KR20160029919A - Method for improving the resistance to the drought stress using ABA receptor, CaRCAR1, in plants - Google Patents

Abstract

The present invention relates to a CaRCAR1 which is a gene encoding a pepper-derived abscisic acid receptor, associated with resistance against desiccation stress in plants. More specifically, the present invention relates to a method for enhancing resistance against desiccation stress in plants, by regulating expression of CaRCAR1 protein. A transgenic plant (CaRCAR1-OX) in which CaRCAR1 of the present invention is over-expressed has shown effects of increasing sensitivity for abscisic acid and enhancing resistance against desiccation stress. Thus the method of the present invention can be useful for improving crops that men can use.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for enhancing dry stress resistance of a plant using an abscisic acid receptor CaRCAR1,

The present invention relates to a method for enhancing the dry stress resistance of a plant using the Caesalin receptor gene CaRCAR1 derived from pepper, and more particularly, to a method for enhancing resistance to the dry stress of a plant by controlling the expression of CaRCAR1 will be.

Environmental stresses, such as drought conditions, directly cause detrimental effects on grain growth and production. Especially, as the desertification progresses today, water shortages cause big problems in agriculture and the environment. Therefore, it is necessary to develop plants that can survive in a dry environment even when water is used less. When these techniques are developed and applied to crops, agricultural production is expected to increase greatly. Especially in dry areas, plants with improved resistance to dryness, that is, plants capable of lowering the transpiration rate, are favorable to survival, And may be useful for environmental purification in areas where the environment is very dry.

Plants, on the other hand, have been developing physiological, biochemical and molecular defense mechanisms to cope with the harmful effects of stress because they are frequently exposed to various environmental stresses such as drought, salt, cold, heat, abscisic acid: ABA) signal transduction regulation allows plants to overcome these stresses. It has been reported that plants pretreated with abscisic acid and stressed are resistant to stress compared to plants that do not produce stress, while mutant plants that fail to produce abscisic acid or fail to respond to abscisic acid are known to be stress-free. Therefore, it is expected that the use of proteins involved in the reaction of abscisic acid will enable to develop plants with improved resistance to environmental stress.

Accordingly, a method for increasing the dry resistance of plants has been the subject of much research, and studies have been conducted on the use of proteins involved in the reaction of abscisic acid (Korean Patent Publication No. 10-2010-0040789) to be.

The present invention has been made to solve the above problems, the present inventors, ABA or CaRCAR1 reduced gene expression effect of drought stress, the Absecon seusan sensitivity enhancement effect of CaRCAR1 a transgenic plant (CaRCAR1 -OX) And the present invention has been completed on the basis thereof.

Accordingly, an object of the present invention is to provide a gene CaRCAR1 comprising the nucleotide sequence of SEQ ID NO: 1 encoding a dry stress-resistant protein derived from pepper.

Another object of the present invention is to provide a peptide of the amino acid sequence of SEQ ID NO: 2 which is encoded by the gene CaRCAR1 .

It is still another object of the present invention to provide a method for enhancing dry stress resistance of a plant, comprising transforming a gene encoding CaRCAR1 protein into a plant, and overexpressing the CaRCAR1 protein in the transformed plant.

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

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

In order to accomplish the object of the present invention as described above, the present invention provides a gene CaRCAR1 comprising the nucleotide sequence of SEQ ID NO: 1 encoding a dry stress resistant protein derived from pepper.

The present invention provides a peptide of the amino acid sequence of SEQ ID NO: 2 which is encoded by the gene CaRCAR1 .

The present invention provides a method for promoting dry stress resistance of a plant, comprising transforming a gene encoding a CaRCAR1 protein into a plant, and overexpressing the CaRCAR1 protein in the transformed plant.

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

The present invention provides a screening method for a dry stress resistance enhancer of a plant, comprising the step of treating the candidate substance and then measuring the expression of the CaRCAR1 protein.

The present invention relates to a gene CaRCAR1 of drying stress tolerance-related origin of pepper plants, the CaRCAR1 bar hayeotneun determine Absecon seusan sensitivity enhancement effect of the transgenic plants (CaRCAR1 -OX), humans available on the expression control CaRCAR1 It is expected that it will be useful for improving crops.

