SlRGLG2 gene and Method for improving the resistance to the drought and salt stresses using SlRGLG2 in plants

Abstract

The present invention relates to the SlRGLG2 (Solanum lycopersicum E3 ubiquitin-protein ligase RGLG2) protein, a gene encoding the same, and a method for enhancing drought stress resistance in plants using the same. It has been confirmed that plants in which the said gene is silenced show reduced drought stress resistance and increased salt stress resistance. Since SlRGLG2 can function as a positive regulator of drought stress resistance and as a negative regulator of salt 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 SlRGLG2 ( Solanum lycopersicum The invention relates to the E3 ubiquitin-protein ligase RGLG2) gene, protein, and a method for enhancing drought and salt stress resistance in plants using the same. Background Technology

[0003] Because higher plants are immobile, they face various environmental stresses throughout their lives, such as drought, high salt, heavy metals, cold damage, heat shock, and ozone. These abiotic stresses act as limiting factors for crop growth and development, and the loss in crop yield caused by abiotic environmental stress is significant, reportedly amounting to more than 50% of potential production. Furthermore, with rapid environmental changes recently being recorded globally, the extent of resulting crop damage is becoming increasingly concerning.

[0004] In particular, global warming causes extreme climate change, inducing environmental stress that hinders the normal growth and development of plants; specifically, water scarcity and high salinity are identified as major stressors that reduce crop yields because they cause the greatest stress to plants.

[0005] Drought stress caused by water scarcity is considered the most serious environmental factor, acting as a major cause of reduced crop production. Globally, water consumption continues to increase, and the availability of clean water is a critical issue not only for humans but also for higher plants. Among environmental stresses, salinity stress has the most direct impact on crop productivity across the widest range; when plants are exposed to high salinity stress, intracellular Na + Osmotic pressure imbalance and intracellular ion concentration imbalance occur due to the accumulation of ions, leading to stunted development and impaired photosynthesis.

[0006] In response to this, plants have evolved sophisticated defense mechanisms to survive in adverse environments such as drought and salinity. For example, plants can employ various defense strategies, such as regulating signaling pathways or inducing stress-responsive genes, to increase tolerance to short-term or long-term water scarcity.

[0007] However, natural evolution alone has limitations in enhancing tolerance to drought and salt stress, and knowledge regarding the biological functions of stress-related genes involved in stress tolerance or sensitivity in higher plants remains insufficient.

[0008] Accordingly, methods to enhance plant resistance to drought and salt stress are becoming a major subject of research. In this regard, studies are being conducted on genes related to drought and salt stress tolerance and growth promotion, as well as the resulting transgenic plants, but the current state of such research is still insufficient. Prior art literature

[0010] Korean Registered Patent 10-2555526 The problem to be solved

[0011] One object of the present invention is SlRGLG2 composed of the amino acid sequence of SEQ ID NO. 1 ( Solanum lycopersicum It provides the E3 ubiquitin-protein ligase RGLG2 protein.

[0012] Another objective of the present invention is to encode the SlRGLG2 protein SlRGLG2 It is to provide genes.

[0013] Another objective of the present invention is the above SlRGLG2 It is to provide a recombinant vector containing a gene.

[0014] Another objective of the present invention is to provide a plant transformed with the recombinant vector.

[0015] Another objective of the present invention is the above SlRGLG2 protein, encoding the same SlRGLG2 Genes, and the above SlRGLG2 The present invention provides a composition for enhancing drought stress resistance of a plant, comprising one or more active ingredients selected from a group consisting of recombinant vectors containing genes.

[0016] Another objective of the present invention is to provide a kit for enhancing drought stress resistance of a plant, comprising the above composition.

[0017] Another objective of the present invention is to provide a method for enhancing drought stress resistance of a plant, comprising the following steps:

[0018] (S1) A step of transforming a plant into a gene encoding the SlRGLG2 protein of the present invention; and

[0019] (S2) A step of overexpressing the SlRGLG2 protein in the transformed plant body.

[0020] Another objective of the present invention is to provide a transgenic plant with enhanced drought stress resistance by the above method.

[0021] Another objective of the present invention is... SlRGLG2 The present invention provides a composition for enhancing salt stress resistance in plants, comprising a protein expression or activity inhibitor as an active ingredient.

