Modified plant body

Abstract

The present invention addresses the problem of providing a technology for controlling the size of seeds. The problem is solved by a modified plant body in which at least one gene selected from the group consisting of callose degradation enzyme genes and callose synthase enzyme genes is introduced or modified, and, by having the introduction or modification, the expression and / or function of the gene is controlled.

Description

Modified plants

[0001] The present invention relates to modified plants and the like.

[0002] Food shortages are becoming increasingly serious due to population explosion, global warming, overdevelopment, and worsening energy issues, so there is a need for more efficient production of edible parts of plants.

[0003] Seeds are rich in nutrients, and some plant seeds are in high demand as food. Therefore, increasing the size of seeds will lead to more efficient production of edible parts. On the other hand, reducing the size of seeds will also reduce the space required for seed storage and, for some plants, reduce the risk of lodging. Therefore, reducing the size of seeds will also lead to more efficient production of edible parts.

[0004] Callose is a polysaccharide synthesized in plants using glucose as a substrate and is known as a constituent of pollen and pollen tube cell walls. Non-Patent Document 1 reports that callose controls the initiation of meiosis. However, the relationship between callose and seed size has not been reported.

[0005] Plant Physiology, Volume 191, Issue 1, January 2023, Pages 400-413.

[0006] An object of the present invention is to provide a technique for adjusting seed size.

[0007] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that the above-mentioned problems can be solved by a modified plant into which at least one gene selected from the group consisting of a callose degrading enzyme gene and a callose synthase gene has been introduced or modified, and the expression and / or function of the gene has been regulated by said introduction or modification. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0008] Item 1. A modified plant into which at least one gene selected from the group consisting of a callose degrading enzyme gene and a callose synthase gene has been introduced or modified, and the expression and / or function of the gene has been regulated by the introduction or modification.

[0009] Item 2. The modified plant according to Item 1, wherein the protein encoded by the callose-degrading enzyme gene is at least one selected from the group consisting of protein (a) and protein (b): (a) a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 49, and (b) a protein consisting of an amino acid sequence with 90% or more identity to the protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 49 and having callose-degrading activity, and / or the protein encoded by the callose synthase gene is at least one selected from the group consisting of protein (c) and protein (d): (c) a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 50 to 61, and (d) a protein consisting of an amino acid sequence with 90% or more identity to the protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 50 to 61 and having callose synthase activity.

[0010] Item 3. The modified plant according to Item 1, wherein (A) (A1) a callose degrading enzyme gene has been introduced or modified, and expression of the callose degrading enzyme gene has been improved by the introduction or modification, and / or (A2) a callose synthase gene has been modified, and expression and / or function of the callose synthase gene has been reduced by the modification, or (B) (B1) a callose degrading enzyme gene has been modified, and expression and / or function of the callose degrading enzyme gene has been reduced by the modification, and / or (B2) a callose synthase gene has been introduced or modified, and expression of the callose synthase gene has been improved by the introduction or modification.

[0011] Item 4. The modified plant according to Item 3, into which (A1) a callose degrading enzyme gene has been introduced or modified, and the introduction or modification has resulted in improved expression of the callose degrading enzyme gene.

[0012] Item 5. The modified plant according to Item 4, wherein the protein encoded by the callose-degrading enzyme gene is at least one selected from the group consisting of protein (a1) and protein (b1): (a1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 31, and (b1) a protein consisting of an amino acid sequence having 90% or more identity to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 31 and having callose-degrading activity.

[0013] Item 6. The modified plant according to Item 1, wherein the genetic mutation caused by the gene modification is at least one type selected from the group consisting of gene disruption, a mutation in a protein coding region, a partial deletion, a mutation in a splicing regulatory region, and a mutation in an expression control region.

[0014] Item 7. The modified plant according to Item 1, which is a modified plant of a cultivated variety.

[0015] Item 8. The modified plant according to Item 1, which is a modified plant of a cereal, pulse, or nut seed plant.

[0016] Item 9. Seeds produced from the modified plant according to any one of Items 1 to 8.

[0017] Item 10. A method for adjusting the size of seeds produced, comprising introducing or modifying at least one gene selected from the group consisting of a callose degrading enzyme gene and a callose synthase gene into a plant, thereby regulating the expression and / or function of the gene.

[0018] Item 11. A method for screening for a substance that regulates plant seed size, using as an index the effect of a test substance on callose degrading enzyme activity and / or callose synthase activity.

[0019] According to the present invention, a technique for adjusting seed size can be provided.

