WO2021070549A1 - Procédé d'édition de génome dans le blé et son utilisation - Google Patents

Procédé d'édition de génome dans le blé et son utilisation Download PDF

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WO2021070549A1
WO2021070549A1 PCT/JP2020/034298 JP2020034298W WO2021070549A1 WO 2021070549 A1 WO2021070549 A1 WO 2021070549A1 JP 2020034298 W JP2020034298 W JP 2020034298W WO 2021070549 A1 WO2021070549 A1 WO 2021070549A1
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wheat
gene
genome
shoot apex
deletion
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PCT/JP2020/034298
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English (en)
Japanese (ja)
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亮三 今井
祐也 熊谷
洋三 柳楽
晴康 濱田
隆二 三木
直明 田岡
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国立研究開発法人農業・食品産業技術総合研究機構
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Publication of WO2021070549A1 publication Critical patent/WO2021070549A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/14—Plant cells

Definitions

  • the present invention relates to a method for editing the genome of wheat, a method for producing wheat having an SD1 gene mutation, and a method for producing wheat having an SD1 gene mutation.
  • the "Green Revolution” introduced the semi-dwarf gene Rht-B1 derived from wheat Norin 10 to breeding varieties around the world, resulting in a significant reduction in culm and the accompanying increase in revenue on a global scale.
  • the Rht-B1 gene encodes a protein involved in the reception of the plant growth hormone gibberellin and is insensitive to gibberellin, resulting in shortening of the culm. After that, the "Green Revolution” occurred in rice, but a gene different from wheat was used.
  • the Rht homologous gene mutation became extremely dwarf and could not be used for breeding.
  • rice another SD1 gene was used.
  • the SD1 gene encodes a gibberellin biosynthetic enzyme, and the mutant SD1 is semi-dwarf because its synthesis is suppressed (see, for example, Patent Document 1). If semi-dwarf wheat varieties with Rht gene mutations can be further shortened, further increase in sales is expected.
  • the SD1 gene is a candidate, but no SD1 mutation has yet been found in wheat. Further, in recent years, as a new transformation technique, a genome editing technique capable of specifically modifying a target gene has been developed.
  • a method for editing a wheat genome that can efficiently introduce a deletion, insertion, or substitution into the SD1 gene of wheat in a short period of time, and a method for efficiently deleting, inserting, or substituting into the SD1 gene of wheat in a short period of time.
  • a method for producing wheat having an SD1 gene mutation capable of obtaining wheat having lodging resistance has not yet been provided, and prompt provision thereof is strongly required.
  • the present invention is a method for editing a wheat genome that can efficiently introduce a deletion, insertion, or substitution into the SD1 gene of wheat in a short period of time, and a method of efficiently deleting, inserting, or substituting into the SD1 gene of wheat in a short period of time. It is an object of the present invention to provide a method for producing wheat having an SD1 gene mutation capable of obtaining wheat having lodging resistance by introducing insertion or substitution.
  • the present inventors adopt a wheat genome editing method including a step of introducing a deletion, insertion, or substitution into the wheat SD1 gene by a genome editing means.
  • a wheat genome editing method capable of efficiently introducing deletion, insertion, or substitution into the wheat SD1 gene in a short period of time, and deletion, insertion into the wheat SD1 gene by genome editing means.
  • a method for producing a wheat having an SD1 gene mutation including a step of introducing a substitution and a step of growing a tissue in which the SD1 gene of the wheat has been deleted, inserted, or substituted to obtain a plant is adopted. By doing so, it was found that it is possible to provide a method for producing wheat that can efficiently introduce deletion, insertion or substitution into the SD1 gene of wheat in a short period of time to obtain wheat having lodging resistance.
  • the present invention is based on the above-mentioned findings by the present inventors, and the means for solving the above-mentioned problems are as follows. That is, ⁇ 1> A wheat genome editing method comprising a step of introducing a deletion, insertion, or substitution into a wheat SD1 gene by a genome editing means. ⁇ 2> A step of introducing a deletion, insertion, or substitution into the SD1 gene of wheat by a genome editing means, and a tissue into which the deletion, insertion, or substitution is introduced into the SD1 gene of wheat are grown to grow a plant. It is a method for producing wheat having an SD1 gene mutation, which comprises a step of obtaining the wheat. ⁇ 3> A wheat having an SD1 gene mutation characterized in that a deletion, insertion, or substitution has been introduced into the SD1 gene.
  • a wheat genome capable of solving the above-mentioned problems in the past, achieving the above-mentioned object, and efficiently introducing a deletion, insertion, or substitution into the SD1 gene of wheat in a short period of time. It is possible to provide an editing method and a method for producing wheat capable of efficiently introducing deletion, insertion or substitution into the SD1 gene of wheat in a short period of time to obtain wheat having lodging resistance.
  • FIG. 1A is a diagram showing a phylogenetic tree of the SD1 gene family in Example 1.
  • FIG. 1B is a diagram showing the amino acid sequences of each SD1 in the A, B, and D genomes of wheat in Example 1.
  • FIG. 1C is a diagram showing a target site for genome editing in Example 2.
  • FIG. 2A is a diagram showing the results of in vitro digestion analysis in Example 3.
  • Lane M is a marker
  • lane 1 is a DNA fragment containing targets 1 and 3
  • lane 2 is a product of target 1
  • lane 3 is a product of target 3
  • lane 4 is a DNA fragment containing target 2
  • lane 5 is a target 2.
  • FIG. 2B is a photograph showing the wheat shoot apex after exposure in Example 4.
  • FIG. 2C is a diagram showing the results of CAPS analysis at the shoot apex stage for mutation introduction by the In planta method in Example 4.
  • Lane M is a marker
  • lanes # 1 to # 5 are DNA derived from the shoot apex of each 5 samples for each target (with restriction enzyme addition)
  • lane WT (+) is DNA derived from the shoot apex without mutation (+).
  • lane WT (-) indicates DNA derived from shoot apex without introduction of mutation (without addition of restriction enzyme).
  • FIG. 3A is a diagram showing the results of CAPS analysis for the A genome (upper row), the B genome (middle row), and the D genome (lower row) at the T0 (five leaf) stage in Example 5.
  • FIG. 3B is a diagram showing the DNA sequence results of each sample (H1, H3, H4, and H7) at the T0 (five leaf) stage in Example 5.
  • FIG. 4A is a diagram showing the results of CAPS analysis at the T1 plant (T1 seed) stage in Example 6.
