JP2007306917A - Method for increasing the resistance of rice plant to pathogenic microorganism and pathogenic microorganism-resistant rice transformant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for increasing the resistance to rice plant pathogenic microorganisms and provide a rice plant transformant that has the resistance to the pathogenic microorganisms. <P>SOLUTION: In rice plant, a method for increasing the resistance to rice plant pathogenic microorganism that includes the increase of the expression level of at least one kind of gene relating to the biological defense bearing a specific base sequence or at least one kind of its mutant genes is provided. Further, the pathogenic microorganism resistant rice plant transformant in which at least one kind of gene such as biological defense relating gene or at least one kind of gene can ecdemicly and expressibly introduced. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for increasing resistance of rice to pathogenic bacteria and a pathogen-resistant rice transformant, and more particularly, to a method for increasing resistance to pathogenic bacteria by increasing the expression level of a biological defense-related gene, and biological defense. The present invention relates to a pathogen-resistant rice transformant into which a related gene has been introduced.

  Conventionally, methods for controlling diseases of agricultural crops include the use of agricultural chemicals that act directly on plant pathogens to control diseases. Pesticides of the type that show bactericidal effects against pathogenic bacteria have the major problem that mutants that are resistant to drugs appear through continuous use. In addition, the effects of residual agricultural chemicals on the human body and the indegradability in the environment are also serious problems.

  Other disease control methods include the use of agricultural chemicals (resistance-inducing pesticides) that control disease of crops by increasing the disease resistance of the plant itself. Resistance-inducing pesticides are known to activate the resistance by recognizing innate immunity mechanisms of plants, that is, pathogens. As resistance-inducing pesticides, propenazole (trade name: orizamate), acibenzoral-S-methyl, and the like have been put into practical use, all of which are chemically synthesized pesticides. Considering the impact on the environment, it is necessary to avoid excessive dependence.

  Therefore, a plant vital agent having a disease control effect that does not contain a chemically synthesized pesticide has been developed (Patent Documents 1 and 2). These plant vital agents are a combination of vinegar, trehalose and plant extract ingredients.

  Trehalose is known to be a so-called signal molecule (elicitor) that enhances the activities of plant phenylalanine ammonia lyase (PAL), peroxidase, jasmonic acid, etc., and induces a plant defense reaction. There is a method for controlling plant diseases using this elicitor. For example, spraying a solution of trehalose onto wheat leaves can reduce powdery mildew infection (Non-patent Document 1). Further, when wheat and grape seedlings are treated with trehalose and further treated with a synergist such as phosphorous acid and fosetyl aluminum, there is a synergistic effect on wheat powdery mildew and grape downy mildew (Patent Document 3). However, when only trehalose is applied to wheat powdery mildew and grape downy mildew, there is no direct effect (Patent Document 3).

  In recent years, crops that are resistant to disease have been produced by genetic recombination. For example, there is a rice transformant having resistance to rice blast disease into which a gene encoding a protein whose expression is induced by a rice blast fungus-derived cerebroside-type elicitor is introduced (Patent Document 4).

  However, there is no comprehensive study of rice proteins and genes whose expression is induced using trehalose, the aforementioned elicitor, and the use of such rice genes to increase resistance to pathogenic bacteria. Has not yet been done.

JP 2001-61344 JP JP 2001-64112 A Special Table 2002-511495 JP 2002-272291 A PH. Reignault et al., (2001) Trehalose induces resistance to powdery meldew in wheat.New Phytologist, Vol.149, pp.519-529

  An object of the present invention is to provide a method for increasing resistance to pathogenic bacteria in rice by clarifying a gene related to biological defense in rice induced by trehalose and increasing the expression level of the gene.

  Another object of the present invention is to provide a rice transformant having resistance to pathogenic bacteria by introducing a biological defense-related gene into rice.

In order to elucidate the signal molecule function of trehalose, the present inventors identified a gene induced by trehalose treatment using a rice microarray, and found that a number of rice defense-related genes are induced by trehalose. The headline and the present invention were completed.
That is, the present invention is summarized as follows.

  In the first aspect, the present invention provides a host defense-related gene having any one of the nucleotide sequences of SEQ ID NOS: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61, or a rice There is provided a method for increasing resistance to pathogens in rice, comprising increasing the expression level of at least one gene of a mutant gene.

  In another embodiment, the expression level of the gene is increased by treating rice or seed with trehalose or a derivative thereof.

  In another embodiment, the expression level of the gene is increased by introducing the gene exogenously and expressively into rice.

  In another embodiment, the expression level of the gene is increased by more than 2-fold relative to control rice.

  In another embodiment, the pathogenic fungus is a blast fungus or a seedling blight fungus.

  In a second embodiment, at least one of the biological defense-related genes having any one of the nucleotide sequences of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 or 61 or mutant genes thereof Provided is a pathogen-resistant rice transformant, wherein a gene is introduced exogenously and in an expressible manner.

  In another embodiment, the pathogenic fungus is a blast fungus or a seedling blight fungus.

  In the third embodiment, the biological defense having the base sequence of any one of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61, for increasing resistance to pathogens of rice Methods of using at least one gene of related genes are provided.

  The method for increasing resistance of rice according to the present invention to pathogenic bacteria uses a natural defense-related gene inherent in rice, does not adversely affect the human body and the environment, and is a natural saccharide trehalose having no persistence such as agricultural chemicals. Thus, the biological defense reaction of rice can be induced safely and inexpensively. In addition, by introducing a biodefense-related gene into rice, a rice transformant having high resistance to pathogenic bacteria can be produced. Since the amount of the synthetic chemical pesticide used according to the present invention can be reduced, the commercial value of rice (rice) can be increased economically and in quality.

Trehalose is a natural carbohydrate widely distributed in mold, yeast, red algae, lichens and many insects, and is contained in large amounts in shiitake mushrooms, fermented foods, etc., and is produced at low cost. It is a non-reducing disaccharide in which two molecules of D-glucose are 1,1-linked and has a molecular formula of C ₁₂ H ₂₂ O ₁₁ and a molecular weight of 342.30. There are three types of isomers, α, α-, α, β-, β, β-, and there are α, α-forms in nature. The trehalose used in the present invention may be of any binding mode. Also, it may be a derivative of trehalose. The trehalose derivative is obtained by esterifying, diesterifying, monoesterifying, etherifying, acylating, cationizing or otherwise condensing trehalose, for example, trehalose fatty acid ester, α, α -Trehalose trimycolate, α, α-trehalose fatty acid tetraester, trehalose fatty acid monoester, α, α-trehalose ether derivative, 2-O- (9,10-epoxystearoyl) -α, α-trehalose derivative, 4 , 4-Di-O-alkyl α, α-trehalose derivatives, 2,3,2-tetraalkyl α, α-trehalose derivatives, α, α-trehalose fatty acid diamide derivatives, neotrehalose (acting α-amylase on starch) Product), lactone trehalose (forms glycosyltransferase in aqueous solution containing lactose and starch) Used product). Trehalose is commercially available (Hayashibara Shoji Co., Ltd.) in the form of water-containing crystal trehalose, for example.

