JP2012055266A - Method for producing plant body having regulated inflorescence pattern, method for producing plant body having regulated flowering time, and plant body obtained by using them - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a plant body having a regulated inflorescence pattern, and a method for producing a plant body having a regulated flowering time, applicable even to a large amount of farm crops without requiring time and cost, and to provide the plant bodies obtained by using these methods.SOLUTION: The method for producing the plant body having the regulated inflorescence pattern includes introducing a recombinant expression vector containing a chimera gene comprising a gene encoding a protein described in the following (i) or (ii), and a polynucleotide encoding a transcription-activating domain or a transcription-controlling domain to a plant cell: wherein (i) is a protein comprising an amino acid sequence represented by sequence number 1, 17 or 19; and (ii) is a protein comprising an amino acid sequence obtained by substituting, deleting, inserting and/or adding one or several amino acids with, from or to the amino acid sequence represented by sequence number 1, 17 or 19, and having functions for retarding the flowering and maintaining the inflorescence meristem.

Description

  The present invention relates to a method for producing a plant body in which the inflorescence form is controlled, a method for producing a plant body in which the flowering time is controlled, and a plant body obtained by using these methods, and particularly to a large amount of crops. The present invention relates to a method for producing a plant body in which the inflorescence form is controlled, a method for producing a plant body in which the flowering time is controlled, and a plant body obtained by using these.

  Plant organs arise from a group of stem cells called meristems. In the above-ground part, various tissues are formed from the shoot apical meristem, but there are many unclear parts regarding the control mechanism. In general, when a plant shifts from vegetative growth after germination to reproductive growth, a flower is formed from the meristem that had previously formed leaves. This transition from vegetative growth to reproductive growth is called “flowering”. In addition, the method of flowering is called “inflorescence”, and in a flowering plant, it has an inflorescence configuration of either an infinite inflorescence or a finite inflorescence. Infinite inflorescence refers to inflorescences that do not have flowers at the end of the inflorescence axis, and finite inflorescence refers to inflorescences that have flowers at the end of the inflorescence axis.

  The TERMINAL FLOWER 1 gene (TFL1 gene, hereinafter referred to as “TFL1 gene” in the present specification) has been found as a causative gene of the tfl1 mutant, which is a flowering premature mutation of Arabidopsis thaliana. Moreover, in the tfl1 mutant of Arabidopsis thaliana, wild-type infinite inflorescence is converted to finite inflorescence. From previous studies, it is known that the TFL1 gene has a function of delaying flowering and maintaining inflorescence meristem. On the other hand, the FLOWERING LOCUS T gene (FT gene) in which 59% of the amino acid sequence is conserved with respect to the TFL1 gene promotes flowering. A gene having homology with the TFL1 gene has also been found in plants other than Arabidopsis thaliana and is referred to as the TFL1 family. Examples of genes belonging to the TFL1 family include the Hd3a gene derived from rice (Oryza sativa). The Hd3a gene is similar to the FT gene and is known to promote flowering.

  Since FT is known to exist in the nucleus in the presence of FD, a model has been proposed in which FT binds to FD, which is a bZIP transcription factor, and performs functional regulation in an antagonistic manner. On the other hand, details of the mechanism of TFL1, which is highly homologous to FT but functionally opposite, have not been clarified. Both may be involved in the transcriptional regulation of genes involved in flowering by different mechanisms.

  In the production of agricultural crops, the control of inflorescence morphology is directly related to the number of flowering, leading to increased crop production. In actual farm work sites, we are working to improve yield and quality by changing the inflorescence form of the ends by cutting the shoot apex called pinching. Usually, since pinching is performed manually, it takes a lot of time and cost to apply it to a large amount of crops. In addition, control of flowering is a very important point in adjusting crop harvest time and yield.

  As described above, some genes involved in the determination of inflorescence morphology and flower formation have been identified, and an invention using them has been published (see, for example, Patent Document 1). However, the detailed mechanism has not been clarified yet, and it is difficult to use in plants where the genome sequence of the homologue of the above gene is unknown.

  By the way, a method of transforming a plant using a transcription activation domain is known (for example, see Non-Patent Document 1). Non-Patent Document 1 reports that a fusion protein of AP1 (APETALA1) known as a flower bud meristem-determining gene and VP16, a transcriptional activation domain, was overexpressed, and the transformant is evaluated. Thus, the result of analyzing the function of AP1 is disclosed. AP1 is known to have a MADS domain that is a transcription factor.

  In addition, a method of transforming a plant using a transcription repression domain is also known. For example, a chimeric protein in which a transcription factor that controls environmental response and morphogenesis in a plant and a transcription repression domain are fused is used. A method for producing a flower bud formation-delayed plant body by producing the plant body and suppressing the expression of a gene involved in flower bud formation has been disclosed (for example, see Patent Document 2).

JP 2007-202441 A (released on August 16, 2007) Japanese Patent Laying-Open No. 2005-295878 (released on October 27, 2005)

The Plant Cell, Vol. 13, 739-753, April 2001

  As described above, in crop production, control of inflorescence morphology and flowering is important for improving yield and quality, or adjusting the harvest time and yield of crops. There is a problem that it takes enormous time and cost.

  The present invention has been made in view of the above-mentioned problems, and the object thereof is a method for producing a plant body with controlled inflorescence morphology, which can be applied to a large amount of agricultural products without taking time and cost, The object is to realize a method for producing a plant body in which the flowering time is controlled, and a plant body obtained by using these methods.

The method for producing a plant according to the present invention is a method for producing a plant whose inflorescence morphology is controlled. In order to solve the above problems, the following protein (i) or (ii):
(I) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 17 or 19,
(Ii) consisting of 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: 1, 17 or 19, and delays flowering; A trait for introducing a recombinant expression vector comprising a gene encoding a protein having a function of maintaining an inflorescence meristem and a polynucleotide encoding a transcriptional activation domain or transcriptional repression domain into a plant cell. It is characterized by including a conversion process.

  According to the above configuration, in contrast to the existing method, the inflorescence morphology can be controlled by introducing the chimeric gene of the present invention even in a biological species whose homologous genomic gene information has the same function as that of TFL1. There is an effect that it becomes possible.

  In the plant production method according to the present invention, the transcription activation domain is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3, and the transcription repression domain is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5. It is preferable that

In the method for producing a plant according to the present invention, as a gene encoding the protein described in (i) or (ii) above, the following gene described in (a) or (b):
(A) a gene having the base sequence represented by SEQ ID NO: 2, 18, or 20 as an open reading frame region,
(B) A function of hybridizing under stringent conditions with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2, 18 or 20, and delaying flowering to maintain the inflorescence meristem It is preferable to use a gene encoding a protein having

  In the method for producing a plant according to the present invention, it is preferable to use a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 or 6 as the polynucleotide encoding the transcription activation domain or the transcription repression domain, respectively.

