JP2005204657A - Method for producing plant body with reduced tannin content, plant body obtained using the same, and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a plant body decreased in a tannin content. <P>SOLUTION: The method for producing the plant body decreased in the tannin content comprises producing a chimeric protein formed by fusing a transcription factor which promotes gene expression of an enzyme participating in a phenylpropanoid synthesis system together with a functional peptide which converts an optional transcription factor into a transcription inhibitory factor in a plant cell, by introducing a chimeric gene composed of a gene which encodes the transcription factor and a polynucleotide which encodes the functional peptide into the plant cell. Thus, the plant body decreased in the tannin content is produced, because the chimeric protein inhibits expression of a gene participating in a tannin synthesis system. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  TECHNICAL FIELD The present invention relates to a method for producing a plant with reduced tannin content, a plant obtained using the same, and use thereof, and in particular, tannin by suppressing transcription of a gene involved in a tannin synthesis system. The present invention relates to a method for producing a plant having a reduced content, a plant obtained using the method, and use thereof.

  Conventionally, PAP1 (Production of Anthocyanin Pigment1) protein of Arabidopsis thaliana is known as a transcription factor that promotes gene expression of enzymes involved in phenylpropanoid synthesis systems in plants. When PAP1 protein is overexpressed, it promotes the expression of chalcone synthase (CHS), dihydroflavonol reductase (DFR), phenylalanine ammonia-lylase (PAL), etc. involved in the phenylpropanoid synthesis system, It has been reported that the concentrations of anthocyanin, hydroxycinnamic acid, and lignin increase (see, for example, Non-Patent Document 3).

  In plants, the Arabidopsis TT2 (TRANSPARENT TESTA2) protein is known as a transcription factor that controls gene expression of an enzyme involved in the tannin synthesis system. Overexpression of TT2 protein promotes the expression of dihydroflavonol reductase (DFR), anthocyanidin reductase, also known as vanilla (BAN), involved in the phenylpropanoid synthesis system in the roots of Arabidopsis germinating plants on the 10th day. (Non-patent Document 8). Banyuras (BAN) is a core enzyme in the biosynthesis of proanthocyanidins, and it is known that when a mutation occurs in the TT2 gene, the promoter activity of Vanyuras (BAN) is lost (Non-patent Document 9). .

  Here, the present inventors have found various peptides that convert an arbitrary transcription factor into a transcriptional repressing factor (see, for example, Patent Documents 1 to 7, Non-Patent Documents 1 and 2). 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 expresses a fusion protein (chimeric protein) in which the PAP1 protein and the peptide are fused in a plant body, converts the PAP1 protein, which is a transcription factor, into a transcription repressor, and suppresses the accumulation of anthocyanins. Has been successful. Specifically, a chimeric gene was constructed by binding a polynucleotide encoding the peptide to a gene encoding a PAP1 protein, and the chimeric gene was incorporated into a vector and introduced into a plant. When a plant body expressing the chimeric gene is grown in the presence of 3% sucrose, which is a stressing condition, the accumulation of red-violet pigment showing the characteristics of anthocyanins observed in the wild type may not occur. It was confirmed (see, for example, Patent Document 7).

  By the way, tannin is known as a kind of plant polyphenol. This tannin is a substance widely present in the plant kingdom, and is classified into a condensed type and a hydrolyzed type. Among them, condensed tannin is a flavonoid polymer and is therefore a phenylpropanoid metabolite, but the exact synthetic route is not well understood.

  Tannin has been known for its usefulness such as being used for tanning since ancient times, and in recent years, its antioxidant action has attracted attention. However, when tannin is seen as a component contained in a plant body, it has one aspect of a component that exerts a food damage protection function. Specifically, animals that eat tannin-containing plants feel the astringent and unpleasant taste of tannins, but not only that, but plant proteins bind to tannin and become unable to digest. Gastrointestinal enzymes and tannin may react. Therefore, for example, there is a report that plants containing a large amount of tannin do not have a good feed effect (see, for example, Non-Patent Documents 4, 5, 6, and 7).

  For the same reason, tannin in tea (especially green tea) is not good for people with weak bodies because it denatures cells, tissues or enzymes in the body. Furthermore, in green tea, fruit liquor, etc., it may cause astringency or cause deterioration in appearance and flavor due to oxidation browning. Furthermore, tannin also causes discoloration and spots in wood.

Thus, in the use using the plant body itself, it is preferable that the tannin content in the plant body is small. Therefore, if the tannin content of the plant body can be reduced by suppressing the synthesis of tannin, these problems can be solved. Thus, there are many areas where it is advantageous to reduce the tannin content.
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) 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 JOBorevitz, Y.Xia, J.Blount, RADixon, C.Lamb, Activation Tagging Identifies a Conserved MYB Regulator of Phenylpropanoid Biosynthesis Hassan IA, Elzubeir EA, El Tinay AH, Growth and apparent absorption of minerals in broiler chicks fed diets with low or high tannin contents, Trop Anim Health Prod. 2003 Apr; 35 (2): 189-96 Van der Poel AF, Dellaert LM, Van Norel A, Helsper JP, The digestibility in piglets of faba bean as affected by breeding towards the absence of condensed tannins, Br J Nutr. 1992 Nov; 68 (3): 793-800 Sarwar, G. et al., 1981, Nutritional evaluation of meals and meal fractions derived from rape and mustard seed, Canadian Journal of Animal Science 61,719-733 Simbaya, J. et al., 1995, Quality characteristics of yellow-seeded Brassica seed meals: protein, carbohydrates, and dietary fiber components, Journal of agricultural and Food Chemistry 43,2062-2066 Nathalie Nesi, Clarisse Jond, Isabelle Debeaujon, Michel Caboche, and Loic lepiniec., The Plant Cell, 13, 2099-2114, 2001 Debeaujon I, Nesi N, Perez P, Devic M, Grandjean O, Caboche M, Lepiniec L, Plant Cell, 15, 2514-2531. 2003

  As described above, it would be very useful if a plant with a reduced tannin content could be obtained. However, the conventional technique has a problem that it cannot produce a plant with a reduced tannin content.

  That is, as described above, it has been reported that the expression of genes such as CHS is promoted in plants overexpressing PAP1 protein, and the expression of genes such as DFR is promoted in plants overexpressing TT2 protein. However, since there is no report that the synthesis of tannin was promoted, it is not clear which reaction in the tannin synthesis pathway can be suppressed to suppress the synthesis of tannin. Conventionally, a technique for reducing the tannin content by suppressing transcription of a gene involved in the tannin synthesis system has not been known. Therefore, it was impossible to produce a plant with a reduced tannin content.

  This invention is made | formed in view of the said subject, The objective provides the method of producing the plant body with which the tannin content was reduced by suppressing the transcription | transfer of the gene involved in the tannin synthesis system. There is.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor can suppress not only anthocyanin synthesis but also tannin synthesis by converting PAP1 protein into a transcriptional repressor, and PAP1 is a tannin. It was revealed for the first time that it is a transcription factor involved in the synthesis system. Moreover, tannin synthesis can be suppressed by converting the TT2 protein into a transcriptional repressing factor, and thus the present invention has been completed.

