KR102524955B1 - CaSIZ1 gene and Method for improving the resistance to the drought stress using CaSIZ1 in plants - Google Patents

Abstract

The present invention is a novel CaSIZ1 derived from red pepper (Capsicum annuum It relates to SAP and Miz1) genes/proteins, and methods for enhancing the drying stress resistance of plants using the same. The present invention identified the CaSIZ1 gene, which encodes a protein that promotes resistance to dry stress, and the CaSIZ1 protein functions as a positive regulator of ABA signaling, and the effect of enhancing dry stress resistance was confirmed in transgenic plants in which the protein was overexpressed. of the CaSIZ1 gene. Through expression control, it can be used to improve crops that can be used by mankind, and it is expected to be usefully used to improve plant productivity.

Description

CaSIZ1 gene and method for improving drought 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 the drying stress resistance of plants using the same.

Global warming causes extreme climate change to induce environmental stress that inhibits the normal growth and development of plants. In response, plants have evolved sophisticated defense mechanisms to survive in adverse environments such as cold, high salinity, and drought. For example, drought is caused by a water shortage environment and seriously affects the normal growth of plants, eventually resulting in a decrease in crop yield. It activates various defense mechanisms such as the synthesis and accumulation of ABA).

ABA is known to be a regulator for protecting plants against abiotic stress, particularly dry stress, and plays a very important role in plant growth and development. ABA signals are known to regulate the expression of various stress-related genes, such as transcription factors and E3 ligase, which are involved in osmotic adaptation and regulation of root hydraulic conductivity, but the complete ABA signaling pathway has not yet been identified. It didn't work. Initiation of ABA signaling in plant cells requires the presence of ABA receptors and transmission of signals to downstream proteins. Until now, it has been known that PYR/PYL/RCARs as ABA receptors play a role in recognizing ABA. When the receptor recognizes ABA, sub-proteins are phosphorylated and dephosphorylated by SnRK2 (non-fermenting 1-related subfamily 2) protein 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 to inhibit their activity. When the 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, enabling plants to adapt to unfavorable environments.

On the other hand, like ubiquitination, protein sumoylation is also determined by the sequential action of three enzymes (E1: SUMO-activating enzyme, E2: SUMO-conjugating enzyme, E3: SUMO ligase). Sumoylation means 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 sumoy protease responsible for SUMO recycling. In plants, hair conjugation to target proteins can occur through a fast-response mechanism that manipulates substrates during abiotic and biotic stress responses. In addition, hydration is genetically related to dry stress, heat stress, salt and cold stress, phosphate deficiency response, and innate immunity. Sumoylation is also 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 sumo ligase. They confer substrate specificity to transfer SUMO from the E2 enzyme to the substrate. Meanwhile, ABI5 and MYB30, which are important transcription factors in the ABA signaling pathway, are affected by SIZ1 (SAP and Miz1). In other words, it has been reported that sumoylation of ABI5 or MYB30 by SIZ1 mediates the ABA response.

In this way, many plants have been used to study whether E3 ligase is involved in the drying stress response through the ABA signaling pathway, 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 the CaSIZ1 (Capsicum annuum SAP and Miz1) gene, an E3 ligase, in pepper while studying the relationship between SUMO E3 ligase and drying stress response through regulation of the ABA signaling pathway, and the gene The present invention was completed by confirming that the CaDRHB1 protein, which is a negative regulator of ABA signaling, functions as a positive regulator of ABA signaling that mediates and stabilizes the ammonia, thereby enhancing the drying stress resistance of plants.

Accordingly, an object of the present invention is to provide a CaSIZ1 (Capsicum annuum SAP and Miz1) protein consisting of 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 containing 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 the drying stress resistance of plants comprising at least one selected from the group consisting of the CaSIZ1 protein, the CaSIZ1 gene encoding the same, and a recombinant vector containing the gene as an active ingredient. .

