Polynucleotides, plant drought stress tolerance enhancers, cells, plants, methods for improving plant drought stress tolerance, and methods for producing plants with drought stress tolerance.

Abstract

To provide novel polynucleotides, and to improve drought stress tolerance in plants using said polynucleotides. [Solution] (a) or (b) below: (a) The coding sequence of the amino acid sequence shown in SEQ ID NOs: 13, 18, or 23, or (b) A coding sequence for a protein having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 13, 18, or 23, and which has the effect of improving the drought stress tolerance of plants. Isolated polynucleotides containing [the specified element].

Description

Technical Field

[0001] The present invention relates to polynucleotides, plant drought stress tolerance enhancers, cells, plants, methods for improving plant drought stress tolerance, methods for producing drought stress-tolerant plants, and the like.

Background Art

[0002] Mongolia is located on highlands with an altitude of 1500 meters or more, characterized by step grasslands, mountains, and deserts without a sea. Summers in the Mongolian grasslands are short, with little rain, winters are cold, and the temperature is below -20°C. In particular, the Gobi Desert area in Mongolia has a continental climate and is very dry, with an annual precipitation of only 50 mm. In the Mongolian grasslands under such harsh conditions, only plants with the ability to grow rapidly and adapt to the short summer and the characteristic of being strong against drought stress can survive.

[0003] Climate change associated with global warming is a troublesome problem for human society. Abnormal weather has fallen into a vicious cycle of reducing the production of crops and vegetables in agriculture, expanding desert areas, and accelerating the destabilization of the climate. Many attempts have been made to improve the growth of crops using plant biotechnology by evaluating drought stress tolerance in experimental plants such as Arabidopsis thaliana and rice and identifying drought stress tolerance genes.

[0004] The Arabidopsis thaliana NCED3 gene encodes 9-cis-epoxycarotenoid dioxygenase, a central enzyme in abscisic acid (ABA) biosynthesis (Non-Patent Documents 1 and 2). Arabidopsis thaliana transformed with NCED3 showed increased endogenous ABA levels, increased expression of drought and ABA-inducible genes, and improved drought stress tolerance (Non-Patent Document 3). Furthermore, overexpression of the NCED3 gene in soybeans improved drought stress tolerance under greenhouse and field conditions (Non-Patent Document 4). In addition, ABA-responsive element-binding protein 1 (AREB1) is a basic leucine zipper (bZIP) transcription factor and plays a central role in the ABA-dependent signaling pathway (Non-Patent Documents 5 and 6). Overexpression of AREB1 has been shown to improve drought resistance in various plant species, including Arabidopsis thaliana, rice, and soybeans (Non-Patent Documents 7-9). Climate change is expected to worsen year by year, and further testing is needed to identify novel drought stress tolerance genes.

[0005] Attempts have been made to identify useful genes not only from experimental plants but also from wild plants not utilized in the field. In Eurasia, RNA-seq analysis of Agropyron mongolicum revealed 41,792 unigenes and 1,104 miRNAs (Non-Patent Literature 10). From Caragana korshinskii, a leguminous shrub with strong stress tolerance, a total of 129,451 cDNA contigs were obtained under drought and salt treatment conditions (Non-Patent Literature 11). Whole-genome sequencing was performed on Chloris gayana, also known as rose grass in Africa, and 46,087 predicted genes were annotated (Non-Patent Literature 12). While identifying these common genes using an omics approach should provide useful information, the specific identification of their effective functions in plants remains limited. Furthermore, our report is the only attempt to identify growth and stress-related genes from Mongolian steppe plants (Non-Patent Literature 13). [Prior art documents] [Non-patent literature]

[0006] [Non-licensed document 1] Behnam et al., DNA Res. 20(4), 315-324 (2013). [Non-licensed document 2] Kalladan et al., Plant Physiol. 179(4), 1620-1631 (2019). [Non-licensed document 3] Iuchi et al., Plant J. 27(4), 325-333 (2001).

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Non-licensed literature 9

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Non-licensed Document 11

Non-licensed Document 12

[0007] The present invention aims to provide a novel polynucleotide and to improve the drought stress tolerance of plants using the polynucleotide. [Means for solving the problem]

[0008] The inventors identified three novel genes derived from Chloris virgata (hereinafter referred to as C. virgata): Mongolian Grassland plant Drought-stress resistance gene 1 (MGD1), Mongolian Grassland plant Drought-stress resistance gene 2 (MGD2), and Mongolian Grassland plant Drought-stress resistance gene 3 (MGD3), which are involved in the drought stress tolerance of Chloris virgata. To investigate the function of these genes, the inventors created transgenic Arabidopsis thaliana plants that overexpressed each gene and examined their drought stress tolerance. Surprisingly, the transgenic Arabidopsis thaliana plants showed significantly greater tolerance to drought stress compared to the wild type. In other words, the inventors found that the novel genes MGD1, MGD2, and MGD3 could solve the above problem. Based on this finding, the inventors furthered their research and completed the present invention.

[0009] The present invention encompasses the following aspects. Section 1. The following (a) or (b): (a) a coding sequence of the amino acid sequence shown in SEQ ID NO: 13, 18 or 23, or (b) a coding sequence of a protein consisting of an amino acid sequence having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, 18 or 23 and having an effect of improving the drought stress tolerance of plants, An isolated polynucleotide comprising the same. Item 2. The polynucleotide according to item 1, wherein the sequence identity is 90% or more. Item 3. The polynucleotide according to item 1, comprising a promoter upstream of the coding sequence. Item 4. The polynucleotide according to item 1, which is a vector. Item 5. A plant drought stress tolerance enhancer comprising the polynucleotide according to any one of items 1 to 4. Item 6. A cell comprising the polynucleotide according to any one of items 1 to 4. Item 7. A plant comprising the cell according to item 6. Item 8. A method for improving the drought stress tolerance of a plant by introducing the polynucleotide according to any one of items 1 to 4 into the plant. Item 9. A method for producing a plant having drought stress tolerance, comprising: The step of introducing the polynucleotide according to any one of items 1 to 4 into a plant. <0000…

Advantages of the Invention

[0010] According to the present invention, a novel polynucleotide can be provided. Further, the drought stress tolerance of plants can be improved by using the polynucleotide. Specifically, a novel polynucleotide, a plant drought stress tolerance enhancer comprising the polynucleotide, a cell, and a plant, and a method for improving the drought stress tolerance of plants using the polynucleotide, and a method for producing a plant having improved drought stress tolerance can be provided.