[0020] A phylogenetic tree of Arabidopsis callose synthases is shown. The codes beginning with AT shown to the right of the phylogenetic tree indicate gene IDs. The numbers 1 to 49 shown to the right of the gene IDs indicate the sequence numbers of the protein's amino acid sequence. A phylogenetic tree of Arabidopsis callose synthases is shown. The codes beginning with AT shown to the right of the phylogenetic tree indicate gene IDs. The numbers 50 to 61 shown to the right of the gene IDs indicate the sequence numbers of the protein's amino acid sequence. Toluidine blue-stained images of sections of fertilized rice pistils are shown (Test Example 1). A comparison image of wild-type rice seeds (left side of each photograph) and callose-degrading enzyme-overexpressing rice seeds (right side of each photograph) is shown (Test Example 2). The results of measuring the seed area when wild-type rice seeds (WT) and callose-degrading enzyme-overexpressing rice seeds (OE_PPAP) were arranged as shown in the photographs in Figure 4 are shown (Test Example 2). Error bars indicate standard deviation. a: Schematic diagram of the anatomy of a rice pistil. After fertilization, rice pistils swell as shown in the left image. The swollen pistil was cut along the center of the image and sectioned, and the interior was observed as shown in the right image (b-d). b: The callose accumulation structure (callose gate) of rice overexpressing callose enzyme after fertilization is stained with aniline blue. The staining level is considered to be the same as that of other tissues, and the gate staining is weak. c: The callose gate of an unfertilized rice overexpressing callose enzyme is stained with aniline blue. The gate stains strongly, and its shape can be clearly observed. d: An enlarged image of the rice callose gate is shown. This is the final form of the rice phloem terminal. A comparison of wild-type rice seeds (left side of each photograph) and seeds of rice overexpressing callose enzyme (right side of each photograph) is shown (Test Example 3). The results of measuring the seed area of ​​wild-type rice seeds (WT) and callose-degrading enzyme-overexpressing rice seeds (OE_PPAP) when they were arranged as shown in the photograph in Figure 7 (Test Example 3) are shown. Error bars indicate standard deviation. ** indicates a P value of less than 0.01.

[0021] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0022] 1. Definitions In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0023] "Identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTP, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0024] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0025] As used herein, the terms "nucleic acid" and "polynucleotide" are not particularly limited and encompass both natural and artificial nucleic acids. Specifically, in addition to DNA, RNA, and the like, known chemical modifications may be used, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be substituted with a chemically modified phosphate residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. Furthermore, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Also usable are BNA (LNA), in which the conformation of the sugar moiety of the nucleotide is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety.

[0026] As used herein, the term "gene" includes not only the regions (exons, introns) on genomic DNA that code for a protein, but also the regions that control the expression of the protein (expression control regions).

[0027] 2. Modified Plants In one aspect, the present invention relates to a modified plant (sometimes referred to herein as a "modified plant of the present invention") into which at least one gene selected from the group consisting of a callose degrading enzyme gene and a callose synthase gene has been introduced or modified, and the expression and / or function of the gene has been regulated by the introduction or modification. The plant from which the modified plant of the present invention is derived (an unmodified plant) is not particularly limited. Examples of plants include a wide range of plants, including the angiosperms Magnolia, monocotyledons, and eudicotyledons (Rosaceae I, Rosaceae II, Chrysanthemum I, Chrysanthemum II, and their outgroups). Cultivated varieties are particularly preferred. More specific examples of cultivated varieties include eggplants such as tomatoes, bell peppers, chili peppers, and eggplants; melons such as cucumbers, pumpkins, melons, and watermelons; vegetables such as cabbage, broccoli, and Chinese cabbage; fresh or spicy vegetables such as celery, parsley, and lettuce; onions such as leeks, onions, and garlic; beans such as soybeans, peanuts, green beans, peas, and adzuki beans; other fruit vegetables such as strawberries; taproots such as radishes, turnips, carrots, and burdock; potatoes such as taro, cassava, potato, sweet potato, and Chinese yam; soft vegetables such as asparagus, spinach, and mitsuba; flowers such as lisianthus, stock, carnations, and chrysanthemums; rice, wheat, barley, oats, and corn. Examples of suitable crops include cereals, turfgrass such as bentgrass and Zoysiagrass, oil crops such as rapeseed and peanut, sugar crops such as sugarcane and sugar beet, fiber crops such as cotton and rush, forage crops such as clover, sorghum and dent corn, deciduous fruit trees such as apples, pears, grapes and peaches, citrus fruits such as Satsuma mandarins, lemons and grapefruit, woody plants such as azalea, azalea and cedar, and nuts and seeds such as almonds, hemp, flax, perilla, cashew, pumpkin, Japanese kaya, ginkgo, chestnut, walnut, poppy, coconut, sesame, Japanese castanea, watermelon, chia, horse chestnut, lotus, water chestnut, pistachio, sunflower, Brazil nut, hazel, pecan, macadamia, pine and peanut. Among these, grains, beans, nuts and seeds are particularly preferred from the viewpoint that the significance of the technology of the present invention is greater because the seeds are relatively large and can be used in a wide variety of ways.

