KR101988712B1 - Method for improving the resistance to drought stress using DEAD-box RNA helicase RH 8 in plants - Google Patents

Abstract

The present invention relates to a gene for RH8 (DEAD-box RNA helicase 8) derived from Arabidopsis thaliana, and a method for enhancing dry stress resistance of a plant using the gene and protein.
The present invention relates to a RH8 (DEAD-box RNA helicase 8) gene encoding a positive regulator protein for drying stress. More specifically, the present invention relates to a RH8 gene encoding a positive regulator protein for drying stress, Interacts with and acts as a positive regulator in the ABA signal transduction pathway and has been shown to be able to regulate the tolerance to dryness in plants using the same, It is expected that it will be used to improve the crops available to mankind and improve the productivity of plants.

Description

[0001] The present invention relates to a method for enhancing dry stress resistance of plants using DEAD-box RNA helicase RH 8,

The present invention relates to a DEAD-box RNA helicase, a RH8 gene, a protein, and a method for enhancing dry stress resistance of a plant using the same.

Since the plants are sticky, they are constantly exposed to various stress conditions such as light, extreme temperature and moisture stress, among which water shortage in the soil affects the yield of crops and the quality of agricultural products worldwide. Plants have relieved the adverse effects of water stress through controlled pore opening and ion transport changes to reduce the loss of water vapor.

On the other hand, abscisic acid (ABA) plays a particularly important role in the reaction of dry stress, which is the key to plant defense mechanisms. For example, when a plant is exposed to dry stress, the plant begins signaling the irony response to the plant by increasing and accumulating ABA biosynthesis in the leaves. Since most stress-associated genes, including plant protection mechanisms, are regulated by ABA, the ABA- and dry-signaling pathways have been studied extensively, but this specific mechanism has not yet been clearly elucidated.

Some protein kinase 2C (PP2C) genes are required for ABA signaling, and 6 to 9 of the A group PP2Cs, including ABI1, ABI2, HAB1, HAB2, AHG1 and PP2CA in Arabidopsis, PP2CA blocks the expression of the ABA-mediated gene, the pp2ca mutant exhibits the ABA hypersensitive phenotype during seed germination and post-germination growth, while the transgenic plant with overexpression of PP2CA exhibits the ABA non-susceptible phenotype and the PP2CA Was further reported to modulate anionic channel activity in protected cells by inhibition of SnRK2.6 kinase. These results suggest that PP2CA plays an important role in ABA signal transduction.

Recent studies have reported that some genes related to the nucleic acid pathway, including RNA helicase, are involved in defense responses to abiotic stress. RNA helicase is a ubiquitous enzyme found in prokaryotes and eukaryotes, Lt; RTI ID = 0.0 > ATP-dependent < / RTI > In addition, at the sequence level, the RNA helicase is divided into five groups of SF1 to SF5, and the SF2 superfamily known as DEAD-box RNA helicase is the largest group of RNA helicases containing 58 members in the Arabidopsis genome. DEAD-box RNA helicase contains 7-8 conserved motifs related to ATP hydrolysis and double strand breaks, and RNA is present in a nonfunctional secondary structure before it is completed to functionally mature. Therefore, DEAD-box RNA helicase is involved in the reconstitution of RNA and ribonucleoprotein structure through RNA unwinding activity.

Several functions such as ribosome biogenesis of DEAD-box RNA helicase, mRNA splicing, siRNA gene silencing and non-sensory mediated mRNA collapse have been identified, and DEAD-box RNA helicase has also been shown to regulate ABA signaling and abiotic stress response Although the importance has been demonstrated (Korean Patent No. 10-1052565), the activity of RNA helicase has not yet been fully understood.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above problems, and the present inventors have found that RH 8 gene interacts with PP2CA in the drying stress of plants and plays a role as a positive regulator in the ABA signal transduction mechanism And confirmed that the gene silences the ABA hypersensitive and drought-susceptible phenotype. Based on this finding, the present invention has been completed.

The object of the present invention is to provide a RH8 (DEAD-box RNA helicase 8) gene comprising the nucleotide sequence of SEQ ID NO: 1, which encodes a positive regulator protein against the dry stress.

Another object of the present invention is to provide a composition for enhancing the dry stress resistance of a plant, which comprises the above gene or a RH8 (DEAD-box RNA helicase 8) protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

It is still another object of the present invention to provide a method for enhancing resistance to dry stress of a plant by transforming a gene encoding a RH8 (DEAD-box RNA helicase 8) protein into a plant.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a RH8 (DEAD-box RNA helicase 8) gene comprising the nucleotide sequence of SEQ ID NO: 1, which encodes a positive regulator protein against dry stress.