Brief Description of Drawings

[0011] [Figure 1] Test Example 1: Diagrams showing the drought stress tolerance of C. virgata. (A) Analysis of drought stress tolerance in C. virgata, rice, oats and wheat. Scale bar = 10 cm. (B) Analysis of osmotic stress tolerance in C. virgata, rice and oats. Scale bar = 5 cm. (C) Endogenous chlorophyll content of plants treated with mannitol. Statistical analysis was performed using Student's t-test (ns = no significant difference, ***P < 0.001). [Figure 2] Test Example 1: Diagrams showing the osmotic stress tolerance of C. virgata. (A, B) Fresh weight (A) and shoot length (B) of plants of C. virgata, rice and oats after mannitol treatment. The three highest values for each treatment were converted to 100% (n = 18). [Figure 3] Test Example 2: Diagrams showing the results of expression analysis of drought stress-inducible genes in C. virgata. (A, B) Expression levels of the CvNCED3 gene (A) and the CvAREB1 gene (B) in C. virgata under drought stress conditions. The relative mRNA expression level is shown as an n-fold change when the value of the control (1 hour) is 1. Error bars represent the mean ± SD of 4 replicate samples. Different letters above the bars indicate that the mean values are significantly different (One-way ANOVA with post-hoc Tukey HSD test (P < 0.05)). [Figure 4] Test Example 4: Diagrams showing the results of GO enrichment analysis. (A) Venn diagram showing genes with increased expression in C. virgata after 3 hours and / or 6 hours of drought treatment (P < 0.05, fold change > 1.5). (B) Enrichment of GO annotations in the biological process category of genes with increased expression in the 3-hour drought treatment group and the 6-hour drought treatment group. The Y-axis represents the pathway name, and the X-axis represents the gene ratio. The p-adjusted value is represented by the color of each dot, and the number of DEGs is represented by the size of each dot. [Figure 5]Example 5-1: Figure showing the results of transgene expression analysis in transformed Arabidopsis thaliana. (AE) The expression levels of transgenes in CvNCED3-OX (A), CvAREB1-OX (B), MGD1-OX (C), MGD2-OX (D), and MGD3-OX (E) were compared with the expression levels in wild-type Arabidopsis thaliana. Error bars represent the mean ± SD of the four replicate samples. Different letters above the bars indicate a significant difference in the mean (One-way ANOVA with post-hoc Tukey HSD test (P < 0.05)). [Figure 6] Test Example 5-2: Figure showing drought stress tolerance of CvNCED3-overexpressing Arabidopsis thaliana and CvAREB1-overexpressing Arabidopsis thaliana. (A, B) Survival rate (A) and fresh weight (B) of wild-type and CvNCED3-overexpressing plants under drought stress conditions. (C, D) Survival rate (C) and fresh weight (D) of wild-type and CvAREB1-overexpressing plants under drought stress conditions. Survival rate was calculated from the number of surviving plants 2 days after re-irrigation (n=12). Fresh weight was measured 2 days after re-irrigation (n=12). [Figure 7]Examples 6-1-1 to 6-1-3: Figures showing drought stress tolerance in Arabidopsis thaliana overexpressing MGD1. (A) Expression levels of the MGD1 gene under drought stress conditions in C. virgata. Error bars represent the mean ± SD of 4 replicate samples. Different letters above the bars indicate significantly different mean values ​​(One-way ANOVA with post-hoc Tukey HSD test (P < 0.05)). (B) Phylogenetic tree of MGD1 and related proteins in other species (Pp, Physcomitrium patens; Sm, Selaginella moellendorffii; Bn, Brassica napus; At, Arabidopsis thaliana; Nt, Nicotiana tabacum; Pn, Populus nigra; Gm, Glycine max; Ob, Oryza brachyantha; Og, Oryza glaberrima; Os, Oryza sativa; Bd, Brachypodium distachyon; Ta, Triticum aestivum; Lr, Lolium rigidum; Cv, Chloris virgata; Si, Setaria italica; Pv, Panicum virgatum; Sb, Sorghum bicolor; Zm, Zea mays). The numbers at branch sites indicate the posterior probability values ​​of node support. (C, D) Survival rate (C) and fresh weight (D) of wild-type and MGD1 overexpressing plants under drought stress conditions. Four-week-old plants were dried for 14 days and then rewatered for 2 days. Survival rate was calculated from the number of surviving plants 2 days after rewatering (n=12). Fresh weight was measured 2 days after rewatering (n=12). [Figure 8]Test Example 6-1-4: Figure showing sequence conservation and intracellular localization of MGD1. (A) Amino acid sequences of MGD1 and homologs of other plant species. Accession numbers: C. virgata (ChlorisST30368(MGD1)), O. sativa (XP_015622376.1), B. distachyon (XP_003567507.1), T. aestivum (XP_044345791.1), A. thaliana (NP_001330291.1), and N. tabacum (XP_016510532.1). Highly conserved amino acids are shown on a red background. Predicted α-helix regions are shown with black lines. Predicted transmembrane regions are shown with blue lines. Predicted coiled-coil regions are shown with green lines. (B) Intracellular localization of 35S:GFP-MGD1 in root cells of 4-day-old Arabidopsis thaliana. Scale bar = 20 μm. [Figure 9] Example 6-1-4: Figure showing structural prediction results for the MGD1 protein. (A) Hydropathy plot of MGD1. The plot was generated using the NovoPro server (https: / / www.novoprolabs.com / tools / protein897hydropathy) and the Kyte & Doolittle scale (Kyte & Doolittle, J Mol Biol., 157(1), 105-32 (1982)). (B, C) Structural models and predicted alignment errors of monomer (B) and dimer (C) of MGD1 generated using the AlphaFold3 server. The N-terminus and C-terminus are indicated as N and C, respectively. [Figure 10]Examples 6-2-1 to 6-2-3: Figures showing drought stress tolerance in Arabidopsis thaliana overexpressing MGD2. (A) Expression levels of the MGD2 gene under drought stress conditions in C. virgata. Error bars represent the mean ± SD of 4 replicate samples. Different letters above the bars indicate significantly different mean values ​​(One-way ANOVA with post-hoc Tukey HSD test (P < 0.05)). (B) Phylogenetic tree of MGD2 and related proteins in other species (Vv, Vitis vinifera; Nt, Nicotiana tabacum; Pa, Populus alba; Bn, Brachypodium distachyon; At, Arabidopsis thaliana; Bd, Brachypodium distachyon; Lr, Lolium rigidum; Hv, Hordeum vulgare; Ta, Triticum aestivum; Ob, Oryza brachyantha; Os, Oryza sativa; Cv, Chloris virgata; Si, Setaria italica; Pv, Panicum virgatum; Zm, Zea mays; Sb, Sorghum bicolor). The numbers at branching points indicate the posterior probability values ​​of node support. (C, D) Survival rate (C) and fresh weight (D) of wild-type and MGD2 overexpressing plants under drought stress conditions. 4-week-old plants were dried for 14 days and then re-irrigated for 2 days. The survival rate was calculated from the number of surviving plants two days after re-irrigation (n=12). Fresh weight was measured two days after re-irrigation (n=12). [Figure 11]Test Example 6-2-4: Figure showing sequence conservation and intracellular localization of MGD2. (A) Amino acid sequence of MGD2 and homologs of other plant species. Accession numbers: C. virgata (ChlorisST6804 (Cv. MGD2)), O. sativa (NP_001410341.1), B. distachyon (XP_003577510.1), T. aestivum (XP_044420749.1), A. thaliana (NP_196350.1), and N. tabacum (XP_016443868.1). Highly conserved amino acids are shown on a red background. Predicted α-helix regions are shown with black lines. Predicted transmembrane regions are shown with blue lines. (B) Intracellular localization of 35S:GFP-MGD2 in root cells of 4-day-old Arabidopsis thaliana. Scale bar = 20 μm. [Figure 12] Example 6-2-4: Figure showing the structural prediction results for the MGD2 protein. (A) Hydropathy plot of MGD2. The plot was created using the NovoPro server and Kyte & Doolittle scale. (B) Structural model of MGD2 and predicted alignment error created using the AlphaFold3 server. The N-terminus and C-terminus are indicated by N and C, respectively. [Figure 13]Examples 6-3-1 to 6-3-3: Figures showing drought stress tolerance in Arabidopsis thaliana overexpressing MGD3. (A) Expression levels of the MGD3 gene under drought stress conditions in C. virgata. Error bars represent the mean ± SD of 4 replicate samples. Different letters above the bars indicate significantly different mean values ​​(One-way ANOVA with post-hoc Tukey HSD test (P < 0.05)). (B) Phylogenetic trees of MGD3 and related proteins in other species (Pp, Physcomitrium patens; Sm, Selaginella moellendorffii; Cr, Capsella rubella; At, Arabidopsis thaliana; Nt, Nicotiana tabacum; Pa, Populus alba; Gm, Glycine max; Sb, Sorghum bicolor; Zm, Zea mays; Pv, Panicum virgatum; Si, Setaria italica; Cv, Chloris virgata; Ob, Oryza brachyantha; Os, Oryza sativa; Og, Oryza glaberrima; Ta, Triticum aestivum; Lr, Lolium rigidum; Bd, Brachypodium distachyon). The numbers at branching points indicate the posterior probability values ​​of node support. (C, D) Survival rate (C) and fresh weight (D) of wild-type and MGD3 overexpressing organisms under drought stress conditions. Four-week-old plants were dried for 14 days, then rewatered for 2 days. Survival rate was calculated from the number of surviving plants 2 days after rewatering (n=12). Fresh weight was measured 2 days after rewatering (n=12). [Figure 14]Test Example 6-3-4: Figure showing sequence conservation and intracellular localization of MGD3. (A) Amino acid sequences of MGD3 and homologs of other plant species. Accession numbers: C. virgata (ChlorisST36889 (Cv. MGD3)), O. sativa (AAK43505.1), B. distachyon (XP_003572048.1), T. aestivum (XP_044335483.1), A. thaliana (NP_568771.1), and N. tabacum (XP_016501775.1). Highly conserved amino acids are shown on a red background. Predicted α-helix regions are shown with black lines. Predicted transmembrane regions are shown with blue lines. (B) Intracellular localization of 35S:GFP-MGD3×35S:HDEL-RFP in root cells of 4-day-old Arabidopsis thaliana. Scale bar = 20 μm. [Figure 15] Example 6-3-4: Figure showing the structural prediction results for the MGD3 protein. (A) Hydropathy plot of MGD3. The plot was created using the NovoPro server on a Kyte & Doolittle scale. (B) Structural model of MGD3 and predicted alignment error created using the AlphaFold3 server. The N-terminus and C-terminus are indicated by N and C, respectively. [Figure 16] Test Example 7: Figure showing the results of drought-induced gene expression analysis in Arabidopsis thaliana overexpressing MGD1, MGD2, and MGD3. (A-E) Expression analysis of stress-induced genes under drought stress conditions in transformants (MGD1-OX (A), MGD2-OX (B), MGD3-OX (C), CvNCED3-OX (D), and CvAREB1-OX (E)) and wild-type plants. 10-day-old plants were treated for specified times, with or without dehydration. Error bars represent the mean ± SD of 4 replicate samples. Statistical analysis was performed using Student's t-test (ns = no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001). [Modes for carrying out the invention]

[0012] 1.Definition In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0013] In this specification, amino acids / amino acid residues may sometimes be represented by a single letter.

