JP2002281979A - Stress tolerant plant - Google Patents

Abstract

(57) [Summary] (Modifications) [Problem] To provide a stress-tolerant transgenic plant caused by active oxygen. SOLUTION: The transformed plant contains a gene encoding the following protein (a) or (b). (a) a protein consisting of a specific amino acid sequence isolated from paraquat-resistant tobacco callus; (b) one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and the protein has a peroxidase activity. A stress-tolerant transgenic plant into which a gene encoding a protein having
The production method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a transgenic plant (transgenic plant) having a high resistance to active oxygen generated under the conditions of environmental stress (high / low temperature, drying, strong light, salt, air pollutant gas, pathogenic bacteria, etc.). Plant).

[0002]

2. Description of the Related Art Plants are exposed to various environmental stresses such as high and low temperatures, dryness, strong light, salt, air polluting gas, and pathogenic bacteria. However, if useful plants that can grow sufficiently under such environmental stress, such as cereals, can be developed, food production will be possible even in areas where cereals cannot grow due to environmental stress, and it will be necessary to prepare for the serious food crisis expected in the future. Likely to be. For this reason, production of plants with improved tolerance to these environmental stresses is being carried out worldwide. For example, low temperature tolerance (Natu
re, 356, 710-703, 1992, Plant Physiol. , 105,601-
605, 1994), drought resistance (Plant Physiol., 107, 125-1)
30, 1995, Nature, 379, 683-684, 1996, Nature Biote
ch., 17, 287-291, 1999), salt tolerance (Science, 259, 508)
-510, 1993, Biotechnology, 14, 177-180, 1996, Plan
t J., 12, 133-142, 1997), air pollutant resistance (Plant
Cell Physiol., 34, 129-135, 1993, Biotechnology,
12, 165-168, 1994), disease resistance (Chemistry and Biology, 37,
295-305, 385-392, 1999) have been produced by genetic recombination techniques. Pesticide resistance by genetic recombination technology (Nature, 317, 741-744, 1985, Pro
c. Natl. Acad. Sci. USA, 85, 391-395, 1988, EMBO J.,
6, 2513-2518, 1987, EMBO J., 7, 1241-1248, 1988)
Some plants provided with are commercially available.

[0003] These environmental stresses are reactive oxygen species in vivo (superoxide radical: O ^2-, hydrogen peroxide: H ₂ O _2, hydroxyl radical: OH ^-) are closely related to the occurrence of. Reactive oxygen species are generated by respiration, photosynthesis, environmental stress, and the like, and cause fatal damage to cells by excessively oxidizing proteins, nucleic acids, membrane structures, and the like. There are also reports that active oxygen-tolerant plants created by genetic recombination technology have led to the improvement of these environmental stress tolerances (Plant Physiol., 111, 1177-1181, 1
996, FEBS Letters, 428, 47-51, 1998).

[0004] Active oxygen-tolerant plants are produced mainly by introducing a gene for an enzyme for eliminating reactive oxygen species (such as superoxide dismutase, peroxidase, catalase, and glutathione reductase) into plants. Studies have also been conducted on the mechanism of paraquat tolerance in plants that are resistant to paraquat (a herbicide that generates active oxygen and has a herbicidal effect) (Pestic. Biochem. Physiol.,
26, 22-28, 1986, Theor. Appl. Genet. 75, 850-856,
1988, Plant Physiol., 91, 1174-1178, 1989). However, there has been no report that a plant regenerated from paraquat-resistant cultured cells has resistance to paraquat, and that a gene considered to be involved in the resistance was isolated. If such a gene involved in paraquat resistance is isolated, it will be resistant to active oxygen generated under various environmental stress conditions (high and low temperatures, dryness, strong light, salt, air pollutant gas, pathogenic bacteria, etc.). It seems to be useful for the development of high plants.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a stress-tolerant plant and to provide a method for imparting resistance to stress caused by active oxygen in a plant.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have focused on genes present in paraquat-resistant plants, and have found that paraquat-resistant tobacco callus (Plant Cell Physiol., 25, 1247-1254, 1984)
, A method for measuring superoxide dismutase activity using McCord and Fridovich (J. Biol. Chem., 246, 6049-
6055, 1969), and the protein was partially purified using the reoxidation pattern of cytochrome C which was specifically observed in the above as an index.

[0007] Then, they found that this protein is peroxidase, and completed the present invention by introducing a gene encoding this protein into a plant. That is, the present invention is as follows.

(1) A transformed plant containing a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) a protein having one or several amino acids deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having peroxidase activity The gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 includes the following (c) or (d)
And those containing DNA. (c) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 (d) DNA hybridizing with the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions, and encoding a protein having peroxidase activity

[0009] Plants include plant bodies, plant organs,
A plant tissue or a plant culture cell can be exemplified. The above plants include Solanaceae, Brassicaceae, Gramineae, Legumes, Cucurbitaceae, Convolvulaceae, Lilies, Lamiaceae, Asteraceae, Rosaceae, Rutaceae, Lamiaceae, Myrtaceae, Salixaceae, Rhododendronaceae, Gentian And those belonging to any of the families selected from the group consisting of the family A.

(2) A stress-tolerant plant containing a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) a protein having one or several amino acids deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having a peroxidase activity As an example, a stress caused by active oxygen can be exemplified. The stress caused by the active oxygen includes environmental stress (for example, at least one selected from the group consisting of high temperature stress, low temperature stress, drought stress, high light stress, salt stress, air pollution stress, pesticide stress and disease stress). Is included.

(3) A method for imparting stress tolerance to a plant, or a method for producing a stress-tolerant plant, comprising incorporating a gene encoding the following protein (a) or (b) into a plant. (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4; (b) one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and a peroxidase activity In the above method, examples of a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 include a DNA containing the following DNA (c) or (d). (c) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 (d) DNA that hybridizes with the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions and encodes a protein having peroxidase activity Is the same as above. Hereinafter, the present invention will be described in detail.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a transgenic plant incorporating a callus-derived peroxidase gene which is resistant to paraquat which is a herbicide which generates active oxygen and has a herbicidal effect. This is a method for imparting active oxygen tolerance of a plant, which is characterized by being incorporated into a plant gene.

As an example, the peroxidase gene of the genus Tobacco is shown. Similar to plants belonging to the genus Tobacco, other plant species encode a peroxidase derived from plant callus that is resistant to paraquat, and it is considered that a peroxidase gene induced by paraquat treatment also exists in plants. Can be

The transformed plant of the present invention has a gene encoding the amino acid sequence shown in SEQ ID NO: 2 incorporated therein. However, there may be some difference in amino acid sequence between plants depending on the cultivar or the like. Also, even in the same plant variety, amino acids may change due to mutation or the like. Therefore, in the present invention, a trait containing a gene encoding an amino acid sequence in which a plurality (one or several, for example, 1 to 10) of the amino acid sequences shown in SEQ ID NO: 2 have been substituted, deleted or added Converted plants are also included in the present invention.

The transformed plant of the present invention is, for example, SEQ ID NO: 1.
Has the peroxidase gene shown in FIG. However, the present invention is not limited to the gene, and includes all genes encoding the amino acid sequence shown in SEQ ID NO: 2.

1. (1) Preparation and Screening of cDNA Library In the present invention, the gene to be introduced into a plant is McCo
The protein is partially purified based on the reoxidation pattern of cytochrome C, which is specifically observed in the superoxide dismutase activity measurement method by rd and Fridovich, and cloned from a library prepared from paraquat-resistant tobacco callus based on its amino acid sequence. be able to. Paraquat-resistant tobacco callus means tobacco callus that can grow and proliferate even when paraquat, a herbicide, is added to a medium at 125 μM or more (PlantCell Physiol., 25, 1).
247-1254, 1984).

[0017] Extraction of mRNA from tobacco callus and preparation of a cDNA library can be performed according to a conventional method. mR
Sources of NA include, for example, paraquat-resistant tobacco callus, but are not limited thereto. mR
Preparation of NA can be performed by a commonly used technique. For example, from the above-mentioned source, after extracting total RNA by a guanidium thiocyanate-cesium trifluoroacetate method or the like, an affinity column method using oligo dT-cellulose or poly-U-sepharose, or a poly (batch method) A) + RNA (mRNA) can be obtained.
Furthermore, poly (A) + RNA may be further fractionated by sucrose density gradient centrifugation or the like.

Using the mRNA thus obtained as a template, a single-stranded DNA using oligo dT primer and reverse transcriptase
After synthesizing the cDNA, a double-stranded cDNA is synthesized from the single-stranded cDNA. Next, the obtained double-stranded cDNA is inserted into an appropriate cloning vector to prepare a recombinant vector. Then, Escherichia coli or the like is transformed with the recombinant vector,
By selecting a transformant using tetracycline resistance, ampicillin resistance or the like as an index, a cDNA library can be obtained. Here, E. coli was transformed by the method of Hanahan (J. Mol. Biol. 166: 557-580, 198).
3) That is, the method can be carried out by a method of adding a recombinant vector to competent cells prepared in the presence of calcium chloride, magnesium chloride or rubidium chloride. When a plasmid is used as a vector, it is necessary to contain a drug resistance gene such as tetracycline or ampicillin. In addition, cloning vectors other than plasmids, for example, λ phage (λgt11 and the like) can also be used.

To select a strain having the desired DNA from the transformants obtained as described above, for example, a degenerate sense primer and a degenerate antisense primer corresponding to the amino acid sequence of the peroxidase protein family are synthesized. PCR is performed using the obtained fragment, and the obtained fragment is used as a probe to screen from a cDNA library. Alternatively, when λ phage (λgt11 or the like) is used, a primer for amplifying λgt11 insert is used.
A method of performing PCR can be employed. However, the present invention is not limited to these primers. The primer can be prepared by chemical synthesis.

The amplified DNA fragment thus obtained is
^The probe can be screened by labeling with ³² P, ³⁵ S, biotin, or the like, hybridizing it to a nitrocellulose filter on which the DNA of the transformant has been denatured and fixed, and searching for the obtained positive strain.

(2) Preferably, the oligonucleotide having the sequence shown in SEQ ID NO: 5 among the cloned partial sequences and
PCR is performed between the cDNA population ligated to A and the oligonucleotide having the sequence of the λ phage DNA to obtain a longer partial sequence. It is desirable to prepare a probe by PCR with reference to the obtained sequence and to screen the gene from a cDNA library.

