KR101625749B1 - BrDSR Protein Implicated in Drought Stress Tolerance, Gene Encoding the Protein and Transformed Plants with the Same - Google Patents

Abstract

The present invention relates to Chinese cabbage rapa- derived BrDSR ( Brassica rapa Drought Stress Resistance genes and proteins. Of the present invention BrDSR (Brassica rapa Drought Stress Resistance genes and proteins are involved in tolerance to dry stress in plants, and transgenic plants overexpressing them are highly resistant to dry stress. Therefore, the transgenic plants described above can be usefully used as novel functional crops having excellent resistance to drying stress caused by changes in climatic conditions due to global warming and the like and thus being adaptable to dry cultivation conditions.

Description

The present invention also relates to a method for producing the same, which comprises the steps of: (a) culturing the host cell with a culture medium containing the BrandsRad protein BrDSR, a gene encoding the protein, and a plant transformed with the gene,

The present invention relates to Chinese cabbage rapa ), a gene encoding the protein, and a plant transformed with the gene.

Growth of plants is greatly affected by drying stress. Therefore, plants must adapt to dry stress for survival. In this study, we investigated the effect of ABA-responsive element binding protein 1 (AREB1) / ABA-responsive (ABA) on the production of abscisic acid (ABA) related protein kinase 2C (SRK2C), calcium-dependent protein (DREB2A), receptor-like kinase 1 (RPK1), dehydration-responsive element (DRE) -binding factor 2 kinases (CDPKs) CPK3 and CPK6 have been identified as important signals. Dry stress also causes a variety of biochemical and physiological responses in plants. When dry stress occurs, plants accumulate sugars, trehalose, sorbitol, sugar, mannitol, amino acids (proline, etc.) and amines (glycine betaine, polyamine etc). These metabolites have been found to act as osmolytes, antioxidants, and scavengers that can help plants tolerate dry stress.

However, many plants are under the threat of growth and survival due to their inability to synthesize specific metabolites that can tolerate dry stress. Genetic engineering studies using genes involved in dry stress-related metabolic machinery are very useful in developing dry stress-resistant transgenic plants, and many transgenic plants developed by genetic engineering techniques have improved resistance to dry stress . So far, various resistance genes have been found in various crops, and research on the expression network has been actively carried out.

For example, in corn, NPK1 (tobacco MAPKKK), ZmNF-YB2 (maize nuclear factor YB2), ZmPLC1 (phospholipase C1) and TsVP (vacuolar-H ⁺ -pyrophosphatase) ( Datura (DRE-binding protein 1), MnSOD (pea Mn superoxide dismutase), SNAC1 (rice stress responsive NAC1), OsDREB1 (rice DRE-binding protein 1), stramonium arginine decarboxylase (ABF3), Arabidopsis ABRE- , HvCBF4 (barley C-repeat binding factor), OsCIPK12 (rice calcineurin B-like protein-interacting protein kinase 12), and ZFP252 (rice TFIIIA-type zinc finger protein).

On the other hand, Chinese cabbage is one of the four major vegetables of Korea as the main ingredient of kimchi and is included in the lacquer ( Brassica ). Rice ( Brassica ) is an important crop in foreign countries because it includes crops with excellent industrial economics such as oilseed rape, mustard. Brassica crops are susceptible to damage from the drying environment, resulting in reduced crop yields, which can be devastating to farmers and consumers.

The degradation of agricultural productivity caused by such a drying environment is serious, and development of good varieties resistant to such environment is urgently needed to secure stable food resources.

Accordingly, the inventors of the present invention have made extensive efforts to discover a gene capable of improving the plant tolerance of a plant. As a result, it has been found that when a gene having the nucleotide sequence of SEQ ID NO: 5 is involved in the above-described trait of a plant and the gene is transformed into a plant , And that it is possible to improve the durability of the plant, thereby completing the present invention.

An object of the present invention is to provide a Brassica rapa Drought Stress Resistance (BrDSR) having an amino acid sequence of SEQ ID NO: 6, a BrDSR ( Brassica rapa Drought Stress Resistance) gene encoding the protein, And to provide a transformed plant.

It is still another object of the present invention to provide a method for enhancing the plant tolerance by increasing expression of the gene.