Another object of the present invention is to provide a method for enhancing the 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 with enhanced drying stress resistance 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 containing the CaSIZ1 gene.

In one embodiment of the present invention, the recombinant vector may be used to improve dry 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 the 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 dry stress resistance of a plant, comprising the following steps.

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

(b) overexpressing the CaSIZ1 protein in the transformed plant.

In addition, the present invention provides a transgenic plant with enhanced drying stress resistance by the above method.

In addition, the present invention provides a use of a composition 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 to enhance the drying stress resistance of plants.

In addition, the present invention is a drying stress resistance of plants comprising the step of administering to the plant a composition 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 Provides a method of enhancement.

The present invention identified the CaSIZ1 gene, which encodes a protein that promotes resistance to drying stress, and the CaSIZ1 protein functions as a positive regulator of ABA signaling, and the effect of enhancing drying stress resistance was confirmed in transgenic plants overexpressing the protein. Bar, of the CaSIZ1 gene. Through expression control, it can be used to improve crops that can be used by mankind, and it is expected to be usefully used to improve plant productivity.

Figure 1a shows the results of cell-free degradation analysis of CaDRHB1 using a crude extract extracted from dehydrated red pepper leaves through immunoblot analysis.
Figure 1b shows the results of cell-free degradation analysis of CaDRHB1 using the crude extract extracted from ABA-treated red pepper leaves through immunoblot analysis.
Figure 2a shows the result of confirming the conjugation of hair follicles promoted by drought of the CaDRHB1 protein in vivo.
Figure 2b shows the result 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 crude extracts from CaSIZ1- silenced pepper leaves.
Figure 2d shows the results of cell-free degradation of CaDRHB1 using crude extracts from CaMMS21 silenced pepper leaves.
Figure 2e shows the results of confirming CaSIZ1 expression in the leaves of pepper plants after drought stress treatment.
Figure 2f shows the result of confirming CaMMS21 expression in the leaves of pepper plants after drought stress treatment.
Figure 3a shows the results of predicting the sumo site of CaDRHB1.
Figure 3b shows the results of Co-IP analysis of the CaDRHB1 fragment using CaSUMO 1-5.
Figure 3c shows the result of confirming that K138 is the site of in vivo sumoization.
Figure 4a shows the results of pull-down analysis of direct interactions between MBP-CaDRHB1 and GST-CaSIZ1 fusion proteins.
Figure 4b shows the results of Co-IP analysis of CaSIZ1 and CaDRHB1.
Figure 4c shows the results of BiFC analysis of CaDRHB1 and CaSIZ1 .
Figure 4d shows the results of in vitro sumoization analysis of CaDRHB1.
Figure 4e shows the result of in vivo sumoization analysis of CaDRHB1.
Figure 5a shows the result of confirming the expression of CaSIZ1 according to drought, NaCl, ABA and cold treatment by qRT-PCR analysis.
Figure 5b shows the organ-specific expression of CaSIZ1 in various pepper organs at seedling and mature stages (LF, leaf; RT, root; CT, cotyledon; ST, stem; YLF, young leaf; FEL, fully grown leaf; PT, petiole; TR, primary root; LR, secondary root; FL, flower; GF, green fruit).
Figure 5c shows the results of intracellular localization of CaSIZ1 based on transient expression of GFP fusion protein in N. benthamiana.
Figure 6a shows the results of confirming the expression of the CaSIZ1 gene in the leaves of TRV2:CaSIZ1 and TRV2:00 pepper plants by RT-PCR analysis.
Figure 6b shows the results of confirming the sensitivity to drought of TRV2: CaSIZ1 and TRV2:00 pepper plants.
Figure 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 leaf temperature according to ABA treatment in TRV2:CaSIZ1 and TRV2:00 pepper plants.
Figure 6e shows the results of measuring leaf temperature according to ABA treatment in TRV2:CaSIZ1 and TRV2:00 pepper plants.
Figure 6f shows the results of measuring stomatal porosity according to ABA treatment in TRV2:CaSIZ1 and TRV2:00 pepper plants.
Figure 6g shows the results of measuring stomatal porosity according to ABA treatment in TRV2:CaSIZ1 and TRV2:00 pepper plants.
Figure 6h shows the results of qRT-PCR analysis of the expression of stress response genes (CaOSR1, CaRD29B, CaRAB18, CaLOX1) in dehydrated leaves of TRV2:CaSIZ1 and TRV2:00 pepper plants.
Figure 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 and wild-type plants in 0.5X MS medium supplemented with various concentrations of ABA.
Figure 7c shows the growth rates of transgenic and wild-type plants exposed to ABA.
Figure 7d shows the rate of cotyledon greening of transgenic plants and wild-type plants exposed to ABA.
Figure 7e shows the degree 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 from the leaves of CaSIZ1-OX transgenic plants and wild-type plants.
8C shows thermal 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 stomatal views of CaSIZ1-OX transgenic plants and wild-type plants.
Figure 8f shows the results of measuring stomatal porosity of CaSIZ1-OX transgenic plants and wild-type plants.

