Plant having increased yield of storage tissue and method for producing same

Abstract

The purpose of the present invention is to provide: a plant having an increased yield of storage tissue; and a method for producing the plant. It was discovered that the yield of storage tissue in a plant can be increased by expressing, in the storage tissue of the plant, a sucrose synthase having a substrate specificity to adenosine diphosphate.

Description

Plants with increased yield of storage tissue and methods for producing the same

[0001] The present invention relates to a plant in which the yield of storage tissue has been increased, and to a method for producing the same, and more particularly to a plant in which the yield of storage tissue has been increased by expressing a sucrose synthase having substrate specificity for adenosine diphosphate (ADP) in the storage tissue, and to a method for producing the plant including the expression step.

[0002] Strengthening the productivity of major grains such as rice, wheat, and corn has become increasingly important in recent years due to changes in the surrounding environment, including global population growth, reduced yields due to climate change, and disruptions in distribution due to international conflicts. Similarly, for potatoes and other crops that store starch and become edible, there is a need to increase their yield due to the aforementioned global situation.

[0003] However, the increase in yield of these storage tissues through conventional crossbreeding-based breeding has plateaued in recent years, and the development of unprecedented new breeding techniques is needed.

[0004] Special Publication No. 2007-520228

[0005] Margo Diricks et al., Protein Eng Des Sel., March 1, 2017, Vol. 30, No. 3, pp. 141-148. Carlos M. Figueroa et al., FEBS Lett., January 16, 2013, Vol. 587, No. 2, pp. 165-169. Journal of the Japanese Society for Applied Glycoscience, published August 20, 2014, Vol. 4, No. 3, Abstracts of Presentations at the 63rd Annual Meeting of the Japanese Society for Applied Glycoscience and the Symposium on Applied Glycoscience. General Presentations. Ba-8 Qiyan Jiang et al., Function Integr Genomics, March 2011, Vol. 11, No. 1, pp. 49-61.

[0006] This invention has been made in view of the problems of the prior art described above, and aims to provide a plant with increased yield of storage tissue and a method for producing the same.

[0007] The inventors of this invention have diligently conducted research and studies to achieve the above objectives and have focused on sucrose synthase. In plants, sucrose synthase has substrate specificity for uridine diphosphate (UDP) and catalyzes the synthesis reaction of UDP-glucose (Glc). Furthermore, in plants, UDP-Glc is used to synthesize GIP, GIP is used to synthesize adenosine diphosphate (ADP)-Glc, and ADP-Glc is used to synthesize starch, which is stored in tissues such as endosperm (storage tissue) and becomes grains, etc.

[0008] On the other hand, sucrose synthases from some microorganisms exhibit substrate specificity for ADP rather than UDP (Non-Patent Documents 1-3). In other words, sucrose synthases derived from such microorganisms can directly catalyze the synthesis reaction of ADP-Glc, a substrate for starch biosynthesis. While plants require three steps in the synthesis reaction, ADP-Glc can be synthesized in one step.

[0009] Therefore, the inventors hypothesized that if the substrate specificity of sucrose synthase expressed in plant storage tissues were modified from UDP to ADP, efficient starch biosynthesis would be induced, and the yield of the storage tissues would increase. Based on this hypothesis, the following experiments were conducted using wheat.

[0010] First, we searched the Ensemble genome database for wheat sucrose synthase genes and extracted seven clades based on amino acid sequence homology, namely Wheat sucrose synthase 1 (WSUS1) to WSUS7. Next, we performed tissue-specific genetic analysis on these WSUS genes and identified WSUS3 as a gene specifically expressed in immature seeds during ripening.

[0011] Then, using WSUS3 as the target gene, we attempted to create wheat that expresses WSUS3 in immature seeds in which the substrate specificity of WSUS3 was modified to ADP by introducing an amino acid substitution (A650I / Q651R / M652L / N657S) into the nucleoside diphosphate (NDP) recognition region of the protein encoded by this gene. As a result, we succeeded in creating two genome-edited lines (201-1, 701-3) that express this modified WSUS3.

[0012] Analysis of the seed yields of these two strains revealed, as per the above concept, a tendency for increased seed width and length compared to the wild type was observed, likely reflecting high starch accumulation. A significant increase in seed weight per grain was also observed (1.21 times and 1.26 times compared to the wild type). Furthermore, it became clear that not only seed size and weight, but also the number of seeds per panicle and per plant, which was difficult to predict from high starch accumulation, also significantly increased in these two strains (1.60 times and 1.55 times compared to the wild type). Finally, it was found that the total seed weight, which corresponds to the yield per plant, was approximately twice that of the wild type (1.92 times and 1.94 times), thus completing the present invention.

[0013] In other words, the present invention provides the following embodiments.

[0014] [1] A method for producing a plant with increased storage tissue yield, comprising the step of expressing a sucrose synthase having substrate specificity for adenosine diphosphate (ADP) in the storage tissue of the plant.

[0015] [2] The method according to [1], wherein the nucleoside diphosphate (NDP) recognition region in the sucrose synthase is a region containing the amino acid sequence described in Sequence ID No. 1.

[0016] [3] The method according to [1], wherein the expression is modified to change the substrate specificity of sucrose synthase, which is naturally expressed in plant storage tissues, to ADP.

[0017] [4] The method according to [1], wherein the expression is achieved by modifying the NDP recognition region of sucrose synthase, which is naturally expressed in plant storage tissues, to a region containing the amino acid sequence described in Sequence ID No. 1.

[0018] [5] The method according to any one of [1] to [4], wherein the storage tissue is at least one tissue selected from the group consisting of endosperm, cotyledons, ovary, tuber, rhizome and scales.

[0019] [6] The method according to any one of [1] to [5], wherein the plant whose yield of storage tissue has increased is the plant whose number of storage tissues has increased.

[0020] [7] The method according to any one of [1] to [5], wherein the plant that has increased the yield of storage tissue is the plant that has increased the size and / or weight of storage tissue.

[0021] [8] Plants in which a sucrose synthase with substrate specificity for ADP is expressed in the storage tissue, and in which the yield of the storage tissue is increased.

[0022] [9] The plant according to [8], wherein the NDP recognition region of the sucrose synthase is a region containing the amino acid sequence described in Sequence ID No. 1.

[0023]

[10] The plant according to [8] or [9], wherein the storage tissue is at least one tissue selected from the group consisting of endosperm, cotyledons, ovary, tuber, rhizome and scales.

[0024]

[11] A plant that has an increased number of storage tissues, as described in any one of items [8] to

[10] .

[0025]

[12] A plant according to any one of items [8] to

[10] , wherein the size and / or weight of the storage tissue has increased.

[0026] Regarding WSUS3, it has been suggested that there are nucleotide polymorphisms between varieties, and that these are related to wheat yield (Non-Patent Literature 4). However, in the WSUS3 gene, these nucleotide polymorphisms are completely separate from the sequence encoding the NDP recognition region, and are entirely different from the amino acid substitutions described in the present invention. Furthermore, there is no disclosure or suggestion whatsoever as to how these nucleotide polymorphisms alter the function of WSUS3. In addition, regarding the sucrose synthase gene, a method has been disclosed in which overexpression of the gene increases the starch content in storage tissue (Patent Literature 1). However, there is no disclosure or suggestion whatsoever as increasing not only the amount of storage tissue but also its number by modifying the substrate specificity of sucrose synthase, as in the present invention.