The inventors of the present invention have made intensive researches to discover genes and proteins capable of improving the plant tolerance. As a result, it has been found that a protein having the amino acid sequence of SEQ ID NO: 6 and a gene encoding the protein are involved in the drying stress of the plant, It was confirmed that when the plant was transfected and overexpressed, the dry stress resistance of the plant could be improved.

In some embodiments for achieving the above object, the present invention naegeonseong has the amino acid sequence of SEQ ID NO: 6 protein BrDSR (Brassica rapa Drought Stress Resistance.

In the present invention, the protein is not limited to the protein consisting of the amino acid sequence of SEQ ID NO: 6 described in the attached Sequence Listing, and may be a functional equivalent to the resistant protein having the amino acid sequence of SEQ ID NO: 6. The "functional equivalents" have a sequence homology of at least 60%, preferably 70%, more preferably 80% or more, with the amino acid sequence of SEQ ID NO: 6 as a result of amino acid addition, substitution or deletion Quot; means a protein exhibiting substantially the same activity as the BrDSR of the present invention.

Such functional equivalents include, for example, amino acid sequence variants in which a portion of the amino acid sequence of SEQ ID NO: 6 has been substituted, deleted or added. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of the amino acid is preferably located at a site that is not directly involved in the activity of the BrDSR of the present invention. The functional equivalents also include protein derivatives in which the basic structure of BrDSR and its chemical structure has been modified while maintaining its physiological activity. These include, for example, fusion proteins made by fusion with other proteins such as GFP (Green Fluorescent Protein) while retaining the structural modification and physiological activity to change the stability, storage stability, volatility or solubility of the protein of the present invention .

In another embodiment of the present invention, the present invention provides a gene encoding the protein.

According to one embodiment of the present invention, first, a 438 bp ORF (open reading frame) in the KBGP-24K oligo chip and a Arabidopsis thaliana ( Brassica rapa EST and Microarray Database) Arabidopsis (AT1G16850) which had homology in the thaliana (Example 1). Thereafter, my Chinese cabbage ( Brassica rapa ssp. pekinensis inbred line were isolated total RNA from "CT001") (Example 2), after the above-obtained RNA to synthesize cDNA using oligo dT primer (primer) and a reverse transcriptase (reverse transcriptase) as a template, the above-described Using the primer obtained in Example 1, a 438 bp PCR product was obtained and cloned into a vector pGEM-T easy vector for sequencing analysis to construct a recombinant vector (Example 3). Subsequently, to analyze the inserted 438 bp PCR product, the nucleotide sequence was examined using T7 and SP6 primers close to the insertion site, and the sequence analysis was performed using the GenBank BLAST program. The identified nucleotide sequence (SEQ ID NO: 5) and then to determine the start codon (codon) and the stop codon, based on determining the amino acid sequence (SEQ ID NO: 6), it naegeonseong gene of the full-length (full length) isolated from Chinese cabbage BrDSR (Brassica rapa Drought Stress Resistance) (Experimental Example 3).

The genes of the present invention BrDSR (Brassica with a gene encoding the naegeonseong protein of amino acid sequence of SEQ ID NO: 6, the nucleotide sequence of SEQ ID NO: 5 rapa Drought Stress Resistance) gene, Brassica rapa ), preferably from the Chinese cabbage ( Brassica rapa ssp. pekinensis . In addition, when the dry stress is applied to the plant, the expression of the BrDSR gene is increased, and when the expression of the gene is increased, resistance to the dry stress is increased. Thus, it is confirmed that the gene is involved in the vegetative resistance of the plant Respectively.

According to one experimental example of the present invention, the Brassica rapa Drought Stress Resistance) gene was inserted into a transfection vector, pH2GW7 (VIB-Ghent University, Belgium) to prepare pDSR as a transformation vector (Experimental Example 4). Tobacco transformants were prepared using this method (Experimental Example 5). In case of inducing dry stress in this experiment, unlike the control, all of the transformants were kept the same as before the drying treatment, (Experimental Example 8), the Brassica rapa Drought Stress Resistance (BrDSR) gene was involved in the drying stress of the plant. When the gene was transfected and overexpressed in the plant, the dry stress resistance of the plant was enhanced Respectively.

As described above, Brassica ( Brassica rapa Drought Stress Resistance) gene has the nucleotide sequence shown in SEQ ID NO: 5, which gene is not limited to the nucleotide sequence of SEQ ID NO: 5 described in the attached Sequence Listing, and may be a functionally equivalent codon or a codon , Or a nucleotide sequence comprising a codon that encodes a biologically equivalent amino acid. Considering a mutation having biologically equivalent activity, the nucleotide sequence used in the present invention is interpreted to include a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.