[0014] In this specification, "identity" of amino acid sequences refers to the degree of agreement between two or more comparable amino acid sequences. Therefore, the higher the agreement between two amino acid sequences, the higher their identity or similarity. The level of amino acid sequence identity can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin, S., & Altschul, SF, Proc. Natl. Acad. Sci. USA., 87(6), 2264-2268 (1990), Karlin, S., & Altschul, SF, Proc. Natl. Acad. Sci. USA., 90(12), 5873-5877 (1993)). A program called BLASTX has been developed based on this BLAST algorithm. The specific methods for these analyses are publicly known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). Furthermore, the "identity" of the base sequence is defined in accordance with the above.

[0015] In this specification, "conservative substitution" means that an amino acid residue is substituted for an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, are considered conservative substitutions. Other examples of conservative substitutions include amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0016] In this specification, "nucleic acids" and "polynucleotides" are not particularly limited and include both natural and artificial ones. Specifically, in addition to DNA, RNA, etc., known chemically modified nucleotides may also be used, as exemplified below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues of each nucleotide can be replaced with chemically modified phosphate residues such as phosphorothioates (PS), methylphosphonates, or phosphorodithionates. Furthermore, the hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may be replaced with -OR (where R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). In addition, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at position 5 of the pyrimidine base, or by substituting the carbonyl group at position 2 with a thiocarbonyl group. Furthermore, examples include, but are not limited to, those in which the phosphate or hydroxyl portion is modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group. In addition, BNA (LNA), in which the conformation of the sugar portion of the nucleotide is fixed to the N-type by cross-linking the 2' oxygen and 4' carbon atoms of the sugar portion, can also be used.

[0017] In this specification, "gene" includes not only the protein-coding regions (exons, introns) on genomic DNA, but also the regions that control the expression of said protein (expression regulatory regions).

[0018] In this specification, “isolated” means a state in which it has been separated from its natural state.

[0019] In this specification, "plant drought stress tolerance" refers to the tolerance of the target plant to drought stress. In this specification, "drought stress" refers to the stress that occurs when a plant becomes deprived of water due to drought. When a plant becomes deprived of water due to drought, physiological responses occur such as progression of drought, inhibition of growth, inhibition of photosynthesis, abscisin (ABA) synthesis, stomatal closure, increased respiration, and, in severe cases of water deprivation due to drought, death of the plant. Therefore, "plant drought stress tolerance" more specifically refers to the ability of the target plant to prevent the above physiological responses from occurring under drought conditions, to reduce the degree of the above physiological responses, or to delay the progression of the above physiological responses.

[0020] In this specification, "vector" means a carrier used to introduce a target gene into a cell or nucleus.

[0021] In this specification, "plant body" means the entire plant, including all its tissues (roots, stems, leaves).

[0022] 2. Polynucleotides In one embodiment, the present invention relates to (a) or (b) below: (a) The coding sequence of the amino acid sequence shown in SEQ ID NOs: 13, 18, or 23, or (b) A coding sequence for a protein having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 13, 18, or 23, and which has the effect of improving the drought stress tolerance of plants. This invention relates to isolated polynucleotides containing (which may be referred to herein as "the polynucleotides of the present invention").

[0023] In (b) above, the identity is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

[0024] (b) An example of a code array is: (b1) A coding sequence for a protein having the effect of improving the drought stress tolerance of plants, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted to the amino acid sequence shown in Sequence ID No. 13. (b2) A coding sequence for a protein having the effect of improving the drought stress tolerance of plants, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted to the amino acid sequence shown in Sequence ID No. 18, and (b3) A coding sequence for a protein having the effect of improving the drought stress tolerance of plants, consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted to the amino acid sequence shown in Sequence ID No. 23. These are some examples.

[0025] In (b1) above, "multiple" means, for example, 2 to 84, preferably 2 to 56, more preferably 2 to 42, even more preferably 2 to 28, even more preferably 2 to 14, particularly preferably 2 to 5, and most preferably 2.

[0026] In (b2) above, "multiple" means, for example, 2 to 52, preferably 2 to 35, more preferably 2 to 26, even more preferably 2 to 17, even more preferably 2 to 8, particularly preferably 2 to 3, and most preferably 2.

[0027] In (b3) above, "multiple" means, for example, 2 to 84, preferably 2 to 56, more preferably 2 to 42, even more preferably 2 to 28, even more preferably 2 to 14, particularly preferably 2 to 5, and most preferably 2.

[0028] The above amino acid substitutions are preferably conservative substitutions.

[0029] The presence and extent of an effect that improves the drought stress tolerance of plants can be evaluated by examining the survival of the target plants when subjected to drought stress. Specifically, this can be evaluated by subjecting at least 10 target plants to a water deprivation treatment of 1 to 4 weeks, followed by a re-irrigation treatment of 1 to 5 days, and then examining the survival rate of the target plants afterward. For example, in the case of Arabidopsis thaliana, at least 10 4-week-old plants can be subjected to a water deprivation treatment of 2 weeks and a re-irrigation treatment of 2 days, and the survival rate can be calculated from the number of surviving plants afterward. The duration of the water deprivation treatment and re-irrigation treatment can be appropriately determined according to the target plant species and the growth stage of the target plants, within a range where the survival rate in the control group is 35% or less.

[0030] The polynucleotides of the present invention are derived from the genus Chloris, preferably from Chloris virgata.

[0031] The polynucleotides of the present invention exhibit high expression in heterologous plants. In this specification, "high expression in heterologous plants" means that when a polynucleotide is introduced into a plant of a different species (heterologous plant) from which it originates, the polynucleotide is highly expressed in the heterologous plant. Specifically, this means that the expression level of the introduced polynucleotide in the transformed Arabidopsis thaliana obtained by introducing the polynucleotide into wild-type Arabidopsis thaliana according to the method for producing transformed Arabidopsis thaliana shown in Test Example 5-1 below is 100 times or more, preferably 500 times or more, more preferably 1000 times or more, even more preferably 2000 times or more, even more preferably 3000 times or more, and particularly preferably 4000 times or more, the expression level of the polynucleotide in wild-type Arabidopsis thaliana.

[0032] The polynucleotide-encoded proteins of the present invention have the effect of improving the drought stress tolerance of plants. This effect is due, for example, to the fact that the polynucleotide-encoded proteins of the present invention directly and / or indirectly activate signaling related to drought stress tolerance, and / or directly and / or indirectly activate (promote) ABA biosynthesis and / or ABA signaling, etc., although this is not limited to the following logic.

[0033] In one embodiment, the polynucleotide of the present invention preferably includes a promoter upstream of the coding sequence of (a) or (b).

[0034] The promoters are not particularly limited and include, for example, promoters for constitutive gene expression in plant cells, such as the cauliflower mosaic virus (CaMV) 35S promoter, the nopalin synthase (NOS) gene promoter, and the ubiquitin promoter. In addition, promoters for transient gene expression in plant cells can also be used, such as drought-induced promoters, such as the rd29A gene promoter, the Oshox2 promoter, the LIP9 promoter, and the OsNAC6 promoter, as well as promoters for tissue-specific and / or time-specific gene expression.

[0035] The polynucleotide of the present invention may contain other elements in addition to the promoter described above. Other elements include, for example, terminators (e.g., CaMV-derived terminators, NOS gene-derived terminators, etc.), intron sequences that have the function of enhancing gene expression (e.g., intron sequences near the promoter, etc.), and selection markers for efficiently selecting the cells or plants of the present invention as described later (e.g., drug resistance genes).

[0036] In one embodiment, the polynucleotide of the present invention is preferably a vector.

[0037] The morphology of the vector is not particularly limited and may be linear or circular. Examples of specific vector forms include plasmid vectors, Agrobacterium vectors, and plant virus vectors.

[0038] The base length of the polynucleotide of the present invention is not particularly limited. From the viewpoint of enhancing the functional stability of the polynucleotide of the present invention, the base length of the polynucleotide of the present invention is, for example, 372 bases or more, preferably 423 bases or more, more preferably 450 bases or more, even more preferably 477 bases or more, even more preferably 504 bases or more, particularly preferably 519 bases or more, and most preferably 522 bases or more. Furthermore, from the viewpoint of enhancing the introduction efficiency to the target, the base length of the polynucleotide of the present invention is, for example, 55,000 bases or less, more preferably 50,000 bases or less, even more preferably 45,000 bases or less, even more preferably 40,000 bases or less, particularly preferably 35,000 bases or less, and most preferably 30,000 bases or less.

