JP2004041209A - Method for creating plant resistant to two or more diseases using thionine gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To create a plant expressing resistance to at least one disease damage. <P>SOLUTION: The plant contains thionine gene and a method for creating the plant includes a process for constituting an expression vector containing an expression cassette for expressing thionine, a process for transforming cells with the expression vector and a process for regenerating the plant from the transformed cells. The plant created by the method expresses resistance to at least one disease damage out of disease damages caused by plant pathogenic bacteria such as Xanthomonas oryzae, Burkholderia plantarii or the like or plant pathogenic molds such as Magnaporthe grisea or the like. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a transformed plant that is resistant to at least one disease. More particularly, the present invention relates to a transgenic plant which contains an thionin gene and has an expression cassette capable of expressing this thionin, and which is resistant to at least one disease.

Generally, in crop production, stable production of high-quality plants and reduction of dependence on pesticides are demanded. Therefore, improvement, breeding, and development of varieties of plants showing resistance to pests and pathogens are actively performed by utilizing useful plant biotechnology such as plant cell fusion technology and recombinant DNA technology. . Transformed plants already exhibiting herbicide resistance (JP-A-2-186925), transformed plants exhibiting virus resistance (JP-A-4-330233), and transformed plants exhibiting pest resistance (JP-A-3-247220) It has been created using recombinant DNA technology. In addition, the introduction of a gene for an enzyme that inactivates toxins produced by pathogenic filamentous fungi enables transgenic plants that show resistance to pathogenic filamentous fungi (Plant Physiology, 104: 109-118) and insect-derived antibacterial properties Due to the introduction of a protein gene, several transformed plants exhibiting resistance to phytopathogenic bacteria have been produced, such as transformed plants exhibiting resistance to at least one pathogenic bacterium (Japanese Patent Application Laid-Open No. 7-250687).

The present inventors isolated a novel thionin which is an antibacterial protein from oat leaves, and sequenced a gene encoding thionin using a known technique. Then, transformation of tobacco was attempted using the thionin gene (JP-A-8-266).
279). This attempt has resulted in transformed and regenerated tobacco.

However, not all transformed plants are practical and useful plants that exhibit high disease resistance. This is because the disease resistance can be changed or the growth can be inhibited by factors such as the plant species, the transformation method, the expression level and expression site of the transgene, and the effect of the product on the plant. further,
It is known that whether or not the obtained disease resistance is stably inherited in future generations also differs depending on the situation. For these reasons, it is desired to produce a practical and useful plant exhibiting high disease resistance.

An object of the present invention is to solve the above-mentioned conventional problems. It is an object of the present invention to produce a plant containing a thionin gene and having an expression cassette capable of expressing this thionin, and to produce at least one disease. The purpose is to provide a plant that shows high resistance to the plant. Another object of the invention is to provide plants and seeds whose properties are stably preserved in progeny. Still another object of the present invention is to provide a method for obtaining a plant showing high resistance to the above-mentioned diseases.

The disease-resistant plant of the present invention is a plant that contains a thionin gene and has an expression cassette capable of expressing this thionin, and is resistant to at least one disease.

The method for producing a disease-resistant plant of the present invention comprises a step of constructing an expression vector containing a thionin gene and an expression cassette capable of expressing the thionin, a step of transforming a plant cell with the expression vector, and a method of producing the disease-resistant plant. Regenerating a plant from the converted plant cells.

に お い て In one embodiment of the present invention, the thionin is derived from oat leaves.

In another embodiment of the present invention, the disease resistant plant is a monocotyledonous or dicotyledonous plant.

In still another embodiment of the present invention, wherein the disease resistant plant is a plant of the Gramineae or a plant of the Solanaceae family.

In yet another embodiment of the present invention, the disease of the disease resistant plant is a disease derived from a phytopathogenic fungus, for example, a plant pathogenic bacterium such as rice leaf blight or rice seedling wilt, or potato blight. It is a disease derived from phytopathogenic fungi such as fungi.

The present invention also includes seeds obtained from the above disease resistant plants.

According to the present invention, a transgenic plant which is resistant to at least one disease, usually a plurality of diseases, is produced by introducing a gene for the antibacterial protein thionin into a plant. A method for producing such a plant is also provided by the present invention. Many high-quality plant varieties which have been vulnerable to diseases have become resistant to diseases by implementing the present invention, so that high-quality plant varieties can be stably produced. Furthermore, since the present invention exhibits resistance to diseases, it is possible to reduce the dependence on pesticides that kill pathogenic bacteria, and the present invention greatly contributes to agricultural crop production.

To facilitate understanding of the present invention, terms used in the present specification will be described.

As used herein, the term “disease” refers to a disease derived from a phytopathogenic bacterium (a phytopathogenic bacterium and / or a phytopathogenic fungus). Diseases derived from plant pathogenic bacteria include rice leaf blight, rice seedling blight, rice blight, tobacco cavity disease, tobacco wildfire, soft rot of various vegetables, spot bacterial disease, and bacterial wilt. Disease and the like,
Diseases derived from phytopathogenic fungi include rice blast, rice sheath blight, rice seedling rot, rice seedling wilt, rice stunt seedling, rice yellowing dwarf, rice sesame leaf blight, wheat Rust, wheat powdery mildew, potato blight, tobacco blight, tobacco gray mold,
Tobacco disease, rust, rust, blight, snow rot, vegetable plague, downy mildew, powdery mildew, gray mold, anthracnose, seedling wilt, clubroot, Carnation wilt, chrysanthemum rust and the like. In the present invention, particularly effective diseases are rice white leaf blight, bacterial seedling blight, and potato blight.