In another aspect of the present invention, the present invention provides a recombinant vector comprising the BrDSR gene.

In the present invention, the recombinant vector includes a recombinant vector for transformation.

The plasmids derived from Escherichia coli (pBR322, pBR325, pUC118, pUC119, pET30a, pET30c and pGEX-GST), Bacillus subtilis plasmids (pUB110 and pTP5), yeast- (YEp13, YEp24 and YCp50) and Ti plasmids. Animal viruses such as retroviruses, adenoviruses or vaccinia viruses, insect viruses such as baculoviruses, and plant viruses can be used, and pPZP, pGA and pCAMBIA family You can use the same binary vector. Those skilled in the art can select a vector suitable for introducing the BrDSR gene of the present invention. In the present invention, any vector can be used as long as it can introduce the gene into a host cell. Preferably, however, vectors designed to facilitate induction of protein expression and separation of expressed proteins can be used. More preferably, pGEM-T, pGEX 4T-1 and pCAMBIA1300 vectors can be used.

In one experimental example of the present invention, the wild-type resistance-related gene amplified from cabbage was inserted into pGEM-T easy vector in order to analyze the base sequence, and the restriction enzyme EcoR was confirmed by treating the isolated recombinant DNA Experimental Example 2). The nucleotide sequence of the PCR fragment cloned into the pGEM-T easy vector for sequencing was determined using the GenBank BLAST program. Based on the confirmed nucleotide sequence (SEQ ID NO: 5), the start codon and the stop codon After determining the amino acid sequence (SEQ ID NO: 6), it was named full length wild-type gene BrDSR isolated from Chinese cabbage (Experimental Example 3).

In the present invention, a "transgenic recombinant vector" is a gene construct containing an essential regulatory element operatively linked to the expression of a gene insert, and includes all plasmid vectors, cosmid vectors, bacteriophage vectors, .

"A recombinant vector for transformation" of the present invention, the BrDSR (Brassica rapa Drought Stress Resistance) gene is expressed. For example, the vector may comprise a signal sequence or leader sequence for membrane targeting or secretion in addition to an expression regulatory element such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, an enhancer, . In addition, the vector may comprise a selectable marker, and the vector may be self-replicating or integrated into the host DNA. The vector of the present invention can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage are performed using enzymes generally known in the art

According to one experimental example in the present invention, the BrDSR (Brassica In order to confirm whether the rapa Drought Stress Resistance gene has resistance to the dry strain, a 438 bp BrDSR gene was inserted into a transfection vector, pH2GW7 (VIB-Ghent University, Belgium) (Experimental Example 4).

In another aspect of the present invention, the present invention provides a plant transformed with said recombinant vector.

As used herein, the term "plant" is understood to include not only mature plants but also plant cells, plant tissues and plant seeds that develop into mature plants. Transformation of a plant with the vector in the present invention can be carried out by a transformation technique known to a person skilled in the art. The microorganism is preferably transformed with Agrobacterium, microprojectile bombardment, electroporation, PEG-mediated fusion, microinjection, liposome (liposome) method, mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method, and Agrobacteria spraying method can be used. And more preferably, a transformation method using Agrobacterium can be used.

In the present invention, the plant is a food crop including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats or millet; Vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions or carrots; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut or rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots or bananas; Roses, gladioluses, gerberas, carnations, mums, lilies or tulips; And feed crops including raglass, red clover, orchardgrass, alpha-alpha, tall fescue or perennial rice, preferably ginseng, tobacco, cotton, sesame, sugarcane, beet, Perilla, peanut or rapeseed, more preferably cigarette, but not limited thereto.