Examples from the partial purification of the protein to the determination of the amino acid sequence are described in Examples 1 and 2. Extraction of mRNA and preparation of cDNA library are described in Example 3.
A specific example is shown in FIG. The production of the probe is described in Example 4.
Specific examples of screening of the gene from the cDNA library are shown in Example 5. The induction of this gene by tobacco treatment in tobacco plants is based on the expression analysis technique Northern blot analysis or RT.
-It can be confirmed by the PCR method or the like. A specific example is shown in Example 6 for the confirmation.

Next, a full-length cDNA is cloned from the obtained clone. For cloning cDNA, for example, RA
The CE (Rapid Amplification of cDNA ends) method is used. The RACE method is a method for rapidly recovering the 5 'or 3' deletion site of cDNA by PCR. Incidentally, RACE method, commercially available kit (Marathon ^TM cDNA Amplification Kit (Clonetech
)).

(2) Determination of Nucleotide Sequence In the present invention, the cDNA base sequence of the isolated cDNA clone obtained in the above screening is determined using the PCR product as a template. The nucleotide sequence can be determined by a known method such as the Maxam-Gilbert chemical modification method or the dideoxynucleotide chain termination method using M13 phage. Usually, an automatic nucleotide sequencer (for example, ABI373 sequencer manufactured by Applied Biosystems, etc.) ) Is used for sequencing.

SEQ ID NO: 1 or 3 contains the nucleotide sequence of the peroxidase gene (hereinafter referred to as peroxidase gene) contained in the transformed plant of the present invention, and SEQ ID NO: 2 or 4 contains the nucleotide sequence of the peroxidase contained in the transformed plant of the present invention. Although the amino acid sequence is exemplified, as long as a protein containing this amino acid sequence has peroxidase activity, mutations such as deletion, substitution, and addition may occur in a plurality, preferably one or several amino acids 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 or 4 may be deleted. -10, preferably 1-5 amino acids may be added, or 1-10, preferably 1-5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 are replaced by other amino acids May be.

In the present invention, peroxidase activity means the activity of catalyzing the reaction of donor + H ₂ O ₂ → oxd. Donor + 2H ₂ O, and guaiacol and pyrogallol are used as donor electron donors. , Ascorbic acid, cytochrome C, glutathione, FAD, NADH, NADPH
And others. oxd. donor means donor oxide. The activity of the protein of the present invention can be confirmed by adding the electron donor and H ₂ O ₂ to a reaction solution containing the protein, and measuring the change in absorbance of the reaction solution to determine the presence or absence thereof. It is possible (Koji Asada, Minoru Nakano, Katsuko Kakinuma, ed., Manual for active oxygen measurement, 1992, 205-222pp,
Kodansha Scientific, Tokyo).

[0028] The present invention also includes a transformed plant which is a DNA capable of hybridizing with the above-mentioned peroxidase gene under stringent conditions and which contains a DNA encoding a protein having peroxidase activity. Stringent conditions refer to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, nucleic acids with high homology,
That is, the condition is such that complementary strands of DNA having homology of 60% or more, preferably 80% or more hybridize, and complementary strands of nucleic acids having lower homology do not hybridize. More specifically, the sodium concentration is 150 to 900 mM,
Preferably, it is 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.

In order to introduce a mutation into the peroxidase gene, a known method such as the Kunkel method or the gapped duplex method or a method similar thereto can be adopted. For example, using a mutagenesis kit using site-directed mutagenesis (for example, Mutan-K (manufactured by TAKARA) or Mutan-G (manufactured by TAKARA)) or the like, or using TAKARA's LA PCR in v
Mutations are introduced using the itro Mutagenesis series kit.

Once the nucleotide sequence of the peroxidase gene is determined, it is subsequently synthesized by chemical synthesis, by PCR using a cloned cDNA as a template, or by hybridization using a DNA fragment having the nucleotide sequence as a probe. A peroxidase gene can be obtained. Furthermore, a modified DNA encoding peroxidase can be synthesized by site-directed mutagenesis or the like.

2. Preparation of Recombinant Vector and Transformant (1) Preparation of Recombinant Vector A recombinant vector containing a peroxidase gene can be obtained by ligating (inserting) a peroxidase gene into an appropriate vector. The vector for inserting the peroxidase gene is not particularly limited as long as it can be replicated in the host, and examples thereof include plasmid DNA and phage DNA.

As the plasmid DNA, plasmids derived from E. coli (for example, pBR322, pBR325, pUC118, pUC119, pUC
18, pUC19, etc.) and plasmids derived from Bacillus subtilis (for example, pUB11
0, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp
24, YCp50, etc.), and the phage DNA is λ
Phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt1
0, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.

In order to insert a peroxidase gene into a vector, first, purified DNA is cut with an appropriate restriction enzyme, inserted into an appropriate vector DNA restriction enzyme site or a multiple cloning site, and ligated to the vector. Is adopted.

The peroxidase gene needs to be incorporated into a vector so that the function of the gene is exerted. Therefore, vectors include cis-elements such as enhancers, splicing signals, poly-
A Additional signal, selectable marker, ribosome binding sequence (S
D sequence) can be linked. In addition, examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.

(2) Preparation of Transformed Plant A transformed plant containing a peroxidase gene can be obtained by introducing a recombinant vector containing a peroxidase gene into a plant so that the gene of interest can be expressed. The transformed plant of the present invention has a peroxidase gene incorporated into its own gene,
It is produced as follows.

In the present invention, a plant to be transformed may be a whole plant or a plant organ (eg, leaves, petals, stems, roots,
Seeds), plant tissue (e.g., epidermis, phloem, soft tissue, xylem,
Vascular bundle, palisade tissue, spongy tissue, etc.) or cultured plant cells. Plants used for transformation include, for example, Solanaceae, Brassicaceae, Poaceae, Legumes, Cucurbitaceae, Convolvulaceae, Lilies, Lamiaceae, Compositae,
Examples include plants belonging to the family Rosaceae, Rutaceae, Labiatae, Myrtaceae, Salixaceae, Rhododendronaceae, Gentianaceae, and Rhododendronaceae (see below), but are not limited to these plants.

Solanaceae: tobacco (Nicotiana tabacum L.),
Eggplant (Solanum melongena L.), tomato (Lycopersicon es)
culentum Mill), peppers (Capsicum annuum L. var.gro.
ssum), potato (Solanum tuberosum L.), petunia (Petunia hybrida Vilm.) Brassicaceae: Arabidopsis thaliana
, Brassica (Brassica campestris L.), Cabbage (Bras
sica oleracea L.var.capitata L.), broccoli (Bra
ssica oleracea L.var.botrytis L.), Chinese cabbage (Brassica c
ampestris L.var.amplexicaulis), wasabi (Eutrema was
abi) Gramineae: rice (Oryza sativa L.), corn (Zea ma
ys L.), wheat (Triticum aestivum L.), barley (Hordeum
vulgare L.) Legumes: soybean (Glycine max L.), adzuki beans (Vigna angu
laris Willd.), cowpea (Vigna unguiculata), Lotus japonicus (Lotus corniculatus L.var.japonicus Regel), acacia (Acacia mangium Willd.) Cucurbitaceae: Cucumber (Cucumis sativus L.), melon (Cucu)
mis melo L.) Convolvulaceae: Sweet potato (Ipomoea batatas) Lily family: Leek (Allium fistulosum L.) Sericaceae: Carrot (Daucus carota L.) Asteraceae: Chrysanthemum morifolium, lettuce (Lac)
tuca sativa L.) Rosaceae: Rose (Rose hybrida Hort.), Peach (Prunus pers.
ica), apple (Malus pumila Mill) Rutaceae: Citrus unshiu Lamiaceae: Lamiaceae (Perilla frutescens Britt.var.crispa) Futomomolidae: Eucalyptus (Eucalyptus globulus Labill) Salix: Poplar (Populas nigra L. var. italica Koehn
e) Acalyptaceae: Spinach (Spinacia oleracea L.) Gentianaceae: Gentian (Gentiana scabra Bunge var.bue)
rgeri Maxim.) Carabaceae: Carnation (Dianthus caryophyllus)
L.) The above recombinant vector can be introduced into a plant by a conventional transformation method, for example, Agrobacterium method, particle gun method, PEG method, electroporation method and the like. For example, when the Agrobacterium method is used, the constructed plant expression vector is introduced into an appropriate Agrobacterium, for example, Agrobacterium tumefaciens, and this strain is transformed into a leaf disk method (by Hirofumi Uchimiya). , Plant Gene Manipulation Manual, 1990, 27-31pp, Kodansha Scientific, Tokyo)
A transgenic plant can be obtained by infecting aseptically cultured leaf pieces of tobacco according to the method described above.

When the particle gun method is used, plants, plant organs, and plant tissues themselves may be used as they are, may be used after preparing sections, or may be used after preparing protoplasts. Good. The sample prepared in this manner is transferred to a gene transfer device (for example, PDS-1000 (BIO-RAD)
Etc.). The treatment conditions vary depending on the plant or the sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

When a plant culture cell is used as a host,
For transformation, the recombinant vector is introduced into cultured cells by a particle gun method, an electroporation method, or the like. The tumor tissue, shoot, hairy root, etc. obtained as a result of the transformation can be used for cell culture, tissue culture, or organ culture as it is. Plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene,
(Eg, brassinolide) can be regenerated into plants.

Whether or not the gene has been incorporated into the plant can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from the transformed plant, PCR is carried out by a DNA-specific primer is designed. PCR can be performed under the same conditions as those used for preparing the plasmid. Thereafter, subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, or the like amplification product, ethidium bromide, SYBR Gre
Transformation can be confirmed by staining with an en solution or the like, and detecting the amplified product as a single band. Alternatively, PCR can be performed using a primer that has been labeled with a fluorescent dye or the like in advance to detect an amplification product. Furthermore, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence or an enzymatic reaction can also be adopted.

3. Stress-tolerant plant When the peroxidase gene or a recombinant vector incorporating the gene is introduced into a plant, the resulting transformed plant acquires resistance to stress. Therefore, the transformed plant can be used as a stress-tolerant plant. Here, examples of the stress include stress based on active oxygen. The stress based on active oxygen means a stress based on environmental stress that generates active oxygen in a plant body, and means a degree of growth inhibition or death when a wild-type plant is left in this environment. For example, artificially, there is a stress state when a plant is cultured in a medium containing 1 μM or more of an active oxygen generating herbicide (paraquat) or the like. The stress-tolerant plant of the present invention can grow even under the above-described conditions of active oxygen.

Active oxygen is generated when exposed to various environmental stresses. As environmental stress, high temperature stress,
Examples include low-temperature stress, drought stress, high-light stress, salt stress, air pollution stress, pesticide stress, disease stress, and the like, and these stresses may be used alone or in combination.