As used herein, the term "thionine" refers to a basic protein that has various pharmacological actions such as antibacterial action and blood pressure lowering action, and is stable to acids and heat.
Thionine is generally derived from plants (eg, wheat, barley, rye, oats, and oats) and contains, for example, 46 amino acid residues and a molecular weight of about 5 kDa.
Cysteine-rich proteins, but include proteins having at least one amino acid addition, deletion, and substitution as long as they have the above-mentioned properties. In the present invention, it preferably refers to thionin derived from oat leaves.

As used herein, the term "transformed plant" refers to a gene product (eg, a polypeptide such as an enzyme) having a gene introduced from the outside and having a biologically more specific function than this gene. Refers to plants that can be expressed stably or temporarily. Expression may be constitutive or inducible (in response to a particular stimulus such as temperature, drug, etc.). The introduced gene may be derived from a plant to be transformed, from a plant of the same or a different species as the plant to be transformed, or may be derived from other species of organisms (eg, animals such as insects, microorganisms). Derived from. This also includes genes reconstructed by combining some of these genes with one another, as long as gene products such as enzymes do not lose their biological functions.

As used herein, the term “gene” is a polymer in which nucleotides encoding amino acids constituting a polypeptide such as an enzyme are linked linearly in a direction. A gene may be single-stranded (eg, RNA) or double-stranded (eg, DNA). DNA is, for example, transcribed RNA (mRN
It can be cDNA prepared enzymatically from A), genomic DNA from chromosomes, or chemically synthesized DNA. These genes include, in addition to a sequence (coding region or translation region) encoding a polypeptide such as an enzyme, a promoter region for controlling transcription of the coding region, an enhancer region acting on the promoter region, and other regulatory regions. (Eg, terminator and poly A region), and introns and the like. It is known in the art that these genes can be modified such as additions, deletions, and substitutions as long as they do not lose the activity of each of the above regions.

The term "expression cassette" as used herein refers to a protein, e.g.,
A DNA fragment in which a gene sequence encoding thionin and various regulatory elements such as a promoter and an enhancer that regulate its expression are operably linked in a host cell.

As used herein, the term "vector" refers to a DNA that transfers the above expression cassette into a host cell. It is known in the art that the type of the above-described vector and the type of each regulatory element used can be changed depending on the host cell. A preferred type of the vector of the present invention is a plasmid vector, which is a vector that can transfer the above expression cassette to microorganisms, animal cells, or plant cells. Those that can be transmitted to plant cells are particularly preferred.

Hereinafter, the present invention will be described in detail. The practice of the present invention may be practiced, unless otherwise indicated, using conventional techniques of molecular biology, biochemistry, genetics, genetic engineering, and plant breeding.

Plants transformed according to the method of the present invention may be monocots or dicots. Preferred monocotyledonous plants in the present invention are plants of the Poaceae family. Examples of plants of the Poaceae family include, but are not limited to, oak mushrooms, chishimasa, suzutake, medaka, rice, strawberry hornwort, mudwort eel, komegaya, oak wheat, nukabo, barley, kamojigusa, barley, bread wheat, tarho wheat, rye, reed, kazekusa, and oak grass. , Shiva, millet, millet, millet, crabgrass, sugarcane, pampas grass,
Tigaya, sorghum, barley, corn and the like. Particularly, rice is preferable. Preferred dicotyledonous plants in the present invention are plants of the Solanaceae family. Examples of plants of the Solanaceae family include wolfberry, hasidoro, physalis, eggplant, potato, tomato, capsicum, tobacco, datura and tsukubanasaagao. In particular, tobacco is preferred.

Thionine gene used in the present invention, wheat, barley, rye,
It is a gene for thionine derived from endosperm or leaves of wheat such as oats and oats. Alternatively, a gene of a protein similar to the above-mentioned thionin, for example, a mistletoe-derived biscotoxin gene, an abyssinian mustard oil seed-derived crambin gene, or Pyrularia pubera (parasitic plant of the sandalwood family) thionin derived from leaves and berries Similar protein genes (Wada and Ozaki, Plant Cell Engineering, 3 (3), pp. 200-207, (1991)) can be used. In the present invention, preferably,
The thionin gene derived from oat leaves is used.

The thionin gene described above has been isolated and its nucleotide sequence is known. Their nucleotide sequences have been registered with gene sequence registrars such as GenBank®. Those skilled in the art can obtain the nucleotide sequence of the thionin gene by searching the gene sequence repository or various databases (for example, DNASIS ^™ ) based on the sequence registered by the gene sequence repository.

The thionin gene useful in the present invention can be directly isolated from various plants based on a known nucleotide sequence using a method known to those skilled in the art. Or
The thionine gene, already isolated and cloned into a vector, is also used. Alternatively, a chemically synthesized gene based on a known nucleotide sequence is also used.