According to one experimental example of the present invention, in Example 5, to produce a transformant of tobacco overexpressing the BrDSR gene of Chinese cabbage, Agrobacterium tumefaciens ) was used to construct a transformant of tobacco. The expression level of the BrDSR gene in the body during the development of dry stress was quantitatively measured using five quantitative real- time RT) PCR showed that the expression level was about 1.7 to 2.6 times higher than that of the non-transformant (CON) (Experimental Example 7), and a PCR assay using a hygromycin resistance gene (RT-PCR) analysis of quantitative real-time RT-PCR analysis of 5 transgenic plants with higher expression levels than non-transformants. Was observed under natural drying conditions. As a result, when the non-transformant was in the 5th day of the drying treatment, the non-transformant lost its elasticity of leaves and stems and began to fall down. On the 7th day, And or showed a state also can not recover, the transformants are all confirmed to maintain the same state as before the drying treatment by not show significant differences of expression geometry (Example 8), the BrDSR (Brassica rapa Drought Stress Resistance) gene Is involved in the dry stress of the plant and it is confirmed that when the gene is transfected and overexpressed in the plant, the dry stress resistance of the plant can be improved.

Therefore, the plants transformed by the present invention can increase the intracellular expression level of the BrDSR gene having tolerance to dry stresses, so that the plant is able to suppress the dry stress of the cultivated land caused by the change in the climatic condition due to global warming and the like It can be used effectively as a new functional crop which is excellent in tolerance and excellent in adaptability to dry cultivation conditions.

According to another aspect of the present invention, there is provided a method for enhancing plant tolerance by increasing the expression of the gene.

The term "expression of a gene" in the present invention means transcription of a gene, and specifically, transcription into mRNA. Said "transcription" involves the transcription of DNA and the processing of the resulting mRNA product. In the present invention, the term " enhancement "is intended to mean at least 5% or 10%, preferably at least 20% or 40%, more preferably 50%, 60%, 70% Means 80% or more resistant to weathering, and means that the resistance to harshness is increased by introducing the gene as compared with the original resistance of the plant.

In the present invention, the term " mud-resistant "can be defined as maintaining growth and production by a comprehensive response effort of various morphological, physiological or biochemical traits under dry stress conditions. To date, the known trait-related traits related to mycorrhizae are myocardial, leaf senescence, pore opening and closing, osmotic control, cell membrane structure, and ability to maintain good moisture and productivity in dry conditions.

According to the present invention, the BrDSR (Brassica When the expression of rapa Drought Stress Resistance gene is increased, the resistance of the plant is increased. Therefore, in order to improve the resistance of the plant to weathering, BrDSR rapa Drought Stress Resistance) gene may be introduced into a vector as described above, and then the expression of the gene may be increased by transforming the plant or by treating with an artificial substance, thereby improving the resistance of the plant, Do not.

The Brassica rapa Drought Stress Resistance gene of the present invention is involved in tolerance to dry stress of a plant, and the transgenic plant overexpressing it is excellent in tolerance to drying stress caused by changes in climatic conditions due to global warming and the like It can be used as a new functional crop with excellent adaptability to dry cultivation conditions.

1 is a photograph of a fragment of a gene encoding BrDSR amplified from Chinese cabbage by PCR using each pair of BrDSR-F and BrDSR-R primers.
Fig. 2 is a photograph showing the gene encoding BrDSR amplified from Chinese cabbage by PCR using each pair of BrDSR-F and BrDSR-R primers, which was inserted into pGEM-T easy vector and treated with EcoRI enzyme to confirm correct insertion .
Fig. 3 shows the nucleotide sequence and amino acid sequence of a gene encoding BrDSR of Chinese cabbage separated by PCR.
FIG. 4 is a diagram showing a transcriptional overexpression vector (pDSR) overexpressing the BrDSR gene of Chinese cabbage separated by PCR.
[Note] LB, left border
T-35S, Cauliflower Mosaic Virus 35S Termination site
hpt , hygromycin resistance gene
P-35S, cauliflower mosaic virus 35S promoter
RB, right border
FIG. 5 shows the results of PCR analysis of the transformation of tobacco transformants produced using the BrDSR overexpression vector.
FIG. 6 shows the results of quantitative real-time RT PCR analysis of the expression level of the BrDSR gene in tobacco transgenic plants transfected with the PCR assay.
FIG. 7 shows the results of analysis of phenotype after dry stress treatment of tobacco transformants showing resistance to drying.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Investigation of resistance to cabbage-derived genes

The information of Brassica rapa EST and Microarray Database was used to identify the novel resistance genes of Chinese cabbage. The above-mentioned database was prepared by cultivating a Chinese cabbage ( Brassica rapa ssp. Pekinensis inbred line "Chiifu") for 3 weeks in a growth phase (luminosity: 290 占 퐉 m ^-2 sec ^-1 , (24 hrs), growth temperature (25 ℃), growth humidity (40-70%)), dry stress (natural drying) was treated for 48 hours, total RNA was extracted by treatment time and expression of each gene was analyzed using KBGP-24K oligo chip And provides comparative analysis information.