[0039] When the coding sequence in (b) above is the coding sequence in (b1) above, the polynucleotide of the present invention is, from the viewpoint of enhancing the functional stability of the polynucleotide of the present invention, for example, 594 bases or longer, preferably 678 bases or longer, more preferably 720 bases or longer, even more preferably 762 bases or longer, even more preferably 804 bases or longer, particularly preferably 831 bases or longer, and most preferably 840 bases or longer. Furthermore, from the viewpoint of enhancing the introduction efficiency to the target, the base length of the polynucleotide of the present invention is, for example, 55,000 bases or less, more preferably 50,000 bases or less, even more preferably 45,000 bases or less, even more preferably 40,000 bases or less, particularly preferably 35,000 bases or less, and most preferably 30,000 bases or less.

[0040] When the coding sequence in (b) above is the coding sequence in (b2) above, the polynucleotide of the present invention is, from the viewpoint of enhancing the functional stability of the polynucleotide of the present invention, for example, 372 bases or longer, preferably 423 bases or longer, more preferably 450 bases or longer, even more preferably 477 bases or longer, even more preferably 504 bases or longer, particularly preferably 519 bases or longer, and most preferably 522 bases or longer. Furthermore, from the viewpoint of enhancing the introduction efficiency to the target, the base length of the polynucleotide of the present invention is, for example, 55,000 bases or less, more preferably 50,000 bases or less, even more preferably 45,000 bases or less, even more preferably 40,000 bases or less, particularly preferably 35,000 bases or less, and most preferably 30,000 bases or less.

[0041] When the coding sequence in (b) above is the coding sequence in (b3) above, the polynucleotide of the present invention is, from the viewpoint of enhancing the functional stability of the polynucleotide of the present invention, for example, 588 bases or longer, preferably 672 bases or longer, more preferably 714 bases or longer, even more preferably 756 bases or longer, even more preferably 798 bases or longer, particularly preferably 825 bases or longer, and most preferably 834 bases or longer. Furthermore, from the viewpoint of enhancing the introduction efficiency to the target, the base length of the polynucleotide of the present invention is, for example, 55,000 bases or less, more preferably 50,000 bases or less, even more preferably 45,000 bases or less, even more preferably 40,000 bases or less, particularly preferably 35,000 bases or less, and most preferably 30,000 bases or less.

[0042] 3. Plant drought stress tolerance enhancers In one embodiment, the present invention relates to a plant drought stress tolerance enhancer comprising the polynucleotide of the present invention (which may also be referred to herein as "the drought stress tolerance enhancer of the present invention").

[0043] One of the features of the drought stress tolerance enhancer of the present invention is that when the protein encoded by the polynucleotide of the present invention is expressed in cells, the drought stress tolerance of plants containing those cells is enhanced.

[0044] The drought stress tolerance enhancer of the present invention may consist solely of the polynucleotide (essential component) of the present invention, or it may contain various other components in addition to the essential component, depending on how the essential component is used. The content ratio of the essential component (dry weight) in the drought stress tolerance enhancer of the present invention can be appropriately determined depending on the manner of use, etc., but for example, a range of 0.0001 to 100% by mass can be exemplified. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc. The form of the drought stress tolerance enhancer of the present invention is not particularly limited and can be, for example, in a dry form, a solution form, etc., or it may also be in kit form. The kit may optionally contain nucleic acid introduction reagents, buffers, and other materials, reagents, equipment, etc. necessary for the production of plant bodies.

[0045] 4.Cells In one embodiment, the present invention relates to cells containing the polynucleotide of the present invention (which may be referred to herein as "cells of the present invention").

[0046] In the cells of the present invention, the polynucleotides of the present invention are exogenous. Here, "exogenous" means that the polynucleotides of the present invention are base sequences that are not present in the wild-type genomic DNA of the cells of the present invention.

[0047] In the cells of the present invention, "containing" the polynucleotide of the present invention means that the polynucleotide of the present invention may be incorporated into the cell genome, or it may exist separately from the cell genome.

[0048] The cells of the present invention may be eukaryotic cells or prokaryotic cells. Specific examples of the cells of the present invention include bacterial cells such as Escherichia coli, plant cells, and fungal cells.

[0049] In one embodiment, the cells of the present invention are plant cells capable of regenerating plant bodies with enhanced drought stress tolerance. Plant cells include, for example, cultured cells, callus, protoplasts, and leaf sections.

[0050] 5.Plants In one embodiment, the present invention relates to a plant containing the cells of the present invention (which may be referred to herein as "the plant of the present invention").

[0051] The proportion of cells of the present invention in the plant of the present invention is not particularly limited, and from the viewpoint of increasing the drought stress tolerance of the plant of the present invention, for example, it is 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90%, particularly preferably 95%, and most preferably 100%, relative to 100% of the total number of cells in the plant of the present invention.

[0052] In the plant of the present invention, the tissue containing the cells of the present invention is not particularly limited, but from the viewpoint of increasing the drought stress tolerance of the plant of the present invention, leaves, stems, roots, vascular bundles, etc., are preferred.

[0053] The plants of the present invention are not particularly limited. Examples of plants include a wide range of plants, such as angiosperms (magnolias), monocots, and eudicots (roses I, roses II, chrysanthemums I, chrysanthemums II, and their outgroups). Cultivated varieties are particularly preferred as plants. Specific examples of plants include tomatoes, bell peppers, chili peppers, eggplants, cucumbers, pumpkins, melons, watermelons, and other gourds, cabbage, broccoli, Chinese cabbage and other leafy greens, celery, parsley, lettuce and other raw or spicy vegetables, leeks, onions, garlic and other alliums, soybeans, peanuts, green beans, peas, adzuki beans and other legumes, strawberries and other fruit vegetables, taproots such as radishes, turnips, carrots, and burdock, tubers such as taro, cassava, potatoes, sweet potatoes, and yams, tender vegetables such as asparagus, spinach, and Japanese parsley, flowers such as lisianthus, stock, carnations, and chrysanthemums, and grains such as rice, wheat, barley, oats, and corn. Examples include grasses such as bentgrass and Korean lawn grass, oil crops such as rapeseed and peanuts, sugar crops such as sugarcane and sugar beets, fiber crops such as cotton and rushes, fodder crops such as clover, sorghum and dent corn, deciduous fruit trees such as apples, pears, grapes and peaches, citrus fruits such as Satsuma mandarins, lemons and grapefruits, woody plants such as azaleas, rhododendrons and cedars, and nuts such as almonds, hemp, flax, perilla, cashews, pumpkins, nutmeg, ginkgo, chestnuts, walnuts, poppies, coconuts, sesame, oak, watermelon, chia, horse chestnut, lotus, watermelon, pistachios, sunflowers, Brazil nuts, hazelnuts, pecans, macadamia nuts, pine and peanuts.

[0054] The plants of the present invention include plants containing cells into which the polynucleotides of the present invention have been introduced by the method described later, and plants into which the polynucleotides of the present invention have been introduced at target sites in the genome by genome editing technology.

[0055] 6. Methods to improve plant drought stress tolerance In one embodiment, the present invention relates to a method for improving the drought stress tolerance of a plant by introducing the polynucleotide of the present invention into the plant (which may be referred to herein as "the method of the present invention").

[0056] In the method of the present invention, the method for introducing polynucleotides into plants is not particularly limited as long as the introduced substance can reach the plant cells, and can be appropriately selected depending on the introduced substance and the target of introduction. Examples of introduction methods include the Agrobacterium method, polyethylene glycol method, electroporation method, particle gun method, and the like.

[0057] The target of introduction is not particularly limited and may be the entire plant, some organs of a mature plant (e.g., leaves, petals, stems, roots, seeds, etc.), some tissues of a plant (e.g., meristematic tissue such as the shoot apex), or undifferentiated plant tissue (e.g., callus, etc.).

[0058] 7. Method for producing plants with drought stress tolerance In one embodiment, the present invention relates to a method for producing drought-stress-tolerant plants, comprising the step of introducing the polynucleotide of the present invention into the plants (which may be referred to herein as "the production method of the present invention").

[0059] In the manufacturing method of the present invention, the method for introducing polynucleotides into plants is as described above.

