JP2005087129A - Metallothionein gene derived from seashore panalum to which resistance to salt stress is imparted - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new gene capable of imparting resistance to salt stress to plants over a long period and a salt stress-resistant transgenic plant to which the gene is introduced. <P>SOLUTION: The gene encodes (a) a protein composed of a specific amino acid sequence and (b) a protein composed of an amino acid sequence in which one or several amino acids are lost, substituted or added in a specific amino acid sequence and having activity imparting resistance to salt stress. The salt stress-resistant transgenic plant is obtained by introducing the gene. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a gene that imparts salt stress tolerance, a transformed plant into which the gene has been introduced, and the like.

There are salt, dryness, high temperature, low temperature, strong light, air pollution, and the like as environmental stresses to plants, but salt damage and dryness are the most problematic from the viewpoint of agricultural production. Salt damage occurs not only in areas with high salinity, but also in farmland that had no problems until now due to irrigation. Currently, 12 million hectares of cultivated land in Asia have been damaged by salt damage and drought, and 9.5 million hectares of arable land are actually left unused due to salt damage. In particular, rice is a major grain in Asia, so if salt tolerance can be imparted to rice, unused farmland can be converted into a place for food production, contributing to the stabilization of global grain production. Until now, various attempts to produce environmental stress-resistant plants that can be cultivated even in poor environments or in poor soils using gene recombination techniques have been studied. For example, it is considered that a salt stress-tolerant plant can be produced by isolating a gene induced by salt stress and expressing the gene. Soybean-derived betaine synthase gene (patent document 1), arthrobacter globiformis-derived choline oxidase gene (non-patent document 1), rice-derived chloroplast glutamine synthase (non-patent document 2), Rice-derived transcriptional activator (Os DREB) (Non-patent Document 3), Na ⁺ / H ⁺ antiporter gene (Patent Document 2) derived from Hosowana Akaza and the like are known. The enzyme genes involved in sugar metabolism and synthesis are trehalose synthesis-related genes derived from Escherichia coli (Non-patent Document 4), and the galactinol synthase (AtGolS) gene that synthesizes galactinol from UDP galactose contains water such as dry, salt, and low temperature. It has been reported in Arabidopsis that it is involved in imparting stress tolerance (Non-Patent Documents 5 and 6). In the case of dicotyledonous plants, salt stress tolerance was evaluated for 10 weeks at 200 mM in tomato (Non-patent Document 7) and Brassica plant (Non-patent Document 8) transformed with Na ⁺ / H ⁺ antiporter gene derived from Arabidopsis thaliana There have been reports of fruit and seed harvesting. However, there is no gene that imparted long-term salt stress tolerance from transplanting to harvesting seeds in monocotyledonous transformed plants. For example, the conditions of salt stress tolerance in transformed rice are as short as 13 days at 100 mM, 2 weeks at 150 mM, and 3 days at 300 mM. Therefore, it is difficult to judge that the above gene group can impart a salt stress tolerance trait corresponding to an actual production process from several weeks to several months from seedling transplantation to seed harvesting.
Therefore, it is desired to produce plants that exhibit salt stress tolerance over a long period of time.

  On the other hand, metallothionein is a cysteine-rich low molecular weight protein capable of binding heavy metals, and the metallothionein gene is known to exist in a wide range of animals and plants and forms a gene family. Conventionally, it has been suggested that nuclei may be protected from active oxygen by metallothionein in animal cells (Non-Patent Documents 9 to 11), but salt stress tolerance in plants has not been reported.

JP 2001-309789 JP 2000-157287 A Mohanty A., et al., Theor. Appl. Genet., 106, pp. 51-57 (2002) Hoshida H., et al., Plant Mol. Biol., 43, pp.103-111 (2000) Dobouzet J. G., et al., Plant J. 33, pp.751-763 (2003) Jang I.C., et al., Plant Physiol., 131, pp. 516-524 (2003) Cell Engineering, Vol.21, No.12, pp.1455-1459 (2002) Teruaki T., et al., The Plant Journal, 29 (4), pp.417-426 (2002) Zhang H.X., Blumwald E., Nature Biotechnol., 19, pp.765-768 (2001) Zhang H.X., et al., Proc. Natl. Acad. Sci USA., 98, pp.12832-12836 (2001) Chubatsu, L.S. and R. Meneghini, Biochem. J. 291, pp.193-198 (1993) Sogawa, C.A. et al., Brain Res. 853, pp.310-316 (2000) Miura, T. et al., Life Sci. 60, pp. 301-309 (1997)

  An object of the present invention is to provide a novel gene capable of imparting salt stress tolerance to a plant over a long period of time, a salt stress resistant transformed plant into which the gene is introduced, and the like.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have focused on the fact that there is a gene induced by salt stress in seashore paspalum and attempted cloning thereof. Has been found to be a gene that encodes metallothionein and has an activity of imparting salt stress tolerance, thereby completing the present invention.