Briefly, the isolation of a thionin gene from various plants is, for example, isolation of mRNA from cells expressing thionin in the plant, construction of a gene (cDNA) library from the isolated mRNA, hybridization. It can be carried out by screening a gene library by the method. For hybridization, a polynucleotide chemically synthesized based on a known nucleotide sequence, or the entire cloned thionin gene or a fragment thereof can be used as a hybridization probe. Some of these operations can also be performed using various commercially available kits corresponding to each operation step. Conditions and procedures in this series of operations are described, for example, in Molecular Clonin
g A Laboratory Manual 2nd edition (ed. by Sambrook, J. et al., Cold Spring Harborlabora)
tory Press, 1989) and Current Protocols in Molecular Biology (Ausubel,
FM et al., John Wiley & Sons, 1987), and standard laboratory manuals in the field, and instructions for the kit used.

The thionine gene thus obtained may be modified, if necessary, in part or in its sequence to alter its activity and specificity, or to facilitate subsequent genetic recombination operations. Can be. Such modifications can be made using methods well known to those skilled in the art, such as site-directed mutagenesis or the polymerase chain reaction (PCR). It will be apparent to those skilled in the art that such alterations in the nucleotide sequence will alter one or more amino acids or amino acid sequence regions. These conditions are within the skill of the art. These standard methods are described in the above experimental manual.

An expression cassette for efficiently expressing thionin in plant cells includes, in addition to the coding region of the thionin gene, a promoter for transcribing the thionin gene to mRNA, and, if necessary, acting on the promoter to reduce its transcription activity. It consists of a regulatory enhancer and a terminator region that is involved in the stability of the transcribed mRNA.

The promoter is not particularly limited as long as it can transcribe the thionin gene into mRNA in the introduced plant cell. Such a promoter is not a plant-derived promoter (for example, cauliflower mosaic virus 35S (CaMV35S)).
RNA, a nopaline synthase (nos), or an octopine synthase (ocs) promoter, or a plant-derived promoter (eg, a secondary metabolic enzyme promoter such as chalcone synthase, or glycinin) Storage protein promoter). By properly selecting a promoter, a plant showing high disease resistance can be produced. Suitable promoters for enabling the expression of thionin in plants include, for example, CaMV35S
Promoter. The CaMV35S promoter is, for example, pBI121 (Clontech), p
It has been cloned into vectors such as BI221 (Clontech) and pCaMVCN (Pharmacia). In the present invention, the CaMV35S promoter is preferably used for the expression of the thionin gene in the cells of the transformed plant.

The expression level of the introduced thionin gene is increased by introducing an enhancer region. The enhancer region is a non-translated region such as an intron or a specific region within the promoter region, and may be located 5 ′ or 3 ′ of the promoter region to be used, or both. The enhancer region is, for example, a region of -343 to -90 in the CaMV35S promoter (Kar, R. et al., Science, 236: 1299-).
1302, 1987), the 5 'untranslated region of the tobacco mosaic virus called the Ω sequence (Gal
lie, DR et al., Nucl. Acids. Res., 15: 3257-3272, 1987), 5 'untranslated region of alfalfa mosaic virus (Jobling, SA et al., Nature, 325: 622-625, 19).
87), and the intron sequence of kidney bean phaseolin (Slightom, JL et al.,
Proc. Natl. Acad. Sci. USA 80: 1897-1901, 1983). These enhancer regions may be used alone or in combination with each other.
Further, a plurality of these enhancer regions can be used. In the present invention, preferably, the Ω sequence of the tobacco mosaic virus, the region within the CaMV35S promoter, or the intron sequence of common bean phaseolin, and more preferably all of them are used. When using these enhancer sequences in combination with the CaMV35S promoter, preferably the Ω sequence and the intron sequence of kidney bean phaseolin are 3 to the core region (-90 to -1) of the CaMV35S promoter.
On the 'side, the enhancer region of the region within the CaMV35S promoter is located on the 5' side. The number of enhancer sequences depends on the plant to be transformed.

The expression level (translation level) of thionin as an enzyme varies depending on the 3 ′ untranslated region (ie, terminator) present in 3 ′ of the thionin gene (Ow, DW et al., Supra). A terminator containing a polyadenylation signal site was added to the 3rd region of the coding region.
In this case, the transcribed mRNA is stabilized, and as a result, the amount of thionin translated is changed. Such terminators include, but are not limited to, CaMV35S terminator, nopaline synthase and octopine synthase terminators. In the present invention, a nopaline synthase terminator is preferably used.

The thionin expression vector used in the present invention contains, in addition to the thionin gene, a gene that imparts a trait as a selectable marker that allows easy selection of transformed plant cells (or plants). Such genes are, for example, the neomycin phosphotransferase (NPTII) gene for resistance to both kanamycin and neomycin antibiotics, and the hygromycin phosphotransferase (HPT) gene for hagromycin resistance. These selectable marker genes may be used alone or in combination of two or more. In the present invention, the thionin expression vector preferably contains an NPTII gene and an HPT gene as selectable marker genes. These genes are also under the control of the CaMV35S promoter and the nos terminator
Have to '. As a result, the resulting transformed plants are resistant to both kanamycin and hygromycin.

