JP2001231574A - Low-temperature-resistant oryza sativa transferred with glutathione-s-transferase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain Oryza sativa imparted with various stress resistances by overexpression of an isolated glutathione-S-transferase(GST) gene involved in the stress resistances in a monocotyledonous plant such as Oryza sativa. SOLUTION: 1. The subject gene encoding the protein described in (a) or (b) below: (a) a protein composed of a specific amino acid sequence and having glutathione-S-transferase(GST) activity or (b) a protein composed of an amino acid sequence where one or several amino acids are deleted, substituted or added to the amino acid sequence in (a) and having GST activity, 2. a vector containing the GST gene described in 1. and intended for transfer into plant, 3. a recombinant protein from the protein described in (a) or (b), and 4. a method for creating GST gene-transferred Oryza sativa comprising culturing on a solid medium dividing cells of the germ layer callus of Oryza sativa transferred with the vector described in 2. and the resultant GST gene-transferred Oryza sativa, are provided respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to rice glutathione S.
-A gene encoding a transferase (hereinafter referred to as GST), a recombinant GST, a vector for introducing the gene, a method for producing a rice with enhanced stress resistance such as low-temperature resistance into which the GST gene has been introduced, and the method for producing rice. About rice.

[0002]

2. Description of the Related Art In recent years, attempts have been made to introduce useful genes into plants using genetic engineering techniques to produce plants resistant to various environmental stresses. For example, low-temperature resistance (Japanese Patent Laid-Open No. 10-179167), drying resistance (Science, 2
59: 508-510, 1993), pesticide resistance (Trend Biotechnol., 8:
61, 1990).

Under the above technical background, there is a report that the gene encoding GST isolated from tobacco, a dicotyledonous plant, is overexpressed in tobacco to enhance growth under stress. (Nature Biot
ech., 15: 988-991,1997). However, there has been no report on the GST gene isolated from rice, a monocotyledonous plant, or the stress tolerance imparted by overexpressing the GST gene in rice.

GST has a function of detoxifying peroxide by metabolizing xenobiotics such as herbicides and endogenous substrates (Annu. Rev. Plant
Physiol.Plant Mol.Biol., 47: 127-158, 1996), and is known to have an active oxygen scavenging ability (Plant
J., 18, 285-292, 1999), which is extremely important as an enzyme that protects cells against various stresses.

[0005]

An object of the present invention is to provide a means for imparting various stress tolerances by isolating a GST gene involved in stress tolerance and overexpressing this gene in monocotyledonous plants. It is in. Another object is to provide a means for efficiently producing wood.

[0006]

Means for Solving the Invention As a result of intensive studies to solve such problems, the present inventors have obtained efficiently transformed cells into which a GST gene has been introduced by genetic engineering techniques. By regenerating, it was found that GST transgenic rice can be obtained reliably, and furthermore, it was found that it was possible to provide a means for imparting excellent resistance to low temperature stress to rice, and the present invention was completed. .

That is, the present invention resides in a gene encoding the following protein (a) or (b). (A) a protein having glutathione S-transferase activity consisting of the amino acid sequence represented by SEQ ID NO: 4 (b) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in (a) , And a protein having glutathione S-transferase activity

Further, the present invention relates to the glutathione S-
It is in a plant introduction vector containing a transferase gene. Further, the present invention resides in the following recombinant protein (a) or (b). (A) a protein having glutathione S-transferase activity consisting of the amino acid sequence represented by SEQ ID NO: 4 (b) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in (a) , And a protein having glutathione S-transferase activity

Furthermore, the present invention relates to the above glutathione S-
A method for producing a rice plant into which glutathione S-transferase gene has been introduced, comprising culturing, on a solid medium, dividing cells of a rice embryoid body callus into which a vector containing a transferase gene has been introduced. Further, the present invention resides in a rice plant into which a glutathione S-transferase gene has been introduced, obtained by the above method.

[0010]

Embodiments of the present invention will be described below. A. The present invention relates to a specific GST gene (hereinafter, also simply referred to as GST gene). The GST gene of the present invention is a gene capable of producing a rice having particularly excellent low-temperature resistance by transforming the rice by introducing the GST gene into the rice.

The cloning of the GST gene can be carried out by a generally known method. That is, first, GST
The cells of the organism from which the gene is derived are separated, and total RNA is extracted from the cells. This extraction of total RNA can be performed by a generally known method. Next, only mRNA is separated from the total RNA by a generally known method. cDNA
Can also be synthesized by a generally known method. That is, a desired cDNA can be prepared by using the obtained mRNA as a template, oligo dT or a random primer as a primer, and synthesizing a cDNA using a reverse transcriptase.

Next, a fragment group of this cDNA is prepared, inserted into a cloning vector, and further amplified in an appropriate host to prepare a cDNA library of an organism from which the GST gene is derived. can do. Whether or not a cDNA fragment has been incorporated into this cloning vector can be confirmed by a drug resistance marker or the like possessed by this vector. The vector for cloning is not particularly limited, for example,
Known cloning vectors used for normal cloning, such as pUC8, pUC9, pBR322, pBluescriptII, and λZAPII, can be used.

[0013] These cloning vectors can be produced by generally known methods, but commercially available products can also be used. The cDN synthesized above
In A, it is also possible to amplify a cDNA fragment related to the base sequence of the GST gene by PCR using a DNA primer having a base sequence related to the GST gene, and to clone this. The preparation of the above gene library can also be easily carried out using a commercially available kit. Also,
Depending on the type of organism, the following clones can be screened as they are using a commercially available gene library.

