KR102598907B1 - HDA6 gene from Arabidopsis thaliana for regulating regeneration efficiency of plant and uses thereof - Google Patents

Abstract

The present invention relates to the Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) gene, which regulates the redifferentiation efficiency of plants, and its use. More specifically, the expression of the Arabidopsis-derived HDA6 gene is inhibited in plant cells to increase the callus of the plant compared to the wild type. It relates to a method of increasing callus formation and shoot regeneration efficiency.

Description

HDA6 gene from Arabidopsis thaliana for regulating regeneration efficiency of plant and uses thereof}

The present invention relates to the Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) gene, which regulates the re-differentiation efficiency of plants, and its use.

Plant regeneration is an essential technology for genetic engineering applications such as crop genome editing and transformation, and low regeneration efficiency is known to be a major obstacle to genome editing. In order to breed high value-added crops through agricultural biotechnology, tissue dedifferentiation (callus formation) and aerial part regeneration (shoot regeneration) are performed sequentially after genome editing or transformation. At this time, the efficiency of the plant regeneration process is very low, so trait improvement in major crops is very limited.

Recently, with the development of CRISPR technology, the demand and market for precision breeding has grown explosively, and plant redifferentiation technology is essential in the market, and efforts are being focused around the world to innovatively improve problems. . In general, efforts have been made to promote plant redifferentiation by mixing various hormones and additives into redifferentiation inducing media to improve plant redifferentiation efficiency, but are facing distinct limitations.

It is known that the low redifferentiation efficiency of plants is due to genetic barriers. Since the redifferentiation efficiency is already determined by the plant's genetic factors, removal of genes that interfere with the redifferentiation process is known to be a key technology that enables improved redifferentiation. there is. Therefore, it is necessary to secure genes involved in controlling replanting efficiency and research methods to improve crop replanting efficiency by regulating the expression of the genes.

In the present invention, we attempted to propose a method to improve redifferentiation efficiency by selecting important genes related to redifferentiation and controlling the expression of these genes.

Meanwhile, Korean Patent No. 1898392 discloses ‘Method for increasing redifferentiation efficiency of plants using ATX4 gene derived from Arabidopsis thaliana and resulting plants’, and Korean Patent No. 2125714 discloses ‘Arabidopsis thaliana that regulates redifferentiation efficiency of plants’. Although the 'DEMETER gene derived from and its use' is disclosed, there is no description of the 'Arabidopsis-derived HDA6 gene that regulates the re-differentiation efficiency of plants and its use' of the present invention.

The present invention was derived from the above needs. In the present invention, key genes accompanied by expression changes during the process of dedifferentiation and redifferentiation were selected, and knock-out mutants of key genes were selected to promote dedifferentiation and redifferentiation. As a result of analyzing the effect on the process, it was discovered that the HDA6 gene functions to suppress both dedifferentiation and redifferentiation processes, and the knockout mutant of the gene promoted both dedifferentiation and redifferentiation processes, significantly improving redifferentiation efficiency. By confirming this, the present invention was completed.

In order to solve the above problem, the present invention provides a method for regeneration of plants, which includes the step of regulating in plant cells the expression of a gene encoding an Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) protein consisting of the amino acid sequence of SEQ ID NO: 2. ) Provides a method of controlling efficiency.

In addition, the present invention provides a method for producing a transgenic plant with controlled redifferentiation efficiency, comprising the step of transforming a plant cell with a recombinant vector containing a gene encoding an Arabidopsis thaliana-derived HDA6 protein consisting of the amino acid sequence of SEQ ID NO: 2. provides.

In addition, the present invention includes the steps of (a) correcting the genome by introducing guide RNA and endonuclease protein specific to the target base sequence of the Arabidopsis-derived HDA6 protein coding gene into plant cells; and (b) redifferentiating a plant from the genome-corrected plant cell.

In addition, the present invention provides a mutant plant in which the expression of the gene encoding the Arabidopsis thaliana-derived HDA6 protein consisting of the amino acid sequence of SEQ ID NO: 2 is inhibited, and the redifferentiation efficiency is increased.

In addition, the present invention provides a composition for controlling re-differentiation efficiency of plants, which contains as an active ingredient a gene encoding an Arabidopsis thaliana-derived HDA6 protein consisting of the amino acid sequence of SEQ ID NO: 2.