The term "high temperature stress" means a stress when a condition of 35 ° C. or more is continuously or temporarily applied for several minutes or more. “Low-temperature stress” means stress when a condition of 10 ° C. or less is continuously or temporarily applied for several minutes or more.

"Drying stress" refers to stress when water depletion is sustained or temporarily applied. "High light stress" means stress when plants are irradiated with strong light exceeding photosynthetic ability, for example, 20%.
This corresponds to the case where light of 00 μmol / s / m ² or more is continuously or temporarily irradiated.

The term "salt stress" refers to stress when the physiological function of a plant is damaged, for example, the salt accumulated in the soil lowers the water potential of the soil and the plant cannot absorb water. "Air pollution stress"
The term means the stress when air pollutants (such as ozone, PAN: peroxyacetyl nitrate, sulfur dioxide, hydrogen fluoride and ethylene) are continuously or temporarily loaded. "Pesticide stress" means the stress when a plant body is in continuous or temporary contact with a pesticide. The pesticides include herbicides that generate active oxygen such as paraquat and exhibit a herbicidal effect. "Disease stress" means the stress when harmed by mold or the like,
Examples of the harm include soybean purpura.

A stress-tolerant plant can be produced by breeding a transgenic plant into which a peroxidase gene has been incorporated (transgenic plant) to such an extent that it can be used as the stress-tolerant plant. In this case, under the conditions in which the above-mentioned various stresses are generated for the plant, a plant that does not damage the physiological function and that is resistant without causing growth inhibition or withering may be selected. The time to start using the plant as a stress-tolerant plant may be any time after selecting a plant exhibiting tolerance.

[0047]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention should not be limited by these Examples.

Example 1 Purification of Peroxidase from Paraquat-Resistant Tobacco Callus (1) Propagation of Paraquat-Resistant Tobacco Callus Paraquat-resistant tobacco callus was obtained from α-naphthaleneacetic acid (NA
A) (10 μM), benzylaminopurine (BAP) (1 μM), paraquat (125 μM) and 0.8% agar (or 0.25% GELLAN GUM)
Murashige & Skoog (MS) (Physiol. Plant. 15,
473-497, 1962) and cultured at 25 ° C. under illumination of about 1000 lx (light 16 hours, dark 8 hours). Calli were transplanted and grown approximately every 20 days. The grown calli were stored at -80 ° C until use. As the paraquat, a commercially available product name “Gramoxone” (manufactured by Takeda Pharmaceutical Co., Ltd.) was used.

(2) Activity measurement A method for measuring the activity of superoxide dismutase (SOD) by McCord and Fridovich (J. Biol. Chem., 246, 6049-6)
055, 1969), a xanthine-xanthine oxidase system was used as the O ²⁻ generation system, and a cytochrome C reduction system was used as the O ²⁻ reaction system. The peroxidase protein was purified using the reoxidation pattern of cytochrome C specifically observed in the above-mentioned SOD activity measurement method as an index.

(3) Partial Purification of Peroxidase from Paraquat-Resistant Tobacco Callus 500 ml of 50 mM PEM (phosphate buffer, pH 7.8; 1 mM EDTA; 1 mM β-mercaptoethanol)
With Waring Blender (DX-11: Nippon Seiki)
The milled liquid is filtered with gauze, and the filtrate is filtered at 6000 rpm, 1
The mixture was centrifuged at 4 ° C for 0 minutes (J-6M: manufactured by BECKMAN). Acetone was added to the supernatant 3 at a ratio of 7 at −20 ° C., allowed to stand in ice for 1 hour, and then centrifuged in the same manner as above to precipitate 40 m
and suspended in 5 l of the same buffer overnight. After dialysis, this was centrifuged at 10,000 rpm for 10 minutes at 4 ° C. (J2-21M / E: manufactured by BECKMAN), and the supernatant was subjected to 10 mM PEM.
Was added to a DEAE-Sephadex (manufactured by Pharmacia) column equilibrated in, and eluted with 10 mM PEM.

(NH ₄ ) ₂ SO ₄ was added to the unadsorbed fraction (60 g / 100 g).
The mixture was salted out overnight, centrifuged as above, and the precipitate was suspended in 10 mM PEM and dialyzed against 5 L of PEM overnight.
This was centrifuged as above to obtain a supernatant (10 ml).
The above operation was performed under low temperature conditions (4 ° C.).

For purification of peroxidase, Pharmacia's FPLC system was used, and the column was used as hydrophobic chromatography using Hi Trap Phenyl Sepharose HP (5 ml).
And Phenyl Superose HR 5/5, copper (0.1M CuSO ₄ ) was retained as affinity chromatography
Hi Trap Chelating (5 ml) was used.

The supernatant obtained above was replaced with 50 mM phosphate buffer, pH 7.0; 1.7 M (NH 4) ₂ SO ₄ and Hi Trap Phenyl
Run on Sepharose HP (5 ml), 50 mM phosphate buffer, pH 7.0
After eluted linearly with, the active fraction was collected.

After the active fraction was desalted and concentrated, it was replaced with 20 mM phosphate buffer, pH 8.5; 1 M NaCl. Hi Trap Chelating with the substituted active fraction holding copper (0.1 M CuSO ₄ )
(5 ml), and eluted linearly with 20 mM phosphate buffer, pH 8.5; 1 M NH ₄ Cl. Then, the active fraction as an unadsorbed fraction was collected. Next, the active fraction was again subjected to 50 mM phosphate buffer, pH 7.0;
After substituting with 1.7 M (NH ₄ ) ₂ SO ₄ and applying to Phenyl Superose HR 5/5 and eluting stepwise with 50 mM phosphate buffer, pH 7.0, the active fraction was collected. The collected active fraction is concentrated
SDS-PAGE (general Laemmli system, gel concentration: 12%) was performed, but was not completely purified. Therefore, the active fraction was used as a partially purified product. For protein quantification, use Brad
The ford method (PROTEIN ASSAY kit: manufactured by BIO-RAD) was used.

Example 2 Determination of Amino Acid Sequence of Partially Purified Product (1) Preparation of Sample for Enzymatic Digestion Trichloroacetic Acid (TCA) Precipitation The partially purified product (about 50 μg) obtained in Example 1 was reduced to about 50 μg.
After desalting and concentrating to a concentration of g / 100 μl, a final concentration of 10% TCA aqueous solution was prepared, and the mixture was allowed to stand on ice for 30 minutes. Thereafter, the mixture was centrifuged with a microcentrifuge (manufactured by TOMY), the supernatant was removed, and the precipitate was washed twice with 100 μl of cold acetone and air-dried.

Carboxamidomethylation 50 μl of an 8 M urea-0.4 M ammonium bicarbonate solution was added to the dried protein, and the pH was confirmed (pH: 7.5 to 8.5).
5 μl of dithiothreitol was added and incubated at 50 ° C. for 15 minutes. After the incubation, the temperature was returned to room temperature, 5 μl of 100 mM iodoacetamide was added, the mixture was further incubated at room temperature for 15 minutes, and 140 μl of water was added.

(2) Decomposition into peptides by trypsin and lysyl endopeptidase Trypsin treatment Trypsin (Sequencing Grade Modified Trypshin: Pr
omega) 20 μg (9300 units / mg protein)
It was dissolved in 0.1N HCl, 1 μl (9.3 units) of the solution was added to the sample prepared in (1), and the mixture was incubated at 37 ° C. for 24 hours. After incubation, the samples were stored frozen until use.

Lysyl endopeptidase treatment 0.71 g of lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.)
(2.8 AU / mg) was dissolved in 1 ml of Tris-HCl buffer (pH 9.0), 5 μl (0.01 AU) was added to the sample prepared in (1), and the mixture was incubated at 37 ° C. for 24 hours. After incubation, the samples were stored frozen until use.

(3) Separation of peptide by HPLC A reversed phase column HighPore RP-304 (4.6 mm id x 250 m) was connected to a Waters HPLC system (Waters 600E, 484, 741: manufactured by Waters).
m: manufactured by BIO-RAD), 50 μl of each sample decomposed by trypsin and lysyl endopeptidase treatment
Was applied to separate the peptides. The separation conditions are shown below.

Buffer A = 0.06% trifluoroacetic acid (TFA), water Buffer B = 0.052% TFA, 80% acetonitrile UV detection = 210 nm Gradient: 0-60 min 0-40% B 60-90 min 40-75 % B 90-105 min 75-100% B Flow rate: 0.5ml / min

(4) Determination of Amino Acid Sequence of Peptide The amino acid sequence of each centrifuged peptide was determined by an automatic gas phase protein sequencer (model 473A, manufactured by Applied Biosystems) (SEQ ID NOs: 6 to 13). When this amino acid sequence was searched in the protein amino acid database, peanut peroxidase PNC2
(Proc. Natl. Acad. Sci. USA 87, 8874-8878, 1990).

Example 3 Preparation of Paraquat-Resistant Callus cDNA Library (1) Extraction and Purification of mRNA from Paraquat-Resistant Callus Preparation of RNA from Paraquat-Resistant Tobacco Callus RNA was prepared by cloning and sequencing / plant biotechnology experiments. The phenol-SDS method combining sodium acetate precipitation and ultracentrifugation with a cesium chloride solution described in a manual (Rural Bunkasha, 1989) was used.

Paraquat-resistant calli (about 20 g) were frozen with liquid nitrogen and powdered using a mortar pestle. Liquid nitrogen was appropriately added to the mortar so that the callus did not melt during the operation.
Extraction buffer (40 ml), phenol / chloroform (8
0 ml) The powdered callus was placed in the mixed solution, and crushed and extracted with a polytron.

The crushed extract was dispensed into a centrifuge tube (50 ml, manufactured by Corning) and centrifuged at 3000 rpm for 30 minutes at room temperature (GP: manufactured by BECKMAN). The aqueous layer (upper part) was collected in a centrifuge tube, phenol / chloroform (20 ml) was added, and the mixture was shaken for about 10 minutes. After shaking, centrifuge as above (15
Min) to collect the aqueous layer. Add 2 more drops until there is no white precipitate between the aqueous and phenol / chloroform layers.
The same operation was performed once.

The collected aqueous layer was further mixed with chloroform (20 m
After l) was added and mixed, the mixture was centrifuged in the same manner as above to collect an aqueous layer. Add 1/10 volume 3M sodium acetate to the collected aqueous layer,
The mixture was allowed to stand at -80 ° C for 10 minutes or more to precipitate polysaccharide. 600 after standing still
The mixture was centrifuged at 0 rpm for 15 minutes at 4 ° C (J2-21M / E: manufactured by BECKMAN), and the supernatant was recovered. Centrifugation was performed again to remove the polysaccharide. Add 2.5 volumes of ethanol to the collected aqueous layer,
Left at ℃ for more than 30 minutes.