The preparation of the desired gene library is carried out by selecting a clone containing the desired GST gene from the gene library of the organism from which the GST gene of the present invention obtained as described above is obtained. As the screening method, a generally known method depending on the form of the gene library can be used.

Use as a probe for the above GST gene
It is necessary that the DNA fragment contains at least a part of the base sequence of the desired GST gene (SEQ ID NO: 3). A probe having such a sequence is obtained by, for example, extracting a DNA fragment obtained by chemically synthesizing a sequence of a portion common to known GST genes or a genomic DNA of a plant from which the GST gene of the present invention is derived from a cell nucleus portion. Using genomic DNA as a template,
A DNA fragment obtained by a PCR method using an oligonucleotide fragment obtained by chemically synthesizing a sequence of a region conserved between GST genes as a primer can be used.

From the clone thus obtained, the desired base sequence of the GST gene can be determined using an automatic base sequence analyzer or the like. The GST gene of the present invention itself can be obtained. In other words, using the cDNA of the organism from which the GST gene was prepared in the same manner as above as a template, a DNA fragment containing the 5'-terminal and 3'-terminal sequences of the nucleotide sequence of the GST gene determined as described above was used as a primer. The GST gene can be amplified by the PCR method described above.

Incidentally, by taking measures for altering the nucleotide sequence of a generally known gene, such as a so-called site-directed mutagenesis method, a part of the nucleotide sequence of the GST gene of the present invention prepared in the above-mentioned step is altered, The inventor of the present invention recognizes that the GST gene in which a part of the amino acid sequence encoded by the gene is artificially changed or the GST including the amino acid sequence is included in the technical scope of the present invention.

B. One purpose of the present invention is to introduce a GST gene into a plant such as rice to produce a transformed plant having low temperature resistance or the like by introducing the GST gene into a plant. As a prerequisite for introducing the GST gene into plants such as rice, it is necessary to create a gene transfer vector having excellent GST gene transfer efficiency of the present invention (hereinafter, simply referred to as a transfer vector). .
That is, it is necessary to construct a recombinant DNA in which the GST gene and, if necessary, a marker gene have been incorporated into a plasmid. Examples of the vector used in the present invention include commercially available pUC-based vectors such as pUC19.

The vector for introduction includes, in addition to the GST gene, a promoter sequence as a gene expression regulating mechanism used in the present invention, for example, cauliflower mosaic virus
(CaMV) -derived transcript [EMBO J. 6: 3901-3907, 198
7] and corn ubiquitin [Plant Mol. Bio
l., 18: 675-689, 1992] (SEQ ID NO: 1)
Examples of the terminator sequence include a terminator derived from a cauliflower mosaic virus and a nopaline synthase gene (SEQ ID NO: 2). However, any terminator that is known to function in plants can be used. However, they are not limited to these (see FIG. 4).

If necessary, a promoter sequence and G
Between the ST genes, an intron sequence having a function of enhancing the expression of the GST gene, for example, an intron sequence of corn alcohol dehydrogenase (Adh 1) [Gene &
Dev., 1: 1183-1200, 1987].

C. Introduction of GST gene into rice A vector containing the GST gene prepared as described above was introduced into rice embryoid body calli by the Agrobacterium method [Pl
ant J., 6: 271-282, 1994]. Transgenic cells carrying the GST gene can be selected from rice cells into which the GST gene has been introduced by analyzing the GST gene or its expression product, but the target transformed cells can be selected more efficiently. For this purpose, it is preferable to use an effective selection marker gene in combination with the GST gene. The selection markers used at this time include a hygromycin phosphotransferase (hpt) gene that confers resistance to the antibiotic hygromycin to plants and phosphinothricin (Phosphinothricin) acetyl that confer resistance to bialaphos One or more genes selected from transferase (bar) genes and the like can be used. In the present invention, in order to enhance the efficiency of selecting transformed cells using these selectable marker genes, a cauliflower mosaic virus-derived 3
A binary vector pEKH in which the hpt gene was linked to the 5S promoter was constructed and used (see FIG. 5).

As will be described in detail in Examples, gene transfer by the Agrobacterium method requires transformation of Agrobacterium with the vector for introducing the gene of the present invention. In the present invention, a vector obtained by incorporating a vector containing a GST gene into a binary vector containing the above-mentioned selection marker gene by restriction enzyme treatment can be used.

D. Recombinant DN containing GST gene of the present invention
Introduction of A into Rice The embryoid body callus of rice in the present invention can be obtained as follows. Refers to the species. Representative cultivars include, but are not limited to, Japanese varieties such as Koshihikari and Indian varieties such as IR36.

Rice (Oryza sativa L. var. Sasanishik)
After disinfecting the mature seeds of i) with hypochlorous acid, place them on ND2 agar solid medium containing 2,4-dichloroacetic acid and culture them. After 3 weeks, the callus generated from the scutellum tissue is transplanted to ND2 agar solid medium and cultured for 3 days or more to obtain an embryoid body callus that can be used for the Agrobacterium method.

Introduction of Recombinant DNA into Embryoid Callus Using the callus obtained above as a material, introduction is performed by the Agrobacterium method. Agrobacterium is known
EHA101, LBA4404 and the like can be used. Invention G
A method for introducing a recombinant DNA containing the ST gene into Agrobacterium is usually a known electroporation method, and specific examples of the apparatus include Gene Pulser (BioRad).

By culturing the Agrobacterium obtained above in a medium containing a drug for selection, it is possible to grow only Agrobacterium into which the GST gene of the present invention and the selection marker gene have been introduced. By infecting the embryoid body callus obtained above with the Agrobacterium obtained above, the GST of the present invention
Genes and selectable marker genes can be incorporated.

