KR20210063235A - Glucoraphanin rich plants and preparation method thereof - Google Patents

Abstract

The present invention relates to a plant having a high glucoraphanin content. More particularly, by a composition comprising a guide RNA including a sequence complementary to at least a portion of SEQ ID NO: 1, and Cas9 protein, it is possible to efficiently prepare a plant with a high glucoraphanin content.

Description

Plants with a high glucoraphanin content and a method for producing the same {GLUCORAPHANIN RICH PLANTS AND PREPARATION METHOD THEREOF}

The present invention relates to a plant with a high glucoraphanin content.

Glucosinolate and its derivatives are anionic natural products rich in sulfur ions, and are one of the secondary metabolites abundantly present in Brassicaceae and plants. Glucosinolate is decomposed into isothiocyanates and nitriles by myrosinases, an endogenous hydrolytic enzyme, when plant tissues are damaged. These hydrolysis products are harmful to pests. It has several other biological activities, such as a defense agent and an attractant. As a representative plant having a high glucosinolate content, Brassica oleracea , which includes broccoli and cabbage as sub-cultivars, is known.

The prior art used a method of introducing a species with a high glucoraphanin content into a breeding program and performing continuous crossbreeding for several decades in order to cultivate a variety having a higher content of glucosinolate. There is a technical difficulty in that the breeding becomes possible only by continuously performing crossbreeding for a long period of time by using the wild resources of other species with a high glucoraphanin content as a material for breeding.

Korean Patent No. 1616570

An object of the present invention is to provide a composition for producing a plant having a high glucoraphanin content.

An object of the present invention is to provide a method for producing a plant having a high glucoraphanin content.

An object of the present invention is to provide a plant with a high glucoraphanin content.

1. A guide RNA comprising a sequence complementary to at least a portion of SEQ ID NO: 1; And Cas9 protein; glucoraphanin high content plant composition comprising a.

2. The composition of the above 1, wherein the guide RNA has the sequence of SEQ ID NO: 4, glucoraphanin high content plant preparation.

3. The plant according to 1 above, wherein the plant is at least one selected from the group consisting of collard greens, gairan, cauliflower, cabbage, red cabbage, bell pepper, kohlrabi, broccoli, savoy cabbage, cauliflower and kale, glucoraphanin A composition for producing a high content plant.

4. A guide RNA comprising a sequence complementary to at least a portion of SEQ ID NO: 1; And Cas9 protein; A method for producing a plant having a high glucoraphanin content, comprising the step of treating plant cells.

5. The method of 4 above, wherein the guide RNA has the sequence of SEQ ID NO: 4, glucoraphanin high content plant production method.

6. The plant according to 4 above, wherein the plant is at least one selected from the group consisting of collard greens, cauliflower, cabbage, red cabbage, bell pepper, kohlrabi, broccoli, savoy cabbage, cauliflower and kale, glucoraphanin A method for producing high content plants.

7. A plant with a high glucoraphanin content comprising a sequence in which at least one of the nucleotides at positions corresponding to positions 896 to 901 in SEQ ID NO: 2 is deleted.

8. The plant with a high glucoraphanin content according to the above 7, comprising a sequence in which the nucleotides at positions corresponding to positions 896 to 900 in SEQ ID NO: 2 are deleted.

9. The plant with a high glucoraphanin content according to the above 7, comprising a sequence in which at least one nucleotide is inserted between the nucleotides at positions corresponding to positions 900 and to 901 in SEQ ID NO: 2.

10. The above 7, wherein the plant is at least one selected from the group consisting of collard greens, cauliflower, cabbage, red cabbage, cherry radish, kohlrabi, broccoli, savoy cabbage, cauliflower and kale, glucoraphanin high content plants.

The guide RNA of the present invention can specifically target a portion of the gene sequence encoding MYB28 of Brassica oleracea.

By using the composition for producing a plant having a high glucoraphanin content of the present invention, the gene encoding MYB28 can be edited within a short period of time, and thus, a plant having a high glucoraphanin content can be efficiently produced.