[0022] Another objective of the present invention is to provide a method for enhancing salt stress resistance of a plant, comprising the step of inhibiting the expression or activity of a SlRGLG2 protein composed of the amino acid sequence of SEQ ID NO. 1.

[0024] 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

[0026] To achieve the above-mentioned objective of the present invention, the present invention comprises SlRGLG2 composed of the amino acid sequence of SEQ ID NO. 1 ( Solanum lycopersicum It provides the E3 ubiquitin-protein ligase RGLG2 protein.

[0027] In addition, the present invention relates to the above-mentioned SlRGLG2 protein encoding SlRGLG2 Provides genes.

[0028] In one embodiment of the present invention, the above SlRGLG2 The gene may consist of the nucleotide sequence of SEQ ID NO. 2, but is not limited thereto.

[0029] In another embodiment of the present invention, the above SlRGLG2 The gene may be derived from tomatoes, but is not limited to them.

[0030] In addition, the present invention is the present invention SlRGLG2 Provides a recombinant vector containing a gene.

[0031] In one embodiment of the present invention, the recombinant vector of the present invention may be used to enhance drought stress resistance of a plant, but is not limited thereto.

[0032] In addition, the present invention provides a plant transformed with the recombinant vector of the present invention.

[0033] In addition, the present invention relates to the SlRGLG2 protein of the present invention, encoding the same SlRGLG2 Genes, and the above SlRGLG2 A composition for enhancing drought stress resistance of a plant is provided, comprising one or more active ingredients selected from a group of recombinant vectors containing genes.

[0034] In addition, the present invention provides a kit for enhancing drought stress resistance of a plant, comprising the composition of the present invention.

[0035] In addition, the present invention provides a method for enhancing drought stress resistance of a plant, comprising the following steps:

[0036] (S1) A step of transforming a plant into a gene encoding the SlRGLG2 protein of the present invention; and

[0037] (S2) A step of overexpressing the SlRGLG2 protein in the transformed plant body.

[0038] In addition, the present invention provides a transgenic plant with enhanced drought stress resistance by the method of the present invention.

[0039] In one embodiment of the present invention, the transgenic plant of the present invention may be a tomato, but is not limited thereto.

[0040] In addition, the present invention provides a composition for enhancing salt stress resistance of a plant, comprising an inhibitor of the expression or activity of the SlRGLG2 protein as an active ingredient.

[0041] In one embodiment of the present invention, the SlRGLG2 protein may consist of the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0042] In addition, the present invention provides a method for enhancing salt stress resistance of a plant, comprising the step of inhibiting the expression or activity of a SlRGLG2 protein composed of the amino acid sequence of SEQ ID NO. 1. Effects of the invention

[0044] The present invention is SlRGLG2 ( Solanum lycopersicumThe invention relates to an E3 ubiquitin-protein ligase RGLG2 protein, a gene encoding the same, and a method for enhancing drought stress resistance in plants using the same. It was confirmed that plants in which the said gene is silenced have reduced drought stress resistance and increased salt stress resistance. Since SlRGLG2 can function as a positive regulator of drought stress resistance and as a negative regulator of salt 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

[0046] Figures 1a and 1b show the results of phylogenetic tree analysis and multiple sequence alignment of the SlRGLG gene group. Figures 2a to 2c are drawings showing the results of analyzing the molecular characteristics of SlRGLG2, respectively showing the expression pattern of SlRGLG2 under abiotic stress conditions (Figure 2a), the expression pattern within plant organs (Figure 2b), and the intracellular location (Figure 2c). Figure 2d is a diagram showing the results of confirming the ubiquitination activity of SlRGLG2 through ubiquitination analysis. Figures 3a to 3f show the results of confirming the reduction in drought stress resistance of SlRGLG2-silenced tomato plants through qRT-PCR analysis (Figure 3a), phenotypic analysis (Figure 3b), transpiration loss analysis (Figure 3c), leaf temperature measurement (Figure 3d), electrolyte leakage and lipid peroxidation measurement (Figure 3e), and drought stress response gene expression analysis of tomatoes following SlRGLG2 silencing (Figure 3f). Figures 4a to 4g show the results of confirming the increased salt stress resistance of tomato plants silenced by SlRGLG2 through phenotypic analysis (Figure 4a), chlorophyll content analysis (Figure 4b), electrolyte leakage (Figure 4c), lipid peroxidation measurement (Figure 4d), and proline content measurement (Figure 4e). Specific details for implementing the invention