After standing, the mixture was left standing at room temperature until the liquid temperature reached 15 ° C. or higher to dissolve it. After dissolution, the precipitate was collected by centrifugation at 6000 rpm for 15 minutes at 4 ° C. 20 ml of 70% ethanol was added to the precipitate, and the precipitate was rinsed and centrifuged at 6000 rpm for 5 minutes at 4 ° C. The collected precipitate was dried under reduced pressure (about 3 minutes). The precipitate dried under reduced pressure was dissolved in TE / HPRI (8 ml). Dissolve 60
Centrifugation was performed at 00 rpm for 5 minutes at 4 ° C to remove dust and collect the supernatant. 10 M lithium chloride (2 ml) was added to the collected supernatant, and left at −80 ° C. for 1 hour or more to precipitate RNA. When the precipitation was poor, ethanol precipitation was performed again, and the above operation was performed again with the scale of lithium precipitation reduced to 1/4. The collected precipitate (RNA) was dissolved in TE / HPRI (3 ml). Dissolve 6
Centrifugation was performed at 000 rpm for 5 minutes at 4 ° C to remove dust and collect the supernatant (RNA solution). Electrophorese a portion of the RNA solution (1
% Agarose, TBE (89 mM Tris-borate, pH 8.3; 2 mM EDT
A) Buffer) and RNA were not degraded. In addition, the concentration and purity were assayed using a spectrophotometer (DU-70: manufactured by BECKMAN). Next, ultracentrifugation was performed as follows to increase the purity of RNA.

Ultracentrifuge tube (13.5 ml: manufactured by BECKMAN)
A cesium chloride solution (3.6 ml) was added to the mixture, and the RNA solution was overlaid. After the upper layer of the RNA solution, the tube was filled with TE / HPRI and sealed. After sealing the tube, use the angle rotor (90TI:
Ultracentrifugation was performed at 48,000 rpm for 16 hours at 16 ° C. using a BECKMAN (XL-90: BECKMAN). The tube contains
It is marked so that the direction of centrifugation can be identified.

After ultracentrifugation, about half of the cesium chloride solution was withdrawn with a syringe. The tube was cut in half with a scalpel, and the remaining cesium chloride solution was carefully removed with care for unprecipitated walls. Unsettled walls were wiped off with an autoclaved Kimwipe. The RNA pellet was quickly washed with sterile water (4 ° C) (twice). The RNA pellet was dissolved in TE / HPRI (0.8 ml). A part of the RNA solution was subjected to electrophoresis, and it was confirmed that the RNA was not degraded. In addition, the concentration and purity were examined using a spectrophotometer. Except for immediate use, it was stored in an ethanol precipitated state (-80 ° C). Finally, about 1.7 mg of RNA was obtained.

Extraction buffer: 200 mM Tris-HCl, pH 9.0;
100 mM NaCl; .10 mM EDTA; 0.5% SDS; 14 mM β-mercatorethanol TE / HPRI: 10 mM Tris-HCl, pH7.5; .1 mM EDTA; 5 units / m
l RNase inhibitor; 1mMDTT cesium chloride solution: TE / HPRI containing 5.7M cesium chloride

Preparation of Poly (A) ⁺ RNA Using Oligo (dT) Cellulose Column Poly (A) ⁺ RNA was prepared using mRNA Purification Kit (Pharm
acia Biotech). After precipitation of RNA (about 850 μg) with ethanol, elution buffer (Elution buffer) (1m
l), treated at 65 ° C. for 5 minutes, placed on ice, added with a sample buffer (Samplebuffer) (0.2 ml), and gently mixed.

An RNA sample was applied to the column and soaked in the gel. With the column set in a centrifuge tube,
The mixture was centrifuged at 350 × g for 2 minutes (GP: manufactured by BECKMAN). After centrifugation, a high-salt buffer (0.25 ml) was added and centrifuged as above. This operation was performed again. Add low-salt buffer (0.25ml),
Centrifuge as above. This operation was performed three times. A new centrifuge tube was set, an elution buffer (0.25 ml) was added, and the mixture was centrifuged as above. This operation was performed four times, and the eluate was collected. The elution buffer was previously warmed at 65 ° C.

The eluate was subjected to ethanol precipitation and dissolved in sterilized water. A portion was electrophoresed and the concentration was determined by a spot test using ethidium bromide. After purification, about 10
μg of poly (A) ⁺ RNA was recovered.

[0073] High-salt buffer: 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.5 M NaCl Sample buffer: 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 3.0 M NaCl Low-salt buffer: 10 mM Tris-HCl ( pH 7.4), 1mM EDTA, 0.1M NaCl Elution buffer: 10mM Tris-HCl (pH 7.4), 1mM EDTA

(2) Synthesis of cDNA from poly (A) ⁺ RNA For the synthesis of cDNA, ZAP-cDNA SYNTHESIS KIT (STRATAGENE
Was used. The poly (A) ⁺ RNA (5
μg), first-strand synthesis, second-strand synthesis, blunting of synthesized cDNA, ligation with EcoRI adapter, phosphorylation of EcoR I-terminal, restriction enzyme (Xho I) treatment, and cDNA Spun Column (Pharma
cDNA was purified using cia.

After purification, phenol / chloroform extraction and ethanol precipitation were performed, and the precipitate was dissolved in 6.0 μl of sterilized water. A part of this was electrophoresed, and the concentration was determined by a spot test using EtBr. The rest was stored at -20 ° C.
Finally, the cDNA had a concentration of about 50 ng / μl (about 300 ng in total).

(3) Preparation of cDNA Library To prepare a cDNA library, use the ZAP-cDNA SYNTHESIS KIT
(Manufactured by STRATAGEN), Gigapack II Gold Packaging Extr
act (manufactured by STRATAGENE) was used.

Ligation of cDNA and Vector The cDNA (100 ng) obtained in (2) was ligated at a ratio of 1 μg of Uni-ZAP XR vector.

In vitro packaging 1 μl of the ligation solution (5 μl) obtained above
Was used for Gigapack II Gold Packaging Extract. Ligation solution to Freeze / Thaw extract immediately after dissolution
One microliter was added and thawed on ice. Immediately, 15 μl of Sonic extract was added, pipetted and mixed well. After gently centrifuging, it was left at room temperature for 2 hours. 500 μl of phage dilution buffer (SM buffer) was added, and 20 μl of chloroform was further added and mixed. Storage was performed at 4 ° C.

Phage plating E. coli (XL1-Blue and SURE) were prepared using LB / tetracycline.
(12.5 [mu] g / ml) and incubated 37 ° C. overnight in agar medium, single colonies were 30 ° C. shaking culture in LB medium supplemented with 0.2% maltose and 10 mM MgSO _4. The cells were collected by centrifugation at 1000 × g for 10 minutes (J-6M: manufactured by BECKMAN) and suspended in 10 mM MgSO ₄ (when used, diluted to OD666 = 1.0).

The packaging solution obtained in the in vitro packaging was diluted 10-fold with a phage dilution buffer, and the diluted solution (1 μl) and the host cells (200 μl) were incubated at 37 ° C. for 15 times.
The cells were cultured separately. Add the above bacteria to 3ml Top Agar (48 ℃)
Immediately plated on NZY Agar Plate warmed to 37 ° C. After overnight culture at 37 ° C., the plaque forming ability of the phage was determined from the number of plaques after the culture. Also Top Ag
ar to 15 μl of 0.5 M IPTG (in H ₂ O) and 50 μl of 250 mg / ml Xg
al (in DMF) was added, and the above operation was performed to determine the recombination frequency of the library (color selection). In this example, SURE was used for determining plaque forming ability, and XL1-Blue was used for color selection. As a result of phage titration, a cDNA library of 1.6 × 10 ⁶ pfu / ml was prepared. The recombination frequency by color selection was about 99%.

Example 4 Preparation of Probe (1) Acquisition of a Partial Fragment Containing the 3 'End The 3'-RACE (Rapid Amplification of cDNA Ends) method (Pro
c. Natl. Acad. Sci. USA 85, 8998, 1988) to obtain a partial fragment containing the 3 'end of the gene.

That is, poly (A) ⁺ RNA (1 μg) obtained from paraquat-resistant callus and dT11-adapter primer having an adapter sequence at the 5 ′ end (RD29 primer: NEB
Reverse Transcriptase SuperScript II
(GIBCO BRL) to perform a reverse transcription reaction to synthesize first-strand cDNA. Further, a degenerate primer was prepared based on the amino acid sequence obtained in Example 2, and this was used as a sense primer.
A PCR reaction was performed with the adapter sequence inside the cDNA.

Degenerate primer 1 (sense): 5'-gaycaraart
trtggac-3 ′ (SEQ ID NO: 14) TaKaRa Ex Taq (manufactured by Takara Shuzo) was used for the PCR reaction, the primer concentrations were each 0.5 μM, and the temperature condition of the PCR reaction was 94
3 cycles of ℃ (2 minutes) / 37 ° C (0.5 minutes) / 72 ° C (3 minutes), 9
35 cycles of 4 ° C (0.5 minutes) / 50 ° C (0.5 minutes) / 72 ° C (1 minute) and one cycle of 94 ° C (0.5 minutes) / 50 ° C (0.5 minutes) / 72 ° C (15 minutes) were performed. .

After the PCR reaction product was electrophoresed on a low-melting point agarose gel, the amplified band was cut out of the gel and purified. The purified PCR amplification product is the Original TA Cloning Kit
(Invitrogen) and cloned into pCRII (Invitrogen) vector. Each of the clones cloned into the pCRII vector was transformed into E. coli, and the plasmid was recovered by a conventional method.
I, HindIII) treatment, and screening was performed based on the size and orientation of the insert fragment.

As a result, a cDNA fragment of about 400 bp was obtained. A clone containing this fragment is called ABI PRISM Dye Terminat
Perform a sequencing reaction with the er Cycle Sequencing Kit (PerkinElmer) and use Genetic Analyzer A
The nucleotide sequence was determined using BI PRISM 310 (Perkin Elmer). As a result, a 334 bp DNA fragment (SEQ ID NO: 15) at the 3 ′ end including the sequence of the degenerate primer was obtained,
Approximately 75% homology was found with the amino acid sequence of peanut peroxidase (PNC2).