  • FIG. 4B is a diagram showing the results of DNA sequencing at the T1 plant (T1 seed) stage in Example 6.
  • FIG. 4C is a diagram showing the results of analysis of the expression level of the SD1 gene by RT-PCR in the T1 plant (T1 seed) stage in Example 7.
  • FIG. 5A is a photograph showing the plant height of the wild-type strain and the T2 plant in Example 8.
  • FIG. 5B is a diagram showing the results of measuring the plant heights of the wild-type strain and the T2 plant in Example 8.
  • FIG. 5C is a diagram showing the results of measuring the number of seeds harvested from the wild-type strain and the T2 plant and their weights in Example 8.
  • the wheat genome editing method of the present invention includes a step of introducing a deletion, insertion, or substitution into the SD1 gene of wheat by a genome editing means, and can further include other steps.
  • the step of introducing deletion, insertion, or substitution into the SD1 gene of wheat by the genome editing means is not particularly limited, and a known genetic engineering method can be used, for example, Agrobacterium method, electroporation. Examples thereof include steps by the poration method, the particle gun method, the PEG-calcium phosphate method, the liposome method, the microinjection method, the whisker method, the plasma method, the laser injection method and the like.
  • the Agrobacterium method is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a genome editing means is introduced into Agrobacterium by an electroporation method, and wheat shoot apex, callus, etc. Examples thereof include a method of infecting the scutellum and the like with the Agrobacterium.
  • the genome editing means is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a guide RNA or a nucleic acid encoding the guide RNA, a nucleic acid metabolizing enzyme, or the nucleic acid metabolizing enzyme can be selected.
  • examples include a combination with any of the encoding nucleic acids.
  • a guide RNA targeting the SD1 gene or a nucleic acid encoding a guide RNA targeting the SD1 gene, a nucleic acid metabolizing enzyme, or the nucleic acid metabolizing enzyme is encoded.
  • a combination with any of the nucleic acids is preferable, and a combination of a guide RNA targeting the SD1 gene and a nucleic acid-metabolizing enzyme is more preferable.
  • the amino acid sequence of SD1 is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include wheat ortholog of the amino acid sequence of rice SD1 (SEQ ID NO: 1).
  • Examples of the wheat ortholog of the amino acid sequence of rice SD1 include TRIAE CS42 3AL TGACv1 194412 AA0632590.1 (SEQ ID NO: 2), TRIAE CS42 3B TGACv1 223804 AA0787660.1 (SEQ ID NO: 3), and TRIAE2 1 (SEQ ID NO: 4), and homologs thereof and the like can be mentioned.
  • the homolog is not particularly limited and may be appropriately selected depending on the intended purpose, but the sequence identity with the sequence of SEQ ID NO: 1 is preferably 70% or more, and 75% or more of the sequence identity.
  • the sex is more preferable, 80% or more of the sequence identity is more preferable, 85% or more of the sequence identity is particularly preferable, and the sequence identity with any of the sequences of SEQ ID NOs: 2 to 4 is 80% or more of the sequence identity.
  • the sex is preferable, 85% or more of sequence identity is more preferable, 90% or more of sequence identity is further preferable, and 95% or more of sequence identity is particularly preferable.
  • the lower limit of the length of the guide RNA is not particularly limited and may be appropriately selected depending on the intended purpose, but 15 nucleotides or more is preferable, 16 nucleotides or more is more preferable, 17 nucleotides or more is further preferable, and 18 Nucleotides and above are particularly preferred, 19 nucleotides and above are even more preferred, and 20 nucleotides and above are most preferred.
  • the upper limit of the length of the guide RNA is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 30 nucleotides or less, more preferably 25 nucleotides or less, further preferably 22 nucleotides or less, and 20 Nucleotides and below are particularly preferred.
  • the guide RNA may contain a guide sequence fused to the tracr sequence.
  • the guide RNA that targets the SD1 gene is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a guide RNA produced using the following primer set or a sequence of the following primer set can be used.
  • those produced using a primer set having sequence identity, the sequence represented by any of the following target sequences 1 to 3, and the sequences of the following target sequences 1 to 3 have sequence identity. Examples thereof include sequences having.
  • those produced using the primer set 2 those produced using a primer set having sequence identity with respect to the sequence of the primer set 2, the target sequence 2 and the sequence of the target sequence 2 Sequence identity is preferred.
  • sequence identity is not particularly limited and may be appropriately selected depending on the intended purpose, but 80% or more of sequence identity is preferable, 85% or more of sequence identity is more preferable, and 90% or more of sequences is preferable. Identity is even more preferred, with 95% or greater sequence identity being particularly preferred.
  • Target sequence 1 CGCGGTGTACCGACTCCGGGAGGG (SEQ ID NO: 11)
  • Target sequence 2 GGGCTGGAGGTCCTCGTCGACGG (SEQ ID NO: 12)
  • Target sequence 3 GGGCTGGAGGTCCTCGTCGACGG (SEQ ID NO: 13)
  • the SD1 gene may be mutated by inserting or substituting foreign DNA by recombination between the cleaved sites.
  • the nucleic acid-metabolizing enzyme is not particularly limited and may be appropriately selected depending on the intended purpose, and examples thereof include nuclease and deaminase.
  • the nucleic acid metabolizing enzyme may contain one or more nuclear localization signals (NLS).
  • the nuclease is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the nuclease may be derived from a different species, and for example, genes such as animals, plants, microorganisms, and viruses, or artificially synthesized genes can be used.
  • the CASnuclease is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include type I CRISPR enzyme, type II CRISPR enzyme, type III CRISPR enzyme, and type II CRISPR enzyme. Cas9 is preferable.
  • the Cas nuclease may be codon-optimized for expression in eukaryotic cells. The Cas nuclease can direct the cleavage of one or two strands in the localization of the target sequence. In another aspect of the invention, the expression of the gene product is reduced and the gene product is a protein.
  • the Cas9 is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the deaminase is not particularly limited as long as it has deamination enzyme activity, and can be appropriately selected depending on the intended purpose. It is used by adding it to a CRISPR-CAS system from which nuclease activity has been removed. can do.
  • nucleic acid encoding the guide RNA or the nucleic acid encoding the nucleic acid metabolizing enzyme one incorporated in a vector can be used.
  • the type of the vector may be a circular plasmid, and may be a linear DNA obtained by cutting the plasmid with a restriction enzyme or the like, a linear plasmid, a nucleic acid cassette fragment obtained by cutting out only the DNA fragment to be introduced, or one or both of the cassette fragments. It may be a DNA fragment to which a nucleic acid of 0.8 kb or more and 1.2 kb or less is added to the end. These DNA fragments may be DNA fragments amplified by PCR.
