Developing of late-bolting lettuce and method using genome editing

Abstract

The present invention relates to a method for producing a genome-edited lettuce plant having late-blooming traits by editing the FT (Flowering locus T) gene, and to a plant produced thereby.

Description

Technology Field

[0001] The present invention relates to a method for producing a genome-edited lettuce plant having late-blooming traits by editing the FT (Flowering locus T) gene, and to a plant produced thereby. Background Technology

[0002] CRISPR / Cas9 (gene editing technology) has recently emerged as an alternative technology to circumvent GMO regulations. Gene editing technologies include methods involving the introduction of Agrobacterium into plant tissues (explants) and the introduction of RNPs (complexes of sgRNA and Cas9 proteins) into plant protoplasts. While Agrobacterium-mediated gene editing involves the insertion of foreign genes, the method utilizing protoplasts and RNPs is currently recognized as a DNA-free gene editing technology.

[0004] Various lettuce varieties are distributed domestically, varying by leaf type and growing season. Domestic consumers tend to prefer lettuce varieties that are commonly available in spring, autumn, and winter. From a producer's perspective, selecting varieties for summer cultivation is the most difficult. The biggest problem with summer cultivation is bolting caused by high temperatures, which leads to a decline in quality and yield; therefore, late-bolting varieties must be selected. However, among the lettuce varieties currently on the market, it is difficult to find any that possess late-bolting characteristics satisfactory to farmers.

[0006] Accordingly, the inventors conducted research to solve the problem of reduced quality and yield of lettuce due to early flowering under high temperature and long day conditions during summer lettuce cultivation. As a result, they designed a guide RNA (sgRNA) based on the nucleotide sequence of the FT (Flowering locus T) gene of lettuce, and confirmed that flowering of lettuce was suppressed when plants were redifferentiated from plant cells edited with the FT gene, thereby completing the present invention. Prior art literature

[0007] (Patent Document 001) Republic of Korea Registered Patent Publication 10-2113500 The problem to be solved

[0008] The objective of the present invention is that the inventors completed the invention by introducing a gene editing system targeting the FT (Flowering locus T) gene into plant cells to introduce late autumn characteristics into lettuce, and confirming that the flowering time of plants with the FT gene edited was significantly delayed compared to the control group that did not edit the genome. means of solving the problem

[0010] To achieve the above objective, the present invention provides a method for producing a genome-edited lettuce plant having a delayed bolting trait, comprising the steps of: introducing into a plant cell a DNA sequence encoding a guide RNA specific to target DNA composed of the nucleotide sequence of SEQ ID NO. 1 among lettuce-derived FT (Flowering locus T) genes to edit the genome; and redifferentiating the plant from the food cell in which the genome has been edited.

[0012] The term 'genome / gene editing' refers to a technology capable of introducing targeted mutations into the genomic sequences of animal and plant cells, including human cells, and involves knocking out or knocking in specific genes by the deletion, insertion, or substitution of one or more nucleic acid molecules through DNA cutting, or introducing mutations into non-coding DNA sequences that do not produce proteins. For the purposes of the present invention, the genome editing may specifically involve introducing mutations into a plant using Cas protein and guide RNA.

[0014] The term "target gene" refers to a portion of DNA within the genome of a plant to be edited through the present invention. That is, in principle, it is not limited to the type of gene and may include both coding and non-coding regions. A person skilled in the art may select the target gene according to the desired variation in the genome-edited plant to be manufactured, depending on the purpose.

[0016] The above target gene may be a lettuce-derived FT (Flowering locus T) gene, preferably a gene containing the nucleotide sequence of SEQ ID NO. 1, but is not limited thereto.

[0018] The term 'guide RNA' refers to RNA specific to DNA encoding a target gene, and is a ribonucleic acid that binds wholly or partially complementarily to a target DNA sequence to guide an endonuclease protein to that target DNA sequence.

[0020] The guide RNA described above refers to a dual RNA comprising two RNAs, namely crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA), as components; or a single-stranded guide RNA (sgRNA) comprising a first region having a sequence wholly or partially complementary to a sequence within the target DNA and a second region having a sequence that interacts with an RNA-guide nuclease; however, any form capable of having activity at the target sequence may be included within the scope of the present invention without limitation. The guide RNA according to the present invention may preferably be in the form of a single-stranded guide RNA, but is not limited thereto, and may be appropriately selected depending on the type of endonuclease used or the microorganism from which it originates. The single-stranded guide RNA (sgRNA) may be a gene comprising the nucleotide sequence (5′-CACCTCCAATATCAACCCT-3) disclosed in Example 2, but is not limited thereto.