The present invention provides a composition for promoting dry stress resistance of a plant, which comprises the above gene or a RH8 (DEAD-box RNA helicase 8) protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

The present invention relates to a method for producing a plant comprising the steps of: (a) transforming a gene of SEQ ID NO: 1 encoding a RH8 (DEAD-box RNA helicase 8)

(b) over-expressing the RH8 (DEAD-box RNA helicase 8) protein in the transformed plant.

In one embodiment of the present invention, the RH8 protein may comprise the amino acid sequence of SEQ ID NO: 2.

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

In one embodiment of the present invention, the plant is a Arabidopsis thaliana.

The present invention relates to a RH8 (DEAD-box RNA helicase 8) gene encoding a positive regulator protein for drying stress. More specifically, the present invention relates to a RH8 gene encoding a positive regulator protein for drying stress, Interacts with and acts as a positive regulator in the ABA signal transduction pathway and has been shown to be able to regulate the tolerance to dryness in plants using the same, It is expected that it will be used to improve the crops available to mankind and improve the productivity of plants.

FIG. 1A shows a schematic diagram of the DEAD-box RNA helicase core domain for RH8, and FIG. 1B shows the results of analysis of the RH8 sequence by DEAD-box RNA helicase protein from other organisms.
FIG. 2A shows results obtained by a yeast two-hybrid assay (Y2H). FIG. 2B shows the fluorescence signals of the pp2ca-GFP fusion protein observed with a confocal microscope. FIG. , And Bimolecular Fluorescence Complementation (BiFC) analysis.
FIG. 3A shows the result of observing the intracellular position of the RH8 protein with a confocal microscope (white bar = 10 mu m) of the fluorescent signal of the RH8-GFp fusion protein, and FIG. 3B shows the presence or absence of the expression (T: total cells, C: cytoplasm, and N: nucleus).
Figure 4a shows the results of RH8 expression confirmation by RT-PCR analysis of wild-type (WT) and RH8 mutants and Figure 4b shows the results of RT-PCR analysis of wild type (WT) plants and mutants in rh8, pp2ca, rh8 / pp2ca mutants (WT) and RH8 mutants at ABA treatment conditions, Fig. 3d shows the results of RT-PCR analysis of wild-type (WT) and RH8 mutants at ABA treatment conditions 3E shows the root lengths of the wild type (WT) and RH8 mutants under ABA treatment conditions and FIG. 3F shows the results of comparing the root length growth rates of the wild type (WT) and RH8 mutants under ABA treatment conditions .
Figure 5a shows the dry weight of the wild-type (WT) and RH8 mutants at ABA treatment conditions, Figure 5b shows the wild type (WT) and RH 8 The leaf temperature of the mutant and Figure 5d show the results of comparing the pore openings of the wild-type (WT) and RH8 mutants under ABA treatment conditions.
Figure 6a shows the wild-type (WT), rh8, pp2ca and rh8 / pp2ca mutant phenotypes in ABA or dry stress conditions, Figure 6b shows wild type (WT) 6c and 6d show the root lengths of the wild type (WT), rh8, pp2ca and rh8 / pp2ca mutants in ABA or dry stress conditions, Fig. 6e shows the root lengths of ABA or dry In stress conditions, the live weight of the wild type (WT), rh8, pp2ca and rh8 / pp2ca mutants and Figure 6f shows the phenotype of the wild type (WT), rh8, pp2ca and rh8 / pp2ca mutants in ABA or dry stress conditions and Survival rates were compared.
Fig. 7 shows the results of confirming the phosphatase activity in wild-type (WT), RH8 and pp2ca mutants in the PP2C cells under ABA treatment conditions.

The inventors of the present invention have found that RH8 (DEAD-box RNA helicase 8) gene interacts with PP2Cs in the drying stress of plants and plays a role as a positive regulator in the ABA signal transduction mechanism, Thereby completing the invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a RH8 (DEAD-box RNA helicase 8) gene encoding a pogitive regulator protein for dry stress.

The RH8 gene preferably comprises the nucleotide sequence of SEQ ID NO: 1, and the protein encoded by the RH8 gene may desirably be SEQ ID NO: 2, but is not limited thereto.

As used herein, a "positive regulator protein" refers to a protein that acts in an increasing direction in the regulation of life events. That is, when the gene of the present invention, PIR, is overexpressed, resistance to ABA sensitivity increase and dry stress may be increased.