(2) Obtaining a partial fragment containing the 5 ′ end From (1), a partial fragment containing the 3 ′ end could be obtained. However, a longer fragment was obtained and used as a reference for preparing a probe. A partial fragment including the end was obtained. Of the partial fragment containing the 3 'end obtained from (1), primer 1D which is a sequence 3' end from the termination codon and λZAP II Insert Screening Amplim
a ligation solution in which the cDNA obtained in (2) of Example 3 was ligated to a Uni-ZAP XR vector with a 5 ′ primer contained in the er Set (manufactured by CLONTECH) as a template,
A PCR reaction was performed. Primer 1D: 5'-caagagtagtaagtacataaaaggg-3 '(SEQ ID NO: 16) 5' primer: 5'-tctagaactagtggatcccccg-3 '(SEQ ID NO: 17) TaKaRa Taq (manufactured by Takara Shuzo) was used for the PCR reaction, and the primer concentration was 0.4 μM each. , The temperature condition of the PCR
35 cycles of 5 minutes) / 50 ° C (0.5 minutes) / 72 ° C (1 minute), 72 ° C
(7 minutes) in one cycle.

After the PCR reaction product was electrophoresed on an agarose gel, the amplified band was excised from the gel, and
Purification was performed using II Kit (manufactured by BIO 101). Purified PCR
The amplified product was cloned into a pCRII (Invitrogen) vector using the Original TA Cloning Kit (Invitrogen). After transforming each clone cloned into the pCRII vector into Escherichia coli, the plasmid was recovered by a conventional method, treated with a restriction enzyme (EcoRII), and screened by the size of the insert fragment.

As a result, a cDNA fragment of about 1 kbp was obtained.
A clone containing this fragment was subjected to ABI PRISM Dye Terminater Cycle Sequencing Ki
Perform a sequence reaction with t (PerkinElmer), and
The nucleotide sequence was determined by NA sequencer ABI 373A (Perkin Elmer). As a result, a 1090 bp cDNA fragment (SEQ ID NO: 16) containing the sequence at the 3 'end (SEQ ID NO: 16)
18) was obtained. PER4, pCRI containing PER4
The I vector was called pPER4.

(3) Preparation of Probe Used for Screening of cDNA Library For synthesis of the probe, a PCR DIG probe synthesis kit (Boehringer Mannheim) was used. That is,
Based on PER4 obtained in (2), prepare the following primers, perform a PCR reaction using pPER4 as a template, DIG-11
Prepared by labeling with -dUTP. Primer PER4PCR1: 5'-atggagtactatcaccattc-3 '(SEQ ID NO: 19) Primer PER4U: 5'-tcagctgctctcgaaactcg-3' (SEQ ID NO: 20)

Example 5 Cloning of cDNA Encoding Full Length (1) Screening of cDNA Library Escherichia coli (XL1-Blue MRF ') was prepared using LB / tetracycline (12.
5 [mu] g / ml) and incubated 37 ° C. overnight in agar medium, 30 single colonies in LB medium supplemented with 0.2% maltose and 10 mM MgSO ₄
The cells were cultured with shaking at about 7 ° C for about 7 hours. The cells were collected by centrifugation at 1000 × g for 10 minutes (J-6M: BECKMAN) and suspended in 10 mM MgSO ₄ (when used, diluted to OD600 = 0.5).

In Example 3, 3 μl (about 5,0) of the cDNA library (λZAPII) prepared from paraquat-resistant tobacco callus was used.
Host bacterium (OD600 of 00Pfu) and was suspended in the 10 mM MgSO ₄ =
0.5) 200 μl were cultured at 37 ° C. for 15 minutes. 3ml Top Agar
(48 ° C), and immediately warmed to 37 ° C
Plated on NZY Agar Plate. Plate is 37
The cells were cultured at about 12 hours at about ℃ to form plaques. The plate on which the plaque was formed was refrigerated at 4 ° C. for about 2 hours, and then nitrocellulose membrane (BA83: manufactured by Schleicher & Schuell)
Was adhered for about 2 minutes, and a hole was made with an injection needle to perform marking. Thereafter, the nitrocellulose membrane was carefully peeled off the plate, and the phage DNA was copied. This nitrocellulose membrane is treated with an alkaline denaturing solution (1.5 M NaCl, 0.5 M NaO
H) for 1 minute, followed by neutralization solution (1.5 M NaCl, 0.5 M Tr
is-HCl pH7.5) for 1 minute and air-dried on filter paper. After air-drying the nitrocellulose membrane, the DNA was fixed by baking at 80 ° C. for 2 hours under reduced pressure. Hereinafter, the screening of the cDNA library used the DIG nucleic acid detection kit (Boehringer Mannheim) and the DIG-11-dUTP-labeled probe prepared in Example 4.

The nitrocellulose membrane on which the phage DNA was immobilized was placed on a hybridization solution [5 × SSC (15 mM
Sodium citrate, 150mM NaCl; pH7.0), 1% blocking reagent (nonfat dry milk fraction), 0.1% N-laurorysarcosine
sodium salt, 0.02% SDS] at 68 ° C. for 1 hour with shaking. Next, the hybridization solution was exchanged and heat-denatured DIG-11-dUTP
A labeled probe was added, and hybridization was performed at 68 ° C. for 12 hours with shaking.

After hybridization, 2 × SSC; 0.1
Wash twice with% SDS at room temperature for 5 minutes, then 0.1x SSC;
The plate was washed twice with 0.1% SDS at 68 ° C. for 15 minutes. The detection of the signal was performed using a DIG nucleic acid detection kit (coloring).

As a result, 24,000 plaques were screened, and as a result of the secondary screening, 9 positive phage clones were obtained. Also, λZAP II Insert Scre
When the insert DNA was confirmed using the ening Amplimer Set (manufactured by CLONTECH), the insert DNA of the desired size was confirmed in eight phage clones.

(2) Positive phage clone (λZAPII phag
Conversion from e) to phagemid (pBluescript) Conversion to pBluescript is performed using the ZAP-cDNA SYNTHESIS KIT (STR
ATAGENE) (The ExAssist-SOLR system) was used. XL1-Blue MRF '(OD600 = 1.0) 250 μl, λZAPII phage 100 μl (including 1 × 10 ⁵ or more phage particles), E
xAssist helper phage 1 μl (1 × 10 ⁶ pfu / ml or more)
Were mixed and cultured at 37 ° C. for 15 minutes. 2 × YT (3m
l) was added and the cells were cultured with shaking at 37 ° C. for about 4 hours.

The mixture was heat-treated at 70 ° C. for 20 minutes and centrifuged at 4000 × g for 15 minutes (J2-21M / E: manufactured by BECKMAN). The supernatant was recovered after centrifugation, and the recovered phage was stored at 4 ° C. 100 μl and 200 μl of the collected phage SOLR (OD600 = 1.0)
Were mixed and cultured at 37 ° C. for 15 minutes. 20 of the cultured bacteria
0 μl (100-fold dilution) of LB / ampicillin plate (50 μg / m
l), and cultured at 37 ° C. overnight. The colonies that appeared on the plate were collected. Five pBluescripts could be recovered from one positive phage clone (eight).

(3) Confirmation of Insert DNA and Determination of Nucleotide Sequence Restriction enzyme (X
The insert cDNA was cut out using hoI and EcoRI or KpnI and SacI), and the length of the insert cDNA was confirmed by electrophoresis. The nucleotide sequence was determined by the method described in Example 4. As a result, two types of clones were obtained, one of which was PER4
This cDNA fragment contains the same nucleotide sequence as NTPX.
C2 (SEQ ID NO: 1), and the pBluescript vector containing NTPXC2 was pNTPXC2. In addition, another cDNA fragment was
XC1 (SEQ ID NO: 3) and the pBluescript vector containing NTPXC1 was pNTPXC1.

Comparison of the homology of the two clones with the peroxidases of peanut and other plants showed that the homology with the amino acid sequence of peanut peroxidase (PNC2) was high, and that the two types of histidines present in the active center of peroxidase were used. The two types of clones were presumed to be Cationic-type peroxidases from the fact that they were conserved and the amino acids in the vicinity were also highly homologous.

Furthermore, the two clones NTPXC2 and NTPX
Amino acid sequence of C1 (SEQ ID NO: 2 and SEQ ID NO: 4, respectively)
Is the partial amino acid sequence of the peroxidase isozyme accumulated in the culture medium of tobacco suspension cultured cells (Plant Phys.
iol., 120, 371-381, 1999).
The amino acid sequence encoded by NTPXC2 (SEQ ID NO: 2) contains a peroxidase (Plant Ce) that is rapidly induced by injury.
Physiol. 41, 165-170, 2000).

Example 6 Confirmation of Induction of Expression by Paraquat Whether the peroxidase gene derived from paraquat-resistant tobacco callus was induced by paraquat in plants was confirmed by RT-PCR.

Paraquat-resistant tobacco regenerated from paraquat-resistant tobacco callus (protein nucleic acid enzyme, 33, 3184-318)
6, 1988) and its parent, Samsung (Nicotiana tabacum)
L. cv. Samsun) seeds are sown in trays containing vermiculite, grown in Coittron (manufactured by Koito Kogyo Co., Ltd.) (16 hours photoperiod), and plants 2 months after sowing (size: 15c)
m-20 cm) was sprayed with paraquat (625 μM). After paraquat spraying, return the plant body to Coittron, 500 μmol
m ^-2 sec ^-1 light was applied. After paraquat treatment,
After 3, 6, 9, 12, and 24 hours, 1 to 3 leaves (leaves 1 cm or more in size larger than the upper leaves) of each tobacco (only paraquat-resistant tobacco after 12 hours) are collected, and immediately It was frozen in liquid nitrogen (if stored, stored at -80 ° C).
Frozen leaves are finely ground in a mortar under liquid nitrogen,
Total RNA using RNeasy Plant Mini Kit (Qiagen)
Was extracted. The total RNA extracted this time and the total RNA extracted from the paraquat-resistant callus shown in Example 3 (without ultracentrifugation) were treated with DNaseI (manufactured by Amersham Pharmacia Biotech) to remove the contaminated genomic DNA and re-
Total RNA was purified using the asy Plant Mini Kit. Using the GeneAmp RNA PCR Kit (manufactured by PerkinElmer), the purified total RNA (0.4 μg) was subjected to RT-PCR reaction between the following primers, which are internal sequences of NTPXC1. Primer PER6D: 5'-cgtatttctccaacctcagg-3 '(SEQ ID NO: 21) Primer RT3D-11: 5'-atttggttaccacacaattg-3' (SEQ ID NO: 22)

Similarly, an RT-PCR reaction was performed between the following primers which are internal sequences of NTPXC2. Primer PER6D: 5'-cgtatttctccaacctcagg-3 '(SEQ ID NO: 21) Primer RT3D-51: 5'-gttggatatcacaacaattc-3'
(SEQ ID NO: 23)

After the RT-PCR reaction, a part of the reaction solution was subjected to electrophoresis [0.8% agarose, TAE (40 mM Tris-acetate, pH 7.6;
1 mM EDTA) buffer]. DNA markers include Perfect DNA
Markers (Novagen) were used. As a result, the peroxidase gene obtained this time by paraquat treatment:
It was confirmed that NTPXC1 and NTPXC2 were induced (Fig. 1
And FIG. 2).

