Tomato SlDRHB12 gene and method for improving drought stress resistance in plants using it

Abstract

The present invention relates to the SlDRHB12 (Solanum lycopersicum Drought Responsive Homeobox Gene 12) protein, a gene encoding the same, and a method for enhancing drought stress resistance in plants using the same. The invention was completed by confirming that drought stress resistance decreases in plants with overexpression of the said gene and increases in plants with silenced expression of the said gene. Since SlDRHB12 can function as a negative regulator of drought stress resistance, it can be utilized for the improvement of crops available to humans through the regulation of the expression of the said protein, and is expected to be particularly useful for improving plant productivity.

Description

Technology Field

[0001] The present invention SlDRHB12 ( Solanum lycopersicum Drought Responsive Homeobox Gene 12) This invention relates to a gene, a protein, and a method for enhancing drought stress resistance in plants using the same. Background Technology

[0003] Global warming causes extreme climate change, inducing environmental stress that hinders the normal growth and development of plants. In response, plants have evolved sophisticated defense mechanisms to survive in adverse environments such as cold, high salinity, and drought. For instance, drought, caused by water scarcity, severely impacts normal plant growth and ultimately leads to reduced crop yields; to adapt to this drought stress, plants activate various defense mechanisms, such as stomatal closure and the synthesis and accumulation of abscisic acid (ABA).

[0004] ABA is known to be a regulatory factor that protects plants against abiotic stress, particularly drought stress, and plays a crucial role in plant growth and development. It has been reported that plants treated with ABA before stress exhibit better stress resistance compared to those that are not, whereas mutant plants that cannot produce ABA or respond to it are known to be vulnerable to stress. Therefore, it is expected that utilizing proteins involved in the response to ABA will enable the development of plants with enhanced resistance to environmental stress.

[0005] Although ABA signaling is known to regulate the expression of various stress-related genes, such as transcription factors and E3 ligases involved in osmotic adaptation and the regulation of root hydraulic conductivity, the complete ABA signaling pathway has not yet been elucidated. For the initiation of ABA signaling in plant cells, an ABA receptor must be present to transmit the signal to downstream proteins. To date, PYR / PYL / RCARs are known to play the role of recognizing ABA as ABA receptors. When the receptor recognizes ABA, it promotes the expression of specific genes and the activation of ion channels through the phosphorylation of target proteins, thereby enabling the plant to adapt to adverse environments.

[0006] As desertification progresses today, water scarcity is causing significant problems for agriculture and the environment, necessitating the development of plants capable of surviving in dry environments with minimal water usage. It is expected that the development and application of such technologies to crops will lead to a substantial increase in agricultural production. Particularly in arid regions, plants with enhanced drought resistance—specifically those capable of reducing transpiration—are advantageous for survival; thus, they can not only contribute to improved agricultural productivity but also be useful for environmental remediation in extremely dry areas.

[0007] Accordingly, methods to enhance plant resistance to drought stress have become a major subject of research. In this regard, studies on genes related to drought stress tolerance and growth promotion, as well as the resulting transgenic plants, are being conducted, but the current state of such research is still insufficient. Prior art literature

[0009] Korean Registered Patent 10-1894179 The problem to be solved

[0010] While researching to investigate the relationship between drought stress responses through the regulation of the ABA signaling pathway, the inventors discovered that tomatoes possessing a homeodomain leucine zipper domain SlDRHB12 The present invention was completed by identifying a gene and determining that the gene functions as a negative regulator in ABA signaling and response to dryness stress.

[0011] Accordingly, the object of the present invention is SlDRHB12 composed of the amino acid sequence of SEQ ID NO. 1 ( Solanum lycopersicum Drought Responsive Homeobox Gene 12) provides a protein.

[0012] Another objective of the present invention is the above SlDRHB12 Encoding proteins SlDRHB12 It is to provide genes.

[0013] Another objective of the present invention is the above SlDRHB12 It is to provide a recombinant vector containing a gene.

[0014] Another objective of the present invention is to... SlDRHB12 The present invention provides a composition for enhancing drought stress resistance of a plant, comprising a protein expression or activity inhibitor as an active ingredient.

[0015] Another object of the present invention is SlDRHB12 composed of the amino acid sequence of SEQ ID NO. 1 The present invention provides a method for enhancing drought stress resistance of a plant, comprising a step of inhibiting the expression or activity of a protein.

[0016] Another objective of the present invention is to provide a transgenic plant with enhanced drought stress resistance by the above method.

[0018] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0020] To achieve the above-mentioned objective of the present invention, the present invention comprises SlDRHB12 composed of the amino acid sequence of SEQ ID NO. 1 ( Solanum lycopersicum Drought Responsive Homeobox Gene 12) provides a protein.

[0021] In addition, the present invention relates to the above SlDRHB12 Encoding proteins SlDRHB12 Provides genes.

[0022] In one embodiment of the present invention, the above SlDRHB12 The gene may consist of the nucleotide sequence of SEQ ID NO. 2, but is not limited thereto.

[0023] In another embodiment of the present invention, the above SlDRHB12 The gene may be derived from tomatoes, but is not limited to them.

