WO2024063076A1 - Production method, broccoli, and seed - Google Patents

Production method, broccoli, and seed Download PDF

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WO2024063076A1
WO2024063076A1 PCT/JP2023/034032 JP2023034032W WO2024063076A1 WO 2024063076 A1 WO2024063076 A1 WO 2024063076A1 JP 2023034032 W JP2023034032 W JP 2023034032W WO 2024063076 A1 WO2024063076 A1 WO 2024063076A1
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broccoli
aco3
gene
seeds
harvest
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French (fr)
Japanese (ja)
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信也 肥塚
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学校法人玉川学園
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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
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • 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
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • 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

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  • the present invention relates to a production method, broccoli, and seeds.
  • Genome editing technology has conventionally existed (for example, see Patent Document 1).
  • the present invention was made in view of this situation, and aims to provide crops with more suitable properties using genome editing and to promote understanding of genome editing.
  • a method for producing broccoli includes: Genetically manipulate the target broccoli to knock out at least one of ACO2 and ACO3, which generate ethylene that degrades within a predetermined period of time after harvest, among the genes of the target broccoli.
  • the broccoli or seeds of one embodiment of the present invention are It is produced from broccoli that has been genetically engineered to knock out at least one of ACO2 and ACO3, which generate ethylene that deteriorates over a predetermined period of time after harvest, or from said broccoli.
  • FIG. 1 is a diagram showing an example of the difference in broccoli senescence depending on whether or not the ACO3 gene is knocked out, including a method for producing broccoli according to one embodiment of the present invention.
  • FIG. 2 is a diagram showing an example of the influence of knockout of the ACO3 gene of FIG. 1 on aging of broccoli. It is a diagram showing the resynthesis route of ethylene.
  • FIG. 4 is a diagram showing the ethylene resynthesis route of FIG. 3 in more detail.
  • FIG. 2 is a diagram showing the flow of the entire experiment. It is a diagram showing an image of gene introduction. It is a figure showing an example of Ti plasmid and CFRISPR/Cas9 vector in Agrobacterium method.
  • FIG. 2 is a diagram showing primer sequence information used for amplifying the ACO3 gene fragment. This is an example of the base sequence of an ACO3 transformant.
  • FIG. 3 is a diagram showing the results of direct sequence analysis.
  • FIG. 2 is a diagram showing a flow of the broccoli production method of the present invention. It is a figure showing the result of a flower bud deterioration test using T1 generation.
  • FIG. 1 shows the ACO3 mutant line (1) 210611-6.
  • FIG. 1 shows the ACO3 mutant line (2) 210226-1.
  • FIG. 2 shows ACO2 mutant line (1) 230306-1.
  • FIG. 3 shows ACO2 mutant line (2) 230306-3.
  • FIG. 3 shows ACO2 mutant line (3) 230516-1.
  • FIG. 2 shows ACO2 mutant line (4) 230516-2.
  • broccoli is used as an example of a crop.
  • the background and purpose of broccoli being adopted will be explained below.
  • broccoli is a green-yellow vegetable whose edible parts are stems and flower buds. It is harvested and commercialized when the inflorescence structure is immature and growing rapidly. Moreover, as mentioned above, broccoli turns yellow about three days after being harvested. Specifically, yellowing of florets occurs after harvest due to lack of chlorophyll. This tissue degeneration seen after broccoli is harvested is not a typical aging process, but is caused by strongly disrupted metabolic processes. It has been revealed that this yellowing after harvest is caused by ethylene. Thus, the lifespan of fresh broccoli is short, and yellowing has been a cause of discarding broccoli.
  • ACO ACC oxidases
  • the first ACO gene (hereinafter appropriately referred to as "ACO1 gene") is a gene that contributes to basic ethylene synthesis. Ethylene generated through involvement of the ACO1 gene contributes to the aging of vegetative tissues.
  • the second ACO gene (hereinafter appropriately referred to as "ACO2 gene”) is a gene that contributes to ethylene synthesis in reproductive organs. Ethylene generated through the involvement of the ACO2 gene contributes to early senescence after harvest.
  • the third ACO gene (hereinafter appropriately referred to as "ACO3 gene”) is a gene whose expression is synchronized with etiolation, and is a gene that contributes to the synthesis of ethylene after harvest. Ethylene generated through the involvement of the ACO3 gene contributes to late senescence after harvest.
  • FIG. 1 is a diagram showing an example of the difference in aging of broccoli depending on the presence or absence of knockout of the ACO3 gene, including the broccoli production method of one embodiment of the present invention.
  • FIG. 2 is a diagram showing an example of the influence of knockout of the ACO3 gene in FIG. 1 on aging of broccoli.
  • flower buds appear about 40 days after sowing and planting. When these flower buds grow to a certain size, they are harvested. For example, broccoli harvested from late at night to early morning is displayed in stores on the same day as harvest as "morning-picked broccoli.” The number written in the upper left corner of the broccoli indicates the number of days since harvest. Broccoli on the 0th or 1st day after harvest is green, as indicated by dark hatching. Then, for example, on the 2nd day after harvest, some of the broccoli flower buds turn yellow, as indicated by light hatching. Then, for example, on the 3rd day after harvest, all of the broccoli flower buds turn yellow. Broccoli with some or all of its flower buds turning yellow has reduced commercial value.
  • the ACO2 gene and the ACO3 gene are knocked out.
  • some or all of the broccoli flower buds can be displayed on store shelves without yellowing, for example, even on the second or third day after harvest.
  • the ACO2 gene and ACO3 gene are knocked out while leaving the ACO1 gene intact. That is, as shown in FIG. 2, the ACO1 gene is expressed in the same manner as in the conventional FIG. 1(A). As a result, the flower buds grow (senescence) as usual.
  • the ACO2 gene is conventionally expressed 2 hours after harvest. It also contributes to ethylene generation in the early period after harvest. However, in this embodiment, since the ACO2 gene is knocked out, the ACO2 gene is not expressed, and ethylene is not generated due to the contribution of the ACO2 gene in the early stage after harvest. This prevents yellowing of broccoli florets in the early stage after harvest.
  • the ACO2 gene has conventionally been triggered by harvest, and is expressed from about the second or third day after harvest. It also contributes to ethylene generation in the late period after harvest. However, in this embodiment, since the ACO3 gene is knocked out, the ACO3 gene is not expressed, and ethylene is not generated due to the contribution of the ACO3 gene in the late period after harvest. This prevents yellowing of broccoli florets in the late stage after harvest.
  • FIG. 3 is a diagram showing the resynthesis route of ethylene.
  • FIG. 4 is a diagram showing the ethylene resynthesis route of FIG. 3 in more detail.
  • Ethylene is synthesized in the order of methionine, S-adenosylmethionine, 1-aminocyclopropane-1-carboxylic acid (hereinafter appropriately abbreviated as "ACC"), and ethylene.
  • ACS is an enzyme that catalyzes the reaction of S-adenosylmethionine to ACC.
  • ACO is an enzyme that catalyzes the reaction of ethylene from ACC.
  • ACC which is the enzyme in the last step of ethylene biosynthesis, includes ACC Ox1 (cDNA caused by the ACO1 gene), ACC Ox2 (cDNA caused by the ACO2 gene), and Bo-ACO3 (cDNA caused by the ACO3 gene). cDNA) exists.
  • Translation levels of ACO1 are low in whole inflorescences at harvest, but increase markedly after harvest. It also increases in sepals after harvest and in yellowed leaves that are cut. It is hardly expressed in reproductive organs. Translation of ACO2 is detected only in the reproductive organs and is not expressed at the time of harvest, but within 2 hours it begins to increase and accumulates in large amounts. That is, there is no change in the translation level of ACC Ox1 by IAA and ABA treatment or wound treatment. ACO2 increases with abscisic acid and propylene treatment. Expression of the ACO3 gene was observed on the third day after harvest.
  • ACO1 is involved in basic ethylene production and senescence in vegetative tissues (such as leaves), as described above.
  • ACO2 since ACO2 is expressed only in the reproductive organs of harvested florets, it is involved in the production of ethylene in the reproductive organs when stimulated at the time of harvest. Involved in early degeneration in other organs. ACO3 is involved in senescence not through early ethylene synthesis after harvest but through late ethylene synthesis.
  • the present invention aims to introduce knockout mutations targeting ACO3 in addition to ACO2, and performs genome editing using the Agrobacterium method. As a result, it is possible to verify that broccoli is improved by not expressing ACO3, and to commercialize broccoli in which these knockouts have been performed.
  • ACO 1-aminocyclopropane-1-carboxylic acid oxidase
  • FIG. 5 is a diagram showing the flow of the entire experiment.
  • the hypocotyls were sectioned. Next, it was immersed in Agrobacterium culture solution. As a result, Cas9, gRNA, and marker genes were introduced. Next, culture, sterilization, and callus induction are performed. Next, we trained the shoots. Next, total DNA was isolated. Next, PCR amplification of the target gene fragment was performed. Then, the mutant type was identified by restriction enzyme digestion. More specifically, for the ACO gene, CRISPR-Cas9 genome editing constructs were constructed, and Agrobacterium carrying them was infected with hypocotyl sections of broccoli green ridges to generate transformants.
  • ACO2 and ACO3 are the genes whose expression levels increase after harvesting, so we created constructs that can edit ACO2 and ACO3 individually. .
  • the mutation introduction site of the target gene was amplified by PCR, the base sequence of the fragment was determined, and the presence or absence of mutation introduction was analyzed.
  • FIG. 6 is a diagram showing an image of gene introduction.
  • the binary vector used in this experiment has a structure containing gRNA, Cas9 (transgene), and hygromycin regime gene (selection marker gene).
  • gRNA, Cas9 (transfer gene), and hygromycin regime gene selection marker gene
  • gRNA, Cas9 (transfer gene), and hygromycin regime gene selection marker gene
  • Ru The binary vector pZH_gYSA_FFCas9 used in this experiment was constructed based on the one provided by the National Agriculture and Food Research Institute under a material provision agreement (MTA).
  • FIG. 7 is a diagram showing an example of a Ti plasmid and a CFRISPR/Cas9 vector in the Agrobacterium method.
  • the Ti plasmid used in this experiment contains a plant hormone production-related gene (T-DNA), a group of genes involved in DNA transfer, and an origin of replication (Agrobacterium).
  • T-DNA plant hormone production-related gene
  • Agrobacterium an origin of replication
  • the CRISPR/Cas9 vector used in this experiment contained T-DNA consisting of gRNA, Cas9 (transgene), and hygromycin regime gene (selection marker gene), and an origin of replication (Agrobacterium Contains E. coli).
  • FIG. 8 is a diagram showing the flow of the CRISPR-Cas9 method.
  • the hypocotyl is sectioned and immersed in Agrobacterium culture solution to infect it with Agrobacterium and introduce the CRISPR/Cas9 vector. That is, in the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 method, Cas9 and sgRNA are expressed and the guide RNA binds to the gene, thereby inducing Cas9 and causing genetic modification (CRISPR/Cas9 introduction of vectors) It will be done. After that, shoots are created through culturing, sterilization, and callus induction.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Broccoli seeds were transformed using Ryokurei, an F1 variety available from Sakata Seed.
  • the equipment to be prepared is as follows. (1) Sterilized filter paper A (advantech quantitative filter paper No. 2, ⁇ 125 mm, wrapped in foil, autoclaved, and dried) (2) Sterile filter paper B (Whatman 5 quantitative filter paper, ⁇ 70mm, autoclaved in a petri dish and dried) (3) Luce tweezers (17 cm, various manufacturers) (4) Razor (feather blue box double-edged) (5) Leather blade folder (Kenneth etc.) Scalpel (feather, stainless steel surgical blade scalpel 11) (6) 50 mL disposable tube (Corning, 430290, etc.) (7) Plastic petri dish (IWAKI sterile petri dish deep 90 mm x 20 mm, etc.) (8) Surgical tape (3M Micropore code: 1530-0, etc.) (9) 5 mL disposable pipette (Falcon, #357543)
  • the specifications of the culture medium for this experiment are as follows. Kohno-Murase et al. Broccoli was transformed by partially modifying the method for transforming Brassica napus reported by.
  • Agrobacterium culture solution YEB: 0.1% yeast extract, 0.5% beef extract, 0.5% peptone, 0.5% sucrose, 0.05% MgSO4 ⁇ 7H20 (pH 7)
  • BY2 culture liquid medium D0.2: MS medium containing 30 g/L sucrose, 0.2 mg/L 2,4-D, dispensed into 30 mL in a 100 mL Kolben. pH was adjusted from 5.6 to 5.8.
  • Seeding medium MS medium containing 30 g/L sucrose, 4 g/L Gelrite (Fuji Film Wako, 075-05655). pH was adjusted from 5.6 to 5.8. After autoclaving, dispense 50 mL into a magenta box (Sigma, GA-7).
  • Liquid medium for infection MSH: MS medium containing 30 g/L sucrose. pH was adjusted from 5.6 to 5.8.
  • Selection medium 1 MSB5 medium containing 10 g/L sucrose, 3 mg/L BAP, 500 mg/L carbenicin, 10 mg/L hygromycin, and 0.6% Agarose. pH was adjusted from 5.6 to 5.8.
  • MB1 containing AgNO3 is added after sterilization so that AgNO3 is 5 mg/L.
  • Selection medium 2 B5BZ: B5 containing 30 g/L sucrose, 3 mg/L BAP, 1 mg/L zeatin, 500 mg/L carbenicin, 10 mg/L hygromycin, 0.6% Agarose Culture medium. pH was adjusted from 5.6 to 5.8.
  • MS medium so that the MS I to IV storage solution is 1:1.
  • MSB5 prepare the MS I-III stock solution and the B5 Vitamin stock solution in a 1:1 ratio.
  • B5 medium is prepared using B5 vitamin preservation solution and B5 Inorganic preservation solution in a ratio of 1:1.
  • Zeatin, carbenicin, and hygromycin are added after the medium has sufficiently cooled down after autoclaving. Furthermore, after dispensing 20 mL of MS3D medium and 25 mL of other media per petri dish, they were wrapped in plastic wrap and stored in the refrigerator.
  • the method for producing transformants is as follows.
  • Seed sterilization The Midorine seeds used for transformation are used after sterilization as follows. Midorine seeds are sold as coated seeds. This sterilization washes off the coating agent from the seeds. When sowing seeds that have been refrigerated, do not remove the seal until the seeds return to room temperature. The germination rate will be maintained for about one year, but if the germination rate decreases, it is better to sterilize it again. Add about 2 to 5 g of seeds and 70% ethanol to a 50 mL disposable tube, and shake for 1 minute at room temperature.