[0047] In one embodiment of the present invention, through phylogenetic tree and multiple sequence analysis, the SlRGLG2 of tomato ( Solanum lycopersicum It was confirmed that the E3 ubiquitin-protein ligase RGLG2) protein exhibits similarity to a homologous protein of a pepper line, and multiple sequence analysis was performed using the amino acid sequence of the tomato-derived SlRGLG gene group, confirming that the von Willebrand factor type A (VWA) domain and the RING domain are conserved (Example 1).

[0048] In another embodiment of the present invention, by abiotic stress SlRGLG2 It was confirmed that the expression of the gene showed a significant change, and that the gene exhibited the highest expression level in the flesh of the tomato. Upon examining its intracellular location, it was confirmed that it is located in the cytoplasm and possesses ubiquitination activity (Example 2).

[0049] In another embodiment of the present invention, qRT-PCR analysis, phenotypic analysis, transpiration loss analysis, leaf temperature measurement, electrolyte leakage measurement, lipid peroxidation measurement, and changes in tomato drought stress response gene expression following SlRGLG2 silencing were performed on tomato plants in which SlRGLG2 was artificially silenced, and it was confirmed that silencing of SlRGLG2 reduces resistance to drought stress (Example 3).

[0050] In another embodiment of the present invention, it was confirmed that the silencing of SlRGLG2 enhances resistance to salt stress through phenotypic analysis, chlorophyll content measurement, electrolyte leakage measurement, lipid peroxidation measurement, and proline content measurement of tomato plants in which SlRGLG2 was artificially silenced (Example 4).

[0052] Accordingly, the present invention relates to a SlRGLG2 protein comprising the amino acid sequence of SEQ ID NO. 1, and a component encoding the same SlRGLG2 Provides genes.

[0053] In one embodiment of the present invention, the above SlRGLG2 The gene may consist of the nucleotide sequence of SEQ ID NO. 2, but is not limited thereto. In addition, the above SlRGLG2 The peptide encoded by the gene may preferably consist of the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0054] In another embodiment of the present invention, the above SlRGLG2 The gene may be derived from tomatoes, but is not limited to them.

[0055] 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.

[0056] 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 SlRGLG2Since it codes for a protein that functions as a positive regulator of drought stress tolerance and a negative regulator of salt stress, SlRGLG2 When gene expression is suppressed, it induces a decrease in drought stress tolerance and an increase in salt stress tolerance, and conversely SlRGLG2 Overexpression of the gene can lead to increased drought stress tolerance and decreased salt stress tolerance.

[0057] In this invention, the term “drought stress resistance” refers to the property of a plant to withstand dry conditions, signifying the plant’s resistance to drought. When prolonged drought occurs, varieties with strong stress resistance are required, and a crop’s drought stress resistance means that the reduction in plant growth or productivity is suppressed or delayed under drought stress conditions.

[0058] In this invention, the term “salt stress resistance” or “salt stress resistance” refers to the property of a plant to withstand conditions of high salt concentration, signifying the plant’s resistance to high salt concentrations. In reclaimed land, varieties with strong salt stress resistance are required. A crop’s salt stress resistance is the maximum salt concentration it can withstand without losing productivity, and higher salt concentrations have a negative impact on crop productivity. The aforementioned salt concentration generally refers to soil salinity or the salinity of irrigation water.

[0059] In the present invention, the term “resistance” may broadly include the meaning of “tolerance.”

[0060] 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.

[0061] While researching genes involved in the stress response of plants, the inventors, from tomatoes SlRGLG2 We identified the gene and sought to determine the association between the gene and the plant's tolerance to drought and salt stress.

[0062] Accordingly, the present invention SlRGLG2 Provides a recombinant vector containing a gene.

[0063] 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.

[0064] In the present invention, SlRGLG2 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 SlRGLG2 It has successfully silenced gene expression.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] In the present invention, the recombinant vector of the present invention may preferably be a recombinant plant expression vector.

