KR20220029224A - CaSIZ1 gene and Method for improving the resistance to the drought stress using CaSIZ1 in plants - Google Patents

Abstract

The present invention relates to a novel Capsicum annuum SAP and Miz1 (CaSIZ1) gene/protein derived from red pepper and a method for increasing resistance to drought stress in plants using the same. According to the present invention, a function of the CaSIZ1 gene, which encodes a protein dephosphorylation enzyme regulating resistance to drought stress, is proved. It is confirmed that the CaSIZ1 protein functions as a negative regulator of abscisic acid (ABA) signaling and resistance to drought stress was increased in transgenic plants in which the protein was overexpressed. Accordingly, the CaSIZ1 gene can be used to increase crops capable of being used by mankind through regulation of expression of the gene and, in particular, is expected to be usefully used to increase productivity of plants.

Description

CaSIZ1 gene and method for improving drying stress resistance of plants using the same {CaSIZ1 gene and Method for improving the resistance to the drought stress using CaSIZ1 in plants}

The present invention relates to a novel CaSIZ1 ( Capsicum annuum SAP and Miz1) gene/protein derived from red pepper, and a method for enhancing drying stress resistance of plants using the same.

Global warming causes extreme climate change and causes environmental stress that inhibits the normal growth and development of plants, and plants have evolved sophisticated defense mechanisms to survive in adverse environments such as cold, high salt, and drought. For example, drought is caused by a moisture-deprived environment and severely affects the normal growth of plants, which eventually leads to a decrease in crop yield. It activates various defense mechanisms such as synthesis and accumulation of ABA).

ABA is a regulator for protecting plants against abiotic stress, particularly drying stress, and is known to play a very important role in plant growth and development. It is known that ABA signaling regulates the expression of various stress-related genes such as transcription factors and E3 ligase involved in osmotic adaptation and regulation of root hydraulic conductivity, but the complete ABA signaling pathway has yet to be elucidated. It didn't happen. In order to initiate ABA signaling in plant cells, the ABA receptor must be present and the signal must be transmitted to a subprotein. So far, it is known that PYR/PYL/RCARs play a role in recognizing ABA as ABA receptors. When the receptor recognizes ABA, the sub-protein is phosphorylated and dephosphorylated by SnRK2 (non-fermenting 1-related subfamily 2) protein kinase (kinase) and PP2Cs (type 2C protein phosphatases), respectively. SnRK2 kinases such as SnRK2.2, SnRK2.3, and SnRK2.6 (OST1) function as positive regulators of ABA signaling, whereas group A PP2Cs phosphatases function as negative regulators of ABA. , It has been reported that PYR/PYL/RCAR proteins, which are ABA receptors, interact with group A PP2Cs and inhibit their activity. When an increase in ABA expression is recognized by PYR/PYL/RCAR, protein phosphatase-kinase interaction is inhibited, and SnRK2 type kinase secretion is induced. SnRK2 kinase promotes expression of specific genes and activation of ion channels through phosphorylation of target proteins including transcription factors and SLAC1 anion channels, which enables plants to adapt to unfavorable environments.

On the other hand, like ubiquitination, protein sumoization is also determined by the sequential action of three enzymes (E1: SUMO-activating enzyme, E2: SUMO-conjugating enzyme, E3: SUMO ligase). Sumoization refers to a covalent bond through an isopeptide bond between a C-terminal glycine and a specific lysine in a target protein. Deconjugation (desumoylation) can occur by the sumo proteases responsible for SUMO recycling. In plants, medulla conjugation to target proteins can occur through rapid response mechanisms that manipulate substrates during abiotic and biotic stress responses. Sumohwa is also genetically associated with drying stress, heat stress, salt and cold stress, phosphate deficiency response and innate immunity. Sumoization has also been implicated in the salicylic acid (SA) and abscisic acid (ABA) signaling pathways that regulate the stress response. SUMO E3 ligase is known to play an important role in the in vivo sumoization pathway. They confer substrate specificity to transfer SUMO from the E2 enzyme to the substrate. On the other hand, ABI5 and MYB30, which are important transcription factors in the ABA signaling pathway, are affected by SIZ1 (SAP and Miz1). That is, it has been reported that ABI5 or MYB30 Sumoization by SIZ1 mediates the ABA reaction.

As such, studies have been made on whether E3 ligase is involved in the drying stress response through the ABA signaling pathway using many plants, but the results are still incomplete.

Catala, R., Ouyang, J., Abreu, I.A., Hu, Y., Seo, H., Zhang, X. and Chua, N.H. (2007) The Arabidopsis E3 SUMO ligase SIZ1 regulates plant growth and drought responses. The Plant cell, 19, 2952-2966.

The present inventors identified CaSIZ1 (Capsicum annuum SAP and Miz1) gene, which is an E3 ligase in red pepper, while studying to investigate the relationship between SUMO E3 ligase and the drying stress response through regulation of the ABA signaling pathway, and the gene was The present invention was completed by stating that it functions as a positive regulator of ABA signaling that mediates and stabilizes CaDRHB1 protein, which is a negative regulator of ABA signaling, and thereby promotes drying stress resistance of plants.

Accordingly, an object of the present invention is to provide a CaSIZ1 (Capsicum annuum SAP and Miz1) protein comprising the amino acid sequence of SEQ ID NO: 1, which is a positive regulator for drying stress.

Another object of the present invention is to provide a CaSIZ1 gene encoding the CaSIZ1 protein.

Another object of the present invention is to provide a recombinant vector comprising the CaSIZ1 gene.

Another object of the present invention is to provide a plant transformed with the recombinant vector.

Another object of the present invention is to provide a composition for enhancing drying stress resistance of plants comprising, as an active ingredient, at least one selected from the group consisting of the CaSIZ1 protein, the CaSIZ1 gene encoding it, and a recombinant vector containing the gene. .

Another object of the present invention is to provide a method for improving drying stress resistance of plants by transforming and overexpressing the CaSIZ1 gene in plants.

Another object of the present invention is to provide a transgenic plant having improved resistance to drying stress by the above method.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems 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 provides a CaSIZ1 (Capsicum annuum SAP and Miz1) protein consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention The CaSIZ1 gene encoding the CaSIZ1 protein is provided.

In one embodiment of the present invention, the CaSIZ1 gene may consist of the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

In another embodiment of the present invention, the CaSIZ1 gene may be isolated from pepper, but is not limited thereto.

In addition, the present invention provides a recombinant vector comprising the CaSIZ1 gene.

In one embodiment of the present invention, the recombinant vector may be for promoting drying stress resistance of plants, but is not limited thereto.

In addition, the present invention provides a plant transformed with the recombinant vector.

In addition, the present invention provides a composition for enhancing drying stress resistance of a plant comprising at least one selected from the group consisting of the protein, the CaSIZ1 gene encoding the same, and a recombinant vector containing the gene as an active ingredient.

