KR102132276B1 - Mutated dehydrin from the Arctic mouse-ear chickweed Cerastium arcticum and use thereof - Google Patents

Abstract

The present invention is an arctic point herb ( Cerastium) arcticum ) and a mutant dihydrin protein encoded thereby. In addition, the present invention relates to a composition for enhancing abiotic stress tolerance, comprising the mutant dihydrin protein derived from the arctic point nadonace. The present invention significantly increases resistance to abiotic stress by regulating the dimerization of CaDHN, an intrinsically disordered protein, to develop environmental stress-resistant crops, and to transform microorganisms and plants using molecular biology and genetic manipulation techniques. It can be widely used throughout plant biotechnology, including basic research on improvement.

Description

Mutant dehydrin from the Arctic mouse-ear chickweed Cerastium arcticum and use thereof}

The present invention is an arctic point herb ( Cerastium) arcticum ) and a mutant dihydrin protein encoded thereby. In addition, the present invention relates to a composition for enhancing abiotic stress tolerance, comprising the mutant dihydrin protein derived from the arctic point nadonace.

Plants are always exposed to a variety of biological and abiotic stresses, especially abiotic stresses such as lack of moisture in the soil, high salinity levels, cold seas, heavy metals, thermal shocks, and ozone, which contribute to the growth and development of terrestrial plants. It is known to be a major factor in inhibiting and reducing quality. Functional studies on abiotic stress have been conducted so far, but knowledge of the biological functions of stress-related genes involved in resistance or sensitivity to abiotic stress, including cold sea (cold) stress, is still lacking. Accordingly, research to increase the resistance of plants and to improve crop yields has become an important task.

Meanwhile, plants activate specific and unique stress responses when faced with abiotic stress. For example, when there is insufficient moisture in a cold environment, plants defend themselves by activating genes that make the Late embryogenesis Abundant (LEA) protein. The LEA gene is significantly induced by abiotic stress and is known to increase resistance of transgenic plants to abiotic stress when overexpressed. A common feature of LEA proteins is that they have an intrinsically disordered protein structure according to high glycine content and high hydrophilicity, and are classified into three groups according to sequence similarity: LEAI, LEAII and LEAIII. In addition, the dihydrin protein DHN (dehydrins) is a hydrophilic, thermostable protein of 9-200 kDa present in various plants, algae, and cyanobacteria. It is very rich in glycine residues and lacks cysteine and tryptophan. Or very small amount. The structural feature of the dihydrin protein is the presence of highly conserved sequence motifs named K-, S- and Y- segments. In addition to the highly conserved domain, the dihydrin protein has a φ-segment located between the Y-, S- and K-segments, and the sequence and length of the φ-segment are very irregular compared to other segments.

Among plants, polar plants have unique physiological mechanisms that enable them to adapt to harsh environmental conditions. Arctic Point Cannabis ( Cerastium arcticum , Arctic mouse-ear chickweed or Arctic mouse-ear ) is a plant that thrives in tundra in the Arctic. CaDHN (Arctic), a stress-reactive dehydrin gene derived from the North Pole sprouts Cerastium arcticum Lange) is a dihydrin gene belonging to the SK ₅ type, and unlike most dihydrin proteins do not have a cysteine residue, it specifically includes a cysteine residue as a single residue.

Graether SP, Boddington KF. Disorder and function: a review of the dehydrin protein family. Front Plant Sci. 2014;5. Cuevas-Velazquez CL, Rendon-Luna DF, Covarrubias AA. Dissecting the cryoprotection mechanisms for dehydrins. Front Plant Sci. 2014;5. Kovacs D, Kalmar E, Torok Z, Tompa P. Chaperone activity of ERD10 and ERD14, two disordered stress-related plant proteins. Plant Physiol. 2008;147(1):381-90.

The inventors of the North Pole ( Cerastium) arcticum ) confirms that the recombinant CaDHN coexists in two forms, a monomer and a dimer, and pays attention to the dimerization to participate in resistance to abiotic stress such as low temperature stress. I tried to identify the mechanism. As a result, it is confirmed that dimerization of CaDHN is a result of intermolecular disulfide bonds between cysteine molecules, and that a single cysteine residue of CaDHN regulates dimerization of CaDHN and enhances resistance to abiotic stress. It was confirmed that it can be a target.

Therefore, the object of the present invention is the Arctic point sprouts ( Cerastium arcticum ) to provide a mutant dihydrin peptide and a polynucleotide encoding the same.

Another object of the present invention is to provide a composition for enhancing abiotic stress resistance comprising the peptide or polynucleotide.

Another object of the present invention is to provide a method for enhancing abiotic stress tolerance, comprising introducing the polynucleotide into a host cell.

Another object of the present invention is the North Pole sprouts ( Cerastium arcticum ) to provide a composition for promoting high temperature stress resistance, and a composition for enhancing protein homeostasis, comprising a wild-type dihydrin peptide or a polynucleotide encoding the same.

The present invention is an arctic point herb ( Cerastium) arcticum ), and a polynucleotide encoding the mutant dihydrin peptide.

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

In addition, the present invention provides a host cell having enhanced abiotic stress resistance transformed with the recombinant vector.

In addition, the present invention provides a composition for enhancing abiotic stress resistance comprising the peptide or polynucleotide.

In addition, the present invention provides a method for enhancing abiotic stress resistance, comprising introducing the polynucleotide into a host cell.

In addition, the present invention is an arctic point sprout ( Cerastium) arcticum ) It provides a composition for promoting high temperature stress resistance comprising a wild-type dihydrin peptide or a polynucleotide encoding the same.

In addition, the present invention is an arctic point sprout ( Cerastium) arcticum ) provides a composition for enhancing protein homeostasis comprising a wild-type dihydrin peptide or a polynucleotide encoding the same.

The present invention significantly increases resistance to abiotic stress by regulating the dimerization of CaDHN, an intrinsically disordered protein, to develop environmental stress-resistant crops, and to transform microorganisms and plants using molecular biology and genetic manipulation techniques. It can be widely used throughout plant biotechnology, including basic research on improvement.