We observed protein-protein interaction proteins with all A-type PP2Cs (PP2CA) and found that protein RH8 (DEAD-box RNA helicase 8), a protein that interacts with PP2CA, a negative regulator of dry stress And to determine whether the protein is related to the ABA drying stress response (see Example 2).

In one embodiment of the present invention, it was confirmed that the RH8 protein was located in the nucleus and cytoplasm using the 35S: RH8-GFP fusion protein (see Example 3). Further, when the RH8 gene was silenced, It was confirmed that the sensitivity to ABA was decreased at the germination stage and the weaning stage (see Example 4).

In another embodiment of the present invention, leaf temperature and pore openness were measured in leaves of a mutant and wild type (WT) plant in which the RH8 gene was knocked out under dry conditions, and the effect of AB8 susceptibility by RH8 gene knockout was confirmed (See Example 5), functional interactions between PP2CA in ABA signal transduction and drought stress response were confirmed. As a result, RH8 regulated PP2CA to be an upstream regulator of PP2CA, and RH8 showed PP2CA phosphatase activity And activating ABA signaling (see Examples 6 and 7).

Accordingly, by enhancing the expression or activity of the RH8 protein, the resistance to the dry stress of the plant can be enhanced. Accordingly, in another aspect of the present invention, the present invention provides a method for producing the RH8 protein, The present invention provides a composition for promoting dryness resistance of a plant.

The enhancer may be a small interference RNA (siRNA), a shRNA (short hairpin RNA), or a miRNA (complementary RNA) that complementarily binds to mRNA for a sequence complementary to the RH8 gene, microRNA, ribozyme, DNAzyme, PNA (peptide nucleic acids), and antisense nucleotide, and is preferably a compound that binds complementarily to RH8 protein to inhibit activity, a peptide, a peptide But is not limited to, any one of Peptide Minetics, an antibody, and an aptamer.

In the present invention, the term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. Recombinant cells can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used herein to refer to a DNA fragment (s), a nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. A preferred example of the recombinant vector in the present invention may be, but is not limited to, a plasmid vector, pTRV1 or pTRV2, capable of transferring a part of itself to a plant cell when present in a suitable host such as Agrobacterium tumefaciens , And other suitable vectors that can be used to introduce the DNA of the present invention into a plant host include viruses such as those that can be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, Vector, e. G., An incomplete plant viral vector. The vector can be advantageously used when it is difficult to transform a plant host properly. Further, in the recombinant vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. The term "promoter " refers to a DNA upstream region from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.

In another aspect of the present invention, the present invention provides a plant transformed with the above recombinant vector and having improved dry resistance and / or seeds thereof. In addition, the present invention provides a method for enhancing the dry stress resistance of a plant comprising the step of over-expressing the RH8 gene by transforming a plant with the vector.

In the present invention, the plant (sieve) is a food crop including rice, wheat, barley, corn, soybean, potato, red bean, oats, sorghum; Vegetable crops including Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops including ginseng, tobacco, cotton, sesame seed, sugar cane, beet, perilla, peanut, and rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots, and bananas; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, tulips; And feed crops including rice, red clover, orchardgrass, alpha-alpha, tall fescue, perennialla grass, and the like, most preferably, but not limited to, Arabidopsis thaliana or red pepper.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example  1. Experimental Preparation and Experimental Methods

1-1. Yeast double-hybrid assay (Y2H)

For yeast library screening, the N-terminal 19 amino acid deletion construct was amplified from PP2CA cDNA and cloned into the pGBKT7 vector. The partial PP2CA BD-construct was transformed by introduction into yeast strain Y2Hgold using the lithium acetate-regulated transfer method (Ito et al. 1983). At this time, Y2H analysis was performed according to a conventionally known method.

More specifically, each construct was prepared by cloning full length RH8 and PP2Cs into the pGBKT7 and pGADT7 vectors, respectively. The bait and prey structures were constructed using the lithium acetate-controlled transfer method (Ito et al. 1983) And co-transformed into yeast strain AH109.

Transformants were selected as SC-lucisin-tryptophan medium and transferred onto interation selection medium (SC-adenine-histidine-leucine-tryptophan) to measure the growth rate as an indicator of protein-protein interaction. For serial dilution analysis, exponentially growing yeast cells were harvested and the OD600 value was adjusted to 0.5 with sterile double-distilled water. 5 [mu] l of each yeast cell culture was incubated with 10 [mu] M ABA added or not added SC-luisin-tryptophan and SC-adenine-histidine-leucine-tryptophan plates.