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

(a) CaSIZ1 (Capsicum annuum SAP and Miz1) transforming the gene encoding the protein into a plant; and

(b) overexpressing CaSIZ1 protein in the transformed plant.

In addition, the present invention provides a transgenic plant having improved resistance to drying stress by the above method.

In addition, the present invention provides the use of a composition comprising, as an active ingredient, at least one selected from the group consisting of the protein, the CaSIZ1 gene encoding it, and a recombinant vector containing the gene, as an active ingredient, to enhance resistance to drying stress in plants.

In addition, the present invention provides a drying stress resistance of a plant comprising administering to the plant a composition comprising, as an active ingredient, at least one selected from the group consisting of the protein, the CaSIZ1 gene encoding the same, and a recombinant vector containing the gene. It provides a method of promotion.

The present invention has identified the CaSIZ1 gene encoding the drying stress resistance enhancing protein, the CaSIZ1 protein functions as a positive regulator of ABA signaling, and the drying stress resistance enhancing effect was confirmed in the transgenic plant in which the protein was overexpressed. of the CaSIZ1 gene It can be used for the improvement of crops that can be used by mankind through expression control, and is expected to be usefully used to improve the productivity of plants in particular.

1a shows the results of a cell-free degradation assay for CaDRHB1 using a crude extract extracted from dehydrated pepper leaves through immunoblot analysis.
Figure 1b shows the results of cell-free analysis of CaDRHB1 using a crude extract extracted from ABA-treated pepper leaves through immunoblot analysis.
Figure 2a shows the results of confirming the medulla conjugation promoted by the drought of the CaDRHB1 protein in vivo.
Figure 2b shows the results of cell-free degradation of CaDRHB1-SUMO (SUMO-conjugation-mimic) protein. M: MG132
Figure 2c shows the results of cell-free degradation of CaDRHB1 using a crude extract extracted from CaSIZ1 silent pepper leaves.
2d shows the results of cell-free degradation of CaDRHB1 using a crude extract extracted from CaMMS21 silenced pepper leaves.
Figure 2e shows the results of confirming the CaSIZ1 expression in the leaves of pepper plants after drought stress treatment.
Figure 2f shows the results of confirming CaMMS21 expression in the leaves of pepper plants after drought stress treatment.
Figure 3a shows the results of predicting the sumoization site of CaDRHB1.
3b shows the results of Co-IP analysis of CaDRHB1 fragments using CaSUMO 1-5.
Figure 3c shows the result of confirming that K138 is the in vivo sumohwa position.
Figure 4a shows the results of pull-down analysis of the direct interaction between MBP-CaDRHB1 and GST-CaSIZ1 fusion protein.
Figure 4b shows the results of Co-IP analysis of CaSIZ1 and CaDRHB1.
Figure 4c shows the BiFC analysis results of CaDRHB1 and CaSIZ1 .
Figure 4d shows the results of the in vitro Sumoization analysis of CaDRHB1.
Figure 4e shows the results of the in vivo Sumoization analysis of CaDRHB1.
Figure 5a shows the results of confirming the expression of CaSIZ1 according to drought, NaCl, ABA and low-temperature treatment by qRT-PCR analysis.
Figure 5b shows the organ-specific expression of CaSIZ1 in various pepper organs at seedling and maturation stages (LF, leaf; RT, root; CT, cotyledon; ST, stem; YLF, young leaf; FEL, fully grown leaf; PT, petiole; TR, primary root; LR, side root; FL, flower; GF, green fruit).
Figure 5c shows the results of analysis of the intracellular localization of CaSIZ1 based on the transient expression of the GFP fusion protein in N. benthamiana.
Figure 6a shows the results of RT-PCR analysis of the expression of CaSIZ1 gene in the leaves of TRV2:CaSIZ1 and TRV2:00 pepper plants.
Figure 6b shows the results of confirming the sensitivity to drought of TRV2:CaSIZ1 and TRV2:00 pepper plants.
6c shows the results of confirming the water loss of TRV2:CaSIZ1 and TRV2:00 at various time points after leaf separation.
Figure 6d shows the results of measuring the leaf temperature according to the ABA treatment in TRV2:CaSIZ1 and TRV2:00 pepper plants.
Figure 6e shows the results of measuring the leaf temperature according to the ABA treatment in TRV2:CaSIZ1 and TRV2:00 pepper plants.
Figure 6f shows the results of measuring pore opening according to ABA treatment in TRV2:CaSIZ1 and TRV2:00 pepper plants.
Figure 6g shows the results of measuring the pore opening according to the ABA treatment in TRV2:CaSIZ1 and TRV2:00 pepper plants.
6h shows the results of confirming the expression of stress response genes (CaOSR1, CaRD29B, CaRAB18, CaLOX1) in the dehydrated leaves of TRV2:CaSIZ1 and TRV2:00 pepper plants by qRT-PCR analysis.
7a shows the results of confirming CaSIZ1 expression in the leaves of CaSIZ1-OX transgenic plants and wild-type (WT) plants by RT-PCR analysis.
Figure 7b shows the germination rates of transgenic plants and wild-type plants in 0.5X MS medium supplemented with various concentrations of ABA.
Figure 7c shows the growth rate of transgenic plants and wild-type plants exposed to ABA.
7D shows the rate of cotyledon greening of transgenic plants and wild-type plants exposed to ABA.
Figure 7e shows the extent of root elongation of transgenic plants and wild-type plants exposed to ABA.
Figure 8a shows the drought susceptibility of CaSIZ1-OX transgenic plants and wild-type (WT) plants.
Figure 8b shows evaporative water loss in the leaves of CaSIZ1-OX transgenic plants and wild-type plants.
FIG. 8c shows thermal image images of CaSIZ1-OX transgenic plants and wild-type plants treated with 50 μM ABA.
Figure 8d shows the average leaf temperature of the three largest leaves of CaSIZ1-OX transgenic plants and wild-type plants.
Figure 8e shows the open air diagram of CaSIZ1-OX transgenic plants and wild-type plants.
Figure 8f shows the results of measuring the air opening degree of CaSIZ1-OX transgenic plants and wild-type plants.

The present inventors have identified the CaSIZ1 gene, which is a SUMO E3 ligase that functions as a positive regulator of ABA signaling, and confirmed the increase in resistance to drying stress in the transgenic Arabidopsis thaliana plant overexpressed with the gene, thereby completing the present invention.

Accordingly, the present invention provides a CaSIZ1 (Capsicum annuum SAP and Miz1) protein comprising the amino acid sequence of SEQ ID NO: 1, which is a positive regulator for drying stress, and a gene encoding the same.

CaSIZ1 , a gene of the present invention, preferably consists of the nucleotide sequence of SEQ ID NO: 2, and the peptide encoded by the CaSIZ1 gene may preferably consist of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

CaSIZ1 gene of the present invention may be isolated from red pepper, but is not limited thereto.

