135.

  Of course, the SPW1 gene used in the present invention or a gene encoding a transcription factor is not limited to the above example, and consists of positions 216 to 890 of the base sequence shown in SEQ ID NO: 135. It may be a gene having homology with the base sequence or the base sequence shown in SEQ ID NO: 135. Specifically, for example, a gene and a string consisting of a base sequence consisting of positions 216 to 890 of the base sequence shown in SEQ ID NO: 135 or a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 135 Examples thereof include a gene that hybridizes under a gentle condition and encodes the above transcription factor. Here, hybridizing under stringent conditions means binding at 60 ° C. under 2 × SSC washing conditions.

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

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

(I-2) Functional peptide that converts a transcription factor into a transcriptional repressing factor As used in the present invention, a functional peptide that converts a transcription factor into a transcriptional repressing factor (referred to as a transcription repressing conversion peptide for convenience of explanation) There is no particular limitation, and a peptide capable of suppressing transcription of a target gene controlled by the transcription factor by forming a chimeric protein fused with the transcription factor (Patent Documents 1 to 7, Non-Patent Documents) (See 5-6, etc.). 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.

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

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

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

  In the transcriptional repressor converting peptide of the above formula (1), specific sequences of pentamers (5mers) consisting of 5 amino acid residues excluding X1 and X3 are shown in SEQ ID NOs: 40 and 41. The amino acid sequence when X2 is Asn is the amino acid sequence shown in SEQ ID NO: 40, and the amino acid sequence when X2 is Glu is the amino acid sequence shown in SEQ ID NO: 41.

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

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

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

  In the transcription repressor converting peptide of the above formula (2), specific sequences of pentamers (5mers) consisting of 5 amino acid residues excluding Y1 and Y3 are shown in SEQ ID NOs: 42 and 43. The amino acid sequence when Y2 is Phe is the amino acid sequence shown in SEQ ID NO: 42, and the amino acid sequence when Y2 is Ile is the amino acid sequence shown in SEQ ID NO: 43. A specific sequence of a tetramer (4mer) consisting of 4 amino acid residues excluding Y2 is shown in SEQ ID NO: 44.

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

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

  The amino acid residue represented by Z3 should be as short as possible in view of the ease of synthesizing the transcriptional repression 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 from the viewpoint of ease of synthesis, it may be 20 amino acids or less. preferable.

  In the transcriptional repression converting peptide of the above formula (3), the specific sequence of the oligomer consisting of 7 to 10 amino acid residues excluding Z3 is shown in SEQ ID NOs: 45 to 53. The amino acid sequence when Z1 is Leu and Z2 is Glu, Gln or Asp is the amino acid sequence shown in SEQ ID NO: 45, 46 or 47, respectively, and Z1 is Asp-Leu and Z2 is Glu, Gln or Asp. Is the amino acid sequence shown in SEQ ID NO: 48, 49 or 50, respectively, and the amino acid sequence when Z1 is Leu-Asp-Leu and Z2 is Glu, Gln or Asp is SEQ ID NO: 51, respectively. The amino acid sequence shown in 52 or 53.

(I-2-4) Transcriptional Repression Conversion Peptide of Formula (4) The transcriptional repression conversion peptide of the above formula (4) is a hexamer (6mer) consisting of 6 amino acid residues, and its specific sequence is: SEQ ID NOs: 7, 16, and 54 are shown. The amino acid sequence when Z4 is Glu is the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence when Z4 is Asp is the amino acid sequence shown in SEQ ID NO: 16, and the amino acid sequence when Z4 is Gln. The sequence is the amino acid sequence shown in SEQ ID NO: 54.

  In particular, the transcriptional repression converting peptide used in the present invention may be a peptide consisting of a minimal sequence such as a hexamer represented by the above formula (4). For example, the amino acid sequence shown in SEQ ID NO: 7 corresponds to the 196th to 201st amino acid sequence of Arabidopsis SUPERMAN protein (SUP protein), and as described above, the present inventor newly found as the above transcription repressor conversion peptide. It is.

(I-2-5) More specific example of transcriptional repression converting peptide As a more specific example of the transcriptional repressing converting peptide represented by each formula described above, for example, any one of SEQ ID NOs: 3 to 19 is shown. And a peptide consisting of the amino acid sequence. These oligopeptides have been found by the present inventor to be the above-described transcriptional repression converting peptide (see, for example, Patent Document 7).

