KR101431124B1 - Transgenic plant with enhanced tolerance to cold stress and salt stress by introducing nrdH gene and preparation method thereof - Google Patents

Abstract

The present invention relates to a composition for improving cold stress or high salt stress resistance of a plant containing an nrdH gene, and a plant cell or plant transformed with said composition. In addition, the present invention relates to a method for producing a transgenic plant in which a nrdH gene is transformed into a plant to thereby enhance resistance to cold stress or high salt stress. The present invention also relates to a method for transforming a nrdH gene into a plant to promote resistance to cold stress or high salt stress of the plant.

Description

[0001] The present invention relates to a transgenic plant having enhanced cold tolerance and salt tolerance and a method for producing the same,

The present invention relates to a composition for improving cold stress or high salt stress resistance of a plant containing an nrdH gene, and a plant cell or plant transformed with said composition. In addition, the present invention relates to a method for producing a transgenic plant in which a nrdH gene is transformed into a plant to thereby enhance resistance to cold stress or high salt stress. The present invention also relates to a method for transforming a nrdH gene into a plant to promote resistance to cold stress or high salt stress of the plant.

Reactive oxygen species (ROS) generated through the activity of various oxidase and peroxidase enzymes act as an intracellular signaling substance in response to an internal developmental signal or an external environmental stimulus, The expression of genes necessary for defense is induced, but the increase of ROS concentration causes physiological disorder. ROS in plants may accumulate excessively due to various abiotic environmental stresses such as drought, low temperature, salinity, dryness, pollution, scratches, ozone, excessive UV exposure and osmotic shock, whereby macromolecules ) And can lead to cell death.

Recently, interest in ROS-mediated signal transduction pathways has been increasing in plants. Regulation of these pathways may increase the expression of antioxidant enzymes and bio-defense proteins in cells, and ultimately resistance to various environmental stresses It is expected to be able to develop plants.

On the other hand, the physiological role of glutaredoxin (Grx) in plant antioxidant systems is not well known. Grx is known as a glutathione-dependent reductase present in almost all animals, plants and microorganisms and catalyzes glutathione-dependent reactions to protect specific cellular proteins from damage by reactive oxygen species. E. coli has five Grx ( EcGrxA , EcGrxB , EcGrxC , EcGrxD , and EcNrdH ), and Arabidopsis has 31 Grx.

In studying the effect of the Grx genes of E. coli on the resistance to environmental stress of plants, the present inventors confirmed that the nrdH gene exhibits tolerance to cold stress and salt stress in the transformed plant, Completed.

It is an object of the present invention to provide a composition for improving cold stress or high salt stress resistance of a plant, which comprises the nrdH gene.

It is another object of the present invention to provide a plant cell transformed with the above composition.

It is another object of the present invention to provide a plant transformed with said composition.

It is another object of the present invention to provide a method for producing a transgenic plant having enhanced resistance to cold stress or high salt stress, comprising the step of transforming a nrdH gene into a plant.

It is another object of the present invention to provide a method for promoting resistance to cold stress or high salt stress of a plant, including the step of transforming a nrdH gene into a plant.

In order to achieve the above object, the present invention relates to a composition for improving cold stress or high salt stress resistance of a plant, which comprises an nrdH gene.

In a preferred embodiment, the nrdH gene may be derived from E. coli.

Preferably, the nrdH gene may consist of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1, and more preferably, the nrdH gene may comprise the nucleic acid sequence of SEQ ID NO: 2.

Also preferably, the nrdH gene may be in the form of a recombinant vector for plant expression.

In another aspect, the present invention relates to a plant cell transformed with the above composition and having enhanced cold stress or high salt stress resistance.

In another aspect, the present invention relates to a plant transformed with the composition and having enhanced cold stress or high salt stress resistance.

In another aspect, the present invention relates to a method for producing a transgenic plant having enhanced resistance to cold stress or high salt stress, comprising the step of transforming a nrdH gene into a plant.

In another aspect, the present invention relates to a method for enhancing resistance to cold stress or high salt stress of a plant, including the step of transforming the nrdH gene into a plant.

Hereinafter, the present invention will be described in more detail.

Glutaredoxin (Grx) is known as a glutathione-dependent reductase present in almost all animals, plants and microorganisms. It has CXXC and CXXS as active sites and catalyzes the glutathione-dependent reaction, Protects specific cellular proteins. E. coli has five Grx ( EcGrxA , EcGrxB , EcGrxC , EcGrxD , and EcNrdH ).