[0024] In addition, the present invention is the present invention SlDRHB12 Provides a recombinant vector containing a gene.

[0025] In addition, the present invention is SlDRHB12 A composition for enhancing drought stress resistance of a plant is provided, comprising a protein expression or activity inhibitor as an active ingredient.

[0026] In one embodiment of the present invention, the SlDRHB12 Proteins may consist of the amino acid sequence of SEQ ID NO. 1, but are not limited thereto.

[0027] In one embodiment of the present invention, the composition is SlRGLG2 ( Solanum lycopersicum E3 ubiquitin-protein ligase RGLG2) protein, encoding this SlRGLG2 It may further include one or more active ingredients selected from the group consisting of a gene and a recombinant vector containing the said gene, but is not limited thereto.

[0028] In one embodiment of the present invention, the composition may overexpress SlRGLG2, but is not limited thereto.

[0029] In addition, the present invention relates to SlDRHB12 composed of the amino acid sequence of SEQ ID NO. 1. A method for enhancing drought stress resistance of a plant is provided, comprising the step of inhibiting the expression or activity of a protein.

[0030] In one embodiment of the present invention, the method is SlDRHB12 The method may further include a step of overexpressing the protein, but is not limited thereto.

[0031] In addition, the present invention provides a transgenic plant with enhanced drought stress resistance by the method for enhancing drought stress resistance of the plant.

[0032] In one embodiment of the present invention, the transgenic plant of the present invention may be a tomato, but is not limited thereto. Effects of the invention

[0034] The present invention is SlDRHB12 ( Solanum lycopersicumThe invention relates to a Drought Responsive Homeobox Gene 12) protein, a gene encoding the same, and a method for enhancing drought stress resistance in plants using the same. It was completed by confirming that drought stress resistance decreases in plants with overexpression of the said gene and increases in plants with silenced expression of the said gene. Since SlDRHB12 can function as a negative regulator of drought stress resistance, it can be utilized for the improvement of crops available to humans through the regulation of the expression of the said protein, and is expected to be particularly useful for improving plant productivity. Brief explanation of the drawing

[0036] Figures 1a and 1b are SlDRHB12 This is a diagram showing the results of performing phylogenetic tree analysis and multiple sequence alignment of genes. Figures 2a to 2c are drawings showing the results of analyzing the molecular characteristics of SlDRHB12, respectively showing the expression pattern of SlDRHB12 under abiotic stress conditions (Figure 2a), the expression pattern within plant organs (Figure 2b), and the intracellular location (Figure 2c). Figures 3a to 3d show the results of confirming the interaction between SlDRHB12 and SlRGLG2 by Y2H analysis (Figure 3a), pull-down analysis (Figure 3b), and bimolecular fluorescence complementarity analysis (Figure 3c), respectively, and Figure 3d shows the results of confirming through ubiquitination analysis that the interaction is that SlRGLG2 ubiquitinated SlDRHB12. Figures 4a to 4g show the results of confirming the increased drought stress resistance of SlDRHB12-silenced tomato plants through qRT-PCR analysis (Figure 4a), phenotypic analysis (Figure 4b), viability analysis (Figure 4c), transpiration loss analysis (Figure 4d), leaf temperature measurement (Figure 4e), electrolyte leakage and lipid peroxidation measurement (Figure 4f), and drought stress response gene expression analysis of tomatoes following SlDRHB12 silencing (Figure 4g). Specific details for implementing the invention

[0037] In one embodiment of the present invention, it was confirmed through phylogenetic tree and multiple sequence analysis that the SlDRHB12 protein of tomato exhibits similarity to a homologous protein of a potato lineage, and as a result of performing multiple sequence analysis using the sequences of homologous proteins between different plant species, it was confirmed that the Homeodomain leucine zipper (HD-ZIP), Homeodomain (HOX), and Homeobox associated leucine zipper (HALZ) domains are conserved (Example 1).

[0038] In another embodiment of the present invention, by abiotic stress SlDRHB12 It was confirmed that the expression of the gene showed a significant change, and it was confirmed that the gene showed the highest expression level in the leaves of the tomato, and upon confirming its location within the cell, it was confirmed that it is located in the nucleus (Example 2).

[0039] In another embodiment of the present invention, it was confirmed that tomato-derived SlDRHB12 interacts with SlRGLG2, an E3 ligase of tomato, and that the interaction is the ubiquitination of SlDRHB12 through SlRGLG2 (Example 3).

[0040] In another embodiment of the present invention, qRT-PCR analysis, phenotypic analysis, viability analysis, transpiration loss analysis, leaf temperature measurement, electrolyte leakage measurement, lipid peroxidation measurement, and changes in tomato drought stress response gene expression following SlDRHB12 silencing were performed on tomato plants in which SlDRHB12 was artificially silenced, and it was confirmed that silencing of SlDRHB12 enhances resistance to drought stress (Example 4).

[0042] Accordingly, the present invention relates to SlDRHB12 composed of the amino acid sequence of SEQ ID NO. 1 ( Solanum lycopersicum Drought Responsive Homeobox Gene 12) protein, and encoding it SlDRHB12 Provides genes.