  In the present invention, trehalose is a substance (signal molecule) that induces a host defense reaction of rice. However, the relationship between trehalose signal and a host defense-related gene has not been clarified so far in rice. Therefore, to elucidate the signal molecule function of trehalose, rice was treated with trehalose and the induced gene was identified. Identification can be performed, for example, by the following method.

  Rice seeds are germinated and grown into rice seedlings, the seedling roots are treated with a trehalose solution (for example, concentration 5 mM), and the root tissue is treated with liquid nitrogen to obtain powder, thereby preparing total RNA. Examples of RNA preparation methods include, but are not limited to, the CTAB method, the guanidine method, and the SDS / phenol method. A commercially available RNA extraction reagent (for example, TRIzol reagent manufactured by Invitorogen) may be used.

  Next, labeled RNA is synthesized by the one-color fluorescence method or the two-color fluorescence method. For example, reverse transcription is performed from the prepared RNA to synthesize a single-stranded cDNA, and Cyanine3 CTP or Cyanine5 CTP is added and transcribed to synthesize labeled RNA. A commercially available kit (eg, Agilent's Low RNA input liner amp kit) can be used for this synthesis. Alternatively, the synthesized labeled RNA may be purified using a commercially available RNA extraction kit (for example, RNeasy mini kit manufactured by QIAGEN). Alternatively, the synthesized cDNA may be labeled.

  Next, a hybridization solution is prepared from the purified labeled RNA or labeled cDNA, and hybridization is performed using, for example, an Agilent 22K rice microarray. Hybridization is performed, for example, at about 60 ° C. for about several hours to 20 hours. After hybridization, the microarray is washed, water is removed, gene expression level information is read with a microarray scanner (for example, Agilent), and it is compared with trehalose treatment with analysis software (for example, Agilent, Feature Extraction Software). The difference in the expression level of each gene in the root tissue is analyzed.

  Table 1 shows rice genes induced by treatment with 5 mM trehalose more than twice as compared with the control (rice not treated with trehalose).

  The gene numbers in the table are GenBank accession numbers accessible from the NCBI (National Center for Biotechnology Information) website, and the genes are rice gene names or homolog gene names corresponding to the gene numbers. In addition, fold indicates how many times the gene was induced by trehalose treatment compared to the control. That is, the numerical value of the expression level treated with trehalose (Tre) / the numerical value of the expression level of the control (mock).

  As shown in Table 1, it was found that trehalose induces many biological defense-related genes. In the case of plants, in general, the expression of many biological defense-related genes is induced by either salicylic acid (SA), ethylene, or jasmonic acid (JA). Based on the microarray experiment results, trehalose induced ethylene-responsive transcription factor gene (gene number: AK109390 in Table 1), SA biosynthesis gene (AK067801), JA-responsive transcription factor gene (AK069082), etc. 3 It is thought that all signal transduction systems involving two hormones are activated. Thus, the mechanism that recognizes trehalose is thought to be located upstream of the three hormone signaling, and therefore, activates various pathways located downstream.

  From previous studies, it is known that disease resistance is enhanced by increasing the expression level of transcription factors or receptor kinases involved in disease response (Reference: Berrocal-Lobo et al., Plant J. 29:23). -32 (2002), Hu, H et al., Planta 222: 107-117 (2005)). Therefore, nine genes were arbitrarily selected from the biological defense-related genes induced by trehalose in Table 1 (Table 2), and it was confirmed that the gene expression was actually induced by trehalose.

Confirmation of the induction of expression by trehalose is performed, for example, by Northern analysis.
First, a cDNA library from the root tissue of trehalose-treated rice was synthesized by synthesizing probe fragments of each gene to produce synthetic nucleotides for Northern blot probes, and by synthesizing PCR primers to amplify each gene. Amplify the partial length cDNA by PCR. For amplification, a commercially available PCR kit (for example, Takara Shuzo Ex Taq PCR kit) may be used. PCR conditions include, for example, DNA denaturation at about 95 ° C. for 15 seconds to 5 minutes, primer annealing at about 50 to 58 ° C. for 30 seconds to 1 minute, and extension reaction at about 65 ° C. to 72 ° C. for 30 seconds to 10 minutes. This includes 20 to 40 cycles as one cycle, and finally, an extension reaction at about 65 ° C. to 72 ° C. is performed for 1 to 5 minutes.

  Furthermore, the amplified cDNA is cloned using an appropriate vector (for example, pGEM-Teasy, Promega), and the base sequence of the obtained DNA is determined. For example, it can be determined using a sequencer ABI1310 (Applied Biosystem).

Next, rice seeds are germinated and cultivated on rice seedlings. A trehalose solution (for example, a concentration of 5 mM) is brought into contact with the roots, and root tissues are collected every several hours. The control is root tissue collected before contact with the trehalose solution. Total RNA is prepared from the root tissue by the method described above, and used as an RNA sample. The RNA sample is subjected to agarose gel electrophoresis to separate the size, and the RNA is transferred to a nylon or nitrocellulose membrane. The synthetic probe for Northern blot synthesized as described above is labeled with a labeling agent such as a radioisotope such as ³² P, a fluorescent molecule such as fluoresamine, rhodamine, dansyl, and derivatives thereof, and this is used as a probe for hybridization. To detect the target gene (Molecular Biology Experiment Protocol I (1997), co-translated by Nishino and Sano, Maruzen Co., Ltd.). FIG. 1 shows that expression of the biological defense-related genes in Table 2 was induced by trehalose.

  Next, through microarray analysis (Table 1) and confirmation of gene expression as described above, genes with high inducibility were selected from transcription factor genes and receptor kinase genes involved in the pathogenic response induced by trehalose (Table 1). 3).