As used herein, the term "positive regulator protein" refers to a protein that acts in an increasing direction in regulating life phenomena. That is, when CaSIZ1 , which is the gene of the present invention, is overexpressed, ABA sensitivity may be increased, and resistance to drying stress may be increased.

The present inventors identified CaSIZ1, an E3 ligase protein, while studying the genes involved in the drying stress response, and the fact that as the sensitivity of abscisic acid (ABA) increases, it promotes pore closure, thereby enhancing drying resistance. Based on this, we tried to investigate the relationship between abscisic acid or drying stress and the CaSIZ1 gene.

First, in one embodiment of the present invention, it was confirmed through GFP fluorescence that CaSIZ1 protein was expressed in the nucleus, and it was confirmed that the expression of CaSIZ1 gene was increased in ABA or dried pepper plant leaves (see Example 4). .

In another embodiment of the present invention, in the leaves of red pepper plants and normal control pepper plants in which the expression of CaSIZ1 is inhibited under ABA-treated conditions, leaf temperature, stomata, water loss, survival rate, expression of stress response genes, etc. are measured Thus, the effect of reducing ABA sensitivity by CaSIZ1 gene silencing was confirmed, and it was confirmed that drying stress resistance was reduced in plants in which CaSIZ1 expression was actually inhibited compared to the control group (see Example 5).

In another embodiment of the present invention, a transgenic Arabidopsis plant overexpressing CaSIZ1 was prepared and CaSIZ1 was dried by measuring the germination rate, green cotyledon, and root growth rate of a wild-type plant and a CaSIZ1 overexpressing plant under ABA-treated conditions. It was confirmed that it acts as a positive regulator to enhance stress resistance (see Example 6), and in fact, it was confirmed through measurement of leaf temperature, pore openness, and moisture loss that drying resistance was increased when CaSIZ1 gene was overexpressed in Arabidopsis plants (implemented) See Example 7).

As another aspect of the present invention, there is provided a recombinant vector comprising the CaSIZ1 gene.

The term "recombinant" refers to a cell in which the cell replicates, expresses a heterologous nucleic acid, or expresses a peptide, heterologous peptide or protein encoded by the heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the native form of the cell, either in sense or antisense form. Recombinant cells can also express genes found in cells in a natural state, but the genes are modified and re-introduced into cells by artificial means.

The term "recombinant expression vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid and vector can be used as long as it is capable of replication and stabilization in the host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.

An expression vector comprising the above gene sequence and appropriate transcriptional/translational control signals can be constructed by methods well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a part of itself, the so-called T-region, into a plant cell when present in a suitable host such as Agrobacterium tumefaciens. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is currently used to transfer hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert the hybrid DNA into the genome of the plant and there is. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors such as those that can be derived from double-stranded plant viruses (eg CaMV) and single-stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous, especially when it is difficult to adequately transform a plant host.

The expression vector may include one or more selectable markers. The marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, Bleomycin, hygromycin, chloramphenicol There is a resistance gene, but is not limited thereto.

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 region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in a plant cell. A “constitutive promoter” is a promoter that is active under most environmental conditions and developmental states or cell differentiation.

In the recombinant vector of the present invention, a conventional terminator may be used, for example, nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens) ) of octopine (Octopine) gene terminator, etc., but is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells.

Transformation of a plant refers to any method of transferring DNA into a plant. Such transformation methods need not necessarily have a period of regeneration and/or tissue culture. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the hybrid DNA according to the invention into suitable progenitor cells. Methods include the calcium/polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts. electroporation (Shillito RD et al., 1985 Bio/Technol. 3, 1099-1102), microinjection with plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185) ), particle bombardment (DNA or RNA-coated) of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumapsis by infiltration of plants or transformation of mature pollen or vesicles In ence-mediated gene transfer, it can be appropriately selected from (incomplete) virus infection (EP 0 301 316) and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA transfer.

In the present invention, the recombinant vector is a recombinant plant expression vector.

As another aspect of the present invention, there is provided a plant transformed with the recombinant vector.

As another aspect of the present invention, there is provided a composition for promoting drying stress resistance of plants comprising, as an active ingredient, at least one selected from the group consisting of a CaSIZ1 protein, a CaSIZ1 gene encoding the same, and a recombinant vector containing the gene.

As another aspect of the present invention, the present invention provides a method for enhancing drying stress resistance of a plant, comprising the following steps.

(a) CaSIZ1 (Capsicum annuum SAP and Miz1) transforming the gene encoding the protein into a plant; and

(b) overexpressing CaSIZ1 protein in the transformed plant.

As another aspect of the present invention, there is provided a transgenic plant having improved resistance to drying stress by the method.

In the present invention, the plant (sieve) is rice, wheat, barley, corn, soybean, potato, red bean, oats, food crops including sorghum; vegetable crops including Arabidopsis thaliana, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion and carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, poplars, grapes, tangerines, persimmons, plums, apricots, and bananas; flowers including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And may be ryegrass, red clover, orchard grass, alpha wave, tall fescue, feed logistics including perennial ryegrass, and most preferably Arabidopsis thaliana or red pepper, but is not limited thereto.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental preparation and experimental method

1-1. Plant materials and growing conditions

Pepper ( Capsicum annuum L., cv. Nockwang ), and tobacco ( Nicotiana benthamiana ) seeds were steam-sterilized mixed soil [peat moss: perlite: vermiculite = 5:3:2, v/v /v], sand, and loam soil were mixed at a ratio of 1:1:1 (v/v/v) and sown. Thereafter, pepper plants were grown under a white fluorescent lamp (80 μmol photons m ^-2 ㆍS ^-1 ) in a growth room at 27±1° C. with a light/dark cycle of 16 hours. Tobacco plants were maintained in a growth chamber at 25±1° C. with a 16 hour light/8 hour dark cycle. Arabidopsis thaliana (ecological type Col-0) plant seeds were germinated in Murashige and Skoog (MS) salt containing 1% sucrose and Microagar (Duchefa Biochemistry). All seeds were allowed to flower and fruit for 2 days at 4°C before being placed in a growth chamber, and the plates were incubated at 24°C in a growth chamber for 16 hours light/8 hours dark cycle. Arabidopsis seedlings are white in steam-sterilized mixed soil [peat moss:perlite:vermiculite=9:1:1, v/v/v] at 24°C for 16 hours light/8 hours dark cycle. It was maintained at 60% relative humidity under fluorescent light (130 μmol photons m ^-2 ㆍS ^-1 ).

1-2. Cell-free degradation assay

The raw protein extract was prepared using extraction buffer [25 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 5 mM dithiothreitol (DTT), 0.1% Triton X-100, 10 mM ATP, and 10 mM NaCl] for 4 weeks. It was prepared from the leaves of the pepper plant. GST-labeled CaDRHB1 or GST-CaDRHB1-SUMO protein (500 ng) was incubated with the original protein extract (50 μg of total protein), and at the same time incubated in the presence of 50 μM of MG132 for 3 hours. To measure the protein degradation, it was analyzed by immunoblotting using an anti-GST antibody.