[0027] According to the present invention, it is possible to provide plants with increased storage tissue yield. According to the present invention, by expressing sucrose synthase having substrate specificity for ADP in the storage tissue of plants, it is possible not only to increase the size and weight of the storage tissue but also to increase its number, and consequently to significantly increase the total weight of the storage tissue in the plant.

[0028] This figure shows a phylogenetic tree classifying sucrose synthase genes from plants (monocotyledonous and dicotyledonous) based on amino acid sequence homology. The sucrose synthase from Thermosynechococcus elongatus (RefSeq ID: BAC08600.1, SEQ ID NO: 135) is also shown in this phylogenetic tree. This figure shows the results of alignment analysis of the NDP recognition regions of sucrose synthases from plants (monocotyledonous and dicotyledonous). The sucrose synthase from Thermosynechococcus elongatus (RefSeq ID: BAC08600.1, SEQ ID NO: 135) is also shown in this alignment. The sequences shown in the figure are the amino acid sequences from positions 635 to 672 of WSUS3 (Sequence IDs: 70-72) and the sequences corresponding to the aforementioned amino acid sequences in Sequence IDs: 64-69 and 73-135. A phylogenetic tree is shown classifying wheat-derived sucrose synthases based on amino acid sequence homology. For comparison, the sequence of rice sucrose synthase (OsSUS) is also shown. The graph shows the results of analyzing the expression of wheat-derived sucrose synthase genes (WSUS1-WSUS7) in various tissues. In the figure, the vertical axis represents the relative expression level of each WSUS gene, with the expression level in the root set to 1. In the horizontal axis notation, "Endosperm1" to "Endosperm3" represent the endosperm at 7 days after flowering, 14 days after flowering, and 21 days after flowering, respectively. This photograph shows the results of measuring and comparing the width (short axis) and length (long axis) of wheat genome-edited lines (201-1, 701-3) in which the substrate specificity of the NDP recognition region of WSUS3 was modified to ADP, and wild-type (WT) wheat. This box plot shows the results of measuring and comparing the number of seeds per plant (total number of seeds), weight per grain, and total weight of seeds per plant for genome-edited lines (201-1, 701-3) and wild-type (WT). The number of plants analyzed was 8 for each, and their average values ​​are indicated by the "X" marks in the box plots. Furthermore, for "a" and "b", it is indicated that there is a significant difference (p < 0.01) between different letters. This graph shows the results of analyzing the number of effective tillers for genome-edited lines (201-1, 701-3) and wild-type (WT).The number of individuals analyzed was 12 for each group, and the average values ​​are shown in each bar graph. The bars on each graph indicate the standard error. This figure shows the results of analyzing panicles in genome-edited lines (201-1, 701-3) and wild-type (WT). In the figure, (a) shows a photograph showing the results of observing each panicle, and (b) is a box plot showing the results of analyzing the number of spikelets per panicle. In (b), the number of panicles analyzed was 18 for wild-type (WT), 17 for genome-edited line (201-1), and 18 for genome-edited line (701-3), and the average values ​​are shown by the cross marks in the box plots. The dots on each box indicate outliers. The notation "a" and "b" indicate a statistically significant difference (p < 0.01) between different letters. (c) is a graph showing the results of analyzing the ratio of fertile seeds per spikelet. In (c), the number of spikelets for each analysis was 265 for the wild type (WT), 318 for the genome-edited line (201-1), and 324 for the genome-edited line (701-3).

[0029] <Method for producing plants with increased storage tissue yield> As shown in the examples described below, it has become clear that by expressing sucrose synthase, which has substrate specificity for adenosine diphosphate (ADP), in the storage tissue of plants, it is possible not only to increase the size and weight of the storage tissue, but also to increase its number, and consequently to significantly increase the total weight of the storage tissue in the plant. Therefore, the present invention relates to a method for producing plants with increased storage tissue yield, which includes the step of expressing sucrose synthase, which has substrate specificity for ADP, in the storage tissue of plants.

[0030] In this invention, "storage tissue" refers to tissue that stores (accumulates) nutrients within the plant body, and is also called nutrient storage tissue. In this invention, it particularly refers to tissue that stores at least starch (starch storage tissue), but it may also be tissue that stores other nutrients (sugars, fats, proteins, inulin, mannan, etc.) or other substances (water, etc.) in addition to starch. There are no particular limitations on such storage tissue, but examples include seeds, stems, roots, and leaves, and more specifically, endosperm (in endospermic seeds), cotyledons (in non-endospermic seeds), ovary (fruit), tuber, rhizome, and scale leaves.

[0031] In the present invention, "storage tissue yield" may refer not only to the total weight of storage tissue per plant, but also to at least one numerical value selected from the group consisting of the number of storage tissues per plant, the size of one storage tissue, and the weight of one storage tissue that contribute to it. Furthermore, in the present invention, "weight" may refer to fresh weight (raw weight) or dry weight. "Size" may refer to at least one numerical value selected from the group consisting of the long axis, short axis, and thickness of the storage tissue, or it may refer to the area and volume obtained from these. In the present invention, "increased storage tissue yield" means that the aforementioned storage tissue yield has increased compared to plants that do not express sucrose synthase having substrate specificity for ADP in their storage tissue, for example, being 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, or 2 times or more compared to the aforementioned plants.

[0032] In the present invention, there are no particular restrictions on the plants that are targeted for increased yield of storage tissue, as long as they possess such tissue, but examples include seed plants. Seed plants include angiosperms and gymnosperms, and angiosperms include monocots and dicots, and the present invention can target these plants.

[0033] "Monocotyledonous plants" can be any type, but examples include grasses, lilies, bananas, pineapples, and orchids. Examples of "grasses" include wheat, barley, rice, corn, oats, grass, sorghum, rye, millet, and sugarcane. Examples of "lilies" include onions and asparagus. Examples of "bananas" include bananas. Examples of "pineapples" include pineapples.

[0034] Examples of "dicotyledonous plants" include Brassicaceae, Fabaceae, Solanaceae, Cucurbitaceae, Convolvulaceae, Rosaceae, Moraceae, Malvaceae, Asteraceae, Amaranthaceae, and Polygonaceae. Examples of "Brassicaceae" include Arabidopsis thaliana, Chinese cabbage, rapeseed, cabbage, cauliflower, and radish. Examples of "Fabaceae" include soybeans, adzuki beans, kidney beans, peas, cowpeas, and alfalfa. Examples of "Solanaceae" include tomatoes, eggplants, potatoes, tobacco, and chili peppers. Examples of "Cucurbitaceae" include cantaloupes, cucumbers, melons, and watermelons. Examples of "Convolvulaceae" include morning glories, sweet potatoes, and bindweed. Examples of "Rosaceae" include roses, strawberries, and apples. Examples of plants in the Moraceae family include mulberry, fig, and rubber tree. Examples of plants in the Malvaceae family include cotton and kenaf. Examples of plants in the Asteraceae family include lettuce. Examples of plants in the Amaranthaceae family include sugar beet. Examples of plants in the Polygonaceae family include buckwheat.