  • the pre-cultured hypocotyl sections are collected using the handle of a medicine spoon, placed into the infection solution in the Petri dish, and gently shaken for 20 minutes at room temperature.
  • the infection solution is removed with a pipette, and the hypocotyl sections are spread on sterile filter paper A to remove excess infection solution and returned to the MB3D medium used in the preculture. At this time, lay the cut ends flat so that they do not dry out.
  • Selection 2 (3 weeks after infection treatment) Two weeks after transplanting selection 1, transplant to MB1 (use 3 to 4 medium plates per 2 sowing boxes).
  • Rooting Transplant the cut out shoots to MSNB. Rooting is often promoted by removing excess tissue from the base with a scalpel. Transplant while adjusting the shape of the shoot every 2 to 3 weeks until rooting takes place. Here, it is also possible to divide the plants by transplanting the branched shoots into NSNB medium. Furthermore, if no rooting is observed, the plants may be cultured in a 1/2 MS medium containing 10 g/L sucrose and 4 g/L Gelrite. Once the roots have grown sufficiently in the magenta box, transplant them to vermiculite medium.
  • FIG. 9 is a diagram showing primer sequence information used for ACO3 gene fragment amplification.
  • the sequence information shown in FIG. 9 is the genome sequence of the Bo-ACO3 gene.
  • the registration number on the database is GenBank: LR031877.1. Starts with “GGT” from the 6th character from the end of the 6th line, ends with “ACA” up to the 16th character of the 8th line, and starts with “AGG” from the 26th character of the 12th line, ends with “AAC”
  • the 5th character from the end of the 14th line is an intron.
  • "CCA" shown in the center of the 8th line is the PAM array.
  • the black inverted region from the 5th line to the 6th line and the black inverted region in the 9th line are the primer pairs used for amplifying the ACO3 gene fragment.
  • DW per sample 26 ⁇ L
  • 5 ⁇ PrimeSTAR GXL Buffer (TAKARA): 10 ⁇ L, 2.5 mM dNTP Mixture: 4 ⁇ L, 10 ⁇ M Forward primer: 2.5 ⁇ L, 10 ⁇ M Reverse Primer: 2.5 ⁇ L, Prime STAR GXL DNA Polymerase (TAKARA, 1.25 U/ ⁇ L) 1.0 ⁇ L.
  • TAKARA 5 ⁇ PrimeSTAR GXL Buffer
  • the PCR amplification cycle consisted of the first step at 95°C for 1 minute, the second step at 95°C for 20 seconds, the third step at 60°C for 20 seconds, the fourth step at 68°C for 30 seconds, and the second step at 60°C for 30 seconds. The fourth step was repeated for 35 cycles. Then, after 3 minutes at 68°C, it was stored at 4°C.
  • the amplified products were analyzed by electrophoresis on a 1.2% agarose gel prepared with TAE or TBE.
  • the Forward primer sequence is AGAGAGAGGGACTCACGATGGAG
  • the Reverse primer sequence is TGAATCTGTCTTCCATGCACTT.
  • the PCR product was purified using QIAquick PCR Purification kit (Qiqgen), and then sequenced using Sanger sequencing. Furthermore, after cloning the purified PCR product, the sequence was similarly determined by Sanger sequencing.
  • FIG. 10 is an example of the base sequence of an ACO3 transformant.
  • FIG. 11 shows the results of direct sequence analysis.
  • the broccoli base sequence in this experiment is shown as "210611-9 ⁇ A.”
  • the bases are numbered at the top of Figures 10 and 11. The following description will use these numbers.
  • the amino acids are arranged as follows, IPHELLDR..., except for the broccoli used in this experiment.
  • the base sequence of broccoli in this experiment has amino acids arranged as follows: DSTRAT ⁇ Q . . .
  • FIG. 12 is a diagram showing the flow of the broccoli production method of the present invention.
  • step S11 the target broccoli is knocked out.
  • the knockout method for the target broccoli is as described above.
  • step S12 seeds are produced from the target broccoli.
  • step S13 this trait is introduced into other broccoli varieties using the target broccoli line as the mother plant. That is, by crossing the broccoli grown from the target broccoli seeds in step S12 with broccoli of another variety, broccoli having the above-mentioned traits and the traits of broccoli of another variety is produced, and the seeds of the broccoli are to make. In addition, this trait may be introduced not only into the seeds in step S12 but also into other broccoli varieties by direct crossing with the target broccoli in step S11.
  • step S14 the seeds are sown and broccoli for shipping is grown. This allows broccoli with the target gene knocked out to be produced for shipment.
  • Ethylene biosynthesis in plants involves the production of 1-aminocyclopropane-1-carboxylic acid (hereinafter abbreviated as ACC), a cyclic compound, from the precursor methionine via S-adenosylmethionine. is synthesized, and ethylene is produced by the catalytic action of ACC oxidase (hereinafter abbreviated as ACO) using this compound as a substrate.
  • ACC 1-aminocyclopropane-1-carboxylic acid
  • ACO ACC oxidase
  • Bo-ACO1 is involved in basal ethylene synthesis and ethylene synthesis in vegetative tissues.
  • Bo-ACO2 is involved in ethylene synthesis in reproductive organs and ethylene biosynthesis in the early stage after harvest, and is not expressed immediately after harvest, but increases within 2 hours after harvest. It has been reported that Bo-ACO3 is involved in ethylene synthesis in the late stage after harvest, and is expressed on the third day after harvest.
  • Bo-ACO2 and Bo-ACO3 are subjected to the above-mentioned genome editing to create yellowing suppressed lines. Specifically, when we attempted to create broccoli with a mutant ACO gene, we obtained ACO2 with a different sequence from ACO3 and ACO1.
  • Genome-edited T0 generation (lineage 210611-9) broccoli was grown and enlarged, self-fertilized, and T1 generation broccoli seeds were used to conduct a flower bud deterioration test.
  • the date is just an example, but the sowing date is June 25, 2022, and the growing conditions are germination (23°C) using soil pots that can be planted as is. Raise seedlings in 9cm polyester pots (23°C light condition/18°C dark condition until September 18th). Vernalization treatment (10 hours of light at 12°C/14 hours of darkness at 8°C from September 18th).
  • the T1 generation includes ACO3 * /ACO3 * , a strain in which ACO3 has been knocked out and both chromosomes are recessively homozygous, and a combination of strains with and without ACO3 knockout (ACO3 * / ACO3 ) was used, and Midorine (ACO3/ACO3) was used.
  • ACO3 * / ACO3 * a strain in which ACO3 has been knocked out and both chromosomes are recessively homozygous
  • ACO3 * / ACO3 was used
  • Midorine ACO3/ACO3
  • the flower bud deterioration test is a flower bud aging experiment, and is a test conducted at room temperature using a wild type (Midorine), an ACO3 mutant hetero line, and an ACO3 recessive homo line (loss-of-function type).
  • FIG. 13 shows the results of a flower bud deterioration test using the T1 generation.
  • Figure 13 shows the state 10 days after the flower bud deterioration test, and as a result, after 10 days, flower bud aging and yellowing/falling off had progressed in the wild type and mutant heterozygous lines.
  • the loss-of-function mutant homozygous line had a significantly higher number of surviving flower buds (confirming the above item (3)), and some even bloomed.
  • Midorirei confirming the above item (2).
  • the flower buds fall off as yellowing progresses, and no enlarged flower buds can be seen.
  • the flower buds fall off as yellowing progresses, and some of the flower buds only become slightly larger.
  • the ACO3 recessive homozygous line ACO3 * homo
  • the number of surviving flower buds is significantly larger, although there is some yellowing, and the surviving flower buds are larger and some even bloom (not shown in the figure). Appeared. Therefore, the ACO3 mutant homologous line was able to suppress senescence of flower buds. Through the real flower bud deterioration test, we were able to confirm that the gene was properly inherited from the T0 generation to the T1 generation.
  • FIG. 14 is a diagram showing ACO3 mutant line (1) 210611-6.
  • a mutant strain that knocks out ACO3 one in which a single nucleotide of A has been inserted was obtained.
  • a stop codon was generated, and as a result, a loss-of-function mutant strain (no full-length enzyme protein was produced) was obtained.
  • one with a 22 base deletion and a different amino acid sequence was also obtained.
  • a stop codon was created, and a loss-of-function mutant strain was obtained.
  • FIG. 15 is a diagram showing ACO3 mutant line (2) 210226-1.
  • ACO3 mutant line (2) 210226-1 As a mutant strain that knocks out ACO3, one in which a single base of T has been inserted was obtained. As shown in FIG. 15, a stop codon was created, and as a result, a loss-of-function mutant strain was obtained. From the explanation of FIGS. 14 and 15, the content of (4) above was confirmed.
  • FIG. 16 is a diagram showing ACO2 mutant line (1) 230306-1. Mutant lines in which ACO2 is knocked out include those in which a single base of A or G has been inserted. As shown in FIG. 16, a stop codon was created, and as a result, a functional deletion mutant strain was obtained.
  • FIG. 17 shows ACO2 mutant line (2) 230306-3.
  • a mutant strain in which ACO2 was knocked out was one in which a single nucleotide of C was inserted. As shown in FIG. 17, a stop codon was created, and as a result, a functional deletion mutant strain was obtained.
  • FIG. 18 shows ACO2 mutant line (3) 230516-1. Mutant lines in which ACO2 was knocked out were those in which a single G or T base was inserted. As shown in FIG. 18, a stop codon was created, and as a result, a functional deletion mutant strain was obtained.
  • FIG. 19 shows ACO2 mutant line (4) 230516-2.
  • ACO2 mutant line (4) 230516-2 As a mutant strain that knocks out ACO2, one in which a single base of T has been inserted was obtained. As shown in FIG. 19, a stop codon was created, and as a result, a mutant strain lacking function was obtained. From the explanation of FIGS. 16 to 19, the content of (5) above was confirmed.
  • the broccoli production method to which the present invention is applied only needs to have the following characteristics, and can take various embodiments. That is, the method for producing broccoli (for example, the broccoli shown in FIG. 1) to which the present invention is applied is as follows: Genetically manipulate the target broccoli to knock out at least one of ACO2 and ACO3, which generate ethylene that degrades within a predetermined period of time after harvest, among the genes of the target broccoli. A first step (for example, step S11 in FIG. 12), a second step of producing seeds from the target broccoli after the genetic manipulation (for example, step S12 in FIG. 12); A third step (for example, step S13 in FIG. 12) of growing broccoli for shipping using the seeds or seeds sown from the seeds; including. This makes it possible to use genome editing to provide crops with more suitable properties and to promote understanding of genome editing.
  • the seeds to which the present invention is applied only need to have the following characteristics, and can take various embodiments. That is, broccoli (for example, the broccoli shown in FIG. 1) or seeds (for example, the above-mentioned T1 generation broccoli seeds, etc.) to which the present invention is applied, It is produced from broccoli that has been genetically engineered to knock out at least one of ACO2 and ACO3, which generate ethylene that deteriorates over a predetermined period of time after harvest, or from said broccoli. This makes it possible to use genome editing to provide crops with more suitable properties and to promote understanding of genome editing.

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Abstract

The present invention provides a crop having more suitable properties by using genome editing, and promotes understanding of genome editing.　The present invention uses, as target broccoli, broccoli to be subjected to genetic manipulation, and involves performing genetic manipulation for knocking out, among the genes of the target broccoli, ACO3 that causes production of ethylene which deteriorates the broccoli in a predetermined post-harvesting period. Next, seeds are produced from the target broccoli after the genetic manipulation. The seeds are sown, and grown into broccoli to be shipped. As a result, broccoli to be shipped is produced.

Description

生産方法、ブロッコリ、及び種子Production methods, broccoli, and seeds
　本発明は、生産方法、ブロッコリ、及び種子に関する。 The present invention relates to a production method, broccoli, and seeds.
　従来より、ゲノム編集技術が存在する（例えば、特許文献１参照）。 Genome editing technology has conventionally existed (for example, see Patent Document 1).
国際公開第２０１３／１７６７７２号パンフレットInternational Publication No. 2013/176772 pamphlet
　上述の特許文献１を含む従来のゲノム編集技術は日々発達している。このようなゲノム編集技術を利用して、例えば、作物の耐病性、高品質性、栽培適応性を向上させるためのより具体的なゲノム編集が試みられている。
　日本国においては、このようなゲノム編集を施された作物については、安全性の審査や各種届出などの上で販売が可能となる。しかしながら、一部の消費者には、ゲノム編集技術に対する理解が得られず、ゲノム編集が施された作物も忌避されてしまうことがあった。 Conventional genome editing techniques, including that described in the above-mentioned Patent Document 1, are being developed day by day. Using such genome editing techniques, more specific genome editing has been attempted for improving, for example, disease resistance, high quality, and cultivation adaptability of crops.
In Japan, such genome-edited crops can be sold only after undergoing safety screening and various notifications, etc. However, some consumers do not understand genome editing technology and tend to avoid genome-edited crops.
　ここで、アブラナ科には、ダイコン、キャベツ、ブロッコリ、ハクサイなど国内の主要野菜（作物）として重要なものが多く、その品種改良には、雑種強勢、種間交雑、Ｆ１採種系の確立などの様々な育種学の成果が利用されてきた。
　当然ながら、収穫後に野菜（作物）は劣化する。特に、ブロッコリにおいては、収穫から３日程度で黄化が始まってしまい、商品価値が低下してしまっていた。
　本発明者は、このような商品価値の低下に影響する性質をゲノム編集により改良することにより、作物の廃棄などを削減することに想到した。 Here, there are many important vegetables (crops) in the Brassicaceae family, such as radish, cabbage, broccoli, and Chinese cabbage, which are important domestically. A variety of breeding studies have been utilized.
Naturally, vegetables (crops) deteriorate after they are harvested. In particular, broccoli begins to yellow about three days after harvesting, reducing its commercial value.
The present inventors came up with the idea of reducing crop waste by improving the properties that affect such reduction in commercial value through genome editing.
　本発明は、このような状況に鑑みてなされたものであり、ゲノム編集を用いてより好適な性質の作物を提供し、ゲノム編集への理解を促進することを目的とする。 The present invention was made in view of this situation, and aims to provide crops with more suitable properties using genome editing and to promote understanding of genome editing.
