JP2012235774A - Nucleic acid construct showing responsiveness to disease stress and disease resistance inducing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To clarify the responsiveness of a WRKY45 gene in a rice plant, and to provide a novel nucleic acid construct that shows the responsiveness to a disease stress and a disease resistance inducing agent.SOLUTION: The nucleic acid construct comprises connecting a polynucleotide consisting of a base sequence of a sequence number 1 and consisting of a region from a transcription start point upstream 1965 to just before 3'-UTR of a WRKY45 gene in a rice plant with a polynucleotide encoding a target protein.

Description

  The present invention relates to a nucleic acid construct exhibiting responsiveness to disease stress and disease resistance inducers, an expression vector having the nucleic acid construct, a transformant having the expression vector, a transgenic plant, a method for inducing gene expression due to disease stress, and the like. The present invention relates to a screening method for disease resistance inducers.

Terrestrial plants are exposed to infections with various plant pathogens. It is known that when a plant is exposed to disease stress, biochemical, physiological and molecular reactions such as expression of specific genes are induced to show resistance to the disease stress. This reaction proceeds as a result of stimulus recognition and signal transduction in plant cells. On the other hand, compounds that induce such plant specific reactions are called disease resistance inducers, such as benzothiadiazole S-methyl ester (BTH) and dichloroisonicotinic acid (INA). (Non-Patent Document 1: Hideo Ishii, Plant Protection, 53, 10, 393-397 (1999)). Although details of plant resistance induction mechanisms caused by disease stress and disease resistance inducers have not yet been fully elucidated, substances that can be used as indicators of plant resistance induction include development of disease resistance inducers, etc. Can be used effectively.

By the way, disease resistance inducers such as benzothiadiazole (BTH) act on plant defense response signal transduction pathway (salicylic acid pathway), and disease is caused by the preparatory effect (priming effect) on rapid resistance expression at the time of pathogen infection It is known to increase resistance. BTH
WRKY45 (Warky 45) is known as a transcription factor whose expression is induced in rice by treatment. WRKY45 regulates the transcription of nearly 300 of the genes that are transcriptionally induced by BTH, and plays a very important role in resistance induction via the salicylic acid pathway (Non-Patent Document 2: Shimono et al.). al., The Plant Cell, 19, 2064 (2007), Non-Patent Document 3:
Takatsuki Hiroshi et al., Proceedings of the Plant Infection Physiology Discourse Meeting, 44, 1-10 (2008) and Patent Document 1: WO2006 / 126671).

  However, the WRKY45 gene expression mechanism resulting from treatment with a disease resistance inducer or the addition of disease stress is not yet known.

Hideo Ishii, Plant Protection, 53, 10, 393-397 (1999) Shimono et al., The Plant Cell, 19, 2064 (2007) Takashi Hiroshi et al., Proceedings of the Plant Infection Physiology Conference, 44, 1-10 (2008)

WO2006 / 126671

  Accordingly, the present invention aims to clarify the responsiveness of the WRKY45 gene in rice, and to provide a novel nucleic acid construct exhibiting responsiveness to disease stress and disease resistance inducers and use thereof.

  As a result of intensive studies to solve the above problems, the present inventor has clarified one end of the responsive mechanism of the WRKY45 gene, which shows a specific expression pattern to disease stress, and responds to disease stress and disease resistance inducers. The present inventors have succeeded in isolating a nucleic acid construct exhibiting sex and completed the present invention.