Furthermore, the other oligopeptide of the following (f) or (g) description can be mentioned as another specific example of the said transcription repression conversion peptide.
(F) A peptide consisting of any amino acid sequence shown in SEQ ID NO: 20 or 21.
(G) In any one of the amino acid sequences shown in SEQ ID NO: 20 or 21, one or several amino acids are substituted, deleted, inserted, and / or added, and a transcription factor is transcribed. A peptide having a function of converting to a suppressor.

  The peptide consisting of the amino acid sequence shown in SEQ ID NO: 20 is a SUP protein. In addition, “one or several in the amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in any one of the amino acid sequences shown in SEQ ID NO: 20 or 21”. The range of “pieces” is not particularly limited, but for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3 means.

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

  The functional peptide is not limited to the peptide consisting of the full-length sequence of the amino acid sequence shown in SEQ ID NO: 20, and may be a peptide consisting of a partial sequence thereof.

  Examples of the peptide consisting of the partial sequence include a peptide consisting of the amino acid sequence shown in SEQ ID NO: 21 (the 175th to 204th amino acid sequence of the SUP protein). Examples of the peptide consisting of the partial sequence include the above (3 ).

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

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

  As more specific examples of the transcriptional repression converting peptide consisting of the amino acid sequence represented by the above formulas (5) to (9), SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, Peptides having the amino acid sequence shown by 31, 32, 33, 34, 35, 36, 37, 133, 59 or 60 can be mentioned. Among these, the peptide of SEQ ID NO: 29, 30, 32, 34, 133, 59, or 60 corresponds to the peptide represented by the general formula (6), and the peptide of SEQ ID NO: 22, 25, 35, 36, or 37 is The peptide of SEQ ID NO: 26, 27, 28, 31, or 33 corresponds to the peptide of general formula (8), and the peptide of SEQ ID NO: 23 or 24. Corresponds to the peptide represented by the general formula (9).

  In addition to the peptides represented by the general formulas (5) to (9), a transcription repressing / converting peptide consisting of the amino acid sequence represented by SEQ ID NO: 38 or 39 can also be used.

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

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

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

  Specific examples of the transcription repression conversion polynucleotide include, for example, SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, Poly, consisting of the base sequence shown in 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 1, 55, or 57 Mention may be made of nucleotides. Also, SEQ ID NOs: 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 2, 56, or 58 are each a polynucleotide complementary to the polynucleotide exemplified above. It is a nucleotide. These polynucleotides correspond to the amino acid sequences shown in SEQ ID NOs: 3-39, 133, 59, 60 as shown in Table 1 below. 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: 20 and 21, as shown in Table 1 below.

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

(II) An example of a method for producing a plant according to the present invention The method for producing a plant according to the present invention comprises producing the chimeric protein described in (I) above in a plant and expressing a gene involved in flower organ formation. Although it is not particularly limited as long as it includes a process of suppressing, if the production method of the plant according to the present invention is shown in a specific process, for example, an expression vector construction process, a transformation process, a selection process, etc. It can be mentioned as a production method including a process. Among these, in the present invention, at least the transformation step may be included. Hereinafter, each step will be specifically described.

(II-1) Expression vector construction step The expression vector construction step carried out in the present invention comprises the gene encoding the transcription factor described in (I-1) above and the transcriptional repression conversion poly described in (I-4) above. There is no particular limitation as long as it is a step of constructing a recombinant expression vector containing a nucleotide and a promoter.

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

  The promoter is not particularly limited as long as it is a promoter capable of expressing a gene in a plant body, and a known promoter can be suitably used. Examples of such promoters include actin promoter, cauliflower mosaic virus 35S promoter (CaMV35S), 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, the actin promoter or the cauliflower mosaic virus 35S promoter can be more preferably used. When each of the above promoters is used, the resulting recombinant expression vector can strongly express any gene when introduced into a plant cell.

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

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

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

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

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

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

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

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

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

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

  The method (transformation method) for introducing the recombinant expression vector into a plant cell is not particularly limited, and any conventionally known method suitable for the plant cell can be used. Specifically, for example, a method using Agrobacterium or a method of directly introducing into plant cells can be used. Examples of the method using Agrobacterium include Goto, F., Yoshihara, T., Shigemoto, N., Toki, S., and Takaiwa, F., (1999) Iron fortification of rice seed by the soybean ferritin gene. , Nature biotechnology 17,282-286.

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

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

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

(II-3) Other Steps and Other Methods In the method for producing a plant according to the present invention, it is sufficient if the transformation step is included, and further the recombinant expression vector construction step is included. However, other steps may be included. Specifically, the selection process etc. which select an appropriate transformant from the plant body after transformation can be mentioned.

