KR20200000271A - CaDIL1 gene and the method for improving the resistance to the drought stress using CaDIL1 gene - Google Patents

Abstract

The present invention relates to a novel Capsicum annuum drought induced late embryogenesis abundant 1 (CaDIL1) gene derived from a variety of green chili peppers, proteins, and a method for improving the dry stress resistance of plants using the same. The present invention identifies CaDIL1 genes which encrypt proteins for improving resistance to dry stress, CaDIL1 proteins can serve as a positive regulator of abscisic acid (ABA) signal transmittance, and it is confirmed that transgenic plants in which the proteins are overexpressed have improved dry stress resistance. Accordingly, the adjustment of the expression of CaDIL1 genes can be used in improvements of crops available for humankinds, and in particular, can be expected to be useful in increasing productivity of plants.

Description

CaDIL1 gene and the method for improving the resistance to the drought stress using CaDIL1 gene}

The present invention relates to a novel gene, protein, and method for enhancing the dry stress resistance of plants using the same.

Drought is one of the major abiotic stresses that limits plant growth and significantly affects crop productivity. Plants are frequently exposed to drought stress, which causes osmotic stress and serious damage to plant tissue. To adapt to drought stress, plants exhibit physiological and molecular changes, including stomatal closure and reprogramming of gene expression. Although the adaptive response to drought stress has been revealed, the exact mechanism of functional transformation is not clear because of the complexity and diversity at the whole plant level as well as at the cellular level.

Late embryogenesis abundant (LEA) proteins are present in bacteria, yeasts, fungi and many species of vascular plants. LEA proteins play a role in a variety of biological processes, from growth and development to stress responses. In vascular plants, LEA proteins are highly expressed in embryos in plant organs under late fetal origin and drought stress conditions, indicating that these proteins play an adaptive role in water restriction. Previous studies have shown that some LEA proteins are involved in the transmission of abscisic acid (ABA) and protect cells against stress, especially osmotic pressure. For example, overexpression of OsLEA5 confers resistance to drought and high salinity, while LEA3 and RAB16D are involved in adaptive responses to water deprivation. These proteins consist of hydrophilic amino acids and constitute a multiple gene family classified into seven groups based on amino acid sequence similarity and corresponding mRNA homology. Group 3 contains typical LEA proteins, which exhibit hydrophilic properties and are significantly more diverse than other groups of LEA proteins. Although the exact function of the Group 3 LEA protein has not been fully identified, previous studies have shown that its expression is induced by drought stress in plants as well as in some non-plant organisms.

Abscisic acid (ABA) is a plant hormone that plays an important role in the cellular adaptation to drought stress. Under drought stress conditions, the biosynthesis and accumulation of absic acid (ABA) increases and signal transduction pathways associated with adaptive responses begin. A large number of genes are regulated by absic acid (ABA) and are involved in the ABA signaling pathway. For example, more than 10% of Arabidopsis genes are induced by Abbasic Acid (ABA). The best known reaction is Abs Acid (ABA) mediated pore closure through the release of cations and anions from protective cells. . This reduces the amount of evaporation necessary for plant survival under drought stress conditions. Abs acid (ABA) deficient mutations therefore exhibit a phenotype that is hypersensitive to drought stress. In contrast, mutations exhibiting the absic acid (ABA) hypersensitivity phenotype show drought tolerance. In addition, many genes involved in the adaptive response to drought stress are regulated by absic acid (ABA). Abbic acid (ABA) levels increase in plant tissues under drought stress conditions to induce the expression of many stress related genes such as NCED3, RD29B, RAB18 and RD20. Until now, a method for improving the resistance to dry stress of plants has been the main research subject, but related studies on genes related to Abs acid (ABA) mediated dry stress resistance and growth promotion and the resulting transgenic plants It is happening, but it is still inadequate.

Korean Patent Publication No. 10-1678859 (Notice Date: 2016.11.17)

The present inventors identified CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) gene in red pepper, while studying to investigate the relationship between dry stress response through the regulation of the ABA signaling pathway. The present invention was completed by elucidating that it functions as a positive regulator of absic acid (ABA) signaling that enhances dry stress resistance.

Accordingly, an object of the present invention is to encode a protein that enhances resistance to dry stress, CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) gene consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: encoded by the CaDIL1 gene It is an object to provide a CaDIL1 protein consisting of the amino acid sequence of 2.

Another object of the present invention is to provide a novel CaDIL1 gene / protein and a composition for enhancing the dry stress resistance of a plant comprising the gene or protein as an active ingredient.

It is another object of the present invention to provide a method of enhancing the dry stress resistance of plants by transforming and overexpressing the CaDIL1 gene in plants.

Still another object of the present invention is to provide a transgenic plant having improved dry stress resistance by the above method.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention encodes a protein that enhances resistance to dry stress, CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) gene consisting of the nucleotide sequence of SEQ ID NO: 1, the Provided is a CaDIL1 protein consisting of the amino acid sequence of SEQ ID NO: 2 encoded by the CaDIL1 gene.