  In order to isolate the genes listed in Table 3, first, primers for each gene are designed with reference to rice genome sequence information registered in GenBank, and the full-length gene is amplified by RT-PCR. Total RNA is prepared from rice root tissue treated with trehalose as described above, and single-stranded cDNA is synthesized from the RNA by reverse transcription. Using this as a template, each gene fragment is amplified by PCR. The amplified gene fragment is incorporated into the appropriate cloning vector described above, and the nucleotide sequence is determined.

  The nucleotide sequences of the identified genes are shown in SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 and 61. The amino acid sequences of the proteins encoded by these genes are shown in SEQ ID NOs: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62, respectively.

  In the present specification, the biological defense-related gene of the present invention is a gene involved in a disease response induced by trehalose in rice, and includes genomic DNA, RNA, cDNA, variants thereof, or functional fragments thereof.

  In the present specification, the functional fragment is an arbitrary fragment having a biological defense ability against pathogenic bacteria.

  In the present specification, the mutated gene is 1 in the base sequence (SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 or 61) of any of the biological defense-related genes of the present invention. Or more, preferably having one or several base substitutions, deletions or additions, or base sequence and 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more homology. Further, the term “several” means 2 to about 10, preferably 2 to 5. Since various types and varieties of rice exist, some of the above genes have polymorphisms and mutations between the types and varieties of rice. In the present invention, these are included as mutant genes. .

  By highly expressing the biological defense-related genes identified as described above in rice, the resistance of rice to pathogenic bacteria can be increased. Here, high expression means expression that is 2 times or more, for example, 2 to 7 times higher than the expression level of the gene in rice (that is, control rice) when trehalose is not treated. In the present invention, there are at least one biologically-related gene that is highly expressed, that is, one or two or more, preferably three or more, and more preferably five or more.

  In rice, the method for high expression of a biodefense-related gene is not limited to the following. For example, a method of introducing the gene into rice exogenously and in an expressible manner, rice or its seeds in trehalose or The method of processing with the derivative | guide_body etc. can be mentioned.

  Rice used herein is a plant that produces rice, and examples of rice include japonica rice and indica rice, but are not limited thereto. Rice shall include all varieties.

  In a method of introducing a host defense-related gene into rice exogenously and in an expressible manner, the gene of the host defense-related gene is increased by introducing the gene into rice exogenously and in a expressible manner by genetic recombination technology. Can do. Accordingly, the present invention also provides a rice transformant imparted with resistance to pathogenic bacteria by exogenously enhancing the expression of the gene.

  In the present specification, exogenous means that a foreign gene is artificially introduced into a plant cell or tissue. In the same specification, the expression possible means that the introduced foreign gene is autonomously replicated / expressed in the plant under the control of the control sequence, or is incorporated into the chromosome to be inducibly or non-inducible. It refers to a condition that can be expressed in an automated manner.

  As the above method, for example, a foreign gene can be incorporated into an appropriate vector and introduced into rice cells. A control sequence such as a promoter and a promoter / enhancer can be inserted into the vector so that the gene can be expressed.

  Examples of promoters include, but are not limited to, rice actin gene (Act1) promoter, cauliflower mosaic virus-derived 35S promoter, Agrobacterium Ti plasmid-derived nopaline synthase gene promoter, corn-derived ubiquitin promoter, and the like. . Examples of further promoters include rice lip19 gene promoter induced by low temperature, rice hsp80 gene or hsp72 gene promoter induced by high temperature, Arabidopsis rab16 gene promoter induced by dryness, anaerobic conditions The promoter of corn alcohol dehydrogenase gene induced, the promoter of parsley chalcone synthase gene induced by ultraviolet irradiation, the promoter of rice chitinase gene expressed by infection with pathogenic bacteria, and the like can also be included.

  Examples of vectors include, but are not limited to, intermediate vectors such as pGV3850, pLGV23Neo, pNCAT, and pMON200, binary vectors such as pBI121, pBI101, pBI2113Not, pBI2113, pGA482, pGAH, pBIG, and pActZH2 used in Example 6. , Etc.

  The intermediate vector is a cloning vector containing T-DNA, in which the target gene is inserted together with a selection marker and the like. A set in which the target gene is incorporated into the Ti plasmid by homologous recombination of T-DNA by conjugation of bacterial cells such as Escherichia coli transformed with this vector and Agrobacterium containing the T-DNA-containing Ti plasmid. Since the transformed Agrobacterium is produced, plant cells can be infected with this to transform the plant.

  The binary vector lacks T-DNA but has a right border (RB) and a left border (LB) in the region, where the target gene is inserted between LB and RB, Further included are markers, promoters and the like for selection in bacteria. The transformed Agrobacterium produced by joining E. coli containing this vector and Agrobacterium containing Ti plasmid has two vectors, and the Ti plasmid contains the vir gene. The vir gene has an effect of increasing transformation efficiency, but in the transformation of monocotyledonous plants, it is preferable to add acetosyringone to the medium in order to induce the vir gene.

  The biological defense-related genes of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61 introduced by the present invention may be at least one, that is, one or more, and are not particularly limited. However, the number is preferably 3 or more, more preferably 5 or more and all types. These genes can be introduced into the same vector continuously via a control sequence such as a promoter, or separately into different vectors.

  Examples of Agrobacterium include, but are not limited to, Agrobacterium tumefaciens, such as EHA105, EHA101, ASE1, Gv3101, C58, LBA4404, C58C1RifR strains, and the like.

  Examples of methods for producing pathogen-resistant rice transformants in which the level of expression of biological defense-related genes is increased by gene recombination are given below.

  First, the target biological defense related gene is inserted into an appropriate expression vector. The expression vector may be one usually used for producing a transformant of a plant, and the above-mentioned vector can be used, and a commercially available vector (Clontech, etc.) is preferable. In addition to the target biological defense-related gene, the vector includes the above-described control sequences that control the expression of the biological defense-related gene, the hygromycin resistance gene, the carbenicillin resistance gene, kanamycin to facilitate the selection of transformants. Resistance genes, ampicillin resistance genes, drug resistance genes such as phosphinothricin acetyltransferase, selectable markers such as neomycin phosphotransferase II (NPTII), dihydrofolate reductase, terminators derived from cauliflower mosaic virus or nopaline synthase genes, replication initiation Dots, ribosome binding sites, polyadenylation sites, and the like can be inserted.