  The selection method is not particularly limited. For example, selection may be performed on the basis of drug resistance such as hygromycin resistance, and normal pollen formation may occur in a plant that has grown after growing the transformant. You may select based on what you cannot do.

  In the method for producing a plant according to the present invention, since the chimeric gene is introduced into a plant, progeny from which expression of a gene involved in flower organ formation is suppressed by sexual reproduction or asexual reproduction is selected from the plant. Can be obtained. 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.

  Moreover, the production method of the plant body concerning this invention is not limited to the method of transforming with a recombinant expression vector, You may use another method. Specifically, for example, the chimeric protein itself may be administered to a plant body. At that time, the administration method of the chimeric protein is not particularly limited, and various known methods may be used.

(III) Plant obtained by the present invention, its usefulness, and use thereof The method for producing a plant according to the present invention is based on expressing a gene encoding the chimeric protein in the plant. A DNA binding domain derived from a transcription factor in the chimeric protein binds to a target gene presumed to be involved in flower organ formation. The transcription factor is converted to a transcription repressor, and transcription of the target gene is repressed. As a result, a male sterile plant can be obtained in which variation occurs in flower organ formation and pollen formation is not normally performed. Therefore, the present invention also includes a male sterile plant obtained by the method for producing a plant described above.

(III-1) Specific Examples of Plants According to the Present Invention The specific types of male sterile plants according to the present invention are not particularly limited, and may be angiosperms or angiosperms. It may be a dicotyledonous plant or a monocotyledonous plant, and among these, the male sterile plant according to the present invention is more preferably a monocotyledonous plant. Among them, the male sterile plant according to the present invention is more preferably a plant whose usefulness is enhanced by acquiring male sterility. Examples of such plants include plants used for food, feed, and industry. Specifically, for example, plants such as rice, barley, bread wheat, rye, oats, pearl barley, sugar cane, corn, sorghum, duckweed, pineapple, ginger, sorghum, yam, rush, taro, onion, konjac and the like can be mentioned. .

  Further, the male sterile plant according to the present invention may be a foliage plant or a floret plant using a flower or the plant itself as a product. Therefore, specific examples of the male sterile plant according to the present invention further include various plants such as agave, iris, iris, lily, tulip, janohi, yablanc, narcissus, water hyacinth, hyacinth, crocus, orchidaceae Can be mentioned.

  Alternatively, the male sterile body according to the present invention may be a weed that can function as a biopesticide by itself by acquiring male sterile. Examples of such plants include weeds such as upland field weeds and paddy field weeds. Specific examples include plants such as Inobie, Enokorogusa, Kinenokoro, Vulgaridae, Vulgaris, Niwadokoro, Akihishiba, Ashishiba, Kamogaya, Susuki, Chigaya, Chikarashiba, Reed, and other plants.

(III-2) Usefulness of the Present Invention The present invention has utility in a field having certain effects by imparting male sterility to a plant. Some specific examples are given below, but the usefulness of the present invention is not limited thereto.

  First, by the technique of the present invention, a male sterile plant that cannot form normal pollen can be produced, and can be used for breeding by crossing using hybrid stress. In the male sterile plant of the present invention, normal pollen formation is not possible, and therefore self-pollination is not performed even for self-propagating plants such as rice. Therefore, crossing between species can be performed simply by pollinating pollen of other species. Thereby, the search for the primary hybrid of the excellent variety using the hybrid strength can be easily and efficiently performed. Therefore, for example, by using the technique of the present invention for transgenic rice, it is possible to produce male sterile rice useful for crop breeding with high reproducibility and high frequency. Therefore, it is possible to efficiently produce rice having a breeding useful character. Furthermore, by applying this technology to important crops such as wheat and barley, it is possible to breed useful crops using male sterile plants.

  The technique of the present invention can also be applied to other bred plants such as corn. In other breeding plants, self-pollination is avoided by manual harvesting of male stamens (male removal work), and varieties are improved by pollinating pollen of other varieties. On the other hand, if male sterile plants are produced by the technique of the present invention, such labor is not required, so the time and cost required for breed improvement, or the labor required for cultivation of excellent varieties, As compared with the above, it can be greatly reduced.