1 is a schematic diagram of a conserved site of the CaDHN of the present invention, and a unique single cysteine residue of CaDHN is represented by Cys143. K: K-segment, S: S-segment, Φ: Φ- segment.
2 is a diagram showing the results of SDS-PAGE of wild-type CaDHN and mutant C143A proteins. WT: wild-type CaDHN, M: mutant C143A protein, β-ME: 10 mM β-mercaptoethanol treatment, BC: before cleavage of the N-terminal tag by TEV protease, AC: after cleavage of the N-terminal tag.
3 is a diagram showing the results of electrophoresis of wild-type CaDHN and mutant C143A proteins to confirm whether dimerization through purified disulfide bonds of CaDHN occurs. 3A is a diagram showing the results of treatment of wild-type CaDHN and C143A with various concentrations of DTT and CuSO ₄ . FIG. 3B is a diagram showing the results of treating wild-type CaDHN with DTT (0-20mM).
4 is a view showing the results of analyzing the chaperone (chaperone) activity of CaDHN against green fluorescent protein (GFP) protein refolding. The acid-modified GFP was refolded in regeneration buffer containing wild type CaDHN (orange) or mutant C143A (blue). Wild-type CaDHN (green) treated with 10 mM DTT was also used for the analysis of chaperone activity. Spontaneous refolding of GFP protein (black) was used as a control.
5 is a view showing the results of analyzing the metal binding capacity of wild-type CaDHN and mutant C143A by performing metal ion affinity chromatography (A in FIG. 5) and SDS-PAGE (B in FIG. 5). .
6 is a diagram showing the results of analyzing the resistance of transformed yeast cells to oxidative stress. Yeast cells transformed with an empty vector (EV) were used as a control.
7 and 8 are diagrams showing the results of analyzing the resistance of transformed yeast cells to cold and hot stress. Yeast cells transformed with an empty vector (EV) were used as a control.
9 is a diagram showing the results of a metal ion homeostasis experiment of transformed yeast cells. Yeast cells transformed with an empty vector (EV) were used as a control.

Hereinafter, the present invention will be described in detail.

The present invention provides a polynucleotide encoding a mutant dihydrin peptide derived from an arctic agonist and the same.

Dehydrin (DHN) is a group of proteins expressed in plants under conditions of low temperature or drought stress, has hydrophilicity, and has heat resistance.

The mutant dihydrin peptide of the present invention is an arctic point-nadenum ( Cerastium ; Cerastium ) obtained from Dasan Korean Arctic Research Station. arcticum L.; Caryophyllaceae) derived from mutations in the dihydrin peptide, characterized in that the 143 th amino acid of the wild type dihydrin peptide CaDHN (cysteine) consisting of the amino acid sequence of SEQ ID NO: 1 substituted with alanine (alanine) do.

In addition, the wild-type dihydrin peptide of the present invention is a peptide derived from the arctic point-nadonum that does not cause mutation, and is characterized by comprising the amino acid sequence of SEQ ID NO: 3.

In the present invention, the term “polynucleotide” is a deoxyribonucleotide or a polymer of ribonucleotides present in single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified.

The polynucleotide encoding the mutant dihydrin peptide may preferably consist of the nucleotide sequence of SEQ ID NO: 2. In addition, the polynucleotide encoding the wild-type dihydrin peptide may preferably consist of the nucleotide sequence of SEQ ID NO:4. The amino acid sequences of the wild-type dihydrin peptides and mutant dihydrin peptides used in the present invention and the polynucleotide sequences encoding each are shown in Table 1 below.

order Sequence number Mutation
Dihydrin Peptide
( C143A , M) MSNLHPTGGETETTNTDRGLFGFGKKTETPVATPTEPPHKPTLTEKLHRSGSSSSSSSDEEVIENGAKIRKPRRKGIAGKIKDKLPGGHKDEYETATPIAAGEYYHQEPNKHASDRGVAEKIKEKLPGGHKDEYGTAPTGDH A HQEQNKHASEMGAAEKIKDKLPGGQKDEYGTAPTGDHYHRDQNKHTAAGNTHYPEGNKGFVGNVKDKLPGNQTDVNHHQQQQQHPAVHVSQQAVQEPHHEKKGLLDNIKDKMPGGHKDDADKAHKGY One Mutation
Dihydrin gene ATGTCGAACTTACACCCAACCGGAGGCGAAACCGAGACCACTAATACTGATAGAGGCCTGTTTGGCTTCGGTAAGAAGACGGAAACCCCCGTCGCCACCCCGACTGAGCCACCACACAAGCCTACTCTCACGGAGAAGCTTCACCGCTCGGGCAGTAGTAGCTCTAGCTCGTCAGATGAGGAGGTAATAGAAAATGGTGCGAAGATTAGGAAGCCGAGGAGGAAGGGAATAGCAGGGAAAATCAAGGACAAGCTTCCGGGTGGTCACAAGGATGAATACGAGACTGCGACTCCTATTGCTGCGGGTGAGTACTATCACCAGGAGCCGAACAAGCACGCTTCAGACAGGGGAGTAGCGGAGAAGATCAAGGAAAAGTTACCAGGCGGTCACAAGGATGAATACGGGACAGCGCCTACCGGTGACCAC GCC CACCAGGAGCAGAACAAGCACGCTTCTGAGATGGGTGCGGCGGAGAAAATCAAGGACAAGTTGCCAGGCGGACAGAAGGATGAATACGGGACTGCGCCTACTGGCGACCACTACCACCGGGACCAGAACAAGCATACTGCGGCAGGAAACACGCATTACCCCGAGGGAAACAAGGGGTTTGTTGGCAATGTTAAGGATAAATTGCCGGGAAATCAGACAGACGTGAACCATCACCAGCAGCAGCAACAGCACCCGGCTGTTCATGTTTCACAACAGGCGGTCCAGGAGCCGCATCATGAGAAGAAAGGGCTCTTAGACAACATTAAGGACAAGATGCCCGGCGGCCACAAGGATGATGCTGATAAAGCTCACAAGGGTTACTAA 2 Wild type
Dihydrin Peptide
( CaDHN , WT) MSNLHPTGGETETTNTDRGLFGFGKKTETPVATPTEPPHKPTLTEKLHRSGSSSSSSSDEEVIENGAKIRKPRRKGIAGKIKDKLPGGHKDEYETATPIAAGEYYHQEPNKHASDRGVAEKIKEKLPGGHKDEYGTAPTGDH C HQEQNKHASEMGAAEKIKDKLPGGQKDEYGTAPTGDHYHRDQNKHTAAGNTHYPEGNKGFVGNVKDKLPGNQTDVNHHQQQQQHPAVHVSQQAVQEPHHEKKGLLDNIKDKMPGGHKDDADKAHKGY 3 Wild type
Dihydrin gene ATGTCGAACTTACACCCAACCGGAGGCGAAACCGAGACCACTAATACTGATAGAGGCCTGTTTGGCTTCGGTAAGAAGACGGAAACCCCCGTCGCCACCCCGACTGAGCCACCACACAAGCCTACTCTCACGGAGAAGCTTCACCGCTCGGGCAGTAGTAGCTCTAGCTCGTCAGATGAGGAGGTAATAGAAAATGGTGCGAAGATTAGGAAGCCGAGGAGGAAGGGAATAGCAGGGAAAATCAAGGACAAGCTTCCGGGTGGTCACAAGGATGAATACGAGACTGCGACTCCTATTGCTGCGGGTGAGTACTATCACCAGGAGCCGAACAAGCACGCTTCAGACAGGGGAGTAGCGGAGAAGATCAAGGAAAAGTTACCAGGCGGTCACAAGGATGAATACGGGACAGCGCCTACCGGTGACCAC TGC CACCAGGAGCAGAACAAGCACGCTTCTGAGATGGGTGCGGCGGAGAAAATCAAGGACAAGTTGCCAGGCGGACAGAAGGATGAATACGGGACTGCGCCTACTGGCGACCACTACCACCGGGACCAGAACAAGCATACTGCGGCAGGAAACACGCATTACCCCGAGGGAAACAAGGGGTTTGTTGGCAATGTTAAGGATAAATTGCCGGGAAATCAGACAGACGTGAACCATCACCAGCAGCAGCAACAGCACCCGGCTGTTCATGTTTCACAACAGGCGGTCCAGGAGCCGCATCATGAGAAGAAAGGGCTCTTAGACAACATTAAGGACAAGATGCCCGGCGGCCACAAGGATGATGCTGATAAAGCTCACAAGGGTTACTAA 4