1-2. Plant materials and growth conditions

Arabidopsis thaliana (Ecological Col-0) plants were routinely cultivated in blended soil (peat moss: perlite: vermiculite = 9: 1: 1, v / v / v). The plants were grown under the conditions of 24 ℃ and 60% humidity at the light intensity of 130 ĩmol photons m ^-2 s ^{- 1} with a white fluorescence intensity of 16 hours / day / 8 hours / night. Were sterilized with 70% ethanol for 1 min and treated with 2% sodium hydroxide for 10 min. Next, the seeds were washed 10 times with sterilized distilled water and inoculated into Murashige and Skoog (1962) (MS) agar medium (pH 5.7) containing 1% sucrose. After incubation at 4 ° C for 2 days, the seeds were transferred to a growth chamber And grown under the same conditions as described above. At this time, T-DNA insertion line strains rh8-1 (SALK_016830), rh8-2 (SALK_029698) and pp2ca (SALK_124564) were obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org/).

Nicotiana benthamiana plants were cultivated in soil at 22 ° C for 16 hours / 8 hours at night for analysis of Bimolecular Fluorescence Complementation (BiFC) described below.

1-3. Biological tests of Stomatal apearture

For biological assays of pore openings, 5-week-old Arabidopsis rosette leaves were harvested and stomatal opening solution (SOS: 50 mM KCl, 10 mM MES-KOH, pH 6.15, 10 μM CaCl 2 ₂ ) and incubated for 3 hours, then the solution was replaced with SOS solution containing 10 [mu] M and 20 [mu] M ABA and further incubated for 2.5 hours. Next, pores were measured in each sample, and each experiment was performed three times independently.

1-4. Dehydration stress treatment

For dry tolerance analysis, water loss rates of wild-type (col-0), rh 8 knockout mutants and pp2ca / rh 8 double knockout mutants were measured.

More specifically, three weeks old Arabidopsis plants were removed from the soil, transferred to a culture dish, and the fresh weight was measured at the designated time to determine the water loss rate. Dry tolerance was performed using 4 weeks as previously described and dehydration stress was given water dehydration stress through re-watering for 2 days without water for 10 days until re-watering.

1-5. Analysis of Bimolecular Fluorescence Complementation (BiFC)

BiFC methods were used in plant cells to further confirm the protein-protein interactions identified in the yeast double hybridization assay.

More specifically, in order to prepare a BIFC construct, full-length cDNAs of RH8 and PP2CA having no stop codon were subcloned into 35S-CYCE and 35S-VYNE vectors via Spel / sall, The Agrobacterium tumefaciens strain GV3101 with the construct was mixed with the p19 strain to avoid gene silencing. Using a 1 ㎖ needle - less syringe, 5 - week - old tobacco ( Nicotiana benthamiana ) After 3 days of infiltration, the epidermal cells were cut and the epidermal cells were analyzed with a confocal microscope equipped with LSM Image Browser software (Zeiss 710 UV / Vis Meta, Oberkochen, Germany).

1-6. PP2C activity assay

Type 2C protein phosphatase assays were performed using the RediPlate 96 EnzChek Serine / Threonine Phosphatase Assay kit (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's instructions.

More specifically, seedlings grown in MS medium for 12 days were treated with 100 [mu] M ABA for 3 hours and seedlings to be used as control were left without ABA treatment. The harvested seedlings were pulverized in buffer [100 mM Tris-HCl, pH 7.9, 100 mM NaCl, 20 mM MgCl ₂ , 1 mM EGTA, 5 μM okadaic acid, 2 mM dithiothreitol, and 1% protease inhibitor cocktail] The homogenate was centrifuged at 14,000 x g for 10 minutes and the supernatant was collected and the total protein was quantitated using a BCA assay kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions.

Phosphatase assays were performed using 100 μg total protein and the reaction was initiated by the administration of the same volume of protein on 99-well plates pre-coated with substrate 6,8-difluoro-4-methylumberlliferyl phosphate (DiFMUP) , The substrate is catalyzed by protein phosphatase, which produces DiFMUs with excitation and emission maxima of 355 nm and 460 nm, respectively. After 15 min incubation at room temperature, fluorescence was monitored using an ELISA reader.

1-7. RNA isolation and qRT - PCR  (quantitative reverse transcription- polymerage  chain reaction analysis

Total RNA was extracted from Arabidopsis leaf tissues dehydrated or infected with bacterial pathogens using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). All RNA samples were digested with RNA-free DNase to remove genomic DNA.