In FIG. 1 and FIG.
The band observed at the following position (about 0.3 kb) was the peroxidase gene, and it was confirmed that the band was induced 1 hour after paraquat treatment.

Example 7 Peroxidase (NTPXC2)
Preparation of Antibody to Protein In order to confirm the expression of the peroxidase (NTPXC2) protein shown in SEQ ID NO: 2, a peptide was synthesized based on the internal amino acid sequence of peroxidase (NTPXC2) shown in SEQ ID NO: 24, and the synthesized peptide was used as an antigen. The antibody was prepared. That is, the internal amino acid sequence (SEQ ID NO: 24: Val As
p Ile Gln Lys Arg Lys Phe Leu Thr Lys Gly Leu Asn
A peptide in which Cys has been added to the carboxyl terminal of Thr) is synthesized, purified by HPLC, and then subjected to MBS (m-maleimid) via Cys.
The carrier protein (KLH: keyhole limpe) was obtained by the obenzoyl-N-hydroxysuccinimide ester) method (covalently bonding the SH group of the Cys residue on the peptide side and the amino group on the KLH side).
hemocyanin (PIERCE) was bound to the carboxyl terminus of the synthetic peptide. This synthetic peptide was administered to a rabbit as an antigen to prepare an antiserum, which was used as an antibody.

Example 8 Construction of Plant Expression Vector and Production of Transformed Plant Primer GEXPE to which the following restriction enzyme BamHI site was added
Primer pBIPER4 with R4U and restriction enzyme SacI site added
Prepare D and use pPER4 obtained in Example 4 as a template
A PCR reaction was performed. Primer GEXPER4U: 5'-cgcggatccatggagtactatcacca
ttc -3 '(SEQ ID NO: 25) Primer pBIPER4D: 5'-cgagctctcagttgatagcagagcaa
a-3 ′ (SEQ ID NO: 26) TaKaRa Ex Taq (manufactured by Takara Shuzo) was used for the PCR reaction, the primer concentration was 0.4 μM each, and the temperature condition of the PCR reaction was 95 ° C. (7
Min) for 1 cycle, 95 ° C (45 seconds) / 60 ° C (1 minute /) 72 ° C (2 minutes)
Min) in 30 cycles.

The PCR reaction product was treated with phenol / chloroform, precipitated with ethanol, and then treated with restriction enzymes BamHI and SacI. After restriction enzyme treatment, perform electrophoresis on agarose gel,
The target band is cut out of the gel and purified (QIAquick Gel
Extraction Kit: Qiagen) and NTPXC2 as an insert gene.

On the other hand, pBIN19-derived β-glucuronidase (GUS) expression binary vector pBI121 (manufactured by CLONTECH) was treated with restriction enzymes BamHI and SacI, and after the restriction enzyme treatment, electrophoresis was carried out on an agarose gel. After electrophoresis, a target band was cut out of the gel and purified, and a fragment from which the GUS gene had been removed was recovered.

[0109] Both of the above fragments were used in the Takara DNA Ligation Kit.
After ligation using (Takara Shuzo) and transformation of Escherichia coli (HB101: Takara Shuzo), a recombinant plasmid was selected to obtain a plant expression vector (pBINTPXC2).

Also, pBI121 was treated with restriction enzymes SmaI and SacI, and similarly treated with restriction enzymes, followed by electrophoresis on an agarose gel. After electrophoresis, a target band was cut out of the gel and purified, and a fragment from which the GUS gene had been removed was recovered. The recovered DNA fragment was blunt-ended using Takara DNA Blunting Kit (Takara Shuzo). The blunted DNA fragment is
After self-circularization using akara DNA Ligation Kit (Takara Shuzo) and transforming Escherichia coli (HB101: Takara Shuzo), a recombinant plasmid was selected, and pBI121 was removed from the GUS gene for plant expression. The vector (pBI121-GUS) was obtained.

Further, the expression vector for each plant constructed by the triparental mating method (Gus Gene Fusion System: manufactured by CLONTECH) was used to convert Agrobacterium tumefaciens LB.
Introduced into A4404 strain. Agrobacterium tumefaciens LBA44
The 04 strain was tobacco (Nicotiana tabacum L. cv.) By the leaf disk method (by Hirofumi Uchimiya, Plant Gene Manipulation Manual, 1990, 27-31pp, Kodansha Scientific, Tokyo).
Petit Havana SR1) Infected the aseptically cultured leaf pieces to obtain transformed tobacco (the transformed day is called T1). The resulting transformed tobacco was self-pollinated to obtain seeds (T2), the seeds were selected on a medium containing kanamycin, and similarly self-pollinated to obtain seeds (T3). Further, the seeds were selected on a medium containing kanamycin, and the transgene (NTPXC2) was one copy, a strain considered to be homozygous, and a kanamycin resistance gene (NP
Only TII) was one copy, and a line considered to be homozygous was selected. In the line into which NTPXC2 was introduced, protein expression was confirmed by an antibody that recognizes the transgene product (Example 7). Only one kanamycin resistance gene (NPTII)
A line that was a copy and appeared homozygous (transformed with pBI121-GUS) was used as a transformation control line.

Example 9 Confirmation of Paraquat Resistance of Transformed Plant The seed (T3) obtained in Example 8 was converted to paraquat (1.29 μM).
Was aseptically inoculated into an MS medium containing, and cultured for about 1.5 months in the same manner as in the growth of paraquat-resistant tobacco callus in Example 1. Untransformed plants and transformed control lines were used as controls. As a result, in one of the transgenic plants, a line having better growth than the non-transformed plant and the transgenic control line was obtained (FIG. 3).

In FIG. 3, A and E represent transformed plants (35
-5 lines), B and F are transformed plants (7-3 lines), C and G
Is a transformed control line, and D and H are untransformed plants (after 48 days of culture). The 35-5 strain is the generation T
The 35th individual at 1 means the 5th individual at T2, and the 7-3 strain is the 7th individual at the transformed T1 and 3 at the T2.
Means individual eyes.

[0114]

Industrial Applicability According to the present invention, there is provided a transgenic plant having high resistance to active oxygen generated under various environmental stress conditions (high and low temperatures, drying, strong light, salt, air polluting gas, pathogenic bacteria, etc.). You. The method of introducing a peroxidase gene contained in the present invention into a plant is resistant to active oxygen generated under various environmental stress (high / low temperature, drying, strong light, salt, air pollutant gas, pathogen, etc.). Useful for high plant development.

[0115]