[0035] Examples of "gymnosperms" include pine, cedar, ginkgo, and cycad.

[0036] Furthermore, the plants used in this invention may be wild species or cultivated species. In addition, these plants may be genetically modified or genome-edited (for example, disease-resistant crops, herbicide-resistant crops, insect-resistant crops, crops with improved taste, crops with improved storage life, or crops with improved yield).

[0037] In the present invention, the expression of sucrose synthase having substrate specificity for ADP can be achieved by modifying the substrate specificity of sucrose synthase that is constitutively expressed in the storage tissues of plants to ADP. The sucrose synthase whose substrate specificity is modified in this way is not particularly limited, but is preferably a sucrose synthase derived from plants. Examples thereof include genes derived from monocotyledonous plants (wheat, barley, corn, rice) and dicotyledonous plants (Arabidopsis thaliana, buckwheat, potato, soybean) shown in the following table.

[0038]

[0039]

[0040]

[0041] In Table 1, the CDS and genomic nucleotide sequences of sucrose synthase derived from wheat, and the amino acid sequences of the proteins encoded thereby are shown by the indicated sequence numbers. In Table 2, the amino acid sequences of sucrose synthase derived from other monocotyledonous plants (barley, corn, rice) are shown by the indicated sequence numbers. In Table 3, the amino acid sequences of sucrose synthase derived from dicotyledonous plants (Arabidopsis thaliana, buckwheat, potato, soybean) are shown by the indicated sequence numbers.

[0042] It should be noted that mutations in nucleotide sequences can occur in nature. Consequently, the encoded amino acids can also change. Furthermore, the same protein derived from plants belonging to the same family exhibits high amino acid sequence identity. Therefore, the sucrose synthase modified in the present invention to have substrate specificity for ADP is not limited to the above-mentioned typical sequences, but may include amino acid sequences that have high homology (high similarity), preferably high identity, with these (for example, the amino acid sequences described in SEQ ID NOs: 70-72 (WSUS3)). Here, "high" means at least 40% (for example, 46% or more), more preferably 50% or more, even more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and even more preferably 85% or more (for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more).

[0043] Sequence homology can be determined using the BLAST program (Altschul et al. J. Mol. Biol., 215:403-410, 1990). This program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). For example, when analyzing amino acid sequences using BLAST, the parameters are, for example, score=50 and wordlength=3. Furthermore, when analyzing amino acid sequences using the Gapped BLAST program, the procedure can be carried out as described by Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using BLAST and the Gapped BLAST program, the default parameters of each program should be used. The specific methods for these analysis techniques are publicly known.

[0044] Furthermore, Figure 1 shows a phylogenetic tree classifying these sucrose synthases based on the homology of their amino acid sequences. Figure 1 also shows the amino acid sequence alignment of the nucleoside diphosphate (NDP) recognition regions of these sucrose synthases.

[0045] In the present invention, a sucrose synthase modified to have substrate specificity for ADP is more preferably a sucrose synthase in which the nucleoside diphosphate (NDP) recognition region is a region containing the amino acid sequence described in XQXXXXXXN (in the sequence, the first X represents A or S, the next X represents M or T, and the other X represents any amino acid), even more preferably a sucrose synthase in which the NDP recognition region is a region containing the amino acid sequence described in AQMXXXXXXN (Sequence ID: 3, in the sequence, X represents any amino acid), and even more preferably a sucrose synthase in which the nucleoside diphosphate (NDP) recognition region is a region containing the amino acid sequence described in AQMNRVRN (Sequence ID: 4). More specifically, as such sucrose synthase, examples include sucrose synthases derived from monocotyledonous plants belonging to the clade containing WSUS2, the clade containing WSUS3, or the clade containing WSUS1, as shown in Figure 1. In the NDP recognition region, XQXXXXXXN, AQMXXXXN (Sequence ID: 3), and AQMNRVRN (Sequence ID: 4) correspond to positions 650-657 of WSUS3 (described in Sequence IDs: 70-72) in sucrose synthase. Furthermore, in the present invention, it is desirable that the sucrose synthase modified to have substrate specificity for ADP is expressed in the above-mentioned storage tissue.

[0046] In the present invention, a modified wild-type sucrose synthase (sucrose synthase variant) is used in which the substrate specificity of the wild-type sucrose synthase is made to ADP. Such a modification may include, for example, the substitution of the sequence corresponding to positions 650-657 of WSUS3 (described in SEQ ID NOs: 70-72) in the NDP recognition region with the amino acid sequence described in any of SEQ ID NOs: 1-3. Among these, substitution with the amino acid sequence described in SEQ ID NO: 1 is preferred from the viewpoint of originating from an organism that has photosynthetic activity similar to higher plants.

[0047] In the present invention, "corresponding" means a relationship in which the amino acid sequence of WSUS3 (described in SEQ ID NOs: 70-72) and the corresponding amino acid sequence (such as the amino acid sequence of another sucrose synthase) are aligned (applied to alignment) using nucleotide and amino acid sequence analysis software (such as GENETYX, Sequencher, etc.) or BLAST (http: / / blast.ncbi.nlm.nih.gov / Blaster.cgi), and are in the same order.

[0048] The amino acid sequence (IRLXXXXXS) described in Sequence ID No. 1 is derived from the NDP recognition region of sucrose synthase (RefSeq ID: BAC08600.1, Sequence ID No. 135) of cyanobacteria (Thermosynethococcus elongatus). The amino acid sequence (ALLXXXXA) described in Sequence ID No. 2 is derived from the NDP recognition region of sucrose synthase of Acidithiobacillus caldus.

[0049] In sequence numbers 1 to 3, amino acids other than those corresponding to positions 650, 651, 652, and 657 of WSUS3 are "X", i.e., any amino acid. For example, the amino acid corresponding to position 653 of WSUS3 could be N, D, E, A, or P. For example, the amino acid corresponding to position 654 of WSUS3 could be a basic amino acid (R, K, etc.) or D. For example, the amino acid corresponding to position 653 of WSUS3 could be N, an acidic amino acid (D, E), A, or P. For example, the amino acid corresponding to position 654 of WSUS3 could be a basic amino acid (R, K, etc.) or D. For example, the amino acid corresponding to position 655 of WSUS3 could be V, A, H, Y, or T. For example, the amino acid corresponding to position 656 of WSUS3 could be R, D, or V. Furthermore, the table below shows specific examples of the sequences of the regions corresponding to positions 653 to 656 of WSUS3.

[0050]

[0051] The amino acid sequence described in NRVR (Sequence ID: 5) corresponds to the sequence at positions 653-656 of WSUS3. The amino acid sequences described in Sequence IDs: 6-19 are plant-derived sequences corresponding to positions 653-656 of WSUS3 in the sucrose synthases shown in Tables 1-3 or Figure 2. Furthermore, the amino acid sequence described in PKAD (Sequence ID: 20) corresponds to the sequence at positions 653-656 of WSUS3 in the sucrose synthase from Thermosynechococcus elongatus, and the amino acid sequence described in DKTV (Sequence ID: 21) corresponds to the sequence at positions 653-656 of WSUS3 in the sucrose synthase from Acidithiobacillus caldus.