[0060] In the manufacturing method of the present invention, confirmation that the polynucleotide of the present invention has been introduced into a plant can be performed by extracting DNA from the cells, tissues, plant body, etc. of the plant according to a conventional method and detecting the introduced polynucleotide using a known PCR method. [Examples]

[0061] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0062] 1. The Mongolian grassland plant Chloris virgata exhibits drought stress tolerance. 1-1. Drying process To analyze the drought stress tolerance of C. virgata, the plants were subjected to drought treatment, and the effects were investigated. Rice (Oryza sativa L. cv. Nipponbare), oats (Avena sativa L. Niron 61), and wheat (Triticum aestivum L. cv. No.61) were used as control plants.

[0063] The drying treatment of C. virgata, rice, oats, and wheat was carried out as follows: C. virgata seeds were collected from grasslands native to Dornogovi Province, Mongolia. The seeds of C. virgata, rice, oats, and wheat were sterilized with 5% sodium chloride and 0.07% Tween®-20 for 20 minutes, and then washed three times with sterile water for 10 minutes each. After sterilization, the seeds were sown on 1 / 2 Murashige and Skoog basal (Duchefa-Biochemie, Netherlands) plates containing 1.5% sucrose and 0.9% phytoagar (Duchefa-Biochemie, Netherlands). After culturing in the dark at 4°C for 2 days, the seeds were cultured at 22°C with a photoperiod of 16 hours light / 8 hours dark to grow plants. Seedlings grown in 1 / 2 MS medium at 7 days old were transplanted into soil and grown for 3 weeks with sufficient watering. The seedlings that were transplanted into the soil were withheld water for three weeks, and then watered again for five days.

[0064] Figure 1A shows the results of subjecting C. virgata to a drying treatment. After water withholding, almost all leaves of rice, oats, and wheat wilted severely and turned brown, while the leaves of C. virgata remained fresh and green. After re-irrigation, leaf growth and greening of rice, oats, and wheat were limited, while only C. virgata recovered and produced further green leaves.

[0065] 1-2. Mannitol treatment To analyze the osmotic stress tolerance of C. virgata, the plants were subjected to mannitol treatment, and its effects were investigated. Rice and oats were used as control plants.

[0066] Mannitol treatment was carried out as follows: Seven-day-old seedlings of C. virgata, rice, and oats, grown using the method described in 1-1 above, were transplanted into 1 / 2 MS medium with or without mannitol at the concentrations shown in Figure 1B, and grown for three weeks.

[0067] Figure 1B shows the results of treating C. virgata with mannitol. Similar to the drying treatment results shown in 1-1 above, the leaves of mannitol-treated rice and oats were brown, while the leaves of C. virgata were green.

[0068] Since mannitol treatment affected the leaf color of the plants, the endogenous chlorophyll content of the plants was measured. The endogenous chlorophyll content of the plants was measured as follows: The above-ground parts of the plants were homogenized in liquid nitrogen using a BEADS CRUSHER μT-01 (Tytec Co., Ltd., Japan), added to 10 mL of 80% v / v acetone, incubated on ice in the dark for 10 minutes, and then centrifuged at 13,000 rpm for 10 minutes at 4°C. The chlorophyll content of the centrifugated supernatant was measured at 646.6 nm, 663.6 nm, and 750 nm using a UV / VIS spectrophotometer Biochrom Ultrospec® 2100 Pro (Harvard Bioscience, USA).

[0069] Figure 1C shows the results of measuring the endogenous chlorophyll content of plants. The chlorophyll content of rice and oats grown in media supplemented with 300 mM or 350 mM mannitol was significantly reduced compared to the chlorophyll content of rice and oats grown in control media without mannitol (0 mM) (Figure 1C). Although the growth of C. virgata was slightly inhibited by mannitol treatment (Figure 2), C. virgata maintained a high chlorophyll content even in 300 mM or 350 mM mannitol media, which was comparable to the chlorophyll content of C. virgata in the control medium (Figure 1C).

[0070] 2. Expression analysis of drought-induced genes in C. virgata To investigate the molecular function of C. virgata under drought stress conditions, C. virgata was subjected to drought treatment, and the expression levels and duration of known drought-induced genes in C. virgata were analyzed. The genes used as known drought-induced genes were the gene encoding NCED3 (CvNCED3), an abscisic acid (ABA) biosynthesis enzyme, and the gene encoding AREB1 / ABF2 (CvAREB1), an ABA-responsive transcription factor.

[0071] The drying treatment of C. virgata was performed as follows: Seven-day-old C. virgata seedlings grown using the method described in 1-1 above were transplanted into empty Parafilm or 1 / 2 MS medium and treated for 1 hour, 3 hours, 6 hours, 12 hours, or 24 hours. Samples transplanted into empty Parafilm were used as the drying treatment samples, and samples transplanted into 1 / 2 MS medium were used as the control samples.

[0072] The expression levels of drought-induced genes were measured as follows: Total RNA was extracted from approximately 50 mg of plant tissue using the Rneasy® Plant Mini Kit (Qiagen, Germany). First-strand cDNA was synthesized from 0.5 μg of isolated RNA using the PrimeScript® RT Reagent Kit (Takara Bio Inc., Japan). qRT-PCR was performed using TB Green® Premix Ex Taq II (Takara Bio Inc., Japan) according to the instructions for the Thermal Cycler Dice Real Time System III. The primer pairs shown in SEQ ID NOs: 1-4 were used for qRT-PCR.

[0073] The results are shown in Figure 3. CvNCED3 expression induction began 1 hour after drying, and the expression level of CvNCED3 was highest 6 hours after drying. The expression level of CvNCED3 6 hours after drying was approximately 28 times higher than that of the control (Figure 3A). Similarly, CvAREB1 expression induction began 1 hour after drying, and the expression level of CvAREB1 was highest 6 hours after drying. The expression level of CvAREB1 6 hours after drying was approximately 74 times higher than that of the control (Figure 3B). The expression levels of both CvNCED3 and CvAREB1 genes decreased 12 hours after drying (Figures 3A, B).

[0074] 3. De novo transcriptome assembly of dried C. virgata To analyze the detailed molecular mechanisms of drought stress tolerance in C. virgata and to identify key genes contributing to drought stress tolerance in C. virgata, we performed de novo RNA-seq analysis.

[0075] De novo RNA-seq analysis was performed as follows: Only shoots were collected from dried samples (hereinafter referred to as the "3-hour drying group" and the "6-hour drying group") and control samples obtained using the method shown in Test Example 2 (drying time of 3 hours or 6 hours), and the roots were removed. Total RNA was extracted from the obtained plant samples using the method shown in Test Example 2. Three independent RNA samples were used as biological replicates. Approximately 1.5-2 μg of RNA was used to construct an mRNA-Seq library using the NextFlex® Rapid Directional RNA-seq Library Prep Kit (Perkin-Elmer, USA), and the library was sequenced using DNBSEQ-T7 (MGI, China). The raw read dataset was submitted to the DNA Data Bank of Japan Sequence Read Archive (DDJB SRA) with accession number PRJDB18577. Raw RNA-seq reads were trimmed using Trimomatic (v0.39) with the parameters: PE -threads 70 -phred33 SLIDINGWINDOW:4:15 LEADING:20 TRAILING:20 MINLEN:50. Quality-checked reads were de novo assembled using Trinity (v2.8.5) with default parameters. The assembled sequences were deredacted using the pcap module of PBSuite (v15.8.24) with the parameters: autopcap -m 1900 -y 80 -t 80. Furthermore, clustering was performed using CD-HIT-EST (v4.8.1) with the parameters: -T 50 -c 0.8 -d 50 -M 1500, and the longest sequence was selected as representative of each cluster. Sequences with high coding ability were identified using Transdecoder (v5.5.0).

[0076] A total of 1,307,170,602 raw sequence reads were generated from the cDNA of C. virgata, with 1,281,678,077 being filtered reads. The quality-checked reads were newly assembled using Trinity, resulting in 256,330 contigs with an N50 value of 2,366 bp. These contigs were assembled into 46,628 supercontigs with an N50 value of 2,723 bp using the PCAP tool, and clustering using the CD-HIT-EST tool yielded 31,149 transcript clusters with an N50 value of 2,681 bp. Next, the longest transcripts in each cluster were subjected to open reading frames, identifying 25,469 protein-coding transcripts, which were used as a reference transcript dataset for downstream analysis. Of the reference transcripts, 22,683 and 21,501 were homologous to protein-coding genes in rice and Arabidopsis thaliana, respectively (Table 1).

[0077] [Table 1]

[0078] 4. Functional annotation of genes expressed during the drying period in C. virgata To analyze the genetic characteristics of up-expression genes identified by RNA-seq analysis of dried C. virgata, reference transcripts obtained from C. virgata transcriptome assemblies were evaluated using homology searches and Gene Ontology (GO) enrichment analysis.

