KR102542508B1 - α1-COP gene controlling abiotic stress of plant and uses thereof - Google Patents

Abstract

The present invention relates to an α1-COP gene that regulates abiotic stress resistance of plants and its use. Since the α1-COP gene according to the present invention can be used to develop multi-stress resistant crops, it is used as an important material for breeding new varieties. It will be possible.

Description

α1-COP gene controlling abiotic stress resistance of plants and its uses {α1-COP gene controlling abiotic stress of plant and uses thereof}

The present invention relates to an α1-COP (Coatomer subunit alpha-1) gene that regulates abiotic stress resistance of plants and uses thereof.

Plants live by exchanging signals constantly under biotic stress, such as pathogen infection and attack by herbivores and insects, and abiotic stress, such as drought, heat, cold, and nutrient deficiency. Plants with low resistance to abiotic stress require a lot of energy and consume a lot of water and fertilizer when adapting to the isolated environment, which can put a great burden on the environment. Among abiotic stresses, high salinity, drought, and temperature changes have a great influence on the growth and development of plants that live a sedentary lifestyle.

The movement of various cellular materials through plasmodesma affects plant development as well as plant resistance to external stress such as disease resistance. Through various experiments, the present inventors have found that α1-COP (Coatomer subunit alpha-1) is required for cell-to-cell movement of PDBG2 (plasmodesmata-associated b-1,3-glucanase), an enzyme that decomposes callose accumulated in plasma liaisons. confirmed that

On the other hand, Korean Patent Publication No. 2015-0010299 discloses 'a method for controlling environmental stress tolerance of plants using ARR22 gene and plants accordingly', and Korean Patent No. 2101757 discloses 'accumulation of protoplasmic ligature callose in plants' and Arabidopsis-derived SPHK1 gene and its use for regulating plant growth' have been disclosed, but the α1-COP gene and its use for regulating abiotic stress resistance of plants of the present invention have not been described.

The present invention was derived from the above needs, and as a result of securing and analyzing mutations related to the accumulation of callose, which plays a key role in regulating protoplast liaisons, the present inventors found a dominant mutation in the α1-COP gene. It was established that it happened. In addition, as a result of preparing α1-COP knockout and overexpressing plants and treating stresses such as drought, high temperature, low temperature, high salt and various heavy metals, the α1-COP knockout mutant Although it showed resistance to stress, it was confirmed that it did not show heavy metal resistance. In addition, it was found that α1-COP overexpressing organisms had increased sensitivity to drought, high temperature, low temperature and high salt stress compared to non-transformants. Through the above results, the present invention was completed by confirming the possibility of the α1-COP gene as a multi-stress resistant crop development target.

In order to solve the above problems, the present invention provides a method for controlling the resistance of plants to abiotic stress, comprising the step of regulating the expression of a plant-derived α1-COP (Coatomer subunit alpha-1) protein coding gene. do.

In addition, the present invention (a) correcting the genome by introducing guide RNA and endonuclease proteins specific to the target nucleotide sequence of the plant-derived α1-COP protein coding gene into plant cells; and (b) regenerating plants from the genome-corrected plant cells.

In addition, the present invention comprises the steps of transforming a plant cell with a recombinant vector containing a plant-derived α1-COP protein coding gene; and regenerating the transformed plant from the transformed plant cell.

In addition, the present invention provides a transgenic plant with controlled resistance to abiotic stress prepared by the above production method and a transformed seed thereof.

In addition, the present invention provides a composition for regulating the resistance of plants to abiotic stress, comprising a plant-derived α1-COP protein coding gene as an active ingredient.

The present invention suggests the possibility of the α1-COP gene as a target for the development of multi-stress resistant crops. Since securing multi-stress resistant crops is a very important characteristic in the agricultural field, the Arabidopsis-derived α1-COP gene of the present invention is expected to be highly utilized. In addition, it has been confirmed that the α1-COP gene is related to various abiotic stress-related genes, and it can be used as a very important material for gene editing-based precision breeding.

Figure 1 shows the plant growth results of wild-type, mutant, and overexpressed Arabidopsis thaliana plants after water was cut off for 21 days and then water supply was resumed for 4 days in order to confirm the function of the α1-COP gene for drought stress. Col-0: wild type, α1-cop-1 and α1-cop-5 : mutant, HA: α1-COP-OE#2, HA: α1-COP-OE#3 and GFP: α1-COP-OE#1 : α1-COP overexpressing transformant.
Figure 2 shows the plant growth results of wild-type, mutant, and overexpressors after perfusion with a 300 mM NaCl solution to confirm the function of the α1-COP gene for high salt stress.
Figure 3 shows that in order to confirm the function of the α1-COP gene for high temperature stress, it was grown at 22 ° C in MS medium for 1 week and 22 ° C in soil for 2 weeks, treated at 37 ° C for 5 days, and then recovered at 22 ° C for 1 week. It shows the plant growth results of wild type, mutant and overexpressing products after incubation. Col-0: wild type, α1-cop-5 : mutant, HA: α1-COP-OE#2: α1-COP overexpressing transformant.
Figure 4 shows the plant growth results of wild-type, mutant and overexpressors after culturing in normal MS medium at 22 ° C for 14 days and then treatment at 4 ° C for 9 days to confirm the function of the α1-COP gene against cold stress. .