  The technology of the present invention can also be applied to plants that use rhizomes as commodities, such as onion, lily, tulip, hyacinth, crocus, konjac, and taro. In this type of plant, it is known that when pollination occurs, the growth of rhizomes is significantly inhibited and the commercial value is lowered. Therefore, at present, a male removal work is required to avoid pollination, and the labor and cost for that purpose are very large. According to the technique of the present invention, a male sterile body of a plant having a rhizome as a product can be obtained, so that male removal work is not required and pollination can be avoided. Therefore, the cost and time for producing a product by growing a plant body can be significantly reduced compared to the current situation.

  The technology of the present invention can also be suitably applied to plants that do not use fruits and flowers as commodities. One example is the prevention of hay fever. That is, if a male sterile body is produced by the technique of the present invention in a plant that disperses a large amount of pollen that causes hay fever, for example, weeds such as gramineous plants such as duckweed, blue-necked frog, and nagahagusa, normal sterility is produced. Since pollen cannot be formed, there is no fear of pollen scattering from these plants. Therefore, if these male sterile bodies are replaced with natural wild-type plants, the pollen scattering that causes hay fever can be suppressed, and thus hay fever can be prevented.

  Further, the technique of the present invention can prevent plant diseases caused by virus infection via pollen. Certain plant viruses are known to be present in the pollen of diseased plants and transmitted to healthy plants through stamens causing disease. By producing a plant that does not form normal pollen by the technique of the present invention, viral infection that mediates pollen is not performed, and thus diseases of such plants can be prevented.

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

  However, if the genetically modified plant body is transformed into a male sterile body that cannot form normal pollen using the technique of the present invention, the genetically modified plant body will not diffuse into the natural world by pollen distribution. . Therefore, the verification test of the genetically modified plant can be performed under conditions closer to those in the actual outdoor environment as compared with the current situation. Thereby, the character introduced into the genetically modified plant can be verified in a more natural environment.

  In addition, if the technique of the present invention is applied to weeds such as upland field weeds and paddy field weeds, weeds that become male sterile can be produced. Such weeds pollinate pollen of natural wild-type weeds, but part of the next generation becomes male-sterile, so that the propagation of wild-type weeds can be suppressed. Thus, if the technique of the present invention is used for weeds, it can be used as a biopesticide.

  Moreover, if the technique of this invention is used, the plant body from which a part of flower organs, such as a cocoon and a cocoon, was lose | deleted or deform | transformed can be produced. Thereby, since the plant body which has the flower container of the peculiar shape which did not exist conventionally can be produced, the new ornamental plant which has not existed until now can be obtained.

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

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

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and FIG. 1 thru | or FIG. 5, this invention is not limited to a following example.

  In this example, a polynucleotide encoding a 12 amino acid peptide LDLDLELRLGFA (SRDX) (SEQ ID NO: 19), which is one of transcription repressor conversion peptides, is located between the rice actin promoter and the transcription termination region of the nopaline synthase gene. Rice was transformed by constructing a recombinant expression vector incorporating a polynucleotide bound downstream of the rice SPW1 gene and introducing it into rice using the Agrobacterium method.

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

  (1) Each region of attL1 and attL2 on the pENTR vector manufactured by Invitrogen Co., Ltd. is expressed as primers attL1-F (SEQ ID NO: 138), attL1-R (SEQ ID NO: 139), attL2-F (SEQ ID NO: 140), attL2-R ( Amplified by PCR using SEQ ID NO: 141). The obtained attL1 fragment was digested with restriction enzymes HindIII and attL2 fragment with EcoRI and purified. The PCR reaction conditions were 25 cycles, with a denaturation reaction at 94 ° C. for 1 minute, an annealing reaction at 47 ° C. for 2 minutes, and an extension reaction at 74 ° C. for 1 minute. All subsequent PCR reactions were carried out under the same conditions.

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

(3) A DNA fragment consisting of the following SEQ ID NOs: 142 and 143 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 consisting of the sequences of SEQ ID NOs: 142 and 143 includes a BamHI restriction enzyme site at the 5 ′ end, an omega sequence derived from tobacco mosaic virus that enhances translation efficiency, and restriction enzyme sites SmaI, SalI, and SstI in this order.
5'-ctagaggatccacaattaccaacaacaacaaacaacaaacaacattacaattacagatcccgggggtaccgtcgacgagctc-3 '(SEQ ID NO: 142)
5'-cgtcgacggtacccccgggatctgtaattgtaatgttgtttgttgtttgttgttgttggtaattgtggatcct-3 '(SEQ ID NO: 143)
(4) The p35S-Nos plasmid was digested with the restriction enzyme HindIII, and the attL1 fragment was inserted. This was further digested with EcoRI and the attL2 fragment was inserted to complete the vector p35SG.