  Insertion of the gene of interest into an intermediate vector or binary vector is performed at the cloning site present on the vector. In particular, in the case of a binary vector, the target gene is inserted between LB and RB of the T-DNA region.

  A series of gene manipulations can be performed by conventional gene recombination techniques. Such techniques include, for example, Sambrook et al., Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, Ausbel et al., Short Protocols in Molecular Biology, A Compendium of Methods from Current Protocols in Molecular Biology, Third Edition, WILEY, 1995, etc.

  Methods for introducing recombinant DNA containing the gene of interest into rice include the method using particle gun, the method using protoplasts (PEG method, electroporation method), freeze-thawing in addition to the method using Agrobacterium. A method using Agrobacterium, which is a routine method for producing rice transformants, will be described.

Remove the ripened rice seeds and sterilize with 70% ethanol, 20-30% sodium hypochlorite aqueous solution (effective chlorine 5%). After washing, the seeds are placed on a callus induction medium (N6D, N6Cl medium, etc.), for example, at 25-30 ° C. under conditions of 60 μmol / m ² s, 16 hours light period / 8 hours dark period (hereinafter referred to as light) Incubate for several days to 3 weeks. The seed endosperm and shoot part after induction may be cut out, and only the scutellum-derived callus may be transplanted to a new callus culture medium and cultured at 25 ° C. for 3 days. Separately, transformed Agrobacterium is applied to AB medium and cultured in the dark at 28 ° C. for 2-3 days. The transformed Agrobacterium grown in AB medium is suspended in Agrobacterium suspension medium (AAM, AA medium, etc.) containing acetosyringone. The pre-cultured callus is immersed in the suspension, and then excess water is removed from the callus. The callus is placed on a coculture medium (2N6AS, N6CO medium, etc.) and cultured at 28 ° C. in the dark for 3 days. Next, the callus is washed with sterilized purified water or a sterilized water washing solution containing carbenicillin to remove Agrobacterium. Excess water is removed from the callus, and placed on a selective medium (N6D, N6SE medium, etc.) containing, for example, hygromycin and carbenicillin, and cultured at 25 to 28 ° C. in the light for 2 to 3 weeks. After culturing, the cells are transplanted to a new selection medium, the selection is continued, and the callus is transplanted to a redifferentiation medium (RE-III, MSRE medium, etc.) and cultured at 25-28 ° C. until it is redifferentiated in the light. The redifferentiated solid is transplanted into HF medium to regenerate roots. Alternatively, it may be placed on an assay medium (MSHF) and assayed for drug resistance, for example. If it is resistant, it roots and shows the same growth as the wild type. In non-transformants, new roots do not grow and die in about a week. Once the plant has grown, it is acclimated and transplanted to a pot in a closed greenhouse (Reference: Cell Engineering, Separate Plant Cell Engineering Series 15, Experimental Protocol for New Model Plants (2001), Supervised by Shimamoto, Okada, Shujunsha, S Toki, N. Hara, K. Ono, H. Onodera, A. Tagiri, S. Oka, H. Tanaka, 2006. Plant Journal 47: 969-976).

  Whether a rice transformant has resistance to pathogenic bacteria can be easily confirmed by a test method described in, for example, Methods for Isolation, Cultivation, Inoculation of Plant Pathogens, Japan Plant Protection Association. For example, in the case of rice blast, the degree of lesion formation and the rate of lesion area when rice is inoculated with a race of rice blast fungus that is susceptible to the rice cultivar is compared between the original cultivar and the recombinant. However, the present invention is not limited to this. For example, in the rice blast resistance test, a punch inoculation method in which a rice blast spore paste is made on a rice leaf with a punch for inoculation and then the blast spore paste is placed on the rice leaf, and the blast spore paste is applied to the tip of the needle. A needle inoculation method that pierces leaves and a spray inoculation method in which a suspension of blast fungus spores is sprayed on rice. In these inoculation methods, lesions develop so as to spread from the wounds of the leaves. Therefore, the resistance strength is tested by measuring the extension length of the lesions.

  In the present invention, as another method for high expression of biological defense-related genes, high expression of the biological defense-related genes of the present invention is induced by treatment with trehalose or a derivative thereof. To that end, rice or seeds can be treated with an aqueous solution containing trehalose. The trehalose concentration is a concentration that induces the biological defense-related gene, and is not limited to the following range, but is preferably 0.5 to 50 mM, and more preferably 1 to 5 mM. Although processing time is not specifically limited, 1-48 hours are preferable and 2-6 hours are more preferable. The trehalose treatment can be performed any number of times regardless of the rice growth stage, but it is preferably performed before heading. The treatment method is not particularly limited, and for example, the entire seedling may be immersed in a trehalose aqueous solution or irrigated. In the case of seed (regardless of seed pod, brown rice, etc.), it is immersed in the aqueous solution for 6 hours to 2 days, for example.

  The method for increasing resistance to pathogenic fungi of rice of the present invention and the pathogenic fungi targeted by the pathogenic fungus-resistant rice transformants are not limited to the following. ), Blight blight (Rhizoctonia solani, AG-1), white leaf blight (Xanthomonas oryzae pv.oryzae), and blight blight (Burkholderia glumae). The method and the rice transformant of the present invention have excellent resistance particularly against blast fungus and seedling blight.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the scope of the present invention is not limited to these examples.

Microarray expression analysis of trehalose-inducible rice gene Rice (variety: Yukihikari) seeds were sterilized with 70% alcohol for 5 minutes and then with a 2-fold dilution of hypochlorous acid (Wako Pure Chemical Industries) for 30 minutes. It was allowed to stand at 25 ° C. in the dark for a day to absorb water and induce germination. Thereafter, germinated seeds were sown on a plastic mesh, a plastic container filled with water was placed under the mesh, and hydroponically cultivated for 2 weeks under continuous light at 25 ° C. Root tissues were collected from rice seedlings immediately before the start of treatment and designated as “C0”, and root tissues were collected from rice seedlings 2 hours after 5 mM trehalose treatment and designated as “T2”. Samples were snap frozen in liquid nitrogen simultaneously with collection and stored at -80 ° C until RNA extraction. The trehalose treatment was performed by directly contacting the root with a 5 mM trehalose solution that had been kept warm at 25 ° C. in advance. Samples C0 and T2 were powdered in liquid nitrogen and then stored at -80 ° C.