That is, the present invention includes the following.
(1) A nucleic acid construct obtained by linking the following polynucleotide (a), (b) or (c) and a polynucleotide encoding a target protein.
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1
(b) a polynucleotide comprising a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 1 and exhibiting responsiveness to disease stress and disease resistance inducers
(c) a polynucleotide that hybridizes under stringent conditions with a DNA fragment comprising a base sequence complementary to the base sequence of SEQ ID NO: 1 and exhibits responsiveness to disease stress and disease resistance inducers (2) The nucleic acid construct according to (1), wherein the polynucleotide encoding the target protein is a coding region of a reporter gene.
(3) An expression vector comprising the nucleic acid construct of (1) or (2) above.
(4) A transformant comprising the expression vector according to (3) above.
(5) A transgenic plant comprising the expression vector according to (3) above.
(6) The transgenic plant according to (5), which is a plant body, plant organ, plant tissue or plant cultured cell.
(7) A method for producing a plant for culturing or cultivating the transgenic plant according to (5) or (6) above.
(8) A method for inducing gene expression, wherein the transgenic plant according to (5) or (6) is contacted with a disease resistance inducer or added with disease stress to induce expression of a gene encoding the target protein.
(9) The gene expression induction method according to (8), wherein the disease resistance inducer is benzothiadiazole S-methyl ester (BTH) or dichloroisonicotinic acid.
(10) a step of bringing a compound to be examined into contact with a transformant into which an expression vector containing the nucleic acid construct according to (2) has been introduced; and a step of detecting the expression of the reporter gene in the transformant; A method for screening a disease resistance inducer, wherein the compound is determined to be a disease resistance inducer when the expression level of the reporter gene is increased by contacting the compound to be examined.

  ADVANTAGE OF THE INVENTION According to this invention, the completely novel nucleic acid construct which shows the responsiveness with respect to disease stress and a disease resistance inducer can be provided. Moreover, disease stress and disease resistance inducers can be screened by analyzing the expression of a reporter gene using the nucleic acid construct according to the present invention.

It is a figure which shows typically the structure of the nucleic acid construct which drove the genome structure and GUS gene of OsWRKY45. FIG. 3 is a diagram showing a construct of a pSAMHDN627-M2GUS vector plasmid. It is the photograph before the leaf blade sampling of WRKY45-P4R3 :: GUS recombinant rice treated with BTH. It is the photograph before the sampling of WRKY45-P4R3 :: GUS recombinant rice grown in BTH-containing gellan gum medium. It is a figure which shows the salicylic acid non-responsiveness of WRKY45-P4R1 :: GUS recombinant rice, and the salicylic acid responsiveness of WRKY45-P4R3 :: GUS recombinant rice. It is a figure which shows the rice blast fungus infection responsiveness of WRKY45-P4R3 :: GUS recombinant rice. It is a figure which shows the white leaf blight infection responsiveness of WRKY45-P4R3 :: GUS recombinant rice. It is a figure which shows BTH and INA responsiveness of WRKY45-P4R3 :: GUS recombinant rice grown in an unglazed pot. It is a figure which shows the BTH responsiveness of WRKY45-P4R3 :: GUS recombinant rice grown on a drug-containing gellan gum medium.

  Hereinafter, the present invention will be described in detail.

1. Nucleic acid construct The nucleic acid construct according to the present invention is obtained by linking the following polynucleotide (a), (b) or (c) and a polynucleotide encoding a target protein.
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1
(b) a polynucleotide comprising a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 1 and exhibiting responsiveness to disease stress and disease resistance inducers
(c) a polynucleotide that hybridizes with a DNA fragment consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1 under stringent conditions and exhibits responsiveness to disease stress and disease resistance inducers

  Here, the nucleotide sequence of SEQ ID NO: 1 is the region from 1965 upstream of the transcription start point of rice WRKY45 (OsWRKY45) gene to immediately before 3'-UTR (however, it does not include the stop codon in the coding region of WRKY45 gene) It is a base sequence containing. That is, the base sequence of SEQ ID NO: 1 consists of 1965 bases upstream of the transcription start point of the WRKY45 gene in rice and 3749 bases including the WRKY45 gene.

In the above (b), the “plurality of bases” means, for example, 1 to 300 bases, preferably 1 to 200 bases, more preferably 1 to 100 bases, and further preferably 1 to 50 bases. Of bases, more preferably 1 to 10 bases, more preferably 1 to 5 bases. These “plurality of bases” may be a region where the above number of bases are continuous, or may be a discontinuous plurality of regions. In addition, the region in which one or more bases can be deleted, substituted or added in the base sequence of SEQ ID NO: 1 is not particularly limited, but a motif sequence involved in transcription contained in the base sequence of SEQ ID NO: 1 It is preferable that the region is excluded. Examples of motif sequences include WRKY transcription factor binding site motifs (regions 771 to 775 and 1665 to 1669 in SEQ ID NO: 1), ACGT sequence motifs (101 to 104 and 1270 to 1273 in SEQ ID NO: 1). Region), TGA sequence motif (regions 42 to 44 and 1452 to 1454 in SEQ ID NO: 1), TGACGT / C motif (SEQ ID NO: 1) which is the core sequence of activation sequence-1 elements 1st to 1184th and 1254-1259th regions), MYB motif (1645th to 1650th region in SEQ ID NO: 1) and MYC motif (240th to 245th and 1220th to 1225th regions in SEQ ID NO: 1) be able to.