[0052] Furthermore, the substrate specificity being ADP can be determined, for example, by measuring the reaction rate in an enzyme activity test, as shown in the examples described later. More specifically, the kcat / Km of the modified sucrose synthase for ADP-glucose is preferably 1 s. －１ mM －１as described above, more preferably 2 s －１ mM －１ as described above, more preferably 5 s －１ mM －１ as described above, more preferably 7 s －１ mM －１ as described above, more preferably 9 s －１ mM －１ as described above. Further, as the sucrose synthase variant according to the present invention, the kcat / Km for ADP-glucose is preferably 2-fold or more, more preferably 5-fold or more, further preferably 10-fold or more, more preferably 20-fold or more, and further preferably 23-fold or more, as compared with the sucrose synthase before modification (for example, wild type). On the other hand, the kcat / Km for UDP-glucose of the sucrose synthase variant according to the present invention is preferably 0.5-fold or less, more preferably 0.25-fold or less, further preferably 0.1-fold or less, more preferably 0.05-fold or less, more preferably 0.025-fold or less, further preferably 0.01-fold or less, more preferably 0.005-fold or less, and more preferably 0.0025-fold or less, as compared with the sucrose synthase before modification (for example, wild type).

[0053] As described above, the expression of sucrose synthase having substrate specificity for ADP in storage tissues can be achieved by modifying the substrate specificity of sucrose synthase that is constitutively expressed in storage tissues to ADP. For example, it can be achieved by introducing a mutation involving the above amino acid substitution into the sucrose synthase gene that is constitutively expressed in storage tissues. The introduction of such a mutation can be achieved by methods known to those skilled in the art. Known methods for introducing mutations include, but are not limited to, genome editing methods, homologous recombination methods, physical mutation introduction methods, methods using chemical mutagens, and methods for introducing transposons etc. into genomic DNA.

[0054] Genome editing is a method of modifying target genes using site-specific nucleases (for example, zinc finger nucleases (ZFNs), transcription-activating effector nucleases (TALENs), and DNA double-strand cleavage enzymes such as CRISPR-Cas). For example, fusion proteins such as ZFNs (US Patents 6,265,196, 8,524,500, 7,888,121, European Patent No. 1,720,995), TALENs (US Patents 8,470,973, 8,586,363), PPR (pentatricopeptiderepeat) with a fused nuclease domain (Nakamura et al., Plant Cell Physiol 53:1171-1179 (2012)), CRISPR-Cas9 (US Patent No. 8,697,359, International Publication 2013 / 176772), CRISPR-Cpf1 (Zetsche B. et al.) Examples include using guide RNA-protein complexes, or protein complexes, such as K. Nishida et al., Cell, 163(3):759-71, (2015) or Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokalyotic and vertebrate adaptive immunosystems, Science, DOI:10.1126 / science.aaf8729, (2016)).

[0055] There are no particular restrictions on the "Cas enzyme," and it can be appropriately selected according to the purpose. More specifically, it can be appropriately selected and used from class 1 CRISPER-related enzymes (e.g., type I and type IV such as Cas3, type III such as Cas10), class 2 CRISPER-related enzymes (e.g., type II such as Cas9, Cas12 (Cas12a (Cpf1), Cas12b (C2c1), Cas12e (CasX), and Cas12f1), type V such as Cas14, type VI such as Cas13), etc.

[0056] Furthermore, as shown in the examples described later, genome editing can also be used in combination with donor DNA containing a sequence encoding a desired amino acid mutation (for example, DNA in which homology arms corresponding to the endogenous sucrose synthase gene are positioned at both ends of the sequence encoding the desired amino acid substitution) to introduce a mutation into the endogenous sucrose synthase gene via homologous recombination (SDN-2 type genome editing). The length of such homology arms can be any number that allows homologous recombination to occur, for example, 50 to 200 nucleotides.

[0057] Furthermore, regarding genome editing methods, methods that do not involve double-strand breaks can also be used in this invention. For example, a method that modifies only one base in a target gene (base editing) can be used, using a mutant Cas that lacks nuclease activity but has a deaminase attached, and a guide RNA. In addition, a method that modifies a base or inserts a DNA sequence in a target gene (prime editing) can also be used in this invention, using a mutant Cas that lacks double-strand break ability but has a reverse transcriptase attached, and a guide RNA (pegRNA) to which the sequence of the primer binding site and the template sequence of the reverse transcriptase have been attached.

[0058] In the "homologous recombination method," by using a donor sequence containing a sequence encoding a desired amino acid mutation (for example, a vector in which homology arms corresponding to an endogenous sucrose synthase gene are positioned at both ends of a sequence encoding a desired amino acid substitution), the endogenous sucrose synthase gene can be converted into a gene encoding sucrose synthase into which the target mutation has been introduced through homologous recombination. The length of such homology arms can be any number that allows homologous recombination to occur, for example, 500 to 7000 nucleotides (preferably 1000 to 5000 nucleotides, more preferably 2000 to 4000 nucleotides).

[0059] Examples of "physical mutation induction methods" include heavy ion beam (HIB) irradiation, fast neutron irradiation, gamma ray irradiation, and ultraviolet irradiation (see Hayashi et al., Cyclotrons and Their Applications, 2007, 18th International Conference, pp. 237-239, and Kazama et al., Plant Biotechnology, 2008, Vol. 25, pp. 113-117).

[0060] Examples of "methods using chemical mutagens" include methods of treating seeds, etc., with chemical mutagens (see Zwar and Chandler, Planta, 1995, Vol. 197, pp. 39-48, etc.). There are no particular restrictions on the chemical mutagens, but examples include N-methyl-N-nitrosourea (MNU), ethylmethanesulfate (EMS), N-ethyl-N-nitrosourea (ENU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitroguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrite, formic acid, and nucleotide analogs.

[0061] "Methods for introducing transposons, etc., into genomic DNA" include, for example, T ＯＳ Examples include methods for inserting transposons such as 17, T-DNA, etc., into the plant's genomic DNA (see Kumar et al., Trends Plant Sci., 2001, Vol. 6, No. 3, pp. 127-134, and Tamara et al., Trends in Plant Science, 1999, Vol. 4, No. 3, pp. 90-96).

[0062] In plants into which mutations have been introduced using the methods described above, the presence of mutations in the sucrose synthase gene can be confirmed by known methods. Examples of known methods include DNA sequencing (next-generation sequencing, etc.), PCR, microarray analysis, and Southern blotting. Using these methods, it is possible to determine whether or not a mutation has been introduced into the sucrose synthase gene by comparing the sequence of the sucrose synthase gene before and after the introduction of the mutation.

[0063] Another method for confirming the introduction of mutations into the sucrose synthase gene is TILLING (Targeting Induced Local Lessons in Genomes) (see Slade et al., Transgenetic Res., 2005, Vol. 14, pp. 109-115, and Comai et al., Plant J., 2004, Vol. 37, pp. 778-786). In particular, when non-selective mutations are introduced into the plant genome using heavy ion beam irradiation or chemical mutagens, the sucrose synthase gene or a part thereof can be amplified by PCR, and then individuals with mutations in the amplified product can be selected by TILLING or the like.

