JP2002272292A - DISEASE INJURY-RESISTANT PLANT TRANSFERRED WITH WASABI gamma-THIONINE GENE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a disease injury-resistant rice transferred with the wasabi γ-thionine gene. SOLUTION: A method for creating the objective disease injury-resistant rice is characterized by comprising transferring the wasabi γ-thionine gene into rice.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a disease resistant plant and a method for producing the same.

[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. Already, low-temperature resistance [JP-A-10-179167], drying resistance [Science, 25
9: 508-510, 1993], pesticide resistance [Trend Biotechnol. 8: 6
1, 1990], a variety of stress-tolerant plants have been produced. In rice, a gene encoding chitinase isolated from rice was overexpressed in rice to impart resistance to blast, a rice pathogen, [Theor. Appl. Ge.
net. 99: 383, 1999], and reports that the overexpression of the thaumatin-like protein gene enhances resistance to sheath blight fungi [Theor. Appl. Genet. 98: 1138, 1999]. In addition, there is no report that resistance to blast was maintained in the T3 line of rice.

[0003] By the way, γ-thionine is one of protective proteins that protect plants against invasion from pathogenic bacteria by making a hole in the cell membrane of the pathogenic bacteria and dissolving intracellular substances [Appl. Envir. Micro. 65: 5451, 1999]. However, there is no report that overexpression of γ-thionine in rice imparts resistance to rice pathogens such as blast.

[0004]

An object of the present invention is to provide a disease resistant plant and a method for producing the same.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have been able to improve the disease resistance of plants by introducing the wasabi γ-thionin gene into plants. Successfully, the present invention has been completed.

That is, the present invention is a disease-resistant plant into which a wasabi γ-thionin gene has been introduced, and a method for producing a disease-resistant plant, which comprises introducing a wasabi γ-thionin gene into a plant. Here, examples of the plant include rice, wheat, gentian, apple, and lettuce. Examples of the disease include potato disease, blight bacterial rot, gray mold, sheath blight, rot, and spot leaf spot.

The wasabi γ-thionine gene includes those encoding the following proteins. (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4; (b) a protein having one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 or 4 Having the physiological activity of thionine

Further, the wasabi γ-thionine gene includes the following DNA of (c) or (d). (c) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3 (d) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3
DNA that encodes a protein that hybridizes under stringent conditions and has a physiological activity of thionin is introduced into a plant by introducing the wasabi γ-thionin gene into an embryoid body callus derived from the plant. It can be performed by introducing a recombinant vector containing a thionin gene. Hereinafter, the present invention will be described in detail.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The disease resistant plant of the present invention is a transgenic plant into which a wasabithionine gene has been introduced. The disease resistant plant of the present invention can be produced as follows. 1. Preparation of Wasabi γ-Thionine Gene First, the wasabi γ-thionine gene used in the present invention is prepared. Wasabi γ-thionine gene is obtained according to a standard method.
It can be prepared from wasabi ( Wasabia japonica ). That is, mRNA is prepared from the above source. For example, plant tissues such as stems, leaves, and roots are treated with a guanidine reagent, a phenol reagent, or the like to separate total RNA. Next, poly-U-sepharose using oligo-dT-cellulose or Sepharose 2B as a carrier from total RNA
(A) ⁺ RNA (mRNA) is isolated. Single-stranded cD using oligo dT primer and reverse transcriptase with the resulting mRNA as template
After synthesizing NA, double-stranded cDNA is synthesized. Using the obtained double-stranded cDNA as a template, a polymerase chain reaction (also referred to as PCR) is performed using sense primers and antisense primers designed based on the base sequence of a known horseradish γ-thionin gene, thereby obtaining wasabi γ-thionin. Can be obtained. The resulting amplified DNA fragment was subcloned into an appropriate vector such as pBlueScriptSK (+) (manufactured by STRATAGENE), pCR2.1 (manufactured by Invitrogen), and the nucleotide sequence was determined, whereby the wasabi γ-thionin gene of interest was obtained. Make sure that The base sequence can be determined by a known method such as the dideoxynucleotide chain termination method.