In addition, the 771st to 1669th regions in SEQ ID NO: 1 are considered to be regions that are deeply involved in the responsiveness to disease stress and disease resistance inducers in the nucleic acid construct according to the present invention. Therefore, the region where one or more bases can be deleted, substituted or added is preferably a region excluding the 771 to 1669th regions in SEQ ID NO: 1.

Furthermore, in the above (c), stringent conditions mean conditions under which a pair of polynucleotides having high sequence similarity can specifically hybridize. A pair of polynucleotides having high sequence similarity means, for example, a polynucleotide having 90%, preferably 95%, more preferably 97% identity. The identity between a pair of polynucleotides can be calculated using homology search software such as Blast with default settings. Such stringent conditions include, for example, a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68 ° C., preferably 42 to 65 ° C. More specifically, 5 × SSC (83 mM NaCl, 83 mM sodium citrate), temperature 42 ° C.

That is, the requirement (c) is 90% with respect to the nucleotide sequence of SEQ ID NO: 1, preferably 95
%, More preferably 97%, and can be said to be a polynucleotide exhibiting responsiveness to disease stress and disease resistance inducers.

On the other hand, the polynucleotide encoding the target protein linked to the polynucleotide (a), (b) or (c) described above is not particularly limited, and may be any polynucleotide. That is, any protein may be used as the target protein. The polynucleotide encoding the target protein means both the sense strand and the antisense strand encoding the protein. An example of a polynucleotide encoding a target protein is a reporter gene. Examples of the reporter gene include β-glucuronidase (GUS) gene, green fluorescent protein (GFP) gene, luciferase (LUC) gene and the like. By measuring the expression level of these reporter genes, it is possible to analyze the expression inducing activity of the polynucleotide (a), (b) or (c) described above.

By the way, whether or not a given polynucleotide is “showing responsiveness to disease stress and disease resistance inducer” can be determined by analyzing the expression pattern of the target protein. Specifically, a so-called reporter gene is used as the polynucleotide encoding the target protein. And a transformed plant is produced using this nucleic acid construct. The expression level of the reporter gene in the obtained transformed plant is measured under the presence or absence of addition of disease stress or the contact / non-contact conditions of the disease resistance inducer. The expression level of the reporter gene in the additional condition of disease stress and the contact condition of the disease resistance inducer is significantly larger than the expression level of the reporter gene in the condition of no disease stress and the non-contact condition of the disease resistance inducer. If there is, it can be determined that the polynucleotide "shows responsiveness to disease stress and disease resistance inducer".

  At this time, disease stress means stress that infects plant disease-causing microorganisms such as molds, bacteria, viruses, or viruses. As a disease resistance inducer, benzothiadiazole S-methyl ester (BTH), dichloroisonicotinic acid (INA), probenazole, thiadianyl, isothianyl, and the like are meant.

2. Expression vector The expression vector of the present invention can be obtained by ligating (inserting) the nucleic acid construct into an appropriate vector. The vector for inserting the above-described nucleic acid construct is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, shuttle vectors, and helper plasmids.

  As plasmid DNA, plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YCp50, etc.) And λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.

To insert a nucleic acid construct into a vector, first, a method is used in which the purified nucleic acid construct is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of an appropriate vector DNA, and linked to the vector. Is done.

3. Production of transformant The transformant of the present invention can be obtained by introducing the expression vector of the present invention into a host. Here, the host is not particularly limited as long as it can express a promoter or a target gene, but a plant is preferable. When the host is a plant, a transformed plant (transgenic plant) can be obtained as follows.

Plants to be transformed in the present invention include whole plants, plant organs (e.g. leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g. epidermis, phloem, soft tissue, xylem, vascular bundles, etc.) ) Or plant cultured cells. Examples of plants used for transformation include plants belonging to the Brassicaceae, Gramineae, Solanum, Legumes, etc. (see below), but are not limited to these plants.