[0043] In one embodiment of the present invention, the above SlDRHB12 The gene may consist of the nucleotide sequence of SEQ ID NO. 2, but is not limited thereto. In addition, the above SlDRHB12 The peptide encoded by the gene may preferably consist of the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0044] In another embodiment of the present invention, the above SlDRHB12 The gene may be derived from tomatoes, but is not limited to them.

[0045] In the present invention, as a result of continuous global climate anomalies and extreme weather phenomena, plant growth and development are affected by various abiotic stresses, which ultimately impact survival, and among these abiotic stresses, drought stress related to water scarcity can significantly reduce crop yields.

[0046] In the present invention, a “negative regulator” of resistance (tolerance) to abiotic stress or environmental stress, etc., refers to a gene and / or protein that reduces the stress resistance of a plant as its expression or activity increases, and a “positive regulator” refers to a gene and / or protein that improves the stress resistance of a plant as its expression or activity increases. That is, the gene of the present invention SlDRHB12 Is Since it codes for a protein that functions as a negative regulator of drought stress tolerance, SlDRHB12 If gene expression is suppressed, resistance to drought stress increases, and conversely SlDRHB12 If the gene is overexpressed, resistance to drought stress may decrease.

[0047] In the present invention, gene expression mediated by transcription factors in plants plays an essential role in regulating drought stress responses and abscisic acid (ABA) signaling. That is, as a strategy to adapt to adverse environments, plants can regulate the expression of various abiotic stress-related genes, and in this regard, the plant hormone ABA can play several important roles in defense mechanisms induced in response to abiotic stresses such as drought.

[0048] While researching genes involved in the drought stress response of plants, the inventors, from tomatoes SlDRHB12 We identified the gene and sought to determine the association between the gene and the plant's tolerance to drought stress.

[0049] Accordingly, the present invention SlDRHB12 Provides a recombinant vector containing a gene.

[0050] In the present invention, virus-induced gene silencing (VIGS), a gene silencing technique induced by a virus, is also referred to as virus-induced RNA silencing. It is a type of post-transcriptional gene silencing phenomenon well known in various plants, fungi, insects, nematodes, fish, and mice. It can be described as a phenomenon in which the expression and replication of both the resistance gene and the viral genome are suppressed when a resistance gene homologous to the viral genome is expressed in the infected plant during the process of a virus invading and replicating a plant. In other words, if a part or the whole of a specific gene derived from a host is inserted into the cDNA of the viral genome and produced as infectious RNA to infect a plant, the corresponding host RNA becomes the target, and the expression of the target gene is suppressed or lost in the infected host plant. As a result, the function of the target gene can be indirectly estimated. Virus-induced gene silencing (VIGS) is known to be a type of RNA-mediated plant defense mechanism against viruses and has the characteristics of 1) post-transcriptional gene silencing, 2) RNA conversion, and 3) nucleotide sequence specificity.

[0051] In the present invention, SlDRHB12 A transgenic vector TRV (Tobacco Rattle Virus) for VIGS, into which a portion of the ORF (open reading frame) is inserted, is used against Agrobacterium ( Agrobacterium By inserting into ) and infiltrating the above Agrobacterium into the plant body SlDRHB12 It has successfully silenced gene expression.

[0052] In the present invention, the term "recombinant" refers to a cell that replicates a heterogeneous nucleic acid, expresses said nucleic acid, or expresses a protein encoded by a peptide, a heterogeneous peptide, or a heterogeneous nucleic acid. A recombinant cell may express a gene or gene fragment not found in the natural form of said cell in either a sense or antisense form. Additionally, a recombinant cell may express a gene found in a cell in its natural state, provided that said gene is modified and reintroduced into the cell by artificial means.

[0053] The term "vector" above is used to refer to DNA fragment(s) or nucleic acid molecules delivered into a cell. Vectors replicate DNA and can be reproduced independently in the host cell. A preferred example of a recombinant vector in the present invention is Agrobacterium tumefaciens ( Agrobacterium tumefaciens The plasmid vector, pTRV1 or pTRV2, which can transfer a portion of itself into plant cells when present in a suitable host such as a plant host, may be used, but is not limited thereto. Other suitable vectors that can be used to introduce the DNA according to the present invention into a plant host may be selected from viral vectors, such as those derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, Gemini viruses, etc., such as incomplete plant viral vectors. Such vectors may be advantageously used when it is difficult to properly transform the plant host. Furthermore, in the recombinant vector of the present invention, the promoter may be a CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. The term "promoter" refers to a DNA upstream region from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.

[0054] The term "recombinant vector" or "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. Generally, any plasmid or vector may be used if it can replicate and stabilize within a host. An important characteristic of the expression vector is that it possesses a replication origin, a promoter, a marker gene, and a translation control element.

[0055] An expression vector comprising the above gene sequence and a suitable transcription / translation regulatory signal can be constructed by the following method. The method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be effectively linked to a suitable promoter within the expression vector to lead mRNA synthesis. Additionally, the expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site.