<Construction of Transformation Vector Construction Vector pActG>
Using the obtained p35SG, pActG, a vector construction vector for transformation, was constructed as shown in the following steps (1) to (3) as shown in FIG.

  (1) The vector p35SG was digested with restriction enzymes HindIII and SmaI, the CaMV35S region was removed by electrophoresis, and the attL1 fragment was recovered.

  (2) Plasmid pActF1 / TA (N. Sentoku, Y. Sato, M. Matsuoka, Develop. Biol. 220, 358-364 (2000)) transferred from the University of Tokyo was digested with restriction enzymes HindIII and SmaI and subjected to electrophoresis. Thus, a DNA fragment containing the rice actin promoter region was recovered.

  (3) The obtained DNA fragment was inserted into the HindIII-SmaI site of the p35SG plasmid fragment from which CaMV35S was removed. This was further digested with HindIII, and the recovered attL1 fragment was inserted to complete the vector pActG.

<Construction of a construction vector incorporating a polynucleotide encoding a transcriptional repressor conversion peptide>
As shown in FIG. 3, pActSRDXG, a construction vector incorporating a polynucleotide encoding a transcription repressing conversion peptide, was constructed as shown in the following steps (1) to (2).

(1) DNAs consisting of the following sequences, which were designed to encode the 12 amino acid transcription repressor conversion peptide LDLDLELRLGFA (SRDX) and have a stop codon TAA at the 3 ′ end, were respectively synthesized and heated at 70 ° C. for 10 minutes. Thereafter, it was annealed by natural cooling to obtain double-stranded DNA.
5'-gggcttgatctggatctagaactccgtttgggtttcgcttaag-3 '(SEQ ID NO: 144)
5'-tcgacttaagcgaaacccaaacggagttctagatccagatcaagccc-3 '(SEQ ID NO: 145)
(2) pActG was digested with restriction enzymes SmaI and SalI, and double-stranded DNA encoding the above SRDX was inserted into this region to construct pActSRDXG.

<Construction of transformation vector>
As shown in FIG. 4, 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 SPW1 gene into construction vector>
A gene encoding the rice-derived transcription factor SPW1 protein was incorporated into the construction vector pActSRDXG as shown in the following steps (1) to (3).

(1) From a cDNA library prepared using mRNA prepared from rice, a DNA fragment containing only the coding region of the SPW1 gene excluding the stop codon was amplified by PCR using the following primers.
Primer 1 (SPW1-F) 5′-ggggatggggaggggcaagatcga-3 ′ (SEQ ID NO: 136)
Primer 2 (SPW1-R) 5'-tcaaccgaggcgcaggtcgtggt-3 '(SEQ ID NO: 137)
The cDNA and amino acid sequence encoding the SPW1 gene are shown in SEQ ID NOs: 135 and 134, respectively.

  (2) The obtained DNA fragment of the SPW1 coding region was ligated to the SmaI site of the construction vector pActSRDXG 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 an actin promoter, a chimeric gene, Nos-ter and the like on the construction vector into a 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, pACTSPW1SRDXG in which the coding region of the rice SPW1 gene is inserted in the forward direction, LR buffer 4.0 μL and TE buffer (10 mM TrisCl) diluted 5-fold to 1.5 μL (about 300 ng) and pBIGCKH 4.0 μL (about 600 ng) 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 (5), rice is transformed with pBIG-SPW1SRDX, which is a plasmid in which the DNA fragment containing the chimeric gene is incorporated into pBIGCKH, and transformed plants are obtained. Produced.

  (1) First, the obtained plasmid, pBIG-SPW1SRDX, was introduced into Agrobacterium EHA105, which was washed with 10% glycerol after culturing, using an electroporation method. After electroporation, Agrobacterium EHA105 (pBIG-SPW1SRDX) was obtained by culturing on an LB agar medium containing rifampicillin 100 mg / ml and kanamycin 20 mg / ml at 28 ° C. for 2 days.

  (2) Rice seeds were soaked in 70% ethanol for 1 minute and further sterilized by soaking in 2% sodium hypochlorite for 1 hour. After washing with sterile water, N6D solid medium (per liter: CHU [N6] Basal Salt Mixture (Sigma) 3.98 g, sucrose 30 g, myo-inositol 100 mg, casamino acid 300 mg, L-proline 2878 mg, glycine 2 mg, nicotine Nine grains were seeded on plates of 0.5 mg of acid, 0.5 mg of pyridoxine hydrochloride, 1 mg of thiamine hydrochloride, 2 mg of 2,4-D, 4 g of gellite, pH 5.8), and cultured for 24 days to induce callus. The callus of about 20 formed seeds was transplanted to a new N6D solid medium and further cultured for 3 days.