Brassicaceae: Arabidopsis thaliana
Solanum: Tobacco (Nicotiana tabacum)
Gramineae: maize (Zea mays), rice (Oryza sativa)
Legumes: Soybean (Glycine max)

The recombinant vector can be introduced into a plant by a conventional transformation method such as electroporation (electroporation), Agrobacterium method, particle gun method, PEG method and the like.

For example, when the electroporation method is used, the gene is introduced into the host by treatment with an electroporation apparatus equipped with a pulse controller under conditions of a voltage of 500 to 1600 V, 25 to 1000 μF, and 20 to 30 msec.

  When the particle gun method is used, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The sample thus prepared can be processed using a gene introduction apparatus (for example, PDS-1000 / He manufactured by Bio-Rad). The treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 1000-1800 psi and a distance of about 5-6 cm.

  Moreover, a target gene can be introduced into a plant body by using a plant virus as a vector. Examples of plant viruses that can be used include cauliflower mosaic virus. That is, first, a virus genome is inserted into a vector derived from E. coli to prepare a recombinant, and then these target genes are inserted into the virus genome. The gene of interest can be introduced into a plant host by excising the viral genome thus modified from the recombinant with a restriction enzyme and inoculating the plant host.

In the method using the Agrobacterium Ti plasmid, when a bacterium belonging to the genus Agrobacterium infects a plant, it takes advantage of the property of transferring a part of the plasmid DNA that it has into the plant genome. The target gene is introduced into the plant host. Among the bacteria belonging to the genus Agrobacterium, Agrobacterium tumefaciens infects plants to form a tumor called crown gall, and Agrobacterium rhizogenes infects plants. To generate hairy roots. These are because the region called T-DNA region (Transferred DNA) on the plasmid that is present in each bacterium called Ti plasmid or Ri plasmid at the time of infection is transferred into the plant and integrated into the genome of the plant. This is due to

If the DNA to be incorporated into the plant genome is inserted into the T-DNA region on the Ti or Ri plasmid, the target DNA is transferred into the plant genome when Agrobacterium bacteria infect the plant host. Can be incorporated.

  Tumor tissue, shoots, hairy roots, and the like obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture, and can be used at an appropriate concentration using a conventionally known plant tissue culture method. Of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).

  By the way, the compound to be examined is brought into contact with a transformant transformed with an expression vector having the DNA fragment according to the present invention and a reporter gene incorporated in a form in which expression can be controlled by the DNA fragment. By detecting the expression of a reporter gene in, a compound showing disease resistance-inducing activity can be screened.

The vector of the present invention is not only introduced into the plant host, but also Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, or Pseudomonas putida such as Pseudomonas putida. Transformants are introduced into yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, animal cells such as COS cells and CHO cells, or insect cells such as Sf9. It can also be obtained. When a bacterium such as E. coli or yeast is used as a host, the recombinant vector of the present invention can autonomously replicate in the bacterium, and at the same time comprises the DNA fragment of the present invention, a ribosome binding sequence, a target gene, and a transcription termination sequence. It is preferable that Moreover, the gene which controls DNA of this invention may be contained.

The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. Examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.

When insect cells are used as hosts, Sf9 cells and the like are used. Examples of the method for introducing a recombinant vector into insect cells include the calcium phosphate method, lipofection method, electroporation method and the like.

Whether or not the gene has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization or the like. For example, DNA is prepared from the transformant, PCR is performed by designing a DNA-specific primer. PCR is performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, Confirm that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like 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 enzyme reaction may be employed.

4). Manufacture of a plant In this invention, it can reproduce | regenerate to a transformed plant body from the said transformed plant cell. As a regeneration method, a method is employed in which callus-like transformed cells are transferred to a medium with different hormone types and concentrations and cultured to form somatic embryos to obtain complete plants. Examples of the medium to be used include LS medium and MS medium. Moreover, a plant seed can be obtained from the obtained transformed plant body, and a plant body can also be produced from this plant seed. In order to obtain plant seeds from transformed plants, for example, the transformed plants are collected from the rooting medium, transplanted to pots containing water-containing soil, and grown at a constant temperature to form flowers. And finally seeds are formed. In order to produce a plant from seeds, for example, when the seed formed on the transformed plant has matured, it is isolated, sown in water-containing soil, and grown under constant temperature and illuminance. To produce a plant.