The present invention also provides a composition for enhancing the dry stress resistance of plants, comprising the gene or the protein as an active ingredient.

In addition, the present invention provides a method for enhancing dry stress resistance of a plant, comprising the following steps.

(a) transforming a plant with a gene of SEQ ID NO: 1 encoding a CaDIL1 protein; And

(b) overexpressing CaDIL1 protein in the transformed plant.

The present invention also provides a transgenic plant having improved dry stress resistance by the above method.

In another aspect, the present invention provides a method for screening a dry stress resistance enhancer of plants, comprising measuring the overexpression of CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) protein after treating the candidate substance.

The present invention has identified a novel CaDIL1 gene encoding a protein that enhances resistance to dry stress, and the CaDIL1 protein functions as a positive regulator of absic acid (ABA) signaling and is resistant to dry stress in transgenic plants expressing the protein. As it was confirmed that the enhancement effect, the expression of the CaDIL1 gene can be used to improve the crops and the like that can be used by humans, and is expected to be particularly useful for improving the productivity of plants.

Figure 1a shows the results of multiple sequence alignment of CadIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) and LEA3 protein derived from red pepper green varieties.
FIG. 1B shows the result of Phylogenetic tree analysis of CaDIL1 protein.
FIG. 2A shows the expression level of CaDIL1 gene after drought and absic acid (ABA) treatment.
Figure 2b shows the results of confirming the position of the CaDIL1 protein in the plant cell.
Figure 3a shows the difference in transcription accumulation between CaDIL1-silenced pepper plants and the internal control Actin 1 (Catin1).
FIG. 3B shows observation images of drought susceptibility of CaDIL1-silenced pepper plants (2 days after rewatering before dehydration (left), after dehydration (middle) and after dehydration (right) ).
3C shows the water loss rate of CaDIL1-silenced pepper plants (TRV2: CaDIL1) and control leaves (TRV2: 00).
3D and 3E show pore openings of CaDIL1-silenced pepper plants and control leaves.
3F and 3G show the leaf temperatures of CaSIBZ1-silenced pepper plants and controls.
4A shows germination rates of CaDIL1 overexpressed (CaDIL1-OX) mutants and wild-type (WT) plants in 0.5 × MS (Murashige and Skoog) medium with varying concentrations of absic acid (ABA).
4B and 4C show the results of seeding development of wild-type plants exposed to CaDIL1 overexpressed (CaDIL1-OX) mutants and absic acid (ABA) (taken at 5 days after plating) b) Record the number of seedlings in each line with expanded cotyledons (c)).
4D and 4E show primary root elongation results of wild-type and transgenic lines exposed to absic acid (ABA).
4F and 4G show the results of confirming the effect of absic acid (ABA) on seed germination with the altered absic acid (ABA) sensitivity of CaDIL1 overexpressed (CaDIL1-OX) plants in the seedling stage.
Figure 5a shows the survival rate of plants as a result of drought tolerance of transgenic Arabidopsis overexpressed CaDIL1 (CaDIL1-OX).
Figure 5b shows the rate of Transpirational water obtained from the leaves of wild type plants and transgenic plants.
5C and 5D show microscopic images and size of pore holes of wild-type and CaDIL1-overexpressed (CaDIL1-OX) plants treated with absic acid (ABA).
Figures 5e and 5f show the results of measuring the image and leaf temperature of wild-type and CaDIL1 overexpressed (CaDIL1-OX) plants exposed to ABA treatment.
Figure 6 shows the results confirmed by the quantitative reverse transcription polymerase chain reaction (qRT-PCR) expression of the drought-induced gene expression of CaDIL1 overexpressed (CaDIL1-OX) plants exposed to 3 hours drought stress.
7 shows the results of reverse transcription-polymerase chain reaction (RT-PCR) analysis of CaDIL1 expression in wild-type (WT) and CaDIL1 overexpressed (CaDIL1-OX) transgenic lines, Actin8 being used as an internal control gene.

The inventors have found that increased expression of CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) under dry stress conditions and reduced resistance to dry stress in CaDIL1-silenced pepper, loss of pore closure, and loss of evaporated water, CaDIL1 In this overexpressed transgenic Arabidopsis, the effect of enhancing the sensitivity of abscisic acid (ABA), and the effect of enhancing the resistance to drying were confirmed, and based on this, the present invention was completed.

Accordingly, the present invention provides a CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) gene consisting of a nucleotide sequence of SEQ ID NO: 1, which encodes a protein that enhances resistance to dry stress, and an amino acid sequence of SEQ ID NO: 2 encoded thereby. It provides a peptide consisting of.

CaDIL1, which is a gene of the present invention, preferably consists of the nucleotide sequence of SEQ ID NO: 1, and the peptide encoded by the CaDIL1 gene may be preferably composed of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The present invention provides a method for enhancing the dry resistance of a plant, comprising the step of transforming a gene encoding a CaDIL1 protein into a plant and overexpressing the CaDIL1 protein in the transformed plant.