[0064] Furthermore, by crossing a plant into which a mutation has been introduced using the method described above with a wild-type plant and then performing a backcross, it is possible to remove mutations introduced into sequences other than the target sucrose synthase gene.

[0065] Plants into which a mutation has been introduced into the sucrose synthase gene may be heterozygotes (for example, heterozygotes consisting of a wild type and a sucrose synthase gene mutant) or homozygotes, as long as the yield of storage tissue increases, as shown in the examples described below. Homozygotes can be selected, for example, by crossing the heterozygotes with each other to obtain F1 plants, and then selecting homozygotes from the F1 plants that have the mutated sucrose synthase gene. Furthermore, in polyploid plants such as wheat, as shown in the examples described below, at least one of the multiple alleles may be heterozygote or homozygote. Moreover, all of the multiple alleles may be heterozygote or homozygote.

[0066] In the present invention, the introduction of mutations into the sucrose synthase gene can be carried out in plants, seeds, or plant cells according to the methods described above. Plant cells include cultured cells as well as cells within the plant body. Furthermore, various forms of plant-derived cells are included, such as shoot apex, suspension culture cells, protoplasts, leaf sections, callus, immature embryos, pollen, etc.

[0067] Furthermore, in the present invention, the above-mentioned site-specific nucleases, fusion proteins, or DNA encoding a complex of guide RNA and protein, DNA encoding a transposon, etc., may be introduced into plant cells in a form inserted into a vector.

[0068] The vector into which the DNA for introducing a mutation into the sucrose synthase gene is inserted is not particularly limited as long as it is capable of expressing the inserted gene in plant cells, but it may contain a promoter for constitutive or inductive expression of the DNA. Examples of promoters for constitutive expression include the rice ubiquitin promoter, the cauliflower mosaic virus 35S promoter, the rice actin promoter, and the maize ubiquitin promoter. Examples of promoters for inductive expression include promoters known to be expressed by external factors such as infection or invasion by filamentous fungi, bacteria, or viruses, low temperature, high temperature, drought, ultraviolet irradiation, and spraying of specific compounds. Furthermore, as a promoter for expressing DNA encoding a short RNA such as guide RNA as the DNA of the present invention, poll III-type promoters such as the U6 promoter are preferably used.

[0069] As a method for introducing the DNA or a vector into which the DNA is inserted into plant cells, various methods known to those skilled in the art can be used, such as particle bombardment, the Agrobacterium method (Agrobacterium method), the polyethylene glycol method, and electroporation. Furthermore, as shown in the examples described below, by using genome editing technology that directly introduces a genome editing system into the meristem tissue such as the shoot apex of a plant (in-plant particle bombardment (iPB) method), it becomes possible to produce plants with desired mutations introduced into various plants without the need for cultivation (Japanese Patent Publication No. 2017-205104, Japanese Patent Publication No. 2022-058497 (Patent No. 7236121), Japanese Patent Publication No. 2023-056018 (Patent No. 7321477)).

[0070] Furthermore, even without taking the form of DNA, the aforementioned site-specific nucleases, fusion proteins, and transposons can introduce mutations into plant cells as proteins, and the aforementioned guide RNAs can introduce mutations as RNA.

[0071] Thus, in the present invention, the yield of plant storage tissue can be increased by using substances that target the sucrose synthase gene, such as the DNA, the vector into which the DNA is inserted, the protein, and the RNA. Accordingly, the present invention may also provide a drug for increasing the yield of plant storage tissue, which contains as an active ingredient at least one substance that targets the sucrose synthase gene, selected from the group consisting of the DNA, the vector into which the DNA is inserted, the protein, and the RNA.

[0072] Such a drug may be configured to contain two active ingredients in a single composition, or it may be configured to contain two active ingredients in separate compositions (a so-called kit). In addition, the drug of the present invention may contain other components such as buffer solutions, stabilizers, preservatives, and antiseptics in addition to the above-mentioned substances.

[0073] Furthermore, by regenerating a plant from cells into which a mutation has been introduced into the sucrose synthase gene according to the present invention using the methods described above, it is possible to obtain a plant with increased storage tissue yield.

[0074] For example, methods for producing transgenic plants related to wheat include those described by Tingay et al. (Tingay S. et al. Plant J. 11:1369-1376, 1997), Murray et al. (Murray F et al. Plant Cell Report 22:397-402, 2004), Travalla et al. (Travalla S et al. Plant Cell Report 23:780-789, 2005), Vasil et al. (Vasil V. et al. Nat Biotechnology 10:667-674, 1992), and Ishida et al. (Ishida Y. et al. Methods in The method described in Molecular Biology 1223:189-193, 2015) can be cited.

[0075] For maize, examples include the methods described by Shillito et al. (Bio / Technology, 7:581, 1989) and Golden-Kamm et al. (Plant Cell 2:603, 1990).

[0076] For example, in rice, methods for producing transgenic plants include a method of regenerating plants by introducing genes into protoplasts using polyethylene glycol (Datta, S.K. In Gene Transfer To Plants (Potrykus I and Spagenberg Eds.) pp66-74, 1995), a method of regenerating plants by introducing genes into protoplasts using electrical pulses (Toki et al. Plant Physiol. 100, 1503-1507, 1992), a method of regenerating plants by directly introducing genes into cells using the particle gun method (Christou et al. Bio / technology, 9:957-962, 1991), and a method of regenerating plants by introducing genes via Agrobacterium (Hiei et al. Several techniques have already been established and are widely used in the technical field of the present invention, such as al. Plant J. 6:271-282, 1994.

[0077] Suitable methods for regenerating sorghum plants include, for example, the Agrobacterium method or the particle gun method, which involves gene transfer into immature embryos or callus to regenerate the plant, and pollination using pollen genetically modified by ultrasound (J.A. Able et al., In Vitro Cell. Dev. Biol. 37:341-348, 2001; A.M. Casas et al., Proc. Natl. Acad. Sci. USA 90:11212-11216, 1993; V. Girijashankar et al., Plant Cell Rep 24:513-522, 2005; J.M. JEOUNG et al., Hereditas 137:20-28, 2002, V Girijashankar et al. , Plant Cell Rep 24(9):513-522, 2005, Zuo-yu Zhao et al. , Plant Molecular Biology 44:789-798, 2000, S. Gurel et al. , Plant Cell Rep 28(3):429-444, 2009, ZY Zhao, Methods Mol Biol, 343:233-244, 2006, AK Shrawat and H Lorz, Plant Biotechnol J, 4(6): 575-603, 2006, D Syamala and P Devi Indian J Exp Biol, 41(12): 1482-1486, 2003, Z Gao et al. , Plant Biotechnol J, 3(6):591-599, 2005).

[0078] For soybeans, one example is the method described in the U.S. Patent Publication No. 5,416,011.

[0079] For tomatoes, examples include the methods described by Matsukura et al. (J. Exp. Bott., 44: 1837-1845, 1993), Sun et al. (Plant cell physics, 2006; 47(3): 426-431), and Sonia Hamza et al. (J. Exp. Bott., 44: 1837-1845, 1993).