E. Regeneration of plant body from GST-introduced embryoid body callus Remove residual Agrobacterium from GST-introduced embryoid body callus and induce regeneration including selection agents such as hygromycin A GST transgenic plant can be obtained by culturing by placing it on a medium for use.

As a basal medium used for plant regeneration, an N6SE agar solid medium containing a selective agent such as hygromycin was used. Hygromycin-resistant calli grown on the selection medium were placed on MSRE agar solid medium containing hygromycin and the like to induce regeneration. The regenerated shoots are transferred to an MSHF agar solid medium for acclimation and promote rooting to grow recombinant plants.

The callus transplanted to the regeneration medium is 20-30
C., preferably 25-28 ° C., 500-2,000 lux per day, preferably 800-1,000 lux per day, when cultured for 20-60 days, preferably 30-40 days, retained the GST gene introduced from each resistant callus. Rice plants regenerate.

The GST of interest in the hygromycin-resistant cell mass (callus) obtained above and the regenerated plant body
To confirm that the gene has been incorporated, DNA can be extracted from these cells and tissues according to a standard method, and a known PCR method can be used.
It can be carried out by detecting the introduced gene using the method or the Southern method.

The obtained rice plant can be grown by cultivation in a container filled with soil or a material that can substitute for soil such as vermiculite, or a hydroponic solution, and dividing the strain into strains. GST transgenic rice grown in this manner is also included in the scope of the present invention. In the present invention, GST transgenic rice refers to callus and other cells into which the gene of the present invention has been introduced, roots and stems of plants,
It is a generic term for leaves, flowers, and seeds (seeds).

The transgenic rice of the present invention can produce a progeny plant by self-pollination, and the gene of the present invention is stably inherited in progeny. In addition, the transgenic plant of the present invention can be used to produce a new variety by crossing with another rice. Obtained by the present invention
The GST transgenic rice is resistant to various stresses such as low temperature germination and low temperature elongation.

[0033]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The embodiments of the present invention will be specifically described below. However, the technical scope of the present invention is not limited to these examples. A. Acquisition of rice GST gene Preparation of cDNA Library Spores of blast fungus race 037 were sprayed on young seedlings of three weeks after sowing of rice cultivar Sasanishiki having low blast field resistance. After spraying, the seedlings were placed in a vinyl moist chamber for 24 hours, and the leaves were collected and used as a material for preparing a cDNA library. Extract mRNA from blast-infected leaves, create cDNA library using Stratagene λZAPII system,
In vitro using helper phage from λZAPII vector
The plasmid was introduced into E. coli by the ro excision method. The transformed E. coli was ampicillin (100 mg /
ml) on a MacConkey agar plate containing
Colonies were formed and used as a cDNA plasmid library.

2. Extraction / purification / denaturation / preservation of plasmids Select an E. coli colony and inoculate 2 ml of 2 × YT (1.6% Bacto tryptone, 1% yeast extract, 0.5% NaCl) medium. After culturing at 37 ° C, Promega's Wizard DNA
The plasmid was purified using the iniprep kit. The purified plasmid was purified by agarose gel electrophoresis.
The amount of DNA was quantified and about 30 μg of plasmid DNA was taken and subjected to an alkali denaturation treatment. Alkali denaturation is 0.2N N
The reaction was carried out in an aOH solution for 5 minutes, a 1/4 amount of 5M ammonium acetate was added, and ethanol precipitation was performed, followed by drying under reduced pressure. The remaining plasmid DNA was stored at -20 ° C and a portion of the E. coli culture was stored at -80 ° C as a glycerol stock.

3. Analysis of partial nucleotide sequence The partial nucleotide sequence of each clone obtained above was analyzed using an alkaline-denatured plasmid as a template.
It was determined using the ABI373 automated sequencer according to the Biosystems Dye Terminator Cycle Sequencer method. Using the T3 and T7 primers as sequencing primers, the nucleotide sequence of the cDNA was determined from both directions. By this analysis, 10 clones that seemed to contain full-length cDNA were selected,
The nucleotide sequence of the full-length cDNA clone was determined by the following method.

4. Determination of base sequence of full-length cDNA clone Plasmid DNA containing full-length cDNA is cut with several types of restriction enzymes that recognize 6 bases, and rough restriction enzyme maps are prepared, and appropriate size (200 to 400 bp) Was subcloned into a pUC vector. Extraction, purification, denaturation, storage and sequencing of these plasmids were performed in the same manner as described above, and bidirectional sequencing was performed using M13 and RV primers.

5. Homology search for full-length cDNA Clones whose full-length cDNA sequence was determined as above
PSL029 was found to consist of a total of 1032 bases.
About the homology of PSL029, NCBI (NationalCenter f
or Biotechnology Information) to search. As a result, the full-length GST cDNA fragment contained in clone PSL029 was
It was found to have a translation site at the base and encode a polypeptide consisting of 212 amino acids (SEQ ID NO: 4 and SEQ ID NO: 5) (FIG. 1). The amino acid sequence encoded by complete CDS is a known wheat GST (Triticum aestivu
mglutathione S-transferase mRNA, complete cds.Sub
ramaniam, et. al., 1997) and 83% homology,
The homology was found to be very high.