　上記目的を達成するために、本発明の一態様のブロッコリの生産方法は、
　遺伝子操作の対象となるブロッコリを対象ブロッコリとして、当該対象ブロッコリの遺伝子のうち、収穫後所定期間で劣化させるエチレンを発生させるＡＣＯ２とＡＣＯ３のうち少なくとも一方に対して、ノックアウトさせるための遺伝子操作を行う第１ステップと、
　前記遺伝子操作の後の前記対象ブロッコリから種子をつくる第２ステップと、
　前記種子又は当該種子から播種された種子を用いて、出荷用のブロッコリを生育する第３ステップと、
　を含む。 In order to achieve the above object, a method for producing broccoli according to one embodiment of the present invention includes:
Genetically manipulate the target broccoli to knock out at least one of ACO2 and ACO3, which generate ethylene that degrades within a predetermined period of time after harvest, among the genes of the target broccoli. The first step and
a second step of producing seeds from the target broccoli after the genetic manipulation;
a third step of growing broccoli for shipping using the seeds or seeds sown from the seeds;
including.
　また、本発明の一態様のブロッコリ又は種子は、
　収穫後所定期間で劣化させるエチレンを発生させるＡＣＯ２とＡＣＯ３のうち少なくとも一方に対して、ノックアウトさせるための遺伝子操作が行われたブロッコリ又は当該ブロッコリを起源として作られる。 Furthermore, the broccoli or seeds of one embodiment of the present invention are
It is produced from broccoli that has been genetically engineered to knock out at least one of ACO2 and ACO3, which generate ethylene that deteriorates over a predetermined period of time after harvest, or from said broccoli.
　本発明によれば、ゲノム編集を用いてより好適な性質の作物を提供し、ゲノム編集への理解を促進することができる。 According to the present invention, it is possible to use genome editing to provide crops with more suitable properties and to promote understanding of genome editing.
本発明の一実施形態のブロッコリの生産方法を含む、ＡＣＯ３遺伝子のノックアウトの有無によるブロッコリの老化の違いの例を示す図である。FIG. 1 is a diagram showing an example of the difference in broccoli senescence depending on whether or not the ACO3 gene is knocked out, including a method for producing broccoli according to one embodiment of the present invention. 図１のＡＣＯ３遺伝子のノックアウトによるブロッコリの老化への影響の例を示す図である。FIG. 2 is a diagram showing an example of the influence of knockout of the ACO3 gene of FIG. 1 on aging of broccoli. エチレンの再合成経路を示す図である。It is a diagram showing the resynthesis route of ethylene. 図３のエチレンの再合成経路について、より詳細に示す図である。FIG. 4 is a diagram showing the ethylene resynthesis route of FIG. 3 in more detail. 実験全体の流れを示す図である。FIG. 2 is a diagram showing the flow of the entire experiment. 遺伝子導入のイメージを示す図である。It is a diagram showing an image of gene introduction. アグロバクテリウム法における、ＴｉプラスミドとＣＦＲＩＳＰＲ／Ｃａｓ９ベクターの例を示す図である。It is a figure showing an example of Ti plasmid and CFRISPR/Cas9 vector in Agrobacterium method. ＣＲＩＳＰＲ－Ｃａｓ９法の流れを示す図である。It is a diagram showing the flow of the CRISPR-Cas9 method. ＡＣＯ３遺伝子断片増幅に利用したプライマー配列情報を示す図である。FIG. 2 is a diagram showing primer sequence information used for amplifying the ACO3 gene fragment. ＡＣＯ３形質転換体の塩基配列の一例である。This is an example of the base sequence of an ACO3 transformant. ダイレクトシークエンス解析の結果を示す図である。FIG. 3 is a diagram showing the results of direct sequence analysis. 本発明のブロッコリの生産方法のフローを示す図である。FIG. 2 is a diagram showing a flow of the broccoli production method of the present invention. Ｔ１世代を用いた花蕾劣化試験の結果を示す図である。It is a figure showing the result of a flower bud deterioration test using T1 generation. ＡＣＯ３変異体系統（１）２１０６１１－６を示す図である。FIG. 1 shows the ACO3 mutant line (1) 210611-6. ＡＣＯ３変異体系統（２）２１０２２６－１を示す図である。FIG. 1 shows the ACO3 mutant line (2) 210226-1. ＡＣＯ２変異体系統（１）２３０３０６－１を示す図である。FIG. 2 shows ACO2 mutant line (1) 230306-1. ＡＣＯ２変異体系統（２）２３０３０６－３を示す図である。FIG. 3 shows ACO2 mutant line (2) 230306-3. ＡＣＯ２変異体系統（３）２３０５１６－１を示す図である。FIG. 3 shows ACO2 mutant line (3) 230516-1. ＡＣＯ２変異体系統（４）２３０５１６－２を示す図である。FIG. 2 shows ACO2 mutant line (4) 230516-2.
　以下、本発明の実施形態について図面を用いて説明する。 Hereinafter, embodiments of the present invention will be described using the drawings.
　本発明では、作物の一例としてブロッコリを採用している。
　以下、ブロッコリが採用された背景と目的等について説明する。 In the present invention, broccoli is used as an example of a crop.
The background and purpose of broccoli being adopted will be explained below.
　近年、アブラナ科の各作物のゲノムの構造が明らかとなり、その栽培化の起源などについての理解が進むとともに、ＤＮＡマーカーを利用した分子育種が活発に進んでいる。
　今後の温暖化等による栽培様式の多様化や消費者の食嗜好等の多様化に対応するためには、それら分子育種技術を活かして耐病性、高品質性、栽培適応性などの新たな形質を導入することが重要である。
　更に、効率的かつ迅速な標的変異導入法としてのゲノム編集技術の利用とその評価法の確立も不可欠な課題である。 In recent years, the genome structure of each crop of the Brassicaceae family has been clarified, and as the understanding of the origins of their domestication has progressed, molecular breeding using DNA markers is actively progressing.
In order to respond to the diversification of cultivation styles due to future global warming and the diversification of consumer food preferences, we will utilize these molecular breeding techniques to develop new traits such as disease resistance, high quality, and cultivation adaptability. It is important to introduce
Furthermore, the use of genome editing technology as an efficient and rapid method for introducing targeted mutations and the establishment of evaluation methods are also essential issues.
　ここで、ブロッコリは、茎と花蕾を可食部とする、緑黄色野菜である。花序の構造が未成熟で急激に成長しているときに収穫されて商品化される。
　また、上述したように、ブロッコリは、収穫後３日程度で黄化する。具体的には、クロロフィルの欠失による収穫後の小花の黄化が起きる。
　このような、ブロッコリの収穫後に見られる組織の退化は典型的な老化過程ではなく、代謝過程が強く乱されたことによって起きている。この収穫後の黄化は、エチレンの影響であることが明らかにされている。
　このように、新鮮なブロッコリの寿命は短く、黄化はブロッコリの廃棄の原因となっていた。 Here, broccoli is a green-yellow vegetable whose edible parts are stems and flower buds. It is harvested and commercialized when the inflorescence structure is immature and growing rapidly.
Moreover, as mentioned above, broccoli turns yellow about three days after being harvested. Specifically, yellowing of florets occurs after harvest due to lack of chlorophyll.
This tissue degeneration seen after broccoli is harvested is not a typical aging process, but is caused by strongly disrupted metabolic processes. It has been revealed that this yellowing after harvest is caused by ethylene.
Thus, the lifespan of fresh broccoli is short, and yellowing has been a cause of discarding broccoli.
　詳しくは後述するが、ブロッコリのエチレンの生合成には、３つのＡＣＣ　ｏｘｉｄａｓｅ（以下、「ＡＣＯ」と適宜略記する）が関与している。 As will be described in detail later, three ACC oxidases (hereinafter appropriately abbreviated as "ACO") are involved in the biosynthesis of ethylene in broccoli.
　第１のＡＣＯ遺伝子（以下、「ＡＣＯ１遺伝子」と適宜呼ぶ）は、基礎的なエチレンの合成に寄与する遺伝子である。ＡＣＯ１遺伝子が関与することで発生したエチレンは栄養組織の老化に寄与する。 The first ACO gene (hereinafter appropriately referred to as "ACO1 gene") is a gene that contributes to basic ethylene synthesis. Ethylene generated through involvement of the ACO1 gene contributes to the aging of vegetative tissues.
　第２のＡＣＯ遺伝子（以下、「ＡＣＯ２遺伝子」と適宜呼ぶ）は、生殖器官でのエチレンの合成に寄与する遺伝子である。ＡＣＯ２遺伝子が関与することで発生したエチレンは、収穫後の初期老化に寄与する。 The second ACO gene (hereinafter appropriately referred to as "ACO2 gene") is a gene that contributes to ethylene synthesis in reproductive organs. Ethylene generated through the involvement of the ACO2 gene contributes to early senescence after harvest.
　第３のＡＣＯ遺伝子（以下、「ＡＣＯ３遺伝子」と適宜呼ぶ）は、黄化と発現が同期している遺伝子であり、収穫後のエチレンの合成に寄与する遺伝子である。ＡＣＯ３遺伝子が関与することで発生したエチレンは、収穫後の後期老化に寄与する。 The third ACO gene (hereinafter appropriately referred to as "ACO3 gene") is a gene whose expression is synchronized with etiolation, and is a gene that contributes to the synthesis of ethylene after harvest. Ethylene generated through the involvement of the ACO3 gene contributes to late senescence after harvest.
　本発明者は、上述したように、特にＡＣＯ３遺伝子をノックアウトすることにより、ブロッコリの収穫後の後期老化を抑制し、ブロッコリの商品価値を維持できるようになることを想到した。
　図１は、本発明の一実施形態のブロッコリの生産方法を含む、ＡＣＯ３遺伝子のノックアウトの有無によるブロッコリの老化の違いの例を示す図である。
　図２は、図１のＡＣＯ３遺伝子のノックアウトによるブロッコリの老化への影響の例を示す図である。 As described above, the present inventors have come up with the idea that by knocking out the ACO3 gene in particular, it is possible to suppress late senescence of broccoli after harvest and maintain the commercial value of broccoli.
FIG. 1 is a diagram showing an example of the difference in aging of broccoli depending on the presence or absence of knockout of the ACO3 gene, including the broccoli production method of one embodiment of the present invention.
FIG. 2 is a diagram showing an example of the influence of knockout of the ACO3 gene in FIG. 1 on aging of broccoli.
　図１（Ａ）に示すように、播種ののち定植し４０日前後で花蕾が発生する。この花蕾の成長中に所定の大きさになったところで、収穫が行われる。例えば、深夜から朝にかけて収穫されたブロッコリは、収穫の当日中に朝採れのブロッコリとして店頭に陳列される。ブロッコリの左上に記された数字は、収穫後の日数を示している。収穫後０日目や１日目のブロッコリは、濃いハッチングで示される緑色である。その後、例えば、収穫後２日目には、ブロッコリの花蕾の一部が薄いハッチングで示されるように黄化する。そして、例えば、収穫後３日目には、ブロッコリの花蕾の全部が黄化する。花蕾の一部又は全部が黄化したブロッコリは、商品価値が低下してしまう。 As shown in Figure 1 (A), flower buds appear about 40 days after sowing and planting. When these flower buds grow to a certain size, they are harvested. For example, broccoli harvested from late at night to early morning is displayed in stores on the same day as harvest as "morning-picked broccoli." The number written in the upper left corner of the broccoli indicates the number of days since harvest. Broccoli on the 0th or 1st day after harvest is green, as indicated by dark hatching. Then, for example, on the 2nd day after harvest, some of the broccoli flower buds turn yellow, as indicated by light hatching. Then, for example, on the 3rd day after harvest, all of the broccoli flower buds turn yellow. Broccoli with some or all of its flower buds turning yellow has reduced commercial value.
　ここで、本実施形態では、図１（Ａ）に示すように、ＡＣＯ２遺伝子及びＡＣＯ３遺伝子をノックアウトする。これにより、収穫まではそのままに、例えば、収穫後２日目や３日目においても、ブロッコリの花蕾の一部又は全部が黄化せず、店頭に並べられるようになるのである。 Here, in this embodiment, as shown in FIG. 1(A), the ACO2 gene and the ACO3 gene are knocked out. As a result, some or all of the broccoli flower buds can be displayed on store shelves without yellowing, for example, even on the second or third day after harvest.
　具体的には、本実施形態のブロッコリは、ＡＣＯ１遺伝子をそのままに、ＡＣＯ２遺伝子及びＡＣＯ３遺伝子をノックアウトする。
　即ち、図２に示すように、ＡＣＯ１遺伝子は、従来の図１（Ａ）と同様に発現する。これにより、花蕾は従来通り成長（老化）する。 Specifically, in the broccoli of this embodiment, the ACO2 gene and ACO3 gene are knocked out while leaving the ACO1 gene intact.
That is, as shown in FIG. 2, the ACO1 gene is expressed in the same manner as in the conventional FIG. 1(A). As a result, the flower buds grow (senescence) as usual.
　また、ＡＣＯ２遺伝子は、従来、収穫２時間後から発現する。そして、収穫後初期のエチレン発生に寄与する。しかしながら、本実施形態では、ＡＣＯ２遺伝子はノックアウトされているため、ＡＣＯ２遺伝子は発現せず、収穫後初期のＡＣＯ２遺伝子の寄与によるエチレンの発生がない。
　これにより、ブロッコリの花蕾の収穫後初期における黄化が防がれる。 Furthermore, the ACO2 gene is conventionally expressed 2 hours after harvest. It also contributes to ethylene generation in the early period after harvest. However, in this embodiment, since the ACO2 gene is knocked out, the ACO2 gene is not expressed, and ethylene is not generated due to the contribution of the ACO2 gene in the early stage after harvest.
This prevents yellowing of broccoli florets in the early stage after harvest.
　また、ＡＣＯ２遺伝子は、従来、収穫をトリガとして、収穫後２日又は３日目頃から発現する。そして、収穫後後期のエチレン発生に寄与する。しかしながら、本実施形態では、ＡＣＯ３遺伝子はノックアウトされているため、ＡＣＯ３遺伝子は発現せず、収穫後後期のＡＣＯ３遺伝子の寄与によるエチレンの発生がない。
　これにより、ブロッコリの花蕾の収穫後後期における黄化が防がれる。 Furthermore, the ACO2 gene has conventionally been triggered by harvest, and is expressed from about the second or third day after harvest. It also contributes to ethylene generation in the late period after harvest. However, in this embodiment, since the ACO3 gene is knocked out, the ACO3 gene is not expressed, and ethylene is not generated due to the contribution of the ACO3 gene in the late period after harvest.
This prevents yellowing of broccoli florets in the late stage after harvest.