SEQ ID NOs: 1 and 3 show the base sequence of the horseradish γ-thionine gene, and SEQ ID NOs: 2 and 4 show the amino acid sequence of the horseradish γ-thionine, as long as the protein has the physiological activity of thionin. In the present invention, a gene encoding a protein in which at least one amino acid has a mutation such as deletion, substitution, or addition can also be used in the present invention. Here, “physiological activity of thionin” refers to an antibacterial activity expressed by making a hole in the cell membrane of a plant pathogenic bacterium such as a fungus. For example, at least one, preferably about 1 to 10, and more preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 or 4 is deleted, substituted, and DNA encoding a protein in which As long as the protein does not lose the physiological activity of thionine, it can be suitably used in the present invention. Further, a DNA that can hybridize under stringent conditions with a horseradish γ-thionine gene consisting of a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3 is not limited as long as it encodes a protein having the physiological activity of thionin. It can be used in the present invention.
Stringent conditions include, for example, a sodium concentration of 300 to 2000 mM, preferably 600 to 900 mM, and a temperature of 4 to 4 mM.
The condition at 0 to 75 ° C, preferably at 65 ° C. The mutation can be introduced into the gene by a known method such as the Kunkel method or the Gapped duplex method or a method equivalent thereto, for example, a mutagenesis kit using a site-directed mutagenesis method (for example, Mutant manufactured by TAKARA). -K or TAKARA Mutant-G) or TAKARA LA PCR in vitro Mutage
This can be done using the nesis series kit.

2. Production of Disease-Resistant Plants Disease-resistant plants can be produced by introducing the above horseradish γ-thionin gene into a plant host using a genetic engineering technique. Introduction of the wasabi γ-thionine gene into plants was carried out using Agrobacterium
m ) The method can be carried out by using the infection method or the direct introduction method of DNA. Methods utilizing Agrobacterium infection include co-integrative vectors (Co-integ
rative vector) and binary vectors (Binary vect)
or) direct injection methods include microinjection method, electroporation method, liposome method and the like, and their detailed procedures are known in the art.

For example, when a binary vector system of Agrobacterium is used, the above-mentioned 1 is located between the boundary sequences (LB, RB) of the binary vector such as pBI101, pBI121, pGA482.
Insert the wasabi γ-thionine gene obtained in the above.
Here, in order to express the wasabi γ-thionin gene in the plant into which the wasabi γ-thionin gene is introduced, it is necessary to arrange a promoter and a terminator for a plant before and after the wasabi γ-thionin gene in the binary vector. Available promoters include the cauliflower mosaic virus (CaMV) 35S transcript [Jeffer
son, RA et al .: The EMBO J 6: 3901-3907 (1987)],
Corn ubiquitin gene [Christensen, AHe
18: 675-689 (1992)], the promoter of the nopaline synthase (NOS) gene, the promoter of the octopine (OCT) synthase gene, and the like. But the terminator is not limited thereto. If necessary, an intron sequence having a function of enhancing gene expression, for example, an intron of corn alcohol dehydrogenase (Adh1) [Genes & Development 1:11] may be inserted between the promoter sequence and the horseradish γ-thionin gene.
83-1200 (1987)]. Furthermore, in order to efficiently select a desired transformed cell, it is preferable to arrange a selectable marker gene on a vector. The selection markers used at this time include a kanamycin resistance gene (NPTII), a hygromycin phosphotransferase (htp) gene that confers resistance to the antibiotic hygromycin to plants, and a phosphine that confers resistance to bialaphos. Nosurisin acetyltransferase (bar) gene and the like.

Next, the obtained recombinant vector is amplified in Escherichia coli, and then subjected to electroporation, freeze-thawing, tripartite contact with Agrobacterium tumefaciens EHA101, LBA4404, and the like. Agrobacterium for plant infection containing a horseradish γ-thionine gene is prepared by introduction by a legal method [Nucleic Acids Research, 12: 8711 (1984)] or the like. For example, introduction by the electroporation method can be performed by Gene Pulser (BioRad) or the like. Selection of Agrobacterium into which the recombinant vector has been introduced, that is, Agrobacterium containing the wasabi γ-thionine gene, can be performed by selecting a strain that can grow on a medium containing the drug for selection.

Next, Agrobacterium containing the horseradish γ-thionine gene is infected to a desired plant. Here, the plant body refers to a plant cultured cell (for example, an embryoid body callus), a whole plant of a cultivated plant, a plant organ (for example, a leaf, a petal, a stem, a root, a rhizome, a seed, or the like), or a plant tissue (for example, Epidermis, phloem, parenchyma, xylem, vascular bundle, etc.). The embryoid callus is, for example, rice ( Or
In the case of yza sativa L. var. Sasanishiki), it can be obtained as follows: a rice mature seed is disinfected with hypochlorous acid and then placed on ND2 agar solid medium containing 2,4-dichloroacetic acid. And culture. After about 4 to 5 weeks, the callus generated from the scutellum tissue is transplanted to ND2 agar solid medium and cultured for about 3 days or more to obtain embryoid body callus. Plant species to which the wasabi γ-thionin gene is introduced include, but are not limited to, monocotyledonous plants such as rice, wheat, barley, and corn, and dicotyledonous plants such as tobacco, apple, gentian, and lettuce. Infection of the plant with the Agrobacterium can be carried out by immersing the plant in a culture solution of Agrobacterium and leaving it at room temperature for several minutes.