1 shows a gene sequence encoding wild-type MYB2 8 and a portion in which a mutation occurred in the MYB28 sequence of a gene-edited individual (Examples 1 to 3).
2 shows data comparing the glucoraphanin content of genetically edited broccoli individuals (Examples 1 to 3) and several commercial varieties.
3 is a wild-type Brassica oleracea broccoli variety ( Brassica oleracea var. italica ) to some sequences including SEQ ID NO: 1 among the gene sequences encoding MYB28 of other species of cruciferous plants Corresponding to the gene sequence encoding MYB28 This is the result of sorting.

The present invention provides a guide RNA comprising a sequence complementary to at least a portion of SEQ ID NO: 1; And Cas9 protein; provides a composition for producing a plant containing a high glucoraphanin content.

The sequence of SEQ ID NO: 1 (CAGAGATGATGACCACAATGAGG) is a sequence included in the gene sequence encoding MYB28 of wild-type Brassica oleracea.

The guide RNA is complementary to at least a portion of SEQ ID NO: 1, completely complementary to the entire sequence of SEQ ID NO: 1 or complementary to some of the sequences of SEQ ID NO: 1, that is, a hybrid-complex. may be complementary enough to form. For example, in the sequence of SEQ ID NO: 1, 23, 22 or more, 21 or more, 20 or more, 19 or more, 18 or more, 17 or more, 16 or more, 15 or more, 14 or more, 13 or more, 12 or more, 11 or more, 10 or more, 9 or more, 8 or more, 7 or more, 6 or more, or 5 or more.

The length of the guide RNA is not particularly limited as long as it forms a hybrid complex with the sequence of SEQ ID NO: 1 and is sufficient to guide gene editing, for example, 5 or more, 8 or more, 10 or more, 13 or more, 15 or more, 18 or more, 20 or more, 22 or more, 23 or more. The upper limit may be, for example, 30, 25, 23, etc., but is not limited thereto.

Preferably, the guide RNA may consist of a sequence completely complementary to the sequence of SEQ ID NO: 1.

Guide RNA refers to RNA that complementarily binds to a specific part of DNA and serves to guide the cleavage of a specific part of DNA. The guide RNA can form a complex with Cas9, an enzyme that cuts the double helix of DNA.

Cas9 protein is a Cas9 protein from Streptococcus pyogenes , a Cas9 protein from Campylobacter jejuni , a Cas9 protein from Streptococcus thermophilus , Staphylococcus aureus. It may be selected from the group consisting of Cas9 protein derived from Staphylococcus aureus and Cas9 protein derived from Neisseria meningitidis.

The Cas9 protein may include, for example, the sequence of SEQ ID NO: 3, and specifically, may consist of the sequence of SEQ ID NO: 3.

At least one of the guide RNA and the Cas9 protein of the composition of the present invention may be loaded into a vector in the form of a nucleic acid.

For example, the composition of the present invention may include a vector loading a nucleic acid encoding a guide RNA; and a Cas9 protein.

For another example, the composition of the present invention may include a guide RNA; and a vector loading a nucleic acid encoding a Cas9 protein.

For another example, the composition of the present invention may include a first vector loading a nucleic acid encoding a guide RNA; and a first vector loading a nucleic acid encoding a Cas9 protein.

For another example, the composition of the present invention may include a vector loading both a nucleic acid encoding a guide RNA and a nucleic acid encoding a Cas9 protein.

The vector may be a plasmid or a viral vector.

The viral vector may be one or more viral vectors selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vacciniavirus, poxvirus and herpes simplex virus.

The guide RNA and Cas9 protein may be included in the composition of the present invention in the form of a guide RNA-Cas9 protein complex. The guide RNA-Cas9 protein complex may be formed by interaction between some nucleic acids of the guide RNA and some amino acids of the Cas9 protein.