The polynucleotide includes not only the nucleotide sequence encoding the dihydrin peptide, but also a sequence complementary to the sequence. The complementary sequence includes not only perfectly complementary sequences, but also substantially complementary sequences. This means a sequence that can hybridize under stringent conditions known in the art, for example, with a polynucleotide sequence encoding the amino acid of SEQ ID NO: 1.

In addition, the polynucleotide can be modified. Such modifications include addition, deletion or non-conservative substitutions or conservative substitutions of nucleotides. It is understood that the polynucleotide encoding the amino acid sequence also includes a nucleotide sequence showing substantial identity to the nucleotide sequence. The substantial identity is 70% or more when aligning the nucleotide sequence with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. It means that the base sequence is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more. . Meanwhile, a part of the polynucleotide sequence in the comparison region may include addition or deletion (gap) compared to a reference sequence (not including addition or deletion) for optimal alignment of the two sequences.

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

In the present invention, the term "vector" means a means for expressing a gene of interest in a host cell. For example, viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors. Vectors that can be used as the recombinant vector are plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14) , pGEX series, pET series, pUC19 and p426GPD, etc.), phage (e.g., λgt4λB, λ-Charon, λΔz1 and M13, etc.) or viruses (e.g., CMV, SV40, etc.) It is not limited thereto. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention.

In the recombinant vector, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 may be operably linked to a promoter. In the present invention, the term "operably linked" means that the promoter sequence and the gene sequence to initiate and mediate the transcription of the polynucleotide encoding the protein of interest of the present invention are functionally linked.

The recombinant vector can typically be constructed as a vector for cloning or for expression. The expression vector can be used in the art, conventional ones used to express foreign proteins in plants, animals or microorganisms. The recombinant vector can be constructed through various methods known in the art.

The recombinant vector can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is a host, a strong promoter (eg, pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) capable of undergoing transcription, translation It is common to include a ribosome binding site for the initiation and transcriptional/detox termination sequences. When eukaryotic cells are used as a host, replication origins that operate on eukaryotic cells included in vectors include f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, CMV replication origin, and BBV replication origin. However, it is not limited thereto. In addition, a promoter derived from the genome of a mammalian cell (eg, a metallothionine promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, Cytomegalovirus (CMV) promoter and HSV's tk promoter) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

In addition, the present invention provides a host cell having enhanced abiotic stress resistance transformed with the recombinant vector.

The host cell may be selected from the group consisting of animal cells, plant cells, fungi, bacteria, and viruses, but is not limited thereto, and any host cell known in the art may be used. Prokaryotic cells include, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis ( Bacillus subtilis ), Bacillus thuringiensis , Bacillus subtilis , Salmonella typhimurium , Serratia marsense marcescens), and when an jangnaegyun and the like, and strains such as various Pseudomonas (Pseudomonas) species, transformed with a eukaryotic cell as a host cell, yeast (Saccharomyce cerevisiae ), insect cells, plant cells and animal cells, e.g. SP2/0, Chinese hamster ovary (CHO) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3 , RIN and MDCK cell lines can be used. In one embodiment of the present invention, it was confirmed that yeast was transformed with the recombinant vector according to the present invention to improve abiotic stress resistance.

For insertion of a polynucleotide or a recombinant vector containing the same into a host cell, methods well known in the art can be used. For the transport method, for example, when the host cell is a prokaryotic cell, a calcium chloride (CaCl ₂ ) method or an electroporation method may be used, and when the host cell is a eukaryotic cell, a micro-injection method, a calcium phosphate precipitation method, and electricity Perforation, liposome-mediated transfection and gene bombardment may be used, but are not limited thereto. When using microorganisms such as E. coli , productivity is higher than that of animal cells, but due to glycosylation problems, it is not suitable for producing intact Ig type antibodies, and production of antigen-binding fragments such as Fab and Fv Can be used for

The method for selecting the transformed host cell can be easily performed according to a method well known in the art using a phenotype expressed by a selection label. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

In addition, the present invention provides a composition for promoting abiotic stress resistance comprising the dihydrin peptide or a polynucleotide encoding the same.

The stress may be abiotic stress selected from the group consisting of moisture, salt, low temperature, osmotic pressure, oxidation, reduction, alcohol and starvation.

In the present specification, the term “plant (body)” is understood to mean not only a mature plant but also a plant cell, a plant tissue, and a plant seed capable of developing into a mature plant.

Plants subject to transformation in the present invention include rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and food crops including sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion and carrot; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rapeseed; Fruit trees, including apple trees, pears, date palms, peaches, spears, grapes, tangerines, persimmons, plums, apricots and bananas; Flowers including roses, gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; And feed crops, including, but not limited to, ryegrass, red clover, orchardgrass, alpha alpha, tolpescu, and perennial lyegrass.