[0075] In the present invention, the recombinant vector of the present invention may be for enhancing drought stress resistance of a plant, but is not limited thereto.

[0076] In addition, the present invention provides a plant transformed with the recombinant vector of the present invention.

[0077] In addition, the present invention provides a method for enhancing drought stress resistance of a plant, comprising the following steps:

[0078] (S1) A step of transforming a plant into a gene encoding the SlRGLG2 protein of the present invention; and

[0079] (S2) A step of overexpressing the SlRGLG2 protein in the transformed plant body.

[0080] In the present invention, the step of overexpressing the SlRGLG2 protein of the present invention may mean not only transforming a plant that does not have a gene encoding the SlRGLG2 protein into a plant that has a gene encoding the SlRGLG2 protein, but also expressing SlRGLG2 at a higher level than the level of SlRGLG2 expressed in a plant (wild) that has a gene encoding the SlRGLG2 protein, but is not limited thereto.

[0081] In addition, the present invention provides a transgenic plant with enhanced drought stress resistance by the method of the present invention.

[0082] In the present invention, the transgenic plant of the present invention may be a tomato, but is not limited thereto.

[0083] In another aspect of the present invention, the present invention provides a transgenic plant in which drought stress resistance is enhanced by a method for enhancing drought stress resistance of the plant.

[0084] 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.

[0086] In addition, the present invention relates to the SlRGLG2 protein of the present invention, encoding the same SlRGLG2 Genes, and the above SlRGLG2 A composition for enhancing drought stress resistance of a plant is provided, comprising one or more active ingredients selected from a group of recombinant vectors containing genes.

[0087] In the composition according to the present invention, the SlRGLG2 protein may be composed of the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0088] 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.

[0089] In addition, the present invention provides a kit for enhancing drought stress resistance of a plant, comprising the composition and instructions of the present invention.

[0090] 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.

[0091] 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.

[0093] In another aspect of the present invention, the present invention provides a composition for enhancing salt stress resistance of a plant, comprising an expression or activity inhibitor of the SlRGLG2 protein as an active ingredient.

[0094] The above inhibitor is SlRGLG2It 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 SlRGLG2 It is preferably one of a compound that binds complementarily to a protein to inhibit its activity, a peptide, peptide mimetics, an antibody, or an aptamer, but is not limited thereto.

[0095] In the composition of the present invention, the SlRGLG2 protein may consist of the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0096] In another aspect of the present invention, the present invention provides a method for enhancing salt stress resistance of a plant, comprising the step of inhibiting the expression or activity of a SlRGLG2 protein composed of the amino acid sequence of SEQ ID NO. 1. The step of inhibiting the expression or activity of the SlRGLG2 protein may be performed without limitation using the aforementioned siRNA, shRNA, miRNA, ribozyme, DNAzyme, PNA, antisense nucleotide, compound, peptide, peptide mimetics, antibody, or aptamer.

[0098] 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.

[0100] Plant materials and growth conditions

[0101] 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 -1 They 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°C and 60% relative humidity.

[0103] Virus-induced gene silencing

[0104] To elucidate the biological function of SlRGLG2, a tobacco rattle virus (TRV)-induced gene silencing (VIGS) system was used (Lim et al., 2021). Plant J , 107 (4), 1148-1165).

[0105] Using the SGN VIGS tool (http: / / solgenomics.net) SlRGLG2 A 300-bp fragment corresponding to the target sequence of the gene was designed, and then this fragment was inserted into the pTRV2 vector. Carrying pTRV1 and pTRV2:00 as negative control vectors A . tumefaciensAlternatively, pTRV2:SlRGLG2 was co-inoculated into the cotyledons of tomato plants using a needleless syringe (OD 600 = 0.25).

[0107] RNA isolation and real-time transcription polymerase chain reaction (qRT-PCR)

[0108] 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.).

[0110] Intracellular localization analysis

[0111] The full-length coding region of SlRGLG2 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).

[0113] Ubiquitination assay

[0114] To confirm the E3 ubiquitin ligase activity of SlRGLG2, in vitro Autoubiquitination analysis was performed. Specifically, purified MBP-tagged SlRGLG2 and SlRGLG2 C394 / 397S It 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, and it was incubated at 30°C for 12 hours. Afterward, it was analyzed by Western blot using anti-MBP (New England Biolabs) and anti-ubiquitin (Santa Cruz Biotechnology) antibodies.