That is, the present invention includes the following inventions.
(1) A gene encoding a protein shown in (a) or (b) below.
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing
(b) a 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 in the sequence listing and having an activity to impart salt stress tolerance (2) A gene comprising the DNA shown in (c) or (d).
(c) DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing
(d) encodes a protein that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing and has an activity to impart salt stress tolerance DNA
(3) A recombinant vector comprising the gene of (1) or (2) above.
(4) A salt stress-tolerant transformed plant into which the gene according to (1) or (2) above or the recombinant vector of (3) above is introduced.
(5) The salt stress-tolerant transformed plant of (4) above, wherein the plant is a monocotyledonous plant.
(6) The salt stress resistant transformed plant according to (5) above, wherein the monocotyledonous plant is a plant belonging to the family Gramineae, Lilyaceae, or Gingeraceae.
(7) The salt stress-tolerant transformed plant according to (6) above, wherein the plant belonging to the family Gramineae is selected from the group consisting of rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet and millet.
(8) A method for producing a salt stress tolerant plant, comprising introducing the gene of (1) or (2) above or the recombinant vector of (3) above into a plant.
(9) The production method according to (8) above, wherein the plant is a monocotyledonous plant.
(10) The production method according to (9) above, wherein the monocotyledonous plant is a plant belonging to the family Gramineae, Lilyaceae, or Gingeraceae.
(11) The production method according to the above (10), wherein the plant belonging to the family Gramineae is selected from the group consisting of rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet and millet.

  According to the present invention, a gene capable of imparting salt stress tolerance is provided. By introducing this gene into plants such as rice, it is possible to produce a salt-stress-tolerant plant that can grow for a long time in a salt-stressed environment and can be seeded.

Hereinafter, the present invention will be described in detail.
1. Gene Cloning (1) Preparation and Screening of cDNA Library The gene conferring salt stress tolerance of the present invention can be obtained, for example, as follows. First, seawater resistant sea buckwheat (Seashore Paspalum; Duedck AE and Peacock CH, Agronomy Journal vol. 77, pp. 47) cultivated with seawater or salt stress applied (salt-treated) and not (untreated), respectively. -50 (1985)), total RNA is prepared, and PCR is performed using a single-stranded cDNA prepared using oligo dT as a template to prepare a salt-treated probe and a control probe.

  Next, using these two types of probes (salt treatment probe and control group probe), a clone that is specifically detected only with the salt treatment group probe is selected from the seashore paspalum cDNA library by the differential screening method. Next, Northern analysis is performed using a probe prepared on the basis of the partial sequence of cDNA contained in the clone, and a clone that is induced in seawater-resistant shiba by salt stress but not in rice is secondarily selected. Finally, using this clone as a probe, a clone containing the gene of interest is obtained from a cDNA library prepared from seawater-resistant shiba with salt stress.

  Extraction of mRNA from seawater-resistant shiba and preparation of a cDNA library can be performed according to conventional methods. Examples of mRNA supply sources include, but are not limited to, seawater-resistant shiba adult leaves. mRNA can be prepared by a commonly used technique. For example, after extracting total RNA from the above-mentioned source by guanidinium thiocyanate-cesium trifluoroacetate method or the like, by an affinity column method using oligo dT-cellulose or poly U-sepharose or by a batch method, A) + RNA (mRNA) can be obtained. Furthermore, poly (A) + RNA may be fractionated by sucrose density gradient centrifugation or the like.

  Next, using the obtained mRNA as a template, a single-stranded cDNA was synthesized using oligo dT primer and reverse transcriptase, and then double-stranded from the single-stranded cDNA using DNA synthase I, DNA ligase, RnaseH, etc. Synthesize strand cDNA. The synthesized double-stranded cDNA is smoothed by T4DNA synthase, then ligated with an adapter (for example, EcoRI adapter), phosphorylated, etc., and incorporated into a vector such as λgt11 to create a cDNA library in vitro. can do. In addition to λ phage, a cDNA library can also be prepared using a plasmid.

  To select a strain having the target DNA from the transformant obtained as described above, for example, when λ phage (λgt11 or the like) is used, PCR is performed using a primer for λgt11 insert amplification. Can be adopted.

  Examples of the template DNA used here include cDNA synthesized from the mRNA by a reverse transcription reaction. Moreover, a commercially available random hexamer etc. can be used as a primer.

  Specific examples of mRNA extraction, cDNA library preparation, probe preparation, and cDNA library differential screening are shown in Example 1. In addition, it can be confirmed that the cloned gene is induced by salt stress treatment in a barn (paspalum) by Northern blot analysis or RT-PCR, which is an expression analysis technique. Specific examples of the confirmation are shown in Examples 1 and 3.

(2) Determination of base sequence The cDNA base sequence of the cDNA clone obtained above is determined using the PCR product as a template. The base sequence can be determined by a known technique such as a chemical modification method of Maxam-Gilbert or a dideoxynucleotide chain termination method using M13 phage. Usually, an automatic base sequencer (for example, ABI373 sequencer manufactured by Applied Biosystems, Sequencing is performed using the company's 310 DNA sequencer). The obtained base sequence can be analyzed by DNA analysis software such as DNASIS (Hitachi Software Engineering Co., Ltd.), and the protein coding portion encoded in the obtained DNA strand can be found.