In one embodiment of the present invention, in the case of the yeast transformed with the polynucleotide encoding the dihydrin of the present invention, enhancement of abiotic stress compared to yeast expressing wild-type CaDHN in the presence of H ₂ O ₂ and cold stress It was confirmed that the exhibited resistance.

In addition, the present invention provides a method for enhancing abiotic stress resistance, comprising introducing a polynucleotide encoding the dihydrin peptide into a host cell. The stress may be abiotic stress selected from the group consisting of moisture, salt, low temperature, osmotic pressure, oxidation, reduction, alcohol and starvation. The introduction does not limit the method as long as it is a method capable of delivering the polynucleotide encoding the dihydrin to a host cell, and examples thereof are as described above.

In addition, the present invention is a dihydrin peptide derived from Arctic point nadonum ( Cerastium arcticum ) consisting of the amino acid sequence of SEQ ID NO: 3; Or it provides a composition for promoting high temperature stress resistance comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 encoding the peptide. The high temperature stress resistance may be due to molecular chaperone activity, and in one embodiment of the present invention, wild type CaDHN exhibits chaperone activity related to protein folding, and the chaperone activity is a unique single cysteine of wild type CaDHN. It was confirmed that dimerization through intermolecular disulfide bonds by residues plays a decisive role.

In addition, the present invention is a dihydrin peptide derived from Arctic point nadonum ( Cerastium arcticum ) consisting of the amino acid sequence of SEQ ID NO: 3; Or it provides a composition for enhancing protein homeostasis comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 encoding the peptide.

In the present specification, the term “proteostasis” results in a lack of normal proteins, or suppresses misfolding of proteins that increase toxicity by accumulating abnormal proteins, and thus despite changes in external and internal environments. It means the regulating ability to keep the physiological state constant.

The composition can be used to control overall protein quality control, such as accumulation of misfolded proteins or use in diseases associated with protein homeostasis disorders. The term “Protein Quality Control” refers to the mechanism by which organisms remove or denature proteins denatured by external stress. The protein homeostasis may be due to molecular chaperone activity, and in one embodiment of the present invention, as a result of experiments using acid-modified GFP, wild-type CaDHN not only has a protective effect against thermal denaturation, but also of denatured proteins. It was confirmed that refolding is possible.

Accordingly, the present invention establishes a defense system for adapting to extreme environments in the polar regions by controlling the dimerization of CaDHN, a unique single cysteine residue present in CaDHN, which is an intrinsically disordered protein, through which abiotic stress The biggest characteristic is that it is confirmed that it strengthens resistance to.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples. All experiments were performed independently at least 3 times, and the results were expressed as mean and standard deviation. Spotting analysis was conducted at least twice independently under the same conditions.

Example 1. Cloning and analysis of CaDHN gene

C. arcticum L. ( Caryophyllaceae ) was obtained from Brogger-Halvoya (N75 55″, E11 54″) of Svalbard Islands at Dasan Korean Arctic Research Station in Korea. Total RNA was isolated from the leaves of C. arcticum using an RNeasy Plant Mini kit (Qiagen, Hilden, Germany). The cDNA probe was synthesized using an RT-PCR (reverse transcription-PCR) premix kit (Bioneer, Daejeon, Korea). The CaDHN coding region was amplified from cDNA by PCR, and the PCR product was purified and inserted into the pET-30a(+)(Novagen, USA) vector EcoRI and XhoI restriction sites. The TEV protease recognition sequence was inserted before the CaDHN gene. All primer sequences used in the present invention are shown in Table 2 below (F: forward, R: reverse).

primer order Restriction enzyme site Sequence number pET30a/WT(F) CTGATATCGGATCC GAATTC ATGTCGAACTTACACCCAAC EcoRI 5 pET30a/WT(R) TGGTGGTGGTGGTG CTCGAG TTAGTAACCCTTGTGAGCTT XhoI 6 pGPD426/WT(F) CCCCCGGGCTGCAG GAATTC ATGTCGAACTTACACCCAAC EcoRI 7 pGPD426/WT(R) AACTAATTACATGA CTCGAG TTAGTAACCCTTGTGAGCTT XhoI 8 C143A(F) CCTACCGGTGACCAC GCC CACCAGGAGCAG - 9 C143A(R) CTGCTCCTGGTG GGC GTGGTCACCGGTAGG - 10

On the other hand, as a result of confirming the base sequence through the sequencing reaction with respect to cDNA, unlike other dihydrin proteins, the arctic point nadonamul ( Cerastium Arcticum; Cerastium) arcticum L.; It was confirmed that CaDHN, a gene encoding a dihydrin protein derived from Caryophyllaceae), has a single cysteine residue (Cys143). As shown in FIG. 1, the cysteine residue was located in the phi-segment between the second and third K-segments, and specifically, was confirmed to be the 143th residue.

Example 2. Expression and purification of CaDHN protein

For the expression and purification of CaDHN protein, the recombinant plasmid prepared in Example 1 was transformed into E. coli BL21 (DE3) strain. Cell pellets were suspended in 20 mM Tris-HCl, pH 8.0, 200 mM NaCl and 10 mM imidazole and disrupted by sonication. After centrifugation of the lysate to remove cellular debris, the supernatant was recovered and loaded using gravity to a column containing Ni-NTA affinity resin (Qiagen, Hilden, Germany), and the column was lysine-free lysis buffer. It was pre- homogenized with (preequilibrate). Then, the column was washed with a washing buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 30 mM imidazole), and the CaDHN protein bound to the Ni-NTA resin was eluted with a buffer (20 mM Tris-HCl, pH 8.0, 200). Elution with mM NaCl, 300 mM imidazole). Then, TEV protease was added and the eluted CaDHN protein was filtered using a dialysis buffer at 4°C overnight. Nickel-charged affinity chromatograpy was performed once again to remove uncleaved CaDHN and N-terminal peptide tags. The molecular weight of the CaDHN protein was predicted to be 29,509 Da (about 29.5 kDa).

Meanwhile, site-directed mutagenesis was induced in a single cysteine (Cys143) of CaDHN using PCR to obtain a C143A mutant gene in which cysteine was substituted with alanine. Then, a recombinant plasmid was prepared in the same manner as described above, and transformed into an E. coli BL21 (DE3) strain. For the mutant protein expressed in E. coli BL21 (DE3) cells, protein purification was performed as in the wild-type (WT) protein described above.