Since the Arabidopsis-derived HDA6 gene can effectively improve the dedifferentiation and redifferentiation efficiency of plants, it is expected to contribute to improving crop transformation efficiency, shorten the breeding period, and make the breeding process more efficient.

Figure 1 shows the appearance and live weight analysis of callus induced when leaf explants of wild type (Col-0) and HDA6 knockout mutant ( hda6-6 ) were cultured in callus induction medium for the same period of time. *** P <0.001.
Figure 2 shows the appearance and number of shoots formed when callus derived from root explants of the wild type (Col-0) and the HDA6 knockout mutant ( hda6-6 ) were cultured in shoot induction medium for the same period of time. ** P <0.01.

In order to achieve the purpose of the present invention, the present invention is a redifferentiation of plants, comprising the step of regulating the expression of the gene encoding the Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) protein consisting of the amino acid sequence of SEQ ID NO: 2 in plant cells. (regeneration) Provides a method of controlling efficiency.

The Arabidopsis thaliana-derived HDA6 protein of the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 2 and functional equivalents of the protein. “Functional equivalent” means at least 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 90% or more, as a result of addition, substitution or deletion of amino acids, compared to the amino acid sequence shown in SEQ ID NO: 2. refers to a protein that has more than 95% sequence homology and exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 2. “Substantially homogeneous physiological activity” refers to the activity that regulates the regeneration efficiency of plants.

Additionally, the present invention provides a gene encoding the Arabidopsis thaliana-derived HDA6 protein. The gene includes both genomic DNA and cDNA encoding the Arabidopsis thaliana-derived HDA6 protein. The gene encoding the Arabidopsis thaliana-derived HDA6 protein of the present invention may include the base sequence represented by SEQ ID NO: 1. Additionally, homologs of the above base sequence are included within the scope of the present invention. Specifically, the gene encoding the Arabidopsis-derived HDA6 protein is at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the base sequence of SEQ ID NO: 1. It may include a base sequence having sequence homology. The “% sequence homology” for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, where a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to those that do not contain .

In the method of controlling the redifferentiation efficiency of plants according to one embodiment of the present invention, the expression of the gene encoding the Arabidopsis thaliana-derived HDA6 protein is preferably controlled by inhibiting the expression of the HDA6 protein encoding gene, and the HDA6 protein in plant cells is preferably controlled by inhibiting the expression of the gene encoding the HDA6 protein. Inhibition of the expression of the coding gene increased the efficiency of plant redifferentiation compared to the wild type.

In addition, in the present invention, the increased regeneration efficiency may be callus formation and shoot regeneration, and more specifically, increasing callus formation from leaf-derived tissue or root-derived tissue. It may be an increase in above-ground redifferentiation including shoot formation from tissues, but is not limited to this.

In one embodiment of the present invention, the expression of the gene encoding the HDA6 protein is suppressed by T-DNA insertion into the HDA6 protein encoding gene, endogenous transposon, mutagenesis through X-ray or γ-ray irradiation, and VIGS. (Virus-induced gene silencing) may be, but is not limited to, inhibiting (suppressing) the expression of the HDA6 protein coding gene using vectors, RNAi (RNA interference), antisense RNA, or the CRISPR/Cas9 gene editing system. Any method common in the art to inhibit expression may be possible.

The present invention also provides a trait with controlled redifferentiation efficiency, comprising the step of transforming a plant cell with a recombinant vector containing a gene encoding an Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) protein consisting of the amino acid sequence of SEQ ID NO: 2. A method for producing a converted plant is provided.

The production method according to one embodiment of the present invention is characterized in that it increases the efficiency of redifferentiation of plants compared to the wild type by inhibiting the expression of the gene encoding the Arabidopsis thaliana-derived HDA6 protein in plant cells, but is not limited thereto.

As used herein, the term “recombinant” refers to a cell that replicates a heterologous nucleic acid, expresses a heterologous nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the natural form of the cell, either in sense or antisense form. Additionally, recombinant cells can express genes found in cells in their native state, but the genes have been modified and reintroduced into the cell by artificial means.