(3) On the other hand, Agrobacterium EHA105 (pBIG-SPW1SRDX) containing 5 ml of rifampicillin 100 mg / ml and kanamycin 20 mg / ml of YEP medium (per liter: Bacto peptone 10 g, Bacto yeast extract 10 g, NaCl 5 g, MgCl _2. The cells were cultured at 28 ° C. for 24 hours with 406 mg of 6H ₂ O, pH 7.2). This Agrobacterium is AAM medium containing 20 mg / l acetosyringone (per liter: MnSO ₄ · 5H ₂ O 10 mg, H ₃ BO ₃ 3 mg, ZnSO ₄ · 7H ₂ O 2 mg, Na ₂ MoO ₄ · 2H ₂ O 250μg, CuSO ₄ · 5H ₂ O 25μg, CoCl ₂ · 6H ₂ O 25μg, KI 750μg, CaCl ₂ · 2H ₂ O 150mg, MgSO ₄ · 7H ₂ O 250mg, Fe-EDTA 40mg, NaH ₂ PO4 · 2H ₂ O 150 mg, nicotinic acid 1 mg, thiamine hydrochloride 10 mg, pyridoxine hydrochloride 1 mg, myo-inositol 100 mg, L-arginine 176.7 mg, glycine 7.5 mg, L-glutamine 900 mg, aspartic acid 300 mg, KCl 3 g, pH 5.2). D. 660 was diluted to 0.1 to prepare a 20 ml Agrobacterium suspension.

  (4) The callus cultured for 3 days was mixed with the Agrobacterium suspension for 1 minute. The callus was then placed on a sterilized paper towel to remove excess Agrobacterium suspension. The callus was then sterilized with filter paper and 2N6-AS solid medium (per liter: CHU [N6] Basal Salt Mixture 3.98 g, sucrose 30 g, glucose 10 g, myo-inositol 100 mg, casamino acid 300 mg, glycine 2 mg, nicotine Acid 0.5 mg, pyridoxine hydrochloride 0.5 mg, thiamine hydrochloride 1 mg, 2,4-D 2 mg, acetosyringone 10 mg, gellite 4 g, pH 5.2), and cultured in the dark at 25 ° C. for 3 days. After culturing for 3 days, the cells were thoroughly washed with a 3% sucrose aqueous solution containing 500 mg / l carbenicillin until they became no cloudy, and cultured for 1 week on an N6D solid medium containing 500 mg / l carbenicillin and 10 mg / l hygromycin. Thereafter, the cells were transplanted to an N6D solid medium containing 500 mg / l carbenicillin and 50 mg / l hygromycin and cultured for 18 days.

  (5) Further, this callus was regenerated into a redifferentiation medium (per liter: mixed salt for Murashige-Skoog medium (manufactured by Nippon Pharmaceutical) 4.6 g, sucrose 30 g, sorbitol 30 g, casamino acid 2 g, myo-inositol 100 mg, glycine 2 mg, nicotinic acid 0.5 mg, pyridoxine hydrochloride 0.5 mg, thiamine hydrochloride 0.1 mg, NAA 0.2 mg, kinetin 2 mg, carbenicillin 250 mg, hygromycin 50 mg, agarose 8 g, pH 5.8). Replanted to a new medium every week, and redifferentiated and buds grown to about 1 cm are hormone-free medium (per liter: mixed salt for Murashige-Skoog medium (made by Nippon Pharmaceutical) 4.6 g, sucrose 30 g Sorbitol 30 g, casamino acid 2 g, myo-inositol 100 mg, glycine 2 mg, nicotinic acid 0.5 mg, pyridoxine hydrochloride 0.5 mg, thiamine hydrochloride 0.1 mg, hygromycin 50 mg, gellite 2.5 g, pH 5.8). The plant grown to about 8 cm on the hormone-free medium was transferred to a flower pot containing a synthetic granular soil bonsol No. 1 (manufactured by Sumitomo Chemical Co., Ltd.) to grow a transformed plant. In this way, seven lines of T1 plants obtained by redifferentiating and growing callus infected with Agrobacterium were obtained.

  (4) For each line, total RNA was prepared from these plants, and it was confirmed that the SPW1SRDX gene was introduced using RT-PCR.

