Composition for diagnosing plant drought stress and method for diagnosing plant drought stress using high-expression factor

Abstract

The present invention relates to a composition, a diagnostic kit, and a diagnostic method for diagnosing drought stress in plants. Specifically, the present invention provides a composition comprising a biomarker consisting of one or more genes selected from the gene group listed in Table 1, and a kit and method capable of diagnosing the drought stress state of a plant using the same. The composition and diagnostic method according to the present invention have the effect of rapidly and accurately determining whether a plant is under drought stress by quantitatively measuring the expression level of a specific biomarker gene, thereby enabling the early identification of the growth status and appropriate countermeasures.

Description

Technology Field

[0001] The present invention relates to a composition for diagnosing drought stress in plants and a method for diagnosing drought stress in plants using high-expression factors. Background Technology

[0003] Climate change caused by global warming leads to environmental stresses such as rising temperatures and drought, which negatively affect plant growth and cause growth retardation and developmental disorders. These environmental stresses impair the structural stability of proteins, cell membranes, RNA, and the cytoskeleton within plants, resulting in metabolic imbalances.

[0004] Plants adjust their transcriptome, protein, metabolite, and lipid composition to adapt to environmental changes, and heat shock proteins are known to play a crucial role in plant metabolic processes, particularly in response to high temperatures. Plant responses to such stress manifest as changes in gene expression, manifesting tolerance to various stressors such as high temperature, drought, and salinity. Therefore, gene expression analysis at the transcriptome level can be effectively utilized to understand these tolerance mechanisms.

[0005] Korean fir (Abies koreana) is a species endemic to Korea, inhabiting the alpine regions of southern the Korean Peninsula. Recently, collective leaf blight has been occurring due to environmental stresses such as rising temperatures and drought. Although Korean fir is currently classified as a climate change-sensitive species, there has been little research on its molecular adaptation mechanisms to environmental stress.

[0006] Therefore, research on technologies capable of effectively detecting and diagnosing drought stress is urgently required for the ecological maintenance and protection of Korean fir. Prior art literature

[0008] Republic of Korea Published Patent Application No. 10-2019-0136631 (December 10, 2019)

[0009] Mauri A, De Rigo D, Caudullo G. Abies alba in Europe: distribution, habitat, usage and threats. In: European Atlas of forest tree species. 2016;48-49.Mαtyαs C, Beran F, Dostαl J, Θαp J, Ful n M, Vejpustkovα M, Bo쪥i G, Balαzs P, Fr dl J. Surprising drought tolerance of fir (Abies) species between past climatic adaptation and future projections reveals new chances for adaptive forest management. Forests. 2021;12:821.Tanaka N, Nakao K, Tsuyama I, Higa M, Nakazono E, Matsui T. Predicting the impact of climate change on potential habitats of fir (Abies) species in Japan and on the East Asian continent. Procedia Environ Sci. 2012;13:455-466.Nelson KN, O'Dean E, Knapp EE, Parker AJ, Bisbing SM. Persistent yet vulnerable: resurvey of an Abies ecotone reveals few differences but vulnerability to climate change. Ecology. 2021;102:e03525.Navarro-Cerrillo RM, Gonzαlez-Moreno P, Ruiz-Gσmez FJ, Sαnchez-Cuesta R, Gazol A, Camarero JJ. 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Transcriptomic analysis of drought stress responses in Ammopiptanthus mongolicus leaves using the RNA-Seq technique. PLOS ONE. 2015;10:e0124382.George S, Manoharan D, Li J, Britton M, Parida A.Transcriptomic responses to drought and salt stress in desert tree Prosopis juliflora. Plant Gene. 2017;12:114-122.Du M, Ding G, Cai Q. The transcriptomic responses of Pinus massoniana to drought stress. Forests. 2018;9:326.Fox H, Doron-Faigenboim A, Kelly G, Bourstein R, Attia Z, Zhou J, Moshe Y, Moshelion M, David-Schwartz R. Transcriptome analysis of Pinus halepensis under drought stress and during recovery. Tree Physiol. 2018;38:423-441.Zhao D, Zhang X, Fang Z, Wu Y, Tao J. Physiological and transcriptomic analysis of tree peony (Paeonia section Moutan DC.) in response to drought stress. Forests. 2019;10:135.Kim T-L, Lim H, Denison MIJ, Oh C. Transcriptomic and physiological analysis reveals genes associated with drought stress responses in Populus alba x Populus glandulosa. Plants. 2023;12:3238.Seleiman MF, Al-Suhaibani N, Ali N, Akmal M, Alotaibi M, Refay Y, Dindaroglu T, Abdul-Wajid HH, Battaglia ML.Drought stress impacts on plants and different approaches to alleviate its adverse effects. Plants (Basel). 2021;10:259.Lozano-Elena F, F. bregas N, Coleto-Alcudia V, Caρo-Delgado AI. Analysis of metabolic dynamics during drought stress in Arabidopsis plants. Sci Data. 2022;9:90.Joshi R, Wani SH, Singh B, Bohra A, Dar ZA, Lone AA, Pareek A, Singla-Pareek SL. Transcription factors and plants response to drought stress: current understanding and future directions. Front Plant Sci. 2016;7:1029.Hu Y, Chen X, Shen X. Regulatory network established by transcription factors transmits drought stress signals in plant. Stress Biol. 2022;2:26.Jia D, Jiang Q, van Nocker S, Gong X, Ma F. An apple (Malus domestica) NAC transcription factor enhances drought tolerance in transgenic apple plants. Plant Physiol Biochem. 2019;139:504-512.Liu Y, Yang T, Lin Z, Gu B, Xing C, Zhao L, Dong H, Gao J, Xie Z, Zhang S, Huang X. A WRKY transcription factor PbrWRKY53 from Pyrus betulaefolia is involved in drought tolerance and AsA accumulation. Plant Biotechnol J. 2019;17:1770-1787.Gao Y, Wang K, Wang R, Wang L, Liu H, Wu M, Xiang Y.Identification and expression analysis of LBD genes in moso bamboo (Phyllostachys edulis). J Plant Growth Regul. 2022;41:2798-2817.Liang B, Wan S, Ma Q, Yang L, Hu W, Kuang L, Xie J, Huang Y, Liu D, Liu Y. A novel bHLH transcription factor PtrbHLH66 from trifoliate orange positively regulates plant drought tolerance by mediating root growth and ROS scavenging. Int J Mol Sci. 2022;23:15053.Song Q, Kong L, Yang J, Lin M, Zhang Y, Yang X, Wang X, Zhao Z, Zhang M, Pan J, Zhu S, Jiao B, Xu C, Luo K. The transcription factor PtoMYB142 enhances drought tolerance in Populus tomentosa by regulating gibberellin catabolism. 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[0010] The present invention aims to solve the aforementioned problem and other related problems.