In order to achieve the object of the present invention, the present invention is a method for controlling the resistance of plants to abiotic stress, comprising the step of regulating the expression of a plant-derived α1-COP (Coatomer subunit alpha-1) protein coding gene provides

In the control method according to one embodiment of the present invention, the α1-COP protein may preferably be an Arabidopsis-derived α1-COP protein consisting of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The scope of the Arabidopsis-derived α1-COP protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of the protein. In the present invention, the term "functional equivalent" is at least 70% or more, preferably 80% or more, more preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 2 as a result of addition, substitution or deletion of amino acids. More preferably, it refers to a protein having a sequence homology of 95% or more, and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" means an activity that regulates the abiotic stress resistance of plants.

The method for regulating the abiotic stress resistance of plants according to the present invention inhibits the expression of the plant-derived α1-COP protein-encoding gene in plant cells to increase the resistance of plants to abiotic stress compared to non-transformed plants. It may be to increase, or it may be to increase the sensitivity to abiotic stress compared to non-transformed plants by overexpressing the expression of the α1-COP protein coding gene in plant cells, but is not limited thereto.

In one embodiment of the present invention, the suppression of expression of the gene encoding the α1-COP protein is performed by T-DNA insertion into the α1-COP protein encoding gene, endogenous transposon, X-ray or γ-ray irradiation. Mutagenesis, RNAi or antisense RNA, CRISPR/Cas9 gene editing system may be used to inhibit (suppress) the expression of an α1-COP protein-encoding gene, but is not limited thereto. Any method of can be possible.

In the abiotic stress resistance control method of plants according to the present invention, the abiotic stress may be at least one selected from the group consisting of drought, high temperature, low temperature and salt stress, but is not limited thereto.

The present invention also includes a gene encoding a plant-derived α1-COP protein, and the scope of the gene includes both genomic DNA, cDNA and synthetic DNA encoding the α1-COP protein. Preferably, the gene encoding the α1-COP protein of the present invention may include the Arabidopsis-derived α1-COP nucleotide sequence represented by SEQ ID NO: 1. In addition, homologues of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene includes a base sequence having a sequence homology of at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% with the base sequence of SEQ ID NO: 1. can do. The "% sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences, wherein a portion of the polynucleotide sequence in the region of comparison is a reference sequence (not containing additions or deletions) to the optimal alignment of the two sequences. may include additions or deletions (i.e., gaps) compared to

As used herein, the term "recombinant" refers to a cell that replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a protein encoded by a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the cell's native form, either in sense or antisense form. A recombinant cell may also express a gene found in the cell in its natural state, but the gene has been reintroduced into the cell by artificial means as a modified one.

In this specification, the term "vector" is used to refer to DNA fragment(s) or nucleic acid molecules that are delivered into cells. Vectors replicate DNA and can reproduce independently in host cells. The term “delivery vehicle” is often used interchangeably with “vector”.

The vectors of the present invention may typically be constructed as vectors for cloning or expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (eg, pLλ promoter, Trp promoter, Lac promoter, T7 promoter, Tac promoter, etc.), It typically includes a ribosome binding site for initiation of translation and a transcription/translation termination sequence. When Escherichia coli is used as the host cell, the E. coli tryptophan biosynthesis pathway promoter and operator regions and the leftward promoter of phage λ (pLλ promoter) can be used as control regions.

In the recombinant vector of the present invention, the promoter may be promoters suitable for transformation, preferably CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter, preferably CaMV 35S promoter. However, it is not limited thereto.

In the present invention, the term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in a plant cell. A “constitutive promoter” is a promoter that is active under most environmental conditions and states of development or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of transformants may be made by various tissues at various stages. Thus, constitutive promoters do not limit selection possibilities.

The recombinant vector of the present invention can be constructed by methods well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The DNA sequence can be effectively linked to a suitable promoter in an expression vector to direct mRNA synthesis. In addition, the vector may include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a part of itself, the so-called T-region, into plant cells when present in a suitable host such as Agrobacterium tumefaciens . Another type of Ti-plasmid vector (see EP 0 116 718 B1) is 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. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce DNA according to the present invention into plant hosts include viral vectors, such as those that can be derived from double-stranded plant viruses (eg, CaMV) and single-stranded viruses, gemini viruses, and the like. For example, it may be selected from incomplete plant viral vectors. The use of such vectors can be particularly advantageous when properly transforming a plant host is difficult.

Recombinant expression vectors may preferably contain one or more selectable markers. The marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing transformed cells from non-transformed cells. The marker gene may be a dominant drug resistance gene, but is not limited thereto.

Any host cell known in the art, including microalgae and microorganisms, can be used as the host cell capable of stably and continuously cloning and expressing the vector of the present invention. For example, E. coli JM109, E. coli BL21, Bacillus genus strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis , Bacillus thuringiensis , and Enterobacteriaceae and strains such as Salmonella typhimurium , Serratia marcescens and various Pseudomonas species.

In addition, when the vector of the present invention is transformed into a eukaryotic cell, as a host cell, yeast (eg, Saccharomyce cerevisiae ), insect cells, human cells (eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7 , 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells, etc. may be used, preferably plant cells.