  (5) The flower was observed about the obtained T1 plant body. As a result, it was confirmed that in 5 lines of T1 plants excluding 2 lines currently under investigation, stamens became feminized in almost all T1 plant flowers. FIG. 5 shows the results of observing spikes and flowers for 5 lines of T1 plants. FIG. 5 (a) shows, as a control, a rice flower into which a vector similar to that used in the examples was introduced except that the chimeric gene of SPW1 gene and a polynucleotide encoding a transcription repressing conversion peptide was not included (b) ) Indicates the ear. FIG. 5 (c) shows a flower obtained from line # 1 of the obtained T1 plant body, and (d) shows its ear. FIG. 5 (e) shows a flower obtained from line # 4 of the obtained T1 plant body, and (f) shows its ear. FIG. 5 (g) shows a flower obtained from line # 5 of the obtained T1 plant, and (h) shows its ear. FIG. 5 (i) shows the flower obtained from line # 6 of the obtained T1 plant, and (j) shows its ear. FIG. 5 (k) shows a flower obtained from line # 7 of the obtained T1 plant body. The flowers shown in the figure are obtained by removing half of the outer pod and inner pod (or the outer pod and inner pod-like organs in the transformant) so that the inside can be seen. As shown in FIG. 5, in the obtained T1 plant body, it was confirmed that six stamens in the wild type (control) became feminine in any example, and the formation of stamens was inhibited. All six stamens were feminized.

  In addition, as shown in FIG. 5 (b), in the control, the buds are closed with the buds left outside after flowering. Thus, the flowering of rice is a series of movements in which the spikelets open, the spikes protrude outward, and the spikelets close while leaving the spikes outside. On the other hand, in the obtained T1 plant body, as shown in FIGS. 5 (d), (f), (h), and (j), no wrinkles left outside are observed. This is because stamens (flower threads and cocoons) are not formed in the T1 plant.

  Table 2 shows the number of spikes in the T1 plant, the number of spikes in which no wrinkles and pollen were formed and no fruiting, and the presence or absence of a flower abnormality. Here, “the wrinkles and pollen were not formed and there was no fruiting” means “because the male camellia became female and the wrinkles did not remain outside after flowering and did not bear fruit”. As shown in Table 2, in the lines # 1, # 4, # 5, # 6, and # 7 of the T1 plant body, no wrinkles and pollen were formed in all ears, and no fruit was formed. Moreover, abnormality was seen in all the flowers. Thus, when the expression of SPW1 was suppressed by RNAi in Non-Patent Document 6, the number of flowers in which at least one stamen changed into the heart skin was 56, 2% of the total number of flowers of the transformant. However, in the method of the present invention, all stamens changed to female pods in approximately 100% of the total number of flowers in the transformant.

  Thus, the plant transformed with pBIG-SPW1SRDX was a mutant in which normal pollen formation was not performed, in which formation of stamens was inhibited. In addition, when the pollen of the wild type plant body was pollinated by the female pod of this plant body, seeds were formed. From this, it was confirmed that the plant body transformed with pBIG-SPW1SRDX was a male sterile body in which the pistil had fertility.

  According to the present invention, normal pollen formation is not performed, but so-called male sterile plants that have fertility can be produced in a wide range of plants. Therefore, the present invention can be used for various agriculture, 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 vector p35SG used in an Example. It is process drawing which shows the construction method of vector pActG used in an Example. FIG. 4 is a process diagram for incorporating a gene encoding the transcription repressor conversion peptide SRDX and the SPW1 gene into the vector pActG used in the Examples. It is process drawing which shows the construction method of vector pBIGCKH for transformation. FIG. 5 (a) is a diagram showing a rice flower into which a vector not containing a chimeric gene of SPW1 gene and a polynucleotide encoding a transcription repressing conversion peptide is introduced, and (b) is a diagram showing its ears, (C) is a figure which shows the flower obtained from line # 1 of the obtained T1 plant body, (d) is a figure which shows the ear, (e) is line # of the obtained T1 plant body (F) is a figure showing its ears, (g) is a figure showing flowers obtained from line # 5 of the obtained T1 plant body, h) is a diagram showing the ear, (i) is a diagram showing a flower obtained from line # 6 of the obtained T1 plant body, (j) is a diagram showing the ear, and (k ) Is a diagram showing a flower obtained from line # 7 of the obtained T1 plant body.