  Total RNA was prepared from frozen tissues using TRIzol reagent (Invitrogen). 10 ml of TRIzol was added to 1 g of the sample, and after stirring well, it was allowed to stand at room temperature for 5 minutes. Next, 2 ml of chloroform was added and stirred well for 30 seconds, allowed to stand at room temperature for 3 minutes, and centrifuged at 12000 × g for 10 minutes at room temperature. The aqueous phase was taken, and 5 ml of 2-propanol was added thereto and allowed to stand at room temperature for 10 minutes. Centrifugation at 12000 × g was performed for 10 minutes to precipitate RNA. This precipitate was washed with 5 ml of 70% ethanol, dissolved in 500 μl of sterilized water, and 500 μl of PCI solution (phenol: chloroform: isoamyl alcohol, 25: 24: 1) was added and stirred. Centrifugation at 15000 × g was performed at room temperature for 10 minutes, the aqueous phase was taken, 500 μl of PCI solution was added thereto and stirred, and centrifugation at 15000 × g was further performed for 10 minutes. Take the aqueous phase, add 50 μl of 3M sodium acetate (pH 5.2) and 1 ml of 100% ethanol, incubate at -80 ° C for 2 hours, and then centrifuge at 15000 xg at 4 ° C to precipitate RNA. . After the precipitate was dissolved in about 300 μl of sterilized water, the concentration was measured.

  After the purity was tested with Agilent Bioanalyzer 2100, single-stranded cDNA was synthesized using the Low RNA input liner amp kit as shown below. 1.2 μl of T7 promoter primer was added to 0.4 μg of RNA, and sterilized water was further added to make 5.75 μl. This was treated at 65 ° C. for 10 minutes, then placed in ice and rapidly cooled. To this was added 4.25 μl of cDNA synthesis mix (see below for composition) and incubated at 40 ° C. for 2 hours to synthesize cDNA. The reaction was stopped by treatment at 65 ° C. for 15 minutes, and the mixture was left on ice for 5 minutes. To this was added 1.2 μl of 10 mM Cyanine3 CTP or Cyanine5 CTP, and further 28.8 μl of a transcription mix (see below for composition) was added and incubated at 40 ° C. for 2 hours to synthesize labeled RNA. Thereafter, purification was performed using RNeasy mini kit. The purification procedure is as follows. 60 μl of sterilized water was added to the RNA solution after the incubation, 350 μl of RLT-buffer was further added, 250 μl of ethanol was added, and the mixture was transferred to an RNeasy mini column equipped with a collection tube. This was centrifuged at 11000 × g for 30 seconds, the filtrate was discarded, and 500 μl of fresh RPE-buffer was added to the column and centrifuged in the same manner. After repeating this operation, 25 μl of sterilized water was poured onto the column and centrifuged at 11000 × g for 30 seconds. This operation was repeated, and the filtrate obtained two times was collected in a new tube, and the concentration and dye uptake rate were measured.

  The hybridization solution was prepared as follows. Sterile water was added to 1 μg of fluorescently labeled RNA and 50 μl of 10 × control target, respectively, to a final volume of 250 μl, and further 10 μl 25 × fragmentation buffer was added and treated at 60 ° C. for 30 minutes. 250 μl of 2 × hybridization buffer was added thereto to obtain a hybridization solution. Using 1 μg each, a hybridization solution was prepared according to the instructions of the company's Hybridization kit Plus, and hybridization was performed at 60 ° C. for 17 hours using an Agilent 22K rice microarray. The microarray was washed with 2 x SSC (SSC; 150 mM NaCl, 15 mM Tri-sodium Citrate, pH 7.0), 0.05% Triton X-102 for 10 minutes at room temperature, then with 0.2 x SSC, 0.05% Triton X-102 Washed for 5 minutes at 4 ° C. Remove moisture remaining on the microarray with nitrogen gas, read the gene expression level information with an Agilent microarray scanner, and analyze the difference in the expression level of each gene in C0 and T2 using the company's analysis software Feature Extraction Software did. Table 1 shows the genes induced by trehalose treatment from the top using the difference in expression level as an index.

[Composition of cDNA synthesis mix]
5 x First strand buffer 2.0 μl
0.1 M DTT 1.0 μl
10 mM dNTP mix 0.5 μl
MMLV-RT 0.5 μl
RNaseOUT 0.25μl
Total 4.25μl

[Composition of transcription mix]
7.65μl water
4 x transfer buffer 10.0 μl
0.1 M DTT 3.0 μl
NTP mix 4.0 μl
50% PEG 3.2 μl
RNaseOUT 0.25μl
Inorganic pyrophosphatase 0.3 μl
T7 RNA polymerase 0.4 μl
28.8 μl total

Northern analysis of genes induced by trehalose Among the trehalose-inducible genes identified in the microarray experiment, several genes considered to be involved in disease response were selected as shown in Table 2. For the purpose of examining the expression of these genes in response to trehalose by Northern analysis, first, probe fragments of each gene were synthesized. The following nucleotides that specifically amplify the partial sequence of the gene were synthesized, and the partial length cDNA was amplified and cloned using the Ex TaqPCR kit (Takara Shuzo) in the same manner as described above, and the base sequence was determined. The synthetic nucleotides used for PCR conditions and Northern blot probe amplification are shown in SEQ ID NOs: 1-18.

[PCR reaction solution]
Takara Ex Taq (5u / μl) 0.1 μl
10 x Ex Taq buffer 2.5 μl
dNTP mix (2.5 mM each) 2.0 μl
Template cDNA 0.2 μl
20 μM Fw primer 0.5 μl
20 μM Rv primer 0.5 μl
Sterile water 19.2 μl
25.0 μl total

[PCR conditions]
Step 1 95 ° C 3 minutes Step 2 95 ° C 30 seconds Step 3 52 ° C 30 seconds Step 4 68 ° C 30 seconds Step 5 68 ° C 3 minutes Step 2 to Step 4 were repeated 30 times.

  5 mM trehalose was treated on the roots of rice seeds hydroponically cultivated as described above, and root tissues were collected from seedlings before treatment, 1, 2, 4, and 6 hours after treatment. From the collected tissue, total RNA was prepared according to the method described above, 10 μg was weighed and dried by a centrifugal evaporator. To the dried RNA, add 20 μl of electrophoresis buffer (MOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0) containing 42% formalin, 14% formaldehyde), dissolve completely, and add 5 μl at 95 ° C. Heat-denatured for minutes and quenched. The denatured RNA was electrophoresed in a 0.8% agarose gel and then transferred to a Hybond-N + nylon membrane (Amersham) overnight. The transferred membrane was air-dried for 2 hours and exposed to ultraviolet light for 3 minutes to immobilize RNA on the membrane.