B. Preparation of the Vector for Introduction of the Present Invention The SpUC vector and pBI221 vector containing the spectinomycin resistance gene were treated with the restriction enzymes HindIII and EcoRI, respectively, and the cauliflower mosaic virus 35S promoter (35S), β-glucuronidase gene (GUS ) And the nopaline synthase terminator (nos) were introduced to create the pS221 vector (Fig. 2).
reference). Subsequently, a SalI linker was introduced into the SacI site of pS221 to create pS221s. Then, the PSL029 cDNA fragment cloned in the BS vector was
It was cut with XbaI and XhoI and inserted into pS221s by replacing the GUS gene. GST that enables strong expression in dicots such as tobacco
The transfection vector pS221s 29PS was obtained. (See Fig. 3)

PS221s 29PS 35S promoter and pAHC
The following operations were performed to replace the 17 ubiquitin promoters. 100 ng each for vector cloned as pS221s 29PS and plasmid pAHC17
Was treated with restriction enzymes XbaI and PstI, respectively. Specifically, a plasmid DNA solution as a vector 5 ng / μ
l 2 μl, TE buffer 6 μl, 5 × H buffer 2 μl,
1,000 units of PstI and XbaI were added in 1 μl each, mixed and reacted at 37 ° C. for 1 hour. After reaction, pS221s 29PS
1 μl of restriction enzyme treatment solution, pAHC17 restriction enzyme treatment solution 3
Take 10 μl of DW, 6 μl of DW, 10 μl of s solution I of TAKARA DNA ligation kit (ver.2), mix at 16 ° C.
Ligation performed overnight

After 50 μl of competent cell DH5α stocked at −80 ° C. in a 1.5 ml tube was thawed for 5 minutes on ice, 2 μl of the above ligation product was added, and the mixture was further left on ice for 15 minutes. Then, a heat shock was applied at 42 ° C. for 2 minutes, followed by rapid cooling in ice. Then, add SOC medium (bacto trypton (20 g / L), Bacto yeast extract (5 g / L), NaCl (0.5 g / L), 2.5 mM
500 μl of KCl, 10 mM MgCl 2, 20 mM glucose, pH 7.0) was added, and the mixture was cultured at 37 ° C. for 2 hours. 80 μl of the culture was mixed with 2 × YT agar solid medium containing 1.6 ppm of spectinomycin (1.6% bacto
trypton, 1% yeast extract, 0.5% NaCl, 1.5% agar)
It was spread on a plate and cultured overnight at 37 ° C.

From a large number of spectinomycin-resistant colonies generated on the plate, 16 were randomly selected, and a part of each colony was scraped off with a sterilized toothpick, and a 2 × YT liquid medium (1.6% bactotrypton, 1% yeast
extract, 0.5% NaCl) and cultured with shaking overnight at 37 ° C.

The culture was centrifuged at 15,000 rpm at 4 ° C. for 5 minutes, and the supernatant was removed. Add cell resuspension solution (50 mM Tris (pH
7.5); 10mM EDTA; and 100μg / ml RNase A) Add 50μl, suspend with a micro tube mixer, and put cell lysis solution (0.2M NaOH and 1%
(SDS) was added, and the mixture was gently stirred until it became clear. Next, add 75 μl of neutralization solution (2.55 M potassium acetate), stir well while turning it upside down, and then
After centrifugation at rpm for 5 minutes, the supernatant (cleared lysate) was transferred to a new tube. After adding 2.5 times the volume of ethanol to the supernatant and thoroughly stirring, centrifuged at 4 ° C and 15,000 rpm for 20 minutes to remove the supernatant, and then added 1 ml of 70% ethanol for washing.
The mixture was centrifuged at 15,000 rpm for 5 minutes. After repeating the 70% ethanol washing once again, the supernatant was discarded, the precipitate as a plasmid DNA was dried in a desiccator, and 10 μl of TE buffer was added to dissolve at 65 ° C. for 10 minutes.

16 plasmids prepared by the above method
The DNA solution was subjected to electrophoresis on a 0.7% agarose gel in order to confirm whether or not the target ubiquitin promoter was inserted. From the results of electrophoresis,
A larger clone was selected than the pS221s 29PS plasmid, and this plasmid DNA was transferred to the restriction enzymes PstI and XbaI.
Processed. The treated solution was confirmed by electrophoresis, and the 2.0 kbp position of the ubiquitin promoter fragment and pS221s 29PS
4.7 kbp which is a fragment from which the 35S promoter has been deleted
, A plasmid in which the GST gene was normally linked downstream of the ubiquitin promoter was obtained as the vector for introduction of the present invention, Sub29PS (see FIG. 4).

C. Introduction and expression of GST gene in rice Preparation of Binary Vector The vector containing the GST gene prepared as described above was used for the Agrobacterium method [Plant J., 6: 271-282,
1994], a binary vector pEKH in which the hpt gene was linked to the cauliflower mosaic virus-derived 35S promoter was constructed and used (see FIG. 5). This binary vector also includes a kanamycin resistance gene (npt) linked downstream of the nos promoter. Vector containing the GST gene Sub29PS and binary vector p
EKH was cut at a predetermined position with the restriction enzyme Sse8387I, and ligated to
EKH-Sub29PS (FIG. 5) was obtained. In addition, binary vector
The direction of Sub29PS inserted into pEKH may be either forward or reverse.