[0079] Homology searches and GO enrichment analyses were performed as follows: To assess coding potential, BLASTp searches (using BLAST v2.10.1+) were performed against the Swiss-Prot database, and domain searches (using HMMER v3.3.1) were performed against the Pfam-A database. Gene expression levels were quantified by mapping quality-checked reads to this reference set using Salmon (v1.9.0), and the number of reads per transcript was calculated using the salmon quant function to determine expression levels. In differential expression analysis, upregulatory genes were defined as genes with an absolute change greater than 1.5 times, and p-values ​​of full statistical tests were converted to adjusted p-values ​​less than 0.05 using the DEseq2 package (version 1.40.2). A Venn diagram was created using the Gplots package (version 3.1.3), GO enrichment analysis was performed using the ClusterProfiler package (version 4.8.1, based on the org.At.tair.db annotation), and the results were visualized in the R studio environment (version 2023.09.0+463).

[0080] The results are shown in Figure 4. Compared to the control sample, 1182 genes and 1198 genes were highly expressed in the 3-hour drying group and the 6-hour drying group, respectively. Of these, 1161 genes were expressed in both the 3-hour and 6-hour drying groups (Figure 4A). For detailed analysis of expressed genes, the expressed genes in the drying samples were divided into three clusters: genes that were relatively highly expressed in the 3-hour drying group (Cluster 1, 21 upregulatory genes), genes that were relatively highly expressed in the 6-hour drying group (Cluster 2, 37 upregulatory genes), and genes that were expressed in both the 3-hour and 6-hour drying groups (Cluster 3, 1161 upexpressed genes) (Figure 4A). As a result of GO enrichment analysis, 139 GO terms were annotated in Cluster 3 and 2 in Cluster 1. No GO terms were annotated in Cluster 2 due to the small number of identified genes. The top 25 GO terms were identified in cluster 3 (Figure 4B). In cluster 3, several GO terms related to the ABA pathway and direct drought stress responses were identified, including "cellular response to ABA stimulation," "abscisic acid-activated signaling pathway," "seed germination," "stomatal movement," and "response to drought." Therefore, ABA may be highly regulated after 3 and 6 hours of drought treatment.

[0081] For all 25,469 ORFs of C. virgata predicted by de novo RNA-seq analysis, the expression levels of each gene were compared in dried samples and control samples, and the expression induction ratios for all genes were ranked in a list. The highest log2-fold changes were identified as approximately 22 log2-fold to 7 log2-fold changes for the top 60 genes in the 3-hour treatment group, and approximately 30 log2-fold to 10 log2-fold changes for the top 60 genes in the 6-hour treatment group.

[0082] 5. Arabidopsis thaliana overexpressing drought-inducible and drought-stress-tolerant genes derived from *Arabidopsis virgata* exhibits drought-stress tolerance. 5-1. Creation of transgenic Arabidopsis thaliana overexpressing drought-inducible and drought-stress-tolerant genes derived from C. virgata. To confirm the technical feasibility of genetic manipulation of Arabidopsis thaliana using genes derived from C. virgata and to analyze the functions of C. virgata homologs (CvNCED3 and CvAREB1) of drought-inducible and drought-stress-tolerant genes in Arabidopsis thaliana, the following experiments were conducted. The cDNAs of CvNCED3 and CvAREB1 were fused under the cauliflower mosaic virus 35S promoter (CaMV35Spro), and the constructed vectors were transformed into wild-type Arabidopsis thaliana (Arabidopsis thaliana ecotype Columbia) using the Agrobacterium system. The expression of the transgenes in transformed Arabidopsis thaliana (CvNCED3 overexpression (CvNCED3-OX) and CvAREB1 overexpression (CvAREB1-OX)) was examined by qRT-PCR (Figures 5A and 5B). Gene expression levels were measured using the same method as in Experiment 2.

[0083] The following procedure was used to create transformed Arabidopsis thaliana. The full-length coding region of the target gene was amplified using the primer set shown in Sequence IDs 5-8. The amplified fragment was introduced into the pENTR / D-TOPO vector (Thermo Fisher Scientific, USA), and subsequently cloned according to Gateway® (Thermo Fisher Scientific, USA) into the binary vector pGWB2 containing the CaMV35S promoter (Nakagawa et al., Biosci. Biotechnol. Biochem., 71(8), 2095-2100 (2007)). To create GFP-transformed plants, the amplified fragment was cloned, inserted into the pENTR / D-TOPO vector, and subsequently cloned according to Gateway into the binary vector pGWB6 containing the CaMV35S promoter. Plasmid fusion constructs were transformed into Arabidopsis thaliana via the Agrobacterium tumefaciens strain GV3101:pMP90 using the floral dip method (Clough & Bent, Plant J., 16(6), 735-743 (1998)). Transformants were screened in 1 / 2 MS medium supplemented with 25 mg / L hygromycin. T3 homozygous plants were used in all experiments.

[0084] 5-2. Drying treatment of transformed Arabidopsis thaliana Transgenic Arabidopsis thaliana produced in 5-1 was subjected to a drying treatment in soil. Wild-type Arabidopsis thaliana (hereinafter sometimes referred to as "wild-type") was used as a control. The drying treatment of Arabidopsis thaliana in soil was carried out as follows: Seedlings at 4 weeks old were grown in sufficiently moist soil, then watering was withheld for 14 days, followed by rewatering for 2 days. Survival rate, chlorophyll content, and fresh weight were calculated after rewatering. All pots were placed in a growth chamber in a random order, and the position of each pot was changed randomly daily to avoid the influence of position. Each experiment was repeated at least three times.

[0085] The results are shown in Figure 6. After drying, the rosette leaves of the wild type turned yellow and withered, and some died. Compared to the brown or yellow leaves of the wild type, the CvNCED3 overexpression and CvAREB1 overexpression groups exhibited relatively green and fresh rosette leaves. After re-irrigation, 67-75% of the CvNCED3 overexpression groups and 100% of the CvAREB1 overexpression groups survived, while 0% of the wild type survived (Figure 6A, D). Quantitative analysis showed that the proportion of plants with a fresh weight of 500 mg or more was significantly higher in the CvAREB1 overexpression groups than in the wild type (Figure 6D), while the proportion of plants with a fresh weight of 500 mg or more was slightly lower in the CvNCED3 overexpression groups than in the wild type (Figure 6B). This result is thought to be due to the CvNCED3 overexpression groups slowing growth under normal conditions. These results suggest that overexpression of the CvNCED3 gene or the CvAREB1 gene in Arabidopsis thaliana can enhance its tolerance to drought stress.

[0086] 6. Identification and analysis of novel genes involved in drought stress tolerance in C. virgata To identify novel genes contributing to improved drought stress tolerance in C. virgata, ten C. virgata cDNAs were selected from the 60 genes that were most highly induced 3 and 6 hours after drought treatment in Test Example 4. These C. virgata cDNAs were fused under the CaMV35S promoter, and the constructed vectors were used to transform wild-type Arabidopsis thaliana. The T3 generation of transformed Arabidopsis thaliana introduced with C. virgata cDNA was subjected to drought treatment, and the drought stress tolerance of each transformant was observed. The drought treatment of Arabidopsis thaliana was performed as follows: 10-day-old seedlings grown in 1 / 2 MS medium were transferred to empty Parafilm and treated for the specified time. As a result, three novel genes were identified: Mongolian Grassland plant Drought-stress resistance gene 1 (MGD1), Mongolian Grassland plant Drought-stress resistance gene 2 (MGD2), and Mongolian Grassland plant Drought-stress resistance gene 3 (MGD3). The results of the analysis of these three novel genes are shown below.

[0087] 6-1. Analysis of MGD1 6-1-1. Analysis of MGD1 mRNA expression levels under drought stress conditions To analyze the mRNA expression level of MGD1 under drought stress conditions in C. virgata, C. virgata was dried using the method shown in Test Example 2, and the mRNA expression level of MGD1 was measured. The primer pairs shown in SEQ ID NOs: 9 and 10 were used to measure the mRNA expression level of MGD1. The results are shown in Figure 7A. The mRNA expression level of MGD1 in dried C. virgata was approximately 7 times higher under 3 hours of drying and approximately 12 times higher under 6 hours of drying compared to undried C. virgata (control) (Figure 7A).