For the plant cells into which the wasabi γ-thionine gene has been introduced, transformed cells having the wasabi γ-thionine gene can be selected by analyzing the wasabi γ-thionine gene or its expression product, but more efficiently. In order to select a desired transformed cell, effective selection markers include a hygromycin phosphotransferase (hpt) gene that confers resistance to the antibiotic hygromycin to a plant and bialaphos (bia
One or more genes selected from phosphinothricin (Phosphinothricin) acetyltransferase (bar) gene and the like, which confer resistance to laphos, can be used. In Examples of the present invention, a binary vector pEKH2 in which the hpt gene was linked to a cauliflower mosaic virus-derived 35S promoter was constructed and used in order to enhance the selection efficiency of transformed cells using these selection marker genes ( (Fig. 1).

[0016] After removing the remaining Agrobacterium from the plant subjected to the Agrobacterium infection treatment, the plant is placed in a regeneration induction medium containing a selection agent such as hygromycin and cultured to obtain wasabi γ-thionin. A plant into which the gene has been introduced can be obtained. An agar medium containing a selective agent such as hygromycin (for example, N6SE agar solid medium) is used as a basic medium for plant regeneration.
And the like. In Examples of the present invention using rice as a plant, hygromycin-resistant calli grown on a selection medium were placed on an N6SE agar solid medium containing hygromycin and the like to induce regeneration. The callus transplanted to the regeneration medium is 20 to 30 ° C., preferably 25 to 28 ° C., under light irradiation of 500 to 2,000 lux, preferably 800 to 1,000 lux per day, for 20 to 60 days, preferably 30 to 30 lux. By culturing for up to 40 days, it is possible to redifferentiate into a rice plant containing the wasabi γ-thionine gene. From the regenerated shoots, the shoots are transferred to an MSHF agar solid medium for acclimation to promote rooting and to grow recombinant plants.

It was confirmed that the wasabi γ-thionine gene of interest was incorporated into the hygromycin-resistant cell mass (callus) and the regenerated plant obtained above.
DNA can be extracted from these cells and tissues according to a conventional method, and the introduced gene can be detected using a known PCR method or Southern method.

In general, when a target gene is introduced into the genome of a plant, a difference (position effect) in the expression level of the transgene depending on the location of the gene may be observed. By performing a test using the Northern method using a DNA fragment of the transgene as a probe, a transformant in which the transgene is more strongly expressed can be selected.
Further, it was confirmed that the gene was incorporated into the transgenic plant into which the wasabi γ-thionine gene was introduced and the next-generation plant thereof, by DNA from the plant according to a conventional method.
, And directly detecting the transgene using PCR, Southern analysis, or the like.

The obtained wasabi γ-thionine transgenic plant is cultivated in a container filled with soil or a material capable of substituting for soil such as vermiculite or a hydroponic culture solution, and divided into strains. Can be propagated. The wasabi γ-thionin transgenic plant thus grown is also included in the scope of the present invention. In the present invention, a wasabi γ-thionin transgenic plant is wasabi γ.
It is a generic term for cells such as callus into which a thionin gene has been introduced, roots, leaf sheaths, leaves, inflorescences, and seeds (seed rice) of plants.

The wasabi γ-thionin 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. Wasabi γ-thionin transgenic rice obtained by the present invention,
It has resistance to various diseases. Such diseases include, but are not limited to, blast, blight bacterial rot, gray mold, sheath blight, rot, spot leaf spot, seedling blight, striped leaf blight, and the like.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The embodiments of the present invention will be specifically described below. However, the scope of the present invention is not limited to these examples. Example 1 Construction of Wasabi γ-Thionine Gene Transfer Vector A wasabi γ-thionine gene transfer vector was constructed as follows. First, the SpUC vector and pBI221 vector containing the spectimycin resistance gene were treated with restriction enzymes HindIII and EcoRI, respectively, and the cauliflower mosaic virus 35S promoter (CaMV3
5S), β-glucuronidase gene (GUS), and nopaline synthase terminator (nosT) were introduced to prepare a pS221 vector. Next, the SacI site of the pS221 vector
pS221s was created by introducing an alI linker. There, p
CDNA encoding wasabi γ-thionine gene (SEQ ID NO: 1) cloned into BlueScriptSK + vector
The fragment was cut with restriction enzymes XbaI and XhoI and inserted into pS221s by replacing the GUS gene. As a result, a horseradish γ-thionin gene transfer vector pS221s-WT capable of strong expression in dicotyledonous plants such as tobacco was obtained.