[0045] 2. Induction of rice callus embryoid body Callus (Oryza sativa L. var. Sasanishiki) ripe seeds were disinfected with 1% hypochlorous acid, and ND2 medium (KNO ₃ (2830mg /
L), (NH ₄ ) ₂ SO ₄ (463 mg / L), KH ₂ PO ₄ (400 mg / L), CaCl ₂
/ 2H ₂ O (166 mg / L), MgSO ₄ / 7H ₂ O (185 mg / L), MnSO ₄ / 4H ₂ O
_{(4.4mg / L), H 3} BO 3 (1.6mg / L), ZnSO 4 / 7H 2 O (1.5mg /
L), KI (0.8mg / L ), FeSO 4 / 7H 2 O (27.8mg / L), Na 2 / EDTA
(37.3mg / L), glycine (2.0mg / L), nicotinic acid (0.5m
g / L), pyridoxine hydrochloride (0.5 mg / L), thiamine hydrochloride
(1.0 mg / L), myo-inositol (100 mg / L), shoe rose (20 g / L), 2,4-D (2 mg / L), 0.9% agar solid medium) and place at 25 ° C in the light. Incubate. After 3 weeks, callus with a good 2-3 mm state produced from scutellum tissue is selected and
By culturing in ND2 medium in the light for 3 days, embryoid body calli that can be used for the Agrobacterium method are obtained.

3. Introduction of Binary Vector Containing GST Gene into Agrobacterium Commercially available Agrobacterium EHA101, which was dispensed in 40 μl aliquots and stored at -80 ° C, was dissolved in ice, and a binary vector containing GST gene was introduced. Plasmid DNA
Add 2 μl of the solution (0.1 ng / μl), pipette gently and leave on ice for 5 minutes. Then, place the Bio-Ra in ice.
Transfer to c-vette for Gene Pulser of d company, 25μF, 200
A pulse was applied at Ω, 2.5 kV for 4.3 seconds. Thereafter, 1 ml of SOC liquid medium was immediately added, and the cells were cultured at 28 ° C. for 1 hour for expression of a drug resistance gene. The present invention is obtained by applying the cultured Agrobacterium to an AB agar solid medium containing 25 ppm of chloramphenicol, 50 ppm of kanamycin and 50 ppm of spectinomycin and culturing at 25 ° C. for 3 days.
Only Agrobacterium into which the GST gene and the selectable marker gene had been introduced could be grown.

4. Introduction of Recombinant DNA into Embryoid Callus The Agrobacterium obtained above is applied to AB agar solid medium and cultured at 25 ° C. for 3 days. The grown Agrobacterium is scraped with a spoon, and AA medium containing acetosyringone
(AA inorganic salts, amino acids, B5 vitamins, cream rose (2
0g / L), 2,4-D (2mg / L), kinetin (0.2mg / L), acetosyringone (acetosyringone) (10mg / L), Muller et a
l. 1978) and absorbance at a wavelength of 600 nm.
Adjust so that it is 0.15 to 0.20.

The embryoid body callus is infected with Agrobacterium by immersing the embryoid body callus obtained above in the prepared suspension for 1.5 to 2 minutes with gentle shaking. After immersion, the callus of the embryoid body is removed with a sterilized paper towel or the like to remove excess water, and the N6CO agar solid medium (KNO ₃ (28
_{30mg / L), (NH 4} ) 2 SO 4 (463mg / L), KH 2 PO 4 (400mg / L), Ca
Cl ₂ / 2H ₂ O (166 mg / L), MgSO ₄ / 7H ₂ O (185 mg / L), MnSO ₄ / 4H
₂ O (4.4 mg / L), H3BO3 (1.6 mg / L), ZnSO4 / 7H ₂ O (1.5 mg / L
L), KI (0.8mg / L ), FeSO4 / 7H 2 O (27.8mg / L), Na 2 / EDTA
(37.3 mg / L), glycine (2.0 mg / L), nicotinic acid (0.5 mg / L
L), pyridoxine hydrochloride (0.5mg / L), thiamine hydrochloride
(1.0 mg / L), shoe-close (30 g / L), glucose (1
0g / L), 2,4-D (2mg / L), acetosyringone (10mg / L),
Gelrite (2 g / L)) and culture in a dark place at 25 to 28 ° C. for 3 days. This allows the GST gene of the present invention and the selectable marker gene to be integrated into the embryoid callus genome.

5. Regeneration of Plant from Embryoid Callus Introduced with GST Gene In order to remove the remaining Agrobacterium from the embryoid body callus cultured above, washing is performed with sterilized water containing claforan (500 mg / L). The washed callus is treated with a sterilized paper towel to remove excess water, and a N6SE agar solid medium (KN
O ₃ (2830 mg / L), (NH4) ₂ SO ₄ (463 mg / L), KH ₂ PO ₄ (400 mg / L
_{L), CaCl 2 / 2H 2} O (166mg / L), MgSO 4 / 7H 2 O (185mg / L), Mn
_{SO 4 / 4H 2 O (4.4mg} / L), H 3 BO 3 (1.6mg / L), ZnSO 4 / 7H 2 O (1.
5mg / L), KI (0.8mg / L), FeSO 4 / 7H 2 O (27.8mg / L), Na 2 / E
DTA (37.3 mg / L), glycine (2.0 mg / L), nicotinic acid (0.
5 mg / L), pyridoxine hydrochloride (0.5 mg / L), thiamine hydrochloride (1.0 mg / L), shoe rose (30 g / L), 2,4-D (2 mg / L
L), gellite (2 g / L), claforan (500 mg / L), hygromycin (50 mg / L)). 3 weeks after placing, 2
By culturing in the dark at 5 ° C., a drug-resistant callus that grows can be obtained. If Agrobacterium grows during this time, claforan
(500 mg / L), wash again with sterile water, and continue culturing on N6SE agar solid medium.