Herein, the nrdH gene codes for the Glutaredoxin-like protein NrdH, which is one of the Grx family. The nrdH gene derived from Escherichia coli has the nucleic acid sequence of SEQ ID NO: 2, and the protein encoded thereby has the amino acid sequence of SEQ ID NO: 1.

Preferably, the nrdH gene in the present invention comprises a nucleic acid molecule comprising a functionally equivalent codon or a codon coding for the same amino acid (by codon degeneracy), or a codon coding for a biologically equivalent amino acid. In addition, considering a mutation having biological equivalent activity, the gene 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 arranging the above-mentioned sequence of the present invention and any other sequence to correspond to each other as much as possible, and analyzing sequences arranged using algorithms commonly used in the art, at least 60% , More preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Arrangement methods for sequence comparison are known in the art.

In a situation where it is not known how the Grx affects environmental stress resistance in the plant antioxidant system, the inventors have for the first time demonstrated that the nrdH gene transformed plants have greatly improved resistance to cold stress and high salt stress. Thus, plants with enhanced cold stress or high salt stress resistance can be produced using the nrdH gene.

The introduction of the nrdH gene into the plant can be accomplished by a variety of methods known in the art, and preferably an expression vector for plant transformation can be used. Suitable promoters to be included in the vector may be any of those conventionally used in the art for transgenic introduction of plants, such as SP6 promoter, T7 promoter, T3 promoter, PM promoter, ubiquitin promoter of corn, (CaMV) 35S promoter, nopaline synthase (nos) promoter, Piguet mosaic virus 35S promoter, water crane basil lipid viral promoter, comeline yellow mothball virus promoter, ribulose-1,5- (TPI) promoter of Arabidopsis, adenine phospholiposyl transferase (APRT) promoter of Arabidopsis, a promoter of rice papilloma synthase An azeotropic promoter and a blue copper binding protein (BCB) promoter. .

The vector may also comprise a poly A signal sequence which results in a 3'-terminal polyadenylation, for example from the nopaline synthase gene of Agrobacterium tumefaciens (NOS 3 ' end of the protease inhibitor I or II gene of the tomato or potato, the CaMV 35S terminator and the octopine synthase terminator sequence. The term " endoprotein synthase terminator " can do.

In addition, the vector may optionally further comprise a gene encoding a reporter molecule (e.g., luciferase and beta -glucuronidase), and may further comprise an antibiotic (such as neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) or a herbicide (e.g., Basta) resistance gene (e.g., neomycin phospho-transferase dehydratase (nptⅡ), hygromycin phospho transferase dehydratase comprise the like) (hpt) .

According to a preferred embodiment of the present invention, the recombinant vector for plant expression of the present invention does not contain the Agrobacterium binary vector, the cointegration vector or the T-DNA site but is designed so as to be expressed in plants Can be used. Of these, the binary vector refers to a vector having two plasmids containing a plasmid having a left border (RB) and a right border (RB) necessary for movement in a Ti (tumor inducible) plasmid and a gene necessary for transferring a target nucleotide , A vector containing a promoter region and a polyadenylation signal sequence for expression in a plant can be used.

When a binary vector or a cointegration vector is used, it is preferable to use Agrobacterium - mediated transformation as a transformation strain for introducing the recombinant vector into a plant ( Agrobacterium - mediated transformation) Agrobacterium tumefaciens ) Or Agrobacterium ( Agrobacterium < RTI ID = 0.0 > rhizogenes ) Can be used. In the case of using a vector not containing a T-DNA region, electroporation, microparticle bombardment, polyethylene glycol-mediated uptake, and the like are used to introduce a recombinant plasmid into a plant .

Plants to be transformed herein are understood to include both plant cells, plant tissues, and plant seeds that develop into adult plants as well as mature plants.

Plants to which the method of the present invention can be applied are not particularly limited. The plants to be transformed in the present invention include food crops selected from the group consisting of rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats and sorghum; Vegetable crops selected from the group consisting of Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops selected from the group consisting of Ryegrass, Red Clover, Orchardgrass, Alpha Alpha, Tall Fescue, and Fereniallaigrus. Preferably, the plant is selected from the group consisting of Arabidopsis, potato, eggplant, cigarette, pepper, tomato, burdock, ciliaceae, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, parsley, parsley, cabbage, cabbage, Can be a dicotyledon such as melon, cucumber, amber, pak, strawberry, soybean, mung bean, kidney bean, or pea, and most preferably it can be a rice cob.

The nrdH gene can be introduced into plants to enhance tolerance to cold stress and tolerance to overtreatment stress. Therefore, it is possible to utilize the nrdH gene to tolerate abnormal low temperature due to climate change or to increase the productivity of crops or flower crops Promotion can be promoted.