Further, the following operation was performed to replace the 35S promoter of the pS221s-WT vector with the maize ubiquitin promoter of the pAHC17 vector. Chain 100 ng of each of pS221s-WT and plasmid pAHC17 obtained by the above-described operation were respectively restricted with restriction enzymes Xba
Treated with I and PstI. Specifically, DNA solution 5ng / μl
2 μl, TE buffer 6 μl, 5xH buffer 2 μl
1 and 10 units / μl of PstI and XbaI were added to 1 μl each, mixed, and reacted at 37 ° C. for 1 hour. After the reaction, pS221s-W
Take 1 μl of the T restriction enzyme treatment solution and 3 μl of the pAHC17 restriction enzyme treatment solution, add 6 μl of sterile distilled water, and 10 μl of solution I of DNA ligation kit (ver. 2) manufactured by TAKARA, and mix.
Ligation was performed by reacting at 6 ° C. overnight.

After dissolving 50 μl of competent cells DH5α stored in a 1.5 ml tube at −80 ° C. in ice, 10 μl of the above-ligated reaction solution was added, and the mixture was left on ice for 30 minutes. Next, the cells were subjected to heat shock at 42 ° C. for 2 minutes and then kept on ice for 2 minutes to perform transformation. Then add 50 g of SOC medium (20 g / L Bacto tryptone, 5 g / L Bacto yeast extract, 0.5 g / L NaCl) to this tube.
After adding 0 μl, L containing 100 mg / L of spectinomycin
B agar solid medium (1% bactotryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar)
Cultured overnight.

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 2xLB liquid medium (2% bactotryptone, 1% yeast extract) was used. , 2% NaCl, 1.5% agar) at 37 ° C overnight.

The culture was centrifuged at 15,000 rpm for 5 minutes at 4 ° C., and the supernatant was removed. Add the cell RNase solution (50 mM TrisHCl (pH 7.5), 10 mM EDTA, 100 μg / ml RNase
A), add 50 μl, suspend, and add an alkaline solution (0.2 M NaOH,
(1% SDS) was added and gently suspended. Further, 75 μl of a neutralization solution (2.55 M potassium acetate) was added thereto, and the mixture was gently suspended, followed by centrifugation at 4 ° C. and 15,000 rpm for 5 minutes, and the supernatant was transferred to a new tube. After adding 2.5 times the volume of ethanol to the supernatant and thoroughly stirring, centrifuge at 15,000 rpm for 20 minutes at 4 ° C, remove the supernatant, add 70% ethanol, and add 4% at 15,000 rpm at 4 ° C.
Centrifuged for 5 minutes. After further washing with 70% ethanol, the supernatant was discarded, and the DNA precipitate was dried in a desiccator and dissolved in 10 µl of TE buffer.

The plasmid DNA prepared by the above method
The solution was subjected to electrophoresis on a 1% agarose gel to confirm whether or not the target ubiquitin promoter was inserted. PS221s-W from electrophoresis results
A clone is selected from the T plasmid and this plasmid DNA
Was treated with restriction enzymes PstI and XbaI. The treatment solution was confirmed by electrophoresis, and the ubiquitin promoter fragment of 2.0 k
A clone in which a band was confirmed at the position b, that is, a plasmid pSubWT in which the wasabi γ-thionin gene was normally ligated downstream of the ubiquitin promoter was isolated. Next, a vector pSubWT containing a wasabi γ-thionine gene was prepared.
And binary vector pEKH with restriction enzyme Sse8
A predetermined position was cut at one site with 387I, and these were ligated to obtain pEKH-SubWT (FIG. 1).

Example 2 Introduction and Expression of Wasabi γ-Thionine Gene into Rice (1) Induction of Embryoid Callus in Rice Rice (variety: Sasanishiki; Oryza sativa L. var. Sasan)
After disinfection of ripe seeds of ishiki) with 1% hypochlorous acid, ND2 medium [KNO ₃ (2830 mg / L), (NH ₄ ) ₂ SO ₄ (463 mg / L), KH ₂ PO ₄ (400 m
g / L), CaCl ₂ / 2H ₂ O (166 mg / L), MgSO ₄ / 7H ₂ O (185 mg / L), Mn
_{SO 4 / 4H 2 O (4.4mg} / L), H 3 BO 3 (1.6mg / L), ZnSO 4 / 7H 2 O (1.5
mg / L), KI (0.8 mg / L), FeSO ₄ / 7H ₂ O (27.8 mg / L), Na ₂ EDTA
(37.3mg / L), glycine (2.0mg / L), nicotinic acid (0.5mg / L
L), pyridoxine hydrochloride (0.5mg / L), thiamine hydrochloride
(1.0mg / L), myo-inositol (100mg / L), sucrose (20g / L
L), 2,4-D (2 mg / L), 0.9% agar] and cultured at 25 ° C. in the light. After 3 weeks, callus of 2-3 mm in good condition derived from the scutellum tissue is selected and cultured in an ND2 medium for 3 days in a light place at 25 ° C. to obtain an embryoid body callus that can be used for the Agrobacterium method. Was.