As used herein, the term “vector” is used to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. Vectors replicate DNA and can reproduce independently in host cells. The term “vector” is often used interchangeably with “vector”. The term “expression vector” refers to a recombinant DNA molecule containing a coding sequence of interest and an appropriate nucleic acid sequence necessary to express the operably linked coding sequence in a particular host organism. The promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

An expression vector containing the gene sequence encoding the Arabidopsis thaliana-derived HDA6 protein of the present invention and appropriate transcription/translation control signals can be constructed by methods well known to those skilled in the art. The methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be effectively linked to an appropriate promoter within an expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

Preferred examples of recombinant vectors of the present invention are VIGS vectors or RNAi vectors. VIGS refers to a phenomenon in which when a plant is infected after introducing a plant gene into a viral vector, the expression of the endogenous plant gene of the introduced gene is suppressed. This is a type of PTGS (Post-transcriptional gene silencing) and has the characteristics of post-transcriptional, RNA turnover, and nucleotide sequence-specific. The VIGS vector can be used as a transient expression vector that can temporarily express a foreign gene in a plant into which the foreign gene has been introduced, and a plant expression vector that can permanently express the foreign gene in a plant that has been introduced.

A preferred example of a plant expression vector is the Ti-plasmid vector, which is capable of transferring part of itself, the so-called T-region, into plant cells when present in a suitable host such as Agrobacterium tumefaciens . Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences into plant cells or protoplasts from which new plants can be produced that properly integrate the hybrid DNA into the plant's genome. there is. Particularly preferred forms of Ti-plasmid vectors are the so-called binary vectors as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host include viral vectors, such as those that may be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses, etc. For example, it may be selected from non-intact plant virus vectors. The use of such vectors can be particularly advantageous when it is difficult to properly transform plant hosts.

The expression vector will preferably include one or more selectable markers. The marker is a nucleic acid sequence that has characteristics that can be generally selected by chemical methods, and includes all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate, phosphinothricin and glufosinate, kanamycin, G418, bleomycin, hygromycin, There are antibiotic resistance genes such as chloramphenicol, but it is not limited to this.

In the plant expression vector of the present invention, the promoter may preferably be, but is not limited to, a T7 promoter, SP6 promoter, CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter. The term “promoter” refers to the region of DNA upstream from a structural gene and refers to the DNA molecule to which RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. A “constitutive promoter” is a promoter that is active under most environmental conditions and developmental states or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of transformants can be accomplished at various stages and by various tissues. Therefore, constitutive promoters do not limit selection possibilities.

In the recombinant vector of the present invention, common terminators can be used, examples of which include nopaline synthase, rice α-amylase RAmy1 A terminator, Phaseolin terminator, Agrobacterium tumefaciens ( A tumefaciens ) terminator of the Octopine synthase gene, the rrnB1/B2 terminator of E. coli, etc., but are not limited thereto. Regarding the necessity of terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly preferred in the context of the present invention.

The method for transporting the vector of the present invention into a host cell is the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), electroporation of protoplasts (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), microscanning of plant elements (Crossway A. et al., 1986, Mol Gen. Genet. 202, 179-185), particle bombardment method (DNA or RNA-coated) of various plant elements (Klein T.M. et al., 1987, Nature 327, 70), infiltration or maturation of pollen or spores of plants. It can be appropriately selected from Agrobacterium tumefaciens-mediated gene transfer by transformation (incomplete), infection by virus (EP 0 301 316), etc.

Additionally, the production method of the present invention includes the step of redifferentiating the transformed plant cells into pre-transformed plants. Any method known in the art can be used to redifferentiate a transgenic plant from a transgenic plant cell.

The “plant cell” used for plant transformation can be any plant cell. A plant cell is a cultured cell, cultured tissue, cultured organ, or whole plant. “Plant tissue” refers to differentiated or undifferentiated plant tissues, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used in culture, such as single cells and protoplasts. (protoplast), shoot and callus tissue. Plant tissue may be in planta or in organ culture, tissue culture, or cell culture.

In addition, the present invention provides transgenic plants and seeds thereof with controlled regeneration efficiency prepared by the above production method. Preferably, the transgenic plant and its seeds are transgenic plants and seeds with increased regeneration efficiency compared to the wild type.

The plants include Arabidopsis thaliana, potatoes, eggplants, tobacco, peppers, tomatoes, burdock, mugwort, lettuce, bellflower root, spinach, Swiss chard, sweet potatoes, celery, carrots, water parsley, parsley, Chinese cabbage, cabbage, leaf radish, watermelon, melon, cucumber, It may be a dicotyledonous plant such as pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, or pea, or a monocotyledonous plant such as rice, barley, wheat, rye, corn, sugarcane, oats, or onion, and is preferably a dicotyledonous plant. Preferably, it may be an Arabidopsis thaliana plant, but is not limited thereto.

