Methods for producing genetically modified plant

Abstract

To provide a novel technology for producing a genetically modified plant that is modified so that flowering times can be artificially modified.SOLUTION: Provided is a method for producing a genetically modified plant, comprising using a genome editing technology to replace an endogenous flowering-inducing gene of a target plant with an expression cassette containing an inducible promoter and an exogenous flowering-inducing gene operably linked to the inducible promoter.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a genetically modified plant, specifically, a method for producing a genetically modified plant that has been modified so that its flowering time can be artificially regulated. [Background technology]

[0002] In crop cultivation, it is necessary to carry out the entire reproductive process, starting with flower bud differentiation, which then involves flowering, fruiting, and seed collection, safely and reliably at the desired time to produce high-quality, high-yield crops.

[0003] Conventionally, flowering time has been regulated by subjecting plants to long-day conditions through electric lighting for a certain period of time, or by subjecting plants to short-day conditions through complete shading for a certain period of time (Non-Patent Document 1). Such conventional methods require the installation of treatment facilities and the carrying out of various manual tasks during the treatment period. For this reason, conventional methods require large-scale equipment and manpower, and are very time-consuming.

[0004] Meanwhile, it is known that flower bud differentiation in plants occurs as a result of the expression of the FT (Flowering Locus T) gene in response to photoperiod (Non-Patent Document 2). Specifically, flower bud differentiation occurs when the leaf senses that a certain photoperiod is suitable for flower bud differentiation, and the FT gene is expressed in cells adjacent to the vascular bundles of the leaf, producing the FT protein. The FT protein travels through the vascular bundles to reach cells at the shoot apex, where it binds to the FD (Flowering Locus D) protein. This initiates flower bud differentiation in the shoot apical meristem.

[0005] Therefore, if the function of the endogenous FT gene is destroyed by mutation or artificial genetic manipulation, or under conditions that are not suitable for flower bud differentiation, such as under inappropriate day length conditions, it becomes difficult to harvest crops of the desired quality at the desired time. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Kotobank "Flowering regulation", https: / / kotobank.jp / word / %E9%96%8B%E8%8A%B1%E8%AA%BF%E7%AF%80-1515349 [Non-Patent Document 2] Kobayashi, Y. et al., “A pair of related genes with antagonistic roles in mediating flowering signals.” Science (1999) 286, 1960-1962 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a new technique for producing genetically modified plants that have been modified so that their flowering time can be artificially regulated. [Means for solving the problem]

[0008] According to one aspect, there is provided a method for producing a genetically modified plant, comprising using genome editing technology to replace an endogenous flowering-inducing gene (e.g., an endogenous FT gene) of a target plant with an expression cassette comprising an inducible promoter and an exogenous flowering-inducing gene (e.g., an exogenous FT gene) operably linked to the inducible promoter.

[0009] According to another aspect, there is provided a genetically modified plant that has been modified to replace an endogenous flowering-inducing gene (e.g., an endogenous FT gene) of a subject plant with an expression cassette comprising an inducible promoter and an exogenous flowering-inducing gene (e.g., an exogenous FT gene) operably linked to the inducible promoter. Effect of the Invention

[0010] According to the present invention, a new technique is provided for producing genetically modified plants that have been modified so that their flowering time can be artificially controlled. [Brief description of the drawings]

[0011] [Figure 1] 1A and 1B are diagrams showing an overview of a method according to a first embodiment and a method according to a second embodiment. [Diagram 2] FIG. 1 shows an example in which the target sequence of a target gene is designed to contain an intron. [Diagram 3] A diagram showing how to design the sequence of the modified FT gene. [Figure 4] FIG. 13 is a diagram showing an outline of a method according to a modified example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, embodiments of the present invention will be described. The embodiments described below are more specific embodiments of any of the above aspects. The following items can be incorporated into each of the above aspects alone or in combination.

[0013] The following description will be given by taking the case where the FT (Flowering Locus T) gene is used as the flowering induction gene as an example. However, the present invention is not limited to the FT gene, and is widely applicable to "flowering induction genes" other than the FT gene. According to one embodiment of the present invention, a genetically modified plant is produced by replacing the endogenous FT gene of a target plant with an expression cassette containing an inducible promoter and an exogenous FT gene operably linked to the inducible promoter using genome editing technology. When the inducible promoter of the obtained genetically modified plant is artificially activated, the expression of the exogenous FT gene is induced, and flower bud formation can be caused at a desired timing. This allows the flowering time to be artificially regulated.

[0014] <1. Genome editing technology> In the present invention, genome editing technology is used. First, genome editing technology will be described. Genome editing technology is known as a technology that uses site-specific nucleases to modify target genes. A representative example of genome editing technology is CRISPR / Cas9 technology. CRISPR / Cas9 is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats / Crispr Associated protein 9.