The present inventors identified the CaSIZ1 gene, a SUMO E3 ligase that functions as a positive regulator of ABA signaling, and completed the present invention by confirming increased resistance to drying stress in transgenic Arabidopsis plants overexpressing the gene.

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

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

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

While studying genes involved in the drying stress response, the inventors identified CaSIZ1, an E3 ligase protein, and the fact that as the sensitivity of abscisic acid (ABA) increases, stomatal closure can be promoted and drying resistance can be improved. Based on this, we tried to identify the association between abscisic acid or dry stress and the CaSIZ1 gene.

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

In another embodiment of the present invention, leaf temperature, stomatal opening degree, water loss, survival rate, stress response gene expression, etc. were measured in leaves of pepper plants in which CaSIZ1 expression was inhibited and normal control pepper plants treated with ABA. By doing so, the effect of reducing ABA sensitivity by CaSIZ1 gene silencing was confirmed, and it was confirmed that drying stress resistance was actually reduced in plants in which CaSIZ1 expression was inhibited compared to the control group (see Example 5).

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

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

The term “recombinant” refers to a cell that replicates, expresses a heterologous nucleic acid, or expresses a peptide, a protein encoded by a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the cell's native form, either in sense or antisense form. A recombinant cell may also express a gene found in the cell in its natural state, but the gene has been reintroduced into the cell by artificial means as a modified one.

The term “recombinant expression vector” refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In general, any plasmid and vector may be used provided that it is capable of replicating and stabilizing 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 gene sequence and suitable 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, in vivo recombinant technology, and the like. The DNA sequence can be effectively linked to a suitable promoter in an expression vector to direct mRNA synthesis. In addition, the expression vector may 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 the Ti-plasmid vector, which is capable of transferring a part of itself, the so-called T-region, into plant cells 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 integrate the hybrid DNA into the plant's genome. 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 DNA according to the present invention into plant hosts include viral vectors, such as those that can be derived from double-stranded plant viruses (eg, CaMV) and single-stranded viruses, gemini viruses, and the like. For example, it may be selected from incomplete plant viral vectors. The use of such vectors can be particularly advantageous when properly transforming a plant host is difficult.

Expression vectors may contain 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 transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. There is a resistance gene, but is not limited thereto.

In the recombinant vector of the present invention, the promoter may be a 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 states of development or cell differentiation.

In the recombinant vector of the present invention, common terminators can be used, such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, 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.

Plant transformation 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 dicotyledonous as well as monocotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. Methods include the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts of electroporation (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), particle bombardment of various plant elements (DNA or RNA-coated) (Klein T.M. et al., 1987, Nature 327, 70), Agrobacterium tumefacies by infiltration of plants or transformation of mature pollen or microspores infection by a (incomplete) virus in ens-mediated gene transfer (EP 0 301 316) and the like. Preferred methods according to the present invention include Agrobacterium mediated DNA delivery.