Example 3. SDS-PAGE

3-1. Confirmation of Dimer Formation of Wild-type CaDHN(WT) and Mutant C143A(M)

SDS-PAGE was performed on the wild-type CaDHN (WT) purified in Example 2. Specifically, after protein purification, the N-terminal tag of recombinant CaDHN was removed using TEV protease, and wild-type CaDHN(WT) native gel electrophoresis was performed. Electrophoresis was performed using 10% polyacrylamide gel and electrophoresis buffer (25 mM Tris base, 19 mM glycine), and the gel electrophoresis was stained with Coomassie Brilliant Blue R-250. . The results are shown in FIG. 2.

As shown in FIG. 2 (WT lane), two types of protein bands were identified as a result of SDS-PAGE (-β-ME), and the upper band disappeared after treatment with β-ME (β-mercaptoethanol), a reducing agent of about 80 kDa, On the other hand, it was confirmed that the lower band corresponding to 40 kDa did not move before and after β-ME treatment (+β-ME). Through the above results, the present inventors speculated that the cysteine residue of CaDHN formed an intermolecular disulfide bond between proteins in solution.

To specifically confirm the above speculation, SDS-PAGE was performed on mutant C143A(M) in which alanine residue was introduced instead of the cysteine purified in Example 2, and the results are similarly shown in FIG. 2. As shown in FIG. 2 (M lane), mutant C143A showed only a single protein band in the non-reducing SDS-PAGE gel, and it was confirmed that no protein band of 80 kDa was found (-β-ME). Through the above results, it was confirmed that the sub-type CaDHN coexists in two forms, a monomer and a dimer having a disulfide bond between the two monomers, as it has only one cysteine residue.

3-2. Wild type CaDHN (WT) and C143A(M) Dimer Determine the effect of metal ions on formation

For Citrus dihydrin protein, the interaction of histidine residues and metal ions has been reported (Hara M et al, J Exp Bot. 2005;56(420):2695-703), OpsDHN1 from Opuntia streptacantha Lem ( In the case of SK ₃ type) dihydrin protein, dimerization has been reported. Among them, in the case of OpsDHN1, a histidine-rich motif located in an unconserved segment between the first and second K-segments has been reported to play an important role in dimer formation (Hernandez-Sanchez IE et. al, Front Plant Sci. 2014, 5:520; Rahman LN et al, Biochim Biophys Acta. 2013, 1828(3):967-80). Thus, to investigate the effect of metal ions on the formation of dimers of wild-type CaDHN(WT) and C143A(M), SDS-PAGE was performed in the same manner as in Example 3-1 to confirm the presence of intermolecular disulfide bonds. To this end, different concentrations of dithiothreitol (DTT) and copper ions (CuSO ₄ ) were added to both proteins (DTT is a reducing agent that prevents the formation of disulfide bonds between cysteine residues, and copper oxide ions are between two cysteine residues). It was used for the purpose of forming disulfide bonds). The results are shown in FIG. 3.

As shown in Figure 3, it was confirmed that the wild-type CaDHN (WT) and C143A (M) appear as protein bands of different sizes. Specifically, in the case of WT, as the DTT concentration increased, the upper band decreased in a concentration-dependent manner, and it was confirmed that most of the proteins migrated to the lower band above 8 mM (FIG. 3B). On the other hand, when the concentration of copper ions increased, some of the lower bands moved to the upper bands, and it was confirmed that copper ions can induce disulfide bond formation of wild-type CaDHN (A in FIG. 3, left panel).

On the other hand, in the case of C143A without a cysteine residue, despite the presence of copper ions, the lower band did not move, and through this, CaDHN forms a dimer according to disulfide bonds, and between protein molecules through amino acid residues such as histidine It was confirmed that the dimer formation mediated by copper ions was not performed (A in FIG. 3, right panel). In addition, it was confirmed that C143A has no band shift due to DTT. Through the above results, it was confirmed that the wild type CaDHN exists in a dimer form by an intermolecular disulfide bond mediated by a unique single cysteine residue.

Example 4. Analysis of the chaperone activity of CaDHN

To investigate the effect of dimerization of CaDHN on the chaperone activity of CaDHN, acid-denatured GFP was used as a model substrate, and wild-type CaDHN(WT) and C143A chaperone activity Was measured. Specifically, about 100 μM of green fluorescent protein (GFP) was denatured in 120 mM HCl for 60 minutes. Spontaneous refolding was initiated by 100-fold dilution with regeneration buffer (20 mM Tris-Hcl, pH 8.0, 200 mM NaCl). GFP refolding was then initiated by adding 3 μM of wild-type CaDHN (WT) or C143A protein to the denatured GFP in regeneration buffer. GFP refolding was monitored for 20 minutes (400 nm excitation, 510 nm emissioin). All experiments were performed at 25°C. The results are shown in FIG. 4.

As shown in FIG. 4, it was confirmed that refolding of the acid-modified GFP increased by about 40% in the presence of wild-type CaDHN(WT) in a regeneration buffer. On the other hand, in the presence of C143A, there was no change in the refolding activity of the acid-modified GFP, and further, when the regeneration buffer containing CaDHN(WT) and DTT was simultaneously processed, there was no change in the refolding activity of CaDHN. Did. On the other hand, in the case of the regeneration buffer containing CuSO ₄ , it was impossible to measure the chaperone activity of CaDHN due to the reduced fluorescence of GFP. Through the above results, it was confirmed that only the dimer form of wild-type CaDHN exhibits chaperone activity to refold the denatured protein.

As can be seen from the above results, the present inventors confirmed that wild-type CaDHN not only has a protective effect against thermal denaturation, but also enables refolding of the denatured protein. That is, it was confirmed that the wild-type CaDHN exhibits true chaperone activity with respect to the protein folding process, and the dimerization through intermolecular disulfide bonds plays a decisive role in the chaperone activity of CaDHN.

Example 5. Analysis of CaDHN metal ion binding capacity

It is known that some dihydrin proteins exhibit the ability to bind metal ions with scavenging activity against cytotoxic hydroxyl radicals caused by metal catalytic reactions (Hara M et al, J Exp Bot. 2005;56( 420):2695-703). Thus, in order to investigate the metal binding capacity of CaDHN(WT) and mutant C143A and thus ROS scavenging activity, metal ion affinity chromatography was performed. After filling the column with 100mM of each metal ion (Fe ^{3 +} , Co ^{2 +} , Ni ^{2 +} , Cu ²⁺ , Zn ^{2 +} , Mg ^{2 +} , Ca ^{2 +} and Mn ^{2 +} ), about 200 μg of CaDHN (WT ) Or mutant C143A was loaded onto a column equilibrated with an equilibration buffer (20 mM Tris-HCl, pH 8.0 and 200 mM NaCl; equilibration buffer, EQ) to analyze metal-binding activity (MBA). After washing the column sufficiently with an equilibration buffer to remove unbound proteins, the bound proteins were eluted with 100 mM EDTA. The eluted protein was collected, SDS-PAGE was performed, and the electrophoretic gel was stained with Coomassie Brilliant Blue R-250. The results are shown in FIG. 5.