FIG. 1 shows the amino acid sequence comparison between pepper CaRCAR1 and SlPYL8, MtPYL9, PsATF, AhPIP, MdPYL9 and AtRCAR3 .
FIG. 2 shows phylogenetic tree analysis results between pepper CaRCAR1 and SlPYL8, MtPYL9, PsATF, AhPIP, MdPYL9 and AtRCAR3 .
FIG. 3 shows the results of qRT-PCR for the change of CaRCAR1 expression level under (A) ABA or (B) dry stress conditions.
FIG. 4 shows the results of confirming expression of CaRCAR1- GFP in order to confirm intracellular expression and action site of pepper CaRCAR1 .
FIG. 5 shows the ratio of germination (A) to green cotyledons ( CaRCAR1- OX # 4, # 9) and wild type control (WT) germination rate of CaRCAR1 overexpressed CaRCAR1 overexpressed in ABA stress condition As a result of observation, (C) the ratio of green cotyledon was numerically determined, (D) the root length was quantified, and (E) the root length was observed with naked eyes.

The present inventors have confirmed that Absecon seusan sensitivity enhancement effect on the drying ABA or stress conditions, and decreased expression CaRCAR1 CaRCAR1 the transgenic Arabidopsis thaliana in, and have accomplished the present invention based thereon.

Hereinafter, the present invention will be described in detail.

The present invention provides a peptide encoded by the gene CaRCARl or the gene CaRCARl encoding a dry stress resistant protein from pepper. Gene CaRCAR1 of the present invention were isolated from the pepper through Differential hybridization analysis, CaRCAR1 genes of the present invention preferably consists of the nucleotide sequence of SEQ ID NO: 1, peptides encoded by the CaRCAR1 gene is preferably SEQ ID NO: 2, but is not limited thereto.

The inventors of the present invention have been studying to reveal a novel function of the CaRCAR1 gene against non-biological stress, and it has been found that the expression of CaRCAR1 gene is suppressed in pepper leaves exposed to abscisic acid (ABA) and dry (dehydration) (See Example 2), and based on the fact that as the sensitivity of abscisic acid increases, pore closure is promoted and drying resistance can be enhanced, the relationship between abscisic acid or dry stress and the CaRCAR1 gene is identified .

Thus, in one embodiment of the present invention, the homology of the amino acid sequence between the CaRCAR1 protein and the other ABA receptor protein (see Example 1) was confirmed. In addition, by regulating the expression of CaRCARl by preparing transgenic Arabidopsis thaliana overexpressing CaRCARl (see Example 3) and confirming the abscisic acid sensitization enhancing effect (see Example 4), resistance to dry stress can be enhanced .

Accordingly, the present invention provides a method for enhancing dry stress resistance of a plant, comprising transforming a gene encoding CaRCAR1 protein into a plant, and overexpressing the CaRCAR1 protein in the transformed plant.

In another aspect, the present invention provides a transgenic plant in which CaRCARl protein is overexpressed to enhance dry stress resistance.

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

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

Example 1. CaRCAR1  Sequence homology analysis

The present inventors performed multiple alignment analysis and phylogenetic tree analysis in order to determine the degree of similarity of pepper CaRCAR1 amino acid sequence with other ABA receptor proteins. The amino acid sequences of pepper CaRCAR1 and SlPYL8, MtPYL9, PsATF, AhPIP, MdPYL9 and AtRCAR3 were compared.

As shown in FIGS. 1 and 2, the amino acid sequence of pepper CaRCAR1 has high homology with ABA receptor proteins such as PYL and RCAR of various species.

Example 2 < RTI ID = 0.0 > CaRCAR1  Induction of gene

To determine if it was promoted by ABA or drought stress, the expression pattern of CaRCARl gene was confirmed under the above conditions through qRT-PCR.