　その結果、収穫後２日目や３日目においても、黄化がおきないため、商品価値が維持されるのである。
　このように、収穫後２日目や３日目においても商品価値が維持されることにより、収穫後２日目や３日目以降のブロッコリも店頭に陳列が可能となる。これにより、食品ロスを削減することができる。
　また、ブロッコリが購入された後、消費者の手元にある間においても、保管中のブロッコリの黄化が発生しない。即ち、消費者は、ゲノム編集の成果物たるブロッコリがどのように改良されていたのかを実際に感じ取ることが出来る。
　これにより、このような商品価値の低下に影響する性質をゲノム編集により改良することにより、作物の廃棄などを削減するとともに、ゲノム編集技術がこのような消費者の目に触れる性質の改良に寄与することにより、ゲノム編集技術を用いた育種法の有用性を示す効果を奏する。 As a result, even on the second or third day after harvest, yellowing does not occur and the product value is maintained.
In this way, since the product value is maintained even on the second or third day after harvesting, it becomes possible to display broccoli on the second or third day after harvesting in stores. This makes it possible to reduce food loss.
Further, even after the broccoli is purchased, yellowing of the broccoli does not occur during storage even while the broccoli is in the hands of the consumer. In other words, consumers can actually feel how broccoli, a product of genome editing, has been improved.
By using genome editing to improve these properties that affect the decline in product value, we will be able to reduce crop waste, and at the same time, genome editing technology will contribute to improving such properties that are visible to consumers. This has the effect of demonstrating the usefulness of breeding methods using genome editing technology.
　これに対して、従来のゲノム編集においては、作物の耐病性、高品質性、栽培適応性を向上させることが行われてきた。一部の消費者の目線においては、測定も検証も基本的な困難である。そして、その効果（例えば、栽培コスト削減）を実感することが出来なかった。その結果、一部の消費者にとっては、ゲノム編集技術に対する理解が得られず、ゲノム編集が施された作物も忌避されてしまうことがあったといえる。
　本実施形態のブロッコリは、これを解消するものである。 In contrast, conventional genome editing has been carried out to improve the disease resistance, high quality, and cultivation adaptability of crops. From the perspective of some consumers, both measurement and verification are fundamental difficulties. Moreover, it was not possible to realize the effects (for example, reduction in cultivation costs). As a result, it can be said that for some consumers, there was a lack of understanding of genome editing technology, and they sometimes avoided crops that had undergone genome editing.
The broccoli of this embodiment solves this problem.
　以下、図３乃至図１１を用いて上述のＡＣＯ１乃至ＡＣＯ３遺伝子の性質や、本実施形態のブロッコリのノックアウト及びその結果のテストについてより詳細に説明する。
　ここで、収穫後に花の生殖器官で１－ａｍｉｎｏｃｙｃｌｏｐｒｏｐａｎｅ－１－ｃａｒｂｏｘｙｌｉｃ　ａｃｉｄ　（ＡＣＣ）　ｏｘｉｄａｓｅの活性とｍＲＮＡ発現量が大きく増加することがエチレンの生産に大きく関与していることはよく知られている。
　図３は、エチレンの再合成経路を示す図である。
　図４は、図３のエチレンの再合成経路について、より詳細に示す図である。
　エチレンは、メチオニン、Ｓ－アデノシルメチオニン、１－アミノシクロプロパン１－カルボン酸（以下、「ＡＣＣ」と適宜略記する）、エチレンの順に合成される。
　ここで、ＡＣＳは、Ｓ－アデノシルメチオニンから、ＡＣＣの反応の触媒となる酵素である。
　また、ＡＣＯは、ＡＣＣからエチレンの反応の触媒となる酵素である。 Hereinafter, the properties of the above-mentioned ACO1 to ACO3 genes, the broccoli knockout of this embodiment, and the test of the results will be explained in more detail using FIGS. 3 to 11.
It is well known that the activity and mRNA expression level of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase greatly increase in the reproductive organs of flowers after harvest, which is greatly involved in ethylene production. .
FIG. 3 is a diagram showing the resynthesis route of ethylene.
FIG. 4 is a diagram showing the ethylene resynthesis route of FIG. 3 in more detail.
Ethylene is synthesized in the order of methionine, S-adenosylmethionine, 1-aminocyclopropane-1-carboxylic acid (hereinafter appropriately abbreviated as "ACC"), and ethylene.
Here, ACS is an enzyme that catalyzes the reaction of S-adenosylmethionine to ACC.
Furthermore, ACO is an enzyme that catalyzes the reaction of ethylene from ACC.
　本実施形態では、エチレンが生合成される１段階前の酵素であるＡＣＳは、対象としていないが、ＡＣＳについて以下の知見が存在する。
　ブロッコリにおいては、エチレン生合成の１段階前の酵素であるＡＣＳとして、ＢＲＯＣＡＣＳ１、ＢＲＯＣＡＣＳ２、ＢＲＯＣＡＣＳ３の３種のｃＤＮＡが存在する。これらのｃＤＮＡ間のホモロジーは比較的低く、パラログである。
　ＢＲＯＣＡＣＳ１の翻訳レベルは、傷か機械的ストレスにより誘導され収穫後に高い水準に達し、その後検出できなくなる。
　ＢＲＯＣＡＣＳ２の翻訳レベルは、老化の全過程で一定していて、最後に上昇することから、主要なＡＣＣ合成酵素であろう。
　ＢＲＯＣＡＣＳ　３は、ほとんど検出できないレベルである。 Although the present embodiment does not target ACS, which is an enzyme at one stage before ethylene biosynthesis, the following knowledge exists regarding ACS.
In broccoli, there are three types of cDNAs, BROCACS1, BROCACS2, and BROCACS3, as ACS, which is an enzyme that is one step before ethylene biosynthesis. Homology between these cDNAs is relatively low and they are paralogs.
Translation levels of BROCACS1 are induced by wounding or mechanical stress and reach high levels after harvest, after which they become undetectable.
The translation level of BROCACS2 is constant throughout the aging process and increases at the end, so it may be the major ACC synthase.
BROCACS 3 is at an almost undetectable level.
　本実施形態では、エチレン生合成の最後の段階の酵素であるＡＣＣとして、ＡＣＣ　Ｏｘ１（ＡＣＯ１遺伝子に起因するｃＤＮＡ）、　ＡＣＣ　Ｏｘ２（ＡＣＯ２遺伝子に起因するｃＤＮＡ）、　Ｂｏ－ＡＣＯ３（ＡＣＯ３遺伝子に起因するｃＤＮＡ）が存在する。
　ＡＣＯ１の翻訳レベルは、収穫時には全花序で低いが、収穫後に著しく増加する。収穫後のガク（ｓｅｐａｌ）と刈り取った黄色くなった葉でも増加する。生殖器官では、ほとんど発現していない。
　ＡＣＯ２の翻訳は、生殖器官でのみ検出され収穫時には発現していないが、２時間以内に増加し始め、多く蓄積するようになる。
　即ち、ＩＡＡとＡＢＡ処理や傷処理でＡＣＣ　Ｏｘ１の翻訳レベルに変化はない。ＡＣＯ２はアブシジン酸とプロピレン処理により増加する。
　そして、ＡＣＯ３遺伝子は収穫後３日目に発現が見られた。 In this embodiment, ACC, which is the enzyme in the last step of ethylene biosynthesis, includes ACC Ox1 (cDNA caused by the ACO1 gene), ACC Ox2 (cDNA caused by the ACO2 gene), and Bo-ACO3 (cDNA caused by the ACO3 gene). cDNA) exists.
Translation levels of ACO1 are low in whole inflorescences at harvest, but increase markedly after harvest. It also increases in sepals after harvest and in yellowed leaves that are cut. It is hardly expressed in reproductive organs.
Translation of ACO2 is detected only in the reproductive organs and is not expressed at the time of harvest, but within 2 hours it begins to increase and accumulates in large amounts.
That is, there is no change in the translation level of ACC Ox1 by IAA and ABA treatment or wound treatment. ACO2 increases with abscisic acid and propylene treatment.
Expression of the ACO3 gene was observed on the third day after harvest.
　これらの事実から、上述したように、ＡＣＯ１は、基礎的なエチレンの生産と栄養組織（葉など）での老化に関与していることがわかる。
　また、ＡＣＯ２は、収穫した小花の生殖器官でのみ発現していることから、収穫時の刺激により生殖器官でのエチレンの生産に関与していて、そのことで、収穫後のガクなどの花の他の器官における初期の退化に関与している。
　ＡＣＯ３は、収穫後初期のエチレン合成ではなく後期のエチレン合成により老化に関与している。 These facts indicate that ACO1 is involved in basic ethylene production and senescence in vegetative tissues (such as leaves), as described above.
In addition, since ACO2 is expressed only in the reproductive organs of harvested florets, it is involved in the production of ethylene in the reproductive organs when stimulated at the time of harvest. Involved in early degeneration in other organs.
ACO3 is involved in senescence not through early ethylene synthesis after harvest but through late ethylene synthesis.
　また、アンチセンス（発現抑制系）を導入してＡＣＯ２を阻害することにより、エチレン生合成が阻害され、収穫後初期のエチレン生合成がかなり減少し、ブロッコリ小花の黄化が抑えられるという研究も存在する。 Furthermore, there is research showing that by inhibiting ACO2 through the introduction of an antisense (expression suppression system), ethylene biosynthesis is inhibited, significantly reducing ethylene biosynthesis in the early stages after harvest, and suppressing the yellowing of broccoli florets.
　本発明は、ＡＣＯ２に加えて、ＡＣＯ３をターゲットとしたノックアウト変異導入を狙い、アグロバクテイリウム法でゲノム編集を行う。これにより、ＡＣＯ３が発現しないことによるブロッコリの改良が行われることの検証と、これらのノックアウトが行われたブロッコリの製品化を実現するものである。
　実験においては、Ｂｏ－ＡＣＯ３に一塩基挿入され、不完全なＡＣＯ３タンパク質が生合成されると考えられる形質転換体を１系統作出に成功している。この系統の自殖後代と検定交雑後代を育成することで、収穫後の花蕾の老化の程度を評価する。 The present invention aims to introduce knockout mutations targeting ACO3 in addition to ACO2, and performs genome editing using the Agrobacterium method. As a result, it is possible to verify that broccoli is improved by not expressing ACO3, and to commercialize broccoli in which these knockouts have been performed.
In experiments, we succeeded in creating one line of transformants in which a single nucleotide was inserted into Bo-ACO3 and an incomplete ACO3 protein was thought to be biosynthesized. By raising self-fertilized progeny and test-cross progeny of this line, the degree of senescence of flower buds after harvesting will be evaluated.
　そこで、アブラナ科野菜のブロッコリ（Ｂｒａｓｓｉｃａ　ｏｌｅｒａｃｅａ　ｖａｒ．　ｉｔａｌｉｃａ）の鮮度保持に関与する１－アミノシクロプロパン－１－カルボン酸酸化酵素（ＡＣＯ）遺伝子の機能破壊およびプラスチドゲノムにコードされるｐｓｂＡ　遺伝子へのゲノム編集による変異導入をそれぞれ試みたので、この結果について報告する。 Therefore, we aim to disrupt the function of the 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) gene, which is involved in maintaining the freshness of broccoli (Brassica oleracea var. italica), a cruciferous vegetable, and to disrupt the psbA gene encoded in the plastid genome. We have attempted to introduce mutations through genome editing, and will report on the results.
　図５は、実験全体の流れを示す図である。
　まず、胚軸を切片化した。つぎに、アグロバクテリウム培養液に浸した。これにより、Ｃａｓ９、ｇＲＮＡ、マーカー遺伝子を導入した。次に、培養、除菌、カルス誘導を行う。次に、シュート育成を行った。次に、Ｔｏｔａｌ　ＤＮＡの単離を行った。次に、標的遺伝子断片のＰＣＲ増幅を行った。そして、制限酵素消化による変異型の同定を行った。
　より具体的には、ＡＣＯ　遺伝子については、ＣＲＩＳＰＲ－Ｃａｓ９のゲノム編集用コンストラクトを構築し、それらを保持するアグロバクテリムをブロッコリの緑嶺の胚軸切片に感染させ、形質転換体を作出した。ブロッコリにおけるＡＣＯ　遺伝子は、ゲノム中に３コピー存在するが、収穫後にその発現量が増加する遺伝子は、ＡＣＯ２　およびＡＣＯ３　と報告されているため、ＡＣＯ２　およびＡＣＯ３　をそれぞれ単独に編集可能なコンストラクトを作成した。得られた形質転換体のシュートから簡易法でｔｏｔａｌ　ＤＮＡを単離後、ターゲット遺伝子の変異導入部位をＰＣＲ法で増幅し、その断片の塩基配列を決定し、変異の導入の有無を解析した。 FIG. 5 is a diagram showing the flow of the entire experiment.
First, the hypocotyls were sectioned. Next, it was immersed in Agrobacterium culture solution. As a result, Cas9, gRNA, and marker genes were introduced. Next, culture, sterilization, and callus induction are performed. Next, we trained the shoots. Next, total DNA was isolated. Next, PCR amplification of the target gene fragment was performed. Then, the mutant type was identified by restriction enzyme digestion.
More specifically, for the ACO gene, CRISPR-Cas9 genome editing constructs were constructed, and Agrobacterium carrying them was infected with hypocotyl sections of broccoli green ridges to generate transformants. There are three copies of the ACO gene in broccoli in the genome, but it has been reported that ACO2 and ACO3 are the genes whose expression levels increase after harvesting, so we created constructs that can edit ACO2 and ACO3 individually. . After total DNA was isolated from the shoots of the obtained transformants by a simple method, the mutation introduction site of the target gene was amplified by PCR, the base sequence of the fragment was determined, and the presence or absence of mutation introduction was analyzed.
　図６は、遺伝子導入のイメージを示す図である。
　図６に示すように、本実験のバイナリーベクターは、ｇＲＮＡ及びＣａｓ９（導入遺伝子）及び、ハイグロマイシン体制遺伝子（選抜マーカー遺伝子）を有する構造をしている。
　このようなバイナリーベクターを用いることにより、植物細胞（本例ではブロッコリの細胞）の核に含まれる染色体に、ｇＲＮＡ及びＣａｓ９（導入遺伝子）及び、ハイグロマイシン体制遺伝子（選抜マーカー遺伝子）とが導入される。
　なお、本実験のバイナリーベクターｐＺＨ＿ｇＹＳＡ＿ＦＦＣａｓ９は、国立研究開発法人農業・食品産業技術総合研究機構から資料提供契約（ＭＴＡ）を結び分譲されたものを基に構築されている。 FIG. 6 is a diagram showing an image of gene introduction.
As shown in FIG. 6, the binary vector used in this experiment has a structure containing gRNA, Cas9 (transgene), and hygromycin regime gene (selection marker gene).
By using such a binary vector, gRNA, Cas9 (transfer gene), and hygromycin regime gene (selection marker gene) are introduced into the chromosome contained in the nucleus of a plant cell (broccoli cell in this example). Ru.
