JP5019505B2 - Transgenic plants with tolerance to low temperature stress - Google Patents

Description

  For plants, environmental stress such as low temperature, drying, and salt greatly affects growth. This is directly related to crop production in agriculture, and research and development aimed at imparting environmental stress tolerance to crops is underway. Conventionally, stress tolerance enhancement by breeding hybridization methods has been promoted, but genetic resources that can be introduced by hybridization are limited, and it is difficult to significantly improve tolerance. Therefore, new approaches such as overexpression of endogenous or other species-derived genes are required by techniques such as gene recombination.

  In recent years, new methods such as gene recombination have been developed, and it has become possible to introduce various genes into plants. In fact, plants introduced with an enzyme gene that synthesizes proline, an amino acid, have been reported to be resistant to drought and salt stress because of the ability to regulate osmotic pressure by proline. Reference 1). In addition, it has been reported that by introducing a transcription factor gene that controls the stress response, the stress response can be activated and resistance can be imparted to drought, salt, and low-temperature stress (Non-patent Document 2).

  However, for example, when a transcription factor is overexpressed, all the genes induced by the transcription factor are overexpressed, resulting in dwarfing of the plant in addition to acquiring stress tolerance. In addition, an example in which an adverse effect on growth becomes apparent has been reported (Non-patent Document 3). Searches for new resistance-conferring genes that do not have an adverse effect due to overexpression, and improved transgene expression methods are required.

Kishor P, Hong Z, Miao GH, Hu C, Verma D. Plant Physiol. 1995 Aug; 108 (4): 1387-1394. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Plant Cell. 1998 Aug; 10 (8): 1391-406 .; Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF. Science. 1998 Apr 3; 280 (5360): 104-6. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Plant Cell. 1998 Aug; 10 (8): 1391-406.

  Therefore, the present invention finds an excellent gene that has little influence on the growth of a plant and can impart low-temperature stress tolerance to the plant, and imparts low-temperature stress tolerance to a novel transgenic plant having low temperature stress tolerance and a plant. It aims to provide a method.

  As a result of intensive studies by the present inventors in order to achieve the above-described object, the inventors have found that a gene called AtCSP3 gene can be overexpressed in Arabidopsis thaliana, thereby imparting low-temperature stress tolerance to plants. It came to be completed. In addition, the AtCSP3 gene in Arabidopsis thaliana is known to show low-temperature responsive expression (Karlson, D. and Imai, R. Plant Physiol. 131, 12-15, 2003), but RNA with a low-temperature shock domain The finding that it encodes a binding protein and can impart low temperature stress tolerance to a plant body by overexpression is a novel discovery.

The present invention includes the following.
(1) A transgenic plant modified so that a gene corresponding to the AtCSP3 gene in Arabidopsis thaliana is overexpressed.
(2) The transgenic plant according to (1), wherein the gene encodes an RNA-binding protein having a cold shock domain.
(3) The transgenic plant according to (1), wherein the gene is at least one of the following genes (a) to (d):
(a) a gene consisting of DNA consisting of the base sequence shown in SEQ ID NO: 1
(b) a gene comprising a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 under a stringent condition and encodes an RNA-binding protein having a low-temperature shock domain
(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
(d) a gene encoding an RNA-binding protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a cold shock domain (4) The transgenic plant according to (1), which is linked to an overexpression promoter.
(5) A method for imparting low-temperature stress tolerance to a plant, which is modified so that a gene corresponding to the AtCSP3 gene in Arabidopsis thaliana is overexpressed.
(6) The method according to (5), wherein the gene encodes an RNA-binding protein having a cold shock domain.
(7) The method according to (5), wherein the gene is at least one of the following genes (a) to (d):
(a) a gene consisting of DNA consisting of the base sequence shown in SEQ ID NO: 1
(b) a gene comprising a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 under a stringent condition and encodes an RNA-binding protein having a low-temperature shock domain
(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
(d) a gene encoding an RNA-binding protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a cold shock domain (8) The method according to (5), wherein is linked to an overexpression promoter.

  Since the transgenic plant according to the present invention overexpresses a gene corresponding to the AtCSP3 gene in Arabidopsis thaliana, it has resistance to low temperature stress. In addition, according to the method for imparting low temperature stress tolerance to plants according to the present invention, by overexpressing a gene corresponding to the AtCSP3 gene in Arabidopsis thaliana, excellent low temperature stress tolerance is imparted without the plant becoming dwarfed. be able to.

Hereinafter, the present invention will be described in detail with reference to the drawings.
The transgenic plant according to the present invention is modified so that a gene corresponding to the AtCSP3 gene in Arabidopsis thaliana is overexpressed. In the present invention, the AtCSP3 gene in Arabidopsis thaliana encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (hereinafter referred to as AtCSP3). In the process of elucidating the function of a cold shock domain RNA-binding protein in acquiring low-temperature tolerance of plants (see Examples below), Arabidopsis lacking the AtCSP3 gene shows a phenotype that significantly reduces freezing tolerance. This finding indicates that the product of AtCSP3 gene is essential for obtaining freezing tolerance in Arabidopsis thaliana.