The thionin expression vector used in the present invention further comprises, if necessary, a region for integrating a region containing a thionin gene and a selectable marker gene that can be stably expressed in plant cells into the chromosome of plant cells (for example, Agrobacterium tu soil bacteria
RB sequence region and LB sequence region derived from Ti plasmid which are involved in tumor induction by infection with mefaciens), and regions required for replication in Agrobacterium tumefaciens (used for introducing an expression vector into plant cells). In addition. In addition, such expression vectors may have a marker (eg, ampicillin resistance) gene region that enables replication and selectivity in E. coli to facilitate genetic recombination operations on the vector. May be included. The expression vector in the present invention is preferably a thionin gene, a drug resistance gene for selection, an RB and LB sequence region enabling integration into a chromosome of a plant cell, Agrobacterium
It contains regions required for replication in ium tumefaciens, as well as replication and selectable marker gene regions in E. coli.

Such an expression vector can be constructed using a plant expression vector already containing these sequence regions, such as pBI121 (Clontech) derived from the Ti plasmid. pBI121 (Clontech) contains β-glucuronidase (GU) under the control of the CaMV35S promoter in the region between the LB and RB sequences.
S) and two expression units of the NPTII gene.

The thionin expression vector that can be used in the present invention is obtained, for example, by replacing the GUS region of pBI121 (Clontech) with a thionin coding region. Briefly, this expression vector was transformed into GU by restriction enzyme (BamHI and SacI) digestion.
It is prepared by inserting the thionin coding region obtained by digestion with restriction enzymes (BamHI and SacI) into pBI121 (Clontech) from which the S region has been removed. Enhancer sequences are described, for example, in pL ₁₁ A-A25 (Nishiguchi, M et al., Nucl. Acids Res., 13:55).
85-5590, 1985) and pBI121 (Clontech) described above, by restriction enzyme digestion, or by using these as templates by polymerase chain reaction (PCR) using appropriate primers. Subsequently, these sequences are, for example, Hi
It can be introduced 5 'to the CaMV35S promoter using an ndIII site and / or 3' to it using a BamHI site.

Alternatively, the thionin expression vector used in the present invention may be obtained by, on another cloning vector (for example, pUC18 and pBluescript ^™ (Stratagene)), a specific part of a necessary region (for example, a promoter region having an enhancer sequence, a coding region). And a region combining the promoter region and / or the terminator region, and a part thereof). By substituting such a partial region into a plant cell expression vector (for example, pBI121 (Clontech)) by substitution or the like, a desired final thionin expression vector for plant cell introduction can be constructed. Thus, the method of constructing an expression vector is not limited to the above procedure, and is within the purview of those skilled in the art.

Introduction of the thionin expression cassette into the plant cells of the constructed thionin expression vector is performed by infecting Agrobacterium tumefaciens or Agrobacteri
The method can be carried out using a method such as a method using soil bacteria such as um rizogenes or a method using electroporation.

Agrobacterium tumefaciens-mediated method, prior to infection of plant cells,
It requires introduction of the constructed expression vector into Agrobacterium tumefaciens. Agr
Introduction of the constructed expression vector into bacterium tumefaciens is performed by a method similar to the method for E. coli, for example, introduction by calcium treatment, freeze-thaw method, or electroporation method. Agrobacterium tumefaci required for introduction
The culture of ens can be performed using a medium such as an LB medium or a YEB medium under appropriate conditions (for example, at 25 to 30 ° C. for 24 to 36 hours).

Introduction of an expression vector into a plant cell by infection with Agrobacterium tumefaciens is performed by introducing a plant seed or tissue fragment (eg, leaf and / or stem section) and a cell (or culture solution) of Agrobacterium tumefaciens containing the expression vector. And for a predetermined time. Methods using leaf sections are known to those skilled in the art as the leaf disk method (eg, Horsch, RB et al., Science 227).
: 1229,1985). The range of plants infected by Agrobacterium tumefaciens and their efficiency vary depending on the species. For example, Agrobacterium tumefaciens LBA440
The four strains easily infect dicotyledonous plants, particularly tobacco, but are difficult to infect monocotyledonous plants such as rice. In contrast, for example, Agrobacterium tumefaciens EH
The A101 strain has a sufficiently high infection efficiency even for monocotyledonous plants as compared to the LBA4404 strain.

To ensure the infection of Agrobacterium tumefaciens, the plant seeds or explants are then placed on a solid medium. To this medium, a combination of cytokinin and auxin is added for the differentiation / induction and growth of plant cells into plants. As the cytokinin, for example, kinetin, benzyladenine, zeatin and 2-isopentenyladenine are usually used at a concentration of 0.1 to 10 ppm. As the auxin, for example, indoleacetic acid, indolebutyric acid, 2-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid are usually used at a concentration of 1 to 10 ppm. These combinations, and their concentrations, will vary depending on the plant and tissue used, but those skilled in the art can determine them, for example, with reference to the following experimental manual.

Specifically, Agrobacterium tumefaciens containing an expression vector (for example, E.
The culture solution of HA 101 strain) and the callus obtained by culturing the seeds are mixed and slowly stirred (for example, for 15 minutes). Then, in order to differentiate / induce the plant from the callus, for example, a plant tissue culture medium (for example, Murashige-Skoog (MS)) supplemented with 2,4-dichlorophenoxyacetic acid and sugar (usually 3% sucrose)
The callus is placed on an agar medium such as a medium, Rinsmeyer-Skoog (LS) medium, or Gamborg B5 medium, and then cultured, for example, at 27 ° C. for 3 days. It will be apparent to those skilled in the art that the composition of the medium used here will vary depending on the plant to be transformed and its tissue.