Plant transformation refers to any method of transferring DNA into a plant. Such transformation methods need not necessarily have a period of regeneration and/or tissue culture. Transformation of plant species is now common for plant species including both dicotyledonous as well as monocotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., 1987, Plant Mol. Biol. 8, 363-373), Electroporation (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185) , Agrobacterium tumefaciens by particle bombardment of various plant elements (DNA or RNA-coated) (Klein T.M. et al., 1987, Nature 327, 70), infiltration of plants or transformation of mature pollen or microspores It can be suitably selected from infections by (incomplete) viruses in mediated gene transfer (EP 0 301 316) and the like. Preferred methods according to the present invention include Agrobacterium mediated DNA delivery.

The present invention also

(a) Guide RNA and endonuclease proteins specific to the target sequence of the plant-derived α1-COP (Coatomer subunit alpha-1) protein coding gene are introduced into plant cells to correct the genome. step; and

(b) regenerating plants from the genome-corrected plant cells; a method for producing a genome-corrected plant with increased resistance to abiotic stress is provided.

The term "genome/gene editing" refers to a technology capable of introducing target-directed mutations into genomic sequences of animal and plant cells, including human cells, and deletion of one or more nucleic acid molecules by DNA cleavage ( Deletion, insertion, substitution, etc., knock-out or knock-in of a specific gene, or non-coding DNA that does not produce a protein It refers to a technology that can introduce mutations into sequences. For the purposes of the present invention, the genome editing may be to introduce mutations into plants using an endonuclease, such as Cas9 (CRISPR associated protein 9) protein and guide RNA. In addition, 'gene editing' may be used interchangeably with 'gene editing'.

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

The term "guide RNA" refers to an RNA specific to DNA encoding a nucleotide sequence of a target gene, which is complementary in whole or in part to a target DNA nucleotide sequence, thereby forming an endonuclease into the corresponding target DNA nucleotide sequence. It refers to ribonucleic acid that guides the first protein. The guide RNA is a dual RNA comprising two RNAs, that is, 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 complementary to a base sequence in a target gene in whole or in part and a second region comprising a sequence interacting with an RNA-guided nuclease, , RNA-guided nucleases can be included in the scope of the present invention without limitation if they are in the form that can have activity in the target sequence, and considering the type of endonuclease used together or the microorganism derived from the endonuclease, etc. It can be selected appropriately according to known techniques in the art.

In addition, the guide RNA may be a guide RNA transcribed from a plasmid template, transcribed in vitro (eg, oligonucleotide duplex), or synthesized guide RNA, but is not limited thereto.

In addition, in the production method of the genome editing plant according to the present invention, the endonuclease protein is Cas9, Cpf1 (CRISPR from Prevotella and Francisella 1), TALEN (Transcription activator-like effector nuclease), ZFN (Zinc Finger Nuclease) Or it may be one or more selected from the group consisting of functional analogs thereof, preferably a Cas9 protein, but is not limited thereto.

In addition, the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Cas9 protein derived from Streptococcus thermophilus ( S. thermophilus ) or Streptococcus aureus ( S. aureus ) derived Cas9 protein, Neisseria meningitidis ( Neisseria meningitidis ) derived Cas9 protein, Pasteurella multocida ( Pasteurella multocida ) derived Cas9 protein, Francisella novicida ( 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 genetic information thereof can be obtained from known databases such as GenBank of 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 sequence and cut the DNA strand, a short sequence consisting of three bases known as PAM (Protospacer Adjacent Motif) must be present next to the target sequence, and the Cas9 protein has a PAM sequence (NGG) It is cut by estimating between the 3rd and 4th base pairs from .

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

The CRISPR/Cas9 system used in the present invention introduces a double helix break at a specific position of a specific gene to be corrected, thereby inducing insertion-deletion (InDel) mutations caused by incomplete repair induced in the DNA repair process. (non-homologous end joining) is a gene editing method.

In the manufacturing method according to the present invention, introducing the guide RNA and endonuclease protein of step (a) into plant cells is a guide RNA specific to the target sequence of the plant-derived α1-COP gene and endonuclease. complexes of the first proteins (ribonucleoprotein); Or a recombinant vector containing DNA encoding a guide RNA specific to the target sequence of the α1-COP gene and a nucleic acid sequence encoding an endonuclease protein; may be used, but is not limited thereto.

The present invention also includes the steps of transforming plant cells with a recombinant vector containing a plant-derived α1-COP (Coatomer subunit alpha-1) protein coding gene; and regenerating the transformed plant from the transformed plant cell.

In the production method of the present invention, the plant-derived α1-COP protein may preferably include Arabidopsis-derived α1-COP protein consisting of the amino acid sequence of SEQ ID NO: 2 and a functional equivalent of the protein. Same as above.

In addition, in the production method of the present invention, the method of transforming the plant cells is as described above, and the method of regenerating the transformed plants from the transformed plant cells can be any method known in the art. can Transformed plant cells must regenerate into whole plants. Techniques for regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vols. 1-5, 1983-1989 Momillan, N.Y.).

In the manufacturing method according to one embodiment of the present invention, the abiotic stress may be one or more selected from the group consisting of drought, high temperature, low temperature and salt stress, but is not limited thereto.