[0011] One exemplary objective of the present invention is to provide a composition for diagnosing drought stress in plants comprising a biomarker consisting of one or more genes selected from the gene group listed in Table 1.

[0012] Another exemplary objective of the present invention is to provide a kit for diagnosing drought stress in plants, comprising a composition for diagnosing drought stress in plants, comprising one or more genes selected from the gene group listed in Table 1.

[0013] Another exemplary objective of the present invention is to provide a method for diagnosing drought stress in plants, comprising the step of measuring the expression level of one or more genes selected from the gene group listed in Table 1.

[0014] The technical problems to be solved according to the technical concept of the invention disclosed in this specification are not limited to those for solving the problems mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below. means of solving the problem

[0016] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0018] In order to solve the above problem, one aspect of the present invention provides a composition for diagnosing drought stress in plants comprising a biomarker consisting of one or more genes selected from the gene group listed in Table 1.

[0019] In the present invention, the gene group described in Table 1 functions as an optimal test gene capable of determining whether a plant has been subjected to drought stress, and as a genetic indicator capable of diagnosing the drought stress state of a plant by measuring the expression level of the gene. Specifically, the gene group is composed of genes whose expression levels change in response to drought stress, and thus has a direct correlation with the physiological state of the plant, thereby enabling accurate diagnosis of whether the plant is under drought stress.

[0020] In the present invention, the biomarker is a biological molecule that serves as an indicator of an in vivo state or disease, and is used for disease diagnosis, prognosis evaluation, and prediction of treatment response; it mainly includes genes, proteins, metabolites, etc., but is not limited thereto.

[0021] In the present invention, the plant is a fir tree ( Abies koreanaAs such, it is an evergreen conifer native to the Korean Peninsula. Korean fir exhibits characteristics of responding sensitively to environmental changes and is utilized as an indicator for ecosystem conservation. In particular, Korean fir is vulnerable to high temperatures and drought, making it suitable for identifying candidate genes that play a crucial role in drought and high-temperature stress responses.

[0022] In the present invention, the drought stress refers to a state in which a plant's physiological functions deteriorate due to a lack of internal water, which can cause reduced photosynthesis, cell damage, tissue necrosis, etc. Drought stress is primarily caused by environmental factors such as insufficient precipitation, reduced soil moisture, and high temperatures. The present invention provides a composition and a method that contribute to maintaining and protecting the health of plants by diagnosing this condition at an early stage.

[0023] In the present invention, the diagnosis refers to evaluating the physiological state of a plant through specific indicators or biomarkers, and in particular, to the process of confirming the presence of drought stress. The primary purpose of this is to identify the state of drought stress early by measuring the expression level of target genes or specific biochemical changes, but it is not limited thereto.

[0024] In the present invention, the composition refers to a substance in which two or more components are mixed to achieve a specific purpose, and each component is configured to perform a function suitable for that purpose. In particular, the present invention aims to perform the function of diagnosing a dry stress state by mixing specific components, and is configured so that each component cooperates to maximize the effect.

[0025] In the present invention, the composition further comprises a preparation for detecting the biomarker.

[0026] In the present invention, the detection agent may be a gene-specific nucleic acid or oligopolypeptide, such as a primer or probe, and may have a sequence complementary to the gene, such as DNA and / or RNA, or be capable of hybridization.