The resistant callus obtained above was added to MSRE agar solid medium (MS inorganic salt, MS vitamin, puffed rose (30 g / L),
Sorbitol 1 (30 g / L), casamino acid (2 g / L), NAA (1 m
g / L), BAP (2 mg / L), claforan (250 mg / L), hygromycin (50 mg / L), gelrite (4 g / L)) and cultured at 25 ° C in the light to induce regeneration did. The redifferentiated shoots were prepared on MSHF agar solid medium (MS inorganic salts, MS vitamins, shoe rose (30 g / L), hygromycin (50 mg / L), agar
(8 g / L)) to promote rooting and cultivate recombinant plants.

6. Gene analysis of transformed plants Hygromycin-resistant cell mass obtained above (callus)
To confirm that the target GST gene has been incorporated into the regenerated plant, the DNA can be extracted from these cells and tissues according to a conventional method, and the gene introduced using a known PCR method or Southern method can be used. It can be performed by detecting.

Selection of Plants Containing GST Gene 0.1 g of the leaf blade of each of the 87 redifferentiated rice lines was collected, frozen and crushed with liquid nitrogen, and then subjected to Nucleon PHYTOPURE for PLANT.
Genomic DNA was extracted using DNA EXTRACTION KIT (Amersham LIFESCIENCE).

A PCR reaction was carried out using about 100 ng of the genomic DNA obtained above as a template to try to amplify the introduced gene sequence. PCR reaction conditions are PCR Amplificatio
n kit (Takara Co.). Specifically, 10 mM Tris-HCl, 2.5 mM MgCl2, 50 mM KCl, 0.01%
Gelatin, pH 8.3, dNTP (dATP, dGTP, dCTP, dTTP) each
A reaction solution was prepared by mixing a 2.5 mM mixture, 1.25 units of Ex-Taq DNA polymerase, and 2 μM of each of two primers (oligonucleotides having the sequences shown in SEQ ID NOS: 1 and 2). Transfer 25 mL of the reaction solution to DNA Engine PTC-20.
0 (manufactured by MJ Research) at 95 ° C for 1 minute, at 53 ° C
Amplification was carried out by repeating the temperature condition for 30 minutes at 72 ° C. for 1 minute.

When the PCR product was analyzed by a 0.7% agarose gel electrophoresis method according to a conventional method, about 1.2 kb was amplified.
A DNA band was confirmed. From this band pattern, it was confirmed that the GST gene was incorporated into 77 lines out of 86 lines obtained from regenerated plant individuals, excluding individuals that died during breeding (see FIG. 6).

7. Analysis of Transformed Rice Transduced GST Gene Transcripts The purpose of this study was to confirm that mRNA, which is the transcript of the transgene, was expressed in the transgenic rice GST gene. 0.1 g each of the leaves of the above-mentioned second generation of the recombinant rice inbred line numbers 1, 6, 16 and the non-recombinant plant
Collect, freeze with liquid nitrogen, pulverize, RNeasy Plant Mini
Total RNA was extracted using Kit (manufactured by QIAGEN). Total RNA 10μg
Was used for Northern blot analysis. From hybridization to detection, GeneImage ™ (Amersham) was used. Stringency conditions include 1 × SSC, 0.1% SDS solution at 65 ° C. for 20 minutes, followed by 65 ° C.
The plate was washed with 0.1 × SSC and 0.1% SDS at 20 ° C. for 20 minutes. As the probe, an amplified fragment using two kinds of primers shown in SEQ ID NOS: 1 and 3 was used. The result is as shown in FIG. As is clear from FIG. 7, the GST transgenic rice showed a much higher mRNA expression level than the non-recombinant rice.

8. GST activity of transformed rice leaves The above-mentioned second generation of the recombinant rice inbreds, strain numbers 1, 6, and 16,
And 0.1 g of each leaf of a non-recombinant plant were collected, and a protein extraction buffer (100 mM Tris buffer (pH 8.5), 10 mM E
Triturate with 5 ml of DTA, 1 mM mercaptoethanol)
Obtain an extract. The extract is centrifuged at 15000 rpm for 5 minutes (twice) to obtain a supernatant. The total protein concentration in the obtained extract was determined by protein assay (Bio-Ra
d), and adjusted with DW so that the total protein concentration in each extract was 0.5 mg / ml.

Using the protein extract described above, leaves
GST activity was measured. The measurement method was described by Habig et al. (J. Biol.
Chem., 56: 7130-7139, 1974).
Specifically, 90 μl of a reaction solution (100 mM dipotassium hydrogen phosphate (pH 6.5), 0.5 mM GSH, 0.5 mM CDNB as a substrate)
10 μl of the above protein extract (0.5 mg / ml)
It was calculated from the absorbance at 340 nm after incubation at 25 ° C for 15 minutes. The result is as shown in FIG. G
ST transgenic rice is 20-40% higher than non-recombinant rice
It showed a high activity value.

9. The following describes the germination test under low-temperature flooding conditions and the elongation test under low-temperature flooding conditions. The seeds subjected to these tests are commonly treated as follows. Inbred progeny seeds (T1) of untransformed Sasanishiki and transformed rice used as controls were cultivated under the same conditions in a closed greenhouse to obtain inbred seeds. The harvested seeds are dried naturally in a greenhouse for 3 weeks and then at 50 ° C
Dormancy was broken by treatment for 3 days. For seed disinfection before testing, Sportac Emulsion (Nissan Chemical Industries, Ltd.)
) Immersion treatment was performed for 10 minutes with a 100-fold solution.