  That is, a plant having the nucleic acid construct according to the present invention can be produced by the method described above. The obtained plant body has the characteristic of expressing the target protein in response to disease stress and disease resistance inducers. When a protein that imparts or enhances disease resistance to a plant body is applied as a target protein, a disease stress resistant plant is obtained.

5. Screening method A screening method for a disease resistance inducer can be constructed by using the above-described transformed plant. In this screening method, a polynucleotide encoding the target protein linked to the polynucleotide (a), (b) or (c) described above is used as a reporter gene.

  In this screening method, first, the compound to be examined is brought into contact with the above-described transformed plant. Here, the compound to be examined is not particularly limited and may be any substance. The compound to be examined may be a single substance or a mixture composed of a plurality of components. As a compound to be examined, for example, an unidentified substance such as an extract from a plant may be included, or a known composition may be included at a predetermined composition ratio. Good. The compound to be examined may be any of protein, nucleic acid, lipid, polysaccharide, organic compound and inorganic compound.

  Next, the expression of the reporter gene in the transformed plant is detected. When the GUS gene is used as the reporter gene, the GUS activity in the transformed plant can be measured by a known technique. When another gene is used as the reporter gene, its expression level may be measured by a known method according to the reporter gene.

  When the expression level of the reporter gene is increased when the compound to be tested is brought into contact, the compound can be determined as a disease resistance inducer. As used herein, “determining as a disease resistance inducer” refers to determining that the compound has the ability to induce expression of the WRKY45 (OsWRKY45) gene, as with disease resistance inducers such as BTH and INA. It is synonymous with.

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

[Example 1]
Construction of a vector plasmid having a reporter gene under the control of the promoter sequence of the WRKY45 gene In this example, as shown in FIG. The region up to '-UTR, 2077 bases (SEQ ID NO: 2)), and between P4-R3 consisting of upstream region and transcription region (region from upstream transcription start point -1965 to just before 3'-UTR, 3749 bases (sequence) No. 1)) was PCR amplified from rice genomic DNA. Then, as shown in FIG. 2, the amplified fragment obtained by PCR was inserted into the NcoI restriction site of DNA vector pSAMHDN627-M2GUS to prepare WRKY45-P4R1 :: GUS vector plasmid and WRKY45-P4R3 :: GUS vector plasmid. . In the WRKY45-P4R3 :: GUS vector plasmid, the GUS gene which is a reporter gene is expressed as a fusion protein with WRKY45.

Introduction of WRKY45-P4R1 :: GUS and WRKY45-P4R3 :: GUS into A. tumefaciens EHA101 strain To introduce the above two types of plasmids into Agrobacterium tumefaciens EHA101 strain, 0.1% yeast extract, 0.5% meat extract, 0.5% peptone, 0.5% sucrose, 0.05% MgSO ₄ .7H ₂ O) were shaken and cultured at 30 ° C. overnight. Then add 5 ml of the above culture to 200 ml YEB medium in a 1 liter flask,
Further, the cells were cultured at 30 ° C. for 5 to 6 hours. The cells were collected by centrifugation (5 minutes at 4000 rpm) and suspended in 100 ml of 10 mM Tris-HCl (pH 8.0). Centrifugation was performed again, and the obtained cells were suspended in 2 ml of YEB medium. In a microcentrifuge tube, 200 μl of this suspension and 100 μl of DNA solution containing 0.5 μg of pBE7133H-Thionin were mixed and frozen in a dry ice / ethanol bath (5 minutes). Next, it was melted in a 37 ° C. warm bath and left for 25 minutes. Then, 2 ml of YEB medium was added and cultured with shaking at 30 ° C. for 1 hour. 100 μl of the culture solution was spread on a YEB medium containing kanamycin (50 μg / ml) and hygromycin (50 μg / ml), and a resistant bacterium of Agrobacterium tumefaciens EHA101 strain was obtained by culturing at 30 ° C. for about 36 hours. The GUS reporter cassette in this Agrobacterium tumefaciens EHA101 strain was isolated by alkali-SDS method, and its presence and structure were confirmed by restriction enzyme analysis.