  Therefore, not only in Arabidopsis but also in various plants, modification to overexpress the gene corresponding to the AtCSP3 gene in Arabidopsis can improve low temperature stress tolerance.

Gene to be overexpressed in transgenic plant In the transgenic plant according to the present invention, the gene to be overexpressed is a gene corresponding to the AtCSP3 gene in Arabidopsis thaliana or the AtCSP3 gene in plants other than Arabidopsis thaliana.

  The AtCSP3 gene encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. An example of the AtCSP3 gene is a polynucleotide having the base sequence shown in SEQ ID NO: 1.

Cloning of the AtCSP3 gene can be performed, for example, as follows.
(1) Preparation of Arabidopsis mRNA and cDNA libraries
Examples of mRNA supply sources include parts of plants such as Arabidopsis leaves, stems, roots, and flowers, or whole plants. In addition, plants obtained by seeding Arabidopsis seeds on a solid medium such as GM medium, MS medium or # 3 medium and growing under aseptic conditions can also be used. Since the mRNA level of the gene used in the present invention increases in Arabidopsis plants by exposure to low-temperature stress (for example, -10 to 10 ° C), Arabidopsis plants exposed to these stresses may be used. .

  For preparation of mRNA, for example, Arabidopsis plants grown in GM medium are exposed to low temperature stress and then frozen in liquid nitrogen. Thereafter, it can be performed by a commonly performed technique. For example, after the frozen plant body is ground in a mortar or the like, crude RNA is obtained from the obtained ground product by the glyoxal method, guanidine thiocinate-cesium chloride method, lithium chloride-urea method, proteinase K-deoxyribonuclease method, etc. Extract and prepare the fraction. For RNA extraction, a commercially available kit (Total RNA Extraction Kit; manufactured by Amersham) may be used.

  Using the crude RNA fraction thus obtained as a template, a single-stranded cDNA was synthesized using an oligo dT primer and reverse transcriptase, and then the genome information of Arabidopsis thaliana was used using the single-stranded cDNA as a template. The AtCSP3 gene can be amplified by PCR using a pair of primers designed in the above. The pair of primers can be designed, for example, so as to be able to amplify from the 5 'UTR to the stop codon of the nucleotide sequence of AtCSP3 gene (Genebank accession number AY035133).

  However, even in Arabidopsis thaliana, there may be some differences in amino acid sequence or base sequence depending on the variety. Even in the same plant variety, the amino acid sequence or base sequence may change due to mutation or the like. Therefore, the AtCSP3 gene in Arabidopsis thaliana is a gene encoding an RNA binding protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a cold shock domain. Is also included.

  Here, the number of amino acids that may be deleted, substituted or added is preferably 1 to several. For example, 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence shown in SEQ ID NO: 2 may be deleted, and 1 to 10, preferably 1 to 5 amino acid sequences shown in SEQ ID NO: 2. May be added, or 1 to 10, preferably 1 to 5 amino acids in the amino acid sequence shown in SEQ ID NO: 2 may be substituted with other amino acids.

  In addition, amino acid deletion, addition, and substitution can also be performed by artificially modifying the gene encoding the protein by a technique known in the art. Mutation can be introduced into a gene by a known method such as Kunkel method or Gapped duplex method or a method equivalent thereto, for example, a mutation introduction kit (for example, Mutant-K using site-directed mutagenesis). (TAKARA) or Mutant-G (TAKARA)) or the like, or TAKARA's LA PCR in vitro Mutagenesis series kit can be used to introduce mutations.

  In addition, the AtCSP3 gene includes a gene that has a homology of 70% or more with the amino acid sequence shown in SEQ ID NO: 2 and encodes an RNA-binding protein having a cold shock domain. The 70% or higher homology is preferably 80% or higher, more preferably 90% or higher, and most preferably 95% or higher. Here, the homology means a numerical value obtained by analyzing general homology analysis software (BLAST, FASTA, CLUSTAL W, etc.) with default settings.

  Furthermore, the AtCSP3 gene in Arabidopsis thaliana includes a gene consisting of a DNA that hybridizes with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 under a stringent condition and encodes an RNA-binding protein having a low-temperature shock domain.

  Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a nucleic acid having high homology, that is, a DNA comprising a nucleotide sequence having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the nucleotide sequence represented by SEQ ID NO: 1. In which the complementary strands of the nucleic acids hybridize and the complementary strands of nucleic acids having lower homology do not hybridize. More specifically, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.