The binary vector pZH_gYSA_FFCas9 used in this experiment was constructed based on the one provided by the National Agriculture and Food Research Institute under a material provision agreement (MTA).
　図７は、アグロバクテリウム法における、ＴｉプラスミドとＣＦＲＩＳＰＲ／Ｃａｓ９ベクターの例を示す図である。
　図７（Ａ）に示すように、本実験のＴｉプラスミドは、植物ホルモン生産関連遺伝子（Ｔ－ＤＮＡ）と、ＤＮＡ転移に関わる遺伝子群と、複製起点（アグロバクテリウム）とを含んでいる。
　また、図７（Ｂ）に示すように、本実験のＣＲＩＳＰＲ／Ｃａｓ９ベクターは、ｇＲＮＡ及びＣａｓ９（導入遺伝子）及びハイグロマイシン体制遺伝子（選抜マーカー遺伝子）からなるＴ－ＤＮＡと、複製起点（アグロバクテリウム／大腸菌）戸を含んでいる。 FIG. 7 is a diagram showing an example of a Ti plasmid and a CFRISPR/Cas9 vector in the Agrobacterium method.
As shown in FIG. 7(A), the Ti plasmid used in this experiment contains a plant hormone production-related gene (T-DNA), a group of genes involved in DNA transfer, and an origin of replication (Agrobacterium).
In addition, as shown in Figure 7 (B), the CRISPR/Cas9 vector used in this experiment contained T-DNA consisting of gRNA, Cas9 (transgene), and hygromycin regime gene (selection marker gene), and an origin of replication (Agrobacterium Contains E. coli).
　図８は、ＣＲＩＳＰＲ－Ｃａｓ９法の流れを示す図である。
　本実験においては、上述したように、胚軸を切片化し、アグロバクテリウム培養液に浸すことにより、アグロバクテリウムに感染させ、ＣＲＩＳＰＲ／Ｃａｓ９ベクターの導入がなされる。
　即ち、ＣＲＩＳＰＲ（Ｃｌｕｓｔｅｒｅｄ　Ｒｅｇｕｌａｒｌｙ　Ｉｎｔｅｒｓｐａｃｅｄ　Ｓｈｏｒｔ　Ｐａｌｉｎｄｒｏｍｉｃ　Ｒｅｐｅａｔｓ）－Ｃａｓ９法においては、Cas9及びｓｇＲＮＡが発現し、ガイドＲＮＡが遺伝子に結合することにより、Ｃａｓ９が誘導され、遺伝子改変（ＣＲＩＳＰＲ／Ｃａｓ９ベクターの導入）がなされる。
　その後、培養、除菌、カルス誘導を経て、シュートの作成が行われる。 FIG. 8 is a diagram showing the flow of the CRISPR-Cas9 method.
In this experiment, as described above, the hypocotyl is sectioned and immersed in Agrobacterium culture solution to infect it with Agrobacterium and introduce the CRISPR/Cas9 vector.
That is, in the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 method, Cas9 and sgRNA are expressed and the guide RNA binds to the gene, thereby inducing Cas9 and causing genetic modification (CRISPR/Cas9 introduction of vectors) It will be done.
After that, shoots are created through culturing, sterilization, and callus induction.
　以下、本実験の全体の流れについて、より具体的なパラメータを用いて、説明する。 The overall flow of this experiment will be explained below using more specific parameters.
　前提として、アブラナ科野菜のブロッコリ（Ｂｒａｓｓｉｃａ　ｏｌｅｒａｃｅａ　Ｌ．．　ｖａｒ．　ｉｔａｌｉｃａ，　２ｎ＝１８）は地中海東部で、紀元前から栽培されたケールに起源があるといわれている。その特徴的な大きな花蕾には、多くのビタミン類、カロテノイド、ポリフェノールなど含み、機能性成分を多く含む野菜として世界的に生産量が増加している。日本国内でも、１９９０年代以降は生産量が増え続け、さらに、年はスプラウトとしての需要も高まっている。ブロッコリを含むＢｒａｓｓｉｃａ　ｏｌｅｒａｃｅａは、アグロバクテリウム法による形質転換法が報告されている。また、シロイヌナズナとの遺伝子相同性も高いことから、様々な形質に関与する標的遺伝子を同定可能である。さらに、ゲノム情報などのリソースも充実していることから、オフターゲット変異を最小にするｇＲＮＡの設定が可能であり、ゲノム編集利用をした育種の展開が期待できる。
　ここでは、本実験では、以下のように、ブロッコリのゲノム編集による変異系統の作出について、アグロバクテリウム法による形質転換、編集系統の同定、及び後代を得るための採種までをおこなった。 The premise is that broccoli (Brassica oleracea L. var. italica, 2n=18), a cruciferous vegetable, is said to have its origins in kale, which has been cultivated since BC in the eastern Mediterranean. Its characteristic large flower buds contain many vitamins, carotenoids, polyphenols, etc., and its production is increasing worldwide as a vegetable rich in functional ingredients. In Japan, production has continued to increase since the 1990s, and demand for sprouts has also increased in 2019. A transformation method using the Agrobacterium method has been reported for Brassica oleracea, which includes broccoli. Furthermore, since it has high genetic homology with Arabidopsis, it is possible to identify target genes involved in various traits. Furthermore, since there are abundant resources such as genome information, it is possible to design gRNAs that minimize off-target mutations, and we can expect the development of breeding using genome editing.
Here, in this experiment, to create a mutant line by genome editing of broccoli, we performed transformation using the Agrobacterium method, identification of the edited line, and seed collection to obtain progeny, as described below.
　本実験で使用した材料、器具、培地の仕様は以下のとおりである。
　ブロッコリ種子は、サカタのタネより販売されているＦ１品種の緑嶺（リョクレイ）を利用して形質転換を行った。 The specifications of the materials, instruments, and media used in this experiment are as follows.
Broccoli seeds were transformed using Ryokurei, an F1 variety available from Sakata Seed.
　準備する器具は、以下の通りである。
　（１）滅菌濾紙Ａ（アドバンテック定量濾紙Ｎｏ．２，φ１２５ｍｍの２枚一組でホイルに包んでオートクレーブ後、乾燥したもの）
　（２）滅菌濾紙Ｂ（Ｗｈａｔｍａｎ　５定量濾紙，φ７０ｍｍ，シャーレに入れてオートクレーブ後、乾燥したもの）
　（３）ルーツェピンセット（１７　ｃｍ，各種メーカー）
　（４）カミソリ（フェザーの青函両刃）
　（５）レザーブレードフォルダ（ケニスなど）
　　　　メス（フェザー，ステンレス外科用替え刃メス１１）
　（６）５０　ｍＬ　ディスポーサブルチューブ（Ｃｏｒｎｉｎｇ，　４３０２９０など）
　（７）プラスチックシャーレ（ＩＷＡＫＩ　滅菌シャーレ深型９０　ｍｍ　Ｘ　２０　ｍｍなど）
　（８）サージカルテープ（３Ｍマイクロポアｃｏｄｅ：　１５３０－０など）
　（９）５　ｍＬディスポーザブルピペット（ファルコン，　＃３５７５４３） The equipment to be prepared is as follows.
(1) Sterilized filter paper A (advantech quantitative filter paper No. 2, φ125 mm, wrapped in foil, autoclaved, and dried)
(2) Sterile filter paper B (Whatman 5 quantitative filter paper, φ70mm, autoclaved in a petri dish and dried)
(3) Luce tweezers (17 cm, various manufacturers)
(4) Razor (feather blue box double-edged)
(5) Leather blade folder (Kenneth etc.)
Scalpel (feather, stainless steel surgical blade scalpel 11)
(6) 50 mL disposable tube (Corning, 430290, etc.)
(7) Plastic petri dish (IWAKI sterile petri dish deep 90 mm x 20 mm, etc.)
(8) Surgical tape (3M Micropore code: 1530-0, etc.)
(9) 5 mL disposable pipette (Falcon, #357543)
　本実験の培地の仕様は以下のとおりである。
　Ｋｏｈｎｏ－Ｍｕｒａｓｅ　ｅｔ　ａｌ．が報告したセイヨウナタネ（Ｂｒａｓｓｉｃａ　ｎａｐｕｓ）の形質転換法を一部改変して、ブロッコリの形質転換を行った。
　（１）アグロバクテリウム培養液（ＹＥＢ）：　０．１％　ｙｅａｓｔ　ｅｘｔｒａｃｔ，　０．５％　ｂｅｅｆ　ｅｘｔｒａｃｔ，　０．５％　ｐｅｐｔｏｎｅ，　０．５％　ショ糖，　０．０５％　ＭｇＳＯ４・７Ｈ２０　（ｐＨ７）
　（２）ＢＹ２培養用液体培地（Ｄ０．２）：３０　ｇ／Ｌショ糖，０．２　ｍｇ／Ｌ　２，４－Ｄを含むＭＳ培地，１００　ｍＬのコルベンに３０　ｍＬに分注したもの。ｐＨは５．６から５．８に調整。
（３）播種培地：３０　ｇ／Ｌショ糖，４　ｇ／Ｌ　Ｇｅｌｒｉｔｅ　（富士フイルムワコー，０７５－０５６５５）を含むＭＳ培地。ｐＨは５．６から５．８に調整。オートクレーブ後にマゼンタボックス（シグマ，ＧＡ－７）に５０　ｍＬに分注。
（４）感染用液体培地（ＭＳＨ）：３０　ｇ／Ｌショ糖を含むＭＳ培地。ｐＨは５．６から５．８に調整。
（５）前培養・共存培養・除菌培地（ＭＢ３Ｄ）：３０　ｇ／Ｌショ糖，１　ｍｇ／Ｌ　２，４－Ｄ，　０．６％　Ａｇａｒｏｓｅ　（シグマ，タイプ１，Ａ０１６９）を含むＭＳＢ５培地。ｐＨは５．６から５．８に調整。除菌の際は，５００　ｍｇ／Ｌのカルベニシンを含む培地とする。
（６）選抜培地１　（ＭＢ１）：１０　ｇ／Ｌショ糖，３　ｍｇ／Ｌ　ＢＡＰ，　５００　ｍｇ／Ｌカルベニシン，　１０ｍｇ　／Ｌハイグロマイシン，０．６％　Ａｇａｒｏｓｅを含むＭＳＢ５培地。ｐＨは５．６から５．８に調整。ＡｇＮＯ３を含むＭＢ１は、ＡｇＮＯ３を５　ｍｇ／Ｌになるように滅菌後に添加する。
（７）選抜培地２（Ｂ５ＢＺ）：３０　ｇ／Ｌショ糖，３　ｍｇ／Ｌ　ＢＡＰ，　１　ｍｇ／Ｌ　ｚｅａｔｉｎ，　５００　ｍｇ／Ｌカルベニシン，　１０　ｍｇ／Ｌハイグロマイシン，０．６％　Ａｇａｒｏｓｅを含むＢ５培地。ｐＨは５．６から５．８に調整。
（８）シュート育成培地（Ｂ５Ｐ）：３０　ｇ／Ｌショ糖，０．１　ｍｇ／Ｌ　ＢＡＰ，　５００　ｍｇ／Ｌカルベニシン，　１０　ｍｇ／Ｌハイグロマイシン，０．６％　Ａｇａｒｏｓｅを含むＢ５培地。ｐＨは５．６から５．８に調整。
（９）発根培地　（ＭＳＮＢ）：１０　ｇ／Ｌショ糖，０．１　ｍｇ／Ｌ　ＮＡＡ，　０．０１　ｍｇ／Ｌ　ＢＡＰ，　４　ｇ／Ｌ　Ｇｅｌｒｉｔｅを含むＭＳ培地。ｐＨは５．６から５．８に調整。オートクレーブ後にマゼンタボックス（シグマ，ＧＡ－７）に５０　ｍＬに分注。
（１０）順化用マゼンダボックス：マゼタボックスにバーミキュライト３号（ベルミテック株式会社）を１００　ｍＬ程度入れ、１０００倍希釈したハイポネックスを５０　ｍＬ程度加えオートクレーブ滅菌する。
（１１）ＭＳ培地は、ＭＳ　Ｉ～ＩＶ保存液を１倍になるように作成する。ＭＳＢ５は、ＭＳ　Ｉ～ＩＩＩ保存液と　Ｂ５　Ｖｉｔａｍｉｎ保存液を１倍になるように作成する。Ｂ５培地は、Ｂ５　ｖｉｔａｍｉｎ保存液とＢ５　Ｉｎｏｒｇａｎｉｃ保存液を１倍になるように作成する。
（１２）Ｚｅａｔｉｎ、　カルベニシン、ハイグロマイシンは、オートクレーブ後、十分に培地が冷めてから添加する。また、ＭＳ３Ｄ培地は、シャーレあたり２０　ｍＬ、それ以外の培地は２５　ｍＬを分注後、ラップに包んで冷蔵庫に保存している。 The specifications of the culture medium for this experiment are as follows.
Kohno-Murase et al. Broccoli was transformed by partially modifying the method for transforming Brassica napus reported by.
(1) Agrobacterium culture solution (YEB): 0.1% yeast extract, 0.5% beef extract, 0.5% peptone, 0.5% sucrose, 0.05% MgSO4・7H20 (pH 7)
(2) BY2 culture liquid medium (D0.2): MS medium containing 30 g/L sucrose, 0.2 mg/L 2,4-D, dispensed into 30 mL in a 100 mL Kolben. pH was adjusted from 5.6 to 5.8.
(3) Seeding medium: MS medium containing 30 g/L sucrose, 4 g/L Gelrite (Fuji Film Wako, 075-05655). pH was adjusted from 5.6 to 5.8. After autoclaving, dispense 50 mL into a magenta box (Sigma, GA-7).
(4) Liquid medium for infection (MSH): MS medium containing 30 g/L sucrose. pH was adjusted from 5.6 to 5.8.
(5) Preculture/coculture/sterilization medium (MB3D): MSB5 medium containing 30 g/L sucrose, 1 mg/L 2,4-D, 0.6% Agarose (Sigma, Type 1, A0169) . pH was adjusted from 5.6 to 5.8. When sterilizing, use a medium containing 500 mg/L of carbenicin.
(6) Selection medium 1 (MB1): MSB5 medium containing 10 g/L sucrose, 3 mg/L BAP, 500 mg/L carbenicin, 10 mg/L hygromycin, and 0.6% Agarose. pH was adjusted from 5.6 to 5.8. MB1 containing AgNO3 is added after sterilization so that AgNO3 is 5 mg/L.