[0056] Another preferred example of a recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a portion of itself, the so-called T-region, into a plant cell 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 by appropriately inserting the hybrid DNA into the plant genome. A particularly preferred form of a Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce DNA according to the present invention into a plant host may be selected from viral vectors, such as those derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, Gemini viruses, etc., such as incomplete plant viral vectors. The use of such vectors can be advantageous, especially when it is difficult to properly transform plant hosts.

[0057] The expression vector may include one or more selectable markers. The markers are nucleic acid sequences having characteristics that can typically be selected by chemical methods, and include all genes that can distinguish transformed cells from non-transformed cells. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, and antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol.

[0058] In the recombinant vector of the present invention, the promoter may be, but is not limited to, a CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter. 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, developmental states, or cell differentiation.

[0059] In the recombinant vector of the present invention, conventional terminators may be used, examples of which include nophalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, and Agrobacterium tumefaciens. (Agrobacterium tumefaciens) Examples include the terminator of the octopine gene, but are not limited to this. Regarding the necessity of terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells.

[0060] Transformation of a plant refers to any method of transferring DNA into a plant. Such transformation methods do not necessarily require a period of regeneration and / or tissue culture. 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 hybrid DNA according to the present invention into a suitable progenitor cell. The methods include the calcium / polyethylene glycol method on protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), electroporation of protoplasts (Shillito RD et al., 1985, Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), particle impaction of various plant elements (DNA or RNA-coated) (Klein TM et al., 1987, Nature 327, 70), and Agrobacterium tumefaciens by plant infiltration or transformation of mature pollen or microspores. In mediated gene transfer ( Incompleteness) It may be suitably selected from infection by a virus (EP 0 301 316), etc. A preferred method according to the present invention comprises Agrobacterium-mediated DNA delivery.

[0062] As another aspect of the present invention, the present invention is SlDRHB12 A composition for enhancing drought stress resistance of a plant is provided, comprising a protein expression or activity inhibitor as an active ingredient.

[0063] The above inhibitor is SlDRHB12It is preferable that it be any one of siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, PNA (peptide nucleic acids), and antisense nucleotide, which binds complementarily to mRNA for a gene sequence, a sequence complementary to said sequence, or a fragment of said gene sequence to inhibit expression, and SlDRHB12 It is preferable that the compound, peptide, peptide mimetics, antibody, or aptamer binds complementarily to a protein to inhibit its activity, but is not limited thereto.

[0064] In the composition of the present invention, the SlDRHB12 Proteins may consist of the amino acid sequence of SEQ ID NO. 1, but are not limited thereto.

[0065] In another aspect of the present invention, the composition is SlRGLG2 ( Solanum lycopersicum E3 ubiquitin-protein ligase RGLG2) protein, encoding this SlRGLG2 It may further include one or more active ingredients selected from the group consisting of a gene and a recombinant vector containing the said gene, but is not limited thereto.

[0066] In another aspect of the present invention, the composition may overexpress SlRGLG2, but is not limited thereto. That is, the SlRGLG2 (Solanum lycopersicum E3 ubiquitin-protein ligase RGLG2) protein of the composition, the SlRGLG2 gene encoding the same, and a recombinant vector containing said gene are additional active ingredients of a composition for enhancing plant drought stress resistance, wherein the additional active ingredients may be used for overexpressing SlRGLG2. Accordingly, in another aspect of the present invention, the composition comprises the SlRGLG2 (Solanum lycopersicum E3 ubiquitin-protein ligase RGLG2) protein, and said gene encoding the same SlRGLG2 One or more active ingredients selected from the group consisting of a gene and a preparation that overexpresses the said gene may be further included, but are not limited thereto.

[0067] In the present invention, the preparation that overexpresses SlRGLG2 is SlRGLG2 It may be a recombinant vector containing a gene.

[0068] In the present invention, overexpression refers to a level of expression higher than the level at which the corresponding gene is activated or expressed in a cell in a wild state, and is a concept that includes increasing the expression amount by replacing the promoter of a gene present on the genome with a potent promoter, or by cloning the corresponding gene into an expression vector and transforming and / or introducing it into a cell.

[0070] In another aspect of the present invention, the present invention relates to SlDRHB12 composed of the amino acid sequence of SEQ ID NO. 1 A method for enhancing drought stress resistance of a plant is provided, comprising the step of inhibiting the expression or activity of a protein. The above SlDRHB12 The step of inhibiting the expression or activity of a protein may be performed without limitation using the aforementioned siRNA, shRNA, miRNA, ribozyme, DNAzyme, PNA, antisense nucleotide, compound, peptide, peptide mimetics, antibody, or aptamer. That is, the inhibiting step may include, but is not limited to, the step of treating a plant with an inhibitor of the expression or activity of the SlDRHB12 protein.

[0071] In another aspect of the present invention, the method may further include the step of overexpressing the SlRGLG2 protein, but is not limited thereto.