Thereafter, the infected cells containing the thionin gene are selected together with the eradication treatment of the infected Agrobacterium tumefaciens.

Eradication of Agrobacterium tumefaciens is performed by adding antibiotics (for example, 5
The above callus is cultured using a medium further supplemented with (00 ppm carbenicillin). The callus into which the expression vector has been integrated is selected in a medium to which an antibiotic corresponding to the selectable marker gene present in the expression vector has been added. For example, in the case of canine machine or hygromycin resistance, for example, 100 ppm, respectively
Kanai machine, 30 ppm hygromycin is used. Every 2-3 weeks, they are transplanted onto a fresh selection medium to allow eradication and selection while at the same time allowing adventitious shoots to differentiate / induce. In order to complete the eradication of Agrobacterium tumefaciens, carbenicillin may be further added at the beginning of the culture in the selective medium. Culture temperature is usually 20 to
30 ° C. The light-dark conditions are typically 16 hours light / 8 hours dark. It is obvious to those skilled in the art that the culture conditions at this time vary depending on the plant. The adventitious buds that have formed are cut off, transferred to a selective medium without auxin and cytokinin, and further cultured under the same conditions as above to allow rooting. The seedlings obtained in this way are adapted and allowed to grow on ordinary soil in a natural or artificial environment.

In another embodiment of the present invention, introduction (infection) of an expression cassette containing a thionin gene into a plant cell can be performed using protoplasts prepared from plant tissues (eg, leaves) instead of seeds. (See, for example, Marton, L. et al., Nature 2
77: 1229, 1979). When using protoplasts, an expression vector containing a thionin gene can be directly introduced into plant cells by electroporation. The protoplast into which the expression vector has been introduced is cultured under appropriate conditions to regenerate the cell wall. Subsequent procedures differentiate / induce plants using the same procedure as for callus obtained from seeds described above.

Procedures for such plant transformation include, for example, Plat Genetic Transformation
and Gene Expression A Laboratory Manual (edited by Draper, J. et al., Blackwell Scient
ific Publications, 1988) and DNA Cloning, apractical approach (Glover
, DM et al., Vol. 2, IRL Press, 1985).

The transgene of the transformed plant of the present invention can be qualitatively and quantitatively analyzed by the following method. For example, a method comprising recovering the leaves of a plant into which a thionin gene has been introduced, freezing in liquid nitrogen and pulverizing (Verwoerd et al., (1989)
MRNA is recovered by Nucl. Acids Res. 17, 2362). The collected mRNA can be separated by electrophoresis, transferred to a nitrocellulose membrane or the like, and examined by Northern blot analysis in which the mRNA is reacted with a labeled DNA or RNA probe that specifically hybridizes to the mRNA of the thionin gene. It is understood by those skilled in the art that the conditions for the method for collecting mRNA and the method for Northern blot analysis can be changed depending on the situation. In addition, proteins are recovered from the transformed plants by a known method, separated by electrophoresis, transferred to a nitrocellulose membrane or the like, bound to an antibody against thionin, and labeled with a specific antibody ( ^125I- labeled antibody, It can also be determined by Western blot analysis, which detects with peroxidase-conjugated anti-IgG). The qualitative and quantitative analysis of the transgene of the transformed plant is not limited to the above method.

The disease resistance of the transformed plant of the present invention can be assayed by the following method. For example, the leaves of the obtained transformed plant are scratched with a needle, blade, etc. (Mock treatment)
The resistance of the transformed plant can be assayed by inoculating a bacterial solution containing the pathogenic bacteria and comparing the area of the part damaged by the infection with that of a non-transformed plant similarly treated. It is understood by those skilled in the art that the assay conditions can be varied depending on the plant species and pathogen species to be assayed. During the assay, the tissues or cells are fixed or alive, aniline blue (Eschrich W. and Currier HB
, (1964) Stain Technology 39, 303-307) for comparison. Alternatively, seeds obtained from the transformed plant are immersed in the pathogen solution, then sown on the soil, germinated and grown, and the number of individuals growing normally and the number of individuals growing from the seeds of the non-transformed plant treated in the same manner. It can also be checked by comparing with a number. In addition, the assay can also be performed by culturing transformed plant cells and non-transformed plant cells in a medium supplemented with a pathogenic bacterium for a predetermined period (for example, 3 days) and comparing the numbers of surviving cells. It is understood by those skilled in the art that the method for assaying disease resistance of a transformed plant of the present invention is not limited to the above.

The transformed plants of the present invention can form seeds for propagation by crossing (eg, self-pollination, pollination between transformed plants, or pollination between transformed and non-transformed plants).

In addition, tissue culture of transformed plant tissues (eg, roots, stems, leaves) or organs (eg, growing points, pollen) using the procedures for plant differentiation / induction from infected cells described above. Thereby, a further transformed plant can be obtained without going through a reproductive process (seed). Such techniques and procedures are known to those skilled in the art. General methods of tissue culture are described in various laboratory manuals.