  SEQ ID NO: 1 exemplifies the nucleotide sequence of a gene imparting salt stress tolerance of the present invention (hereinafter also referred to as a metallothionein gene), and SEQ ID NO: 2 illustrates the amino acid sequence of the protein encoded by the metallothionein gene of the present invention. As long as the protein comprising an amino acid sequence has an activity to impart salt stress tolerance, a plurality of, preferably one or several amino acids may have mutations such as deletion, substitution and addition in the amino acid sequence.

  For example, 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 may be deleted, and 1 to 10, preferably 1 may be deleted from the amino acid sequence represented by SEQ ID NO: 2. ˜5 amino acids may be added, or 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 may be substituted with other amino acids.

  Further, a protein encoding a protein having 65% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and having an activity of imparting salt stress tolerance is also included in the scope of the present invention. The homology of 65% or more preferably means 70% or more, more preferably 80% or more.

  The amino acid deletion, addition, and substitution can be performed by modifying a gene encoding the protein by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or the 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 a TAKARA LA PCR in vitro Mutagenesis series kit is used to introduce the mutation.

  Here, the “activity that imparts salt stress tolerance” refers to an activity that can impart salt stress tolerance to a plant that grows in the presence of salt stress and that can be attached in some cases. Here, “salt stress” refers to a stress that damages the physiological function of a plant body, such as the salt water accumulated in the soil lowers the water potential of the soil and prevents the plant body from absorbing water. Salts include all salts that cause plant growth inhibition, yield reduction, and death, such as sodium salts and magnesium salts. Whether a gene (for example, a mutated gene) has an “activity that imparts salt stress tolerance” depends on whether the plant into which the gene is introduced grows or granulates in the presence of salt stress that cannot be grown in a wild type plant. Judgment can be made by confirming whether or not it is possible.

  The above “having an activity of imparting salt stress tolerance” means that the activity is substantially equivalent to the activity of the protein having the amino acid sequence set forth in SEQ ID NO: 2.

  The gene of the present invention also has an activity of hybridizing with a DNA comprising a base sequence complementary to the DNA comprising the base sequence shown in SEQ ID NO: 1 in the sequence listing under stringent conditions and imparting salt stress tolerance. Contains DNA encoding the protein. Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a complementary strand of a nucleic acid having a high homology, that is, a DNA comprising a base sequence having a homology of about 65% or more, preferably about 75% or more, more preferably about 85% or more with the base sequence represented by SEQ ID NO: 1. May be hybridized, and a complementary strand of a nucleic acid having a lower homology than that may not be hybridized. 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.

  Once the base sequence of the gene of the present invention has been determined, it is obtained by chemical synthesis, by PCR using the cloned cDNA as a template, or by hybridizing with a DNA fragment having the base sequence as a probe. be able to. Furthermore, modified DNA encoding the gene can be synthesized by site-specific induction or the like.

2. Production of Recombinant Vector and Transformed Plant (1) Production of Recombinant Vector The recombinant vector of the present invention can be prepared by inserting the above gene of the present invention into an appropriate vector. As vectors for introducing and expressing the gene of the present invention into plant cells, pBI vectors, pUC vectors, and pTRA vectors are preferably used. The pBI and pTRA vectors can introduce a target gene into a plant via Agrobacterium. A pBI binary vector or an intermediate vector system is preferably used, and examples thereof include pBI121, pBI101, pBI101.2, pBI101.3 and the like. A pUC vector can directly introduce a gene into a plant, and examples thereof include pUC18, pUC19, and pUC9. Plant virus vectors such as calflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV), and tobacco mosaic virus (TMV) can also be used.

  In order to insert the gene of the present invention into a vector, first, the purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector. Adopted.

  The gene of the present invention needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, in addition to a promoter and a metallothionein gene, an enhancer, a splicing signal, a poly A addition signal, a 5′-UTR sequence, a selection marker gene, and the like can be linked to the vector as desired.

  The “promoter” may be a plant-derived or plant-derived DNA as long as it functions in plant cells and can induce expression in a specific plant tissue or in a specific developmental stage. It does not have to be. Specific examples include a cauliflower mosaic virus (CaMV) 35S promoter, a promoter of nopaline synthase gene (Pnos), a corn-derived ubiquitin promoter, a rice-derived actin promoter, a tobacco-derived PR protein promoter, and the like.

  The “terminator” may be any sequence that can terminate the transcription of the gene transcribed by the promoter. Specific examples include terminator (Tnos) of nopaline synthase gene, cauliflower mosaic virus poly A terminator, and the like.

  The “enhancer” is used to increase the expression efficiency of the target gene. For example, an enhancer region containing an upstream sequence in the CaMV35S promoter is preferable.