It is known that glucoraphanin is a type of glucosinolates and is hydrolyzed by myrosinase to be converted into isothiocyanate derivatives exhibiting anticancer activity. Glucosinolate and its derivatives are anionic natural products rich in sulfur ions, and are one of the secondary metabolites abundantly present in cruciferous plants. Glucosinolate and its derivatives are decomposed into isothiocyanate and nitrile by myrosinase, an endogenous hydrolytic enzyme, when plant tissues are damaged. These hydrolysis products are protective substances and attractants against pests. It has several other biological activities such as substances.

The plant may be a species of Brassica oleracea ( B. oleracea ), and specific sub-cultivars include collard ( B. oleracea ' Acephala' group), gairan (B. oleracea ' Alboglabra' group), and cauliflower ( B. oleracea 'Botrytis' group), cabbage or red cabbage ( B. oleracea 'Capitata' group), bell pepper ( B. oleracea 'Gemmifera' group), kohlrabi (B. oleracea 'Gongylodes' group), broccoli ( B. oleracea 'Gongylodes' group) oleracea 'Italica' group), savoy cabbage ( B. oleracea 'Sabauda' group), cauliflower ( B. oleracea 'Sabellica' group), and kale ( B. oleracea 'Viridis' group) can

Plants are Brassica Juncea ( brassica juncea ; B. juncea ) species, Brassica nigra ( B. nigra ) species, Brassica napus ( B. napus ) species, Brassica rapa ( Brassica rapa ) species ; B. rapa ) may be a species, but is not limited thereto.

In addition, the present invention provides a guide RNA comprising a sequence complementary to at least a portion of SEQ ID NO: 1; And Cas9 protein; provides a method for producing a plant having a high glucoraphanin content comprising the step of treating plant cells.

Since the guide RNA, Cas9 protein and plant have been described above, a detailed description thereof will be omitted.

The cell may be a protoplast from which the cell wall has been removed. Since protoplasts do not have a cell wall, artificial manipulations such as cell fusion, genetic manipulation and artificial somatic mutation are easy.

At least one of the guide RNA and the Cas9 protein may be loaded into a vector in the form of a nucleic acid and processed into a cell.

For example, the method of the present invention may include treating plant cells with a vector loading a nucleic acid encoding a guide RNA; and a Cas9 protein.

For another example, the method of the present invention may include treating a plant cell with a guide RNA; and a vector loading a nucleic acid encoding a Cas9 protein.

For another example, the method of the present invention may include treating a plant cell with a first vector loading a nucleic acid encoding a guide RNA; and a first vector loading a nucleic acid encoding a Cas9 protein.

For another example, the method of the present invention may include treating plant cells with a vector loading both a nucleic acid encoding a guide RNA and a nucleic acid encoding a Cas9 protein.

Since the content of the vector has been described above, a detailed description thereof will be omitted.

In order to process guide RNA and/or Cas9 protein into plant cells, Agrobacterium mediated, gene gun (particle bombardment), virus, electroporation, direct injection, polyethylene glycol (PEG) ), Liposome, Imbibition method, Pollen-tube pathway method, In-planta transformation, etc. may be used.

The virus may be cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV), but is not limited thereto.

The direct injection method may be a microinjection method or a macroinjection method, but is not limited thereto.

When the guide RNA and the Cas9 protein are treated in a cell by the method of the present invention, the guide RNA may be complementarily bound to SEQ ID NO: 1 included in the gene of the cell, and the Cas9 protein can cleave the strand comprising SEQ ID NO: 1 can After that, various indels (insertion and deletion) may occur at the cleaved site.

The term ‘indel’ refers to a mutation in which some nucleotides are inserted or deleted in the middle of the nucleotide sequence of DNA. As described above, when the guide RNA and Cas9 protein complex cuts the target sequence in the transcriptional regulatory region of the target gene, the indel is subjected to homology directed repairing (HDR) or non-homologous end-binding (Non-homologous end). It may be introduced into the target sequence in the process of repair by the joining; NHEJ) mechanism.

The term 'Non-homologous end joining (NHEJ)' refers to a method of repairing or repairing double-stranded breaks in DNA by joining both ends of a cleaved double-stranded or single-stranded together. Two compatible ends formed by breakage (eg, cleavage) of a strand may refer to a mechanism of repairing a broken double-strand as the two ends are completely joined by repeating frequent contact.