After quantification with a spectrophotometer, 1 μg of total RNA was used for cDNA synthesis using the Transcript First Strand cDNA synthesis kit (Roche, Indianapolis, IN, USA). Incidentally, cDNA was synthesized without reverse transcriptase and qRT-PCR was performed to exclude possible contamination of genomic DNA in cDNA samples.

For qRT-PCR analysis, the synthesized cDNA was amplified using the specific primers shown in Table 1 below. At this time, all reactions were repeated three times and the Arabidopsis actin8 (AtACT8) gene was used for normalization.

1-8. Confirmation of migration to nucleus using nuclear fraction

Nuclear fraction was performed according to the previously described method (Kinkema et al. (2000)).

More specifically, the tissue at the Honda buffer [2.5% Ficoll 400, 5% dextran T40, 0.4 M sucrose, 25 mM Tris-HCl pH 7. 4, 10 mM MgCl 2, 10 mM b-mercaptoethanol, and proteinase inhibitor cocktail] The mixture was homogenized using mortar and pestle, filtered using a 62 μm nylon mesh, and then Triton X-100 was added to a final concentration of 0.5%. The homogenate was then cultured on ice for 15 minutes And centrifuged at 1,500 x g for 5 minutes. Then, the supernatant was collected (cytoplasmic fraction), and the pellet was resuspended in 1 ml of Honda buffer, transferred to a microcentritube, and the concentrate was centrifuged at 100 xg for 1 minute to pellet starch and cell debris, The supernatant was centrifuged at 1,800 x g for 5 minutes to pellet the nuclei.

At this time, the separated proteins were identified using Western blot analysis with anti-GFP, anti-histone H3 and anti-heat shock protein 90 antibodies.

Example  2. RH 8 and Of PP2CA  Identify physical interaction

In order to identify the interacting proteins affecting the degradation of PP2CA, Y2H screening was performed using the PP2CA protein as a bait and the Arabidopsis cDNA library as a prey, as shown in Example 1-1 above. At this time, to remove the self-activation, PP2CA in the N-terminal truncation form was used, and several PP2CA interacting protein candidates were isolated and the RNA helicase RH8 (AT4G00660) containing the DEAD- (See Fig. 1). At this time, the genomic sequence of the RH8 protein was obtained by TAIR (Arabidopsis Information Resource), which contains a 1562-bp open reading frame encoding a protein containing 505 amino acid residues.

In addition, the protein domain searched using the EMBL-EBI (pfam.xfam.org/) pFAM tool revealed the presence of DEAD-box and helicase C-terminal domain, 8 gene has RNA helicase properties and can function as a functional helicase.

To confirm the interaction between AtRH8 and PP2CA, one-one Y2H analysis was performed with RH8 and nine Group A PP2Cs as shown in Fig. 2A. As a result, AHG1, PP2CA and AIP 2. ≪ / RTI >

In addition, to confirm the interaction between PP2CA and RH8 in plant cells, Bimolecular Fluorescence Complementation (BiFC) analysis was performed as shown in Examples 1-5.

As a result, as shown in FIG. 2B, previous studies have shown that the intracellular location of PP2CA is in the nucleus, and co-expression of PP2CA and RH8: VYNE, as shown in FIG. 2C, Fluorescence (YFP) was induced, which mainly showed nuclei consistent with DAPI staining.

Based on the above results, it was confirmed that there is a physical interaction between PP2CA and RH8 protein.

Example  3. Analysis of subcellular localization of RH8 protein

In order to confirm the intracellular location of the RH8 protein, the epithelial cells of Nicotiana benthamiana leaves were transiently expressed in Agrobacterium tumefaciens strain GV3101 having a 35S: RH 8-GFP (green fluorescent protein) structure, GFP fluorescence was observed under a microscope, and DAPI staining was performed to stain nuclei.

As a result, as shown in FIG. 3A, GFP fluorescence was mainly found in the nucleus, and it was confirmed that it was weakly expressed in the P-body in accordance with DCP2 as a P-body control.

For each of the nucleus and cytoplasm, cell division analysis was performed using immunoblotting with histone H3 and heat shock protein 90 as fractional markers.

As a result, as shown in FIG. 3B, RH8 was detected only in the purified nucleus portion when ABA was not treated, but it was confirmed that the RH8 protein was weakly detected in cytoplasm 2 hours and 12 hours after ABA treatment.