  The “selection marker” is used for facilitating selection of transformants, and examples thereof include a hygromycin resistance gene, a bialaphos resistance gene, and a neomycin resistance gene.

(2) Production of transformed plant The transformed plant of the present invention is the introduction of the recombinant vector of the present invention into the plant so that the target gene (metallothionein gene) can be incorporated and expressed in its own gene. Can be obtained.

  Plants used for transformation in the present invention include monocotyledonous plants, for example, Gramineae (rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet, barnyard etc.), lily family (asparagus, lily, onion, leek). ), Plants belonging to the family Ginger (ginger, ginger, turmeric, etc.), but are not limited thereto.

  Plants to be transformed in the present invention include whole plants, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, sieve parts, soft tissues, xylem, vascular bundles, A fence-like tissue, a spongy tissue, etc.) or a plant cultured cell is meant. 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.

  Examples of methods for introducing the gene or recombinant vector of the present invention into plants include the Agrobacterium method, the PEG-calcium phosphate method, the electroporation method, the liposome method, the particle gun method, and the microinjection method. For example, when the Agrobacterium method is used, a protoplast may be used or a tissue piece may be used. When using protoplasts, co-culture with Agrobacterium with Ti plasmid, fusing with spheroplasted Agrobacterium (spheroplast method), or when using tissue pieces, use leaf disc for target plant The method can be carried out by infecting a sterile cultured leaf piece (leaf disc method) or infecting callus (undifferentiated cultured cells).

  Whether or not a gene has been incorporated into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing DNA-specific primers. After PCR, the amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplification 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.

  The transformed plant obtained as described above acquires resistance to salt stress. That is, salt stress tolerance can be imparted to a plant by introducing the metallothionein gene of the present invention. Therefore, the transformed plant can be used as a salt stress tolerant plant.

  A salt stress-tolerant plant can be produced by breeding a transformed plant (transgenic plant) into which a metallothionein gene has been incorporated to such an extent that it can be used as the salt stress-tolerant plant. In this case, it is only necessary to select a plant exhibiting tolerance without causing damage to physiological functions and without causing growth inhibition or death under the conditions that cause salt stress. The start of use as a stress-tolerant plant may be any time after selecting a plant exhibiting resistance.

  According to the present invention, a salt stress-tolerant plant that can grow in a salt stress environment that could not be grown conventionally is provided. For example, among the rice used in Example 2, the varieties that can survive in 50 mM NaCl are very limited, and Koshihikari dies at this salt concentration. On the other hand, the salt stress tolerance of Koshihikari introduced with the gene of the present invention was improved. Thus, when the metallothionein gene of the present invention is introduced, a long-term salt stress tolerance improving effect can be achieved throughout the entire growth period (except for the germination period). The plant (especially rice) can be cultivated in a brackish water area of 3000 ppm, and the vegetation in the salt-contaminated soil can be restored (environmental restoration).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited by the following Example.

(Example 1) Cloning of Shiba Gene Induced Under Salt Stress General RNA and DNA experimental methods are based on Molecular cloning-a laboratory manual-second edition, Cold Spring Harbor Laboratory Press New York (1989). did. Moreover, the usage method of the kit used during experiment followed the protocol which a manufacturer shows.

(1) Preparation of plant material Seashore Papalum (Duedck AE and Peacock CH, Agronomy Journal vol. 77, 47-50 (1985)) was planted in a 1 / 5000a Wagner pot containing sand and collected from Tokyo Bay. Water was irrigated with seawater having a salt concentration of 2.3 to 2.7% and cultivated in a greenhouse for 3 to 6 months. Once a day, seawater was sprinkled from the top of the pot and irrigated until the seawater oozed from the drain of the Wagner pot. This seawater irrigation treatment maintained and maintained the seawater-resistant shiba growth for at least 5 years.

  From the branches of this material, sections (Fig. 1) including the first to third leaves, stems, and nodes were collected, sprouting into the sand, rooted, and cloned seedlings were grown in the greenhouse. . This clone seedling was used for the following seawater stress or salt stress experiment by hydroponics.

  Seawater stress was applied as follows. First, the grown seedlings were cultivated in a 1 / 5000a Wagner pot (soil depth 15 cm) for 4 months in a greenhouse with tap water. The soil used was a 1: 1 mixture of rice soil and red balls. Seawater irrigation treatment was started by the above-described method at the stage where seedlings grew smoothly in pot cultivation (4 months after planting).

  The material used as a control group was sampled before starting the seawater irrigation treatment (tap water cultivation) and stored at -80 ° C. The material under salt stress conditions was sampled at 14 days of seawater irrigation and stored at −80 ° C. The sampling site was the above-ground leaves. The cDNA library for differential screening and RNA for probe preparation (3) described later were extracted from this seawater-grown material.