The term 'homologous recombination repair (HDR)' refers to a method that can be corrected without error by using a homologous sequence as a template to repair or repair the DNA of a damaged transcriptional regulatory region. It repairs or repairs damaged DNA using information on the unmodified complementary nucleotide sequence or information on sister chromatids to repair or repair the DNA. The most common form of HDR is homologous recombination (HR). HDR is a repair or repair method that usually occurs during the S or G2/M phase of actively dividing cells.

Further, the present invention provides a glucoraphanin-rich plant comprising a sequence in which at least one of the nucleotides at positions corresponding to positions 896 to 901 in the gene sequence encoding MYB28 of wild-type Brassica oleracea is deleted. to provide.

The gene sequence encoding MYB28 of wild-type Brassica oleracea may be SEQ ID NO:2.

SEQ ID NO: 2: ATGTCAAGAAAGCCATGTTGTGTCGGAGAAGGGCTGAAGAAAGGGGCATGGACCACCGAGGAAGATAAGAAACTCATCTCTTACATCCATGAACATGGAGAAGGAGGCTGGCGTGACATTCCTCAAAAAGCTGGATTGAAAAGGTGTGGAAAGAGTTGTAGACTGCGATGGACTAACTACCTAAAACCTGAAATCAAAAGAGGCGAGTTTAGTTCAGAGGAGGAACAGATTATCATCATGCTTCATGCTTCTCGTGGAAACAAGTGGTCGGTCATAGCGAGACATTTACCTAGAAGAACAGACAATGAGATCAAGAACTACTGGAACACACATCTCAAGAAACGTTTGATCGAACAGGGTACTGATCCCGTGACTCACAAGCCACTAGCTTCTAATACAAACCCTACTGTACCTGAGAATTTGCATTCCCTAGATGCATCTAGTTCCGACAAGCAATACTCCCGGTCAAGCTCAATGCCTTCCATGTCTTGTACTCCTTCCTCCGGTTTCAACACGGTTTTCGAGAATACCAGCAAAGATGGGACACCAGTTCGTGAGGACGATTCCTTGAGTCGCAAGAAACGTTTGAAGAAATCAAGTTCTACATCAAGGCTTTTGAACAAAGTTGCGGCTAAGGCCACTTCCATGAAAGAAGCTTTGTCTGCTTCCATGGAAGGTAGCTTGAATGCTAATACAAGCTTTTCCAATGGCTACTCTGAGCAGATTCTCAATGAAGATGATAGTCCTAATGCATCCCTCATAAACACTCTCGCCGAGTTCGATCCCTTCCTCCAAACAACGTTTTACCCTGAGAATGAAATGAATACTACTTCTGATCTCGATATAGATCAGGACTACTTCTCACATTTTCTCGAAAATTTCGGCAGAGATGATGACCACAATGAGGAGCACTACATGAATCATAACTATGGTCATGATCTTCTTATGTCCGATGTGTCCCAAGAAGTCTCATCAACTAGCGTTGATG ATCAAGACAATACTAATGAGGGTTGGTCAAATTATCTTCTTGACCATGCTGATTTTATACATGACATGGATTCTGATTCCCTCGGAAAGCATATCATATGA.

The term 'a position corresponding to a specific position in a specific gene of a specific species' refers to a position corresponding to the specific position in a specific gene of a specific species when the specific gene sequence of the specific species is aligned with the specific gene of another species can do.

The positions of positions 896 to 901 correspond to the case of the wild-type Brassica oleracea broccoli variety ( Brassica oleracea var. italica ), and the position corresponding to the position may vary depending on the detailed variety, plant type, etc., and the The position can be confirmed by aligning the sequence of the plant with the sequence of SEQ ID NO:2. This can be equally applied to the nucleotide positions to be described later.

Of the gene sequence (SEQ ID NO: 2) encoding MYB28 of the wild-type Brassica oleracea broccoli variety ( Brassica oleracea var. ), a gene sequence encoding MYB28 of a cruciferous plant of a different species was aligned with each other, as shown in FIG. 3 .