  In the method for producing a plant according to the present invention, a recombinant expression vector comprising a chimeric gene comprising a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription activation domain, The infinite inflorescence form may be changed to a finite inflorescence form by including a transformation step to be introduced into the plant cell.

  With the above configuration, a new variety having a finite inflorescence can be created by applying the present invention to a plant having an infinite inflorescence. The change in the inflorescence morphology has the further effect that the number of flowering is increased and an improvement in yield can be expected.

  In the method for producing a plant according to the present invention, a recombinant expression vector comprising a chimeric gene comprising a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription repressing domain, A finite inflorescence form may be changed to an infinite inflorescence form by including a transformation step to be introduced into a plant cell.

  With the above configuration, by changing a plant that is originally a finite inflorescence into an infinite inflorescence, the plant has the effect of being able to continue vegetative growth.

The plant production method according to the present invention is a plant production method in which the flowering time is controlled, and in order to solve the above problems, the following protein (i) or (ii):
(I) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 17 or 19,
(Ii) consisting of 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: 1, 17 or 19, and delays flowering; A trait for introducing a recombinant expression vector comprising a gene encoding a protein having a function of maintaining an inflorescence meristem and a polynucleotide encoding a transcriptional activation domain or transcriptional repression domain into a plant cell. It is characterized by including a conversion process.

  According to the above configuration, unlike the existing method, the flowering time can be controlled by introducing the chimeric gene of the present invention even in a biological species whose genomic gene information of a homolog having the same function as TFL1 is unknown. There is an effect that it becomes possible.

  In the plant production method according to the present invention, the transcription activation domain is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3, and the transcription repression domain is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5. It is preferable that

In the method for producing a plant according to the present invention, as a gene encoding the protein described in (i) or (ii) above, the following gene described in (a) or (b):
(A) a gene having the base sequence represented by SEQ ID NO: 2, 18, or 20 as an open reading frame region,
(B) A function of hybridizing under stringent conditions with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2, 18 or 20, and delaying flowering to maintain the inflorescence meristem It is preferable to use a gene encoding a protein having

  In the method for producing a plant according to the present invention, it is preferable to use a polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 or 6 as the polynucleotide encoding the transcription activation domain or the transcription repression domain, respectively.

  In the method for producing a plant according to the present invention, a recombinant expression vector comprising a chimeric gene comprising a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription activation domain, The flowering time may be advanced by including a transformation step for introduction into plant cells.

  By the said structure, there exists an additional effect that the flowering time can be advanced and the harvest time and yield of a crop can be adjusted.

  In the method for producing a plant according to the present invention, a recombinant expression vector comprising a chimeric gene comprising a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription repressing domain, The flowering time may be delayed by including a transformation step to be introduced into the plant cell.

  By the said structure, there exists the further effect that the flowering time can be delayed and the harvest time and yield of a crop can be adjusted.

  The plant according to the present invention is preferably produced by the production method described above.

The chimeric gene according to the present invention includes the following protein (i) or (ii):
(I) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 17 or 19,
(Ii) consisting of 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: 1, 17 or 19, and delays flowering; It is characterized by comprising a gene encoding a protein having a function of maintaining an inflorescence meristem, and a polynucleotide encoding a transcriptional activation domain or a transcriptional repression domain.

  In the chimeric gene according to the present invention, the transcription activation domain is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3, and the transcription repression domain is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5. Is preferred.

  As described above, the production method of a plant body with controlled inflorescence morphology and the production method of a plant body with controlled flowering time according to the present invention encode the protein described in (i) or (ii) above. Since the method includes a transformation step of introducing a recombinant expression vector comprising a gene and a polynucleotide encoding a transcription activation domain or transcription repression domain into a plant cell, the existing method is In contrast, even in a biological species whose homologous genomic gene information has the same function as that of TFL1, the inflorescence morphology can be controlled by introducing the chimeric gene of the present invention.

In Example 1, it is a figure which shows the form of the plant which introduce | transduced TFL1: VP16M and TFL1: SRDX. In Example 1, it is a figure which shows the number of rosette leaves and the number of flower buds when the flower stalk of the plant which introduce | transduced TFL1: VP16M and TFL1: SRDX is drawn (drawing). It is the figure which showed the amino acid substitution site | part of the Arabidopsis thaliana tfl1 mutant used in the reference example. It is a figure which shows the result of a reference example, A is a figure which shows the number of rosette leaves when the flower stem in a wild type plant and a tfl1 mutant is lottery, B is a figure of the flower bud in a wild type plant and a tfl1 mutant. It is a figure which shows a number. It is process drawing which shows the preparation method of the expression vector used in an Example. It is a figure which shows the base sequence of the polynucleotide which codes VP16. It is a figure which shows the structure of the vector used in an Example.

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

  In order to solve the above-mentioned problems of the present invention, the present inventors overexpressed Arabidopsis thaliana TFL1 gene or tobacco TFL1 gene homologue in tobacco. However, TFL1 gene alone almost changed the flowering time and inflorescence morphology. I couldn't make it. Thus, conventionally, as a fusion protein with a transcription factor, a polynucleotide encoding a transcriptional activation domain and a transcriptional repression domain used for transformation, and a TFL1 protein not having a characteristic domain as a transcription factor are encoded. When a chimeric gene comprising a gene to be introduced was introduced into a plant cell, it was surprisingly found that a transformant with controlled inflorescence morphology could be obtained. Furthermore, by introducing the chimeric gene, it was found that the flowering time can be controlled, and the present invention has been completed.

  As described above, since the transcription factor is not included in the TFL1 gene, there was no precedent for fusing the TFL1 gene with a polynucleotide that encodes a transcriptional activation domain and a transcriptional repression domain. Although the reason is not clear, the flowering time and inflorescence morphology can be controlled by introducing a chimeric gene consisting of the TFL1 gene and a polynucleotide that encodes a transcription activation domain and a transcription repression domain.

From the above findings, it is possible to obtain the same effect when using a gene encoding a protein having homology with the TFL1 protein and having the same function, that is, the function of delaying flowering and maintaining the inflorescence meristem. It is considered possible. That is, the method for producing a plant according to the present invention is a method for producing a plant having a controlled inflorescence morphology and a plant having a controlled flowering time. The protein described in (i) or (ii) below:
(I) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 17 or 19,
(Ii) consisting of 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: 1, 17 or 19, and delays flowering; A trait for introducing a recombinant expression vector comprising a gene encoding a protein having a function of maintaining an inflorescence meristem and a polynucleotide encoding a transcriptional activation domain or transcriptional repression domain into a plant cell. Includes conversion process.

  According to the present invention, unlike existing methods, even in a biological species whose homologous genomic gene information has the same function as that of TFL1, the chimeric gene of the present invention is introduced to produce a function-deficient strain, It is possible to control the flowering time and the inflorescence morphology.