1-3. GST pull-down assay

Bacterial lysates were prepared from BL21 DE3 E. coli expressing GST-CaSIZ1, GST, MBP-CaDRHB1 or MBP for pull-down analysis. Lysates containing GST-CaSIZ1 or GST were incubated with Glutathione S-transferase (GST) resin at 4° C. for 2 hours, and then incubated with MBP-CaDRHB1 or MBP for additional 2 hours. The protein eluted from the resin was subjected to immunoblot analysis with an anti-MBP antibody (New England Biolabs) to detect MBP-CaDRHB1.

1-4. Co-immunoprecipitation assay

Agrobacterium tumefaciens cells containing 35S:HA-CaSIZ1 and 35S:CaDRHB1-GFP constructs were infiltrated into 4-week-old N. benethamiana leaves. Tobacco leaves were homogenized in liquid nitrogen and total protein was purified in protein extraction buffer (150 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM EDTA, 150 mM NaCl, 10 mM DTT, 0.2% Triton X-100, 2% PVPP, and 1X complete cocktail of protease inhibitors). After protein extraction, 5% of the extract was used as an input control. The protein extract was incubated with Pierce anti-HA agarose (Thermo Scientific) at 4°C for 3 hours. The precipitated protein was washed three or more times and then eluted with 4X SDS-PAGE loading buffer. The eluted samples were subjected to immunoblot analysis with anti-GFP (Santa Cruz Biotechnology) and anti-HA (Agrisera) antibodies.

1-5. Bimolecular fluorescence complementation assay (BiFC)

The full-length cDNA of CaSIZ1 or CaSIBZ1 without a stop codon was subcloned into a 35S-VYNE vector for generation of a bimolecular fluorescence complementation (BiFC) construct (Waadt et al., 2008). For transient expression, the Agrobacterium tumefaciens strain GV3101 harboring the above structure was mixed with the p19 strain to avoid gene silencing, and then using a 1 mL needleless syringe to 5 week old tobacco ( N. benthamiana ) plants (OD600) = 0.5) infiltrated into the hypoaxial side of the leaf. Three days after infiltration, leaf discs were excised and inferior epidermal cells were examined under a confocal microscope (510 UV/Vis Meta; Zeiss) equipped with LSM Image Browser software.

1-6. in vitro sumoylation assay

Recombinant proteins MBP-CaDRHB1 and GST-CaSIZ1 were purified from E. coli. 50 ng purified E1 His-CaSAE1, 50 ng purified E2 His-CaSCE1, 1 μg purified His-CaSUMO1, 0.5 μg MBP-CaDRHB1 and increasing amounts of GST-CaSIZ1 were added to 30 μl of reaction buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl ₂ , 2 mM ATP). Proteins were separated by SDS-PAGE and Sumo-conjugated MBP-CaDRHB1 was detected with anti-MBP (New England Biolabs) and anti-Sumo1 (Agrisera) antibodies. An increase in the protein level of GST-CaSIZ1 was detected with an anti-GST (Santa Cruz Biotechnology) antibody.

1-7. RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was extracted from 4-week-old pepper plants using TRI REAGENT (MRC) and reverse transcription was performed using the iScript cDNA synthesis kit (BIO RAD). Then, quantitative real-time PCR analysis was performed with IQ SYBR green SuperMix (BIO RAD) to detect specific genes. The expression of CaACT1 was used as a standard control. The experiment was repeated three times. PCR was performed with a program of 45 cycles at 95° C. for 5 minutes, 95° C. for 20 seconds, 60° C. for 20 seconds, and 72° C. for 20 seconds. The relative expression level of each gene was calculated by the ΔΔCt method.

1-8. Subcellular localization analysis

The CaSIZ1 coding region without a stop codon 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 into fully expanded tobacco plant leaves of 5 weeks. After 2 days of infiltration, the nucleus was stained by infiltrating with a DAPI (4',6-diamidino-2-phenylindole) solution (1 μg/ml), and was analyzed with a confocal microscope (510 UV/Vis Meta; Zeiss) equipped with LSM Image Browser software. Microscopic analysis was performed.

1-9. Virus-induced gene silencing (VIGS)

For loss-of function analysis of CaSIZ1, virus-induced gene silencing (VIGS) was performed in pepper plants. A 726-1025-bp fragment of CaSIZ1 cDNA was inserted into the pTRV2 vector. In addition, the Agrobacterium tumefaciens strain GV3101 carrying pTRV1 and pTRV2: CaSIZ1 or pTRV2:00 was infiltrated into fully expanded cotyledons of pepper (OD ₆₀₀ = 0.2 for each strain). Plants were placed in a growth room at 25°C with a photoperiod set to 16 hours daytime/8 hours night for growth and virus proliferation.

1-10. Production of CaSIZ1-OX transgenic Arabidopsis plants

ml^-1의 카나마이신이 함유된 MS 한천 플레이트에 파종하였다.The full-length CaSIZ1 cDNA was bound to the pENTR/D-TOPO vector (Invitrogen) and then inserted into the pK2GW7 binary vector using the LR reaction for constitutive expression of CaSIZ1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. in Arabidopsis. The construct was transformed into A. tumefaciens strain GV3101 using the floral dip method (Clough and Bent, 1998). To select transgenic lines, 50 μg of seeds harvested from transformed plants

It was sown on MS agar plates containing ml ^-1 of kanamycin.

1-11. ABA, drying stress, NaCl treatment and morphology analysis

To investigate the expression pattern of CaSIZ1 in red pepper, 4-week-old pepper plants were treated with 100 μM ABA, drying stress, 200 mM NaCl, or low temperature (4° C.). For NaCl treatment, pepper plants were irrigated with 200 mM NaCl solution. Harvested at 0, 0.5, 1, 3, 6, 9 and 12 hours (ABA, dry) or 0, 2, 6, 12 and 24 hours (NaCl, low temperature) after each treatment. For qRT-PCR analysis, 4-leaf stage CaSIZ1 silenced pepper plants prior to drought stress were carefully removed from the soil and harvested at designated time points after treatment.

For germination and seedling growth tests, 100 seeds of wild-type and CaSIZ1-OX Arabidopsis lines were sown on plates containing MS agar medium supplemented with various concentrations of ABA. The number of seeds with roots and seeds with green cotyledons were counted every day until 4 days after seeding. For ABA-inhibited primary root growth analysis, MS agar plates were placed vertically and root length was measured on day 6 after dispensing.

Dry stress tolerance analysis was performed using 4-leaf stage CaSIZ1 silent pepper plants and 3-week-old CaSIZ1-OX Arabidopsis plants. To measure transpiration water loss, leaves were separated from 4-leaf pepper plants and 3-week-old Arabidopsis plants, and weight loss was measured at the indicated time points. All experiments were performed at least three times.