[0028] The plant body means the entire plant including all of the plant tissues (roots, stems, leaves, etc.).

[0029] As shown in Test Example 1 below, it is believed that structures formed by the accumulation of callose exist at the ends of the phloem, and that these structures inhibit the passage of nutrients from the plant body to the seeds. As demonstrated in Test Example 2 below, by adjusting the breakdown or synthesis of callose, it is possible to adjust the amount of nutrients delivered to seeds during seed formation, thereby controlling seed size.

[0030] Callose-degrading enzyme genes encode enzymes that cleave the glycosidic bonds of callose to degrade it. One or more callose-degrading enzyme genes exist in plants. In Arabidopsis, the following have been reported: AT5G42100 (protein amino acid sequence: SEQ ID NO: 31), AT3G57270 (protein amino acid sequence: SEQ ID NO: 1), AT3G57260 (protein amino acid sequence: SEQ ID NO: 2), AT3G57240 (protein amino acid sequence: SEQ ID NO: 3), AT5G20330 (protein amino acid sequence: SEQ ID NO: 8), and AT5G20340 (protein amino acid sequence: SEQ ID NO: 7). Based on sequence similarity, phylogenetic analysis suggests that there are a total of 49 callose synthase genes (protein amino acid sequences: SEQ ID NOs: 1 to 49) (Figure 1). Callose degrading enzyme genes of plants other than Arabidopsis thaliana are either already known or can be easily identified based on identity / homology analysis based on the sequence information of the callose degrading enzyme gene of Arabidopsis thaliana.

[0031] The callose-degrading enzyme gene also includes functionally normal mutants that can occur naturally or artificially. The callose-degrading enzyme gene may have base mutations such as substitutions, deletions, additions, and insertions, as long as the callose-degrading activity is not significantly impaired. Preferred mutations are those that do not result in amino acid substitutions in the encoded protein or those that result in conservative amino acid substitutions.

[0032] The protein encoded by the callose-degrading enzyme gene is preferably at least one selected from the group consisting of protein (a) and protein (b): (a) a protein consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 49, and (b) a protein consisting of an amino acid sequence having 80% or more identity to a protein consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 49 and having callose-degrading activity.

[0033] In the above (b), the identity is more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more.

[0034] An example of protein (b) is (b') a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 1 to 49, and which has callose-degrading activity.

[0035] In the above (b'), "plurality" means, for example, 2 to 10, preferably 2 to 5, more preferably 2 to 3, and even more preferably 2.

[0036] Callose synthase genes are genes encoding enzymes that can synthesize callose by catalyzing glycosidic bonds using glucose as a substrate, and one or more types exist in plants. Twelve callose synthase genes (protein amino acid sequences: SEQ ID NOS: 50 to 61) have been identified in Arabidopsis thaliana. The results of a phylogenetic analysis of these genes based on sequence similarity are shown in Figure 2. Callose synthase genes in plants other than Arabidopsis thaliana are either already known or can be easily identified based on identity / homology analysis using the sequence information of the Arabidopsis callose synthase gene.

[0037] The callose synthase gene also includes functionally normal mutants that can occur naturally or artificially. The callose synthase gene may have base mutations such as substitutions, deletions, additions, and insertions, as long as the callose synthase activity is not significantly impaired. Preferred mutations are those that do not result in amino acid substitutions in the encoded protein or those that result in conservative amino acid substitutions.

[0038] The protein encoded by the callose synthase gene is preferably at least one selected from the group consisting of protein (c) and protein (d): (c) a protein consisting of an amino acid sequence shown in any one of SEQ ID NOs: 50 to 61, and (d) a protein consisting of an amino acid sequence having 80% or more identity to a protein consisting of an amino acid sequence shown in any one of SEQ ID NOs: 50 to 61 and having callose synthase activity.