[Sequence List] SEQUENCE LISTING <110> TOYOTA JIDOSHA KABUSHIKI KAISHA <120> a stress-tolerant plant <130> P00-0867 <140> <141> <160> 26 <170> PatentIn Ver. 2.1 <210> 1 <211 > 1230 <212> DNA <213> Nicotiana tabacum <220> <221> CDS <222> (61) .. (1050) <400> 1 aaaatagtag aaatttactt actcaaatct cagaattcgt ttatactgtt aacacgtacc 60 atg gag tac tat cac cat tca att aac aaa atg gcc atg ttt atg gtt 108 Met Glu Tyr Tyr His His Ser Ile Asn Lys Met Ala Met Phe Met Val 1 5 10 15 att cta gtg cta gcc ata gat gtg act atg gtt tta ggc caa gga acc 156 Ile Leu Val Leu Ala Ile Asp Val Thr Met Val Leu Gly Gln Gly Thr 20 25 30 cgt gtt gga ttt tat tcc agt acg tgc cca agg gcc gaa tcc ata gtt 204 Arg Val Gly Phe Tyr Ser Ser Thr Cys Pro Arg Ala Glu Ser Ile Val 35 40 45 caa tca acg gtg agg gct cat ttt cag tct gat cca acg gtg gca cca 252 Gln Ser Thr Val Arg Ala His Phe Gln Ser Asp Pro Thr Val Ala Pro 50 55 60 gga atc ctg aga atg cac ttc cat gat tgt ttt gta cta ggt tgt gat 300 Gly Ile Leu Arg Met His Phe His Asp Cys P he Val Leu Gly Cys Asp 65 70 75 80 ggt tct atc ctc att gaa ggt tca gac gct gag aga act gct atc cca 348 Gly Ser Ile Leu Ile Glu Gly Ser Asp Ala Glu Arg Thr Ala Ile Pro 85 90 95 aac aga aat ttg aaa gga ttc gac gtt att gag gat gct aag acg caa 396 Asn Arg Asn Leu Lys Gly Phe Asp Val Ile Glu Asp Ala Lys Thr Gln 100 105 110 att gaa gct atc tgt cct gga gtt gtt tcc tgt gct gat att ctt gct 444 Ile Glu Ala Ile Cys Pro Gly Val Val Ser Cys Ala Asp Ile Leu Ala 115 120 125 cta gct gct cgt gat tcc gtt gtt gcg act agg gga ctg aca tgg tct 492 Leu Ala Ala Arg Asp Ser Val Val Ala Thr Arg Gly Leu Thr Trp Ser 130 135 140 gtg cct acg gga cgt aga gat ggg cga gtt tcg aga gca gct gat gcg 540 Val Pro Thr Gly Arg Arg Asp Gly Arg Val Ser Arg Ala Ala Asp Ala 145 150 155 160 ggt gac ctg cca gct ttt ttt gat tct gtg gat att caa aag cga aag 588 Gly Asp Leu Pro Ala Phe Phe Asp Ser Val Asp Ile Gln Lys Arg Lys 165 170 175 ttt ctt aca aag ggt ctc aac act caa gat ctt gtc gcc ctt act ggt 636 Phe Leu Thr Lys Gly Leu Asn Thr Gln Asp Leu Val Ala Leu Thr Gly 180 185 190 gcg cac act att gga acc gca ggt tgc gca gtc atc aga gac agg cta 684 Ala His Thr Ile Gly Thr Ala Gly Cys Ala Val Ile Arg Asp Arg Leu 195 200 205 ttc aat ttc aac tcc act ggt ggc cct gac cct tca ata gat gca acc 732 Phe Asn Phe Asn Ser Thr Gly Gly Pro Asp Pro Ser Ile Asp Ala Thr 210 215 220 ttt ctt cct cag ctt cga gca tta tgt cca caa aat ggg gat gcc tca 780 Phe Leu Pro Gln Leu Arg Ala Leu Cys Pro Gln Asn Gly Asp Ala Ser 225 230 235 240 agg cgt gtg gga cta gac act gga agc gta aac aat ttc gac act tcg 828 Arg Arg Val Gly Leu Asp Thr Gly Ser Val Asn Asn Phe Asp Thr Ser 245 250 255 tat ttc tcc aac ctc agg aat ggt cgt gga gtt ctg gaa tca gac cag 876 Tyr Phe Ser Asn Leu Arg Asn Gly Arg Gly Val Leu Glu Ser Asp Gln 260 265 270 aag ctg tgg acc gat gct tct aca cag gtg ttt gtc caa agg ttt tta 924 Lys Leu Trp Thr Asp Ala Ser Thr Gln Val Phe Val Gln Arg Phe Leu 275 280 285 ggt att agg gga tta ctt gga ttg aca ttc ggc gtg gag ttt ggc agg 972 Gly Ile Arg Leu Gly Leu Thr Phe Gly Val Glu Phe Gly Arg 290 295 300 tcc atg gtt aaa atg agc aat att gaa gtt aag act ggc act aat ggt 1020 Ser Met Val Lys Met Ser Asn Ile Glu Val Lys Thr Gly Thr Asn Gly 305 310 315 320 gaa att cgt aaa gtt tgc tct gct atc aac tgatacagat attgtaccct 1070 Glu Ile Arg Lys Val Cys Ser Ala Ile Asn 325 330 tttatgtact tactactctt gtacttagtt tctttaaaaaacat 2 211> 330 <212> PRT <213> Nicotiana tabacum <400> 2 Met Glu Tyr Tyr His His Ser Ile Asn Lys Met Ala Met Phe Met Val 1 5 10 15 Ile Leu Val Leu Ala Ile Asp Val Thr Met Val Leu Gly Gln Gly Thr 20 25 30 Arg Val Gly Phe Tyr Ser Ser Thr Cys Pro Arg Ala Glu Ser Ile Val 35 40 45 Gln Ser Thr Val Arg Ala His Phe Gln Ser Asp Pro Thr Val Ala Pro 50 55 60 Gly Ile Leu Arg Met His Phe His Asp Cys Phe Val Leu Gly Cys Asp 65 70 75 80 Gly Ser Ile Leu Ile Glu Gly Ser Asp Ala Glu Arg Thr Ala Ile Pro 85 90 95 Asn Arg Asn Leu Lys Gly Phe Asp Val Ile Glu Asp Ala Lys Thr Gln 100 105 110 Ile Glu Ala Ile Cys Pro Gly Val Val Ser Cys Ala Asp Ile Leu Ala 115 120 125 Leu Ala Ala Arg Asp Ser Val Val Ala Thr Arg Gly Leu Thr Trp Ser 130 135 140 Val Pro Thr Gly Arg Arg Asp Gly Arg Val Ser Arg Ala Ala Asp Ala 145 150 155 160 Gly Asp Leu Pro Ala Phe Phe Asp Ser Val Asp Ile Gln Lys Arg Lys 165 170 175 Phe Leu Thr Lys Gly Leu Asn Thr Gln Asp Leu Val Ala Leu Thr Gly 180 185 190 Ala His Thr Ile Gly Thr Ala Gly Cys Ala Val Ile Arg Asp Arg Leu 195 200 205 Phe Asn Phe Asn Ser Thr Gly Gly Pro Asp Pro Ser Ile Asp Ala Thr 210 215 220 Phe Leu Pro Gln Leu Arg Ala Leu Cys Pro Gln Asn Gly Asp Ala Ser 225 230 235 240 Arg Arg Val Gly Leu Asp Thr Gly Ser Val Asn Asn Phe Asp Thr Ser 245 250 255 Tyr Phe Ser Asn Leu Arg Asn Gly Arg Gly Val Leu Glu Ser Asp Gln 260 265 270 Lys Leu Trp Thr Asp Ala Ser Thr Gln Val Phe Val Gln Arg Phe Leu 275 280 285 Gly Ile Arg Gly Leu Leu Gly Leu Thr Phe Gly Val Glu Phe Gly Arg Two 90 295 300 Ser Met Val Lys Met Ser Asn Ile Glu Val Lys Thr Gly Thr Asn Gly 305 310 315 320 Glu Ile Arg Lys Val Cys Ser Ala Ile Asn 325 330 <210> 3 <211> 1191 <212> DNA <213> Nicotiana tabacum <220> <221> CDS <222> (49) .. (1038) <400> 3 atttacttac tcaaatctca gaattcgttt atactgttaa cacgtacc atg gag tac 57 Met Glu Tyr 1 tat cac cat tca att aac aaa atg gcc atg ttt atg gtt att cta gtg 105 Tyr His His Ser Ile Asn Lys Met Ala Met Phe Met Val Ile Leu Val 5 10 15 cta gcc ata gat gtg act atg gtt tta ggc caa ggt acc cgt gtt gga 153 Leu Ala Ile Asp Val Thr Met Val Leu Gly Gln Gly Thr Arg Val Gly 20 25 30 35 ttt tat tcc agt acg tgc cca agg gcc gaa tcc ata gtt caa tca acg 201 Phe Tyr Ser Ser Thr Cys Pro Arg Ala Glu Ser Ile Val Gln Ser Thr 40 45 50 gtg agg gct cat ttt cag tct gat cca acg gtg gca cca gga atc ctg 249 Val Arg Ala His Phe Gln Ser Asp Pro Thr Val Ala Pro Gly Ile Leu 55 60 65 aga atg cac ttc cat gat tgt ttt gta cta ggt tgt gat ggt tcc atc 297 Arg Met His Phe His Asp Cys Phe Val Leu Gly Cys Asp Gly Ser Ile 70 75 80 ctc att gaa ggt tca gac gct gag aga act gct atc cca aat aga aat 345 Leu Ile Glu Gly Ser Asp Ala Glu Arg Thr Ala Ile Pro Asn Arg Asn 85 90 95 ttg aga gga ttc gac gtt att gag gat gct aag aag caa att gaa gct 393 Leu Arg Gly Phe Asp Val Ile Glu Asp Ala Lys Lys Gln Ile Glu Ala 100 105 110 115 atc tgt cct gga gtt gtt tcc tgc gct gac att ctt gct ctt gct gct 441 Ile Cys Pro Gly Val Val Ser Cys Ala Asp Ile Leu Ala Leu Ala Ala 120 125 130 cgt gat tct gtt gtc gcg act agg gga ctg aca tgg tct gtg ccc acg 489 Arg Asp Ser Val Val Ala Thr Arg Gly Leu Thr Trp Ser Val Pro Thr 135 140 145 gga cgt aga gat ggg cga gtt tct aga gca gct gat gcg ggt aat ctg 537 Gly Arg Arg Asp Gly Arg Val Ser Arg Ala Ala Asp Ala Gly Asn Leu 150 155 160 cca gct ttt ttt gat tcc gtg gat gtt caa aacaa aag ttt act gca 585 Pro Ala Phe Phe Asp Ser Val Asp Val Gln Lys Gln Lys Phe Thr Ala 165 170 175 aag ggt ctc aac act caa gat ctt gtc gct ctt act ggt gcg cac act 633 Lys Gly Leu Asn Thr Gln Asp Leu Val Ala Le u Thr Gly Ala His Thr 180 185 190 195 att gga acc gca ggt tgc gca gtc atc aga ggc agg cta ttc aat ttc 681 Ile Gly Thr Ala Gly Cys Ala Val Ile Arg Gly Arg Leu Phe Asn Phe 200 205 210 aac tcc act ggc ggc cct gac cct tca ata gat gca acc ttt ctt cct 729 Asn Ser Thr Gly Gly Pro Asp Pro Ser Ile Asp Ala Thr Phe Leu Pro 215 220 225 cag ctt caa gca tta tgt cca caa aat ggg gac gcc gca agg cgt gtg 777 Gln Leu Gln Ala Leu Cys Pro Gln Asn Gly Asp Ala Ala Arg Arg Val 230 235 240 gca cta gac act gga agc gca aac aat ttc gac acc tcg tat ttc tcc 825 Ala Leu Asp Thr Gly Ser Ala Asn Asn Phe Asp Thr Ser Tyr Phe Ser 245 250 255 aac ctc agg aat ggt cgt gga gtt ctg gaa tca gac cag aag ctg tgg 873 Asn Leu Arg Asn Gly Arg Gly Val Leu Glu Ser Asp Gln Lys Leu Trp 260 265 270 275 acc gat gct tct aca aag gtg ttt caa agg ttc ttg ggt att agg 921 Thr Asp Ala Ser Thr Lys Val Phe Val Gln Arg Phe Leu Gly Ile Arg 280 285 290 gga ttg ctt ggg ttg aca ttc ggc gtc gag ttt ggc agg tcc atg gtt 969 Gly Leu Leu Ph e Gly Val Glu Phe Gly Arg Ser Met Val 295 300 305 aaa aaa atg agc aat att gag gtt aag act ggc act aat ggt gaa att cgt 1017 Lys Met Ser Asn Ile Glu Val Lys Thr Gly Thr Asn Gly Glu Ile Arg 310 315 320 aaa gtt tgc tct gcc atc aac taatgcagat attgtaccct tttatgaact 1068 Lys Val Cys Ser Ala Ile Asn 325 330 tgctactctt gtacttggtt ttttcagtc agcgcaagct ggaaaatcca <ttgtgtgtaaat < Nicotiana tabacum <400> 4 Met Glu Tyr Tyr His His Ser Ile Asn Lys Met Ala Met Phe Met Val 1 5 10 15 Ile Leu Val Leu Ala Ile Asp Val Thr Met Val Leu Gly Gln Gly Thr 20 25 30 Arg Val Gly Phe Tyr Ser Ser Thr Cys Pro Arg Ala Glu Ser Ile Val 35 40 45 Gln Ser Thr Val Arg Ala His Phe Gln Ser Asp Pro Thr Val Ala Pro 50 55 60 Gly Ile Leu Arg Met His Phe His Asp Cys Phe Val Leu Gly Cys Asp 65 70 75 80 Gly Ser Ile Leu Ile Glu Gly Ser Asp Ala Glu Arg Thr Ala Ile Pro 85 90 95 Asn Arg Asn Leu Arg Gly Phe Asp Val Ile Glu Asp A la Lys Lys Gln 100 105 110 Ile Glu Ala Ile Cys Pro Gly Val Val Ser Cys Ala Asp Ile Leu Ala 115 120 125 Leu Ala Ala Arg Asp Ser Val Val Ala Thr Arg Gly Leu Thr Trp Ser 130 135 140 Val Pro Thr Gly Arg Arg Asp Gly Arg Val Ser Arg Ala Ala Asp Ala 145 150 155 160 Gly Asn Leu Pro Ala Phe Phe Asp Ser Val Asp Val Gln Lys Gln Lys 165 170 175 Phe Thr Ala Lys Gly Leu Asn Thr Gln Asp Leu Val Ala Leu Thr Gly 180 185 190 Ala His Thr Ile Gly Thr Ala Gly Cys Ala Val Ile Arg Gly Arg Leu 195 200 205 Phe Asn Phe Asn Ser Thr Gly Gly Pro Asp Pro Ser Ile Asp Ala Thr 210 215 220 Phe Leu Pro Gln Leu Gln Ala Leu Cys Pro Gln Asn Gly Asp Ala Ala 225 230 235 240 Arg Arg Val Ala Leu Asp Thr Gly Ser Ala Asn Asn Phe Asp Thr Ser 245 250 255 Tyr Phe Ser Asn Leu Arg Asn Gly Arg Gly Val Leu Glu Ser Asp Gln 260 265 270 lys Leu Trp Thr Asp Ala Ser Thr Lys Val Phe Val Gln Arg Phe Leu 275 280 285 Gly Ile Arg Gly Leu Leu Gly Leu Thr Phe Gly Val Glu Phe Gly Arg 290 295 300 Ser Met Val Lys Met Ser Asn Ile Glu Val Lys Thr GlyThr Asn Gly 305 310 315 320 Glu Ile Arg Lys Val Cys Ser Ala Ile Asn 325 330 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 5 caagagtagt aagtacataa aaggg 25 <210> 6 <211> 10 <212> PRT <213> Nicotiana tabacum <400> 6 Ala Glu Ser Ile Val Gln Ser Thr Val Arg 1 5 10 <210> 7 <211> 15 <212> PRT <213 > Nicotiana tabacum <400> 7 Ala His Phe Gln Ser Asp Pro Thr Val Ala Pro Gly Ile Leu Arg 1 5 10 15 <210> 8 <211> 10 <212> PRT <213> Nicotiana tabacum <400> 8 Gly Phe Asp Val Ile Glu Asp Ala Lys Lys 1 5 10 <210> 9 <211> 10 <212> PRT <213> Nicotiana tabacum <400> 9 Gly Leu Thr Trp Ser Val Pro Thr Gly Arg 1 5 10 <210> 10 <211 > 20 <212> PRT <213> Nicotiana tabacum <400> 10 Gly Leu Asn Thr Gln Asp Leu Val Ala Leu Thr Gly Ala His Thr Ile 1 5 10 15 Gly Thr Ala Gly 20 <210> 11 <211> 8 <212 > PRT <213> Nicotiana tabacum <400> 11 Gly Val Leu Glu Ser Asp Gln Lys 1 5 <210> 12 <211> 8 <212> PRT <213> Nicotiana tabacum <400> 12 Leu Trp Thr Asp Ala Ser Thr Lys 1 5 <210> 13 <211 > 12 <212> PRT <213> Nicotiana tabacum <400> 13 Val Phe Val Gln Arg Phe Leu Gly Ile Arg Gly Leu 1 5 10 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 14 gaycaraart trtggac 17 <210> 15 <211> 334 <212> DNA <213> Nicotiana tabacum <400> 15 gaccagaagt tgtggggaccga tgcttctaca caggtgtttg tccaaaggtt tttaggtatt 60 aggggattac ttggattgag tattcgggag attcgtgag attcgtgag attcgggag attcgggag atg gaggttcgta aagtttgctc tgctatcaac 180 tgatacagat attgtaccct tttatgtact tactactctt gtacttagtt tctttcagtc 240 agcgcaagct gcaaaatcga attgttgtga tatccaacaa ttaataaata aaagcattta 300 cagagtgaaa aaaaaaaaaa aaaaaaaaaa aaaa 334 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400 > 16 caagagtagt aagtacataa aaggg 25 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 17 tctagaacta gtggatcccc cg 22 <210> 18 <211> 1090 <212> DNA <213> Nicotiana tabacum <400> 18 aaatagtaga aatttactta ctcaaatctc agaattcgtt tatactgtta acacgtacca 60 tggagtacta tcaccattca attaacaaaa tggccatgtt tatggttatt ctagtgctag 120 ccatagatgt gactatggtt ttaggccaag gaacccgtgt tggattttat tccagtacgt 180 gcccaagggc cgaatccata gttcaatcaa cggtgagggc tcattttcag tctgatccaa 240 cggtggcacc aggaatcctg agaatgcact tccatgattg ttttgtacta ggttgtgatg 300 gttctatcct cattgaaggt tcagacgctg agagaactgc tatcccaaac agaaatttga 360 aaggattcga cgttattgag gatgctaaga cgcaaattga agctatctgt cctggagttg 420 tttcctgtgc tgatattctt gctctagctg ctcgtgattc cgttgttgcg actaggggac 480 tgacatggtc tgtgcctacg ggacgtagag atgggcgagt ttcgagagca gctgatgcgg 540 gtgacctgcc agcttttttt gattctgtgg atattcaaaa gcgaaagttt cttacaaagg 600 gtctcaacac tcaagatctt gtcgccctta ctggtgcgca cactattgga accgcaggtt 660 gcgcagtcat cagagacagg ctattcaatt tcaactccac tggtggccct gacccttcaa 720 tagatgcaac ctttcttcct cagcttcgag cattatgtcc acaaaatggg gatgcctcaa 780 ggcgtgtggg actagacact ggaagcgtaa acaatttcga cacttcgtat ttctccaacc 840 tcaggaatgg tcgtggagtt ctggaatcag accagaagct gtggaccgat gcttctacac 900 aggtgtttgt ccaaaggttt ttaggtatta ggggattact tggattgaca ttcggcgtgg 960 agtttggcag gtccatggtt aaaatgagca atattgaagt taagactggc actaatggtg 1020 aaattcgtaa agtttgctct gctatcaact gatacagata ttgtaccctt ttatgtactt 1080 actactcttg 1090 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 19 atggagtact atcaccattc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 20 tcagctgctc tcgaaactcg 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 21 cgtatttctc caacctcagg 20 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 22 attggttacc acacaattg 19 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 23 gttggatatc acaacaattc 20 <210> 24 <211> 15 <212> PRT <213> Nicotiana tabacum <400> 24 Val Asp Ile Gln Lys Arg Lys Phe Leu Thr Lys Gly Leu Asn Thr 1 5 10 15 <210> 25 <211> 29 <212> DNA <213> Artificial Sequence <2 20> <223> Synthetic DNA <400> 25 cgcggatcca tggagtacta tcaccattc 29 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 26 cgagctctca gttgatagca gagcaaa 27