The present inventors have found that " Brassica Rapa EST and Microarray Database "were used to classify the genes whose expression was strongly increased in response to dry stress treatment, and selected genes with complete intestine and unknown function.

In order to identify the novel resistance genes in Chinese cabbage, based on the 438 bp ORF (Open Reading Frame) in KBGP-24K oligo chip and the cDNA sequence (AT1G16850) having homology in Arabidopsis thaliana , a pair of primers And the sequence thereof is shown in Table 1 below.

primer Base sequence BrDSR-F (SEQ ID NO: 1) 5-ATG ACT TTG CAA GTT TTCA-3 BrDSR-R (SEQ ID NO: 2) 5-TTA GAG CTC CTC AGT ACCA-3

Example 2: RNA isolation from Chinese cabbage

The Chinese cabbage ( Brassica rapa ssp. pekinensis inbred line 'CT001') was used for RNA isolation by collecting samples from aseptically grown cabinets. Chinese cabbage rapa ssp. The leaves of pekinensis were disrupted by using liquid nitrogen, and 1 ml of trizol (Gibco BRL) solution was added thereto. The mixture was allowed to stand at room temperature for 5 minutes. After adding 200 μl of chloroform, the mixture was centrifuged at 13,000 rpm for 10 minutes at 4 ° C, and the supernatant was transferred to a new tube. Isopropanol (500 μl) was added and centrifuged at 13,000 rpm for 15 minutes to precipitate. Isopropanol was removed and the precipitate was washed with 75% ethanol. Then, it was well dried without ethanol and dissolved in sterilized distilled water treated with DEPC (diethyl pyrocarbonate).

Example 3: Isolation and recombinant vector production

To isolate the resistance-to-resistance-related genes, cDNA was synthesized using the oligo dT primer and reverse transcriptase (reverse transcriptase) as the template, and PCR was performed with the primers prepared in the above Table 1 Respectively.

First, 1 μg of RNA was added to oligo dT primer and stored at 70 ° C to remove the RNA secondary structure. The cDNA was reverse-transcribed at 45 ° C for 30 minutes and then inactivated using reverse transcriptase at 94 ° C For 5 minutes. Then, 1 μg of the synthesized cDNA was subjected to PCR using the primers shown in Table 1 and tag polymerase (Taq polymerase). The PCR program was run for 40 cycles at 94 ° C for 30 seconds, at 61 ° C for 30 seconds and at 72 ° C for 30 seconds, followed by final extension at 72 ° C for 10 minutes , And the whole reaction was terminated at 4 ° C.

1 is a photograph of a fragment of a gene encoding BrDSR amplified from Chinese cabbage by PCR using each pair of BrDSR-F and BrDSR-R primers. As a result of the experiment, a piece of amplified anti-aridity related gene was confirmed by electrophoresis (Fig. 1). As a result, a PCR product corresponding to 438 bp was confirmed.

Then, to analyze the base sequence of the fragment amplified with the primers of SEQ ID NO: 1 and SEQ ID NO: 2, a recombinant vector was constructed by cloning into a vector pGEM-T easy vector for sequencing.

Experimental Example  1. E. coli using recombinant vector. coli DH10 Transformation

Escherichia coli DH10 was inoculated into LB 50, and OD ₅₇₀ = 0.375-0.400, and the culture broth was centrifuged at 3,000 rpm for 10 minutes. The resulting precipitate was dissolved in 0.1 M CaCl ₂ solution and then stored in ice for 30 minutes. Thereafter, the mixture was centrifuged at 3,000 rpm for 7 minutes, and the precipitate was dissolved in 0.1 M CaCl _2. The recombinant vector of Example 3 was mixed with ice for 30 minutes, and then thermally shocked at 42 ° C for 90 seconds. Thereafter, the cells were allowed to stand in ice for 10 minutes, and then LB medium 700 was added thereto, followed by shaking culture at 37 ° C for 1 hour. After centrifuging the culture at 12000 rpm for several seconds, only 100 μl was left, the supernatant was removed, and the precipitate was dissolved with the remaining supernatant. Subsequently, the precipitate dissolved in solid LB medium supplemented with ampicillin (50 mg / L) and X-gal (200 mg / L in NN dimethylformamide) was developed and cultured at 37 ° C. for 12 hours. Transgenic colonies forming visible and white colonies were selected.