In the present invention, the CaDIL1 gene encoding the CaDIL1 protein shows that it is an important regulator of the loss of evaporation of capsicum annuum. The expression of CaDIL1 in red pepper leaves was upregulated after exposure to abscisic acid (ABA) and drought. Phenotypic analysis showed that CaDIL1-silenced peppers and CaDIL1-overexpressed plants (CaDIL1-overexpressing, CaDIL1-OX) had drought resistance reduced and enhanced with varying rates of increase. Absitic acid (ABA) sensitivity was also significantly lower in CaDIL1-silenced from red pepper, but CaDIL1 was higher than control in CaDIL1-OX overexpressed plants.

Accordingly, the present invention provides a transgenic plant in which CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) protein is overexpressed to enhance dry stress resistance.

In one embodiment of the present invention, while studying the gene involved in the dry stress response, CaDIL1, a late embryogenesis abundant (LEA) protein, was identified (see Example 2), and the sensitivity of abscisic acid (ABA) Based on the fact that the increase of porosity can be promoted by increasing porosity, dryness resistance was investigated, the association between Abs acid (ABA) or dry stress and CaDIL1 gene.

In one embodiment of the present invention, it was confirmed by GFP fluorescence that CaDIL1 protein is expressed in the nucleus (see Example 3).

In another embodiment of the present invention, a decrease in drought tolerance of CaDIL1-silenced pepper plants was confirmed. As a result, the high evaporation rate of CaDIL1-silenced pepper plants, which increases drought susceptibility, is mainly caused by abscides (ABA). ) Due to a decrease in sensitivity (see Example 4).

In another embodiment of the present invention, it was confirmed through ectopic expression of CaDIL1 overexpressed transgenic Arabidopsis plants that ectopic expression of CaDIL1 imparts Absmic Acid (ABA) hypersensitivity in Arabidopsis (see Example 5). ). In addition, by measuring the leaf temperature, pore opening, and water loss rate in the leaves of CaDIL1 overexpressed red peppers and normal control pepper plants under the condition of ABA treatment, Abs acid (ABA) hypersensitivity due to overexpression of CaDIL1 gene was drought resistant. The increase effect of was confirmed (see Example 6).

In the present invention, virus-induced gene silencing (VIGS), which is a gene silencing phenomenon induced by a virus, is also called virus-induced RNA silencing. It is a kind of post-transcriptional gene silencing that is well known in various plants, fungi, insects, nematodes, fish, and rats. It is resistant to the expression of resistance genes homologous to the viral genome in infected plants during virus invasion and replication. Expression and replication of genes and viral genomes are suppressed together. That is, when a part or all of a specific gene derived from the host is inserted into the cDNA of the viral genome, and the infective RNA is made to infect the plant, the RNA of the corresponding host is targeted, and the expression of the target gene is suppressed in the infected host plant. Lost. As a result, the function of the target gene can be estimated indirectly. Virus-induced gene silencing (VIGS) mechanism is known to be a type of RNA-mediated plant defense mechanism against viruses, characterized by 1) gene silencing after transcription 2) RNA conversion 3) nucleotide sequence specificity.

In the present invention, by inserting the transforming vector TRV (Tobacco Rattle Virus) for VIGS into which the ORF region of CaDIL1 is inserted into Agrobacterium, the expression of CaDIL1 gene is successfully inhibited by infiltrating the Agrobacterium into a plant. I've done it.

As another aspect of the present invention, the present invention provides a composition for enhancing the dry resistance of plants comprising an inhibitor of the expression or activity of CaDIL1 protein as an active ingredient.

By vector is meant bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host. Preferred examples of the recombinant vector of the present invention are preferably Ti-plasmid vectors capable of transferring a portion of itself, a so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumerfaciens, but are not limited thereto. It is not.

The inhibitor may be siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), which complementarily bind to mRNA of a sequence of CaDIL1 gene, a sequence complementary to the sequence, or a fragment of the gene sequence to inhibit expression. ), Ribozyme, DNAzyme, peptide nucleic acids (PNA), antisense nucleotides, and compounds that bind to CaDIL1 protein complementarily to inhibit activity, peptides, peptide mimetics (Peptide Minetics), an antibody, or an aptamer (preferably any one), but is not limited thereto.

As another aspect of the present invention, the present invention provides a method for enhancing dry stress resistance of a plant comprising the step of inhibiting the expression or activity of CaDIL1 protein.