[0015] In the CRISPR / Cas9 technique, DNA encoding a guide RNA containing a sequence (e.g., 17 to 20 bases) complementary to the target sequence of a target gene and a Cas9 gene encoding a site-specific nuclease called a Cas9 protein are introduced into the nucleus of a cell of a target organism. A guide RNA is generally a single-stranded RNA in which a sequence (crRNA) complementary to the target sequence of a target gene is linked to a sequence (tracrRNA) that functions as a scaffold for binding the Cas9 protein to the crRNA. In the CRISPR / Cas9 technique, a PAM (protospacer adjacent motif) sequence must be present downstream of the target sequence of a target gene. The PAM sequence is a specific base sequence recognized by the Cas9 protein, and in the case of the Cas9 protein derived from Streptococcus pyogenes, it is 5'-NGG-3' (where N refers to any base (i.e., A, T, G, or C)). The Cas9 protein recognizes the PAM sequence and cleaves the target gene (double-stranded DNA) between the base 3 bases upstream of the PAM sequence and the base 4 bases upstream of the PAM sequence. When inserting an exogenous gene into this cleavage site, the exogenous gene is introduced into the target cell in addition to the DNA encoding the guide RNA and the Cas9 gene. By arranging arm sequences at the 5' and 3' ends of the exogenous gene that are homologous to the upstream and downstream sequences of the cleavage site of the target gene, homologous recombination occurs and the target gene is replaced by the exogenous gene.

[0016] <2. Explanation of terms> In one embodiment of the present invention, the endogenous FT gene of a target plant is replaced by genome editing with "an expression cassette comprising an inducible promoter and an exogenous FT gene operably linked to the inducible promoter." The replacement here includes both cases where the expression cassette is introduced into the same position on the genome as the endogenous FT gene while disrupting the endogenous FT gene, and where the expression cassette is introduced into a position different from the position on the genome of the endogenous FT gene while disrupting the endogenous FT gene.

[0017] As used herein, the term "target plant" refers to a plant that is genetically modified by genome editing. As used herein, a "plant" generally refers to a plant that produces flowers (i.e., a flowering plant), specifically, a plant that is known to have a flowering-inducing gene (e.g., a FT (Flowering Locus T) gene) in its genome. The FT gene is also called a flowering-inducing gene, and expression of the FT gene induces flower bud formation.

[0018] The "plant" is preferably an angiosperm, more preferably a dicotyledonous plant. The "plant" may be an annual plant or a perennial plant. In this specification, the term "plant" refers to any form that the plant can take, regardless of the growth stage or the presence or absence of differentiation. The term "plant" includes, for example, a plant body, a seed, a fruit, an adventitious embryo, an adventitious bud, a dedifferentiated cell mass, a protoplast, and the like. In this specification, the term "plant body" refers to an individual plant composed of roots, stems, leaves, and the like.

[0019] Additionally, as used herein, the term "endogenous gene" refers to a gene that is naturally present in the genome of a target plant, and the term "exogenous gene" refers to a gene that is to be introduced into the genome of a target plant by genome editing (e.g., a gene in a vector) or a gene that has been introduced into the genome of a target plant by genome editing.

[0020] 3. First embodiment According to one embodiment, replacement of the FT gene can be achieved by introducing into a target plant "an expression cassette comprising an inducible promoter and an exogenous FT gene operably linked to the inducible promoter," "a coding region of a guide RNA comprising a sequence complementary to at least a portion of the endogenous FT gene," and "a Cas9 gene encoding a Cas9 protein."

[0021] According to a preferred embodiment, the FT gene can be replaced by introducing into a target plant "an expression cassette comprising an inducible promoter and an exogenous FT gene operably linked to the inducible promoter," "a coding region of a guide RNA comprising a sequence complementary to at least a portion of the endogenous FT gene," "a Cas9 gene encoding a Cas9 protein," and "a gene encoding a transcriptional activator that binds to the inducible promoter to activate transcription of the exogenous FT gene."

[0022] For example, the replacement of the FT gene can be carried out by introducing a vector containing the above expression cassette (hereinafter also referred to as the expression cassette of the foreign FT gene), a coding region of a guide RNA, a Cas9 gene, and (if necessary, a gene encoding a transcription activator) into a target plant. An example of a vector containing an expression cassette of the foreign FT gene, a coding region of a guide RNA, a Cas9 gene, and a gene encoding a transcription activator is shown in FIG. 1.

[0023] The abbreviations in Figure 1 are as follows: LB Left Border Kan R Kanamycin resistance gene Arm Arrangement FTcds FT gene coding sequence (foreign FT gene) Terminator Gvg The gene encoding the transcriptional activator GVG crRNA CRISPR RNA coding region tracrRNA The coding region for transactivating crRNA gRNA Guide RNA coding region Cas9 Cas9 gene RB Right Border FT locus The locus of the endogenous FT gene.

[0024] The vector shown in FIG. 1 includes, from the 5' end, a selection marker gene, an expression cassette for the exogenous FT gene, a gene encoding a transcription activator, and a genome editing cassette. The "selection marker gene" is a marker gene for selecting plant cells into which the vector has been introduced. The "expression cassette for the exogenous FT gene" includes, from the 5' end, an arm sequence homologous to the upstream sequence of the cleavage site of the endogenous FT gene (target gene), an inducible promoter, the exogenous FT gene, a terminator, and an arm sequence homologous to the downstream sequence of the cleavage site of the endogenous FT gene (target gene). The "gene encoding the transcription activator" is selected according to the type of inducible promoter, since it encodes a factor that binds to the inducible promoter to activate the transcription of the exogenous FT gene. The "genome editing cassette" includes, from the 5' end, a coding region for the crRNA, a coding region for the tracrRNA, and a Cas9 gene.