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

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

As another aspect of the present invention, a composition for enhancing drying stress resistance of plants comprising 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 an active ingredient is provided.

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

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

(b) overexpressing the CaSIZ1 protein in the transformed plant.

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

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

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Experiment preparation and experiment method

1-1. Plant material and growing conditions

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

1-2. Cell-free degradation assay

Raw protein extracts were 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. Prepared from the leaves of the pepper plant. GST-tagged CaDRHB1 or GST-CaDRHB1-SUMO protein (500 ng) was incubated with raw protein extract (50 μg total protein) and simultaneously incubated for 3 hours in the presence of 50 μM MG132. To measure 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 assays. The lysate containing GST-CaSIZ1 or GST was incubated with glutathione S-transferase (GST) resin at 4°C for 2 hours and then incubated with MBP-CaDRHB1 or MBP for an additional 2 hours. Proteins eluted from the resin were 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 the 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 collected 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. Protein extracts were incubated with Pierce anti-HA agarose (Thermo Scientific) at 4°C for 3 hours. Precipitated proteins were washed at least three 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 the stop codon was subcloned into the 35S-VYNE vector for the creation of a bimolecular fluorescence complementation (BiFC) construct (Waadt et al., 2008). For transient expression, Agrobacterium tumefaciens strain GV3101 having the above construct was mixed with the p19 strain to avoid gene silencing, and then 5-week-old tobacco ( N. benthamiana ) plants (OD600 = 0.5) was infiltrated into the abaxial side of the leaf. After 3 days of infiltration, leaf discs were cut and lower 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) for 3 h at 30 °C. Tris-HCl pH 7.5, 5 mM MgCl ₂ , 2 mM ATP). Proteins were separated on 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. Expression of CaACT1 was used as a standard control. The experiment was repeated three times. PCR was performed with a program of 95°C for 5 minutes, 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 20 seconds for 45 cycles. The relative expression level of each gene was calculated by the ΔΔCt method.

1-8. Subcellular localization analysis

The CaSIZ1 coding region without the 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 coprecipitated onto 5 weeks old fully expanded tobacco plant leaves. After 2 days of infiltration, DAPI (4', 6-diamidino-2-phenylindole) solution (1 μg/ml) was permeabilized to stain the nuclei, and confocal microscopy (510 UV/Vis Meta; Zeiss) equipped with LSM Image Browser software was used. Microscopic analysis was performed.

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

For CaSIZ1 loss-of-function analysis, 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, 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). The plants were placed in a growth chamber at 25° C. with a photoperiod of 16 hours day/8 hours night for growth and virus propagation.

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

ml^-1의 카나마이신이 함유된 MS 한천 플레이트에 파종하였다.The full-length CaSIZ1 cDNA was ligated into 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). For selection of transgenic lines, 50 μg of seeds harvested from transformed plants

They were seeded on MS agar plates containing ml ^-1 of kanamycin.

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

To investigate the CaSIZ1 expression pattern in pepper, 4-week-old pepper plants were treated with 100 μM ABA, desiccation stress, 200 mM NaCl or low temperature (4°C). For NaCl treatment, pepper plants were irrigated with 200 mM NaCl solution. They were harvested at 0, 0.5, 1, 3, 6, 9 and 12 h (ABA, dry) or 0, 2, 6, 12 and 24 h (NaCl, cold) after each treatment. For qRT-PCR analysis, four-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 emerging from roots and seeds with green cotyledons was counted every day up to 4 days after division. For the ABA-inhibited primary root growth analysis, the MS agar plate was placed vertically and the root length was measured on the 6th day after seeding.