[0072] In the present invention, the step of overexpressing the above-mentioned SlRGLG2 protein may mean not only transforming a plant that does not possess a gene encoding the SlRGLG2 protein into a plant that has such a gene, but also causing SlRGLG2 to be expressed at a level higher than the level of SlRGLG2 expressed in a plant (wild) having a gene encoding the SlRGLG2 protein, but is not limited thereto. That is, the above-mentioned step of overexpressing the SlRGLG2 (Solanum lycopersicum E3 ubiquitin-protein ligase RGLG2) protein, and encoding the same SlRGLG2 The method may include, but is not limited to, a step of treating a plant with one or more selected from the group consisting of a gene and a preparation that overexpresses the said gene.

[0074] In addition, the present invention provides a transgenic plant with enhanced drought stress resistance by the method of the present invention.

[0075] In the present invention, the transgenic plant of the present invention may be a tomato, but is not limited thereto.

[0076] In the present invention, the plant (body) may be food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; special crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanuts, and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, kiwifruit, grapes, citrus fruits, persimmons, plums, apricots, and bananas; flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, and tulips; and fodder crops including ryegrass, red clover, orchardgrass, alpha-alpha, tall fescue, and perennial ryegrass, and most preferably tomato, but is not limited thereto.

[0078] In addition, the present invention provides a kit for enhancing drought stress resistance of a plant, comprising the composition of the present invention.

[0079] In the present invention, the “kit” may include a container and instructions. The container may serve to package the formulation and may also serve to store and secure it. The material of the container may take the form, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., and may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may be equipped with a cap that is initially part of the container or can be attached to the container by mechanical, adhesive, or other means and may also be equipped with a stopper that allows access to the contents by a needle. The kit may include an outer package, and the outer package may include instructions regarding the use of the components.

[0080] In one embodiment of the present invention, the kit may be one or more selected from the group consisting of a microarray, an aptamer chip kit, an ELISA (Enzyme Linked Immunosorbent Assay) kit, a blotting kit, an immunoprecipitation kit, an immunofluorescence test kit, a protein chip kit, a reverse transcription polymerase chain reaction (RT-PCR) kit, and a qRT-PCR kit, but is not limited thereto.

[0081] The present invention provides a kit for enhancing drought stress resistance of a plant, comprising the composition and instructions of the present invention.

[0082] In the present invention, the above description may be characterized by describing a method for enhancing the drought stress resistance of a plant body according to the present invention, but is not limited thereto.

[0084] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0086] Plant materials and growth conditions

[0087] tomato ( Solanum lycopersicum ) was used for the experiment. Tomato seeds were soaked for 5 days under dark conditions at 28°C, and then cultured in MS (Murashige and Skoog) medium containing 1% Surcorse and 0.8% Microagar (Duchefa Biochemie). The culture medium was operated under a 16-hour light / 8-hour dark cycle using white fluorescent lights (80 μmol photons·m²). -2 ·s -1They were cultured in a growth chamber for 7 days under ). Tomato seedlings were sown in steam-sterilized compost mixture (peat moss, perlite, vermiculite, 5:3:2, v / v / v) and sand, loam soil (1:1:1, v / v / v). The seedlings were grown under white fluorescent lights (130 μmol photons·m²) under a 16-hour light / 8-hour dark cycle. -2 ·s -1 It was grown in a growth chamber at 24℃ and 60% relative humidity.

[0089] Virus-induced gene silencing

[0090] To elucidate the biological function of SlDRHB12, a tobacco rattle virus (TRV)-induced gene silencing (VIGS) system was used (Lim et al., 2021). Plant J , 107 (4), 1148-1165).

[0091] Using the SGN VIGS tool (http: / / solgenomics.net), 300-bp fragments corresponding to the target sequences of each gene were designed, and then these fragments were inserted into the pTRV2 vector. Carrying pTRV1 and pTRV2:00 as negative control vectors A . tumefaciens Alternatively, pTRV2: SlDRHB12 was co-inoculated into the cotyledons of tomato plants using a needleless syringe (OD 600 = 0.25).

[0093] RNA isolation and real-time transcription polymerase chain reaction (qRT-PCR)

[0094] RNA was isolated from tomatoes using the Ribospin Plant RNA isolation kit (GeneAll) according to the manufacturer's instructions. RNA samples were treated with RNase-free DNase to remove genomic DNA contamination, after which cDNA was synthesized using the Transcript First Strand cDNA Synthesis kit (Roche). The generated cDNA was used as a template for semi-quantitative and quantitative RT-PCR analysis, and tomato SlACT and SlEF1α were used as internal control genes. In qRT-PCR analysis, relative gene expression levels are determined by the ΔΔCt method (Livak & Schmittgen, 2001, Methods It was calculated using , 25, 402-408.).

[0095] To analyze gene expression under abiotic stress, tomato leaves were treated with dry or saline solution (NaCl, 250 mM).

[0097] Intracellular localization analysis

[0098] The full-length coding region of SlDRHB12 was cloned into the GFP fusion binary vector p326GFP, with the termination codon omitted. Agrobacterium tumefaciens The GV3101 strain was used to introduce a gene tagged with green fluorescent protein (GFP) into plants, and the plants A tumefaciens It was infiltrated into p19 strain cells at a 1:1 ratio, and the culture medium of each strain OD 600 This was 0.5. Two days after penetration, the GFP signal was visualized using a confocal microscope (510 UV / Vis Meta, Carl Zeiss, Jena, Germany) and LSM Image Browser software (Carl Zeiss).