[0039] In the above (d), the identity is more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more.

[0040] An example of protein (d) is (d') a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in any of SEQ ID NOs: 1 to 49, and which has callose-degrading activity.

[0041] In the above (d'), "plurality" means, for example, 2 to 10, preferably 2 to 5, more preferably 2 to 3, and even more preferably 2.

[0042] The modified plant of the present invention includes the following aspects: (A) (A1) a callose degrading enzyme gene has been introduced or modified, and the expression of the callose degrading enzyme gene has been improved by the introduction or modification, and / or (A2) a callose synthase gene has been modified, and the expression and / or function of the callose synthase gene has been reduced by the modification, or (B) (B1) a callose degrading enzyme gene has been modified, and the expression and / or function of the callose degrading enzyme gene has been reduced by the modification, and / or (B2) a callose synthase gene has been introduced or modified, and the expression of the callose synthase gene has been improved by the introduction or modification.

[0043] In embodiment (A), the amount of functionally normal callose in the plant is reduced, and the modification results in larger seeds (for example, 103% or more, 104% or more, 106% or more, 108% or more, or 110% or more, or for example, 150% or less, 130% or less, or 120% or less, compared to 100% of unmodified seeds).

[0044] Embodiment (B) is a modified plant in which the amount of functionally normal callose in the plant is improved, and the modification results in smaller seeds (for example, 98% or less, 96% or less, 94% or less, 92% or less, or 90% or less, or for example, 50% or more, 70% or more, or 80% or more, compared to 100% of seeds from an unmodified plant).

[0045] Therefore, in one aspect, the present invention relates to a method for adjusting the size of seeds produced, which comprises regulating the expression and / or function of at least one gene selected from the group consisting of callose degrading enzyme genes and callose synthase genes by introducing or modifying the gene into a plant body.

[0046] The "function" of a gene refers to callose degradation activity / callose synthesis activity. Furthermore, the "expression" of a gene encompasses both the expression of gene mRNA and the expression of gene protein, but preferably refers to the expression of gene protein. The term "decreased" refers to a sample obtained from a plant of the present invention in which the gene protein activity and / or gene expression level (index values ​​of gene function and / or expression) is, for example, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.02% or less, or 0.01% or less of the gene protein activity and / or gene expression level before mutation introduction (index values ​​of function and / or expression in the absence of gene modification). "Improvement" means that, for a sample obtained from a plant body of the present invention, the activity of the gene protein and / or the expression level of the gene (index value of the gene function and / or expression) is, for example, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 200% or more, 300% or more, 400% or more, 500% or more, 1000% or more, or, for example, 10000% or less, 5000% or less, 3000% or less, 2000% or less, relative to 100% of the activity of the gene protein and / or the expression level of the gene before the mutation was introduced (index value of the function and / or expression when the gene is not modified).

[0047] Specifically, when introducing a gene, for example, an insert containing a polynucleotide including an expression cassette of the gene is introduced into plant cells. The expression cassette is not particularly limited as long as the gene is incorporated in an expressible state. Typically, the expression cassette contains a polynucleotide including a promoter sequence and a gene coding sequence (and, if necessary, a transcription termination signal sequence). The expression cassette can also be in the form of a vector.

[0048] The promoter is not particularly limited, and examples thereof include the RPS5A promoter, the UBQ promoter, the CaMV35S promoter, the NOS promoter, etc. Furthermore, promoters of genes that are expressed in a tissue-specific and / or stage-specific manner can also be used.

[0049] When modifying a gene, a specific example is a method of introducing an introducer containing at least one selected from the group consisting of a target-specific nuclease, an expression cassette for the nuclease, and mRNA for the nuclease into plant cells.

[0050] Examples of gene mutations due to such modifications include gene disruption, mutations in protein coding regions, partial deletions, mutations in splicing regulatory regions, mutations in expression control regions (e.g., promoters, activators, enhancers, repressor-binding elements, etc.), etc. Mutations in the expression control regions of the callose degrading enzyme gene and the callose synthase gene can also be used to improve or reduce the expression of the callose degrading enzyme gene and the callose synthase gene.

[0051] The target-specific nuclease is not particularly limited as long as it is a nuclease that can specifically cleave a specific site on genomic DNA to induce a mutation. Examples of target-specific nucleases include Cas proteins, TALEN proteins, and ZFN proteins.

[0052] The CRISPR / Cas system uses a Cas protein, a nuclease (RGN; RNA-guided nuclease), and a guide RNA. By introducing this system into cells, the guide RNA binds to the target site, and the Cas protein is recruited to the binding site and can cleave the DNA.