[0080] For Arabidopsis thaliana, methods include the floral dip method (Clough SJ & Bent AF, Plant J 16:735-743, 1998) and the method by Akama et al. (Akama et al. Plant Cell Reports 12:7-11, 1992).

[0081] Furthermore, transformation and regeneration into plants can be carried out using methods such as those described in Tabei et al. (ed., Yutaka Tabei, "Transformation Protocols [Plant Edition]", Kagaku Dojin Co., Ltd., published September 20, 2012).

[0082] While preferred embodiments of the present invention have been described above, the manufacturing method of the present invention is not limited to the above embodiments. For example, the expression of sucrose synthase having substrate specificity for ADP in plant storage tissue is not limited to modifying the substrate specificity of the endogenous sucrose synthase to ADP as described above, but can also be achieved by exogenously introducing DNA encoding sucrose synthase having substrate specificity for ADP. Such introduction of an exogenous gene can be achieved, for example, by inserting DNA encoding sucrose synthase into a vector and introducing it into plant cells. The vector may contain a promoter for constitutive or inducible expression, similar to the DNA encoding site-specific nucleases described above. Furthermore, storage tissue-specific promoters can also be used in such vectors. The "storage tissue-specific promoter" can be any sequence that allows the gene linked downstream to be expressed in each storage tissue, and examples include seed-specific promoters such as the WSUS3 gene endosperm-specific promoter, the 27kD gammazein promoter, and the Waxy promoter. Furthermore, the introduction of the sucrose synthase into plant cells can be carried out appropriately using methods known to those skilled in the art, similar to the aforementioned site-specific nuclease-encoding DNA. Examples of "sucrose synthase having substrate specificity for ADP" include the aforementioned sucrose synthase variants modified to have substrate specificity for ADP in vitro, and sucrose synthase derived from microorganisms that are naturally substrate-specific for ADP (e.g., Thermosynechococcus elongatus, Acidithiobacillus caldus).

[0083] (Plants with increased storage tissue yield) By the methods described above, plants with increased storage tissue yield can be obtained. Therefore, this relates to plants with increased storage tissue yield in which sucrose synthase having substrate specificity for ADP is expressed in the storage tissue.

[0084] As described above, the sucrose synthase according to the present invention and the plants in which the yield of storage tissue increases due to the expression of said sucrose synthase are as described above. Furthermore, once a plant body in which the yield of storage tissue has increased due to the artificial introduction of a mutation in the sucrose synthase gene is obtained, it is possible to obtain offspring from said plant body through sexual or asexual reproduction. Moreover, it is possible to obtain reproductive materials (e.g., seeds, cuttings, stems, callus, protoplasts, etc.) from said plant body, its offspring, or clones, and mass-produce said plant bodies based on these. Therefore, the present invention includes offspring and clones of plants in which the yield of storage tissue has increased, as well as their reproductive materials. Examples of reproductive materials include seeds, stems, callus, and protoplasts.

[0085] The present invention will be described more specifically below based on examples, but the present invention is not limited to the following examples. Furthermore, these examples were carried out using the following materials and methods.

[0086] (Experimental material and search for sucrose synthase genes) The wheat variety "Norin 61" was used as the experimental material. First, using the Ensembl genome database (https: / / plants.ensembl.org / index.html), genes encoding sucrose synthase were searched for in the genome information of Norin 61, and 21 candidate genes were found. The proteins encoded by these genes were classified into seven clades based on the homology of their amino acid sequences, and it was thought that many of the genes within each clade were related as homogeneous genes located in subgenomes A, B, and D (Figure 3). These groups of homogeneous genes were named from Wheat sucrose synthase 1 (WSUS1) to Wheat sucrose synthase 7 (WSUS7).

[0087] (Gene Expression Analysis) Next, gene expression analysis was performed on each WSUS gene in different tissues. Dried seeds were sown in mixed potting soil (Hokusan Co., Ltd.) and grown in an artificial climate chamber. The environmental conditions in the artificial climate chamber were set to light period (16h, 22°C) / dark period (8h, 16°C). RNA extracted from roots, leaf sheaths, and leaf blades at 21 days after germination, main stems, flag leaves, and immature seeds at 7 days after flowering, immature seeds at 14 days after flowering, and immature seeds at 21 days after flowering was used for gene expression analysis. RNeasy Plant Mini Kit (Qiagen) was used for total RNA extraction, and PrimeScript RT reagent kit (Takara) was used for single-stranded cDNA synthesis. Real-time PCR expression analysis was performed using cDNA diluted 10-fold with nuclease-free water as a template. The PCR enzyme used was TB Green Premix Ex Taq II (Takara), and the thermal cycler was the AriaMx Real-time PCR System (Agilent). The PCR conditions consisted of 30 cycles of denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, and extension at 68°C for 5 seconds. The primer sequences used are shown in Table 5 below.

[0088]

[0089] (Determination of target gene) Based on the results of the gene expression analysis described above, WSUS3 was identified as a gene specifically expressed in immature seeds during ripening (Figure 4). The WSUS3 gene was selected as the target gene for genome editing.

[0090] (Cloning of the WSUS3 gene) Using cDNA prepared from immature seeds as a template, the CDS of the WSUS3 gene (WSUS-B3) located in the B subgenome was cloned. PrimeSTAR GXL (Takara Corporation) was used as the PCR enzyme, and the PCR conditions were 98°C for 10 seconds of heat denaturation, 60°C for 15 seconds of annealing, and 68°C for 3 minutes of extension, repeated for 30 cycles. The following sequences were used as the primer set: WSUS-B3_Full_Fw1:5'-ATGACTGCTTCCCAAAAGCAC-3' (Sequence ID: 152) and WSUS-B3_Full_Rv1:5'-TCATTTGCCCGAGGTCTCCG-3' (Sequence ID: 153). The augmented byproduct was then introduced into the pCR Blunt II-TOPO vector included with the Zero Blunt TOPO PCR Cloning Kit (Invitrogen).

[0091] (Detection of substrate specificity modification of WSUS3 by enzyme activity test) From previous research by the present inventors, it was expected that introducing amino acid substitutions into the sugar nucleotide recognition motif of the WSUS3 protein would change its substrate specificity from UDP to ADP. Therefore, this was confirmed by an in vitro enzyme activity test. WSUS-B3 in the pCR Blunt II-TOPO vector was amplified by PCR and introduced into pET-23a. Using this plasmid DNA as a template, base substitutions were introduced using the Primestar mutagenesis basal kit (Takara Bio Inc.). The base substitutions were made so that the amino acid sequence of the WSUS-B3 protein became A650I / Q651R / M652L / N657S. Expression plasmids of the wild-type enzyme and the mutant enzyme were introduced into E. coli strain BL21 (DE3), respectively, to produce recombinant enzymes. Each transformant was cultured in 1 L of LB medium at 37°C. When A600 reached 0.5, 1 mL of 0.1 M IPTG was added and the cells were cultured at 18°C ​​for 24 hours. The cells were recovered by centrifugation, disrupted by sonication, and then centrifuged again to obtain the supernatant. Recombinant proteins were purified from this supernatant by nickel affinity column chromatography using chelating Sepharose fast flow. In the enzyme activity test, the reaction rate to 50–1000 μM UDP or ADP in the presence of 125 mM sucrose was measured at 30°C. The reaction buffer was 1 mM MgCl ２ The solution used was a 100 mM MES-NaOH buffer (pH 6.5) containing [the substance]. The fructose produced was quantified using F-Kit D-glucose / fructose. The determined reaction rate was regression-regressed to the Michaelis-Menten equation to obtain the rate parameters (kcat and Km). As a result, it was revealed that in the modified WSUS-B3 with the amino acid substitution (A650I / Q651R / M652L / N657S), the kcat / Km for UDP-glucose was 0.00250 times lower and the kcat / Km for ADP-glucose was 23.5 times higher compared to the wild-type WSUS-B3 (Table 6).