(7) Selection medium 2 (B5BZ): B5 containing 30 g/L sucrose, 3 mg/L BAP, 1 mg/L zeatin, 500 mg/L carbenicin, 10 mg/L hygromycin, 0.6% Agarose Culture medium. pH was adjusted from 5.6 to 5.8.
(8) Shoot growth medium (B5P): B5 medium containing 30 g/L sucrose, 0.1 mg/L BAP, 500 mg/L carbenicin, 10 mg/L hygromycin, and 0.6% Agarose. pH was adjusted from 5.6 to 5.8.
(9) Rooting medium (MSNB): MS medium containing 10 g/L sucrose, 0.1 mg/L NAA, 0.01 mg/L BAP, 4 g/L Gelrite. pH was adjusted from 5.6 to 5.8. After autoclaving, dispense 50 mL into a magenta box (Sigma, GA-7).
(10) Magenta box for acclimatization: Put about 100 mL of Vermiculite No. 3 (Vermitec Co., Ltd.) in a mazeta box, add about 50 mL of Hyponex diluted 1000 times, and sterilize it in an autoclave.
(11) Prepare the MS medium so that the MS I to IV storage solution is 1:1. For MSB5, prepare the MS I-III stock solution and the B5 Vitamin stock solution in a 1:1 ratio. B5 medium is prepared using B5 vitamin preservation solution and B5 Inorganic preservation solution in a ratio of 1:1.
(12) Zeatin, carbenicin, and hygromycin are added after the medium has sufficiently cooled down after autoclaving. Furthermore, after dispensing 20 mL of MS3D medium and 25 mL of other media per petri dish, they were wrapped in plastic wrap and stored in the refrigerator.
　形質転換体の作出法は、以下のとおりである。
　（１）種子滅菌
形質転換に用いる緑嶺の種子については、以下のように滅菌後に使用している。緑嶺の種子は、コート種子として販売されている。本滅菌により種子からコート剤が洗い落とされる。冷蔵保存した種子を播種する際は、室温に戻るまでシールは外さない。１年程度は、発芽率が保たれるが、発芽率が低下するようなら、新たに滅菌するほうが好適である。
　５０　ｍＬディスポーザブルチューブに種子２～５　ｇ程度と７０％エタノールを加え室温で１分間振とうする。
　次に、７０％エタノールを駒込ピペットで取り除き、あらかじめ作成した５倍希釈次亜塩素酸ナトリウム４０　ｍＬ程度に対しＴｗｅｅｎ２０または家庭用中性洗剤を２滴ほど加えた溶液を加え、さらに室温で２５分間振とうする。
　次に、次亜塩素酸ナトリウム溶液を駒込ピペットで取り除き、滅菌水で洗浄を５～６回程度繰り返す。
　次に、クリーンベンチ内で滅菌濾紙Ａの上に種子を広げ完全に乾くまで風乾する。筆者らはほぼ１日かけて完全に乾燥させている。その後、３　ｃｍ滅菌シャーレ数個に分けて、パラフィルム等でシールし冷蔵保存する。 The method for producing transformants is as follows.
(1) Seed sterilization The Midorine seeds used for transformation are used after sterilization as follows. Midorine seeds are sold as coated seeds. This sterilization washes off the coating agent from the seeds. When sowing seeds that have been refrigerated, do not remove the seal until the seeds return to room temperature. The germination rate will be maintained for about one year, but if the germination rate decreases, it is better to sterilize it again.
Add about 2 to 5 g of seeds and 70% ethanol to a 50 mL disposable tube, and shake for 1 minute at room temperature.
Next, remove the 70% ethanol with a Komagome pipette, add a solution of about 2 drops of Tween 20 or household neutral detergent to about 40 mL of the 5-fold diluted sodium hypochlorite prepared in advance, and incubate for another 25 minutes at room temperature. Shake.
Next, remove the sodium hypochlorite solution with a Komagome pipette, and repeat washing with sterile water about 5 to 6 times.
Next, spread the seeds on sterile filter paper A in a clean bench and air dry until completely dry. The authors took almost a day to dry completely. Thereafter, divide into several 3 cm sterile petri dishes, seal with parafilm, etc., and store in the refrigerator.
　（２）播種（感染７日前）
　種子を１マゼンタボックスあたり２５粒ずつピンセットで播種培地に埋め込まないように置いていく。本実験では、１実験回あたり２ボックス分播種している。 (2) Seeding (7 days before infection)
Place 25 seeds per magenta box using tweezers so as not to embed them in the seeding medium. In this experiment, seeds were sown in two boxes per experiment.
　（３）アグロバクテリウムの培養（感染前日）
　バイナリベクターを保持しているアグロバクテリウムのグリセロールストックからマイクロスパーテル１杯分を、抗生物質を含む１０　ｍＬのＹＥＢを入れた５０　ｍＬのディスポ－ザブルチューブに加え２８℃で一晩振とう培養する。 (3) Cultivation of Agrobacterium (the day before infection)
Add one microspatial of the glycerol stock of Agrobacterium containing the binary vector to a 50 mL disposable tube containing 10 mL of YEB containing antibiotics, and culture with shaking at 28°C overnight. .
　（４）ブロッコリ胚軸切片化（感染前日）
約０．５　ｍＬのＢＹ２細胞（植継ぎ後７日目）を、先折の５　ｍＬディスポーザブルピペットを用いてＭＢ３Ｄ培地上に加え、その上に滅菌濾紙Ｂを空気が入らないようにのせる。
次に、無菌播種後７日目の胚軸をブレードホルダに装着した片刃カミソリを用いて切り出し、ＭＢ３Ｄ培地にのせた濾紙上で２～５　ｍｍの長さの切片にする。子葉近くの胚軸切片については、茎頂分裂組織が含まれないように注意する。乾かないように切片を濾紙の上に平坦に並べ翌日まで培養する。（２３℃，　１６ｈＬ／８ｈＤ，　培養棚において５０Ｗの蛍光灯２本から３０　ｃｍ程度離れた程度の照度） (4) Sectioning of broccoli hypocotyls (the day before infection)
About 0.5 mL of BY2 cells (7 days after subculture) is added onto the MB3D medium using a bent-tipped 5 mL disposable pipette, and sterile filter paper B is placed on top of the cells to prevent air from entering.
Next, cut out the hypocotyls 7 days after aseptic sowing using a single-edged razor attached to a blade holder, and cut them into pieces 2-5 mm long onto filter paper placed on MB3D medium. When cutting the hypocotyls near the cotyledons, care should be taken not to include the shoot apical meristem. To prevent the pieces from drying out, arrange them flat on the filter paper and culture until the next day. (23°C, 16hL/8hD, illumination approximately 30 cm away from two 50W fluorescent lights on a culture shelf)
　（５）感染処理（感染０日）
　一晩培養したアグロバクテリウム培養液を２６００　ｘ　ｇで１５分遠心分離後、沈殿にＭＳＨを１０　ｍＬ加え、沈殿したアグロバクテリウムを懸濁する。
　次に、同じ条件で遠心分離後、沈殿にＭＳＨ　１０　ｍＬを加えて再懸濁する。再懸濁液の一部を取り置き、ＭＳＨで１０倍程度に希釈しＯＤ６００を測定する。この値から、ＯＤ６００　＝　０．１に調製した感染溶液を２０　ｍＬ作成し、９　ｃｍの滅菌シャーレに入れておく。
　次に、前培養した胚軸切片を薬さじの柄を使ってかき集めてシャーレ内の感染溶液に投入し、室温で２０分間緩やかに振とうする。
　次に、感染溶液をピペットで取り除き、胚軸切片を滅菌濾紙Ａに広げ余分な感染液を取り除き前培養で使用したＭＢ３Ｄ培地上に戻す。この際、切り口が乾燥しないように平らに並べる。
　次に、サージカルテープでシャーレの側面をシール後、暗箱に入れ、２３℃で３日間共存培養する。 (5) Infection treatment (infection day 0)
After centrifuging the overnight Agrobacterium culture at 2600 x g for 15 minutes, 10 mL of MSH is added to the precipitate to suspend the precipitated Agrobacterium.
Next, after centrifugation under the same conditions, 10 mL of MSH is added to the precipitate to resuspend it. A portion of the resuspension is set aside, diluted approximately 10 times with MSH, and the OD600 is measured. From this value, prepare 20 mL of an infection solution adjusted to OD600 = 0.1 and place it in a 9 cm sterile petri dish.
Next, the pre-cultured hypocotyl sections are collected using the handle of a medicine spoon, placed into the infection solution in the Petri dish, and gently shaken for 20 minutes at room temperature.
Next, the infection solution is removed with a pipette, and the hypocotyl sections are spread on sterile filter paper A to remove excess infection solution and returned to the MB3D medium used in the preculture. At this time, lay the cut ends flat so that they do not dry out.
Next, after sealing the sides of the Petri dish with surgical tape, it is placed in a dark box and co-cultured at 23°C for 3 days.
　（６）除菌（感染処理後３日目）
　胚軸切片を５００　ｍｇ／Ｌのカルベニシンを含むＭＢ３Ｄに移植する。次の植え継ぎまで４日間ほどなので切片どうしの間隔はそれ程広く取らないで良い。これ以降２３℃、明期１６時間暗期８時間の条件で培養する。光量については、５０　Ｗの蛍光灯２本ぐらいの照度で行っている（播種２ボックスあたり２～３枚の培地使用）。 (6) Sterilization (3rd day after infection treatment)
Transfer hypocotyl sections into MB3D containing 500 mg/L carbenicin. It will take about 4 days until the next transplant, so there is no need to space the sections so far apart. Thereafter, the cells are cultured at 23° C. with a light period of 16 hours and a dark period of 8 hours. Regarding the amount of light, the illuminance is about the same as two 50 W fluorescent lights (2 to 3 plates of culture medium are used per 2 boxes of sowing).
　（７）選抜１（感染処理後１週間目）
ＡｇＮＯ３を含むＭＢ１に胚軸切片を移植する。カルスの形成で少し大きくなることを想定して切片どうしの間隔を少し空ける（播種２ボックスあたり３～４枚の培地使用）。以後、不定芽が出たら適宜切り出してＢ５Ｐへ移植する。 (7) Selection 1 (1st week after infection treatment)
Transfer hypocotyl sections to MB1 containing AgNO3. Leave some space between the sections to allow for them to grow slightly due to callus formation (use 3 to 4 medium plates per 2 boxes of seeding). Thereafter, when adventitious buds appear, they are cut out and transplanted to B5P.
　（８）選抜２（感染処理後３週間目）
　選抜１の植継ぎから２週間後ＭＢ１に移植する（播種２ボックスあたり３～４枚の培地使用）。 (8) Selection 2 (3 weeks after infection treatment)
Two weeks after transplanting selection 1, transplant to MB1 (use 3 to 4 medium plates per 2 sowing boxes).
　（９）選抜３以降（感染処理後５週間目）
　選抜２から２週間後、Ｂ５ＢＺに植継ぐ。これ以降は２週間おきにＢ５ＢＺに移植する。Ｂ５ＢＺへの植え継ぎ３回を目安にそれ以降の移植の有無を判断する。不定芽を形成しない切片は、黒ずんでくるので適宜移植をせず廃棄する。 (9) Selection 3 and after (5 weeks after infection)
Two weeks after selection 2, transplant to B5BZ. After this, transplant to B5BZ every two weeks. After three transplants to B5BZ, decide whether to transplant thereafter. Cuttings that do not form adventitious buds will turn black and should be discarded without transplanting.
　（１０）シュート育成
　不定芽が出たらメスで余分な組織を取り除きＢ５Ｐ培地へ移植する。移植の目安は２週間だが、シュートの生育状況（数枚の本葉が展開するなど）によりＭＳＮＢへの移植を判断する。 (10) Shoot cultivation When adventitious shoots appear, remove excess tissue with a scalpel and transplant to B5P medium. The standard time for transplanting is two weeks, but the decision to transplant to MSNB will be made based on the growth of the shoot (several true leaves have developed, etc.).
　（１１）発根
　切り出したシュートをＭＳＮＢへ移植する。余分な基部の組織片などをメスで取り除くことで発根が促進する場合が多い。発根するまで２～３週間おきにシュートの形を整えながら植え継ぐ。ここで、分枝したシュートをＮＳＮＢ培地に移植することで株分けする事も可能である。また、発根が見られない場合は、１０　ｇ／Ｌショ糖，４　ｇ／Ｌ　Ｇｅｌｒｉｔｅを含む１／２ＭＳ培地で培養してもよい。マゼンタボックス内で十分に発根したらバーミキュライト培地へ移植する。 (11) Rooting Transplant the cut out shoots to MSNB. Rooting is often promoted by removing excess tissue from the base with a scalpel. Transplant while adjusting the shape of the shoot every 2 to 3 weeks until rooting takes place. Here, it is also possible to divide the plants by transplanting the branched shoots into NSNB medium. Furthermore, if no rooting is observed, the plants may be cultured in a 1/2 MS medium containing 10 g/L sucrose and 4 g/L Gelrite. Once the roots have grown sufficiently in the magenta box, transplant them to vermiculite medium.
　（１２）順化
　アガロースを丁寧に取り除き、バーミキュライト培地に移植する。バーミキュライト培地は、ビニール袋をかぶせて輪ゴムで止めておく。マゼンタボックスの底面から根が確認出来たら、ビニール袋の角の片側を切り、外気を入れて順化を進める。１週間後ぐらいに、根の発達が見られたら、もう一方の角を切り、順化を進める。こまめに枯れた葉を除き、カビの発生を防ぐとよい。ビニール袋の両端を落としても、萎れることなく生育すれば鉢上げする。灌水は滅菌した１０００倍希釈のハイポネックス液を与える。 (12) Acclimation Carefully remove the agarose and transfer to vermiculite medium. Cover the vermiculite medium with a plastic bag and secure it with a rubber band. Once roots can be seen from the bottom of the magenta box, cut one side of the corner of the plastic bag and let outside air in to acclimate. After about a week, when you see root development, cut the other corner and acclimatize. It is a good idea to remove dead leaves frequently to prevent mold from forming. If the plant grows without wilting even if you drop both ends of the plastic bag, pot it up. For irrigation, give a sterile 1000-fold diluted Hyponex solution.