Example  4. Confirm ABA Sensitivity Analysis

To study the in vivo function of RH8, phenotypic analysis of the T-DNA insert mutants (rh8-1: SALK_016830C and rh8-2: SALK_029698C) obtained from the Arabidopsis Biological Resource Center was performed.

As a result, as shown in Fig. 4A, no semi-qRT-PCR analysis revealed RH8 expression in the mutant.

In order to investigate whether knockout of RH8 gene affects ABA response, germination rate and primary root growth for ABA were measured.

More specifically, as a result of germination of wild type and rh8 mutants in MS medium containing 0.0 μM, 0.75 μM or 1.0 μM ABA, as shown in FIGS. 4B and 4C, when ABA was not treated, wild type (WT ) And rh 8 knockout mutants, but the germination rate of rh 8 knockout mutants was higher than that of wild type (WT) plants after ABA treatment, , And as shown in FIGS. 4D and 4E, it was confirmed that the primary roots of rh 8 knockout mutants appeared longer than the wild type (WT) plants.

Based on the above results, it was confirmed that RH 8 affects sensitivity to ABA in the germination and growth stages of plants.

Example  5. Identification of drought tolerance reduction of RH 8 gene knockout mutants

In order to confirm the RH 8 function in the defense reaction against the drought stress, the wild type (WT) and RH 8 mutants were stopped for 10 days and then re-watered for 2 days according to the method of Example 1-4, Lt; / RTI >

As a result, no phenotypic difference was observed between the wild-type (WT) and RH8 mutants under sufficient watering conditions, as shown at the top of FIG. 5A. However, after the drought stress, (WT) plants were more susceptible than wild-type (WT) plants. Then, after 2 days of re-watering for recovery, the survival rate of the wild type (WT) The survival rate was higher than the survival rate.

Next, the fresh weight after the dehydration treatment of the separated rosette leaves was measured to evaluate the water holding ability.

As a result, as shown in FIG. 5B, no significant difference was found in the leaf weight of the leaves between wild type (WT) and RH 8 mutants under sufficient water supply conditions. However, after drought stress, RH 8 Showed significantly higher live weight than mutants.

The results show that mutants with RH8 silencing have a phenotype sensitive to dry stress.

In addition, the water loss was affected by pore size, and the pore size was determined by ABA sensitivity. The leaf temperature and pore size before and after ABA treatment were measured to investigate the role of RH8 in ABA signal. More specifically, when the pore is opened, the rate of transpiration increases, and thus the temperature of the leaves is lowered. To confirm that the low rate of RH 8-silenced plants is due to a decrease in stomatal aperture, ABA And the pore size was measured.

As a result, as shown in Figs. 5C and 5D, there was no significant difference in leaf temperature and pore size between the wild-type (WT) and RH8 mutants when ABA was not treated, , The leaf temperature of the RH8 mutant was significantly lower than the leaf temperature of the wild type (WT) plants, as shown in Fig. 5C. Further, as shown in Fig. 5D, after 20 [mu] M ABA treatment, The pore size of the wild type (WT) leaf was significantly larger than that of the wild type (WT) leaf.

Taken together, these results indicate that RH 8 affects drought tolerance by controlling pore size in ABA mediated signal transduction.

Example 6. Identification of functional relationship between RH8 and PP2CA in ABA signal transduction and drought stress

To elucidate the functional relationship between RH8 and PP2CA in ABA signal transduction and drought stress response, rh8 / pp2ca double mutants were crossed with two independent single mutants and green cotyledon percentage and primary root By measuring the growth rate, ABA susceptibility levels were analyzed in single and double mutants.

As a result, as shown in FIGS. 6A to 6D, in the absence of ABA, no difference in phenotype was observed between wild type (WT) and mutant plants. However, after ABA treatment, mutations of pp2ca and rh8 / While the rh 8 single mutation showed the ABA-susceptibility phenotype.

In addition, the evaporation water loss was evaluated by measuring the fresh weight of the rosette leaves to determine whether the enhanced or reduced ABA susceptibility in the seeding stage was maintained at the growth stage.

As a result, as shown in Fig. 6 (e), it was confirmed that the fresh weight was higher in leaf tissues of mutants of pp2ca and rh8 / pp2ca than those of wild type (WT) plants.

In addition, as a result of a study on the response to drought stress of wild type (WT) plants and rh 8, pp2ca, rh 8 / pp2ca mutants, it was found that, at a sufficient water supply condition, But no phenotypic differences were observed in the rats. As shown in the middle of Fig. 6f, after stopping watering for 10 days, the pp2ca and rh8 / pp2ca mutants showed a drought resistant phenotype. As shown on the right side of Fig. Survival rates of pp2ca and rh8 / pp2ca were 86.4% and 83.7% higher than those of wild type (WT) plants after rehydration.