  That is, the method for producing a plant body with reduced tannin content according to the present invention encodes an enzyme involved in a phenylpropanoid synthesis system in order to reduce the tannin content of the plant body in order to solve the above-mentioned problems. It is characterized by inhibiting the production of tannin by producing a chimeric protein in a plant that fuses a transcription factor that promotes gene expression and a functional peptide that converts any transcription factor into a transcriptional repressor. It is said.

  According to the above configuration, the transcription factor can be converted into a transcription repressor by binding the DNA binding domain derived from the transcription factor in the chimeric protein to a target gene presumed to be involved in the tannin synthesis system. In the plant body, transcription of the target gene of the transcription factor is suppressed. Therefore, the production of tannin, which is the final substance of the tannin synthesis system, is reduced, and as a result, the tannin content of the obtained plant can be reduced.

  Further, the production method may include 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. Good.

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

  According to the above configuration, the chimeric protein can be expressed in a plant cell simply by incorporating the transcription factor gene into a cassette vector to which the functional peptide is added and introducing the gene into the plant cell. Thus, transcription of a transcription factor target gene can be easily suppressed. Therefore, the tannin content of the obtained plant can be reduced.

  The transcription factor is a protein described in the following (a) or (b). (A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1. (B) an amino acid sequence represented by SEQ ID NO: 1 comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and an enzyme involved in a phenylpropanoid synthesis system; A protein having a function of promoting expression of a gene to be encoded.

  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 shown in SEQ ID NO: 2 as an open reading frame region. (D) a gene that hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2 under stringent conditions and encodes an enzyme involved in the phenylpropanoid synthesis system A gene encoding a transcription factor that promotes expression.

  The transcription factor is a protein described in the following (e) or (f). (E) a protein comprising the amino acid sequence represented by SEQ ID NO: 145; (F) an amino acid sequence represented by SEQ ID NO: 145 comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and an enzyme involved in a phenylpropanoid synthesis system; A protein having a function of promoting expression of a gene to be encoded.

  Moreover, it is preferable to use the gene of the following (g) or (h) as a gene which codes the said transcription factor. (G) A gene having the base sequence represented by SEQ ID NO: 146 as an open reading frame region. (H) 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: 146 and encodes an enzyme involved in the phenylpropanoid synthesis system A gene encoding a transcription factor that promotes expression.

  According to the said structure, the said transcription factor is converted into a transcriptional repression factor by the said functional peptide, and transcription | transfer of the target gene of the said transcription factor is suppressed. Therefore, a plant body in which the tannin content is efficiently reduced can be obtained.

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 (i) or (j). (I) A peptide having the amino acid sequence shown in SEQ ID NO: 20 or 21. (J) A peptide having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 20 or 21.

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

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

  The functional peptide is represented by SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 133, 134, 135, or 136. It may be a peptide having the amino acid sequence shown.

  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, and most of them are extremely short peptides, so that synthesis is easy, Transcriptional repression of a gene involved in tannin synthesis, which is a target gene of the transcription factor, can be efficiently performed. Therefore, the expression of the target gene can be effectively suppressed.

  Moreover, the plant body concerning this invention is produced by the said production method, The tannin content is reduced, 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.

  As described above, the plant body includes various forms such as a grown plant individual, a plant cell, and a seed, all of which are considered to have a reduced tannin content compared to the wild-type plant body. It is done. Therefore, in various fields such as agriculture and forestry, it is possible to use a plant body with a reduced tannin content in a desired form.

  A plant tannin content reduction kit according to the present invention is a kit for carrying out the above production method, comprising a transcription factor that promotes the expression of a gene encoding an enzyme involved in a phenylpropanoid synthesis system. It is characterized by comprising at least a recombinant expression vector comprising a gene to be encoded, a polynucleotide encoding a functional peptide that converts any transcription factor into a transcription repressor, and a promoter. The tannin content reduction kit may further include a reagent group for introducing the recombinant expression vector into plant cells.

  Since the said tannin content reduction kit is equipped with the structure required in order to implement the production method of the plant body concerning this invention previously, the said production method can be implemented easily. Therefore, according to the said tannin content reduction kit, the plant body in which the tannin content was reduced can be obtained efficiently.

  As described above, the plant production method according to the present invention converts a transcription factor that promotes the expression of a gene encoding an enzyme involved in a phenylpropanoid synthesis system and an arbitrary transcription factor into a transcription repressor. It has a configuration in which a chimeric protein fused with a functional peptide is produced in a plant body. The chimeric protein can suppress transcription of genes involved in the tannin synthesis system. Therefore, the method for producing a plant according to the present invention has an effect that the tannin content of the obtained plant can be 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 reducing the tannin content of a plant, and converts a transcription factor that promotes the expression of a gene encoding an enzyme involved in a phenylpropanoid synthesis system and an arbitrary transcription factor into a transcriptional repressor. A chimeric protein fused with a functional peptide is produced in a plant. In the plant obtained by this, since the production of tannin is suppressed, a plant with a reduced tannin content can be produced.

  Here, the production of tannin is suppressed 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 the tannin synthesis system. The transcription factor is converted into a transcription repressing factor, and transcription of the target gene is repressed. Thereby, the production | generation of the tannin which is the final substance of a tannin synthesis type | system | group reduces, As a result, the tannin content of the plant body obtained can be reduced.

  As described above, it has been reported that in PAP1 protein overexpressing plants, the expression of PAL, CHS and DFR genes is promoted and the concentrations of anthocyanin, hydroxycinnamic acid and lignin are increased. FIG. 4 (a) is an overview diagram of a phenylpropanoid metabolite, and the PAP1 transcription factor promotes gene expression of PAL, CHS, and DFR, and the pathway through which the synthesis of anthocyanins is promoted is indicated by a bold line. However, there is no report that the synthesis of the tannin (condensed tannin, shown as Condensed tannins in the figure) located downstream is promoted in spite of the enhanced enzyme expression involved in the initial steps of phenylpropanoid synthesis. This suggests that there are unique regulatory factors other than PAP1 protein in the tannin synthesis pathway.

  In addition, as described above, when TT2 protein is overexpressed, dihydroflavonol reductase (DFR), anthocyanidin reductase, also known as BANURAS (BAN) involved in the phenylpropanoid synthesis system in the roots of plants on the 10th day of germination of Arabidopsis thaliana ) Has been reported to be promoted.

  However, there is no report that the synthesis of anthocyanins and tannins (condensed tannins, described as “Condensed tannins” in the figure) has been promoted (Non-patent Document 8). This suggests that there are unique regulatory factors other than TT2 protein in the tannin synthesis pathway.