ABA (100 μM), and drought (dehydrated), respectively, and RNA was extracted from the sampled pepper leaves. Extracted RNA samples were treated with RNA-free DNase to remove genomic DNA and stored at -20 ° C. For reverse transcription PCR, Transcript First Strand cDNA synthesis kit (Roche) was used. At the same time, PCR was performed without reverse transcriptase, and PCR products were used for qRT-PCR to reaffirm genomic DNA contamination in cDNA samples. The synthesized cDNA was amplified using a CFX96 Touch ™ real-time PCR detection system (Bio-Rad, Hercules, CA, USA) together with iQ ^™ SYBR Green Supermix and the specific primers shown in Table 1 below. All reactions were repeated three times. The PCR conditions were as follows: After heating for 5 minutes at 95 ° C, 45 cycles (95 ° C for 20 seconds, 60 ° C for 20 seconds, and 72 ° C for 20 seconds) repeat. qRT-PCR was performed at least twice each in biological and technical replicates. The relative expression level of each gene was calculated by the ΔΔCt method (Livak and Schmittgen 2001), and an internal control gene (18S rRNA) was used for normalization to accurately measure target gene expression.

As a result, as shown in Fig. 3, expression of CaRCARl gene gradually decreased under ABA or dry stress conditions. These results indicate that expression of the CaRCAR1 gene is closely related to ABA and dry stress.

Example  3. CaRCAR1  Protein Intracellular  Expression and action location investigation

In order to investigate the intracellular expression and action site of the CaRCAR1 protein, the fluorescent protein Green flourescent protein (GFP) was bound to the C-terminus of the CaRCAR1 full-length cDNA having no stop codon to be expressed under the cauliflower mosaic virus (CaMV) 35S promoter Vector. For the transient expression, Agrobacterium tumefaciens strain GV3101 with the CaRCAR1- GFP inserted vector was mixed with p19 strain to avoid gene silencing and injected into the hypocotyl of 5 week old Nicotiana benthamiana leaf using a 1 ml needless syringe. Leaf sections were cut 2 days after injection for microscopic analysis. The lower epidermal cells were analyzed with a confocal microscope (model Zeiss 510 UV / Vis Meta) operated with LSM Image Browser software.

As shown in Fig. 4, CaRCAR1 was found to be expressed or acting in both nucleus and cytoplasm of cells.

Example 4. CaRCAR1  Production of overexpressed Arabidopsis mutant plants

The CaRCAR1 gene was transformed into Arabidopsis thaliana, a model plant, to confirm its function in the plant. To under CaMV 35S promoter to drive expression of the constant CaRCAR1 gene, it was inserted into the entire cDNA sequence of the binary vector CaRCAR1 pK2GW7. The binary vector was then introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. CaRCAR1 gene transfer into Arabidopsis plants was performed using floral dipping method. For CaRCAR1 gene overexpression ( CaRCAR1- OX) transgenic selection, harvested seeds from plants suspected of being transformed were selected by inoculation on MS solid medium containing 50 μg / ml kanamycin.

Example 5. CaRCAR1  Promoting increased sensitivity to ABA stress in overexpressed Arabidopsis mutant plants

The CaRCAR1 the transgenic To investigate the role of CaRCAR1 genes for ABA sensitivity of Arabidopsis plants (CaRCAR1 -OX # 4, # 9 ) , and percentage of germination percentage, green cotyledons between the wild-type controls (WT), and the length of the roots Were compared.

The seeds were planted on MS medium containing ABA (0, 0.50, 0.75, 1.00 μM) at each concentration, and the germination rate was measured on the 2nd and the 4th day. Were performed independently and repeated three times. The seeds were planted in MS medium containing ABA (0, 0.75 μM), and the percentage of green cotyledons was measured on the 7th day. The experiment was carried out on 40 seeds, And repeated three times independently. In addition, seeds were planted on MS medium containing ABA (0, 0.5 μM), and the length of roots was measured on day 7, and the experiment was repeated 3 times independently.

As shown in Figure 5, as the concentration of ABA is increased, the CaRCAR1 the transgenic and wild-type Arabidopsis germination of the rod the seedlings, the rate of green cotyledons and root length was reduced, CaRCAR1 the transgenic Arabidopsis Showed a low germination rate and green cotyledon ratio compared to the wild type control, and the root length was greatly reduced.