Fig. 1 shows the constitution (A) of the vector for expressing the EcNrdH gene and the production process (B) for the vector.
FIG. 2 shows the results of electrophoresis on the expression of the EcNrdH gene in the Arabidopsis thaliana # 2-7, # 4-11, # 5-9 transformed with the EcNrdH gene.
Figure 3 compares the phenotypes of plants expressing Col-0 (Columbia-0 ecotype) and EcNrdH under freezing conditions. Two weeks after the freezing treatment, representative plants were shown.
FIG. 4 shows a schedule in which cold stress is processed to evaluate low-temperature resistance.
FIG. 5 shows a schedule for treating high salt stress to evaluate high salt resistance.
FIG. 6 shows phenotypes when transformed plants expressing Col-0 ecotype and EcNrdH were grown for 17 days and then frozen and then restored for 2 weeks under normal conditions.
Fig. 7 shows the survival rate of each plant as a result of freezing assay for Arabidopsis plants grown for 17 days. Each experiment was repeated 3 times independently, and the bars represent mean ± standard deviation.
FIG. 8 shows the phenotype when transformed plants expressing Col-0 ecotype and EcNrdH were grown for 17 days and treated with NaCl 500 mM for 24 hours and then restored for 1 week. Representative plants were shown, and each experiment was performed independently 3 times.
Fig. 9 shows the survival rate of each plant as a result of salt tolerance assay for the Arabidopsis plants grown for 17 days. Each experiment was repeated 3 times independently, and the bars represent mean ± standard deviation.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Plant material and growth conditions

Arabidopsis thaliana ecotype Columbia seeds were sterilized for 5 minutes in 5% sodium hypochlorite and 0.05% Triton-X 100, washed five times with distilled water, and incubated with 0.8% agag and 0.5x MS (Murashige-Skoog salts, Duchefa ) in 1% sucrose concentration, and pH 5.7 medium 110 μmolm ^-2 s ^- were grown under a light intensity of ^one. After 7 days, plants were transferred to soil and used for stress test on the 17th day after sowing. All plants were grown in a 23-degree chamber maintained at 65% humidity with a 16 hr / 8 hr day-night cycle.

Example  2. Plasmid preparation and transgenic plant production

2-1. EcNrdH  Binary vector manufacture

Gateway cloning system was used for the preparation of the entry vector (Fig. 1). The gateway cloning system is based on site-specific recombination of λ phage ( att B × att P → att L × att R). AAA GTC GGC TCC ATG CGC ATT ACT AT-3 '(SEQ ID NO: 3) and reverse priem: 5'-AAA GCT GGG TTT TAT TTC AGC AGG GGA- 3 '(SEQ ID NO: 4) was used to isolate and amplify the grxC gene. PCR was carried out for 30 cycles (94 ° 30 sec; 60 ° 30 sec; 72 ° 30 sec) after 5 cycles (94 ° 30 '; 55 ° 30' and 72 ° 30 ') using one unit of r-Taq Respectively. BP a second PCR for recombinant att B1 primer 5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG CCA TGG-3 '( SEQ ID NO: 5) and att B2 primers 5'-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTA The attB1 attB2 site was made at the rear of the PCR product ( att B1-ORF of EcNrdH - att B2) using CTA GGT CAC C-3 '(SEQ ID NO: 6). PCR was carried out for 30 cycles (94 ° 30 sec; 60 ° 30 sec; 72 ° 30 sec) after 5 cycles (94 ° 30 '; 50 ° 30' and 72 ° 30 ') using one unit of r-Taq Respectively. PYJ3 was used as a donor vector of the BP reaction, pYJ3 had an att P1- ccd B- att P2 cassette, and BP region-specific recombination was performed using BP clonase (Invitrogen). The ccdB gene is removed between attP1 and attP2 when recombination is performed, allowing only the vector to be selected for recombination. BP recombination was performed using 100 ng of PCR product, 40 ng of vector plasmid (pYJ3), 1 μl of 5 × BP Clonase reaction buffer (Invitrogen), 0.2 μl of BP Clonase enzyme mix (Invitrogen) and TE buffer After incubation for 1 hour at 25 ° C, the prepared plasmid was injected into E. coli (DH5α) by electrophoretic transformation and spread on a LB plate containing 100 mg / μl ampicilin to obtain a single colony Respectively. The obtained colonies were cultured in LB liquid medium to obtain a large amount of plasmid and used in the LR recombination method.