(2) Introduction of Binary Vector Containing Wasabi γ-Thionine Gene into Agrobacterium Commercially available Agrobacterium EHA101, which was dispensed in 40 μl portions and stored at −80 ° C., was dissolved in ice. , A binary vector pEKH-SubWT containing the wasabi γ-thionine gene
2 μl of the plasmid DNA solution (0.1 ng / μl) was added, and the mixture was left on ice for 5 minutes by gently pipetting. Then, in ice, transfer to a cuvette for BioRad GnePulser and set at 25 μF, 200 ohm, 2.5 kV at 4.3 kV.
Pulsed for seconds. Immediately afterwards, 1 m of SOC liquid medium
In addition, the cells were cultured at 28 ° C. for 1 hour for expression of the drug resistance gene. The Agrobacterium after the culture was applied to an AB agar solid medium containing 25 ppm of chloramphenicol, 50 ppm of kanamycin, and 50 ppm of spectinomycin, and cultured at 25 ° C. for 3 days to obtain a wasabi γ-thionine gene and a selectable marker gene. Only the Agrobacterium into which was introduced was grown.

(3) 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. Scratch the grown Agrobacterium with a spoonful, AA medium containing acetosyringone (AA inorganic salts, amino acids, B5 vitamins, sucrose (20 g / L),
Suspended in 2,4-D (2 mg / L), kinetin (0.2 mg / L), acetosyringone (10 mg / L); Muller et al. 1978] so that the absorbance at a wavelength of 600 nm is 0.15 to 0.20. did. The embryoid body callus was infected with Agrobacterium by immersing the embryoid body callus obtained in the above (1) in the obtained suspension for 1.5 to 2 minutes with gentle shaking. After immersion, the callus of the embryoid body was removed with a sterilized paper towel or the like to remove excess water, and N6CO agar solid medium (KNO ₃ (2830 mg / L), (NH ₄ ) ₂ SO
₄ (463mg / L), KH ₂ PO ₄ (400mg / L), CaCl ₂ / 2H ₂ O (166mg / L),
MgSO ₄ / 7H ₂ O (185 mg / L), MnSO ₄ / 4H ₂ O (4.4 mg / 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 (2.0m
g / L), nicotinic acid (0.5mg / L), pyridoxine hydrochloride (0.5m
g / L), thiamine hydrochloride (1.0 mg / L), myo-inositol (10
0 mg / L), sucrose (20 g / L), 2,4-D (2 mg / L), acetosyringone (10 mg / L), 0.2% gellite] and placed in a dark place at 25-28 ° C. Cultured for days. As a result, the wasabi γ-thionin gene and the selection marker gene were integrated into the embryoid callus genome.

(4) Regeneration of Plant from Embryoid Callus Introduced with Wasabi γ-Thionine Gene In order to remove the remaining Agrobacterium from the embryoid callus cultured above, clafolan (500 mg / L) was added. Washed with sterile water. The washed callus was washed with a sterilized paper towel to remove excess water, and N6SE agar solid medium [KNO ₃
(2830mg / L), (NH ₄ ) ₂ SO ₄ (463mg / L), KH ₂ PO ₄ (400mg / L),
CaCl ₂ / 2H ₂ O (166mg / L), MgSO ₄ / 7H ₂ O (185mg / L), MnSO ₄ / 4H
₂ O (4.4 mg / L), H ₃ BO ₃ (1.6 mg / L), ZnSO ₄ / 7H ₂ O (1.5 mg / L),
KI (0.8 mg / L), FeSO ₄ / 7H ₂ O (27.8 mg / L), Na ₂ EDTA (37.3 mg
/ L), glycine (2.0mg / L), nicotinic acid (0.5mg / L), pyridoxine hydrochloride (0.5mg / L), thiamine hydrochloride (1.0mg / L),
Myo-inositol (100mg / L), sucrose (20g / L), 2,4-D (2
mg / L), acetosyringone (10mg / L), 0.2% gellite,
Claforan (500 mg / L), Hygromycin (50 mg / L))
Was placed on the floor. After 3 weeks from the implantation, drug-resistant callus was obtained by culturing in the dark at 25 ° C.