[0053] The TALEN system using TALEN protein uses artificial nuclease (TALEN) that comprises DNA cleavage domain (for example, FokI domain) and the DNA binding domain of transcription activator-like (TAL) effector.By introducing this system into cells, TALEN binds to target site through DNA binding domain and cuts DNA there.The DNA binding domain that binds to target site can be designed according to known schemes (for example, Zhang F et al. (2011) Nature Biotechnology 29 (2); this paper is incorporated herein by reference).

[0054] The ZFN system using ZFN protein uses artificial nuclease (ZFN) that comprises nucleic acid cutting domain conjugated with DNA binding domain that comprises zinc finger array.By introducing this system into cells, ZFN binds to target site through DNA binding domain and cuts DNA there.The DNA binding domain that binds to target site can be designed according to known scheme.

[0055] Among target-specific nucleases, Cas proteins are preferred from the viewpoint of being able to more freely determine the cleavage site, and a preferred example of the Cas protein is the Cas9 protein.

[0056] The target-specific nuclease expression cassette is not particularly limited as long as it is DNA capable of expressing a target-specific nuclease in the cells of the subject of the plant production method of the present invention. A typical example of a target-specific nuclease expression cassette is DNA comprising a promoter and a target-specific nuclease coding sequence placed under the control of the promoter. The target-specific nuclease expression cassette may comprise a vector alone or together with other sequences (e.g., a drug resistance gene, a replication origin, etc.). The type of vector is not particularly limited.

[0057] When the target-specific nuclease is a Cas protein, the introduced material in the plant production method of the present invention further includes at least one selected from the group consisting of a guide RNA expression cassette and a guide RNA.

[0058] The guide RNA is not particularly limited as long as it is one that can be used in the CRISPR / Cas system. For example, various types of guide RNAs can be used that can bind to a target site in genomic DNA and bind to a Cas protein, thereby guiding the Cas protein to the target site in genomic DNA.

[0059] It is said that the 12 bases on the 3' side of the crRNA sequence that binds to the target sequence are important for the binding of the guide RNA to the target site. Therefore, if the crRNA sequence that binds to the target sequence is not completely identical to the target strand, it is preferable that the bases that differ from the target strand exist in the crRNA sequence other than the 12 bases on the 3' side of the sequence that binds to the target sequence.

[0060] The introduced product may also contain donor DNA. The use of donor DNA allows for more precise introduction of the desired mutation.

[0061] The target of introduction is not particularly limited, and may be undifferentiated plant tissue (e.g., callus), a part of a seed (e.g., hypocotyl, shoot apex, etc.), or a part of an adult plant (e.g., shoot apex, etc.).

[0062] The introduction method is not particularly limited as long as it allows the introduced substance to reach the plant cells, and can be appropriately selected depending on the type of substance to be introduced and the target of introduction. Examples of introduction methods include the floral dip method, floral spray method, Agrobacterium method, particle gun method, infiltration method, toothpick inoculation method, suction injection method, leaf disc method, inflorescence infiltration method, vacuum filtration method, virus-mediated nucleic acid delivery, etc. Among these, the Agrobacterium method is preferred from the viewpoints of simplicity, safety, etc.

[0063] A more specific example of how to implement it is shown below.

[0064] A first specific example of the introduction method (Introduction Example 1) includes the steps of: preparing a plasmid containing a promoter (e.g., T7 promoter, T3 promoter, 35S promoter, etc.) and a sequence containing an expression cassette downstream of the promoter (step a1); obtaining plant virus genomic RNA from the plasmid obtained in step a1 by in vitro transcription (step b1); and inoculating a plant with the genomic RNA (active ingredient) obtained in step b1 (e.g., friction inoculation, particle gun inoculation, etc.) (step c1). Alternatively, if the plasmid obtained in step a1 is a Ti plasmid containing a promoter capable of activating transcription in plant cells, such as the 35S promoter, instead of steps b1 and c1, the introduction can be carried out by a method including, for example, introducing the plasmid obtained in step a1 into Agrobacterium and culturing it (step b2), and inoculating a plant with the culture solution obtained in step b2 (containing the active ingredient) (e.g., infiltration, toothpick inoculation, suction injection, etc.) (step c2). Alternatively, instead of steps b1 and c1 above, a method including a step (step c3) of inoculating a plant with the plasmid (active ingredient) obtained in step a1 (for example, grinding inoculation, particle gun inoculation, etc.) can be used. Alternatively, instead of step c2 above, a method including a step (step c4) of performing, for example, the leaf disc method, inflorescence infiltration method, vacuum filtration, etc. can be used. The desired protein, peptide, or nucleic acid is produced from the genomic RNA, plasmid, T-DNA, etc. introduced into the plant by these methods.