The present invention also,

(a) correcting the genome by introducing guide RNA and endonuclease protein specific to the target base sequence of the Arabidopsis-derived HDA6 (Histone deacetylase 6) protein coding gene into plant cells; and

(b) redifferentiating a plant from the genome-corrected plant cell; providing a method for producing a genome-edited plant with increased redifferentiation efficiency compared to the wild type.

As used herein, the term "genome/gene editing" refers to a technology that can introduce target-oriented mutations into the genome sequence of animal and plant cells, including human cells, and refers to one or more nucleic acid molecules by cutting DNA. Knock-out or knock-in of specific genes by deletion, insertion, substitutions, etc., or non-coding that does not produce proteins. coding) refers to a technology that can introduce mutations into the DNA sequence. For the purpose of the present invention, the genome editing may be to introduce mutations into plants using endonuclease such as Cas9 (CRISPR associated protein 9) protein and guide RNA. Additionally, ‘gene editing’ can be used interchangeably with ‘gene editing’.

Additionally, the term “target gene” refers to some DNA in the genome of a plant to be corrected through the present invention, is not limited to the type of gene, and may include both coding regions and non-coding regions. A person skilled in the art can select the target gene according to the desired mutation for the genome editing plant to be manufactured, depending on the purpose.

As used herein, the term “guide RNA” refers to an RNA specific to DNA encoding the base sequence of a target gene, and all or part of it binds complementary to the target DNA base sequence to transform into the target DNA base sequence. It refers to a ribonucleic acid that plays the role of guiding the endonuclease protein. The guide RNA is a dual RNA containing two RNAs, namely crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as components; or a single-chain guide RNA (sgRNA) form comprising a first region comprising a sequence that is fully or partially complementary to a base sequence in the target gene and a second region comprising a sequence that interacts with an RNA-guide nuclease. , If the RNA-guide nuclease is in a form that can be active in the target base sequence, it can be included in the scope of the present invention without limitation, taking into account the type of endonuclease used together or the microorganism from which the endonuclease is derived, etc. It can be appropriately selected according to known techniques in the art.

Additionally, the guide RNA may be transcribed from a plasmid template, transcribed in vitro (e.g., oligonucleotide double-stranded), or synthesized guide RNA, but is not limited thereto.

Additionally, in the method for producing a genome-edited plant according to the present invention, the endonuclease protein may be one or more selected from the group consisting of Cas9, Cpf1 (CRISPR from Prevotella and Francisella 1), or a functional analog thereof.

In addition, the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Streptococcus thermophilus ( S. thermophilus ) or a Streptococcus aureus ( S. aureus )-derived Cas9 protein, Neisseria meningitidis -derived Cas9 protein, Pasteurella multocida -derived Cas9 protein, Francisella novicida -derived Cas9 protein, etc. It may be one or more selected from the group consisting of, but is not limited thereto. Cas9 protein or its genetic information can be obtained from known databases such as GenBank of the National Center for Biotechnology Information (NCBI).

The Cas9 protein is an RNA-guided DNA endonuclease enzyme that induces double stranded DNA breaks. In order for the Cas9 protein to accurately bind to the target base sequence and cut the DNA strand, a short base sequence consisting of three bases known as PAM (Protospacer Adjacent Motif) must exist next to the target base sequence, and the Cas9 protein must have a PAM sequence (NGG) It is estimated and cut between the 3rd and 4th base pairs.

In the present invention, the guide RNA and the endonuclease protein form a ribonucleoprotein complex and can operate as RNA-Guided Engineered Nuclease (RGEN).

The CRISPR/Cas9 system used in the present invention is NHEJ, which induces insertion-deletion (InDel) mutations due to incomplete repair induced during the DNA repair process by introducing a double-strand break at a specific position of a specific gene to be corrected. It is a gene editing method using the (non-homologous end joining) mechanism.

In the production method according to the present invention, introducing the guide RNA and endonuclease protein in step (a) into plant cells includes guide RNA and endonuclease specific to the target base sequence of the Arabidopsis-derived HDA6 gene. complex of proteins (ribonucleoprotein); Alternatively, a recombinant vector comprising DNA encoding a guide RNA specific to the target base sequence of the Arabidopsis thaliana-derived HDA6 gene and a nucleic acid sequence encoding an endonuclease protein may be used, but is not limited thereto.