Plants according to the present invention is a food crop including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, millet; Vegetable crops including Arabidopsis, Chinese cabbage, radish, peppers, strawberries, tomatoes, watermelons, cucumbers, cabbages, melons, pumpkins, green onions, onions, carrots; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops including lygras, red clover, orchardgrass, alpha alpha, tolsque cue, perennial lice, and the like, and preferably Arabidopsis, but are not limited thereto.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Experiment Preparation and Experiment Method

1-1. Plant material and growing conditions

Seeds of red pepper (Capsicum annuum L., cv.Nockwang), tobacco (Nicotiana benthamiana), and Arabidopsis thaliana (ecotype Col-0) were steam sterilized soil mixtures (peat moss: perlite) ): Vermiculite, 5: 3: 2, v / v / v), sand, and loam soil (1: 1: 1, v / v / v). Peppers were grown in a growth room at 27 ± 1 ° C. under white fluorescent light (80 μm photons · m ⁻² · s ⁻¹ ; 16 hours / day). Tobacco was maintained in a growth chamber at 25 ± 1 ° C. with a 16 hour light / 8 hour dark cycle. Arabidopsis (ecotype Col-0) seeds were germinated in MS salt (Duchefa Biochemie) with 1% sucrose (1% sucrose) and micro agar (Micro agar, Duchefa Biochemie); The seeded plates were incubated in a 16 hour light / 8 hour dark cycle, 24 ° C. incubation chamber. Arabidopsis seeds were vernalized at 4 ° C. for 2 days before being placed in the culture chamber.

1-2. Preparation of Transgenic Arabidopsis Overexpressed CaDIL1 Gene

The full-length CaDIL1 cDNA was bound to the pENTR / D-TOPO vector (Invitrogen, Carlsbad, CA, USA), and the pK2GW7 binary vector was converted to the Arabidopsis cauliflower mosaic using the LR response for constitutive expression of the CaDIL1 gene. virus (CaMV) was inserted under 35S promoter control. 35S: CaDIL1 structure was transformed with Agrobacterium tumefaciens strain GV3101. Agrobacterium tumefaciens-mediated transformation was performed via the floral dip method (Clough and Bent, 1998). To select transformed lines, seeds harvested from the transformed plants were sown in MS agar plates containing 50 μg · mL ⁻¹ of kanamycin.

1-3. Virus-induced gene silencing and overexpression of CaDIL1

Tobacco rattle virus (TRV) -based virus-induced gene expression suppression for knock-down of CaDIL1 gene in pepper plants for loss-of-function analysis of CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) gene silencing (VIGS) system was used. Protocols previously described for CaDIL1-silenced pepper plants and CaDIL1-transformed Arabidopsis plants, each using 247 bp (116-362 sequences) and CaDIL1 cDNA in its entirety. (Park et al. 2015).

1-4. Subcellular localization analysis

The CaDIL1 coding region without stop codons was inserted into the GFP-fusion binary vector p326GFP. Agrobacterium tumefaciens strain GV3101 was mixed with p19 strain (1: 1 ratio, OD600 = 0.5) and co-precipitated to 5 weeks fully expanded tobacco plant leaves. After 2 days of infiltration, microscopic analysis was performed as described above.

1-5. Abscisic acid (ABA) and drought treatment

Germination rates, root elongation, and seedling establishments were measured to determine Absi acid (ABA) sensitivity. A total of 200 wild-type and CaDIL1 overexpressed (CaDIL1-OX) plants were stratified for 3 days at 4 ° C. and plated in 0.5 × MS agar medium with varying concentrations of abscisic acid (ABA). . For the drought stress treatment in peppers, the control and CaDIL1-silenced pepper plants were stopped for 10 days and rehydrated for 2 days for drought treatment. Survival was measured on each individual sample and each experiment was performed three times with 30 plants. In the case of Arabidopsis, drought stress was treated on three main seedlings with a 14-day hold of watering. The plants were rehydrated for two days for recovery, after which the survival rates of the plants were calculated. Survival was measured in each individual sample and each experiment was performed three times with 50 plants. Drought tolerance is determined by measuring evaporative moisture loss. Fifty leaves were separated from the fourth leaf stage pepper plant and the three week Arabidopsis and placed in Petri dishes. The dish was kept in a culture chamber with a relative humidity of 40% and fresh weight loss was measured at the indicated time points. All experiments were performed at least three times.

1-6. Phenotypic analyses

To test seedling growth, 36 seeds per genotype were sown on plates containing MS agar medium with varying ABA concentrations. One week old seedlings from wild type baby poles and CaDIL1 overexpressed (CaDIL1-OX) transgenic baby poles were randomly planted in bowls containing soil mixture and grown for 2 weeks in wet conditions. In order to provide drought stress, watering was withheld for 9-10 days and re-watered. After 1-2 days, survival rates of plants with rehydrated leaves were calculated. In the case of red pepper, drought stress was applied to four-leaf stage plants with water holding for 10-11 days, and the survival rate of plants with rehydrated leaves was calculated 1 day after the watering again. . Drought resistance was determined quantitatively by measuring evaporative moisture loss. The leaves were separated from four-leaf stage peppers and three main baby pole plants and placed in a Petri dish. The dish was kept at 40% relative humidity in the growth chamber and the loss of raw weight was measured at the indicated time points. All experiments were repeated at least three times independently in biological samples.

1-7. Thermal aperture bioassay and thermal imaging

The measurement of pore pore size and leaf temperature was performed as previously described (Lim and Lee 2016). Pepper and Arabidopsis leaf husks were suspended in a stomatal opening solution (SOS: 50 mM KCl and 10 mM MES-KOH, pH 6.15, 10 mM CaCl ₂ ) under bright conditions for 2.5 hours. Pore closure was induced by replacing the buffer with fresh SOS containing varying concentrations of absic acid (ABA). After an additional 2.5 hours of incubation in each buffer, 100 pores per sample were observed under a Nikon Eclipse 80i microscope.