In LR recombination, a pEarleygate vector with att R- ccd B- att R2 cassette was used. The ccdB gene is removed between attR1 and attR2 when recombination is performed, allowing only the vector to be genetically recombined. LR recombination was performed using 100 ng of recombinant pYJ3 and 40 ng of vector plasmid (pEarleygate), 1 μL of 5 × LR Clonase reaction buffer (Invitrogen), 0.2 μL of LR Clonase enzyme mix (Invitrogen) and TE buffer Lt; RTI ID = 0.0 > 25 C < / RTI > for 1 hour.

After the recombination reaction, the prepared plsmids were injected into E. coli (DH5α) by electrophoretic transformation method, and single colonies were obtained by spreading on an LB plate containing 100 mg / kan kanamycin. The obtained colonies were cultured in LB liquid medium to obtain a large amount of plasmids, which were sequenced after purification and confirmed to be 100% as compared with the E. coli nrdH gene.

2-2. Production of Overexpressed Transgenic Plants

The overexpressed plasmid prepared in Example 2-1 was transformed into a plant by floral dipping in Arabidopsis thaliana. After seeding the obtained T1 seeds, BASTA solution containing 0.01% of ammonium glutocinate Were used to select transformants and T2 seeds were obtained for each individual. T2 seeds were sown on a medium supplemented with 0.5 X MS, 1% sucrose and 50 μg / ml DL-phosphinothricin (PPT) to determine the number of susceptible individuals and susceptible Basta (BASTA) A line with a p-value of less than 0.05 was considered to have inserted a copy of overexpressed T-DNA by the directed Kwai-square test. Table 1 shows the process of selecting the nrdH line in the T2 generation.

(sum: total plant, PPT R: Vasta resistance, PPT S: Vasta sensitivity, X ² value is the expected ratio of 3: 1, X ² = (oe) ² / e (Vasta R) + (oe) ² / e Busta s); P <0.05 when X ² was 3.84 or less;

One more generation of selected T2 plants was harvested, and seeds were re-seeded to select 100% surviving homologous lines in the medium containing PPT. RNA was isolated from the rosette leaves of the plants and the degree of overexpression of each gene was determined And detected by RT-PCR using specific primers (Figure 2). As a result, it was found that each gene was expressed at a certain level or more in each plant, and it was confirmed that the gene was not expressed in normal WT Arabidopsis thaliana (Col-0). Table 2 shows the results of screening in the T3 generation.

Example  3. Cold stress treatment and Freezing Assay

The plants grown for 17 days under the conditions described in Example 1 were used for cold stress treatment as shown in FIG. Specifically, in order to test the effect on phasing sensitivity, plants containing Arabidopsis ecotype Col-0 and E. coli nrdH were grown for 17 days. Transgenic plants grown for 17 days had no morphological differences when compared to wild-type plants (Fig. 3). Then treated at 8 hours -1 degrees in dark conditions; Under light conditions, treatment is carried out from -1 to -12 degrees and then treated at -12 degrees for 2 hours while lowering 1 degree per hour. Again treated at 4 degrees for 12 hours under dark conditions. After freezing, the plants were grown in a normal 23-degree growth chamber for two weeks. After recovery for 2 weeks under normal growth conditions, the low temperature resistance was evaluated by calculating the percentage of living plants among the whole plants at a specific temperature. The experiment was repeated three times independently of each other in 16 plants.

Example  4. High salt  Stress handling and High salt  stress Assay

The plants grown for 17 days under the conditions described in Example 1 were used for salt stress treatment as shown in Fig. Specifically, to test the effect on high salt sensitivity, plants containing Arabidopsis ecotype Col-0 and E. coli nrdH were grown for 17 days. The plants that were 17 days old were treated with 500 mM NaCl for 24 hours. After one week of high salt stress treatment, water was added again, and after one week from the start of the water cycle, the percentage of living plants among the whole plants was calculated to evaluate the salt resistance. All experiments were performed in a 23-degree chamber and repeated three times independently of each other in 16 plants.