  In addition, unlike conventional gene mutants, the functional genes inherent in plants remain in the plants, so that the influence on basic growth can be minimized. Moreover, by expressing the chimeric gene according to the present invention using a space and time-specific promoter, the function of the flowering gene can be deleted in a space and time-specific manner, and more precise control becomes possible.

  Further, since the method according to the present invention is considered to be able to act antagonistically on the endogenous gene product inherent to the plant, the plant in which the mutant of the homolog does not exist, or the gene sequence of the homolog The same effect can be expected even in plants in which the unknown is unknown.

(I) Gene encoding the protein described in (i) or (ii) In the present invention, the protein described in (i) or (ii) above has TFL1 protein of Arabidopsis thaliana, or homology with TFL1, and TFL1 As with proteins, there is no particular limitation as long as the protein has a function of delaying flowering and maintaining an inflorescence meristem.

  Examples of such proteins include, in addition to Arabidopsis TFL1 protein, Snapdragon CEN protein, tomato SP protein, and the like.

  The TFL1 protein of Arabidopsis thaliana used in the present invention is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, the CEN protein of snapdragon is a protein consisting of the amino acid sequence shown in SEQ ID NO: 17, and the SP protein of tomato is It is a protein consisting of the amino acid sequence shown in SEQ ID NO: 19.

  Further, the protein is not limited to the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 17 or 19, and in the amino acid sequence shown in SEQ ID NO: 1, 17 or 19, one or several, respectively. Even a protein having an amino acid sequence in which a single amino acid is substituted, deleted, inserted, and / or added can be used in the present invention as long as it has the above function. In addition, “one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1, 17 or 19 in which one or several amino acids are substituted, deleted, inserted and / or added” ”Is not particularly limited, but means, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 3 To do.

  It is known that the amino acid sequence of a protein having the above function is highly conserved among many plants of different species. Therefore, it is not always necessary to isolate a gene that encodes a protein having homology with the TFL1 protein and having a similar function in an individual plant body in which inflorescence morphology and flowering time are to be controlled. That is, by introducing a chimeric gene using a gene encoding the protein described in (i) or (ii) above into other plants, the inflorescence morphology and flowering time were easily controlled in various types of plants. Plants can be produced.

  The plant production method according to the present invention includes a chimeric gene comprising a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding a transcriptional activation domain or transcriptional repression domain. A transformation step of introducing a recombinant expression vector into a plant cell, and in a plant transformed by the method, the protein described in (i) or (ii) above and a transcription activation domain or transcription A chimeric protein consisting of an inhibitory domain is produced.

  The gene encoding the protein described in (i) or (ii) is not particularly limited, and corresponds to the amino acid sequence of the protein described in (i) or (ii) based on the genetic code. I just need it. As a specific example, for example, when a TFL1 protein is used as the protein, a gene encoding the TFL1 protein (referred to as a TFL1 gene for convenience of description) can be exemplified. As a specific example of the TFL1 gene, for example, a polynucleotide containing the base sequence shown in SEQ ID NO: 2 as an open reading frame (ORF) can be mentioned. In addition, when a CEN protein is used as the protein, a gene encoding the CEN protein (for convenience of explanation, referred to as CEN gene) can be exemplified. As a specific example of the CEN gene, for example, a polynucleotide containing the base sequence shown in SEQ ID NO: 18 as an open reading frame (ORF) can be mentioned. Moreover, when using SP protein as said protein, the gene (it is called SP gene for convenience of explanation) which codes this SP protein can be mentioned. As a specific example of the SP gene, for example, a polynucleotide containing the base sequence shown in SEQ ID NO: 20 as an open reading frame (ORF) can be mentioned.

  Of course, the gene encoding the TFL1 protein, CEN protein or SP protein used in the present invention is not limited to the above example, and is homologous to the nucleotide sequence shown in SEQ ID NO: 2, 18 or 20, respectively. It may be a gene having Specifically, for example, it hybridizes with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2, 18 or 20 under stringent conditions, delays the flowering, and inflorescence A gene encoding a protein having a function of maintaining a meristem can be mentioned. As used herein, “stringent conditions” refers to high only when at least 90% identity, preferably at least 95% identity, most preferably at least 97% identity exists between sequences. It means that hybridization occurs.

  The hybridization is described in J. Org. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory (1989), and the like. Usually, 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 protein described in (i) or (ii) above is not particularly limited, and it can be isolated from many plants by a conventionally known method. For example, a primer pair prepared based on the base sequence of a gene encoding a known TFL1 protein, CEN protein or SP protein 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 TFL1 protein, CEN protein or SP protein can also be obtained by chemical synthesis by a conventionally known method.

(II) Polynucleotide encoding a transcription activation domain, polynucleotide encoding a transcription repression domain The transcription activation domain used in the present invention is particularly limited as long as it is known as a transcription activation domain. In other words, a known transcription activation domain can be used. More specifically, for example, VP16, which is a transcription activation domain of herpesvirus-derived VP16 protein, can be preferably used. In addition, the transcription activation region having a transactivation domain consisting of 9 amino acids commonly found in Gal4, Oaf1, Leu3, Rtg3, Pho4, Gln3, Gcn4, p53, NFAT, NF-κB, etc. Can be used as a domain.

  Specifically, VP16 is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3. The transcription activation domain comprises 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: 3, and transcription activation It may be a peptide having a function as a domain. In addition, the range of “one or several” in the above-mentioned “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: 3” , For example, 1 to 7, more preferably 1 to 5, and still more preferably 1 to 3.

  Further, the transcription repression domain used in the present invention is not particularly limited as long as it is known as a transcription repression domain, and a known transcription repression domain can be used. More specifically, for example, SRDX can be preferably used. In addition, repressor domains contained in the engrailed gene, adenovirus E1A, Lac repressor, and the like can also be used as the transcription repression domain.

  Specifically, SRDX is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5. The transcription repression domain comprises 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: 5, and transcription repression is performed. It may be a peptide having a function as a domain. In addition, the range of “one or several” in the above “amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 5” For example, it means 1 to 3, more preferably 1 to 2.

  The specific base sequences of the polynucleotide encoding the transcription activation domain and the polynucleotide encoding the transcription repression domain are not particularly limited, and based on the genetic code, the transcription activation domain and the transcription repression, respectively. It suffices to include a base sequence corresponding to the amino acid sequence of the domain.