For the thermal imaging analysis, the first and second leaves were treated with 100 μM Abbic Acid (ABA) in four-week pepper plants and three-week Arabidopsis plants. Thermal images were obtained using an infrared camera (T420; FLIR system) and leaf temperatures were measured with FLIR Tools + ver 5.2 software.

1-8. Virus-induced gene silencing (VIGS)

For loss-of function analysis of CaDIL1, virus-induced gene silencing (VIGS) was performed in pepper plants. Briefly, Agrobacterium tumefaciens strain GV3101 carrying pTRV1 and pTRV2: CaDIL1 or pTRV2: 00 as negative controls was infiltrated into fully expanded cotyledons of pepper (OD ₆₀₀ = 0.2 for each strain). ). Plants were placed in a growth room at 24 ° C. with a photoperiod of 16 hours day / 8 hours night for growth and virus propagation.

1-9. Quantitative real-time transcription-polymerase chain reaction

RNA samples are digested with RNA-free DNase to inhibit genomic DNA contamination. CDNA was synthesized using Transcript First Strand cDNA Synthesis kit (Roche). For qRT-PCR analysis, specific primers (Table 1) and CFX96 Touch ^™ Real Time PCR Detection System (Bio-Rad) were used. PCR was programmed as follows: 5 min at 95 ° C; 45 times at 95 ° C. for 20 seconds and 60 ° C. for 20 seconds; And 20 seconds at 72 ° C. The ΔΔCt method (Livak and Schmittgen 2001) was used to determine relative expression levels. Arabidopsis AtACT8 gene was used for normalization.

Example 2. Isolation and Sequence Analysis of Chili CaDIL1 Gene

To identify drought-induced genes, pepper CaDIL1 genes were isolated from cDNA libraries constructed from drought-treated pepper leaf tissues using RNA-seq analysis. Among the gene clones, a drought up-regulated gene was selected. CaDIL1 cDNA consists of an open reading frame (ORF) of 504 bp nucleic acid residues encoding 167-amino acid residues having an isoelectric point of 7.89 and a molecular weight of 17,300 Da. Protein BLAST search and multiple sequence alignment analysis showed relatively high amino acid sequence identity (48.9-81.4%) between CaDIL1 and other LEA proteins (FIG. 1A). FIG. 1A shows cultivated tobacco (Nacotiana tabacum, accession no. XP_016459037.1), tomato genus (Solanum lycopersicum, accession no.NP_001238798.1), carrot (Daucus sativus, accession no. And amino acid sequence of CaDIL1 amino acid sequence (accession no. NP_175678.1) protein. The same amino acid residues are highlighted in black here. Multiple alignments of the CaDIL1 protein sequence and homologous LEA3 protein were performed using ClustalW2 (Multiple Sequence Alignment).

Phylogenetic tree analysis of CaDIL1 protein is shown (FIG. 1B). Basic local alignmnet (BLAST) searches were performed using the amino acid sequence of CaDIL1 and the sequence with the highest similarity was collected from each plant species. Phylogenetic tree analysis was performed using MEGA software (version 7.0).

Example 3 Induction of CaDIL1 in response to drought and ABA treatments and subcellular localization of CaDIL1

CaDIL1 was isolated from the drought-treated red pepper leaves to confirm the expression level of CaDIL1 gene after drought and absic acid (ABA) treatment (FIG. 2A). CaDIL1 was induced in pepper leaves at various time points after drought and 50 μM abscisic acid (ABA) treatment, and pepper Actin1 gene was used as an internal control. Induction of CaDIL1 transcript was first detected 2 hours after drought treatment and peaked at 12 hours. Abs (ABA) plays a role in plant response to drought stress, and Abs (ABA) and drought stress signals share common factors. CaDIL1 transcription started 2 hours after the treatment of absic acid (ABA) and decreased 12 hours after the treatment of absic acid (ABA). This confirmed that CaDIL1 functions in nonbiological stress response.

To investigate the intracellular location of CaDIL1 , the green fluorescent protein (GFP) reporter gene was fused to the C-terminal region of CaDIL1 under the control of the 35S promoter ( 35S: CaDIL1-GFP ). 35S: CaDIL1-GFP fusion protein was expressed in Nicotiana benthamiana epidermal cells and the GFP signal is localized to the cytoplasm and nucleus. Subcellular localization of CaDIL1 protein was performed using transient expression of green fluorescent protein (GFP) in Nicotiana benthamiana (top class tobacco) cells 35S: CaDIL1 The -GFP constructs were expressed using Agroinfiltration of Nicotiana ventamiana leaves and observed with a confocal laser-scanning microscope (FIG. 2B). A blue fluorescence signal for DAPI (4 ', 6-diamidino-2-phenylindole) was detected in the nucleus. These results indicate that CaDIL1 functions in the cytoplasm and nucleus.