[0065] A second specific example of the introduction method (Introduction Example 2) includes a step (step d1) of collecting a plant virus from a plant containing the virus (e.g., obtained by the above-mentioned Introduction Example 1), and a step (step e1) of inoculating a plant with the virus (a virus containing an active ingredient) collected in step d1. The collection in step d1 can be carried out, for example, by grinding a part of the plant containing the plant virus (e.g., a leaf) and recovering the virus solution. The inoculation in step e1 can be carried out, for example, by using an abrasive such as silicon carbide to make a wound in the part of the plant to be inoculated (e.g., a leaf) and contacting the wound with the virus.

[0066] After the introduction, the modified plant of the present invention can be obtained by growing the resulting plant or plant cells, or by growing the resulting plant through callus. Furthermore, after the introduction, the introduced cells, tissues, etc. can be selected with a drug, if necessary.

[0067] The above-mentioned introduced material can be used as an agent for producing the modified plant of the present invention.

[0068] The manufacturing agent of the present invention may consist solely of the above-described introduced substance (essential component), but may also contain various other components in addition to the essential component, depending on the type of essential component contained, the dosage form (described below), and the mode of use. The content of the essential components (dry weight) in the manufacturing agent of the present invention can be determined appropriately depending on the dosage form (described below) and the mode of use, and can range, for example, from 0.0001 to 100% by mass. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, and chelating agents. The form of the manufacturing agent of the present invention is not particularly limited and can be, for example, a dry form, a solution form, or a kit form. The kit may also contain other materials, reagents, and tools necessary for plant production, such as nucleic acid transfer reagents and buffer solutions, as needed.

[0069] In one aspect, the present invention relates to seeds produced from the modified plants of the present invention. The seeds of the present invention can be obtained by harvesting seeds from the modified plants of the present invention.

[0070] 3. Screening Method In one aspect, the present invention relates to a screening method for a substance that regulates plant seed size, using the regulatory effect of a test substance on callose degrading enzyme activity and / or callose synthase activity as an index.

[0071] A wide range of test substances can be used, regardless of whether they are naturally occurring or artificially produced. Furthermore, not only purified compounds but also compositions containing a variety of compounds and animal and plant extracts can be used. Compounds are not limited to low-molecular-weight compounds, but also include high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.

[0072] More specifically, the screening method of the present invention can select a test substance as an active ingredient of a substance / drug that increases plant seeds or as a candidate substance thereof, for example, if the test substance has the effect of improving callose-degrading enzyme activity and / or the effect of reducing callose-synthesizing enzyme activity.

[0073] More specifically, the screening method of the present invention can select a test substance as an active ingredient of a substance / drug that reduces plant seeds or as a candidate substance thereof, for example, if the test substance has the effect of reducing callose-degrading enzyme activity and / or the effect of improving callose-synthesizing enzyme activity.

[0074] The decrease in activity is, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less, relative to 100% activity before contact with the test substance.

[0075] The improvement in activity is, for example, 110% or more, 150% or more, 200% or more, 300% or more, 400% or more, 500% or more, or 1000% or more, relative to 100% activity before contact with the test substance.

[0076] The conditions for measuring the enzyme activity are not particularly limited as long as they allow the enzyme to synthesize / decompose callose and allow the test substance to come into contact with the enzyme. The method may be an in vitro method or an in vivo method using cells or plants.

[0077] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0078] Test Example 1. Observation of rice tissue structure 1 Fertilized rice pistils were embedded in tissue embedding resin (Technovit 7100, Kulzer) and then longitudinal sections 14 μm thick were prepared. The obtained sections were stained with 0.025% toluidine blue and photographed.

[0079] The results are shown in Figure 3. Structures were observed at the ends of the phloem. These were presumed to be structures formed by the accumulation of callose. These results suggest that these structures inhibit the passage of nutrients from the plant body to the seeds.

[0080] Test Example 2. Seed Size Control Test 1 Based on the results of Test Example 1, we came up with the idea that by adjusting the breakdown or synthesis of callose, the amount of nutrients delivered to seeds during seed formation can be adjusted, and thus seed size can be controlled. To verify this hypothesis, we created modified rice plants that overexpressed callose-degrading enzymes, and compared the seed size of these modified rice plants with that of unmodified rice plants. Specifically, this was done as follows.