Experiment result

1. E. coli nrdH  Enhancement of cold tolerance in plants using genes

When stress occurs in plants, reactive oxygen species (ROS) are generated, which can have a devastating effect on plants. To reduce this ROS, a number of reducing enzymes are needed. It is known that about 31 GRXs are present in plants, and more than 80 genes known to have similar functions, such as GRX-like proteins, are known. The nrdH gene of Escherichia coli, which is believed to contain fewer of the functions of the GRX of the plant and is thought to contain the functions of the GRX of the plant, was cloned using a site-specific recombination method, and the expression was expressed by the 35S promoter Four generic resistant Arabidopsis thaliana transformants with the EcnrdH gene were screened at T2 generation and allogeneic splicing lines overexpressing EcNrdH were selected by segregation analysis using a selective antibiotic marker at T3 generation, The last 3 plants were selected. (# 2-7, # 4-11, # 5-9). Treating cold tolerance stress with three nrdH transgenic plants showed that only 25% of the Arabidopsis WT plants survived the freezing test while the EcNrdH overexpressing plants # 2-7, # 4-11, # 5 -9 were 43.8%, 50.0% and 31.3%, respectively. Leaf yellowing occurs as a sign of stress-induced damage in the leaves of most WT plants and the size of surviving WT plants is small, while in the case of EcNrdH overexpressing transgenic plants, (Fig. 6 and Fig. 7). These results indicate that WT plants are severely damaged by ROS caused by freezing stress and affect growth. In the case of EcNrdH transgenic transgenic plants, ROS in E. coli nrdH gene polymorphism Lt; RTI ID = 0.0 &gt; low-temperature &lt; / RTI &gt; tolerance can be increased.

2. E. coli nrdH  Increased salt tolerance in plants using genes

For the three transgenic plants (# 2-7, # 4-11, # 5-9) finally selected for overexpression of EcNrdH, high salt stress treatment resulted in only 46.8% of the Arabidopsis WT plants surviving On the other hand, EcNrdH overexpressed plants # 2-7, # 4-11, # 5-9 survived 74.7%, 74.1% and 64.1%, respectively. Leaf yellowing occurs as a sign of stress-induced damage in the leaves of most WT plants. In the case of EcNrdH overexpression plants, the rate of sulphation was slower than that of WT, and the rate of emergence of the peduncle after stress treatment was faster than that of WT, and it was found that the growth rate was different (Figs. 8 and 9) . This suggests that the nrdH gene in the EcNrdH overexpressed plant reduced the ROS and caused it to survive better under high salt stress.

<110> Ewha University - Industry Collaboration Foundation <120> Transgenic plant with enhanced tolerance to cold stress and salt          stress by introducing nrdH gene and preparation method thereof <130> DPP20128104KR <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 81 <212> PRT <213> Escherichia coli <220> <221> PEPTIDE &Lt; 222 > (1) .. (81) <223> amino acid sequence of NRdH <400> 1 Met Arg Ile Thr Ile Tyr Thr Arg Asn Asp Cys Val Gln Cys His Ala   1 5 10 15 Thr Lys Arg Ala Met Glu Asn Arg Gly Phe Asp Phe Glu Met Ile Asn              20 25 30 Val Asp Arg Val Pro Glu Ala Ala Glu Ala Leu Arg Ala Gln Gly Phe          35 40 45 Arg Gln Leu Pro Val Val Ile Ala Gly Asp Leu Ser Trp Ser Gly Phe      50 55 60 Arg Pro Asp Met Ile Asn Arg Leu His Pro Ala Pro His Ala Ala Ser  65 70 75 80 Ala     <210> 2 <211> 246 <212> DNA <213> Escherichia coli <220> <221> gene &Lt; 222 > (1) <223> nucleic acid sequence of nrdH <400> 2 atgcgcatta ctatttacac tcgtaacgat tgcgttcagt gccacgccac caaacgggcg 60 atggaaaacc ggggctttga ttttgaaatg attaatgtcg atcgcgttcc tgaagcggca 120 gaagcgttgc gtgctcaggg ctttcgtcag ttgccggtag tgattgctgg cgatcttagc 180 tggtctggtt tccgtccgga catgattaac cgtctgcatc cagcgccaca cgcggccagt 240 gcatga 246 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 aaagcaggct ccatgcgcat tactat 26 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 aaagctgggt tttatttcag cagggga 27 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> attB1 primer <400> 5 ggggaccact ttgtacaaga aagctgggtg ccatgg 36 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> attB2 primer <400> 6 ggggacaagt ttgtacaaaa aagcaggcta ctaggtcacc 40

Claims (9)

1. A composition for improving cold stress or high salt stress resistance of a plant, comprising an nrdH gene consisting of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1. delete delete 2. The composition of claim 1, wherein the nrdH gene comprises the nucleic acid sequence of SEQ ID NO: 2. 2. The composition of claim 1, wherein the nrdH gene is included in the form of a recombinant vector for plant expression. 6. A plant cell which is transformed with the composition of any one of claims 1, 4, and 5, or which has enhanced cold stress or high salt stress resistance. A plant with enhanced cold stress or high salt stress resistance transformed with the composition of any one of claims 1, 4 and 5. Comprising transforming a plant with an nrdH gene consisting of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1,
A method of producing a transgenic plant having enhanced resistance to cold stress or high salt stress. Comprising transforming a plant with an nrdH gene consisting of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1,
A method for promoting resistance to cold or high salt stress in plants.

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