The resistant callus obtained above was added to MSRE agar solid medium [MS inorganic salt, MS vitamin, KNO ₃ (2830 mg / L), (NH ₄ ) ₂ S
O ₄ (463mg / L), KH ₂ PO ₄ (400mg / L), CaCl ₂ / 2H ₂ O (166mg / L),
MgSO ₄ / 7H ₂ O (185 mg / L), MnSO ₄ / 4H ₂ O (4.4 mg / L), H ₃ BO
₃ (1.6mg / L), ZnSO ₄ / 7H ₂ O (1.5mg / L), KI (0.8mg / L), FeS
O ₄ / 7H ₂ O (27.8 mg / L), Na ₂ EDTA (37.3 mg / L), glycine (2.0
mg / L), nicotinic acid (0.5 mg / L), pyridoxine hydrochloride (0.5 mg / L)
mg / L), thiamine hydrochloride (1.0 mg / L), myo-inositol (1
00mg / L), sucrose (20g / L), sorbitol (30g / L), casamino acid (2g / L), NAA (1mg / L), 2,4-D (2mg / L), acetosyringone ( 10mg / L), 0.2% gellite, claforan (500
mg / L), hygromycin (50 mg / L)
The cells were cultured in the light to induce regeneration. The redifferentiated shoots were mixed with MSHF agar solid medium (MS inorganic salts, MS vitamins, sucrose
(30 g / L), hygromycin (50 mg / L), agar (8 g / L))
To promote rooting, and cultivated recombinant plants.

A PCR reaction was performed using about 10 ng of genomic DNA prepared from the recombinant plant obtained above as a template to attempt to amplify the introduced gene sequence. PCR reaction conditions were performed according to a PCR amplification kit (TAKARA). Specifically, 10 mM Tris-HCl, 2.5 mM MgCl2, 50
mM KCl, 0.01% gelatin (pH8.3), dNTP (dATP, dGT
(P, dCTP, dTTP) A reaction solution was prepared by mixing a 2.5 mM mixture of each, ExTaq DNA polymerase (1.25 units) and 2 μM of each of two types of primers (oligonucleotides having the sequences shown in SEQ ID NOS: 5 and 6, respectively). 25 mL of reaction solution
95 ℃ using DNAengine PTC-200 (MJ Reseach)
An amplification reaction was carried out by repeating the temperature conditions for −1 minute, 53 ° C.-0.5 minute, and 75 ° C.-1 minute 30 times. PCR products are prepared according to the standard method.
% Agarose gel electrophoresis showed that
The PCR product of b was confirmed. From this band pattern, signals were detected in all test individuals from 20 individuals obtained from the regenerated plant individuals, and it was confirmed that the wasabi γ-thionine gene was incorporated (FIG. 2).

[Example 3] Analysis of transfected wasabi γ-thionine gene into transgenic rice leaves In order to perform Western blot analysis, extraction from leaves of transformed plants was performed in Tris-HCl (pH 7.5). The sample obtained was used for SDS-P
After AGE electrophoresis, it was transferred to Immobilon PSQPVDF membrane. 3%
After treatment with gelatin / PBS, the rice with a wasabi γ-thionine gene introduced was treated with a 250-fold diluted solution of a synthetic peptide (LEGARHGSCNYIFPYHRCICYFPC: SEQ ID NO: 7) prepared from the wasabi γ-thionine gene. Tween20 /
After washing with TBS, the cells were treated with anti-rabbit IgG-AP diluted 2000-fold. After washing with TBS, a color reaction was performed with BCIP and NBT and detected.

FIG. 3 shows the results of Western blot analysis of the T3 strain of rice with the wasabi γ-thionine gene. In all T3 generation plants tested, the signal strength varied, but the signal was confirmed, and the wasabi γ introduced in the T3 progeny was also observed.
Production of a protein derived from the thionin gene was confirmed.

Example 4 Disease Resistance Test of Transformed Rice A disease resistance test against blast fungus was performed as follows. Ten transformant T2 generation plants were grown by pot cultivation. The seeds tested are commonly treated as follows. 3 seeds after inoculation of seeds (T1) of inbred progeny of non-transformant and transformed rice used as control and homozygous line (22 lines) selected with hygromycin
Weekly rice plants were subjected to a blast inoculation test. For the blast fungus strain 007, spray the spore solution onto the plant and leave it in the inoculation box (humidified) for 20 hours.
%).

As another susceptibility test for blast fungus, 007 strain of blast fungus was used for punch inoculation test. The spore solution was dropped on the surface of the plant body with a 5 mm diameter punch on the leaf, left in the inoculation box (humidified) for 20 hours after inoculation, and grown at 25 ° C (humidity 90%) for 3 days. The development of the symptoms after 10 days and 14 days was examined for the resistance to the blast fungus by comparing with the non-transformant Sasanishiki and the varieties of Tsuyuake having the true resistance gene against the 007 strain of blast fungus. The results are shown in FIG. As is clear from FIG. 4, the development of the symptom was remarkably suppressed in the rice cultivated with the wasabi γ-thionine gene, as compared with the rice (Sasanishiki) of the same cultivar not introduced.