In the manufacturing method according to one embodiment of the present invention, when the expression of the gene encoding the α1-COP protein is inhibited in plant cells, resistance to abiotic stress of the plant is increased compared to non-transformed plants, that is, wild-type plants. Alternatively, overexpression of the α1-COP protein-encoding gene in plant cells can increase the sensitivity of plants to abiotic stress compared to non-transformed plants, but is not limited thereto.

Coding the α1-COP protein Inhibiting the expression of the gene may be inhibiting the expression of the α1-COP protein coding gene by transforming plant cells with a recombinant vector containing sense or antisense DNA or microRNA for the α1-COP gene. , It is not limited thereto, and gene expression inhibition techniques known in the art may be used.

The present invention also provides transgenic plants with controlled resistance to abiotic stress prepared by the production method of the present invention and their transformed seeds.

Transgenic plants according to the present invention are characterized by increased resistance to abiotic stress when the expression of the α1-COP protein-encoding gene is inhibited, and when the expression of the α1-COP protein-encoding gene is increased, the transgenic plant is characterized by increased resistance to abiotic stress. It is characterized by a decrease in resistance to biotic stress.

The plants are Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, crown daisy, lettuce, bellflower, spinach, chard, sweet potato, carrot, water parsley, Chinese cabbage, cabbage, mustard, watermelon, melon, cucumber, pumpkin, gourd, Dicotyledonous plants such as strawberries, soybeans, mung beans, kidney beans, and peas, or monocotyledonous plants such as rice, barley, wheat, rye, corn, sugar cane, oats, and onions, preferably dicotyledonous plants, more preferably It may be an Arabidopsis thaliana plant, but is not limited thereto.

The present invention also provides a composition for regulating the resistance of a plant to abiotic stress, comprising a plant-derived α1-COP (Coatomer subunit alpha-1) protein coding gene as an active ingredient.

The composition contains, as an active ingredient, a gene encoding a plant-derived α1-COP protein or a mutation of a gene encoding the α1-COP protein, and the gene encoding the α1-COP protein or a gene encoding the α1-COP protein By transforming plant cells with a recombinant vector containing the vector, the resistance of plants to abiotic stress can be controlled.

In the composition according to the present invention, the plant-derived α1-COP protein may preferably be an Arabidopsis-derived α1-COP protein consisting of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

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

Materials and Methods

1. Plant material

The mutant used in the present invention is the genetic background of the Arabidopsis thaliana Col-O ecotype, and the T-DNA insertion strains are α1-cop-1 (SALK_078465) and α1-cop-5 (SALK_003425). To prepare α1-COP overexpressing transformants (p35S::α1-COP-OE#2, p35S::α1-COP-OE#3 and p35S::GFP:α1-COP-OE#1), α1- PCR products (with or without stop codons) amplified from the entire cDNA of COP were cloned into pDONR207 plasmid (Invitrogen, USA). The resulting entry clone was cloned into the gateway binary vectors pMDC43 and pDEST- ^GW VYCE. Transgenic plants were obtained from wild-type Col-O through Agrobacterium-mediated transformation. Briefly, developing Arabidopsis flowers were immersed in a culture solution containing Agrobacterium transformed with the vector for 5 seconds, and then T ₁ seeds were grown in a selective medium for identifying transformed plants.

2. Dealing with abiotic stress

2-1. Drought stress treatment and resistance verification

Seeds of wild-type, mutant and overexpressors were surface sterilized with 20% (v/v) bleach for 15 minutes, washed 4 times with distilled water, left in the dark at 4°C for 3 days, and then containing 0.6% agar. were cultured on MS medium. For drought (/ mannitol) stress, the above-described seeds were germinated under a photoperiod condition of 16 hours light/8 hours dark and a temperature condition of 22° C., and then, 2 days after germination, the seedlings were treated with a new normal MS medium (mock) or 300 mM After transfer to a medium containing mannitol (drought stress conditions), primary root length analysis was performed at 14 DAG (day after germination). For drought stress in the soil, 14-day-old Arabidopsis plants grown in MS medium were transferred to the soil and treated with drought stress (no water supply) for 21 days. After re-irrigation for 4 days after the stress treatment, the survival rate of the plants was analyzed.

2-2. High salt stress treatment and resistance verification

After germination, 2-day-old seedlings sterilized and grown in the same way as in 2-1. were transferred to a normal MS medium (mock) or a medium containing 300 mM sodium chloride (high salt stress condition), and the length of the main root at 11 DAG and Survival rates were analyzed. In addition, after transferring the 14-day-old Arabidopsis plants grown in MS medium to soil, and placing them in normal soil (control group) or 300 mM salt condition soil (stress group) for 24 days, the live weight of the plants was measured on day 24.

2-3. High temperature stress assay

Sterilization, growth and germination were performed in the same manner as in 2-1. After transferring 7-day-old seedlings to soil, they were grown for 2 weeks in a 22°C growth chamber (16 hours light/8 hours dark photoperiod), and then 3-week-old plants were grown in a 37°C growth chamber (16 hours light/8 hours dark photoperiod). photoperiod of cancer) and treated with high-temperature stress. After 5 days, the plants were transferred back to the growth chamber at 22° C., and the survival rate of the plants was checked after 1 week.