Germination test under low-temperature flooding conditions Non-transformed Sasanishiki was used as a control, and transgenic rice was line numbers 1, 6, and 16. As a reference, a rice cultivar Dunghan Shali having excellent low-temperature germination was also used. Depth 20
The above-disinfected 100 seeds were placed on a petri dish with a diameter of 9 mm and a diameter of 9 cm, DW was added thereto so as to form a submergence of 10 mm, cultured at 13 ° C., and the germination rate was examined over time. The test scale was for untransformed Sasanishiki and Dunghan Shali
For the transformed rice, three repetitions were taken for the transformed rice, and one repetition of the seed of each line number was taken for the sake of the amount of seeds. The results are shown in FIGS. 9 and 10.
Shown in

FIG. 9 shows the transition of the germination rate after placing,
Fig. 10 shows the germination rate of 50% calculated based on the transition of the germination rate.
The number of days to reach was shown. From these results, it was confirmed that the germination of the transformed rice under the submerged condition was lower than that of Dunghan Shali, but was clearly faster than that of the non-transformed rice. This proves that the introduction of the GST gene into rice improves cold germination.

Elongation test under low temperature submergence condition As in the germination test under low temperature submergence condition, non-transformed Sasanishiki as a control, rice cultivar Dunghan Shali having excellent low temperature elongation as a control, and transgenic rice as strain No. 1,
6, 16 were used. These were subjected to seed disinfection as described above, and then prepared to have a germination length of 3 mm at 30 ° C. Three
25 seeds with a uniform germination length of 5 mm on a 0.6% agar medium
mm, and DW was added so that the surface was submerged 10 mm from the surface of the agar medium. FIG. 11 shows leaf height and root length on day 13 from the start of culture.

From FIG. 11, it was found that the leaves of the transformed rice under the low-temperature flooding condition were Dunghan Shali
Although it did not reach, it was confirmed that it was clearly extended from non-transformed rice. Also, for roots, strain numbers
Except for 1, clear elongation was observed. For roots, Dunghan Shali, which has excellent low-temperature extensibility,
It is a remarkable fact that the transgenic rice grows only to the same extent as the untransformed Sasanishiki, while the transformed rice grows. The above results demonstrate that the introduction of the GST gene into rice improves the low-temperature extensibility of leaf blades and roots after budding.

As described above, the results of the test on cold resistance are certain to be a technique that enables direct sowing of brand rice in cold regions, which is particularly difficult, by introducing the GST gene into rice. In addition to direct sowing cultivation, improvement in resistance to various stresses such as low temperature in paddy rice cultivation is expected.

Effects of the Invention According to the present invention, a GST gene involved in stress tolerance was isolated, and overexpression of this gene in monocotyledonous plants could impart various stress tolerances to monocotyledonous plants such as rice. . SEQUENCE LISTING

[Sequence List] <110> Iwate prefecture <120> Glutathione S-transferase Transgenic Rice with Enhanced Cold-tolerance <130> P99-0667 <160> 5 <170> PatentIn Ver. 2.0 <210> 1 <211> 20 <212 > DNA <213> Oryza sativa <400> 1 tggtactgtt tcttttgtcg 20 <210> 2 <211> 20 <212> DNA <213> Oryza sativa <400> 2 caagaccggc aacaggattc 20 <210> 3 <211> 20 <212> DNA <213> Oryza sativa <400> 3 agactctcat cggggcttag 20 <210> 4 <211> 639 <212> DNA <213> Oryza sativa <220> <221> CDS <222> (1) .. (636) <400> 4 atg gcg tcg tca aag cca atc ctg tac ggt gcc tgg atc agc tcc tgc 48 Met Ala Ser Ser Lys Pro Ile Leu Tyr Gly Ala Trp Ile Ser Ser Cys 1 5 10 15 tct cac cga atc cgg atc gtt ctc aat ctc aaa ggt gtg gat tat gag 96 Ser His Arg Ile Arg Ile Val Leu Asn Leu Lys Gly Val Asp Tyr Glu 20 25 30 tac aag tca gtc aat cct cga aca gat cca gac tat gag aaa atc aat 144 Tyr Lys Ser Val Asn Pro Arg Thr Asp Pro Asp Tyr Glu Lys Ile Asn 35 40 45 cca att aag tac ata cca gca tta gta gat gga gac cta gtt gtt tct 192 Pro Ile Lys Tyr Ile Pr o Ala Leu Val Asp Gly Asp Leu Val Val Ser 50 55 60 gac tct ctc gcc att gca ttg tat ctt gaa gat aaa tac cct caa cat 240 Asp Ser Leu Ala Ile Ala Leu Tyr Leu Glu Asp Lys Tyr Pro Gln His 65 70 75 80 gct ctc cta cct aag gat ctc aaa aag aaa gct ctt aat ctt cag att 288 Ala Leu Leu Pro Lys Asp Leu Lys Lys Lys Ala Leu Asn Leu Gln Ile 85 90 95 gca aac ata gtt tgt tca agc atc caa cgt ctc ca g tat gct gta 336 Ala Asn Ile Val Cys Ser Ser Ile Gln Pro Leu Gln Gly Tyr Ala Val 100 105 110 att ggt ttg cat gag ggt aag cta agc ccc gat gag agt ctt cag ata 384 Ile Gly Leu His Glu Gly Lys Leu Ser Pro Asp Glu Ser Leu Gln Ile 115 120 125 gtt cag cat tat att gat aag ggc ttc aaa gca atc gaa aag ctt tta 432 Val Gln His Tyr Ile Asp Lys Gly Phe Lys Ala Ile Glu Lys Leu Leu 130 135 140 gaa gga tcc aac ttt aaa tat gct act ggg gac gaa gtt caa ttg ggg 480 Glu Gly Ser Asn Phe Lys Tyr Ala Thr Gly Asp Glu Val Gln Leu Gly 145 150 155 160 gat gtg ttt cta gca cca cag atc cat gct ggc ata aat cgc ttc caa 528 Val Phe Leu Ala Pro Gln Ile His Ala Gly Ile Asn Arg Phe Gln 165 170 175 att gat atc aca aag tac cca aat ttg gca aga ctc cat gac aca tac 576 Ile Asp Ile Thr Lys Tyr Pro Asn Leu Ala Arg Leu His Asp Thr Tyr 180 185 190 atg gaa att cct gca ttt cag gca gca ctt cca aag aat cag cca gat 624 Met Glu Ile Pro Ala Phe Gln Ala Ala Leu Pro Lys Asn Gln Pro Asp 195 200 205 gca cct tca tgc taa 639 Ala Pro Ser Cys 210 <210 > 5 <211> 212 <212> PRT <213> Oryza sativa <400> 5 Met Ala Ser Ser Lys Pro Ile Leu Tyr Gly Ala Trp Ile Ser Ser Cys 1 5 10 15 Ser His Arg Ile Arg Ile Val Leu Asn Leu Lys Gly Val Asp Tyr Glu 20 25 30 Tyr Lys Ser Val Asn Pro Arg Thr Asp Pro Asp Tyr Glu Lys Ile Asn 35 40 45 Pro Ile Lys Tyr Ile Pro Ala Leu Val Asp Gly Asp Leu Val Val Ser 50 55 60 Asp Ser Leu Ala Ile Ala Leu Tyr Leu Glu Asp Lys Tyr Pro Gln His 65 70 75 80 Ala Leu Leu Pro Lys Asp Leu Lys Lys Lys Ala Leu Asn Leu Gln Ile 85 90 95 Ala Asn Ile Val Cys Ser Ser Ile Gln Pro Leu Gln Gly Tyr Ala Val 100 105 110 Ile Gly Leu His Glu Gly Lys Leu Ser Pro Asp Glu Ser Leu Gln Ile 115 120 125 Val Gln His Tyr Ile Asp Lys Gly Phe Lys Ala Ile Glu Lys Leu Leu 130 135 140 Glu Gly Ser Asn Phe Lys Tyr Ala Thr Gly Asp Glu Val Gln Leu Gly 145 150 155 160 Asp Val Phe Leu Ala Pro Gln Ile His Ala Gly Ile Asn Arg Phe Gln 165 170 175 Ile Asp Ile Thr Lys Tyr Pro Asn Leu Ala Arg Leu His Asp Thr Tyr 180 185 190 Met Glu Ile Pro Ala Phe Gln Ala Ala Leu Pro Lys Asn Gln Pro Asp 195 200 205 Ala Pro Ser Cys 210