  • the nucleic acid to be added is not particularly limited and may be a sequence derived from a vector, but the sequence of the target site for introduction is more preferable.
  • the lower limit of the length of the nucleic acid added to the end of the cassette fragment is preferably 0.5 kb or more, more preferably 0.8 kb or more, still more preferably 1.0 kb or more.
  • the upper limit of the length of the nucleic acid added to the end of the cassette fragment is preferably 3.0 kb or less, more preferably 2.0 kb or less, still more preferably 1.5 kb or less.
  • vectors examples include pAL system (pAL51, pAL156, etc.), pUC system (pUC18, pUC19, pUC9, etc.), pBI system (pBI121, pBI101, pBI221, pBI2113, pBI101.2, etc.), pPZP system, pSMA system, etc.
  • Intermediate vector systems pLGV23Neo, pNCAT, etc.
  • cauliflower mosaic virus CaMV
  • BGMV green bean mosaic virus
  • TMV tobacco mosaic virus
  • the method for inserting the target sequence into the vector is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a purified nucleic acid is cleaved with an appropriate restriction enzyme and an appropriate vector DNA restriction enzyme is used. Examples thereof include a method of inserting into a site or a multi-cloning site and ligating to a vector.
  • the target sequence may be inserted into the intermediate vector by double cross-over, and TA cloning, In-Fusion cloning, or the like may be used.
  • nucleic acid encoding the guide RNA or the nucleic acid encoding the nucleic acid metabolizing enzyme for example, a promoter, an enhancer, an insulator, an intron, a terminator, a poly
  • a promoter, an enhancer, an insulator, an intron, a terminator, a poly is ligated to the vector. Can be done.
  • the promoter may be derived from a plant as long as it is a DNA that functions in a plant or in a plant cell and is constitutively expressed, or can induce expression in a specific tissue of a plant or at a specific developmental stage. It does not have to be. Specific examples include, for example, cauliflower mosaic virus (CaMV) 35S promoter, El2-35S omega promoter, noparin synthase gene promoter (Pnos), corn-derived ubiquitin promoter, rice-derived actin promoter, tobacco-derived PR protein promoter, ADH. Promoters, RuBisco promoters and the like can be mentioned. Translation efficiency can be increased by using a sequence that enhances translation activity, for example, an omega sequence of tobacco mosaic virus.
  • a protein can be translated from a plurality of coding regions by inserting an IRES (internal ribosome entry site) as a translation start region on the 3'-downstream side of the promoter and on the 5'-upstream side of the translation start codon.
  • IRES internal ribosome entry site
  • the terminator may be any sequence that can terminate the transcription of the gene transcribed by the promoter and has a poly A addition signal.
  • the terminator of the nopaline synthase (NOS) gene and the octopine synthase (OCS) gene examples include a terminator and a CaMV 35S terminator.
  • the selection marker gene examples include herbicide resistance genes (biaraphos resistance gene, glyphosate resistance gene (EPSPS), sulfonylurea resistance gene (ALS), etc.), drug resistance genes (tetracycline resistance gene, ampicillin resistance gene, canamycin). Resistance genes, hyglomycin resistance genes, spectinomycin resistance genes, chloramphenicol resistance genes, neomycin resistance genes, etc.), fluorescent or luminescent reporter genes (luciferase, ⁇ -galactosidase, ⁇ -glucuronidase (GUS), green fluorescence Examples include enzyme genes such as protein (GFP), neomycin phosphotransferase II (NPT II), and dihydrofolate reductase. However, according to the present invention, it is possible to prepare a transformant without introducing a selectable marker gene.
  • the target sequence to be inserted into the vector may be a plurality of types per vector, and the recombinant vector to be coated on the fine particles may be a plurality of types per fine particle. Further, a recombinant vector containing a nucleic acid encoding the guide RNA and / or a nucleic acid encoding the nucleic acid metabolizing enzyme and a recombinant vector containing a drug resistance gene are separately prepared, mixed and coated with fine particles. You may shoot into the plant tissue.
  • the target gene for genome editing may be two or more, or a vector may be constructed so that two or more target genes can be cleaved. In that case, two or more kinds of vectors may be prepared, or a plurality of guide RNAs may be inserted so as to be expressed on one plasmid.
  • the guide RNA and the nucleic acid-metabolizing enzyme may be naturally occurring (combinations) or may be non-naturally occurring combinations.
  • the tissue into which a deletion, insertion, or substitution is introduced into the SD1 gene of wheat is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include shoot apex, callus, and scutellum.
  • the method for introducing deletion, insertion, or substitution into the SD1 gene of wheat is not particularly limited and may be appropriately selected depending on the intended purpose. However, fine particles coated with the genome editing means are used as wheat stems. The method of shooting at the top is preferable.
  • the stem apex includes a growth point (stem apex meristem) at the tip of the stem, a growth point, and a tissue consisting of several leaf primordia generated from the growth point.
  • a growth point stem apex meristem
  • tissue consisting of several leaf primordia generated from the growth point.
  • only the hemispherical (dome-shaped) growth point from which the leaf primordia is removed may be used as the shoot apex, and the growth point, the shoot apex containing the leaf primordia, and the plant tissue containing the shoot apex.
  • the cells to be introduced are not particularly limited and may be appropriately selected depending on the intended purpose, but cells of shoot apical meristem are preferable, and L2 cells in the L2 layer in shoot apical meristem are more preferable. , Cells that migrate to the germline in shoot apical meristems are more preferred.
  • the germ cell lineage is a general term for germ cells ranging from primordial germ cells, which are the source of germ cells, to egg cells and sperm cells, which are the final products, and the L2 layer is 2 from the outside of the shoot apical division tissue. The second cell layer.
  • the shoot apex is not particularly limited and may be appropriately selected depending on the intended purpose, but the shoot apex of a ripe seed embryo or the shoot apex of an immature embryo is preferable.
  • the ripe seed embryo (ripe embryo) is an embryo of a ripe seed, and the ripe seed means a seed that has completed the ripening process after pollination and is fully ripe as a seed. Whether or not the ripening process is completed can be determined by whether or not the water content of the seed is 35% or less.
  • the immature embryo is an embryo of an immature seed, and the immature seed means an embryo that is not fully ripe as a seed. Whether or not the seeds are immature can be determined by the degree of ripening of the seeds (the water content of the seeds is greater than 35%) as described above.