Taken together, these results demonstrate that RH8 modulates PP2CA and is an upstream regulator of PP2CA.

Example  7. PP2CA  Confirmation of RH 8-mediated inhibition of phosphatase activity

To determine if RH8 affects ABA signaling through inhibition of PP2CA phosphatase activity, total protein was extracted from 12 day old seedlings and PP2C activity was detected.

As a result, as shown in Fig. 7, it was confirmed that the PP2C activity of the rh8 mutant was higher than that of the wild type (WT) but the PP2C activity of the PP2CA mutant was lower than that of the wild type (WT).

Thus, it was confirmed that RH8 activates ABA signaling by inhibiting PP2CA phosphatase activity.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Method for improving the resistance to drought stress using          DEAD-box RNA helicase RH 8 in plants <130> MP17-037 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 1518 <212> DNA <213> DEAD-box RNA helicase 8_RH 8 <400> 1 atgaacaatc gaggaaggta ccctcctgga attggcgctg gccgtggagc ctttaaccct 60 aaccctaatt accaatcacg gtcgggttat caacaacatc cgccgcccca atacgttcaa 120 cgcggtaatt acgctcagaa tcaccaacaa cagtttcaac aagcgccgtc tcagccgcat 180 caataccaac agcaacagca gcaacaacaa caatggcttc gaagaggtca gatccccggc 240 ggtaacagta acggtgacgc tgttgtagaa gttgagaaaa ctgttcaatc cgaagttatc 300 gacccaaact cggaagattg gaaagcaagg ctaaaactac ctgcacctga tactcgatac 360 agaacagagg atgtgacagc caccaaaggg aacgagtttg aagattactt tttgaaacgg 420 gagcttctca tgggaatata tgagaagggt tttgaaagac cttctcctat tcaggaagag 480 agtattccaa ttgccttaac tggtagagat atccttgcca gagcaaagaa cgggactgga 540 aagaccgccg ccttttgtat tcctgtcttg gaaaaaattg accaagataa caacgttatt 600 caagctgtta taattgttcc gactcgagag ttagcccttc agacatcaca ggtctgtaaa 660 gagctgggta agcatttgaa gattcaagtc atggtgacca ctggtggcac cagtctgaaa 720 gacgatatca tgcgtttgta tcagcctgtt catttactgg taggaactcc tgggagaatt 780 ctggatctta ccaagaaagg tgtttgtgtt ttaaaagatt gttcggtgct tgttatggat 840 gaggctgaca agcttttgtc tcaagagttc caaccttcag tggaacattt gattagcttt 900 cttccagaaa gtcgtcagat tttgatgttc tcagccacct tccctgtcac tgtcaaggat 960 tttaaggata ggtttctgac gaatccttac gtcatcaatc tcatggatga acttacgctc 1020 aagggtatta cccaattcta tgctttcgtt gaagaaagac agaaaattca ttgcttgaat 1080 acactattct ccaagctgca aattaaccaa tcaatcatat tctgcaattc tgtaaatcgt 1140 gtggagctct tagccaagaa aatcacagaa cttgggtatt catgctttta catccatgca 1200 aagatgcttc aagaccaccg aaacagagtg ttccatgact tccggaatgg tgcatgccgt 1260 aatctcgtgt gtactgactt gttcacacgt ggtattgaca ttcaagccgt caatgtggtg 1320 ataaactttg atttcccaaa gaatgctgaa acttatctcc atagggttgg acgatcagga 1380 aggtttggcc atcttggtct agctgtgaat cttatcacct atgaagatcg atttaacctt 1440 taccggattg agcaagagct tggaacggag atcaagcaaa ttcctccaca tatcgatcag 1500 gcaatttatt gccaataa 1518 <210> 2 <211> 505 <212> PRT <213> DEAD-box RNA helicase 8_RH 8 <400> 2 Met Asn Asn Arg Gly Arg Tyr Pro Pro Gly Ile Gly Ala Gly Arg Gly   1 5 10 15 Ala Phe Asn Pro Asn Pro Asn Tyr Gln Ser Ser Ser Gly Tyr Gln Gln              20 25 30 His Pro Pro Pro Gln Tyr Val Gln Arg Gly Asn Tyr Ala Gln Asn His          35 