Experimental Example  2: Recombination DNA  Detection of isolation and gene insertion

The selected transformed colonies were inoculated in 3 ml of LB medium supplemented with antibiotics and cultured at 37 占 폚 for 12 hours. The culture broth was centrifuged at 12,000 rpm for 10 minutes at 4 占 폚. Solution (200 μl / ml) was added to the precipitated microbial cells, followed by vortexing for a few seconds, followed by solution (200 μl / ml) and carefully mixed. For 5 minutes. After that, Solution (200 μl / ml) was added and mixed well. After left on ice for 10 minutes, supernatant was obtained by centrifugation at 12,000 rpm for 10 minutes at 4 ° C. Recombinant DNA was precipitated at -20 ° C for 2 hours by adding 1/10 NaOAc and 2.5-fold 100% ethanol to the obtained supernatant, and centrifuged to obtain a precipitate. The precipitate was washed with 70% ethanol and dissolved in sterile distilled water.

The restriction enzyme EcoR was confirmed to be inserted into the pGEM-T easy vector by amplifying the resistance-to-resistance gene from the Chinese cabbage (FIG. 2). Fig. 2 shows the results of RT-PCR using BrDSR-F and BrDSR-R primer pairs. The gene encoding BrDSR amplified from Chinese cabbage was inserted into pGEM-T easy vector, It is a photograph. As a result, it was confirmed by EcoRI treatment that the gene inserted into the pGEM-T easy vector was separated into two fragments by 438 bp inserted gene and pGEM-T easy vector.

Experimental Example  3. Dry  Identification of the entire sequence of the relevant gene

Next, in order to confirm the nucleotide sequence of the 438 bp inserted gene, nucleotide sequences were examined using T7 and SP6 primers close to the insertion site. Sequencing of the PCR fragments cloned into the pGEM-T easy vector for sequencing was performed using the GenBank BLAST program.

FIG. 3 shows the nucleotide sequence and amino acid sequence of the gene encoding BrDSR of Chinese cabbage separated by RT-PCR. The amino acid sequence (SEQ ID NO: 4) was determined by determining a start codon and a termination codon based on the confirmed nucleotide sequence (SEQ ID NO: 3), and it was named as a full-length resistant gene BrDSR isolated from Chinese cabbage (Fig. 3).

Experimental Example  4. BrDSR  Production of recombinant vector for transformation of gene

In order to over-express the isolated BrDSR gene, pDSR, which is a transformation vector, was constructed by inserting a 438 bp BrDSR gene into pH2GW7 (VIB-Ghent University, Belgium) (FIG. Fig. 4 shows a transcriptional overexpression vector (pDSR) overexpressing the BrDSR gene of Chinese cabbage isolated by RT-PCR.

Experimental Example  5: BrDSR  Genetically transformed tobacco

In order to prepare a tobacco transformant overexpressing the BrDSR gene of Chinese cabbage, an efficient transformation method of Chinese cabbage by Agrobacterium (Patent Registration No. 0523758, October 31, 2005) was modified and carried out. The detailed transformation method is as follows.

1. Culture of Agrobacterium tomafaciens containing the transduction carrier (pDSR)

Transfected with a vector to switch to inoculate tobacco Agrobacterium tumefaciens containing a vector for plant transformation pDSR Solarium mapo-to-Hsien (Agrobacterium tumefaciens LBA4404 / pDSR were used. Hereinafter, Agrobacterium tomafachene refers to Agrobacterium tumefaciens LBA4404 / pDSR.

In order to increase the efficiency of transformation when the tobacco leaf is inoculated with Agrobacterium tomafaciens, Agrobacterium ( Agrobacterium tumefaciens), which firstly contains a transduction carrier (pDSR) tumefaciens LBA4404 / pDSR) was plated on a solid YEP medium containing 50 mg / L of spectinomycin and cultured at 28 ° C for 48 hours. Several colonies of the cultured Agrobacterium tmafaciens cells were photographed Was suspended in 5 mL of liquid YEP medium containing 50 mg / L of spectinomycin and suspension-cultured at 28 DEG C for 48 hours. 700 μl was extracted from the cultured suspension and added to 50 ml of liquid YEP medium to which 0.02 mM Acetosyringone was added and the pH was adjusted to 5.6, and suspension culture was performed at 28 ° C until the OD ₆₀₀ reached 1.0. Finally, the cultured suspension was centrifuged at 4000 rpm for 15 minutes in a centrifuge, resuspended in 25 ml liquid YEP medium, and used for inoculation of tobacco leaves.