[0022] Additionally, the guide RNA may be transcribed from a plasmid template or transcribed in vitro (e.g., an oligonucleotide double strand), but is not limited thereto.

[0024] In a method for producing a genome-edited lettuce plant according to one embodiment of the present invention, the endonuclease protein may be one or more selected from the group consisting of Cas9 (CRISPR associated protein 9), Cpf1 (CRISPR from Prevotella and Francisella 1), TALEN (Transcription activator-like effector nuclease), ZFN (Zinc Finger Nuclease), or functional analogs thereof, and preferably may be a Cas9 protein, but is not limited thereto.

[0026] Cas9 protein or genetic information can be obtained from known databases such as GenBank of the NCBI (National Center for Biotechnology Information). For example, the Cas9 protein may be one or more selected from the group consisting of Cas9 proteins derived from Streptococcus pyogenes, Cas9 proteins derived from Campylobacter jejuni, Cas9 proteins derived from Streptococcus thermophilus or Streptococcus aureus, Cas9 proteins derived from Neisseria meningitidis, Cas9 proteins derived from Pasteurella multocida, and Cas9 proteins derived from Francisella novicida, but is not limited thereto. The above Cas9 protein may be an amino acid containing the nucleotide sequence of SEQ ID NO. 2, but is not limited thereto.

[0028] The Cas9 protein is an RNA-guided DNA endonuclease that induces double-stranded DNA breaks. For the Cas9 protein to accurately bind to the target DNA sequence and cut the DNA strand, a short sequence of three bases known as a Protospacer Adjacent Motif (PAM) must be present next to the target DNA sequence, and the Cas9 protein cuts by estimating the interval between the third and fourth base pairs from the PAM sequence (NGG) (see Fig. 1).

[0030] In a method for producing a genome-edited lettuce plant according to one embodiment of the present invention, the guide RNA and the endonuclease protein form a ribonucleic acid-protein complex and function as an RNA gene scissors (RNA-Guided Engineered Nuclease, RGEN).

[0032] The CRISPR / Cas9 system used in the present invention is a gene editing method based on the NHEJ (non-homologous end joining) mechanism, which induces insertion-deletion (InDel) mutations caused by incomplete repair induced during the DNA repair process by introducing a double helix cut at a specific location of a specific gene to be edited.

[0034] In the method according to the present invention, introducing DNA encoding the guide RNA and a nucleic acid sequence encoding an endonuclease protein into a plant cell constitutes a transformation method. Transformation of plant species is now common for plant species including both dicotyledonous and monocotyledonous plants. In principle, any transformation method can be used to introduce the recombinant vector according to the present invention into a suitable progenitor cell. The methods include the calcium / polyethylene glycol method on protoplasts (Krens et al., 1982, Nature 296: 72-74; Negruti et al., 1987, Plant Mol. Biol. 8: 363-373), electroporation of protoplasts (Shillito et al., 1985, Bio / Technol. 3: 1099-1102), microinjection into plant elements (Crossway et al., 1986, Mol. Gen. Genet. 202: 179-185), particle impaction of various plant elements (DNA or RNA-coated) (Klein et al., 1987, Nature 327: 70), and Agrobacterium tumefaciens-mediated genes by plant infiltration or transformation of mature pollen or microspores. It may be suitably selected from infections caused by viruses (EP 0 301 316) in the course of transmission (incompleteness). A preferred method according to the present invention comprises a calcium / polyethylene glycol method.

[0036] The “plant cell” used for the transformation of a plant may be any plant cell. The plant cell is a cultured cell, cultured tissue, cultured organ, or whole plant. The “plant tissue” includes differentiated or undifferentiated plant tissues, such as, but not limited to, roots, stems, leaves, pollen, seeds, female tissue, and various forms of cells used for culture, namely single cells, protoplasts, buds, and callus tissues. The plant tissue may be in planta or in organ culture, tissue culture, or cell culture.

[0038] The present invention also provides a genome-edited lettuce plant having a delayed bolting trait produced by the method of the present invention, and a seed in which the genome of said plant is edited.

[0040] The genome-edited lettuce plant having a delayed bolting trait according to the present invention is a genome-edited plant in which the FT gene, a major gene related to flowering, is edited using the CRISPR / Cas9 system and the lettuce FT gene is knocked out.

[0042] The present invention also provides a genome editing composition for delaying bolting of a lettuce plant, comprising DNA encoding a guide RNA specific to target DNA consisting of the nucleotide sequence of SEQ ID NO. 1 among the lettuce-derived FT (Flowering locus T) gene, and a nucleic acid sequence encoding an endonuclease protein.