  The salt stress experiment by hydroponics was performed as follows. First, from the above-mentioned clonal seedlings cultivated in tap water, the sections shown in FIG. 1 were collected and sown by hydroponics (distilled water). This is 1 in the plant growing device (Tomy, Cultivation Chamber, CU-251) under the conditions of light period (illuminance 5000 lx, temperature 30 ° C) 16 hours, dark period (illuminance: 0 lx, temperature 22 ° C) 8 hours. Raised and rooted for a week. After rooting, distilled water was added to the modified Yoshida hydroponic medium shown in Table 1 below (S. Yoshida, et al., Laboratory Manual For Physiological Studies of Rice 3rd. Ed., The International Rice Research Institute, pp. 61-66). (1976); Trace elements are DR Hoagland & Arnon, DI, Univ. Calif. Coll. Agri. C. Exp. Sta. Circ., P.347 (1936), iron is Murashige T. & Skoog F., Physiologia Replaced with Plant., Pp.473-497 (1962) FeEDTA), 1 week, light period (illuminance 10000 lx, temperature 30 ° C) 16 hours, dark period (illuminance: 0 lx, temperature 22 ° C) 8 hours It was brought up on the condition of.

  After the growth, salt stress was applied for 4 weeks. The salt stress condition was the above-mentioned modified Yoshida hydroponic medium with 500 mM NaCl added. Four weeks after applying salt stress, leaves on the aerial part were collected. RNA extracted from hydroponic sections was used as a cDNA library for Northern analysis (4) and full length cDNA isolation (5).

  The hydroponic medium was adjusted to pH 5.5 using 5N NaOH. The volume of the hydroponic solution was examined every two days, and when the volume decreased, distilled water was added to restore the initial volume (in the case of Wagner pot, 4 L). The hydroponic solution was replaced with a fresh one once every two weeks.

(2) Preparation of RNA and construction of cDNA library About 1 mg of total RNA from 2 g of leaf by raw weight was added to the Rneasy Mini Kit (Qiagen) or the method of Chang, S. et al. (1993) (Plant Mol. Biol. Report, 11, pp. 113-116). In both cases, treatment with Dnase I (manufactured by Takara Bio Inc., Rnase-free, 5 mM MgSO _{4 in the} presence of 25 ° C., 11 hours) was added. Furthermore, 10 μg of mRNA was purified from the total RNA using BioMag SelectaPure, mRNA Purification System (manufactured by Polysciences, Inc.). From 5 μg of this mRNA, 1st strand cDNA and 2nd strand cDNA were synthesized using Oligo (dT) _12-18 using Time Saver cDNA synthesis kit (Amersham Biosciences). Next, using the Directional Cloning Kit (Amersham Biosciences) to clone the cDNA, inserted into the λgt11 phage vector using the Ready-To-Go Lambda Packaging Kit (Amersham Biosciences) Packaging was done. E.coli Y1088 was used as a host and subjected to differential screening after titer check.

(3) Probe preparation and differential screening The above seawater-resistant shiba was cultivated for 4 months from the state of buds by tap water cultivation (control group), and 2 weeks after seawater was continuously irrigated after 4 months ( Total RNA was prepared from each of the salt treatment groups, and 1st strand cDNA was prepared using Oligo (dT) _12-18 using Time Saver cDNA synthesis kit (Amersham Biosciences). Using this 1st strand cDNA as a template, PCR was performed using the random hexamer (pdN ₆ ) attached to the kit (PCR conditions: Premix ExTaq (manufactured by Takara Bio Inc.) and the cDNA at 94 ° C. for 5 minutes. Thermal denaturation (94 ° C. for 30 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute) × 25 cycles, 72 ° C. for 1 minute, using Gene Amp PCR system 9600). After confirming the obtained PCR product by gel electrophoresis, two types of probes (control group and salt-treated group) were prepared using ECL direct labeling kit (manufactured by Amersham Bioscience).

  The cDNA library was spread on a petri dish with a diameter of 14.5 cm and differentially screened from 2000 plaques. Plaque blotting was performed using Hybond N (Amersham Bioscience), and an ECL detection system (Amersham Bioscience) was used to detect plaque signals. One sheet was detected with the two types of probes described above, and plaques that were detected with the salt-treated group probe but not detected with the control group probe were selected as specific in the salt-treated group.

  In order to transfer the insert region of the selected clone to a plasmid vector, phage DNA was extracted. The phage DNA was treated at 94 ° C for 10 minutes and rapidly cooled, using the template as a template, λgt11 forward primer (5'-GGT GGC GAC GAC TCC TGG AGC CCG-3 ': SEQ ID NO: 3) and reverse primer (5'-TTG PCR was performed with Premix ExTaq (manufactured by Takara Bio Inc.) using ACA CCA GAC CAA CTG GTA ATG-3 ′ (SEQ ID NO: 4) (manufactured by Takara Bio Inc.) (95 ° C. for 1 minute, 94 ° C. for 1 minute, 55 ℃ 2 minutes, 72 ℃ 2 minutes) x 25 cycles, 72 ℃ 2 minutes). The obtained PCR product was subcloned into pT7 Blue T-vector using Perfectly Blunt Cloning Kit (Novagen). The selected 5 clones were sequenced by dRhodamine dye-terminator method (AmpliTaq DNA polymerase FS; manufactured by Applied Biosystems), and then DNA was analyzed from both strands using ABI 310 Genetic Analyzer (Applied Biosystems). The sequence was determined.