1-12. Thermal imaging

For thermal imaging analysis, both first and second leaf-growing 4-week-old pepper plants and 3 or 4-week-old Arabidopsis plants were treated with 50 μM ABA. Thermal images were obtained using an infrared camera (FLIR systems; T420) and leaf temperature was measured with FLIR Tools + ver 5.2 software.

1-13. Bioassay of stomatal aperture

Bioquantification of stomatal aperture was performed as previously described (Lim and Lee, 2016). Briefly, leaf epidermal skins were harvested from rosette leaves of 3-week-old Arabidopsis plants and suspended in stomata opening solution (SOS; 50 mM KCl, 10 mM MES-KOH, 10 μM CaCl ₂ , pH 6.15). After 3 hours of incubation, the buffer was replaced with fresh SOS containing 10 or 20 μM ABA. After an additional 2.5 h incubation, 100 pores in each individual sample were randomly observed with a Nikon Eclipse 80i microscope, and the width and length of the pores were measured using Image J 1.46r (http://imagej.nih.gov/ij). measured. Each experiment was performed three times.

Example 2. Confirmation of improvement in stability of CaDRHB1 protein through Sumoization

In order to confirm that CaDRHB1 acts as a positive regulator of ABA and drying stress, acellular degradation assay was performed using ABA and drying stress-treated pepper plants.

As shown in FIG. 1A , the degradation of CaDRHB1 protein was reduced in the crude extract of red pepper subjected to drought stress for 6 hours compared to the crude extract of healthy pepper.

In addition, as shown in Figure 1b, the degradation of CaDRHB1 protein was also reduced in the crude extract of ABA-treated red pepper. These results indicate that CaDRHB1 protein was stabilized in response to drought and ABA.

On the other hand, CaDRHB1, which is a component of HD-ZIP I, is known to contain a putative motif for sumohwa. Therefore, in order to confirm whether drought is involved in the sumoization of CaDRHB1 protein, in vivo sumoization was performed using Nicotiana benthamiana transiently expressing 35S:CaDRHB1-GFP and 35S:HA-CaSUMO1-5.

As a result, as shown in FIG. 2a , it was confirmed that the Sumoization of CaDRHB1 was higher in the drought-treated leaves than in the healthy leaves.

Then, by performing a cell-free analysis using a sumo-like CaDRHB1 protein made by fusing the sumo-protein sequence to the C-terminus of CaDRHB1, it was confirmed whether sumo-ization and the stability of CaDRHB1 were associated.

As a result, as shown in FIG. 2b , it was found that degradation of Sumo-mimicking CaDRHB1 (CaDRHB1-SUMO) was reduced compared to wild-type CaDRHB1. From the above results, since CaSIZ1 and CaMMS21 are pepper SUMO E3 ligase genes, it was assumed that CaSIZ1 or CaMMS21 could promote SUMO binding of CaDRHB1.

Next, cell-free digestion was performed with the crude extract of CaSIZ1- or CaMMS21 silenced pepper in order to identify the protein mediating the sumoization of CaDRHB1 protein.

As shown in Figure 2c, the GST-CaDRHB1 protein was much more degraded in CaSIZ1 silenced pepper than the control plant, whereas

As shown in Figure 2d, CaMMS21 silenced peppers were not different from control plants.

In addition, as shown in FIGS. 2e and 2f , drought treatment induced the expression of CaSIZ1, but not CaMMS21.

These results suggest that drought-induced sumolation of CaDRHB1 increases the stability of CaDRHB1 protein and that sumolation of CaDRHB1 may be regulated by CaSIZ1 sumo E3 ligase.

Example 3. Confirmation of Sumoization Location of CaDRHB1

In order to confirm that there is a relationship between sumoization and stability of CaDRHB1, the position of sumoization of CaDRHB1 was confirmed.

As shown in Fig. 3a, according to the SUMOplot analysis program, CaDRHB1 was predicted to have two high-confidence SUMO junction sites (K120 and K138).

In addition, five fragments of truncated form of CaDRHB1 were generated to investigate and confirm the sumoization site of CaDRHB1. Each of the five fragments of 35S: HA-CaSUMO1-5 and CaDRHB1 (35S: CaDRHB1 F1 to 5-GFP) was transiently co-expressed in Nicotiana benthamiana, and then in the total protein using anti-HA agarose beads. Immunoprecipitation was performed.

As a result, as shown in FIG. 3B , immunoblot analysis using an anti-GFP antibody showed that SUMO conjugation occurred strongly in the F3 fragment.

In addition, 9 CaDRHB1 F3 mutants were generated in which lysine (K) of the F3 fragment was replaced with arginine (R) in order to find the correct sumoization site.

As a result, as shown in Fig. 3c, SUMO conjugation of CaDRHB1 F3 K138R was significantly reduced compared to intact CaDRHB1 F3 and other mutants.

From the above results, it was confirmed that the K138 residue of CaDRHB1 was a possible site for sumoization.

Example 4. Confirmation of physical interaction of CaDRHB1 and CaSIZ1

Pull-down, co-immunoprecipitation (co-IP) and bimolecular fluorescence complementarity analysis (BiFC) were performed to confirm the direct interaction between CaDRHB1 and CaSIZ1.

As shown in Figure 4a, GST-CaSIZ1 was able to pull down MBP-CaDRHB1 using the purified recombinant protein. On the other hand, it was shown that GST and MBP free proteins failed to interact with MBP-CaDRHB1 and GST-CaSIZ1, respectively.

As shown in Fig. 4b, in the co-IP assay, CaDRHB1-GFP was immunoprecipitated by anti-HA agarose beads from the whole protein of N. benthamiana expressing HA-CaSIZ1/CaDRHB1-GFP, but HA-CaSIZ1/GFP was GFP was not immunoprecipitated from all expressed proteins.

As shown in Fig. 4c, in the BiFC analysis, a yellow fluorescent protein (YFP) signal was generated in N. benthamiana expressing CaDRHB1-VYNE and CaSIZ1-CYCE. These results indicate that CaDRHB1 directly interacts with CaSIZ1 in vitro and in vivo.

On the other hand, in order to determine whether CaSIZ1 promotes Sumoization of CaDRHB1, an in vitro Sumoization analysis was performed. MBP-CaDRHB1, CaSUMO1-5, SUMOylation enzymes E1 (CaSAE1) and E2 (CaSCE1) were co-cultured with increasing amounts of GST-CaSIZ1.

As shown in FIG. 4D , immunoblot results using an anti-MBP antibody and an anti-SUMO1 antibody showed an increase in SUMO-conjugated MBP-CaDRHB1 as the GST-CaSIZ1 level increased.

In addition, to confirm CaSIZ1-mediated sumomorphization of CaDRHB1, we performed an in vivo sumomorphization assay using N. benthamiana transiently expressing 35S:HA-CaSIZ1, 35S:CaDRHB1-GFP and 35S:HA-CaSUMO1-5. did.