2-4. cold stress assay

After germination, 2-day-old seedlings sterilized and grown in the same manner as in 2-1. were transferred to a new MS medium and subjected to photoperiod conditions of 16 hours light/8 hours dark and temperature conditions of 22 ° C. (control group) or 16 hours light. It was cultured for 9 days under photoperiod conditions of / 8 hours cancer and temperature conditions of 4 ° C (low temperature stress conditions). After 9 days, the length of the main root and the number of lateral roots were analyzed.

Example 1. Abiotic stress α1-COP Verification of resistance of gene mutants and overexpressors

In order to analyze the resistance of plants to abiotic stress caused by the α1-COP gene, wild-type, mutant and overexpressing plants were treated with drought, high salt, high and low temperature stress conditions, and plant growth was confirmed.

First, drought stress was analyzed through the level of plant growth after water was cut off for 21 days and water supply was resumed for 4 days, and high salt stress was analyzed using 2-day-old seedlings germinated in normal MS medium with or without 300 mM NaCl. After growing in MS medium for 9 days, the growth level of each plant was analyzed. High-temperature stress was measured by transferring 7-day-old seedlings germinated in normal MS medium to soil, growing them at 22°C for 2 weeks, and then transferring them to a growth chamber at 37°C. After being transferred and allowed to stand for 5 days, the growth level of each plant was analyzed after being transferred to 22 ° C for recovery, and the low temperature stress was cultured in normal MS medium at 22 ° C for 14 days and then treated at 4 ° C for 9 days. was analyzed.

As a result, as shown in Figures 1 to 4, it was observed that the α1-COP mutant had improved resistance to drought, high salt, high temperature and low temperature stress compared to the wild type, and the α1-COP overexpressing transformant was compared to the wild type. Compared to the above, it was confirmed that resistance was reduced in all abiotic stress conditions.