The plant having a high glucoraphanin content of the present invention may include a sequence in which nucleotides at positions corresponding to positions 896 to 900 in SEQ ID NO: 2 are deleted.

The plant having a high glucoraphanin content of the present invention may include a sequence in which a nucleotide at the position corresponding to position 901 in SEQ ID NO: 2 is deleted.

The plant having a high glucoraphanin content of the present invention may include a sequence in which nucleotides at positions corresponding to positions 898 to 900 in SEQ ID NO: 2 are deleted.

The plant having a high glucoraphanin content of the present invention may include a sequence in which nucleotides at positions corresponding to positions 899 to 901 in SEQ ID NO: 2 are deleted.

The plant having a high glucoraphanin content of the present invention may further include a sequence in which at least one nucleotide is inserted between nucleotides at positions corresponding to positions 900 and 901 in SEQ ID NO: 2. The base of the inserted sequence may be adenine.

The plant having a high glucoraphanin content of the present invention may be a plant genetically edited using guide RNA and Cas9.

The plant having a high glucoraphanin content of the present invention is a gene-edited plant, and has a higher glucoraphanin content than a wild-type plant. In addition, the plant having a high glucoraphanin content of the present invention has a glucoraphanin content twice or more higher than that of a commercial variety. For example, Brassica vilosa , which has a high glucoraphanin content, is introduced into a breeding system and has more than twice the glucoraphanin content than the Beneporteran variety developed through continuous crossbreeding for decades.

Hereinafter, examples will be described in detail in order to describe the present invention in detail.

1. Construction of a gene-edited protoplast encoding MYB28

1-1. Gene editing methods

Guide RNA (SEQ ID NO: 4: CAGAGATGATGACCACAATG) of SEQ ID NO: 1 (CAGAGATGATGACCACAATGAGG) and Cas9 protein (SEQ ID NO: 3; ) were synthesized in Brassica oleracea var. Italica protoplasm was introduced by treatment with polyethylene glycol (PEG).

PEG (20% PEG, 0.2M mannitol, 0.1M CaCl ₂ ) was mixed ^{with broccoli protoplasts (5×10 5} ) together with guide RNA (25 μg) and Cas9 protein (25 μg). Guide RNA and Cas9 protein were introduced through the addition of the same amount of culture medium 1 (2mM MES, _{154mM NaCl 2} , 125mM CaCl _{2 , 5mM KCl, pH5.7) as the mixture.} After the gene introduction process, culture solution 1 was removed by centrifugation, and culture solution 2 (4mM MES, 0.5M mannitol, 20mM KCl, pH5.7) was added and cultured at 25°C for 24 hours.

1-2. Sequence confirmation of gene-edited protoplasts

The sequence of the protoplasts of the three gene-edited individuals was confirmed by the method of 1-1, and the results are shown in FIG. 1 .

Different types of sequence mutations occurred in the three gene-edited individuals, and it was confirmed that mutations occurred on both chromosomes when compared with the wild-type sequence. Wild-type Brassica oleracea var. The sequence of the gene encoding MYB28 of italica is shown in SEQ ID NO:2.

1 shows a gene sequence encoding wild-type MYB28 and a portion in which a mutation occurs in the MYB28 sequence of a gene-edited individual (Examples 1 to 3).

As shown in FIG. 1 , in Example 1, 4bp deletions of different types occurred in different homologous chromosomes (see Example 1_Chromosome 1 and Example 1_Chromosome 2 in FIG. 1 ). In Example 2, 6bp deletion and 1bp insertion occurred in different homologous chromosomes (see Example 2_Chromosome 1 and Example 2_Chromosome 2 in FIG. 1). Also, in Example 3, 2bp deletion and 1bp insertion occurred in different homologous chromosomes (see Example 3_chromosome 1 and Example 3_chromosome 2 in FIG. 1 ).