These results indicate that sensitivity to abscisic acid (ABA) is enhanced in the Arabidopsis overexpressing CaRCAR1 gene.

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

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Method for improving the resistance to the drought stress using          ABA receptor, CaRCAR1, in plants <130> CAU20141516KR_PB14-12162 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 561 <212> DNA <213> Artificial Sequence <220> <223> CaRCAR1 <400> 1 atggaggcac agtttgtaga aagataccat agtcatcagc ctagtgacca tcaaagtagt 60 tcatctcttg ttaagcacat caaagcacct gttgatattg tttggtcact cgtaaggaga 120 ttcgatcagc ctcagaagta taagccattt attagcaggt gtactgtgaa gggtgatctt 180 acaattggta gtgttagaga ggtgaatgtc aagtctggtc ttccagccac cactagcacc 240 gaaagattgg aacttcttga tgatgaggag catatccttg gcatcagaat cgttggtggc 300 gatcacaggc ttaagaacta ttcttcggtt attacagtcc atcccgagac acttgatggt 360 agaccgggga cactggtgat tgagtcattt atggtggatg tgcctgaagg gaacactcaa 420 gaggagactt gctactttgt gaaggcctta atcaattgca atctgaaatc actagctgat 480 gtatctgaga gaatggctat gcagggaggt gttgtaccca ccagtgtgaa ctggtcttct 540 agtaaccaga tcgagacatg a 561 <210> 2 <211> 186 <212> PRT <213> Artificial Sequence <220> <223> CaRCAR1 Protein <400> 2 Met Glu Ala Gln Phe Val Glu Arg Tyr His Ser Gln Pro Ser Serp   1 5 10 15 His Gln Ser Ser Ser Ser Leu Val Lys His Ile Lys Ala Pro Val Asp              20 25 30 Ile Val Trp Ser Leu Val Arg Arg Phe Asp Gln Pro Gln Lys Tyr Lys          35 40 45 Pro Phe Ile Ser Arg Cys Thr Val Lys Gly Asp Leu Thr Ile Gly Ser      50 55 60 Val Arg Glu Val Asn Val Lys Ser Gly Leu Pro Ala Thr Thr Ser Thr  65 70 75 80 Glu Arg Leu Glu Leu Leu Asp Asp Glu Glu His Ile Leu Gly Ile Arg                  85 90 95 Ile Val Gly Gly Asp His Arg Leu Lys Asn Tyr Ser Ser Val Ile Thr             100 105 110 Val His Pro Glu Thr Leu Asp Gly Arg Pro Gly Thr Leu Val Ile Glu         115 120 125 Ser Phe Met Val Asp Val Pro Glu Gly Asn Thr Gln Glu Glu Thr Cys     130 135 140 Tyr Phe Val Lys Ala Leu Ile Asn Cys Asn Leu Lys Ser Leu Ala Asp 145 150 155 160 Val Ser Glu Arg Met Ala Met Gln Gly Gly Val Val Pro Thr Ser Val                 165 170 175 Asn Trp Ser Ser Ser Asn Gln Ile Glu Thr             180 185 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> CaRCAR1-Forward <400> 3 atggaggcac agtttgtaga aagatacc 28 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> CaRCAR1-Reverse <400> 4 tcatgtctcg atctggttac tagaagacc 29 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 18S rRNA-Forward <400> 5 tatggtgtgc accggtcgtc tcgt 24 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 18S rRNA-Reverse <400> 6 gcagttgttc gtctttcata aatccaa 27

Claims (4)

A CaRCAR1 gene consisting of the nucleotide sequence of SEQ. ID. NO: 1 encoding a dry stress resistant protein derived from pepper.
A peptide of the amino acid sequence of SEQ ID NO: 2 which is encoded by the CaRCAR1 gene of claim 1.
A method for enhancing dry stress resistance of a plant comprising the steps of:
(a) transforming a gene encoding a CaRCAR1 protein into a plant; And
(b) overexpressing the CaRCARl protein in the transformed plant.
A transgenic plant having increased dry stress resistance by the method of claim 3.

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