[0100] Yeast protein hybrid assay (Yeast two-hybrid assay, Y2H)

[0101] Y2H (yeast two-hybrid) analysis was performed as previously reported (Lim and Lee, 2016). To investigate the interaction between SlDRHB12 and SlRGLG2 SlDRHB12 Replicate the sequence into the pGBKT7 vector, and SlRGLG2 The sequence was replicated into the pGADGH vector. Subsequently, the yeast strain was transformed using the lithium acetate-mediated transformation method (Ito et al., 1983). Saccharomyces cerevisiae Co-transformed with AH109. To evaluate the interaction between SlDRHB12 and SlRGLG2 proteins, AH109 cells carrying the vector construct were selected in selection medium (SC-adenine-histidine-leucine-tryptophan medium containing 2 mM 3-amino-1,2,4-triazole).

[0103] Purification and pull-down assay of recombinant protein

[0104] Escherichia coli To express MBP (maltose-binding protein) or GST (glutathione transferase)-labeled recombinant proteins, SlDRHB12 , and, SlRGLG2 The coding sequence of the gene was inserted into the pMAL-c2X (New England Biolabs) and pGEX-4T-3 (GE Healthcare) vectors. Each vector E. coli Each recombinant protein was introduced into strain BL21 cells. After inducing expression of each protein with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at 37°C for 3 hours, E. coli Cells were harvested and obtained. The collected lysate was purified according to the manufacturer's instructions (New England Biolabs, GE Healthcare Bio-Sciences).

[0105] For protein pull-down analysis, GST-labeled SlDRHB12 protein (GST-SlDRHB12) was reacted with MBP-tagged SlRGLG2 and Glutathione-Sepharose 4 Fast Flow beads at 4°C for 2 hours. The beads were washed 5 times with a washing solution [20 mM Tris-HCl, 150 mM NaCl, 5 mM Ethylenediaminetetraacetic acid (EDTA), and 0.5% Triton X-100] and then boiled. The boiled samples were used for Western blot analysis with anti-GST and anti-MBP antibodies to confirm the presence of the appropriate antibodies.

[0107] bimolecular fluorescence complementation assay

[0108] The full-length coding sequences of SlDRHB12 and SlRGLG2 were cloned into the 35S-VYNE vector and the 35S-CYCE vector, respectively, for the generation of bimolecular fluorescence complementation (BiFC) constructs (Waadt et al., 2008). For transient expression, Agrobacterium possessing the said constructs ( Agrobacterium tumefaciens After mixing strain GV3101 with the p19 strain to avoid gene silencing, tobacco ( N. benthamiana ) Plants (OD 600 = 0.5) was infiltrated into the hypocotyl surface of the leaves. On the third day of infiltration, 1 μg / ml of 4',6-diamidino-2-phenylindole (DAPI) was infiltrated as a nuclear marker 2 hours before signal detection, after which the leaf discs were cut and the lower epidermal cells were examined under a confocal microscope (Carl Zeiss, Germany) equipped with LSM Image Browser software.

[0110] Ubiquitination assay

[0111] To investigate the ubiquitination of SlDRHB12 by SlRGLG2, purified MBP-tagged SlRGLG2 was mixed with reaction buffer [5 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 2 mM ATP, and 2 mM dithiothreitol (DTT)]. Subsequently, recombinant human UBE1 (Boston Biochemicals), His-tagged human UbcH5b (Enzo Life Sciences), and bovine-derived ubiquitin (Sigma-Aldrich) were added to the reaction mixture. After adding GST-SlDRHB12 (500 ng) as a substrate to the mixture, the mixture was incubated at 30°C for 12 hours. The results were then analyzed by Western blot using anti-GST (Santa Cruz Biotechnology), anti-MBP (New England Biolabs), and anti-ubiquitin (Santa Cruz Biotechnology) antibodies.

[0113] Plant Phenotype Analysis

[0114] After treating tomato plant leaves with dry, ABA (100 μM), and saline solution (NaCl, 250 mM), RNA was extracted and qRT-PCR was performed to evaluate the response to abiotic stress.

[0115] To evaluate drought stress tolerance, 3-week-old gene-silenced tomato plants were withheld from water for 20 days and then re-watered for 5 days. Survival rate was determined based on the number of rehydrated leaves after the watering period.

[0116] To record leaf temperature, a FLIR T420 infrared camera was used to monitor the temperature, and analysis was performed using FLIR Tools+ ver5.13 software.

[0117] Tranquil loss was measured by obtaining 20 tomato leaf samples from each strain. The samples were placed in Petri dishes, and the loss of fresh weight was recorded at one-hour intervals.