  In general, it is known that it is difficult to achieve the expected effect when trying to enhance or reduce plant secondary metabolites using genetic engineering (PNAS 2001, vol. 19, 367-372) . The reason is that there are isozymes and duplicate factors in the enzymes and regulatory factors that are the targets of genetic manipulation, and that they are supplemented by another metabolic pathway (the presence of a bypass).

  Therefore, anthocyanin biosynthesis and tannin biosynthesis have regulatory factors specific to the respective pathways, and the presence of duplicate factors is presumed. Therefore, by merely suppressing transcription promoted by PAP1 protein or TT2 protein, It is usually difficult to suppress tannin production. The method according to the present invention succeeded in overcoming such difficulties by fusing the functional protein with PAP1 protein or TT2 protein.

  That is, a chimeric protein in which a PAP1 protein and a functional peptide that converts an arbitrary transcription factor into a transcriptional repressor can fuse two anthocyanin and tannin biosynthetic pathways simultaneously. Moreover, the chimeric protein in which the TT2 protein and the functional peptide were fused was able to suppress at least the tannin biosynthesis pathway.

  In the following description, a chimeric protein used in a method for producing a plant with reduced tannin content according to the present invention (for convenience of explanation, referred to as a tannin-reduced plant), an example of a method for producing a plant according to the present invention, The plant body obtained by this, its usefulness, and its utilization are each demonstrated.

(I) Chimeric protein used in the present invention As described above, the chimeric protein used in the present invention comprises a transcription factor that promotes the expression of a gene encoding an enzyme involved in a phenylpropanoid synthesis system, and an arbitrary transcription. It is a fusion with a functional peptide that converts a factor into a transcriptional repressor. Therefore, each of the transcription factor and the functional peptide will be described.

(I-1) Transcription factor that promotes the expression of a gene encoding an enzyme involved in a phenylpropanoid synthesis system The transcription factor used in the present invention promotes the gene expression of an enzyme involved in a phenylpropanoid synthesis system The transcription factor is not particularly limited. Since the synthesis system of phenylpropanoids is widely conserved in the plant kingdom, such transcription factors 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. Among these, the transcription factor is more preferably a transcription factor having a MYB-like structure that promotes anthocyanin synthesis.

  Examples of such transcription factors include, but are not particularly limited to, PAP1 protein, PAP2 protein, TT2 protein, AN2 protein, C1 protein, P1 protein, P protein, GL1 protein, Mixta protein, and the like. .

  Typical examples of transcription factors used in the present invention include PAP1 protein and TT2 protein. The PAP1 protein is a protein having the amino acid sequence shown in SEQ ID NO: 1, and, as described above, chalcone synthase (CHS), dihydroflavonol reductase (DFR), phenylalanine ammonia- involved in the phenylpropanoid synthesis system. It is known to promote the expression of lylase (PAL) and the like. The TT2 protein is a protein having the amino acid sequence shown in SEQ ID NO: 145. Among the TT2 null mutants, dihydroflavonol reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), anthocyanidin reductase (BAN) And TT2 expression is known to be suppressed.

  In the present invention, for example, a PAP1 protein or TT2 protein, which is a transcription factor, is converted into a transcription repressing factor by fusing a functional peptide described later to the PAP1 protein or TT2 protein.

  The transcription factor used in the present invention is not limited to the PAP1 protein having the amino acid sequence shown in SEQ ID NO: 1 or the TT2 protein having the amino acid sequence shown in SEQ ID NO: 145. Any transcription factor may be used as long as it has a function of promoting the expression of a gene encoding the enzyme involved. Specifically, for example, in the amino acid sequence shown in SEQ ID NO: 1 or 145, even if the protein consists of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, If it has a function, it can be used in the present invention.

  In the above “one or several amino acid sequences in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or 145” Although the range is not particularly limited, for example, it means 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably 1 to 3.

  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 a PAP1 protein is used as the transcription factor, the gene encoding the PAP1 protein (for convenience of explanation, (Referred to as PAP1 gene). As a specific example of the PAP1 gene, for example, a polynucleotide containing the base sequence shown in SEQ ID NO: 2 as an open reading frame (ORF) can be mentioned.

  Further, as another specific example, for example, when TT2 protein is used as a transcription factor, a gene encoding this TT2 protein (referred to as TT2 gene for convenience of description) can be exemplified. As a specific example of the TT2 gene, for example, a polynucleotide containing the base sequence represented by SEQ ID NO: 146 as an open reading frame (ORF) can be mentioned.

  Of course, the PAP1 gene, the TT2 gene, or the gene encoding the transcription factor used in the present invention is not limited to the above example, and is represented by the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 146. It may be a gene having homology with the base sequence. Specifically, for example, a gene that hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2 or SEQ ID NO: 146 under stringent conditions and 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 a known transcription factor base sequence can be used. Using this primer pair, the gene can be obtained by PCR using plant cDNA or genomic DNA as a template. The gene encoding the transcription factor can also be obtained by chemical synthesis by a conventionally known method.

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

  The present inventor has found that a protein obtained by binding an AtERF3 protein, an AtERF4 protein, an AtERF7 protein, or an AtERF8 protein derived from Arabidopsis thaliana, which is one of the Class II ERF gene group, significantly suppresses gene transcription. Obtained. Thus, an effector plasmid containing a gene encoding each of the above proteins and DNA excised therefrom was constructed and introduced into a plant cell, thereby succeeding in actually suppressing the transcription of the gene (for example, Patent Documents 1 to 3). 4).

  In addition, a gene encoding tobacco ERF3 protein (see, for example, Patent Document 5), a rice OsERF3 protein (see, for example, Patent Document 6), which is one of the Class II ERF gene groups, and one of the zinc finger protein gene groups. A similar test as described above was performed for a gene encoding a certain Arabidopsis thaliana ZAT10 and ZAT11, and it was found that transcription of the gene was suppressed. Furthermore, the present inventor has revealed that these proteins have a common motif including aspartic acid-leucine-asparagine (DLN) in the carboxyl group terminal region. As a result of examining 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-2-1) Transcriptional Repression Conversion Peptide of Formula (1) In the transcriptional repression conversion peptide of the above formula (1), the number of amino acid residues represented by X1 should be in the range of 0-10. That's fine. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by X1 is not specifically limited, What kind of thing may be sufficient. In other words, in the transcription repressor converting peptide of the above formula (1), an oligomer consisting of one arbitrary amino acid or 2 to 10 arbitrary amino acid residues may be added to the N-terminal side. However, no amino acid may be added.

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

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

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

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

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

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

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

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

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

  The amino acid residue represented by Z3 should be as short as possible from the viewpoint of ease of synthesis of the transcriptional repressor converting peptide of formula (3). Specifically, it is preferably 10 or less, and more preferably 5 or less. Specific examples of the amino acid residue represented by Z3 include Gly, Gly-Phe-Phe, Gly-Phe-Ala, Gly-Tyr-Tyr, Ala-Ala-Ala and the like. It is not limited.