  • the shoot apex of the immature embryo is not particularly limited and may be appropriately selected depending on the intended purpose, but the shoot apex of the embryo 8 to 35 days after pollination is preferable, and the embryo 10 to 20 days after pollination.
  • the shoot apex of the embryo is more preferable, and the shoot apex of the embryo 15 to 20 days after pollination is further preferable.
  • the seed is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include natural seeds and artificial seeds, but natural seeds are preferable. Examples of the natural seeds include seeds obtained by cultivating (or culturing) natural or similar conditions, seeds obtained by greenhouse cultivation, seeds obtained from tissue culture such as in vitro seedlings, and the like. The artificial seeds include those to be developed in the future as long as a transformable shoot apex can be obtained.
  • the method for exposing the shoot apex is not particularly limited and may be appropriately selected depending on the intended purpose. For example, endosperm, pods, leaf primordia, and excess scutellum are removed from ripe seeds or immature seeds. The method can be mentioned. Specifically, the shoot apex is exposed by removing the sheath leaf and the leaf primordia from the ripe seed or the immature seed, and then the endosperm and the excess scutellum portion are excised, and the embryo containing the exposed shoot apex and the embryo. Place the scutellum on the agar medium with the shoot apex facing up.
  • the means for removing the sheath leaf and the leaf primordia, or the seed coat and the cotyledon is not particularly limited as long as the sheath leaf and the leaf primordia, or the seed coat and the cotyledon can be removed under a stereomicroscope.
  • examples thereof include instruments for piercing such as needles having a diameter of about 0.2 mm, tweezers, pipettes, syringes, and cutting instruments such as scalpels and cutters.
  • a cutting instrument such as a scalpel can be mentioned.
  • a virus-free transformant can be obtained.
  • the fine particles coated with the genome editing means can be obtained by coating the fine particles with the genome editing means.
  • the fine particles (microcarriers) are not particularly limited and may be appropriately selected depending on the intended purpose. Those that are active and do not easily harm the living body are preferable, and examples thereof include metal fine particles, ceramic fine particles, and glass fine particles.
  • the metal fine particles are not particularly limited and may be appropriately selected depending on the intended purpose, and examples thereof include metal simple substance fine particles and alloy fine particles. As the simple metal fine particles, gold particles, tungsten particles and the like are preferable.
  • the lower limit of the average particle size of the fine particles is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.3 ⁇ m or more, more preferably 0.4 ⁇ m or more, and further preferably 0.5 ⁇ m or more. Preferably, 0.6 ⁇ m is particularly preferable.
  • the upper limit of the average particle size of the fine particles is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1.5 ⁇ m or less, more preferably 1.4 ⁇ m or less, still more preferably 1.3 ⁇ m or less. , 1.2 ⁇ m or less is particularly preferable, 1.1 ⁇ m is even more preferable, and 1.0 ⁇ m or less is most preferable.
  • the average particle size is not particularly limited and may be appropriately selected depending on the intended purpose, and examples thereof include a number average particle size.
  • the shape of the fine particles is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include spherical shape, cube shape, rod shape and plate shape, and among these, spherical shape is preferable.
  • the coating method is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the fine particles are washed and sterilized, and the fine particles, nucleic acids (recombinant vector, linear DNA, RNA, etc.) and / Or add protein, CaCl 2 , spermidine, etc. with stirring with a vortex mixer or the like, coat (coat) the nucleic acid and / or protein with fine particles, and wash with ethanol or phosphate buffered saline (PBS, etc.).
  • PBS phosphate buffered saline
  • the fine particles can be applied to the macrocarrier film as uniformly as possible using a pipetman or the like, and then dried in a sterile environment such as a clean bench.
  • a sterile environment such as a clean bench.
  • hydrophilic macrocarrier film a hydrophilic film may be attached to the macrocarrier film, or a hydrophilic coating may be applied.
  • a hydrophilic coating may be applied.
  • the method for making a film hydrophilic include a method using a surfactant, a photocatalyst, and a hydrophilic polymer.
  • the hydrophilic polymer is not particularly limited and may be appropriately selected depending on the intended purpose. Polyethylene glycol, hydroxyethyl methacrylate, hydroxypropyl methacrylate, dihydroxyethyl methacrylate, diethylene glycol methacrylate, triethylene glycol methacrylate, polyethylene glycol methacrylate, etc.
  • Hydrophilic monomers such as vinylpyrrolidone, acrylic acid, acrylamide, dimethylacrylamide, glucoxyoxyethyl methacrylate, 3-sulfopropylmethacryloxyethyldimethylammonium betaine, 2-methacryloyloxyethylphosphorylcholine, 1-carboxydimethylmethacryloyloxyethylmethaneammonium Polymers can be mentioned.
  • the coverage of the fine particles is not particularly limited and may be appropriately selected depending on the intended purpose, and may be the entire surface or a part of the fine particles.
  • the means for shooting the fine particles is not particularly limited as long as the fine particles can be shot into plant cells, and examples thereof include a particle gun (gene gun) in the particle gun method. From the viewpoint of introduction efficiency into a plant, a method of introducing into a ripe seed embryo or an immature seed embryo by using a particle gun method is preferable.
  • the particle gun method is a method in which the fine particles are coated with the genome editing means and shot into a cell tissue, and is effective when the infection efficiency of Agrobacterium is low as in monocotyledonous plants.
  • the genome editing method using the particle gun method is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a macrocarrier film coated with fine particles, a target ripe embryo, or a stem of an immature embryo is an example.
  • An example is a method in which a plate on which the top is placed is installed in a particle gun device, and high-pressure gas is emitted from a gas acceleration tube toward a macrocarrier film.
  • the macrocarrier film stops at the stopping plate, but the fine particles coated on the macrocarrier film pass through the stopping plate and penetrate into the target placed under the stopping plate, and the target gene is introduced.
  • the high-pressure gas is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include helium.
  • the particle gun device is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include Biolistic (registered trademark) PDS-1000 / He Particle Delivery System (BIO-RAD).
  • the upper limit of the distance between the stopping plate and the target shoot apex is, for example, preferably 9 cm or less, more preferably 8 cm or less, further preferably 7 cm or less, and particularly preferably 6 cm or less.
  • As the lower limit of the distance between the stopping plate and the shoot apex as a target for example, 2 cm or more is preferable, 3 cm or more is more preferable, and 4 cm or more is further preferable.