[0119] Phylogenetic tree analysis and multiple sequence alignment

[0120] Phylogenetic tree analysis of the SlDRHB12 gene and amino acid sequence alignment of homologous proteins were performed. The amino acid sequences encoding the said gene and their homologous sequences were obtained using BLAST searches (http: / / www.ncbi.nlm.nih.gov / BLAST), and Arabidopsis thaliana, Solanum tuberosum, Capsicum annuum, Nicotiana tabacum, Populus alba, Vitis vinifera, and Glycine max Phylogenetic tree analysis was performed using the sequences of plant species.

[0121] The phylogenetic tree of the above homologous proteins was constructed using Molecular Evolutionary Genetics Analysis (MEGA) software (version 10.1.7), and multiple sequence alignment was performed using Clustal Omega ( http: / / www.ebi.ac.uk / Tools / msa / clustalo / It was written using ) and ClustalW2 software.

[0123] The primer sequences used for the above VIGS, cloning, and qRT-PCR are listed in Table 1 below.

[0124]

[0126] Example 1. SlDRHB12 Phylogenetic Tree and Sequence Analysis

[0127] As a result of performing phylogenetic tree analysis and multiple sequence alignment using the tomato-derived SlDRHB12 amino acid sequence, as shown in Figure 1a, phylogenetic tree analysis revealed that the tomato SlDRHB1 amino acid is potato ( Solanum tuberosumIt was confirmed that it exhibited similarity to homologous proteins of the lineage. In addition, as shown in Figure 1b, when comparing the sequences of homologous proteins derived from each plant species after performing multiple sequence alignment, it was confirmed that the Homeodomain leucine zipper (HD-ZIP), Homeodomain (HOX), and Homeobox associated leucine zipper (HALZ) domains were conserved.

[0129] Example 2. Molecular properties of SlDRHB12

[0130] SlDRHB12 To evaluate the expression of the gene under abiotic stress conditions, qRT-PCR analysis was performed using dried and NaCl (250 mM) treated tomato plant leaves (Fig. 2a). As an internal control SlRD29 Genes were used, and after drying treatment, SlDRHB12 The expression of decreased sharply at 2 hours and increased at 6 hours, and in the NaCl-treated group after 6 hours SlDRHB12 It was confirmed that the expression of significantly increased.

[0131] Next, SlDRHB12 Analysis of how it is expressed in plant organs confirmed that it shows the highest expression level in tomato leaves (Fig. 2b).

[0132] SlDRHB12 To confirm the intracellular location, a fluorescent protein was tagged and expressed; as shown in Fig. 2c, it was confirmed that the fluorescent signal exhibited the same pattern as the nuclear location signal, and through the above results SlDRHB12 It was possible to see that it was located in the nucleus.

[0134] Example 3. SlDRHB12 and SlRGLG2 Interaction between

[0135] E3 ligases are known to function by interacting with substrate proteins and ubiquitinating them (Hershko and Ciechanover, 1998; Smalle and Vierstra, 2004). Additionally, it is known that some E3 ligases can interact with phosphokinases and ubiquitinate them in response to abiotic stress (Stone et al., 2014).

[0136] Accordingly, the tomato E3 ligase SlRGLG2 ( Solanum lycopersicum To identify factors interacting with E3 ubiquitin-protein ligase RGLG2, Y2H analysis, pull-down analysis, and bimolecular fluorescence complementarity analysis were performed.

[0137] As shown in FIGS. 3a and 3b, through Y2H analysis (Fig. 3a) and pull-down analysis (Fig. 3b) SlDRHB12 and SlRGLG2 It was possible to confirm that this is physically interacting, and through bimolecular fluorescence complementarity analysis (Fig. 3c), it was confirmed that the interaction occurs in the cell nucleus.

[0138] In addition, confirmed through the above analysis SlDRHB12 and SlRGLG2 Based on the interaction between, SlRGLG2 this SlDRHB12 A ubiquitination assay was performed to confirm whether it ubiquitinated (Fig. 3d). As a result of co-incubating MBP-SlRGLG2 and GST-SlDRHB12, smear bands were detected, and these results SlRGLG2 this SlDRHB12 Indicates that it can ubiquitinate.

[0139] Based on the above results, E3 ligase SlRGLG2 this SlDRHB12 We confirmed that it interacts with, and furthermore SlRGLG2 this SlDRHB12 It was found that it ubiquitinated.

[0141] Example 4. SlDRHB12 Increased drought stress resistance in silent tomato plants

[0142] In tomato plants under drought stress SlDRHB12 To study the biological functions of the tobacco stain virus (TRV), a VIGS-based technique was used.

[0143] qRT-PCR analysis results showed that compared to the control group in TRV2:SlDRHB12 plants SlDRHB12 It was confirmed that transcript levels were low (Fig. 4a). In addition, a drought stress resistance experiment was conducted on TRV2:00 and TRV2:SlDRHB12 tomato plants by withholding water for 20 days and then re-watering them for 5 days. As a result, there was no significant difference between TRV2:00 and TRV2:SlDRHB12 tomato plants under normal conditions (Fig. 4b, top). However, after inducing drought stress and rehydration, it was confirmed that TRV2:SlDRHB12 plants exhibited a drought stress resistance phenotype compared to the control group, and as shown in Fig. 4c, the survival rate was also found to be higher than that of the control group.