  In addition, the number of amino acid residues in the entire transcriptional repressor conversion peptide represented by the formula (3) is not particularly limited, but 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 having a minimum sequence such as a hexamer represented by the above formula (4). For example, the amino acid sequence shown in SEQ ID NO: 7 corresponds to the 196th to 201st amino acid sequence of Arabidopsis SUPERMAN protein (SUP protein), and as described above, the present inventor newly found as the above transcription repressor conversion peptide. It is.

(I-2-5) More specific example of transcriptional repression converting peptide As a more specific example of the transcriptional repressing converting peptide represented by each of the above-mentioned formulas, And a peptide having an amino acid sequence. These oligopeptides have been found by the present inventor to be the above-described transcriptional repression converting peptide (see, for example, Patent Document 7).

Furthermore, the other oligopeptide of the following (i) or (j) description can be mentioned as another specific example of the said transcription repression conversion peptide.
(I) A peptide consisting of any amino acid sequence shown in SEQ ID NO: 20 or 21.
(J) A peptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in any one of the amino acid sequences shown in SEQ ID NO: 20 or 21.

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

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

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

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

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

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

  As more specific examples of the transcriptional repression converting peptide having the amino acid sequence represented by the above formulas (5) to (9), SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, Mention may be made of peptides having the amino acid sequence represented by 31, 32, 33, 34, 35, 36, 37, 133, 134, 135 or 136. Among these, the peptide of SEQ ID NO: 29, 30, 32, 34, 133, 134, 135 or 136 corresponds to the peptide represented by the general formula (6), and the peptide of SEQ ID NO: 22, 25, 35, 36 or 37 Corresponds to the peptide represented by the general formula (7), the peptide of SEQ ID NO: 26, 27, 28, 31, or 33 corresponds to the peptide represented by the general formula (8), and The peptide 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-4) Chimeric protein production method The various transcription repressor converting peptides described in (I-2) and (I-3) above are fused with the transcription factor described in (I-1) above to form a chimeric protein. By doing so, the transcription factor can be used as a transcriptional repression factor. Therefore, in the present invention, a chimeric protein can be produced by obtaining a chimeric gene with a gene encoding a transcription factor using the polynucleotide encoding the transcription repressing conversion peptide.

  Specifically, a chimeric gene is constructed by linking a polynucleotide encoding the transcription repressing conversion peptide (referred to as a transcription repressing conversion polynucleotide for convenience of explanation) and a gene encoding the transcription factor, Introduce into cells. Thereby, a chimeric protein can be produced. A specific method for introducing the chimeric gene into plant cells will be described in detail in the section (2) described later.

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

  Specific examples of the transcription repression conversion polynucleotide include, for example, SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 137, 139, 141 or 143 Mention may be made of polynucleotides. Also, SEQ ID NOs: 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 138, 140, 142, 144 are complementary to the polynucleotides exemplified above, respectively. It is a polynucleotide. In addition, other specific examples of the above transcription repressor conversion polynucleotide include the polynucleotides shown in SEQ ID NOs: 95 and 96, for example. These polynucleotides correspond to the amino acid sequences shown in SEQ ID NOs: 3-39 and 133-136 as shown in Table 1 below.

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

(II) An example of a method for producing a plant according to the present invention A method for producing a plant according to the present invention includes a process of producing the chimeric protein described in (I) above in a plant and suppressing tannin production. However, if the production method of the plant body according to the present invention is shown by specific steps, for example, a production method including steps such as an expression vector construction step, a transformation step, and a selection step Can be mentioned. Among these, in the present invention, at least the transformation step may be included. Hereinafter, each step will be specifically described.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  The selection method is not particularly limited. For example, the selection may be performed on the basis of drug resistance such as hygromycin resistance, and after growing the transformant, the plant itself or any organ or tissue may be selected. You may select from the tannin content contained. For example, as an example of selecting from the tannin content, there can be mentioned a method of comparing the color of the seed of the transformant with the color of the seed of the non-transformed plant (see Examples described later). In particular, since the seed contains a large amount of tannin, the selection can be made by simply comparing the colors of the seeds, and the effect of the present invention of reducing the tannin content can be confirmed.

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

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

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

(III) Plant obtained by the present invention, its usefulness, and use thereof The method for producing a plant according to the present invention is based on expressing a gene encoding the chimeric protein in the plant. A transcription factor-derived DNA binding domain in the chimeric protein binds to a target gene presumed to be involved in the tannin synthesis system. The transcription factor is converted to a transcription repressor, and transcription of the target gene is repressed. Thereby, the production | generation of the tannin which is the final substance of a tannin synthesis type | system | group reduces, As a result, the tannin content of the plant body obtained can be reduced. Therefore, the present invention includes a plant obtained by the above-described method for producing a plant.

(III-1) Specific examples of plants according to the present invention The specific types of plants with reduced tannin content according to the present invention are not particularly limited, and their usefulness can be reduced by reducing the tannin content. Mention may be made of plants with increased properties. Such a plant may be an angiosperm or a gymnosperm. As a gymnosperm, for example, a cedar family, a pine family, a cypress family or a asteraceae plant can be mentioned. Further, the angiosperm may be a monocotyledonous plant or a dicotyledonous plant. Examples of the dicotyledonous plants include plants such as Brassicaceae such as Arabidopsis thaliana, Camelliaaceae such as legume, and tea. In addition, examples of monocotyledonous plants include plants such as rice, corn, wheat, etc.

(III-2) Usefulness of the Present Invention In the present invention, the tannin content of the plant can be reduced, but the usefulness of the present invention is not particularly limited, and any field that is effective by low tannins. For example, application to livestock feed, application to food, application to wood and the like can be mentioned.

  First, an application example to livestock feed will be described. For example, there are low tannin and tannin-free sorghum (sorghum, white sorghum) substitute feeds. These have a very good feed effect compared to high tannin sorghum. For example, broilers given low tannin sorghum grow significantly faster than those given high tannin sorghum, such as Ca, P, etc. There is a report that minerals are well absorbed (see Non-Patent Document 4). Sorghum is a sorghum plant. Low tannins and tannin-free sorghum are said to have almost the same feed effect as corn. Major producers are the United States, Argentina, Australia, etc., but especially Australian products are highly evaluated for low tannins and high protein. The present invention is considered to be applicable to low tannin and tannin-free sorghum. In general, sorghum is predominantly used for feed, but due to recent improvements in milling technology, processing technology for bread and cake flour and application technologies for foods such as starch, syrup and alcohol are also considered. Yes.

  In addition, it has been reported that when low tannin broad beans are given to piglets, digestibility is better than that of high tannin (see Non-Patent Document 5). In addition, when non-ruminant livestock and humans use seed foods as feed or nutritional supplements, tannins such as astringent taste, food discoloration, and the production of non-digestible substances by reacting with proteins, etc. It has been reported that undesirable adverse effects are observed (see Non-Patent Documents 6 and 7). If the tannin content of a wide variety of seed foods can be reduced, seed foods such as rape can be used for livestock feeds other than ruminants and for nutritional supplements that do not contain human gluten. It is thought that it becomes existence to compete for the usefulness of.