[0044] The genome editing composition of the present invention comprises DNA encoding a guide RNA specific to the target DNA of a target gene and a nucleic acid sequence encoding an endonuclease protein, so that when the composition is applied to a lettuce plant, the complex of the guide RNA and the RNA-guide nuclease protein acts as an RNA gene scissors to edit the target gene.

[0046] The genome editing composition of the present invention preferably includes a guide RNA specific to the target DNA (Sequence No. 1) of the lettuce FT gene and a Cas9 (CRISPR associated protein 9) protein, so that the FT gene can be knocked out and bolting of the plant can be delayed through the knockout of the FT gene.

[0048] The present invention also provides a method for producing hybrid seeds of a lettuce line having delayed bolting traits, comprising the step of crossing a genome-edited lettuce plant having the delayed bolting trait of the present invention as a parent plant with a male fertile lettuce to be introduced, and hybrid seeds of a lettuce line having the delayed bolting trait produced by the method.

[0050] The male fertile lettuce of the present invention refers to lettuce capable of normal natural mating. Effects of the invention

[0051] The lettuce edited with the FT gene of the present invention has the effect of suppressing the flowering time compared to wild-type plants. It enables stable lettuce production under high-temperature, long-day cultivation conditions during the summer, and has the effect of increasing farm income through increased harvest frequency and the production of high-quality lettuce. Brief explanation of the drawing

[0052] Figure 1 is a result showing the location (location of gene editing) targeted by the gene scissors of the present invention in the lettuce FT gene. Figure 2 is the result of comparing the FT genes of a lettuce plant with an edited FT gene according to the present invention and a wild-type plant without gene editing. Figure 3 shows the results of comparing the flowering times of the redifferentiated gene-edited lettuce plants of Examples 1, 2, 3, and 4 with the unedited wild-type plants. Specific details for implementing the invention

[0053] The present invention will be explained in more detail below through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0055] Experimental Example 1. Design of a target sequence for gene editing tools

[0057] The nucleotide sequence of the lettuce FT gene (LsFT) was obtained by using the NCBI (http: / / www.ncbi.nlm.nih.gov) homology search for the protein sequence of the gene known as the Arabidopsis FT gene (AtFT). Based on the obtained nucleotide sequence information of the lettuce FT gene, total RNA was extracted from commercially available lettuce varieties and cDNA was synthesized. PCR (polymerase chain reaction) was performed using the synthesized cDNA and the Forward (5′-ATGATGCCTAGGGAGAGGG-3′; Sequence No. 3) and Reverse (5′-TTATCTTCTTCGCCCACCAAACC-3′; Sequence No. 4) primer sets to confirm and obtain the nucleotide sequence of the lettuce FT gene (Sequence No. 1). An sgRNA was designed based on the PAM (protospacer adjacent motif) site, which is a Cas9 nuclease recognition sequence located near the 5′ end of the obtained lettuce FT gene sequence, and a single guide RNA (sgRNA) was designed using Cas-Designer (http: / / www.rgenome.net / cas-designer) (Fig. 1).

[0059] Experimental Example 2. Gene editing method

[0061] sgRNA of the lettuce FT gene (5′-CACCTCCAATATCAACCCT-3; SEQ ID NO. 5) and Cas9 protein (SEQ ID NO. 2) were introduced by treating lettuce protoplasts with polyethylene glycol (PEG). Lettuce protoplasts (5×10⁶) were treated with sgRNA (25 μg) and Cas9 (25 μg) together with sgRNA (25 μg) and Cas9 (25 μg). 5 A PEG solution (20% PEG, 0.2 M mannitol, 0.1 M CaCl2) was mixed with the mixture. An equal amount of culture medium (2 mM morpholineethanesulfonic acid, 154 mM NaCl2, 125 mM CaCl2, 5 mM KCl, pH 5.7) was added to the mixture to introduce sgRNA and Cas9 protein.

[0063] Experimental Example 3. Production of Gene-Edited Plants

[0065] Lettuce protoplasts with sgRNA and Cas9 protein introduced (5×10 5After adding 1 ml of primary medium (1×B5 culture medium, 70 g / L D-mannitol, 20 g / L glucose, 0.1 g / L MES, 0.2 mg / L 2,4-D, 0.1 mg / L NAA, 0.5 mg / L BAP, pH 5.7) to the sample, it was cultured for 7 days at 25°C under dark conditions. After that, 2 ml of secondary medium (1×B5 culture medium, 40 g / L D-mannitol, 20 g / L sucrose, 0.1 g / L MES, 0.2 mg / L 2,4-D, 0.1 mg / L NAA, 0.5 mg / L BAP, pH 5.7) was added, and the sample was cultured for 1 to 2 weeks. Afterwards, they were transferred to the 3rd medium (1×B5 culture medium, 40 g / L D-mannitol, 20 g / L sucrose, 0.1 g / L MES, 8 g / L phyto agar, 0.2 mg / L 2,4-D, 0.1 mg / L NAA, 0.5 mg / L BAP, pH 5.7) and cultured for 4 to 5 weeks. They were cultured for 2 to 4 months in the 4th medium (1×B5 culture medium, 10 g / L sucrose, 8 g / L phyto agar, 0.1 mg / L NAA, 0.5 mg / L BAP, pH 5.7), and individuals were produced by changing the medium composition in sequence to the 5th medium (0.5× MS culture medium, 30 g / L sucrose, 8 g / L phyto agar, pH 5.7).