Example 4 Decreased Drought Tolerance of CaDIL1-silenced Pepper Plants That Increase Drought Susceptibility

As CaDIL1 is isolated from drought pepper leaves, it is assumed that CaDIL1 is involved in drought stress response. To test this hypothesis, phenotypic analysis was performed using virus-induced gene silencing (VIGS) analysis in pepper plants, resulting in overexpressed Arabidopsis plants. Virus Induced Gene Expression Inhibition (VIGS) Using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis of Control Tobacco Rattle Virus (TRV2: 00) and CaDIL1-silenced Pepper (TRV2: CaDIL1) Leaves The expression level of was investigated. CaDIL1 in pepper leaves transfected with empty vector control (tobacco rattle virus (TRV2: 00)) or CaDIL1-silenced constructs (TRV2: CaDIL1) immediately after isolation (after 0 hours). Reverse transcription-polymerase chain reaction (RT-PCR) analysis of expression was performed. Actin 1 (CaACT1) was used as an internal control. CaDIL1 expression in the leaves of CaDIL1-silenced pepper was impaired and almost undetectable (FIG. 3A). Later phenotypic analysis used CaDIL1-silenced pepper plants. Drought stress treated CaDIL1-silenced pepper plants (TRV2: CaDIL1) and control plants (TRV2: 00) and compared their phenotypes (FIG. 3B). To determine the drought susceptibility of CaDIL1-silenced pepper plants. Empty vector control and CaDIL1-silenced pepper plants were grown in pots for 3 weeks under normal growth conditions. After that, the plants suffered drought stress for 10 days without seeing water and spent two more days. Representative images were taken before dehydration (left), after dehydration (middle), and 2 days after rewatering after dehydration (right). Survival was measured by counting the number of plants with green and rehydrated leaves two days after watering again. No phenotypic differences were observed between the control and CaDIL1-silenced pepper plants under normal growth conditions (FIG. 3B, top panel). However, when the plant was subjected to drought stress without seeing water for 10 days and then replenished for 2 days, CaDIL1-silenced pepper plants showed a more withered phenotype than control plants (FIG. 3B, middle and lower panels). . The survival rates of CaDIL1-silenced plants and controls were 22.2% and 61.7%, respectively. Based on the drought-sensitive phenotypes exhibited by CaDIL1-silenced pepper plants, it was assumed that the water retention capacity was reduced in CaDIL1-silenced pepper plants. To test this hypothesis, water loss was measured in the leaves of the empty vector control and CaDIL1-silenced pepper plants at various times after the leaves were separated (FIG. 3C). Data represent mean ± standard deviation of three independent experiments. Leaf husks were harvested from two main pepper plants and incubated in pore open solution containing 10 and 20 μM ABA. The pore diameter was then measured under the microscope. At several time points after separation, the evaporated water loss was higher in the CaDIL1-silenced pepper plant leaves than in the control plant leaves. To assess whether the reduced water retention exhibited by CaDIL1-silenced pepper leaves was derived from inconsistencies with the absic acid (ABA) sensitivity, with or without stomatal aperture (ABA) treatment. Leaf temperature was monitored (FIGS. 3D-G). Absmic acid (ABA) susceptibility can be determined by measuring the change in pore size in response to treatment with absic acid (ABA). In the absence of absic acid (ABA), there were no significant differences in the pores between the leaves of the control and the CaDIL1-silenced pepper plants (Figures 3d and e). However, the pores were significantly greater in CaDIL1-silenced pepper plant leaves than control leaves after the treatment with Abs (ABA). Consistent with the pores, the leaf temperature of CaDIL1-silenced pepper plants was significantly lower than that of the control plants after the treatment of Abs (ABA) due to evaporative cooling by open pores. Photographs were taken before (left) and after 2.5 hours (right) after treatment with Absic Acid (ABA). The data represent the mean ± standard error of three independent experiments, with 20 plants each tested (FIGS. 3F and g). Representative images (FIG. 3F) and leaf temperatures were measured (FIG. 3G). Data represent mean ± standard error of three independent experiments. An asterisk (*) indicates a significant difference between the control and CaDIL1-silenced pepper plants (Student's t-test, P <0.05) The high evaporation rate of CaDIL1-silenced pepper plants that increases drought susceptibility as a result of the overall result. It was confirmed that is mainly due to the decrease in the sensitivity of Abs (ABA).