[0081] An expression plasmid was constructed by cloning a sequence consisting of, from the 5' end, the pUbi (maize ubiquitin gene promoter) sequence (SEQ ID NO: 62), the Ω sequence (SEQ ID NO: 63), a linker sequence (derived from the MCS of pCAMBIA1380) (SEQ ID NO: 64), and the AT5G42100 (Arabidopsis thaliana callose degrading gene) CDS sequence (SEQ ID NO: 65) into the AscI-HindIII site of the pCAMBIA1380 vector plasmid. The resulting expression plasmid was then introduced into calli induced from rice (T65: O. sativa, var. Taichung 65) seeds using Agrobacterium tumefaciens (strain EHA105). Regeneration and regeneration were performed to obtain an overexpressor (T0 generation) of the protein (callose degrading enzyme) consisting of the amino acid sequence shown in SEQ ID NO: 31. The overexpressor was cultivated according to standard methods, and seeds were obtained.

[0082] A photograph of the seeds is shown in Figure 4. When the seeds were arranged as shown in the photograph, the area of ​​the seeds was measured and found to be 11.49 mm for wild-type rice seeds (n = 14). 2 (SD: 1.04), and the seeds of overexpressing rice (n = 10) were 12.90 mm 2 The difference between the two groups was 0.0011 (SD: 0.55), and a t-test between the two groups revealed a p-value of 0.0011 (Fig. 5). These results support the idea that regulating callose degradation or synthesis can regulate the amount of nutrients delivered to seeds during seed development, thereby regulating seed size.

[0083] Experimental Example 3. Observation of Rice Tissue Structure 2 and Seed Size Control Experiment 2 <Preparation of Rice Vectors> The pCAMBIA-1380 vector was used to prepare the PPAP-OE construct. The 1986-bp rice UBIQUITIN promoter (pUBI) and the 1275-bp AT5g42100 (Arabidopsis thaliana callosylase gene: PPAP gene) coding sequence were inserted into the vector according to standard procedures. To enhance overexpression, a translational enhancer (ω; 77 bp) was inserted between the UBI promoter and the PPAP gene. Following the transfection cloning protocol, pUBI and the omega fragment amplified using primers were inserted into EcoRI-digested pCAMBIA-1380 vector. Next, the Arabidopsis thaliana PPAP CDS was cloned by PCR and inserted into the previously prepared vector after HindIII digestion, ensuring that the CDS was ligated between the omega sequence and the NOS terminator. <Rice Transformation and Regeneration> Rice transformation was performed as previously reported (Communications Biology 3, 617 (2020)). For regeneration culture, immature embryos were co-cultured and then transferred to 9 cm plastic dishes. For subsequent tissue transplantation, tissues other than the pod were removed with a scalpel. The divided pods were placed face-up in Petri dishes with a divider on the bottom of each immature embryo. The embryos were cultured at 30°C for 6–20 days under bright light without selection. Immature embryos can be divided by scraping off the pod with a scalpel. The six divided pods were further divided into 3–5 sections using a scalpel under a stereomicroscope and transferred to selective medium. The embryos were cultured at 30°C for 7–10 days. The resulting resistant calli were then transferred to regeneration medium. Each divided tissue was treated as an independent transformed callus. After approximately 7 days of culture at 30°C under light, the expanded resistant calli were transferred to regeneration medium. The proliferated resistant callus was transferred to regeneration medium. The excised pieces were transferred to regeneration medium and cultured at 30°C under light conditions for approximately 14 days to obtain regenerated shoots or regenerated seedlings. The regenerated seedlings were then transplanted directly to nursery medium. After 14 days of culture at 30°C under light conditions, the grown plantlets were carefully separated. The entire germinated callus was carefully removed from the roots, wrapped in a cotton ball, and placed in a small pot in a nursery box.The pots containing the seedlings were placed on stainless steel pads filled with 1000x Hyponex solution and placed on a plastic rack at 25°C. After approximately 7 days of growth under light conditions, the pots were transferred to a greenhouse. <Observation of rice pistils> Two to three days after flowering, rice pistils from control (wild-type: Nipponbare) and overexpressors of the PPAP gene (callose-degrading enzyme gene) were fixed in 4% paraformaldehyde, 5% acetic acid, and 50% ethanol. The pistils were vacuum-sealed and dehydrated in an ethanol series (50%, 70%, 80%, 90%, 95%, and 100%) for one hour each, followed by overnight dehydration in 100% ethanol. These were infiltrated for 1 hour in 5:1, 3:1, 2:3, 1:5, or 0:1 ethanol / Technovit 7100 (Kulzer, Wehrheim, Germany) and embedded in Technovit 7100 according to the manufacturer's instructions. The samples were cut into transverse sections (14 μm) using a rotary microtome OSK97LF506R (Ogawa Seiki, Tokyo, Japan). The sections were incubated in 2% K3PO4 (Fujifilm Wako, Osaka, Japan) for 30 minutes and then stained with 0.01% aniline blue (water-soluble, Nacalai Tesque, Kyoto, Japan) on glass slides for 2 hours. The aniline blue signal was imaged using cellSens Ver. 3.0 (OLYMPUS, Sapporo, Japan) with an Olympus BX51 fluorescence microscope equipped with a 10x 0.40 air objective, UPlanSApo, UV filters (excitation filter: 330-385 nm bandpass, emission filter: 420 nm longpass), and an Olympus DP27 camera. The results are shown in Figure 6. A callose-accumulated structure (callose gate) was confirmed. Seeds were harvested from the control (wild-type: Nipponbare) and the overexpressor of the PPAP gene (callose-degrading enzyme gene). The seed area was measured when the seeds were arranged as shown in the photograph in Figure 7. The results are shown in Figure 8. The wild-type rice seeds (n = 32) were 11.86 mm. 2 (SD: ±0.9mm 2 ), the seeds of overexpressing rice (n = 32) were 12.93 mm 2 (SD: ±1.23 mm 2) and a t-test between the two groups showed a p-value of <0.01.