40 45 Gln Gln Gln Phe Gln Gln Ala Pro Ser Gln Pro His Gln Tyr Gln Gln      50 55 60 Gln Gln Gln Gln Gln Gln Gln Trp Leu Arg Arg Gly Gln Ile Pro Gly  65 70 75 80 Gly Asn Ser Asn Gly Asp Ala Val Val Glu Val Glu Lys Thr Val Gln                  85 90 95 Ser Glu Val Ile Asp Pro Asn Ser Glu Asp Trp Lys Ala Arg Leu Lys             100 105 110 Leu Pro Ala Pro Asp Thr Arg Tyr Arg Thr Glu Asp Val Thr Ala Thr         115 120 125 Lys Gly Asn Glu Phe Glu Asp Tyr Phe Leu Lys Arg Glu Leu Leu Met     130 135 140 Gly Ile Tyr Glu Lys Gly Phe Glu Arg Pro Ser Pro Ile Gln Glu Glu 145 150 155 160 Ser Ile Pro Ile Ala Leu Thr Gly Arg Asp Ile Leu Ala Arg Ala Lys                 165 170 175 Asn Gly Thr Gly Lys Thr Ala Ala Phe Cys Ile Pro Val Leu Glu Lys             180 185 190 Ile Asp Gln Asp Asn Asn Val Ile Gln Ala Val Ile Ile Val Pro Thr         195 200 205 Arg Glu Leu Ala Leu Gln Thr Ser Gln Val Cys Lys Glu Leu Gly Lys     210 215 220 His Leu Lys Ile Gln Val Met Val Thr Thr Gly Gly Thr Ser Leu Lys 225 230 235 240 Asp Asp Ile Met Arg Leu Tyr Gln Pro Val His Leu Leu Val Gly Thr                 245 250 255 Pro Gly Arg Ile Leu Asp Leu Thr Lys Lys Gly Val Cys Val Leu Lys             260 265 270 Asp Cys Ser Val Leu Val Met Asp Glu Ala Asp Lys Leu Leu Ser Gln         275 280 285 Glu Phe Gln Pro Ser Val Glu His Leu Ile Ser Phe Leu Pro Glu Ser     290 295 300 Arg Gln Ile Leu Met Phe Ser Ala Thr Phe Pro Val Thr Val Lys Asp 305 310 315 320 Phe Lys Asp Arg Phe Leu Thr Asn Pro Tyr Val Ile Asn Leu Met Asp                 325 330 335 Glu Leu Thr Leu Lys Gly Ile Thr Gln Phe Tyr Ala Phe Val Glu Glu             340 345 350 Arg Gln Lys Ile His Cys Leu Asn Thr Leu Phe Ser Lys Leu Gln Ile         355 360 365 Asn Gln Ser Ile Ile Phe Cys Asn Ser Val Asn Arg Val Glu Leu Leu     370 375 380 Ala Lys Lys Ile Thr Glu Leu Gly Tyr Ser Cys Phe Tyr Ile His Ala 385 390 395 400 Lys Met Leu Gln Asp His Arg Asn Arg Val Phe His Asp Phe Arg Asn                 405 410 415 Gly Ala Cys Arg Asn Leu Val Cys Thr Asp Leu Phe Thr Arg Gly Ile             420 425 430 Asp Ile Gln Ala Val Asn Val Val Ile Asn Phe Asp Phe Pro Lys Asn         435 440 445 Ala Glu Thr Tyr Leu His Arg Val Gly Arg Ser Gly Arg Phe Gly His     450 455 460 Leu Gly Leu Ala Val Asn Leu Ile Thr Tyr Glu Asp Arg Phe Asn Leu 465 470 475 480 Tyr Arg Ile Glu Gln Glu Leu Gly Thr Glu Ile Lys Gln Ile Pro Pro                 485 490 495 His Ile Asp Gln Ala Ile Tyr Cys Gln             500 505

Claims (6)

delete delete A method for increasing the dry stress resistance of a plant comprising the steps of:
(a) transforming the gene of SEQ ID NO: 1 encoding a RH8 (DEAD-box RNA helicase 8) protein into a plant, and
(b) Overexpressing the RH8 (DEAD-box RNA helicase 8) protein in the transformed plant.
4. The method of claim 3, wherein the RH8 protein comprises the amino acid sequence of SEQ ID NO: 2.
A transgenic plant having enhanced dry stress resistance by the method of claim 3.
6. The transgenic plant according to claim 5, wherein the plant is a Arabidopsis thaliana.

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