[0116] Plant Phenotype Analysis

[0117] After treating tomato plant leaves with dry, saline solution (NaCl, 250 mM), or ABA (100 μM), RNA was extracted and qRT-PCR was performed to evaluate the response to abiotic stress.

[0118] 3-week-old control group (TRV2:00) and SlRGLG2 -After planting silent tomato plants (TRV2:SlRGLG2), drought stress was applied by suspending watering for 20 days. Subsequently, the plants were watered again for 5 days to allow the dehydrated plants to recover, after which the number of plants that had grown again was counted to calculate the survival rate. To quantitatively evaluate drought tolerance, transpirational water loss was measured. For this purpose, leaves isolated and recovered from tomato plants at the 6-leaf stage were placed in petri dishes. The petri dishes were placed in a growth chamber at 40% relative humidity, and fresh weight loss was measured at set intervals; the experiment was repeated three times. 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.

[0119] 3-week-old control group (TRV2:00) and SlRGLG2 - After planting silent tomato plants (TRV2:SlRGLG2), salt stress was induced under 400 mM NaCl treatment conditions for 7 days, and the survival rate was calculated by counting the number of plants that grew again. To quantitatively evaluate salt stress tolerance, the amount of chlorophyll was measured.

[0121] Phylogenetic tree analysis and multiple sequence alignment

[0122] SlRGLG Phylogenetic tree analysis of the gene group and amino acid sequence alignment of homologous proteins were performed. The amino acid sequences encoding the above genes and their homologous sequences were obtained using BLAST searches (http: / / www.ncbi.nlm.nih.gov / BLAST), and Arabidopsis thaliana and Capsicum annuum Phylogenetic tree analysis was performed using the sequences of plant species.

[0123] 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.

[0125] The primer sequences used for the above VIGS, cloning, and qRT-PCR are listed in Table 1 below.

[0126]

[0128] Example 1. SlRGLG phylogenetic tree and sequence analysis

[0129] As a result of performing phylogenetic tree analysis and multiple sequence alignment using the amino acid sequences of the tomato-derived SlRGLG gene group, as shown in Figure 1a, through phylogenetic tree analysis, tomato SlRGLG2 Amino acids are chili peppers ( Capsicum annuum It was confirmed that it exhibited similarity to the homologous protein (CA03g26200) of the lineage. In addition, as shown in Figure 1b, multiple sequence alignment was performed using the amino acid sequence of the tomato-derived SlRGLG gene group, and it was confirmed that the von Willebrand factor type A (VWA) domain and the RING domain were conserved.

[0131] Example 2. Molecular properties of SlRGLG2

[0132] SlRGLG2 To evaluate the expression of the gene under abiotic stress conditions, qRT-PCR analysis was performed using tomato plant leaves treated with dryness, NaCl (250 mM), or ABA (100 μM) (Fig. 2a). As an internal control SlACT and SlEF1α Genes were used, and as time passed under abiotic stress conditions SlRGLG2It was confirmed that gene expression increased, and in particular, under dry and ABA conditions, the highest expression was observed 8 hours after treatment.

[0133] Next, SlRGLG2 Analysis of how it is expressed in plant organs confirmed that it shows the highest expression level in mature tomato flesh (red fruit) (Fig. 2b).

[0134] To confirm the intracellular location of SlRGLG2, a fluorescent protein was tagged and expressed, and as shown in Figure 2c, it was confirmed that a fluorescent signal appeared in the cytoplasm.

[0135] As it was confirmed in Example 1 that SlRGLG2 possesses a RING domain, a ubiquitination assay was performed to determine whether it has ubiquitination activity (Fig. 2d). Western blot analysis was performed using anti-MBP and anti-ubiquitin antibodies, and the results showed that the mutant protein (MBP-SlRGLG2 C394 / 397S While smear bands were not detected in the normal protein, smear bands were detected in the normal protein, and these results indicate that SlRGLG2 has ubiquitination activity.

[0137] Example 3. SlRGLG2 Reduced drought stress resistance in silenced tomato plants

[0138] To study the biological function of SlRGLG2 in tomato plants under drought stress, a tobacco stain virus (TRV)-based VIGS technique was used.