[0027] The above primer refers to a nucleic acid sequence having a free 3' hydroxyl group that can bind complementarily to a template and allows reverse transcriptase or DNA polymerase to initiate replication of the template. The primer is a nucleotide having a sequence complementary to the nucleic acid sequence of a specific gene, having a length of about 7 bp to 50 bp, preferably about 10 bp to 30 bp. The primer may also include additional other base sequences that do not alter the basic properties of the primer acting as a starting point for DNA synthesis. The primer can be chemically synthesized using widely known methods, and such nucleic acid sequences can also be modified using many means known in the art.

[0028] The probe used in the present invention is a nucleic acid strand having partial or complete complementarity with a target nucleic acid and is composed of an oligonucleotide capable of base-specific binding to the target nucleic acid. Preferably, it comprises an oligonucleotide having complete complementarity with the target nucleic acid.

[0029] The probe may consist not only of nucleic acids but also of various conventionally known nucleic acid derivatives capable of complementary binding to the target, such as peptide nucleic acids. The binding between the probe and the target nucleic acid (hereinafter referred to as "hybridization") occurs in a sequence-dependent manner and can be performed under various conditions. Generally, the hybridization reaction takes place at a temperature approximately 5°C lower than the Tm of the target sequence at a specific ionic strength and pH. Here, Tm represents the state in which 50% of the probe relative to the total target sequence is bound to the target sequence.

[0030] The length of the probe can be appropriately adjusted to bind to the target nucleic acid in a sequence-specific manner and is not limited to a specific length. For example, the length of the probe may be 10 to 200 nucleotides, 10 to 150 nucleotides, 10 to 100 nucleotides, or 10 to 50 nucleotides, and in some cases, may be a single strand length corresponding to the full-length gene. The above examples are for illustrative purposes only and the present invention is not limited thereto.

[0031] In addition, the probe may be labeled with a detectable label. Examples of such labels include, but are not limited to, fluorescent substances, radioisotopes, and chromogenic enzymes. For example, fluorescent labels may include SYBR Green I, TaqMan probe (containing the fluorescent substance FAM at the 5'-terminus and the quencher TAMRA at the 3'-terminus), Cy3, Cy5, etc. Additionally, enzyme labels may include enzymes that convert a substrate into a chromogenic substance, and the present invention is not limited to these examples, and the user may select various labeling methods as needed.

[0033] In order to solve the above problem, one aspect of the present invention provides a kit for diagnosing dry stress in plants comprising the composition of claim 1.

[0034] The description of genes, biomarkers, plants, drought stress, and diagnosis in the present invention is as described above.

[0035] In the present invention, the kit may further include one or more other components, solutions, or devices suitable for a method of analyzing gene expression levels or expression patterns or a method of analyzing protein abundance or presence patterns, in addition to nucleic acids or antibodies. For example, if the kit is a kit for detecting gene expression levels or expression patterns, it may be a kit containing essential components required to perform RT-PCR, and such RT-PCR kit may include, in addition to each primer specific to the mRNA of the biomarker gene, a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, and a pair of primers specific to the gene used as a quantitative control, depending on the specific embodiment. Meanwhile, if the above kit is a kit for detecting the amount or pattern of presence of a protein, the kit may be, for example, a kit containing essential components necessary to perform an ELISA, and such an ELISA kit may include components capable of detecting conjugated antibodies, for example, a labeled secondary antibody, chromophores, an enzyme (e.g., an enzyme conjugated to an antibody) and its substrate, and an antibody specific to a quantitative control protein. Depending on a specific embodiment, the above kit may include a DNA microarray or a protein microarray.

[0036] The above gene expression levels or expression patterns can be detected not only by DNA microarrays but also by conventional biochemical analysis methods that identify the amount or pattern of mRNA generated by the transcription of the corresponding gene. Analysis methods for identifying such mRNA amounts or patterns include RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay, and Northern blot, and any other method conventionally performed in the industry may also be used.

[0037] Methods for measuring the amount or pattern of presence of protein using antibodies include Western blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc., and any other method commonly performed in the industry may also be used.

[0039] In another aspect of the present invention, a method for diagnosing drought stress in plants is provided, comprising the step of measuring the expression level of one or more genes selected from the gene group listed in Table 1.

[0040] The description of genes, biomarkers, plants, drought stress, and diagnosis in the present invention is as described above.

[0041] In the present invention, the expression level refers to the degree of activity exhibited by a specific gene or protein within a cell, which can be measured by the amount of transcription of the gene or the amount of protein produced. The expression level is regulated by external environmental factors or physiological changes, and by analyzing this, specific states or responses within the body can be quantitatively evaluated. In the present invention, the expression level of a specific gene group listed in Table 1 is measured under drought stress conditions and utilized to diagnose whether a plant is in a state of drought stress.