Example 2. Comparison of homology of plant α1-COP proteins

Arabidopsis α1-COP and α1-COP proteins of other plants were compared for homology. As a result, as shown in Tables 1 to 3 below, it was found that α1-COP protein was conserved to a very high degree in various crops and fruit trees. Through the above results, it was inferred that abiotic stress resistance can be induced as in Arabidopsis thaliana plants when α1-COP is knocked out in other plants other than Arabidopsis thaliana.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> a1-COP gene controlling abiotic stress of plant and uses its <130> PN20211 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 3651 <212> DNA <213> Arabidopsis thaliana <400> 1 atgttgacaa agttcgaaac caaaagtaac agagttaagg gactgagttt tcaccctaaa 60 cgaccatgga ttctcgcgag tttgcacagt ggcgtgatcc agctctggga ttatcgtatg 120 ggtactttga tcgatagatt cgacgaacac gaaggacctg ttcgtggtgt tcatttccac 180 aattctcagc ctctcttcgt ctccggaggg gatgattaca agattaaagt gtggaactac 240 aaaaaccaca ggtgcctttt cactcttcta gggcatcttg attatatccg cacggttcag 300 ttccatcatg agtacccatg gattgtgagt gcaagtgatg atcaaactat ccgtatatgg 360 aactggcaat cacggacttg tgtttctgta ttgactggtc acaatcatta tgttatgtgt 420 gcttctttcc atccgaaaga agacctcgtc gtatcagctt cgttagatca gactgttcgt 480 gtttgggata ttggtgctct taggaagaag actgtatctc ctgctgatga tatcatgagg 540 ttgactcaga tgaatagtga tctttttggt ggagttgatg ccattgtcaa gtacgttctt 600 gaaggtcatg atagaggtgt aaactgggct gcttttcatc ccaccctgcc tcttattgtc 660 720 gtggatacat tgcgaggcca tatgaataac gtttcatctg ttatgttcca tgcaaaacag 780 gacataattg tatcaaactc tgaagataag agcatccgtg tctgggatgc taccaagcga 840 actggacttc agactttccg tcgtgaacat gatagattct ggattcttgc agttcaccct 900 gagatgaatc tactagcagc aggacatgac agtggcatga ttgtgttcaa gcttgagaga 960 gaacgtcctg catttgcttt gagtggggat tctttgttct atgccaagga tagattcttg 1020 cggtactatg aatattcaac tcaaagggat tcccaagtta tccctatacg gcgtccgggt 1080 actccaagct tgaatcaaag tcctaggact ctatcataca gtcccactga aaatgcagtt 1140 ttgatctgct cagatctgga tggtggttcg tatgagttgt acatcatacc gaaagatagt 1200 gttggcagga gtgacgttgt gcaagatgca aagagaggga caggtggctc tgcagtattc 1260 attgcccgta accgatttgc tgttctcgag aaaagcacca gccaagtgct agtcaagaat 1320 ctaaagaatg aggtggttaa aaagagtcct ctgcctattc ccacagatgc tatcttttat 1380 gctgggactg ggaatttgct ttgtagatct gaggataaag tagttatctt tgacctccag 1440 caaaggcttg ttcttggtga gcttcagaca ccatttgtta ggtacgttgt ttggtccagt 1500 gatatggaga gtgttgcttt gctcagcaaa catactatca ttatcgcgag caaaaaactt 1560 gttctccagt gcacgcttca tgagacgata cgtgtgaaaa gtggagcttg ggacgataat 1620 ggtgtcttca tttacacaac tttgaatcat atcaagtact gtcttcctaa tggagatagt 1680 ggaatcatcc gaaccctgga tgtcccaatc tatatcacca aggtgtctgg taatacaatc 1740 ttttgcttgg atcgggatgg caaaaacaag gctatcacca ttaatgcaac cgagtacatt 1800 ttcaagcttt cactgctgag gaagagatat gatcatgtca tgagcatgat aaagaactct 1860 cagctatgtg gacaggctat gattgcttac ctgcagcaga aaggattccc tgaagttgcc 1920 ctccattttg ttgaagatga gaggatccga ttcaatctgg ctcttgaaag tggcaacatc 1980 agtgtggctg ttgcatccgc tacacagatt aacgagaaag accactggta tagacttgga 2040 gtggaggctc ttcgccaagg taattctgga attgttgagt ttgcatacca gcagacaaag 2100 aactttgaga ggttatcttt tctttatctc attactggaa atttggacaa gctttcaaag 2160 ttgatgaaga ttgcagaagt gaagaacaac gtaatgggac agtttcacaa tgctctctac 2220 ctaggagatg ttaaagagcg tgttaagata ctggagaatg ctggtcattt gcctctagct 2280 tatatcacag cttcggttca tggtttaaat gacattgctg agcgactggc aactgagtta 2340 ggagacaacg tgccctcatt gcctgaagga aaaactccat cgcttctaat gccaccgaca 2400 cccatcatgt gtggtggtga ttggcctctt cttagagtga tgaaaggaat atttgaagga 2460 ggacttgaga gtgcagacag aggaggtact gttgatgaag aagatgttga aggggattgg 2520 ggtgaggaac ttgacattaa tgttgatgga atggagaaca gagatattga agacattttg 2580 gcggctgcag aagcaggtga agaagaaaac gatgaggaag gtggatgggg acttgaagac 2640 ttggtgctcc caccagagct tgacactcct aaagcctctg ctaatgcacg ttcatctgtt 2700 tttgtgacac ctccccaagg tatgcccgta agtcagagct ggagccagaa atcttctctt 2760 gctgctgagc aagccgcagc gggaagcttc gacactgcaa tgcgtctgct ccacagacag 2820 cttggtatca agaactttac acctttgaaa tcgatgtttc tagatctgtt caatggcagc 2880 catagctatc tgcgtgcttt ttcttcatgt cctgtggttc cacttgccat tgagcgtgga 2940 tggagcgagt cttccagtcc caacgtccgt agcccaccag ctcttgttta tgatttctct 3000 cagctagacg agaagctcaa atccggctac aaagctacaa caactgggaa attcactgag 3060 gctctccgtc tcttcctttc tatcctgcac accatccctc tagtggtagt cgagacaaga 3120 agagaagtgg atgaggtcaa ggaattaatc gttatcgtta aagaatatgt ccttggtctt 3180 caaatggagc tcaagaggag agagatgaaa gatgatccgg tgaggcagca agagctcgct 3240 gcttacttca ctcactgcaa cctccaaaca cctcacttac ggcttgcgct gcttagtgca 3300 atgggtgtct gctacaaggc aaagaacctc gccactgctt ctaatttcgc cagaagactg 3360 ttggaaacaa gcccagttga tagccaagcc aagatggcta gacaagttgt gcaagccgct 3420 gaacgtaaca tgaccgatga aacaaagctt aactacgact tcaggaaccc ttttgtggta 3480 tgtggttcca catatgtccc gatctacaga ggacagaaag acgtctcatg tccttactgc 3540 accgcccggt ttgtaccaaa ccaggaagga aacatctgca cggtctgtga tctagcagtg 3600 attggcgcag acgcatctgg tttgttatgc tctccatctc aagtccggtg a 3651 <210> 2 <211> 1216 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Leu Thr Lys Phe Glu Thr Lys Ser Asn Arg Val Lys Gly Leu Ser 1 5 10 15 Phe His Pro Lys Arg Pro Trp Ile Leu Ala Ser Leu His Ser Gly Val 20 25 30 Ile Gln Leu Trp Asp Tyr Arg Met Gly Thr Leu Ile Asp Arg Phe Asp 35 40 45 Glu His Glu Gly Pro Val Arg Gly Val His Phe His Asn Ser Gln Pro 50 55 60 Leu Phe Val Ser Gly Gly Asp Asp Tyr Lys Ile Lys Val Trp Asn Tyr 65 70 75 80 Lys Asn His Arg Cys Leu Phe Thr Leu Leu Gly His