[0139] qRT-PCR analysis results showed that compared to the control group in TRV2: SlRGLG2 plants SlRGLG2It was confirmed that transcript levels were low (Fig. 3a). In addition, a drought stress resistance experiment was conducted on TRV2:00 and TRV2:SlRGLG2 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:SlRGLG2 tomato plants under normal conditions (Fig. 3b, top). However, after inducing drought stress and rehydration, it was confirmed that TRV2:SlRGLG2 plants exhibited a more wilting phenotype and lower survival rate compared to the control group.

[0140] In addition, analysis of transpiration loss in the above plants revealed that the TRV2:SlRGLG2 plants exhibited higher transpiration loss compared to the control group (Fig. 3c), and when leaf surface temperature was checked in the presence or absence of ABA, it was confirmed that the leaf temperature of the TRV2:SlRGLG2 plants was lower than that of the control group (Fig. 3d). Through the above results SlRGLG2 It was observed that the decrease in drought stress resistance caused by gene silencing led to increased transpiration loss, which in turn resulted in a decrease in leaf surface temperature, and through this SlRGLG2 It was found that the gene reduces ABA sensitivity, thereby reducing resistance to drought stress.

[0141] The effects of drought stress on electrolyte leakage and lipid peroxidation in the leaves of TRV2:00 and TRV2:SlRGLG2 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 24 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 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.

[0142] As a result, it was confirmed that when SlRGLG2 was silenced, electrolyte leakage was higher than in the control plant after 24 hours of drying (Fig. 3e right), and lipid peroxidation showed the same pattern (Fig. 3e left).

[0143] SlRGLG2 As a result of analyzing the effect of gene silencing on the expression of drought stress response genes in tomatoes using qRT-PCR (Fig. 3f), the drought stress response gene in tomatoes is SlRD29, SlDREB2, SlAREB2, SlNAC4, SlNAC11 and SlNAC6 It was confirmed that gene expression in TRV2:SlRGLG2 plants was lower compared to the normal control group.

[0145] Example 4. Confirmation of excellent salt stress resistance due to SlRGLG2 inhibition

[0146] To confirm excellent salt stress resistance due to SlRGLG2 inhibition, salt stress was applied to the SlRGLG2-silenced tomatoes prepared in Example 3, and the resulting phenotypic changes, survival rate, chlorophyll content, electrolyte leakage, lipid peroxidation, and proline content were analyzed, respectively.

[0147] As shown in Figure 4a, when 400 mM saline (NaCl) was treated for 7 days with the control group (TRV2:00) and the SlRGLG2 silenced tomato (TRV2:SlRGLG2), it was confirmed that the TRV2:SlRGLG2 plants exhibited a salt stress resistance phenotype compared to the control group, and the survival rate was also found to be higher than that of the control group.

[0149] The relative chlorophyll content was analyzed after treating each line with 400 mM NaCl. At this time, the chlorophyll content of the TRV2:00 line was set to 1.0. Specifically, equal amounts of leaves were taken from each line treated with 0 or 400 mM NaCl, placed in tubes, treated with 95% ethanol, sealed, and incubated for 48 hours. The chlorophyll content dissolved in the ethanol was then measured and converted into a relative value.

[0150] As a result, as shown in Figure 4b, it was confirmed that TRV2:SlRGLG2 plants maintained a higher chlorophyll content compared to control plants.

[0152] When subjected to salt stress, lipid components of the cell membrane undergo peroxidation, destabilizing the cell membrane structure and causing internal electrolytes to leak out of the cell. Therefore, in this embodiment, the results of electrolyte leakage and MDA content measurements were used as indicators of sensitivity to high salt stress. Meanwhile, cells synthesize proline as a countermeasure to this peroxidation; proline is a substance that plays a role in maintaining homeostasis by regulating oxidation processes. Therefore, proline was used as an indicator representing the degree of resistance to stress.