[0025] Below, we will explain the "selection marker gene," "exogenous FT gene expression cassette," "genome editing cassette," and "gene encoding a transcription activator," in that order.

[0026] (selection marker gene) As the selection marker gene, any selection marker gene, such as a drug resistance gene, can be used as long as it can function as a marker for selecting plant cells into which the vector has been introduced. In Figure 1, the kanamycin resistance gene is shown as an example of a selection marker gene.

[0027] The nucleotide sequence of the kanamycin resistance gene (SEQ ID NO: 1) is shown below.

[0028] [Table 1]

[0029] The selectable marker gene may contain a constitutive promoter (e.g., CaMV 35S promoter) upstream and a polyadenylation signal (e.g., CaMV polyadenylation signal) downstream. The nucleotide sequence of the CaMV 35S promoter (SEQ ID NO: 2) and the nucleotide sequence of the CaMV polyadenylation signal (SEQ ID NO: 3) are shown below.

[0030] [Table 2]

[0031] [Table 3]

[0032] (Exogenous FT gene expression cassette) 1, the expression cassette for the exogenous FT gene contains, in this order from the 5' end, an inducible promoter, the exogenous FT gene operably linked to the inducible promoter, and a terminator, i.e., the expression of the exogenous FT gene is controlled by the activity of the inducible promoter.

[0033] An "inducible promoter" refers to a promoter that is activated only under specific conditions, i.e., a promoter that induces the expression of an exogenous FT gene only under specific conditions. Known examples of "inducible promoters" include drug-inducible promoters and stimulus-inducible promoters. Known examples of drug-inducible promoters include glucocorticoid-inducible promoters and estrogen-inducible promoters. Known examples of stimulus-inducible promoters include light-inducible promoters (e.g., blue light-inducible promoters).

[0034] The base sequence of the glucocorticoid-inducible promoter (SEQ ID NO: 4) is shown below.

[0035] [Table 4]

[0036] The exogenous FT gene has the coding sequence of the FT gene and does not contain an intron. In the method according to the first embodiment, the exogenous FT gene can have the same base sequence as the coding sequence of the endogenous FT gene. The base sequence (SEQ ID NO: 5) of the exogenous FT gene having the same base sequence as the coding sequence of the endogenous FT gene is shown below.

[0037] [Table 5]

[0038] An example of the base sequence of the terminator of an exogenous FT gene (SEQ ID NO: 6) is shown below.

[0039] [Table 6]

[0040] In FIG. 1, the expression cassette for the exogenous FT gene contains, on the 5' side, an arm sequence (Arm) that is homologous to a sequence in the upstream region of the cleavage site of the endogenous FT gene (target gene), and further contains, on the 3' side, an arm sequence (Arm) that is homologous to a sequence in the downstream region of the cleavage site of the endogenous FT gene (target gene). The arm sequence on the 5' side can be a sequence that is homologous to a sequence in the upstream region of the cleavage site of the endogenous FT gene. The arm sequence on the 3' side can be a sequence that is homologous to a sequence in the downstream region of the cleavage site of the endogenous FT gene. By locating such arm sequences on the 5' and 3' sides of the expression cassette, homologous recombination with the genomic DNA of the target plant occurs, and the exogenous FT gene can be integrated into the genomic DNA of the target plant together with the inducible promoter.

[0041] An example of the 5' arm sequence (SEQ ID NO: 7) and an example of the 3' arm sequence (SEQ ID NO: 8) are shown below.

[0042] [Table 7]

[0043] [Table 8]

[0044] (Genome editing cassette) In Figure 1, the genome editing cassette includes, in order from the 5' end, a coding region of crRNA, a coding region of tracrRNA, and a Cas9 gene. In this specification, the "coding region of RNA" refers to DNA that encodes RNA.

[0045] The crRNA has a sequence complementary to a part of the sequence (target sequence) of the endogenous FT gene (target gene) of the target plant. The crRNA is also called a "guide sequence" and can be, for example, 17 to 20 bases. The tracrRNA functions as a scaffold for binding the Cas9 protein to the crRNA. The crRNA and tracrRNA constitute the guide RNA (see FIG. 1). As the guide RNA, generally, a single-stranded RNA in which a sequence complementary to the target sequence of the endogenous FT gene (crRNA) and a sequence functioning as a scaffold for binding the Cas9 protein to the crRNA (tracrRNA) are linked can be used. Such a single-stranded RNA is known as a single guide RNA (sgRNA).

[0046] An example of the base sequence of the "crRNA coding region" is shown below (SEQ ID NO: 9).

[0047] [Table 9]

[0048] The crRNA (SEQ ID NO: 9) encoded by this sequence has a sequence complementary to at least a portion of an intron contained in the endogenous FT gene of the target plant.

[0049] The base sequence of the "tracrRNA coding region" (sequence number 10) is shown below.

[0050] [Table 10]

[0051] As described above, the guide RNA can be a single guide RNA (sgRNA). An example of the base sequence of the "single guide RNA (sgRNA) coding region" is shown below (SEQ ID NO: 11).

[0052] [Table 11]

[0053] The nucleotide sequence of the Cas9 gene encoding the Cas9 protein (SEQ ID NO: 12) is shown below. The Cas9 gene can contain a constitutive promoter (e.g., CaMV 35S promoter: SEQ ID NO: 2) upstream thereof.