  On the other hand, in plants other than Arabidopsis thaliana, in order to identify a gene corresponding to the AtCSP3 gene, if the genome analysis of the plant is progressing, the base sequence of the AtCSP3 gene (SEQ ID NO: 1) or by searching for a highly homologous gene using the AtCSP3 amino acid sequence (SEQ ID NO: 2) as a key. If genomic information of the plant cannot be obtained, a cDNA library of the plant is constructed, and the AtCSP3 gene is probed using a probe consisting of part or all of the base sequence of the AtCSP3 gene (SEQ ID NO: 1). The corresponding gene can be identified. Once a gene corresponding to the AtCSP3 gene is identified, the gene can be isolated according to a conventional method.

  Whether or not the gene corresponding to the AtCSP3 gene encodes an RNA-binding protein in the same manner as the AtCSP3 gene may be determined by expressing an expression vector incorporating the gene in an appropriate host and examining the same function in the expressed protein. In addition, whether the gene corresponding to the AtCSP3 gene encodes a protein having a low-temperature shock domain as in the AtCSP3 gene is determined by determining the amino acid sequence deduced from the base sequence of the gene of interest, What is necessary is just to investigate whether it has an amino acid sequence characteristic of a shock domain.

  Whether or not it functions as an RNA-binding protein can be examined using so-called gel shift analysis. Specifically, the target protein is added to a solution containing nucleic acid, and then electrophoresis is performed using an agarose gel with an appropriate concentration. When a protein interacts with a nucleic acid molecule, the migration degree of the nucleic acid changes. On the other hand, when the protein does not interact with the nucleic acid molecule, the mobility is constant regardless of whether or not the protein is added. In this case, a plurality of samples in which the protein concentration is appropriately changed may be used, or a known protein that is known not to interact with a nucleic acid can be used as a control. Here, by using single-stranded DNA, double-stranded DNA or mRNA as the nucleic acid molecule, the type of nucleic acid molecule to which the target protein binds can be examined. That is, by performing gel shift analysis using mRNA as a nucleic acid molecule, the RNA binding property of the target protein can be examined.

  In addition, in FIG. 1, the result of the gel shift analysis using the protein which Arabidopsis thaliana AtCSP3 gene codes is shown. In this example, M13ssDNA, M13dsDNA, and luciferase mRNA are used as nucleic acid molecules. These results demonstrated that the protein encoded by the AtCSP3 gene can bind to ssDNA, dsDNA and mRNA.

  Moreover, a cold shock domain can be defined as a domain having an amino acid sequence corresponding to positions 10 to 77 in the amino acid sequence shown in SEQ ID NO: 2, for example. A domain having 50% or more, preferably 60% or more, more preferably 70% or more homology to the 10th to 77th amino acid sequence in SEQ ID NO: 2 can be defined as a cold shock domain.

Production of recombinant vectors and transformed plants
(1) Preparation of Recombinant Vector A recombinant vector can be constructed by introducing the above-described AtCSP3 gene or a gene corresponding to the AtCSP3 gene (hereinafter also referred to as “target gene”) into an appropriate vector. Here, as the vector, pBI, pPZP, and pSMA vectors that can introduce a target gene into a plant via Agrobacterium are preferably used. In particular, pBI binary vectors or intermediate vector systems are preferably used, and examples thereof include pBI121, pBI101, pBI101.2, pBI101.3 and the like. A binary vector is a shuttle vector that can replicate in Escherichia coli and Agrobacterium. When a plant is infected with Agrobacterium holding the binary vector, it is a border sequence consisting of LB and RB sequences on the vector. It is a vector that can incorporate the enclosed DNA into plant nuclear DNA (EMBO Journal, 10 (3), 697-704 (1991)). On the other hand, a pUC vector can directly introduce a target gene into a plant, and examples thereof include pUC18, pUC19, and pUC9. Plant virus vectors such as cauliflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV), and tobacco mosaic virus (TMV) can also be used.

  When a binary vector plasmid is used, the target gene is inserted between the boundary sequences (LB, RB) of the above binary vector, and this recombinant vector is amplified in E. coli. Next, the amplified recombinant vector is introduced into Agrobacterium tumefaciens C58, LBA4404, EHA101, EHA105, etc. by electroporation or the like, and the Agrobacterium is used for transduction of plants.

  In order to insert a target gene into a vector, first, a method in which purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site of a suitable vector DNA or a multicloning site, and ligated to the vector is employed. The In addition, the target gene needs to be incorporated into a vector so as to be overexpressed in the plant to be introduced. Therefore, a promoter, an enhancer, a terminator, a poly A addition signal, a 5′-UTR sequence, a selection marker gene, and the like can be linked to the vector upstream, inside, or downstream of the above gene.

  As the “promoter”, a promoter having a function of controlling expression of a downstream gene in a plant cell is used. For example, the promoter may be one that induces expression in a specific plant tissue or in a particular developmental stage, or that constantly induces expression in all tissues of a plant and in all developmental stages. Also good. The promoter may be derived from a plant or may not be derived from a plant. Specific examples include a cauliflower mosaic virus (CaMV) 35S promoter, a nopaline synthase gene promoter (Pnos), a corn-derived ubiquitin promoter, a rice-derived actin promoter, a tobacco-derived PR protein promoter, and the like.