[0042] Specifically, in the present invention, if the expression level of a specific gene group listed in Table 1 is significantly higher than that of a normal control group, it can be determined that the plant is in a state of drought stress. Here, "normal control group" refers to the gene expression level in a state where external environmental stress, such as high temperature or drought, is not applied, and this is set as the standard expression level in a non-stress state of the plant. Effects of the invention

[0044] The present invention provides a composition and method capable of diagnosing drought stress in plants at an early stage, thereby enabling the rapid and accurate evaluation of the plant's growth status. The diagnostic composition and kit according to the present invention determine the presence of stress by measuring the expression level of specific biomarker genes that respond to drought stress, thereby providing high reliability and reproducibility compared to conventional visual inspection or general physiological evaluation methods. Furthermore, the diagnostic method of the present invention enables real-time monitoring of physiological changes in plants, allowing for the rapid implementation of appropriate measures when drought stress occurs, which can contribute to the optimization of the plant growth environment and increased productivity. Brief explanation of the drawing

[0046] Figure 1 is a figure showing the growth state of a fir tree exposed to dry stress according to the present invention. Figure 2 is a figure showing the soil moisture content of a fir tree exposed to dry stress according to the present invention. Figure 3 is a figure comparing the number of transcripts expressed according to functional classifications (Biological Process, Cellular Component, Molecular Function) of gene expression related to the drought stress response of the present invention. Figure 4 is a figure showing the number of transcriptome sequences of the fir tree related to the drought stress response of the present invention classified by functional classification (A: RNA processing and modification, B: chromatin structure and kinetics, C: energy production and conversion, D: cell cycle regulation, cell division, chromosome distribution, E: amino acid transport and metabolism, F: nucleotide transport and metabolism, G: carbohydrate transport and metabolism, H: cofactor transport and metabolism, I: lipid transport and metabolism, J: translation, ribosome structure and biosynthesis, K: transcription, L: replication, recombination and repair, M: cell wall / membrane / coat biosynthesis, N: cell motility, O: post-translational modification, proteolysis, chaperone, P: inorganic ion transport and metabolism, Q: secondary metabolite biosynthesis, transport and catabolism, R: general function only predictable, S: function unclassified, T: signal transduction mechanism, U: nucleic acid transport and metabolism, V: defense mechanism, W: nuclear structure, X: cytoskeleton, Y: not specifically classified, Z: unclassified). Figure 5 is a figure showing the changes in high-expression and low-expression transcript expression of Korean fir trees over time (7, 10, and 14 days) under the dry stress conditions of the present invention. Figure 6 is a figure showing the functional classification (Biological Process, Cellular Component, Molecular Function) of transcriptome expression of Korean fir trees by elapsed days (7, 10, and 14 days) under the dry stress conditions of the present invention. Figure 7 is a figure showing the high and low expression patterns of transcription factors for fir trees over time (7, 10, and 14 days) under the dry stress conditions of the present invention. Figure 8 is a figure showing the change in the relative expression levels of specific genes (BES1, ERF, HD-ZIP, LBD1, MYB) that are highly expressed in Korean fir under the dry stress conditions of the present invention over the elapsed days (0, 7, 10, and 14 days). Specific details for implementing the invention

[0047] The present invention will be explained in more detail below through the following examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0049] Example 1. Materials and Method

[0050] 1.1. Materials

[0051] fir tree used in the present invention ( Abies koreana The seeds of Wilson (hereinafter A. koreana) were collected from Mt. Halla on Jeju Island, Korea, and were sown in seedling trays containing soil after being stored at a low temperature (4℃) for 3 months. Seedlings that were 1 year old were transplanted into individual pots and cultivated for 3 years in a greenhouse provided with natural sunlight conditions. In this invention, 3-year-old Korean fir trees provided by the National Institute of Forest Science (Jeju) were used as the material.

[0053] 1.2. Induction of Drying Stress and Measurement of Moisture Content

[0054] To induce drought stress, watering of 3-year-old Korean fir trees was stopped for 7, 10, and 14 days. Soil moisture content was measured according to the manufacturer's instructions using a portable soil moisture meter (DM-18) manufactured by Takemura Electric (Japan). The soil moisture content immediately before the drought treatment (day 0) was set as the control group, and moisture content was measured on days 0, 7, 10, and 14 to evaluate changes resulting from the drought treatment.

[0056] 1.3. RNA Extraction and RNA-Seq Library Construction

[0057] RNA was extracted from leaves of Korean fir collected on days 0, 7, 10, and 14 following drought stress treatment. RNA extraction was performed using the TRIzol reagent from GibcoBRL (USA) according to the manufacturer's instructions, and the quality and concentration of the extracted RNA samples were evaluated using the Agilent Technologies 2100 Bioanalyzer (USA). An RNA-seq library was constructed using the Illumina TruSeq RNA Sample Preparation Kit with only high-quality RNA samples, and sequencing was performed using the Illumina HiSeq 2000 platform with a 101-bp pair-terminal read. Three independent biological samples were used for RNA extraction and library construction.