[0054] [Table 12-1]

[0055] [Table 12-2]

[0056] [Table 12-3]

[0057] [Table 12-4]

[0058] (genes encoding transcriptional activators) The vector shown in Figure 1 may further contain, in addition to the "selection marker gene," the "expression cassette for an exogenous FT gene," and the "genome editing cassette," a "gene encoding a transcription activator" depending on the type of inducible promoter.

[0059] For example, when the inducible promoter is a glucocorticoid-inducible promoter, the vector may further include a "gene encoding the transcription activator GVG." The "transcription activator GVG" is a chimeric transcription factor composed of the DNA binding domain of GAL4 derived from yeast, the transcription activation domain of VP16 derived from herpes simplex, and the glucocorticoid hormone binding domain derived from rat. Only when a glucocorticoid hormone such as dexamethasone is bound to the transcription activator GVG, the transcription activator GVG can bind to the glucocorticoid-inducible promoter and activate the transcription of the downstream gene. This transcription induction system is known as the GVG transcription induction system.

[0060] The "gene encoding the transcription activator GVG" can be located, for example, between the expression cassette and the genome editing cassette. In addition, the "gene encoding the transcription activator GVG" can include a constitutive promoter (e.g., CaMV 35S promoter: SEQ ID NO: 2) upstream thereof and a terminator (e.g., terminator of the small subunit of ribulose-1,5-bisphosphate carboxylase derived from pea (RbcS): SEQ ID NO: 14) downstream thereof.

[0061] The base sequence of the "gene encoding the transcription activator GVG" (SEQ ID NO: 13) and an example of the base sequence of the "terminator of the gene encoding the transcription activator GVG" (SEQ ID NO: 14) are shown below.

[0062] [Table 13]

[0063] In the above base sequence, the single underlined sequence indicates the region encoding the DNA-binding domain of yeast-derived GAL4, the non-underlined sequence indicates the region encoding the transcription activation domain of herpes simplex-derived VP16, and the double underlined sequence indicates the region encoding the rat-derived glucocorticoid hormone-binding domain.

[0064] [Table 14]

[0065] (Target sequence design) In the method according to the first embodiment, the target sequence of the endogenous FT gene (target gene) of the target plant can be designed to include an intron of the endogenous FT gene (target gene). The target sequence is a sequence targeted by the crRNA contained in the guide RNA, and the crRNA has a sequence complementary to the target sequence. Thus, the crRNA can have a sequence complementary to at least a portion of the intron contained in the endogenous FT gene of the target plant. This complementary sequence is called a "guide sequence."

[0066] An example of such a design in which the target sequence of the endogenous FT gene (target gene) of a target plant is designed to include an intron of the endogenous FT gene (target gene) is shown in FIG. 2. In FIG. 2, as an example, the boundary region spanning exon 2 (ex2) of the endogenous FT gene and its upstream intron is inserted into the target sequence (5'- NNNNNNNNNNNAG Alternatively, in FIG. 2, as another example, the boundary region spanning exon 1 (ex1) of the endogenous FT gene and the downstream intron is determined as the target sequence (5'-NNNNNN GTNNNNNNNNNN -3'). In these target sequences, N refers to any base (i.e., A, T, G, or C). Underlined bases refer to introns and non-underlined bases refer to exons.

[0067] The design of the target sequence (or the design of the crRNA (guide sequence)) can be performed, for example, using software named SnapGene (GSL Biotech LLC). For example, the target sequence can be designed so that the GC content of the crRNA (guide sequence) is 30-70%.

[0068] In this way, when the target sequence of the endogenous FT gene (target gene) of the target plant is designed to include an intron of the endogenous FT gene (target gene), and the crRNA (guide sequence) has a sequence complementary to at least a part of the intron contained in the endogenous FT gene of the target plant, the following advantages can be obtained. That is, the guide RNA containing such a crRNA can specifically bind to the endogenous FT gene of the target plant (which includes an intron), but cannot bind to the exogenous FT gene in the expression cassette (which does not include an intron because it is the coding sequence of the FT gene). Therefore, in this case, the Cas9 protein can be prevented from cleaving the exogenous FT gene in the expression cassette, and as a result, only the endogenous FT gene of the target plant can be cleaved. This allows the endogenous FT gene of the target plant to be efficiently replaced with the expression cassette of the exogenous FT gene.

[0069] The vector shown in FIG. 1 (containing a selection marker gene, an expression cassette for an exogenous FT gene, a gene encoding a transcription activator, and a genome editing cassette) can be prepared using a commercially available genome editing vector (e.g., pKSE401 (addgene)). The commercially available genome editing vector exemplified already contains a selection marker gene, a coding region for tracrRNA, and a Cas9 gene. Therefore, the vector shown in FIG. 1 can be prepared by introducing a coding region for crRNA (guide sequence), an expression cassette for an exogenous FT gene, and a gene encoding a transcription activator into a commercially available genome editing vector.

[0070] As explained above, genome editing can be performed by introducing the vector shown in Figure 1 into the cells of a target plant. Introduction of the vector into a target plant can be performed by a standard method such as the Agrobacterium method or a callus induction method using a particle gun. As shown in Figure 1, genome editing cuts the endogenous FT gene of the target plant and introduces an exogenous FT gene into the same position on the genome as the endogenous FT gene.