  Examples of the enhancer include an enhancer region that is used to increase the expression efficiency of the target gene and includes an upstream sequence in the CaMV35S promoter. The terminator may be any sequence that can terminate the transcription of a gene transcribed by a promoter. For example, a terminator for a nopaline synthase (NOS) gene, a terminator for an octopine synthase (OCS) gene, a terminator for a CaMV 35S RNA gene, etc. Is mentioned.

  Examples of selection marker genes include ampicillin resistance gene, neomycin resistance gene, hygromycin resistance gene, bialaphos resistance gene, and the like. Alternatively, the selection marker gene may be prepared by ligating the same gene together with the target gene as described above to prepare a recombinant vector, or alternatively, a recombinant vector obtained by linking the selection marker gene to a plasmid, A recombinant vector obtained by ligating the target gene to a plasmid may be prepared separately. When prepared separately, each vector is co-transfected (co-introduced) into the host.

(2) Production of transformed plant The transformed plant of the present invention can be prepared by transforming a target plant using the recombinant vector of (1) above. In preparing a transformed plant body, various methods already reported and established can be used as appropriate. Preferred examples thereof include the Agrobacterium method, the PEG-calcium phosphate method, the electroporation method, Examples include the liposome method, particle gun method, and microinjection method. When the Agrobacterium method is used, there are a case where a protoplast is used, a case where a tissue piece is used, and a case where a plant body itself is used (in planta method). When using protoplasts, co-culture with Agrobacterium with Ti plasmid, fusing with spheroplasted Agrobacterium (spheroplast method), and when using tissue fragments, aseptic culture of the target plant It can be performed by infecting leaf pieces (leaf discs) or infecting callus. In addition, when the in planta method using seeds or plants is applied, that is, in systems that do not involve tissue culture with the addition of plant hormones, for direct treatment of Agrobacterium to water-absorbing seeds, seedlings (plant seedlings), potted plants, etc. Can be implemented.

  When the target gene is introduced by the Agrobacterium infection method, a step of infecting the plant with Agrobacterium having a plasmid containing the target gene is essential, but this can be performed by the reduced pressure infiltration method. That is, Arabidopsis thaliana grown in soil containing equal amounts of vermiculite and perlite was directly immersed in the Agrobacterium culture medium containing the plasmid containing the target gene, and placed in a desiccator until the vacuum pump reached 65-70 mmHg. After aspiration, leave at room temperature for 5-10 minutes. Transfer the bowl to the tray and cover with wrap to keep the humidity. Take the wrap the next day, grow the plant as it is, and harvest the seeds. Subsequently, in order to select an individual carrying the target gene, the seed is sown on an MS agar medium supplemented with an appropriate antibiotic. By transferring Arabidopsis thaliana grown in this medium to a pot and growing it, seeds of a transformed plant introduced with the target gene can be obtained.

  Whether or not the target gene has been incorporated into the plant can be confirmed by PCR, Southern hybridization, Northern hybridization, Western blotting, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing a DNA-specific primer. After PCR, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band. By doing so, it can confirm that it transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, the amplification product may be bound to a solid phase such as a microplate, and the amplification product may be confirmed by fluorescence or enzymatic reaction.

  Alternatively, various reporter genes such as beta glucuronidase (GUS), luciferase (LUC), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), beta galactosidase (LacZ), etc. This can be confirmed by preparing a vector linked to, transforming a plant in the same manner as described above using the Agrobacterium introduced with the vector, and measuring the expression of the reporter gene.

  The plant used for transformation in the present invention may be either a monocotyledonous plant or a dicotyledonous plant. Monocotyledonous plants include, for example, Gramineae (rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet, millet, etc.), liliaceae (asparagus, lily, onion, leek, Japanese chestnut, etc.), ginger (ginger) And dicotyledonous plants such as Brassicaceae (Arabidopsis, cabbage, rapeseed, cauliflower, broccoli, radish, etc.), solanaceae (tomato, eggplant, potato, tobacco, etc.), Legumes (soybeans, peas, green beans, alfalfa, etc.), Cucurbitaceae (cucumbers, melons, pumpkins, etc.), Seriraceae (carrots, celery, honeybees, etc.), Asteraceae (lettuce, etc.) , Red crustaceae (sugar beet, spinach, etc.), myrtaceae (eucalyptus, clove, etc.), willow Plants belonging to the family (Poplar, etc.) can be mentioned, but are not limited thereto. Among these, plants belonging to the Brassicaceae family, tobacco, soybean, wheat and the like are particularly preferably used.

  In the present invention, examples of plant materials to be transformed include roots, stems, leaves, seeds, embryos, ovules, ovaries, shoot tips (growth points at the tips of plant buds), buds, pollen and the like. Plant tissue and its slices, undifferentiated callus, and plant cultured cells such as proplasts from which cell walls have been removed by enzyme treatment. In the case of applying the in planta method, water-absorbing seeds or the entire plant body can be used.