The present invention also provides a mutant plant in which the expression of the gene encoding the Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) protein, which has the amino acid sequence of SEQ ID NO: 2, is inhibited, resulting in increased redifferentiation efficiency. Inhibition of expression of the gene can be done by inserting T-DNA into the gene encoding the HDA6 protein to induce a loss-of-function mutant, or by using a VIGS (Virus-induced gene silencing) vector or RNAi (RNA interference) vector containing the HDA6 gene in the plant. This can be achieved by transforming cells to induce HDA6 mutants, but is not limited thereto. The gene encoding the HDA6 protein may be composed of the base sequence of SEQ ID NO: 1, but is not limited thereto.

The present invention also provides a composition for controlling redifferentiation efficiency of plants, which contains as an active ingredient a gene encoding an Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) protein consisting of the amino acid sequence of SEQ ID NO: 2.

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

1. Plant materials and growth conditions

In the present invention, Columbia-0 ecotype Arabidopsis thaliana (Col-0) was used as the wild type. After sterilizing the surface of Arabidopsis seeds, they were planted on 0.7% agar plates containing 1/2 MS (Murashige and Skoog). Plants were grown under long-day conditions (16 hours light/8 hours dark cycle) using white fluorescent lights (120 μmol photons·m ^-2 ·s ^-1 ) at 22-23°C. HDA6 knockout mutant ( hda6-6 ) was purchased from ABRC (https://abrc.osu.edu/) and used (Stock Number: CS66153).

2. Callus induction

For callus induction, leaf explants of the first and second leaves of 2-week-old plants were cultured in callus-inducing medium (CIM); The cells were cultured in the dark for 2 weeks at 22°C in MS medium supplemented with 0.1 μg/ml 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 μg/ml kinetin. Thirty calli from each genotype were collected and their live weights were measured, and live weights were performed in three independent replicates. Statistical significance between wild type and mutant was determined by Student's t-test.

3. Above-ground regeneration (shoot regeneration)

For above-ground regeneration, root explants of 7-day-old plants were cultured in callus-inducing medium (CIM); The cells were cultured in the dark for 7 days at 22°C in MS medium supplemented with 0.5 μg/ml 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 μg/ml kinetin. Thereafter, the induced callus was pre-cultured in CIM for 7 days and then cultured in shoot-inducing medium (SIM); The cells were cultured in [MS medium supplemented with 0.9 μmol/L 3-indoleacetic acid and 2.5 μmol/L 2-isopentenyladenine] at 25°C under continuous light conditions for 3 weeks.

Example 1. Wild type and hda6-6 Analysis of callus formation ability of mutants

Plant redifferentiation involves a two-step process involving callus induction and de novo aerial organogenesis. Callus formation is the first step in redifferentiation and is similar to the establishment of lateral root primordium. Consistently, the genetic pathways of lateral root initiation and callus formation overlap significantly. For example, key factors regulating lateral root initiation, including ALF4 (ABERRANT LATERAL ROOT FORMATION 4), ARFs (AUXIN RESPONSE FACTORs), and LBDs (LATERAL ORGAN BOUNDARIES DOMAINs), have been reported to also be involved in callus formation. Accumulating evidence has shown that cell fate transitions are accompanied by genome-wide epigenetic changes. To determine whether chromatin regulators regulate callus formation, the present inventors identified the callus phenotypes of genetic mutants of various chromatin regulators and confirmed that HDA6 negatively regulates callus proliferation.

The present inventors induced callus using leaf explants derived from wild-type Arabidopsis and an HDA6 knockout mutant ( hda6-6 ), respectively, and measured the fresh weight. As a result, the fresh weight of callus derived from the hda6-6 mutant was compared to that of wild-type Arabidopsis thaliana (Col- 0) It was confirmed that the live weight of the derived callus was increased by 46% (Figure 1).

Example 2. Wild type and hda6-6 Analysis of above-ground redifferentiation ability of mutants

Plant redifferentiation involves a two-step process including callus induction and de novo aerial part redifferentiation. First, tissue explants are cultured in auxin-rich callus induction medium to stimulate callus formation from differentiated somatic cells. The pluripotent callus is then transferred to a shoot induction medium containing a high ratio of cytokinin to auxin to induce de novo shoot formation.