  • the gas pressure of the particle gun device is preferably 1,100 to 1,600 psi, more preferably 1,200 to 1,500 psi, depending on the type of fine particles.
  • the upper limit of the number of times the fine particles are shot into the shoot apex is preferably 20 times or less, more preferably 15 times or less, still more preferably 10 times or less.
  • the guide RNA, the nucleic acid metabolizing enzyme, the nucleic acid encoding these, and the like contained in the genome editing means are released from the fine particles in the cells into which the fine particles are shot, and in the case of nucleic acids, , Nucleic acid translocates to the nucleus and expresses a nucleic acid-metabolizing enzyme to cleave genomic DNA, and in the process of its repair, mutation occurs to obtain a genome-edited cell.
  • genome editing occurs by translocating to the nucleus (organelle) by a nuclear localization signal (which may be an organelle translocation signal) and cleaving DNA.
  • Transformed cells can also be obtained by releasing the genome editing means from the fine particles and integrating nucleic acids or the like into genomic DNA in the cells into which the fine particles have been shot.
  • a nucleic acid that proliferates in the form of a plasmid such as geminivirus or an artificial chromosome is introduced, it may be transformed without integration.
  • the other steps are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a water absorption step before exposing the shoot apex.
  • Water absorption process before exposing the shoot apex- Ripe seeds or immature seeds can be made to absorb water before the shoot apex is exposed. If necessary, vernalization treatment (breaking dormancy) may be performed before water absorption. Water absorption is performed by soaking the seeds in water and incubating them. Alternatively, the embryo excluding the endosperm and the like may be incubated on an agar medium.
  • the water absorption temperature is preferably 15 to 25 ° C. At this time, the water may be changed at least once.
  • the water absorption period is preferably before the radicles start to grow (roots of 1 mm or less) or before new leaf primordia are formed.
  • water absorption time it is less than 16 hours after water absorption, preferably 12 hours, although it depends on the dormant state of the seed.
  • the vernalization treatment is not particularly limited and may be appropriately selected depending on the purpose.
  • the treatment can be performed at 4 ° C. for 2 days.
  • the method for producing a wheat having an SD gene mutation of the present invention includes a step of introducing deletion, insertion or substitution into the SD1 gene of wheat by genome editing means, and deletion, insertion or substitution in the SD1 gene of the wheat. It is possible to include a step of growing a tissue into which the above-mentioned plant has been introduced to obtain a plant body, and further include other steps.
  • the steps of introducing a deletion, insertion, or substitution into the SD1 gene of wheat by genome editing means are as described above. -The process of growing a tissue in which a deletion, insertion, or substitution has been introduced into the SD1 gene of wheat to obtain a plant body-
  • the growth method is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the introduced-treated ripe embryos or tissues such as shoot apex, callus and scutellum of immature embryos are placed on an agar medium. After growing for about one month, there is a method of transplanting to soil.
  • a transformant can be obtained by growing in a normal medium (without containing antibiotics, plant hormones, etc.) without applying selective pressure with a drug, but a drug resistance gene can be obtained. May be further introduced.
  • a genome editing individual can be selected by simultaneously introducing a nuclease and a drug resistance gene.
  • transformed cells When a drug resistance gene is introduced, transformed cells can be selectively cultured by the drug.
  • a selective drug suitable for shoot apex culture for example, a sulfonylurea herbicide chlorosulfuron (resistance can be acquired by introducing a mutant ALS gene (acetobutyric acid synthase gene)) is known.
  • the drug resistance gene When introducing a drug resistance gene, the drug resistance gene may be on the same vector as the target gene or the nuclease gene, or may be on a different vector. When inserted on separate vectors, when they are integrated into different chromosomes, the plant individual into which the gene of interest has been introduced by self-pollination or backcrossing to progeny, or the genome-edited plant individual It has the advantage that it can be separated into individual plants having a drug resistance gene.
  • the process of selecting a genome-edited plant from the plant- is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the individual after introduction or the growing individual after introduction can be selected.
  • the presence or absence of the RNA can be confirmed by, for example, the RT-PCR method. It may be detected by Northern blotting.
  • the presence or absence of the protein can be confirmed by, for example, staining of plant pieces, electrophoresis, ELISA method, RIA, dot immunobiding assay, and / or Western blotting.
  • the process of growing a genome-edited plant is not particularly limited and may be appropriately selected depending on the intended purpose.
  • seeds after germination are artificially prepared at 20 ° C. in a long-day environment (16 hL / 8 hD). It can be grown in a meteorological room.
  • a genome-edited plant can be produced, and the plant can be further grown.
  • the plant thus produced stably expresses the trait of the target gene or the genome-edited gene, or the expression of the target gene is suppressed, and the plant is normally inherited (transmitted) to progeny. ).
  • DNA is extracted from the individual after introduction or the growing individual after introduction, and the target gene is genome-edited by PCR method and / or electrophoresis, Southern blotting, or CAPS analysis. It is possible to detect whether or not it is present, and the genome editing efficiency is calculated from the number of explants used for introduction and the number of growing individuals whose target gene has been genome-edited.
  • the implanter transformation method is generally a transformation method that does not include the operation of tissue culture, and is a method of transforming cells at a growth point site while the plant is growing.
  • the implanter transformation method is used in the sense that it includes only the tissue culture method using shoot apex culture and does not include other methods including tissue culture operations.
  • the implanter method (In plant method) has the same meaning.
  • the wheat having the SD1 gene mutation of the present invention can be obtained by the above-mentioned method for editing the genome of wheat or the above-mentioned method for producing wheat having the SD1 gene mutation.
  • the wheat having the SD1 gene mutation of the present invention has a deletion, insertion, or substitution introduced into the SD1 gene.
  • As the wheat having the SD1 gene mutation it is preferable that a deletion, insertion, or substitution is introduced into the SD1 gene in the entire ABD genome.
  • the upper limit of the culm length of wheat having the SD1 gene mutation is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 85 cm or less, more preferably 80 cm or less, still more preferably 75 cm or less.
  • the lower limit of the culm length of wheat having the SD1 gene mutation is not particularly limited and may be appropriately selected depending on the intended purpose, but 60 cm or more is preferable.
  • Example 1 Acquisition of wheat SD1 gene sequence>
  • the protein sequence information of wheat GA20ox was obtained using the Gramene database (http://www.gramene.org/) and compared with GA20ox1, GA20ox2, GA20ox3, and GA20ox4 of rice.