2. MYB28 Confirmation of glucoraphanin content of gene-edited individuals encoding

2-1. Genetically Edited Object Production

Broccoli protoplasts introduced with guide RNA and Cas9 protein were treated in primary medium (1×B5 culture medium, 70g/L D-mannitol, 20g/L glucose, 0.25mg/L 2,4-D, 1mg/L NAA, 2mg/L L TDZ, 0.1g/L MES, pH 5.8), dark treatment at 25℃ for 10 days, and cultured for 1 week under long day conditions, secondary medium (1×B5 culture medium, 40g/L D-mannitol, 20g/ L sucrose, 0.25 mg/L 2,4-D, 0.2 mg/L NAA, 2 mg/L TDZ, 0.1 g/L MES, pH 5.8) for 4 to 5 weeks, tertiary medium (1×MS culture medium) , 30g/L sucrose, 0.5mg/L NAA, 2mg/L TDZ, 0.1g/L, 6g/L agarose, pH 5.8) for 2 to 4 months incubation, quaternary medium (0.5×MS culture medium, 10g/L sucrose, 6g/L agarose, pH 5.8) was replaced in the order of the medium composition to produce individuals.

2-2. Comparison of glucoraphanin content of genetically edited individuals

The glucoraphanin contents of Examples 1 to 3, which are genetically edited broccoli individuals, and some commercial varieties (Comparative Examples 1 to 4) were compared.

As Comparative Example 1, a maternal species of a commercially available cultivar kingdom (kingdom maternal, KDM), as Comparative Example 2 a paternal cultivar of a commercially available cultivar kingdom as Comparative Example 2 (kingdom paternal, KDP), Comparative Example 3 a commercially available cultivar Kingdom (KD), Comparative Example 4 As a commercially available variety Beneforete (Beneforete; BF) was used.

Frozen broccoli samples were pulverized, suspended in 100% methanol extraction solvent, sonicated for 1 hour, and then pretreated with a 55°C bath treatment and reduced pressure concentration for 1 hour. The pre-treated samples were extracted and quantitatively analyzed three times using a high performance liquid chromatography (HPLC) system. For HPLC separation analysis, 5% mobile phase A: 5 mM Tetramethylammonium bromide and 95% mobile phase B: 100% acetonitrile were separated at a flow rate of 0.8 ml/min, and the separated material was monitored at UV 230 nm.

As a result of the glucoraphanin content analysis, it was confirmed that Example 2 showed a glucoraphanin content that was more than twice that of other commercial varieties and Examples 1 and 3 (see FIG. 2 ).

Example 2 showing a high glucopanin content includes a sequence in which nucleotides 896 to 900 are deleted in the gene sequence (SEQ ID NO: 2) encoding MYB28 of wild-type Brassica oleracea. In addition, Example 2 includes a sequence in which one nucleotide (base: A) is further inserted between nucleotides 900 and 901 among the gene sequence (SEQ ID NO: 2) encoding MYB28 of wild-type Brassica oleracakea.