[0092]

[0093] (Design of guide RNA and donor DNA for genome editing) Based on the results of enzyme activity tests, it was decided to introduce four amino acid substitutions (A650I / Q651R / M652L / N657S) into the WSUS3 protein of Norin 61 by genome editing. CRISPR / Cas9 was used as the genome editing tool, and the desired base substitutions were to be introduced via homologous recombination by using donor DNA in combination. Target sequences for CRISPR / Cas9 (Target 1 and Target 2) were designed near the sugar nucleotide recognition motif coding sequence within the WSUS3 gene. Donor DNA (Donor 1 and Donor 2) corresponding to each target sequence was also designed. Donor 1 is a single-stranded DNA homologous to the antisense strand of WSUS-B3, and Donor 2 is a 3′-end overhang DNA homologous to WSUS-B3. For each donor DNA, base substitutions corresponding to four amino acid substitutions (A650I / Q651R / M652L / N657S) were designed in the sugar nucleotide recognition motif coding sequence. In addition, multiple synonymous substitutions were designed in the CRISPR / Cas9 target sequence to prevent the genome-edited chromosome from being re-cleaved by CRISPR / Cas9.

[0094] (Preparation of shoot apical samples for genome editing) For genome editing, the in plant Particle Bombardment (iPB) method developed by the National Agriculture and Food Research Organization (NARO) and Kaneka Corporation was used. Shoot apical samples were prepared according to the standard iPB method. Dried seeds of Norin 61 were shaken in a 20% sodium hypochlorite aqueous solution for 30 minutes to sterilize the seed surface. A paper towel soaked in sterile water was placed in a plastic petri dish, and the sterile seeds were sown on it. The sterile seeds were allowed to absorb water overnight in the dark at 4°C and used for the preparation of shoot apical samples. Using a Nanopass Needle II 34G (TERUMO), the cotyledon sheath, first leaf, and second leaf were removed from the embryo of the water-absorbed seed to expose the tip of the shoot apical meristem. Embryos containing shoot apical meristem were separated from the endosperm and placed on MS medium [MS salt (Sigma) 4.3 g / L, Maltose 30 g / L, MES 9.8 g / L, Phytagel 7 g / L, Plant Preservative Mixture (Plant Cell Technology) 3%, pH 5.8]. At this time, 30 to 40 shoot apical samples were arranged in the center of the medium in a donut-shaped circle with a diameter of 1 cm.

[0095] (Preparation of RNP complex) Recombinant SpCas9 protein used for genome editing was supplied by the Advanced Analytical Research Center of the National Agriculture and Food Research Organization. Chemically synthesized sgRNA (Integrated DNA Technologies) was used as the guide RNA (SEQ ID NOs: 154, 155, see Table 7 below). 10 μL of 2 μg / μL sgRNA, 10 μL of 4 μg / μL SpCas9 solution, 5 μL of 10×CutSmart buffer (New England Biolabs), and 4 μL of nuclease-free water were added to a 1.5 mL tube, and allowed to stand for 10 minutes to form the RNP complex.

[0096]

[0097] (Preparation of donor DNA) Chemically synthesized single-stranded DNA (Integrated DNA Technologies) was used (Sequence IDs: 156-158, Table 7 above). Single-stranded donor DNA was prepared by diluting it to 0.8 μg / μL with nuclease-free water. For double-stranded donor DNA, equal amounts of two types of single-stranded DNA (0.8 μg / μL) were mixed and annealed by thermal denaturation. Thermal denaturation was performed using a thermal cycler at 96°C for 10 minutes.

[0098] (Preparation of gold particles adsorbed with RNP complex and donor DNA) 0.6 μm Gold microcarriers (Bio-Rad) were suspended in nuclease-free water at 100 μg / μL to prepare a gold suspension. 5 μL of TransIT-LT1 reagent (Mirus Bio) was added to the prepared RNP complex and allowed to stand for 5 minutes. Subsequently, 16 μL of the gold suspension was added and gently mixed, then allowed to stand for 10 minutes. The mixture was centrifuged at 2,500 G for 15 seconds to precipitate the RNP-bound gold particles. The supernatant was removed, and the RNP-bound gold particles were resuspended in 25 μL of 0.8 μg / μL donor DNA solution and allowed to stand for 5 minutes.

[0099] (Particle bombardment) A 6 μL suspension of gold particles adsorbed with RNP complex and donor DNA was spread onto a hydrophilic film (3M) and dried at room temperature. This was then fired twice onto shoot apical samples on MS medium using a particle gun. A PDS-1000 / He Particle delivery system (Bio-Rad) was used as the particle gun, and the gold particle ejection pressure was set to 1,350 psi.

[0100] (DNA Extraction and Genome PCR) After particle bombardment, the plants were grown to the fifth leaf stage and subjected to genotyping analysis. A 5 mm square tissue sample was collected from the fifth leaf blade in a sampling tube, and 200 μL of DNA extract [100 mM Tris-HCl (pH 9.0), 10 mM EDTA, 1 M KCl] was added. The plant sample was crushed using a multi-bead shocker and then heated at 95°C for 10 minutes. 50 μL of the resulting crude extract was diluted with an equal volume of nuclease-free water and centrifuged at 11,000 G for 5 minutes. The supernatant was used for genome PCR. A partial sequence of WSUS3 containing the sugar nucleotide recognition motif coding sequence was amplified by PCR. The following primer sets were used for amplification. Note that by using the following primer sets, it is possible to amplify the partial sequences of WSUS-A3, B3, and D3 together. WSUS3_screening_Fw1:5'-TGCTGGTTATCTTAACTTGACTACCTC-3' (SEQ ID NO: 159) and WSUS3_screening_Rv1:5'-TAACTTCTCCCTCGATTGCGCTG-3' (SEQ ID NO: 160). The PCR enzyme used was KOD-One (Toyobo). The PCR conditions consisted of 30 cycles of denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, and extension at 68°C for 5 seconds.