FIG. 5 shows the development of lesions 7 days after the inoculation. In the blast spore spray inoculation test of the T2 strain, a strain in which the suppression of the development of the lesion was confirmed as compared with the non-transformed Sasanishiki was confirmed. Further, when a punch inoculation test was performed using a progeny line, it was confirmed that the development of lesions was suppressed as compared with the non-transformed Sasanishiki. In these transformants, production of a protein derived from the introduced horseradish γ-thionine gene was confirmed, indicating that the introduced gene had enhanced resistance to blast fungi.

[0038]

According to the present invention, a disease resistant plant and a method for producing the same are provided.

[0039]

[Sequence List] SEQUENCE LISTING <110> Iwate prefecture <120> Disease tolerance plants transformed by Wasabi γ-thionin gene <130> P00-0797 <160> 7 <170> PatentIn Ver. 2.0 <210> 1 <211> 243 < 212> DNA <213> Wasabia japonica <220> <221> CDS <222> (1) .. (240) <400> 1 atg gct aag ttt gct tct atc atc gct ctt ctc ttc gct gct ctt gtt 4 8 Met Ala Lys Phe Ala Ser Ile Ile Ala Leu Leu Phe Ala Ala Leu Val 1 5 10 15 ctc ttt tct gct ttt gaa gca cca tca atg gtg gaa gcg cag aag ttg 9 6 Leu Phe Ser Ala Phe Glu Ala Pro Ser Met Val Glu Ala Gln Lys Leu 20 25 30 tgc gag aag tca agt ggg aca tgg tca gga gtc tgt gga aac aac aat 14 4 Cys Glu Lys Ser Ser Gly Thr Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45 gcg tgc aag aat cag tgc atc aac ctt gag gga gca cga cat gga tct 19 2 Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Gly Ala Arg His Gly Ser 50 55 60 tgc aac tat atc ttc cca tat cac aga tgt atc tgt tac ttc cca tgt 24 0 Cys Asn Tyr Ile Phe Pro Tyr His Arg Cys Ile Cys Tyr Phe Pro Cys 65 70 75 80 taa 243 <210> 2 <211> 80 <212> PRT <213> Wasabia japonica <400> 2 Met Ala Lys Phe Ala Ser Ile Ile Ala Leu Leu Phe Ala Ala Leu Val 1 5 10 15 Leu Phe Ser Ala Phe Glu Ala Pro Ser Met Val Glu Ala Gln Lys Leu 20 25 30 Cys Glu Lys Ser Ser Gly Thr Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45 Ala Cys Lys Asn Gln Cys Ile Asn Leu Glu Gly Ala Arg His Gly Ser 50 55 60 Cys Asn Tyr Ile Phe Pro Tyr His Arg Cys Ile Cys Tyr Phe Pro Cys 65 70 75 80 <210> 3 <211> 243 <212> DNA <213> Wasabia japonica <220> <221> CDS <222> (1) .. (240) <400> 3 ATG GCT AAG TTT GCT TCT ATC ATC GCT CTT CTC TTC GCT GCT CTT GTT 4 8 Met Ala Lys Phe Ala Ser Ile Ile Ala Leu Leu Phe Ala Ala Leu Val 1 5 10 15 CTC TTT TCT GCT TTT GAA GCA CCA TCA ATG GTG GAA GCG CAG AAG TTG 9 6 Leu Phe Ser Ala Phe Glu Ala Pro Ser Met Val Glu Ala Gln Lys Leu 20 25 30 TGC GAG AAG TCA AGT GGG ACA TGG TCA GGA GTC TGT GGA AAC AAC AAT 14 4 Cys Glu Lys Ser Ser Gly Thr Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45 GCG TGC AAG CAT CAG TGC ATC AAC CTT GAG GGA GCA CGA CAT GGA TCT 19 2 Al a Cys Lys His Gln Cys Ile Asn Leu Glu Gly Ala Arg His Gly Ser 50 55 60 TGC AAC TAT ATC TTC CCA TAT CAC AGA TGT ATC TGT TAC TTC CCA TGT 24 0 Cys Asn Tyr Ile Phe Pro Tyr His Arg Cys Ile Cys Tyr Phe Pro Cys 65 70 75 80 taa 243 <210> 4 <211> 80 <212> PRT <213> Wasabia japonica <400> 4 Met Ala Lys Phe Ala Ser Ile Ile Ala Leu Leu Phe Ala Ala Leu Val 1 5 10 15 Leu Phe Ser Ala Phe Glu Ala Pro Ser Met Val Glu Ala Gln Lys Leu 20 25 30 Cys Glu Lys Ser Ser Gly Thr Trp Ser Gly Val Cys Gly Asn Asn Asn 35 40 45 Ala Cys Lys His Gln Cys Ile Asn Leu Glu Gly Ala Arg His Gly Ser 50 55 60 Cys Asn Tyr Ile Phe Pro Tyr His Arg Cys Ile Cys Tyr Phe Pro Cys 65 70 75 80 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 5 tgtttctttt gtcgatgctc accctgttgt ttggt 35 <210> 6 <211> <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 6 gattgaatcc tgttgccggt cttgcgatga ttatc 35 <210> 7 <211 > 24 <212> PRT <213> Wasabia japonica <400> 7 Leu Glu Gly Ala Arg His Gly Ser Cys Asn Tyr Ile Phe Pro Tyr His 1 5 10 15 Arg Cys Ile Cys Tyr Phe Pro Cys 20