  Specific examples of the polynucleotide encoding the transcription activation domain include, for example, a polynucleotide comprising the base sequence shown in SEQ ID NO: 4. This polynucleotide corresponds to the amino acid sequence shown in SEQ ID NO: 3. The polynucleotide encoding the transcription activation domain 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: 4, and the transcription activation domain It may be a gene encoding a peptide having a function as

  Moreover, as a specific example of the polynucleotide encoding the transcription repressing domain, for example, a polynucleotide comprising the base sequence shown in SEQ ID NO: 6 can be mentioned. This polynucleotide corresponds to the amino acid sequence shown in SEQ ID NO: 5. The polynucleotide encoding the transcription repressing domain 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: 6, and the transcription repressing domain. It may be a gene encoding a peptide having a function as

(III) Chimeric gene The chimeric gene used in the present invention comprises a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding a transcription activation domain or transcription repression domain. The gene may be a chimeric gene, and the binding sequence is upstream of the gene encoding the protein described in (i) or (ii) above, and downstream of the polynucleotide encoding the transcription activation domain or transcription repression domain. Alternatively, the polynucleotide encoding the transcription activation domain or transcription repression domain may be upstream and the gene encoding the protein described in (i) or (ii) may be downstream.

  In the chimeric gene, a polynucleotide that encodes a transcriptional activation domain and a polynucleotide that encodes a transcriptional repression domain and a gene that encodes the protein described in (i) or (ii) above serve as a linker. More preferably, they are linked by a sequence. That is, the chimeric gene includes a polynucleotide encoding a transcription activation domain and a polynucleotide encoding a transcription repression domain, and a gene encoding the protein described in (i) or (ii) above. It is more preferable that a base sequence serving as a linker for linking is included.

  In such a case, the chimeric gene may comprise a gene encoding the protein described in (i) or (ii) above, a polynucleotide encoding a transcription activation domain or a transcription repression domain, and a base sequence serving as a linker. I can say that. The base sequence serving as the linker is not particularly limited, but a linker comprising a polynucleotide encoding GGGT, that is, a peptide comprising an amino acid sequence represented by Gly-Gly-Gly-Thr (SEQ ID NO: 15) Can be suitably used. The polynucleotide encoding the peptide is not particularly limited as long as it corresponds to GGGT based on the genetic code. As a specific example, for example, a polynucleotide comprising the base sequence shown in SEQ ID NO: 16 can be mentioned.

  By using a linker containing a polynucleotide encoding a peptide having an amino acid sequence represented by GGGT, the effects of the present invention can be suitably obtained.

  In addition, the base sequence serving as the linker may further include a restriction enzyme site, a CTC sequence, and the like. Furthermore, the amino acid reading frame of the polynucleotide encoding the transcription activation domain or the transcription repression domain, When the reading frame of the gene encoding the protein described in i) or (ii) does not match, an additional base sequence for matching them may be included.

  In a plant transformed with a recombinant expression vector containing a chimeric gene linked by a base sequence serving as a linker, the protein described in (i) or (ii) above and a transcription activation domain or transcription repression A chimeric protein is produced in which the domains are linked by a peptide serving as a linker. The peptide serving as the linker preferably includes GGGT, that is, an amino acid sequence represented by Gly-Gly-Gly-Thr.

  In the present invention, a transformant with controlled inflorescence morphology or flowering time can be obtained by introducing the chimeric gene into a plant cell. Therefore, the present invention also includes the above chimeric gene.

  The chimeric gene according to the present invention may be any gene comprising the gene encoding the protein described in (i) or (ii) above and the polynucleotide encoding the transcription activation domain or the transcription repression domain. The chimeric gene according to the present invention further comprises a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription activation domain or the transcription repression domain. It is more preferable that they are linked by a base sequence to be

(IV) Plants in which inflorescence morphology is controlled In the present invention, “inflorescence morphology is controlled” means that the infinite inflorescence morphology is changed to a finite inflorescence morphology, and the finite inflorescence morphology is infinite inflorescence. Includes both changing to form.

  Here, changing the infinite inflorescence form to the finite inflorescence form may be any means that converts an inflorescence that does not have a flower at the tip of the inflorescence axis into an inflorescence that has a flower at the tip of the inflorescence axis. Therefore, it is not always necessary to convert the number of flower buds to one, and it is sufficient that the number of flower buds is significantly reduced. Here, the number of flower buds is significantly reduced as long as the number of flower buds is 80% or less before conversion, more preferably 50% or less before conversion, and more preferably before conversion. It is 20% or less, and particularly preferably 10% or less before conversion. The lower limit of the number of flower buds after conversion is one.

  Further, changing the finite inflorescence form to the infinite inflorescence form may be any means as long as it converts an inflorescence with a flower at the tip of the inflorescence axis into an inflorescence without a flower at the tip of the inflorescence axis. Accordingly, the present invention is not necessarily limited to the case where the number of flower buds is originally one, and it is sufficient that the number of flower buds is significantly increased. In addition, here, the number of flower buds is significantly increased. The number of flower buds is preferably 110% or more before conversion, and more preferably 120% or more.

  By applying the present invention to a plant having an infinite inflorescence, a new variety having a finite inflorescence can be created. Changes in inflorescence morphology can also increase the number of flowers and can be expected to improve yield.

  At the agricultural site, pinching is performed for the purpose of high quality and high yield. By introducing and using the chimeric gene according to the present invention, the same effect as that of pinching can be obtained, leading to reduction of farm work and improvement of quality.

  In addition, by changing a plant that is originally a finite inflorescence into an infinite inflorescence, the plant can continue vegetative growth.

  For example, recombination containing a chimeric gene comprising a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription activation domain in a plant cell having an infinite inflorescence morphology By introducing an expression vector, the infinite inflorescence form can be changed to a finite inflorescence form.

  Conversely, a chimeric gene comprising a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription repressing domain is applied to a plant cell having a finite inflorescence form. By introducing a recombinant expression vector containing the finite inflorescence form, the infinite inflorescence form can be changed.

(V) An example of a method for producing a plant with controlled inflorescence morphology A method for producing a plant with controlled inflorescence morphology according to the present invention comprises a gene encoding the protein described in (i) or (ii) above, Although it is not particularly limited as long as it includes a transformation step for introducing a recombinant expression vector containing a chimeric gene comprising a polynucleotide encoding a transcriptional activation domain or transcriptional repression domain into a plant cell, If the production method of the plant body concerning this invention is shown by a specific process, it can mention as a production method including other processes, such as an expression vector construction process, a transformation process, and a selection process, for example. Among these, in the present invention, at least the transformation step may be included. Hereinafter, each step will be specifically described.

(V-1) Expression vector construction step The expression vector construction step that can be included in the plant production method according to the present invention is a gene encoding the protein described in (i) or (ii) described in (I) above. As long as it is a step of constructing a recombinant expression vector comprising a polynucleotide encoding the transcription activation domain or transcription repression domain described in (II) above and a promoter, there is no particular limitation.

  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 vectors such as pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, pCGN, and pBI. In particular, when the method of introducing a vector into a plant is a method using Agrobacterium, a pCGN binary vector such as pCGN1547; for example, a pBI binary vector such as pBIG, pBIN19, pBI101, pBI121, and pBI221 Can be suitably used.

  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), various actin gene promoters, various ubiquitin gene promoters, and the like.