[0101] (Selection of genome-edited contemporary (E0) individuals by CAPS analysis) A partial sequence of WSUS3 amplified by genomic PCR was combined with a CRISPR / Cas9 RNP solution that cleaves the sugar nucleotide recognition motif coding sequence, and an in vitro cleavage reaction was performed. The guide RNA used for cleavage was prepared using the GeneArt Precision gRNA Synthesis Kit (Invitrogen). Purified guide RNA was prepared according to the instructions using the following oligo-DNAs containing the target sequence: sgRNA-template_ANmotif_Fw1:5'-TAATACGAACTCACTATAGTCATCTGAGCAGAGATCCAG-3' (SEQ ID NO: 161) and sgRNA-template_ANmotif_Rv1:5'-TTCTAGCTCTTAAAAACCTGGAATCTCTGCTCCAGATA-3' (SEQ ID NO: 162). The products of the in vitro cleavage reaction were analyzed using a microchip electrophoresis system. Genome-edited contemporary individuals (E0 individuals) in which DNA fragments not cleaved by RNP were observed were designated as genome-edited chimeric individuals. In experiments combining the target sequence "Target 1" with donor DNA "Donor 1," one genome-edited chimeric individual was obtained from the analysis of 698 individuals (Table 8). Similarly, in experiments combining the target sequence "Target 2" with donor DNA "Donor 2," one genome-edited chimeric individual was obtained from the analysis of 564 individuals (Table 8). These individuals were named 201 and 701, respectively.

[0102]

[0103] (Selection of E1 individuals) Genome-edited chimeric individuals (201 and 701) have a mosaic of genome-edited cells and wild-type cells. Therefore, individual selection by genotyping analysis is necessary even in the next generation (E1 individuals). The same genotyping analysis as in the previous section was performed on E1 populations collected from 201 and 701. DNA fragments that were not cleaved by RNPs were observed in some of each E1 individual. These individuals were named 201-1 and 701-3.

[0104] (Acquisition of Genome Editing Lines) Uncleaved fragments found in 201-1 and 701-3 were introduced into the pCR Blunt II-TOPO vector included with the Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and sequencing analysis was performed. BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and SeqStudio Genetic Analyzer (Applied Biosystems) were used for sequencing analysis. The WSUS3 genes in 201-1 and 701-3 had synonymous substitutions near the target sequence derived from donor DNA, as well as base substitutions corresponding to four amino acid substitutions. Furthermore, based on the combination of two SNPs (SNP1 and SNP2) present between the WSUS3 homogeneous genes, it was considered that genome editing mutations were introduced into WSUS-D3 in 201-1 and into WSUS-A3 in 701-3. After fixing the genome editing mutations introduced into 201-1 and 701-3 to a homozygous state, these genome-edited lines were used for the following analyses.

[0105] (Yield Experiment) Dried seeds of Norin 61 and genome-edited lines (201-1 and 701-3) were sown in approximately 1 L of mixed potting soil (Hokusan Co., Ltd.) and grown in an artificial climate chamber. The environmental conditions in the artificial climate chamber were set to light period (16 h, 22°C) / dark period (8 h, 16°C). After ripening, the grains were collected and the total number of seeds and total seed weight per plant were measured. The seed weight per grain was calculated from the total number of seeds and total seed weight. In addition, the number of spikelets per spike and the number of seeds ripened in each spikelet were measured by observing the panicles after ripening. The number of effective tillers per plant was also measured.

[0106] (Seed Yield of Genome-Edited Lines) In genome-edited lines (201-1 and 701-3), a tendency for increased seed width and length compared to the wild type was observed (Figure 5). In addition, the total number of seeds per plant significantly increased to 1.60 times that of the wild type in line 201-1 and to 1.55 times that of the wild type in line 701-3 (Figure 6a). Similarly, the seed weight per grain also significantly increased to 1.21 times that of the wild type in line 201-1 and to 1.26 times that of the wild type in line 701-3 (Figure 6b). Finally, the total seed weight, which corresponds to the yield per plant, increased to 1.92 times that of the wild type in line 201-1 and to 1.94 times that of the wild type in line 701-3 (Figure 6c).

[0107] (Comparison of Effective Tiller Numbers) In the genome-edited lines (201-1 and 701-3), an increase in the total number of seeds was observed in addition to an increase in seed weight per seed. Therefore, the factors contributing to the increase in seed number were investigated. First, the number of effective tillers of the wild-type plant and the genome-edited lines were compared, and the average values ​​were 3.75 for the wild-type plant, 4.00 for the 201-1 line, and 4.08 for the 701-3 line (Figure 7). A tendency for an increase in tiller number was observed in the genome-edited lines, but the minimum P-value between measurements was 0.459 from the results of statistical analysis (Table 9). Therefore, it was determined that there was no significant difference between the wild-type plant and the genome-edited lines.

[0108]

[0109] (Comparison of Inflorescence Morphology) Next, the inflorescence morphology of the wild-type and genome-edited lines was compared. In the genome-edited lines, a tendency for the overall length of the inflorescence to be longer than that of the wild-type line was observed (Figure 8a). In addition, the number of spikelets per inflorescence significantly increased to 1.24 times that of the wild-type line in the 201-1 line and to 1.20 times that of the wild-type line in the 701-3 line (Figure 8b). Furthermore, when spikelets were tallied according to the number of twisted seeds and their proportion to the total number of spikelets was calculated, the proportion of spikelets with 3 twisted seeds increased in the 201-1 and 701-3 lines (Figure 8c). From these results, it was concluded that the total number of seeds increased in the genome-edited lines due to an increase in the number of spikelets per inflorescence and an increase in the number of twisted seeds per spikelet.

[0110] As described above, the present invention makes it possible to provide plants with increased storage tissue yield. According to the present invention, by expressing sucrose synthase with substrate specificity for ADP in the storage tissue of plants, it is possible not only to increase the size and weight of the storage tissue but also to increase its number, thereby significantly increasing the total weight of the storage tissue in the plant. Thus, the present invention is useful in the fields of agriculture, food, biomass, etc., for increasing the storage tissue of plants.

Claims

1. A method for producing a plant with increased storage tissue yield, comprising the step of expressing a sucrose synthase having substrate specificity for adenosine diphosphate (ADP) in the storage tissue of the plant.

2. The method according to claim 1, wherein the nucleoside diphosphate (NDP) recognition region in the sucrose synthase is a region comprising the amino acid sequence described in SEQ ID NO:

1.

3. The method according to claim 1, wherein the expression involves modifying the substrate specificity of sucrose synthase, which is naturally expressed in plant storage tissues, to ADP.

4. The method according to claim 1, wherein the expression is achieved by modifying the NDP recognition region of sucrose synthase, which is naturally expressed in plant storage tissue, to a region containing the amino acid sequence described in SEQ ID NO:

1.

5. The method according to claim 1, wherein the storage tissue is at least one tissue selected from the group consisting of endosperm, cotyledons, ovary, tuber, rhizome and scales.

6. The method according to any one of claims 1 to 5, wherein the plant in which the yield of storage tissue has increased is the plant in which the number of storage tissues has increased.

7. Plants in which sucrose synthase with substrate specificity for ADP is expressed in the storage tissue, resulting in increased storage tissue yield.

8. The plant according to claim 7, wherein the NDP recognition region of the sucrose synthase is a region containing the amino acid sequence described in SEQ ID NO:

1.

9. The plant according to claim 7, wherein the storage tissue is at least one tissue selected from the group consisting of endosperm, cotyledons, ovary, tuber, rhizome and scales.

10. The plant according to any one of claims 7 to 9, which is a plant with an increased number of storage tissues.

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