A DNA fragment (100 ng) serving as a probe was radiolabeled using [α- ³² P] dCTP and BcaBEST DNA labeling kit (Takara Shuzo) according to the protocol. Unreacted dCTP was removed using Microspin S-300 HR (Amersham) according to the manufacturer's instructions. Prehybridization was carried out in Rapid hybri buffer (Amersham) kept at 65 ° C., and then hybridization was carried out by adding a radiolabeled probe to the hybridization solution and incubating at 65 ° C. overnight. The nylon membrane was washed twice by shaking for 15 minutes in 2 x SSC, 0.1% SDS solution kept at 65 ° C, and then shaking in 65 ° C for 15 minutes in 0.2xSSC, 0.1% SDS solution. It was done by doing. After washing, the film was exposed to a nylon membrane X-ray film for an appropriate time, exposed, and developed.

  As shown in FIG. 1, the induction of the expression of any gene was confirmed by trehalose treatment.

CDNA cloning of biological defense-related genes induced by trehalose Among the trehalose-inducible genes, the genes in Table 3 were identified as transcription factors and receptor kinase genes that are thought to be involved in trehalose signaling.
In order to isolate these genes, primers (SEQ ID NOs: 19 to 40) were designed with reference to rice genome sequence information registered in Genbank, and the full-length gene was amplified by RT-PCR. Single-stranded cDNA was synthesized from approximately 0.1 μg of total RNA prepared from rice (cultivar: Nipponbare) root tissue 2 hours after 5 mM trehalose treatment using Applied Biosystem's GeneAmp RNA PCR core kit according to the manufacturer's instructions. (See below for the composition of the single-stranded cDNA synthesis solution). Nucleotides capable of specifically amplifying transcription factors and receptor protein kinases that are important for the control of gene expression were synthesized. Prepare a reaction solution according to the manufacturer's instructions using a set of synthetic nucleotides specific to each gene and the KOD-Plus-DNA polymerase kit (Toyobo) using single-stranded cDNA as a template. The target gene fragment was amplified by Applied Biosystem Cirmal cycler 9700 under the above conditions. After the reaction solution containing the amplified gene fragment was treated at 95 ° C. for 20 minutes, 1.5 μl of 20 mM dATP was added and incubated at 60 ° C. for 20 minutes to add A to the gene fragment. This fragment was incorporated into a pGEM-Teasy cloning vector (Promega), and the nucleotide sequence was confirmed with BigDye sequencing kit ver 1.1 and sequencer ABI310 (Applied Biosystem). The base sequences of these genes are shown in SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 and 61. The reaction conditions used for amplification of each gene are as follows.

[Composition of single-stranded cDNA synthesis solution]
25 mM MgCl ₂ 2 μl
10 x PCR buffer II 1 μl
1 μl of water
dNTP mix 4 μl
RNase inhibitor 0.5 μl
MuLV reverse transcriptase 0.5 μl
Oligo d (T) 16 primer 0.5 μl
RNA solution 0.5 μl
Total 10.0 μl

[Single-stranded cDNA synthesis reaction]
Step 1 25 ° C 10 minutes Step 2 42 ° C 30 minutes Step 3 99 ° C 5 minutes Step 4 4 ° C 5 minutes

[PCR reaction solution]
2.5 μl of 10 x PCR buffer for KOD-Plus
2 mM dNTPs 2.5 μl
25 mM MgSO ₄ 1.0 μl
Primer mix (10 μM each) 0.8 μl
Template cDNA 0.5 μl
KOD-Plus-DNA polymerase 0.5 μl
Sterile water 17.2 μl
25.0 μl total

[PCR conditions]
Step 1 94 ° C 3 minutes Step 2 94 ° C 30 seconds Step 3 58 ° C 30 seconds Step 4 68 ° C 1 minute (2 minutes only for SEQ ID NO: 53)
Step 5 68 ° C. 3 minutes Step 2 to Step 4 were repeated 30 times.

Effect of trehalose treatment on resistance to rice blast fungus
[Cultivation]
The cultivar "Nihonbare" was used and cultivated in a greenhouse that was adjusted so that rice blast was fully developed. Pots used for cultivation were those with an inner diameter of 28 mm and a height of 115 mm, and 5 rices / pot of rice were grown.

[Drug processing]
Individuals in which the fourth leaf of rice, which is susceptible to disease, grew to about 1-3 cm, were irrigated with 1 mM and 5 mM trehalose. As a comparative example, rice treated with 0.5 mM of the agricultural chemical BIT (provenazole, chemical name: 1,2-benzisothiazole-1,1-dioxide) used for rice blast was prepared. Five days after the drug treatment, blast fungus was inoculated.

[Preparation of spore solution of Magnaporthe grisea]
Growing so that the blast fungus grows on an oatmeal medium plate (28 ° C), removes the aerial mycelium, and continues to grow under BLB lamp irradiation to form spores. A spore solution prepared by suspending the grown spores in sterile water was used for the infection test.

[Infection experiment]
Rice was allowed to stand in an inoculation box (60 × 60 × 60 cm) and spray-inoculated with a blast fungus spore solution. Rice was left in the inoculation box under dark conditions at 100% humidity for 24 hours, then removed from the inoculation box and continued to grow in the greenhouse. 5 days after inoculation, the number of lesions appearing on the 4th leaf was measured to evaluate the degree of disease onset, and the rate of disease control against the control group ((number of lesions in control group-disease in trehalose-treated group) The number of spots) ÷ the number of lesions in the control group × 100) was determined as the control value (%).

[result]
As shown in FIG. 2, treatment with 1 mM and 5 mM trehalose induced a level of resistance similar to BIT.