As it is shown in A of Fig 5, and C143A CaDHN all ^{Fe 3 +, Co 2 +,} Ni 2 +, Cu 2 + , and Zn ²⁺ ion has been completely bonded to, Mg ^{+ 2,} Ca ^{+ 2} and Mn ^{+ 2} is It was confirmed that it did not bind. Also,, it was confirmed that no change in size of the band from all seven metal ions other than the Cu ^{+ 2} ion, as shown in Fig. 5 B. Through the above results, CaDHN confirmed that binding is not related to the dimerization of CaDHN for Cu ^{+ 2} such as this has a binding activity to Cu ^{2 +,} but.

Example 6. Preparation of CaDHN gene expression transformed yeast

The amplified CaDHN gene using PCR contains GPD1 (glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase) promoter (Euroscarf, Germany) and CYC (cytochrome-c-oxidase) terminator. Yeast expression vector was constructed by linking to the p426GPD vector. The cloned plasmid was amplified by plasmid DNA using E.coli DH5α, and then sequenced using a CYC terminator primer. The resulting plasmid was named p426GPD::CaDHN_WT.

Then, p426GPD::CaDHN_WT and PCR-dependent site-directed mutagenesis kit (New England Biolabs) were used to induce the mutation of CaDHN_C143A and insert the C143A mutant gene into the vector to express the yeast expression vector. p426GPD::CaDHN_C143A was produced. Each plasmid DNA was isolated using a plasmid separation kit (Nucleogen, Siheung, Korea), and the plasmid was transformed into S. cerevisiae BY4741 strain (Euroscarf). Meanwhile, the strain information used in the present invention is shown in Table 3 below.

Strain Genotype Source Genbank
accession number E. coli BL21 fhuA2 [lon] ompT gal (λ DE3) [ dcm ] ΔhsdS
λ DE3 = λ sBamHIo ΔEcoRI -B int::(lacI::PlacUV5::T7 gene1 ) i21
Δnin5 NEB CP001509.3 S. cerevisiae BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 Euroscarf JRIS00000000

Specifically, S. cerevisiae BY4741 cells were incubated in YPD broth (1% yeast extract, 2% peptone and 2% dextrose) overnight at 28° C., followed by inoculation into fresh YPD broth and culture at ₆₀₀ nm (A ₆₀₀ ). Incubated at 28° C. for 4 hours with stirring (180 rpm) until the light density reached about 1.0 (1×10 ⁷ cells mL ^-1 ). The cells were then transformed with the target plasmids p426GPD::CaDHN_WT and p426GPD::CaDHN_C143A plasmids according to the PEG/LiCl method, respectively. The transformed cells are free of amino acids and contain 0.67% Yeast Nitrogen Base containing ammonium sulfate, 0.192% Yeast Nitrogen Base without uracil (yeast synthetic dropout medium supplement; Sigma, St. Louis) , USA), 2% glucose and 2% agar was placed in a yeast synthetic dropout agar medium. Samples were incubated at 28°C for 3 days. Positive colonies were grown in uracil-free medium and used in subsequent experiments.

Example 7. Oxidation and low temperature stress tolerance experiment

In order to confirm the mutations CaDHN protein, that is, whether an expression of C143A improve transgenic resistance in yeast cells to oxidative and low temperature stress, medium-10 logs group (A ₆₀₀ = 2.5) yeast cells with fresh YPD broth ^{- It was} diluted ⁹ times. Then, after loading on a YPD agar plate containing hydrogen peroxide, they were incubated at 40°C (Heat shock) for 6 days, and at 15°C (Chilling) for 10 days. The results are shown in FIGS. 6 and 7.

As shown in FIG. 6, when the transformed yeast cells were exposed to hydrogen peroxide (H ₂ O ₂ ) for a long time and a short time, both transformed yeast cells (WT, C143A) were more resistant than the empty vector-only cells (EV). In particular, it was confirmed that the resistance to oxidative stress was significantly improved in the case of C143A compared to WT.

In addition, as shown in FIG. 7, the resistance to high temperature stress was higher in CaDHN(WT) than C143A, and the resistance to low temperature stress was higher in C143A than CaDHN(WT).

Meanwhile, in order to test low temperature stress resistance under various cooling conditions, medium-log phase (A ₆₀₀ =2.5) transformed yeast cells were diluted 10 ^-9 times with fresh YPD broth, and then loaded onto a YPD agar plate containing hydrogen peroxide. After treatment, each was treated under the following conditions: iv) incubated at 15°C (Chilling) for 11 days, ii) incubated at 15°C (Chilling) for 10 days, iv) incubated at 4°C for 7 days, and then incubated at 28°C for 3 days. . The results are shown in FIG. 8.

As shown in FIG. 8, as a result of experiments under various cooling conditions, it was confirmed that the resistance to low temperature stress of C143A under all treatment conditions was significantly improved over that of CaDHN(WT).

Example 8. Metal ion homeostasis experiment

Transduced yeast cells grown in YPD broth to mid-log phase (A ₆₀₀ =2.5) with stirring (180 rpm) for 1 hour, 20 and 25 mM hydrogen peroxide (H ₂ O ₂ ), 100 mM zinc chloride (ZnCl ₂ ), 20mM copper sulfate (CuSO ₄ ), 80mM cobalt chloride (CoCl ₂ ), 80mM nickel sulfate (CoSO ₄ ), 80mM iron chloride (FeCl ₂ ), 80mM manganese chloride, 250mM calcium chloride (CaCl ₂ ) and 250mM Magnesium chloride (MgCl ₂ ). It was then diluted 10 ^-4 fold steps with fresh YPD broth. Then, 5 μl of the diluted solution was loaded onto a YPD agar (2% agar+YPD) plate, followed by incubation at 28° C. for 3 days, and then photographed. On the other hand, in order to monitor the low temperature stress, the same medium-log phase (A ₆₀₀ =2.5) transformed yeast cells were diluted 10 ^-9 fold step with fresh YPD broth, loaded into YPD agar plates not containing hydrogen peroxide, respectively. Incubation was performed at 40°C for 6 days, and at 15°C for 10 days. The results are shown in FIG. 9.