  In addition, as a herbivore diet, the advantage of using plants with reduced tannin content is great. Plants containing tannins are not preferred as a herbivore diet. In other words, the plant protein in the diet cannot be digested because it binds to tannin, making it unusable as a nutrient source. Tannins may also react with gastrointestinal digestive enzymes in herbivores. This technique can be used to reduce the tannin content of plants used as feed.

  Next, application examples to food will be described. For example, green tea, sencha, black tea, coffee, etc. with reduced tannin content can be mentioned. It is well known that green tea and sencha are not recommended for people with weak stomachs, but it is thought that the tannins of tea (especially green tea) transform cells, tissues, or enzymes in the body. . By the way, there is a report that human and animal cells are killed immediately when they are cultured in a test tube and added with tea. Therefore, it is considered that the present technology can be used for weak constitution, green tea, sencha and black tea for pregnant women, children and the like.

  Furthermore, in green tea, fruit liquor, tannin can cause astringency and can also cause the appearance and flavor to be impaired by oxidation browning. In particular, in PET bottle drinks and bottled fruit wines, it is desired to reduce the tannin content in order to solve these problems. It is considered that the present invention can be used in such a region.

  Next, application examples to wood will be described. For example, application to redwood and red cedar, which are frequently used for construction, can be given. These timbers are in great demand due to their high durability and beautiful grain finish, but they are said to be easily discolored (mainly black) due to their strong tannin quality, and the tannin component tends to stain. If this invention is used, it will become possible to improve a weak point, maintaining the advantage of these timbers, and it can also be utilized for quality improvement of timber.

(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. A reduction kit can be mentioned.

  As a specific example of this tannin content reduction kit, it suffices to contain at least a recombinant expression vector containing a chimeric gene comprising a gene encoding the above transcription factor and the above transcription repressor conversion polynucleotide. It is more preferable that a reagent group for introduction into plant cells is included. 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.

  Hereinafter, the present invention will be described more specifically based on examples and FIGS. 1 and 2, but the present invention is not limited thereto.

  In the following examples, a cauliflower that binds to a PAP1 gene or a TT2 gene, and that functions in plant cells, binds a polynucleotide that encodes the 12 amino acid peptide LDLDLELRLGFA (SRDX) (SEQ ID NO: 19), which is one of transcriptional repressor conversion peptides. A recombinant expression vector was constructed downstream of the mosaic virus 35S promoter and introduced into Arabidopsis thaliana using the Agrobacterium method to transform Arabidopsis thaliana.

(1) Construction of plant transformation vector pBIG2 Plasmid p35S-GFP from Clontech (Clontech, USA) was cleaved with restriction enzymes HindIII and BamHI, and a DNA fragment containing the cauliflower mosaic virus 35S promoter was subjected to agarose gel electrophoresis. Separated and recovered.

  The plant transformation vector pBIG-HYG (Becker, D. 1990 Nucleic Acid Research, 18: 203) transferred from Michigan State University, USA was cleaved with restriction enzymes HindIII and SstI, and the GUS gene was removed by agarose gel electrophoresis. DNA fragments were obtained.

DNA having the following sequence was synthesized, heated at 70 ° C. for 10 minutes, and then annealed by natural cooling to obtain double-stranded DNA. This DNA fragment has, from the 5 ′ end, a BamHI restriction enzyme site, an omega sequence derived from tobacco mosaic virus that enhances translation efficiency, a restriction enzyme site SmaI, and restriction enzyme sites SalI and SstI in this order.
5'-GATCCACAATTACCAACAACAACAAACAACAAACAACATTACAATTACAGATCCCGGGGGTACCGTCGACGAGCT-3 '(SEQ ID NO: 59)
5'-CGTCGACGGTACCCCCGGGATCTGTAATTGTAATGTTGTTTGTTGTTTGTTGTTGTTGGTAATTGTG-3 '(SEQ ID NO: 60)
Next, the DNA fragment containing the cauliflower mosaic virus 35S promoter region and the synthesized double-stranded DNA were inserted into the HindIII and SstI sites of pBIG-HYG excluding the GUS gene to obtain a plant transformation vector pBIG2. .

(2) Construction of recombinant expression vector pPAP1SRDX or pTT2SRDX <Isolation of PAP1 cDNA>
From an Arabidopsis thaliana cDNA library, a DNA fragment containing only the coding region of PAP1 excluding the stop codon using the following primers was amplified using PCR, and separated and collected by agarose electrophoresis. PCR 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.
5 'primer
5'-AAAATGGAGGGTTCGTCCAAAGGGCTGCGAAAAGG-3 '(SEQ ID NO: 57)
3 'primer
5'-ATCAAATTTCACAGTCTCTCCATCGAAAAGACTC-3 '(SEQ ID NO: 58)
The cDNA of PAP1 gene and the encoded amino acid sequence are shown in SEQ ID NOs: 2 and 1, respectively.
<Isolation of TT2 cDNA>
From an Arabidopsis thaliana cDNA library, a DNA fragment containing only the coding region of TT2 excluding the stop codon was amplified using PCR using the following primers, and separated and collected by agarose electrophoresis. PCR 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.
5 'primer
5′-GATGGGAAAGAGAGCAACTACTAGTGTGAG-3 ′ (SEQ ID NO: 147)
3 'primer
5'-ACAAGTGAAGTCTCGGAGCCAATCTTCATC-3 '(SEQ ID NO: 148)
The cDNA and encoded amino acid sequence of the TT2 gene are shown in SEQ ID NOs: 146 and 145, respectively.

<Synthesis of Polynucleotide Encoding Transcriptional Repression Conversion Peptide LDLDLELRLGFA (SRDX)>
DNAs encoding the 12 amino acid peptide LDLDLELRLGFA (SRDX) and designed to have a stop codon TAA at the 3 ′ end, each having the following sequence were synthesized, heated at 70 ° C. for 10 minutes, and then annealed by natural cooling Thus, double-stranded DNA was obtained.
5'-CTGGATCTGGATCTAGAACTCCGTTTGGGTTTCGCTTAAG-3 '(SEQ ID NO: 55)
5'-CTTAAGCGAAACCCAAACGGAGTTCTAGATCCAGATCCAG-3 '(sequence number 56)
<Construction of recombinant expression vector>
The DNA fragment containing only the protein coding region of the PAP1 gene or TT2 gene obtained above and the DNA fragment containing the coding region of the transcription repressor conversion peptide SRDX are inserted into the pBIG2 cut with the restriction enzyme SmaI and cloned in the forward direction Then, plasmid p35S :: PAP1SRDX which is a recombinant recombinant expression vector was obtained. FIG. 1 (a) shows the structure of p35S :: PAP1SRDX, and FIG. 1 (b) shows the structure of p35S :: TT2SRDX.