Effect of trehalose treatment on resistance to rice seedling blight Bacterium rice seeds were sterilized with 70% ethanol for 3 minutes and then washed thoroughly with sterilized water. The sterilized seeds were transferred to a 90 mm sterilized petri dish, 20 ml of a sterilized 5 mM trehalose solution was added, and the mixture was allowed to stand at 25 ° C. for 24 hours in the dark. As a control, seeds soaked in sterilized water instead of the trehalose solution after sterilization were prepared. After 24 hours, the trehalose solution was discarded and the seeds were thoroughly rinsed with sterile water. PY (Bacto Yeast extract 2g, Bacto Pepton 5g / L) were grown overnight at 30 ° C. in a medium, 10 ⁶ CFU / ml to adjust the rice seedling Burkholderia bacteria fungi (Burkholderia plantarii) added culture solution 20ml petri dish, further It was allowed to stand for 48 hours at 25 ° C in the dark. Vermiculite was laid on a 90 mm sterilized petri dish, and sterilized water was added to the entire surface, and 20 seeds of rice were planted there. In an artificial weather chamber set for 16 hours (25 ° C) light period and 8 hours (25 ° C) dark period After germination, the growth of seedlings was observed after about one week.

[result]
As shown in FIG. 3, the death rate of 5 mM trehalose-treated rice was 25.3% ± 3.3% and the control was 60.1 ± 3%, and clear resistance was induced by trehalose.

Production of rice transformants of pathogenic bacteria Among the genes with high inducibility by trehalose shown in Table 3, transformation of genes with gene numbers AK112056 (SEQ ID NO: 59) and AK108522 (SEQ ID NO: 49) with actually increased expression The body was made by the following method.

[Cloning]
For the purpose of producing a transformant plant, a primer set for newly introducing a restriction enzyme Xba I and Kpn I site (primer set 1 for AK112056 1: SEQ ID NOs: 63 and 64, primer set 2 for AK108522: SEQ ID NOs: 65 and 66) ) Was produced. A single-stranded cDNA synthesized using the same method as in Example 3 was used as a template, and a PCR reaction was performed using the KOD-Plus-DNA polymerase kit (Toyobo) according to the manufacturer's instructions, and AK112056 and AK108522 The gene fragment was amplified. The amplified cDNA fragment was incorporated into a pGEM-Teasy vector and the nucleotide sequence was confirmed.

  A cDNA fragment corresponding to AK112056 (base sequence: SEQ ID NO: 67, amino acid sequence: SEQ ID NO: 68) and a cDNA fragment corresponding to AK108522 (base sequence: SEQ ID NO: 69, amino acid sequence: SEQ ID NO: 70) are restricted to the restriction enzymes Xba I and Kpn. It was excised using I and inserted into the site of the binary vector pActZH2. The binary vector pActZH2 is located between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator in the T-DNA portion of pPZP202 (P. Hajdukiewicz, Z. Svab, P. Maliga, 1994.Plant Molecular Biology 25: 989-994). A cassette containing a hygromycin resistance gene and a gene expression cassette with multiple cloning sites between the rice Act1 gene promoter (Sentoku et al. Developmental Biology Volume 220, 2000, Pages 358-364) and the nopaline synthase terminator Is. This vector was introduced into Agrobacterium EHA101 by the freeze-thaw method.

[Rice transformation]
Transformant plants are produced by the method of Toki et al. (S. Toki, N. Hara, K. Ono, H. Onodera, A. Tagiri, S. Oka, H. Tanaka, 2006. Plant Journal 47: 969-976. ). The specific operation method is shown below.

  Rice (variety: Yukihikari) seeds were removed and sterilized with 70% ethanol for 2 minutes, and then thoroughly washed with sterilized purified water. Next, the mixture was sterilized with 2-fold diluted hypochlorous acid (chlorine concentration: 2.5%) for 30 minutes and thoroughly washed with sterilized purified water. Sterilized seeds were left on N6D medium, and callus was induced for 5 days under light conditions at 30 ° C. The callus was allowed to coexist for 2 minutes in a 30 ml AAM liquid medium (containing 10 mg / l acetosyringone) together with transformed Agrobacterium grown on AB medium for 2 days. Excess water was removed with a sterilized paper towel, and the callus was allowed to stand in a 2N6AS solid medium for 3 days and co-cultured in the dark at 28 ° C. Thereafter, the callus was thoroughly washed with sterilized purified water, left standing in N6D medium containing 50 mg / l hygromycin and 250 mg / l carbenicillin, and transformants were selected at 28 ° C. in the light. After culturing for 2 weeks, the callus was transplanted to a new same medium, and selection was continued. After further 2 weeks of culturing, the callus in which proliferation was observed was placed on RE-III medium and redifferentiated. Plants in which above-ground redifferentiation was observed were transplanted to HF medium to regenerate roots. The fully regenerated plant body was transplanted to soil. RNA was extracted from the leaf tissue of the present plant, and high expression of the transgene was confirmed by RT-PCR using the primer set 1 or 2 described above. Next generation seeds were obtained and used for resistance testing.

The composition of the medium is shown below.
[N6D solid medium (per liter)]
KNO ₃ 2,830 mg, (NH ₄ ) ₂ SO ₄ 463 mg, MgSO ₄・ 7H ₂ O 185 mg, CaCl ₂・ 2H ₂ O 166 mg, KH ₂ PO ₄ 400 mg, Na ₂ EDTA 37.3 mg, FeSO ₄・ 7H ₂ O 27.8 mg , MnSO ₄・ 4H ₂ O 4.4mg, ZnSO ₄・ 7H ₂ O 1.5mg, KI 0.8mg, H ₃ BO ₃ 1.6mg, Casamino acid 300mg, Glycine 2mg, L-Proline 2,878mg, myo-Inositol 100mg, Nicotinic acid 0.5mg, Pyridoxine-HCl 0.5mg, Thiamine-HCl 1mg, 2,4-D 2mg, Sucrose 30g, Gellan Gum 3g (pH 5.8)

[2N6AS solid medium (per liter)]
KNO ₃ 2,830 mg, (NH ₄ ) ₂ SO ₄ 463 mg, MgSO ₄・ 7H ₂ O 185 mg, CaCl ₂・ 2H ₂ O 166 mg, KH ₂ PO ₄ 400 mg, Na ₂ EDTA 37.3 mg, FeSO ₄・ 7H ₂ O 27.8 mg , MnSO ₄・ 4H ₂ O 4.4mg, ZnSO ₄・ 7H ₂ O 1.5mg, KI 0.8mg, H ₃ BO ₃ 1.6mg, Casamino acid 300mg, Glycine 2mg, myo-Inositol 100mg, Nicotinic acid 0.5mg, Pyridoxine-HCl 0.5mg, Thiamine-HCl 1mg, 2,4-D 2mg, Sucrose 30g, Glucose 10g, Gellan Gum 3g, Acetosyringone 10mg (pH 5.2)