[0071] (effect) When a plant body is regenerated from a plant cell into which the above-mentioned vector has been introduced and the inducible promoter of the resulting genetically modified plant is artificially activated, expression of the exogenous FT gene is induced, and flower bud formation can be caused at the desired timing. This allows artificial control of the flowering time. As a result, it is expected that cut flowers and fruits can be produced out of season, seeds can be produced stably, and vegetable yields can be increased due to sustained vegetative growth, which can greatly contribute to stabilizing agricultural production and increasing income.

[0072] As described in the Background Art section, the conventional method requires the installation of a treatment facility and the carrying out of various manual operations during the treatment period. For this reason, the conventional method requires large-scale equipment and manpower, and is very time-consuming. On the other hand, the technology provided by the present invention is extremely simple, since it can induce flowering in plants placed under normal cultivation conditions by inducing the activity of an inducible promoter (for example, in the case of a drug-inducible promoter, spraying a specific drug). Thus, the technology provided by the present invention is superior to the conventional method in that it allows for extremely simple adjustment of the flowering time.

[0073] <4. Second embodiment> The method according to the second embodiment will be described below, but explanations that overlap with the method according to the first embodiment will be omitted, and only the points that differ from the method according to the first embodiment will be described.

[0074] In the method of the second embodiment, genome editing can also be performed by introducing a vector having the structure shown in Figure 1 (i.e., a vector containing a selection marker gene, an expression cassette, a gene encoding a transcriptional activator, and a genome editing cassette) into cells of a target plant.

[0075] As described above, in the method according to the first embodiment, an "exogenous FT gene" having the same base sequence as the coding sequence of the endogenous FT gene (SEQ ID NO: 5) is used as the "exogenous FT gene," and a "coding region of crRNA (guide sequence)" having a sequence complementary to at least a part of an intron contained in the endogenous FT gene of the target plant (SEQ ID NO: 9) is used as the "coding region of crRNA (guide sequence)." Meanwhile, in the method according to the second embodiment, a modified FT gene described below is used as the "exogenous FT gene," and a "coding region of crRNA (guide sequence)" having a sequence complementary to an exon contained in the endogenous FT gene of the target plant is used.

[0076] An example of the base sequence of such a "crRNA coding region" is shown below (SEQ ID NO: 15).

[0077] [Table 15]

[0078] As described above, the guide RNA can be a single guide RNA (sgRNA). An example of the base sequence of the "single guide RNA (sgRNA) coding region" is shown below (SEQ ID NO: 16).

[0079] [Table 16]

[0080] In other words, in the method of the second embodiment, there is no need to use the "crRNA coding region" used in the method of the first embodiment (i.e., one having a sequence complementary to at least a portion of the intron contained in the endogenous FT gene of the target plant) as the "crRNA coding region."

[0081] (Design of modified FT gene sequence) In the method according to the second embodiment, the exogenous FT gene is a nucleotide sequence of the coding sequence of the endogenous FT gene that satisfies the following conditions: (i) the modified FT gene is modified so that it is not cleaved by the Cas9 protein; and (ii) the modified FT gene encodes a protein identical to the protein encoded by the endogenous FT gene (i.e., the endogenous FT protein); It is a modified FT gene that has been modified to satisfy the above.

[0082] The modified FT gene can be designed, for example, as follows. FIG. 3 shows how to design the sequence of the modified FT gene. The PAM (protospacer adjacent motif) sequence present downstream of the target sequence of the endogenous FT gene (target gene) is modified so that it is not recognized by the Cas9 protein. The FT gene modified in this way is referred to as the "modified FT gene." This makes it possible to prevent the modified FT gene from being cleaved by the Cas9 protein. This modification of the PAM sequence makes the modified FT gene have a different base sequence from the endogenous FT gene but does not code for a different amino acid sequence from the endogenous FT protein (see FIG. 3). That is, the modification of the PAM sequence is performed so that the modified FT gene codes for the same amino acid sequence as the endogenous FT protein (i.e., a silent mutation). In FIG. 3, the amino acid sequence of the endogenous FT protein is simplified and shown as X1X2X3X4X5X6X7. It is desirable to modify this PAM sequence based on information on the codon frequency of the target plant so as to change it to a relatively major codon (i.e., not to a rare codon).

[0083] The sequence of the modified FT gene can be designed, for example, using software called SnapGene (GSL Biotech LLC).

[0084] An example of the base sequence of a modified FT gene (SEQ ID NO: 17) is shown below.

[0085] [Table 17]

[0086] In the above modified FT gene, the modified site is underlined. In the above modified FT gene, the PAM sequence GGG is modified to GAG so that it is not recognized by the Cas9 protein. In addition, this modified FT gene can encode the same amino acid sequence as the protein encoded by the unmodified FT gene. Specifically, the codon: A at the modified site of the unmodified FT gene GG G TT and the codon at the modified site of the modified FT gene: A GA G TT can code for the same amino acid.

[0087] As described above, when the above-mentioned modified FT gene is used as the exogenous FT gene in the expression cassette, it is possible to prevent the Cas9 protein from cleaving the exogenous FT gene in the expression cassette, and as a result, it is possible to cleave only the endogenous FT gene of the target plant, thereby enabling efficient replacement of the endogenous FT gene of the target plant with the expression cassette of the exogenous FT gene.