(4) Secondary screening by Northern analysis (comparison with rice)
A probe for secondary selection was prepared from the obtained 5 clones. Using the prepared probe, we compared the salt-inducibility of gene expression in seawater-resistant shiba and rice (variety Pokkari), and in the seawater-resistant shiba, the expression was induced by salt stress but not in rice. Next selected. The seawater-resistant shiba and rice (pokkari) use the above-mentioned modified Yoshida hydroponic medium, the control group (0 mM NaCl), the salt-treated area (400 mM NaCl) for seawater-resistant shiba and the salt-treated area (poccari) In 50 mM NaCl), the cells were grown for 1 week, and total RNA was extracted from the foliage part after 1 week. Total RNA electrophoresis, Northern blotting and hybridization were performed according to conventional methods.

  As a result of Northern analysis of 5 clones, clones that had high homology with ABA-inducible proteins were excluded from the isolation because they were expressed in seawater-resistant shiba and rice. There were two clones that were expressed only in seawater-resistant shiba but not in rice, and we thought that these were likely seawater-resistance related genes. One clone (Ps2) was selected as a gene transfer target in Example 2 below.

(5) Isolation and sequencing of full-length cDNA 10 μg of mRNA was obtained from seawater-resistant shiba that had been subjected to salt stress (400 mM NaCl) for 1 week using Yoshida's hydroponic medium. ZAP Express cDNA Gigapack III Gold Cloning kit ) Was used for cDNA synthesis, cloning into a Zap Express vector, and packaging. The cDNA library was spread on 10 14 × 9 cm square petri dishes and selected from about 100,000 plaques. Plaque blotting using Hybond N (Amersham Biosciences), labeling the Ps2 probe used in Northern analysis in (4) above with ECL direct labeling kit (Amersham Biosciences) ECL detection system (manufactured by Amersham Bioscience) was used for detection. As a result of the 1st to 3rd screening, plaques detected by the Ps2 probe were isolated. Each cDNA was transferred to pBK-CMV phagemid vector (Stratagene) by in vivo excision. The selected cDNA is sequenced by the dRhodamine dye-terminator method (AmpliTaq DNA polymerase FS: Applied Biosystems), and then the DNA sequence is determined from both strands using ABI 310 Genetic Analyzer (Applied Biosystems). did. For sequencing, T7 and T3 primers and primers specific to each cDNA were used. For analysis of the sequence, Genentyx Mac. Ver. 11 (Software Development Co. LTD, 2000) was used.

  As a result of sequencing, a cDNA clone (Ps2) almost identical to the sequence of Ps2 was obtained. The base sequence of Ps2 is shown in SEQ ID NO: 1, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 2. This Ps2 was highly homologous to type II metallothionein (about 85%).

(Example 2) Production of transformant and confirmation of transgene (1) Construction of vector for gene introduction The full-length cDNA of Ps2 obtained in Example 1 is linked to an actin promoter and Nos terminator, and a vector for gene introduction And a transformant was prepared as follows.

(2) Production of Ps2-transformed rice A protoplast was isolated from a liquid culture system capable of redifferentiation derived from a mature seed of rice (variety: Nipponbare, available from Shiga Prefectural Agricultural Cooperative). Protoplast preparation and electroporation were performed by the method of Kyozuka et al. (Kyozuka et al., Mol. Gen. Gnet., 206, pp. 408-413, 1987) and the method of Toriyama et al. (Toriyama et al., Bio / Technology 6, pp. 1072-1074) and Akagi et al. (Akagi et al., Mol. Gen. Gnet., 215, pp. 501-506, 1989).

Suspend in the introduction buffer of 0.4 M mannitol, 70 mM potassium aspartate, 5 mM calcium gluconate, 5 mM MES so that the protoplast is 2 × 10 ⁶ / mL and the vector DNA for gene introduction constructed in (1) above is 50 μg / mL. Turbid and electroporated. The electric pulse was an attenuation wave having an electric field intensity of 450 V / cm and a time constant of about 40 ms. Note that GTE-10 manufactured by Shimadzu Corporation was used for the introduction device, and FTC-54 was used for the introduction chamber.

  Protoplasts embedded in agarose medium based on R2P medium (Kimiko Ito, 1996, Plant Cell Engineering Series, Model Plant Experiment Protocol, Shujunsha, pp.82-88) are cultured with nurse cells by adding liquid medium. did. 14 days after introduction, the selection of transformants was started by adding R2P medium supplemented with hygromycin 50 μg / mL except for nurse cells and liquid medium. After the resistant callus grew to 1 to 2 mm, the selected transformed callus was transplanted into RS2A medium (Kimiko Ito, ibid.) Of hygromycin 50 μg / mL. After growing for 10-14 days, hygromycin 50 μg / mL redifferentiation medium (Tetsu Nishimura and Isao Shimamoto, 1996, Plant Cell Engineering Series, Experimental Protocol for Model Plants, pp. 78-81, Shujunsha) The callus was transplanted to regenerate the plant body.