As shown in FIG. 9, the resistance to stress induced by various metal ions, including Co ^{2 +} , Ni ^{2 +} , Fe ^{3 +} and Zn ^{2 +} ions, is that CaDHN(WT) expressing cells are resistant to metal ions of EV cells. It was excellent compared to. On the other hand, when treated with Mg ^{2 +} , Ca ^{2 +} and Mn ^{2 +} , the metal ion resistance of CaDHN(WT) expressing cells and EV cells is similar, and these results are consistent with those of the in vitro experiment of Example 5 above. Was confirmed. Specifically, under Cu ^{2 +} stress, C143A cells showed more stress tolerance than CaDHN(WT) cells. Through the above results, the metal ion homeostasis by CaDHN was identified and is once again that regardless of the dimerization of CaDHN except for Cu ^{+ 2.}

The present invention induces a mutation in CaDHN, a type of dihydrin protein known as an intrinsic atypical protein, and confirms that when the mutant CaDHN (C143A) is expressed in yeast, resistance to abiotic stress of yeast is significantly improved. It has the greatest significance.

Specifically, the present inventors found that CaDHN has a unique single cysteine residue that plays an important role in dimerization by intermolecular disulfide bonds, and that dimerization of CaDHN is independent of metal ions unlike other dihydrin proteins. It was first identified. Furthermore, the present inventors pay attention to such a single cysteine residue of CaDHN to induce a mutation in which cysteine is substituted with another amino acid (alanine), and perform experiments such as SDS-PAGE, oxidation and cold stress resistance, and metal ion homeostasis. , It was confirmed that the structural resistance of CaDHN can significantly improve the resistance of an organism to abiotic stress. At the same time, in the case of wild-type CaDHN, it was found that CaDHN exists as a dimer by a disulfide bond mediated by a unique single cysteine residue of the CaDHN protein, and that it has excellent high temperature stress resistance and protein homeostasis compared to the mutant C143A.

Accordingly, the present invention significantly increases resistance to abiotic stress by controlling dimerization of CaDHN, an intrinsically disordered protein, to develop environmental stress-resistant crops, microorganisms and plants using molecular biology and genetic manipulation techniques It can be widely used in the field of plant biotechnology, including basic research on the improvement of traits.