Example 5 Identification of Improved Absis Acid (ABA) Susceptibility of CaDIL1 Overexpressed (CaDIL1-OX) Transgenic Arabidopsis Plants

CaDIL1-silenced pepper plants showed a drought sensitive phenotype (FIG. 3). Therefore, further genetic analyzes were performed to elucidate the function of CaDIL1 in abiotic stress responses by generating transgenic Arabidopsis plants that overexpress the CaDIL1 gene in Arabidopsis thaliana (ecotype Col-0). Two independent T3 transgenic lines (CaDIL1-OX # 1 and CaDIL1-OX # 2) were obtained and CaDIL1 (FIG. 7) was relatively high by RT-PCR analysis. The two lines were used in phenotypic analysis. Under normal growth conditions, the phenotype of CaDIL1 overexpressing (CaDIL1-OX) plants was identical to that of wild type plants (FIGS. 4 and 5). To confirm the association of CaDIL1 with absic acid (ABA) signal transduction, phenotypic analysis of CaDIL1 overexpressed (CaDIL1-OX) plants was carried out in the germination and seedling stages in response to abscidic acid (ABA). Without Abs (ABA), it was concluded that there was no significant difference in germination rate between wild-type and CaDIL1-overexpressed (CaDIL1-OX) seeds. However, the germination rate of CaDIL1 overexpressed (CaDIL1-OX) seeds in the presence of absic acid (ABA) was lower than that of wild seeds (FIG. 4A). In order to assess Absmic Acid (ABA) susceptibility at the seedling stage, cotyledon greening and initial root length ratios were determined (Figure 4b-e). The data represent the mean ± standard error of four independent experiments and evaluate 25 seeds each (FIGS. 4B and 4C). Primary root elongation of wild-type and transgenic lines exposed to absic acid (ABA) (FIGS. 4D and e). Representative images (FIG. 4D) were taken and the root length of each plant was measured 8 days after sowing (FIG. 4E). Consistent with the germination rate, Abs acid (ABA) treatment decreased the greening speed of the cotyledon and decreased the root length. Seed germination was altered by the sensitivity of ABA in plants with CaDIL1 overexpression (CaDIL1-OX) at the seedling stage. It was examined whether it was an indirect response to the effect of absic acid (ABA) on or directly from the effect of absic acid (ABA) on seedling growth. To this end, 3 day old seedlings germinated in MS (Murashige and Skoog) medium were transferred to fresh MS medium added with 0 μM or 20 μM absic acid (ABA) (FIGS. 4F and g). The data represent the mean ± standard error of three independent experiments, each of which evaluates 25 seeds. Other letters indicate significant differences between wild type and transgenic lines (Student's t-test; P <0.05) (FIGS. 4F and G). Primary root elongation of wild-type and transgenic plants exposed to germic acid after germination. Five-day seedlings grown in 0.5 × MS medium were transferred to fresh 0.5 × MS medium containing 0um or 20um Abbic Acid (ABA). After 7 days, representative images were taken (FIG. 4F) and the root length of each line was measured (FIG. 4G). The data represents the mean standard error of three independent experiments. Asterisk (*) indicates significant difference between three independent experiments (ANOVA; P <0.05). As shown in FIG. 4F, the roots of CaDIL1 overexpressed (CaDIL1-OX) seedlings were significantly shorter than the roots of wild type seedlings. This data indicates that ectopic expression of CaDIL1 confers absic acid (ABA) hypersensitivity in Arabidopsis.

Example 6 Identification of Enhanced Drought Tolerance of CaDIL1 Overexpressed (CaDIL1-OX) Transgenic Arabidopsis Plants

CaDIL1 overexpressed (CaDIL1-OX) plants were examined for drought tolerance because they exhibited a hypersensitive phenotype to absic acid (ABA) at seed germination and seedling growth stages (FIG. 5). When grown in a well-watered condition, no phenotypic difference was observed between the three-week wild type (WT) and CaDIL1 overexpressed (CaDIL1-OX) plants (FIG. 5A, left panel). On the other hand, plants with 14 days of water retention and 2 days of rewatering, drought stress (transgenic plants in the middle and right panels, respectively) showed slightly less withered phenotypes than wild-type plants, and survival rates were wild-type. Higher than the proportion of plants. The survival rate data of the plants in FIG. 5A represent the mean value ± standard error of three independent experiments, each evaluating 30 plants. Figure 5b shows the results of transpirational water loss from the leaves of wild-type and transgenic plants at the point after leaf separation, and this data represents the mean ± standard error of three independent experiments and evaluates 50 leaves each. .

To investigate whether the drought resistant phenotype represented by adult CaDIL1 overexpressed (CaDIL1-OX) plants was due to changes in water retention, we assessed evaporated water loss by measuring the fresh weight of separated leaves. (FIG. 5C). It was found that the CaDIL1 overexpressed (CaDIL1-OX) plants had lower transpiration rates than those of wild plants and that the drought resistant phenotype was derived from increased water retention. Pore pore of wild type and CaDIL1 overexpressed (CaDIL1-OX) plants treated with Absic acid (ABA). Leaf peels were harvested from three-week old plants in each row and incubated in a stomatal opening solution (SOS) containing 0 μM or 20 μM ABA. Representative images were taken under a microscope (FIG. 5C) and stomatal apertures were measured (FIG. 5D). In general, drought tolerance and sensitivity are determined by at least two cellular or molecular parameters. Measurements of leaf temperature and pore size indicate that abscitic acid (ABA) hypersensitivity increases drought tolerance. 5E and 5F show the leaf temperature of each wild type and CaDIL1 overexpressed (CaDIL1-OX) exposed to Abs acid (ABA) treatment and measured representative images. This data represents the mean ± standard error of three independent experiments, each assessing 10 plants. Asterisks (*) indicate significant differences between wild type and transgenic lines (P <0.05; ANOVA, Fisher's LSD test). The pore diameter and leaf temperature were measured to determine whether the enhanced drought resistance of CaDIL1 overexpressed (CaDIL1-OX) plants was associated with an increase in the sensitivity of Absacid (ABA) (FIGS. 5C-F). Without Abs (ABA), no significant differences were found in pore compartments or leaf temperatures between wild-type and CaDIL1-overexpressed (CaDIL1-OX) plants. However, after exposure to absic acid (ABA), the pore diameter and leaf temperature of CaDIL1 overexpressed (CaDIL1-OX) plants were significantly lower than those of wild type plants (FIGS. 5C-F). These results indicate that heterotopic expression of CaDIL1 overexpressed (CaDIL1-OX) plants alters the response to drought stress.