[0059] 1.4. RNA-seq Data Analysis

[0060] Raw RNA-seq reads were preprocessed using Trimmomatic software (version 0.32) to remove adapter sequences and low-quality reads. The preprocessed reads were de novo assembled using Trinity software (version r20140717), and the assembled unigenes were functionally annotated using BLASTX program (version 2.6.0+) based on NCBI non-redundant protein sequences and the Kyoto Encyclopedia for Genes and Genomes (KEGG) database. Additionally, abundance analyses were performed on molecular functions, biological processes, and cellular components within the Gene Ontology (GO) category.

[0062] 1.5. Identification of Differentially Expressed Genes (DEGs)

[0063] The expression levels of Unigene were calculated based on the number of fragments per kilobase per million fragments (FPKM), and expression analysis was performed using RSEM software based on RNA-seq data. Genes with significant changes in expression were identified as DEGs based on fold change (FC) ≥ 2 and P-value < 0.05, and Venn diagrams were constructed using the Venny tool to analyze DEGs across various drying conditions.

[0065] 1.6. Identification of Drought Stress Response Transcription Factors (TFs)

[0066] To identify transcription factors (TFs) that respond to drought stress, plant-specific TF information was retrieved from the Plant Transcription Factor Database and unigenes were classified based on gene lineage-related information. The BLASTX algorithm was applied to DEG sequences, and only genes with an E-value of 1e^-10 or less were used as classification targets.

[0068] 1.7. DEG Verification using qRT-PCR

[0069] To evaluate the reliability of RNA-seq data, six representative DEGs were randomly selected, and their expression was verified via qRT-PCR. Total RNA was extracted from Korean fir needles subjected to drought stress for 0, 7, 10, and 14 days, and the extracted RNA was synthesized into cDNA and analyzed according to the method of Hwang et al. (2018). Relative expression levels were calculated using the 2^-△△Ct method, and all results were expressed as the standard deviation of three repeated experiments.

[0071] Experimental Example 1. Measurement of Soil Moisture Content (SWC) under Drought Stress

[0072] Three-year-old Korean fir (A. koreana) was subjected to drought stress conditions by suspending water supply for 7, 10, and 14 days, respectively. It was confirmed that the growth of the drought-treated Korean fir was delayed compared to the control group (Fig. 1).

[0073] In addition, the soil moisture content gradually decreased due to the drying treatment, and on the 14th day, it decreased to less than 20% compared to the control group (Fig. 2).

[0075] Experimental Example 2. RNA-Seq Data Assembly and Annotation

[0076] RNA quality was verified in each sample using raw reads obtained from a total of 24 RNA-seq libraries. After trimming the raw reads, the reliability and accuracy of the RNA-seq data were confirmed, and high-quality trimmed reads were stored in the NCBI Sequence Read Archive database (access numbers SAMN40219202–SAMN40219213). Based on this data, a total of 85,403 contigs were assembled using Trinity software; the N50 value of the contigs was found to be 2,087 bp, and the average length was analyzed to be 1,364 bp. A total of 42,839 final assembled unigenes were annotated for future functional analysis.

[0078] Experimental Example 3. Functional Annotation and Classification of Unigine

[0079] Functional annotation was performed on 42,839 assembled unigenes using the GO database and WEGO tool. Of these, 29,739 (69.42%) unigenes were successfully annotated and classified into three major GO categories: biological processes, cellular components, and molecular functions (Fig. 3).

[0080] Specifically, in biological processes, cellular processes (13,805 unigenes), metabolic processes (13,051 unigenes), and responses to stimuli (7,360 unigenes) appeared as major subgroups, and in cellular components, cells (19,410 unigenes), cellular parts (19,326 unigenes), and organelles (15,230 unigenes) were included. In molecular functions, binding (10,158 unigenes), catalytic activity (9,334 unigenes), and transporter activity (1,350 unigenes) were classified as major groups (Fig. 4).

[0082] Experimental Example 4. Analysis and Functional Annotation of Differentially Expressed Genes (DEGs)

[0083] In order to analyze changes in gene expression related to the drought stress response of Korean fir (A. koreana) in the present invention, the gene expression profile of Korean fir was measured under drought treatment conditions. The experiment was conducted at four time points: day 0 (control group), day 7, day 10, and day 14, and genes with prominent changes in expression due to drought stress at each time point were identified (Fig. 5).

[0084] As a result of measuring the number of genes whose expression levels increased or decreased by more than twofold at each treatment time point, 313 genes showed increased expression and 547 genes showed decreased expression on the 7th day of treatment, while 566 genes showed increased expression and 627 genes showed decreased expression on the 10th day of treatment. On the 14th day of treatment, 449 genes showed increased expression and 347 genes showed decreased expression. In particular, the changes in gene expression were most pronounced on the 10th day of treatment, confirming that the response of Korean fir to drought stress was most active on the 10th day.

[0085] In addition, it was confirmed that there were 218 genes that were commonly upregulated and 448 genes that were commonly downregulated under the 7-day and 10-day treatment conditions. Compared to the control group (0 days), it was confirmed that 50 genes were commonly upregulated and 77 genes were commonly downregulated under all treatment conditions.

[0086] The experimental results of the present invention show that the Korean fir regulates specific gene expression in response to drought stress, and confirmed that the genes are particularly actively expressed on the 10th day, thereby contributing to drought stress-related metabolic processes and catalytic activity (Fig. 6).