[0116]

[Sequence List Free Text] SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 14: Synthetic DNA SEQ ID NO: 17: Synthetic DNA SEQ ID NO: 19: Synthetic DNA SEQ ID NO: 20: Synthetic DNA SEQ ID NO: 21: Synthetic DNA SEQ ID NO: 22: Synthetic DNA SEQ ID NO: 23: Synthetic DNA SEQ ID NO: 25: Synthetic DNA SEQ ID NO: 26: Synthetic DNA

[Brief description of the drawings]

FIG. 1 is a photograph showing an agarose electrophoresis image in which a peroxidase gene NTPXC1 induced by paraquat treatment was detected by an RT-PCR reaction.

FIG. 2 is a photograph showing an agarose electrophoresis image in which a peroxidase gene NTPXC2 induced by paraquat treatment was detected by an RT-PCR reaction.

[FIG. 3] Growth of transformed plants (strains 7-3 and 35-5) into which a peroxidase gene (NTPXC2) has been introduced after culturing for about 1.5 months in an MS medium containing paraquat (1.29 μM). It is a photograph showing.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:91) (72) Inventor Iwao Furusawa 1-23 Takano Higashikaimachi Sakyo-ku Kyoto City, Kyoto Prefecture 36-303 F term (reference) 2B030 AA02 AB03 AD20 CA06 CA17 CA19 CB02 CD02 CD03 CD07 CD10 CD13 CD17 CD22 4B024 AA08 BA08 CA04 DA01 EA05 GA11 GA17 GA27 4B050 CC03 DD09 LL10 4B065 AA11X AA89X AA89Y AB01 AC02 AC03 AC04 AC08 AC08

Claims (14)

[Claims] 1. A transformed plant containing a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) a protein having one or several amino acids deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having peroxidase activity 2. A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is represented by the following (c) or (d):
The transformed plant according to claim 1, which comprises the DNA of (1). (c) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 (d) DNA hybridizing with the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions, and encoding a protein having peroxidase activity 3. The transformed plant according to claim 1, wherein the plant is a plant, a plant organ, a plant tissue, or a plant cultured cell. 4. The plant according to claim 1, wherein the plant is a solanaceae, a crucifer, a gramineous plant,
Any of the group consisting of legumes, cucurbitaceae, convolvulaceae, lily family, lamiaceae, asteraceae, rose family, citrus family, lamiaceae family, myrtaceae family, willow family, akazaceae family, gentian family, and dianthus family Claim 1 which belongs to the family of
4. The transformed plant according to any one of claims 1 to 3. 5. A stress-tolerant plant comprising a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) a protein having one or several amino acids deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having peroxidase activity 6. The stress-tolerant plant according to claim 5, wherein the stress is caused by active oxygen. 7. The stress-tolerant plant according to claim 6, wherein the stress caused by active oxygen is environmental stress. 8. The environmental stress according to claim 7, wherein the environmental stress is at least one selected from the group consisting of high temperature stress, low temperature stress, drought stress, high light stress, salt stress, air pollution stress, pesticide stress and disease stress. Stress-tolerant plant. 9. A method for imparting stress tolerance to a plant, comprising incorporating a gene encoding the following protein (a) or (b) into the plant. (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4; (b) one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and a peroxidase activity Protein with 10. A method for producing a stress-tolerant plant, comprising incorporating a gene encoding the following protein (a) or (b) into a plant. (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4; (b) one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and a peroxidase activity Protein with 11. A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is represented by the following (c) or
The method according to claim 9 or 10, which comprises the DNA of (d). (c) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 (d) DNA hybridizing with the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions, and encoding a protein having peroxidase activity 12. The method according to claim 9, wherein the stress is caused by active oxygen. 13. The method according to claim 12, wherein the stress caused by active oxygen is environmental stress. 14. The environmental stress may be a high temperature stress, a low temperature stress, a drought stress, a high light stress, a salt stress,
14. The method according to claim 13, which is at least one selected from the group consisting of air pollution stress, pesticide stress, and disease stress.