The thus obtained transformed plant of the present invention grows normally without being inhibited by the thionin gene introduced by the transformation, and has a high resistance to at least one disease due to the expression of the thionin gene. Is shown. In addition, the seeds obtained from the transformed plants germinate and grow normally and show high resistance to at least one disease. This indicates that the introduced thionin gene is conserved in the next generation, and thus that the above-mentioned disease resistance is stably passed on to progeny. Therefore, according to the present invention, practical and useful plants exhibiting high disease resistance can be obtained.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the examples.

<Example 1>
(1) Preparation of thionin gene Thionine cDNA clone LTH92 (Japanese Patent Application Laid-Open No. 8-266279) and a known conserved region of thionin base sequence (5 ′ region and acidic protein region) The following sequences, represented by SEQ ID NOs: 1 and 2, were synthesized based on:
Forward direction: 5'-AATCAAGCTTCCAAGTAGAAGGCAAGAGTTGC-3 '(SEQ ID NO: 1)
Reverse direction: 5'-AGTCTCTAGAGTCCATGTTGTCACAGACGG-3 '(SEQ ID NO: 2)
BamHI, Sam was added to thionin cDNA by PCR using two sets of primers having
The thionin gene to which an acI restriction enzyme site was added was obtained.

The reaction system of PCR was 0.5 μl of Taq polymerase (5 U / ml), 5 μl of 10 × buffer (100 mM Tris (pH 8.3), 500 mM KCl, 1% Triton X100), 4 μl of 2.5 mM MgCl ₂
4 μl of 10 mM dNTP, 1.25 μl of each of the above primers (10 pmol) and about 5
A system in which 00 ng of cDNA was adjusted to 50 μl with distilled water was used. The PCR reaction conditions include a denaturation reaction at 94 ° C. for 2 minutes, a denaturation reaction at 94 ° C. for 1 minute, an annealing reaction at 50 ° C. for 1 minute, and an elongation reaction at 72 ° C. for 2 minutes. The cycle was repeated 25 times, and conditions were used that lasted an additional extension reaction at 72 ° C. for 5 minutes.
(2) Construction of thionin expression cassette and expression vector pE7133GUS (Mitsuhara et al., Plant Cell Physiol., 37 (1): 49-59 (1996)) was digested with restriction enzymes HindIII / BamHI and separated by agarose electrophoresis. , GeneClean2 (B
IO101) to obtain a promoter cassette. Also, pBI121 (Clontech) was digested with restriction enzymes HindIII / BamHI, separated by agarose electrophoresis, and GeneClean2 (B
IO101) gave the vector portion. These were ligated using Takara Ligation Kit (Takara) to obtain pBE7133GUS.

Next, pFF19 (Timmermans, MCP et al., J. Biotechnol., 14: 333-344, 1990) was used.
By restriction enzyme digestion with BamHI / SacI, CaMV35S promoter and CaMV35S
The hygromycin phosphotransferase (HPT) gene containing the terminator was isolated. After smoothing both ends of the obtained HPT gene fragment, Sm
The aI linker was ligated and digested with the SchiI isoschizomer restriction enzyme Cfr9I. Thereafter, this HPT gene fragment was inserted into the Cfr9I site located 3 ′ of the GUS gene of pBE7133GUS to obtain pBE7133GUS-HPT.