  • the phylogenetic tree was classified into four clades composed of wheat (A, B, and D genomes) and rice protein (Fig. 1A). The bootstrap value was calculated at 1,000.
  • TRIAE CS42 3AL TGACv1 194412 AA06325900.1 SEQ ID NO: 2
  • TRIAE CS42 3B TGACv1 223804 AA0787660.1
  • TRIAE 42 which are the amino acid sequences of each SD1 in the A, B, and D genomes of wheat.
  • TGACv1 249609 AA0852340.1. SEQ ID NO: 4
  • SEQ ID NO: 4 showed 77-78% identity with rice SD1 (SEQ ID NO: 1) (FIG. 1B).
  • SD1 of wheat consists of three exons, of which the following target sequences 1 are used as target sites for genome editing from the sequences common to each SD1 gene in the A, B, and D genomes of wheat.
  • the target sequence 2 and the targets 1 to 3 represented by the target sequence 3 were determined (FIG. 1C).
  • Targets 1 and 3 were designed for exon 1, and target 2 was designed for exon 2.
  • the restriction enzyme recognition sequences were designed so as to be close to the PAM sequences, respectively.
  • Target sequence 1 CGCGGTGTACCGACTCCGGGAGGG (SEQ ID NO: 11)
  • Target sequence 2 GGGCTGGAGGTCCTCGTCGACGG (SEQ ID NO: 12)
  • Target sequence 3 GGGCTGGAGGTCCTCGTCGACGG (SEQ ID NO: 13)
  • Example 3 In vitro digestion analysis> The efficiency of the designed sgRNA was confirmed by in vitro digestion.
  • Example 3-1 Amplification of DNA fragment containing target sequence by PCR >> Using wheat genomic DNA, sequences common to the A, B and D genomes were selected and approximately 300 bp PCR fragments containing each target site were amplified. To the PCR reaction solution, add 20 ng of genomic DNA, TaKaRa LA Taq with GC buffer (registered trademark, TaKaRa) 0.25 U, 2X GC Buffer I 5.0 ⁇ L, 2.5 mM dNTPs, and 2.5 pmol of each of the following primers and sterilize. It was filled up with distilled water to a total of 10 ⁇ L.
  • GC buffer registered trademark, TaKaRa
  • Primer sequence (F) targets 1 and 3): TCCCAGCATTGACCCGGTTG (SEQ ID NO: 14)
  • Primer sequence (R) targets 1 and 3): ACACCTGGAAGAACCCGGTGC (SEQ ID NO: 15)
  • Primer sequence (R) ((Target 2): GGTGTCGCCGATGTTGGATGA (SEQ ID NO: 17)
  • Example 3-2 Preparation of sgRNA >> The sgRNA was prepared using the GeneArtTM Precision gRNA synthesis kit (Thermo Fisher Scientific, USA) according to the protocol of the kit. The following primer sets were used for targets 1, 2 and 3 of sgRNA.
  • Example 3-3 Preparation of CRISPR / Cas9RNP >> Cas9 protein (0.2 ⁇ g) and sgRNA (0.2 ⁇ g) were mixed and incubated at 25 ° C. for 10 minutes. Then, 1,4-bis (3-oleoylamidepropyl) piperazine and histone H1 protein mixture (ratio 3: 1) (1 ⁇ L) were added and incubated at 25 ° C. for 5 minutes.
  • the Cas9 protein a recombinant protein of SpCas9 was produced using Escherichia coli, and then the cells were crushed and purified.
  • Example 3-4 In vitro digestion >> 1.0 ⁇ L of CutSmart buffer (registered trademark, New England BioLabs) was added to the DNA fragment (100-200 ng) containing the CRISPR / Cas9RNP and the target sequences 1, 2, or 3 to make a total of 10 ⁇ L, and 1 at 37 ° C. Incubated for hours. The reaction was stopped by heating at 100 ° C. for 5 minutes. When the product was confirmed by electrophoresis using a 3% agarose gel, the DNA fragment containing the target sequence 1 was hardly decomposed (lane 2 in FIG. 2A), and the DNA fragment containing the target sequence 3 was found. It was slightly disassembled (lane 3 in FIG. 2A). The DNA fragment containing the target sequence 2 was completely degraded (lane 5 in FIG. 2A). From the above, it was found that CRISPR / Cas9RNP, which cleaves target 2, is efficient in in vitro digestion.
  • CutSmart buffer registered trademark, New England BioLabs
  • Example 4 Confirmation of mutation introduction at the shoot apex stage by the In plant method>
  • the introduction of mutations at the shoot apex stage by the in-plat method using a particle gun was examined for the targets 1, 2 and 3.
  • Example 4-1 Preparation of mature embryos >> The ripe seeds of wheat (Haruyo Koi) were immersed in a highter (hypochlorous acid concentration 6%, Kao Corporation), shaken at 25 ° C. for 10 minutes, and then washed with sterile water in a clean bench. After washing, the cells were placed on a Kim towel moistened with sterile water and incubated at 4 ° C. for 2 days to break the dormancy. Then, it was incubated at 25 ° C. for about 12 hours and used in the following experiments.
  • Example 4-2 Exposure of shoot apex in ripe seed embryo >> Under a stereomicroscope, the sheath leaf of the embryo part of the germinated seed and the third leaf primordia were removed from the first leaf primordia using the tip of a needle (diameter 0.20 mm). The endosperm and excess scutellum were then removed using a sterile knife to completely expose the shoot apex.
  • FIG. 2B A photograph showing the apex of wheat after exposure is shown in FIG. 2B.
  • the GFP gene is introduced into the shoot apex.
  • a plasmid DNA pUC-based plasmid
  • S65T fluorescent reporter gene
  • the gene is designed to be expressed under the control of the maize ubiquitin promoter and first intron.
  • a terminator of the nopaline synthase (NOS) gene is added as the terminator.
  • MS-maltose medium (4.3 g / L MS salt, ⁇ 1 MS vitamin (Sigma), 30 g / L) was prepared by completely exposing the shoot apex so as to form a circle with a diameter of 1.0 cm from the center of the plate.
  • the cells were placed in maltose, 0.98 g / L MES, 3% PPM (plat preservative mixture, Nacalai Tesque), 7.0 g / L phytagel (registered trademark, Sigma-Aldrich, pH 5.8) (30 pieces / plate).