　（１３）鉢上げ
　バーミキュライト培地から丁寧に植物体を取り、根についているバーミキュライトを洗い流した後、園芸培土を入れた３号鉢ポリ鉢に植え、十分に灌水する。鉢にビニール袋をかぶせて温室で生育させる。移植後、２から３日目からビニール袋の角を切り順化する。移植後１０から２０日ほどで鉢底から根が観察出来ることが多く、それを目安にビニール袋を外し、十分に葉が展開するまで栽培を続ける。その後、花芽分化のために春化処理を行う。本発明者は、１０時間明期１２℃／１４時間暗期８℃の条件で４０日ほど行い花芽を誘導している。 (13) Raising the pot Carefully remove the plant from the vermiculite medium, wash away the vermiculite attached to the roots, then plant it in a No. 3 plastic pot filled with horticultural soil and water thoroughly. Grow in a greenhouse by covering the pot with a plastic bag. From 2 to 3 days after transplanting, cut the corners of the plastic bag and acclimate. Roots can often be observed from the bottom of the pot about 10 to 20 days after transplanting, at which time the plastic bag can be removed and cultivation continued until the leaves have fully developed. After that, vernalization treatment is performed for flower bud differentiation. The present inventor induces flower buds by conducting the process for about 40 days under the conditions of a 10-hour light period of 12°C and a 14-hour dark period of 8°C.
　ここで、本実験に用いたプライマー配列情報を示す。
　図９は、ＡＣＯ３遺伝子断片増幅に利用したプライマー配列情報を示す図である。
　図９に示す配列情報は、Bo-ACO3遺伝子のゲノム配列である。
　なお、データベース上の登録番号は、GenBank：LR031877.1である。
　６行目末尾から６文字目以降の「GGT」から始まり、「ACA」で終わる８行目１６文字目まで、及び、１２行目２６文字目以降の「AGG」から始まり、「AAC」で終わる１４行目末尾から５文字目までが、イントロンである。
　８行目中央に示す「CCA」がPAM配列である。
　５行目から６行目にかけて黒地で反転している領域、及び、９行目の黒地で反転している領域が、ACO3遺伝子断片増幅に用いたプライマーペアである。 Here, information on the primer sequences used in this experiment is shown.
FIG. 9 is a diagram showing primer sequence information used for ACO3 gene fragment amplification.
The sequence information shown in FIG. 9 is the genome sequence of the Bo-ACO3 gene.
The registration number on the database is GenBank: LR031877.1.
Starts with "GGT" from the 6th character from the end of the 6th line, ends with "ACA" up to the 16th character of the 8th line, and starts with "AGG" from the 26th character of the 12th line, ends with "AAC" The 5th character from the end of the 14th line is an intron.
"CCA" shown in the center of the 8th line is the PAM array.
The black inverted region from the 5th line to the 6th line and the black inverted region in the 9th line are the primer pairs used for amplifying the ACO3 gene fragment.
　標的遺伝子であるＢｏ－ＡＣＯ３遺伝子断片の増幅については以下の手順で行った．ＰＣＲ増幅用のミクスチャーとして，１サンプル当たりＤＷ：　２６　μＬ，５×ＰｒｉｍｅＳＴＡＲ　ＧＸＬ　Ｂｕｆｆｅｒ　（ＴＡＫＡＲＡ）：１０　μＬ，２．５ｍＭ　ｄＮＴＰ　Ｍｉｘｔｕｒｅ：　４　μＬ，１０　μＭ　Ｆｏｒｗａｒｄ　ｐｒｉｍｅｒ：　２．５μＬ，１０　μＭ　Ｒｅｖｅｒｓｅ　ｐｒｉｍｅｒ：　２．５　μＬ，Ｐｒｉｍｅ　ＳＴＡＲ　ＧＸＬ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（ＴＡＫＡＲＡ，　１．２５　Ｕ／　μＬ）　１．０　μＬとした。これに、形質転換体から調製したＤＮＡを４　μＬ加え反応液とした。．ＰＣＲ増幅サイクルは，第１ステップとして９５℃で１分間，第２ステップとして９５℃で２０秒間，第３ステップとして６０℃（で２０秒間，第４ステップとして６８℃で３０秒間とし、第２ステップから第４ステップを３５サイクル繰り返した。その後、６８℃で３分後、４℃で保存した。増幅産物はＴＡＥまたはＴＢＥで作成した１．２％アガロースゲルで電気泳動し分析した．私用したプライマーの配列は、Ｆｏｒｗａｒｄ　ｐｒｉｍｅｒの配列がＡＧＡＧＡＧＡＧＧＡＣＴＣＡＣＧＡＴＧＧＡＧ，Ｒｅｖｅｒｓｅ　ｐｒｉｍｅｒの配列がＴＧＡＡＴＣＴＧＴＣＴＴＣＣＡＴＧＣＡＣＴＴである。
ＰＣＲ産物をＱＩＡｑｕｉｃｋ　ＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　ｋｉｔ（Ｑｉｑｇｅｎ）を用いて精製後、サンガーシークエンス法を用いて配列を決定した。また、精製ＰＣＲ産物をクローニング後、同様にサンガーシーケンス法で配列を決定した。 Amplification of the Bo-ACO3 gene fragment, which is the target gene, was performed according to the following procedure. As a mixture for PCR amplification, DW per sample: 26 μL, 5×PrimeSTAR GXL Buffer (TAKARA): 10 μL, 2.5 mM dNTP Mixture: 4 μL, 10 μM Forward primer: 2.5 μL, 10 μM Reverse Primer: 2.5 μL, Prime STAR GXL DNA Polymerase (TAKARA, 1.25 U/μL) 1.0 μL. To this, 4 μL of DNA prepared from the transformant was added to prepare a reaction solution. ．． The PCR amplification cycle consisted of the first step at 95°C for 1 minute, the second step at 95°C for 20 seconds, the third step at 60°C for 20 seconds, the fourth step at 68°C for 30 seconds, and the second step at 60°C for 30 seconds. The fourth step was repeated for 35 cycles. Then, after 3 minutes at 68°C, it was stored at 4°C. The amplified products were analyzed by electrophoresis on a 1.2% agarose gel prepared with TAE or TBE. Regarding the primer sequences, the Forward primer sequence is AGAGAGAGGGACTCACGATGGAG, and the Reverse primer sequence is TGAATCTGTCTTCCATGCACTT.
The PCR product was purified using QIAquick PCR Purification kit (Qiqgen), and then sequenced using Sanger sequencing. Furthermore, after cloning the purified PCR product, the sequence was similarly determined by Sanger sequencing.
　このような実験により、以下の結果がえられた。
　図１０は、ＡＣＯ３形質転換体の塩基配列の一例である。
　図１１は、ダイレクトシークエンス解析の結果を示す図である。
　本実験のブロッコリの塩基配列は「２１０６１１－９★Ａ」として示されている。
　図１０及び図１１の上部には、塩基の番号が付されている。この番号を用いて説明する。
　１０８番目を基準に、それ以後の塩基配列を参照すると、本実験のブロッコリ以外においては、ＩＰＨＥＬＬＤＲ…とアミノ酸が配列されている。
　これに対して、本実験のブロッコリの塩基配列は、ＤＳＴＲＡＴ★Ｑ…とアミノ酸が配列されている。 From such experiments, the following results were obtained:
FIG. 10 is an example of the base sequence of an ACO3 transformant.
FIG. 11 shows the results of direct sequence analysis.
The broccoli base sequence in this experiment is shown as "210611-9★A."
The bases are numbered at the top of Figures 10 and 11. The following description will use these numbers.
When the base sequences subsequent to the 108th base are referenced, the amino acids are arranged as follows, IPHELLDR..., except for the broccoli used in this experiment.
In contrast, the base sequence of broccoli in this experiment has amino acids arranged as follows: DSTRAT★Q . . .
　本実験の結果、ＡＣＯ３標的コンストラクトの形質転換体については、１１系統取得できた。そのうち１系統には一塩基挿入がバイアレリックに検出された。この系統については、自殖系統および緑嶺との交配を行い、得られた種子を播種し、栽培中である。
　このように、ＡＣＯ３遺伝子について、ゲノム編集が可能である。 As a result of this experiment, 11 lines of transformants of the ACO3 target construct were obtained. A single nucleotide insertion was biallelicly detected in one of the strains. This line has been crossed with the inbred line and Midorine, and the resulting seeds have been sown and are currently being cultivated.
In this way, genome editing is possible for the ACO3 gene.
　図１２は、本発明のブロッコリの生産方法のフローを示す図である。
　ステップＳ１１において、対象ブロッコリのノックアウトを行う。対象ブロッコリのノックアウト方法は上述の通りである。
　ステップＳ１２において、対象ブロッコリから種子をつくる。 FIG. 12 is a diagram showing the flow of the broccoli production method of the present invention.
In step S11, the target broccoli is knocked out. The knockout method for the target broccoli is as described above.
In step S12, seeds are produced from the target broccoli.
　ステップＳ１３において、対象ブロッコリの系統を母本として、他のブロッコリ品種に本形質を導入する。即ち、ステップＳ１２の対象ブロッコリの種子から成長したブロッコリと、他の品種のブロッコリとを交配させることにより、上述の形質と、他の品種のブロッコリの形質とを持つブロッコリとし、当該ブロッコリの種子をつくる。
　なお、ステップＳ１２の種子のみならず、ステップＳ１１の対象ブロッコリと直接交配させることにより他のブロッコリ品種に本形質を導入してもよい。 In step S13, this trait is introduced into other broccoli varieties using the target broccoli line as the mother plant. That is, by crossing the broccoli grown from the target broccoli seeds in step S12 with broccoli of another variety, broccoli having the above-mentioned traits and the traits of broccoli of another variety is produced, and the seeds of the broccoli are to make.
In addition, this trait may be introduced not only into the seeds in step S12 but also into other broccoli varieties by direct crossing with the target broccoli in step S11.
　ステップＳ１４において、種子を播種し出荷用のブロッコリを生育する。
　これにより、対象の遺伝子がノックアウトされたブロッコリが出荷用として生産される。 In step S14, the seeds are sown and broccoli for shipping is grown.
This allows broccoli with the target gene knocked out to be produced for shipment.
　次に、以下（１）乃至（５）に関連する内容について説明する。
　（１）ＡＣＯ３＊／個体は人工気象室内ではコントロールの緑嶺と同じ生育状況を示した。
　（２）高温（２０℃）での劣化試験を行ったところ、４日目では緑嶺と比べて変化はなかった。
　（３）１０日後、ＡＣＯ３＊個体では生存花蕾が多いという観察結果を得た。
　（４）新たなＡＣＯ３＊変異系統を２つ取得した。
　（５）ＡＣＯ２＊変異系統を４つ取得した。 Next, contents related to (1) to (5) below will be explained.
(1) The ACO3 * /individual showed the same growth conditions as the control Midorine in the artificial climate room.
(2) When a deterioration test was conducted at high temperature (20°C), there was no change compared to Midorine on the 4th day.
(3) After 10 days, it was observed that there were many surviving flower buds in ACO3 * individuals.
(4) Two new ACO3 * mutant lines were obtained.
(5) ACO2 * Four mutant lines were obtained.
　植物の黄変の原因は、老化現象の１つでエチレンの作用により、クロロフィルの分解が促進され生じると考えられている。植物におけるエチレンの生合成は（図４参照）、前駆体であるメチオニンからＳ－アデノシルメチオニンを経由して、環状化合物である１－アミノシクロプロパン－１－カルボン酸（以下、ＡＣＣと略す）が合成され、この化合物を基質としたＡＣＣ酸化酵素（以下、ＡＣＯと略す）の触媒作用によりエチレンが生じる。
　ＡＣＯの発現抑制や変異導入が可能になれば、低温処理が従来よりも少なくてもエチレンの生合成量が減り、老化過程が抑制できると考えられる。
　Ｂｏ－ＡＣＯ１は、基礎的なエチレンの合成と栄養組織におけるエチレン合成に関与する。
　Ｂｏ－ＡＣＯ２は、生殖器官におけるエチレン合成と収穫後初期のエチレン生合成に関与し、収穫直後には発現しないが、収穫後２時間以内に増加する。
　Ｂｏ－ＡＣＯ３は、収穫後後期のエチレン合成に関与し，収穫後３日目に発現することが報告されている。 The cause of yellowing in plants is one of the aging phenomena, and it is thought that the decomposition of chlorophyll is promoted by the action of ethylene. Ethylene biosynthesis in plants (see Figure 4) involves the production of 1-aminocyclopropane-1-carboxylic acid (hereinafter abbreviated as ACC), a cyclic compound, from the precursor methionine via S-adenosylmethionine. is synthesized, and ethylene is produced by the catalytic action of ACC oxidase (hereinafter abbreviated as ACO) using this compound as a substrate.
If it becomes possible to suppress ACO expression or introduce mutations, it is thought that the amount of ethylene biosynthesis can be reduced and the aging process can be suppressed even if the low-temperature treatment is less than before.
Bo-ACO1 is involved in basal ethylene synthesis and ethylene synthesis in vegetative tissues.
Bo-ACO2 is involved in ethylene synthesis in reproductive organs and ethylene biosynthesis in the early stage after harvest, and is not expressed immediately after harvest, but increases within 2 hours after harvest.
It has been reported that Bo-ACO3 is involved in ethylene synthesis in the late stage after harvest, and is expressed on the third day after harvest.
　ここでは、Ｂｏ－ＡＣＯ２、Ｂｏ－ＡＣＯ３に対し上述のゲノム編集を施し、黄変抑制系統を作出する。
　具体的には、ＡＣＯ遺伝子の変異ブロッコリ作出の試みをしたところ、ＡＣＯ２については、ＡＣＯ３やＡＣＯ１と異なる配列のものが得られた。 Here, Bo-ACO2 and Bo-ACO3 are subjected to the above-mentioned genome editing to create yellowing suppressed lines.
Specifically, when we attempted to create broccoli with a mutant ACO gene, we obtained ACO2 with a different sequence from ACO3 and ACO1.
　先ず、花蕾劣化試験について説明する。
　ゲノム編集を施したＴ０世代（上述の２１０６１１－９の系統）のブロッコリを育てて大きくし、これを自殖させてＴ１世代のブロッコリの種を用いることにより花蕾劣化試験を行った。
　日付は一例であるが、播種日は２０２２年６月２５日、育成条件としては、そのまま植えられる土ポットを用いて発芽（２３℃）。９ｃｍポリ鉢にてポット育苗（２３℃　明条件／１８℃　暗条件　～９月１８日）。春化処理（９月１８日から１２℃　明条件１０時間／８℃　暗条件１４時間）。 First, the flower bud deterioration test will be explained.
Genome-edited T0 generation (lineage 210611-9) broccoli was grown and enlarged, self-fertilized, and T1 generation broccoli seeds were used to conduct a flower bud deterioration test.
The date is just an example, but the sowing date is June 25, 2022, and the growing conditions are germination (23°C) using soil pots that can be planted as is. Raise seedlings in 9cm polyester pots (23℃ light condition/18℃ dark condition until September 18th). Vernalization treatment (10 hours of light at 12°C/14 hours of darkness at 8°C from September 18th).