Next, pBE7133GUS-HPT was digested with BamHI / SacI restriction enzymes, the GUS coding region was removed, the thionin gene obtained above was inserted, and the thionin expression vector pB
E7133H-Thionin was obtained. CaMV35 regulates the NPTII gene in pBE7133H-Thionin
Nos located 3 'of the HPT gene from the S promoter with the thionin gene in between
The part up to the terminator is called a thionin expression cassette.
(3) Introduction of Thionine Expression Cassette into Agrobacterium tumefaciens EHA101 Strain Agrobacterium tumefaciens EHA101 strain (obtained from Plant Breeding Laboratory, Tohoku University, Faculty of Agriculture) was mixed with 5 ml of YEB medium (0.1% yeast extract, 0.5% meat extract, 0.5% peptone, 0.5% sucrose, and cultured with shaking overnight at 30 ° C. with _{0.05% MgSO 4 · 7H 2 O} ). Then, 5 ml of the above culture was added to 200 ml of YEB medium in a 1 liter flask, and further cultured at 30 ° C. for 5 to 6 hours. The cells were collected by centrifugation (4000 rpm for 5 minutes) 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. 200 μl of this suspension and 100 μl of DNA solution containing 0.5 μg of pBE7133H-Thionin were mixed in a microfuge tube and frozen in a dry ice / ethanol bath (5 minutes). Then 37 ° C
In a warm bath and left for 25 minutes. Thereafter, 2 ml of YEB medium was added, and 3
Shaking culture was performed at 0 ° C. for 1 hour. 100 μl of the culture solution was kanamycin (50 μg / ml)
And Agrobacterium tumefaciens strain EHA101 was obtained by plating on YEB medium containing E. coli and hygromycin (50 μg / ml) and culturing at 30 ° C. for about 36 hours. The thionin expression cassette in this Agrobacterium tumefaciens EHA101 strain was isolated by the alkali-SDS method, and its presence and structure were confirmed by restriction enzyme analysis.
(4) Transformation of rice Sterilized rice seeds were treated with 2 mg / ml of 2.4-dichlorophenoxyacetic acid (hereinafter referred to as 2.4-D
) Containing MS medium (T. Murashige et al., Physiol. Plant., 15, 473 (1962))
At 27 ° C. for 2 weeks to form callus. This callus was added to a co-culture medium (2 mg / m
l-2.4-D and an MS medium containing 1 g / l casamino acid) and cultured at 27 ° C. On the third day of the culture, the Agrobacterium tumefaciens EHA101 strain containing the thionin expression 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 an Agrobacterium tumefaciens EHA101 strain solution for infection. This bacterial solution was kept at 30 ° C. for 20 minutes. Agrobacterium tu for infection on day 4 of culture
The callus mass was added to the mefaciens EHA101 strain solution, and the mixture was gently stirred at 30 ° C. for 15 minutes to infect the callus mass. After 15 minutes, the calli were collected and placed on a co-culture medium. Culture at 27 ° C. for 16 days with a photoperiod of 3 days, then at 30 ° C. for 1 hour in LB medium containing carbenicillin, then collect only callus and select (grow) medium (
(A co-culture medium containing 500 mg / l carbenicillin and 50 mg / l hygromycin). This was cultured at 27 ° C. for 16 hours with a photoperiod of 2 weeks. Newly grown yellow-white callus is selected (pre-regeneration treatment) medium (1 mg / l 2.4-D, 0.5 mg / l BAP, 2 g / l casamino acid, 20 g / l sucrose, 30 g / l sorbitol, 500 mg / l carbenicillin, 100mg /
l) After transplanting to N6 medium containing hygromycin and culturing for about 2 weeks, a selection (regeneration) medium (0.01 mg / l β-naphthaleneacetic acid (NAA), 0.1 mg / l BAP, 1 g / l casamino acid, (N6 medium containing 500 mg / l carbenicillin and 100 mg / l hygromycin). In about 2 to 3 weeks, regenerated transformed rice was obtained.
<Example 2>
Assay for Resistance of Rice (Chiyohonami) to Bacterial Leaf Blight The test for resistance to rice blight was carried out by the first-generation self-lined lines 2-1 and 11 of the transgenic rice obtained by selecting with hygromycin. -2 and 13-3 lines and untransformed rice leaves. Make two wounds on rice leaves with short needles,
A bacterial suspension containing 1 × 10 ⁷ cfu / ml of Bacterial blight of rice was inoculated with Bacterial blight of rice. After cultivation for 2 weeks, the area of the leaves damaged by the infection of the bacterial leaf blight fungus was visually evaluated. Noda et al. ("Hokuriku Agricultural Experiment Report", 1989) was used as an index of resistance to rice leaf blight, using the rice leaf blight disease incidence index inoculated with a needle.

In the non-transformed rice inoculated with the rice Bacterial Blight fungus, the disease index was 4.4, whereas the transformed rice inbred first generation line 2-
The value was 1.8 for 1; 0.8 for 11-2; and 2.2 for 13-3. Transformed rice is
High resistance to rice leaf blight. The results are shown in FIG. 1 and FIG.
FIG. 1 is a graph showing the resistance to Bacterial blight on rice using the Index of Bacterial Blight Onset of Rice. Eight leaves were tested for each individual leaf to determine the rice leaf blight onset index, and the average value was shown in a graph. FIG. 2 is a photograph showing the results of the resistance test. The upper leaf shows the leaves of the first-generation transgenic rice line 2-1 inoculated with the Bacterial Blight Fungus, and the lower leaf shows the leaves of non-transformed rice inoculated with the Bacterial Wilt Blight.
<Example 3>
Assay for Resistance of Rice (Chiyohonami) to Bacterial Seedling Blight Disease The resistance test for rice seedling blight disease was carried out by using a transgenic second-generation transgenic rice line 2-1.
1 and seeds obtained from the same line 11-2.1 and seeds obtained from untransformed rice. The rice seeds were sucked for 1 hour and immersed for 3 hours in 1 × 10 ⁶ cfu / ml suspension of rice seedling bacterial wilt, and then air-dried at room temperature overnight. Soak at 20 ° C
For three days in twice the volume of ion-exchanged water, and germination was carried out at 30 ° C. under high humidity for one day. Thereafter, 24 seeds per minicup were sown on the cultivation soil for paddy rice seedlings,
Embryos were germinated for 3 days at ℃, high humidity and dark, and then cultivated in a closed greenhouse. About 10 days after sowing, the resistance was tested by comparing the number of individuals normally growing from the seeds and the establishment rate of seedlings.

When seeds obtained from non-transformed rice were infected with bacterial seedling blight, all individuals died shortly after emergence. On the other hand, when the seeds obtained from the transformed rice second-generation line 2-1.1 were infected with the bacterial fungus of rice seedling blight, the seeds sown showed a seedling establishment rate of 75%, which was normal. Grew up. Also, in the case of the same line 11-2.1, the sown individual showed a 96% seedling establishment rate and similarly grew normally. The transformed rice showed high disease resistance to rice seedling blight disease. The results are shown in Table 1 and FIG. FIG. 3 is a photograph showing the results of the resistance test. The left plant shows untransformed rice not inoculated with rice seedling bacterial wilt, the middle plant shows transformed rice line 2-1.1 inoculated with rice seedling dead bacillus, and the right plant Shows a non-transformed rice plant inoculated with a rice seedling bacterial wilt disease.