  In the present invention, the transformed plant body means the whole plant body, plant organ (eg, root, stem, leaf, petal, seed, seed, fruit, etc.) plant tissue (eg, epidermis, phloem, soft tissue, xylem, Vascular bundles, etc.) and plant cultured cells. When plant cultured cells are targeted, in order to regenerate a transformant from the obtained transformed cells, an organ or an individual may be regenerated by a known tissue culture method. Such an operation can be easily performed by those skilled in the art by a method generally known as a method for regenerating plant cells from plant cells. Regeneration from plant cells to plants can be performed, for example, as follows.

  First, when plant materials or protoplasts are used as plant materials to be transformed, these are inorganic elements, vitamins, carbon sources, sugars as energy sources, plant growth regulators (plant hormones such as auxin and cytokinin) And so on, and cultured in a sterilized callus formation medium to form dedifferentiated callus that grows irregularly (hereinafter referred to as “callus induction”). The callus thus formed is transferred to a new medium containing a plant growth regulator such as auxin, and further grown (subcultured).

  When callus induction is performed in a solid medium such as agar and subculture is performed in, for example, liquid culture, each culture can be performed efficiently and in large quantities. Next, callus grown by the above subculture is cultured under appropriate conditions to induce organ redifferentiation (hereinafter referred to as “redifferentiation induction”), and finally regenerates a complete plant body. . Redifferentiation induction can be performed by appropriately setting the types and amounts of various components such as plant growth regulators such as auxin and cytokinin, carbon sources, and the like, light, temperature and the like in the medium. By such redifferentiation induction, somatic embryos, adventitious roots, adventitious shoots, adventitious foliage, etc. are formed and further grown into complete plants. Alternatively, storage or the like may be performed in a state before becoming a complete plant body (for example, encapsulated artificial seeds, dried embryos, freeze-dried cells and tissues).

  The transformed plant body of the present invention is not only the “T1 generation” which is the regenerated generation after the transformation treatment, but also the “T2 generation” which is a progeny obtained from the seed of the plant, the drug selection or the Southern method, etc. This includes progeny plants such as the next generation (T3 generation) obtained by self-pollination of "T2 generation" plants that have been found to be transgenic by analysis.

  The transformed plant obtained as described above acquires resistance to low-temperature stress. Therefore, the transformed plant can be used as a low temperature stress resistant plant. Here, “low temperature stress” means that a temperature lower than the optimum temperature of each species is continuously loaded (for example, in the case of Arabidopsis thaliana, a temperature of −10 to 5 ° C. is continuously applied for 1 hour to several weeks. This is the stress that occurs when the load is applied.

Evaluation of the tolerance of the transformed plant to low temperature stress The tolerance of the transformed plant of the present invention to low temperature stress is the plant body when the transformed plant is planted in a flower pot containing soil containing vermiculite, perlite, etc., and the low temperature stress is loaded. Can be evaluated by examining the state (eg, growth rate, survival rate, plant height, weight, yield, or a combination thereof). Here, the load condition of the low temperature stress can be performed as described in the previous section.

Screening Method for Plant Environmental Stress Tolerance Regulatory Substance The above-described AtCSP3 gene and the gene corresponding to the AtCSP3 gene can be used for screening a substance that regulates low temperature stress tolerance of a plant. If the expression level of the gene in a plant body or plant cell increases, the low temperature stress tolerance of the plant increases, and if the gene expression level decreases, the low temperature stress resistance decreases. Therefore, a regulator of low-temperature stress tolerance can be screened by using as an index the increase or decrease in the expression level of the above gene in the plant body or plant cell. Specifically, a candidate substance for an environmental stress tolerance regulator is added to a plant capable of expressing the above-described gene, for example, Arabidopsis thaliana, and the change in the expression level of the above-described gene in the plant cell is determined by quantitative PCR Measure by Northern method. When the expression level of the gene described above is increased by adding a candidate substance, the candidate substance can be a candidate substance for a substance that enhances tolerance to low-temperature stress in plants, and by adding the candidate substance, When the expression level of the gene of the present invention decreases, the candidate substance can be a candidate substance for a substance that weakens the tolerance to low temperature stress of plants.

  The type of candidate substance to be screened is not particularly limited. For example, naturally occurring molecules (eg, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, etc.); lipids, steroids, glycopeptides, glycoproteins, proteoglycans, etc .; or synthetic analogs or derivatives of naturally occurring molecules (eg, , Peptidomimetics, etc.); and non-naturally occurring molecules (eg, low molecular weight organic compounds made using combinatorial chemistry techniques, etc.); and mixtures thereof.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

1. Synthesis of cDNA Total RNA was extracted from rosette leaves of Arabidopsis (Columbia-0) using RNeasy Mini Kit (Qiagen). Using the obtained RNA, cDNA was synthesized using PCR Core Kit (manufactured by Applied Biosystem).