<110> Korea Institute of Ocean Science and Technology <120> Mutated dehydrin from the Arctic mouse-ear chickweed Cerastium arcticum and use thereof <130> KOPRI1-2 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> Mutated dehydrin C143A from Cerastium arcticum <400> 1 Met Ser Asn Leu His Pro Thr Gly Gly Glu Thr Glu Thr Thr Asn Thr 1 5 10 15 Asp Arg Gly Leu Phe Gly Phe Gly Lys Lys Thr Glu Thr Pro Val Ala 20 25 30 Thr Pro Thr Glu Pro Pro His Lys Pro Thr Leu Thr Glu Lys Leu His 35 40 45 Arg Ser Gly Ser Ser Ser Ser Ser Ser Ser Asp Glu Glu Val Ile Glu 50 55 60 Asn Gly Ala Lys Ile Arg Lys Pro Arg Arg Lys Gly Ile Ala Gly Lys 65 70 75 80 Ile Lys Asp Lys Leu Pro Gly Gly His Lys Asp Glu Tyr Glu Thr Ala 85 90 95 Thr Pro Ile Ala Ala Gly Glu Tyr Tyr His Gln Glu Pro Asn Lys His 100 105 110 Ala Ser Asp Arg Gly Val Ala Glu Lys Ile Lys Glu Lys Leu Pro Gly 115 120 125 Gly His Lys Asp Glu Tyr Gly Thr Ala Pro Thr Gly Asp His Ala His 130 135 140 Gln Glu Gln Asn Lys His Ala Ser Glu Met Gly Ala Ala Glu Lys Ile 145 150 155 160 Lys Asp Lys Leu Pro Gly Gly Gln Lys Asp Glu Tyr Gly Thr Ala Pro 165 170 175 Thr Gly Asp His Tyr His Arg Asp Gln Asn Lys His Thr Ala Ala Gly 180 185 190 Asn Thr His Tyr Pro Glu Gly Asn Lys Gly Phe Val Gly Asn Val Lys 195 200 205 Asp Lys Leu Pro Gly Asn Gln Thr Asp Val Asn His His Gln Gln Gln 210 215 220 Gln Gln His Pro Ala Val His Val Ser Gln Gln Ala Val Gln Glu Pro 225 230 235 240 His His Glu Lys Lys Gly Leu Leu Asp Asn Ile Lys Asp Lys Met Pro 245 250 255 Gly Gly His Lys Asp Asp Ala Asp Lys Ala His Lys Gly Tyr 260 265 270 <210> 2 <211> 813 <212> DNA <213> Artificial Sequence <220> <223> the sequence encoding mutated dehydrin C143A from Cerastium arcticum <400> 2 atgtcgaact tacacccaac cggaggcgaa accgagacca ctaatactga tagaggcctg 60 tttggcttcg gtaagaagac ggaaaccccc gtcgccaccc cgactgagcc accacacaag 120 cctactctca cggagaagct tcaccgctcg ggcagtagta gctctagctc gtcagatgag 180 gaggtaatag aaaatggtgc gaagattagg aagccgagga ggaagggaat agcagggaaa 240 atcaaggaca agcttccggg tggtcacaag gatgaatacg agactgcgac tcctattgct 300 gcgggtgagt actatcacca ggagccgaac aagcacgctt cagacagggg agtagcggag 360 aagatcaagg aaaagttacc aggcggtcac aaggatgaat acgggacagc gcctaccggt 420 gaccacgccc accaggagca gaacaagcac gcttctgaga tgggtgcggc ggagaaaatc 480 aaggacaagt tgccaggcgg acagaaggat gaatacggga ctgcgcctac tggcgaccac 540 taccaccggg accagaacaa gcatactgcg gcaggaaaca cgcattaccc cgagggaaac 600 aaggggtttg ttggcaatgt taaggataaa ttgccgggaa atcagacaga cgtgaaccat 660 caccagcagc agcaacagca cccggctgtt catgtttcac aacaggcggt ccaggagccg 720 catcatgaga agaaagggct cttagacaac attaaggaca agatgcccgg cggccacaag 780 gatgatgctg ataaagctca caagggttac taa 813 <210> 3 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> Dehydrin CaDHN from Cerastium arcticum <400> 3 Met Ser Asn Leu His Pro Thr Gly Gly Glu Thr Glu Thr Thr Asn Thr 1 5 10 15 Asp Arg Gly Leu Phe Gly Phe Gly Lys Lys Thr Glu Thr Pro Val Ala 20 25 30 Thr Pro Thr Glu Pro Pro His Lys Pro Thr Leu Thr Glu Lys Leu His 35 40 45 Arg Ser Gly Ser Ser Ser Ser Ser Ser Ser Asp Glu Glu Val Ile Glu 50 55 60 Asn Gly Ala Lys Ile Arg Lys Pro Arg Arg Lys Gly Ile Ala Gly Lys 65 70 75 80 Ile Lys Asp Lys Leu Pro Gly Gly His Lys Asp Glu Tyr Glu Thr Ala 85 90 95 Thr Pro Ile Ala Ala Gly Glu Tyr Tyr His Gln Glu Pro Asn Lys His 100 105 110 Ala Ser Asp Arg Gly Val Ala Glu Lys Ile Lys Glu Lys Leu Pro Gly 115 120 125 Gly His Lys Asp Glu Tyr Gly Thr Ala Pro Thr Gly Asp His Cys His 130 135 140 Gln Glu Gln Asn Lys His Ala Ser Glu Met Gly Ala Ala Glu Lys Ile 145 150 155 160 Lys Asp Lys Leu Pro Gly Gly Gln Lys Asp Glu Tyr Gly Thr Ala Pro 165 170 175 Thr Gly Asp His Tyr His Arg Asp Gln Asn Lys His Thr Ala Ala Gly 180 185 190 Asn Thr His Tyr Pro Glu Gly Asn Lys Gly Phe Val Gly Asn Val Lys 195 200 205 Asp Lys Leu Pro Gly Asn Gln Thr Asp Val Asn His His Gln Gln Gln 210 215 220 Gln Gln His Pro Ala Val His Val Ser Gln Gln Ala Val Gln Glu Pro 225 230 235 240 His His Glu Lys Lys Gly Leu Leu Asp Asn Ile Lys Asp Lys Met Pro 245 250 255 Gly Gly His Lys Asp Asp Ala Asp Lys Ala His Lys Gly Tyr 260 265 270 <210> 4 <211> 813 <212> DNA <213> Artificial Sequence <220> <223> the sequence encoding dehydrin CaDHN from Cerastium arcticum <400> 4 atgtcgaact tacacccaac cggaggcgaa accgagacca ctaatactga tagaggcctg 60 tttggcttcg gtaagaagac ggaaaccccc gtcgccaccc cgactgagcc accacacaag 120 cctactctca cggagaagct tcaccgctcg ggcagtagta gctctagctc gtcagatgag 180 gaggtaatag aaaatggtgc gaagattagg aagccgagga ggaagggaat agcagggaaa 240 atcaaggaca agcttccggg tggtcacaag gatgaatacg agactgcgac tcctattgct 300 gcgggtgagt actatcacca ggagccgaac aagcacgctt cagacagggg agtagcggag 360 aagatcaagg aaaagttacc aggcggtcac aaggatgaat acgggacagc gcctaccggt 420 gaccactgcc accaggagca gaacaagcac gcttctgaga tgggtgcggc ggagaaaatc 480 aaggacaagt tgccaggcgg acagaaggat gaatacggga ctgcgcctac tggcgaccac 540 taccaccggg accagaacaa gcatactgcg gcaggaaaca cgcattaccc cgagggaaac 600 aaggggtttg ttggcaatgt taaggataaa ttgccgggaa atcagacaga cgtgaaccat 660 caccagcagc agcaacagca cccggctgtt catgtttcac aacaggcggt ccaggagccg 720 catcatgaga agaaagggct cttagacaac attaaggaca agatgcccgg cggccacaag 780 gatgatgctg ataaagctca caagggttac taa 813 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pET30a/WT(F) <400> 5 ctgatatcgg atccgaattc atgtcgaact tacacccaac 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pET30a/WT(R) <400> 6 tggtggtggt ggtgctcgag ttagtaaccc ttgtgagctt 40 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pGPD426/WT(F) <400> 7 cccccgggct gcaggaattc atgtcgaact tacacccaac 40 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> pGPD426/WT(R) <400> 8 aactaattac atgactcgag ttagtaaccc ttgtgagctt 40 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> C143A(F) <400> 9 cctaccggtg accacgccca ccaggagcag 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> C143A(R) <400> 10 ctgctcctgg tgggcgtggt caccggtagg 30

Claims (13)

A mutant dihydrin peptide derived from Cerastium arcticum consisting of the amino acid sequence of SEQ ID NO: 1.
A polynucleotide encoding the peptide of claim 1.
According to claim 2, The polynucleotide is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2, polynucleotide.
Recombinant vector comprising the polynucleotide of claim 2.
A host cell having enhanced abiotic stress resistance transformed with the recombinant vector of claim 4.
The host cell of claim 5, wherein the host cell is selected from the group consisting of animal cells, plant cells, fungi, bacteria, and viruses.
The host cell of claim 6, wherein the fungus is yeast.
A composition for promoting abiotic stress resistance, comprising the dihydrin peptide of claim 1 or the polynucleotide of claim 2.
The composition of claim 8, wherein the stress is abiotic stress selected from the group consisting of moisture, salt, low temperature, osmotic pressure, oxidation, reduction, alcohol, and starvation. .
A method for promoting abiotic stress resistance, comprising introducing the polynucleotide of claim 2 into a host cell.
A dihydrin peptide derived from Arcastum arcticum consisting of the amino acid sequence of SEQ ID NO: 3; Or as a composition for promoting high temperature stress resistance comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 encoding the peptide,
The high temperature stress resistance is enhanced by molecular chaperone activity;
Characterized in that it enhances resistance to high-temperature stress than a mutant dihydrin derived from the arctic point nadonum ( Cerastium arcticum ) consisting of the nucleotide sequence of SEQ ID NO: 2 as a control,
A composition for enhancing high temperature stress resistance.
A dihydrin peptide derived from Cerastium arcticum consisting of the amino acid sequence of SEQ ID NO: 3; Or a composition for enhancing protein homeostasis comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 encoding the peptide.
13. The composition of claim 12, wherein the protein homeostasis is due to molecular chaperone activity.

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