[0088] As a result, 83 genes useful for diagnosing drought stress were selected from among the genes that are highly expressed when Korean fir is exposed to drought stress (Table 1 below). These genes are characterized by a significant increase in expression levels under drought stress conditions and can be used as indicators to quantitatively evaluate physiological changes in Korean fir.

[0090] Gene ID E-value Homology Reference Gene ID Transcription factor (TF) name 7d 10d 14d 1 Akoreana1SL005263t0001 8.00E-11 100 MA_98095g0010 AP2 2.07 1.56 1.32 2 Akoreana1SL010605t0001 5.00E-42 100 MA_86195g0010 AP2 1.61 1.61 1.54 3 Akoreana1SL39703t0001 8.00E-17 38.38 MA_67041g0010 AP2 2.37 1.02 2.05 4 Akoreana1SL007270t0001 8.00E-138 78.87 MA_195595g0010 BES1 1.08 2.20 2.48 5 Akoreana1SL010625t0001 4.00E-69 69.05 MA_196213g0010 bHLH 1.55 1.67 1.64 6 Akoreana1SL013561t0001 1.00E-11 76.47 MA_11847g0020 bZIP 1.81 1.91 2.07 7 Akoreana1SL010709t0001 5.00E-33 75 MA_105743g0010 bZIP 2.15 1.75 1.77 8 Akoreana1SL002978t0001 7.00E-86 93.06 MA_35014g0010 bZIP 1.67 1.33 1.81 9 Akoreana1SL011163t0001 3.00E-28 50.47 MA_141733g0010 C2H2 1.90 2.35 1.88 10 Akoreana1SL022084t0001 2.00E-13 53.09 MA_8390711g0010 C2H2 1.76 2.54 1.47 11 Akoreana1SL022942t0001 0 78.48 MA_109421g0010 C2H2 1.09 1.71 1.27 12 Akoreana1SL022141t0001 3.00E-42 84.21 MA_104763g0010 C2H2 1.62 1.67 1.63 13 Akoreana1SL41337t0001 0 78.48 MA_109421g0010 C2H2 1.1 1.73 1.28 14 Akoreana1SL31987t0001 1.00E-13 50.85 MA_910870g0010 C3H 1.68 1.18 1.67 15 Akoreana1SL022657t0001 1.00E-11 50.85 MA_7292g0010 CO-like 1.51 1.25 1.29 16 Akoreana1SL37659t0001 3.00E-11 50.85 MA_7292g0010 CO-like 1.52 1.26 1.3 17 Akoreana1SL026324t0001 0 84.66 MA_944867g0010 Dof 2.12 2.2 2.02 18 Akoreana1SL41654t0001 0 84.36 MA_944867g0010 Dof 2.09 2.23 1.99 19 Akoreana1SL008166t0001 1.00E-49 65.82 MA_203191g0010 ERF 2.49 2.84 2.39 20 Akoreana1SL006588t0001 7.00E-66 74.87 MA_18454g0020 ERF 3.18 3.48 3.25 21 Akoreana1SL022564t0001 4.00E-68 74.87 MA_4032g0010 ERF 1.94 2.21 2.01 22 Akoreana1SL021253t0001 1.00E-82 80.11 MA_1037g0020 ERF 3.90 3.32 1.89 23 Akoreana1SL020920t0001 4.00E-59 78.2 MA_96063g0020 ERF 2.46 3.36 2.30 24 Akoreana1SL008085t0001 1.00E-25 86.05 MA_16778g0010 ERF 1.87 1.97 1.28 25 Akoreana1SL003683t0001 2.00E-52 86.05 MA_10209922g0010 ERF 1.88 1.75 1.80 26 Akoreana1SL025964t0001 6.00E-68 81.55 MA_203191g0010 ERF 3.53 4.29 3.18 27 Akoreana1SL019838t0001 4.00E-24 69.42 MA_16778g0010 ERF 3.74 4.02 4.03 28 Akoreana1SL026052t0001 4.00E-24 53.93 MA_10435354g0010 ERF 1.61 2.43 1.18 29 Akoreana1SL012191t0001 4.00E-32 85.25 MA_9785986g0010 ERF 2.71 3.19 2.05 30 Akoreana1SL024196t0001 4.00E-38 95.08 MA_108992g0010 ERF 1.74 1.05 1.11 31 Akoreana1SL019651t0001 2.00E-18 90.57 MA_168025g0010 ERF 1.31 2.04 2.38 32 Akoreana1SL010388t0001 3.00E-82 64.5 MA_9304g0020 ERF 1.51 1.97 2.01 33 Akoreana1SL010197t0001 2.00E-72 80.54 MA_203191g0010 ERF 3.31 3.66 2.75 34 Akoreana1SL001607t0003 7.00E-67 77.38 MA_10031781g0010 ERF 2.75 2.90 2.38 35 Akoreana1SL022637t0001 4.00E-115 78.7 MA_32651g0010 ERF 3.77 4.00 4.84 36 Akoreana1SL024949t0001 2.00E-20 67.65 MA_10031781g0010 ERF 2.76 2.64 2.31 37 Akoreana1SL026186t0001 2.00E-58 73.43 MA_19854g0020 ERF 4.49 1.72 2.50 38 Akoreana1SL025881t0001 1.00E-13 58.82 MA_67707g0010 ERF 2.11 1.44 1.26 39 Akoreana1SL018214t0001 7.00E-74 