  • Example 4-3 Preparation of gold particles >> 90 mg (InBio Gold, Australia) of gold particles having an average particle size of 0.6 ⁇ m was weighed, 500 ⁇ L of 70% ethanol was added, and the mixture was well suspended in a vortex. Then, the gold particles were precipitated by centrifugation to remove ethanol. Then, 500 ⁇ L of RNA-free water was added to prepare an RNA-free gold particle solution.
  • Example 4-4 Preparation of sgRNA >> This was done in the same manner as in Example 3-2.
  • Example 4-5 CRISPR / Cas9RNP-Preparation of gold particles >> Mix Cas9 protein (12 ⁇ g), sgRNA (5 ⁇ g), RNase Inhibitor (40 U) (registered trademark, TaKaRa), and CutSmart Buffer (5 ⁇ L) (registered trademark, New England BioLabs) and incubate at 25 ° C. for 10 minutes. CRISPR / Cas9RNP was prepared. As the Cas9 protein, a recombinant protein of SpCas9 was produced using Escherichia coli, and then the cells were crushed and purified.
  • 1,4-bis (3-oleoylamidepropyl) piperazine and histone H1 protein mixture (ratio 3: 1) (5 ⁇ L) are added and incubated at 25 ° C. for 5 minutes, and further, the average particle size is 0.6 ⁇ m.
  • Gold particles (270 ⁇ g) were added and incubated on ice for 10 minutes.
  • centrifugation was performed, and RNA-free water (20 ⁇ L) was added again to the gold particles from which the supernatant had been removed.
  • 6 ⁇ L was poured into the center of a macro carrier (manufactured by Bio-rad) and air-dried.
  • Example 4-6 Gene transfer >> Using a particle gun (Biolistic® PDS-1000 / He Particle Delivery System (BIO-RAD)), the gold particles were shot three times per petri dish. The pressure at the time of shooting was about 94.9 kgf / cm2 (1,350 psi), and the distance to the target tissue was 6 cm. After shooting, it was allowed to stand overnight at 22 ° C. in a dark place.
  • a particle gun Biolistic® PDS-1000 / He Particle Delivery System (BIO-RAD)
  • the pressure at the time of shooting was about 94.9 kgf / cm2 (1,350 psi), and the distance to the target tissue was 6 cm. After shooting, it was allowed to stand overnight at 22 ° C. in a dark place.
  • Example 4-7 Examination of introduction of mutation by CRISPR / Cas9 in shoot apical tissue >>
  • genomic DNA was extracted from the shoot apex 2 days after iPB (shooting) and confirmed by CAPS analysis. Five shoot apexes were sampled for each target and analyzed as one sample (eg, # 1) (# 1 through # 5 in FIG. 2C).
  • Example 4-7-1 Extraction of genomic DNA >>> A PCR template is obtained by removing the shoot apex from a plant using a needle, adding 10 ⁇ L of buffer (0.1 M Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA), and heating at 95 ° C. for 10 minutes. Used as.
  • buffer 0.1 M Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA
  • Example 4-7-2 Amplification of DNA fragment containing target sequence by PCR >>> This was done in the same manner as in Example 3-1.
  • Example 4-7-3 Confirmation of mutagenesis by Cleared amplified polymorphic sequences (CAPS) >>> Fill 5 ⁇ L of PCR-amplified DNA fragment 2 ⁇ L, CutSmart Buffet 0.5 ⁇ L, restriction enzyme (Target 1: BspEI; Target 2: SalI; Target 3: FspI) (registered trademark, New England BioLabs) 0.2 ⁇ L with sterile water. It was up and digested at 37 ° C. for 5 hours. When the digested product was confirmed by electrophoresis using a 3% agarose gel, no mutation was confirmed in the samples of Target 1 and Target 3 (upper and lower rows of FIG. 2C). In lane 4 (M4) of target 2, uncut pieces of DNA fragments could be confirmed (middle of FIG. 2C). From the above, it was found that the genome is efficiently edited when the target sequence of target 2 is used in the In plant method.
  • CPS Cleared amplified polymorphic sequences
  • Examples 5-1 to 5-6 Preparation of mature embryos, exposure of shoot apex in ripe seed embryos, preparation of gold particles, preparation of sgRNA, preparation of CRISPR / Cas9RNP-gold particles, gene transfer in the same manner as in Examples 4-1 to 4-6. went.
  • Example 5-7 Examination of introduction of mutation by CRISPR / Cas9 at the T0 (5 leaf) stage >> In order to confirm the mutagenesis in T0 (5 leaves) after the mutagenesis into the shoot apical tissue by the iPB method, the target 2 was used to grow until 5 leaves appeared after iPB, and the genomic DNA was extracted from the growth. CAPS analysis was performed.
  • Examples 5-7-1 to 5-7-2 >> Amplification of the DNA fragment containing the target by PCR was carried out in the same manner as in Examples 4-7-1 and 4-7-2.
  • Example 5-7-3 Confirmation of mutagenesis by Cleared amplified polymorphic sequences (CAPS) >>> 2 ⁇ L of PCR-amplified DNA fragment, 0.5 ⁇ L of CutSmart Buffer, and 0.2 ⁇ L of restriction enzyme (SalI) (registered trademark, New England BioLabs) were filled up to 5 ⁇ L with sterile water and digested at 37 ° C. for 5 hours. The digest was confirmed by electrophoresis on a 3% agarose gel.
  • Confirmation of mutagenesis by Cleared amplified polymorphic sequences (CAPS) >>> 2 ⁇ L of PCR-amplified DNA fragment, 0.5 ⁇ L of CutSmart Buffer, and 0.2 ⁇ L of restriction enzyme (SalI) (registered trademark, New England BioLabs) were filled up to 5 ⁇ L with sterile water and digested at 37 ° C. for 5 hours. The digest was confirmed by electrophoresis on a 3% agarose gel.
  • CRISPR / Cas9RNP was introduced into the shoot apical tissue by the iPB method, genomic DNA was extracted from the 5 leaves after growth, and the following primers specific for the A, B, and D genomes (SEQ ID NOS: 16 and 18 to 18 to D) were extracted. A DNA fragment was prepared using 20), and the introduction of mutations into each genomic DNA was confirmed by the CAPS method. For each genome, 16 samples (H1 to H16) were analyzed. When the A, B, and D genomes were analyzed by CAPS, it was confirmed that there was a residue due to the introduction of the mutation (Fig. 3A). Among them, the DNA sequences of H1, H3, H4, and H7 were confirmed by the Sanger method (FIG.