Assuming that drought tolerance and sensitivity are related to the expression levels of stress-related genes, quantitative RT-PCR analysis was used to investigate the expression of CaDIL1. In Figure 6 the relative expression level (ΔΔCT) of each gene was normalized to the geometric mean of actin 8 as an internal control gene. Data represent mean ± standard error of three independent experiments. Significant differences between wild-type and transgenic lines were shown (P <0.05; ANOVA, Fisher's LSD test). It was confirmed that enhanced expression of CaDIL1 altered the expression of stress related genes in wild-type and CaDIL1-overexpressed (CaDIL1-OX) plants under drought stress conditions (FIG. 6).

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> CaDIL1 gene and the method for improving the resistance to the          drought stress using CaDIL1 gene <130> MP18-111 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 504 <212> DNA <213> CaDIL1 <400> 1 atggcttccc acgagcagag ttacagagct ggtgaaacca agggccagac tcaggcgaag 60 gttggtcaag caatggaagg catgaaggac aaggctcaag aagcaaagga caggacttca 120 gagatggccc attcagccaa aggaacggcc catgagaaga cgggcgcagc caaagacaag 180 acttcagatg cagctcaggc taccaaagat aagacaagtg gagcagctca ggaaaccaaa 240 gataagacag ctggagcagc tcaggctact aaagagaagg ctagtggagc agctcaggct 300 actaaagata aggcttcaga aatgatggag tcggcaaaag agacggctca ggctggtcaa 360 gagaaaactg gtagcatcct ctcaaagacg ggagaacaag tgaagagcat ggctcagggt 420 gctgctgatg cagtgaagca cacttttggc atggcggata ctgctgaaga tcctcatgcc 480 acaaaaacac ccagagaacc atag 504 <210> 2 <211> 167 <212> PRT <213> CaDIL1 <400> 2 Met Ala Ser His Glu Gln Ser Tyr Arg Ala Gly Glu Thr Lys Gly Gln   1 5 10 15 Thr Gln Ala Lys Val Gly Gln Ala Met Glu Gly Met Lys Asp Lys Ala              20 25 30 Gln Glu Ala Lys Asp Arg Thr Ser Glu Met Ala His Ser Ala Lys Gly          35 40 45 Thr Ala His Glu Lys Thr Gly Ala Ala Lys Asp Lys Thr Ser Asp Ala      50 55 60 Ala Gln Ala Thr Lys Asp Lys Thr Ser Gly Ala Ala Gln Glu Thr Lys  65 70 75 80 Asp Lys Thr Ala Gly Ala Ala Gln Ala Thr Lys Glu Lys Ala Ser Gly                  85 90 95 Ala Ala Gln Ala Thr Lys Asp Lys Ala Ser Glu Met Met Glu Ser Ala             100 105 110 Lys Glu Thr Ala Gln Ala Gly Gln Glu Lys Thr Gly Ser Ile Leu Ser         115 120 125 Lys Thr Gly Glu Gln Val Lys Ser Met Ala Gln Gly Ala Ala Asp Ala     130 135 140 Val Lys His Thr Phe Gly Met Ala Asp Thr Ala Glu Asp Pro His Ala 145 150 155 160 Thr Lys Thr Pro Arg Glu Pro                 165

Claims (5)

CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) gene encoding a protein that enhances resistance to dry stress and consists of the nucleotide sequence of SEQ ID NO: 1.
CaDIL1 protein consisting of the amino acid sequence of SEQ ID NO: 2 encoded by the CaDIL1 gene according to claim 1.
Claim 1 gene or a protein of claim 2 comprising the active ingredient, a composition for enhancing the dry stress resistance of the plant.
A method for enhancing dry stress resistance of a plant, comprising the following steps:
(a) transforming a plant with a gene of SEQ ID NO: 1 encoding CaDIL1 (Capsicum annuum Drought Induced Late embryogenesis abundant 1) protein; And
(b) overexpressing CaDIL1 protein in the transformed plant.
A transgenic plant having improved dry stress resistance by the method of claim 4.

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