2. Tobacco Transformation and Chute Induction

Tobacco [ Nicotiana tabacum SR1] was cultivated in an aseptic condition, and the leaves were cut into 10 mm in width and 10 mm in length, and then inoculated by immersing in a suspension of Agrobacterium tomafaciene for 1 minute. Inoculated leaf slices were co-cultured for 3 days in co-culture medium (MS base medium, 3% sucrose, 0.8% plant agar, 1.5 mg / L BA, pH 5.7). The leaf sections were then washed with a liquid co-culture medium to rinse the Agrobacterium tomafaciens, followed by shute induction medium (MS basal medium, 3% sucrose, 0.8% plant agar, 1.5 mg / L BA, 200 mg / L cefotaxime , pH 5.7), so that the shoot was induced only in the transformed tissue.

3. Root induction

The induction transformants were cut and transferred to root induction medium (1/2 MS base medium, 3% sucrose, 0.8% plant agar, 200 mg / L cefotaxime, pH 5.8) to induce root development. Tobacco transgenic plants with roots were transplanted into sterilized vermiculite, purified, and then transplanted into pollen and grown in a greenhouse.

Experimental Example  6. PCR  Selection of transgenic plants through assay

PCR assays were performed to confirm the transformation of tobacco transformants transformed with the pDSR vector as described above.

FIG. 5 shows the results of PCR analysis of the transformation of tobacco transformants produced using the BrDSR overexpression vector. As a result, a PCR assay was performed on the hygromycin resistance gene ( hpt ), and all 5 individuals produced by the test were identified as transformants (FIG. 5). The PCR program was run for 40 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute, after the initial denaturation time of 94 ° C for 2 minutes, After extension, the whole reaction was terminated by maintaining at 4 ° C. The nucleotide sequences of the primers HPT-F (SEQ ID NO: 3) and HPT-R (SEQ ID NO: 4) used for PCR assays are shown in Table 2.

primer Base sequence HPT-F (SEQ ID NO: 3) 5-TTT CCA CT GCG AGT AC-3 HPT-R (SEQ ID NO: 4) 5-TGT CGA GAA GTT TCT GAT CGA-3

Experimental Example  7. Quantitative real -time( Quantitative real - time ) RT PCR  Analysis of expression levels of BrDSR gene in transformants

The expression level of the BrDSR gene in the body during dry stress was analyzed by quantitative real-time RT PCR in 5 individuals identified as transformants in Experimental Example 6.

FIG. 6 shows the results of quantitative real-time RT PCR analysis of expression levels of the BrDSR gene in tobacco transformants transfected with PCR assay. As a result, the tobacco transformants whose transformation was confirmed by the PCR test showed an expression level of about 1.7 to 2.6 times higher than that of the non-transformant (CON) (FIG. 6).

The PCR program was first denatured at 95 ° C for 10 minutes, followed by cDNA synthesis for 30 minutes at 42 ° C. Thereafter, the reaction was repeated 35 times at 95 캜 for 10 seconds, 59 캜 for 15 seconds, and 72 캜 for 20 seconds. In the expression analysis method, fluorescence intensity data according to the amount of amplification was measured at the end of each cycle, and the measured values were analyzed by a Rotor-Gene 6000 analysis software. The BrDSR expression level of each transformant was calculated relative to the expression level of the actin gene. The primers used were SEQ ID NO: 1 and SEQ ID NO: 2.

Experimental Example  8. In case of dry stress, tobacco transformants Dry  black

Transformation was confirmed by PCR assays using the hygromycin resistance gene and quantitative real-time RT PCR analysis revealed 5 transformants with higher expression levels than non-transformants The changes of phenotype (whether or not dryness) were observed under natural drying conditions when actual dry stress occurred. Dry stress may be caused by any non-biological stimuli (moisture, temperature, light, etc.) or biological stimuli (pathogen infection, pest damage, etc.) during the drying process, ) Was phenotyped in a limited condition (luminosity of 250 μmol · m ^-2 · sec ^-1 , photoperiod of 16 hours treatment / 8 hours of cancer treatment, growth temperature of 22 ° C, humidity of 30 - 50% And to the time when there was a clear difference.