As shown in Figure 4e, immunoprecipitation followed by immunoprecipitation using GFP-trap, immunoblot showed that CaDRHB1-GFP was primed and stabilized by HA-CaSIZ1, whereas HA-CaSIZ1C379S/H381Y (CaSIZ1 SP-RING domain mutant) or CaDRHB1K138R -GFP-expressing N. benthamiana showed decreased sumomorphization and stability.

Taken together, these results suggest that CaSIZ1 interacts with CaDRHB1 and promotes SUMO conjugation of CaDRHB1.

Example 4. Confirmation of molecular properties of CaSIZ1

Based on the fact that CaDRHB1 positively modulates the drought response, we speculated that CaSIZ1 might be involved in the stress response because male-hair conjugation of CaDRHB1 is facilitated by CaSIZ1 and CaDRHB1 stability is increased by male-hair bonding. Accordingly, RT-PCR analysis was performed to investigate the expression pattern of CaSIZ1 under stress conditions.

As shown in Figure 5a, in the case of dried (drought) and ABA-treated red pepper, the expression level of CaSIZ1 gradually increased after treatment and was highest at 12 hours. The NaCl-treated red pepper showed the highest expression level at 6 hours and gradually decreased. The expression level was decreased in cold stress.

As shown in FIG. 5B , as a result of examining the expression of CaSIZ1 in each organ of a pepper plant, the expression level in flowers (FL) and green fruit (GF) was higher than in other organs.

As shown in FIG. 5c , the intracellular localization of CaSIZ1 was analyzed by transiently expressing the CaSIZ1-GFP fusion protein in N. benthamiana leaves, and it was confirmed that the CaSIZ1-GFP protein was localized to the nucleus.

Example 5. Confirmation of improvement in sensitivity of CaSIZ1 silenced pepper plants to drying stress

To study the biological function of CaSIZ1 in pepper plants, virus-induced gene silencing (VIGS)-based gene function analysis was used according to Examples 1-9.

First, as shown in Figure 6a, CaSIZ1 expression was not shown in CaSIZ1 silenced (CaSIZ1-silenced) pepper (TRV2:CaSIZ1).

Therefore, in order to investigate the response to drying stress, watering was stopped for 14 days in a 4-week-old control plant (TRV2:00) and CaSIZ1 silent pepper.

As shown in Fig. 6b, CaSIZ1 silenced peppers exhibited a more wilted phenotype under drought stress conditions and re-watering conditions, as shown in the middle and right panels of Fig. 6b, and CaSIZ1 than control plant viability (91.1%). Silent pepper survival rate (23.2%) was much lower, indicating more drought sensitivity compared to control plants.

To evaluate whether the drought-sensitive phenotype exhibited by CaSIZ1-silencing plants was derived from altered water retention capacity, we calculated leaf water loss rates by weighing fresh leaves.

As shown in FIG. 6c , the water loss rate of the leaves was higher in CaSIZ1-silenced plants than in control plants.

Next, the effect of ABA on the drought-sensitive phenotype was confirmed by measuring the leaf surface temperature and stomata after ABA treatment.

As shown in FIGS. 6D and 6E , there was no difference in leaf temperature in the absence of ABA treatment. The leaf temperature of CaSIZ1 silenced peppers after 50 μm ABA treatment was lower than that of control plants. This indicates that relatively more evaporative cooling occurred in CaSIZ1 silenced peppers.

To determine whether the enhanced evaporative cooling was due to the inhibition of ABA-induced pore occlusion, the pore size was measured.

As shown in FIGS. 6f and 6g , in the absence of ABA, stomata diameter did not differ between control plants and CaSIZ1-silent peppers. However, ABA-induced stomatal occlusion was reduced in CaSIZ1 silenced peppers compared to control plants. This means that CaSIZ1 silenced peppers exhibit a drought-sensitive phenotype.

In addition, the expression patterns of stress response genes in control plants and CaSIZ1 silenced peppers were analyzed.

As shown in FIG. 6h , the expression levels of CaOSR1, CaRD29B, CaRAB18 and CaLOX1 were significantly lower in CaSIZ1 silenced pepper plants than in control plants at 3 and 6 hours after drought treatment.

Example 6. Confirmation of improved ABA sensitivity in Arabidopsis thaliana plants overexpressing CaSIZ1

To further investigate the function of CaSIZ1, a CaSIZ1 overexpressing (OX) Arabidopsis mutant was constructed and semi-quantitative RT-PCR analysis was performed.

As shown in Figure 7a, CaSIZ1 transcript expression was detected in two independent T3 homozygous transgenic lines, but not in wild-type (WT) plants.

In addition, the germination rate and ABA sensitivity were investigated at the seedling stage.

7B to 7E , in the absence of ABA, there was no difference in germination rate, napped hair, and root length between wild-type and CaSIZ1-OX plants. On the other hand, when ABA was treated, CaSIZ1-OX plants showed lower germination rate, green cotyledon and root growth rate than wild-type plants.

These results indicate that CaSIZ1 positively regulates the ABA response.

Example 7. Confirmation of improvement in tolerance of CaSIZ1 overexpressing Arabidopsis thaliana to drought stress

To investigate the effect of CaSIZ1 on drought stress response, 4-week-old wild-type plants and CaSIZ1-OX plants were used.

As shown in FIG. 8A , there was no difference from the wild-type control group in the well-watered state (upper panel). In contrast, CaSIZ1-OX plants exhibited a less wilted phenotype than wild-type plants when no water was given for 14 and 15 days (middle panel). After 3 days of rewatering, CaSIZ1-OX plants showed a more drought-tolerant phenotype than wild-type plants, and the survival rate of CaSIZ1-OX plants was higher than that of wild-type plants (lower panel).

As shown in Figure 8b, the water loss of CaSIZ1-OX plants was lower than that of wild-type plants, which is consistent with the drought-tolerant phenotype.

In addition, leaf temperature and stomatal pore size were measured to determine whether the decreased transpiration water loss was due to ABA sensitivity.

As shown in FIGS. 8c to 8f , there was no difference in leaf temperature and stomata before ABA treatment. Specifically, the leaf temperature of CaSIZ1-OX plants after ABA treatment was higher than that of wild-type plants, and the pore size was smaller in CaSIZ1-OX plants than in wild-type plants.

Taken together, these results suggest that CaSIZ1 positively modulates drought tolerance by promoting ABA-mediated stomatal closure.