Leu Asp Tyr Ile 85 90 95 Arg Thr Val Gln Phe His His Glu Tyr Pro Trp Ile Val Ser Ala Ser 100 105 110 Asp Asp Gln Thr Ile Arg Ile Trp Asn Trp Gln Ser Arg Thr Cys Val 115 120 125 Ser Val Leu Thr Gly His Asn His Tyr Val Met Cys Ala Ser Phe His 130 135 140 Pro Lys Glu Asp Leu Val Val Ser Ala Ser Leu Asp Gln Thr Val Arg 145 150 155 160 Val Trp Asp Ile Gly Ala Leu Arg Lys Lys Thr Val Ser Pro Ala Asp 165 170 175 Asp Ile Met Arg Leu Thr Gln Met Asn Ser Asp Leu Phe Gly Gly Val 180 185 190 Asp Ala Ile Val Lys Tyr Val Leu Glu Gly His Asp Arg Gly Val Asn 195 200 205 Trp Ala Ala Phe His Pro Thr Leu Pro Leu Ile Val Ser Gly Ala Asp 210 215 220 Asp Arg Gln Val Lys Leu Trp Arg Met Asn Glu Thr Lys Ala Trp Glu 225 230 235 240 Val Asp Thr Leu Arg Gly His Met Asn Asn Val Ser Ser Val Met Phe 245 250 255 His Ala Lys Gln Asp Ile Ile Val Ser Asn Ser Glu Asp Lys Ser Ile 260 265 270 Arg Val Trp Asp Ala Thr Lys Arg Thr Gly Leu Gln Thr Phe Arg Arg 275 280 285 Glu His Asp Arg Phe Trp Ile Leu Ala Val His Pro Glu Met Asn Leu 290 295 300 Leu Ala Ala Gly His Asp Ser Gly Met Ile Val Phe Lys Leu Glu Arg 305 310 315 320 Glu Arg Pro Ala Phe Ala Leu Ser Gly Asp Ser Leu Phe Tyr Ala Lys 325 330 335 Asp Arg Phe Leu Arg Tyr Tyr Glu Tyr Ser Thr Gln Arg Asp Ser Gln 340 345 350 Val Ile Pro Ile Arg Arg Pro Gly Thr Pro Ser Leu Asn Gln Ser Pro 355 360 365 Arg Thr Leu Ser Tyr Ser Pro Thr Glu Asn Ala Val Leu Ile Cys Ser 370 375 380 Asp Leu Asp Gly Gly Ser Tyr Glu Leu Tyr Ile Ile Pro Lys Asp Ser 385 390 395 400 Val Gly Arg Ser Asp Val Val Gln Asp Ala Lys Arg Gly Thr Gly Gly 405 410 415 Ser Ala Val Phe Ile Ala Arg Asn Arg Phe Ala Val Leu Glu Lys Ser 420 425 430 Thr Ser Gln Val Leu Val Lys Asn Leu Lys Asn Glu Val Val Lys Lys 435 440 445 Ser Pro Leu Pro Ile Pro Thr Asp Ala Ile Phe Tyr Ala Gly Thr Gly 450 455 460 Asn Leu Leu Cys Arg Ser Glu Asp Lys Val Val Ile Phe Asp Leu Gln 465 470 475 480 Gln Arg Leu Val Leu Gly Glu Leu Gln Thr Pro Phe Val Arg Tyr Val 485 490 495 Val Trp Ser Ser Asp Met Glu Ser Val Ala Leu Leu Ser Lys His Thr 500 505 510 Ile Ile Ile Ala Ser Lys Lys Leu Val Leu Gln Cys Thr Leu His Glu 515 520 525 Thr Ile Arg Val Lys Ser Gly Ala Trp Asp Asp Asn Gly Val Phe Ile 530 535 540 Tyr Thr Thr Leu Asn His Ile Lys Tyr Cys Leu Pro Asn Gly Asp Ser 545 550 555 560 Gly Ile Ile Arg Thr Leu Asp Val Pro Ile Tyr Ile Thr Lys Val Ser 565 570 575 Gly Asn Thr Ile Phe Cys Leu Asp Arg Asp Gly Lys Asn Lys Ala Ile 580 585 590 Thr Ile Asn Ala Thr Glu Tyr Ile Phe Lys Leu Ser Leu Leu Arg Lys 595 600 605 Arg Tyr Asp His Val Met Ser Met Ile Lys Asn Ser Gln Leu Cys Gly 610 615 620 Gln Ala Met Ile Ala Tyr Leu Gln Gln Lys Gly Phe Pro Glu Val Ala 625 630 635 640 Leu His Phe Val Glu Asp Glu Arg Ile Arg Phe Asn Leu Ala Leu Glu 645 650 655 Ser Gly Asn Ile Ser Val Ala Val Ala Ser Ala Thr Gln Ile Asn Glu 660 665 670 Lys Asp His Trp Tyr Arg Leu Gly Val Glu Ala Leu Arg Gln Gly Asn 675 680 685 Ser Gly Ile Val Glu Phe Ala Tyr Gln Gln Thr Lys Asn Phe Glu Arg 690 695 700 Leu Ser Phe Leu Tyr Leu Ile Thr Gly Asn Leu Asp Lys Leu Ser Lys 705 710 715 720 Leu Met Lys Ile Ala Glu Val Lys Asn Asn Val Met Gly Gln Phe His 725 730 735 Asn Ala Leu Tyr Leu Gly Asp Val Lys Glu Arg Val Lys Ile Leu Glu 740 745 750 Asn Ala Gly His Leu Pro Leu Ala Tyr Ile Thr Ala Ser Val His Gly 755 760 765 Leu Asn Asp Ile Ala Glu Arg Leu Ala Thr Glu Leu Gly Asp Asn Val 770 775 780 Pro Ser Leu Pro Glu Gly Lys Thr Pro Ser Leu Leu Met Pro Pro Thr 785 790 795 800 Pro Ile Met Cys Gly Gly Asp Trp Pro Leu Leu Arg Val Met Lys Gly 805 810 815 Ile Phe Glu Gly Gly Leu Glu Ser Ala Asp Arg Gly Gly Thr Val Asp 820 825 830 Glu Glu Asp Val Glu Gly Asp Trp Gly Glu Glu Leu Asp Ile Asn Val 835 840 845 Asp Gly Met Glu Asn Arg Asp Ile Glu Asp Ile Leu Ala Ala Ala Glu 850 855 860 Ala Gly Glu Glu Glu Asn Asp Glu Glu Gly Gly Trp Gly Leu Glu Asp 865 870 875 880 Leu Val Leu Pro Pro Glu Leu Asp Thr Pro Lys Ala Ser Ala Asn Ala 885 890 895 Arg Ser Ser Val Phe Val Thr Pro Pro Gln Gly Met Pro Val Ser Gln 900 905 910 Ser Trp Ser Gln Lys Ser Ser Leu Ala Ala Glu Gln Ala Ala Ala Gly 915 920 925 Ser Phe Asp Thr Ala Met Arg Leu Leu His Arg Gln Leu Gly Ile Lys 930 935 940 Asn Phe Thr Pro Leu Lys Ser Met Phe Leu Asp Leu Phe Asn Gly Ser 945 950 955 960 His Ser Tyr Leu Arg Ala Phe Ser Ser Cys Pro Val Val Pro Leu Ala 965 970 975 Ile Glu Arg Gly Trp Ser Glu Ser Ser Ser Pro Asn Val Arg Ser Pro 980 985 990 Pro Ala Leu Val Tyr Asp Phe Ser Gln Leu Asp Glu Lys Leu Lys Ser 995 1000 1005 Gly Tyr Lys Ala Thr Thr Thr Gly Lys Phe Thr Glu Ala Leu Arg Leu 1010 1015 1020 Phe Leu Ser Ile Leu His Thr Ile Pro Leu Val Val Val Glu Thr Arg 1025 1030 1035 1040 Arg Glu Val Asp Glu Val Lys Glu Leu Ile Val Ile Val Lys Glu Tyr 1045 1050 1055 Val Leu Gly Leu Gln Met Glu Leu Lys Arg Arg Glu Met Lys Asp Asp 1060 1065 1070 Pro Val Arg Gln Gln Glu Leu Ala Ala Tyr Phe Thr His Cys Asn Leu 1075 1080 1085 Gln Thr Pro His Leu Arg Leu Ala Leu Leu Ser Ala Met Gly Val Cys 1090 1095 1100 Tyr Lys Ala Lys Asn Leu Ala Thr Ala Ser Asn Phe Ala Arg Arg Leu 1105 1110 1115 1120 Leu Glu Thr Ser Pro Val Asp Ser Gln Ala Lys Met Ala Arg Gln Val 1125 1130 1135 Val Gln Ala Ala Glu Arg Asn Met Thr Asp Glu Thr Lys Leu Asn Tyr 1140 1145 1150 Asp Phe Arg Asn Pro Phe Val Val Cys Gly Ser Thr Tyr Val Pro Ile 1155 1160 1165 Tyr Arg Gly Gln Lys Asp Val Ser Cys Pro Tyr Cys Thr Ala Arg Phe 1170 1175 1180 Val Pro Asn Gln Glu Gly Asn Ile Cys Thr Val Cys Asp Leu Ala Val 1185 1190 1195 1200 Ile Gly Ala Asp Ala Ser Gly Leu Leu Cys Ser Pro Ser Gln Val Arg 1205 1210 1215