(3) Preparation of plant transformed with recombinant expression vector p35S :: PAP1SRDX or p35S :: TT2SRDX Transformation of Arabidopsis thaliana by p35S :: PAP1SRDX or p35S :: TT2SRDX is performed by 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. p35S :: PAP1SRDX or p35S :: TT2SRDX was introduced into the soil bacterium Agrobacterium tumefaciens strain GV3101 (C58C1Rifr) pMP90 (Gmr) (koncz and Schell 1986) by electroporation. The introduced bacteria were cultured in a YEP medium containing 1 liter of antibiotics (kanamycin (Km) 50 μg / ml, gentamicin (Gm) 25 μg / ml, rifampicillin (Rif) 50 μg / ml) until OD600 was 1. Subsequently, the cells were collected from the culture solution and suspended in 1 liter of an infiltration medium (Table 2 below).

  Arabidopsis thaliana grown for 14 days was soaked in this solution for 1 minute, infected, and then grown again and seeded. The collected seeds were sterilized with 50% 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).

  Transformed plants that grow on the hygromycin plate were selected and replanted in soil to obtain next generation seeds. The seeds of the plant transformed with p35S :: PAP1SRDX are shown in FIGS. 2 (b) and (c), the seeds of the wild type plant are shown in FIG. 2 (a), and transformed with p35S :: TT2SRDX. The seeds of the plant are shown in FIG. 3 (b), and the seeds of the wild type plant are shown in FIG. 3 (a).

  As shown in FIG. 2 (a), the wild-type seeds are dark brown indicating the accumulation of tannin, but as shown in FIGS. 2 (b) and (c), the plants transformed with p35S :: PAP1SRDX The seeds were white. Similarly, as shown in FIG. 3 (a), the wild-type seed is dark brown indicating accumulation of tannin, but as shown in FIG. 3 (b), the plant transformed with p35S :: TT2SRDX The seeds were white. From this, it was confirmed that the tannin content was remarkably reduced in the transformed plant body.

  PAP1 transcription factor and TT2 transcription factor are transcription factors that promote gene expression of enzymes involved in the phenylpropanoid synthesis system. In Arabidopsis thaliana plants transformed with p35S :: PAP1SRDX, not only anthocyanin synthesis but also tannin synthesis was suppressed, which revealed for the first time that PAP1 is a transcription factor involved in the tannin synthesis system. It was. Moreover, in the Arabidopsis plant transformed with p35S :: TT2SRDX, synthesis of tannin could be suppressed.

  This indicates that a gene expression suppression system using a chimeric gene of a PAP1 transcription factor gene or TT2 transcription factor gene and a transcriptional repression conversion polynucleotide can also be used for the suppression of tannin synthesis.

  That is, as shown in FIG. 4A, the PAP1 transcription factor promotes gene expression of enzymes PAL, CHS, and DFR involved in the phenylpropanoid synthesis system, thereby promoting anthocyanin synthesis. In contrast, the chimeric protein PAP1SRDX in which this PAP1 transcription factor is fused with the transcription repressor conversion peptide SRDX is involved in phenylpropanoid synthesis as well as PAL, CHS, and DFR as shown in FIG. 4 (b). It suppresses gene expression, thereby suppressing not only anthocyanin but also tannin synthesis.

  In addition, as described above, it has been reported that the gene expression of DFR and BAN is promoted in an overexpressing plant of TT2 transcription factor. However, as described above, there are regulatory factors specific to each pathway in tannin biosynthesis, and the presence of duplicate factors is presumed, so that transcription of DFR and BAN genes promoted by TT2 protein is promoted. It is usually difficult to suppress tannin production only by suppression.

  In the method according to the present invention, it is considered that expression of genes involved in phenylpropanoid synthesis as well as DFR and BAN genes is suppressed by the chimeric protein TT2SRDX. It is thought that synthesis is suppressed.

(4) Quantitative analysis of cyanidin and anthocyanins in Arabidopsis transformed with p35S :: PAP1SRDX The cyanidin content of Arabidopsis transformed with p35S :: PAP1SRDX (hereinafter referred to as “transformed Arabidopsis thaliana”) was determined by De-Yu Xie, Shashi B. The calculation was performed according to the method described in Sharma, Nancy L. Paiva, Daneel Ferreira, Richard A. Dixon., Science, 299, 396-399, 2003. Cyanidin is a pigment belonging to the anthocyanidins shown in FIGS. 4 (a) and 4 (b) (Rikagaku Dictionary 5th edition, page 552, Iwanami Shoten). First, a standard curve was obtained from a dilution series of a standard (commercially available) cyanidin and an absorbance at 550 nm. Next, after measuring the seed or leaf weight of wild-type Arabidopsis thaliana and the transformed Arabidopsis thaliana, the seed or leaf is treated with a 70% acetone solution containing 5.26 mM sodium isomeric bisulfite to extract the seed or leaf. A liquid was prepared. Further, the extract was incubated in a 95% butanol-5% hydrochloric acid solution at 100 ° C. for 1 hour. Subsequently, the absorbance at 550 nm of the incubated extract was measured, and the amount of cyanidin was calculated from the standard curve based on the measured absorbance data.

  FIG. 5 (a) shows the cyanidin content (mg) per gram of the transformed Arabidopsis seeds, and FIG. 5 (b) shows the cyanidin content (μg) per gram fresh weight of the transformed Arabidopsis leaves. Is shown. The horizontal axes of FIGS. 5A and 5B indicate WT wild-type Arabidopsis thaliana, and # 12, # 13, and # 17 indicate the identification numbers of the transformed Arabidopsis thaliana, respectively. That is, cyanidin was quantitatively analyzed for three lines of the transformed Arabidopsis thaliana.

  In any of the above three lines, it was confirmed that the cyanidin content was significantly reduced in both seeds and leaves as compared to the wild type, and it was confirmed that the synthesis of cyanidin was suppressed. In general, in Arabidopsis thaliana (cyanidines) are contained in a large amount in seeds but not in leaves, so the unit display is mg in FIG. 5 (a), and FIG. ) In μg.

  The gray column in FIG. 5 (c) shows the above transformation when the absorbance derived from anthocyanin at 530 nm of wild-type Arabidopsis thaliana grown in MS medium (normal conditions) containing 0.5% sucrose is 1. The relative value of the light absorbency derived from the anthocyanin in 530 nm of Arabidopsis thaliana is represented.

  The relative value of the absorbance derived from the anthocyanins is determined by the method described in Ron J. Laby, M. Sean Kincaid, Donggiun Kim and Susan I. Gibson., The Plant J, 23 (5), 587-596, 2000. Calculated in conformity. First, after measuring the weight of the leaves of the wild type Arabidopsis (2 weeks old) and the transformed Arabidopsis (2 weeks of age), the leaves were treated with a 45% methanol-5% acetic acid solution to obtain the leaf extract. Prepared. Furthermore, the absorbance at 530 nm and 637 nm of the extract was measured, and the value calculated from the absorbance values using the formula described in the above document was the amount of anthocyanin per 1 g of the fresh Arabidopsis leaf weight. (Relative value).