The present inventors were able to confirm that the number of shoots produced from callus derived from root explants of the hda6-6 mutant was more than 1.7 times greater than the number of shoots produced from callus derived from wild-type Arabidopsis thaliana (FIG. 2).

In general, callus formation, that is, dedifferentiation and aerial part redifferentiation processes, are independent processes, and each process requires opposite hormonal signals, so it is known to be difficult to promote dedifferentiation and redifferentiation at the same time. However, since dedifferentiation and redifferentiation can be promoted simultaneously using the HDA6 gene, the gene of the present invention can be usefully used to improve the low redifferentiation efficiency of plants.

<110> Seoul National University R&DB Foundation <120> HDA6 gene from Arabidopsis thaliana for regulating regeneration efficiency of plant and uses thereof <130> PN20358 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1416 <212> DNA <213> Arabidopsis thaliana <400> 1 atggaggcag acgaaagcgg catctctctg ccgtcgggac ccgacggacg taagcggcga 60 gtcagttact tctacgagcc gacgatcgga gactactact acggtcaagg ccacccgatg 120 aagcctcacc ggatccgtat ggctcatagc ctaatcattc actatcacct ccaccgtcgc 180 ttagaaatca gtcgccctag cctcgctgac gcctccgata tcggccgatt ccattcgccg 240 gagtatgttg acttcctcgc ttccgtttcg ccggaatcta tgggcgatcc ttccgctgca 300 cgaaacctaa ggcgattcaa tgtcggtgag gattgtcctg tcttcgacgg actttttgat 360 ttttgccgtg cttccgccgg aggttctatt ggtgctgccg tcaaattaaa cagacaggac 420 gctgatatcg ctatcaattg gggcggtggg cttcaccatg ctaagaaaag cgaggcttct 480 gggttttgct atgtaaacga catcgtgcta gggattctgg agttgctcaa gatgtttaag 540 cgggttctct acatagatat tgatgtccac catggagatg gagtggaaga agcgttttac 600 accactgata gagttatgac tgtttctttc cacaaatttg gggacttttt cccaggaact 660 ggtcacataa gagatgttgg cgctgaaaaa gggaaatact atgctctaaa tgttccacta 720 aacgatggta tggacgatga aagtttccgc agcttgttta gacctcttat ccagaaggtt 780 atggaagtgt atcagccaga ggcagttgtt cttcagtgtg gtgctgactc cttaagtggt 840 gatcggttgg gttgcttcaa cttatcagtc aagggtcacg ctgattgcct tcggttctta 900 agatcttaca acgttcctct catggtgttg ggtggtggag ggtatactat tcgaaatgtt 960 gcccgttgct ggtgttatga gactgcagtt gctgttggag tagagccgga caacaaactc 1020 ccttacaatg agtattttga gtatttcggc ccagattata cgcttcatgt cgacccaagt 1080 cctatggaga atttaaacac gcccaaagat atggagagga taaggaacac gttgctggaa 1140 caactttcgg gactaataca cgcacctagc gtccagtttc agcacacacc accagtcaat 1200 cgagttttgg acgagccgga agatgacatg gagacaagac caaaacctcg catctggagt 1260 ggaactgcga cttatgaatc agacagtgac gatgatgata aacctcttca tggttactca 1320 tgtcgtggtg gcgcaactac ggacagggac tctaccggtg aagatgaaat ggatgacgat 1380 aacccagagc cagacgtgaa tcctccatcg tcttaa 1416 <210> 2 <211> 471 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Glu Ala Asp Glu Ser Gly Ile Ser Leu Pro Ser Gly Pro Asp Gly 1 5 10 15 Arg Lys Arg Arg Val Ser Tyr Phe Tyr Glu Pro Thr Ile Gly Asp Tyr 20 25 30 Tyr Tyr Gly Gln Gly His Pro Met Lys Pro His Arg Ile Arg Met Ala 35 40 45 His Ser Leu Ile Ile His Tyr His Leu His Arg Arg Leu Glu Ile Ser 50 55 60 Arg Pro Ser Leu Ala Asp Ala Ser Asp Ile Gly Arg Phe His Ser Pro 65 70 75 80 Glu Tyr Val Asp Phe Leu Ala Ser Val Ser Pro Glu Ser Met Gly Asp 85 90 95 Pro Ser Ala Ala Arg Asn Leu Arg Arg Phe Asn Val Gly Glu Asp Cys 100 105 110 Pro Val Phe Asp Gly Leu Phe Asp Phe Cys Arg Ala Ser Ala Gly Gly 115 120 125 Ser Ile Gly Ala Ala Val Lys Leu Asn Arg Gln Asp Ala Asp Ile Ala 130 135 140 Ile Asn Trp Gly Gly Gly Leu His His Ala Lys Lys Ser Glu Ala Ser 145 150 155 160 Gly Phe Cys Tyr Val Asn Asp Ile Val Leu Gly Ile Leu Glu Leu Leu 165 170 175 Lys Met Phe Lys Arg Val Leu Tyr Ile Asp Ile Asp Val His His Gly 180 185 190 Asp Gly Val Glu Glu Ala Phe Tyr Thr Thr Asp Arg Val Met Thr Val 195 200 205 Ser Phe His Lys Phe Gly Asp Phe Phe Pro Gly Thr Gly His Ile Arg 210 215 220 Asp Val Gly Ala Glu Lys Gly Lys Tyr Tyr Ala Leu Asn Val Pro Leu 225 230 235 240 Asn Asp Gly Met Asp Asp Glu Ser Phe Arg Ser Leu Phe Arg Pro Leu 245 250 255 Ile Gln Lys Val Met Glu Val Tyr Gln Pro Glu Ala Val Val Leu Gln 260 265 270 Cys Gly Ala Asp Ser Leu Ser Gly Asp Arg Leu Gly Cys Phe Asn Leu 275 280 285 Ser Val Lys Gly His Ala Asp Cys Leu Arg Phe Leu Arg Ser Tyr Asn 290 295 300 Val Pro Leu Met Val Leu Gly Gly Gly Gly Tyr Thr Ile Arg Asn Val 305 310 315 320 Ala Arg Cys Trp Cys Tyr Glu Thr Ala Val Ala Val Gly Val Glu Pro 325 330 335 Asp Asn Lys Leu Pro Tyr Asn Glu Tyr Phe Glu Tyr Phe Gly Pro Asp 340 345 350 Tyr Thr Leu His Val Asp Pro Ser Pro Met Glu Asn Leu Asn Thr Pro 355 360 365 Lys Asp Met Glu Arg Ile Arg Asn Thr Leu Leu Glu Gln Leu Ser Gly 370 375 380 Leu Ile His Ala Pro Ser Val Gln Phe Gln His Thr Pro Pro Val Asn 385 390 395 400 Arg Val Leu Asp Glu Pro Glu Asp Asp Met Glu Thr Arg Pro Lys Pro 405 410 415 Arg Ile Trp Ser Gly Thr Ala Thr Tyr Glu Ser Asp Ser Asp Asp Asp 420 425 430 Asp Lys Pro Leu His Gly Tyr Ser Cys Arg Gly Gly Ala Thr Thr Asp 435 440 445 Arg Asp Ser Thr Gly Glu Asp Glu Met Asp Asp Asp Asn Pro Glu Pro 450 455 460 Asp Val Asn Pro Pro Ser Ser 465 470