Claims (18)

A transcription factor that promotes transcription of a gene involved in flower organ formation, the following (a) or (b)
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 134,
(B) The amino acid sequence shown in SEQ ID NO: 134 consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and promotes transcription of genes involved in flower organ formation. Functional protein,
A chimera protein produced by fusing a transcription factor consisting of the protein described in 1. and a functional peptide that converts a transcription factor into a transcriptional repressor is produced in a plant, and transcription of genes involved in flower organ formation is suppressed. A method for producing male sterile bodies of plants.   The method for producing a male sterile body of a plant according to claim 1, wherein the male sterile body of the plant is at least prevented from forming a male stamen.   2. A transformation step of introducing a recombinant expression vector containing a chimeric gene comprising a gene encoding the transcription factor and a polynucleotide encoding the functional peptide into a plant cell. Or the production method of the male sterile body of the plant of 2.   The method for producing a male sterile body of a plant according to claim 1, further comprising an expression vector construction step for constructing the recombinant expression vector. The method for producing a male sterile body of a plant according to claim 3 or 4, wherein the gene described in (c), (d) or (e) below is used as the gene encoding the transcription factor: ,
(C) a gene consisting of the base sequence represented by SEQ ID NO: 135,
(D) a gene having a base sequence consisting of positions 216 to 890 of the base sequence represented by SEQ ID NO: 135 as an open reading frame region;
(E) encodes a transcription factor that hybridizes with a gene comprising a base sequence complementary to either (c) or (d) above under stringent conditions and promotes transcription of a gene involved in flower organ formation. gene.   The said plant is a monocotyledonous plant, The production method of the male sterile body of the plant of Claims 1-5.   The method for producing a male sterile body of a plant according to claim 1, wherein the plant is rice. The functional peptide is represented by the following formulas (1) to (4):
(1) X1-Leu-Asp-Leu-X2-Leu-X3
(In the formula, X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Wherein, Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)
(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Wherein, Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)
(4) Asp-Leu-Z4-Leu-Arg-Leu
(However, in the formula, Z4 represents Glu, Gln or Asp.)
The method for producing a male sterile body of a plant according to any one of claims 1 to 7, which comprises an amino acid sequence represented by any one of the above.   The production of a male sterile body of a plant according to any one of claims 1 to 7, wherein the functional peptide is a peptide consisting of the amino acid sequence shown in any one of SEQ ID NOs: 3 to 19. Method. The method for producing a male sterile body of a plant according to any one of claims 1 to 7, wherein the functional peptide is a peptide described in (f) or (g) below.
(F) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 20 or 21.
(G) The amino acid sequence shown in SEQ ID NO: 20 or 21 comprises an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and the transcription factor is used as a transcription repressor. A peptide having a function to convert. The functional peptide is represented by the following formula (5)
(5) α1-Leu-β1-Leu-γ1-Leu
(Wherein α1 represents Asp, Asn, Glu, Gln, Thr or Ser, β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, and γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys, or Asp.)
The method for producing a male sterile body of a plant according to any one of claims 1 to 7, wherein the plant is composed of an amino acid sequence represented by the following formula. The functional peptide is represented by the following formulas (6) to (8):
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(Wherein α1 represents Asp, Asn, Glu, Gln, Thr or Ser, α2 represents Asn, Glu, Gln, Thr or Ser, and β1 represents Asp, Gln, Asn, Arg, Glu. , Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, and γ2 represents Gln, Asn, Thr, Ser, His, Lys or Asp.)
The method for producing a male sterile body of a plant according to any one of claims 1 to 7, which comprises an amino acid sequence represented by any one of the above.   The functional peptide is an amino acid sequence represented by SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 133, 59 or 60 The method for producing a male sterile body of a plant according to any one of claims 1 to 7, which is a peptide comprising:   The said functional peptide is a peptide which consists of an amino acid sequence shown by sequence number 38 or 39, The production method of the male sterile body of a plant of any one of Claims 1-7 characterized by the above-mentioned.   A male sterile body of a plant produced by the production method according to claim 1.   The plant male sterile body according to claim 15, wherein the plant body contains 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 14,
A transcription factor that promotes transcription of a gene involved in flower organ formation, the gene encoding a transcription factor comprising the protein described in (a) or (b) above, and a functional peptide that converts the transcription factor into a transcription repressor A kit for producing a male sterile body of a plant, comprising at least a recombinant expression vector comprising a polynucleotide encoding a promoter and a promoter.   The kit for producing a male sterile body according to claim 17, further comprising a reagent group for introducing the recombinant expression vector into a plant cell.

Images