55.88 MA_184464g0010 ERF 2.38 2.15 2.11 40 Akoreana1SL30244t0012 2.00E-44 85.58 MA_10031781g0010 ERF 2.63 2.79 2.18 41 Akoreana1SL023561t0001 2.00E-92 52.17 MA_181986g0010 G2-like 1.34 1.25 1.05 42 Akoreana1SL003569t0001 0 83.28 MA_238039g0010 HD-ZIP 3.20 3.35 2.05 43 Akoreana1SL004904t0001 4.00E-54 74.22 MA_12053g0010 HD-ZIP 2.97 2.47 1.85 44 Akoreana1SL005349t0001 2.00E-104 81.35 MA_91369g0010 LBD 1.50 3.11 1.44 45 Akoreana1SL021978t0001 5.00E-76 73.33 MA_35037g0010 LBD 1.83 1.30 2.18 46 Akoreana1SL011234t0001 8.00E-70 80.41 MA_328535g0010 LBD 2.63 2.66 4.81 47 Akoreana1SL021563t0001 3.00E-94 81.14 MA_11285g0020 LBD 1.99 2.08 1.14 48 Akoreana1SL020734t0001 4.00E-74 83.66 MA_16891g0020 LBD 2.19 3.12 1.63 49 Akoreana1SL018825t0001 4.00E-65 75.89 MA_131805g0010 LBD 2.52 1.85 1.77 50 Akoreana1SL39755t0001 1.00E-127 74.23 MA_91369g0010 LBD 2.47 3.48 1.72 51 Akoreana1SL024935t0001 7.00E-53 79.67 MA_936250g0010 MYB 1.39 2.41 1.13 52 Akoreana1SL000550t0002 0 85.8 MA_4704g0010 MYB 1.00 1.90 1.02 53 Akoreana1SL027596t0001 3.00E-21 66.67 MA_16413g0010 MYB 1.45 2.14 1.09 54 Akoreana1SL019192t0002 0 74.27 MA_139448g0010 MYB 2.33 2.68 1.23 55 Akoreana1SL005911t0001 0 82.46 MA_89683g0010 MYB 3.03 2.51 1.45 56 Akoreana1SL003644t0001 2.00E-40 87.32 MA_10190951g0010 MYB 1.75 1.36 1.98 57 Akoreana1SL003803t0001 0 89.06 MA_82197g0010 MYB 2.82 1.81 2.27 58 Akoreana1SL023779t0001 2.00E-43 55.36 MA_113824g0010 MYB 2.54 2.11 3.96 59 Akoreana1SL012170t0001 2.00E-51 85.06 MA_33964g0010 MYB 1.68 1.17 2.75 60 Akoreana1SL009078t0001 3.00E-120 70.77 MA_82197g0010 MYB 2.75 2.66 1.67 61 Akoreana1SL024704t0001 4.00E-36 80.56 MA_37058g0010 MYB 1.19 1.34 1.27 62 Akoreana1SL008562t0001 2.00E-38 86.36 MA_936250g0010 MYB 1.19 1.68 1.47 63 Akoreana1SL35573t0001 1.00E-123 66.67 MA_21440g0010 MYB 4.71 5.14 1.08 64 Akoreana1SL42713t0001 2.00E-78 74.84 MA_8147g0020 MYB 4.11 4.32 1.2 65 Akoreana1SL018792t0001 7.00E-175 94.41 MA_17466g0010 MYB_related 2.16 2.42 1.75 66 Akoreana1SL025553t0001 9.00E-49 81.4 MA_336002g0010 MYB_related 4.45 3.70 1.38 67 Akoreana1SL011151t0001 5.00E-38 83.33 MA_9856146g0010 MYB_related 2.28 3.38 1.65 68 Akoreana1SL024077t0001 4.00E-39 95.31 MA_86256g0010 NAC 1.65 1.75 1.02 69 Akoreana1SL003819t0001 0 92.78 MA_8980g0010 NAC 1.81 1.30 1.65 70 Akoreana1SL006221t0001 3.00E-164 85.81 MA_75192g0010 NAC 2.68 3.03 2.75 71 Akoreana1SL019679t0001 0 81.75 MA_103386g0010 NAC 2.5 2.94 2.92 72 Akoreana1SL003776t0002 3.00E-132 82.05 MA_9929143g0010 NF-YC 2.76 2.91 3.37 73 Akoreana1SL001545t0001 9.00E-133 82.05 MA_11029g0010 NF-YC 4.04 3.7 3.78 74 Akoreana1SL018678t0001 1.00E-110 75.5 MA_10428500g0010 TALE 2.57 1.85 2.01 75 Akoreana1SL018249t0001 6.00E-22 89.74 MA_303404g0010 TALE 2.41 1.18 2.82 76 Akoreana1SL014363t0001 0 86.08 MA_37369g0010 TCP 2.16 3.28 1.33 77 Akoreana1SL011842t0001 3.00E-40 64.18 MA_16704g0010 TCP 2.28 2.25 1.40 78 Akoreana1SL021762t0001 4.00E-72 50.35 MA_10430713g0010 Trihelix 2.25 1.30 2.57 79 Akoreana1SL008111t0002 9.00E-101 62.26 MA_10426942g0010 WRKY 1.29 2.40 1.12 80 Akoreana1SL025074t0001 1.00E-91 54.44 MA_134559g0010 WRKY 1.08 2.59 2.04 81 Akoreana1SL015106t0001 5.00E-33 58.93 MA_10426942g0010 WRKY 2.56 1.89 1.80 82 Akoreana1SL021787t0001 7.00E-156 85.07 MA_23415g0010 WRKY 1.67 1.76 1.25 83 Akoreana1SL41492t0001 2.00E-154 85.07 MA_23415g0010 WRKY 1.53 1.68 1.21