[0144] In addition, analysis of transpiration loss in the above plants revealed that the TRV2:SlDRHB12 plants exhibited lower transpiration loss compared to the control group (Fig. 4d), and when leaf surface temperature was assessed in the presence or absence of ABA, the leaf temperature of the TRV2:SlDRHB12 plants was found to be higher than that of the control group (Fig. 4e). Based on these results SlDRHB12 It was observed that increased resistance to drought stress due to gene silencing reduced transpiration loss, which in turn increased leaf surface temperature, and through this SlDRHB12 It was found that genes are involved in ABA sensitivity.

[0145] The effects of drought stress on electrolyte leakage and lipid peroxidation in the leaves of TRV2:00 and TRV2:SlDRHB12 tomato plants were analyzed. Specifically, for the electrolyte leakage experiment, shoots from each line were removed to induce dehydration, and equal amounts of leaves were collected after 0 or 12 hours, placed in tubes containing triple-distilled water, and the amount of leaked electrolyte was measured using an ion meter. For the lipid peroxidation experiment, shoots from each line were removed to induce dehydration, and equal amounts of leaves were collected from each line at different time intervals (0, 12, or 24 hours), finely ground using liquid nitrogen, quantified, and placed in tubes. To measure lipid peroxidation, the samples were dissolved in an acidic solution, and the amount of malondialdehyde (MDA) reacting with thiobarbituric acid (TBA) under high-temperature conditions was measured.

[0146] As a result, it was found that in the case where SlDRHB12 was silenced, electrolyte leakage remained at the same level even after 12 hours had elapsed after drying. In addition, lipid peroxidation increased in all plants over time after drying, but in the case of SlDRHB12-silenced tomatoes, the level was found to be lower compared to the control group (Fig. 4f).

[0147] SlDRHB12 As a result of analyzing the effect of gene silencing on the expression of drought stress response genes in tomatoes using qRT-PCR (Fig. 4g), the drought stress response gene in tomatoes is SlRD29, SlAREB and SlDREB2 It was confirmed that gene expression was higher in TRV2:SlDRHB12 plants than in normal control groups.

[0148] Synthesizing the above results, tomato-derived SlDRHB12 The gene is a factor located in the cell nucleus whose expression increases under drought stress, and is degraded by ubiquitination caused by SlRGLG2, a tomato-derived E3 ligase, and SlDRHB12As it was confirmed that drought stress resistance increased in gene-silencing tomato plants, it was found that the tomato-derived SlDRHB12 gene acts as a factor that negatively regulates drought stress, controlling drought stress resistance in tomato plants by regulating ABA sensitivity and stress-responsive gene expression, including transpiration loss.

[0150] In conclusion, the present invention functions as a negative regulator of drought stress in tomato plants. SlDRHB12 They identified and characterized it, thereby confirming a new pathway for ABA signaling and response to drought in tomato plants.

[0152] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 SlDRHB12 consisting of the amino acid sequence of SEQ ID NO. 1 ( Solanum lycopersicum Drought Responsive Homeobox Gene 12) Protein. Claim 2 SlDRHB12 described in paragraph 1 Encoding proteins SlDRHB12 gene. Claim 3 In paragraph 2, the above SlDRHB12 The gene is characterized by being composed of the nucleotide sequence of SEQ ID NO. 2, SlDRHB12 gene. Claim 4 In paragraph 2, the above SlDRHB12 The gene is characterized by being derived from a tomato, SlDRHB12 gene. Claim 5 Paragraph 2 SlDRHB12 A recombinant vector containing a gene. Claim 6 SlDRHB12 ( Solanum lycopersicum A composition for enhancing drought stress resistance of a plant, comprising as an active ingredient an inhibitor of the expression or activity of a Drought Responsive Homeobox Gene 12). Claim 7 In paragraph 6, the above SlDRHB12 A composition for enhancing drought stress resistance of a plant, characterized in that the protein consists of the amino acid sequence of SEQ ID NO.

1. Claim 8 In paragraph 6, the above composition is SlRGLG2 ( Solanum lycopersicum E3 ubiquitin-protein ligase RGLG2) protein, encoding this SlRGLG2 A composition for enhancing drought stress resistance of a plant, comprising one or more active ingredients selected from the group consisting of a gene and a recombinant vector containing said gene. Claim 9 A composition for enhancing drought stress resistance of a plant, characterized in that, in claim 8, the composition overexpresses SlRGLG2. Claim 10 SlDRHB12 consisting of the amino acid sequence of SEQ ID NO. 1 ( Solanum lycopersicum A method for enhancing drought stress resistance of a plant, comprising the step of inhibiting the expression or activity of a Drought Responsive Homeobox Gene 12). Claim 11 A method for enhancing drought stress resistance of a plant, characterized in that, in claim 10, the above method further comprises the step of overexpressing the SlRGLG2 protein. Claim 12 Transgenic plant with enhanced drought stress resistance by the method of paragraph 10. Claim 13 In Clause 12, the plant is characterized in that the transgenic plant is a tomato.

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