A drying stress tolerance assay was performed using 4-leaf stage CaSIZ1 silenced pepper plants and 3-week-old CaSIZ1-OX Arabidopsis thaliana plants. To measure transpirational water loss, leaves were separated from 4 leaf stage 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, 4-week-old pepper plants and 3- or 4-week-old Arabidopsis plants with both first and second leaves growing 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. Biological assay of stomatal aperture

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

Example 2. Confirmation of CaDRHB1 protein stability improvement through sumoization

To confirm that CaDRHB1 acts as a positive regulator of ABA and drying stress, a cell-free degradation assay was performed using pepper plants treated with ABA and drying stress.

As shown in Figure 1a, the degradation of CaDRHB1 protein was reduced in the crude extract of peppers subjected to drought stress for 6 h compared to the crude extract of healthy peppers.

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 the CaDRHB1 protein was stabilized in response to drought and ABA.

On the other hand, CaDRHB1, a component of HD-ZIP I, is known to contain a putative motif for sumoization. Therefore, in order to confirm whether drought is involved in the CaDRHB1 protein sumoization , 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 number of CaDRHB1 was higher in drought-treated leaves than in healthy leaves.

Then, by performing a cell-free degradation assay using a male hair protein sequence fused to the C-terminus of CaDRHB1, a cell-free degradation assay was performed to confirm the association between the male hair protein and the stability of CaDRHB1.

As a result, as shown in Figure 2b, it was found that the degradation of sumo mimic 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 speculated that CaSIZ1 or CaMMS21 could promote the binding of CaDRHB1 to SUMO.

Next, cell-free lysis was performed with crude extracts of CaSIZ1- or CaMMS21-silenced red pepper to identify proteins that mediate the CaDRHB1 protein sumolysis.

As shown in Figure 2c, the GST-CaDRHB1 protein was significantly more degraded in CaSIZ1 silenced peppers than in control plants, whereas

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

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

These results suggest that drought-induced hydrolysis of CaDRHB1 increases CaDRHB1 protein stability and that CaDRHB1 hydrolysis can be regulated by CaSIZ1 supermolar E3 ligase.

Example 3. Confirmation of the localization of CaDRHB1

In order to confirm that there is a relationship between CaDRHB1 sumoylation and stability, the location of CaDRHB1 sumoylation 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 to this, truncated forms of CaDRHB1 were generated in five fragments to investigate and identify the site of CaDRHB1 sumoylation. 35S: HA-CaSUMO1-5 and each of the five fragments of CaDRHB1 (35S: CaDRHB1 F1 ~ 5-GFP) were transiently co-expressed in Nicotiana benthamiana, and then the whole protein was stained 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 strongly occurred in the F3 fragment.

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

As a result, as shown in Figure 3c, the SUMO junction 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 is a site capable of sumoization.

Example 4. Check the physical interaction of CaDRHB1 and CaSIZ1

Pull-down, co-immunoprecipitation (co-IP) and bimolecular fluorescence complementation assay (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, GST and MBP free proteins were shown to fail 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 total protein of N. benthamiana expressing HA-CaSIZ1/CaDRHB1-GFP but not HA-CaSIZ1/GFP. GFP was not immunoprecipitated from the total protein expressed.

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

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

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

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

As shown in Figure 4e, following immunoprecipitation with GFP-trap, immunoblot showed that CaDRHB1-GFP was submodified and stabilized by HA-CaSIZ1, whereas HA-CaSIZ1C379S/H381Y (CaSIZ1 SP-RING domain mutation) or CaDRHB1K138R In N. benthamiana expressing -GFP, submoylation and stability were reduced.

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

Example 4. Confirmation of molecular characteristics of CaSIZ1

Based on the fact that CaDRHB1 positively regulates the drought response, we speculated that CaSIZ1 may be involved in the stress response because male hair junction of CaDRHB1 is promoted by CaSIZ1 and CaDRHB1 stability is increased by male hair junction. Therefore, 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 peppers, the expression level of CaSIZ1 gradually increased after treatment and was highest at 12 hours. NaCl-treated pepper had the highest expression level at 6 hours and gradually decreased. The expression level decreased under cold stress.