  It is known that Arabidopsis thaliana accumulates anthocyanins when grown on MS medium containing 6% sucrose. Therefore, in order to make the difference in anthocyanin content easier to see, the wild-type Arabidopsis thaliana (2 weeks old) and the transformed Arabidopsis thaliana (2 weeks old) are transferred to a 6% sucrose-containing MS medium for 5 days, and the transformed Arabidopsis thaliana The amount of anthocyanin (relative value) per 1 g of fresh leaf weight was calculated, and the result was represented by a black column in FIG.

  In the transformed Arabidopsis thaliana, it was confirmed that the anthocyanin content was remarkably reduced in all of the three lines compared to the wild type, and the synthesis of anthocyanins was suppressed.

(5) Gene Expression Analysis of Phenylpropanoid Synthetic Enzymes and Transcription Factors in Arabidopsis Transformed with p35S :: PAP1SRDX Gene expression of phenylpropanoid synthase enzymes and transcription factors in the above transformed Arabidopsis Analyzed.

  First, total RNA was extracted and purified from immature wild type Arabidopsis and immature soybean of the transformed Arabidopsis, and 1.65 μg of total RNA of the wild type Arabidopsis and 1.65 μg of total RNA of the transformed Arabidopsis. After separation, DNase treatment was performed to remove the mixed DNA. Next, RNA was converted to first strand cDNA using T-Primed First-Strand Kit (Amersham). Using this as a template, PCR was performed using primers for each gene shown below. PCR was carried out for 21 to 38 cycles at 95 ° C. for 2 minutes (1 cycle), followed by 95 ° C. for 30 seconds, 58 ° C. for 30 seconds and 72 ° C. for 1 minute. A certain amount of RT-PCR product was electrophoresed, and the expression level of each gene was examined from the band. As a comparative control, the expression level of β-tubulin was simultaneously detected.

  The real-time RT-PCR for which quantitative analysis was performed was performed for 40 cycles using a LightCycler (Roche) at 95 ° C. for 10 minutes (1 cycle), 95 ° C. for 10 seconds, 62 ° C. for 10 seconds, and 72 ° C. for 7 seconds. .

[Base sequence of primers used for PCR]
<CHS 5 'primer>
5'-TGGTCTCCGTCCTTCCGTCAA-3 '(SEQ ID NO: 149)
<CHS 3 'primer>
5'-CCCTCAAATGTCCGTCTATGGAA-3 '(SEQ ID NO: 150)
<DFR 5 'primer>
5'-AAAAAGATGACAGGATGGGT-3 '(SEQ ID NO: 151)
<DFR 3 'primer>
5'-CCCCTGTTTCTGTCTTGTTA-3 '(SEQ ID NO: 152)
<LDOX 5 'primer>
5'-ATGGTTGCGGTTGAAAGAGTTGAGAG-3 '(SEQ ID NO: 153)
<LDOX 3 'primer>
5'-CCAACTTCTTTCTCTAGACGGTCAGGC-3 '(SEQ ID NO: 154)
<BAN 5 'primer>
5'-ATGGACCAGACTCTTACACACACCGGA-3 '(SEQ ID NO: 155)
<BAN 3 'primer>
5'-GTTTCCGGCTATAAGTGCCGGAATCACG-3 '(SEQ ID NO: 156)
<TT2 5 'primer>
5'-GGGATGGGAAAGAGAGCAACTACTAGTGTGAGG-3 '(SEQ ID NO: 157)
<TT2 3 'primer>
5'-ACAAGTGAAGTCTCGGAGCCAATCTTCATCG-3 '(SEQ ID NO: 158)
<PAP2 5 'primer>
5'-GAAAAACATTATTTCCCCTCCTACAAC-3 '(SEQ ID NO: 159)
<PAP2 3 'primer>
5'-CAATCGCATTAGCTTCTTGGTTTTCCC-3 '(SEQ ID NO: 160)
<PAP1 5 'primer>
5'-GGGATGGAGGGTTCGTCCAAAGGGCTGCGAAAAGG-3 '(SEQ ID NO: 161)
<PAP1 3 'primer>
5'-TACACAAACGCAAACAAATGTTCGAAAC-3 '(SEQ ID NO: 162)
<PAP1SRDX 5 'primer>
5'-GGGATGGAGGGTTCGTCCAAAGGGCTGCGAAAAGG-3 '(SEQ ID NO: 163)
<PAP1SRDX 3 'primer>
5'-AGCGAAACCCAAACGGAGTTCTAG-3 '(SEQ ID NO: 164)
<TUB 5 'primer>
5'-CGTGGATCACAGCAATACAGAGCC-3 '(SEQ ID NO: 165)
<TUB 3 'primer>
5'-CCTCCTGCACTTCCACTTCGTCTTC-3 '(SEQ ID NO: 166)
[Base sequence of primers used for real-time RT-PCR]
<CHS 5 'primer>
5'-CGTGTTGAGCGAGTATGG-3 '(SEQ ID NO: 167)
<CHS 3 'primer>
5′-GCTGTGCAAGACGACTG-3 ′ (SEQ ID NO: 168)
<DFR 5 'primer>
5′-TGACAGGATGGATGTATTTC-3 ′ (SEQ ID NO: 169)
<DFR 3 'primer>
5'-GACCGACCACCAATGTT-3 '(SEQ ID NO: 170)
<LDOX 5 'primer>
5'-ACTGGAAAGATTCAAGGCTA-3 '(SEQ ID NO: 171)
<LDOX 3 'primer>
5'-GTACTCACTCGTTGCTTCTATG-3 '(SEQ ID NO: 172)
<BAN 5 'primer>
5'-ACGAAGAAAACTGGACTGAC-3 '(SEQ ID NO: 173)
<BAN 3 'primer>
5'-GCTATAAGTGCCGGAATCA-3 '(SEQ ID NO: 174)
<TT2 5 'primer>
5'-TTGATGGTTTGGACTGTG-3 '(SEQ ID NO: 175)
<TT2 3 'primer>
5'-GCAATATAGGCTCAACAAGT-3 '(SEQ ID NO: 176)
<PAP1 5 'primer>
5′-GAGGAAAGCCAAGAGGTA-3 ′ (SEQ ID NO: 177)
<PAP1 3 'primer>
5'-CAAACCTATACACAAACGCA-3 '(SEQ ID NO: 178)
<PAP2 5 'primer>
5′-AAACCAAGAAGCTGATGC-3 ′ (SEQ ID NO: 179)
<PAP2 3 'primer>
5'-AAAAGCCACCCACAATCT-3 '(SEQ ID NO: 180)
FIG. 6 is an electrophoresis photograph showing the results of the RT-PCR, and also shows the results of the real-time RT-PCR. In the band shown in FIG. 6, the left column shows the results of wild-type Arabidopsis (WT), and the right column shows the results of the transformed Arabidopsis (PAP1SRDX). As described above, CHS, DFR, LDOX, and BAN described on the left side of the photograph are abbreviations of enzyme names of the phenylpropanoid synthesis system, and TT2, PAP2, and PAP1 are genes of enzymes involved in the phenylpropanoid synthesis system. It is a transcription factor that promotes expression. PAP1SRDX and TUB were used as comparative controls.