[0088] In the method according to the second embodiment, too, the endogenous FT gene of the target plant is cut by genome editing, and an exogenous FT gene can be introduced into the plant at the same position on the genome as the endogenous FT gene.

[0089] <5. Modifications> In the methods according to the first and second embodiments, genome editing can be performed by introducing a vector containing an expression cassette for an exogenous FT gene and a genome editing cassette into cells of a target plant. For such genome editing techniques, see Wolter et al., "Efficient in planta gene targeting in Arabidopsis using egg cell-specific expression of Cas9 nuclease of Staphylococcus aureus", The Plant Journal (2018) 94, 735-746.

[0090] Alternatively, genome editing may be performed by introducing a first vector containing an expression cassette of the Cas9 gene into a target plant, obtaining a plant body (transformant) that overexpresses the Cas9 gene, and then introducing a second vector (see FIG. 4) containing an expression cassette of an exogenous FT gene and a coding region of a guide RNA into the transformant. An example of the second vector containing an expression cassette of an exogenous FT gene, a coding region of a guide RNA, and a gene encoding a transcription activator is shown in FIG. 4. As described above, as the guide RNA, generally, a single-stranded RNA in which a sequence complementary to the target sequence of the endogenous FT gene (crRNA) and a sequence (tracrRNA) that functions as a scaffold for binding the Cas9 protein to the crRNA are linked can be used. Such a single-stranded RNA is known as a single guide RNA (sgRNA).

[0091] The abbreviations in Figure 4 are as follows: LB Left Border Hm R Hygromycin resistance gene Arm Arrangement FTcds FT gene coding sequence (foreign FT gene) Terminator Gvg The gene encoding the transcriptional activator GVG crRNA CRISPR RNA coding region tracrRNA The coding region for transactivating crRNA gRNA Guide RNA coding region RB Right Border FT locus The locus of the endogenous FT gene.

[0092] The vector shown in FIG. 4 includes, from the 5' end, a selection marker gene, an expression cassette for the foreign FT gene, a gene encoding a transcription activator, and a coding region for the guide RNA. The "selection marker gene" is a marker gene for selecting a plant cell into which the vector has been introduced. The "expression cassette for the foreign FT gene" includes, from the 5' end, an arm sequence homologous to the upstream sequence of the cleavage site of the endogenous FT gene (target gene), an inducible promoter, the foreign FT gene, a terminator, and an arm sequence homologous to the downstream sequence of the cleavage site of the endogenous FT gene (target gene). The "gene encoding the transcription activator" encodes a factor that binds to the inducible promoter to activate the transcription of the foreign FT gene, and is selected according to the type of inducible promoter. The "coding region for the guide RNA" includes, from the 5' end, a coding region for the crRNA and a coding region for the tracrRNA.

[0093] For a genome editing method that involves two rounds of transformation, see Miki et al., “CRISPR / Cas9-mediated gene targeting in Arabidopsis using sequential transformation”, Nature Communications (2018) 9, 1967.

[0094] <6. Third embodiment> According to the third embodiment, replacement of the FT gene can be carried out by disrupting the endogenous FT gene of the target plant to obtain an endogenous FT gene knockout strain, and introducing an expression cassette of an exogenous FT gene into the endogenous FT gene knockout strain.

[0095] In the method according to the third embodiment, first, the endogenous FT gene of the target plant is disrupted to obtain an endogenous FT gene-disrupted strain. For example, the endogenous FT gene can be disrupted using genome editing techniques such as CRISPR / Cas9 technology. Specifically, the endogenous FT gene can be disrupted by introducing into the target plant a coding region of a guide RNA containing a sequence complementary to at least a portion of the endogenous FT gene, and a Cas9 gene encoding a Cas9 protein. In this genome editing, the base sequence shown in SEQ ID NO: 15 can be used as the "coding region of the crRNA (guide sequence)". Alternatively, the endogenous FT gene-disrupted strain may be prepared by a mutation treatment (for example, a treatment by irradiation or a treatment by ethyl methanesulfonate) or by natural mutation.

[0096] Next, an expression cassette for an exogenous FT gene is introduced into the genome of the obtained endogenous FT gene disruptant. The expression cassette for the exogenous FT gene contains an inducible promoter and an exogenous FT gene operably linked to the inducible promoter. The expression cassette can be introduced into a target plant by a conventional method such as the Agrobacterium method or a callus induction method using a particle gun. In this transformation, the base sequence shown in SEQ ID NO:5 can be used as the "exogenous FT gene".

[0097] As a result of this transformation, the expression cassette of the exogenous FT gene is inserted at any position on the genome of the endogenous FT gene disruptant, and is not inserted at a specific position. In general, a "position effect" is observed in which the expression level of the exogenous gene differs depending on the position on the genome where the target expression cassette is inserted. Therefore, from the obtained transformants, a plant body with high flower bud formation efficiency may be selected. For example, several lines of transformants are obtained, and the inducible promoter of each transformant is artificially activated to induce the expression of the exogenous FT gene. The flower bud formation efficiency of each transformant can be confirmed, and a plant body with high flower bud formation efficiency can be selected.

[0098] As described above, in the method according to the third embodiment, the endogenous FT gene is disrupted while an expression cassette for an exogenous FT gene is introduced into a position on the genome different from the position of the endogenous FT gene.