Production of transgenic rice Sterilized rice (Oryza sativa, cv. Nipponbare) seeds were prepared in MS medium (T. Murashige et al., Physiol. Plant) containing 2 mg / ml of 2.4-dichlorophenoxyacetic acid (hereinafter 2.4-D). , 15, 473 (1962)) and cultured at 27 ° C. for 2 weeks to form callus. This callus was added to the co-culture medium (2mg / ml
It was transplanted to 2.4-D and 1 g / l casamino acid-containing MS medium) and cultured at 27 ° C. On the third day of culture, the Agrobacterium tumefaciens EHA101 strain containing the GUS reporter cassette was grown in a YEB medium containing kanamycin and hygromycin and centrifuged. After suspending the pelleted cells in LB medium, acetosyringone was added to prepare Agrobacterium tumefaciens EHA101 strain bacterial solution for infection. This bacterial solution was incubated at 30 ° C. for 20 minutes. On the fourth day of the culture, the above callus mass was added to the Agrobacterium tumefaciens EHA101 strain solution for infection and gently stirred at 30 ° C. for 15 minutes to infect this callus mass. After 15 minutes, the callus was collected and placed in a co-culture medium. Cultivate for 3 days at 27 ° C for 16 hours, then incubate in LB medium containing carbenicillin for 1 hour at 30 ° C, then collect only callus and select (grow) culture (500mg / l carbenicillin, 50mg / l Co-culture medium containing hygromycin). This was cultured for 2 weeks at 27 ° C. and 16 hours long. Newly grown yellowish white callus (pre-redifferentiation pretreatment) medium (1 mg / l2.4-D, 0.5 mg / l BAP, 2 g / l casamino acid, 20 g / l sucrose, 30 g / l sorbitol, N6 medium containing 500 mg / l carbenicillin and 100 mg / l hygromycin, cultured for about 2 weeks, then selected (redifferentiation) medium (0.01 mg / l β-naphthalene acetic acid (NAA), 0.1 mg / l BAP N6 medium containing 1 g / l casamino acid, 500 mg / l carbenicillin, 100 mg / l hygromycin (Toki et al., Plant J., 47, 969 (2006)). Transformed rice (T ₀ ) regenerated in about 2-3 weeks was obtained.

Production of progeny of transformed rice Transgenic rice generation (T ₀ ) was cultivated in an isolated greenhouse to obtain T ₁ seeds. Several kinds of T ₂ seeds obtained by further cultivating the T ₁ seeds were sown in a gellan gum medium containing 100 mg / L hygromycin to test the hygromycin resistance. All of the individuals tested showed hygromycin resistance as homozygotes. Gellan gum medium is Hogrant mix 1
A can and 3 g of gellite were suspended in 1 L of distilled water, and then dissolved by heating in a microwave oven. 15 ml of this was dispensed into a tube bottle, and hygromycin was added to a final concentration of 100 mg / L.

Inoculation of rice blast fungus and white leaf blight fungus Suspended rice blast fungus (Magnaporthe grisea, race 007.0) in 0.01% Silwet L77 aqueous solution (1-2x10 ⁵ spores / ml), 28 ℃ (daytime) / 23 ℃ (night) After spraying on the four-leaf rice (T ₀ ) seedlings cultivated in the greenhouse in ( ₂ ), the plants were moisturized for about 24 hours in the inoculation box under dark conditions and then returned to the greenhouse to continue cultivation. To inoculate white leaf blight fungus, white leaf blight fungus (Xanthomonas oryzae pv. Oryzae, strain T7174) is suspended in water so that OD ₆₀₀ = 0.03, and the 6-7 leaf rice Approximately 3 cm from the tip of the topmost fully expanded leaf was cut and infected (Kauffman et al., Plant Dis. Rep., 57, 537 (1973)).