  The numbers shown on the right side of the photograph show the results of the real-time RT-PCR. In the transformed Arabidopsis thaliana, the expression level of each enzyme gene or transcription factor gene in the wild-type Arabidopsis thaliana is 1. It is the relative value of the expression level of each enzyme gene or each transcription factor gene. The relative value was calculated from the copy number of the PCR product, the measurement was performed twice, and the average value was obtained.

  FIG. 6 confirms that in the transformed Arabidopsis thaliana, the genes of CHS, DFR, LDOX, and BAN all have a relative value of less than 0.5, and the expression is suppressed compared to the wild type. In addition, the expression of the PAP1 gene in the transformed Arabidopsis thaliana is due to the expression of the endogenous PAP1 gene. That is, no co-suppression occurs in the transformed Arabidopsis thaliana. In addition, regarding the gene expression of TT2 and PAP2 in the transformed Arabidopsis thaliana, the relative value is 0.5 or more in any case, so that the gene expression of TT2 and PAP2 is suppressed from the biological viewpoint. Is interpreted as not being judged.

  Thus, in the present invention, it is possible to obtain a plant body in which the content of tannin is reduced by suppressing transcription of a gene involved in the tannin synthesis system. 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.

(A) is a schematic diagram showing the structure of the recombinant expression vector p35S :: PAP1SRDX used in the examples, and (b) is a schematic diagram showing the structure of the recombinant expression vector p35S :: TT2SRDX used in the examples. (A) is a view showing the next generation seeds of wild type Arabidopsis thaliana, (b) and (c) are the next generation seeds of Arabidopsis thaliana transformed with recombinant expression vector p35S :: PAP1SRDX in the examples. FIG. (A) is a figure which shows the next generation seed of wild type Arabidopsis thaliana, (b) is a figure which shows the next generation seed of Arabidopsis thaliana transformed by recombinant expression vector p35S :: TT2SRDX in the Example. . (A) is a diagram showing that PAP1 promotes the expression of genes involved in phenylpropanoid synthesis, whereby anthocyanin synthesis is promoted, and (b) shows that a chimeric protein in which PAP1 and SRDX are fused is phenyl It is a figure which shows that not only anthocyanin but the synthesis | combination of a tannin is also suppressed by suppressing the expression of the gene involved in a propanoid synthesis. (A) shows the cyanidin content per gram of Arabidopsis seeds transformed with the recombinant expression vector p35S :: PAP1SRDX in the Examples, and (b) shows the recombinant expression vector p35S :: in the Examples. It is a figure which shows the cyanidin content per gram fresh leaf weight of Arabidopsis thaliana transformed by PAP1SRDX. (C) shows the relative value of the anthocyanin-derived absorbance at 530 nm of Arabidopsis transformed with the recombinant expression vector p35S :: PAP1SRDX in the Examples. It is the electrophoresis photograph which shows the result of RT-PCR, and also shows the result of real-time RT-PCR.

Claims (18)

In order to reduce the tannin content of the plant body,
Produces a chimeric protein in a plant that fuses a transcription factor that promotes the expression of a gene encoding an enzyme involved in the phenylpropanoid synthesis system and a functional peptide that converts any transcription factor into a transcriptional repressor. The production method of the plant body with which the tannin content was reduced characterized by suppressing the production of tannin by making it.   2. A transformation step of introducing into a plant cell a recombinant expression vector comprising a chimeric gene comprising a gene encoding the transcription factor and a polynucleotide encoding the functional peptide. 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 consisting of the amino acid sequence shown in SEQ ID NO: 1.
(B) an amino acid sequence represented by SEQ ID NO: 1 comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and an enzyme involved in a phenylpropanoid synthesis system; A protein having a function of promoting expression of a gene to be encoded. 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 shown in SEQ ID NO: 2 as an open reading frame region.
(D) a gene that hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2 under stringent conditions and encodes an enzyme involved in the phenylpropanoid synthesis system A gene encoding a transcription factor that promotes expression. 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 (e) or (f).
(E) a protein comprising the amino acid sequence represented by SEQ ID NO: 145;
(F) an amino acid sequence represented by SEQ ID NO: 145 comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and an enzyme involved in a phenylpropanoid synthesis system; A protein having a function of promoting expression of a gene to be encoded. The method for producing a plant according to claim 2 or 3, wherein the gene described in (g) or (h) below is used as the gene encoding the transcription factor.
(G) A gene having the base sequence represented by SEQ ID NO: 146 as an open reading frame region.
(H) 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: 146 and encodes an enzyme involved in the phenylpropanoid synthesis system A gene encoding a transcription factor that promotes expression. 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 plant production method according to any one of claims 1 to 7, wherein the plant body has an amino acid sequence represented by any one of the above.   The method for producing a plant according to any one of claims 1 to 7, 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 7, wherein the functional peptide is a peptide according to the following (i) or (j).
(I) A peptide having the amino acid sequence shown in SEQ ID NO: 20 or 21.
(J) A peptide having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 20 or 21. The functional peptide is represented by the following formula (5)
(5) α1-Leu-β1-Leu-γ1-Leu
(Wherein α1 represents Asp, Asn, Glu, Gln, Thr or Ser, β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, and γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys, or Asp are shown.)
It has an amino acid sequence represented by these, The production method of the plant of any one of Claims 1-7 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.)
The plant production method according to any one of claims 1 to 7, wherein the plant body has an amino acid sequence represented by any one 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, 134, 135 or 136. It is a peptide which has an amino acid sequence, The production method of the plant body of any one of Claims 1-7 characterized by the above-mentioned.   The method for producing a plant according to any one of claims 1 to 7, wherein the functional peptide is a peptide having an amino acid sequence represented by SEQ ID NO: 38 or 39.   The plant body with which the tannin content was reduced produced by the production method of any one of Claims 1-14.   The plant body according to claim 15, 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 14,
A gene encoding a transcription factor that promotes the expression of a gene encoding an enzyme involved in a phenylpropanoid synthesis system, a polynucleotide encoding a functional peptide that converts any transcription factor into a transcription repressor, and a promoter A tannin content reduction kit for a plant, comprising at least a recombinant expression vector comprising   The kit for reducing the tannin content of a plant according to claim 17, further comprising a reagent group for introducing the recombinant expression vector into a plant cell.

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