Through the above results, it was confirmed that CaSIZ1 acts as a positive regulator for drying resistance in Arabidopsis as well as pepper plants by inducing the expression of ABA-mediated stomatal occlusion and drying response marker genes.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> CaSIZ1 gene and Method for improving the resistance to the drought stress using CaSIZ1 in plants <130> MP20-146 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 858 <212> PRT <213> Artificial Sequence <220> <223> CaSIZ1 <400> 1 Met Asp Leu Val Thr Ser Cys Lys Asp Lys Leu Thr His Phe Arg Ile 1 5 10 15 Lys Glu Leu Lys Asp Val Leu Thr Gln Leu Gly Leu Ser Lys Gln Gly 20 25 30 Lys Lys Gln Asp Leu Val Asp Arg Ile Leu Ala Ile Leu Ser Asp Glu 35 40 45 Arg Val Ser Gly Met Trp Ala Lys Lys Asn Ser Val Gly Lys Glu Glu 50 55 60 Val Ala Lys Leu Val Asn Gly Ile Tyr Arg Lys Met Pro Leu Ser Gly 65 70 75 80 Ala Thr Asp Leu Ala Ser Lys Ser Gln Ile Val Ser Asp Ser Ser Asn 85 90 95 Val Thr Leu Lys Glu Glu Val Glu Asp Thr Tyr Lys Met Lys Ile Arg 100 105 110 Cys Pro Cys Gly Ser Ser Leu Gln Ala Glu Thr Met Ile Gln Cys Glu 115 120 125 Asp Ile Arg Cys Arg Thr Trp Gln His Ala Cys Cys Val Ile Ile Pro 130 135 140 Lys Lys Pro Met Glu Gly Gly Phe Pro Pro Ile Phe Pro Glu Thr Phe 145 150 155 160 Tyr Cys Glu Leu Cys Arg Leu Ser Arg Ala Asp Pro Phe Trp Val Thr 165 170 175 Met Ala Asn Pro Leu Tyr Pro Val Lys Leu Ala Ile Thr Ser Ala Pro 180 185 190 Ala Asp Ser Thr Asn Pro Met Gln Arg Ile Glu Lys Thr Phe Gln Leu 195 200 205 Thr Arg Ala Asp Met Glu Leu Leu Ala Lys Gln Glu Tyr Asp Val Gln 210 215 220 Ala Trp Cys Met Leu Leu Asn Asp Lys Val Gln Phe Arg Met Gln Trp 225 230 235 240 Pro Gln Cys Ala Asp Leu Gln Val Asn Gly Ala Pro Val Arg Cys Ile 245 250 255 Asn Arg Ser Gly Ser Gln Leu Leu Gly Ala Asn Gly Arg Asp Asp Gly 260 265 270 Pro Ile Ile Thr Thr Cys Ser Arg Asp Gly Ile Asn Lys Ile Leu Leu 275 280 285 Ala Gly Cys Asp Ala Arg Ile Phe Cys Leu Gly Val Arg Leu Val Lys 290 295 300 Arg Arg Thr Ser Gln Gln Ile Leu Asn Ile Ile Pro Lys Glu Ala Asp 305 310 315 320 Gly Glu Val Phe Asp Asp Ala Leu Ala Arg Val Arg Arg Cys Val Gly 325 330 335 Gly Gly Thr Thr Thr Glu Asn Ala Asp Asn Asp Ser Asp Leu Glu Val 340 345 350 Val Ala Asp Cys Ile Pro Val Asn Leu Arg Cys Pro Met Ser Gly Ser 355 360 365 Arg Met Lys Val Ala Gly Arg Phe Lys Pro Cys Val His Met Gly Cys 370 375 380 Phe Asp Leu Glu Val Phe Val Glu Met Asn Gln Arg Ser Lys Lys Trp 385 390 395 400 Gln Cys Pro Ile Cys Leu Lys Asn Tyr Ser Leu Glu His Val Ile Ile 405 410 415 Asp Pro Tyr Phe Asn Arg Ile Ser Ser Gln Leu Arg Thr Tyr Gly Glu 420 425 430 Glu Val Ser Glu Ile Glu Val Lys Pro Asp Gly Ser Trp Arg Ala Lys 435 440 445 Ala Glu Gly Asp Arg Lys Ser Leu Gly Asp Leu Val Arg Trp His Leu 450 455 460 Pro Asp Gly Thr Leu Ile Glu Ser Gln Asp Ile Glu Ser Lys Pro Lys 465 470 475 480 Pro Gly Val Leu Lys His Val Lys Gln Glu Gly Gly Ser Glu Ser Leu 485 490 495 Gly Gly Leu Lys Val Gly Leu Lys Lys Asn Arg Asn Gly Leu Trp Glu 500 505 510 Ile Asn Lys Pro Glu Asp Ile Gln Ala Leu Pro Tyr Arg Asn Gly Leu 515 520 525 Arg Glu Asn Phe Glu Asn His Ile Gln Asp Ile Ile Pro Met Gly Ser 530 535 540 Ser Ala Thr Glu Ser Gly Lys Glu Gly Glu Asp Pro Ser Val Asn Gln 545 550 555 560 Gly Gly Asp Val Ile Phe Asp Phe Pro Thr Asn Gly Phe Glu Leu Glu 565 570 575 Thr Ile Ser Pro Asn Ser Ala Pro Ala Tyr Gly Cys Asn Asp Arg Asn 580 585 590 Pro Pro Ala Pro Ala Gly Asp Ala Glu Ile Ile Val Leu Ser Asp Ser 595 600 605 Asp Glu Glu Asn Glu Pro Leu Ile Ser Ser Ala Ser Ile Tyr Asn Asn 610 615 620 Asn His Thr Asp Glu Pro Ile Val Ser Phe Ala Gly Arg Pro Lys Gly 625 630 635 640 Ile Ser Asp Ser Cys Asn Asp Asp His Ala Leu Val Asn Asp Gly Asn 645 650 655 Ser Cys Leu Gly Leu Phe Phe Gly Met Asn Met Trp Pro Leu Pro Thr 660 665 670 Ile Asn His Gly Arg Pro Gly Phe Gln Leu Phe Gly Ser Asp Met Gln 675 680 685 Gln Gly Pro Ile Asn Cys Ala Ser Ser Ile Asn Gly Tyr Ser Leu Ala 690 695 700 Ala Asp Thr Gly Ile Gly Ser Cys Ser Leu Leu Pro Glu Ser Ser Val 705 710 715 720 Asp Arg Thr Asn Thr Gln Ile Asn Asp Gly Leu Val Asn Asn Pro Leu 725 730 735 Ser Phe Gly Ser Asn Asp Pro Ser Leu Gln Ile Phe Leu Pro Thr Arg 740 745 750 Pro Leu Asn Ala Ser Val Glu Ala Asp Val Gly His Gln Pro Gly Leu 755 760 765 Ser Thr Gly Ile His Thr Glu Asp Trp Phe Ser Leu Ser Leu Gly Ala 770 775 780 Gly Gly Asp Ser Ala Val Ala Ser Gly Leu Arg Ser Gly Lys Pro Ser 785 790 795 800 Gln Thr Lys Asn Ser Ser Leu Asp Ser Leu Ala Asp Thr Ala Ser Phe 805 810 815 Leu Leu Gly Met Asn Gly Gly Val Ser Met Lys Ser Gly Arg Glu Arg 820 825 830 Ser Asp Gly Pro Phe Asn Phe Pro Arg Gln Arg Arg Ser Val Arg Pro 835 840 845 Arg Tyr Leu Asn Ile Asp Ser Asp Thr Asp 850 855 <210> 2 <211> 2577 <212> DNA <213> Artificial Sequence <220> <223> CaSIZ1 <400> 2 atggatttgg taactagctg caaggataaa