2. Isolation of AtCSP3 Gene Part from cDNA PCR was performed using the cDNA synthesized as described above as a template and the following forward primer and reverse primer.
Forward primer:
5'- tctaga cgaacaaagtgcttcg-3 '(underlined portion is XhoI site: SEQ ID NO: 3)
Reverse primer:
5'- gagctc ttaagcaaccgaagta-3 '(underlined part is SacI site: SEQ ID NO: 4)
These forward primer and reverse primer are designed to amplify from the 5 ′ UTR to the stop codon of the known AtCSP3 gene base sequence (Genebank accession number At2g18780). PCR was performed in a 50 μl reaction system.

Ex Taq DNA polymerase (TAKARA BIO INC .; 5 units / μl) 0.2 μl, 10 × polymerase buffer (including MgCl ₂ ) 5 μl, 2.5 mM dNTP solution (2.5 mM) 2.5 μl, each primer (10 pmol) 0.1 μl) and 2 μl of the cDNA synthesized above (about 1 μg / μl) were mixed, and the total reaction volume was adjusted to 50 μl with milliQ water. The PCR reaction conditions and the number of reactions used are shown in Table 1 below.

  When the PCR product was confirmed by electrophoresis after the PCR reaction, a nucleic acid fragment of the expected length (about 900 bases) was amplified. The obtained fragment was cloned using pGEM-Teasy vector system (Promega) to obtain a plurality of positive clones. For the DNA inserts contained in these positive clones, the base sequence was determined using the BigDye Terminator v1.1 cycle Sequencing kit (Amersham Bioscience) using an ABI DNA sequencer (373 DNA sequencer), and further described above. A comparative analysis with a known AtCSP3 gene was performed, and it was confirmed that the cloned DNA insert was the AtCSP3 gene.

3. Transformation of AtCSP3 gene into Arabidopsis using Agrobacterium As described above, the isolated AtCSP3 gene is placed downstream of the CaMV35S promoter in the pBI 121 vector (Clonetech), which is a Ti-based vector, in the sense strand. Introduced in the direction.

First, a plasmid in which the AtCSP3 gene was inserted into the pGEM-Teasy vector obtained above was treated with XhoI and SacI to prepare an XhoI-SacI fragment. The prepared AtCSP3 gene-containing XhoI-SacI fragment was ligated in the sense direction to the pBI121 vector cleaved with XhoI and SacI. The resulting nucleic acid construct was transformed into Agrobacterium (GV3101 / Pmp-90) by freeze-thawing (Hofgen et al., (1998) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res. Oct 25; 16 (20): 9877 ) Was used for transformation. In order to select transformed Agrobacterium, the Agrobacterium into which the gene was introduced was selected for kanamycin resistance and gentamicin resistance on a YEP medium containing kanamycin (50 mg / L) and gentamicin (100 mg / L). The selected colonies were cultured in 5 ml of YEP medium containing kanamycin and gentamicin for 20-24 hours, then inoculated into 100 ml of YEP medium containing kanamycin and gentamicin, and cultured until OD ₆₀₀ = 0.80. The cultured bacterial solution was centrifuged at 5000 × g for 10 minutes at room temperature. The precipitated cells were dissolved in 50 ml of floral dropping medium. The dissolved bacterial solution was injected 3-5 times into the arabidopsis sputum. Plants infused with Agrobacterium were placed in plastic bags and grown overnight in the dark at 22 ° C. The next day, the plants were moved under long day conditions at 22 ° C. Three days after infecting the plant with the fungus, water was started. The composition of the medium used in the above experiments is as shown in Tables 2 and 3.

^*1000 x MSビタミン(1L)：100g myo-inositol, 2g L-glycine, 0.5g Nicotinic acid, 0.5g Pyridoxine・HCl, 0.5g Thiamine・HCl.

^* 1000 x MS vitamin (1L): 100g myo-inositol, 2g L-glycine, 0.5g Nicotinic acid, 0.5g Pyridoxine · HCl, 0.5g Thiamine · HCl.

4). Selection of AtCSP3-transformed plants The seeds obtained from the transformed plants were sterilized with a sterile solution (70% ethanol, 0.5% Triton X-100) for 30 minutes and then sterilized with 100% ethanol for 2 minutes. Thereafter, seeds were seeded on MS medium containing kanamycin (50 mg / L) and vancomycin (200 mg / L). Plants grew normally on the same medium (T ₀ generation) segregation ratio of wild-type strain and transformants T ₁ generation, which is E selfed 3: was confirmed to be 1. Further the transgene in the T ₂ generation were selected plants with a homo. The composition of the medium used in the above experiment is as shown in Table 4.

^*1000 x MSビタミン(1L)：100g myo-inositol, 2g L-glycine, 0.5g Nicotinic acid, 0.5g Pyridoxine・HCl, 0.5g Thiamine・HCl.