Remove the redifferentiated plant from the callus mass, place it on a hormone-free medium (Nishimura and Shimamoto, same as above) containing 50 μg / L hygromycin, select the rooting individuals, open the petri dish lid, and apply sterile water. Pour onto a petri dish medium and acclimatize for 1 week. At this time, sterilized water was supplied once every two days so as not to dry the medium. After acclimatization, selected plant bodies (Nippon Hare To generation) are rice-growing soil (Mitsubishi Chemical Co., Ltd.): Jiffy pot (red x wide x height: 5cm x 5cm x 6cm) containing red balls (1: 1) ), Transplanted to a plastic pot (width x depth x height: 15 cm x 5.5 cm x 9.5 cm) after 1 to 2 months, further grown for 4 to 10 months, self-bred or mated to obtain seeds It was. The cultivation was carried out in an isolated greenhouse. T ₁ generation, out of work by selfing of Ps2 introduced transgenic lines T ₀ generation, also BC ₁ F ₁ generation, the F ₁ generation were crossed and the T ₁ generation and Koshihikari, and backcrossed again Koshihikari Created.

(3) Ps2 gene introduction and confirmation of expression Ps2 gene was confirmed by PCR using the transgenic rice generation (T ₀ ), T ₁ generation and BC ₁ F ₁ generation. To extract genomic DNA from transformed rice, cut young leaves into 1- 3 cm lengths, put them in a sterilized Eppendorf tube, and add 100 μL TE buffer (10 mM Tris HCl, pH 8.0, 1 mM EDTA). This was carried out by grinding with a homogenizer for Eppendorf tubes for 1 to 3 minutes. After grinding, it was stored on ice, centrifuged at 4 ° C. and 15,000 rpm for 2 minutes, and the supernatant was transferred to a new sterile tube. This supernatant was used as a genomic DNA fraction.

In order to confirm the presence of the Ps2 gene, genomic PCR was performed using 5′-CTC ACA AAG CAA AGG CAA TC-3 ′ (SEQ ID NO: 5) as a sense primer, and 5′-GGA TCA GGA GGC TAC TAT TC-3 ′ (sequence). No. 6) as an antisense primer, 1.25 U enzyme KOD-Dash (Toyobo), 10 pmole primer, 0.2 mM dNTP, reaction buffer (final concentration: 20 mM Tris-HCl (pH 7.5), 8 mM) MgCl ₂ , 7.5 mM DTT, 2.5 μg / 50 μL BSA). The genomic DNA described above was used in an amount of 10 μL for a total volume of 20 μL PCR reaction solution. The reaction apparatus uses Gene Amp PCR System 9600 or Gene Amp PCR System 9700 manufactured by ABI, and PCR conditions are 98 ° C for 2 minutes (98 ° C for 30 seconds, 55 ° C for 2 seconds, 74 ° C for 30 seconds) x 30 cycles. , 74 ° C. for 5 minutes and 4 ° C. ∞.

For 47 individuals, RT-PCR was performed to confirm gene expression. RT-PCR uses SEQ ID NO: 5 (same as above) as a sense primer, SEQ ID NO: 6 (same as above) as an antisense primer, 1.25 U enzyme KOD-Dash (manufactured by Toyobo), 10 pmole primer, 0.2 mM dNTP, reaction Buffer solution (final concentration: 20 mM Tris-HCl (pH 7.5), 8 mM MgCl ₂ , 7.5 mM DTT, 2.5 μg / 50 μL BSA). 0.2 to 10 μL of 1st strand cDNA prepared from the above total RNA using a 1st strand cDNA synthesis kit (manufactured by Life Science) was used for 20 μL of the total PCR reaction solution. The reaction apparatus uses Gene Amp PCR System 9600 or Gene Amp PCR System 9700 manufactured by ABI, and PCR conditions are 98 ° C for 2 minutes (98 ° C for 30 seconds, 55 ° C for 2 seconds, 74 ° C for 30 seconds) x 30 cycles. , 74 ° C. for 5 minutes and 4 ° C. ∞.

  As a result of the above PCR and RT-PCR, expression could be confirmed in 87.5% of the tested individuals. Moreover, these individuals were classified into Ps2-bearing individuals (Ps2 +) and Ps2-non-bearing individuals (Ps-) using genomic PCR.

Evaluation salt stress tolerance test (Example 3) Ps2 gene evaluation of the introduced salt stress tolerance of transgenic rice was (1) transgenic lines (T ₁ strain) and the non-recombinant strains of salt stress tolerance, Yokohama I went in an isolated greenhouse in Totsuka Ward. The test period was from October to January. The average temperature was 28 ° C, the maximum temperature was 32 ° C, the minimum temperature was 24 ° C, and the average humidity was 60 to 80%. The day length was 9 hours on average and the illuminance was 5000 to 10,000 lx.