Cultivation and plant hormone treatment of transformed rice
Cut the 4th leaf of rice (T ₀ ) seedlings grown in a greenhouse at 28 ° C (daytime) / 23 ° C (nighttime) into 0.5-1.0cm leaf pieces and contain various plant hormones. It was immersed in% Silwet L77 aqueous solution or solvent only (mock) and treated at 30 ° C. for about 6 hours under bright conditions. The plant hormones used for treatment and their concentrations are as follows. That is, sodium salicylate (SA, 1 mM), Indole-3-acetic acid (IAA, 50 μM), gibberellin A3 (GA ₃ , 50 μM), Kinetin (CK, 50 μM), 1-aminocyclopropane-1-carboxylic acid (ACC, 50 μM), abscisic acid ((±) -cis-trans, ABA, 50 μM), methyl jasmonate (ME-JA, 50 μM) and brassinolide (BR, 2 μM).

Cultivation of transformed rice and treatment with disease resistance inducer 1
Cut the 4th leaf of rice (T ₀ ) seedlings grown in a greenhouse at 28 ° C (daytime) / 23 ° C (nighttime) into 0.5-1.0cm leaf pieces and 0.01% containing 50µM BTH It was immersed in% Silwet L77 aqueous solution or solvent only (mock) and treated at 30 ° C. for about 6 hours under bright conditions.

Cultivation of transformed rice and treatment with disease resistance inducer 2
Recombinant rice homozygote (T ₂ ) seedlings were transplanted into an unglazed pot with a diameter of 6 cm, and 32 ° C (daytime) / 27
Cultivation at ℃ (night) until the late four-leaf stage. This was subjected to 100 ml soil irrigation treatment with 1% acetone aqueous solution containing 400 μM BTH and 333-fold diluted cumene or 1% acetone solution containing 400 μM INA and 333-fold diluted cumene. The untreated group was treated with a 1% acetone solution containing cumene diluted 333 times, and four leaves on the third day after drug treatment were sampled (FIG. 3).

Cultivation of transformed rice and treatment with disease resistance inducers (3)
Five recombinant rice homozygotes (T ₂ ) were lightly embedded in BTH-containing gellan gum medium, placed in a transparent case together with a beaker containing distilled water, and the case mouth was closed with a wrap. Cultivated under fluorescent lighting in a constant temperature room at 27 ℃ until 2-3 leaf stage, and sampled all the above-ground parts (Fig. 4)
. The final concentration of BTH was 10 ⁻⁷ M, 10 ⁻⁶ M, 10 ⁻⁵ M or 10 ⁻⁴ M, and 1% acetone was added as a control.

GUS staining Rice leaves inoculated with the rice blast fungus described above were washed with 90% acetone solution for 2-3 seconds to remove the wax layer on the surface, and then immersed in GUS staining solution at 37 ° C. for 2 to 2 hours. Color was developed for 4 hours. On the other hand, the plant hormone-treated or BTH-treated rice leaf pieces were immersed in a GUS staining solution (below) and developed for 2 to 4 hours at 37 ° C. (Gallagher, GUS protocols, Academic Press, Inc., pp. 103-104). (1992)).

Composition of GUS staining solution: 1.0 mM X-glucuronide, 10 mM EDTA, 0.5 mM Potassium Ferricyanide, 0.5 mM Potassium Ferrocyanide, 0.1% Triton X-100, 20% Methanol, 50 mM Potassium phosphate buffer, pH 7.0

GUS activity measurement Rice grown in the above-mentioned clay pot and sampled after drug treatment and rice leaf grown in drug-containing gellan gum medium were ground in a mortar filled with liquid nitrogen, then placed in an eppendorf tube and 100 μl EX buffer (below) was added and stirred vigorously. Thereafter, centrifugation was performed at 15000 rpm and 4 ° C. for 5 minutes, and the supernatant was collected. 10 μl of the collected supernatant was suspended in 100 μl 4-MUG / EX buffer and incubated at 37 ° C. for 1 hour. As a control, 100 μl 4-MUG / EX buffer alone was also incubated. After the incubation, 1.4 ml of 0.2 M Na ₂ CO ₃ was added to stop the reaction (the control was the same). This was placed in ice protected from light until the GUS activity was measured.

Composition of EX buffer: 5mM EDTA 200μl, 0.1% TritonX-100, 10mM 2-mercaptoethanol, 50mM
NaHPO4 (pH7.0)

GUS activity was measured with EX355 and EM460 after zero correction with 0.2 M Na ₂ CO ₃ as background. A calibration curve was prepared using 4-methyl umbelliferone (4-MU).