[0153] To determine changes in electrolyte leakage, lipid peroxidation, and proline content, plants were treated with NaCl (0 or 400 mM) for each time setting after 3 weeks, and each indicator was investigated (n = 20 plants per line, 3 replicates). Specifically, for the electrolyte leakage experiment, 24 hours after treatment with 0 or 400 mM NaCl, equal amounts of leaves were collected from each line, placed in tubes containing triple-distilled water, and the amount of leaked electrolyte was measured using an ion meter. For the lipid peroxidation and proline content experiments, 24 hours after treatment with 400 mM NaCl, equal amounts of leaves were collected from each line, finely ground using liquid nitrogen, quantified, and placed in tubes. 1) To measure lipid peroxidation, the amount of malondialdehyde (MDA) reacting with thiobarbituric acid (TBA) under high temperature conditions was measured after dissolving in an acidic solution. 2) For the proline content, an acid solution was added to the quantified tube, and the amount of proline reacting with ninhydrin under high temperature conditions was measured.

[0154] As a result, electrolyte leakage in TRV2:SlRGLG2 compared to TRV2:00 was found to be very low even after salt treatment (Fig. 4c). In addition, the level of lipid peroxidation also showed a lower level compared to the control group over time after salt treatment (Fig. 4d). Finally, it was confirmed that the increase in proline content in TRV2:SlRGLG2 was smaller than that of the control plant after 24 hours of salt treatment (Fig. 4e). Through the above results, it was found that the SlRGLG2 gene-silencing plants could respond to salt stress more quickly.

[0156] Synthesizing the above results, tomato-derived SlRGLG2 The gene is a factor with a RING domain located in the cytoplasm that increases in expression under drought stress and exhibits ubiquitination activity, SlRGLG2 It was confirmed that in gene-silencing tomato plants, drought stress resistance decreased, while conversely, salt stress resistance increased. Accordingly, tomato-derived SlRGLG2 It was found that the gene, acting as a factor with positive drought stress and negative salt stress regulatory activity, regulates drought and salt stress resistance in tomato plants by controlling gene expression related to ABA sensitivity and stress responsiveness, including transpiration loss.

[0158] In conclusion, the present invention functions as a drought and salt stress regulator in tomato plants. SlRGLG2 They identified and characterized it, thereby confirming a new pathway for ABA signaling and the response to drought and salt in tomato plants.

[0160] 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 SlRGLG2 composed of the amino acid sequence of SEQ ID NO. 1 ( Solanum lycopersicum E3 ubiquitin-protein ligase RGLG2) protein. Claim 2 Encoding the SlRGLG2 protein described in claim 1 SlRGLG2 gene. Claim 3 In paragraph 2, the above SlRGLG2 The gene is characterized by being composed of the nucleotide sequence of SEQ ID NO. 2, SlRGLG2 gene. Claim 4 In paragraph 2, the above SlRGLG2 The gene is characterized by being derived from a tomato, SlRGLG2 gene. Claim 5 Paragraph 2 SlRGLG2 A recombinant vector containing a gene. Claim 6 In claim 5, the recombinant vector is characterized as being for enhancing drought stress resistance of a plant. Claim 7 A plant transformed with the recombinant vector of paragraph 5. Claim 8 The SlRGLG2 protein of claim 1, encoding the same SlRGLG2 Genes, and the above SlRGLG2 A composition for enhancing drought stress resistance of a plant, comprising one or more active ingredients selected from a group consisting of recombinant vectors containing genes. Claim 9 A kit for enhancing drought stress resistance of a plant, comprising the composition of claim 8. Claim 10 A method for enhancing drought stress resistance of a plant, comprising the following steps: (S1) transforming a plant with a gene encoding the SlRGLG2 protein of claim 1; and (S2) overexpressing the SlRGLG2 protein in the transformed plant. Claim 11 Transgenic plant with enhanced drought stress resistance by the method of paragraph 10. Claim 12 A plant body characterized in that, in Clause 11, the transgenic plant body is a tomato. Claim 13 A composition for enhancing salt stress resistance in plants, comprising an expression or activity inhibitor of the SlRGLG2 protein of claim 1 as an active ingredient. Claim 14 A composition according to claim 13, characterized in that the above-mentioned SlRGLG2 protein consists of the amino acid sequence of SEQ ID NO.

1. Claim 15 A method for enhancing salt stress resistance of a plant, comprising the step of inhibiting the expression or activity of a SlRGLG2 protein composed of the amino acid sequence of SEQ ID NO. 1.

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