As shown in Figure 5b, the results of examining the expression of CaSIZ1 in each organ of pepper plants showed higher expression levels in flowers (FL) and green fruits (GF) than in other organs.

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

Example 5. Confirmation of improved susceptibility of CaSIZ1 silenced pepper plants to drying stress

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

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

Therefore, in order to investigate the response to drying stress, water supply to 4-week-old control plants (TRV2:00) and CaSIZ1 silenced peppers was stopped for 14 days.

As shown in Figure 6b, CaSIZ1 silenced peppers showed a more withered phenotype under drought stress conditions and re-watering conditions, as shown in the middle and right panels of Figure 6b, and the survival rate of control plants (91.1%) was higher than CaSIZ1. Silenced peppers had a much lower survival rate (23.2%), indicating that they were more susceptible to drought compared to control plants.

To assess whether the drought-sensitive phenotype exhibited by CaSIZ1 silenced plants was derived from altered water retention capacity, the rate of leaf water loss was calculated by weighing fresh leaves.

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

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

As shown in Figures 6d and 6e, there was no difference in leaf temperature when ABA was not treated. After 50 μm of ABA treatment, the leaf temperature of CaSIZ1-silenced pepper was found to be 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 is due to inhibition of ABA-induced stomatal closure, pore size was measured.

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

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

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 h after drought treatment.

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

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

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

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

As shown in Figures 7b to 7e, without ABA, there was no difference in germination rate, pili and root length between wild-type and CaSIZ1-OX plants. On the other hand, when treated with ABA, CaSIZ1-OX plants showed lower germination rate, green cotyledon formation and root growth rate than wild-type plants.

These results indicate that CaSIZ1 positively regulates the ABA response.

Example 7. Confirmation of improved tolerance of CaSIZ1 overexpressing Arabidopsis to drought stress

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

As shown in Figure 8a, there was no difference from the wild-type control group in the well-watered state (upper panel). On the other hand, when not watered for 14 and 15 days, CaSIZ1-OX plants showed a less wilted phenotype than wild-type plants (middle panel). After 3 days of re-watering, 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 Fig. 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 confirm that the reduced transpirational water loss was due to ABA sensitivity.

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

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

Through the above results, it was found that CaSIZ1 acts as a positive regulator for drying resistance in not only pepper plants but also Arabidopsis thaliana by inducing the expression of ABA-mediated stomatal closure and desiccation response marker genes.

The description of the present invention described above is for illustrative purposes, and those skilled in the art 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, the embodiments described above should be understood as illustrative in all respects and not limiting.

<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 cctcagtgg 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 gaagattcaa 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 agaatacag 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 cagttattatg 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)

delete delete CaSIZ1 gene encoding the CaSIZ1 protein, consisting of the nucleotide sequence of SEQ ID NO: 2.
According to claim 3,
The CaSIZ1 gene is characterized in that isolated from pepper, CaSIZ1 gene.
A recombinant vector comprising the CaSIZ1 gene of claim 3.
According to claim 5,
The recombinant vector is a recombinant vector, characterized in that for enhancing the drying stress resistance of plants.
A plant transformed with the recombinant vector of claim 5.
A composition for enhancing dry stress resistance of plants, comprising, as an active ingredient, at least one selected from the group consisting of a CaSIZ1 gene encoding the CaSIZ1 protein, and a recombinant vector containing the gene, comprising the nucleotide sequence of SEQ ID NO: 2.
A method for enhancing dry stress resistance of a plant, comprising the following steps:
(a) consisting of the nucleotide sequence of SEQ ID NO: 2, CaSIZ1 (Capsicum annuum SAP and Miz1) transforming a gene encoding a protein into a plant; and
(b) overexpressing the CaSIZ1 protein in the transformed plant.
delete

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