Result: Plant hormone responsiveness
Leaf pieces of WRKY45-P4R1 :: GUS recombinant rice (T ₀ ) showed GUS activity regardless of the presence or absence of plant hormones (FIG. 5). On the other hand, in WRKY45-P4R3 :: GUS recombinant rice (T ₀ ), mock treatment (-SA
) Did not show GUS activity, but SA treatment (+ SA) significantly induced GUS activity (FIG. 5). Induction of GUS activity by other hormone treatments was not observed. From these results, it was revealed that recombinant rice having RKY45-P4R3 :: GUS construct expresses GUS activity in response to SA specifically.

  From this result, it became clear that the WRKY45 gene responsiveness to disease stress and disease resistance inducers is not sufficient only in the upstream region of the WRKY45 gene, and the transcription region of the WRKY45 gene including the intron is required. It was.

Result: Responsiveness to pathogen infection
As a result of inoculating rice blast fungus on WRKY45-P4R3 :: GUS recombinant rice (T ₀ ), GUS activity was detected 2 and 3 days after inoculation in tissues around the infected site (FIG. 6). In rice blades after inoculation with white leaf blight fungus, GUS activity was detected at a site separated from the site of inoculation on the second day after inoculation (FIG. 7). In all cases, GUS activity was not observed in uninfected upper leaves (data not shown).

  In the photograph shown in FIG. 7, the right-sided blade cut surface is the inoculation site of the rice leaf blight fungus, and GUS activity is observed not only in the inoculation site but also on the left in the figure.

Result: Disease resistance inducer responsiveness
In WRKY45-P4R3 :: GUS recombinant rice (T ₀ ), it was revealed from GUS staining that GUS activity was remarkably induced by BTH treatment in the same manner as SA treatment (data not shown). Therefore, GUS activity induced by BTH treatment was quantitatively measured using WRKY45-P4R3 :: GUS recombinant rice homozygote (T ₂ ) as a material. As a result, it was revealed that GUS activity was significantly induced in rice leaf blades cultivated in an unglazed pot and sampled after BTH treatment (FIG. 8). In addition, treatment with dichloroisonicotinic acid (INA), which is known as a disease resistance inducer like BTH, significantly induced GUS activity (FIG. 8). On the other hand, in rice grown on BTH-containing gellan gum medium, the induction of GUS activity was observed with increasing drug treatment concentration, but the induction of GUS activity was smaller in the treatment concentration group where phytotoxicity occurred (FIG. 9).

Claims (10)

A nucleic acid construct obtained by linking the following polynucleotide (a), (b) or (c) and a polynucleotide encoding a target protein.
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1
(b) a polynucleotide comprising a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence of SEQ ID NO: 1 and exhibiting responsiveness to disease stress and disease resistance inducers
(c) a polynucleotide that hybridizes with a DNA fragment consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1 under stringent conditions and exhibits responsiveness to disease stress and disease resistance inducers   The nucleic acid construct according to claim 1, wherein the polynucleotide encoding the target protein is a coding region of a reporter gene.   An expression vector comprising the nucleic acid construct according to claim 1 or 2.   A transformant comprising the expression vector according to claim 3.   A transgenic plant comprising the expression vector according to claim 3.   The transgenic plant according to claim 5, which is a plant body, a plant organ, a plant tissue or a plant cultured cell.   The manufacturing method of the plant which culture | cultivates or grows the transgenic plant of Claim 5 or 6.   A gene expression inducing method, wherein the transgenic plant according to claim 5 or 6 is contacted with a disease resistance inducer or added with disease stress to induce expression of a gene encoding the target protein. The gene expression induction method according to claim 8, wherein the disease resistance inducer is benzothiadiazole S-methyl ester (BTH) or dichloroisonicotinic acid.   A step of bringing a compound to be tested into contact with a transformant into which an expression vector containing the nucleic acid construct according to claim 2 has been introduced; and a step of detecting the expression of the reporter gene in the transformant, A screening method for a disease resistance inducer, wherein when the expression level of the reporter gene is increased by contacting a compound to be examined, the compound is determined as a disease resistance inducer.

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