[0067] Experimental Example 4. Sequence analysis of gene-edited plants

[0069] DNA extracted from plants produced after gene editing using CRISPR / Cas9 gene editing technology and the forward (5′-ATGATGCCTAGGGAGAGGGA-3′; SEQ ID NO. 6) and reverse (5′-AGGAGCAATCCATTGTCTATCAC-3′; SEQ ID NO. 7) primer sets were used to obtain FT gene sequences, which were then sent to an external institution (BIO CORE CENTOR, KAIST) for NGS analysis. By comparing the FT genes of plants with edited lettuce FT genes using CRISPR / Cas9 gene editing technology with those of unedited wild-type plants, gene-edited plants with mutations occurring near the DNA sequence targeted by sgRNA were identified, and the results are shown in Figure 2. Different types of sequence mutations occurred in each of the four gene-edited individuals, and it was confirmed that mutations occurred on both sides of the chromosome when compared to the wild-type sequences. In Example 1, different forms of deletions of 7bp and 4bp occurred on different homologous chromosomes (see Example 1_Chromosome 1 and Example 1_Chromosome 2 in Fig. 2). In Example 2, deletions of 7bp and 4bp occurred on different homologous chromosomes (see Example 2_Chromosome 1 and Example 2_Chromosome 2 in Fig. 2). In Example 3, deletions of 3bp and 1bp occurred on different homologous chromosomes (see Example 3_Chromosome 1 and Example 3_Chromosome 2 in Fig. 2). In Example 4, deletions of 3bp and 1bp occurred on different homologous chromosomes (see Example 4_Chromosome 1 and Example 4_Chromosome 2 in Fig. 2). As a result, it was found that a frame shift occurred due to the deletion mutation in Examples 1 and 2, and that a stop codon was generated in the middle of the gene, so a normal FT protein was not produced.It was found that in Examples 3 and 4, a stop codon was generated due to a frame shift caused by a 1 bp deletion mutation on one chromosome, and a 3 bp deletion on the other chromosome could generate an FT protein in which one amino acid (valline) was deleted in the frame.

[0071] Experimental Example 5. Analysis of late-blooming traits in gene-edited plants

[0073] The flowering time of the redifferentiated gene-edited lettuce plants of Examples 1, 2, 3, and 4 was compared with that of the wild-type plants that were not gene-edited. It was confirmed that the wild-type plants flowered after an average of 20 days, whereas the plants with the FT gene edited did not flower even after more than 3 months (Fig. 3). Through this, it was found that the FT gene-edited lettuce plants of the present invention possess the characteristic of delayed bolting.

Claims

Claim 1 A method for producing a genome-edited lettuce plant having a delayed bolting trait, comprising: a step of editing the genome by introducing into a plant cell DNA encoding a guide RNA specific to target DNA composed of the nucleotide sequence of SEQ ID NO. 1 among lettuce-derived FT (Flowering locus T) genes and a nucleic acid sequence encoding an endonuclease protein; and a step of redifferentiating the plant from the genome-edited plant cell, wherein the DNA encoding the specific guide RNA is composed of SEQ ID NO. 5 and the endonuclease protein is a Cas9 protein. Claim 2 Genome-edited lettuce plants having a delayed bolting trait produced by the method of claim 1. Claim 3 A seed in which the genome of a plant body has been edited according to Paragraph 2. Claim 4 A genome editing composition for delaying bolting of a lettuce plant, comprising DNA encoding a guide RNA specific to target DNA consisting of the nucleotide sequence of SEQ ID NO. 1 among the lettuce-derived FT (Flowering locus T) gene and a nucleic acid sequence encoding an endonuclease protein, wherein the DNA encoding the specific guide RNA consists of SEQ ID NO. 5 and the endonuclease protein is a Cas9 protein. Claim 5 A method for producing hybrid seeds of a lettuce line having a delayed bolting trait, comprising the step of crossing a genome-edited lettuce plant having the delayed bolting trait of claim 2 with a male fertile lettuce to be introduced using the plant as a parent.

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