[0099] <7. Inventions relating to other aspects and generalized inventions> According to another aspect, there is provided a genetically modified plant produced by the above-mentioned "method for producing a genetically modified plant". That is, according to another aspect, there is provided a genetically modified plant that has been modified so that the endogenous FT gene of a target plant is replaced with an expression cassette comprising an inducible promoter and an exogenous FT gene operably linked to the inducible promoter.

[0100] In addition, so far, the invention has been described as "an invention relating to a genetically modified plant in which an endogenous FT gene has been replaced with an expression cassette of an exogenous FT gene by genome editing." However, in the above invention, the target gene is not limited to the FT gene, and can be generalized to a flowering induction gene other than the FT gene. The "flowering induction gene" refers to a gene that, when destroyed, prevents the formation of flower buds or delays the formation of flower buds. In addition to the FT gene, the "flowering induction gene" can be a flowering induction gene known as an FT homolog gene. Examples of flowering induction genes known as FT homolog genes include the Hd3a gene derived from rice, the SFT gene derived from tomato, the CiFT gene derived from citrus, and the LEAFY (LFY) gene derived from Arabidopsis thaliana.

[0101] That is, according to the generalized invention, there is provided a method for producing a genetically modified plant, comprising using genome editing technology to replace an endogenous flowering-inducing gene of a target plant with an expression cassette comprising an inducible promoter and an exogenous flowering-inducing gene operably linked to the inducible promoter.Further, according to the generalized invention, there is provided a genetically modified plant modified so that an endogenous flowering-inducing gene of a target plant is replaced with an expression cassette comprising an inducible promoter and an exogenous flowering-inducing gene operably linked to the inducible promoter.

[0102] <8. Preferred embodiment> Preferred embodiments are summarized below. (A1) A method for producing a genetically modified plant, comprising using genome editing technology to replace the endogenous FT gene of a target plant with an expression cassette comprising an inducible promoter and an exogenous FT gene operably linked to the inducible promoter. (A2) The substitution is The expression cassette, a coding region of a guide RNA comprising a sequence complementary to at least a portion of the endogenous FT gene; and The Cas9 gene encoding the Cas9 protein The method according to (A1), which is carried out by introducing into the target plant. (A3) The substitution is The expression cassette, A coding region of a guide RNA comprising a sequence complementary to at least a portion of the endogenous FT gene; a Cas9 gene encoding the Cas9 protein, and A gene encoding a transcription activator that binds to the inducible promoter and activates the transcription of the exogenous FT gene. The method according to (A1), which is carried out by introducing into the target plant.

[0103] (A4) The method according to (A2) or (A3), wherein the guide RNA comprises a sequence complementary to at least a portion of an intron contained in the endogenous FT gene. (A5) The method according to (A4), wherein the exogenous FT gene has the same nucleotide sequence as the coding sequence of the endogenous FT gene.

[0104] (A6) The exogenous FT gene is a nucleotide sequence of the coding sequence of the endogenous FT gene, which satisfies the following conditions: (i) the modified FT gene has been modified so as not to be cleaved by the Cas9 protein (preferably, the modified FT gene has been modified so that a PAM (protospacer adjacent motif) sequence contained in the endogenous FT gene is not recognized by the Cas9 protein); and (ii) the modified FT gene encodes a protein that is identical to the protein encoded by the endogenous FT gene; The method according to (A2) or (A3), wherein the FT gene is modified so as to satisfy the above. (A7) A method according to any one of (A2) to (A6), wherein the guide RNA is a single-stranded RNA comprising a sequence (crRNA) complementary to at least a portion of the endogenous FT gene and a sequence (tracrRNA) that functions as a scaffold for binding the Cas9 protein to the crRNA.

[0105] (A8) The substitution is disrupting the endogenous FT gene of the target plant using a genome editing technique to obtain an endogenous FT gene disruptant; Introducing the expression cassette into the endogenous FT gene disruptant. The method according to (A1), which is carried out by (A9) The substitution is disrupting the endogenous FT gene of the target plant using a genome editing technique to obtain an endogenous FT gene disruptant; introducing into the endogenous FT gene disruptant the expression cassette and a gene encoding a transcription activator that binds to the inducible promoter to activate transcription of the exogenous FT gene; The method according to (A1), which is carried out by

[0106] (B1) A genetically modified plant that has been modified to replace the endogenous FT gene of a subject plant with an expression cassette comprising an inducible promoter and an exogenous FT gene operably linked to the inducible promoter. (B2) the substitution is The expression cassette, a coding region of a guide RNA comprising a sequence complementary to at least a portion of the endogenous FT gene; and The Cas9 gene encoding the Cas9 protein A genetically modified plant according to (B1), which is carried out by introducing into the target plant. (B3) the substitution is The expression cassette, A coding region of a guide RNA comprising a sequence complementary to at least a portion of the endogenous FT gene; a Cas9 gene encoding the Cas9 protein, and A gene encoding a transcription activator that binds to the inducible promoter and activates the transcription of the exogenous FT gene. A genetically modified plant according to (B1), which is carried out by introducing into the target plant.

[0107] (B4) A genetically modified plant according to (B2) or (B3), wherein the guide RNA comprises a sequence complementary to at least a portion of an intron contained in the endogenous FT gene. (B5) A genetically modified plant according to (B4), wherein the exogenous FT gene has the same base sequence as the coding sequence of the endogenous FT gene.