[0088] 6-1-2. Phylogenetic tree construction of MGD1 and related proteins in other species Figure 7B shows a phylogenetic tree of MGD1 and related proteins in other species. The phylogenetic tree was constructed as follows: Based on the amino acid sequence of MGD1, homologous genes were searched using BLASTP with the NCBI reference protein (refseq_protein) and MarpolBase's MpTak1_v5.1 (Montgomery et al., Curr. Biol., 30(4), 573-588 (2020)). Based on amino acid length and identity, among the candidate genes identified by BLASTP, genes with an e-value of 1e-16 or less were determined to be MGD1 homologous genes. The amino acid sequences of MGD1 homologous genes were aligned using MAFFT (Katoh et al., Brief. Bioinform. 20(4), 1160-1166 (2019)). The "automatic" setting was used in MAFFT. A phylogenetic tree was generated using IQ-TREE (Trifinopoulos et al., Nucleic. Acids. Res., 44(W1), W232-W235 (2016)) from alignment data generated by MAFFT, and the tree file obtained from IQ-TREE was corrected using iTOL (Letunic & Bork, Nucleic. Acids. Res., 49(W1), W293-W296 (2021)). MGD1 encodes a novel gene conserved in homologous genes of various land plants, but detailed functional analyses of these genes have not yet been reported (Figure 7B).

[0089] 6-1-3. MGD1-overexpressing Arabidopsis thaliana exhibits higher drought stress tolerance than the wild type. The drought stress tolerance of two independent transgenic Arabidopsis thaliana plants overexpressing MGD1 (MGD1-OX) OX1 and OX-2 was analyzed compared to that of the wild type. MGD1 overexpressions were created using the same method as in Experiment 5-1. The primer pair shown in SEQ ID NOs. 11 and 12 was used to amplify the full-length coding region of MGD1. qRT-PCR revealed that the expression level of the MGD1 gene was significantly higher in the MGD1 overexpressions (Figure 5C). When the MGD1 overexpressions were cultivated in soil with moderate watering for 4 weeks, the leaf phenotypes of the MGD1 overexpressions and wild types were similar (Figure 7C). Compared to the wild type, the MGD1 overexpressions showed significantly higher drought stress tolerance, with fewer wilted leaves after watering. After re-watering, almost all wild-type leaves wilted and plant growth was restricted, while the MGD1 overexpressions recovered and produced fresh green leaves. While the survival rate of the wild type was 0%, the survival rates of MGD1 overexpressing plants OX-1 and OX-2 were 75% and 100%, respectively (Figure 7C). Furthermore, the MGD1 overexpressing plants showed a larger fresh weight than the wild type after re-irrigation (Figure 7D). These results suggest that the MGD1 gene improves drought stress tolerance in Arabidopsis thaliana.

[0090] 6-1-4. Analysis of the MGD1 protein The MGD1 protein consists of 282 amino acids (Figure 8A, Sequence ID No. 13). Figure 8A shows the results of multiple sequence alignment analysis of the amino acid sequences of MGD1 and homologs of other plant species. The results of the multiple sequence alignment analysis were visualized using ESPript 3.0 (Robert & Gouet, Nucleic. Acids. Res., 42(W1), W320-W324 (2014)). The MGD1 protein showed high similarity to homologs of monocotyledonous plants such as T. aestivum (XP_044345791.1, 64.78%), O. sativa (XP_015622376.1, 64.35%), and B. distachyon (XP_003567507.1, 63.53%). The similarity to homologs of dicotyledonous plants such as N. tabacum (XP_016510532.1, 50.81%) and A. thaliana (NP_001330291.1, 48.91%) was lower than the similarity to homologs of monocotyledonous plants.

[0091] The results of the structural prediction analysis of the MGD1 protein are shown in Figure 8A. For protein sequence structure prediction, AlphaFold3 (Abramson et al., Nature., 630(8016), 493-500 (2024)), XtalPred (Slabinsk et al., Bioinformatics, 23(24), 3403-5 (2007)), InterPro (Paysan-Lafosse et al., Nucleic. Acids. Res., 51(D1), D418-27 (2023)), deepTMHMM(http: / / biorxiv.org / lookup / doi / 10.1101 / 2022.04.08.487609), TOPCONS(Tsirigos et al., Nucleic. Acids. Res., 43(W1), W401-7 (2015)) and SOSUI (Hirokawa et al., Bioinformatics, The analysis was performed using the NovoPro server (14(4), 378-9 (1998)). Predictions using AlphaFold3 were performed using high-performance computing resources to handle the model's enormous computational demands. The quality of the predicted structures was assigned by the Predictive Local Distance Difference Test (plDDT) score (Mariani et al., Bioinformatics, 29(21), 2722-8 (2013)), which indicates the reliability of the model's predictions. The structures were further analyzed and validated to ensure the reliability of the predictions. Protein hydropathy analysis was performed using the NovoPro server, a tool for evaluating the hydrophobicity and hydrophilicity of the entire protein sequence, with default settings (https: / / www.novoprolabs.com / tools / protein897hydropathy). Hydropathy profiles were measured and calculated using the Kyte-Doolittle scale (Kyte & Doolittle, J. Mol. Biol., 157(1), 105-2 (1982)). The results were further analyzed and interpreted, highlighting important hydrophobic and hydrophilic regions.

[0092] The coiled-coil region of the MGD1 protein was predicted by the XtalPred and InterPro servers to be between amino acid residues 56 and 128, and was highly conserved between monocots and dicots (Figure 8A (shown as a green line)). According to the AlphaFold3 server, the MGD1 protein contains one long α-helix structure at the N-terminus and three short α-helix structures at the C-terminus (Figure 8A (shown as a black line), Figure 9B). Furthermore, the deepTMHMM and TOPCONS servers predicted the possibility of a transmembrane region at the N-terminus (Figure 8A (shown as a blue line)). The predicted transmembrane region corresponded to and overlapped with the α-helix structure and the hydrophobic region of MGD1, which were predicted with high confidence (pIDDT score of 70 or higher) (Figure 9A, B). We also modeled the MGD1 dimer using the AlphaFold3 server, and the results suggested that the coiled-coil region functions as a dimer interface, forming a coiled-coil superhelix between two MGD1 molecules (Figure 9C).

[0093] To elucidate the intracellular localization of the MGD1 protein, transformed Arabidopsis thaliana expressing a fusion protein of MGD1 protein and green fluorescent protein (GFP) under the CaMV35S promoter were created. Root cells of 4-week-old transformants were observed, and the GFP fluorescence signal was detected. The GFP fluorescence signal was detected using a Zeiss LSM 700 confocal laser scanning microscope (Zeiss, Germany). The GFP fluorescence signal was observed as a reticular and punctate signal in the cytoplasm, particularly around the nucleus, suggesting localization to the endoplasmic reticulum or Golgi apparatus (Figure 8B).

[0094] 6-2. Analysis of MGD2 6-2-1. Analysis of MGD2 mRNA expression levels under drought stress conditions Similar to Test Example 6-1-1, the mRNA expression level of MGD2 in C. virgata under drought stress conditions was measured. The primer pair shown in SEQ ID NOs. 14 and 15 was used to measure MGD2 mRNA expression. The results are shown in Figure 10A. The mRNA expression level of MGD2 in C. virgata subjected to drought treatment was approximately 10 times higher under 3 hours of drought treatment and 56 times higher under 6 hours of drought treatment compared to C. virgata not subjected to drought treatment (control).

[0095] 6-2-2. Phylogenetic tree construction of MGD2 and related proteins in other species Similar to Test Example 6-1-2, a phylogenetic tree of MGD2 and related proteins in other species was constructed. The results are shown in Figure 10B. Phylogenetic analysis revealed that the MGD2 gene is conserved in homologous genes in monocots and dicots, but the detailed functions of these homologous genes have not yet been reported.

[0096] 6-2-3. MGD2-overexpressing Arabidopsis thaliana exhibits higher drought stress tolerance than the wild type. Similar to Experiment 6-1-3, two independent transgenic Arabidopsis thaliana plants (MGD2-overexpressing plants (MGD2-OX)) OX-1 and OX-2 were created and their drought stress tolerance was analyzed. The primer pairs shown in SEQ ID NOs. 16 and 17 were used to amplify the full-length coding region of MGD2. qRT-PCR revealed that the expression level of the MGD2 gene was significantly higher in the MGD2-overexpressing plants (Figure 5C). Only 17% of the wild-type plants survived, while 100% of the MGD2-overexpressing plants survived and continued to grow after re-irrigation (Figure 10C). The fresh weight of the MGD2-overexpressing plants was higher than that of the wild-type plants after re-irrigation (Figure 10D).

[0097] 6-2-4. Analysis of the MGD2 protein The MGD2 protein contains 177 amino acids (Figure 11A, Sequence ID 18). Sequence alignment analysis was performed to analyze the similarity of MGD2's amino acid sequence to that of other common plant species (Figure 11A). MGD2 showed high similarity to homologs of monocotyledonous plants such as B. distachyon (XP_003577510.1, 76.92%), T. aestivum (XP_044420749.1, 76.24%), and O. sativa (XP_001410341.1, 74.05%). Furthermore, the similarity to dicotyledonous plant proteins such as N. tabacum (XP_016443868.1, 53.89%) and A. thaliana (NP_196350.1, 49.75%) was lower than the similarity to monocotyledonous plant proteins, which was consistent with the results of the constructed polygene tree. These results indicate that the amino acid sequence of MGD2 has high identity with homologous proteins of both monocotyledonous and dicotyledonous plants.