FIG. 7 shows the results of analysis of phenotype after dry stress treatment of tobacco transformants showing resistance to drying. As a result, at the 5th day of the drying treatment, the control group, non-transformant, lost its elasticity of leaves and stems and began to fall down. On the other hand, all of the transformants maintained the same shape as before the drying treatment, so that there was no significant difference in expression shape (FIG. 7).

<110> University-Industry Cooperation Group of Kyung Hee University <120> BrDSR Protein Implicated in Drought Stress Tolerance, Gene          Encoding Thereof Protein and Transformed Plants with the Same <130> KPA140802KR <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer BrDSR-F <400> 1 atgactttgc aagttttcca 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer BrDSR-R <400> 2 ttagagctcc tcagtacca 19 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Primer HPT-F <400> 3 tttccactgc gagtac 16 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer HPT-R <400> 4 tgtcgagaag tttctgatcg a 21 <210> 5 <211> 438 <212> DNA <213> BrDSR <400> 5 atgactttgc aagttttcca cttgctaagc atatctttag tcatcttctt tgtattctct 60 gtgaatgttg tcagtgagga ggattaccag agagcggtgc ctccggaaga taaaacgact 120 acggtttggt tctctaggat caaacagtct ggtaatgatt actgggggaa actcaaagag 180 agtttaggtc gtggtcacgc ccgctttttt cctccaaaca tagaaggaaa ggatgatcca 240 tcaatgggag caggaggaaa gatgaaagag gcggtcacaa ggagcttcga gcacagtaaa 300 gattcggtgg aggaagctgc cagatcagca gcggaggtgg cgtgtgatgc ggcggaagca 360 gttaaagaaa aggtgaagag gagcttttcc ggcagcgaga cgactcagca gcagtcatat 420 ggtactgagg agctctaa 438 <210> 6 <211> 145 <212> PRT <213> BrDSR <400> 6 Met Thr Leu Gln Val Phe His Leu Leu Ser Ile Ser Leu Val Ile Phe   1 5 10 15 Phe Val Phe Ser Val Asn Val Val Ser Glu Glu Asp Tyr Gln Arg Ala              20 25 30 Val Pro Pro Glu Asp Lys Thr Thr Thr Val Trp Phe Ser Arg Ile Lys          35 40 45 Gln Ser Gly Asn Asp Tyr Trp Gly Lys Leu Lys Glu Ser Leu Gly Arg      50 55 60 Gly His Ala Arg Phe Phe Pro Pro Asn Ile Glu Gly Lys Asp Asp Pro  65 70 75 80 Ser Met Gly Ala Gly Gly Lys Met Lys Glu Ala Val Thr Arg Ser Phe                  85 90 95 Glu His Ser Lys Asp Ser Val Glu Glu Ala Ala Arg Ser Ala Ala Glu             100 105 110 Val Ala Cys Asp Ala Ala Glu Ala Val Lys Glu Lys Val Lys Arg Ser         115 120 125 Phe Ser Gly Ser Glu Thr Thr Gln Gln Gln Ser Tyr Gly Thr Glu Glu     130 135 140 Leu 145

Claims (10)

A Brassica rapa Drought Stress Resistance (BrDS) protein consisting of the amino acid sequence of SEQ ID NO: 6.
A gene encoding the protein of claim 1.
3. The gene according to claim 2, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 5.
3. The method according to claim 2, wherein the gene is selected from the group consisting of Chinese cabbage The gene that is derived from rapa .
3. The method according to claim 2, wherein the gene is selected from the group consisting of Brassica rapa ssp. The gene that is derived from pekinensis .
A recombinant vector comprising the gene of any one of claims 2 to 5.
A plant transformed by the recombinant vector of claim 6.
8. The method according to claim 7, wherein the plant is selected from the group consisting of food crops including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats or millet; Vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions or carrots; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut or rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots or bananas; Roses, gladioluses, gerberas, carnations, mums, lilies or tulips; And feed crops including raglass, red clover, orchardgrass, alpha-alpha, tall fescue or perennialla grass.
9. The plant according to claim 8, wherein the plant is tobacco.
A method for enhancing plant tolerance by increasing the expression of the gene of any one of claims 2 to 5.

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