  Furthermore, as the above promoter, in addition to the overexpression promoter, a drug-inducible promoter such as a dexamethasone (DEX) inducible promoter, or a promoter of a gene that is specifically expressed in an inflorescence meristem such as FD is organ-specific. An expression promoter or the like can be used.

  In the present invention, the expression control can be temporally and spatially regulated by using the various promoters described above. This makes it possible to arbitrarily adjust the harvest time of crops and the like.

  The promoter is linked so as to express a chimeric gene in which the gene encoding the protein described in (i) or (ii) above and the polynucleotide encoding the transcription activation domain or transcription repression domain are linked. The specific structure as a recombinant expression vector is not particularly limited as long as it is introduced into the vector.

  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, an enhancer, a selection marker, a base sequence for adjusting translation efficiency, and the like.

  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, a transcription termination region (Nos terminator) of a nopaline synthase gene, a cauliflower mosaic virus 35S terminator (CaMV35S terminator), and the like can be preferably used.

  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 kanamycin, hygromycin, bleomycin, gentamicin, chloramphenicol and the like. Thereby, the transformed plant body can be easily selected by selecting the plant body which grows in the culture medium containing the said antibiotic.

  The method for constructing the recombinant expression vector is not particularly limited, and the promoter, the gene encoding the protein described in (i) or (ii), and the transcription described above are appropriately selected as a parent vector. What is necessary is just to introduce | transduce the polynucleotide which codes an activation domain or a transcription repression domain, and the said linker and said other DNA segment as needed in a predetermined order. For example, a chimeric gene obtained by linking a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription activation domain or transcription repression domain using the linker as necessary. Next, this chimeric gene, a promoter, and a terminator (if necessary) are linked to construct an expression cassette, which is then introduced 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.

(V-2) Transformation Step The transformation step performed in the present invention is to introduce the recombinant expression vector described in (V-1) above into a plant cell to produce the chimeric protein. That's fine.

  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, the floral dipping method described in Plant J. 1998 Dec; 16 (6): 735-43 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.

(V-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 a transformation process can be mentioned.

  The selection method is not particularly limited. For example, selection may be performed based on drug resistance such as kanamycin resistance, or rosettes when the number of flower buds or flower stems are lottered after growing transformants. You may select based on the number of leaves.

  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 progeny having a controlled inflorescence form from the plant. 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.

(VI) Method for producing plant body in which flowering time is controlled According to the method for producing a plant body according to the present invention, a plant body in which the flowering time is controlled can be produced. That is, the method for producing a plant according to the present invention is a method for producing a plant with a controlled flowering time, the gene encoding the protein described in (i) or (ii) above, a transcription activation domain or Also included is a method comprising a transformation step of introducing a recombinant expression vector comprising a chimeric gene comprising a polynucleotide encoding a transcription repression domain into a plant cell.

  A gene encoding the protein according to the above (i) or (ii), a polynucleotide encoding a transcription activation domain, and a transcription repression domain used in the method for producing a plant with controlled flowering time according to the present invention. An example of the method for producing the encoding polynucleotide, the chimeric gene, and the plant is as described in the above (I)-(III) and (V), and therefore the description thereof is omitted here.

  In the present invention, the plant having a controlled flowering time includes both advancing the flowering time and delaying the flowering time. Here, it can be easily confirmed by observing the time from sowing to flowering that the flowering time is controlled to be advanced and the flowering time is controlled to be delayed. The fact that the flowering time was controlled to be earlier can also be confirmed by a significant decrease in the number of rosette leaves when the flower stalks are lottered. This can also be confirmed by a significant increase in the number of rosette leaves.

  For example, a recombinant expression vector containing a chimeric gene comprising a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription activation domain is introduced into plant cells of the plant body. By this, flowering time can be advanced.

  Conversely, a recombinant expression vector comprising a chimeric gene comprising a gene encoding the protein described in (i) or (ii) above and a polynucleotide encoding the transcription repressing domain is applied to a plant cell of the plant body. By introducing, the flowering time can be delayed.

(VII) Plant obtained by the present invention A plant production method according to the present invention encodes a gene encoding the protein described in (i) or (ii) above and a transcription activation domain or transcription repression domain. By introducing a recombinant expression vector comprising a chimeric gene comprising a polynucleotide into a plant cell. Therefore, the present invention includes a plant obtained by the above-described method for producing a plant.

  Here, the specific kind of the plant body which concerns on this invention is not specifically limited, An angiosperm may be sufficient and a gymnosperm may be sufficient. Examples of gymnosperms include pine, cedar and ginkgo. Further, the angiosperm may be a monocotyledonous plant or a dicotyledonous plant.

  Dicotyledonous plants are not limited to Arabidopsis thaliana, tobacco, tomato, petunia, snapdragon, etc. Sweet potatoes) Plants, wasp genus plants, leaf genus plants, clover genus plants, cirrus genus plants, mantema genus plants, genus plants, beetles genus plants, genus genus plants Pepperaceae plant, Pleurophyceae plant, Yanagiaceae plant, Yamamomoaceae plant, Walnut family plant, Crabaceae plant, Beechaceae plant, Ganoderma plant, Nectaraceae plant, Iranaceae plant, Kawakokeso Family plant, Yamamaga family plant, Shabby family plant, Baccariaceae plant, Yadorigi family plant, Uma no Suzukusa family plant, Dendrobaceae plant, Tsuchitorimochi family plant, Bamboo family plant, Redfish family Plants, Hiyariaceae, Oshiroibanana, Yamatogusa, Yamagobo, Tsurunana, Surihiyu, Moleaceae, Tuna, Wig, Spruce Plant, pine family plant, genus genus plant, aceae plant, genus plant, tsuzushi fuji family plant, rosaceae plant, duckweed plant, licorice plant, plantaceae plant, oilseed plant, Another genus plant Utsubo-kazura family plant, Benxiaceae plant, Yukinoaceae plant, Periaceae plant, Mansaceae plant, Suzukake family plant, Rose family plant, Lariceae plant, Cubaceae plant, Diplomataceae plant, Amariaceae, Hamabidae, Mandarinaceae, Phyllaceae, Sudaenaceae, Himehagi, Toudaigusa, Agagogi, Tsurugidae, Ganoderma , Glycaceous plants, Urushiaceae plants, Mochinoaceae plants, Nishikigi family plants, Mitsuba genus plants, Kurotaki kurazura plants, Maple plants, Tochimakiceae plants, Muroguroaceae plants, Abalone family Plants, Ribbonaceae Plants, Kurome Modaceae Plants, Grapeaceae Plants, Horonophyceae Plants, Shinogi Family Plants, Aoi Family Plants, Papilio Family Plants, Monkeys Family Plants, Tsubaki Family Plants, Fairy Ties Rizo family plant, the groove Piperaceae, Phylumaceae, Violetaceae, Limeaceae, Papaveraceae, Pleurosumaceae, Pleurotusaceae, Sabotenaceae, Zanthaceae, Guroaceae, Misohagi Family plant, pomegranate plant, uraceae plant, urchinaceae plant, noctae plant, rhinoceros plant, red plant family plant, arinoto pusaceae plant, sugi family plant, urchinaceae plant, Apiaceae plant, Apricotaceae plant, Iwaceae plant, Rhododendron plant, Ichikuzai plant, Azalea plant, Diplomataceae plant, Sakuraiaceae plant, Isomatsuri plant, Oyster family plant, Yes Oysteraceae plants, Echinoceraceae plants, Cranaceae plants, Fujipiaceae plants, Glycaceae plants, Ganodermaceae plants, Ginger family plants, Hanabinoceae plants, Murasakieaceae plants, Bears Spiraceae plant, Lamiaceae plant, eggplant Plants, sesame seeds, genus plants, sesames, plants, citrus plants, raccoonaceae plants, raccoonaceae plants, foxes, genus plants, hazelsaceae Examples thereof include plants, abalone family, akane family plant, watermelon family plant, lepidopterous plant, seaweed family plant, pine family plant, cucurbitaceae plant, asteraceae plant, asteraceae plant and the like.