[0098] A highly identical conserved amino acid domain may exist in the intermediate region of the MGD2 protein, approximately between amino acid residues 49 and 151. However, this conserved domain has never been identified or reported in previous studies. First, we predicted the α-helix structure of the MGD2 protein on the AlphaFold3 server. As a result, four possible α-helix structures were identified (Figure 11A (shown as a black line), Figure 12B). Furthermore, deepTMHMM, TOPCONS, and SOSUI predicted that MGD2 has one transmembrane region (Figure 11A (shown as a blue line)). The predicted transmembrane region overlapped with the α-helix structure predicted with high reliability (pDDT score of 70 or higher) and the hydrophobic region of MGD2 (Figure 12A). Taken together, these structural features suggest that MGD2 is a membrane protein with a single transmembrane helix.

[0099] To identify the intracellular localization of MGD2, transformed Arabidopsis thaliana expressing a fusion protein of MGD2 protein and GFP under the CaMV35S promoter was created, similar to Test Example 6-1-4. The root cells of the 4-week-old transformants were observed, and the GFP fluorescence signal was detected. The GFP fluorescence signal was observed in the cytoplasm (Figure 11B).

[0100] 6-3.MGD3 6-3-1. Analysis of MGD3 mRNA expression levels under drought stress conditions Similar to Test Example 6-1-1, the mRNA expression level of MGD3 in C. virgata under drought stress conditions was measured. The primer pairs shown in SEQ ID NOs. 19 and 20 were used to measure MGD3 mRNA expression. The results are shown in Figure 13A. The mRNA expression level of MGD3 in C. virgata subjected to drought treatment was approximately 17 times higher under 3 hours of drought treatment and approximately 48 times higher under 6 hours of drought treatment compared to C. virgata not subjected to drought treatment (control).

[0101] 6-3-2. Phylogenetic tree construction of MGD3 and related proteins in other species Similar to Test Example 6-1-2, a phylogenetic tree of MGD3 and related proteins in other species was constructed. The results are shown in Figure 13B. Phylogenetic analysis revealed that MGD3 encodes a novel gene that is conserved among homologous genes in various land plants, but detailed functional analysis of this gene has not yet been reported.

[0102] 6-3-3. MGD3-overexpressing Arabidopsis thaliana exhibits higher drought stress tolerance than the wild type. Similar to Experiment 6-1-3, two independent transgenic Arabidopsis thaliana plants (MGD3-OX) OX-1 and OX-2, which overexpress MGD3, were created and their drought stress tolerance was analyzed. The primer pairs shown in SEQ ID NOs. 21 and 22 were used to amplify the full-length coding region of MGD3. qRT-PCR revealed that the expression level of the MGD3 gene was significantly higher in the MGD3-overexpressing plants (Figure 5C). MGD3-overexpressing plants OX-1 and OX-2 showed resistance to drought stress, with survival rates of 83% and 67%, respectively, which were higher than the wild-type survival rate of 33% (Figure 13C). The MGD3-overexpressing plants had a relatively higher fresh weight after re-irrigation compared to the wild-type (Figure 13D).

[0103] 6-3-4. Analysis of the MGD3 protein MGD3 codes for a 281-amino acid protein (Figure 14A, Sequence ID 23). Homology search analysis using BLAST identified homologous proteins with similar amino acid lengths from monocots and dicots (Figure 14A). Amino acid sequence alignment revealed that the MGD3 protein has high sequence similarity to its monocot homologs: 80% with O. sativa (AAK43505.1), 79.4% with B. distachyon (XP_003572048.1), 58.6% with T. aestivum (XP_044335483.1), 52.3% with N. tabacum (XP_016501775.1), and 52.2% with A. thaliana (NP_568771.1). These results indicate that the amino acid sequence of MGD3 has a high degree of identity with homologous proteins in monocots and dicots.

[0104] The intermediate region of the MGD3 protein, approximately between amino acid residues 120 and 195, contains a highly conserved amino acid domain, which had never been identified or reported before. The α-helix structure of the MGD3 protein was predicted by the AlphaFold3 server. As a result, six antiparallel α-helix structures were predicted, each consisting of more than 20 amino acids (Figure 14A (shown as black lines), Figure 15B). Furthermore, several transmembrane region prediction servers, such as deepTMHMM and TOPCONS, suggested the possibility that MGD3 has six transmembrane regions (Figure 14A (shown as blue lines)). The predicted transmembrane regions corresponded to and overlapped with the α-helix structure and hydrophobic region of MGD3 (Figure 15A). This predicted structural information suggests that MGD3 may be a membrane-bound protein with six putative transmembrane helices.

[0105] To analyze the cellular function of the MGD3 protein, its intracellular localization was observed. Transformed Arabidopsis thaliana expressing a fusion protein of MGD3 and GFP under the CaMV35S promoter were created. These transformed Arabidopsis thaliana were crossed with transformed Arabidopsis thaliana expressing a fusion protein of endoplasmic reticulum retention signal (HDEL) fused to RFP under the CaMV35S promoter. Root cells of the resulting 4-week-old transformed Arabidopsis thaliana were observed, and the fluorescence signals of GFP and RFP were detected. Overlapping fluorescence signals of GFP-MGD3 and HDEL-RFP were observed in the roots (Figure 14B). These results suggest that MGD3 is localized to the endoplasmic reticulum or endoplasmic reticulum membrane.

[0106] 7. Analysis of drought-induced gene expression in Arabidopsis thaliana overexpressing MGD1, MGD2, and MGD3. To investigate the potential involvement of MGD1, MGD2, and MGD3 in drought stress tolerance signaling and ABA biosynthesis, the expression of drought-inducible genes (DREB2A, RD29A, RD29B, and GAPDH) in plants overexpressing these genes was analyzed (Figure 16). Specifically, the procedure was as follows: Transgenic Arabidopsis thaliana (MGD1-OX-1, MGD2-OX-1, MGD3-OX-1, CvNCED3-OX-1, and CvAREB1-OX-1) and wild-type Arabidopsis thaliana were transferred from 1 / 2 MS agar to Parafilm to initiate drought treatment. After 0, 15, 30, or 45 minutes of drought treatment, the mRNA expression levels of the three drought-inducible genes in each plant were measured using qRT-PCR. Primer pairs shown in SEQ ID NOs. 24 to 31 were used to measure the mRNA expression levels of the drought-inducible genes. Before drying (0 minutes after drying), no difference was detected in the expression of the three stress-inducible genes mentioned above between all transformed Arabidopsis thaliana and the wild type. After drying for 15 to 45 minutes, the expression of the drought-related transcription factor DREB2A was higher in MGD1 overexpression plants than in the wild type (Figure 16A). The signaling factor RD29A and the ABA biosynthesis enzyme NCED3 were also induced to be more highly expressed in MGD1 overexpression plants than in the wild type after drying (Figure 16A). In MGD2 overexpression plants, NCED3 was strongly induced, and RD29A was slightly induced compared to the wild type (Figure 16B). In MGD3 overexpression plants, RD29A was slightly induced compared to the wild type (Figure 16C). These results suggest that MGD1, MGD2, and MGD3 strongly or partially activate signaling related to drought stress tolerance, and that the biosynthesis of the drought-resistant plant hormone ABA may be activated by MGD2 and MGD1.

Claims

1. (a) or (b) below: (a) The coding sequence of the amino acid sequence shown in SEQ ID NO: 13, 18, or 23, or (b) A coding sequence for a protein having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 13, 18, or 23, and which has the effect of improving the drought stress tolerance of plants. Isolated polynucleotides containing [the specified element].

2. The polynucleotide according to claim 1, wherein the sequence identity is 90% or more.

3. The polynucleotide according to claim 1, comprising a promoter upstream of the coding sequence.

4. A vector, the polynucleotide according to claim 1.

5. A plant drought stress tolerance enhancer comprising a polynucleotide according to any one of claims 1 to 4.

6. A cell comprising the polynucleotide described in any one of claims 1 to 4.

7. A plant comprising the cells described in claim 6.

8. A method for improving the drought stress tolerance of a plant by introducing a polynucleotide according to any one of claims 1 to 4 into the plant.

9. A method for producing plants that have drought stress tolerance, A method for producing a plant, comprising the step of introducing a polynucleotide according to any one of claims 1 to 4.

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