<110> SEJONG UNIVERSITY INDUSTRY ACADEMY COOPERATION FOUNDATION <120> GLUCORAPHANIN RICH PLANTS AND PREPARATION METHOD THEREOF <130> 20P11020 <150> KR 10-2019-0151645 <151> 2019-11-22 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Brassica oleracea <400> 1 cagagatgat gaccacaatg agg 23 <210> 2 <211> 1089 <212> DNA <213> Brassica oleracea <400> 2 atgtcaagaa agccatgttg tgtcggagaa gggctgaaga aaggggcatg gaccaccgag 60 gaagataaga aactcatctc ttacatccat gaacatggag aaggaggctg gcgtgacatt 120 cctcaaaaag ctggattgaa aaggtgtgga aagagttgta gactgcgatg gactaactac 180 ctaaaacctg aaatcaaaag aggcgagttt agttcagagg aggaacagat tatcatcatg 240 cttcatgctt ctcgtggaaa caagtggtcg gtcatagcga gacatttacc tagaagaaca 300 gacaatgaga tcaagaacta ctggaacaca catctcaaga aacgtttgat cgaacagggt 360 actgatcccg tgactcacaa gccactagct tctaatacaa accctactgt acctgagaat 420 ttgcattccc tagatgcatc tagttccgac aagcaatact cccggtcaag ctcaatgcct 480 tccatgtctt gtactccttc ctccggtttc aacacggttt tcgagaatac cagcaaagat 540 gggacaccag ttcgtgagga cgattccttg agtcgcaaga aacgtttgaa gaaatcaagt 600 tctacatcaa ggcttttgaa caaagttgcg gctaaggcca cttccatgaa agaagctttg 660 tctgcttcca tggaaggtag cttgaatgct aatacaagct tttccaatgg ctactctgag 720 cagattctca atgaagatga tagtcctaat gcatccctca taaacactct cgccgagttc 780 gatcccttcc tccaaacaac gttttaccct gagaatgaaa tgaatactac ttctgatctc 840 gatatagatc aggactactt ctcacatttt ctcgaaaatt tcggcagaga tgatgaccac 900 aatgaggagc actacatgaa tcataactat ggtcatgatc ttcttatgtc cgatgtgtcc 960 caagaagtct catcaactag cgttgatgat caagacaata ctaatgaggg ttggtcaaat 1020 tatcttcttg accatgctga ttttatacat gacatggatt ctgattccct cggaaagcat 1080 atcatatga 1089 <210> 3 <211> 1368 <212> PRT <213> Artificial Sequence <220> <223> Cas9 <400> 3 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 1010 1015 1020 Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser 1025 1030 1035 1040 Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 1045 1050 1055 Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 1060 1065 1070 Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 1075 1080 1085 Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1090 1095 1100 Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile 1105 1110 1115 1120 Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 1125 1130 1135 Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 1140 1145 1150 Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1155 1160 1165 Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 1170 1175 1180 Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 1185 1190 1195 1200 Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 1205 1210 1215 Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 1220 1225 1230 Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 1250 1255 1260 Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val 1265 1270 1275 1280 Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 1285 1290 1295 His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu 1300 1305 1310 Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 1315 1320 1325 Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1330 1335 1340 Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile 1345 1350 1355 1360 Asp Leu Ser Gln Leu Gly Gly Asp 1365 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> guide RNA <400> 4 cagagatgat gaccacaatg 20

Claims (10)

a guide RNA comprising a sequence complementary to at least a portion of SEQ ID NO: 1; And Cas9 protein; glucoraphanin high content plant composition comprising a.
The method according to claim 1, wherein the guide RNA has the sequence of SEQ ID NO: 4, glucoraphanin high content plant production composition.
The method according to claim 1, wherein the plant is at least one selected from the group consisting of collard, kale, cauliflower, cabbage, red cabbage, cherry radish, kohlrabi, broccoli, savoy cabbage, cauliflower and kale, high glucoraphanin content Compositions for the manufacture of plants.
a guide RNA comprising a sequence complementary to at least a portion of SEQ ID NO: 1; And Cas9 protein; glucoraphanin high content plant production method comprising the step of treating plant cells.
The method according to claim 4, wherein the guide RNA has the sequence of SEQ ID NO: 4, glucoraphanin high content plant production method.
The method according to claim 4, wherein the plant is at least one selected from the group consisting of collard greens, cauliflower, cabbage, red cabbage, cherry radish, kohlrabi, broccoli, savoy cabbage, cauliflower and kale. Plant manufacturing method.
A plant having a high glucoraphanin content, comprising a sequence in which at least one of the nucleotides at positions corresponding to positions 896 to 901 in SEQ ID NO: 2 is deleted.
The plant according to claim 7, wherein the glucoraphanin-rich plant comprises a sequence in which the nucleotides at positions corresponding to positions 896 to 900 in SEQ ID NO: 2 are deleted.
The plant according to claim 7, wherein the glucoraphanin high content plant comprises a sequence in which at least one nucleotide is inserted between the nucleotides at positions corresponding to positions 900 to 901 in SEQ ID NO: 2.
The method according to claim 7, wherein the plant is at least one selected from the group consisting of collard, kale, cauliflower, cabbage, red cabbage, cherry radish, kohlrabi, broccoli, savoy cabbage, cauliflower and kale, glucoraphanin high content plant.

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