  In addition, examples of monocotyledonous plants include duckweed plants (duckweed) and duckweed plants (duckweed, hinokimo), duckweed plants, cattleya plants, cymbidium plants, dendrobium plants, phalaenopsis plants, banda Genus plants, paphiopedilum plants, Oncidium plants, etc., orchidaceae plants, urchinaceae plants, cinnamonaceae plants, hiru-raceae plants, thorny family plants, spider family plants, chironomids , Tochika department plant, Japanese cucurbitaceae plant, rice plant (rice, corn), Kasumarigusa plant, Palmiaceae plant, Sugar beet family plant, Hosogusa family plant, Tsukuusa family plant, Mizuaoi Family plant, rush family plant, japaceae plant, liliaceae plant (lily), longhorned plant, mountain potato plant, ayame family plant, Xiang Plants, ginger family plant, planers family plant can be exemplified Hinanoshakujou Plants like.

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

[Reference Example 1]
TFL1 protein is known to be important in the regulation of plant flowering and inflorescence morphology, tfl1 mutants are prematurely blooming, and wild-type infinite inflorescences are converted into finite inflorescences, with flowers at the end of the inflorescence axis. It is known to show a terminal inflorescence phenotype (Bradley et al., 1997). Therefore, first, tfl1-1 mutants were confirmed using tfl1-1, tfl1-11, tfl1-13, tfl1-14, and tfl1-17.

  tfl1 mutant seeds were obtained from the Arabidopsis Biological Resource Center (ABRC). Among the mutant lines, the background of tfl1-1, tfl1-11, tfl1-13, and tfl1-14 is wild type Col-0. These tfl1 mutant lines are generated by substitution of amino acids at positions shown in FIG. Tfl1-17 is a mutant of RNA-null (Mimida et al. 2001. Genes to Cells, 6, 327-336).

  In the tfl1 mutant, the wild-type infinite inflorescence was converted into a finite inflorescence, and all of the tfl1 mutants became terminal inflorescences with flowers at the tip of the inflorescence axis, and the inflorescences of the inflections were shortened. FIG. 4B shows the number of flower buds in wild type plants and tfl1 mutants. In the graph of FIG. 4B, the vertical axis indicates the number of flower buds.

  As shown in FIG. 4B, wild type plants (indicated as “Col” in FIG. 4) have 20 or more flower buds, whereas all tfl1 mutants have 5 or less flower buds, and tfl1-17. Then, the result was less than 1 flower bud. Thus, it was confirmed that the tfl1 mutant showed a terminal inflorescence phenotype by converting a wild-type infinite inflorescence into a finite inflorescence, and the number of flower buds was reduced.

In addition, in order to analyze the flowering time of the tfl1 mutant, the number of rosette leaves when the flower stem was lottered was counted. The results are shown in FIG. In FIG. 4A, the vertical axis indicates the number of rosette leaves when the flower stem is lottery, white indicates the result at 22 ° C., and black indicates the result at 16 ° C.
Here, the lottery refers to a phenomenon in which a plant that has maintained a rosette shape and continues vegetative growth under a non-inducing condition of flower bud formation causes rapid flower stem growth while forming a flower bud under the inducing condition. As shown in FIG. 4A, the tfl1 mutant was prematurely blooming, and thus it was confirmed that the number of rosette leaves was small compared to the wild type plant. In the tfl1 mutant, flower stem lottery occurs early because it blooms early, but at 22 ° C., the difference in lottery time was slight. The early bloom of the tfl1 mutant appears to have increased at 16 ° C. because the wild type lottery is delayed at 16 ° C. At 22 ° C., tfl1-17 bloomed slightly earlier than the wild type, but no significant change was observed between 16 ° C. and 22 ° C. This suggests that the loss of TFL1 function reduces the temperature sensitivity of plants.

[Example 1]
A transformant was prepared by introducing a chimeric gene in which a polynucleotide encoding a transcription activation domain was linked to the TFL1 gene into Arabidopsis thaliana wild-type Col-0 and TFL1 null mutant tfl1-17.

  Similarly, a transformant was prepared by introducing a chimeric gene in which a polynucleotide encoding a transcription repression domain was bound to the TFL1 gene into Col-0 and tfl1-17.

  Specifically, a chimeric gene in which a polynucleotide encoding VP16, which is a transcriptional activation domain, or SRDX, which is a transcriptional repression domain, is linked downstream of the TFL1 gene, respectively, to control a cauliflower mosaic virus (CaMV) 35S promoter. Expression vectors TFL1: VP16M or TFL1: SRDX to be expressed below were prepared. Then, these expression vectors were introduced into Arabidopsis thaliana using the Agrobacterium method to transform Arabidopsis thaliana.

<TFL1: Preparation of VP16M expression vector>
PCGN 35S: TFL1: VP16M NOS was prepared as an expression vector for expressing a chimeric gene in which a polynucleotide encoding a transcriptional activation domain was bound to the TFL1 gene. Hereinafter, a method for producing an expression vector will be described with reference to FIG.

  First, using pVP16C-1 (Novagen) as a template, PCR was performed using a primer set consisting of the following primers VP16 FW SmaI and VP16 M RV, and a primer set consisting of VP16 M FW and VP16 RV SalI, respectively. Fragments amplified by PCR were annealed.

  An annealed fragment of each PCR was used as a template, and further PCR was performed to amplify a VP16M fragment in which the SmaI site of VP16 was replaced from CCCGGG to CCCAGG. FIG. 6 shows the nucleotide sequence of a polynucleotide encoding VP16. In FIG. 6, the underlined portion is the SmaI site. In pVP16C-1, the underlined original sequence is CCCGGG (SmaI site), but the SmaI site was changed to CCCAGG by PCR. This does not change the amino acid sequence. Further, the VP16M fragment amplified by PCR has a SmaI site and a SalI site at both ends thereof. This PCR product was incorporated into Bluescript II SK + plasmid vector (pBluescript II SK +: manufactured by Stratagene) to obtain pBSVP16M.