^* 1000 x MS vitamin (1L): 100g myo-inositol, 2g L-glycine, 0.5g Nicotinic acid, 0.5g Pyridoxine · HCl, 0.5g Thiamine · HCl.

5. Selection of AtCSP3 knockout mutants
In order to obtain a knockout mutant of AtCSP3 gene, a mutant (WiscDsLox353G12) in which T-DNA was inserted into the low temperature shock domain of AtCSP3 was purchased from the TAIR website (http://www.arabidopsis.org/) (FIG. 2). reference). The seeds purchased are sown in soil (mixed in a ratio of 3: 1 vermiculite (manufactured by Kushiro Coal Drying Co., Ltd.) and Geefi Mix (manufactured by Sakata Seed Co., Ltd.)), grown to rosette leaves, and then the Leaf PCR method Genomic DNA was extracted by (Klimyuk et al., (1993) Alkali treatment for rapid preparation of plant material for reliable PCR analysis. Plant J. Mar; 3 (3): 493-4). Specific primer for T-DNA (5'-cgtccgcaatgtgtta-3 ': SEQ ID NO: 5) and specific primer for AtCSP3 gene (5'-tgtacgaacaaagtgcttcga-3': to select mutants having homozygous T-DNA insertions: PCR reaction was performed using SEQ ID NO: 6 and 5′-tacaatccctagcgaaatgtc-3 ′: SEQ ID NO: 7). PCR reaction is 1 μl of Arabidopsis genomic DNA (approximately 1 ng / μl), Ex Taq DNA polymerase (TAKARA BIO INC .; 5 units / μl) 0.1 μl, 10 × polymerase buffer (including MgCl ₂ ) 2 μl, dNTP solution 1 μl of (2.5 mM) and 0.2 μl of each of the above primers (10 pmol / μl) were mixed, and the total amount of the reaction solution was adjusted to 20 μl with milliQ water. The PCR reaction conditions and number of reactions used are shown in Table 5 below.

6). Confirmation of AtCSP3 gene expression in AtCSP3 gene knockout mutants
Total RNA was extracted from rosette leaves of AtCSP3 gene knockout mutants using RNeasy Mini Kit (Qiagen). Using the obtained RNA, cDNA was synthesized using PCR Core Kit (manufactured by Applied Biosystem). PCR was carried out using the cDNA synthesized above as a template and the following forward and reverse primers.
Forward primer: 5'-aagtgcttcgagtttttgtta-3 '(SEQ ID NO: 8)
Reverse primer: 5'-gcagtgaacaggttgcat-3 '(SEQ ID NO: 9)

2 μl of cDNA (about 1 μg), 0.1 μl of Ex Taq DNA polymerase (TAKARA BIO INC .; 5 units / μl), 1 μl of 10 × polymerase buffer (including MgCl ₂ ), 0.4 μl of dNTP solution (2.5 mM), 0.5 μl of each of the above primers (5 pmol / μl) was mixed, and the total amount of the reaction solution was adjusted to 10 μl with milliQ water. The PCR reaction conditions and number of reactions used are shown in Table 6 below.

PCRの結果、AtCSP3遺伝子ノックアウト変異株においては、AtCSP3遺伝子の発現が認めらなかった。よって、本実施例で作製したAtCSP3遺伝子ノックアウト変異株は、AtCSP3遺伝子の機能を欠損した株であって、AtCSP3遺伝子の機能解析に有用であることがわかった。

As a result of PCR, AtCSP3 gene expression was not observed in the AtCSP3 gene knockout mutant. Therefore, the AtCSP3 gene knockout mutant prepared in this Example was found to be a strain lacking the function of AtCSP3 gene and useful for functional analysis of AtCSP3 gene.

7). Evaluation of freezing tolerance of AtCSP3 overexpression strain and AtCSP3 gene knockout mutant
AtCSP3 overexpression strain, AtCSP3 gene knockout mutant strain and Arabidopsis wild strain (Columbia-0) seeds are sterilized with sterilization solution (70% ethanol, 0.5% Triton X-100) for 30 minutes, then sterilized with 100% ethanol for 2 minutes And seeded in Gamborg medium. After 2 weeks of growth at 22 ° C. under continuous light conditions, freeze resistance was evaluated using TEMPERATURE CABINET (manufactured by TABAI ESPEC). Thereafter, the survival rate was measured one week after returning to 22 ° C. and continuous light conditions. Freezing tolerance evaluation steps, temperature, and time are shown in Table 7 below.

  In order to freeze the medium in step 1, ice was added after 2 hours. Further, water was removed from the medium when returning from 4 ° C. to 22 ° C. Table 8 shows the composition of the medium used in the above experiment.