Claims

1. The callose-degrading enzyme gene has been introduced or modified. The expression and / or function of the callose-degrading enzyme gene are regulated by the aforementioned introduction or modification. The proteins encoded by the aforementioned callose-degrading enzyme gene are protein (a) and protein (b): (a) a protein consisting of an amino acid sequence shown in any of Sequence IDs 1 to 49, and (b) a protein consisting of an amino acid sequence having 90% or more identity with the protein consisting of an amino acid sequence shown in any of Sequence IDs 1 to 49, and having callose-degrading activity. A modified plant body which is at least one species selected from the group consisting of the following.

2. The proteins encoded by the aforementioned callose-degrading enzyme gene are protein (a) and protein (b): (a) a protein consisting of an amino acid sequence shown in any of Sequence IDs 1 to 49, and (b) a protein consisting of an amino acid sequence having 95% or more identity with the protein consisting of an amino acid sequence shown in any of Sequence IDs 1 to 49, and having callose-degrading activity. It is at least one selected from the group consisting of the following: , the modified plant body according to claim 1.

3. (A1) The callose-degrading enzyme gene has been introduced or modified. The introduction or modification described above has improved the expression of the callose-degrading enzyme gene, or (B1) The callose-degrading enzyme gene has been modified, and the expression and / or function of the callose-degrading enzyme gene has been reduced as a result of the modification. The modified plant body according to claim 1.

4. (A1) A callose-degrading enzyme gene has been introduced or modified, and the expression of the callose-degrading enzyme gene has been improved by the introduction or modification. The modified plant body according to claim 3.

5. The proteins encoded by the aforementioned callose-degrading enzyme gene are protein (a1) and protein (b1): (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 31, and (b1) A protein consisting of an amino acid sequence having 90% or more identity with the protein consisting of the amino acid sequence shown in SEQ ID NO: 31, and having callose-degrading activity. It is at least one selected from the group consisting of, The modified plant body according to claim 4.

6. The modified plant according to claim 1, wherein the gene mutation resulting from the modification of the callose-degrading enzyme gene is at least one selected from the group consisting of gene disruption, mutation in the protein-coding region, partial deletion, mutation in the splicing regulatory region, and mutation in the expression regulatory region.

7. A modified plant according to claim 1, which is a modified plant of a cultivated variety.

8. The modified plant body according to claim 1, which is a modified plant body of a cereal, legume, or nut plant.

9. Seeds produced from a modified plant according to any one of claims 1 to 8.

10. A method for adjusting the size of seeds produced, comprising regulating the expression and / or function of a callose-degrading enzyme gene by introducing or modifying the callose-degrading enzyme gene into a plant.

11. A screening method for substances that regulate plant seed size, using the effect of the test substance on callose-degrading enzyme activity as an indicator.