Claims (9)

A method of increasing the callus formation efficiency of a plant, comprising the step of inhibiting in plant cells the expression of a gene encoding an Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) protein consisting of the amino acid sequence of SEQ ID NO: 2. delete The method of claim 1, wherein the Arabidopsis thaliana-derived HDA6 protein coding gene is knocked out using a gene editing system to increase the callus formation efficiency of the plant compared to the wild type plant. delete Transforming plant cells with a recombinant vector containing a gene encoding the Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) protein consisting of the amino acid sequence of SEQ ID NO: 2, comprising the step of inhibiting the expression of the gene encoding the HDA6 protein, Method for producing transgenic plants with increased callus formation efficiency. delete (a) correcting the genome by introducing guide RNA and endonuclease protein specific to the target base sequence of the Arabidopsis-derived HDA6 (Histone deacetylase 6) protein coding gene into plant cells; and
(b) redifferentiating a plant from the genome-corrected plant cell; A method for producing a genome-edited plant with increased callus formation efficiency compared to the wild type. A mutant plant in which the expression of the gene encoding the Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) protein, which has the amino acid sequence of SEQ ID NO: 2, is inhibited, resulting in increased callus formation efficiency. A composition for increasing callus formation efficiency in plants, comprising as an active ingredient a gene encoding an Arabidopsis thaliana-derived HDA6 (Histone deacetylase 6) protein consisting of the amino acid sequence of SEQ ID NO: 2.