[0108] (B6) The exogenous FT gene is capable of synthesizing a nucleotide sequence of the coding sequence of the endogenous FT gene under the following conditions: (i) the modified FT gene has been modified so as not to be cleaved by the Cas9 protein (preferably, the modified FT gene has been modified so that a PAM (protospacer adjacent motif) sequence contained in the endogenous FT gene is not recognized by the Cas9 protein); and (ii) the modified FT gene encodes a protein that is identical to the protein encoded by the endogenous FT gene; A genetically modified plant according to (B2) or (B3), which is a modified FT gene modified so as to satisfy the above. (B7) A genetically modified plant according to any one of (B2) to (B6), wherein the guide RNA is a single-stranded RNA linking a sequence (crRNA) complementary to at least a portion of the endogenous FT gene and a sequence (tracrRNA) that functions as a scaffold for binding the Cas9 protein to the crRNA.

[0109] (B8) The substitution is disrupting the endogenous FT gene of the target plant using a genome editing technique to obtain an endogenous FT gene disruptant; Introducing the expression cassette into the endogenous FT gene disruptant. A genetically modified plant according to (B1), which is produced by (B9) The substitution is disrupting the endogenous FT gene of the target plant using a genome editing technique to obtain an endogenous FT gene disruptant; introducing into the endogenous FT gene disruptant the expression cassette and a gene encoding a transcription activator that binds to the inducible promoter to activate transcription of the exogenous FT gene; A genetically modified plant according to (B1), which is produced by

[0110] The present invention is not limited to the above-mentioned embodiment, and can be modified in various ways without departing from the gist of the present invention. The embodiments may be combined as appropriate, and in that case, the combined effect can be obtained. Furthermore, the above-mentioned embodiment includes various inventions, and various inventions can be extracted by combinations selected from the multiple components disclosed. For example, if the problem can be solved and the effect can be obtained even if some components are deleted from all the components shown in the embodiment, the configuration from which the components are deleted can be extracted as an invention. EXAMPLES

[0111] [1] Vector creation The vector shown in FIG. 1 was prepared as follows. First, the guide sequence of the designed guide RNA (SEQ ID NO: 9) was introduced into the genome editing vector pKSE401 (addgene). pKSE401 (addgene) already contains the kanamycin resistance gene (SEQ ID NO: 1 to 3), the coding region of tracrRNA (SEQ ID NO: 10), and the Cas9 gene (SEQ ID NO: 2 and 12). Specifically, the introduction of the guide sequence of the guide RNA was performed by treating pKSE401 (addgene) with a restriction enzyme BsaI to prepare a linearized vector, and introducing an oligonucleotide annealed to the linearized vector by ligation. Next, the expression cassette of the exogenous FT gene (drug-inducible construct; containing the base sequences of SEQ ID NO: 4 to 8) and the transcription activator GVG gene (SEQ ID NO: 2, 13, and 14) were linked to the genome editing cassette (containing the base sequences of SEQ ID NO: 9, 10, and 12) by ligation or seamless cloning such as In-Fusion. As a result, the vector shown in FIG. 1 was prepared.

[0112] [2] Introduction of the vector into the target plant The constructed vectors were introduced into the target plant cells using the Agrobacterium method or particle gun method, and plants were regenerated from the plant cells into which the vectors were introduced. Genome editing and homologous recombination were confirmed by PCR analysis or Southern blot analysis of the target regions.

Claims

1. A method for producing a genetically modified plant, comprising using genome editing technology to replace an endogenous flowering-inducing gene of a target plant with an expression cassette containing an inducible promoter and an exogenous flowering-inducing gene operably linked to the inducible promoter.

2. The method of claim 1, wherein the replacement is carried out by introducing into the target plant the expression cassette, a coding region of a guide RNA comprising a sequence complementary to at least a portion of the endogenous flowering-inducing gene, and a Cas9 gene encoding a Cas9 protein.

3. The method according to claim 2, wherein the guide RNA comprises a sequence complementary to at least a portion of an intron contained in the endogenous flowering-inducing gene.

4. The method according to claim 3 , wherein the exogenous flowering-inducing gene has the same base sequence as the coding sequence of the endogenous flowering-inducing gene.

5. The exogenous flowering induction gene is capable of expressing a base sequence of the coding sequence of the endogenous flowering induction gene under the following conditions: (i) The modified flowering induction gene has been modified so as not to be cleaved by the Cas9 protein; and (ii) The modified flowering induction gene encodes the same protein as the protein encoded by the endogenous flowering induction gene. The method according to claim 2, wherein the modified flowering-inducing gene is modified so as to satisfy the above.

6. The method according to claim 1, wherein the replacement is carried out by disrupting the endogenous flowering-inducing gene of the target plant to obtain an endogenous flowering-inducing gene knockout strain, and introducing the expression cassette into the endogenous flowering-inducing gene knockout strain.

7. The method according to any one of claims 1 to 6, wherein the endogenous flowering-inducing gene is an endogenous FT (Flowering Locus T) gene, and the exogenous flowering-inducing gene is an exogenous FT (Flowering Locus T) gene.

8. A genetically modified plant that has been modified so that an endogenous flowering-inducing gene of a target plant is replaced with an expression cassette comprising an inducible promoter and an exogenous flowering-inducing gene operably linked to the inducible promoter.

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