[0092] Experimental Example 5. Identification of Transcription Factors (TFs) Involved in Drought Stress

[0093] To identify the transcription factors (TFs) of Korean fir responding to drought stress, several transcription factors of the bHLH, ERF, MYB, WRKY, LBD, and NAC families were analyzed (Fig. 7). In general, the expression of the ERF transcription factor increased due to drought, while the expression of the bHLH transcription factor decreased. The TF genes of the MYB family showed diverse expression patterns, confirming that they play an important role in explaining the complex physiological responses of Korean fir to drought.

[0095] Experimental Example 6. Verification of DEG via qRT-PCR

[0096] Next, to verify the reliability of the RNA-seq results, qRT-PCR was performed on six representative DEGs (Akoreana1SL003569t0001 [HD-ZIP], Akoreana1SL011234t0001 [LBD], Akoreana1SL007270t0001 [BES1], Akoreana1SL019838t0001 [ERF], Akoreana1SL006588t0001 [ERF], Akoreana1SL005911t0001 [MYB]) containing the transcription factors HD-ZIP, LBD, BES1, ERF, and MYB. As a result of measuring the expression levels of each DEG under drought stress conditions (days 0, 7, 10, and 14), all DEGs showed higher expression than the control group under drought conditions (Fig. 8), and this pattern was confirmed to be consistent with RNA-seq data, thereby proving the reliability of the data in this study.

[0098] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Claim 1 Korean fir ( Abies koreana A method for diagnosing drought stress of ), comprising:

1. a step of extracting RNA from the leaves of a fir tree to be tested; 2. a step of measuring the expression level of a transcription factor gene group of (a) to (f) below on the mRNA or cDNA obtained from the RNA; and 3. The method comprises the step of determining that the Korean fir is in a state of drought stress if the expression level of the transcription factor gene group of (a) to (f) above increases compared to a normal control group; wherein the transcription factor gene group is classified as a gene with an E-value of 1e^-10 or less when the BLASTX algorithm is applied to plant-specific transcription factor information in the Plant Transcription Factor Database, and comprises: (a) a gene classified as a BES1 transcription factor having homology with the reference gene MA_195595g0010, with an E-value of 8.00E-138 and homology of 78.87%; (b) a gene classified as an ERF transcription factor having homology with the reference gene MA_18454g0020, with an E-value of 7.00E-66 and homology of 74.87%; (c) a gene classified as an ERF transcription factor having homology with the reference gene MA_16778g0010, with an E-value A method for diagnosing dryness stress in Korean fir, characterized by: (d) a gene having 4.00E-24 and 69.42% homology; (e) a gene having homology with reference gene MA_238039g0010 and classified as an HD-ZIP transcription factor, having an E-value of 0 and 83.28% homology; (f) a gene having homology with reference gene MA_328535g0010 and classified as an LBD transcription factor, having an E-value of 8.00E-70 and 80.41% homology; and (f) a gene having homology with reference gene MA_89683g0010 and classified as a MYB transcription factor, having an E-value of 0 and 82.46% homology. Claim 2 A method for diagnosing drought stress in Korean fir trees according to claim 1, characterized in that the expression levels of the transcription factor gene groups of (a) to (f) each increase at one or more of 7, 10, or 14 days after drought stress treatment compared to a normal control group treated at day 0. Claim 3 A method for diagnosing dry stress in Korean fir, characterized in that, in paragraph 2, the step of measuring the expression level is performed by any one of RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, or Northern blot. Claim 4 A composition for diagnosing drought stress in Korean fir trees for use in a diagnostic method according to any one of claims 1 to 3, comprising a primer or probe for measuring the expression level by specifically binding to one or more mRNAs of the transcription factor gene groups of (a) to (f) or cDNA synthesized therefrom. Claim 5 A kit for diagnosing drought stress in Korean fir trees, comprising the composition for diagnosing drought stress in Korean fir trees according to claim 4.

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