  The amplified VP16M fragment was cleaved with restriction enzymes SmaI and SalI and plant biotech J. 2006; 4: 325-332. (Mitsuda N, Hiratsu K, Todaka D, Nakashima K, Yamaguchi-Shinozaki K, Ohme-Takagi M. , Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice.) To create a pENTRY VP16M NOS vector by replacing the SRDX sequence in the SmaI-SalI site of the pENTRY SRDX NOS vector described in did.

VP16 FW SmaI 5'-AGATCCCGGGCTCGCCCCCCCGACCGATGTC-3 '(SEQ ID NO: 7)
VP16 RV SalI 5'-GCTCGTCGACCTACCCACCGTACTCGTCAAT-3 '(SEQ ID NO: 8)
VP16 M FW 5'-CGGGGATTCCCCAGGTCCGGGA-3 '(SEQ ID NO: 9)
VP16 M RV 5′-TCCCGGACCTGGGGAATCCCCG-3 ′ (SEQ ID NO: 10)
For TFL1, a DNA fragment containing only the coding region excluding the stop codon was amplified by PCR using a Arabidopsis thaliana cDNA clone as a template and a primer sequence to which a linker sequence containing a restriction enzyme site was added. The base sequence encoding the cDNA of TFL1 gene is shown in SEQ ID NO: 2. The base sequence and amino acid sequence of the linker used are shown below. In the following base sequence, the 13th base to the 18th base (CCCGGG) from the 5 ′ end is a restriction enzyme site, and the linker further contains a CTC sequence in addition to the restriction enzyme site.

Linker base sequence 5'-GGAGGAGGTACCCCCGGGCTC-3 '(SEQ ID NO: 11)
Linker amino acid sequence GGGTPGL (SEQ ID NO: 12)
Moreover, the used primer is shown below. The amplified DNA fragment was cleaved with XbaI and SmaI and cloned into the SpeI-SmaI site of pENTRY VP16M NOS to generate pENTRY TFL1: VP16M NOS. Here, the linker is linked between TFL1 and VP16M. Thereby, TFL1: VP16M encodes the TFL1: GGGTPGL: VP16M protein.

The TFL1: VP16M region of the entry clone prepared here was transferred to the pCGN 35S: GW: NOS vector by the LR reaction of Gateway (registered trademark) Cloning kit (Invitrogen). Note that pCGN 35S: GW: NOS is a plant expression vector containing a CaMV 35S promoter sequence and a NOS terminator derived from pCGN1547. The structure of pCGN 35S: GW: NOS is shown in FIG.
TFL1XbaI FW 5'-GGATCTAGAATGGAGAATATGGGAACTAGAGTG-3 '(SEQ ID NO: 13)
TFL1 (linker) SmaI RV 5′-CATCCCGGGGGTACCTCCTCCGCGTTTGCGTGCAGCGGTTTCTC-3 ′ (SEQ ID NO: 14)
<TFL1: Preparation of SRDX expression vector>
PCGN 35S: TFL1: SRDX NOS was prepared as an expression vector for expressing a chimeric gene in which a polynucleotide encoding a transcriptional repression domain was bound to the TFL1 gene. Hereinafter, a method for producing an expression vector will be described with reference to FIG.

  TFL1 was amplified in the same manner as in the production of TFL1: VP16M expression vector using a cDNA clone as a template and a primer with a linker sequence containing a restriction enzyme site added to the end.

  The amplified DNA fragment was cleaved with XbaI and SmaI and cloned into the SpeI-SmaI site of the pENTRY SRDX NOS vector to create pENTRY TFL1: SRDX NOS. The TFL1: SRDX region of the entry clone prepared here was transferred to the pCGN 35S: GW: NOS vector by the LR reaction of Gateway (registered trademark) Cloning kit (Invitrogen). Note that pCGN 35S: GW: NOS is a plant expression vector containing a CaMV 35S promoter sequence and a NOS terminator derived from pCGN1547.

<Transformation process>
Next, using the prepared pCGN35S TFL1: VP16M NOS plasmid and pCGN35S TFL1: SRDX NOS plasmid, respectively, it was introduced into an Arabidopsis thaliana wild type plant and a tfl mutant to produce a transformant. The prepared pCGN35S TFL1: VP16M NOS plasmid and pCGN35S TFL1: SRDX NOS plasmid were each introduced into Agrobacterium C58 strain by electroporation. The introduced bacteria were cultured at 30 ° C. Gene transfer to Arabidopsis thaliana was carried out according to Plant J. 1998 Dec; 16 (6): 735-43.Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Ten or more lines were produced.

  Selection of transformed plants was carried out by sowing and germination on Murashige-Skoog agar medium (0.8% Agar) containing 1% sucrose supplemented with 50 μg / ml kanamycin. Plants without antibiotic resistance are markedly inhibited in growth, whereas transformed plants can germinate and grow as usual on antibiotics. Depending on the selected selection marker, other antibiotics such as hygromycin can be used in addition to kanamycin.

  Moreover, it was confirmed by RT-PCR that TFL1: SRDX and TFL1: VP16M were expressed in the transformant.

<Evaluation of transformant>
Using pCGN35S TFL1: VP16M NOS plasmid and pCGN35S TFL1: SRDX NOS plasmid, respectively, the flowering time and inflorescence morphology were evaluated for the transformants obtained by introducing them into wild-type plants and tfl mutants of Arabidopsis thaliana.

  FIG. 1A shows a wild-type plant as a control and tfl1-17 which is a null mutant of TFL1, and a transformant in which TFL1: VP16M or TFL1: SRDX is introduced into each of the wild-type plant and tfl1-17. The state of 6 weeks after sowing is shown. As shown in FIG. 1A, it was found that in the plant introduced with TFL1: SRDX, flowering was delayed with respect to each control. FIG. 1B shows a plant introduced with TFL1: SRDX grown for 2 months. As shown here, when a plant introduced with TFL1: SRDX is continuously grown, a wild type background is obtained. But even in the background of tfl1-17, flower stems with infinite inflorescences were lottery. This indicates that by introducing TFL1: SRDX, the finite inflorescence characteristic of the tfl1 mutant can be converted into an infinite inflorescence and the terminal inflorescence phenotype can be restored to the inflorescence morphology of the wild type plant. Yes.

  In contrast, when TFL1: VP16M is introduced into a wild-type plant, as shown in FIGS. 1A and 1C, the resulting transformant converts the infinite inflorescence characteristic of the wild-type plant into a finite inflorescence, The inflorescence phenotype was shown. In addition, C of FIG. 1 has shown the mode after growing the transformant which introduce | transduced TFL1: VP16M into the wild type plant further. From these results, it became clear that overexpression of TFL1: VP16M becomes a dominant negative of TFL1. Next, it was analyzed how the transformants produced by introducing the pCGN35S TFL1: VP16M NOS plasmid and the pCGN35S TFL1: SRDX NOS plasmid into wild-type plants and tfl mutants of Arabidopsis thaliana have an effect on the flowering time, respectively. did.