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the readable frame of the GST gene and the encoded amino acid sequence

Fig. 2 Schematic diagram of pS221s plasmid vector

Fig. 3 Schematic diagram of pS221s 29PS plasmid vector

Fig. 4 Schematic diagram of Sub29PS plasmid vector

Fig. 5 Schematic diagram of pEKH-Sub29PS binary vector

[Fig. 6] Confirmation of transgene (PC
R method, strain number 1-15)

[FIG. 7] Analysis of transcript expression level in leaf of transgenic progeny (T2) (Northern analysis)

Fig. 8 GST activity in the leaf blade of transgenic progeny (T2)

FIG. 9: Transition of germination rate of GST-introduced rice under 13 ° C flooding condition

[Fig. 10] Number of days to reach 50% germination rate of rice under GST-introduced conditions at 13 ° C

[Fig. 11] Elongation of GST-introduced rice under 15 ° C flooding condition

──────────────────────────────────────────────────続 き Continued on the front page (51) Int. Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:91) C12N 5/00 C (C12N 5/10 C12R 1:91) C12R 1:91) (72 ) Inventor Hiroyuki Kanzaki 16-166-1 Murasakino, Kitakami-shi, Iwate Prefecture Princess No. 201 (72) Inventor Ikuo Nakamura 648, Matsudo, Matsudo-shi, Chiba F-term (reference) 2B030 AB03 AD04 CA14 CA15 4B024 AA08 BA10 CA04 CA09 CA12 CA20 DA01 DA05 DA06 EA04 FA02 GA14 GA17 GA27 HA13 HA14 4B050 CC01 CC03 DD13 EE01 LL05 LL10 4B065 AA11X AA88X AA88Y AB01 AC14 AC16 BA02 BA03 BA25 BB01 BC32 BC48 BC50 CA29 CA53

Claims (5)

[Claims] 1. A gene encoding the following protein (a) or (b): (A) a protein having glutathione S-transferase activity consisting of the amino acid sequence represented by SEQ ID NO: 4 (b) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in (a) , And a protein having glutathione S-transferase activity 2. A vector for introducing a plant, comprising the glutathione S-transferase gene according to claim 1. 3. A recombinant protein of the following (a) or (b): (A) a protein having glutathione S-transferase activity consisting of the amino acid sequence represented by SEQ ID NO: 4 (b) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in (a) , And a protein having glutathione S-transferase activity 4. A rice transgenic for glutathione S-transferase gene, wherein the dividing cells of the rice embryoid body callus into which the vector containing the glutathione S-transferase gene according to claim 2 is introduced are cultured on a solid medium. Production method. 5. A rice plant into which glutathione S-transferase gene has been introduced, obtained by the method according to claim 4.