<Example 4>
Preparation of Transformed Tobacco (Nicotiana tabacum Sammsun NN) Preparation of a thionin gene and construction of a thionin expression cassette and an expression vector were performed in the same manner as in Example 1.
(1) Introduction of a thionine expression cassette into Agrobacterium tumefaciens strain LBA4404 Agrobacterium tumefaciens strain LBA4404 (Clontech) was cultured in an LB medium containing 250 μg / ml streptomycin and 50 μg / ml rifampicin (10 g / l bactotryptone, 5 g / l yeast extract). , 10 g / l NaCl (pH 7.2)) at 28 ° C. Agr according to the method of Nagel et al. (Microbiol. Lett., 67, 325 (1990)).
A bacterial suspension of bacterium tumefaciens strain LBA4404 was prepared, and the thionine expression vector pBE7133H-Thionin prepared in Example 1 (2) was introduced into the above strain by electroporation. The thionine expression cassette in Agrobacterium tumefaciens strain LBA4404 was isolated by the alkali-SDS method, and its presence and structure were confirmed by restriction enzyme analysis.
(2) Transformation of tobacco Agrobacterium tumefaciens LBA4 containing the thionin expression cassette obtained above
The 404 strain was cultured with shaking in a YEB medium at 28 ° C. overnight. Leaves that were completely elongated from tobacco plants were cut off and sterilized. The sterilized leaves were cut out with a paper punch to prepare leaf disks, and the leaf disks were immersed in the above culture solution diluted 20-fold with sterile water, and gently permeated for 1-2 minutes. After infiltration, the leaf disk was washed with MS medium for 2 hours.
The cells were cultured at 7 ° C for 2 days. After the cultivation, the leaf disk was transferred onto an MS agar medium containing carbenicillin, cultured at 26 ° C. for 1 week under light, and
cterium tumefaciens LBA4404 strain was excluded. The disinfected leaf disk was subcultured every two weeks on an MS agar medium containing carbenicillin, kanamycin, and hygromycin, and the transformed leaf disk was selected. Callus was induced from this leaf disk by a conventional method and redifferentiated into tobacco plants.
<Example 5>
Assay for Resistance of Tobacco (Nicotiana tabacum Sammsun NN) to Potato Blight The resistance test to potato blight was carried out on the transgenic tobacco lines obtained above in the same manner as the current lines 4, 5, 15, 18, 41 and 44 of the leaves and non-leaf. This was performed using transformed tobacco leaves. The potato late blight fungus was cultured in a PDA medium (Tifco) at 18 to 20 ° C. for about one week, and the obtained flora was punched out with a cork borer having a diameter of 3 mm to prepare a potato late blight agar block. The agar block was placed on the leaves so that the microflora and the leaves were in contact with each other, inoculated with potato late blight, and kept at 27 ° C. The area of leaf lesions caused by the potato late blight was visually evaluated 2-3 days later.

The non-transformed tobacco inoculated with the potato late blight had a lesion area of 976 mm ² , whereas the transformed tobacco modern line 4 similarly inoculated with the potato late blight
In 48 mm ^2, 57 mm ² in the same strain 44 was 40 mm ² in the same strain 18. Further, in the same strains 5, 15, and 41, almost no damage was caused by the potato late blight. Thus, the transformed tobacco showed high disease resistance against potato blight. The results are shown in FIGS. FIG. 4 is a graph showing resistance to potato late blight by lesion area. Four points were tested on the leaves of each individual, and the average value of the lesion area caused by the potato late blight was shown in a graph. FIG. 5 is a photograph showing the results of the resistance test. The state of each solid 3 days after inoculation of the potato late blight fungus is shown. From left, untransformed tobacco inoculated with potato late blight (
SNN) Leaves, Transgenic tobacco line 4 (M7TH4) inoculated with potato late blight
And 5 (M7TH5) leaves.
(Sequence list)

The graph which showed the resistance with respect to the bacterial leaf blight of the transformed rice into which the thionin gene was introduced by the lesion index. The photograph which shows the result of the resistance test with respect to the bacterial leaf blight of the transformed rice into which the thionin gene was introduced. The photograph which shows the result of the resistance test with respect to the rice seedling blight disease of the transformed rice into which the thionin gene was introduced. The graph which showed the resistance with respect to the potato late blight of the transformed tobacco which introduced the thionin gene by the lesion area. The photograph which shows the result of the resistance test with respect to the potato late blight of the transgenic tobacco which introduced the thionin gene.

Claims (8)

It contains a thionin gene and has an expression cassette capable of expressing the thionin, and is derived from a pathogenic bacterium selected from the group consisting of rice bacterial wilt, rice seedling wilt, rice wilt and potato late blight. A plant or plant showing resistance to one disease. The plant according to claim 1, wherein the thionin is derived from oat leaves. 3. The plant according to claim 1, wherein the plant is a monocotyledonous or dicotyledonous plant. The plant according to claim 3, wherein the monocotyledonous plant is a plant of the Poaceae family. The plant according to claim 3, wherein the dicotyledonous plant is a plant of the Solanaceae family. The plant according to claim 1, wherein the disease is rice leaf blight or rice seedling blight. The plant according to claim 1, wherein the disease is a potato blight. A seed obtained from the plant of claim 1.

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