[AAM liquid medium (per liter)]
MgSO ₄・ 7H ₂ O 250mg, CaCl ₂・ 2H ₂ O 150mg, NaH ₂ PO ₄・ 2H ₂ O 150mg, KCl 3g, Fe-EDTA 40mg, MnSO ₄・ 4H ₂ O 10mg, ZnSO ₄・ 7H ₂ O 2mg, CuSO ₄・ 5H ₂ O 0.025mg, CoCl ₂・ 6H ₂ O 0.025mg, KI 0.75mg, H ₃ BO ₃ 3mg, Na ₂ MoO ₄・ 2H ₂ O 0.25mg, Casamino acid 500mg, Glycine 7.5mg, L-Arginine 176.7mg, L-Glutamine 900mg, L-Aspartic acid 300mg, myo-Inositol 100mg, Nicotinic acid 1mg, Pyridoxine-HCl 1mg, Thiamine-HCl 10mg, Sucrose 68.5g, Glucose 36g, Acetosyringone 10mg (pH 5.2)

[RE-III solid medium (per liter)]
_{KNO 3 1,900mg, NH 4 NO 3} 1,650mg, MgSO 4 · 7H 2 O 370mg, CaCl 2 · 2H 2 O 440mg, KH 2 PO 4 170mg, Na 2 EDTA 37.3mg, FeSO 4 · 7H 2 O 27.8mg, MnSO ₄・ 4H ₂ O 22.3mg, ZnSO ₄・ 7H ₂ O 8.6mg, CuSO ₄・ 5H ₂ O 0.025mg, CoCl ₂・ 6H ₂ O 0.025mg, KI 0.83mg, H ₃ BO ₃ 6.2mg, Na ₂ MoO ₄・ 2H ₂ O 0.25mg, Casamino acid 2g, Glycine 2mg, myo-Inositol 100mg, Nicotinic acid 0.5mg, Pyridoxine-HCl 0.5mg, Thiamine-HCl 0.1mg, NAA 0.02mg, Kinetin 2mg, Sucrose 30g, Sorbitol 30g, Gellan Gum 3g (pH 5.8)

[HF solid medium (per liter)]
_{KNO 3 1,900mg, NH 4 NO 3} 1,650mg, MgSO 4 · 7H 2 O 370mg, CaCl 2 · 2H 2 O 440mg, KH 2 PO 4 170mg, Na 2 EDTA 37.3mg, FeSO 4 · 7H 2 O 27.8mg, MnSO ₄・ 4H ₂ O 22.3mg, ZnSO ₄・ 7H ₂ O 8.6mg, CuSO ₄・ 5H ₂ O 0.025mg, CoCl ₂・ 6H ₂ O 0.025mg, KI 0.83mg, H ₃ BO ₃ 6.2mg, Na ₂ MoO ₄・ 2H ₂ O 0.25mg, Glycine 2mg, myo-Inositol 100mg, Nicotinic acid 0.5mg, Pyridoxine-HCl 0.5mg, Thiamine-HCl 0.1mg, Sucrose 30g, Gellan Gum 3g (pH 5.8)

[AB medium (per liter)]
NH ₄ Cl 1g, MgSO ₄・ 7H ₂ O 296mg, CaCl ₂・ 2H ₂ O 10mg, NaH ₂ PO ₄・ 2H ₂ O 1.3g, K ₂ HPO ₄ 3g, KCl 150mg, FeSO ₄・ 7H ₂ O 2.5mg, Glucose 5g, Agar 15g (pH7.2)

[Resistance test]
The rice transformants produced by the above method were tested for resistance to rice blast by the punch inoculation method.

Seedlings resistant to hygromycin (50 mg / l) were grown to the 4-leaf stage. A filter paper dampened with distilled water was laid in a plastic case with a lid, and the fourth leaves of rice cut out were arranged on the filter paper. Blast spores to be formed on the oatmeal medium were scraped with sterile flat water (containing + 0.2% Tween 20) using a flat brush to prepare 10 ⁵ spore / ml. The tip of a 10 μl pipette tip was pressed against the leaf and lightly damaged, and then 3 μl of a spore suspension was dropped. Cover the plastic and keep the humidity in the plastic at 100% in a 25 ° C greenhouse for about 1 week, measure the length of the blast of the blast that is formed, and develop the blast The degree was calculated (the length of lesions of the transgene high expression strain ÷ the length of lesions of the wild strain × 100). The results are shown in Table 4. Further, FIG. 4 shows the lesions of the wild strain and the transgene highly expressing strain (6 days after the punch inoculation method treatment).

  It was clear that the resistance of rice transformants to blast was higher than that of the wild type. From this, it was shown that the gene induced by trehalose has a function of enhancing resistance to pathogenic bacteria by increasing its expression level.

  The method for increasing resistance to pathogenic bacteria and the pathogen-resistant rice transformant of the present invention are useful for cultivation and harvesting even in an environment susceptible to pathogenic fungi such as cold damage and long rain.

The induction | guidance | derivation of the biological defense related gene by a trehalose process is shown. The effect of trehalose on rice blast fungus is shown. The effect of trehalose on rice seedling bacteriomycetes is shown. The resistance of rice blast disease by punch inoculation with wild rice strains and transformants is shown.

Claims (8)

  In rice, expression of at least one gene of a biological defense-related gene having any one of the nucleotide sequences of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 or 61 or a mutant gene thereof A method of increasing resistance to pathogens in rice, including increasing levels.   The method according to claim 1, wherein the expression level of the gene is increased by treating rice or seed thereof with trehalose or a derivative thereof.   The method according to claim 1 or 2, wherein the expression level of the gene is increased by introducing the gene into rice exogenously and in an expressible manner.   The method according to any one of claims 1 to 3, wherein the expression level of the gene is increased by 2 times or more with respect to control rice.   The method according to any one of claims 1 to 4, wherein the pathogenic fungus is a blast fungus or a seedling bacterial bacterium.   And at least one gene of a biological defense-related gene having any one of the nucleotide sequences of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61 or a mutant gene thereof is exogenous and A pathogen-resistant rice transformant, which has been introduced so that it can be expressed.   The transformant according to claim 6, wherein the pathogenic bacterium is a blast fungus or a seedling bacterial bacterium.   At least one kind of biodefense-related gene having any one of the nucleotide sequences of SEQ ID NOs: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61 for increasing resistance to rice pathogens How to use the gene.

Images