  • Genome A F: CGGACAGCAGCTCCATCATG (SEQ ID NO: 16) Genome A (R): CTGATACGAGCAGCAGTAGC (SEQ ID NO: 18) B Genome (F): CGGACAGCAGCTCCATCATG (SEQ ID NO: 16) B Genome (R): GCAAGGAAGGTGACCCAATT (SEQ ID NO: 19) D Genome (F): CGGACAGCAGCTCCATCATG (SEQ ID NO: 16) D Genome (R): AGCAACGCTGAGAGAGGAGGT (SEQ ID NO: 20)
  • Example 6 Confirmation of mutation introduction at the T1 plant (T1 seed) stage>
  • the T1 seed is germinated and the genomic DNA is extracted from the first leaf generated from the seed, and the same as in Example 6.
  • DNA fragments were prepared using primers specific to the A, B, and D genomes, and the introduction of mutations into each genomic DNA was confirmed by the CAPS method.
  • the plant H7-1 all of the A, B, and D genomes were found to be uncut by CAPS analysis (Fig. 4A).
  • Table 1 shows the mutation introduction efficiency in the wheat genome editing method.
  • CRISPR / Cas9RNP which mutates target 2 of the SD1 gene into 232 ripe seeds, was introduced into the shoot apical tissue. Mutation introduction was confirmed in 16 (6.7%) of the 5 leaves of T0. Mutations were found in 2 (0.9%) of these T1 seeds (Table 1).
  • T2 seeds (sd1 mutant) obtained from the T1 plant of Example 7 and seeds after germination of the original variety "Haruyo Koi" were subjected to about 20 ° C. in a long-day environment (16 hL / 8 hD) in an artificial weather room. After growing for 120 days, the plant height of the T2 plant is shown in FIGS. 5A and 5B.
  • the two strains on the left in FIG. 5A are sd1 mutants (sd1), and the two strains on the right are wild-type strains (WT: Haruyo Koi).
  • the left bar in FIG. 5B is the sd1 mutant (sd1), and the right bar is the wild-type strain (WT: Haruyo Koi).
  • a wheat genome editing method comprising a step of introducing a deletion, insertion, or substitution into a wheat SD1 gene by a genome editing means.
  • the genome editing means comprises either a guide RNA targeting the SD1 gene or a nucleic acid encoding a guide RNA targeting the SD1 gene, and a nucleic acid metabolizing enzyme or a nucleic acid encoding the nucleic acid metabolizing enzyme.
  • the method for editing a wheat genome according to any one of ⁇ 1> to ⁇ 2> which comprises any of the above.
  • ⁇ 4> The wheat genome editing method according to ⁇ 3>, wherein the nucleic acid-metabolizing enzyme is a nuclease or deaminase.
  • ⁇ 5> The wheat genome editing method according to any one of ⁇ 2> to ⁇ 4>, wherein the average particle size of the fine particles is 0.3 ⁇ m or more and 1.5 ⁇ m or less.
  • the shoot apex of the wheat is the shoot apex of the ripe seed embryo, and the shoot apex of the ripe seed embryo removes endosperm, sheath leaves, leaf primordia, and excess scutellum from the ripe seed.
  • the method for editing a wheat genome according to any one of ⁇ 2> to ⁇ 5>, which is an exposed shoot apex.
  • the shoot apex of the wheat is the shoot apex of an immature embryo, and the shoot apex of the immature embryo is from immature seeds 8 to 35 days after pollination to endosperm, sheath leaves, leaf primordia, and extra scutellum.
  • the method for editing the genome of wheat according to any one of ⁇ 2> to ⁇ 5> above, which is the shoot apex exposed by removing the above-mentioned.
  • a step of introducing a deletion, insertion, or substitution into the SD1 gene of wheat by a genome editing means, and a tissue into which the deletion, insertion, or substitution is introduced into the SD1 gene of wheat are grown to grow a plant. It is a method for producing wheat having an SD1 gene mutation, which comprises a step of obtaining the wheat.
  • ⁇ 9> The wheat according to ⁇ 8>, wherein the step of introducing deletion, insertion, or substitution into the SD1 gene of the wheat is a step of shooting fine particles coated with the genome editing means into the shoot apex of the wheat. Genome editing method.
  • ⁇ 11> The method for producing wheat having the SD1 gene mutation according to any one of ⁇ 8> to ⁇ 10>, further comprising the step of growing the genome-edited plant.
  • ⁇ 12> A wheat having an SD1 gene mutation characterized in that a deletion, insertion, or substitution has been introduced into the SD1 gene.
  • ⁇ 13> The wheat according to ⁇ 12> above, wherein a deletion, insertion, or substitution has been introduced into the SD1 gene in the entire ABD genome.
  • ⁇ 14> The wheat according to any one of ⁇ 12> to ⁇ 13>, wherein the culm length is 85 cm or less.

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Abstract

L'invention concerne un procédé d'édition de génome dans le blé comprenant une étape d'introduction d'une délétion, d'une insertion ou d'une substitution dans le gène SD1 dans le blé par un moyen d'édition de génome ; et un procédé de production de blé ayant une mutation dans le gène SD1 comprenant une étape d'introduction d'une délétion, d'une insertion ou d'une substitution dans le gène SD1 dans le blé par un moyen d'édition de génome, et une étape de culture d'un tissu ayant la délétion, l'insertion ou la substitution introduite dans le gène SD1 dans le blé pour produire un corps végétal.
PCT/JP2020/034298 2019-10-10 2020-09-10 Procédé d'édition de génome dans le blé et son utilisation WO2021070549A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017195906A1 (fr) * 2016-05-13 2017-11-16 株式会社カネカ Procédé d'édition du génome d'une plante
JP2019524147A (ja) * 2016-08-17 2019-09-05 モンサント テクノロジー エルエルシー 収穫可能収量を増加させるためのジベレリン代謝操作を介して低草高植物を得るための方法及び組成物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017195906A1 (fr) * 2016-05-13 2017-11-16 株式会社カネカ Procédé d'édition du génome d'une plante
JP2019524147A (ja) * 2016-08-17 2019-09-05 モンサント テクノロジー エルエルシー 収穫可能収量を増加させるためのジベレリン代謝操作を介して低草高植物を得るための方法及び組成物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HARUYASU HAMADA, YUELIN LIU, YOZO NAGIRA, RYUJI MIKI, NAOAKI TAOKA, RYOZO IMAI: "Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat", SCIENTIFIC REPORTS, vol. 8, no. 1, 1 December 2018 (2018-12-01), XP055646508, DOI: 10.1038/s41598-018-32714-6 *

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