ttgacacatt ttcggataaa ggagctcaaa 60 gatgtgctta ctcaacttgg tctatcaaag caaggaaaga agcaggacct tgttgatcgc 120 atattagcta ttctttctga tgaacgagtt tcaggaatgt gggcaaagaa aaatagtgtt 180 ggaaaggaag aggttgcaaa acttgtcaat ggaatttaca gaaaaatgcc attgtctggg 240 gcaactgacc tagcatctaa gtcacagatt gtctcagata gcagtaatgt aacgttaaaa 300 gaggaagttg aagatactta taagatgaag attcgatgtc cctgtggaag ctcgctacaa 360 gctgaaacaa tgattcagtg tgaagatata agatgccgca catggcaaca tgcatgctgt 420 gtcatcattc caaaaaagcc catggagggg ggttttcctc caatttttcc tgaaactttt 480 tactgtgaat tgtgcagatt gagtcgtgca gacccgttct gggtgacaat ggcaaaccct 540 ttatatcctg ttaagttggc tatcactagc gcacctgctg atagcacaaa cccaatgcaa 600 aggattgaga aaacatttca actcactaga gccgacatgg aattattggc aaagcaggaa 660 tatgatgttc aggcttggtg catgcttctc aatgacaagg tacaatttag gatgcagtgg 720 cctcagtgtg ctgatttgca ggtcaatgga gctccagtac gttgcataaa tagatctggc 780 tcacaattat tgggtgcaaa tggtcgagat gatggcccaa taatcacaac atgctccaga 840 gatggaatta acaaaatttt gttagcagga tgtgatgctc gtatcttttg cttgggagtt 900 agacttgtaa agcgccgtac ttcccaacag attctgaata ttatccctaa agaggctgat 960 ggtgaggtat ttgatgatgc tcttgctcgc gttcgtcgtt gcgttggtgg tgggactacc 1020 actgaaaatg ctgataatga cagtgacctc gaagtggttg ctgattgtat cccagtcaat 1080 cttcgatgcc ctatgagtgg ttcaagaatg aaagttgccg gaagatcaa acctagtgta 1140 tatatgggct gctttgatct tgaagtcttt gttgaaatga atcaaaggtc aaagaagtgg 1200 caatgcccta tctgtctgaa gaactactct ctggagcatg tcattataga tccatatttc 1260 aatcggatct cttctcagct gcgaacttat ggagaagaag tatcagaaat tgaagtgaaa 1320 cctgatggtt cttggcgtgc aaaggcagaa ggtgatcgaa agagtcttgg ggatctcgta 1380 aggtggcact tacctgatgg aactctcatt gaatctcagg atatagagtc aaaacccaag 1440 cctggagttc ttaagcatgt taaacaggaa ggaggctctg agagcctcgg tggacttaaa 1500 gttgggttga agaagaacag aaatggtttg tgggaaatta acaaacctga agatatacag 1560 gcattaccat acagaaatgg attaagagag aactttgaaa atcatattca agatattatt 1620 cccatgggca gcagtgccac tgagagtggc aaggaaggtg aagatcccag tgttaatcag 1680 ggtggtgatg ttatctttga cttccctacc aatgggtttg agcttgaaac catttctcca 1740 aattctgccc cagcatatgg ttgtaatgac cgcaatcccc cagcaccagc tggagatgct 1800 gaaattattg tcctaagtga ttccgacgaa gaaaatgagc cacttatttc ttctgcatcc 1860 atttacaata acaaccatac tgatgaacct atagtctctt ttgcgggcag gcctaaagga 1920 atttcagatt cttgcaatga tgaccatgca cttgttaatg atgggaattc atgcctggga 1980 ctgttctttg ggatgaatat gtggcctttg cctactatta accatgggcg cccaggcttt 2040 cagttatttg gttcagatat gcagcagggt cctattaact gcgcgagctc catcaatggt 2100 tactcgttgg ctgcggatac tggcattgga tcttgttctc ttcttcctga atcttctgtt 2160 gatcgtacga atactcaaat aaatgatggt ttggttaata atcccctatc atttggcagt 2220 aatgatccct cacttcagat atttcttcct actagaccat tgaatgcatc agttgaggct 2280 gatgtggggc atcagccagg tttgtcaact ggcattcata ctgaggattg gttttcactt 2340 agtcttgggg caggtgggga ttctgcagtt gctagtgggt tgaggtcagg gaagccgtcg 2400 caaaccaaaa actcttcctt ggattcattg gctgacactg cttctttcct tcttggtatg 2460 aatggtggag tatcaatgaa gagcggtaga gaaagatcag atggtccgtt taattttcct 2520 cgtcaacgtc gttctgtaag accgagatat ctaaatattg attcagacac tgattaa 2577 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CaSIZ1_F for cloning <400> 3 atggatttgg taactagctg caag 24 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CaSIZ1_R for cloning <400> 4 ttaatcagtg tctgaatcaa tatttagata 30 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaSIZ1_F for RT-PCR <400> 5 aggtttgtca actggcattc atact 25 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CaSIZ1_R for RT-PCR <400> 6 caattaccaa gccatgcaga 20

Claims (10)

CaSIZ1 (Capsicum annuum SAP and Miz1) protein consisting of the amino acid sequence of SEQ ID NO: 1.
The CaSIZ1 gene encoding the CaSIZ1 protein of claim 1.
3. The method of claim 2,
The CaSIZ1 gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2, CaSIZ1 gene.
3. The method of claim 2,
The CaSIZ1 gene is characterized in that isolated from pepper, CaSIZ1 gene.
A recombinant vector comprising the CaSIZ1 gene of claim 3.
6. The method of claim 5,
The recombinant vector is characterized in that for promoting drying stress resistance of plants, the recombinant vector.
A plant transformed with the recombinant vector of claim 5 .
A composition for promoting drying stress resistance of a plant comprising at least one selected from the group consisting of the protein of claim 1, the CaSIZ1 gene encoding the same, and a recombinant vector comprising the gene as an active ingredient.
A method for enhancing drying stress resistance of a plant, comprising the steps of:
(a) CaSIZ1 (Capsicum annuum SAP and Miz1) transforming the gene encoding the protein into a plant; and
(b) overexpressing CaSIZ1 protein in the transformed plant.
10. A transgenic plant having improved resistance to drying stress by the method of claim 9.

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