[0040]

[Sequence List Free Text]

 SEQ ID NO: 5: synthetic DNA SEQ ID NO: 6: synthetic DNA

[Brief description of the drawings]

FIG. 1 is a diagram showing the structure of a binary vector pEKH-SubWT for introducing a horseradish γ-thionine gene.

FIG. 2 is an electrophoretic photograph showing the results of PCR showing the presence of the wasabi γ-thionine gene in the rice with the wasabi γ-thionine gene of the T0 generation.

FIG. 3 is an electrophoretic photograph showing the results of PCR showing the presence of the wasabi γ-thionin gene in the rice with the wasabi γ-thionin gene of the T3 generation.

FIG. 4 is a graph showing the resistance of a rice plant into which the wasabi γ-thionine gene has been introduced to rice blast fungus.

FIG. 5 is a set of photographs showing the symptoms of a rice with the wasabi γ-thionine gene introduced thereinto and a rice with no wasabi γ-thionine gene 14 days after inoculation of the blast fungus.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiromasa Saito 4-7-55 Harmonias KB Building 202, Tokiwadai, Kitakami-shi, Iwate Prefecture (72) Inventor Minako Ito 3-2-31 Nakanocho, Kitakami-shi, Iwate M202 (72) Inventor Masahiro Nishihara 16-166-1 Murasakino, Kitakami-shi, Iwate Prefecture Princess 103 (72) Inventor Ryohei Terauchi 8-83 Ritabu, Kitakami-shi, Iwate Prefecture Laplage 301 (72) Inventor Ikuo Nakamura 648 Matsudo, Matsudo, Matsudo-shi, Chiba 204 Matsudo Dormitory, Chiba University 2F030 AA07 AD05 CA14 CA15 CA17 CA19 4B024 AA08 BA38 BA79 CA01 DA01 GA11 4H045 AA10 AA20 AA30 BA10 CA30 DA83 EA05 FA74

Claims (11)

[Claims] 1. A disease-resistant plant into which a wasabi γ-thionine gene has been introduced. 2. The disease-resistant plant according to claim 1, wherein the wasabi γ-thionine gene encodes the following protein. (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4; (b) a protein having one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 or 4 Having the physiological activity of thionine 3. The horseradish γ-thionine gene has the following (c):
Or the disease-resistant plant according to claim 1, which comprises the DNA of (d). (c) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3 (d) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3
Encoding a protein that hybridizes under stringent conditions with thionine and has the physiological activity of thionine 4. The disease-resistant plant according to claim 1, wherein the plant is selected from the group consisting of rice, wheat, gentian, apple, and lettuce. 5. The disease according to any one of claims 1 to 4, wherein the disease is selected from the group consisting of rice blast, rice blast bacterial disease, gray mold, sheath blight, rot, and spot leaf spot. Method of producing disease-resistant plants. 6. A method for producing a disease-resistant plant, comprising introducing a wasabi γ-thionin gene into a plant. 7. The method for producing a disease-resistant plant according to claim 6, wherein the horseradish γ-thionin gene encodes the following protein. (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4; (b) a protein having one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 or 4 Having the physiological activity of thionine 8. The horseradish γ-thionine gene has the following (c):
7. The method for producing a disease-resistant plant according to claim 6, which comprises the DNA of (d). (c) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3 (d) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3
Encoding a protein that hybridizes under stringent conditions with thionine and has the physiological activity of thionine 9. The method according to claim 6, wherein the wasabi γ-thionin gene is introduced into a plant by introducing a recombinant vector containing the wasabi γ-thionin gene into an embryoid body callus derived from the plant. 8. The method for producing a disease-resistant plant according to any one of 8 above. 10. The method for producing a disease-resistant plant according to claim 6, wherein the plant is selected from the group consisting of rice, wheat, gentian, apple, and lettuce. 11. The disease according to any one of claims 6 to 10, wherein the disease is selected from the group consisting of rice blast, rice blight bacterial disease, gray mold, sheath blight, rot, and spot leaf spot. Method of producing disease-resistant plants.