  In order to examine the flowering time, the number of flower buds and the number of rosette leaves when the flower stalks were counted under a temperature condition of 22 ° C. The results are shown in FIG. In FIG. 2, A represents a transformant obtained by introducing pCGN35S TFL1: VP16M NOS plasmid and pCGN35S TFL1: SRDX NOS plasmid into a wild type plant, the number of flower buds and the flower stem in the wild type background. It is a figure which shows the number of rosette leaves at the time of lottery. In FIG. 2, B represents the number of flower buds in the background of tfl1-17 and the transformant obtained by introducing pCGN35S TFL1: VP16M NOS plasmid and pCGN35S TFL1: SRDX NOS plasmid respectively into tfl1-17. FIG. 4 is a diagram showing the number of rosette leaves when flower stems are lottery. 2A and 2B, the vertical axis represents the number of flower buds, and the horizontal axis represents the number of rosette leaves. Further, black circles and black triangles were obtained by introducing the control, squares were obtained by introducing the pCGN35S TFL1: VP16M NOS plasmid, and diamonds were obtained by introducing the pCGN35S TFL1: SRDX NOS plasmid. Transformants are shown.

  As shown in FIG. 2A, in the transformant in which TFL1: SRDX is introduced into the wild type plant, the number of flower buds is almost the same as that of the wild type plant shown in FIG. As the number of rosette leaves increased when lottery ran, it turned out to be late blooming. In contrast, in the transformant in which TFL1: VP16M was expressed in a wild-type plant, the number of flower buds was greatly reduced due to the end inflorescence, but there was no significant change in the flowering time.

  tfl1-17 significantly reduces the number of flower buds compared to the wild type plant, but in the transformant in which Tfl1: SRDX was expressed in tfl1-17, the finite inflorescence became an infinite inflorescence and the number of flower buds increased. . In addition, the flowering time was delayed and was almost the same as when TFL1: SRDX was introduced into a wild type plant. When TFL1: VP16M was introduced into tfl1-17, the number of flower buds remained almost unchanged and the flowering time was advanced.

  From these results, it was found that not only the inflorescence morphology but also the number of flower buds and the flowering time can be regulated by binding a transcriptional activation domain or repression domain to TFL1.

  According to the present invention, it is possible not only to control the inflorescence morphology and flowering time of plants, but also to expect an improvement in the number of fruit. Therefore, in the field of horticulture, it can be expected to create new varieties and increase crop yields. In addition, it has an effect in place of conventional pinching, leading to efficient farming. 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.

Claims (15)

The following proteins (i) or (ii):
(I) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 17 or 19,
(Ii) consisting of 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: 1, 17 or 19, and delays flowering; A protein having a function of maintaining an inflorescence meristem;
A gene encoding
Inflorescence morphology is controlled, comprising a transformation step of introducing a recombinant expression vector comprising a chimeric gene comprising a polynucleotide encoding a transcriptional activation domain or transcriptional repression domain into a plant cell. Production method for the plant. The transcription activation domain is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3,
The method for producing a plant according to claim 1, wherein the transcription repressing domain is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 5. As a gene encoding the protein described in (i) or (ii) above, the following gene described in (a) or (b):
(A) a gene having the base sequence represented by SEQ ID NO: 2, 18, or 20 as an open reading frame region,
(B) A function of hybridizing under stringent conditions with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2, 18 or 20, and delaying flowering to maintain the inflorescence meristem A gene encoding a protein having
The method for producing a plant according to claim 1 or 2, wherein:   The polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4 or 6, respectively, is used as the polynucleotide encoding the transcription activation domain or transcription repression domain. A method for producing a plant according to claim 1.   Including a transformation step of introducing a recombinant expression vector comprising a chimeric gene comprising a gene encoding the protein according to (i) or (ii) above and a polynucleotide encoding the transcription activation domain into a plant cell. The method for producing a plant according to any one of claims 1 to 4, wherein the infinite inflorescence form is changed to a finite inflorescence form.   Including a transformation step of introducing a recombinant expression vector comprising a chimeric gene comprising a gene encoding the protein according to (i) or (ii) above and a polynucleotide encoding the transcription repressing domain into a plant cell. The plant production method according to any one of claims 1 to 4, wherein the finite inflorescence form is changed to an infinite inflorescence form. The following proteins (i) or (ii):
(I) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 17 or 19,
(Ii) consisting of 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: 1, 17 or 19, and delays flowering; A protein having a function of maintaining an inflorescence meristem;
A gene encoding
Control of flowering time characterized by including a transformation step of introducing a recombinant expression vector containing a chimeric gene comprising a polynucleotide encoding a transcription activation domain or transcription repression domain into a plant cell Production method for the plant. The transcription activation domain is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3,
The method for producing a plant according to claim 7, wherein the transcription repressing domain is a peptide having an amino acid sequence represented by SEQ ID NO: 5. As a gene encoding the protein described in (i) or (ii) above, the following gene described in (a) or (b):
(A) a gene having the base sequence represented by SEQ ID NO: 2, 18, or 20 as an open reading frame region,
(B) A function of hybridizing under stringent conditions with a gene consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 2, 18 or 20, and delaying flowering to maintain the inflorescence meristem A gene encoding a protein having
The method for producing a plant according to claim 7 or 8, wherein:   The polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 or 6, respectively, is used as the polynucleotide encoding the transcription activation domain or transcription repression domain, respectively. A method for producing a plant according to claim 1.   Including a transformation step of introducing a recombinant expression vector comprising a chimeric gene comprising a gene encoding the protein according to (i) or (ii) above and a polynucleotide encoding the transcription activation domain into a plant cell. The method for producing a plant according to any one of claims 7 to 10, wherein the flowering time is advanced by.   Including a transformation step of introducing a recombinant expression vector comprising a chimeric gene comprising a gene encoding the protein according to (i) or (ii) above and a polynucleotide encoding the transcription repressing domain into a plant cell. The method for producing a plant according to any one of claims 7 to 10, wherein the flowering time is delayed.   A plant produced by the production method according to claim 1. The following proteins (i) or (ii):
(I) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, 17 or 19,
(Ii) consisting of 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: 1, 17 or 19, and delays flowering; A protein having a function of maintaining an inflorescence meristem;
A gene encoding
A chimeric gene comprising a polynucleotide encoding a transcription activation domain or a transcription repression domain. The transcription activation domain is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3,
The chimeric gene according to claim 14, wherein the transcription repressing domain is a peptide having an amino acid sequence represented by SEQ ID NO: 5.