　Ｔ１世代としては、ＡＣＯ３がノックアウトされた、染色体が２つとも劣勢ホモになっている系統のＡＣＯ３＊／ＡＣＯ３＊のものと、ＡＣＯ３がノックアウトされたもの及びされてないものの組み合わせ（ＡＣＯ３＊／ＡＣＯ３）となるヘテロのものとを用い、さらには、緑嶺（ＡＣＯ３／ＡＣＯ３）を用いた。
　春化処理にて花蕾の発達を促し、１１月１０日時点で生育を観察したところ、問題は見られなかった（Ｔ１個体は、栄養成長の段階では、野生型の緑嶺と生育に大きな差が見られなかった。即ち上述の（１）の内容が確認できた。）。
　１２月２１日に十分に発達した花蕾が形成され、その形成された花蕾を切り取って所定の状態（ここでは切り取った部分をチューブに入れるような状態）にし、２０℃の環境下で劣化試験を開始した。
　花蕾劣化試験は、言い換えると花蕾の老化実験であり、野生型（緑嶺）、ＡＣＯ３変異ヘテロ系統、ＡＣＯ３劣性ホモ系統（機能喪失型）を用いて室温で行った試験となる。 The T1 generation includes ACO3 * /ACO3 * , a strain in which ACO3 has been knocked out and both chromosomes are recessively homozygous, and a combination of strains with and without ACO3 knockout (ACO3 * / ACO3 ) was used, and Midorine (ACO3/ACO3) was used.
We promoted the development of flower buds through vernalization treatment, and when we observed their growth as of November 10, no problems were observed (at the vegetative growth stage, the growth of T1 individuals was significantly different from that of the wild type Midorigei). (In other words, the content of (1) above was confirmed.)
Fully developed flower buds were formed on December 21st, and the formed flower buds were cut out and placed in a specified state (here, the cut portion was placed in a tube), and a deterioration test was conducted in an environment of 20 degrees Celsius. It started.
In other words, the flower bud deterioration test is a flower bud aging experiment, and is a test conducted at room temperature using a wild type (Midorine), an ACO3 mutant hetero line, and an ACO3 recessive homo line (loss-of-function type).
　図１３は、Ｔ１世代を用いた花蕾劣化試験の結果を示す図である。
　図１３は、花蕾劣化試験１０日後の状態を示しており、結果として１０日後においては、野生型、変異ヘテロ系統で花蕾の老化が進み、黄化脱落が進んだ。一方、機能喪失型の変異ホモ系統では、生存花蕾数が著しく多く（上述の（３）の内容が確認できた。）、また開花したものも出現した。なお、４日後では緑嶺と比べて変化はなかった（上述の（２）の内容が確認できた。）。 FIG. 13 shows the results of a flower bud deterioration test using the T1 generation.
Figure 13 shows the state 10 days after the flower bud deterioration test, and as a result, after 10 days, flower bud aging and yellowing/falling off had progressed in the wild type and mutant heterozygous lines. On the other hand, the loss-of-function mutant homozygous line had a significantly higher number of surviving flower buds (confirming the above item (3)), and some even bloomed. However, after 4 days, there was no change compared to Midorirei (confirming the above item (2)).
　具体的に、図１３の上段に示す野生型（緑嶺ＡＣＯ３／ＡＣＯ３）では、黄化が進むと共に花蕾がボロボロと落ちてしまい、花蕾が大きくなったものが見られない。
　また、図１３の中段に示すＡＣＯ３変異ヘテロ系統（ＡＣＯ３＊ヘテロ）も、黄化が進むと共に花蕾がボロボロと落ちてしまい、一部の花蕾が若干大きくなった程度である。
　これに対し、図１３の下段に示すＡＣＯ３劣性ホモ系統（ＡＣＯ３＊ホモ）では、黄化はあるものの生存花蕾数が著しく多く、その生存した花蕾が大きくなって、特に図示しないが開花したものも出現した。
　従って、ＡＣＯ３変異ホモ系統は、花蕾の老化を抑制することができた。
　本花蕾劣化試験を通じては、Ｔ０世代からＴ１世代へと、きちんと遺伝したという事実が確認できた。 Specifically, in the wild type (Midorine ACO3/ACO3) shown in the upper row of FIG. 13, the flower buds fall off as yellowing progresses, and no enlarged flower buds can be seen.
Furthermore, in the ACO3 mutant heterozygous line (ACO3 * hetero) shown in the middle row of FIG. 13, the flower buds fall off as yellowing progresses, and some of the flower buds only become slightly larger.
On the other hand, in the ACO3 recessive homozygous line (ACO3 * homo) shown in the lower row of Figure 13, the number of surviving flower buds is significantly larger, although there is some yellowing, and the surviving flower buds are larger and some even bloom (not shown in the figure). Appeared.
Therefore, the ACO3 mutant homologous line was able to suppress senescence of flower buds.
Through the real flower bud deterioration test, we were able to confirm that the gene was properly inherited from the T0 generation to the T1 generation.
　次に、ゲノム編集による変異導入に係る新たな結果について、図１４乃至図１９を参照しながら報告する。なお、ゲノム編集に関しては上述の通りであるので、ここでは説明を省略する。 Next, we will report new results regarding mutation introduction by genome editing with reference to FIGS. 14 to 19. Note that genome editing is as described above, so the explanation will be omitted here.
　図１４は、ＡＣＯ３変異体系統（１）２１０６１１－６を示す図である。
　ＡＣＯ３をノックアウトする変異体系統としては、Ａの一塩基挿入がされているものがとれた。図１４に示すように終止コドンができて、結果、機能喪失型（完全長の酵素タンパク質が生産されない）の変異系統がとれた。
　また、２２塩基欠失でアミノ酸配列が異なるものもとれた。終止コドンができて、機能喪失型の変異系統がとれた。 FIG. 14 is a diagram showing ACO3 mutant line (1) 210611-6.
As a mutant strain that knocks out ACO3, one in which a single nucleotide of A has been inserted was obtained. As shown in FIG. 14, a stop codon was generated, and as a result, a loss-of-function mutant strain (no full-length enzyme protein was produced) was obtained.
In addition, one with a 22 base deletion and a different amino acid sequence was also obtained. A stop codon was created, and a loss-of-function mutant strain was obtained.
　図１５は、ＡＣＯ３変異体系統（２）２１０２２６－１を示す図である。
　ＡＣＯ３をノックアウトする変異体系統としては、Ｔの一塩基挿入がされているものがとれた。図１５に示すように終止コドンができて、結果、機能喪失型の変異系統がとれた。
　図１４及び図１５の説明から、上述の（４）の内容が確認できた。 FIG. 15 is a diagram showing ACO3 mutant line (2) 210226-1.
As a mutant strain that knocks out ACO3, one in which a single base of T has been inserted was obtained. As shown in FIG. 15, a stop codon was created, and as a result, a loss-of-function mutant strain was obtained.
From the explanation of FIGS. 14 and 15, the content of (4) above was confirmed.
　一方、図１６は、ＡＣＯ２変異体系統（１）２３０３０６－１を示す図である。
　ＡＣＯ２をノックアウトする変異体系統としては、Ａ又はＧの一塩基挿入がされているものがとれた。図１６に示すように終止コドンができて、結果、機能欠失型の変異系統がとれた。 On the other hand, FIG. 16 is a diagram showing ACO2 mutant line (1) 230306-1.
Mutant lines in which ACO2 is knocked out include those in which a single base of A or G has been inserted. As shown in FIG. 16, a stop codon was created, and as a result, a functional deletion mutant strain was obtained.
　図１７は、ＡＣＯ２変異体系統（２）２３０３０６－３を示す図である。
　ＡＣＯ２をノックアウトする変異体系統としては、Ｃの一塩基挿入がされているものがとれた。図１７に示すように終止コドンができて、結果、機能欠失型の変異系統がとれた。 FIG. 17 shows ACO2 mutant line (2) 230306-3.
A mutant strain in which ACO2 was knocked out was one in which a single nucleotide of C was inserted. As shown in FIG. 17, a stop codon was created, and as a result, a functional deletion mutant strain was obtained.
　図１８は、ＡＣＯ２変異体系統（３）２３０５１６－１を示す図である。
　ＡＣＯ２をノックアウトする変異体系統としては、Ｇ又はＴの一塩基挿入がされているものがとれた。図１８に示すように終止コドンができて、結果、機能欠失型の変異系統がとれた。 FIG. 18 shows ACO2 mutant line (3) 230516-1.
Mutant lines in which ACO2 was knocked out were those in which a single G or T base was inserted. As shown in FIG. 18, a stop codon was created, and as a result, a functional deletion mutant strain was obtained.
　図１９は、ＡＣＯ２変異体系統（４）２３０５１６－２を示す図である。
　ＡＣＯ２をノックアウトする変異体系統としては、Ｔの一塩基挿入がされているものがとれた。図１９に示すように終止コドンができて、結果、機能欠失型の変異系統がとれた。
　図１６乃至図１９の説明から、上述の（５）の内容が確認できた。 FIG. 19 shows ACO2 mutant line (4) 230516-2.
As a mutant strain that knocks out ACO2, one in which a single base of T has been inserted was obtained. As shown in FIG. 19, a stop codon was created, and as a result, a mutant strain lacking function was obtained.
From the explanation of FIGS. 16 to 19, the content of (5) above was confirmed.
　以上、図１３乃至図１９を参照しながら説明してきた。ＡＣＯ２変異系統及びＡＣＯ３との二重変異系統が作出出来、老化の抑制の程度がさらに高いものを取得することができる。 The above has been explained with reference to FIGS. 13 to 19. An ACO2 mutant strain and a double mutant strain with ACO3 can be created, and strains with even higher levels of inhibition of aging can be obtained.
　ここまで本発明の一実施形態について説明したが、本発明は、上述の実施形態に限定されるものではなく、本発明の目的を達成できる範囲での変形、改良等は本発明に含まれるものとみなす。 Although one embodiment of the present invention has been described so far, the present invention is not limited to the above-described embodiment, and modifications, improvements, etc. are included in the present invention within the scope of achieving the purpose of the present invention. regarded as.
　本発明が適用されるブロッコリの生産方法は、次のような特徴を有していれば足り、各種各様な実施の形態を取ることができる。
　即ち、本発明が適用されるブロッコリ（例えば図１に示すブロッコリ等）の生産方法は、
　遺伝子操作の対象となるブロッコリを対象ブロッコリとして、当該対象ブロッコリの遺伝子のうち、収穫後所定期間で劣化させるエチレンを発生させるＡＣＯ２とＡＣＯ３のうち少なくとも一方に対して、ノックアウトさせるための遺伝子操作を行う第１ステップ（例えば図１２のステップＳ１１）と、
　前記遺伝子操作の後の前記対象ブロッコリから種子をつくる第２ステップ（例えば図１２のステップＳ１２）と、
　前記種子又は当該種子から播種された種子を用いて、出荷用のブロッコリを生育する第３ステップ（例えば図１２のステップＳ１３）と、
　を含む。
　これにより、ゲノム編集を用いてより好適な性質の作物を提供し、ゲノム編集への理解を促進することができる。 The broccoli production method to which the present invention is applied only needs to have the following characteristics, and can take various embodiments.
That is, the method for producing broccoli (for example, the broccoli shown in FIG. 1) to which the present invention is applied is as follows:
Genetically manipulate the target broccoli to knock out at least one of ACO2 and ACO3, which generate ethylene that degrades within a predetermined period of time after harvest, among the genes of the target broccoli. A first step (for example, step S11 in FIG. 12),
a second step of producing seeds from the target broccoli after the genetic manipulation (for example, step S12 in FIG. 12);
A third step (for example, step S13 in FIG. 12) of growing broccoli for shipping using the seeds or seeds sown from the seeds;
including.
This makes it possible to use genome editing to provide crops with more suitable properties and to promote understanding of genome editing.
　また、本発明が適用される種子は、次のような特徴を有していれば足り、各種各様な実施の形態を取ることができる。
　即ち、本発明が適用されるブロッコリ（例えば図１に示すブロッコリ等）又は種子（例えば上述のＴ１世代のブロッコリの種等）は、
　収穫後所定期間で劣化させるエチレンを発生させるＡＣＯ２とＡＣＯ３のうち少なくとも一方に対して、ノックアウトさせるための遺伝子操作が行われたブロッコリ又は当該ブロッコリを起源として作られる。
　これにより、ゲノム編集を用いてより好適な性質の作物を提供し、ゲノム編集への理解を促進することができる。 Moreover, the seeds to which the present invention is applied only need to have the following characteristics, and can take various embodiments.
That is, broccoli (for example, the broccoli shown in FIG. 1) or seeds (for example, the above-mentioned T1 generation broccoli seeds, etc.) to which the present invention is applied,
It is produced from broccoli that has been genetically engineered to knock out at least one of ACO2 and ACO3, which generate ethylene that deteriorates over a predetermined period of time after harvest, or from said broccoli.
This makes it possible to use genome editing to provide crops with more suitable properties and to promote understanding of genome editing.
　ＳＴ１、ＳＴ２、ＳＴ３・・・ステップ ST1, ST2, ST3...step

Claims (2)

  1. 　遺伝子操作の対象となるブロッコリを対象ブロッコリとして、当該対象ブロッコリの遺伝子のうち、収穫後所定期間で劣化させるエチレンを発生させるＡＣＯ２とＡＣＯ３のうち少なくとも一方に対して、ノックアウトさせるための遺伝子操作を行う第１ステップと、
    　前記遺伝子操作の後の前記対象ブロッコリから種子をつくる第２ステップと、
    　前記種子又は当該種子から播種された種子を用いて、出荷用のブロッコリを生育する第３ステップと、
    　を含むブロッコリの生産方法。     Genetically manipulate the target broccoli to knock out at least one of ACO2 and ACO3, which generate ethylene that degrades within a predetermined period of time after harvest, among the genes of the target broccoli. The first step and
    a second step of producing seeds from the target broccoli after the genetic manipulation;
    a third step of growing broccoli for shipping using the seeds or seeds sown from the seeds;
    Broccoli production method including.
  2. 　収穫後所定期間で劣化させるエチレンを発生させるＡＣＯ２とＡＣＯ３のうち少なくとも一方に対して、ノックアウトさせるための遺伝子操作が行われたブロッコリ又は当該ブロッコリを起源として作られた種子。 Broccoli or seeds derived from said broccoli that have been genetically engineered to knock out at least one of ACO2 and ACO3, which produce ethylene that causes degradation over a specified period after harvest.
PCT/JP2023/034032 2022-09-21 2023-09-20 Production method, broccoli, and seed WO2024063076A1 (en)

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