Claims (11)

A method for increasing the resistance of a plant to abiotic stress, comprising inhibiting the expression of a plant-derived α1-COP (Coatomer subunit alpha-1) protein coding gene,
The abiotic stress is a method for increasing the resistance of plants to abiotic stress, characterized in that at least one selected from the group consisting of drought, high temperature, low temperature and salt stress. delete The method according to claim 1, wherein the plant-derived α1-COP protein coding gene is knocked out using a gene editing system to increase resistance to abiotic stress. delete (a) Guide RNA and endonuclease proteins specific to the target sequence of the plant-derived α1-COP (Coatomer subunit alpha-1) protein coding gene are introduced into plant cells to correct the genome. step; and
A method for producing a genome-corrected plant with increased resistance to abiotic stress, including (b) regenerating a plant from the genome-corrected plant cell,
The abiotic stress is a method for producing a genome corrected plant, characterized in that at least one selected from the group consisting of drought, high temperature, low temperature and salt stress. Transforming plant cells with a recombinant vector containing a plant-derived α1-COP (Coatomer subunit alpha-1) protein-encoding gene to inhibit expression of the α1-COP protein-encoding gene; and
A method for producing a transgenic plant with increased resistance to abiotic stress compared to a non-transformed plant comprising: regenerating the transformed plant from the transformed plant cell,
The abiotic stress is a method for producing a transgenic plant, characterized in that at least one selected from the group consisting of drought, high temperature, low temperature and salt stress. delete delete A transgenic plant with increased resistance to abiotic stress prepared by the method of claim 6. Transformed seeds of plants according to claim 9. A composition for increasing the resistance of plants to abiotic stress, comprising a plant-derived α1-COP (Coatomer subunit alpha-1) protein coding gene as an active ingredient,
The abiotic stress is a composition for increasing resistance to abiotic stress of plants, characterized in that at least one selected from the group consisting of drought, high temperature, low temperature and salt stress.

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