In the salt stress tolerance evaluation, artificial seawater (NaCl 500 mM) was diluted 10 times and used as resistance against 50 mM NaCl corresponding to the death of Koshihikari. 2 to 3 leaf stages of Ps2 transgenic lines (T ₁ line) with Ps2 expressing and non-expressing lines and non-recombinant lines (Pokkari, IR28, and Nipponbare) Normal cultivation (flood paddy field situation). Transplanted by individual and line at the 2nd leaf stage and cultivated under fresh water flooded condition for one week. The cultivation soil was planted in a jiffy pot (3 cm x 3 cm) using rice seedling soil (Makiki soil) containing fertilizer. In one week of curing cultivation, fresh water was changed to 10-fold diluted seawater and flooded cultivation was continued. The stainless steel tank containing the pot was submerged with diluted seawater, and the salt content in the water tank was kept at 0.3% (50 mM NaCl) by pump stirring.

  The result is shown in FIG. In FIG. 2, the non-recombinant lines (1 to 3) had more portions withered in the presence of salt stress than the control. On the other hand, Ps2 transgene recombinant line-expressed (4) had more parts that were not dead compared to the non-expressed line (5) and could grow even in the presence of salt stress.

In addition, using 6 genetically modified lines (T ₁ generation) of 33 individuals and 3 non-recombinant lines of 30 individuals (Koshihikari, Nipponbare and Pokkari) The results of the resistance evaluation are shown in Table 2.

  In Table 2, Koshihikari died completely at the 10th week (survival rate 0%), but the Ps2 (+) line showed various salt stress tolerance levels ranging from 25.0% to 50.0%. It was suggested that the salt stress tolerance improvement effect by.

(2) Evaluation of salt stress tolerance of genetically modified line BC ₁ F ₁ generation Subsequently, salt stress resistance evaluation was performed using 13 BC ₁ F ₁ generation 13 lines of T ₁ // Koshihikari. The BC ₁ F ₁ population was 82 individuals in the Ps2 (+) group and 67 individuals in the Ps2 (−) group, which matched a 1: 1 ratio. The survival rates at the second, fourth and eighth weeks after the diluted seawater treatment are shown in FIG. In both Ps2 (-) and Ps2 (+) groups, the survival rate was 10-20% at 4 weeks when Koshihikari was completely dead, whereas the Ps2 (-) group was almost dead at 8 weeks, The survival rate of Ps (+) group was significantly high at 11% (χ ² = 4.95).

  Further, Table 3 summarizes the plant granulation in the presence of salt stress. In the recombinant line (Ps2 +) into which Ps2 was introduced, heading was observed in 6 out of 9 individuals that survived up to the 8th week, and showed a fertilization rate of 50 to 100%.

  According to the present invention, a gene capable of imparting salt stress tolerance is provided. By introducing this gene into plants such as rice, it is possible to produce a salt-stress-tolerant plant that can grow for a long time in a salt-stressed environment and can be seeded.

It is a figure which shows the section | slice containing the 1st to 3rd leaf of a seawater tolerance shiba, and a stem and a node. It is a photograph which shows the result of the salt stress tolerance evaluation test of Ps2 gene recombination system. It is a graph showing the results of a salt stress tolerance evaluation test in Ps2 transgenic lines of breeding populations (BC ₁ F ₁ generation).

SEQ ID NOs: 3-6: Synthetic oligonucleotides

Claims (11)

A gene encoding the protein shown in the following (a) or (b).
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing
(b) a 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 in the sequence listing and having an activity of imparting salt stress tolerance The gene consisting of DNA shown in the following (c) or (d).
(c) DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing
(d) encodes a protein that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing and has an activity to impart salt stress tolerance DNA   A recombinant vector comprising the gene according to claim 1 or 2.   A salt stress-tolerant transformed plant into which the gene according to claim 1 or 2 or the recombinant vector according to claim 3 has been introduced.   The salt stress-tolerant transformed plant according to claim 4, wherein the plant is a monocotyledonous plant.   The salt stress resistant transformed plant according to claim 5, wherein the monocotyledonous plant is a plant belonging to the family Gramineae, Lilyaceae, or Gingeraceae.   The salt stress-tolerant transformed plant according to claim 6, wherein the plant belonging to the family Gramineae is selected from the group consisting of rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet, and barnyard millet.   A method for producing a salt stress tolerant plant, comprising introducing the gene according to claim 1 or 2 or the recombinant vector according to claim 3 into a plant.   The production method according to claim 8, wherein the plant is a monocotyledonous plant.   The production method according to claim 9, wherein the monocotyledonous plant is a plant belonging to the family Gramineae, Lilyaceae, or Gingeraceae.   The production method according to claim 10, wherein the plant belonging to the family Gramineae is selected from the group consisting of rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet and millet.

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