  The results of freezing treatment without acclimatization performed on wild type and AtCSP3 gene knockout mutants are shown in FIGS. FIG. 3 (A) is a photograph of the wild type and AtCSP3 gene knockout mutant before freezing treatment, and FIG. 3 (B) is a photograph of the wild type and AtCSP3 gene knockout mutant after freezing treatment. FIG. 4 is a characteristic diagram showing the relationship between the freezing temperature without acclimatization and the survival rate. The results of freezing with acclimatization are shown in FIGS. Fig. 5 (A) is a photograph of wild type and AtCSP3 gene knockout mutants after freezing at -5 ° C, and Fig. 5 (B) is a wild type and AtCSP3 gene knockout mutation after freezing at -7 ° C. FIG. 5 (C) is a photograph of wild-type and AtCSP3 gene knockout mutants after freezing at −9 ° C. FIG. 6 is a characteristic diagram showing the relationship between the freezing temperature with low temperature acclimation and the survival rate. These results revealed that AtCSP3 gene knockout mutants were difficult to grow by freezing treatment, and AtCSP3 gene was directly involved in cold stress tolerance.

  The results of the freezing treatment performed on the wild type and AtCSP3 overexpressing strains are shown in FIGS. FIG. 7 shows the result of observation of mRNA of AtCSP3 gene. FIG. 8 shows the results of observing the protein encoded by the AtCSP3 gene. FIG. 9 is a photograph of wild-type and AtCSP3 overexpressing strains after freezing treatment. FIG. 10 is a photograph of a plant when wild-type and AtCSP3 gene knockout mutants are grown under normal conditions. 7-10, S3 shows an AtCSP3 overexpression strain, and W shows a wild strain. From these results, it can be seen that the AtCSP3-overexpressing strain has significantly improved low-temperature stress tolerance compared to the wild-type strain. In particular, as can be seen from FIG. 10, in the AtCSP3 overexpressing strain, no adverse traits such as hatching are observed.

  From the above results, it has been clarified that by overexpressing the AtCSP3 gene or a gene corresponding to the AtCSP3 gene in plants, it is possible to improve low-temperature stress tolerance without exhibiting bad traits such as hatching.

It is an electrophoresis photograph which shows the result of having examined the binding activity with respect to the nucleic acid molecule of the protein which AtCSP3 gene codes by gel shift analysis. It is a figure which shows typically the AtCSP3 gene in the AtCSP3 gene knockout mutant strain used in the Example. It is a photograph which shows the result of the freezing process experiment performed without the acclimatization process about a wild type and an AtCSP3 gene knockout mutant. It is a characteristic view which shows the result of the freezing process experiment performed without the acclimatization process about a wild type and an AtCSP3 gene knockout mutant. It is a photograph which shows the result of the freezing process experiment after low-temperature acclimatization performed about the wild type and the AtCSP3 gene knockout mutant. It is a photograph which shows the result of the freezing process experiment after low-temperature acclimatization performed about the wild type and the AtCSP3 gene knockout mutant. It is a photograph which shows the result of having measured the mRNA of AtCSP3 gene about a wild type and an AtCSP3 overexpression strain. It is a photograph which shows the result of having detected the protein which AtCSP3 gene codes about a wild type and an AtCSP3 overexpression strain. It is a photograph which shows the result of the freezing process experiment performed about the wild type and the AtCSP3 overexpression strain. FT, Freezing treatment; W, Wild strain; S3, AtCSP3 overexpression strain It is a photograph showing that there is no difference in growth when wild-type and AtCSP3 overexpressing strains are grown in a normal environment. FT, Freezing treatment; W, Wild strain; S3, AtCSP3 overexpression strain

Claims (6)

A transgenic plant having a low-temperature stress tolerance modified so that at least one of the following genes (a) to (d) is overexpressed :
(a) a gene consisting of DNA consisting of the base sequence shown in SEQ ID NO: 1
(b) a DNA comprising an RNA-binding protein having a homology of 80% or more with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 and having a low-temperature shock domain; Genes that confer stress tolerance
(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
(d) a transgenic gene encoding and introducing an RNA-binding protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a cold shock domain Genes that confer tolerance to low temperature stress on plants   The transgenic plant according to claim 1, wherein the gene encodes an RNA-binding protein having a cold shock domain.   The transgenic plant according to claim 1, wherein the gene is linked to an overexpression promoter. A method for imparting low temperature stress tolerance to a plant, which is modified so that at least one of the following genes (a) to (d) is overexpressed :
(a) a gene consisting of DNA consisting of the base sequence shown in SEQ ID NO: 1
(b) a DNA comprising an RNA-binding protein having a homology of 80% or more with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 and having a low-temperature shock domain; Genes that confer stress tolerance
(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
(d) a transgenic gene encoding and introducing an RNA-binding protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a cold shock domain Genes that confer tolerance to low temperature stress on plants 5. The method of claim 4 , wherein the gene encodes an RNA binding protein having a cold shock domain. The method according to claim 4 , wherein the gene is linked to an overexpression promoter.

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